Medieval Equine Medicine from Armenia

*Jasmine Dum-Tragut*

#### **Abstract**

The Armenian medieval and early modern equine medicine has rarely been noticed or researched by veterinarians, historians of science, philologists, or medieval researchers. As Armenia represents both a geographical border and cultural corridor between Muslim East and Christian West, a consideration of its hippiatric texts and their integration into the general history of veterinary medicine can only lead to a deeper understanding of equine medicine from the medieval to the early modern period. They could also contribute toward tracing the paths of knowledge diffusion and transmission across political, linguistic, and religious-cultural boundaries in the time of the Crusades. The role of Armenian manuscripts bridging the traditions of equine medicine from the Muslim East and the Christian West is examined by revealing the complicated history of Armenian horse treatises that traveled the long way from Baghdad via Sis to Tbilisi.

**Keywords:** medieval horse medicine, Cilician Kingdom of Armenia, knowledge transfer, cultural encounter between East and West, Crusades

#### **1. Introduction**

In recent years, much research has been dedicated to the cross-cultural and cross-religious aspects of the encounters of peoples from the Christian West and the predominantly Muslim East during the Crusades. The role of the small Oriental-Christian kingdoms and principalities both geographically and culturally located between Europe and Asia was, however, very rarely taken into account.

Equine medicine occupies a prominent role: hippiatric treatises were widely circulated, translated, and adapted. According to tradition, Greco-Roman and Byzantine works lost in Europe in the Middle Ages were preserved in Arabic libraries in translations from the original Greek to Arabic. These texts returned to Europe in the Renaissance, with a greater or lesser influence of Muslim/Eastern knowledge on European. Detailed analyses show that even if European writings provided the basis for Arabic hippiatric books, the Arabs also referred to the knowledge of Indian medicine and their own local practices. Yet none of these studies question how and where European and Eastern traditions met, and whether there were any cultural intermediaries.

Only gradually, researchers began to understand the historical significance of a corridor country and significant negotiator with the peoples of the Far East: the Armenian principality and later Kingdom of Cilicia between the eleventh and fourteenth centuries. Despite religious and linguistic differences, mutual influences were revealed, which help to identify socio-cultural parallels, particularly concerning royal courts.

In addition to the royal treasury, manuscripts, noble horses, and court physicians were among the most respected possessions in many royal and noble courts.

#### **2. Medieval Armenian equine medicine**

Medical works first appear in Armenian literature with the early translations from Greek into Armenian of the works of Galen (199 AD) by the Armenian philosopher David Anhałt (fifth century). During the age of Hunayn (prior to the ninth century), a translation followed from a Syriac version and the most comprehensive version of Galen's text was made from Arabic to Armenian in the period covered by the ninth to tenth centuries.

Genuinely Armenian medical literature only emerged in the medieval period, in the Armenian principality and the later Kingdom of Cilicia (1081–1375). Veterinary medicine developed later, initially under the influence of the secondary Armenian translation of the Byzantine compilation on agriculture, known as *Geoponica*. In this translation, we find the first chapters on horse medicine ever written in the Armenian language, in book 16, chapters 284–310.

From the eleventh century onward, Armenians experienced a striking acceleration of medical activities thanks to the patronage of the Cilician rulers and later royal dynasties. The number of surviving or known Armenian horse treatises is modest [1].


**5**

*Medieval Equine Medicine from Armenia DOI: http://dx.doi.org/10.5772/intechopen.91379*

(professional) medical book on horses.

named Saračyan from Los Angeles.

and the neighboring traditions.

*3.1.1 The time of the manuscript*

**3.1 The production**

and mount" [2–7].

**3. The Cilician medical book for horses**

Medieval manuscripts—remarkable books painstakingly written and decorated by hand—are regarded some of the most precious objects produced in medieval times. Another precious object was the horse in the medieval aristocratic society. The horse cannot be dissociated from knighthood. The horses of the high-ranking knights and kings have been considered representatives of royal power, physical strength, beauty, and elegance. They were precious and expensive creatures; so, their training, care, and health were of paramount importance for their owners. Kings very often employed their own horse specialists, farriers, horse doctors or mounted militarists, often named by the rank of marshal or constable (**Table 1**). And thus, it is not really surprising that kings and nobles combined the esteemed values of a medieval aristocratic society, namely manuscripts, royal steeds, and horse experts serving the king into one single prestigious object: a

The Armenian institute for ancient manuscripts, Matenadaran, in Yerevan holds a unique, illustrated manuscript, codex M 10975, called "Medical book for horse

At the beginning of the twentieth century, this manuscript was still found on the "List of Armenian Manuscripts of Tabriz" as MS 74, owned by tailor Širmazani [8]. It was donated to the Yerevan collection only in April 1987, by a private owner

The manuscript is a hippological and hippiatric book on 184 folios, containing 182 chapters and some illuminations. It was written in 1296–98 on behalf of the Cilician King Smbat, as can be read in the manuscript's main colophon (f.184a):

*Well, this medical book for horses and mounts was written to recognize the good and the bad [in a horse], on behalf of the Christ-loving 184a // and wise, thoughtful, witty, God-fearing King of the Armenians, Smbat.*

*And thus, I, the humble physician Farayč, on behalf of my lord, the holy king, took this [task] on me with great difficulty and translated this into a correct and clear language, for I was very well versed and have been trained in the art of healing in the big (city) Baghdad for many years. And I am a Syrian by origin and by faith, and by piety completely orthodox. And I worked on the translation of this medical book in the capital Sis [9].*

Before we unfold the general history of this manuscript, its production, reception, and provenance, the socio-historical contexts of its time of production as well as the persons involved must be investigated. The efficacy and importance of this horse book will be tracked in the subsequent horse treatises both in the Armenian

Toward the end of the thirteenth century, after the Armenian Kingdom of Cilicia had already fought on the side of the European Crusaders, the small Christian Kingdom was still under the protection of the Mongolian Ilkhanids. The Armenians fought on the side of the Mongols and the European Crusaders against

**Table 1.** *Overview of known Armenian manuscripts on horse medicine.* *Medieval Equine Medicine from Armenia DOI: http://dx.doi.org/10.5772/intechopen.91379*

*Equine Science*

**2. Medieval Armenian equine medicine**

covered by the ninth to tenth centuries.

About the treatment of horses

book for horse and mount

from the Medical book for mount

book for horses

book for horse, mule and donkey

diseases and ulcers of the horse

*Overview of known Armenian manuscripts on horse medicine.*

M10975 Medical

V2385 Copied

M11161 Medical

M459 Medical

M550 On the

Armenian language, in book 16, chapters 284–310.

185 1296– 96

21 1696 Hogac'

In addition to the royal treasury, manuscripts, noble horses, and court physicians were among the most respected possessions in many royal and noble courts.

Medical works first appear in Armenian literature with the early translations from Greek into Armenian of the works of Galen (199 AD) by the Armenian philosopher David Anhałt (fifth century). During the age of Hunayn (prior to the ninth century), a translation followed from a Syriac version and the most comprehensive version of Galen's text was made from Arabic to Armenian in the period

Genuinely Armenian medical literature only emerged in the medieval period, in the Armenian principality and the later Kingdom of Cilicia (1081–1375). Veterinary medicine developed later, initially under the influence of the secondary Armenian translation of the Byzantine compilation on agriculture, known as *Geoponica*. In this translation, we find the first chapters on horse medicine ever written in the

From the eleventh century onward, Armenians experienced a striking acceleration of medical activities thanks to the patronage of the Cilician rulers and later royal dynasties. The number of surviving or known Armenian horse treatises is modest [1].

**MS Title Folios Date Place Scribe State of Art Comment**

? 1263? Baghdad Step'anos Not

Sis T'oros & Farač the Syrian

6 — — — Edited 1867.

287 1504 Sivas — Unedited.

2 1710? — — Unedited.

Łazar Amt'ec'i

Vank'

preserved. Only mentioned in colophon of F278, BNF

Edited 1984. Translated and analyzed

Translated and analyzed

Superficially analyzed

Unedited; interlinear translation, tentative analysis

Superficially analyzed

Commissioned by King Het'um I

Commissioned by King Smbat

> Copy of M10975

> Copy of M10975

Obviously not related to M10975

> Unclear relation to M109

**4**

**Table 1.**

Medieval manuscripts—remarkable books painstakingly written and decorated by hand—are regarded some of the most precious objects produced in medieval times. Another precious object was the horse in the medieval aristocratic society. The horse cannot be dissociated from knighthood. The horses of the high-ranking knights and kings have been considered representatives of royal power, physical strength, beauty, and elegance. They were precious and expensive creatures; so, their training, care, and health were of paramount importance for their owners. Kings very often employed their own horse specialists, farriers, horse doctors or mounted militarists, often named by the rank of marshal or constable (**Table 1**).

And thus, it is not really surprising that kings and nobles combined the esteemed values of a medieval aristocratic society, namely manuscripts, royal steeds, and horse experts serving the king into one single prestigious object: a (professional) medical book on horses.

#### **3. The Cilician medical book for horses**

The Armenian institute for ancient manuscripts, Matenadaran, in Yerevan holds a unique, illustrated manuscript, codex M 10975, called "Medical book for horse and mount" [2–7].

At the beginning of the twentieth century, this manuscript was still found on the "List of Armenian Manuscripts of Tabriz" as MS 74, owned by tailor Širmazani [8]. It was donated to the Yerevan collection only in April 1987, by a private owner named Saračyan from Los Angeles.

The manuscript is a hippological and hippiatric book on 184 folios, containing 182 chapters and some illuminations. It was written in 1296–98 on behalf of the Cilician King Smbat, as can be read in the manuscript's main colophon (f.184a):

*Well, this medical book for horses and mounts was written to recognize the good and the bad [in a horse], on behalf of the Christ-loving 184a // and wise, thoughtful, witty, God-fearing King of the Armenians, Smbat.*

*And thus, I, the humble physician Farayč, on behalf of my lord, the holy king, took this [task] on me with great difficulty and translated this into a correct and clear language, for I was very well versed and have been trained in the art of healing in the big (city) Baghdad for many years. And I am a Syrian by origin and by faith, and by piety completely orthodox. And I worked on the translation of this medical book in the capital Sis [9].*

Before we unfold the general history of this manuscript, its production, reception, and provenance, the socio-historical contexts of its time of production as well as the persons involved must be investigated. The efficacy and importance of this horse book will be tracked in the subsequent horse treatises both in the Armenian and the neighboring traditions.

#### **3.1 The production**

#### *3.1.1 The time of the manuscript*

Toward the end of the thirteenth century, after the Armenian Kingdom of Cilicia had already fought on the side of the European Crusaders, the small Christian Kingdom was still under the protection of the Mongolian Ilkhanids. The Armenians fought on the side of the Mongols and the European Crusaders against

the Islamic Middle East. The growing conflict with the Mamluks can be seen in this context of the Armenians' involvement with the Mongols [10]. This alley brought the Armenians into first direct contact with the forces of the Mamluk Sultanate: the Mongol conquest of the Middle East in 1259 was heavily supported by the Armenian King Het'um I. The Mamluks, steadily replacing the Ayyubid masters in the Middle East, by 1250 already controlled Egypt after having it defended from the crusaders. They had succeeded in re-uniting the Muslim Middle East.

The conflict between Mamluks and Armenians broke out with the request of the Mameluk Sultan Baybars to the Armenian ruler Het'um I to break the alliance with the Mongols and give those territories and fortresses to the Mamluks that had been granted to the Cilician king through the alliance with the Mamluks [11]. From the first invasion of the Mamluks in Cilicia in 1266, decades of constant threat to the Christian Armenians began. In 1285, the Armenians had to sign a 10-year armistice under harsh conditions, but the Mamluks did not actually keep it. Already in 1292 another Mamluk invasion forced King Het'um II to abandon many towns. He abdicated in favor of his brother T'oros III and entered the monastery Mamistra. After a short interregnum of his younger brother Smbat, King Het'um II returned to the throne in the summer of 1299. Faced with a new attack by the Mameluks, he asked the Mongol Khan of Persia, Ghâzân, for support. In response, Ghâzân marched toward Syria with the support of Franks of Cyprus. The allied Armenians and Mongols defeated the Mamluks in the battle of Wadi al-Khazandar, 1299. In 1303, the Mongols tried to conquer Syria once again in larger numbers (approximately 80,000) along with the Armenians, but they were defeated at Homs on March 30, 1303. At this time, the Mongol leaders had already turned to Islam, and this put also the Armenian-Mongol alleys to the end [12].

The Armenian royal family of the Het'umids continued ruling the unstable Cilicia until the midst of the fourteenth century, but it could not resist attacks from the Mamluks any longer. The Armenian capital Sis fell to the Mamluks in 1375, and the final king, Levon V, died in exile in Paris in 1393 [13, 14].

#### *3.1.2 King Smbat, the commissioner of the horse treatise*

Smbat was the king of the Armenian Kingdom of Cilicia from 1296 to 1298. He was born in 1277 as one of the 16 children of king Levon II of Armenia and his wife Ker̥an of Lambron and was a representant of the Hetumid noble family.

Upon the death of King Levon II on February 6, 1289 AD, his surviving 11 children fought for control of the kingdom, and three of his sons managed to obtain the throne, mostly for relatively brief periods at a time. First to emerge as king was his son Het'um II [15]. Smbat seized the throne with the aid of his younger brother Kostandin while his brothers King Het'um II and prince T'oros visited Constantinople. In 1297, on a journey to the court of the Mongol ruler of Persia, Ghazan, Smbat received recognition of his position as king from Ghazan, which was necessary to legitimate his usurpation. He also received a bride from the Mongol Khan in order to form a matrimonial alliance. During his return to Cilicia, he came across his two brothers in the region of Caesarea and imprisoned them in the fortress of Barjraberd. In early 1298, Smbat even ordered T'oros to be strangled and Het'um to be blinded with a hot iron. This cruel action resulted in the rebellion of his former ally, Kostantin. Smbat was imprisoned, and Het'um was freed [16]. Smbat plotted again to resume the throne of his brother Het'um, meanwhile a Francisan monk, but he was imprisoned for the rest of his life.

Thus, Smbat reigned only for a period of 2 years; he, however, left essential objects to posterity that make him unforgettable: King Smbat had his own smaller bronze

**7**

*Medieval Equine Medicine from Armenia DOI: http://dx.doi.org/10.5772/intechopen.91379*

adjusted and updated version.

*3.2.1 The horse treatises of King Het'um I*

these had been brought to Cilicia in the 1260s.

coins, called *p'oł,* minted, showing him on horseback and he commissioned a medical book for horses. And this makes him unforgettable in Armenian cultural history.

The reception of any manuscript can be measured by its output in the form of later copies and translations. In the later Armenian tradition, we do have at least two

In 1867, a Mekhitarist father published the text of a horse book fragment with the title "I grastu bžškaranēn p'oxac" (copied/taken form the medical book for mount) in the armenological journal Bazmavep [17], which we could identify with manuscript Ms 2385 of the Mekhitharist Library in Venice [18]. This text consists only of three folios and was attached to a (human) medical book that was commissioned by King Het'um II and copied by a Vardapet Mkrtič' in 1294–1295, before Smbat seized his brother's throne. It is still unclear whether these three folios were written by the same scribe and in the same time or whether they were copied later and just bound into this book. Thus, at the current state of research it is uncertain whether these three folios are a later copy of Smbat's or of Het'ums horse book; the title, however, and obvious textual parallel speak for Smbat's horse book [19]. In 2008, the Institute for Ancient Armenian Manuscripts was given a voluminous, damaged codex from the private property of the Nazumlean family of Isfahan. It was catalogued as MS 11161 and contains various medical treatises and a treatise on the care of horses. It has not been explored yet, but in our first analysis after the manuscript's restoration in 2014 we discovered that the compilation's third stratum, a medical book for horses, consisting of folios 210a–261b, was copied in 1504 in the town of Sebaste, present-day Sivas (Turkey). It represents definitely a reproduction of Smbat's Cilician horse book, not an accurate copy, rather an

If we dig deeper into Armenian medieval equine medicine, we see that the other surviving texts and fragments in Armenian language suggest that there was even an

In the main colophon of an Armenian chemistry and pharmacology treatise (*Girk' arvesti k'imiakan*), codex 248, kept in the Bibliotheque Nationale de Paris, one

"King Het'um, who marched again the enemy sultan with an army of [corrupt writing] horsemen, massacred and destroyed everything, and he went with big honour to Baghdad […] and there a certain wise man, a deacon named Step'anos, with the king. This man had learnt plenty of languages and writings just like the former philosophers, and there was no writing, this man could not find. He was much loved by the Armenian king because of his knowledge and thus was asked by the King. And he translated three writings about the farriery (i.e. medical treatment) of horses and about how to make a sword…and he took them to the Armenian lands" [21]. These few lines lead us to believe that King Het'um I, one of the most colorful figures in the Armenian Kingdom of Cilicia and grandfather of Smbat, had commissioned one or even more horse treatises based on an Arabic source, and that

Unfortunately, there is no trace of these Hetumian horse books; we have to rely only on the information from this colophon. But this leaves another question: are Hetum's horse book and Smbat's horse book related? Since there is no preserved copy of Het'ums horse books, a meticulous text comparison cannot answer this

older Armenian horse treatise, written some decades before Smbat's text.

gets the following information about another horse treatise on Ff. 34v [20]:

**3.2 Reception of the Cilician medical book(s) for horses**

preserved Armenian copies of Smbat's horse book.

*Equine Science*

the Islamic Middle East. The growing conflict with the Mamluks can be seen in this context of the Armenians' involvement with the Mongols [10]. This alley brought the Armenians into first direct contact with the forces of the Mamluk Sultanate: the Mongol conquest of the Middle East in 1259 was heavily supported by the Armenian King Het'um I. The Mamluks, steadily replacing the Ayyubid masters in the Middle East, by 1250 already controlled Egypt after having it defended from the crusaders.

The conflict between Mamluks and Armenians broke out with the request of the Mameluk Sultan Baybars to the Armenian ruler Het'um I to break the alliance with the Mongols and give those territories and fortresses to the Mamluks that had been granted to the Cilician king through the alliance with the Mamluks [11]. From the first invasion of the Mamluks in Cilicia in 1266, decades of constant threat to the Christian Armenians began. In 1285, the Armenians had to sign a 10-year armistice under harsh conditions, but the Mamluks did not actually keep it. Already in 1292 another Mamluk invasion forced King Het'um II to abandon many towns. He abdicated in favor of his brother T'oros III and entered the monastery Mamistra. After a short interregnum of his younger brother Smbat, King Het'um II returned to the throne in the summer of 1299. Faced with a new attack by the Mameluks, he asked the Mongol Khan of Persia, Ghâzân, for support. In response, Ghâzân marched toward Syria with the support of Franks of Cyprus. The allied Armenians and Mongols defeated the Mamluks in the battle of Wadi al-Khazandar, 1299. In 1303, the Mongols tried to conquer Syria once again in larger numbers (approximately 80,000) along with the Armenians, but they were defeated at Homs on March 30, 1303. At this time, the Mongol leaders had already turned to Islam, and this put also

The Armenian royal family of the Het'umids continued ruling the unstable Cilicia until the midst of the fourteenth century, but it could not resist attacks from the Mamluks any longer. The Armenian capital Sis fell to the Mamluks in 1375, and

Smbat was the king of the Armenian Kingdom of Cilicia from 1296 to 1298. He was born in 1277 as one of the 16 children of king Levon II of Armenia and his wife

Upon the death of King Levon II on February 6, 1289 AD, his surviving 11 children fought for control of the kingdom, and three of his sons managed to obtain the throne, mostly for relatively brief periods at a time. First to emerge as king was his son Het'um II [15]. Smbat seized the throne with the aid of his younger brother Kostandin while his brothers King Het'um II and prince T'oros visited Constantinople. In 1297, on a journey to the court of the Mongol ruler of Persia, Ghazan, Smbat received recognition of his position as king from Ghazan, which was necessary to legitimate his usurpation. He also received a bride from the Mongol Khan in order to form a matrimonial alliance. During his return to Cilicia, he came across his two brothers in the region of Caesarea and imprisoned them in the fortress of Barjraberd. In early 1298, Smbat even ordered T'oros to be strangled and Het'um to be blinded with a hot iron. This cruel action resulted in the rebellion of his former ally, Kostantin. Smbat was imprisoned, and Het'um was freed [16]. Smbat plotted again to resume the throne of his brother Het'um, meanwhile a Francisan monk, but he was imprisoned for the

Thus, Smbat reigned only for a period of 2 years; he, however, left essential objects

to posterity that make him unforgettable: King Smbat had his own smaller bronze

Ker̥an of Lambron and was a representant of the Hetumid noble family.

They had succeeded in re-uniting the Muslim Middle East.

the Armenian-Mongol alleys to the end [12].

the final king, Levon V, died in exile in Paris in 1393 [13, 14].

*3.1.2 King Smbat, the commissioner of the horse treatise*

**6**

rest of his life.

coins, called *p'oł,* minted, showing him on horseback and he commissioned a medical book for horses. And this makes him unforgettable in Armenian cultural history.

#### **3.2 Reception of the Cilician medical book(s) for horses**

The reception of any manuscript can be measured by its output in the form of later copies and translations. In the later Armenian tradition, we do have at least two preserved Armenian copies of Smbat's horse book.

In 1867, a Mekhitarist father published the text of a horse book fragment with the title "I grastu bžškaranēn p'oxac" (copied/taken form the medical book for mount) in the armenological journal Bazmavep [17], which we could identify with manuscript Ms 2385 of the Mekhitharist Library in Venice [18]. This text consists only of three folios and was attached to a (human) medical book that was commissioned by King Het'um II and copied by a Vardapet Mkrtič' in 1294–1295, before Smbat seized his brother's throne. It is still unclear whether these three folios were written by the same scribe and in the same time or whether they were copied later and just bound into this book. Thus, at the current state of research it is uncertain whether these three folios are a later copy of Smbat's or of Het'ums horse book; the title, however, and obvious textual parallel speak for Smbat's horse book [19].

In 2008, the Institute for Ancient Armenian Manuscripts was given a voluminous, damaged codex from the private property of the Nazumlean family of Isfahan. It was catalogued as MS 11161 and contains various medical treatises and a treatise on the care of horses. It has not been explored yet, but in our first analysis after the manuscript's restoration in 2014 we discovered that the compilation's third stratum, a medical book for horses, consisting of folios 210a–261b, was copied in 1504 in the town of Sebaste, present-day Sivas (Turkey). It represents definitely a reproduction of Smbat's Cilician horse book, not an accurate copy, rather an adjusted and updated version.

If we dig deeper into Armenian medieval equine medicine, we see that the other surviving texts and fragments in Armenian language suggest that there was even an older Armenian horse treatise, written some decades before Smbat's text.

#### *3.2.1 The horse treatises of King Het'um I*

In the main colophon of an Armenian chemistry and pharmacology treatise (*Girk' arvesti k'imiakan*), codex 248, kept in the Bibliotheque Nationale de Paris, one gets the following information about another horse treatise on Ff. 34v [20]:

"King Het'um, who marched again the enemy sultan with an army of [corrupt writing] horsemen, massacred and destroyed everything, and he went with big honour to Baghdad […] and there a certain wise man, a deacon named Step'anos, with the king. This man had learnt plenty of languages and writings just like the former philosophers, and there was no writing, this man could not find. He was much loved by the Armenian king because of his knowledge and thus was asked by the King. And he translated three writings about the farriery (i.e. medical treatment) of horses and about how to make a sword…and he took them to the Armenian lands" [21].

These few lines lead us to believe that King Het'um I, one of the most colorful figures in the Armenian Kingdom of Cilicia and grandfather of Smbat, had commissioned one or even more horse treatises based on an Arabic source, and that these had been brought to Cilicia in the 1260s.

Unfortunately, there is no trace of these Hetumian horse books; we have to rely only on the information from this colophon. But this leaves another question: are Hetum's horse book and Smbat's horse book related? Since there is no preserved copy of Het'ums horse books, a meticulous text comparison cannot answer this

question. Therefore, we have to take a deeper look into the literature of the contemporary royal courts to see whether we find any traces of Het'um's or any other Armenian horse book [22].

#### *3.2.2 The Arabic-Armenian horse treatises*

A certain Abū l-Faraǧ is named the author of an *Aqra'bādīn (al-hayl)* "Treatise about horses," which is kept in the Dār-al-Kutub Library in Cairo [23].

Its introduction says that this treatise was translated from Armenian to Arabic by a certain Mahbub (al Armani) and his friend Abū l-Fara ̥ ǧ, who knew Arabic thoroughly and was versed in many languages. It was commissioned by Mahmud b. ̥ Khalīfah Ya'qūb and the philosopher Sa'd Al-Dīn b. Zāhir al-'Ajamī, during the reign ̥ of Sultan Baybars (i.e., 1260–1277). The colophon tells us that the Armenian king had removed the Arabic original from the school of Baghdad during the reign of Sultan Baybars [24]. This treatise most likely refers to the lost horse book of Het'um I. The mentioned manuscript in Cairo has not been analyzed yet—also because of the complicated access— but the information given in the collection's catalogue states that many expressions to be found in the text are given in Armenian [25].

The Arabic equine literature also provides further information about several copies of this Arabic translation of Het'um's horse treatise in London [26], Bethesda USA [27], and Gotha, Germany [28].

The Manuscript or.3133 of British Library was analyzed, literally translated and compared with the existing copy of the Armenian horse book of King Smbat.

In the introduction to the Arabic text, one reads that this text is a treatise on equine medicine:

*"*The Armenian king took the treatise out of the Dār al-<sup>c</sup> ilm of the Caliph treasures in Baghdād ……. And it was an Arabic manuscript, which he brought to Armenia*"* [29].

In the first chapter the text continues,

"the one who translated the treatise from Armenian was called Mağbūb, the name of his friend was Abū l-Farağ, who spoke excellent Arabic ... .. And when the Armenian king took this treatise away from Bagdād, this was in the realm of Baybars, the ruler of Egypt ... " [29].

The colophon gives an exact date of completion of the manuscript:

"The writing of the book was finished, with God's help, on Thursday, the 11th ğumādā I of 1270*"* [29, 30].

This means, that the mentioned Arabic horse treatises kept in collections in Cairo, London, Bethesda, and Gotha are all based on the translation from the Armenian horse book of King Het'um I [31]. Historical sources additionally confirm this story. In 1258, joint Mongol-Armenian forces led by Het'um captured Bagdad.

The comparison of the Arabic translations of King Het'um's horse book with the text of Smbat's horse book of 1296–98 provokes an even greater confusion: the texts have striking similarities and textual parallels. Thus, a reverse conclusion is obvious: Smbat's horse book was probably created on the basis of the Arabic translation of Het'um's horse book.

#### *3.2.2.1 A multilingual Syrian physician: key to the puzzle?*

The conclusion mentioned above could be supported by the key person in the production of the equine manuscripts: the compiler and translator of the Arabic and Armenian copies, the wise Abū l-Faraǧ of the Arabic and the Syrian

**9**

*Medieval Equine Medicine from Armenia DOI: http://dx.doi.org/10.5772/intechopen.91379*

Abu(l) Faraǰ.

died in Maraga, Persia, in 1286.

*3.2.3 The Georgian-Armenian horse treatise*

Georgian horse treatise copied in 1791 [34].

book in Sebaste 2004, M11161 (**Figure 1**).

The copy was finished on August 23rd, 1791" [36].

The colophons tell us that

other.

physician Farač in the Armenian copies. Of course, it could be a simple similarity of names, but the description of a learned Syrian named Farač, who had an excellent command of Arabic and other languages, is found both in the Armenian and Arabic colophons and allows the conclusion that this is a single person. Thus, the Armenian designation as "wise Syrian Farač," and, especially, the Arabic form Abū l-Faraǧ point to one of the most famous Syrian scholars at the time: Gregory Bar-Hebraeus [32, 33]. The famous Syrian polymath and Bishop Gregory Bar-Hebraeus (1226–1286) wrote and compiled in his numerous and elaborate treatises research in theology, philosophy, medicine, science, and history. Being in general proficient in several languages, he was also known as gifted translator from and into Arabic.

The speculations about Bar-Hebraeus' involvement in the Armenian horse books could be confirmed not only by the name form Abū l-Faraǧ Ibn al-ʿIbrī commonly used in the Arabic sources, but also by the fact that the learned Syrian had studied medicine and also worked as a personal physician of the Ilkhan Hulegu Khan. He also had contact with the Cilician royal house and was ordained Primate of the East in 1264 in the Cilician capital Sis, in the presence of the Cilician King Het'um I. Moreover, the information provided by the Arabic colophons allows a dating of a translation of an Armenian horse book into Arabic during the reign of both King Het'um I and Mamluk Sultan Baybars and to the lifetime of Bar-Hebraeus. The argument in favor of Bar-Hebraeus may be reinforced by the fact that he was known in the Armenian tradition also very often by the Arabized form of his name, as

Strong counterarguments are, however, the fact that there is no indication in Syrian, Arabic, Persian, Armenian, or other sources that Bar-Hebraeus ever translated from Armenian into Arabic on the one hand, and veterinary treatises, on the

The assumption that the same Abū l-Faraǧ was also responsible for the translation of an Arabic horse book into Armenian on behalf of King Smbat, 1296–98, is difficult to sustain due to the biographical data of Bar-Hebraeus, who had already

One can even argue that the Armenian priest T'oros may have been working alone on an already existing, earlier translation by the Syrian Farač/ Abū l-Faraǧ particularly taking the fact into account that Arabic terms describing horse coat colors, diseases, and remedies are very often rendered completely corrupt in the Armenian horse book of king Smbat. A person who is said to have an excellent com-

Another further proof of the importance of this horse book is a late translation into Georgian. The national library of Georgia holds the manuscript T 3467, a

"On May 18th, 1788, we, the High priest of Sioni in Tiflis, Ioane Osedze and the Armenian priest Ter-Petros, were asked by Giorgi [35], the first born son and heir of the King of Georgia Irakli II, to translate this treatise from Armenian into Georgian.

From the colophon we also learn that a certain "Parač'i" had translated the original Armenian treatise from Arabic in the Armenian year 953 (1503) in Sebaste (Sivas) and that the exact name of the treatise is "Medical book for horse and mount." The Georgian translation was obviously based on the copy of Smbat's horse

mand of Arabic would not use such corrupt Arabic terms in Armenian.

#### *Medieval Equine Medicine from Armenia DOI: http://dx.doi.org/10.5772/intechopen.91379*

*Equine Science*

Armenian horse book [22].

*3.2.2 The Arabic-Armenian horse treatises*

USA [27], and Gotha, Germany [28].

In the first chapter the text continues,

Baybars, the ruler of Egypt ... " [29].

ğumādā I of 1270*"* [29, 30].

Het'um's horse book.

equine medicine:

Armenia*"* [29].

Bagdad.

question. Therefore, we have to take a deeper look into the literature of the contemporary royal courts to see whether we find any traces of Het'um's or any other

A certain Abū l-Faraǧ is named the author of an *Aqra'bādīn (al-hayl)* "Treatise

Its introduction says that this treatise was translated from Armenian to Arabic by a certain Mahbub (al Armani) and his friend Abū l-Fara ̥ ǧ, who knew Arabic thoroughly and was versed in many languages. It was commissioned by Mahmud b. ̥ Khalīfah Ya'qūb and the philosopher Sa'd Al-Dīn b. Zāhir al-'Ajamī, during the reign ̥ of Sultan Baybars (i.e., 1260–1277). The colophon tells us that the Armenian king had removed the Arabic original from the school of Baghdad during the reign of Sultan Baybars [24]. This treatise most likely refers to the lost horse book of Het'um I. The mentioned manuscript in Cairo has not been analyzed yet—also because of the complicated access— but the information given in the collection's catalogue states that many expressions to be found in the text are given in Armenian [25]. The Arabic equine literature also provides further information about several copies of this Arabic translation of Het'um's horse treatise in London [26], Bethesda

The Manuscript or.3133 of British Library was analyzed, literally translated and

ilm of the Caliph

compared with the existing copy of the Armenian horse book of King Smbat. In the introduction to the Arabic text, one reads that this text is a treatise on

treasures in Baghdād ……. And it was an Arabic manuscript, which he brought to

"the one who translated the treatise from Armenian was called Mağbūb, the name of his friend was Abū l-Farağ, who spoke excellent Arabic ... .. And when the Armenian king took this treatise away from Bagdād, this was in the realm of

"The writing of the book was finished, with God's help, on Thursday, the 11th

This means, that the mentioned Arabic horse treatises kept in collections in Cairo, London, Bethesda, and Gotha are all based on the translation from the Armenian horse book of King Het'um I [31]. Historical sources additionally confirm this story. In 1258, joint Mongol-Armenian forces led by Het'um captured

The comparison of the Arabic translations of King Het'um's horse book with the text of Smbat's horse book of 1296–98 provokes an even greater confusion: the texts have striking similarities and textual parallels. Thus, a reverse conclusion is obvious: Smbat's horse book was probably created on the basis of the Arabic translation of

The conclusion mentioned above could be supported by the key person in the production of the equine manuscripts: the compiler and translator of the Arabic and Armenian copies, the wise Abū l-Faraǧ of the Arabic and the Syrian

The colophon gives an exact date of completion of the manuscript:

*"*The Armenian king took the treatise out of the Dār al-<sup>c</sup>

*3.2.2.1 A multilingual Syrian physician: key to the puzzle?*

about horses," which is kept in the Dār-al-Kutub Library in Cairo [23].

**8**

physician Farač in the Armenian copies. Of course, it could be a simple similarity of names, but the description of a learned Syrian named Farač, who had an excellent command of Arabic and other languages, is found both in the Armenian and Arabic colophons and allows the conclusion that this is a single person. Thus, the Armenian designation as "wise Syrian Farač," and, especially, the Arabic form Abū l-Faraǧ point to one of the most famous Syrian scholars at the time: Gregory Bar-Hebraeus [32, 33]. The famous Syrian polymath and Bishop Gregory Bar-Hebraeus (1226–1286) wrote and compiled in his numerous and elaborate treatises research in theology, philosophy, medicine, science, and history. Being in general proficient in several languages, he was also known as gifted translator from and into Arabic.

The speculations about Bar-Hebraeus' involvement in the Armenian horse books could be confirmed not only by the name form Abū l-Faraǧ Ibn al-ʿIbrī commonly used in the Arabic sources, but also by the fact that the learned Syrian had studied medicine and also worked as a personal physician of the Ilkhan Hulegu Khan. He also had contact with the Cilician royal house and was ordained Primate of the East in 1264 in the Cilician capital Sis, in the presence of the Cilician King Het'um I. Moreover, the information provided by the Arabic colophons allows a dating of a translation of an Armenian horse book into Arabic during the reign of both King Het'um I and Mamluk Sultan Baybars and to the lifetime of Bar-Hebraeus. The argument in favor of Bar-Hebraeus may be reinforced by the fact that he was known in the Armenian tradition also very often by the Arabized form of his name, as Abu(l) Faraǰ.

Strong counterarguments are, however, the fact that there is no indication in Syrian, Arabic, Persian, Armenian, or other sources that Bar-Hebraeus ever translated from Armenian into Arabic on the one hand, and veterinary treatises, on the other.

The assumption that the same Abū l-Faraǧ was also responsible for the translation of an Arabic horse book into Armenian on behalf of King Smbat, 1296–98, is difficult to sustain due to the biographical data of Bar-Hebraeus, who had already died in Maraga, Persia, in 1286.

One can even argue that the Armenian priest T'oros may have been working alone on an already existing, earlier translation by the Syrian Farač/ Abū l-Faraǧ particularly taking the fact into account that Arabic terms describing horse coat colors, diseases, and remedies are very often rendered completely corrupt in the Armenian horse book of king Smbat. A person who is said to have an excellent command of Arabic would not use such corrupt Arabic terms in Armenian.

#### *3.2.3 The Georgian-Armenian horse treatise*

Another further proof of the importance of this horse book is a late translation into Georgian. The national library of Georgia holds the manuscript T 3467, a Georgian horse treatise copied in 1791 [34].

The colophons tell us that

"On May 18th, 1788, we, the High priest of Sioni in Tiflis, Ioane Osedze and the Armenian priest Ter-Petros, were asked by Giorgi [35], the first born son and heir of the King of Georgia Irakli II, to translate this treatise from Armenian into Georgian. The copy was finished on August 23rd, 1791" [36].

From the colophon we also learn that a certain "Parač'i" had translated the original Armenian treatise from Arabic in the Armenian year 953 (1503) in Sebaste (Sivas) and that the exact name of the treatise is "Medical book for horse and mount." The Georgian translation was obviously based on the copy of Smbat's horse book in Sebaste 2004, M11161 (**Figure 1**).

#### **Figure 1.**

*Presentation of the assumed transfer of knowledge and reception of Armenian equine medicine manuscripts between the thirteenth and nineteenth centuries.*

#### **3.3 Provenance of the Armenian horse books**

Three of the given Armenian horse treatises, the Cilician horse book of king Smbat 1296–98, its not clearly dated Venice copy, and the voluminous Sivas reproduction of 1504, had been kept by private owners in the Armenian-inhabited settlements of Persia before they were donated to the Armenian manuscript collection in Yerevan. It is still one of the many unsolved mysteries of these Armenian horse books, how and when they ended up in Persia.

#### **4. To the west of Cilician Armenia, far beyond the Bosporus**

The Cilician noble families have been under the constant cultural influence of the Frankish kings for many decades, particularly due to the close relations with the European noble crusaders and the Staufer kings. It was Prince Levon II (1150–1219) who profited from this situation by improving relations with the Europeans. Cilician Armenia's prominence in the region is attested by letters sent in 1189 by Pope Clement III to Levon and to Catholicos Gregory IV, in which he asks Armenian military and financial assistance to the crusaders. On January 6, 1199, Prince Levon II was crowned with great solemnity in the cathedral of Tarsus, in the presence of the Syrian patriarch, the Greek metropolitan of Tarsus, and numerous church dignitaries and military leaders. While he was crowned by the Catholicos Gregory VI of Cilicia, Levon received a banner with the insignia of a lion from Archbishop Conrad of Mainz in the name of Henry VI, Holy Roman Emperor. By securing his crown, he became the first King of Armenian Cilicia as King Levon I [13].

**11**

*Medieval Equine Medicine from Armenia DOI: http://dx.doi.org/10.5772/intechopen.91379*

was much oriented toward Europe?

diseases, but hardly noticeable in farriery [37, 38].

**5. Outlook: galloping from east to west?**

into Georgian (18th c).

medicine.

coming years.

Cilician Armenians were attracted by European culture and art. It did not take long for them to absorb the findings and texts of European science and medicine. In addition to their local knowledge and that of the Muslim East they also started to include European knowledge, in particular also in horse breeding and training.

King Het'um I was a major player in the political struggles and shifting alliances during the Crusades, trying to keep ties with all sides, both in the West and the East. Perhaps he was not only fascinated by the Arabic equine treatises from Baghdad but also somewhat inspired by his European counterpart's hippiatric book? In Europe, Jordanus Ruffus, chief marshal and close associate of Staufer emperor Frederick II, completed his very influential "Medicina Equorum" around 1250, which was commissioned by and dedicated to his emperor. We know that this work spread quite quickly from Italy through Europe as a result of the Italian equestrian schools. Can the mere fact, that one of the most important contemporary European monarchs has commissioned a horse book, have affected the Armenian King Het'um I who

The growing influence of European equestrian art and equine knowledge cannot be investigated in the Het'um's horse treatises, only guessed. Some 30 years later, however, this influence is clearly presented in the influential horse book of king Smbat, especially regarding breeding, training, and chivalrous tournaments (such as buhurt and jousting) [6]. The European influence was also increasingly reflected in some newly adopted Frankish terms in horsemanship, anatomy, and names of

In order to understand the history and interrelation of Armenian and Arabic horse treatises, not only the person of the "producer and translator," the wise Syrian Farač/Abū l-Faraǧ, must be investigated, but also the socio-historical and scientific historical context. Moreover, the efficacy and importance of the Cilician Horse Book 1296–98 will be tracked in all subsequent texts—both Armenian copies and foreign translations. A range of local and foreign treatises will be checked: these are the supposed translations of an Armenian text into Arabic (13th c to 14th c) and

The meticulous comparison of all texts in question will perhaps also prove that the main source for all texts was an unknown Arabic text, which King Het'um I had discovered in Baghdad and which he had translated into Armenian. Further investigation of Armenian equine manuscripts and fragments will clarify what can be regarded as the actual starting point of the reception history of Armenian horse

This will be the goal of an interdisciplinary, international research project in the

#### *Medieval Equine Medicine from Armenia DOI: http://dx.doi.org/10.5772/intechopen.91379*

*Equine Science*

**Figure 1.**

**3.3 Provenance of the Armenian horse books**

*between the thirteenth and nineteenth centuries.*

books, how and when they ended up in Persia.

Three of the given Armenian horse treatises, the Cilician horse book of king Smbat 1296–98, its not clearly dated Venice copy, and the voluminous Sivas reproduction of 1504, had been kept by private owners in the Armenian-inhabited settlements of Persia before they were donated to the Armenian manuscript collection in Yerevan. It is still one of the many unsolved mysteries of these Armenian horse

*Presentation of the assumed transfer of knowledge and reception of Armenian equine medicine manuscripts* 

The Cilician noble families have been under the constant cultural influence of the Frankish kings for many decades, particularly due to the close relations with the European noble crusaders and the Staufer kings. It was Prince Levon II (1150–1219) who profited from this situation by improving relations with the Europeans. Cilician Armenia's prominence in the region is attested by letters sent in 1189 by Pope Clement III to Levon and to Catholicos Gregory IV, in which he asks Armenian military and financial assistance to the crusaders. On January 6, 1199, Prince Levon II was crowned with great solemnity in the cathedral of Tarsus, in the presence of the Syrian patriarch, the Greek metropolitan of Tarsus, and numerous church dignitaries and military leaders. While he was crowned by the Catholicos Gregory VI of Cilicia, Levon received a banner with the insignia of a lion from Archbishop Conrad of Mainz in the name of Henry VI, Holy Roman Emperor. By securing his crown, he became the first King of Armenian Cilicia as

**4. To the west of Cilician Armenia, far beyond the Bosporus**

**10**

King Levon I [13].

Cilician Armenians were attracted by European culture and art. It did not take long for them to absorb the findings and texts of European science and medicine. In addition to their local knowledge and that of the Muslim East they also started to include European knowledge, in particular also in horse breeding and training.

King Het'um I was a major player in the political struggles and shifting alliances during the Crusades, trying to keep ties with all sides, both in the West and the East. Perhaps he was not only fascinated by the Arabic equine treatises from Baghdad but also somewhat inspired by his European counterpart's hippiatric book? In Europe, Jordanus Ruffus, chief marshal and close associate of Staufer emperor Frederick II, completed his very influential "Medicina Equorum" around 1250, which was commissioned by and dedicated to his emperor. We know that this work spread quite quickly from Italy through Europe as a result of the Italian equestrian schools. Can the mere fact, that one of the most important contemporary European monarchs has commissioned a horse book, have affected the Armenian King Het'um I who was much oriented toward Europe?

The growing influence of European equestrian art and equine knowledge cannot be investigated in the Het'um's horse treatises, only guessed. Some 30 years later, however, this influence is clearly presented in the influential horse book of king Smbat, especially regarding breeding, training, and chivalrous tournaments (such as buhurt and jousting) [6]. The European influence was also increasingly reflected in some newly adopted Frankish terms in horsemanship, anatomy, and names of diseases, but hardly noticeable in farriery [37, 38].

#### **5. Outlook: galloping from east to west?**

In order to understand the history and interrelation of Armenian and Arabic horse treatises, not only the person of the "producer and translator," the wise Syrian Farač/Abū l-Faraǧ, must be investigated, but also the socio-historical and scientific historical context. Moreover, the efficacy and importance of the Cilician Horse Book 1296–98 will be tracked in all subsequent texts—both Armenian copies and foreign translations. A range of local and foreign treatises will be checked: these are the supposed translations of an Armenian text into Arabic (13th c to 14th c) and into Georgian (18th c).

The meticulous comparison of all texts in question will perhaps also prove that the main source for all texts was an unknown Arabic text, which King Het'um I had discovered in Baghdad and which he had translated into Armenian. Further investigation of Armenian equine manuscripts and fragments will clarify what can be regarded as the actual starting point of the reception history of Armenian horse medicine.

This will be the goal of an interdisciplinary, international research project in the coming years.

*Equine Science*

### **Author details**

Jasmine Dum-Tragut Department of Armenian Studies, Center for the Study of the Christian East, University of Salzburg, Austria

\*Address all correspondence to: jasmine.dum-tragut@sbg.ac.at

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**13**

*Medieval Equine Medicine from Armenia DOI: http://dx.doi.org/10.5772/intechopen.91379*

[1] Dum-Tragut J. The Armenian manuscripts of horse medicine. Journal of the Society for Armenian Studies.

[2] Dum-Tragut J. Kilikische Heilkunst für Pferde. Olms: Hildesheim; 2005

[11] Dadoyan S. The Armenians in the Medieval Islamic World. Paradigms of Interaction. Seventh to Fourteenth Centuries. Vol. 2. New Brunswick, London: Transaction Publishers; 2013

[12] Bournoutian A. Cilician Armenia. In: Hovannisian R, editor. The Armenia People from Ancient to Modern Times. Vol. 1. New York: St. Martins Press;

[13] Le Mutafian C. Royaume Arménien de Cilicie XIIe-XIVe siècle. Paris: CNRS

[14] Der Nersessian S. The kingdom of Cilician Armenia. In: Setton K, Wolff R, Hazar H, editors. History of the Crusades. Vol. 2. The later Crusades. Madison: University of Wisconsin Press;

1969. pp. 1189, 630-1131, 659

[15] Het'um II. the brother of King Smbat was also known as Hayton of Corycus ( *Het'um*; c.1240 - c.1310- 1320). Hayton is also the author of *La Flor des Estoires d'Orient* ("Flower of the Histories of the East", in Latin *Flos historiarum partium Orientis*), in which he narrates the history of Asia particularly relating to Muslim conquests and the Mongol invasion. He dictated the text in Old French to a certain Nicolas Faulcon at the request of Pope Clement V in 1307, while he was at Poitiers. Het'ums work was widely known in the Late Middle Ages and was influential in shaping western European views of the Orient. It was translated into Armenian only in the 19th c. Mutafian, C. Le Royaume Arménien de Cilicie XIIe-XIVe siècle. Paris: CNRS

1997. pp. 273-290

éditions; 1993

éditions; 1993

[16] There is also a report of the Muslim writer Abu'l Fida', personally being involved in the invasion: Smbat was ruling on June 20, but on July 30th it was already Constantin do lead negotiations with the Mamluk army. Abu al-Fida

[4] Č'ugazsian B. Le Traite'd'hippiatrie du XIIIe siècle. In: Kouymjian D, editor. Armenian Studies in Memoriam Haig Berberian. Lisboa: Calouste Gulbenkian

[5] Dum-Tragut J. "The Cilician Medical Book for Horses" on terminological work and interdisciplinary research. In: Handes Amsorya. Vienna: Mekhitarist

[6] Dum-Tragut J. Horse and Knighthood in Cilician Armenia. In: Proceedings of the First international Conference on Cilician Armenia. Antelias: HASK; 2009

[7] C'uc'ak Jer̻agrac' Maštoc'i anuan Matenadarani. Hator III. Ter-Step'anyan A. Erewan: Erewani hamalasaran hratarakč'ut'yun; 2007. p. 156. (Catalogue of manuscripts of

[8] Adjarian H. Katalog der Armenischen

Handschriften in Täbris. Wien: Mechitharisten-Buchdruckerei; 1910

[9] The given quotation from the colophon f.184a of manuscript M10965 was translated from the medieval Cilician-Armenian into English by J.

[10] Stewart A. Reframing the Mongols in 1260. The Armenians, the Mongols and the Magi. Journal of the Royal Asiatic Society. 2018;**28**(1):55-76. DOI:

10.1017/S1356186317000414

Foundation; 1986. pp. 105-123

Order; 2005. pp. 1-12

Matenadaran, Vol. 3.)

Dum-Tragut.

[3] Č'ugazsian B. Bžškaran jioy ew arhasarak grastnoy. Erevan: Haykakan SSH GA Hratarakč'ut'yun; 1980

**References**

2014;**23**:149-157

*Medieval Equine Medicine from Armenia DOI: http://dx.doi.org/10.5772/intechopen.91379*

#### **References**

*Equine Science*

**12**

**Author details**

Jasmine Dum-Tragut

University of Salzburg, Austria

provided the original work is properly cited.

Department of Armenian Studies, Center for the Study of the Christian East,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: jasmine.dum-tragut@sbg.ac.at

[1] Dum-Tragut J. The Armenian manuscripts of horse medicine. Journal of the Society for Armenian Studies. 2014;**23**:149-157

[2] Dum-Tragut J. Kilikische Heilkunst für Pferde. Olms: Hildesheim; 2005

[3] Č'ugazsian B. Bžškaran jioy ew arhasarak grastnoy. Erevan: Haykakan SSH GA Hratarakč'ut'yun; 1980

[4] Č'ugazsian B. Le Traite'd'hippiatrie du XIIIe siècle. In: Kouymjian D, editor. Armenian Studies in Memoriam Haig Berberian. Lisboa: Calouste Gulbenkian Foundation; 1986. pp. 105-123

[5] Dum-Tragut J. "The Cilician Medical Book for Horses" on terminological work and interdisciplinary research. In: Handes Amsorya. Vienna: Mekhitarist Order; 2005. pp. 1-12

[6] Dum-Tragut J. Horse and Knighthood in Cilician Armenia. In: Proceedings of the First international Conference on Cilician Armenia. Antelias: HASK; 2009

[7] C'uc'ak Jer̻agrac' Maštoc'i anuan Matenadarani. Hator III. Ter-Step'anyan A. Erewan: Erewani hamalasaran hratarakč'ut'yun; 2007. p. 156. (Catalogue of manuscripts of Matenadaran, Vol. 3.)

[8] Adjarian H. Katalog der Armenischen Handschriften in Täbris. Wien: Mechitharisten-Buchdruckerei; 1910

[9] The given quotation from the colophon f.184a of manuscript M10965 was translated from the medieval Cilician-Armenian into English by J. Dum-Tragut.

[10] Stewart A. Reframing the Mongols in 1260. The Armenians, the Mongols and the Magi. Journal of the Royal Asiatic Society. 2018;**28**(1):55-76. DOI: 10.1017/S1356186317000414

[11] Dadoyan S. The Armenians in the Medieval Islamic World. Paradigms of Interaction. Seventh to Fourteenth Centuries. Vol. 2. New Brunswick, London: Transaction Publishers; 2013

[12] Bournoutian A. Cilician Armenia. In: Hovannisian R, editor. The Armenia People from Ancient to Modern Times. Vol. 1. New York: St. Martins Press; 1997. pp. 273-290

[13] Le Mutafian C. Royaume Arménien de Cilicie XIIe-XIVe siècle. Paris: CNRS éditions; 1993

[14] Der Nersessian S. The kingdom of Cilician Armenia. In: Setton K, Wolff R, Hazar H, editors. History of the Crusades. Vol. 2. The later Crusades. Madison: University of Wisconsin Press; 1969. pp. 1189, 630-1131, 659

[15] Het'um II. the brother of King Smbat was also known as Hayton of Corycus ( *Het'um*; c.1240 - c.1310- 1320). Hayton is also the author of *La Flor des Estoires d'Orient* ("Flower of the Histories of the East", in Latin *Flos historiarum partium Orientis*), in which he narrates the history of Asia particularly relating to Muslim conquests and the Mongol invasion. He dictated the text in Old French to a certain Nicolas Faulcon at the request of Pope Clement V in 1307, while he was at Poitiers. Het'ums work was widely known in the Late Middle Ages and was influential in shaping western European views of the Orient. It was translated into Armenian only in the 19th c. Mutafian, C. Le Royaume Arménien de Cilicie XIIe-XIVe siècle. Paris: CNRS éditions; 1993

[16] There is also a report of the Muslim writer Abu'l Fida', personally being involved in the invasion: Smbat was ruling on June 20, but on July 30th it was already Constantin do lead negotiations with the Mamluk army. Abu al-Fida

(November 1273 – October 27, 1331), fully Abu Al-fida' Isma'il Ibn 'ali ibn Mahmud Al-malik Al-mu'ayyad 'imad Ad-din and better known in English as Abulfeda, was a Kurdish historian, geographer and local governor of Hama. He was a prince of the Ayyubid dynasty and the author of *The memoirs of a Syrian prince: Abu'l-Fidā*'*, Sultan of Ḥamāh*

[17] Bazmavep. The Horse Book Fragment. Vol. 25. Venice: Mekhitarist Order; 1867. pp. 353-359

[18] Čemčemean S. Mayr c'uc'ak hayēren jeragrac' matenadarin mxit'aryanc' i Venetik. Hator 8. Targmanut'iwnk' naxneac', Vark' haranc', Bžškaran. Venetik: Surb Lazar; 1998. p. 719-722. (Catalogue of Armenian Manuscripts in the Mekhitarist Library in Venice, Vol. 8)

[19] Dum-Tragut J. Die jahrhundertelange Tradierung antiken und mittelalterlichen pferde(heil)kundlichen Wissens. Eine fragmentarische armenische Pferdehandschrift. Übersetzung. Analyse. Besprechung. Wien: Veterinärumedizinische Universität; 2014

[20] Kévorkian R, Ter-Stépanos A. Manuscrits arméniens de la Bibliotèque natonale des France. Paris: Fondation Calouste Gulbenkian; 1998. pp. 814-816

[21] The given quotation from the colophon ff.34v of the manuscript 234 (BNF) was translated from the Modern Armenian original into English by J. Dum-Tragut.

[22] Firsts hints at the entangled relationships between Armenian and Arabic horse treatises can be found in short essay published by Č'ugaszyan and Ter-Łevondyan in 1985. They have neither been scientifically reviewed nor have the alleged Arabic texts ever been examined or analyzed. Č'ugaszyan, B. Ter-Łevondyan, A. Bžskaran jioy erkri norahayt araberen targmanut'yuně. Lraber hasarakakan gitut'yunneri, 11. Erevan; 1985. pp. 63-68

[23] Sayyid F. Fihrist al-makht̻ūt̻āt. Vol. 1. Cairo: Dār al-Kutub al-Mis̻riyya; 1961. p. 305

[24] Sbath P. Manuscrit Arabe sur la pharmacopée hippiatrique. Le Caire: L'institut francais; 1932. pp. 80-81

[25] Shehada H. Mamluks and Animals. Leiden, Boston: Brill; 2012. pp. 100-101

[26] Rieu C. Supplement to the Catalogue of the Arabic manuscripts in the British Museum. London: British Museum; 1894. pp. 532-533

[27] Schullian D, Sommer FA. Catalogue of Incunabula and Manuscripts in the Army Medical Library. Bethesda: Army Medical Library; 1950. p. 298

[28] Pertsch W. Die orientalischen Handschriften der herzoglichen Bibliothek zu Gotha, 3 Teil, die arabischen Handschriften. Gotha: Perthes; 1883. p. 110

[29] The given quotations of manuscript or.3133 were translated from Arabic to English by L. Nigst and J. Dum-Tragut.

[30] The year 1270 in Islamic calendar corresponds to 1834 A.D.

[31] There is another alleged Arabic translation of an Armenian horsebook in Armenian medical literature. Some scholars, never having thoroughly analysed the manuscript, claimed that it was a translation of Smbat's horsebook, commissioned in 1298-99 by Sultan al-Malik al-Mansour Hossam ad-Din Lajin al-Mansuri, Sultan of Egypt (1296-1299). Our research has however revealed that the text in question is nothing else than another copy of the Arabic translation of Het'um's treatise now kept in the library of Bethesda. Č'ugaszyan B. Ter-Łevondyan A. Bžskaran jioy erkri norahayt araberen targmanut'yuně. Lraber hasarakakan gitut'yunneri, 11. Erevan; 1985. pp. 63-68

**15**

*Medieval Equine Medicine from Armenia DOI: http://dx.doi.org/10.5772/intechopen.91379*

Lexikon für Theologie und Kirche. 3. Auflage. Band 4. Freiburg: Herder;

[33] Wright W. A Short History of Syriac Literature. London: Cambridge University Press; 1894. pp. 265-281

[35] Giorgi XII (1746 – December 28, 1800) was the son of Heraclius II (also known as Erekle), the king of Kartli and Kakheti, and his second wife Anna Abašidze. In 1766 he was recognized as crown prince and Lord of Pambak and Lori (now Northern Armenia). After the death of this father in 1798, he reigned Kartli and Kakheti until 1800. Anchabadze, G. History of Georgia. Short sketch. Tbilissi: Causasian House;

[36] This unique specimen has not yet been examined in detail and compared with the Cilician horse book, M10975, Sis of 1296-98 and M11161, Sivas 1504 in terms of structure, content and terminology vocabulary. The translation of the given quotation from the introduction in the Georgian manuscript 3467 was translated from Georgian to English by M. Topadze and

[37] Dum-Tragut J. Äpfel und Birnen - Reflektionen über pferdeanatomische

Niederreiter S, editors. Diachronie und Sprachvergleich. Innsbruck: Universität

Terminologie. In: Krisch T,

Innsbruck; 2015. pp. 102-111

[38] Dum-Tragut J. Bewer̊has, Asahar und R̊aysay (Nageltritt) – Lehnübersetzung, Neologismus und korrupte Entlehnung. Das Tohuwabohu von Krankheitsbezeichnungen in armenischen pferdeheilkundlichen

Ibri*.*

Manuskripten. Commentaria Classica, Studi di filologia greca e latina; 2018.

pp. 359-382

[32] Brock S. Gregor ibn al-<sup>c</sup>

[34] Catalogue of Georgian Manuscript in National Library Tiflis, Fund S. Available at: http:// www.manuscript.ge/index.

php?m=11&cid=13

2005

J. Dum-Tragut.

1995. p. 1001f

*Medieval Equine Medicine from Armenia DOI: http://dx.doi.org/10.5772/intechopen.91379*

*Equine Science*

(November 1273 – October 27, 1331), fully Abu Al-fida' Isma'il Ibn 'ali ibn Mahmud Al-malik Al-mu'ayyad 'imad Ad-din and better known in English as Abulfeda, was a Kurdish historian, geographer and local governor of Hama. He was a prince of the Ayyubid dynasty and the author of *The memoirs of a Syrian prince: Abu'l-Fidā*'*, Sultan of Ḥamāh*

[23] Sayyid F. Fihrist al-makht̻ūt̻āt. Vol. 1. Cairo: Dār al-Kutub al-Mis̻riyya;

[24] Sbath P. Manuscrit Arabe sur la pharmacopée hippiatrique. Le Caire: L'institut francais; 1932. pp. 80-81

[25] Shehada H. Mamluks and Animals. Leiden, Boston: Brill; 2012. pp. 100-101

[27] Schullian D, Sommer FA. Catalogue of Incunabula and Manuscripts in the Army Medical Library. Bethesda: Army

[29] The given quotations of manuscript or.3133 were translated from Arabic to English by L. Nigst and J. Dum-Tragut.

[30] The year 1270 in Islamic calendar

[31] There is another alleged Arabic translation of an Armenian horsebook in Armenian medical literature. Some scholars, never having thoroughly analysed the manuscript, claimed that it was a translation of Smbat's horsebook, commissioned in 1298-99 by Sultan al-Malik al-Mansour Hossam ad-Din Lajin al-Mansuri, Sultan of Egypt (1296-1299). Our research has however revealed that the text in question is nothing else than another copy of the Arabic translation of Het'um's treatise now kept in the library of Bethesda. Č'ugaszyan B. Ter-Łevondyan A. Bžskaran jioy erkri norahayt araberen targmanut'yuně. Lraber hasarakakan gitut'yunneri, 11. Erevan; 1985.

[26] Rieu C. Supplement to the Catalogue of the Arabic manuscripts in the British Museum. London: British

Museum; 1894. pp. 532-533

Medical Library; 1950. p. 298

Perthes; 1883. p. 110

corresponds to 1834 A.D.

pp. 63-68

[28] Pertsch W. Die orientalischen Handschriften der herzoglichen Bibliothek zu Gotha, 3 Teil, die arabischen Handschriften. Gotha:

1961. p. 305

[17] Bazmavep. The Horse Book Fragment. Vol. 25. Venice: Mekhitarist

[18] Čemčemean S. Mayr c'uc'ak hayēren jeragrac' matenadarin mxit'aryanc' i Venetik. Hator 8. Targmanut'iwnk' naxneac', Vark' haranc', Bžškaran. Venetik: Surb Lazar; 1998. p. 719-722. (Catalogue of Armenian Manuscripts in the Mekhitarist Library in Venice, Vol. 8)

[19] Dum-Tragut J. Die jahrhundertelange Tradierung antiken und mittelalterlichen

pferde(heil)kundlichen Wissens. Eine fragmentarische armenische Pferdehandschrift. Übersetzung. Analyse. Besprechung. Wien: Veterinärumedizinische Universität;

[20] Kévorkian R, Ter-Stépanos A. Manuscrits arméniens de la Bibliotèque natonale des France. Paris: Fondation Calouste Gulbenkian; 1998. pp. 814-816

[21] The given quotation from the colophon ff.34v of the manuscript 234 (BNF) was translated from the Modern Armenian original into English by J.

[22] Firsts hints at the entangled relationships between Armenian and Arabic horse treatises can be found in short essay published by Č'ugaszyan and Ter-Łevondyan in 1985. They have neither been scientifically reviewed nor have the alleged Arabic texts ever been examined or analyzed. Č'ugaszyan, B. Ter-Łevondyan, A. Bžskaran jioy erkri norahayt araberen targmanut'yuně. Lraber hasarakakan gitut'yunneri, 11.

Erevan; 1985. pp. 63-68

Order; 1867. pp. 353-359

**14**

2014

Dum-Tragut.

[32] Brock S. Gregor ibn al-<sup>c</sup> Ibri*.* Lexikon für Theologie und Kirche. 3. Auflage. Band 4. Freiburg: Herder; 1995. p. 1001f

[33] Wright W. A Short History of Syriac Literature. London: Cambridge University Press; 1894. pp. 265-281

[34] Catalogue of Georgian Manuscript in National Library Tiflis, Fund S. Available at: http:// www.manuscript.ge/index. php?m=11&cid=13

[35] Giorgi XII (1746 – December 28, 1800) was the son of Heraclius II (also known as Erekle), the king of Kartli and Kakheti, and his second wife Anna Abašidze. In 1766 he was recognized as crown prince and Lord of Pambak and Lori (now Northern Armenia). After the death of this father in 1798, he reigned Kartli and Kakheti until 1800. Anchabadze, G. History of Georgia. Short sketch. Tbilissi: Causasian House; 2005

[36] This unique specimen has not yet been examined in detail and compared with the Cilician horse book, M10975, Sis of 1296-98 and M11161, Sivas 1504 in terms of structure, content and terminology vocabulary. The translation of the given quotation from the introduction in the Georgian manuscript 3467 was translated from Georgian to English by M. Topadze and J. Dum-Tragut.

[37] Dum-Tragut J. Äpfel und Birnen - Reflektionen über pferdeanatomische Terminologie. In: Krisch T, Niederreiter S, editors. Diachronie und Sprachvergleich. Innsbruck: Universität Innsbruck; 2015. pp. 102-111

[38] Dum-Tragut J. Bewer̊has, Asahar und R̊aysay (Nageltritt) – Lehnübersetzung, Neologismus und korrupte Entlehnung. Das Tohuwabohu von Krankheitsbezeichnungen in armenischen pferdeheilkundlichen

Manuskripten. Commentaria Classica, Studi di filologia greca e latina; 2018. pp. 359-382

**17**

**Chapter 2**

**Abstract**

Gene Therapy as a Modern Method

of Treating Naturally Occurring

*Elena Zakirova, Kovac Milomir, Margarita Zhuravleva,* 

*Catrin Sian Rutland and Albert Rizvanov*

trials and the cellular effects and potential mechanisms of actions.

tendon injuries, suspensory ligament

3–12 months after the first injury [3].

**1. Introduction**

Tendinitis and Desmitis in Horses

Tendon and ligament injuries have always been complex to treat, with recovery often taking many months, if successful at all. This chapter looks at recent work undertaken using regenerative medicine, specifically gene therapy and the advances that have been made in equine therapy. It looks at the process from plasmid construction, in vitro testing through to trialing the equine-specific plasmid construct in horses with superficial digital flexor tendon (tendinitis) and suspensory ligament branch injuries. It also looks at the rationale for utilizing vascular endothelial growth factor (VEGF164) and a basic fibroblast growth factor (FGF2) for these

**Keywords:** horse, gene therapy, tissue regeneration, superficial digital flexor tendon,

Tendon and ligament injuries are the most common traumas in horses (*Equus caballus*) irrespective of the age and breed [1]. Based on the statistics, injuries in sport horses can achieve 86% of a total morbidity rate, with 37% of them accounting for muscle, tendon and ligament pathologies. As a rule, musculoskeletal injuries require long-term recovery for 9–12 months [2]. Tendon and ligament injuries result in a loss of performance of the horses and frequently cause discomfort or pain. Therefore, the animals are unable to participate in competitions for a long period of time. Complications of these injuries include chronic musculoskeletal diseases. They result in degenerative-dystrophic damage of collagen fibers of tendons as well as adjacent and underlying tissues. Incomplete tissue recovery leads to recurrent injuries in 80% of horses with treated tendon micro- and macroruptures within

Methods of regenerative medicine are used for appropriate regeneration of damaged tissue in animals. These include the administration of stem cells [4, 5] and recombinant proteins, as well as gene therapy. These methods are presently the most advanced and promising approaches to manage musculoskeletal disorders [6]. However, regenerative medicine is mainly targeted toward the treatment of human disorders. Animals are mostly considered as models to test drugs and devices intended for human use. Drugs developed for human use can be ineffective for the

#### **Chapter 2**

## Gene Therapy as a Modern Method of Treating Naturally Occurring Tendinitis and Desmitis in Horses

*Elena Zakirova, Kovac Milomir, Margarita Zhuravleva, Catrin Sian Rutland and Albert Rizvanov*

#### **Abstract**

Tendon and ligament injuries have always been complex to treat, with recovery often taking many months, if successful at all. This chapter looks at recent work undertaken using regenerative medicine, specifically gene therapy and the advances that have been made in equine therapy. It looks at the process from plasmid construction, in vitro testing through to trialing the equine-specific plasmid construct in horses with superficial digital flexor tendon (tendinitis) and suspensory ligament branch injuries. It also looks at the rationale for utilizing vascular endothelial growth factor (VEGF164) and a basic fibroblast growth factor (FGF2) for these trials and the cellular effects and potential mechanisms of actions.

**Keywords:** horse, gene therapy, tissue regeneration, superficial digital flexor tendon, tendon injuries, suspensory ligament

#### **1. Introduction**

Tendon and ligament injuries are the most common traumas in horses (*Equus caballus*) irrespective of the age and breed [1]. Based on the statistics, injuries in sport horses can achieve 86% of a total morbidity rate, with 37% of them accounting for muscle, tendon and ligament pathologies. As a rule, musculoskeletal injuries require long-term recovery for 9–12 months [2]. Tendon and ligament injuries result in a loss of performance of the horses and frequently cause discomfort or pain. Therefore, the animals are unable to participate in competitions for a long period of time. Complications of these injuries include chronic musculoskeletal diseases. They result in degenerative-dystrophic damage of collagen fibers of tendons as well as adjacent and underlying tissues. Incomplete tissue recovery leads to recurrent injuries in 80% of horses with treated tendon micro- and macroruptures within 3–12 months after the first injury [3].

Methods of regenerative medicine are used for appropriate regeneration of damaged tissue in animals. These include the administration of stem cells [4, 5] and recombinant proteins, as well as gene therapy. These methods are presently the most advanced and promising approaches to manage musculoskeletal disorders [6]. However, regenerative medicine is mainly targeted toward the treatment of human disorders. Animals are mostly considered as models to test drugs and devices intended for human use. Drugs developed for human use can be ineffective for the

treatment of animal diseases due to partial homology of physiological processes. When given to animals, such products can cause long-term immunological disorders, decrease the efficacy of a subsequent treatment or even cause adverse side effects including anaphylactic shock.

In a veterinary practice, an autologous graft rejection can be avoided in 85% of cases [7]. The likelihood of immune responses in animals to the administration of allogenic or autologous species-specific stem cells is also low [8–10]. However, full homology can be of vital importance when applying more advanced therapeutic approaches such as gene therapy.

Gene therapy is a novel, rapidly developing trend in regenerative medicine and veterinary, which can provide continuous stimulation of regeneration. When this approach is used, a recipient's body constantly synthesizes its own substances instead of a multiple drug (pharmaceuticals, recombinant proteins and so on) delivery. Gene therapy has been successful in the treatment of various human disorders [11, 12], and it can be used to treat animals [13]. However, species-specific recombinant genes that would provide biological activity and at the same time have no immunological side effects should be developed for this purpose. A therapeutic potential of gene therapy for the treatment of tendinitis and desmitis in sport horses will be discussed in detail in this review, especially those related to a series of papers recently covering gene therapy in horses [14–17].

#### **2. Use of a species-specific plasmid construct in the treatment of traumas in horses**

#### **2.1 Description of the plasmid construct**

A group of scientists from Russia and Great Britain developed and tested a drug for gene therapy of soft tissue injuries in horses. This gene construct is plasmid DNA (pDNA), encoding animal-specific genes (**Figure 1**). A plasmid construct pBUDK-ecVEGF164-ecFGF2 based on a pBudCE4.1 vector contained codon-optimized sequences of horse genes, a vascular endothelial growth factor (VEGF164) and a basic fibroblast growth factor (FGF2) under eukaryotic promoters (EF-1α and CMV promoters, respectively) [14].

These genes were selected with good reason as VEGF stimulates synthesis of DNA and proliferation of cells involved in antiapoptotic signaling pathways. It promotes the proliferation and migration of endothelial cells, stimulates angiogenesis and attracts endothelial progenitor cells from bone marrow, stimulates the activity of pericytes and stabilizes newly formed blood vessels. VEGF is also a

**19**

**Figure 2.**

*Gene Therapy as a Modern Method of Treating Naturally Occurring Tendinitis and Desmitis…*

chemoattractant for smooth muscle cells, monocytes, macrophages and granulocytes. All of these are involved in the process of wound healing. VEGF also increases vessel wall permeability at the site of injury that enhances the formation of granula-

In turn, FGF2 exerts a wide range of mitogenic and angiogenic activities and is a neurotrophic factor. In intact tissues, it is present in a basement membrane of the epithelium and in the subendothelial extracellular matrix of blood vessels. It stimulates cell proliferation, regeneration of nervous, muscle and connective tissues. Also, FGF2 activates de novo formation of blood vessels by triggering the process of

Thus, a mechanism of action of gene therapy comprising VEGF and FGF2 is to stimulate synthesis of proteins in a recipient that enhances the vascularization of damaged tissues. This, in turn, leads to a higher regeneration rate. Both VEGF and FGF2 are well-known growth factors with a wide range of mitogenic and angiogenic activity. They also contribute to regeneration of muscle and connective tissues. What is more important is that in combination these factors demonstrate synergis-

This gene product has been tested for identification and functional activity in mammal cells in the laboratory. Full genetic sequencing and restriction analysis with subsequent agarose gel electrophoresis demonstrated a complete compliance

Biosynthesis of recombinant VEGF164 and FGF2 in transfected immortalized HEK293FT cells was confirmed by an immunofluorescence assay with anti-VEGF and anti-FGF2 antibodies (**Figure 3**), which confirmed co-expression of recombi-

The biological activity of the рBUDK-ecVEGF164-ecFGF2 DNA plasmid was evaluated during *in vitro* experiments in horse stem cells. For this purpose, horse MSCs were isolated under a standard procedure by incubating a subcutaneous adipose tissue homogenate with crab collagenase. The cells obtained were identified as MSCs with flow cytofluorometry-more than 80% of them expressed MSC-specific markers (Thy-1 in 99.8% and CD44 in 83% of the cells) and no CD34 or CD45 was

*Analysis of VEGF164 and FGF2 biosynthesis by immunoblotting in HEK293FT cells after transfection. Electrophoresis in 12% SDS-PAGE gel was performed in Laemmli system. Antibodies against human actin, VEGF and FGF2 were used. Bands correspond to human actin (42 kDa), horse VEGF164 (22.3 kDa) and horse FGF2 (17.2 kDa). M-molecular weight protein marker (GE LifeSciences RPN756E); Ec-HEK293FT cells* 

*transfected with pBUDK-ecVEGF164-ecFGF2; control nontransfected cells [14].*

tic effects that surpass those of therapy with just one growth factor [19].

with the claimed structure of pBUDK-ecVEGF164-ecFGF2 (**Figure 2**).

*DOI: http://dx.doi.org/10.5772/intechopen.92352*

nant proteins in transgenic cells [14, 14].

tion tissue [14].

angiogenesis [18].

**Figure 1.** *Map of recombinant plasmid pBUDK-ecVEGF164-ecFGF2 [14].*

*Gene Therapy as a Modern Method of Treating Naturally Occurring Tendinitis and Desmitis… DOI: http://dx.doi.org/10.5772/intechopen.92352*

chemoattractant for smooth muscle cells, monocytes, macrophages and granulocytes. All of these are involved in the process of wound healing. VEGF also increases vessel wall permeability at the site of injury that enhances the formation of granulation tissue [14].

In turn, FGF2 exerts a wide range of mitogenic and angiogenic activities and is a neurotrophic factor. In intact tissues, it is present in a basement membrane of the epithelium and in the subendothelial extracellular matrix of blood vessels. It stimulates cell proliferation, regeneration of nervous, muscle and connective tissues. Also, FGF2 activates de novo formation of blood vessels by triggering the process of angiogenesis [18].

Thus, a mechanism of action of gene therapy comprising VEGF and FGF2 is to stimulate synthesis of proteins in a recipient that enhances the vascularization of damaged tissues. This, in turn, leads to a higher regeneration rate. Both VEGF and FGF2 are well-known growth factors with a wide range of mitogenic and angiogenic activity. They also contribute to regeneration of muscle and connective tissues. What is more important is that in combination these factors demonstrate synergistic effects that surpass those of therapy with just one growth factor [19].

This gene product has been tested for identification and functional activity in mammal cells in the laboratory. Full genetic sequencing and restriction analysis with subsequent agarose gel electrophoresis demonstrated a complete compliance with the claimed structure of pBUDK-ecVEGF164-ecFGF2 (**Figure 2**).

Biosynthesis of recombinant VEGF164 and FGF2 in transfected immortalized HEK293FT cells was confirmed by an immunofluorescence assay with anti-VEGF and anti-FGF2 antibodies (**Figure 3**), which confirmed co-expression of recombinant proteins in transgenic cells [14, 14].

The biological activity of the рBUDK-ecVEGF164-ecFGF2 DNA plasmid was evaluated during *in vitro* experiments in horse stem cells. For this purpose, horse MSCs were isolated under a standard procedure by incubating a subcutaneous adipose tissue homogenate with crab collagenase. The cells obtained were identified as MSCs with flow cytofluorometry-more than 80% of them expressed MSC-specific markers (Thy-1 in 99.8% and CD44 in 83% of the cells) and no CD34 or CD45 was

#### **Figure 2.**

*Equine Science*

effects including anaphylactic shock.

approaches such as gene therapy.

**of traumas in horses**

recently covering gene therapy in horses [14–17].

**2.1 Description of the plasmid construct**

and CMV promoters, respectively) [14].

*Map of recombinant plasmid pBUDK-ecVEGF164-ecFGF2 [14].*

treatment of animal diseases due to partial homology of physiological processes. When given to animals, such products can cause long-term immunological disorders, decrease the efficacy of a subsequent treatment or even cause adverse side

In a veterinary practice, an autologous graft rejection can be avoided in 85% of cases [7]. The likelihood of immune responses in animals to the administration of allogenic or autologous species-specific stem cells is also low [8–10]. However, full homology can be of vital importance when applying more advanced therapeutic

Gene therapy is a novel, rapidly developing trend in regenerative medicine and veterinary, which can provide continuous stimulation of regeneration. When this approach is used, a recipient's body constantly synthesizes its own substances instead of a multiple drug (pharmaceuticals, recombinant proteins and so on) delivery. Gene therapy has been successful in the treatment of various human disorders [11, 12], and it can be used to treat animals [13]. However, species-specific recombinant genes that would provide biological activity and at the same time have no immunological side effects should be developed for this purpose. A therapeutic potential of gene therapy for the treatment of tendinitis and desmitis in sport horses will be discussed in detail in this review, especially those related to a series of papers

**2. Use of a species-specific plasmid construct in the treatment** 

A group of scientists from Russia and Great Britain developed and tested a drug

for gene therapy of soft tissue injuries in horses. This gene construct is plasmid DNA (pDNA), encoding animal-specific genes (**Figure 1**). A plasmid construct pBUDK-ecVEGF164-ecFGF2 based on a pBudCE4.1 vector contained codon-optimized sequences of horse genes, a vascular endothelial growth factor (VEGF164) and a basic fibroblast growth factor (FGF2) under eukaryotic promoters (EF-1α

These genes were selected with good reason as VEGF stimulates synthesis of DNA and proliferation of cells involved in antiapoptotic signaling pathways. It promotes the proliferation and migration of endothelial cells, stimulates angiogenesis and attracts endothelial progenitor cells from bone marrow, stimulates the activity of pericytes and stabilizes newly formed blood vessels. VEGF is also a

**18**

**Figure 1.**

*Analysis of VEGF164 and FGF2 biosynthesis by immunoblotting in HEK293FT cells after transfection. Electrophoresis in 12% SDS-PAGE gel was performed in Laemmli system. Antibodies against human actin, VEGF and FGF2 were used. Bands correspond to human actin (42 kDa), horse VEGF164 (22.3 kDa) and horse FGF2 (17.2 kDa). M-molecular weight protein marker (GE LifeSciences RPN756E); Ec-HEK293FT cells transfected with pBUDK-ecVEGF164-ecFGF2; control nontransfected cells [14].*

#### **Figure 3.**

*Immunofluorescence analysis of VEGF164 and FGF2 biosynthesis in HEK293FT cells, 48 h after transfection. (A) Negative control: HEK293FT cells without pDNA transfection, nuclei-stained DAPI (blue). (B)–(D) HEK293FT cells transfected with pBUDK-ecVEGF164-ecFGF2. b) Staining with primary antibody against VEGF and secondary antibody, conjugated with a fluorescent label Alexa Fluor 555 (red). (C) Staining with primary antibody against FGF2 and secondary antibody, conjugated with a fluorescent label Alexa Fluor 488 (green). (D) Overlay image of a, c and d: VEGF (red), FGF2 (green), cell nuclei stained with DAPI (blue) [14].*

expressed (data not provided). Thus, according to literature comparisons and based upon the laboratory data, the cells obtained were equine MSCs [20].

Genetic modification of horse MSCs with pDNA рBUDK-ecVEGF164-ecFGF2 showed that transfected cells possess a higher ability to form a capillary-like networks on the Matrigel™ matrix as compared to intact cells (р < 0.005) (**Figure 4**).

#### **2.2 Use of the plasmid construct in vivo**

Due to a high incidence of tendon and ligament injuries in horses, a high rate of recurrent traumas and a prolonged period of recovery that normally lasts for several months and up to 15 months with severe injuries, these injuries are a medical and surgical challenge. Even when modern technologies are applied, in many cases, damaged tendons and ligaments demonstrate biochemical and ultrastructural abnormalities after 12 months and preinjury biomechanical properties are not completely restored [21].

A total of 12 horses were given gene therapy [15, 16] through in vivo trials of the treatment. Out of them, eight horses had naturally occurring injuries of the superficial digital flexor tendon (SDFT; tendinitis) and four horses had suspensory ligament branch (SLB) desmitis. All the horses had spontaneous SDFT and SLB injuries and were included into the study from 2015 to 2017 undergoing treatment in the veterinary clinic "New Century" at the Moscow State Academy of Veterinary and Biotechnologies, Moscow.

**21**

**Figure 5.**

**Figure 4.**

*modification of mesenchymal stem cells.*

*Gene Therapy as a Modern Method of Treating Naturally Occurring Tendinitis and Desmitis…*

Gene therapy of the four horses with injured SLBs showed that before treatment all horses had pain in the injured leg. By day 40 after treatment, no animals had any sings of inflammation at the site of injury, nor was there a change in the skin surface temperature within the area of injury, swelling or tenderness when palpated. By day 20 after treatment, lameness significantly reduced as compared to the baseline. By 12 weeks and during subsequent follow-up examinations, no horses were lame. Ultrasound parameters in damaged SLB began to improve 20 days after the onset of treatment, this positive tendency remaining thereafter. Parameters such as changes of the zone of damage, echogenicity and fiber alignment made this especially evident. When the treated horses started doing a program of physical exercise, the ligament architecture constantly improved, as indicated by their

Based on the examination results, only one horse had no significant ultrasound improvements in the first 90 days after pDNA injection. On days 20 and 40, this horse had new hypoechoic lesions that indicate a nonstable healing process. By 120–180 days after treatment, this horse had a noticeable ultrasound improvement

Color Doppler ultrasonography (CDU) demonstrated evidently increased blood supply by day 20 after pDNA injection. This tendency remained up to day 40 and

*Ultrasound images prior to plasmid DNA encoding VEGF164 and FGF2 genes on day 0 (A), 20 (B), 40 (C), 90 (D), 180 (E) and 300 (F) after administration in horse with SLB desmopathy. Arrows indicate lesion.*

In vitro *angiogenesis assay using Matrigel to characterize the proangiogenic effect mediated by genetic* 

*DOI: http://dx.doi.org/10.5772/intechopen.92352*

longitudinal alignment and length (**Figure 5**).

in the site of injury.

*Gene Therapy as a Modern Method of Treating Naturally Occurring Tendinitis and Desmitis… DOI: http://dx.doi.org/10.5772/intechopen.92352*

Gene therapy of the four horses with injured SLBs showed that before treatment all horses had pain in the injured leg. By day 40 after treatment, no animals had any sings of inflammation at the site of injury, nor was there a change in the skin surface temperature within the area of injury, swelling or tenderness when palpated. By day 20 after treatment, lameness significantly reduced as compared to the baseline. By 12 weeks and during subsequent follow-up examinations, no horses were lame.

Ultrasound parameters in damaged SLB began to improve 20 days after the onset of treatment, this positive tendency remaining thereafter. Parameters such as changes of the zone of damage, echogenicity and fiber alignment made this especially evident. When the treated horses started doing a program of physical exercise, the ligament architecture constantly improved, as indicated by their longitudinal alignment and length (**Figure 5**).

Based on the examination results, only one horse had no significant ultrasound improvements in the first 90 days after pDNA injection. On days 20 and 40, this horse had new hypoechoic lesions that indicate a nonstable healing process. By 120–180 days after treatment, this horse had a noticeable ultrasound improvement in the site of injury.

Color Doppler ultrasonography (CDU) demonstrated evidently increased blood supply by day 20 after pDNA injection. This tendency remained up to day 40 and

**Figure 4.**

*Equine Science*

expressed (data not provided). Thus, according to literature comparisons and based

*Immunofluorescence analysis of VEGF164 and FGF2 biosynthesis in HEK293FT cells, 48 h after transfection. (A) Negative control: HEK293FT cells without pDNA transfection, nuclei-stained DAPI (blue). (B)–(D) HEK293FT cells transfected with pBUDK-ecVEGF164-ecFGF2. b) Staining with primary antibody against VEGF and secondary antibody, conjugated with a fluorescent label Alexa Fluor 555 (red). (C) Staining with primary antibody against FGF2 and secondary antibody, conjugated with a fluorescent label Alexa Fluor 488 (green). (D) Overlay image of a, c and d: VEGF (red), FGF2 (green), cell nuclei stained with DAPI (blue)* 

showed that transfected cells possess a higher ability to form a capillary-like networks on the Matrigel™ matrix as compared to intact cells (р < 0.005)

Genetic modification of horse MSCs with pDNA рBUDK-ecVEGF164-ecFGF2

Due to a high incidence of tendon and ligament injuries in horses, a high rate of recurrent traumas and a prolonged period of recovery that normally lasts for several months and up to 15 months with severe injuries, these injuries are a medical and surgical challenge. Even when modern technologies are applied, in many cases, damaged tendons and ligaments demonstrate biochemical and ultrastructural abnormalities after 12 months and preinjury biomechanical properties are not

A total of 12 horses were given gene therapy [15, 16] through in vivo trials of the treatment. Out of them, eight horses had naturally occurring injuries of the superficial digital flexor tendon (SDFT; tendinitis) and four horses had suspensory ligament branch (SLB) desmitis. All the horses had spontaneous SDFT and SLB injuries and were included into the study from 2015 to 2017 undergoing treatment in the veterinary clinic "New Century" at the Moscow State Academy of Veterinary

upon the laboratory data, the cells obtained were equine MSCs [20].

**20**

(**Figure 4**).

**Figure 3.**

*[14].*

completely restored [21].

and Biotechnologies, Moscow.

**2.2 Use of the plasmid construct in vivo**

In vitro *angiogenesis assay using Matrigel to characterize the proangiogenic effect mediated by genetic modification of mesenchymal stem cells.*

#### **Figure 5.**

*Ultrasound images prior to plasmid DNA encoding VEGF164 and FGF2 genes on day 0 (A), 20 (B), 40 (C), 90 (D), 180 (E) and 300 (F) after administration in horse with SLB desmopathy. Arrows indicate lesion.*

was high until day 90. By day 180 after plasmid injection, CDU parameters reduced to baseline values in most horses (with the baseline set at values within an intact limb of the same animal). There was no significant correlation between the soft tissue damage severity prior to treatment and post-treatment CDU parameters.

Ultrasound parameters of SDFT lesions in most horses began to improve 20 days after treatment [15]. This positive tendency remained during the follow-up period. With the onset of training, healing of the damaged tissue increased in the tendon treated. This manifested as a longitudinal alignment of fibers and an increase in their length [16].

After treatment, the echogenicity of the damaged SDFT constantly and significantly decreased from day 0 to day 60 in all horses except one. In 3 months after the beginning of treatment, the echostructure was more uniform in most horses, with collagen fibers arranged in parallel to the longitudinal axis.

A linear fiber pattern in horses with SDFT injuries also improved during the study, but this was happening more slowly when compared to the echogenicity. Within nine months, there were scarcely any signs of tendon damage in most of the horses with the SDFT injury. They had correct alignment and a well-arranged longitudinal pattern of fibers.

Doppler ultrasonography demonstrated a significant improvement in blood supply of the affected areas by day 20 [16]. This tendency continued for 90–120 days, with a peak that was reached on day 40. After postinjection day 180, the vascularization decreased to baseline levels (as in healthy limbs of the same animal). There was no significant correlation between the injury severity before treatment and CDU parameters afterwards. There were no significant differences in CDU images between horses with SDFT and SLB injuries after treatment; CDU changes were strictly individual. CDU changes can be due to hypervascularity being natural in the process of healing. Normally, tendons and ligaments are hypovascular [22]. A short-term increase in blood flow results in response to damage-associated tissue hypoxia. We propose that the gene therapy enhanced this effect markedly.

To identify possible side effects, all horses were constantly examined by a veterinarian in the clinic from the time of plasmid administration until 12 months later. Horses did not have any side effects to the pDNA administration, and horse age, gender and the duration of lameness had no effect on the outcome of gene therapy. The main differences in clinical outcomes were determined by the extent and site of the animal's soft tissue damage sustained before treatment. The study results showed that only one horse with a serious injury of the SLB and body did not respond to treatment, and it was lame for the first 3 months after the onset of therapy. Only one horse that recovered after gene therapy (initially with SDFT tendonitis) suffered a repeated injury at the same site 6 months after treatment [15, 16]. In the 12-month follow-up after treatment, owners of the other horses rated gene therapy results as good or excellent in terms of sporting success.

#### **3. Discussion on the use of gene therapy in horses**

One should emphasize that the disappearance of lameness with treated tendinitis or desmitis in a horse does not mean absolute tissue regeneration. In these studies, rapid and mostly complete regeneration of both the tendon and ligament occurred within 2–3 months of treatment, which included a single injection of pBUDK-ecVEGF164-ecFGF2. This was confirmed by increased echogenicity and homogeneity at the site of injury, as well as an increased percentage of parallel collagen fibers.

**23**

*Gene Therapy as a Modern Method of Treating Naturally Occurring Tendinitis and Desmitis…*

Thus, the study data are encouraging and demonstrated a positive effect of using pDNA encoding horse-specific proteins at early stages of healing of traumatic tendinitis and desmitis, when injected into the site of injury. In part, this can be explained by coincidence with conditions and stages of normal tendon healing. However, the horses included into the study had moderate or severe tendon injuries. It is well known that such injuries are associated with a poor prognosis in response

A drawback of these clinical studies is that they did not identify an exact mecha-

Therefore, gene therapy, as one of the most advanced technologies in medicine, is a promising treatment for hereditary diseases and in addition offers new possibilities for a clinical management of numerous orthopedic disorders, including tendon and ligament injuries [23–25]. The use of direct gene therapy with speciesspecific growth factors is quite promising for the treatment of orthopedic disorders not only in horses but also in other animal species and in people [17]. The successful use of direct gene therapy with a similar plasmid construct based on dog-specific VEGF164 genes and bone morphogenetic protein (BMP2) to treat an anterior cruciate ligament injury in large dogs has been previously reported [26]. Moreover, there is a case report on using gene therapy to treat patients with critical lower limb ischemia [27]. Finally, plasmid DNA pl-VEGF165 (approved as Neovasculgen), encoding human VEGF165, has demonstrated its safety and efficacy in the treatment of atherosclerotic peripheral arterial disease in patients with chronic lower limb ischemia without side effects [28]. The high efficacy and safety of direct gene therapy have been demonstrated in all of these cases. There are also numerous benefits of using pDNA rather than recombinant viruses. Plasmids are relatively easy to construct, can be produced in large quantities and provide a safe method of delivery with low levels of immunogenicity associated with delivery. They can often be kept at room temperature for long periods of time, which is especially useful in clinical settings. Although they have lower levels of gene transfer, the studies carried out in the horse show that delivery is appropriate and efficient in these circumstances as it

VEGF and FGF2 gene therapy's direct effects on the regeneration of tendon and ligament injuries in horses should be further evaluated in a larger number of experimental animals, for a longer follow-up period and in a randomized controlled clinical study. Complete and more detailed results could also be obtained by histological examination and immunohistochemistry of samples and biopsy materials given the right conditions. Factors such as gene expression levels in tissues, collagen analysis, identification and quantification, the functional and intracellular distribution of

nism of action of direct gene therapy with pBUDK-ecVEGF164-ecFGF2 on the regeneration of damaged horse tendons and ligaments. Since the horses had fully recovered, the investigators considered possible histological interventions to take tissue samples as inappropriate. If histological samples could be taken looking at the cell types, checking for inflammatory reactions and cells associated with inflammation and immune responses would be advantageous for confirming the lack of immune response at a cellular level. In addition, investigating the healing mechanism via histology by looking at collagen type and wound repair would further the knowledge in this area. Adding RNA and protein expression studies would also help understand the mechanisms involved in this therapy. The pDNA administration used also avoids possible side effects associated with vector-mediated insertional mutagenesis when integration into the patient's genome is the long-term aim. This is not necessary in these disorders as long-term correction/replacement is not required. As previous reports of treatment results of such tendinitis and desmitis in horses are lacking, results of this gene therapy cannot be compared with those of

*DOI: http://dx.doi.org/10.5772/intechopen.92352*

to standard treatments.

other treatment methods.

was delivered directly to the injured area.

#### *Gene Therapy as a Modern Method of Treating Naturally Occurring Tendinitis and Desmitis… DOI: http://dx.doi.org/10.5772/intechopen.92352*

Thus, the study data are encouraging and demonstrated a positive effect of using pDNA encoding horse-specific proteins at early stages of healing of traumatic tendinitis and desmitis, when injected into the site of injury. In part, this can be explained by coincidence with conditions and stages of normal tendon healing. However, the horses included into the study had moderate or severe tendon injuries. It is well known that such injuries are associated with a poor prognosis in response to standard treatments.

A drawback of these clinical studies is that they did not identify an exact mechanism of action of direct gene therapy with pBUDK-ecVEGF164-ecFGF2 on the regeneration of damaged horse tendons and ligaments. Since the horses had fully recovered, the investigators considered possible histological interventions to take tissue samples as inappropriate. If histological samples could be taken looking at the cell types, checking for inflammatory reactions and cells associated with inflammation and immune responses would be advantageous for confirming the lack of immune response at a cellular level. In addition, investigating the healing mechanism via histology by looking at collagen type and wound repair would further the knowledge in this area. Adding RNA and protein expression studies would also help understand the mechanisms involved in this therapy. The pDNA administration used also avoids possible side effects associated with vector-mediated insertional mutagenesis when integration into the patient's genome is the long-term aim. This is not necessary in these disorders as long-term correction/replacement is not required. As previous reports of treatment results of such tendinitis and desmitis in horses are lacking, results of this gene therapy cannot be compared with those of other treatment methods.

Therefore, gene therapy, as one of the most advanced technologies in medicine, is a promising treatment for hereditary diseases and in addition offers new possibilities for a clinical management of numerous orthopedic disorders, including tendon and ligament injuries [23–25]. The use of direct gene therapy with speciesspecific growth factors is quite promising for the treatment of orthopedic disorders not only in horses but also in other animal species and in people [17]. The successful use of direct gene therapy with a similar plasmid construct based on dog-specific VEGF164 genes and bone morphogenetic protein (BMP2) to treat an anterior cruciate ligament injury in large dogs has been previously reported [26]. Moreover, there is a case report on using gene therapy to treat patients with critical lower limb ischemia [27]. Finally, plasmid DNA pl-VEGF165 (approved as Neovasculgen), encoding human VEGF165, has demonstrated its safety and efficacy in the treatment of atherosclerotic peripheral arterial disease in patients with chronic lower limb ischemia without side effects [28]. The high efficacy and safety of direct gene therapy have been demonstrated in all of these cases. There are also numerous benefits of using pDNA rather than recombinant viruses. Plasmids are relatively easy to construct, can be produced in large quantities and provide a safe method of delivery with low levels of immunogenicity associated with delivery. They can often be kept at room temperature for long periods of time, which is especially useful in clinical settings. Although they have lower levels of gene transfer, the studies carried out in the horse show that delivery is appropriate and efficient in these circumstances as it was delivered directly to the injured area.

VEGF and FGF2 gene therapy's direct effects on the regeneration of tendon and ligament injuries in horses should be further evaluated in a larger number of experimental animals, for a longer follow-up period and in a randomized controlled clinical study. Complete and more detailed results could also be obtained by histological examination and immunohistochemistry of samples and biopsy materials given the right conditions. Factors such as gene expression levels in tissues, collagen analysis, identification and quantification, the functional and intracellular distribution of

*Equine Science*

their length [16].

effect markedly.

longitudinal pattern of fibers.

good or excellent in terms of sporting success.

**3. Discussion on the use of gene therapy in horses**

was high until day 90. By day 180 after plasmid injection, CDU parameters reduced to baseline values in most horses (with the baseline set at values within an intact limb of the same animal). There was no significant correlation between the soft tissue damage severity prior to treatment and post-treatment CDU parameters.

Ultrasound parameters of SDFT lesions in most horses began to improve 20 days after treatment [15]. This positive tendency remained during the follow-up period. With the onset of training, healing of the damaged tissue increased in the tendon treated. This manifested as a longitudinal alignment of fibers and an increase in

After treatment, the echogenicity of the damaged SDFT constantly and significantly decreased from day 0 to day 60 in all horses except one. In 3 months after the beginning of treatment, the echostructure was more uniform in most horses, with

A linear fiber pattern in horses with SDFT injuries also improved during the study, but this was happening more slowly when compared to the echogenicity. Within nine months, there were scarcely any signs of tendon damage in most of the horses with the SDFT injury. They had correct alignment and a well-arranged

To identify possible side effects, all horses were constantly examined by a veterinarian in the clinic from the time of plasmid administration until 12 months later. Horses did not have any side effects to the pDNA administration, and horse age, gender and the duration of lameness had no effect on the outcome of gene therapy. The main differences in clinical outcomes were determined by the extent and site of the animal's soft tissue damage sustained before treatment. The study results showed that only one horse with a serious injury of the SLB and body did not respond to treatment, and it was lame for the first 3 months after the onset of therapy. Only one horse that recovered after gene therapy (initially with SDFT tendonitis) suffered a repeated injury at the same site 6 months after treatment [15, 16]. In the 12-month follow-up after treatment, owners of the other horses rated gene therapy results as

One should emphasize that the disappearance of lameness with treated tendinitis or desmitis in a horse does not mean absolute tissue regeneration. In these studies, rapid and mostly complete regeneration of both the tendon and ligament occurred within 2–3 months of treatment, which included a single injection of pBUDK-ecVEGF164-ecFGF2. This was confirmed by increased echogenicity and homogeneity at the site of injury, as well as an increased percentage of parallel

Doppler ultrasonography demonstrated a significant improvement in blood supply of the affected areas by day 20 [16]. This tendency continued for 90–120 days, with a peak that was reached on day 40. After postinjection day 180, the vascularization decreased to baseline levels (as in healthy limbs of the same animal). There was no significant correlation between the injury severity before treatment and CDU parameters afterwards. There were no significant differences in CDU images between horses with SDFT and SLB injuries after treatment; CDU changes were strictly individual. CDU changes can be due to hypervascularity being natural in the process of healing. Normally, tendons and ligaments are hypovascular [22]. A short-term increase in blood flow results in response to damage-associated tissue hypoxia. We propose that the gene therapy enhanced this

collagen fibers arranged in parallel to the longitudinal axis.

**22**

collagen fibers.

proteins and further studies of pathological biochemistry will help identify the main mechanisms of action.

### **4. Conclusions**

The introduction of gene therapy in veterinary clinics becomes ever more possible; however, there are issues that require solutions. The future of veterinary gene therapy seems promising thanks to the studies described, and many other therapies are likely to be approved for use in both human and animal medicine [17].

### **Acknowledgements**

This study was supported by the Russian Government Program of Competitive Growth of Kazan Federal University. Albert A. Rizvanov (https://orcid.org/0000- 0002-9427-5739) was supported by state assignments 20.5175.2017/6.7 and 17.9783.2017/8.9 of the Ministry of Science and Higher Education of Russian Federation. Catrin S. Rutland (https://orcid.org/0000-0002-2009-4898) was funded by the University of Nottingham.

### **Conflicts of interest**

The authors declare no conflicts of interest.

### **Author details**

Elena Zakirova1 , Kovac Milomir2 , Margarita Zhuravleva1 , Catrin Sian Rutland3 and Albert Rizvanov1,3\*

1 OpenLab Gene and Cell Technologies, Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia

2 Moscow State Academy of Veterinary Medicine and Biotechnology, Moscow, Russia

3 School of Veterinary Medicine and Science, Faculty of Medicine, University of Nottingham, Nottingham, United Kingdom

\*Address all correspondence to: albert.rizvanov@kpfu.ru

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**25**

*Gene Therapy as a Modern Method of Treating Naturally Occurring Tendinitis and Desmitis…*

cat for tibial bone pseudoarthrosis therapy (case report). BioNanoScience.

[9] Gomes IS, de Oliveira VC, Pinheiro AO, Roballo KCS, de Araujo GSM, Veronezi JC, et al. Bone marrow stem cell applied in the canine veterinary clinics. Pesquisa Veterinaria Brasileira. 2017;**37**(10):1139-1145

[10] Zakirova EY, Shalimov DV, Garanina EE, Zhuravleva MN, Rutland CS, Rizvanov AA. Use of biologically active 3D matrix for extensive skin defect treatment in veterinary practice: Case report. Frontiers in Veterinary Science. 2019;**6**

[11] Masgutov R, Chekunov M,

2017;**7**(1):194-198

2016;**10**(1):15-21

Zhuravleva M, Masgutova G, Teplov O, Salikhov R, et al. Use of gene-activated demineralized bone allograft in the therapy of ulnar Pseudarthrosis. Case report. BioNanoScience.

[12] Lee JH, Wang JH, Chen JY, Li F, Edwards TL, Hewitt AW, et al. Gene therapy for visual loss: Opportunities and concerns. Progress in Retinal and

[13] Soboka G, Kemal J, Kefale M. Gene therapeutic enhancement of animal health and performance: Review. Advances in Biological Research.

Generation of plasmid DNA expressing

Eye Research. 2019;**68**:31-53

[14] Litvin YA, Zakirova EY, Zhuravleva MN, Rizvanov AA.

species-specific horse VEGF164 and FGF2 factors for gene therapy. BioNanoScience. 2016;**6**(4):550-553

[15] Kovac M, Litvin YA, Aliev RO, Zakirova EY, Rutland CS, Kiyasov AP, et al. Gene therapy using plasmid DNA encoding vascular endothelial growth factor 164 and fibroblast growth factor

2017;**7**(1):207-211

*DOI: http://dx.doi.org/10.5772/intechopen.92352*

[1] Thorpe CT, Clegg PD, Birch HL. A review of tendon injury: Why is the equine superficial digital flexor tendon most at risk? Equine Veterinary Journal.

[2] O'Meara B, Bladon B, Parkin TDH, Fraser B, Lischer CJ. An investigation of the relationship between race performance and superficial digital flexor tendonitis in the thoroughbred racehorse. Equine Veterinary Journal.

[3] Dowling BA, Dart AJ, Hodgson DR, Smith RKW. Superficial digital flexor tendonitis in the horse. Equine

Veterinary Journal. 2000;**32**(5):369-378

[4] De Schauwer C, van de Walle GR, Van Soom A, Meyer E. Mesenchymal stem cell therapy in horses: Useful beyond orthopedic injuries? The Veterinary Quarterly. 2013;**33**(4):234-241

[5] Zakirova EY, Azizovа DA, Rizvanov AA, Khafizov RG. Case of applying allogenic mesenchymal stem cells of adipogenic origin in veterinary dentistry. Journal of Animal and Veterinary Advances. 2015;**14**:140-143

Springer; 2005. p. 307

2011;**33**(3):298-304

[6] Martinek V, Huard J, Fu FH. Gene therapy in tendon ailments. In:

[7] Patel S, Fanshawe T, Bister D, Cobourne MT. Survival and success of maxillary canine autotransplantation:

A retrospective investigation. European Journal of Orthodontics.

[8] Zakirova EY, Valeeva AN, Masgutov RF, Naumenko EA,

adipose-derived multipotent mesenchymal stromal cells from

Rizvanov AA. Application of allogenic

Maffulli N, Renström P, Leadbetter WB, editors. Tendon Injuries. London:

**References**

2010;**42**(2):174-180

2010;**42**(4):322-326

*Gene Therapy as a Modern Method of Treating Naturally Occurring Tendinitis and Desmitis… DOI: http://dx.doi.org/10.5772/intechopen.92352*

#### **References**

*Equine Science*

**4. Conclusions**

**Acknowledgements**

**Conflicts of interest**

main mechanisms of action.

**24**

**Author details**

Elena Zakirova1

Russia

and Albert Rizvanov1,3\*

, Kovac Milomir2

The authors declare no conflicts of interest.

funded by the University of Nottingham.

Biology, Kazan Federal University, Kazan, Russia

Nottingham, Nottingham, United Kingdom

provided the original work is properly cited.

\*Address all correspondence to: albert.rizvanov@kpfu.ru

, Margarita Zhuravleva1

1 OpenLab Gene and Cell Technologies, Institute of Fundamental Medicine and

proteins and further studies of pathological biochemistry will help identify the

The introduction of gene therapy in veterinary clinics becomes ever more possible; however, there are issues that require solutions. The future of veterinary gene therapy seems promising thanks to the studies described, and many other therapies

This study was supported by the Russian Government Program of Competitive Growth of Kazan Federal University. Albert A. Rizvanov (https://orcid.org/0000- 0002-9427-5739) was supported by state assignments 20.5175.2017/6.7 and 17.9783.2017/8.9 of the Ministry of Science and Higher Education of Russian Federation. Catrin S. Rutland (https://orcid.org/0000-0002-2009-4898) was

are likely to be approved for use in both human and animal medicine [17].

2 Moscow State Academy of Veterinary Medicine and Biotechnology, Moscow,

3 School of Veterinary Medicine and Science, Faculty of Medicine, University of

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Catrin Sian Rutland3

[1] Thorpe CT, Clegg PD, Birch HL. A review of tendon injury: Why is the equine superficial digital flexor tendon most at risk? Equine Veterinary Journal. 2010;**42**(2):174-180

[2] O'Meara B, Bladon B, Parkin TDH, Fraser B, Lischer CJ. An investigation of the relationship between race performance and superficial digital flexor tendonitis in the thoroughbred racehorse. Equine Veterinary Journal. 2010;**42**(4):322-326

[3] Dowling BA, Dart AJ, Hodgson DR, Smith RKW. Superficial digital flexor tendonitis in the horse. Equine Veterinary Journal. 2000;**32**(5):369-378

[4] De Schauwer C, van de Walle GR, Van Soom A, Meyer E. Mesenchymal stem cell therapy in horses: Useful beyond orthopedic injuries? The Veterinary Quarterly. 2013;**33**(4):234-241

[5] Zakirova EY, Azizovа DA, Rizvanov AA, Khafizov RG. Case of applying allogenic mesenchymal stem cells of adipogenic origin in veterinary dentistry. Journal of Animal and Veterinary Advances. 2015;**14**:140-143

[6] Martinek V, Huard J, Fu FH. Gene therapy in tendon ailments. In: Maffulli N, Renström P, Leadbetter WB, editors. Tendon Injuries. London: Springer; 2005. p. 307

[7] Patel S, Fanshawe T, Bister D, Cobourne MT. Survival and success of maxillary canine autotransplantation: A retrospective investigation. European Journal of Orthodontics. 2011;**33**(3):298-304

[8] Zakirova EY, Valeeva AN, Masgutov RF, Naumenko EA, Rizvanov AA. Application of allogenic adipose-derived multipotent mesenchymal stromal cells from

cat for tibial bone pseudoarthrosis therapy (case report). BioNanoScience. 2017;**7**(1):207-211

[9] Gomes IS, de Oliveira VC, Pinheiro AO, Roballo KCS, de Araujo GSM, Veronezi JC, et al. Bone marrow stem cell applied in the canine veterinary clinics. Pesquisa Veterinaria Brasileira. 2017;**37**(10):1139-1145

[10] Zakirova EY, Shalimov DV, Garanina EE, Zhuravleva MN, Rutland CS, Rizvanov AA. Use of biologically active 3D matrix for extensive skin defect treatment in veterinary practice: Case report. Frontiers in Veterinary Science. 2019;**6**

[11] Masgutov R, Chekunov M, Zhuravleva M, Masgutova G, Teplov O, Salikhov R, et al. Use of gene-activated demineralized bone allograft in the therapy of ulnar Pseudarthrosis. Case report. BioNanoScience. 2017;**7**(1):194-198

[12] Lee JH, Wang JH, Chen JY, Li F, Edwards TL, Hewitt AW, et al. Gene therapy for visual loss: Opportunities and concerns. Progress in Retinal and Eye Research. 2019;**68**:31-53

[13] Soboka G, Kemal J, Kefale M. Gene therapeutic enhancement of animal health and performance: Review. Advances in Biological Research. 2016;**10**(1):15-21

[14] Litvin YA, Zakirova EY, Zhuravleva MN, Rizvanov AA. Generation of plasmid DNA expressing species-specific horse VEGF164 and FGF2 factors for gene therapy. BioNanoScience. 2016;**6**(4):550-553

[15] Kovac M, Litvin YA, Aliev RO, Zakirova EY, Rutland CS, Kiyasov AP, et al. Gene therapy using plasmid DNA encoding vascular endothelial growth factor 164 and fibroblast growth factor 2 genes for the treatment of horse tendinitis and Desmitis: Case reports. Frontiers in Veterinary Science. 2017;**4**

[16] Kovac M, Litvin YA, Aliev RO, Zakirova EY, Rutland CS, Kiyasov AP, et al. Gene therapy using plasmid DNA encoding VEGF164 and FGF2 genes: A novel treatment of naturally occurring tendinitis and Desmitis in horses. Frontiers in Pharmacology. 2018;**9**

[17] Rizvanov AA, Kovac M, Rutland CS. Advancing modern equine medicine using gene therapy. Equine Veterinary Education. 2018;**30**(10):516-517

[18] Sahni A, Khorana AA, Baggs RB, Peng H, Francis CW. FGF-2 binding to fibrin(ogen) is required for augmented angiogenesis. Blood. 2006;**107**(1):126-131

[19] Kano MR, Morishita Y, Iwata C, Iwasaka S, Watabe T, Ouchi Y, et al. VEGF-A and FGF-2 synergistically promote neoangiogenesis through enhancement of endogenous PDGF-B-PDGFR beta signaling. Journal of Cell Science. 2005;**118**(16):3759-3768

[20] Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;**8**(4):315-317

[21] Yang G, Rothrauff BB, Tuan RS. Tendon and ligament regeneration and repair: Clinical relevance and developmental paradigm. Birth Defects Research Part C. 2013;**99**(3):203-222

[22] Carvalho AD, Badial PR, Alvarez LEC, Yamada ALM, Borges AS, Deffune E, et al. Equine tendonitis therapy using mesenchymal stem cells and platelet concentrates: A randomized controlled trial. Stem Cell Research & Therapy. 2013;**4**

[23] Evans CH, Ghivizzani SC, Robbins PD. Orthopedic gene therapy in 2008. Molecular Therapy. 2009;**17**(2):231-244

[24] Bosch G, Moleman M, Barneveld A, van Weeren PR, van Schie HTM. The effect of platelet-rich plasma on the neovascularization of surgically created equine superficial digital flexor tendon lesions. Scandinavian Journal of Medicine & Science in Sports. 2011;**21**(4):554-561

[25] Tang JB, Wu YF, Cao Y, Chen CH, Zhou YL, Avanessian B, et al. Basic FGF or VEGF gene therapy corrects insufficiency in the intrinsic healing capacity of tendons. Scientific Reports-UK. 2016;**6**

[26] Zakirova EY, Vasin NN, Zhuravleva MN, Rizvanov AA. Case report of application gene construction with VEGF and BMP2 in restoration of tear in the anterior cruciate ligament of a large breed dog. Genes to Cells. 2014;**9**:93-95

[27] Plotnikov MV, Rizvanov AA, Masgutov RF, Mavlikeev MO, Salafutdinov II, Gazizov IM, et al. The first clinical experience of direct gene therapy using VEGF and bFGF in treatment patients with critical lower limb ischemia. Cellular Transplantion and Tissue Engineering. 2012;**7**:180-184

[28] Deev R, Plaksa I, Bozo I, Mzhavanadze N, Suchkov I, Chervyakov Y, et al. Results of 5-year follow-up study in patients with peripheral artery disease treated with PL-VEGF165 for intermittent claudication. Therapeutic Advances in Cardiovascular Disease. 2018;**12**(9):237-246

**27**

**Chapter 3**

**Abstract**

adeno-associated virus

**1. Introduction**

Gene Therapy for the Treatment

Osteoarthritis (OA) is the predominant cause of lameness in horses. As in humans, the clinical symptoms of equine OA are persistent pain and dysfunction of the affected joint. Its pathology is similarly marked by progressive deterioration of the articular cartilage, subchondral bone sclerosis, marginal osteophytes, soft tissue inflammation and joint effusion. Disease pathogenesis is mediated by elevated levels of inflammatory cytokines and proteolytic enzymes in the articular tissues and synovial fluid. Existing pharmacologic agents can alleviate OA joint pain; none are able to inhibit erosive disease progression. As several gene-based treatments for human disease have received approval by the Food and Drug Administration (FDA), the transition to veterinary medicine will almost certainly follow. Several viral vector systems have demonstrated highly efficient gene transfer to the equine joint, enabling expression of therapeutic transgenes at efficacious levels for well over a year. Because of its large size, the equine joint is well suited to studies of genebased therapies for arthritic disease. The forelimb joints are vulnerable to OA onset, and treatment and diagnostic modalities are the same in humans and horses. Here, we discuss the various gene-transfer approaches under investigation and the current

progress toward the development an effective gene therapy for equine OA.

Osteoarthritis (OA) is a chronic, painful, degenerative, often debilitating condition common in weight-bearing joints of both humans and horses. In humans the knees and hips are predominately affected, while in the horse the metacarpophalangeal and carpal joints of the forelimb are the primary sites of onset. In both species, the pathology of OA is marked by the gradual, persistent erosion of the articular cartilage, development of osteophytes at the joint margins, sclerotic growth of subchondral bone, synovitis and joint effusion [1]. Biochemical analyses reveal that the signaling molecules and pathways that drive the inflammatory and degenerative processes in both species are identical [2]. OA is incurable, difficult to manage and often progresses to disabling joint failure. It is estimated that over 50 million people in the US alone have symptomatic OA. Spontaneous joint disease is a common

**Keywords:** osteoarthritis, lameness, interleukin-1, IL-1Ra, gene therapy,

*Rachael Levings, Andrew Smith, Padraic P. Levings,* 

*Glyn D. Palmer, Anthony Dacanay, Patrick Colahan* 

of Equine Osteoarthritis

*and Steven C. Ghivizzani*

#### **Chapter 3**

*Equine Science*

2 genes for the treatment of horse tendinitis and Desmitis: Case reports. Frontiers in Veterinary Science. 2017;**4** [23] Evans CH, Ghivizzani SC, Robbins PD. Orthopedic gene therapy in 2008. Molecular Therapy.

[24] Bosch G, Moleman M, Barneveld A, van Weeren PR, van Schie HTM. The effect of platelet-rich plasma on the neovascularization of surgically created equine superficial digital flexor tendon lesions. Scandinavian Journal of Medicine & Science in Sports.

[25] Tang JB, Wu YF, Cao Y, Chen CH, Zhou YL, Avanessian B, et al. Basic FGF or VEGF gene therapy corrects insufficiency in the intrinsic healing capacity of tendons. Scientific

2009;**17**(2):231-244

2011;**21**(4):554-561

Reports-UK. 2016;**6**

2014;**9**:93-95

[26] Zakirova EY, Vasin NN,

Zhuravleva MN, Rizvanov AA. Case report of application gene construction with VEGF and BMP2 in restoration of tear in the anterior cruciate ligament of a large breed dog. Genes to Cells.

[27] Plotnikov MV, Rizvanov AA, Masgutov RF, Mavlikeev MO, Salafutdinov II, Gazizov IM, et al. The first clinical experience of direct gene therapy using VEGF and bFGF in treatment patients with critical lower limb ischemia. Cellular Transplantion and Tissue Engineering. 2012;**7**:180-184

[28] Deev R, Plaksa I, Bozo I, Mzhavanadze N, Suchkov I,

in Cardiovascular Disease.

2018;**12**(9):237-246

Chervyakov Y, et al. Results of 5-year follow-up study in patients with peripheral artery disease treated with PL-VEGF165 for intermittent claudication. Therapeutic Advances

[16] Kovac M, Litvin YA, Aliev RO, Zakirova EY, Rutland CS, Kiyasov AP, et al. Gene therapy using plasmid DNA encoding VEGF164 and FGF2 genes: A novel treatment of naturally occurring tendinitis and Desmitis in horses. Frontiers in Pharmacology. 2018;**9**

[17] Rizvanov AA, Kovac M, Rutland CS. Advancing modern equine medicine using gene therapy. Equine Veterinary Education. 2018;**30**(10):516-517

[18] Sahni A, Khorana AA, Baggs RB, Peng H, Francis CW. FGF-2 binding to fibrin(ogen) is required for augmented angiogenesis. Blood.

[19] Kano MR, Morishita Y, Iwata C, Iwasaka S, Watabe T, Ouchi Y, et al. VEGF-A and FGF-2 synergistically promote neoangiogenesis through enhancement of endogenous PDGF-B-PDGFR beta signaling. Journal of Cell Science. 2005;**118**(16):3759-3768

[20] Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;**8**(4):315-317

[21] Yang G, Rothrauff BB, Tuan RS. Tendon and ligament regeneration and repair: Clinical relevance and developmental paradigm. Birth Defects Research Part C. 2013;**99**(3):203-222

Alvarez LEC, Yamada ALM, Borges AS, Deffune E, et al. Equine tendonitis therapy using mesenchymal stem cells and platelet concentrates: A randomized controlled trial. Stem Cell Research &

[22] Carvalho AD, Badial PR,

2006;**107**(1):126-131

**26**

Therapy. 2013;**4**

## Gene Therapy for the Treatment of Equine Osteoarthritis

*Rachael Levings, Andrew Smith, Padraic P. Levings, Glyn D. Palmer, Anthony Dacanay, Patrick Colahan and Steven C. Ghivizzani*

#### **Abstract**

Osteoarthritis (OA) is the predominant cause of lameness in horses. As in humans, the clinical symptoms of equine OA are persistent pain and dysfunction of the affected joint. Its pathology is similarly marked by progressive deterioration of the articular cartilage, subchondral bone sclerosis, marginal osteophytes, soft tissue inflammation and joint effusion. Disease pathogenesis is mediated by elevated levels of inflammatory cytokines and proteolytic enzymes in the articular tissues and synovial fluid. Existing pharmacologic agents can alleviate OA joint pain; none are able to inhibit erosive disease progression. As several gene-based treatments for human disease have received approval by the Food and Drug Administration (FDA), the transition to veterinary medicine will almost certainly follow. Several viral vector systems have demonstrated highly efficient gene transfer to the equine joint, enabling expression of therapeutic transgenes at efficacious levels for well over a year. Because of its large size, the equine joint is well suited to studies of genebased therapies for arthritic disease. The forelimb joints are vulnerable to OA onset, and treatment and diagnostic modalities are the same in humans and horses. Here, we discuss the various gene-transfer approaches under investigation and the current progress toward the development an effective gene therapy for equine OA.

**Keywords:** osteoarthritis, lameness, interleukin-1, IL-1Ra, gene therapy, adeno-associated virus

#### **1. Introduction**

Osteoarthritis (OA) is a chronic, painful, degenerative, often debilitating condition common in weight-bearing joints of both humans and horses. In humans the knees and hips are predominately affected, while in the horse the metacarpophalangeal and carpal joints of the forelimb are the primary sites of onset. In both species, the pathology of OA is marked by the gradual, persistent erosion of the articular cartilage, development of osteophytes at the joint margins, sclerotic growth of subchondral bone, synovitis and joint effusion [1]. Biochemical analyses reveal that the signaling molecules and pathways that drive the inflammatory and degenerative processes in both species are identical [2]. OA is incurable, difficult to manage and often progresses to disabling joint failure. It is estimated that over 50 million people in the US alone have symptomatic OA. Spontaneous joint disease is a common

clinical problem in the horse as well where it is estimated that OA accounts for up to 60% of lameness [3], and is among the leading causes of debilitation and wastage of athletic horses. As with humans, the need for an effective treatment for equine OA is immense.

In this chapter we describe progress with an experimental gene-based therapy for OA in parallel development for both humans and horses. The concept of a genetic therapy was initially put forth as a method to replace defective genes and associated protein deficiencies from monogenic diseases, such as cystic fibrosis, severe combined immunodeficiency and hemophilia. In the present application direct intra-articular gene transfer is used as an improved system for sustained local delivery of biologic agents with anti-arthritic potential [4]. By providing for highlevel, persistent production of therapeutic gene products in chronically diseased joints, long-standing obstacles impeding effective drug delivery are overcome to provide stable production of gene products with activities capable of inhibiting not only pain and inflammation, but also the progression underlying the degenerative process. We discuss various approaches for intra-articular gene delivery and promising gene products. We also discuss progress toward clinical application and remaining challenges.

#### **2. Osteoarthritis pathogenesis**

The pathogenesis of OA is complex and can be initiated by a wide range of factors. It is most commonly linked with aging and accumulating degradation of the cartilage matrix from the loss of cellularity and reduced metabolic activity of the chondrocytes [5–7]. In younger individuals, OA most frequently occurs as a secondary consequence of joint injury (post-traumatic OA: PTOA) either from repetitive trauma to the joint surfaces due to overloading and overuse, or acute damage to the structural tissues.

Although cartilage damage and traumatic loading are considered initiating factors, a consensus in the literature indicates that inflammatory cross-talk between the synovium and cartilage is instrumental in driving the erosive progression of OA [8, 9]. Under normal conditions, the chondrocytes, which inhabit the articular cartilage at low density, maintain the integrity and quality of the matrix through slow continual remodeling through degradation and new matrix protein synthesis. Disruption of this homeostasis from chondrocyte dysfunction or depletion from apoptosis or necrosis, leads to a reduction in matrix quality, damage to the articular surface and pathologic load distribution. The increased compressive forces among weight-bearing regions, activates stress signaling pathways in regional chondrocytes and a phenotypic shift to an activated phenotype. Stress-induced activation of nuclear factor-kappa B (NF-κB), and p38 MAPK and c-Jun N-terminal kinases and their downstream signaling cascades halts the synthesis of key extracellular matrix (ECM) proteins, stimulates the release of inflammatory cytokines and chemokines and expression of matrix metalloproteinases and aggrecanases [1, 10]. The release of cellular debris and matrix molecules from eroding cartilage stimulates cytokine and toll-like receptors in the synovial lining cells and an inflammatory response in the synovium [11, 12]. The resulting synovitis, marked by hyperplasia and hypertrophy of synovial fibroblasts, infiltrating macrophages, T cells, and mast cells, is a common feature of both early and late-stage disease. Inflammatory activation of the synovium stimulates production of enzymes and inflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α) that feeds back in a self-perpetuating cycle to further alter chondrocyte metabolism and the balance of cartilage matrix synthesis and degradation [9]. With increasing loss of the

**29**

*Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

of the subchondral bone.

bone articulation [13].

**3. Treatment limitations**

carry increased risk of infection [14].

**4. OA gene therapy principles**

tissue in the joint is not based on scientific evidence [15, 16].

protective cartilage cushion the increased mechanical forces stimulate a compensatory reaction in the calcified cartilage resulting in increased thickening and stiffness

As an avascular tissue, injured cartilage has no mechanism for self-repair or regeneration. There is no influx of exogenous cells from ruptured blood vessels to generate space-filling tissue. Although local chondrocytes attempt proliferate and form chondrocyte clusters in an apparent regenerative, reparative response, the dense ECM limits the migration of the limited number of chondrocytes. In cases of significant damage, cartilage lesions are essentially permanent and progress to fibrillation, formation of fissures, and ultimately complete loss of the cartilage surface. Cumulatively, the slow insidious processes cause fibrillation, fissures, ulceration and over time the full thickness loss of cartilage and painful bone on

Existing medications for OA, such as analgesics and non-steroidal antiinflammatory (NSAID) agents are palliative and only provide temporary relief of joint pain without significantly altering disease progression or restoring cartilage integrity. While there are a variety of biologic agents with activities known to inhibit pathologic signaling pathways, due to the unique anatomy and physiology of synovial joints, conventional methods of drug delivery are unable to achieve or maintain effective concentrations of therapeutic molecules in chronically diseased joints [14]. The synovial fluid which serves to lubricate the articulating surfaces and nourish the chondroctyes is a dialysate of blood plasma that enters the joint through fenestrated capillaries in the subsynovium. This "sieving effect" restricts the entry of proteins and other large molecules into the joint space from the circulation [14]. While intra-articular (IA) injection circumvents physical barriers to systemic delivery, elevated pressure causes rapid turnover of synovial fluid through the lymphatics. Continuous circulation of the synovial fluid causes injected molecules to be rapidly cleared from the joint, often with a half-life of less than 4–5 hours, depending on the size. Repeated intra-articular injection is not a useful clinically as frequent repeated needle sticks are painful, can exacerbate joint pathology and

Local intra-articular injection of corticosteroids can provide temporary relief of joint pain, but the broad spectrum anti-inflammatory effects are transient. Despite the short residence time of intra-articular therapies, studies frequently report positive effects from a number of patient-derived preparations, such as platelet rich plasma, autologous conditioned serum and various formulations of "mesenchymal stem cells" (MSCs). However due to inconsistent methods of preparation and characterization, conflicts of interest and investigator bias, the efficacy of these treatments in both human and equine medicine remains highly controversial. Indeed, the assertion that MSCs injected in suspension have an intrinsic capacity to sense and address whatever is needed for the repair and regeneration of cartilaginous

Arthritis gene therapy was conceived as a novel protein-drug delivery system capable of exploiting the anti-arthritic properties of endogenous soluble gene products for treatment of chronic joint disease [17]. By delivering cDNAs encoding *Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

protective cartilage cushion the increased mechanical forces stimulate a compensatory reaction in the calcified cartilage resulting in increased thickening and stiffness of the subchondral bone.

As an avascular tissue, injured cartilage has no mechanism for self-repair or regeneration. There is no influx of exogenous cells from ruptured blood vessels to generate space-filling tissue. Although local chondrocytes attempt proliferate and form chondrocyte clusters in an apparent regenerative, reparative response, the dense ECM limits the migration of the limited number of chondrocytes. In cases of significant damage, cartilage lesions are essentially permanent and progress to fibrillation, formation of fissures, and ultimately complete loss of the cartilage surface. Cumulatively, the slow insidious processes cause fibrillation, fissures, ulceration and over time the full thickness loss of cartilage and painful bone on bone articulation [13].

#### **3. Treatment limitations**

*Equine Science*

is immense.

remaining challenges.

structural tissues.

**2. Osteoarthritis pathogenesis**

clinical problem in the horse as well where it is estimated that OA accounts for up to 60% of lameness [3], and is among the leading causes of debilitation and wastage of athletic horses. As with humans, the need for an effective treatment for equine OA

In this chapter we describe progress with an experimental gene-based therapy for OA in parallel development for both humans and horses. The concept of a genetic therapy was initially put forth as a method to replace defective genes and associated protein deficiencies from monogenic diseases, such as cystic fibrosis, severe combined immunodeficiency and hemophilia. In the present application direct intra-articular gene transfer is used as an improved system for sustained local delivery of biologic agents with anti-arthritic potential [4]. By providing for highlevel, persistent production of therapeutic gene products in chronically diseased joints, long-standing obstacles impeding effective drug delivery are overcome to provide stable production of gene products with activities capable of inhibiting not only pain and inflammation, but also the progression underlying the degenerative process. We discuss various approaches for intra-articular gene delivery and promising gene products. We also discuss progress toward clinical application and

The pathogenesis of OA is complex and can be initiated by a wide range of factors. It is most commonly linked with aging and accumulating degradation of the cartilage matrix from the loss of cellularity and reduced metabolic activity of the chondrocytes [5–7]. In younger individuals, OA most frequently occurs as a secondary consequence of joint injury (post-traumatic OA: PTOA) either from repetitive trauma to the joint surfaces due to overloading and overuse, or acute damage to the

Although cartilage damage and traumatic loading are considered initiating factors, a consensus in the literature indicates that inflammatory cross-talk between the synovium and cartilage is instrumental in driving the erosive progression of OA [8, 9]. Under normal conditions, the chondrocytes, which inhabit the articular cartilage at low density, maintain the integrity and quality of the matrix through slow continual remodeling through degradation and new matrix protein synthesis. Disruption of this homeostasis from chondrocyte dysfunction or depletion from apoptosis or necrosis, leads to a reduction in matrix quality, damage to the articular surface and pathologic load distribution. The increased compressive forces among weight-bearing regions, activates stress signaling pathways in regional chondrocytes and a phenotypic shift to an activated phenotype. Stress-induced activation of nuclear factor-kappa B (NF-κB), and p38 MAPK and c-Jun N-terminal kinases and their downstream signaling cascades halts the synthesis of key extracellular matrix (ECM) proteins, stimulates the release of inflammatory cytokines and chemokines and expression of matrix metalloproteinases and aggrecanases [1, 10]. The release of cellular debris and matrix molecules from eroding cartilage stimulates cytokine and toll-like receptors in the synovial lining cells and an inflammatory response in the synovium [11, 12]. The resulting synovitis, marked by hyperplasia and hypertrophy of synovial fibroblasts, infiltrating macrophages, T cells, and mast cells, is a common feature of both early and late-stage disease. Inflammatory activation of the synovium stimulates production of enzymes and inflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α) that feeds back in a self-perpetuating cycle to further alter chondrocyte metabolism and the balance of cartilage matrix synthesis and degradation [9]. With increasing loss of the

**28**

Existing medications for OA, such as analgesics and non-steroidal antiinflammatory (NSAID) agents are palliative and only provide temporary relief of joint pain without significantly altering disease progression or restoring cartilage integrity. While there are a variety of biologic agents with activities known to inhibit pathologic signaling pathways, due to the unique anatomy and physiology of synovial joints, conventional methods of drug delivery are unable to achieve or maintain effective concentrations of therapeutic molecules in chronically diseased joints [14]. The synovial fluid which serves to lubricate the articulating surfaces and nourish the chondroctyes is a dialysate of blood plasma that enters the joint through fenestrated capillaries in the subsynovium. This "sieving effect" restricts the entry of proteins and other large molecules into the joint space from the circulation [14].

While intra-articular (IA) injection circumvents physical barriers to systemic delivery, elevated pressure causes rapid turnover of synovial fluid through the lymphatics. Continuous circulation of the synovial fluid causes injected molecules to be rapidly cleared from the joint, often with a half-life of less than 4–5 hours, depending on the size. Repeated intra-articular injection is not a useful clinically as frequent repeated needle sticks are painful, can exacerbate joint pathology and carry increased risk of infection [14].

Local intra-articular injection of corticosteroids can provide temporary relief of joint pain, but the broad spectrum anti-inflammatory effects are transient. Despite the short residence time of intra-articular therapies, studies frequently report positive effects from a number of patient-derived preparations, such as platelet rich plasma, autologous conditioned serum and various formulations of "mesenchymal stem cells" (MSCs). However due to inconsistent methods of preparation and characterization, conflicts of interest and investigator bias, the efficacy of these treatments in both human and equine medicine remains highly controversial. Indeed, the assertion that MSCs injected in suspension have an intrinsic capacity to sense and address whatever is needed for the repair and regeneration of cartilaginous tissue in the joint is not based on scientific evidence [15, 16].

#### **4. OA gene therapy principles**

Arthritis gene therapy was conceived as a novel protein-drug delivery system capable of exploiting the anti-arthritic properties of endogenous soluble gene products for treatment of chronic joint disease [17]. By delivering cDNAs encoding therapeutic products to cells resident in the articular tissues, and providing for high levels of independent expression, the biosynthetic machinery of the modified cells is directed to overproduce and continuously secrete the transgenic protein into the synovial fluid and surrounding tissue. In this manner, the diseased joint becomes an endogenous site of sustained, elevated drug production, eliminating the need for repeated application, while providing the greatest concentration of the protein specifically at the site of disease. While originally envisaged for delivery of secreted proteins, similar principles can be applied to gene products that function intracellularly, including transcription factors and interfering RNAs among others. OA is an excellent candidate for a local gene-based therapy, as only one or two joints are affected in most patients, and there is an absence of significant extra-articular disease. Distinct from any existing treatment for OA, this approach has the capacity for continuous local delivery of therapeutic molecules that block painful symptoms and erosive progression of disease from a single intra-articular injection [4].

The distinct advantage of using a secreted protein is that overproduction from a relatively small number of cells can treat the entire joint. While at least in theory, gene delivery provides the opportunity to explore the application of cDNAs whose products that function intra-cellularly (e.g. transcription factors and interfering RNAs), practical application is far more challenging than it may initially appear. In order to alter the biology of a diseased tissue, a substantial portion of the cells must be modified, requiring extraordinarily high levels of gene transfer in vivo.

A variety of methods can be used deliver therapeutic gene products to joints. Once a candidate cDNA is identified, the delivery vehicle that provides efficient targeting and modification of the desired cell types in vivo and robust transgene expression that persists for a prolonged period of time. For chronic joint diseases, such as RA and OA, a minimum of 6 months to a year or more of benefit following a single injection would likely be the minimum standard for efficacy. Such a profile requires metabolically active target cells with limited turnover [18]. Additionally, the vector and genetically modified cells must avoid recognition and elimination by the immune system whose central function is to eradicate infectious viruses and virally infected cells expressing non-self, surface antigens and stress-induced signaling molecules. The immune stealth of the vector, transgene product and modified cells are essential for effective gene delivery and prolonged, functional transgene expression.

#### **5. Parallel development of gene therapy for human and equine OA**

Early preclinical studies showed that local intra-articular delivery of certain cDNAs could inhibit experimental arthritis in the joints of laboratory animals. Although rodents and rabbits are useful for proof-of-concept studies, their small size does not accurately reflect the environment of the human or equine OA joint. Following intra-articular injection of a gene delivery vehicle, ensuing patterns of transgene expression are dictated by the biophysical interactions between the vector and the target tissues. In the case of a recombinant virus for example, dispersion in the joint space through the viscous synovial fluid, and its subsequent penetration in the ECM of the various tissues, determines the locations, phenotype, number and density of the cells that are physically encountered by the vector and genetically modified. The composition of the cell population modified by the virus at the time of injection, determines the level and duration of therapeutic transgene expression -and, in turn, the efficacy of treatment. In this respect, the small joints of a 100– 200 g quadruped rodent cannot duplicate the complex milieu of the knee of a 75 kg bipedal human, much less a 500 kg horse. The vastly greater size and internal fluid

**31**

disease.

*Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

able to OA secondary to trauma and excessive training [2, 3].

monitored over time within the same animal.

of human and equine medicines in parallel.

**6. Ex vivo gene delivery**

volume, the differences in cellularity within the dramatically larger and thicker connective tissues, as well as the compressive forces generated during locomotion, have a profound influence on the biodistribution of the virus following injection [19]. To model the efficacy of gene delivery in joints of clinically relevant proportions and better assess its utility for treatment of OA, the carpal and metacarpophalangeal (MCP) joints of the equine forelimb provide highly useful targets. These joints are similar in size, function, and tissue composition to the human knee, and since they carry 60–65% of the horse's weight during locomotion, they are highly vulner-

Because of its large size, the equine system is particularly well suited for preclinical studies of joint disease. The horse can readily perform controlled exercise, and clinical treatment and diagnostic modalities are the same in humans and horses [2, 20]. The large joints facilitate joint function analyses, examination of internal structures using magnetic resonance imaging (MRI) and radiograph, and minimally invasive arthroscopy for visual assessment and biopsy of joint tissues. The capacity to aspirate undiluted synovial fluid permits analysis of transgenic protein content by enzyme linked immunosorbent assay (ELISA) [21], and since, joint fluids can be aspirated serially without adverse effect, patterns of transgenic expression can be

By examining the efficacy of OA gene therapy in joints of similar proportion to the human knee and with similar disease, results representative of the human and equine response should be obtainable. Further, the use of the horse as an experimental subject allows practical experience with gene delivery in a relevant context and on an appropriate scale. This provides the ability to identify and troubleshoot technical and logistical problems in a clinical setting and refine working parameters for safety and efficacy prior to entering phase I human or field trials in client horses. Moreover, since OA is a significant health issue in both humans and horses, findings generated in this system can be applied to both species, allowing the development

The initial proof-of-concept was demonstrated using an ex vivo method whereby autologous synovial fibroblasts isolated from surgically harvested joint tissues, were stably modified with recombinant oncoretroviral vector (Moloney murine leukemia virus) to overexpress a secreted IL-1 inhibitor (IL-1Ra) [22, 23]. After expansion in culture the cells were injected into the diseased joint where they engraft in the synovial lining and continuously secrete the transgene product. This method demonstrated the feasibility of intra-articular gene delivery and was used successfully in a phase I human trial [24]. However, the procedure proved to be labor intensive, time consuming and tedious; its exorbitant cost made the procedure impractical for widespread clinical application, especially for a common, non-fatal

It is important to note that cell in suspension (regardless the tissue of origin), following injected into the joint space, consistently engraft in the synovial lining; they do not adhere to or colonize articular cartilage. Cells surgically implanted in cartilage defects within a support matrix will remain localized, but lacking a method of physical containment loose cells will disperse throughout the capsular lining. Along these lines, much has been made in the literature of the anti-arthritic potential of so-called "mesenchymal stem cells" or MSCs [15, 16]. These cells are amenable to genetic modification and can be used as a vehicle for ex vivo gene transfer. However, it is our experience that MSCs in and of themselves are not

#### *Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

*Equine Science*

therapeutic products to cells resident in the articular tissues, and providing for high levels of independent expression, the biosynthetic machinery of the modified cells is directed to overproduce and continuously secrete the transgenic protein into the synovial fluid and surrounding tissue. In this manner, the diseased joint becomes an endogenous site of sustained, elevated drug production, eliminating the need for repeated application, while providing the greatest concentration of the protein specifically at the site of disease. While originally envisaged for delivery of secreted proteins, similar principles can be applied to gene products that function intracellularly, including transcription factors and interfering RNAs among others. OA is an excellent candidate for a local gene-based therapy, as only one or two joints are affected in most patients, and there is an absence of significant extra-articular disease. Distinct from any existing treatment for OA, this approach has the capacity for continuous local delivery of therapeutic molecules that block painful symptoms

and erosive progression of disease from a single intra-articular injection [4].

be modified, requiring extraordinarily high levels of gene transfer in vivo.

**5. Parallel development of gene therapy for human and equine OA**

Early preclinical studies showed that local intra-articular delivery of certain cDNAs could inhibit experimental arthritis in the joints of laboratory animals. Although rodents and rabbits are useful for proof-of-concept studies, their small size does not accurately reflect the environment of the human or equine OA joint. Following intra-articular injection of a gene delivery vehicle, ensuing patterns of transgene expression are dictated by the biophysical interactions between the vector and the target tissues. In the case of a recombinant virus for example, dispersion in the joint space through the viscous synovial fluid, and its subsequent penetration in the ECM of the various tissues, determines the locations, phenotype, number and density of the cells that are physically encountered by the vector and genetically modified. The composition of the cell population modified by the virus at the time of injection, determines the level and duration of therapeutic transgene expression -and, in turn, the efficacy of treatment. In this respect, the small joints of a 100– 200 g quadruped rodent cannot duplicate the complex milieu of the knee of a 75 kg bipedal human, much less a 500 kg horse. The vastly greater size and internal fluid

The distinct advantage of using a secreted protein is that overproduction from a relatively small number of cells can treat the entire joint. While at least in theory, gene delivery provides the opportunity to explore the application of cDNAs whose products that function intra-cellularly (e.g. transcription factors and interfering RNAs), practical application is far more challenging than it may initially appear. In order to alter the biology of a diseased tissue, a substantial portion of the cells must

A variety of methods can be used deliver therapeutic gene products to joints. Once a candidate cDNA is identified, the delivery vehicle that provides efficient targeting and modification of the desired cell types in vivo and robust transgene expression that persists for a prolonged period of time. For chronic joint diseases, such as RA and OA, a minimum of 6 months to a year or more of benefit following a single injection would likely be the minimum standard for efficacy. Such a profile requires metabolically active target cells with limited turnover [18]. Additionally, the vector and genetically modified cells must avoid recognition and elimination by the immune system whose central function is to eradicate infectious viruses and virally infected cells expressing non-self, surface antigens and stress-induced signaling molecules. The immune stealth of the vector, transgene product and modified cells are essential for effective gene delivery and prolonged, functional

**30**

transgene expression.

volume, the differences in cellularity within the dramatically larger and thicker connective tissues, as well as the compressive forces generated during locomotion, have a profound influence on the biodistribution of the virus following injection [19].

To model the efficacy of gene delivery in joints of clinically relevant proportions and better assess its utility for treatment of OA, the carpal and metacarpophalangeal (MCP) joints of the equine forelimb provide highly useful targets. These joints are similar in size, function, and tissue composition to the human knee, and since they carry 60–65% of the horse's weight during locomotion, they are highly vulnerable to OA secondary to trauma and excessive training [2, 3].

Because of its large size, the equine system is particularly well suited for preclinical studies of joint disease. The horse can readily perform controlled exercise, and clinical treatment and diagnostic modalities are the same in humans and horses [2, 20]. The large joints facilitate joint function analyses, examination of internal structures using magnetic resonance imaging (MRI) and radiograph, and minimally invasive arthroscopy for visual assessment and biopsy of joint tissues. The capacity to aspirate undiluted synovial fluid permits analysis of transgenic protein content by enzyme linked immunosorbent assay (ELISA) [21], and since, joint fluids can be aspirated serially without adverse effect, patterns of transgenic expression can be monitored over time within the same animal.

By examining the efficacy of OA gene therapy in joints of similar proportion to the human knee and with similar disease, results representative of the human and equine response should be obtainable. Further, the use of the horse as an experimental subject allows practical experience with gene delivery in a relevant context and on an appropriate scale. This provides the ability to identify and troubleshoot technical and logistical problems in a clinical setting and refine working parameters for safety and efficacy prior to entering phase I human or field trials in client horses. Moreover, since OA is a significant health issue in both humans and horses, findings generated in this system can be applied to both species, allowing the development of human and equine medicines in parallel.

#### **6. Ex vivo gene delivery**

The initial proof-of-concept was demonstrated using an ex vivo method whereby autologous synovial fibroblasts isolated from surgically harvested joint tissues, were stably modified with recombinant oncoretroviral vector (Moloney murine leukemia virus) to overexpress a secreted IL-1 inhibitor (IL-1Ra) [22, 23]. After expansion in culture the cells were injected into the diseased joint where they engraft in the synovial lining and continuously secrete the transgene product. This method demonstrated the feasibility of intra-articular gene delivery and was used successfully in a phase I human trial [24]. However, the procedure proved to be labor intensive, time consuming and tedious; its exorbitant cost made the procedure impractical for widespread clinical application, especially for a common, non-fatal disease.

It is important to note that cell in suspension (regardless the tissue of origin), following injected into the joint space, consistently engraft in the synovial lining; they do not adhere to or colonize articular cartilage. Cells surgically implanted in cartilage defects within a support matrix will remain localized, but lacking a method of physical containment loose cells will disperse throughout the capsular lining. Along these lines, much has been made in the literature of the anti-arthritic potential of so-called "mesenchymal stem cells" or MSCs [15, 16]. These cells are amenable to genetic modification and can be used as a vehicle for ex vivo gene transfer. However, it is our experience that MSCs in and of themselves are not

immune privileged and have no more regenerative or anti-inflammatory value than any other cell type injected into the joint. Most investigators have found that allogeneic MSCs are cleared very rapidly from the joints of experimental animals, with few cells remaining beyond 1–2 weeks.

#### **7. Direct intra-articular gene transfer**

Relative to the ex vivo approach, direct injection into the joint of recombinant vectors dramatically streamlines the gene delivery procedure [4]. A broad range of vector systems, both viral and non-viral have been evaluated for their efficiency of gene transfer to the joint tissues in situ [25–30]. While the use of non-viral gene delivery vehicles has certain theoretical advantages (larger payload, increased perception of safety, straightforward vector production, reduced costs) extensive *in vivo* testing in our laboratory, as well as in others, has shown that non-viral delivery of nucleic acids is currently not suitable for treating chronic articular diseases; the efficiency of delivery is exceedingly low and typically persists in the joint cells for no greater than 2–3 days. While non-viral formulations are often effective transfection reagents in the context of monolayer cell culture, efficacy in vitro does not equate to performance in vivo [31]. Despite claims in the literature regarding the treatment of OA, the use of these systems should be avoided as their pharmacokinetic profile is incompatible with the pathologic progression of OA.

Similarly, there are dozens of published papers that report remarkable efficacy following intra-articular injection of shRNAs, miRNAs, and circRNAs into the joints of animals either in suspension or complexed in nano- or micro-particles. As mentioned above, for an intracellular approach to be effective in OA, an extraordinarily high efficiency of delivery is required to the cells in target tissues in vivo. Moreover as gene expression is an ongoing process, interfering RNAs must be maintained at exceptionally high levels in a large proportion of cells and be continuously replenished to sustain gene silencing. While achievable when delivered in an exogenous expression cassette, it is not possible with the delivery of soluble or complexed inhibitory RNAs. These reports should be regarded with a healthy degree of skepticism.

Viral-based vector systems exploit the natural ability of a virus to deliver its genetic payload to a target cell with high efficiency. For the generation of a viralbased vector system, the coding sequences for viral proteins essential for replication are removed from the viral genome and the products are supplied in *trans* during vector propagation in permissive engineered cell lines. Several recombinant viral vector systems have shown the capacity to deliver exogenous genes to joint tissues and enable expression of therapeutic transgene products at levels sufficient to inhibit arthritic pathologies in laboratory animals. Among these are recombinant adenovirus [27], herpes simplex virus [28], adeno-associated virus (AAV) [32, 33], and lentivirus [29, 34] among others. Each of these systems has inherent advantages and limitations that dictate the applications for which they are best suited. Currently only two viral vectors, recombinant adenovirus and AAV, are in serious preclinical development for equine or human OA.

#### **7.1 Recombinant adenovirus**

First generation recombinant adenovirus provides highly efficient transduction of target cells in various connective tissues both in culture and in vivo [25, 26]. Several years ago this system was the workhorse vector of the field of musculoskeletal gene therapy [35]. Adenoviral vectors showed that the concept of direct

**33**

*Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

exogenous expression cassette [36].

**7.2 Adeno-associated virus**

transgene expression cassette.

intra-articular gene transfer was capable of providing functional levels of transgene expression in the joints of animal models. In the first generation vectors the E1 and E3 genes required for immediate early stage gene expression and initiation of viral replication were deleted from the genome to prohibit viral replication in cells infected by the vector. Their removal also provided room for the insertion of an

The relative ease of production reduced the barrier to entry and provided gene transfer technology to any laboratory with basic molecular biology capabilities. Although viral replication was crippled in non-permissive cells, the vector still retained the majority of the native coding sequences. Leaky expression of viral proteins by transduced cells caused them to be eliminated in 2–3 weeks by adaptive cellular immune responses [27, 37]. Despite its transient nature, adenoviral gene delivery provided a burst of high level transgene expression sufficient to examine the biological activity of a specific gene product in vivo. Adenovirus has the reputation of causing acute toxicity from innate inflammatory responses, but much of this is due to low quality, inconsistent vector preparations containing high levels of cellular debris. Advances in adenoviral technology include the development of helper dependent systems in which the coding sequences for all viral proteins have been removed and are supplied during propagation by a second "helper" adenoviral vector, which is removed by differential centrifugation during purification [38]. These modifications allow for increased immune avoidance and long-term transgene expression without significant reduction in infection efficiency [39–41].

Of the well-characterized viral systems, AAV offers many advantages that favor its use for the treatment of arthritis: (1) The wild type virus is not associated with any pathologic human condition. (2) The recombinant form does not contain native viral coding sequences, which reduces its immunogenicity. (3) AAV can infect both dividing and quiescent cells. (4) Persistent transgenic expression in vivo has been observed in many applications, and (5) the recombinant form does not integrate

A further potential advantage is the relative simplicity of the AAV vector, which is comprised of an ~5000 nucleotide single-stranded DNA genome packaged in a small (20–30 nm), non-enveloped icosahedral particle by three capsid proteins, differing only at their N termini [43]. The only required *cis* elements on the vector DNA are 145 nucleotide-long inverted terminal repeats (ITRs) that flank the

Fortuitously, with regard to veterinary medicine, in head to head comparisons

Additionally, humans are natural hosts to wild type AAV infection and often have high circulating titers of neutralizing antibodies (NAb) to AAV capsids of several serotypes (primarily AAV2, AAV1, and to lesser extents AAV5) from prior infections with wild type virus. Horses, however, are not common hosts for wild type AAV infection, and distinct from humans, have low circulating NAb titers to most AAV vector serotypes. While NAb to AAV5 appears relatively frequently among horses, and one report describes increased NAb titers to AAV2 capsid in a small test sample, pre-existing NAb do not appear to be prevalent in the equine population nor at sufficient titer to prohibit effective gene delivery [44, 45].

equine synovial fibroblasts in culture are significantly more receptive to AAV transduction than their human counterparts. Preliminary evidence suggests increased expression of surface receptors between the two species in culture. How this discrepancy translates to the in vivo situation is unclear, as phase I testing in humans has just begun, but transgene expression is robust in equine joints.

into the genome of the target cell with significant frequency [42].

#### *Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

*Equine Science*

with few cells remaining beyond 1–2 weeks.

**7. Direct intra-articular gene transfer**

immune privileged and have no more regenerative or anti-inflammatory value than any other cell type injected into the joint. Most investigators have found that allogeneic MSCs are cleared very rapidly from the joints of experimental animals,

Relative to the ex vivo approach, direct injection into the joint of recombinant vectors dramatically streamlines the gene delivery procedure [4]. A broad range of vector systems, both viral and non-viral have been evaluated for their efficiency of gene transfer to the joint tissues in situ [25–30]. While the use of non-viral gene delivery vehicles has certain theoretical advantages (larger payload, increased perception of safety, straightforward vector production, reduced costs) extensive *in vivo* testing in our laboratory, as well as in others, has shown that non-viral delivery of nucleic acids is currently not suitable for treating chronic articular diseases; the efficiency of delivery is exceedingly low and typically persists in the joint cells for no greater than 2–3 days. While non-viral formulations are often effective transfection reagents in the context of monolayer cell culture, efficacy in vitro does not equate to performance in vivo [31]. Despite claims in the literature regarding the treatment of OA, the use of these systems should be avoided as their pharmacoki-

Similarly, there are dozens of published papers that report remarkable efficacy following intra-articular injection of shRNAs, miRNAs, and circRNAs into the joints of animals either in suspension or complexed in nano- or micro-particles. As mentioned above, for an intracellular approach to be effective in OA, an extraordinarily high efficiency of delivery is required to the cells in target tissues in vivo. Moreover as gene expression is an ongoing process, interfering RNAs must be maintained at exceptionally high levels in a large proportion of cells and be continuously replenished to sustain gene silencing. While achievable when delivered in an exogenous expression cassette, it is not possible with the delivery of soluble or complexed inhibitory RNAs. These reports should be regarded with a healthy

Viral-based vector systems exploit the natural ability of a virus to deliver its genetic payload to a target cell with high efficiency. For the generation of a viralbased vector system, the coding sequences for viral proteins essential for replication are removed from the viral genome and the products are supplied in *trans* during vector propagation in permissive engineered cell lines. Several recombinant viral vector systems have shown the capacity to deliver exogenous genes to joint tissues and enable expression of therapeutic transgene products at levels sufficient to inhibit arthritic pathologies in laboratory animals. Among these are recombinant adenovirus [27], herpes simplex virus [28], adeno-associated virus (AAV) [32, 33], and lentivirus [29, 34] among others. Each of these systems has inherent advantages and limitations that dictate the applications for which they are best suited. Currently only two viral vectors, recombinant adenovirus and AAV, are in serious

First generation recombinant adenovirus provides highly efficient transduction

of target cells in various connective tissues both in culture and in vivo [25, 26]. Several years ago this system was the workhorse vector of the field of musculoskeletal gene therapy [35]. Adenoviral vectors showed that the concept of direct

netic profile is incompatible with the pathologic progression of OA.

**32**

degree of skepticism.

preclinical development for equine or human OA.

**7.1 Recombinant adenovirus**

intra-articular gene transfer was capable of providing functional levels of transgene expression in the joints of animal models. In the first generation vectors the E1 and E3 genes required for immediate early stage gene expression and initiation of viral replication were deleted from the genome to prohibit viral replication in cells infected by the vector. Their removal also provided room for the insertion of an exogenous expression cassette [36].

The relative ease of production reduced the barrier to entry and provided gene transfer technology to any laboratory with basic molecular biology capabilities. Although viral replication was crippled in non-permissive cells, the vector still retained the majority of the native coding sequences. Leaky expression of viral proteins by transduced cells caused them to be eliminated in 2–3 weeks by adaptive cellular immune responses [27, 37]. Despite its transient nature, adenoviral gene delivery provided a burst of high level transgene expression sufficient to examine the biological activity of a specific gene product in vivo. Adenovirus has the reputation of causing acute toxicity from innate inflammatory responses, but much of this is due to low quality, inconsistent vector preparations containing high levels of cellular debris. Advances in adenoviral technology include the development of helper dependent systems in which the coding sequences for all viral proteins have been removed and are supplied during propagation by a second "helper" adenoviral vector, which is removed by differential centrifugation during purification [38]. These modifications allow for increased immune avoidance and long-term transgene expression without significant reduction in infection efficiency [39–41].

#### **7.2 Adeno-associated virus**

Of the well-characterized viral systems, AAV offers many advantages that favor its use for the treatment of arthritis: (1) The wild type virus is not associated with any pathologic human condition. (2) The recombinant form does not contain native viral coding sequences, which reduces its immunogenicity. (3) AAV can infect both dividing and quiescent cells. (4) Persistent transgenic expression in vivo has been observed in many applications, and (5) the recombinant form does not integrate into the genome of the target cell with significant frequency [42].

A further potential advantage is the relative simplicity of the AAV vector, which is comprised of an ~5000 nucleotide single-stranded DNA genome packaged in a small (20–30 nm), non-enveloped icosahedral particle by three capsid proteins, differing only at their N termini [43]. The only required *cis* elements on the vector DNA are 145 nucleotide-long inverted terminal repeats (ITRs) that flank the transgene expression cassette.

Fortuitously, with regard to veterinary medicine, in head to head comparisons equine synovial fibroblasts in culture are significantly more receptive to AAV transduction than their human counterparts. Preliminary evidence suggests increased expression of surface receptors between the two species in culture. How this discrepancy translates to the in vivo situation is unclear, as phase I testing in humans has just begun, but transgene expression is robust in equine joints.

Additionally, humans are natural hosts to wild type AAV infection and often have high circulating titers of neutralizing antibodies (NAb) to AAV capsids of several serotypes (primarily AAV2, AAV1, and to lesser extents AAV5) from prior infections with wild type virus. Horses, however, are not common hosts for wild type AAV infection, and distinct from humans, have low circulating NAb titers to most AAV vector serotypes. While NAb to AAV5 appears relatively frequently among horses, and one report describes increased NAb titers to AAV2 capsid in a small test sample, pre-existing NAb do not appear to be prevalent in the equine population nor at sufficient titer to prohibit effective gene delivery [44, 45].

Typically, following intra-articular injection of a recombinant virus, the overwhelming majority of genetically modified cells are found in the synovium, subsynovium and supporting capsular and ligamentous tissues. Chondrocytes, while receptive to genetic modification in culture, are not efficiently transduced in vivo due to the inability of most vector particles to effectively penetrate the dense cartilage ECM. The only exception is AAV whose small particle size permits its entry and diffusion through the dense cartilage ECM enabling interaction and transduction of chondrocytes deep within the cartilage. As chondrocyte dysfunction and cartilage degeneration are the characteristic pathologies of OA, the capacity to deliver therapeutic genes to chondrocytes is a clear advantage to this vector technology. Moreover, since these cells are highly stable, their modification with AAV provides the prospect of enduring transgenic expression [46].

#### **8. Therapeutic strategies**

Two complementary gene-based strategies have been investigated for OA. The first is geared toward chondroprotection, and involves delivery of gene products that enhance joint lubrication or block the activities of specific inflammatory cytokines that stimulate inflammation and the subsequent degeneration of cartilage ECM by articular chondrocytes [47–49]. Most studies of OA gene therapy have involved the delivery of the cDNA for interleukin-1 receptor antagonist (IL-1Ra) a competitive inhibitor of IL-1 signaling [50–55].

The second strategy is directed toward cartilage repair or regeneration using various anabolic, proliferative or chondrogenic agents to stimulate regional chondrocytes to proliferate and elaborate cartilage ECM. While these strategies appear attractive at the outset, unfortunately, as vectors (and cells) injected into the joint primarily interact with synovial fibroblasts, many growth factors that may stimulate cartilage repair or matrix synthesis by chondrocytes will likewise stimulate the abundant fibroblast populations in the synovium to generate undesirable, often dramatic adverse side effects. For example, intra-articular delivery of adenovirus containing the cDNA for TGF-β1 induces an extraordinarily potent fibrotic, chondro-osseous response in the synovium and joint capsule [56, 57]. Systemic pathologies such as pulmonary fibrosis in rats and death in rabbits occurred when TGF-β1 was expressed intra-articularly at high levels. Overexpression of TGF-β1 and BMP-2 has also been shown to induce the formation of osteophytes, ectopic cartilage and bone formation [56, 58]. Of the growth factor genes tested thus far, only IGF-1 has not been associated with an overt pathologic response [45, 59], but it has not been evaluated extensively. Concerns over potential side effects have generally limited the use of growth factor genes to localized applications in tissue engineering for cartilage repair, whereby chondrocytes or MSCs are modified in culture to express a specific growth factor before surgical implantation into focal cartilage lesions, In this manner, the expressed protein is localized to the defect, reducing exposure to adjacent tissues.

#### **8.1 Interleukin-1 receptor antagonist**

A consensus in the literature indicates that IL-1, synthesized locally by chondrocytes and synovial cells, is instrumental in driving OA progression [60, 61]. Found at increased levels in OA joints, IL-1 is the most potent physiological inducer of chondrocytic chondrolysis (the major route to cartilage loss in OA) [62]. Even at trace levels, IL-1 strongly inhibits ECM production in cartilage by blocking collagen type II and proteoglycan synthesis and enhancing chondrocyte apoptosis. At

**35**

**9. Preclinical studies**

synovitis [21].

*Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

slightly higher concentrations proteolytic enzyme synthesis is induced in chondrocytes, driving enhanced production of matrix metalloproteinases (MMPs) and aggrecanases that degrade the cartilaginous matrix [63]. As a primary mediator of the inflammatory cascade, IL-1 stimulates articular cells to produce a full complement of OA effector molecules, including cyclooxygenases I and II, nitric oxide, phospholipase A2, prostaglandin E2, reactive oxygen species as well as inflammatory cytokines and chemokines. Release of these molecules further stimulates cartilage matrix degradation, bone erosion, synovitis and fibrosis. IL-1 is also suspected to mediate pain in OA, the most common reason for consulting a physician [64–67]. Traditional pharmacologic approaches have failed to produce clinically useful molecules for inhibiting IL-1 activity intra-articularly [68]. However, two naturally occurring proteins exist specifically for this purpose: IL-1Ra and the soluble IL-1 type II receptor (sIL-1RII) [69, 70]. IL-1Ra functions as a competitive inhibitor by binding to the type I IL-1 signaling receptor and preventing subsequent interaction with IL-1. Once bound, IL-1Ra fails to recruit the IL-1R accessory protein (IL-1-AcP) to the complex and prevents intracellular activation and signaling. The sIL-1RII molecule, in contrast, titrates IL-1 activity by binding directly to soluble IL-1 molecules and blocking interaction with the type I receptor [71, 72]. Despite differences in their modes of action, the two molecules inhibit IL-1 signaling with equal potency. In the context of gene therapy, IL-1Ra is a smaller protein and easier to express as a transgene product. The recombinant protein (anakinra/Kineret®) is well characterized and is approved for clinical use in humans for RA and other conditions in which IL-1 is known to pay a significant role [71, 72]. As anakinra is administered daily by subcutaneous injections of 150 mg, the risk of adverse

response from overproduction intra-articularly is extremely small.

Commercially available ELISAs with specificity for IL-1Ra orthologs in human,

It is possible that a dual therapy combining elements of chondroprotection and regeneration could both inhibit degeneration and stimulate cartilage repair [48, 74]. Such a strategy, though, would likely require gene delivery via separate vectors to account for differences in their expression patterns for safe, effective application.

Following a series of preclinical successes in small laboratory animals demonstrating the proof of concept for direct viral-mediated gene delivery to joints, studies were performed to evaluate direct viral mediated gene delivery to equine joints using a first-generation adenoviral vector containing the cDNA for equine IL-1Ra. Administration of Ad.eqIL-1Ra in the joints of healthy horses, produced dose-dependent increases in IL-1Ra levels in synovial fluid aspirates. However, the highest viral dose tested, 5 × 1011 viral particles (vp) produced an acute

To explore the capacity of Ad.eqIL-1Ra to inhibit OA pathologies, an osteochondral fragment (OCF) model of OA was used [21, 75]. In this system, a small osteochondral chip is surgically generated off the distal radial carpal bone of the midcarpal joint. Following a brief interval to recover from surgery, animals in the

mouse and horse permit sensitive quantitation in culture media and biological fluids. Analysis of synovial fluid permits the use of IL-1Ra as a quantitative reporter of total gene transfer and therapeutic gene expression, allowing direct comparison of various delivery platforms. With respect to OA, IL-1Ra does not require sophisticated regulation. The goal is simply to express IL-1Ra at levels 10–100 fold over IL-1, where it completely inhibits IL-1 signaling activity. Once the threshold for efficacy has been achieved, expression beyond this has no adverse effect [73].

#### *Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

*Equine Science*

Typically, following intra-articular injection of a recombinant virus, the overwhelming majority of genetically modified cells are found in the synovium, subsynovium and supporting capsular and ligamentous tissues. Chondrocytes, while receptive to genetic modification in culture, are not efficiently transduced in vivo due to the inability of most vector particles to effectively penetrate the dense cartilage ECM. The only exception is AAV whose small particle size permits its entry and diffusion through the dense cartilage ECM enabling interaction and transduction of chondrocytes deep within the cartilage. As chondrocyte dysfunction and cartilage degeneration are the characteristic pathologies of OA, the capacity to deliver therapeutic genes to chondrocytes is a clear advantage to this vector technology. Moreover, since these cells are highly stable, their modification with AAV provides

Two complementary gene-based strategies have been investigated for OA. The first is geared toward chondroprotection, and involves delivery of gene products that enhance joint lubrication or block the activities of specific inflammatory cytokines that stimulate inflammation and the subsequent degeneration of cartilage ECM by articular chondrocytes [47–49]. Most studies of OA gene therapy have involved the delivery of the cDNA for interleukin-1 receptor antagonist (IL-1Ra) a

The second strategy is directed toward cartilage repair or regeneration using various anabolic, proliferative or chondrogenic agents to stimulate regional chondrocytes to proliferate and elaborate cartilage ECM. While these strategies appear attractive at the outset, unfortunately, as vectors (and cells) injected into the joint primarily interact with synovial fibroblasts, many growth factors that may stimulate cartilage repair or matrix synthesis by chondrocytes will likewise stimulate the abundant fibroblast populations in the synovium to generate undesirable, often dramatic adverse side effects. For example, intra-articular delivery of adenovirus containing the cDNA for TGF-β1 induces an extraordinarily potent fibrotic, chondro-osseous response in the synovium and joint capsule [56, 57]. Systemic pathologies such as pulmonary fibrosis in rats and death in rabbits occurred when TGF-β1 was expressed intra-articularly at high levels. Overexpression of TGF-β1 and BMP-2 has also been shown to induce the formation of osteophytes, ectopic cartilage and bone formation [56, 58]. Of the growth factor genes tested thus far, only IGF-1 has not been associated with an overt pathologic response [45, 59], but it has not been evaluated extensively. Concerns over potential side effects have generally limited the use of growth factor genes to localized applications in tissue engineering for cartilage repair, whereby chondrocytes or MSCs are modified in culture to express a specific growth factor before surgical implantation into focal cartilage lesions, In this manner, the expressed protein is localized to the defect, reducing exposure to

A consensus in the literature indicates that IL-1, synthesized locally by chondrocytes and synovial cells, is instrumental in driving OA progression [60, 61]. Found at increased levels in OA joints, IL-1 is the most potent physiological inducer of chondrocytic chondrolysis (the major route to cartilage loss in OA) [62]. Even at trace levels, IL-1 strongly inhibits ECM production in cartilage by blocking collagen type II and proteoglycan synthesis and enhancing chondrocyte apoptosis. At

the prospect of enduring transgenic expression [46].

competitive inhibitor of IL-1 signaling [50–55].

**8. Therapeutic strategies**

**34**

adjacent tissues.

**8.1 Interleukin-1 receptor antagonist**

slightly higher concentrations proteolytic enzyme synthesis is induced in chondrocytes, driving enhanced production of matrix metalloproteinases (MMPs) and aggrecanases that degrade the cartilaginous matrix [63]. As a primary mediator of the inflammatory cascade, IL-1 stimulates articular cells to produce a full complement of OA effector molecules, including cyclooxygenases I and II, nitric oxide, phospholipase A2, prostaglandin E2, reactive oxygen species as well as inflammatory cytokines and chemokines. Release of these molecules further stimulates cartilage matrix degradation, bone erosion, synovitis and fibrosis. IL-1 is also suspected to mediate pain in OA, the most common reason for consulting a physician [64–67].

Traditional pharmacologic approaches have failed to produce clinically useful molecules for inhibiting IL-1 activity intra-articularly [68]. However, two naturally occurring proteins exist specifically for this purpose: IL-1Ra and the soluble IL-1 type II receptor (sIL-1RII) [69, 70]. IL-1Ra functions as a competitive inhibitor by binding to the type I IL-1 signaling receptor and preventing subsequent interaction with IL-1. Once bound, IL-1Ra fails to recruit the IL-1R accessory protein (IL-1-AcP) to the complex and prevents intracellular activation and signaling. The sIL-1RII molecule, in contrast, titrates IL-1 activity by binding directly to soluble IL-1 molecules and blocking interaction with the type I receptor [71, 72]. Despite differences in their modes of action, the two molecules inhibit IL-1 signaling with equal potency. In the context of gene therapy, IL-1Ra is a smaller protein and easier to express as a transgene product. The recombinant protein (anakinra/Kineret®) is well characterized and is approved for clinical use in humans for RA and other conditions in which IL-1 is known to pay a significant role [71, 72]. As anakinra is administered daily by subcutaneous injections of 150 mg, the risk of adverse response from overproduction intra-articularly is extremely small.

Commercially available ELISAs with specificity for IL-1Ra orthologs in human, mouse and horse permit sensitive quantitation in culture media and biological fluids. Analysis of synovial fluid permits the use of IL-1Ra as a quantitative reporter of total gene transfer and therapeutic gene expression, allowing direct comparison of various delivery platforms. With respect to OA, IL-1Ra does not require sophisticated regulation. The goal is simply to express IL-1Ra at levels 10–100 fold over IL-1, where it completely inhibits IL-1 signaling activity. Once the threshold for efficacy has been achieved, expression beyond this has no adverse effect [73].

It is possible that a dual therapy combining elements of chondroprotection and regeneration could both inhibit degeneration and stimulate cartilage repair [48, 74]. Such a strategy, though, would likely require gene delivery via separate vectors to account for differences in their expression patterns for safe, effective application.

#### **9. Preclinical studies**

Following a series of preclinical successes in small laboratory animals demonstrating the proof of concept for direct viral-mediated gene delivery to joints, studies were performed to evaluate direct viral mediated gene delivery to equine joints using a first-generation adenoviral vector containing the cDNA for equine IL-1Ra. Administration of Ad.eqIL-1Ra in the joints of healthy horses, produced dose-dependent increases in IL-1Ra levels in synovial fluid aspirates. However, the highest viral dose tested, 5 × 1011 viral particles (vp) produced an acute synovitis [21].

To explore the capacity of Ad.eqIL-1Ra to inhibit OA pathologies, an osteochondral fragment (OCF) model of OA was used [21, 75]. In this system, a small osteochondral chip is surgically generated off the distal radial carpal bone of the midcarpal joint. Following a brief interval to recover from surgery, animals in the treatment group are injected in the OCF joint with the vector, while control animals receive saline [21]. The horses are then exercised 5 days/week on a high-speed treadmill, which, in the context of the osteochondral fracture, generates predictable pathologic lesions that mimic the onset of equine disease [21]. ELISA analysis of joint aspirates showed a peak in eqIL-1Ra expression at 7 days post injection which gradually diminished over a period of 28 days. Clinical examinations indicated that the expression of IL-1Ra decreased joint pain and synovial effusion relative to untreated horses, and protected the cartilage from the loss of proteoglycans.

These findings provided strong support for local gene delivery of IL-1Ra in large mammalian joints. A central limitation, however, was the use of the first generation adenovirus. In later work, we found articular tissues to be highly immune sensitive to the expression of foreign proteins, such that cells expressing foreign non-homologous transgene products or viral proteins are recognized by cell-mediated immune responses which lead to abbreviated persistence of transgenic expression in vivo [37]. Thus while the results showed promise, they indicated an intense need for an improved, immune stealthy vector system.

#### **9.1 AAV-mediated gene delivery to equine joints**

The results of studies of other gene therapy applications, such as hemophilia, indicated that long-term transgene expression was achievable following direct delivery of AAV vectors. The results of exploratory experiments in joints were disappointing. Transgene expression from conventional single-strand AAV vectors required several days or weeks to onset with marginal levels of protein production intra-articularly, a pattern that prevented testing in experimental disease models.

AAV transduction efficiency is known to be enhanced by mechanisms associated with intracellular stress. Certain stimuli, such as UV radiation, which increase the production of DNA synthesis and repair enzymes, significantly enhance intra-articular transgene expression from conventional AAV vectors [33, 76, 77], which indicates that second strand DNA synthesis is rate-limiting in joint tissues. Accordingly, AAV vectors that are self-complementary (sc) (i.e. double stranded, containing both + and – DNA strands) generated through the use of half-genome sized vector plasmids, or those containing a mutation in one of the terminal resolution sequences of the AAV ITRs [78, 79], provided ~20-fold enhancement of gene expression, with rapid onset in synovial and capsular cells in vitro and in vivo [80]. This adaptation was found to provide transduction and transgene expression profiles comparable to that provided by adenovirus. The requirement for a halfsized genome, however, limits the size of the transgene to about 1000–1200 base pairs [79].

Following encouraging results with scAAV vectors in the joints of laboratory animals [80], studies of AAV gene transfer shifted to the equine model to assess more clearly its utility for therapeutic gene delivery in large OA joints [19, 81]. As before, the carpal and MCP joints of the equine forelimbs were targeted for injection. In pilot studies, AAV gene delivery to healthy joints was examined using vectors containing the cDNAs for human IL-1Ra (AAV.hIL-Ra) and green fluorescent protein (AAV.GFP) [19]. In the animals receiving AAV.hIL-1Ra, synovial fluids were aspirated periodically over a period of several weeks. Animals receiving AAV.GFP were euthanized 14 days after injection and the distribution of fluorescence in the joint tissues was used to determine the number and locations of the cells modified by the AAV virus following intra-articular injection.

AAV gene delivery in the equine joints was capable of elevating the steady state hIL-1Ra in synovial fluid to levels equivalent to or greater than observed previously in rodents [19]. Analysis of GFP fluorescence showed that the vast majority of the

**37**

*Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

expressing the xenogeneic human IL-1Ra protein [37].

**9.2 Codon optimization of the equine IL-1Ra cDNA**

highly error prone.

tor dose of 105

vg/cell [18].

levels (<100 pg/ml) throughout [18].

**9.3 AAV gene delivery in naturally occurring OA**

transgene expression originated from the fibroblasts resident in the synovial lining. Fluorescent cells in the articular cartilage, though visible, were sparse, and GFP expression was faint. Peak levels of hIL-1Ra occurred at 1–2 weeks post-injection, but steadily declined over a period of 5 weeks. Studies in nude rats indicated that the abbreviated transgene expression was due to immune elimination of the cells

The commercial release (R&D Systems, Minneapolis, MN) of an ELISA kit specific for the equine IL-1Ra ortholog in 2010 proved to an enabling technology, allowing for the first time definitive quantification of the equine transgene intraarticularly [82]. Prior to this point, expression data relied on inter-species crossreactivity between human and murine ELISAs, results which were inconsistent and

To examine AAV-mediated transgene expression in the absence of immune interference, the human cDNA was replaced with the homologous equine IL-1Ra. To overcome initial problems with low production levels, codon-optimization resulted in >50-fold amplification in IL-1Ra secretion [18, 82]. For use in safety studies for the FDA, the cDNAs for human and rat IL-1Ra were also codon-optimized using the same algorithm [55]. After packaging in the AAV capsid, infection of synovial fibroblast cultures over a range of doses generated exceptionally high levels of IL-1Ra protein in conditioned medium, which exceeded 10 µg/ml at a vec-

Using the optimized AAV.eqIL-1Ra vector, a series of pharmacokinetic studies were performed to establish vector dose expression profiles following intra-articular injection [18]. In each of six horses, the midcarpal and MCP joints of both forelimbs were injected with AAV.eqIL-1Ra at doses ranging from 5 × 1010 to 5 × 1012 vg; the remaining joint received an equivalent volume of saline and served as a negative control. Analysis of synovial fluid, peripheral blood and urine collected periodically over a period of 6 months showed dose-related increases in the eqIL-1Ra content in synovial fluid at 2 weeks of injection, with peak production between 4 and 8 weeks. At the highest vector dose synovial fluid IL-1Ra levels exceeded 40 ng/ml, ~400 fold higher than endogenous synthesis. Importantly IL-1Ra production remained at these levels for the duration of the 6-month study. Despite simultaneous injection of recombinant AAV in three forelimb joints, no adverse effects were observed. IL-1Ra in blood serum, urine and synovial fluid of control joints remained at pre-injection

As discussed previously, the pathologic progression of OA induces sweeping changes in the architecture, cellularity and activation of the articular tissues. In conjunction with vector dosing, a series of tracking studies were performed to examine the impact of the OA environment on transgene expression and the biodistribution of the vector DNA and transduced cells [18]. Using a dose of 5 × 1012 vg, AAV.GFP was injected into one forelimb joint of several healthy horses, and horses with advanced naturally-occurring OA. Analysis of tissue samples 2 weeks later showed GFP fluorescence in healthy joints was concentrated in the synovial lining, with only a handful of GFP+ cells visible in cartilage shavings (**Figure 1**). In joints with advanced OA, there was a striking increase in GFP expression in all joint tissues particularly in articular cartilage. In synovium, enhanced GFP expression was due to the increased cellularity from local inflammation. Although fluorescence *Equine Science*

improved, immune stealthy vector system.

**9.1 AAV-mediated gene delivery to equine joints**

by the AAV virus following intra-articular injection.

treatment group are injected in the OCF joint with the vector, while control animals receive saline [21]. The horses are then exercised 5 days/week on a high-speed treadmill, which, in the context of the osteochondral fracture, generates predictable pathologic lesions that mimic the onset of equine disease [21]. ELISA analysis of joint aspirates showed a peak in eqIL-1Ra expression at 7 days post injection which gradually diminished over a period of 28 days. Clinical examinations indicated that the expression of IL-1Ra decreased joint pain and synovial effusion relative to untreated horses, and protected the cartilage from the loss of proteoglycans.

These findings provided strong support for local gene delivery of IL-1Ra in large mammalian joints. A central limitation, however, was the use of the first generation adenovirus. In later work, we found articular tissues to be highly immune sensitive to the expression of foreign proteins, such that cells expressing foreign non-homologous transgene products or viral proteins are recognized by cell-mediated immune responses which lead to abbreviated persistence of transgenic expression in vivo [37]. Thus while the results showed promise, they indicated an intense need for an

The results of studies of other gene therapy applications, such as hemophilia, indicated that long-term transgene expression was achievable following direct delivery of AAV vectors. The results of exploratory experiments in joints were disappointing. Transgene expression from conventional single-strand AAV vectors required several days or weeks to onset with marginal levels of protein production intra-articularly, a pattern that prevented testing in experimental disease models. AAV transduction efficiency is known to be enhanced by mechanisms associated with intracellular stress. Certain stimuli, such as UV radiation, which increase the production of DNA synthesis and repair enzymes, significantly enhance intra-articular transgene expression from conventional AAV vectors [33, 76, 77], which indicates that second strand DNA synthesis is rate-limiting in joint tissues. Accordingly, AAV vectors that are self-complementary (sc) (i.e. double stranded, containing both + and – DNA strands) generated through the use of half-genome sized vector plasmids, or those containing a mutation in one of the terminal resolution sequences of the AAV ITRs [78, 79], provided ~20-fold enhancement of gene expression, with rapid onset in synovial and capsular cells in vitro and in vivo [80]. This adaptation was found to provide transduction and transgene expression profiles comparable to that provided by adenovirus. The requirement for a halfsized genome, however, limits the size of the transgene to about 1000–1200 base

Following encouraging results with scAAV vectors in the joints of laboratory animals [80], studies of AAV gene transfer shifted to the equine model to assess more clearly its utility for therapeutic gene delivery in large OA joints [19, 81]. As before, the carpal and MCP joints of the equine forelimbs were targeted for injection. In pilot studies, AAV gene delivery to healthy joints was examined using vectors containing the cDNAs for human IL-1Ra (AAV.hIL-Ra) and green fluorescent protein (AAV.GFP) [19]. In the animals receiving AAV.hIL-1Ra, synovial fluids were aspirated periodically over a period of several weeks. Animals receiving AAV.GFP were euthanized 14 days after injection and the distribution of fluorescence in the joint tissues was used to determine the number and locations of the cells modified

AAV gene delivery in the equine joints was capable of elevating the steady state hIL-1Ra in synovial fluid to levels equivalent to or greater than observed previously in rodents [19]. Analysis of GFP fluorescence showed that the vast majority of the

**36**

pairs [79].

transgene expression originated from the fibroblasts resident in the synovial lining. Fluorescent cells in the articular cartilage, though visible, were sparse, and GFP expression was faint. Peak levels of hIL-1Ra occurred at 1–2 weeks post-injection, but steadily declined over a period of 5 weeks. Studies in nude rats indicated that the abbreviated transgene expression was due to immune elimination of the cells expressing the xenogeneic human IL-1Ra protein [37].

#### **9.2 Codon optimization of the equine IL-1Ra cDNA**

The commercial release (R&D Systems, Minneapolis, MN) of an ELISA kit specific for the equine IL-1Ra ortholog in 2010 proved to an enabling technology, allowing for the first time definitive quantification of the equine transgene intraarticularly [82]. Prior to this point, expression data relied on inter-species crossreactivity between human and murine ELISAs, results which were inconsistent and highly error prone.

To examine AAV-mediated transgene expression in the absence of immune interference, the human cDNA was replaced with the homologous equine IL-1Ra. To overcome initial problems with low production levels, codon-optimization resulted in >50-fold amplification in IL-1Ra secretion [18, 82]. For use in safety studies for the FDA, the cDNAs for human and rat IL-1Ra were also codon-optimized using the same algorithm [55]. After packaging in the AAV capsid, infection of synovial fibroblast cultures over a range of doses generated exceptionally high levels of IL-1Ra protein in conditioned medium, which exceeded 10 µg/ml at a vector dose of 105 vg/cell [18].

Using the optimized AAV.eqIL-1Ra vector, a series of pharmacokinetic studies were performed to establish vector dose expression profiles following intra-articular injection [18]. In each of six horses, the midcarpal and MCP joints of both forelimbs were injected with AAV.eqIL-1Ra at doses ranging from 5 × 1010 to 5 × 1012 vg; the remaining joint received an equivalent volume of saline and served as a negative control. Analysis of synovial fluid, peripheral blood and urine collected periodically over a period of 6 months showed dose-related increases in the eqIL-1Ra content in synovial fluid at 2 weeks of injection, with peak production between 4 and 8 weeks. At the highest vector dose synovial fluid IL-1Ra levels exceeded 40 ng/ml, ~400 fold higher than endogenous synthesis. Importantly IL-1Ra production remained at these levels for the duration of the 6-month study. Despite simultaneous injection of recombinant AAV in three forelimb joints, no adverse effects were observed. IL-1Ra in blood serum, urine and synovial fluid of control joints remained at pre-injection levels (<100 pg/ml) throughout [18].

#### **9.3 AAV gene delivery in naturally occurring OA**

As discussed previously, the pathologic progression of OA induces sweeping changes in the architecture, cellularity and activation of the articular tissues. In conjunction with vector dosing, a series of tracking studies were performed to examine the impact of the OA environment on transgene expression and the biodistribution of the vector DNA and transduced cells [18]. Using a dose of 5 × 1012 vg, AAV.GFP was injected into one forelimb joint of several healthy horses, and horses with advanced naturally-occurring OA. Analysis of tissue samples 2 weeks later showed GFP fluorescence in healthy joints was concentrated in the synovial lining, with only a handful of GFP+ cells visible in cartilage shavings (**Figure 1**). In joints with advanced OA, there was a striking increase in GFP expression in all joint tissues particularly in articular cartilage. In synovium, enhanced GFP expression was due to the increased cellularity from local inflammation. Although fluorescence

#### **Figure 1.**

*Representative fluorescence activity in synovium (top row) and cartilage (bottom row) following intraarticular injection of an AAV vector containing the cDNA for green fluorescent protein (GFP) into healthy carpal joints or those with naturally-occurring OA.*

was greater in all cartilage shavings, GFP expression in regions containing visibly damaged cartilage was markedly increased. GFP+ chondrocytes appeared throughout the damaged regions, particularly in clusters of proliferating chondrocytes, characteristic of OA cartilage.

In both healthy and OA joints receiving virus, no GFP+ cells were detected in extra-articular tissues, and qPCR analyses showed that 99.7% of the vector DNA was localized within the cartilage and synovium of the injected joint. Similar patterns of biodistribution were noted by Goodrich et al. [81]. While the enhanced transduction of OA chondrocytes might be presumed to arise from increased vector access from ECM degradation, qPCR analyses showed the vector DNA content in the chondrocytes of healthy and OA cartilage was essentially the same, suggesting that the increased transgene expression arose from increased metabolism due to inflammatory and stress induced activation [18].

Transcription from the CMV immediate early promoter, which drives the scAAV vector expression cassette, is induced in response to NF-κB activation and signal transduction from p38 and other stress-activated protein kinases. Similar stressrelated induction of this promoter serves to re-activate human CMV from latency, and is required for expression of genes necessary for DNA replication [83–87]. In the same manner, inflammation and cellular stress can significantly increase the transcription and expression of transgenes under control of the CMV immediate early promoter [87–90]. In these respects, OA cartilage is highly enriched with stress-activated chondrocytes [91], especially at sites of cartilage degradation, where GFP expression was greatest.

Although several reports describe the generation of synthetic inflammationinducible promoter systems for gene therapy applications, the AAV vector discussed here, at least within the context of a large mammalian joint, appears to be highly responsive to the OA environment and innately disease activated. The regional

**39**

capsid serotype.

**9.5 Long-term efficacy**

*Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

repair of the osteochondral fragment [92].

ous effect on transgene expression.

**9.4 AAV.eqIL-1Ra delivery and expression in the OCF model**

regeneration.

differences in GFP expression seen in OA cartilage indicate the potential to direct therapeutic transgene expression preferentially to areas of articular cartilage under the greatest pathologic stress. This perhaps lays the groundwork for development of vectors for disease-targeted anabolic stimulation of cartilage repair and

The OCF model was adapted to determine if the levels of equine IL-1Ra generated by AAV gene transfer were sufficient to mediate an appropriate biologic response [92]. Following creation of the osteochondral fracture, the OCF joints in the treated animals were injected with 5 × 1012 vg AAV.eqIL-1Ra, while controls animals received an equal volume of saline. One week later the animals were placed on a 5 day/week athletic training protocol for a period of 10 weeks. In this acute injury model, mean eqIL-1Ra expression was initially ~4-fold higher than observed in healthy joints at the same viral dose and correlated directly with the severity of joint pathology at the time of vector delivery. Over the 10 week training period, eqIL-1Ra expression gradually diminished to ~60 ng/ml, similar to that seen with AAV gene transfer in normal joints. Despite variable expression among animals the steadystate eqIL-1Ra in synovial fluids exceeded that of IL-1 by >500-fold in all animals. In agreement with increased IL-1Ra, the treated horses showed a reduction synovial fluid PGE2 levels and a progressive reduction in joint pain. Improved joint function was accompanied by significant reduction in joint pathology by both arthroscopy and MRI. By both diagnostics, the treated animals showed significant reductions in synovial effusion and marrow edema, local protection of cartilage and enhanced

Consistent with the findings of others [45, 93], we observed an increase in AAV capsid-specific neutralizing antibody (NAb) titer over time in both the blood serum and synovial fluid of the horses receiving the AAV vector [92]. The NAb titer in synovial fluids was consistently several-fold higher than in blood but had no obvi-

The primary advantage of a gene-based therapy for OA lies with the capacity for sustained local delivery of anti-arthritic agents and the promise long term therapeutic benefit. To address this, we recently completed a series of studies to assess the safety and efficacy of AAV.eqIL-1Ra delivery in a chronic model of joint disease over the course of a year. For the chronic model, the 10 weeks OCF protocol was used to induce joint pathology consistent with early symptomatic disease. At the

Humans are natural hosts to wild type AAV infection and often have high circulating titers of neutralizing antibodies (NAb) to AAV capsids of several serotypes (primarily AAV2, AAV1, and to lesser extents AAV5) from prior infections with wild type virus. Horses, however, are not common hosts for wild type AAV infection and distinct from humans, have low circulating titers to most AAV vector serotypes. While NAb to AAV5 appears to be relatively common among horses, and one report describes increased NAb titers to AAV2 capsid in a small test sample, pre-existing NAb do not appear to be prevalent in the equine population nor at sufficient titer to prohibit effective gene delivery. As indicated above, high levels of capsid serotype specific NAb will arise from prior treatment with an AAV vector, which can inhibit the efficacy of subsequent vector administration. There is evidence that vector neutralization can be averted through the use of an alternate

*Equine Science*

characteristic of OA cartilage.

*carpal joints or those with naturally-occurring OA.*

**Figure 1.**

inflammatory and stress induced activation [18].

where GFP expression was greatest.

was greater in all cartilage shavings, GFP expression in regions containing visibly damaged cartilage was markedly increased. GFP+ chondrocytes appeared throughout the damaged regions, particularly in clusters of proliferating chondrocytes,

*Representative fluorescence activity in synovium (top row) and cartilage (bottom row) following intraarticular injection of an AAV vector containing the cDNA for green fluorescent protein (GFP) into healthy* 

In both healthy and OA joints receiving virus, no GFP+ cells were detected in extra-articular tissues, and qPCR analyses showed that 99.7% of the vector DNA was localized within the cartilage and synovium of the injected joint. Similar patterns of biodistribution were noted by Goodrich et al. [81]. While the enhanced transduction of OA chondrocytes might be presumed to arise from increased vector access from ECM degradation, qPCR analyses showed the vector DNA content in the chondrocytes of healthy and OA cartilage was essentially the same, suggesting that the increased transgene expression arose from increased metabolism due to

Transcription from the CMV immediate early promoter, which drives the scAAV vector expression cassette, is induced in response to NF-κB activation and signal transduction from p38 and other stress-activated protein kinases. Similar stressrelated induction of this promoter serves to re-activate human CMV from latency, and is required for expression of genes necessary for DNA replication [83–87]. In the same manner, inflammation and cellular stress can significantly increase the transcription and expression of transgenes under control of the CMV immediate early promoter [87–90]. In these respects, OA cartilage is highly enriched with stress-activated chondrocytes [91], especially at sites of cartilage degradation,

Although several reports describe the generation of synthetic inflammationinducible promoter systems for gene therapy applications, the AAV vector discussed here, at least within the context of a large mammalian joint, appears to be highly responsive to the OA environment and innately disease activated. The regional

**38**

differences in GFP expression seen in OA cartilage indicate the potential to direct therapeutic transgene expression preferentially to areas of articular cartilage under the greatest pathologic stress. This perhaps lays the groundwork for development of vectors for disease-targeted anabolic stimulation of cartilage repair and regeneration.

#### **9.4 AAV.eqIL-1Ra delivery and expression in the OCF model**

The OCF model was adapted to determine if the levels of equine IL-1Ra generated by AAV gene transfer were sufficient to mediate an appropriate biologic response [92]. Following creation of the osteochondral fracture, the OCF joints in the treated animals were injected with 5 × 1012 vg AAV.eqIL-1Ra, while controls animals received an equal volume of saline. One week later the animals were placed on a 5 day/week athletic training protocol for a period of 10 weeks. In this acute injury model, mean eqIL-1Ra expression was initially ~4-fold higher than observed in healthy joints at the same viral dose and correlated directly with the severity of joint pathology at the time of vector delivery. Over the 10 week training period, eqIL-1Ra expression gradually diminished to ~60 ng/ml, similar to that seen with AAV gene transfer in normal joints. Despite variable expression among animals the steadystate eqIL-1Ra in synovial fluids exceeded that of IL-1 by >500-fold in all animals. In agreement with increased IL-1Ra, the treated horses showed a reduction synovial fluid PGE2 levels and a progressive reduction in joint pain. Improved joint function was accompanied by significant reduction in joint pathology by both arthroscopy and MRI. By both diagnostics, the treated animals showed significant reductions in synovial effusion and marrow edema, local protection of cartilage and enhanced repair of the osteochondral fragment [92].

Consistent with the findings of others [45, 93], we observed an increase in AAV capsid-specific neutralizing antibody (NAb) titer over time in both the blood serum and synovial fluid of the horses receiving the AAV vector [92]. The NAb titer in synovial fluids was consistently several-fold higher than in blood but had no obvious effect on transgene expression.

Humans are natural hosts to wild type AAV infection and often have high circulating titers of neutralizing antibodies (NAb) to AAV capsids of several serotypes (primarily AAV2, AAV1, and to lesser extents AAV5) from prior infections with wild type virus. Horses, however, are not common hosts for wild type AAV infection and distinct from humans, have low circulating titers to most AAV vector serotypes. While NAb to AAV5 appears to be relatively common among horses, and one report describes increased NAb titers to AAV2 capsid in a small test sample, pre-existing NAb do not appear to be prevalent in the equine population nor at sufficient titer to prohibit effective gene delivery. As indicated above, high levels of capsid serotype specific NAb will arise from prior treatment with an AAV vector, which can inhibit the efficacy of subsequent vector administration. There is evidence that vector neutralization can be averted through the use of an alternate capsid serotype.

#### **9.5 Long-term efficacy**

The primary advantage of a gene-based therapy for OA lies with the capacity for sustained local delivery of anti-arthritic agents and the promise long term therapeutic benefit. To address this, we recently completed a series of studies to assess the safety and efficacy of AAV.eqIL-1Ra delivery in a chronic model of joint disease over the course of a year. For the chronic model, the 10 weeks OCF protocol was used to induce joint pathology consistent with early symptomatic disease. At the

completion of the athletic training period, 5 × 1012 vg of the AAV.eqIL-1Ra vector was injected into the OCF joint, and a 3 day/week training regimen was instituted to maintain a slow but progressive degenerative condition for the following 12 months.

Immediately prior to injection, and then at 2 weeks and monthly thereafter, peripheral blood, urine and synovial fluids were collected, and joint pain and kinematic assessments were performed. Radiographic and MR imaging of both midcarpal joints was performed prior to injection and then at 6- and 12-month time points. Arthroscopic examination of the joints was performed at endpoint and digitally recorded, and synovial and articular cartilage biopsies were taken for histologic examination. At the conclusion of the year-long protocol all animals in the treatment group and five animals from the Control group were euthanized for biodistribution and toxicology analysis.

Analysis of synovial fluids showed that high IL-1Ra levels of 40–50 ng/ml were sustained over the 12-month course of the study. In the chronic OCF model transgenic IL-1Ra expression was far more consistent among individual animals than in joints with an acute osteochondral fracture. Relative to arthritic controls, the treated animals showed a ~40% reduction in lameness, indicative of reduced joint pain and improved mobility. By MRI assessment, joint pathology in the was reduced by ~28% relative to baseline disease, while in control joints the overall pathology was largely unchanged. Relative to pretreatment levels the treated group showed ~28% improvement in all major OA pathologies relative to baseline while in arthritic controls pathologic scores remained unchanged or increased in severity.

#### **9.6 Toxicology and biodistribution**

To establish a qualified biosafety profile for AAV.IL-1Ra gene transfer in a large mammalian joint, formal preclinical toxicology and biodistribution studies were performed addressing the acute and long-term phases of vector delivery. In the Acute Phase studies, early stage disease was induced in one midcarpal joint using the OCF protocol. Three horses each were injected with 5 × 1012 vg AAV.eqIL-1Ra, 1× anticipated clinical dose, and three horses with 5 × 1013, 10× clinical dose, intended to represent a "worst case scenario." For the long-term toxicology studies, each of the 10 horses from the treated group in the 12-month study and 5 horses randomly selected from the control group were euthanized for necropsy. Following euthanasia, samples from more than 50 tissues were collected for histopathologic evaluation or DNA extraction and PCR analysis of vector genome content.

In all animals injected with AAV.eqIL-1Ra at the 1× dose, high vector genome copies (104 –106 ) were detected by qPCR in the synovium and cartilage, which were equivalent in animals euthanized at 2 weeks and 12 months post-injection. No vector DNA was detected in extra-articular tissues. No pathologic response associated with vector injection was observed in any tissue.

The cumulative data from these pharmacokinetic, toxicology and efficacy studies in the equine model demonstrate that a gene-based therapy using recombinant AAV can provide safe, long-term, effective delivery of anti-arthritic proteins, such as IL-1Ra, in large mammalian joints. The results of these safety and efficacy studies in horses formed the bases for a successful IND application and the initiation of a phase I trial of AAV-IL-1Ra delivery for knee OA.

#### **9.7 Gene delivery with high-capacity adenovirus**

Recently, studies involving gene delivery with HD.Ad have begun to move toward clinical studies, at least in human OA. Initial studies in mice using

**41**

**10. Conclusions**

*Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

intra-articular HD.Ad-mediated delivery of the cDNA for lubricin (Prg4) showed a marked chondroprotective effect, maintaining matrix volume and prevention of

Progressing from these results, studies were performed to examine the therapeutic capacity of IL-1Ra gene delivery via HD.Ad in mice and the forelimb joints of horses [94]. In most gene therapy applications a strong, constitutively active promoter sequence, such as the CMV promoter/enhancer or the eukaryotic translation initiation factor 1α (EIF-1α) promoter is used. In this case, however, transgene expression was driven by an inflammation-inducible promoter comprised of a minimal endothelial cell leukocyte adhesion molecule (ELAM1) promoter linked to multiple upstream NF-κB recognition elements. The rationale being that therapeutic transgene expression would be delimited specifically to inflammatory flares. Similar to prior studies with Prg4, HD.Ad delivery of the homologous murine IL-1Ra transgene was found to inhibit osteophyte formation and cartilage erosion in the CLT defect model [94]. Importantly, vector delivery was also associated with a significant reduction in pain sensitivity. Following preliminary dosing studies in healthy equine joints, HD.Ad was used to deliver the eqIL-1Ra cDNA into the carpal joints of horses following surgical generation of the osteochondral lesion in the OCF model. IL-1Ra levels in the injected joints rose to ~20 ng/ml within the first week, but then dropped about 10 fold by week 2 and were near endogenous background by week 3. Despite the relative brevity of expression, the treated animals showed significant improvement in lameness, reduced joint effusion, synovitis and osteo-

Due its relatively large size, the adenoviral particle cannot penetrate the ECM of the synovium or cartilage and remains constrained to cells residing in superficial regions [39]. Interestingly in both reports where intra-articular transgene expression was quantified, a 90–99% loss in therapeutic transgene expression was observed within 1–2 weeks of vector injection, regardless of whether an inducible promoter was used [39, 94]. While transgene expression seems to persist long-term, therapeutic protein levels appear at trace levels. Given this profile, it will be inter-

These data altogether show that in large mammalian joints, local gene transfer can provide persistent IL-1Ra transgene expression at therapeutically relevant levels. Despite variable expression among treated joints in the context of acute inflammation, sustained IL-1Ra expression provides meaningful benefit, such that a single injection reduces joint pain and intra-articular inflammation, and improves repair of the damaged bone and protects cartilage against degradation. No adverse response to the vectors or transgene have been observed with either AAV or HD.Ad, and at least within the equine system local overexpression of IL-1Ra provides no

Having established safety and efficacy of IL-1Ra gene delivery in the equine joint, the next stage in development would be the move into field testing with client animals. Given the limited resources available for equine research, such a large and costly undertaking is likely feasible only through support from partners in the veterinary pharmaceutical industry or private investors looking to advance the treatment methods toward commercialization. In this respect, questions of market size, cost of goods and profitability move to the forefront. Currently human genebased therapies come with a substantial price tag, and range at the high end from \$450,000 per eye for Luxterna® for congenital retinal degeneration to \$2,100,000

degeneration in a cruciate ligament transection (CLT) PTOA model [39].

cyte formation and improved cartilage matrix integrity [94].

esting to see how this platform moves ahead in the future.

apparent risk of systemic immunosuppression.

#### *Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

*Equine Science*

12 months.

biodistribution and toxicology analysis.

**9.6 Toxicology and biodistribution**

PCR analysis of vector genome content.

with vector injection was observed in any tissue.

phase I trial of AAV-IL-1Ra delivery for knee OA.

**9.7 Gene delivery with high-capacity adenovirus**

–106

completion of the athletic training period, 5 × 1012 vg of the AAV.eqIL-1Ra vector was injected into the OCF joint, and a 3 day/week training regimen was instituted to maintain a slow but progressive degenerative condition for the following

Immediately prior to injection, and then at 2 weeks and monthly thereafter, peripheral blood, urine and synovial fluids were collected, and joint pain and kinematic assessments were performed. Radiographic and MR imaging of both midcarpal joints was performed prior to injection and then at 6- and 12-month time points. Arthroscopic examination of the joints was performed at endpoint and digitally recorded, and synovial and articular cartilage biopsies were taken for histologic examination. At the conclusion of the year-long protocol all animals in the treatment group and five animals from the Control group were euthanized for

Analysis of synovial fluids showed that high IL-1Ra levels of 40–50 ng/ml were sustained over the 12-month course of the study. In the chronic OCF model transgenic IL-1Ra expression was far more consistent among individual animals than in joints with an acute osteochondral fracture. Relative to arthritic controls, the treated animals showed a ~40% reduction in lameness, indicative of reduced joint pain and improved mobility. By MRI assessment, joint pathology in the was reduced by ~28% relative to baseline disease, while in control joints the overall pathology was largely unchanged. Relative to pretreatment levels the treated group showed ~28% improvement in all major OA pathologies relative to baseline while in arthritic

controls pathologic scores remained unchanged or increased in severity.

To establish a qualified biosafety profile for AAV.IL-1Ra gene transfer in a large mammalian joint, formal preclinical toxicology and biodistribution studies were performed addressing the acute and long-term phases of vector delivery. In the Acute Phase studies, early stage disease was induced in one midcarpal joint using the OCF protocol. Three horses each were injected with 5 × 1012 vg AAV.eqIL-1Ra, 1× anticipated clinical dose, and three horses with 5 × 1013, 10× clinical dose, intended to represent a "worst case scenario." For the long-term toxicology studies, each of the 10 horses from the treated group in the 12-month study and 5 horses randomly selected from the control group were euthanized for necropsy. Following euthanasia, samples from more than 50 tissues were collected for histopathologic evaluation or DNA extraction and

In all animals injected with AAV.eqIL-1Ra at the 1× dose, high vector genome

equivalent in animals euthanized at 2 weeks and 12 months post-injection. No vector DNA was detected in extra-articular tissues. No pathologic response associated

Recently, studies involving gene delivery with HD.Ad have begun to move toward clinical studies, at least in human OA. Initial studies in mice using

The cumulative data from these pharmacokinetic, toxicology and efficacy studies in the equine model demonstrate that a gene-based therapy using recombinant AAV can provide safe, long-term, effective delivery of anti-arthritic proteins, such as IL-1Ra, in large mammalian joints. The results of these safety and efficacy studies in horses formed the bases for a successful IND application and the initiation of a

) were detected by qPCR in the synovium and cartilage, which were

**40**

copies (104

intra-articular HD.Ad-mediated delivery of the cDNA for lubricin (Prg4) showed a marked chondroprotective effect, maintaining matrix volume and prevention of degeneration in a cruciate ligament transection (CLT) PTOA model [39].

Progressing from these results, studies were performed to examine the therapeutic capacity of IL-1Ra gene delivery via HD.Ad in mice and the forelimb joints of horses [94]. In most gene therapy applications a strong, constitutively active promoter sequence, such as the CMV promoter/enhancer or the eukaryotic translation initiation factor 1α (EIF-1α) promoter is used. In this case, however, transgene expression was driven by an inflammation-inducible promoter comprised of a minimal endothelial cell leukocyte adhesion molecule (ELAM1) promoter linked to multiple upstream NF-κB recognition elements. The rationale being that therapeutic transgene expression would be delimited specifically to inflammatory flares.

Similar to prior studies with Prg4, HD.Ad delivery of the homologous murine IL-1Ra transgene was found to inhibit osteophyte formation and cartilage erosion in the CLT defect model [94]. Importantly, vector delivery was also associated with a significant reduction in pain sensitivity. Following preliminary dosing studies in healthy equine joints, HD.Ad was used to deliver the eqIL-1Ra cDNA into the carpal joints of horses following surgical generation of the osteochondral lesion in the OCF model. IL-1Ra levels in the injected joints rose to ~20 ng/ml within the first week, but then dropped about 10 fold by week 2 and were near endogenous background by week 3. Despite the relative brevity of expression, the treated animals showed significant improvement in lameness, reduced joint effusion, synovitis and osteocyte formation and improved cartilage matrix integrity [94].

Due its relatively large size, the adenoviral particle cannot penetrate the ECM of the synovium or cartilage and remains constrained to cells residing in superficial regions [39]. Interestingly in both reports where intra-articular transgene expression was quantified, a 90–99% loss in therapeutic transgene expression was observed within 1–2 weeks of vector injection, regardless of whether an inducible promoter was used [39, 94]. While transgene expression seems to persist long-term, therapeutic protein levels appear at trace levels. Given this profile, it will be interesting to see how this platform moves ahead in the future.

#### **10. Conclusions**

These data altogether show that in large mammalian joints, local gene transfer can provide persistent IL-1Ra transgene expression at therapeutically relevant levels. Despite variable expression among treated joints in the context of acute inflammation, sustained IL-1Ra expression provides meaningful benefit, such that a single injection reduces joint pain and intra-articular inflammation, and improves repair of the damaged bone and protects cartilage against degradation. No adverse response to the vectors or transgene have been observed with either AAV or HD.Ad, and at least within the equine system local overexpression of IL-1Ra provides no apparent risk of systemic immunosuppression.

Having established safety and efficacy of IL-1Ra gene delivery in the equine joint, the next stage in development would be the move into field testing with client animals. Given the limited resources available for equine research, such a large and costly undertaking is likely feasible only through support from partners in the veterinary pharmaceutical industry or private investors looking to advance the treatment methods toward commercialization. In this respect, questions of market size, cost of goods and profitability move to the forefront. Currently human genebased therapies come with a substantial price tag, and range at the high end from \$450,000 per eye for Luxterna® for congenital retinal degeneration to \$2,100,000

for Zolgensma®, a gene correction therapy for spinal muscular atrophy in infants. Both of these "drugs" employ AAV as a vector. As the popularity of gene-based therapies continues to advance, production costs will likely fall considerably as the field grows and therapies with greater efficacy emerge. Among these business issues, questions regarding genetic enhancement in the racing industry will need to be addressed and resolved.

### **Acknowledgements**

This work was supported in part by grants AR048566 and AR048566-S from the National Institute of Arthritis Musculoskeletal and Skin Diseases (NIAMS) of the National Institutes of Health, and grants PR130999 and PR180272 from the Congressionally Directed Medical Research Programs of the US Department of Defense.

### **Conflict of interest**

Steve Ghivizzani is a founder and share-holder in Genascence Inc., a company pursuing development and commercialization of gene therapies for inflammatory conditions.

### **Author details**

Rachael Levings1 , Andrew Smith2 , Padraic P. Levings1 , Glyn D. Palmer1 , Anthony Dacanay1 , Patrick Colahan2 and Steven C. Ghivizzani1 \*

1 Department of Orthopaedics and Rehabilitation, University of Florida College of Medicine, Gainesville, Florida, United States

2 Department of Large Animal Medicine, University of Florida College of Veterinary Medicine, Gainesville, Florida, United States

\*Address all correspondence to: ghivisc@ortho.ufl.edu

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**43**

*Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

cartilage degradation and chondrocyte

[11] Scanzello CR, Goldring SR. The role of synovitis in osteoarthritis pathogenesis. Bone. 2012;**51**(2):249-257

Harris P, Gent T, Freeman S, et al. The contribution of the synovium, synovial derived inflammatory cytokines and neuropeptides to the pathogenesis of osteoarthritis. Veterinary Journal.

differentiation. RMD Open. 2015;**1**(Suppl 1):e000061

[12] Sutton S, Clutterbuck A,

[13] Ghivizzani SC, Oligino TJ,

Robbins PD, Evans CH. Cartilage injury and repair. Physical Medicine and Rehabilitation Clinics of North America.

[14] Evans CH, Kraus VB, Setton LA. Progress in intra-articular therapy. Nature Reviews Rheumatology.

[15] Marks PW, Witten CM, Califf RM. Clarifying stem-cell therapy's benefits and risks. The New England Journal of Medicine. 2017;**376**(11):1007-1009

[16] Robey P. "Mesenchymal stem cells": Fact or fiction, and implications in their therapeutic use. F1000Research. 2017;**6**(F1000 Faculty Rev):524

[17] Evans CH, Robbins PD. The interleukin-1 receptor antagonist and its delivery by gene transfer. Receptor.

[18] Watson Levings R, Broome TA, Smith AD, Rice BL, Gibbs EP, et al. Gene therapy for osteoarthritis: Pharmacokinetics of intra-articular scAAV.IL-1Ra delivery in an equine model. Human Gene Therapy. Clinical Development. 2018;**29**(2):90-100

[19] Watson RS, Broome TA, Levings PP, Rice BL, Kay JD, et al. scAAV-mediated

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Cicuttini FM, Conaghan PG, Cooper C, et al. Osteoarthritis. Nature Reviews. Disease Primers. 2016;**2**:16072

[2] Goodrich LR, Nixon AJ. Medical treatment of osteoarthritis in the horse—A review. Veterinary Journal.

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Musculoskeletal disease in aged horses and its management. The Veterinary Clinics of North America. Equine Practice. 2016;**32**(2):229-247

[8] Loeser RF, Goldring SR, Scanzello CR, Goldring MB. Osteoarthritis: A disease of the joint as an organ. Arthritis and Rheumatism. 2012;**64**(6):1697-1707

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[10] Olivotto E, Otero M, Marcu KB, Goldring MB. Pathophysiology of osteoarthritis: Canonical NF-kappaB/ IKKbeta-dependent and kinaseindependent effects of IKKalpha in

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*Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

#### **References**

*Equine Science*

be addressed and resolved.

**Acknowledgements**

**Conflict of interest**

Defense.

conditions.

**42**

**Author details**

Rachael Levings1

Anthony Dacanay1

, Andrew Smith2

Medicine, Gainesville, Florida, United States

provided the original work is properly cited.

, Patrick Colahan2

Veterinary Medicine, Gainesville, Florida, United States

\*Address all correspondence to: ghivisc@ortho.ufl.edu

, Padraic P. Levings1

1 Department of Orthopaedics and Rehabilitation, University of Florida College of

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

for Zolgensma®, a gene correction therapy for spinal muscular atrophy in infants. Both of these "drugs" employ AAV as a vector. As the popularity of gene-based therapies continues to advance, production costs will likely fall considerably as the field grows and therapies with greater efficacy emerge. Among these business issues, questions regarding genetic enhancement in the racing industry will need to

This work was supported in part by grants AR048566 and AR048566-S from the National Institute of Arthritis Musculoskeletal and Skin Diseases (NIAMS) of the National Institutes of Health, and grants PR130999 and PR180272 from the Congressionally Directed Medical Research Programs of the US Department of

Steve Ghivizzani is a founder and share-holder in Genascence Inc., a company pursuing development and commercialization of gene therapies for inflammatory

2 Department of Large Animal Medicine, University of Florida College of

and Steven C. Ghivizzani1

, Glyn D. Palmer1

\*

,

[1] Martel-Pelletier J, Barr AJ, Cicuttini FM, Conaghan PG, Cooper C, et al. Osteoarthritis. Nature Reviews. Disease Primers. 2016;**2**:16072

[2] Goodrich LR, Nixon AJ. Medical treatment of osteoarthritis in the horse—A review. Veterinary Journal. 2006;**171**(1):51-69

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2005. p. 2613

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1999;**6**(10):1713-1720

2002;**5**(4):397-404

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2005;**64**(12):1677-1684

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[32] Apparailly F, Khoury M, Vervoordeldonk MJ, Adriaansen J, Gicquel E, et al. Adeno-associated virus pseudotype 5 vector improves gene transfer in arthritic joints. Human Gene

Therapy. 2005;**16**(4):426-434

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mediated gene transfer to rabbit synovium in vivo. The Journal of Clinical Investigation.

1993;**92**(2):1085-1092

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[23] Bandara G, Robbins PD, Georgescu HI, Mueller GM,

Biology. 1992;**11**(3):227-231

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2005;**102**(24):8698-8703

1996;**39**(5):820-828

gene delivery to synovium. An

Glorioso JC, Evans CH. Gene transfer to synoviocytes: Prospects for gene treatment of arthritis. DNA and Cell

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**44**

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[36] Robbins PD, Ghivizzani SC. Viral vectors for gene therapy. Pharmacology & Therapeutics. 1998;**80**(1):35-47

[37] Gouze E, Gouze JN, Palmer GD, Pilapil C, Evans CH, Ghivizzani SC. Transgene persistence and cell turnover in the diarthrodial joint: Implications for gene therapy of chronic joint diseases. Molecular Therapy. 2007;**15**(6):1114-1120

[38] Suzuki M, Cela R, Clarke C, Bertin TK, Mourino S, Lee B. Largescale production of high-quality helperdependent adenoviral vectors using adherent cells in cell factories. Human Gene Therapy. 2010;**21**(1):120-126

[39] Ruan MZ, Erez A, Guse K, Dawson B, Bertin T, et al. Proteoglycan 4 expression protects against the development of osteoarthritis. Science Translational Medicine. 2013;**5**(176):176ra34

[40] Brunetti-Pierri N, Ng T, Iannitti D, Cioffi W, Stapleton G, et al. Transgene expression up to 7 years in nonhuman primates following hepatic transduction with helper-dependent adenoviral

vectors. Human Gene Therapy. 2013;**24**(8):761-765

[41] Rosewell A, Vetrini F, Ng P. Helperdependent adenoviral vectors. Journal of Genetic Syndromes and Gene Therapy. 2011;Suppl 5:001

[42] Choi VW, McCarty DM, Samulski RJ. AAV hybrid serotypes: Improved vectors for gene delivery. Current Gene Therapy. 2005;**5**(3):299-310

[43] Berns KI, Linden RM. The cryptic life style of adeno-associated virus. BioEssays. 1995;**17**(3):237-245

[44] Martino AT, Markusic DM. Immune response mechanisms against AAV vectors in animal models. Molecular Therapy - Methods and Clinical Development. 2020;**17**:198-208

[45] Ortved K, Wagner B, Calcedo R, Wilson J, Schaefer D, Nixon A. Humoral and cell-mediated immune response, and growth factor synthesis after direct intraarticular injection of rAAV2-IGF-I and rAAV5-IGF-I in the equine middle carpal joint. Human Gene Therapy. 2015;**26**(3):161-171

[46] Calcedo R, Franco J, Qin Q, Richardson DW, Mason JB, et al. Preexisting neutralizing antibodies to adeno-associated virus capsids in large animals other than monkeys may confound in vivo gene therapy studies. Human Gene Therapy Methods. 2015;**26**(3):103-105

[47] Zhang X, Mao Z, Yu C. Suppression of early experimental osteoarthritis by gene transfer of interleukin-1 receptor antagonist and interleukin-10. Journal of Orthopaedic Research. 2004;**22**(4):742-750

[48] Haupt JL, Frisbie DD, McIlwraith CW, Robbins PD, Ghivizzani S, et al. Dual transduction of insulin-like growth factor-I and

interleukin-1 receptor antagonist protein controls cartilage degradation in an osteoarthritic culture model. Journal of Orthopaedic Research. 2005;**23**(1):118-126

[49] Ortved KF, Begum L, Stefanovski D, Nixon AJ. AAV-mediated overexpression of IL-10 mitigates the inflammatory cascade in stimulated equine chondrocyte pellets. Current Gene Therapy. 2018;**18**(3):171-179

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[53] Pelletier JP, Caron JP, Evans C, Robbins PD, Georgescu HI, et al. In vivo suppression of early experimental osteoarthritis by interleukin-1 receptor antagonist using gene therapy. Arthritis and Rheumatism. 1997;**40**(6):1012-1019

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[56] Mi Z, Ghivizzani SC, Lechman E, Glorioso JC, Evans CH, Robbins PD. Adverse effects of adenovirus-mediated gene transfer of human transforming growth factor beta 1 into rabbit knees. Arthritis Research & Therapy. 2003;**5**(3):R132-R139

[57] Watson RS, Gouze E, Levings PP, Bush ML, Kay JD, et al. Gene delivery of TGF-beta1 induces arthrofibrosis and chondrometaplasia of synovium in vivo. Laboratory Investigation. 2010;**90**(11):1615-1627

[58] Gelse K, Jiang QJ, Aigner T, Ritter T, Wagner K, et al. Fibroblastmediated delivery of growth factor complementary DNA into mouse joints induces chondrogenesis but avoids the disadvantages of direct viral gene transfer. Arthritis and Rheumatism. 2001;**44**(8):1943-1953

[59] Mi Z, Ghivizzani SC, Lechman ER, Jaffurs D, Glorioso JC, et al. Adenovirusmediated gene transfer of insulin-like growth factor 1 stimulates proteoglycan synthesis in rabbit joints. Arthritis and Rheumatism. 2000;**43**(11):2563-2570

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[62] Hubbard JR, Steinberg JJ, Bednar MS, Sledge CB. Effect of purified human interleukin-1 on cartilage

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degradation. Journal of Orthopaedic Research. 1988;**6**(2):180-187

*Equine Science*

2005;**23**(1):118-126

[50] Arend WP, Malyak M,

1998;**16**:27-55

Guthridge CJ, Gabay C. Interleukin-1 receptor antagonist: Role in biology. Annual Review of Immunology.

[51] Howard RD, McIlwraith CW, Trotter GW, Nyborg JK. Cloning of equine interleukin 1 receptor antagonist and determination of its full-length cDNA sequence. American Journal of Veterinary Research. 1998;**59**(6):712-716

[52] Kato H, Ohashi T, Matsushiro H, Watari T, Goitsuka R, et al. Molecular cloning and functional expression of equine interleukin-1 receptor antagonist. Veterinary Immunology

[53] Pelletier JP, Caron JP, Evans C, Robbins PD, Georgescu HI, et al. In vivo suppression of early experimental osteoarthritis by interleukin-1 receptor antagonist using gene therapy. Arthritis and Rheumatism. 1997;**40**(6):1012-1019

Martel-Pelletier J, Lascau-Coman V, Dupuis M, et al. In vivo transfer of interleukin-1 receptor antagonist gene in osteoarthritic rabbit knee joints: Prevention of osteoarthritis progression. The American Journal of Pathology.

[55] Wang G, Evans CH, Benson JM, Hutt JA, Seagrave J, et al. Safety and biodistribution assessment of

and Immunopathology. 1997;**56**(3-4):221-231

[54] Fernandes J, Tardif G,

1999;**154**(4):1159-1169

interleukin-1 receptor antagonist protein controls cartilage degradation in an osteoarthritic culture model. Journal of Orthopaedic Research.

[49] Ortved KF, Begum L, Stefanovski D, Nixon AJ. AAV-mediated overexpression of IL-10 mitigates the inflammatory cascade in stimulated equine chondrocyte pellets. Current Gene Therapy. 2018;**18**(3):171-179

sc-rAAV2.5IL-1Ra administered via intra-articular injection in a monoiodoacetate-induced osteoarthritis rat model. Molecular Therapy - Methods and Clinical Development. 2016;**3**:15052

[56] Mi Z, Ghivizzani SC, Lechman E, Glorioso JC, Evans CH, Robbins PD. Adverse effects of adenovirus-mediated gene transfer of human transforming growth factor beta 1 into rabbit knees. Arthritis Research & Therapy.

[57] Watson RS, Gouze E, Levings PP, Bush ML, Kay JD, et al. Gene delivery of TGF-beta1 induces arthrofibrosis and chondrometaplasia of synovium in vivo. Laboratory Investigation.

2003;**5**(3):R132-R139

2010;**90**(11):1615-1627

2001;**44**(8):1943-1953

[58] Gelse K, Jiang QJ, Aigner T, Ritter T, Wagner K, et al. Fibroblastmediated delivery of growth factor complementary DNA into mouse joints induces chondrogenesis but avoids the disadvantages of direct viral gene transfer. Arthritis and Rheumatism.

[59] Mi Z, Ghivizzani SC, Lechman ER, Jaffurs D, Glorioso JC, et al. Adenovirusmediated gene transfer of insulin-like growth factor 1 stimulates proteoglycan synthesis in rabbit joints. Arthritis and Rheumatism. 2000;**43**(11):2563-2570

[60] Goldring MB, Berenbaum F. Emerging targets in osteoarthritis therapy. Current Opinion in Pharmacology. 2015;**22**:51-63

[62] Hubbard JR, Steinberg JJ,

human interleukin-1 on cartilage

[61] Goldring MB, Otero M, Plumb DA, Dragomir C, Favero M, et al. Roles of inflammatory and anabolic cytokines in cartilage metabolism: Signals and multiple effectors converge upon MMP-13 regulation in osteoarthritis. European Cells & Materials. 2011;**21**:202-220

Bednar MS, Sledge CB. Effect of purified

**46**

[63] Smith RL. Degradative enzymes in osteoarthritis. Frontiers in Bioscience. 1999;**4**:D704-D712

[64] Geng Y, Blanco FJ, Cornelisson M, Lotz M. Regulation of cyclooxygenase-2 expression in normal human articular chondrocytes. Journal of Immunology. 1995;**155**(2):796-801

[65] Gouze JN, Gouze E, Popp MP, Bush ML, Dacanay EA, et al. Exogenous glucosamine globally protects chondrocytes from the arthritogenic effects of IL-1beta. Arthritis Research & Therapy. 2006;**8**(6):R173

[66] Mengshol JA, Vincenti MP, Coon CI, Barchowsky A, Brinckerhoff CE. Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor kappaB: Differential regulation of collagenase 1 and collagenase 3. Arthritis and Rheumatism. 2000;**43**(4):801-811

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[68] Cohen SB, Proudman S, Kivitz AJ, Burch FX, Donohue JP, et al. A randomized, double-blind study of AMG 108 (a fully human monoclonal antibody to IL-1R1) in patients with osteoarthritis of the knee. Arthritis Research & Therapy. 2011;**13**(4):R125

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Functional genomic analysis of type II IL-1beta decoy receptor: Potential for gene therapy in human arthritis and inflammation. Journal of Immunology. 2002;**168**(4):2001-2010

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[78] McCarty DM, Monahan PE, Samulski RJ. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Therapy. 2001;**8**(16):1248-1254

[79] McCarty DM, Fu H, Monahan PE, Toulson CE, Naik P, Samulski RJ. Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo. Gene Therapy. 2003;**10**(26):2112-2118

[80] Kay JD, Gouze E, Oligino TJ, Gouze JN, Watson RS, et al. Intraarticular gene delivery and expression of interleukin-1Ra mediated by selfcomplementary adeno-associated virus. The Journal of Gene Medicine. 2009;**11**(7):605-614

[81] Goodrich LR, Grieger JC, Phillips JN, Khan N, Gray SJ, et al. scAAVIL-1ra dosing trial in a large animal model and validation of long-term expression with repeat administration for osteoarthritis therapy. Gene Therapy. 2015;**22**(7):536-545

[82] Goodrich LR, Phillips JN, McIlwraith CW, Foti SB, Grieger JC, et al. Optimization of scAAVIL-1ra in vitro and in vivo to deliver high levels of therapeutic protein for treatment of osteoarthritis. Molecular Therapy - Nucleic Acids. 2013;**2**:e70

[83] Johnson RA, Huong SM, Huang ES. Activation of the mitogen-activated protein kinase p38 by human cytomegalovirus infection through two distinct pathways: A novel mechanism

for activation of p38. Journal of Virology. 2000;**74**(3):1158-1167

[84] Liu XF, Wang X, Yan S, Zhang Z, Abecassis M, Hummel M. Epigenetic control of cytomegalovirus latency and reactivation. Viruses. 2013;**5**(5):1325-1345

[85] Löser P, Jennings GS, Strauss M, Sandig V. Reactivation of the previously silenced cytomegalovirus major immediate-early promoter in the mouse liver: Involvement of NFkappaB. Journal of Virology. 1998;**72**(1):180-190

[86] Meier JL, Keller MJ, McCoy JJ. Requirement of multiple cis-acting elements in the human cytomegalovirus major immediate-early distal enhancer for viral gene expression and replication. Journal of Virology. 2002;**76**(1):313-326

[87] Svensson RU, Barnes JM, Rokhlin OW, Cohen MB, Henry MD. Chemotherapeutic agents up-regulate the cytomegalovirus promoter: Implications for bioluminescence imaging of tumor response to therapy. Cancer Research. 2007;**67**(21):10445-10454

[88] Bruening W, Giasson B, Mushynski W, Durham HD. Activation of stress-activated MAP protein kinases up-regulates expression of transgenes driven by the cytomegalovirus immediate/early promoter. Nucleic Acids Research. 1998;**26**(2):486-489

[89] Ramanathan M, Haskó G, Leibovich SJ. Analysis of signal transduction pathways in macrophages using expression vectors with CMV promoters: A cautionary tale. Inflammation. 2005;**29**(2-3):94-102

[90] Simpson AJ, Cunningham GA, Porteous DJ, Haslett C, Sallenave JM. Regulation of adenovirus-mediated elafin transgene expression by bacterial

**49**

*Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

lipopolysaccharide. Human Gene Therapy. 2001;**12**(11):1395-1406

[92] Watson Levings R, Smith AD, Broome TA, Rice BL, Gibbs EP, et al. scAAV-mediated IL-1Ra gene delivery for the treatment of osteoarthritis: Test of efficacy in an equine model. Human Gene Therapy. Clinical Development.

[93] Ishihara A, Bartlett JS, Bertone AL. Inflammation and immune response of intra-articular serotype 2 adenoassociated virus or adenovirus vectors in a large animal model. Art.

[94] Nixon AJ, Grol MW, Lang HM, Ruan MZC, Stone A, et al. Diseasemodifying osteoarthritis treatment with interleukin-1 receptor antagonist gene therapy in small and large animal models. Arthritis & Rhematology.

NF-κB signalling pathway in osteoarthritis. The International Journal of Biochemistry & Cell Biology.

2013;**45**(11):2580-2584

2018;**29**(2):101-112

2012;**2012**:735472

2018;**70**(11):1757-1768

[91] Rigoglou S, Papavassiliou AG. The

*Gene Therapy for the Treatment of Equine Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.93000*

lipopolysaccharide. Human Gene Therapy. 2001;**12**(11):1395-1406

*Equine Science*

2004;**22**(4):726-734

2001;**8**(16):1248-1254

[79] McCarty DM, Fu H,

2003;**10**(26):2112-2118

2009;**11**(7):605-614

[81] Goodrich LR, Grieger JC, Phillips JN, Khan N, Gray SJ, et al. scAAVIL-1ra dosing trial in a large animal model and validation of long-term expression with repeat administration for osteoarthritis

therapy. Gene Therapy. 2015;**22**(7):536-545

[82] Goodrich LR, Phillips JN, McIlwraith CW, Foti SB, Grieger JC, et al. Optimization of scAAVIL-1ra in vitro and in vivo to deliver high levels of therapeutic protein for treatment of osteoarthritis. Molecular Therapy -

Nucleic Acids. 2013;**2**:e70

[83] Johnson RA, Huong SM, Huang ES. Activation of the mitogen-activated protein kinase p38 by human

cytomegalovirus infection through two distinct pathways: A novel mechanism

Monahan PE, Toulson CE, Naik P, Samulski RJ. Adeno-associated virus terminal repeat (TR) mutant generates

self-complementary vectors to overcome the rate-limiting step to transduction in vivo. Gene Therapy.

[80] Kay JD, Gouze E, Oligino TJ, Gouze JN, Watson RS, et al. Intraarticular gene delivery and expression of interleukin-1Ra mediated by selfcomplementary adeno-associated virus. The Journal of Gene Medicine.

Pedersen FS. In vivo gene delivery to articular chondrocytes mediated by an adeno-associated virus vector. Journal of Orthopaedic Research.

for activation of p38. Journal of Virology. 2000;**74**(3):1158-1167

control of cytomegalovirus latency and reactivation. Viruses.

2013;**5**(5):1325-1345

1998;**72**(1):180-190

2002;**76**(1):313-326

[84] Liu XF, Wang X, Yan S, Zhang Z, Abecassis M, Hummel M. Epigenetic

[85] Löser P, Jennings GS, Strauss M, Sandig V. Reactivation of the previously

silenced cytomegalovirus major immediate-early promoter in the mouse liver: Involvement of NFkappaB. Journal of Virology.

[86] Meier JL, Keller MJ, McCoy JJ. Requirement of multiple cis-acting elements in the human cytomegalovirus

major immediate-early distal enhancer for viral gene expression and replication. Journal of Virology.

[87] Svensson RU, Barnes JM,

[88] Bruening W, Giasson B,

[89] Ramanathan M, Haskó G, Leibovich SJ. Analysis of signal

using expression vectors with CMV promoters: A cautionary tale. Inflammation. 2005;**29**(2-3):94-102

[90] Simpson AJ, Cunningham GA, Porteous DJ, Haslett C, Sallenave JM. Regulation of adenovirus-mediated elafin transgene expression by bacterial

Mushynski W, Durham HD. Activation of stress-activated MAP protein kinases up-regulates expression of transgenes driven by the cytomegalovirus immediate/early promoter. Nucleic Acids Research. 1998;**26**(2):486-489

transduction pathways in macrophages

Rokhlin OW, Cohen MB, Henry MD. Chemotherapeutic agents up-regulate the cytomegalovirus promoter: Implications for bioluminescence imaging of tumor response to therapy. Cancer Research. 2007;**67**(21):10445-10454

[78] McCarty DM, Monahan PE, Samulski RJ. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Therapy.

**48**

[91] Rigoglou S, Papavassiliou AG. The NF-κB signalling pathway in osteoarthritis. The International Journal of Biochemistry & Cell Biology. 2013;**45**(11):2580-2584

[92] Watson Levings R, Smith AD, Broome TA, Rice BL, Gibbs EP, et al. scAAV-mediated IL-1Ra gene delivery for the treatment of osteoarthritis: Test of efficacy in an equine model. Human Gene Therapy. Clinical Development. 2018;**29**(2):101-112

[93] Ishihara A, Bartlett JS, Bertone AL. Inflammation and immune response of intra-articular serotype 2 adenoassociated virus or adenovirus vectors in a large animal model. Art. 2012;**2012**:735472

[94] Nixon AJ, Grol MW, Lang HM, Ruan MZC, Stone A, et al. Diseasemodifying osteoarthritis treatment with interleukin-1 receptor antagonist gene therapy in small and large animal models. Arthritis & Rhematology. 2018;**70**(11):1757-1768

**51**

Section 2

Reproduction,

Locomotion and Skin

Section 2
