**Recent Advances in Hematopoietic Stem Cell Gene Therapy**

## Toshihisa Tsuruta

ventricular function and remodeling after myocardial infarction. J Cell Mol Med.

[51] Dawn B, Tiwari S, Kucia MJ, Zuba-Surma EK, Guo Y, Sanganalmath SK, Abdel-Latif A, Hunt G, Vincent RJ, Taher H, Reed NJ, Ratajczak MZ, Bolli R. Transplantation of bone marrow-derived very small embryonic-like stem cells attenuates left ventricular dysfunction and remodeling after myocardial infarction. Stem Cells. 2008;26 :1646–

[52] Tendera M, Wojakowski W, Ruzyłło W, Chojnowska L, Kepka C, Tracz W, Musiałek P, Piwowarska W, Nessler J, Buszman P, Grajek S, Breborowicz P, Majka M,Ratajc‐ zak. Intracoronary infusion of bone marrow-derived selected CD34+CXCR4+ cells and non-selected mononuclear cellsin patients with acute STEMI and reduced left ventricular ejection fraction: results of randomized, multicentreMyocardial Regener‐ ation by Intracoronary Infusion of Selected Population of Stem Cells in Acute Myo‐

[53] Danova-Alt R, Heider A, Egger D, Cross M, Alt R. Very small embryonic-like stem cells purified from umbilical cord blood lack stem cell characteristics. PLoS One.

cardialInfarction (REGENT) Trial. Eur Heart J. 2009;30 :1313-21.

2011 15:1319-28.

106 Innovations in Stem Cell Transplantation

2012;7:e34899.

1655.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53587

## **1. Introduction**

Hematopoietic stem cell transplantation (HSCT) has a half-century history. It is currently an indispensable treatment for not only incurable blood diseases such as aplastic anemia and severe hemolytic anemia, but also malignant hematological diseases such as leukemia and lymphoma. Although allergenic HSCT is also used to treat hereditary diseases, its indica‐ tions are restricted because of critical complications including regimen-related toxicities in‐ volving conditioning, infection, and graft-versus-host disease.

Studies in recent decades have shown that HSCT can have a long-term effect in the treat‐ ment of hereditary diseases involving a responsible gene in hematogenous cells. Although the first successful gene therapy using lymphocytes or bone marrow cells for a patient with adenosine deaminase (ADA) deficiency inspired great hope in the future of gene therapy [1-3], subsequent gene therapy using HSCs for patients with X-linked severe combined im‐ munodeficiency (SCID-X1) resulted in tumorigenesis [4]. In addition to the self-renewal and multilineage differentiation capacities of tissue stem cells, HSCs exhibit cell-cycle dormancy, which complicates their use in gene therapy.

However, as technological advances have increased the safety and efficiency of introduc‐ ing genes into HSCs, gene therapy with HSCs is attracting attention again. In this chapter, advances in the technology of HSC gene therapy, e.g., vector design to avoid genotoxicity and increase transgenic efficiency by taking advantage of the special characteristics of HSCs, are reviewed. In addition, recent studies on HSC gene therapy for various heredita‐ ry diseases, such as thalassemia, Fanconi anemia, hemophilia, primary immunodeficiency, mucopolysaccharidosis, Gaucher disease, and X-linked adrenoleukodystrophy (X-ALD) are discussed.

© 2013 Tsuruta; licensee InTech. This is an open access article 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. © 2013 The Author(s). Licensee InTech. 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.

## **2. Characteristics of HSCs and gene therapy**

The concept of the HSC was introduced by Till and McCulloch in 1961 [5]. Although a healthy adult produces approximately 1 trillion blood cells each day, they are considered to originate from a single HSC which can potentially be transplanted into a mouse [6, 7]. Generally stem cells are defined as cells capable of self-renewal and multilineage differentiation. In addition to these two characteristics, HSCs have the capability of cell-cycle dormancy, i.e. to enter a state of dormancy (G0 phase) in the cell cycle and can continue blood cell production over a lifetime while protecting themselves from various kinds of stress [8].

While making a HSC with few opportunities for cell division into a transgenic target, it is important to design a safe and efficient vector for inserting a gene into the host chromo‐ some. Furthermore, since a hematogenous cell also has many cells which exhibit its function in the specialization process to a mature effector cell, it is also important to select differentia‐ tion-specific or non-specific promoters or enhancers during the vector design process.

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109

Vectors derived from the Retroviridae family, RNA viruses with reverse transcriptase activi‐ ty, are widely used for inserting genes in host chromosomes. Although adeno-associated vi‐ rus (AAV) vectors can also insert genes into host chromosomes, this process is inefficient and partial. Gammaretroviruses and lentiviruses are members of the Retroviridae family that are commonly used as vectors in HSC gene therapy. Generally, the former is called sim‐ ply a retroviral vector and the latter is called a lentiviral vector. When a gene is inserted in

Retroviral vectors are commonly constructed from the Moloney murine leukemia virus (MoMLV) genome. Retroviral genomes have a *gag/pol* gene that codes for viral structure proteins, protease and reverse transcriptase, an *env* gene that codes for the envelope glyco‐ protein and the packaging signal. These genes are flanked by long terminal repeats (LTR) which contain enhancers and promoters. A retroviral vector consists of a packaging plasmid that does not have the packaging signal but does include the *gag/pol* gene, a transfer vector with the packaging signal, and the target gene cDNA. After transfection of these plasmids into producer cells (e.g., 297T cells, NIH3T3 cell, etc.), a target vector is obtained by collect‐

Expression of a target gene can be inhibited by mechanisms such as methylation of CpG is‐ lands in the promoter region, insertion of a negative control region (NCR) into the LTR, and the presence of a repressor binding site (RBS) downstream of the 5′ LTR. Other vectors, such as the murine stem cell virus (MSCV) vector [21], the myeloproliferative sarcoma virus vec‐ tor, the negative control region deleted (MND) vector [22], and the MFG-S vector [23] were developed to improve the efficiency of transgene expression; they are widely used in clinical

Since the retroviral viral genome cannot cross the nuclear membrane, it can be incorporated into a chromosome only during the phase of mitosis when the nuclear membrane has disas‐ sembled. Since many HSCs are thought to exist in a dormant phase, insertions into the HSC genome with a retroviral vector require a proliferation stimulus by cytokines. Although var‐ ious combinations of cytokines to suppress the decrease in HSC self-renewal have been studied, stem cell factor (SCF), fms-related tyrosine kinase-3 (Flt-3) ligand, interleukin-3

the chromosome of an HSC with a Retroviridae vector, genotoxicity can occur.

**3. Vectors for HSC gene therapy**

**3.1. Gammaretroviral (Retroviral) vectors**

applications of gene therapy involving HSCs.

(IL-3), TPO, among others, are commonly used [24, 25].

ing the culture solution.

Fig. 1 shows HSC surface markers and the typical cytokines regulating HSCs. Stem cell factor (SCF) and thrombopoietin (TPO) are important direct cytokine regulators of HSCs. Although SCF promotes the proliferation and differentiation of hematopoietic progenitor cells, it is thought to not be essential for the initiation of hematopoiesis and HSC self-renewal [9]. TPO and its receptor, c-Mpl, are thought to play important roles in early hematopoiesis from HSCs. In contrast to the CD34+ CD38 c-Mpl population, CD34+ CD38 c-Mpl+ cells show significantly better HSC engraftment [10]. Mice lacking either TPO or c-Mpl have deficiencies in progenitor cells of multiple hematopoietic lineages [11]. TPO-mediated signal transduction for the self-re‐ newal of HSCs is negatively regulated by the intracellular scaffold protein Lnk [12, 13]. A sig‐ nal from angiopoietin-1 via Tie2 regulates HSC dormancy by promoting the adhesion of HSCs to osteoblasts in the bone marrow niche and maintains long-term repopulating activity [14]. Although cytokine-induced lipid raft clustering of the HSC membrane is essential for HSC reentry into the cell cycle, transforming growth factor-β (TGF-β) inhibits lipid raft clustering and induces p57Kip2 expression, leading to HSC dormancy [15, 16]. Recently, the hypoxic niche of HSCs has been demonstrated. It, along with the osteoblastic and vascular niches, are important for HSC dormancy [17-19]. They are targets in HSC gene therapy [20].

**Figure 1. Hematopoietic stem cell (HSC) surface markers and typical cytokines that regulate HSCs.** Stem cell fac‐ tor (SCF) promotes the proliferation and differentiation of HSCs. Thrombopoietin (TPO) and its receptor, c-Mpl, play important roles in early hematopoiesis, especially self-renewal. Signals from angiotensin-1 via Tie2 and transforming growth factor -β via its receptors regulate HSC dormancy. (This figure is based on the illustration by BioLegend, Inc. San Diego, CA, U.S.A. http://www.biolegend.com/cell\_markers)

While making a HSC with few opportunities for cell division into a transgenic target, it is important to design a safe and efficient vector for inserting a gene into the host chromo‐ some. Furthermore, since a hematogenous cell also has many cells which exhibit its function in the specialization process to a mature effector cell, it is also important to select differentia‐ tion-specific or non-specific promoters or enhancers during the vector design process.

## **3. Vectors for HSC gene therapy**

**2. Characteristics of HSCs and gene therapy**

while protecting themselves from various kinds of stress [8].

CD38-

San Diego, CA, U.S.A. http://www.biolegend.com/cell\_markers)

c-Mpl-

for HSC dormancy [17-19]. They are targets in HSC gene therapy [20].

In contrast to the CD34+

108 Innovations in Stem Cell Transplantation

The concept of the HSC was introduced by Till and McCulloch in 1961 [5]. Although a healthy adult produces approximately 1 trillion blood cells each day, they are considered to originate from a single HSC which can potentially be transplanted into a mouse [6, 7]. Generally stem cells are defined as cells capable of self-renewal and multilineage differentiation. In addition to these two characteristics, HSCs have the capability of cell-cycle dormancy, i.e. to enter a state of dormancy (G0 phase) in the cell cycle and can continue blood cell production over a lifetime

Fig. 1 shows HSC surface markers and the typical cytokines regulating HSCs. Stem cell factor (SCF) and thrombopoietin (TPO) are important direct cytokine regulators of HSCs. Although SCF promotes the proliferation and differentiation of hematopoietic progenitor cells, it is thought to not be essential for the initiation of hematopoiesis and HSC self-renewal [9]. TPO and its receptor, c-Mpl, are thought to play important roles in early hematopoiesis from HSCs.

population, CD34+

better HSC engraftment [10]. Mice lacking either TPO or c-Mpl have deficiencies in progenitor cells of multiple hematopoietic lineages [11]. TPO-mediated signal transduction for the self-re‐ newal of HSCs is negatively regulated by the intracellular scaffold protein Lnk [12, 13]. A sig‐ nal from angiopoietin-1 via Tie2 regulates HSC dormancy by promoting the adhesion of HSCs to osteoblasts in the bone marrow niche and maintains long-term repopulating activity [14]. Although cytokine-induced lipid raft clustering of the HSC membrane is essential for HSC reentry into the cell cycle, transforming growth factor-β (TGF-β) inhibits lipid raft clustering and induces p57Kip2 expression, leading to HSC dormancy [15, 16]. Recently, the hypoxic niche of HSCs has been demonstrated. It, along with the osteoblastic and vascular niches, are important

**Figure 1. Hematopoietic stem cell (HSC) surface markers and typical cytokines that regulate HSCs.** Stem cell fac‐ tor (SCF) promotes the proliferation and differentiation of HSCs. Thrombopoietin (TPO) and its receptor, c-Mpl, play important roles in early hematopoiesis, especially self-renewal. Signals from angiotensin-1 via Tie2 and transforming growth factor -β via its receptors regulate HSC dormancy. (This figure is based on the illustration by BioLegend, Inc.

CD38-

c-Mpl+

cells show significantly

Vectors derived from the Retroviridae family, RNA viruses with reverse transcriptase activi‐ ty, are widely used for inserting genes in host chromosomes. Although adeno-associated vi‐ rus (AAV) vectors can also insert genes into host chromosomes, this process is inefficient and partial. Gammaretroviruses and lentiviruses are members of the Retroviridae family that are commonly used as vectors in HSC gene therapy. Generally, the former is called sim‐ ply a retroviral vector and the latter is called a lentiviral vector. When a gene is inserted in the chromosome of an HSC with a Retroviridae vector, genotoxicity can occur.

#### **3.1. Gammaretroviral (Retroviral) vectors**

Retroviral vectors are commonly constructed from the Moloney murine leukemia virus (MoMLV) genome. Retroviral genomes have a *gag/pol* gene that codes for viral structure proteins, protease and reverse transcriptase, an *env* gene that codes for the envelope glyco‐ protein and the packaging signal. These genes are flanked by long terminal repeats (LTR) which contain enhancers and promoters. A retroviral vector consists of a packaging plasmid that does not have the packaging signal but does include the *gag/pol* gene, a transfer vector with the packaging signal, and the target gene cDNA. After transfection of these plasmids into producer cells (e.g., 297T cells, NIH3T3 cell, etc.), a target vector is obtained by collect‐ ing the culture solution.

Expression of a target gene can be inhibited by mechanisms such as methylation of CpG is‐ lands in the promoter region, insertion of a negative control region (NCR) into the LTR, and the presence of a repressor binding site (RBS) downstream of the 5′ LTR. Other vectors, such as the murine stem cell virus (MSCV) vector [21], the myeloproliferative sarcoma virus vec‐ tor, the negative control region deleted (MND) vector [22], and the MFG-S vector [23] were developed to improve the efficiency of transgene expression; they are widely used in clinical applications of gene therapy involving HSCs.

Since the retroviral viral genome cannot cross the nuclear membrane, it can be incorporated into a chromosome only during the phase of mitosis when the nuclear membrane has disas‐ sembled. Since many HSCs are thought to exist in a dormant phase, insertions into the HSC genome with a retroviral vector require a proliferation stimulus by cytokines. Although var‐ ious combinations of cytokines to suppress the decrease in HSC self-renewal have been studied, stem cell factor (SCF), fms-related tyrosine kinase-3 (Flt-3) ligand, interleukin-3 (IL-3), TPO, among others, are commonly used [24, 25].

#### **3.2. Lentiviral vectors**

Human immunodeficiency virus type 1 (HIV-1), the representative lentivirus, differs from gammaretroviruses in that it can be incorporated during a non-mitotic phase. This is one ad‐ vantage of lentiviral vectors in HSC gene therapy.

Although first-generation lentiviral vectors included modification genes, they were re‐ moved in the second generation because it was discovered that the modification genes are not required for infection during non-mitotic phases. In the third generation, further modifi‐ cations included the deletion of *tat*, use of multiple vector plasmids, and introduction of selfinactivating (SIN) vectors. The structure of HIV-1 and a typical third-generation lentiviral vector system are shown in Fig. 2 [26]. Approximately one-third of the HIV-1 genome has been deleted, and the vector system has been divided into four plasmids, namely, the pack‐ aging plasmid, *rev* plasmid, SIN vector plasmid and envelope plasmid. To prevent produc‐ tion of wild type HIV-1, *tat,* a regulatory gene indispensable to viral reproduction was deleted, and the *rev* gene was moved to a separate plasmid. Moreover, since the HIV-1 LTR promoter is weak in the absence of *tat*, it was replaced with the cytomegalovirus (CMV) pro‐ moter in the packaging plasmid. Since an envelope plasmid can only infect CD4 positive cells with a HIV-1 envelope, the envelope gene was replaced with the vesicular stomatitis virus G glycoprotein (VSV-G) envelope. The SIN vector further improved safety by replac‐ ing the enhancer / promoter portion of the LTR, suppressing the activation of unnecessary

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111

**Figure 3. Mechanism of gene activation induced by vector insertion.** The genomic integration site of an MLVbased retroviral vector is depicted. With this MLV vector design, the enhancer and promoter within the U3 region (blue rectangle) of the long terminal repeat (LTR) drive transcription of the transgene (indicated by the parallel arrow arising from the blue rectangle). Vector integration near Gene X is shown in the top panel. The enhancer elements located in the U3 region (blue rectangle) of the vector can interact with the regulatory elements upstream of Gene X to increase its basal transcription rate to inappropriately high levels, potentially altering the growth of the cell. Two alternatives for eliminating the use of the powerful enhancer in the U3 region include (1) middle panel: use of a selfinactivating (SIN) MLV-based vector in which the U3 region has been deleted. An internal cellular promoter is used to drive transgene expression and (2) bottom panel: use of a SIN lentiviral vector in which U3 (yellow rectangle) has been eliminated. This system also uses an internal cellular promoter to drive transgene expression (Reproduced with modifi‐

To improve the gene transfer into HSCs, Verhoeyen and colleagues designed lentiviral vec‐ tors displaying "early-acting cytokines" such as TPO and SCF. This vector can promote sur‐ vival of CD34 positive HSCs and achieve selective transduction of long-term repopulating

genes with the integrated gene (Fig. 3) [27].

cells in a humanized mouse model (Fig. 4) [28, 29].

cation from [27]).

Both lentiviruses and gammaretroviruses have *gag, pol*, and *env* genes sandwiched between LTRs with promoter activity at both ends. In addition, lentiviruses have accessory genes (*vif, vpr, vpu, nef*) and regulatory genes (*tat, rev*). Double-stranded cDNA produced from the viral genome enters the cell, and a pre-integration complex is formed with a host protein. This complex can pass through the pores of the nuclear membrane during non-mitotic phases, al‐ lowing the viral genome to be inserted into the host cell chromosome.

**Figure 2. HIV provirus (A) and the four plasmids of a third-generation lentiviral vector (B).** The viral long termi‐ nal repeats (LTRs), reading frames of the viral genes, splice donor site (SD), splicing acceptor site (SA), packaging signal (Ψ), and rev-responsive element (RRE) are indicated. The packaging plasmid contains the *gag* and *pol* genes under the influence of the CMV promoter, intervening sequences, and the polyadenylation site (polyA) of the human β-globin gene. As the transcripts of the *gag* and *pol* genes contain cis-repressive sequences, they are expressed only if rev pro‐ motes their nuclear export by binding to the RRE. All *tat* and *rev* exons have been deleted, and the viral sequences upstream of the *gag* gene have been replaced. The rev plasmid expresses *rev* cDNA. The SIN vector plasmid contains HIV-1 cis-acting sequences and an expression cassette for the transgene. It is the only portion transferred to the target cells and does not contain wild-type copies of the HIV LTR. The 5′ LTR is chimeric, with the RSV enhancer and promoter replacing the U3 region to rescue transcriptional dependence on *tat*. The 3′ LTR has an almost completely deleted U3 region, which includes the TATA box. As the latter is the template used to generate both copies of the LTR in the inte‐ grated provirus, transduction of this vector results in transcriptional inactivation of both LTRs; thus, it is a self-inactivat‐ ing (SIN) vector. The envelope plasmid encodes a heterologous envelope to pseudotype the vector, here shown coding for vesicular stomatitis virus (VSV)-G. Only the relevant parts of the constructs are shown (Reproduced with modifications from [26]).

Although first-generation lentiviral vectors included modification genes, they were re‐ moved in the second generation because it was discovered that the modification genes are not required for infection during non-mitotic phases. In the third generation, further modifi‐ cations included the deletion of *tat*, use of multiple vector plasmids, and introduction of selfinactivating (SIN) vectors. The structure of HIV-1 and a typical third-generation lentiviral vector system are shown in Fig. 2 [26]. Approximately one-third of the HIV-1 genome has been deleted, and the vector system has been divided into four plasmids, namely, the pack‐ aging plasmid, *rev* plasmid, SIN vector plasmid and envelope plasmid. To prevent produc‐ tion of wild type HIV-1, *tat,* a regulatory gene indispensable to viral reproduction was deleted, and the *rev* gene was moved to a separate plasmid. Moreover, since the HIV-1 LTR promoter is weak in the absence of *tat*, it was replaced with the cytomegalovirus (CMV) pro‐ moter in the packaging plasmid. Since an envelope plasmid can only infect CD4 positive cells with a HIV-1 envelope, the envelope gene was replaced with the vesicular stomatitis virus G glycoprotein (VSV-G) envelope. The SIN vector further improved safety by replac‐ ing the enhancer / promoter portion of the LTR, suppressing the activation of unnecessary genes with the integrated gene (Fig. 3) [27].

**3.2. Lentiviral vectors**

110 Innovations in Stem Cell Transplantation

modifications from [26]).

vantage of lentiviral vectors in HSC gene therapy.

lowing the viral genome to be inserted into the host cell chromosome.

Human immunodeficiency virus type 1 (HIV-1), the representative lentivirus, differs from gammaretroviruses in that it can be incorporated during a non-mitotic phase. This is one ad‐

Both lentiviruses and gammaretroviruses have *gag, pol*, and *env* genes sandwiched between LTRs with promoter activity at both ends. In addition, lentiviruses have accessory genes (*vif, vpr, vpu, nef*) and regulatory genes (*tat, rev*). Double-stranded cDNA produced from the viral genome enters the cell, and a pre-integration complex is formed with a host protein. This complex can pass through the pores of the nuclear membrane during non-mitotic phases, al‐

**Figure 2. HIV provirus (A) and the four plasmids of a third-generation lentiviral vector (B).** The viral long termi‐ nal repeats (LTRs), reading frames of the viral genes, splice donor site (SD), splicing acceptor site (SA), packaging signal (Ψ), and rev-responsive element (RRE) are indicated. The packaging plasmid contains the *gag* and *pol* genes under the influence of the CMV promoter, intervening sequences, and the polyadenylation site (polyA) of the human β-globin gene. As the transcripts of the *gag* and *pol* genes contain cis-repressive sequences, they are expressed only if rev pro‐ motes their nuclear export by binding to the RRE. All *tat* and *rev* exons have been deleted, and the viral sequences upstream of the *gag* gene have been replaced. The rev plasmid expresses *rev* cDNA. The SIN vector plasmid contains HIV-1 cis-acting sequences and an expression cassette for the transgene. It is the only portion transferred to the target cells and does not contain wild-type copies of the HIV LTR. The 5′ LTR is chimeric, with the RSV enhancer and promoter replacing the U3 region to rescue transcriptional dependence on *tat*. The 3′ LTR has an almost completely deleted U3 region, which includes the TATA box. As the latter is the template used to generate both copies of the LTR in the inte‐ grated provirus, transduction of this vector results in transcriptional inactivation of both LTRs; thus, it is a self-inactivat‐ ing (SIN) vector. The envelope plasmid encodes a heterologous envelope to pseudotype the vector, here shown coding for vesicular stomatitis virus (VSV)-G. Only the relevant parts of the constructs are shown (Reproduced with

**Figure 3. Mechanism of gene activation induced by vector insertion.** The genomic integration site of an MLVbased retroviral vector is depicted. With this MLV vector design, the enhancer and promoter within the U3 region (blue rectangle) of the long terminal repeat (LTR) drive transcription of the transgene (indicated by the parallel arrow arising from the blue rectangle). Vector integration near Gene X is shown in the top panel. The enhancer elements located in the U3 region (blue rectangle) of the vector can interact with the regulatory elements upstream of Gene X to increase its basal transcription rate to inappropriately high levels, potentially altering the growth of the cell. Two alternatives for eliminating the use of the powerful enhancer in the U3 region include (1) middle panel: use of a selfinactivating (SIN) MLV-based vector in which the U3 region has been deleted. An internal cellular promoter is used to drive transgene expression and (2) bottom panel: use of a SIN lentiviral vector in which U3 (yellow rectangle) has been eliminated. This system also uses an internal cellular promoter to drive transgene expression (Reproduced with modifi‐ cation from [27]).

To improve the gene transfer into HSCs, Verhoeyen and colleagues designed lentiviral vec‐ tors displaying "early-acting cytokines" such as TPO and SCF. This vector can promote sur‐ vival of CD34 positive HSCs and achieve selective transduction of long-term repopulating cells in a humanized mouse model (Fig. 4) [28, 29].

when transcription from the provirus 5′ LTR creates a novel truncated isoform of a cellular proto-oncogene via splicing. Fifth, an inserted provirus can disrupt transcription by causing premature polyadenylation. The same mechanisms can occur in cellular oncogenesis when a

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113

**Figure 5. Retroviral mechanisms of oncogenesis.** The detailed mechanisms are shown in the text. The integrated provirus is indicated by two LTRs. Cellular proto-oncogene promoter and exons are indicated by black and grey boxes

Even if a gene is inserted into a HSC similarly, it is also known that there are diseases which may develop a tumor, and diseases a tumor is not accepted to be. Each type of virus has a unique integration profile, and the following observations have been made [30]: (a) Different retroviral vectors have distinct integration profiles. (b) The route of entry does not appear to strongly affect distribution of integration sites. VSV-G–pseudotyped HIV vectors have an in‐ tegration profile similar to HIV virions with the native HIV envelope despite differences in the route of entry. (c) The integration profile is largely independent of the target cell type,

gene is inserted by a retroviral vector [30].

respectively (Reproduced from [30]).

**Figure 4.** Lentiviral vector particles (HIV-1) display recombinant membrane envelope proteins such as stem cell factor (SCF), thrombopoietin (TPO), and vesicular stomatitis virus G glycoprotein (VSV-G). This vector can specifically target vector particles to hematopoietic stem cells (HSCs) expressing c-kit and c-mpl receptors for SCF and TPO, respectively. VSV-G envelope protein can bind to phospholipids in the HSC cell membrane. (Karlsson S, Gene therapy: efficient tar‐ geting of hematopoietic stem cells. Blood. 2005;106(10):3333)

#### **3.3. Genotoxicity of viral vectors**

The most serious problem with using viral vectors to incorporate a gene into a chromosome is the potential development of clonal proliferative diseases such as leukemia, which was observed in clinical trials involving gene therapy for SCID-X1 and chronic granulomatous disease (CGD). Although this problem of genotoxicity represents a great hurdle in the devel‐ opment of clinical applications for gene therapy, there is promising ongoing research on the mechanisms underlying genotoxicity and how to avoid it.

The mechanisms of retrovirus-induced oncogenesis are shown in Fig. 5 [30]. In oncogene capture, an acute transforming replication-competent retrovirus captures a cellular protooncogene and mediates transformation. This mechanism does not occur in replication-in‐ competent vectors. Second, the provirus 3′ LTR can trigger increased transcription of a cellular proto-oncogene. Third, enhancers in the provirus LTRs can activate transcription from nearby cellular proto-oncogene promoters. Fourth, a novel isoform can be expressed when transcription from the provirus 5′ LTR creates a novel truncated isoform of a cellular proto-oncogene via splicing. Fifth, an inserted provirus can disrupt transcription by causing premature polyadenylation. The same mechanisms can occur in cellular oncogenesis when a gene is inserted by a retroviral vector [30].

**Figure 4.** Lentiviral vector particles (HIV-1) display recombinant membrane envelope proteins such as stem cell factor (SCF), thrombopoietin (TPO), and vesicular stomatitis virus G glycoprotein (VSV-G). This vector can specifically target vector particles to hematopoietic stem cells (HSCs) expressing c-kit and c-mpl receptors for SCF and TPO, respectively. VSV-G envelope protein can bind to phospholipids in the HSC cell membrane. (Karlsson S, Gene therapy: efficient tar‐

The most serious problem with using viral vectors to incorporate a gene into a chromosome is the potential development of clonal proliferative diseases such as leukemia, which was observed in clinical trials involving gene therapy for SCID-X1 and chronic granulomatous disease (CGD). Although this problem of genotoxicity represents a great hurdle in the devel‐ opment of clinical applications for gene therapy, there is promising ongoing research on the

The mechanisms of retrovirus-induced oncogenesis are shown in Fig. 5 [30]. In oncogene capture, an acute transforming replication-competent retrovirus captures a cellular protooncogene and mediates transformation. This mechanism does not occur in replication-in‐ competent vectors. Second, the provirus 3′ LTR can trigger increased transcription of a cellular proto-oncogene. Third, enhancers in the provirus LTRs can activate transcription from nearby cellular proto-oncogene promoters. Fourth, a novel isoform can be expressed

geting of hematopoietic stem cells. Blood. 2005;106(10):3333)

mechanisms underlying genotoxicity and how to avoid it.

**3.3. Genotoxicity of viral vectors**

112 Innovations in Stem Cell Transplantation

**Figure 5. Retroviral mechanisms of oncogenesis.** The detailed mechanisms are shown in the text. The integrated provirus is indicated by two LTRs. Cellular proto-oncogene promoter and exons are indicated by black and grey boxes respectively (Reproduced from [30]).

Even if a gene is inserted into a HSC similarly, it is also known that there are diseases which may develop a tumor, and diseases a tumor is not accepted to be. Each type of virus has a unique integration profile, and the following observations have been made [30]: (a) Different retroviral vectors have distinct integration profiles. (b) The route of entry does not appear to strongly affect distribution of integration sites. VSV-G–pseudotyped HIV vectors have an in‐ tegration profile similar to HIV virions with the native HIV envelope despite differences in the route of entry. (c) The integration profile is largely independent of the target cell type, although the transcriptional program and epigenetic status of the target cell can influence integration site selection. (d) For lentiviruses, which can integrate independently of mitosis, the cell-cycle status of the target cell has only a modest effect on the distribution of integra‐ tion sites.

autosomal hemoglobin disorder caused by decreased β-globin chain synthesis. Although individuals with β-thalassemia minor (heterozygote) may be asymptomatic or have mild to moderate microcytic anemia, β-thalassemia major (homozygote) progresses to serious anemia by one or two years of age, and hemosiderosis, iron overload caused by transfu‐ sion or increased iron absorption, develops. Since most patients develop life-threatening complications such as heart failure by adolescence, HSCT has been performed in patients with advanced disease [32]. In recent years, gene therapy using a lentiviral vector con‐ taining a functional β-globin gene has been performed in an HbE/ β-thalassemia (βE/ β<sup>0</sup>

transfusion-dependent adult male, who subsequently did not require transfusions for

The human β-globin locus is located in a large 70kb area which also contains some β-like globulin genes (ε, Gγ, Aγ, δ, β). Gene switching takes place according to the development stage, and the β-globin gene is transcribed and expressed specifically after birth. A powerful enhancer called the LCR (locus control region) exists on the 5′ side of the promoter. The LCR contains five DNase I hypersensitive sites, referred to as HS5 to HS1 starting from the 5′

The structure of the lentiviral SIN vector used in gene therapy for β-thalassemia is shown in Fig. 6. To improve safety, two stop codons were inserted into the packaging signal (ψ) of GAG, the HS5 portion with insulator activity was deleted, and two copies of the 250 base pair (bp) core of the cHS4 chromatin insulators (chicken β-globin insulators) were inserted in the U3 region of the HIV 3′ LTR. Furthermore, the amino acid at the 87th position of βglobin was changed from threonine to glutamine. This altered β-globin can be distinguished from normal adult β-globin by high performance liquid chromatography (HPLC) analysis in

**Figure 6. Diagram of the human β-globin gene in a lentiviral vector.** HIV LTR, human immunodeficiency type-1 virus long terminal repeat; Ψ+, packaging signal; cPPT/flap, central polypurine tract/DNA flap; RRE, rev-responsive ele‐ ment; βp, human β-globin promoter; ppt, polypurine tract; HS, DNase I Hypersensitive Sites (Reproduced with color

A clinical study using this vector was performed in two β-thalassemia patients. As with au‐ tologous bone marrow transplantation, some of the patients' marrow cells were cryopre‐ served as a backup. The lentiviral vector particles containing a functional β-globin were


Recent Advances in Hematopoietic Stem Cell Gene Therapy

http://dx.doi.org/10.5772/53587

side. Furthermore, HS5 contains CCCTC-binding factor (CTCF)-dependent insulator.

individuals receiving red blood cell transfusion and β<sup>+</sup>

over 21 months [33].

modification from [33])

)

115

In order to avoid genotoxicity, various SIN vectors have been developed and improved. In general, lentiviral vectors are considered to have a lower risk of oncogenesis than ret‐ roviral vectors [31]. However, when a HSC is the target cell, more attention should be required because tumorigenesis can occur when the cell with the inserted gene under‐ goes differentiation.

## **4. Clinical applications of HSC gene therapy**

Diseases in which gene therapy using HSCs are being studied are shown in Table 1. They are roughly divided into hematological disorders, immunodeficiencies, and meta‐ bolic diseases. Most are congenital or hereditary diseases. The characteristic clinical fea‐ tures and recent basic science or clinical studies on HSC gene therapy for each disease are discussed below.


**Table 1.** Clinical applications of hematopoietic stem cell gene therapy.

#### **4.1. β-thalassemia**

Hemoglobin A (HbA), comprising 98% of adult human hemoglobin, is a tetramer with two α-globin and two β-globin chains combined with a heme group. β-thalassemia is an autosomal hemoglobin disorder caused by decreased β-globin chain synthesis. Although individuals with β-thalassemia minor (heterozygote) may be asymptomatic or have mild to moderate microcytic anemia, β-thalassemia major (homozygote) progresses to serious anemia by one or two years of age, and hemosiderosis, iron overload caused by transfu‐ sion or increased iron absorption, develops. Since most patients develop life-threatening complications such as heart failure by adolescence, HSCT has been performed in patients with advanced disease [32]. In recent years, gene therapy using a lentiviral vector con‐ taining a functional β-globin gene has been performed in an HbE/ β-thalassemia (βE/ β<sup>0</sup> ) transfusion-dependent adult male, who subsequently did not require transfusions for over 21 months [33].

although the transcriptional program and epigenetic status of the target cell can influence integration site selection. (d) For lentiviruses, which can integrate independently of mitosis, the cell-cycle status of the target cell has only a modest effect on the distribution of integra‐

In order to avoid genotoxicity, various SIN vectors have been developed and improved. In general, lentiviral vectors are considered to have a lower risk of oncogenesis than ret‐ roviral vectors [31]. However, when a HSC is the target cell, more attention should be required because tumorigenesis can occur when the cell with the inserted gene under‐

Diseases in which gene therapy using HSCs are being studied are shown in Table 1. They are roughly divided into hematological disorders, immunodeficiencies, and meta‐ bolic diseases. Most are congenital or hereditary diseases. The characteristic clinical fea‐ tures and recent basic science or clinical studies on HSC gene therapy for each disease

Hemoglobin A (HbA), comprising 98% of adult human hemoglobin, is a tetramer with two α-globin and two β-globin chains combined with a heme group. β-thalassemia is an

tion sites.

goes differentiation.

114 Innovations in Stem Cell Transplantation

are discussed below.

**Primary immunodeficiencies**

**Congenital metabolic diseases**

Gaucher disease

**4.1. β-thalassemia**

β-thalassaemia Fanconi anemia Hemophilia

**Congenital hematopoietic disorders**

X-linked severe combined immunodeficiency (SCID-X1)

Adenosine deaminase deficiency (ADA-SCID) Chronic granulomatous disease (CGD) Wiskott-Aldrich syndrome (WAS) Janus kinase 3 (JAK3) deficiency

Purine nucleoside phosphorylase (PNP) deficiency Leukocyte adhesion deficiency type 1 (LAD-1)

Mucopolysaccharidosis (MPS) types I, II, III, VII

**Table 1.** Clinical applications of hematopoietic stem cell gene therapy.

X-linked adrenoleukodystrophy (X-ALD)

**4. Clinical applications of HSC gene therapy**

The human β-globin locus is located in a large 70kb area which also contains some β-like globulin genes (ε, Gγ, Aγ, δ, β). Gene switching takes place according to the development stage, and the β-globin gene is transcribed and expressed specifically after birth. A powerful enhancer called the LCR (locus control region) exists on the 5′ side of the promoter. The LCR contains five DNase I hypersensitive sites, referred to as HS5 to HS1 starting from the 5′ side. Furthermore, HS5 contains CCCTC-binding factor (CTCF)-dependent insulator.

The structure of the lentiviral SIN vector used in gene therapy for β-thalassemia is shown in Fig. 6. To improve safety, two stop codons were inserted into the packaging signal (ψ) of GAG, the HS5 portion with insulator activity was deleted, and two copies of the 250 base pair (bp) core of the cHS4 chromatin insulators (chicken β-globin insulators) were inserted in the U3 region of the HIV 3′ LTR. Furthermore, the amino acid at the 87th position of βglobin was changed from threonine to glutamine. This altered β-globin can be distinguished from normal adult β-globin by high performance liquid chromatography (HPLC) analysis in individuals receiving red blood cell transfusion and β<sup>+</sup> -thalassemia patients [33].

**Figure 6. Diagram of the human β-globin gene in a lentiviral vector.** HIV LTR, human immunodeficiency type-1 virus long terminal repeat; Ψ+, packaging signal; cPPT/flap, central polypurine tract/DNA flap; RRE, rev-responsive ele‐ ment; βp, human β-globin promoter; ppt, polypurine tract; HS, DNase I Hypersensitive Sites (Reproduced with color modification from [33])

A clinical study using this vector was performed in two β-thalassemia patients. As with au‐ tologous bone marrow transplantation, some of the patients' marrow cells were cryopre‐ served as a backup. The lentiviral vector particles containing a functional β-globin were introduced into the remaining cells. After the transfected cells were cultured for one week *ex vivo*, some were also cryopreserved. The patients were conditioned with intravenous busul‐ fan (3.2 mg/kg/day for four days) without the addition of cyclophosphamide, before trans‐ plantation using the autologous gene-modified cryopreserved cells (Fig. 7) [34].

els of Aγ-globin protein produced by erythroid progenitors derived from thalassemic HSCs [35]. Both lentiviral-mediated γ-globin gene addition and genetic reactivation of endoge‐ nous γ-globin genes are considered potentially capable of providing therapeutic levels of hemoglobin F to patients with β-globin deficiency [36]. In addition, a trial of γ-globin induc‐ tion with β-globin production using mithramycin, an inducer of γ-globin expression, to re‐ move excess α-globin proteins in β-thalassemic erythroid progenitor cells was reported [37].

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117

Fanconi anemia is a hereditary disease characterized by cellular hypersensitivity to DNA crosslinking agents. It leads to bone marrow failure, such as aplastic anemia, by approxi‐ mately eight years of age. Since there is a high risk of developing malignancy, HSCT has been performed as a curative treatment for bone marrow insufficiency. Although the tenyear probability of survival after transplant from an Human leukocyte antigen (HLA) -iden‐ tical donor is over 80%, results with other donors are not satisfactory. HSC gene therapy is considered an alternative in cases where there is no HLA-identical donor available [38-40].

There are currently 13 discovered Fanconi anemia complement groups and 13 distinct genes (*FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN*) have been cloned. Mutations in *FANCB* are associated with an Xlinked form of Fanconi anemia; mutations in the other genes are associated with autosomal recessive transmission. Although frequencies vary by geographical region, *FANCA* gene ab‐ normalities are found in more than half of all Fanconi anemia patients [41]. Although one of the major hurdles in the development of gene therapy for Fanconi anemia is the increased sensitivity of Fanconi anemia stem cells to free radical-induced DNA damage during *ex vivo* culture and manipulation, retroviral and lentiviral vectors have been successfully employed to deliver complementing Fanconi anemia cDNA to HSCs with targeted disruptions of the *FANCA* and *FANCC* genes [20, 42-44]. In a phase I trial of *FANCA* gene therapy, gene trans‐

vector [38]. Whether sufficient HSCs can be obtained is a potential problem in Fanconi ane‐ mia patients due to possible bone marrow insufficiency, but in this study, sufficient target

Engraftment efficiency of *FANCA*-modified cells using a lentiviral vector was studied in a mouse model. Rapid transduction with four hours of culture using only SCF and megakar‐ yocyte growth and development factor and minimal differentiation of gene-induced cells is better than standard 96-hour culture using a variety of cytokines, including SCF, interleu‐ kin-11, Flt-3 ligand, and IL-3 [44]. Moreover, a recent trial demonstrated enhanced viability and engraftment of gene-corrected cells in patients with *FANCA* abnormalities with short

 cells were obtained from most patients. Two patients had *FANCA*-transduced cells successfully infused. The procedure was safe, well tolerated, and resulted in transient im‐ provements in hemoglobin and platelet counts [39]. However, transduced cell products were not obtained in one patient who required cryopreserved bone marrow. The first clini‐ cal study of *FANCC* gene therapy using a retroviral vector involved four patients. Although functional *FANCC* gene expression was observed in peripheral blood and bone marrow

cells and the MSCV retroviral

fer was performed with patient bone marrow-derived CD34+

**4.2. Fanconi anemia**

CD34+

cells, the results were transient [43].

The first patient failed to engraft because the HSCs had been compromised by how they were handled, not because of any issues with the gene therapy vector, and ultimately used backup bone marrow. The second patient, as described previously, achieved long-term βglobin production; one-third of the patient's hemoglobin was produced by the genetically modified cells [33].

Furthermore, the detailed examination of the transgenic cells showed significantly increased expression of high mobility group AT-hook 2 (HMGA2), which interacts with transcription factors to regulate gene expression, in the clones where gene insertion occurred in the *HMGA2* gene. The proportion of the HMGA2 overexpressing clones increased with time, to over 50% of transgenic cells at 20 months after gene therapy. In this patient, the HMGA2 overexpressing cells were only 5% of all circulating hematopoietic cells and there was no evidence of malignant transformation. However, researchers point out that there was ex‐ pressive production of a truncated form of the HMGA2 protein. Since truncated or overex‐ pressed HMGA2 is observed with some blood cancers and non-malignant expansions of blood cells, caution is recommended with this therapy [34].

**Figure 7. Gene-therapy procedure for patient with b-thalassemia. a.** Hematopoietic stem cells (HSCs) are collected from the bone marrow of a patient with β-thalassemia and maintained them in culture. **b**, Lentiviral-vector particles con‐ taining a functional β-globin gene were then introduced into the cells and allowed them to expand further in culture. **c.** To eradicate the patient's remaining HSCs and make room for the geneticaaly modified cells, the patient underwent chemo‐ therapy. **d.** The genetically modified HSCs were then transplanted into the patient (Reproduced from [34]).

Recently, researchers generated a LCR-free SIN lentiviral vector that combines two heredita‐ ry persistence of fetal hemoglobin (HPFH)-activating elements, resulting in therapeutic lev‐ els of Aγ-globin protein produced by erythroid progenitors derived from thalassemic HSCs [35]. Both lentiviral-mediated γ-globin gene addition and genetic reactivation of endoge‐ nous γ-globin genes are considered potentially capable of providing therapeutic levels of hemoglobin F to patients with β-globin deficiency [36]. In addition, a trial of γ-globin induc‐ tion with β-globin production using mithramycin, an inducer of γ-globin expression, to re‐ move excess α-globin proteins in β-thalassemic erythroid progenitor cells was reported [37].

#### **4.2. Fanconi anemia**

introduced into the remaining cells. After the transfected cells were cultured for one week *ex vivo*, some were also cryopreserved. The patients were conditioned with intravenous busul‐ fan (3.2 mg/kg/day for four days) without the addition of cyclophosphamide, before trans‐

The first patient failed to engraft because the HSCs had been compromised by how they were handled, not because of any issues with the gene therapy vector, and ultimately used backup bone marrow. The second patient, as described previously, achieved long-term βglobin production; one-third of the patient's hemoglobin was produced by the genetically

Furthermore, the detailed examination of the transgenic cells showed significantly increased expression of high mobility group AT-hook 2 (HMGA2), which interacts with transcription factors to regulate gene expression, in the clones where gene insertion occurred in the *HMGA2* gene. The proportion of the HMGA2 overexpressing clones increased with time, to over 50% of transgenic cells at 20 months after gene therapy. In this patient, the HMGA2 overexpressing cells were only 5% of all circulating hematopoietic cells and there was no evidence of malignant transformation. However, researchers point out that there was ex‐ pressive production of a truncated form of the HMGA2 protein. Since truncated or overex‐ pressed HMGA2 is observed with some blood cancers and non-malignant expansions of

**Figure 7. Gene-therapy procedure for patient with b-thalassemia. a.** Hematopoietic stem cells (HSCs) are collected from the bone marrow of a patient with β-thalassemia and maintained them in culture. **b**, Lentiviral-vector particles con‐ taining a functional β-globin gene were then introduced into the cells and allowed them to expand further in culture. **c.** To eradicate the patient's remaining HSCs and make room for the geneticaaly modified cells, the patient underwent chemo‐

Recently, researchers generated a LCR-free SIN lentiviral vector that combines two heredita‐ ry persistence of fetal hemoglobin (HPFH)-activating elements, resulting in therapeutic lev‐

therapy. **d.** The genetically modified HSCs were then transplanted into the patient (Reproduced from [34]).

plantation using the autologous gene-modified cryopreserved cells (Fig. 7) [34].

blood cells, caution is recommended with this therapy [34].

modified cells [33].

116 Innovations in Stem Cell Transplantation

Fanconi anemia is a hereditary disease characterized by cellular hypersensitivity to DNA crosslinking agents. It leads to bone marrow failure, such as aplastic anemia, by approxi‐ mately eight years of age. Since there is a high risk of developing malignancy, HSCT has been performed as a curative treatment for bone marrow insufficiency. Although the tenyear probability of survival after transplant from an Human leukocyte antigen (HLA) -iden‐ tical donor is over 80%, results with other donors are not satisfactory. HSC gene therapy is considered an alternative in cases where there is no HLA-identical donor available [38-40].

There are currently 13 discovered Fanconi anemia complement groups and 13 distinct genes (*FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN*) have been cloned. Mutations in *FANCB* are associated with an Xlinked form of Fanconi anemia; mutations in the other genes are associated with autosomal recessive transmission. Although frequencies vary by geographical region, *FANCA* gene ab‐ normalities are found in more than half of all Fanconi anemia patients [41]. Although one of the major hurdles in the development of gene therapy for Fanconi anemia is the increased sensitivity of Fanconi anemia stem cells to free radical-induced DNA damage during *ex vivo* culture and manipulation, retroviral and lentiviral vectors have been successfully employed to deliver complementing Fanconi anemia cDNA to HSCs with targeted disruptions of the *FANCA* and *FANCC* genes [20, 42-44]. In a phase I trial of *FANCA* gene therapy, gene trans‐ fer was performed with patient bone marrow-derived CD34+ cells and the MSCV retroviral vector [38]. Whether sufficient HSCs can be obtained is a potential problem in Fanconi ane‐ mia patients due to possible bone marrow insufficiency, but in this study, sufficient target CD34+ cells were obtained from most patients. Two patients had *FANCA*-transduced cells successfully infused. The procedure was safe, well tolerated, and resulted in transient im‐ provements in hemoglobin and platelet counts [39]. However, transduced cell products were not obtained in one patient who required cryopreserved bone marrow. The first clini‐ cal study of *FANCC* gene therapy using a retroviral vector involved four patients. Although functional *FANCC* gene expression was observed in peripheral blood and bone marrow cells, the results were transient [43].

Engraftment efficiency of *FANCA*-modified cells using a lentiviral vector was studied in a mouse model. Rapid transduction with four hours of culture using only SCF and megakar‐ yocyte growth and development factor and minimal differentiation of gene-induced cells is better than standard 96-hour culture using a variety of cytokines, including SCF, interleu‐ kin-11, Flt-3 ligand, and IL-3 [44]. Moreover, a recent trial demonstrated enhanced viability and engraftment of gene-corrected cells in patients with *FANCA* abnormalities with short transduction (overnight), low oxidative stress (5% oxygen), and the anti-oxidant N-acetyl-Lcysteine [20]. Lentiviral transduction of unselected Fanconi anemia bone marrow cells medi‐ ates efficient phenotypic correction of hematopoietic progenitor cells and CD34 mesenchymal stromal cells, with increased efficacy in hematopoietic engraftment [45]. In *Fancg* -/- mice, the wild-type mesenchymal stem and progenitor cells play important roles in the reconstitution of exogenous HSCs *in vitro* [46]. Recently, a new approach that directly injects lentiviral vector particles into the femur for *FANCC* gene transfer in mice was able to successfully introduce the *FANCC* gene to HSCs. This result provides evidence that target‐ ing the HSCs directly in their native environment enables efficient and long-term correction of bone marrow defects in Fanconi anemia [47].

Recently, human factor VIII variant genes were successfully introduced into the HSCs of a mouse with hemophilia A resulting in therapeutic levels of factor VIII variant protein ex‐ pression. This variant factor VIII has changes in the B and A2 domain in addition to the A1 domain for improved secretion and reduced immunogenicity (wild-type factor VIII has six domains, A1, A2, B, A3, C1, and C2) [54]. To ameliorate the symptoms of hemophilia A, par‐ tial replacement of the mutated liver cells by healthy cells in hemophilia A mice was chal‐ lenged with allogeneic bone marrow progenitor cell transplantation. In this study, the bone marrow progenitor cell-derived hepatocytes and sinusoidal endothelial cells synthesized factor VIII, showing that autologous gene-modified bone marrow progenitor cells have the

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119

Although HSCT has been widely performed as curative treatment for primary immunodefi‐ ciencies, gene therapy has been considered when there is no HLA-identical donor available. As previously shown, the first successful gene therapy was performed in a patient with ADA deficiency in the U.S. in 1990. Since the gene was introduced into T lymphocytes, fre‐ quent treatment was required. However, this treatment was associated with an unacceptable level of toxicity. Since transfected vector and normal ADA gene expression in T lympho‐ cytes continued for two years after the cessation of treatment [1], gene therapy attracted at‐ tention. With advances in HSC gene-transfer technology, gene therapy for many primary

SCID-X1 is an X-linked disease caused by deficiency of the common γ (γc) chain in the IL-2 receptor. Because the γc chain is common to the IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, in SCID-X1 patients, there are defects in T and natural killer (NK) cells, and B cell dysfunction are usually observed [57]. Patients begin suffering from various infections starting several

In SCID-X1, since T cells are lacking, engraftment of the gene-transduced cells can be ach‐ ieved without pre-conditioning therapy. In the clinical studies of SCID-X1 patients in France and the U.K., the MFG retroviral vector was used with HSCs obtained from the patient. Af‐ ter gene therapy, many patients had improvements in immune function. However, since the genes regulating lymphocyte proliferation, such as *LIM domain only 2 (LMO2)*, *Bmi1*, c*yclin D2 (CCND2)* are near the gene insertion region, there was a high frequency of T-cell leuke‐ mia after treatment. Furthermore, in the patients who developed leukemia, additional chro‐ mosomal changes, including activating mutations of *Notch1*, changes in the T cell receptor β region, and deletion of tumor suppressor genes, e.g. cyclin-dependent kinase-2A (*CDKN2A*) were observed [58]. Almost gene integration sites by the retroviral vector were inside or near genes that are highly expressed in CD34 positive stem cells. Furthermore, the activity of protein kinases or transferases coded by these activated genes was stronger in CD3 posi‐ tive T cells than CD34 positive cells [59]. Thus, gene integration mediated by a retrovirus in‐ fluences the target cell's dormant capacity for survival, engraftment, and proliferation.

weeks after birth. Without curative treatment, such as HSCT, patients die in infancy.

potential to treat hemophilia [55].

**4.4. Primary immunodeficiencies**

*4.4.1. SCID-X1*

immunodeficiencies can now be considered [56].

In recent years, the design of lentiviral vectors used for gene therapy in Fanconi anemia has improved. Although the vav and phosphoglycerate kinase (PGK) promoters are relatively weak, physiological levels of *FANCA* gene expression can be obtained in lymphoblastoid cells. CMV and spleen focus-forming virus (SFFV) promoters result in overexpression of *FANCA*. The PGK-*FANCA* lentiviral vectors with either a wild-type woodchuck hepatitis vi‐ rus posttranscriptional regulatory element (WPRE) or a mutated WPRE in the 3′ region have higher levels of *FANCA* gene expression. In conclusion, lentiviral vectors with a mutated WPRE and a PGK promoter are considered the most suitable with respect to safety and effi‐ ciency for Fanconi anemia gene therapy [48].

There was a recent interesting report on the use of induced pluripotent stem cells (iPS cell). Instead of introducing a repaired gene into the HSCs of a patient with a *FANCA* gene abnor‐ mality, the modified gene was introduced into more stable somatic cells, e.g. fibroblasts, and iPS cells were derived from the genetically modified somatic cells. If HSCs can be produced from genetically modified iPS cells, hematological function can be efficiently reconstructed in patients with hematologic disorders [49].

#### **4.3. Hemophilia**

Hemophilia is a common congenital coagulopathy caused by coagulation factor VIII (hemo‐ philia A) or IX (hemophilia B) deficiency. Although the genes encoding both factor VIII (Xq28) and factor IX (Xq27) are located on the X chromosome and most cases are X-linked, many sporadic variations have been reported. Factor substitution therapies have been used to treat hemophilia for many years. However, there is great hope for gene therapy with he‐ mophilia because coagulation factors have short half-lives (factor VIII, 8 to 12 hours; factor IX, 18 to 24 hours), and an inhibitor is produced in many cases. Furthermore, it is possible for gene therapy to suppress immunogenicity by introducing a mutant protein that lacks the domain with which the inhibitor interacts. Since both coagulation factors are usually pro‐ duced in the liver, there are few studies involving HSCs. In addition to hepatocytes, trials introducing the modified gene directly into splenic cells, endothelial cells, myoblasts, fibro‐ blasts, etc. have been reported [50-52]. Since the factor IX gene (34 kb) is smaller than the factor VIII gene (186 kb), hemophilia B gene therapy can be possible with an adenovirus vec‐ tor or an AAV vector. Therefore, hemophilia B is progressing more as a field of gene therapy research even through there are five times more patients with hemophilia A [51-53].

Recently, human factor VIII variant genes were successfully introduced into the HSCs of a mouse with hemophilia A resulting in therapeutic levels of factor VIII variant protein ex‐ pression. This variant factor VIII has changes in the B and A2 domain in addition to the A1 domain for improved secretion and reduced immunogenicity (wild-type factor VIII has six domains, A1, A2, B, A3, C1, and C2) [54]. To ameliorate the symptoms of hemophilia A, par‐ tial replacement of the mutated liver cells by healthy cells in hemophilia A mice was chal‐ lenged with allogeneic bone marrow progenitor cell transplantation. In this study, the bone marrow progenitor cell-derived hepatocytes and sinusoidal endothelial cells synthesized factor VIII, showing that autologous gene-modified bone marrow progenitor cells have the potential to treat hemophilia [55].

#### **4.4. Primary immunodeficiencies**

Although HSCT has been widely performed as curative treatment for primary immunodefi‐ ciencies, gene therapy has been considered when there is no HLA-identical donor available. As previously shown, the first successful gene therapy was performed in a patient with ADA deficiency in the U.S. in 1990. Since the gene was introduced into T lymphocytes, fre‐ quent treatment was required. However, this treatment was associated with an unacceptable level of toxicity. Since transfected vector and normal ADA gene expression in T lympho‐ cytes continued for two years after the cessation of treatment [1], gene therapy attracted at‐ tention. With advances in HSC gene-transfer technology, gene therapy for many primary immunodeficiencies can now be considered [56].

#### *4.4.1. SCID-X1*

transduction (overnight), low oxidative stress (5% oxygen), and the anti-oxidant N-acetyl-Lcysteine [20]. Lentiviral transduction of unselected Fanconi anemia bone marrow cells medi‐ ates efficient phenotypic correction of hematopoietic progenitor cells and CD34 mesenchymal stromal cells, with increased efficacy in hematopoietic engraftment [45]. In *Fancg* -/- mice, the wild-type mesenchymal stem and progenitor cells play important roles in the reconstitution of exogenous HSCs *in vitro* [46]. Recently, a new approach that directly injects lentiviral vector particles into the femur for *FANCC* gene transfer in mice was able to successfully introduce the *FANCC* gene to HSCs. This result provides evidence that target‐ ing the HSCs directly in their native environment enables efficient and long-term correction

In recent years, the design of lentiviral vectors used for gene therapy in Fanconi anemia has improved. Although the vav and phosphoglycerate kinase (PGK) promoters are relatively weak, physiological levels of *FANCA* gene expression can be obtained in lymphoblastoid cells. CMV and spleen focus-forming virus (SFFV) promoters result in overexpression of *FANCA*. The PGK-*FANCA* lentiviral vectors with either a wild-type woodchuck hepatitis vi‐ rus posttranscriptional regulatory element (WPRE) or a mutated WPRE in the 3′ region have higher levels of *FANCA* gene expression. In conclusion, lentiviral vectors with a mutated WPRE and a PGK promoter are considered the most suitable with respect to safety and effi‐

There was a recent interesting report on the use of induced pluripotent stem cells (iPS cell). Instead of introducing a repaired gene into the HSCs of a patient with a *FANCA* gene abnor‐ mality, the modified gene was introduced into more stable somatic cells, e.g. fibroblasts, and iPS cells were derived from the genetically modified somatic cells. If HSCs can be produced from genetically modified iPS cells, hematological function can be efficiently reconstructed

Hemophilia is a common congenital coagulopathy caused by coagulation factor VIII (hemo‐ philia A) or IX (hemophilia B) deficiency. Although the genes encoding both factor VIII (Xq28) and factor IX (Xq27) are located on the X chromosome and most cases are X-linked, many sporadic variations have been reported. Factor substitution therapies have been used to treat hemophilia for many years. However, there is great hope for gene therapy with he‐ mophilia because coagulation factors have short half-lives (factor VIII, 8 to 12 hours; factor IX, 18 to 24 hours), and an inhibitor is produced in many cases. Furthermore, it is possible for gene therapy to suppress immunogenicity by introducing a mutant protein that lacks the domain with which the inhibitor interacts. Since both coagulation factors are usually pro‐ duced in the liver, there are few studies involving HSCs. In addition to hepatocytes, trials introducing the modified gene directly into splenic cells, endothelial cells, myoblasts, fibro‐ blasts, etc. have been reported [50-52]. Since the factor IX gene (34 kb) is smaller than the factor VIII gene (186 kb), hemophilia B gene therapy can be possible with an adenovirus vec‐ tor or an AAV vector. Therefore, hemophilia B is progressing more as a field of gene therapy

research even through there are five times more patients with hemophilia A [51-53].

of bone marrow defects in Fanconi anemia [47].

118 Innovations in Stem Cell Transplantation

ciency for Fanconi anemia gene therapy [48].

in patients with hematologic disorders [49].

**4.3. Hemophilia**

SCID-X1 is an X-linked disease caused by deficiency of the common γ (γc) chain in the IL-2 receptor. Because the γc chain is common to the IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, in SCID-X1 patients, there are defects in T and natural killer (NK) cells, and B cell dysfunction are usually observed [57]. Patients begin suffering from various infections starting several weeks after birth. Without curative treatment, such as HSCT, patients die in infancy.

In SCID-X1, since T cells are lacking, engraftment of the gene-transduced cells can be ach‐ ieved without pre-conditioning therapy. In the clinical studies of SCID-X1 patients in France and the U.K., the MFG retroviral vector was used with HSCs obtained from the patient. Af‐ ter gene therapy, many patients had improvements in immune function. However, since the genes regulating lymphocyte proliferation, such as *LIM domain only 2 (LMO2)*, *Bmi1*, c*yclin D2 (CCND2)* are near the gene insertion region, there was a high frequency of T-cell leuke‐ mia after treatment. Furthermore, in the patients who developed leukemia, additional chro‐ mosomal changes, including activating mutations of *Notch1*, changes in the T cell receptor β region, and deletion of tumor suppressor genes, e.g. cyclin-dependent kinase-2A (*CDKN2A*) were observed [58]. Almost gene integration sites by the retroviral vector were inside or near genes that are highly expressed in CD34 positive stem cells. Furthermore, the activity of protein kinases or transferases coded by these activated genes was stronger in CD3 posi‐ tive T cells than CD34 positive cells [59]. Thus, gene integration mediated by a retrovirus in‐ fluences the target cell's dormant capacity for survival, engraftment, and proliferation.

Although continuous T cell production was founded in many cases, there was little recon‐ struction of myeloid cells and B cells, and some patients required continuous immunoglobu‐ lin substitution therapy. The use of conditioning therapy is also related to immunological reconstruction after γc chain gene therapy. There is decreased NK cell reconstruction with‐ out conditioning therapy, so conditioning chemotherapy is required for the engraftment of undifferentiated stem cells [58]. A trial of SCID-X1 gene therapy in the U.S. involved three patients ranging from 10 to 14 years of age. They had poor immunological recovery after al‐ lergenic HSCT and T cell recovery was only observed in the youngest patient, suggesting there is a limit to the recovery of the function of the thymus in older children [60].

In a joint Italian-Israeli study started in 2000, ten ADA-SCID children were infused with CD34 positive cells transduced with a MoMLV retroviral vector containing the *ADA* gene after nonmyeloablative conditioning with busulfan (2mg/kg/day for two days). T cell counts or function were improved in nine out of the ten patients, and PEG-ADA was dis‐ continued in eight. Many patients also had improvements in B or NK cell function, and immunoglobulin substitution therapy was discontinued in five patients. Although some patients had serious adverse events including prolonged neutropenia, hypertension, Ep‐ stein-Barr virus infection, and autoimmune hepatitis, there were no cases of treatment-in‐

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As with SCID-X1, the retroviral vector gene insertion region is also near genes that control cell proliferation or self-duplication, such as *LMO2*, or proto-oncogenes [66]. In clinical stud‐ ies performed in France, the U.S., and the U.K., none of the ADA-SCID patients had adverse events related to insertional mutagenesis, such as leukemia [67, 68]. Thus, HSC gene therapy for ADA-SCID using a lentiviral vector [69] is expected to become the alternative therapy in cases without a suitable donor for HSCT [70]. As an alternative to HSC-based gene therapy, a study using an AAV vector has reported ADA gene expression in various tissues, includ‐

CGD is a disease caused by an abnormality in nicotinamide dinucleotide phosphate (NADPH) oxidase expressed in phagocytes, resulting in failure to produce reactive oxygen species and decreased ability to kill bacteria or fungi after phagocytosis. NADPH oxidase consists of gp91phox (Nox2) and p22 phox which together constitute the membrane-spanning component flavocytochrome b558 (CYBB), and the cytosolic components p47phox, p67phox, p40phox, and Rac. CGD is caused by a functional abnormality in any of these components. Mutations in *gp91phox* on the X chromosome account for approximately 70% of CGD cases. CGD patients are afflicted with recurrent opportunistic bacterial and fungal infections, lead‐ ing to the formation of chronic granulomas. Although lifelong antibiotic prophylaxis re‐ duces the incidence of infections, the overall annual mortality rate remains high (2%–5%) and the success rate of HSCT is limited by graft-versus-host-disease and inflammatory flare-

In the initial trials of CGD gene therapy without any conditioning therapy, *p47phox* or *gp91phox* gene was inserted using a retroviral vector. The inserted gene was expressed in peripheral blood granulocytes three to six weeks after re-infusion and mobilization by granulocyte col‐ ony-stimulating factor (G-CSF), but there was no clinical effect within six months [72-74]. In a German study where *gp91phox* was inserted with busulfan conditioning (8mg/kg), there were fewer infections after gene therapy. Gene expression was observed in 20% of leuko‐ cytes in the first month, rising to 80% at one year. However, in the gene insertion region there are genes related to myeloid cell proliferation, such as *myelodysplastic syndrome 1-eco‐ tropic virus integration site 1 (MDS1/EVI1)*, *PR domain containing protein 16 (PRDM16)*, *SET binding protein 1 (SETBP1)*. Two patients developed myelodysplasia [75]. These two patients had monosomy 7, considered to be related to *EVI1* activation. One died of severe sepsis 27

duced leukemia [25].

*4.4.3. CGD*

ups at infected sites [56].

ing heart, skeletal muscle, and kidney [71].

To study whether activation of genes near the region of gene insertion or inserted γc chain gene expression itself induces oncogenicity during SCID-X1 gene therapy, a study of the hu‐ man γc chain gene being expressed under the control of the human CD2 promoter and LTR (CD2- γc chain gene) was performed in mice. When the CD2- γc chain gene was expressed in transgenic mice, a few abnormalities involving T cells were observed, but tumorigenesis was not observed and T and B cell functions were recovered in γc chain-gene deficient mice. This study demonstrated that when the γc c chain gene is expressed externally, SCID-X1 may be treated safely [61].

Although SIN vectors were developed from earlier retroviral [62] or lentiviral vectors [63] to reduce the risk of oncogenicity in SCID-X1 gene therapy, genotoxicity unrelated to muta‐ tions in gene insertion regions or γc chain gene overexpression have been reported with len‐ tiviral vectors in recent years, and it seems that more sophisticated vector development is required [64].

#### *4.4.2. ADA-SCID*

ADA is an enzyme that catalyzes the conversion of purine metabolism products adenosine and deoxyadenosine into inosine or deoxyinosine. ADA-SCID is an autosomal recessive dis‐ ease that results in the accumulation of adenosine, deoxyadenosine, and deoxyadenosinetri‐ phosphate (dATP). Accumulated phosphorylated purine metabolism products act on the thymus and cause the maturational or functional disorder of lymphocytes. Because ADA-SCID patients have both T and B cell production fail, patients have a severe combined im‐ munodeficiency disease with a clinical presentation similar to SCID-X1 results, but unlike SCID-X1, many patients have a low level of T cells. Although enzyme replacement therapy with polyethylene glycol–modified bovine ADA (PEG-ADA) was developed to treat ADA-SCID, it is limited by the development of neutralizing antibodies and the cost of lifelong treatment.

In ADA-SCID, since T cell counts are increased by PEG-ADA, gene therapy to increase pe‐ ripheral T cell counts was attempted during the early stages of gene therapy. Although ad‐ verse events were not observed and continuous expression of ADA was achieved in many patients, reconstruction of immune function was not obtained and substitution therapy with PEG-ADA remained necessary. Therefore, HSCs were no longer the target of gene therapy for ADA-SCID. Since ADA-SCID patients have T cells, nonmyeloablative conditioning was performed to achieve gene-transduced HSC engraftment [25, 65].

In a joint Italian-Israeli study started in 2000, ten ADA-SCID children were infused with CD34 positive cells transduced with a MoMLV retroviral vector containing the *ADA* gene after nonmyeloablative conditioning with busulfan (2mg/kg/day for two days). T cell counts or function were improved in nine out of the ten patients, and PEG-ADA was dis‐ continued in eight. Many patients also had improvements in B or NK cell function, and immunoglobulin substitution therapy was discontinued in five patients. Although some patients had serious adverse events including prolonged neutropenia, hypertension, Ep‐ stein-Barr virus infection, and autoimmune hepatitis, there were no cases of treatment-in‐ duced leukemia [25].

As with SCID-X1, the retroviral vector gene insertion region is also near genes that control cell proliferation or self-duplication, such as *LMO2*, or proto-oncogenes [66]. In clinical stud‐ ies performed in France, the U.S., and the U.K., none of the ADA-SCID patients had adverse events related to insertional mutagenesis, such as leukemia [67, 68]. Thus, HSC gene therapy for ADA-SCID using a lentiviral vector [69] is expected to become the alternative therapy in cases without a suitable donor for HSCT [70]. As an alternative to HSC-based gene therapy, a study using an AAV vector has reported ADA gene expression in various tissues, includ‐ ing heart, skeletal muscle, and kidney [71].

#### *4.4.3. CGD*

Although continuous T cell production was founded in many cases, there was little recon‐ struction of myeloid cells and B cells, and some patients required continuous immunoglobu‐ lin substitution therapy. The use of conditioning therapy is also related to immunological reconstruction after γc chain gene therapy. There is decreased NK cell reconstruction with‐ out conditioning therapy, so conditioning chemotherapy is required for the engraftment of undifferentiated stem cells [58]. A trial of SCID-X1 gene therapy in the U.S. involved three patients ranging from 10 to 14 years of age. They had poor immunological recovery after al‐ lergenic HSCT and T cell recovery was only observed in the youngest patient, suggesting

To study whether activation of genes near the region of gene insertion or inserted γc chain gene expression itself induces oncogenicity during SCID-X1 gene therapy, a study of the hu‐ man γc chain gene being expressed under the control of the human CD2 promoter and LTR (CD2- γc chain gene) was performed in mice. When the CD2- γc chain gene was expressed in transgenic mice, a few abnormalities involving T cells were observed, but tumorigenesis was not observed and T and B cell functions were recovered in γc chain-gene deficient mice. This study demonstrated that when the γc c chain gene is expressed externally, SCID-X1

Although SIN vectors were developed from earlier retroviral [62] or lentiviral vectors [63] to reduce the risk of oncogenicity in SCID-X1 gene therapy, genotoxicity unrelated to muta‐ tions in gene insertion regions or γc chain gene overexpression have been reported with len‐ tiviral vectors in recent years, and it seems that more sophisticated vector development is

ADA is an enzyme that catalyzes the conversion of purine metabolism products adenosine and deoxyadenosine into inosine or deoxyinosine. ADA-SCID is an autosomal recessive dis‐ ease that results in the accumulation of adenosine, deoxyadenosine, and deoxyadenosinetri‐ phosphate (dATP). Accumulated phosphorylated purine metabolism products act on the thymus and cause the maturational or functional disorder of lymphocytes. Because ADA-SCID patients have both T and B cell production fail, patients have a severe combined im‐ munodeficiency disease with a clinical presentation similar to SCID-X1 results, but unlike SCID-X1, many patients have a low level of T cells. Although enzyme replacement therapy with polyethylene glycol–modified bovine ADA (PEG-ADA) was developed to treat ADA-SCID, it is limited by the development of neutralizing antibodies and the cost of lifelong

In ADA-SCID, since T cell counts are increased by PEG-ADA, gene therapy to increase pe‐ ripheral T cell counts was attempted during the early stages of gene therapy. Although ad‐ verse events were not observed and continuous expression of ADA was achieved in many patients, reconstruction of immune function was not obtained and substitution therapy with PEG-ADA remained necessary. Therefore, HSCs were no longer the target of gene therapy for ADA-SCID. Since ADA-SCID patients have T cells, nonmyeloablative conditioning was

performed to achieve gene-transduced HSC engraftment [25, 65].

there is a limit to the recovery of the function of the thymus in older children [60].

may be treated safely [61].

120 Innovations in Stem Cell Transplantation

required [64].

treatment.

*4.4.2. ADA-SCID*

CGD is a disease caused by an abnormality in nicotinamide dinucleotide phosphate (NADPH) oxidase expressed in phagocytes, resulting in failure to produce reactive oxygen species and decreased ability to kill bacteria or fungi after phagocytosis. NADPH oxidase consists of gp91phox (Nox2) and p22 phox which together constitute the membrane-spanning component flavocytochrome b558 (CYBB), and the cytosolic components p47phox, p67phox, p40phox, and Rac. CGD is caused by a functional abnormality in any of these components. Mutations in *gp91phox* on the X chromosome account for approximately 70% of CGD cases. CGD patients are afflicted with recurrent opportunistic bacterial and fungal infections, lead‐ ing to the formation of chronic granulomas. Although lifelong antibiotic prophylaxis re‐ duces the incidence of infections, the overall annual mortality rate remains high (2%–5%) and the success rate of HSCT is limited by graft-versus-host-disease and inflammatory flareups at infected sites [56].

In the initial trials of CGD gene therapy without any conditioning therapy, *p47phox* or *gp91phox* gene was inserted using a retroviral vector. The inserted gene was expressed in peripheral blood granulocytes three to six weeks after re-infusion and mobilization by granulocyte col‐ ony-stimulating factor (G-CSF), but there was no clinical effect within six months [72-74].

In a German study where *gp91phox* was inserted with busulfan conditioning (8mg/kg), there were fewer infections after gene therapy. Gene expression was observed in 20% of leuko‐ cytes in the first month, rising to 80% at one year. However, in the gene insertion region there are genes related to myeloid cell proliferation, such as *myelodysplastic syndrome 1-eco‐ tropic virus integration site 1 (MDS1/EVI1)*, *PR domain containing protein 16 (PRDM16)*, *SET binding protein 1 (SETBP1)*. Two patients developed myelodysplasia [75]. These two patients had monosomy 7, considered to be related to *EVI1* activation. One died of severe sepsis 27 months after gene therapy. Although the gene-inserted cells remained expressed in this pa‐ tient, methylation of the CpG site in the LTR of the viral vector was observed and the ex‐ pression of the inserted *gp91phox* gene was decreased. Interestingly, methylation was restricted to the promoter region of the LTR; the enhancer region was not methylated. Therefore, although *gp91phox* gene expression was decreased, the activation of *EVI1* near the inserted region occurred, leading to clonal proliferation [76]. Since there is a possibility that the transcription activity of genes related to myeloid cell proliferation near the gene inser‐ tion site will be increased, there remains a concern about tumorigenesis with peripheral stem cells mobilization by G-CSF in CGD patients, as with X-SCID [74].

SIN lentiviral vectors using the minimal domain of the WAS promoter or other ubiquitous promoters, such as the PGK promoter, are currently being developed for WAS gene therapy. Preclinical studies using the HSCs obtained from mice or human patients have yield good

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Since a study using human embryonic stem cells (hESCs) and WAS-promoter–driven lenti‐ viral vectors labeled by green fluorescent protein (GFP) showed highly specific gene expres‐ sion in hESCs-derived HSCs, the WAS promoter will be used specifically in the generation

JAK3 deficiency is characterized by the absence of T and NK cells and impaired function of B cells, similar to SCID-X1. Treatment consists of HSCT with an HLA-identical or HLA-hap‐ lo-identical donor, often the parents of the patient, with T cell depletion. Engraftment is suc‐

Although the recovery of T cell function is usually observed after HSCT, there are usually no improvements in B or NK cell function [92]. One case report involved introduction of *JAK3* into the patient's bone marrow CD34 positive cells using the MSCV retroviral vector. In this study, immunological recovery was not achieved although gene expression was ob‐ served for seven months [93]. Since JAK activation can cause T-cell lymphoma, tumorigene‐

PNP metabolizes adenosine into adenine, inosine into hypoxanthine, and guanosine into guanine. PNP deficiency is an autosomal recessive metabolic disorder characterized by le‐ thal T cell defects resulting from the accumulation of products from purine metabolism.

In PNP-deficient mice, transplantation of bone marrow cells transduced with a lentiviral vector containing human *PNP* resulted in human PNP expression, improved thymocyte ma‐ turation, increased weight gain, and extended survival. However, 12 weeks after transplant, the benefit of *PNP*-transduced cells and the percentage of engrafted cells decreased [94].

LAD-1 is a primary immunodeficiency disease caused by abnormalities in the leukocyte in‐ tegrin CD11/CD18 heterodimer due to mutations in the *CD18* gene. It is similar to canine leukocyte adhesion deficiency (CLAD). LAD-1 patients begin experiencing repeated serious

In order to suppress gene activation near the gene insertion region in CLAD and to obtain the sufficient expression of the *CD18* gene, researches have used various promoters with a lentiviral vector or foamy virus, a retroviral vector. *In vivo* animal experiments using a PGK or an elongation factor 1α promoter did not lead to symptom improvement [95-97], but im‐

results in terms of gene expression and genotoxicity [86-90].

of hESC-derived HSCs [91].

cessful in most cases.

*4.4.5. Janus Kinase 3 (JAK3) deficiency*

sis remains a concern with JAK gene therapy [92].

*4.4.7. Leukocyte Adhesion Deficiency type 1 (LAD-1)*

bacterial infections immediately after birth.

*4.4.6. Purine Nucleoside Phosphorylase (PNP) deficiency*

Recently, next-generation gene therapy for CGD using lineage- and stage-restricted lenti‐ viral vectors to avoid tumorigenesis [77] and novel approaches involving iPSs derived from CGD patients using zinc finger nuclease (ZFN)-mediated gene targeting were stud‐ ied [78]. Specific gene targeting can be performed in human iPSs using ZFNs to induce se‐ quence-specific double-strand DNA breaks that enhance site-specific homologous recombination. A single-copy of *gp91phox* was targeted into one allele of the "safe harbor" AAVS1 locus in iPSs [79].

#### *4.4.4. Wiskott-Aldrich Syndrome (WAS)*

WAS is a severe X-linked immunodeficiency caused by mutations in the gene encoding the WAS protein (WASP), a key regulator of signaling and cytoskeletal reorganization in hematopoietic cells. Mutations in *WAS* gene result in a wide spectrum of clinical manifes‐ tations ranging from relatively mild X-linked thrombocytopenia to the classic WAS pheno‐ type characterized by thrombocytopenia, immunodeficiency, eczema, high susceptibility to developing tumors, and autoimmune manifestations [80]. Preclinical and clinical evidence suggest that WASP-expressing cells have a proliferative or survival advantage over WASP-deficient cells, supporting the development of gene therapy [56]. Furthermore, up to 11% of WAS patients have somatic mosaicism due to spontaneous *in vivo* reversion to the normal genotype, and in WAS patients, accumulation of normal T-cell precursors are sometimes seen [81].

In one preclinical study introducing the *WAS* gene into human T and B cells or mouse HSCs using a retroviral vector, recovery of T cell function and immune reactions to infection were observed [82, 83]. The first clinical study of WAS using HSCs involved two young boys in Germany. The WASP-expressing retroviral vector was transfected into CD34 positive cells obtained by apheresis of peripheral blood. Busulfan was used for conditioning therapy (4mg/kg/day for two days). Over two years, WASP gene expression by HSCs, lymphoid and myeloid cells, and platelets was sustained, and the number and function of monocytes, T, B, and NK cells normalized. Clinically, hemorrhagic diathesis, eczema, autoimmunity, and the predisposition to severe infections were diminished. Since comprehensive insertion-site analysis showed vector integration near multiple genes controlling growth and immunolog‐ ic responses in a persistently polyclonal hematopoiesis, careful monitoring for tumorigene‐ sis is necessary, as with SCID-X1 and CGD [84, 85].

SIN lentiviral vectors using the minimal domain of the WAS promoter or other ubiquitous promoters, such as the PGK promoter, are currently being developed for WAS gene therapy. Preclinical studies using the HSCs obtained from mice or human patients have yield good results in terms of gene expression and genotoxicity [86-90].

Since a study using human embryonic stem cells (hESCs) and WAS-promoter–driven lenti‐ viral vectors labeled by green fluorescent protein (GFP) showed highly specific gene expres‐ sion in hESCs-derived HSCs, the WAS promoter will be used specifically in the generation of hESC-derived HSCs [91].

#### *4.4.5. Janus Kinase 3 (JAK3) deficiency*

months after gene therapy. Although the gene-inserted cells remained expressed in this pa‐ tient, methylation of the CpG site in the LTR of the viral vector was observed and the ex‐ pression of the inserted *gp91phox* gene was decreased. Interestingly, methylation was restricted to the promoter region of the LTR; the enhancer region was not methylated. Therefore, although *gp91phox* gene expression was decreased, the activation of *EVI1* near the inserted region occurred, leading to clonal proliferation [76]. Since there is a possibility that the transcription activity of genes related to myeloid cell proliferation near the gene inser‐ tion site will be increased, there remains a concern about tumorigenesis with peripheral

Recently, next-generation gene therapy for CGD using lineage- and stage-restricted lenti‐ viral vectors to avoid tumorigenesis [77] and novel approaches involving iPSs derived from CGD patients using zinc finger nuclease (ZFN)-mediated gene targeting were stud‐ ied [78]. Specific gene targeting can be performed in human iPSs using ZFNs to induce se‐ quence-specific double-strand DNA breaks that enhance site-specific homologous recombination. A single-copy of *gp91phox* was targeted into one allele of the "safe harbor"

WAS is a severe X-linked immunodeficiency caused by mutations in the gene encoding the WAS protein (WASP), a key regulator of signaling and cytoskeletal reorganization in hematopoietic cells. Mutations in *WAS* gene result in a wide spectrum of clinical manifes‐ tations ranging from relatively mild X-linked thrombocytopenia to the classic WAS pheno‐ type characterized by thrombocytopenia, immunodeficiency, eczema, high susceptibility to developing tumors, and autoimmune manifestations [80]. Preclinical and clinical evidence suggest that WASP-expressing cells have a proliferative or survival advantage over WASP-deficient cells, supporting the development of gene therapy [56]. Furthermore, up to 11% of WAS patients have somatic mosaicism due to spontaneous *in vivo* reversion to the normal genotype, and in WAS patients, accumulation of normal T-cell precursors are

In one preclinical study introducing the *WAS* gene into human T and B cells or mouse HSCs using a retroviral vector, recovery of T cell function and immune reactions to infection were observed [82, 83]. The first clinical study of WAS using HSCs involved two young boys in Germany. The WASP-expressing retroviral vector was transfected into CD34 positive cells obtained by apheresis of peripheral blood. Busulfan was used for conditioning therapy (4mg/kg/day for two days). Over two years, WASP gene expression by HSCs, lymphoid and myeloid cells, and platelets was sustained, and the number and function of monocytes, T, B, and NK cells normalized. Clinically, hemorrhagic diathesis, eczema, autoimmunity, and the predisposition to severe infections were diminished. Since comprehensive insertion-site analysis showed vector integration near multiple genes controlling growth and immunolog‐ ic responses in a persistently polyclonal hematopoiesis, careful monitoring for tumorigene‐

stem cells mobilization by G-CSF in CGD patients, as with X-SCID [74].

AAVS1 locus in iPSs [79].

122 Innovations in Stem Cell Transplantation

sometimes seen [81].

*4.4.4. Wiskott-Aldrich Syndrome (WAS)*

sis is necessary, as with SCID-X1 and CGD [84, 85].

JAK3 deficiency is characterized by the absence of T and NK cells and impaired function of B cells, similar to SCID-X1. Treatment consists of HSCT with an HLA-identical or HLA-hap‐ lo-identical donor, often the parents of the patient, with T cell depletion. Engraftment is suc‐ cessful in most cases.

Although the recovery of T cell function is usually observed after HSCT, there are usually no improvements in B or NK cell function [92]. One case report involved introduction of *JAK3* into the patient's bone marrow CD34 positive cells using the MSCV retroviral vector. In this study, immunological recovery was not achieved although gene expression was ob‐ served for seven months [93]. Since JAK activation can cause T-cell lymphoma, tumorigene‐ sis remains a concern with JAK gene therapy [92].

#### *4.4.6. Purine Nucleoside Phosphorylase (PNP) deficiency*

PNP metabolizes adenosine into adenine, inosine into hypoxanthine, and guanosine into guanine. PNP deficiency is an autosomal recessive metabolic disorder characterized by le‐ thal T cell defects resulting from the accumulation of products from purine metabolism.

In PNP-deficient mice, transplantation of bone marrow cells transduced with a lentiviral vector containing human *PNP* resulted in human PNP expression, improved thymocyte ma‐ turation, increased weight gain, and extended survival. However, 12 weeks after transplant, the benefit of *PNP*-transduced cells and the percentage of engrafted cells decreased [94].

#### *4.4.7. Leukocyte Adhesion Deficiency type 1 (LAD-1)*

LAD-1 is a primary immunodeficiency disease caused by abnormalities in the leukocyte in‐ tegrin CD11/CD18 heterodimer due to mutations in the *CD18* gene. It is similar to canine leukocyte adhesion deficiency (CLAD). LAD-1 patients begin experiencing repeated serious bacterial infections immediately after birth.

In order to suppress gene activation near the gene insertion region in CLAD and to obtain the sufficient expression of the *CD18* gene, researches have used various promoters with a lentiviral vector or foamy virus, a retroviral vector. *In vivo* animal experiments using a PGK or an elongation factor 1α promoter did not lead to symptom improvement [95-97], but im‐ provement was seen with CD11b and CD18 promoters, respectively, with a SIN lentiviral vector in one animal study [98].

**4.6. Gaucher disease**

**4.7. X-ALD**

obtained with HSCT [107].

**5. Conclusion**

Gaucher disease is the most common lysosomal storage disorder. It is caused by deficiency of glucocerebroside-cleaving enzyme (β-glucocerebrosidase), resulting in the accumulation of glucocerebroside in the reticuloendothelial system [102]. This autosomal recessive disease presents with hepatosplenomegaly, anemia, thrombocytopenia, and convulsions with or with‐ out mental retardation. It is classified into three types based on the clinical course or existence of neurological symptoms: type I (non-neuropathic, adult type), type II (acute neuropathic, in‐ fantile type), and type III (chronic neuropathic, juvenile type). Enzyme replacement therapy has been established in type I. As with MPS, since it is difficult to improve CNS symptoms with enzyme replacement therapy, HSCT is used, especially with type III. Gene therapy is consid‐

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For Gaucher disease without CNS symptoms, a animal model using an AAV vector to pro‐ duce enzyme in hepatocytes yielded good results [103]. HSC gene therapy using a retroviral vector was attempted in type I mice. The treated cells had higher β-glucocerebrosidase activ‐ ity than the HSCs from wild-type mice. Glucocerebroside levels normalized five to six months after treatment and no infiltration of Gaucher cells could be observed in the bone marrow, spleen, and liver [104]. In recent years, development of lentiviral vectors including the human glucocerebrosidase gene [105] and low-risk HSCT with nonmyeloablative doses

X-ALD is a peroxisomal disease in which a lipid metabolism abnormality causes demyelina‐ tion of CNS tissues and dysfunction of the adrenal gland. It results from mutations in the ATP-binding cassette sub-family D (*ABCD1*) gene that codes for the adrenoleukodystrophy (ALD) protein. Behavioral disorders, mental retardation, or both occur by the age of five or six. Once symptoms appear, they progress to gait disorder and visual impairment within several months and the prognosis is poor. Increased levels of very long chain fatty acids (VLCFA), such as C25:0 or C26:0, are observed in the CNS, plasma, erythrocytes, leucocytes, etc. If the neurological defects are not severe, arrest of or improvement in symptoms can be

One study has reported the introduction of wild-type *ABCD1* using a lentiviral vector into pe‐ ripheral blood CD34 positive cells of two patients with no HLA-identical donor. The patients received a transfusion of autologous gene-modified cells after myeloablative conditioning therapy. At three years of follow-up, ALD proteins were expressed in approximately 7–14% of neutrophils, monocytes, and T cells. Clinically, cerebral demyelination stopped 14 and 16 months after gene therapy, respectively, similar to results with allergenic HSCT [108, 109].

Gene therapy using HSCs was outlined. HSCT with HSCs can replace all of the patient's original HSCs with donor HSCs. Therefore, gene therapy using HSCs is an alternative if the

ered in cases with little improvement with enzyme replacement therapy [103].

of busulfan (25mg/kg) and no radiation therapy have been attempted in mice [106].

#### **4.5. Mucopolysaccharidosis (MPS)**

MPS is a general term for diseases characterized by glycosaminoglycan (GAG) accumula‐ tion into lysosomes as a result of deficiencies in lysosomal enzymes that degrade GAG. Although there are more than ten enzymes that are known to degrade GAG, MPS is div‐ ided into seven types: type I (α-L-iduronidase deficiency, Hurler syndrome, Sheie syn‐ drome, Hurler-Sheie syndrome), type II (iduronate sulfatase deficiency, Hunter syndrome), type III (heparan N-sulfatase deficiency, α-N-acetylglucosaminidase deficien‐ cy, α-glucosaminidase acetyltransferase deficiency, N-acetylglucosamine 6-sulfatase defi‐ ciency, Sanfilippo syndrome), type IV (galactose 6-sulfatase deficiency, Morquio syndrome), type VI (N-acetylgalactosamine 4-sulfatase deficiency, Maroteaux-Lamy syn‐ drome), type VII (β-glucuronidase deficiency, Sly syndrome), and type IX (hyaluronidase deficiency). Type II is X-linked; the other types are autosomal recessive. Although lyso‐ somes are found in almost all cells, MPS mainly affects internal organs such as the brain, heart, bones, joints, eyes, liver, and spleen. The extent of disease, including mental retar‐ dation, varies with MPS type.

In types I, II, and VI, enzyme replacement therapy is performed. HSCT is performed in types I, II, IV, and VII. Gene therapy for types I, II, III, and VII type have been investigated. There are trials using an AAV or adenovirus vector to insert the modified gene into various cell types, including hepatocytes, muscle cells, myoblasts, and fibroblasts [99].

The first study of HSC gene therapy for MPS using a retroviral vector was performed on type VII mice in 1992, resulting in decreased accumulation of GAG in the liver and spleen but not in the brain and eyes [100]. Subsequent studies in type I and III animal models showed decreases in GAG accumulation in the kidneys and brain. Introductory efficiency and immunological reactions are considered challenges in HSC gene therapy for MPS [99].

Restoring or preserving central nervous system (CNS) function is one of the major chal‐ lenges in the treatment of MPS. Since replaced enzymes easily cannot pass the bloodbrain barrier (BBB), a high dose of enzyme is needed to improve CNS function. Gene therapy faces the same challenge. Even with high expression of enzyme by, for exam‐ ple, hepatocytes, the BBB prevents efficient delivery into the CNS. When a lentiviral vector is directly injected into the body, gene expression in brain tissue is observed, al‐ though the underlying mechanism is unknown. There are also trials where AAV vec‐ tors are directly injected into the CNS of mice or dogs and gene expression was observed in brain tissue [99].

Recently, a lentiviral vector using an ankyrin-1-based erythroid-specific hybrid promoter/ enhancer (IHK) was used with HSCs to obtain gene expression only in erythroblasts for type I MPS. This approach resulted in decreased accumulation of GAG in the liver, spleen, heart, and CNS via enzyme expression in erythroblasts [101].

#### **4.6. Gaucher disease**

provement was seen with CD11b and CD18 promoters, respectively, with a SIN lentiviral

MPS is a general term for diseases characterized by glycosaminoglycan (GAG) accumula‐ tion into lysosomes as a result of deficiencies in lysosomal enzymes that degrade GAG. Although there are more than ten enzymes that are known to degrade GAG, MPS is div‐ ided into seven types: type I (α-L-iduronidase deficiency, Hurler syndrome, Sheie syn‐ drome, Hurler-Sheie syndrome), type II (iduronate sulfatase deficiency, Hunter syndrome), type III (heparan N-sulfatase deficiency, α-N-acetylglucosaminidase deficien‐ cy, α-glucosaminidase acetyltransferase deficiency, N-acetylglucosamine 6-sulfatase defi‐ ciency, Sanfilippo syndrome), type IV (galactose 6-sulfatase deficiency, Morquio syndrome), type VI (N-acetylgalactosamine 4-sulfatase deficiency, Maroteaux-Lamy syn‐ drome), type VII (β-glucuronidase deficiency, Sly syndrome), and type IX (hyaluronidase deficiency). Type II is X-linked; the other types are autosomal recessive. Although lyso‐ somes are found in almost all cells, MPS mainly affects internal organs such as the brain, heart, bones, joints, eyes, liver, and spleen. The extent of disease, including mental retar‐

In types I, II, and VI, enzyme replacement therapy is performed. HSCT is performed in types I, II, IV, and VII. Gene therapy for types I, II, III, and VII type have been investigated. There are trials using an AAV or adenovirus vector to insert the modified gene into various

The first study of HSC gene therapy for MPS using a retroviral vector was performed on type VII mice in 1992, resulting in decreased accumulation of GAG in the liver and spleen but not in the brain and eyes [100]. Subsequent studies in type I and III animal models showed decreases in GAG accumulation in the kidneys and brain. Introductory efficiency and immunological reactions are considered challenges in HSC gene therapy for MPS [99].

Restoring or preserving central nervous system (CNS) function is one of the major chal‐ lenges in the treatment of MPS. Since replaced enzymes easily cannot pass the bloodbrain barrier (BBB), a high dose of enzyme is needed to improve CNS function. Gene therapy faces the same challenge. Even with high expression of enzyme by, for exam‐ ple, hepatocytes, the BBB prevents efficient delivery into the CNS. When a lentiviral vector is directly injected into the body, gene expression in brain tissue is observed, al‐ though the underlying mechanism is unknown. There are also trials where AAV vec‐ tors are directly injected into the CNS of mice or dogs and gene expression was

Recently, a lentiviral vector using an ankyrin-1-based erythroid-specific hybrid promoter/ enhancer (IHK) was used with HSCs to obtain gene expression only in erythroblasts for type I MPS. This approach resulted in decreased accumulation of GAG in the liver, spleen, heart,

cell types, including hepatocytes, muscle cells, myoblasts, and fibroblasts [99].

vector in one animal study [98].

124 Innovations in Stem Cell Transplantation

dation, varies with MPS type.

observed in brain tissue [99].

and CNS via enzyme expression in erythroblasts [101].

**4.5. Mucopolysaccharidosis (MPS)**

Gaucher disease is the most common lysosomal storage disorder. It is caused by deficiency of glucocerebroside-cleaving enzyme (β-glucocerebrosidase), resulting in the accumulation of glucocerebroside in the reticuloendothelial system [102]. This autosomal recessive disease presents with hepatosplenomegaly, anemia, thrombocytopenia, and convulsions with or with‐ out mental retardation. It is classified into three types based on the clinical course or existence of neurological symptoms: type I (non-neuropathic, adult type), type II (acute neuropathic, in‐ fantile type), and type III (chronic neuropathic, juvenile type). Enzyme replacement therapy has been established in type I. As with MPS, since it is difficult to improve CNS symptoms with enzyme replacement therapy, HSCT is used, especially with type III. Gene therapy is consid‐ ered in cases with little improvement with enzyme replacement therapy [103].

For Gaucher disease without CNS symptoms, a animal model using an AAV vector to pro‐ duce enzyme in hepatocytes yielded good results [103]. HSC gene therapy using a retroviral vector was attempted in type I mice. The treated cells had higher β-glucocerebrosidase activ‐ ity than the HSCs from wild-type mice. Glucocerebroside levels normalized five to six months after treatment and no infiltration of Gaucher cells could be observed in the bone marrow, spleen, and liver [104]. In recent years, development of lentiviral vectors including the human glucocerebrosidase gene [105] and low-risk HSCT with nonmyeloablative doses of busulfan (25mg/kg) and no radiation therapy have been attempted in mice [106].

#### **4.7. X-ALD**

X-ALD is a peroxisomal disease in which a lipid metabolism abnormality causes demyelina‐ tion of CNS tissues and dysfunction of the adrenal gland. It results from mutations in the ATP-binding cassette sub-family D (*ABCD1*) gene that codes for the adrenoleukodystrophy (ALD) protein. Behavioral disorders, mental retardation, or both occur by the age of five or six. Once symptoms appear, they progress to gait disorder and visual impairment within several months and the prognosis is poor. Increased levels of very long chain fatty acids (VLCFA), such as C25:0 or C26:0, are observed in the CNS, plasma, erythrocytes, leucocytes, etc. If the neurological defects are not severe, arrest of or improvement in symptoms can be obtained with HSCT [107].

One study has reported the introduction of wild-type *ABCD1* using a lentiviral vector into pe‐ ripheral blood CD34 positive cells of two patients with no HLA-identical donor. The patients received a transfusion of autologous gene-modified cells after myeloablative conditioning therapy. At three years of follow-up, ALD proteins were expressed in approximately 7–14% of neutrophils, monocytes, and T cells. Clinically, cerebral demyelination stopped 14 and 16 months after gene therapy, respectively, similar to results with allergenic HSCT [108, 109].

## **5. Conclusion**

Gene therapy using HSCs was outlined. HSCT with HSCs can replace all of the patient's original HSCs with donor HSCs. Therefore, gene therapy using HSCs is an alternative if the patient does not have an HLA-identical donor or cannot tolerate the conditioning regimen or other HSCT-related side effects. Fully myeloablative or nonmyeloablative conditioning regimens are still necessary to eliminate potential competition within the bone marrow com‐ partment, in an attempt to increase the number of gene-modified HSCs or progenitors that produce the therapeutic enzyme or protein. With gene therapy, eliminating the risk of im‐ mune reactions against the transgene is necessary. Lentiviral vectors in clinical use must not be contaminated by replication-competent recombinant vectors related to the parent HIV-1 virus. The main risk of retrovirus- or lentivirus-mediated gene therapy may prove to be in‐ sertional mutagenesis caused by random retroviral integration leading to activation of pro‐ to-oncogenes or inactivation of tumor-suppressor genes, ultimately leading to malignancy [107]. However, with advances in gene introduction technology, such as the development of the SIN vector and advances in cell or gene-region targeting, gene therapy can be done more safely and efficiently. Furthermore, since cells more immature than HSCs, i.e., iPS cells, are available, further advances in HSC gene therapy are expected in the future.

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## **Author details**

#### Toshihisa Tsuruta

Department of Clinical Examination, National Hospital Organization Kumamoto Medical Center, Japan

#### **References**


[6] Abe T, Masuya M, Ogawa M. An efficient method for single hematopoietic stem cell engraftment in mice based on cell-cycle dormancy of hematopoietic stem cells. Exp Hematol. 2010;38(7):603-8.

patient does not have an HLA-identical donor or cannot tolerate the conditioning regimen or other HSCT-related side effects. Fully myeloablative or nonmyeloablative conditioning regimens are still necessary to eliminate potential competition within the bone marrow com‐ partment, in an attempt to increase the number of gene-modified HSCs or progenitors that produce the therapeutic enzyme or protein. With gene therapy, eliminating the risk of im‐ mune reactions against the transgene is necessary. Lentiviral vectors in clinical use must not be contaminated by replication-competent recombinant vectors related to the parent HIV-1 virus. The main risk of retrovirus- or lentivirus-mediated gene therapy may prove to be in‐ sertional mutagenesis caused by random retroviral integration leading to activation of pro‐ to-oncogenes or inactivation of tumor-suppressor genes, ultimately leading to malignancy [107]. However, with advances in gene introduction technology, such as the development of the SIN vector and advances in cell or gene-region targeting, gene therapy can be done more safely and efficiently. Furthermore, since cells more immature than HSCs, i.e., iPS cells, are

Department of Clinical Examination, National Hospital Organization Kumamoto Medical

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Center, Japan

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**Section 2**

**Clinical Aspects of Stem Cell Transplantation**

**Clinical Aspects of Stem Cell Transplantation**

**Chapter 6**

**Progress in Hematopoietic Stem Cell Transplantation**

Transplantation of autologous or allogeneic hematopoietic stem cells is a method currently used to treat many malignant and nonmalignant hematological diseases. The indications, methods, goals of therapy have evolved since the introduction of transplantation to the clini‐ cal practice. Progress that has been achieved allowed for the improvement of results. Thanks to the availability of various conditioning regimens, various hematopoietic cells sources as well as variable possibilities of anti-GvHD prophylaxis the individualization of the trans‐ plantation procedure has been more and more widely used in the recent years. This chapter summarizes current clinical practices and presents major clinical problems that have to be

Autologous peripheral hematopoietic stem cells transplantation (auto-HSCT) was for the first time performed at Hammersmith Hospital in London in 1981 to treat the patient in ac‐ celerated phase of CML. Although auto-HSCT does not play any role in the treatment of CML nowadays, indications for this valuable therapeutic method have evolved for many years. In acute leukemia auto-HSCT should be recommended only in the context of clinical studies. Auto-HSCT after myeloablative chemotherapy or radiotherapy has originally been developed as an alternative to allogeneic hematopoietic stem cell transplantation for pa‐ tients with AML with no suitable donor. Several randomized studies in patients with AML

and reproduction in any medium, provided the original work is properly cited.

© 2013 Markiewicz et al.; licensee InTech. This is an open access article 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.

© 2013 The Author(s). Licensee InTech. 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,

optimally managed in order to improve the outcomes of transplantation.

**2. Autologous hematopoietic stem cells transplantation**

Miroslaw Markiewicz,

http://dx.doi.org/10.5772/53086

**1. Introduction**

Malgorzata Sobczyk-Kruszelnicka,

Monika Dzierzak Mietla, Anna Koclega,

Patrycja Zielinska and Slawomira Kyrcz-Krzemien

Additional information is available at the end of the chapter

## **Progress in Hematopoietic Stem Cell Transplantation**

Miroslaw Markiewicz, Malgorzata Sobczyk-Kruszelnicka, Monika Dzierzak Mietla, Anna Koclega, Patrycja Zielinska and Slawomira Kyrcz-Krzemien

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53086

## **1. Introduction**

Transplantation of autologous or allogeneic hematopoietic stem cells is a method currently used to treat many malignant and nonmalignant hematological diseases. The indications, methods, goals of therapy have evolved since the introduction of transplantation to the clini‐ cal practice. Progress that has been achieved allowed for the improvement of results. Thanks to the availability of various conditioning regimens, various hematopoietic cells sources as well as variable possibilities of anti-GvHD prophylaxis the individualization of the trans‐ plantation procedure has been more and more widely used in the recent years. This chapter summarizes current clinical practices and presents major clinical problems that have to be optimally managed in order to improve the outcomes of transplantation.

#### **2. Autologous hematopoietic stem cells transplantation**

Autologous peripheral hematopoietic stem cells transplantation (auto-HSCT) was for the first time performed at Hammersmith Hospital in London in 1981 to treat the patient in ac‐ celerated phase of CML. Although auto-HSCT does not play any role in the treatment of CML nowadays, indications for this valuable therapeutic method have evolved for many years. In acute leukemia auto-HSCT should be recommended only in the context of clinical studies. Auto-HSCT after myeloablative chemotherapy or radiotherapy has originally been developed as an alternative to allogeneic hematopoietic stem cell transplantation for pa‐ tients with AML with no suitable donor. Several randomized studies in patients with AML

© 2013 Markiewicz et al.; licensee InTech. This is an open access article 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. © 2013 The Author(s). Licensee InTech. 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.

in first complete remission (CR1) subsequently suggested reduced relapse rates after auto-HSCT [1]. Auto-HSCT is also widely used to consolidate first remission in AML. The novel molecular and cytogenetic stratification methods may allow the identification of AML enti‐ ties which could benefit from autografting. The overall survival of patients receiving auto-HSCT in ALL in first remission is around 40%. The high-dose therapy followed by auto-HSCT can be an alternative treatment in patients in whom allo-HSCT is precluded.

thalidomide in patients not achieving the remission or a very good partial response after the

Progress in Hematopoietic Stem Cell Transplantation

http://dx.doi.org/10.5772/53086

141

Auto-HSCT constitutes an important treatment option for patients with solid tumors. Select‐ ed subgroups of oncological patients may benefit from high-dose chemotherapy supported by auto-HSCT. High-dose chemotherapy for refractory germ cell tumors is considered a standard therapy. Conditioning regimen in this case incorporates carboplatin and etoposide. Auto-HSCT after conditioning regimen aimed to increase the immunosuppression is being considered in clinical protocols for selected patients with severe multiple sclerosis [13], rheumatoid arthritis [14], systemic lupus erythromatous [15], systemic sclerosis [16], im‐ mune cytopenias and Crohn's disease [17]. Auto-HSCT for other autoimmune disorders is being considered on a developmental basis. Steroid dependency with Cushing threshold

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) constitutes a standard treat‐ ment of hematological malignant and nonmalignant disorders. The possibility of finding a donor has been increased by use of unrelated donors, with similar results of transplantation when compared to results of sibling donor transplants. The use of peripheral blood stem cells, instead of bone marrow, results in faster engraftment, but also in the increased risk of chronic GvHD (Graft versus Host Disease). Reduced-intensity conditioning is used instead of high–dose myeloablative conditioning for older patients and those with comorbidities. Disease relapse is a major problem and thus it should be detected as early as possible, at the stage of the minimal residual disease or recurrent recipient chimerism and managed by im‐ munotherapy with donor lymphocyte infusions. Novel diagnostic tools and anti-microbial

Allogeneic hematopoietic stem cell transplantation connected with application of high-dose chemo- and radiotherapy was first carried out by Thomas et al. in 1957 to treat leukemia pa‐ tient in advanced stage [18]. The concept of treatment at that time was based on the previous observations conducted during the second world war, referring to destructive activity of ra‐ diation on the function of bone marrow, as well as further research conducted in the 1950's, which showed that it was possible to avoid irreversible pancytopenia thanks to the bone marrow cells transplantation in the irradiated animals. The discovery of Human Leucocyte Antigen (HLA) enabled to match appropriately the donor and the recipient, what contribut‐ ed to the significant increase in overall survival after transplantation which has been ob‐ served since 1968 [19]. The improvement of the results was undoubtedly also influenced by other factors: performing the transplantation in the optimal phase- remission of the disease, GvHD prevention, the improvement of adjunctive treatment. Nowadays more that 25.000

The main indication for allo-HSCT is acute myeloblastic leukemia (AML) and acute lympho‐ blastic leukemia (ALL). In high risk ALL and AML, when favorable prognostic genetic

first transplant [12].

and skeletal damage could be an indication.

allo-HSCTs are being performed each year.

**3. Allogeneic hematopoietic stem cell transplantation**

drugs have reduced the morbidity and mortality from infections.

The results of a large European study showed that auto-HSCT can be recommended in pa‐ tients with good-risk cytogenetic characteristics of myelodysplastic syndrome [2]. Auto-HSCT can be recommended as post-remission therapy to reduce the risk of relapse. The longer remission was observed in patients who undergo auto-HSCT.

In myeloproliferative disorders auto-HSCT can induce responses in patients with primary myelofibrosis, but this procedure cannot be recommended out of clinical protocols.

In chronic lymphocytic leukemia auto-HSCT can be considered for patients with poor-risk disease in complete or good partial remission able to withstand high-dose therapy, but it should be performed preferably in the context of clinical protocols.

Auto-HSCT is the standard therapy for patients with Hodgkin's lymphoma (HL) in first che‐ mosensitive relapse or second complete remission as shown by two prospective randomized clinical trials [3,4]. There is no indication for auto-HSCT in first remission, even in patients with poor prognosis at diagnosis [5,6]. Patients refractory to first-line therapy but sensitive to salvage therapy might benefit from auto-HSCT [7]. Auto-HCT might be considered as a part of a clinical protocol for patients with resistant Hodgkin's lymphoma, as an initial de‐ bulking therapy to be followed by an allo-HSCT as consolidation therapy [8].

In many non-Hodgkin's lymphomas auto-HSCT is a standard therapy. In diffuse large B-cell lymphoma (DLBCL) auto-HSCT is a standard therapy for patients with chemosensitive re‐ lapse [9]. The role of auto-HSCT is being re-evaluated with the advance of monoclonal anti‐ bodies and use of chemo-immunotherapy as first-line treatment. Auto-HSCT remains also the standard approach for early relapsing patients with follicular lymphoma (FL) [10]. In both DLBCL and FL, auto-HSCT does not provide any clinical benefit in patients with re‐ fractory disease. Otherwise, most patients with mantle cell lymphoma are being offered an early intensification with an auto-HSCT, owing it to the inherent poor prognosis of the dis‐ ease. The retrospective analysis indicates that the results of auto-HSCT performed beyond the first remission are inferior [11]. Few studies showed an improved survival in patients with T-cell non-Hodgkin's lymphoma (NHL) who received auto-HSCT as a first line treat‐ ment, compared to those who did not.

Patients with multiple myeloma form a large group of patients being transplanted. Auto-HSCT is clearly indicated for patients <70 years of age with satisfactory general health and fitness who respond to the first-line treatment. Although new agents change the place of au‐ to-HSCT in MM, this procedure still has an established position in treatment. Best results are observed in patients achieving good response before the auto-HSCT, but some non-respond‐ ing patients also may benefit from this approach. Double auto-HSCT (or tandem auto-HSCT) has been shown to be superior to consolidation and maintenance with agents such as thalidomide in patients not achieving the remission or a very good partial response after the first transplant [12].

Auto-HSCT constitutes an important treatment option for patients with solid tumors. Select‐ ed subgroups of oncological patients may benefit from high-dose chemotherapy supported by auto-HSCT. High-dose chemotherapy for refractory germ cell tumors is considered a standard therapy. Conditioning regimen in this case incorporates carboplatin and etoposide.

Auto-HSCT after conditioning regimen aimed to increase the immunosuppression is being considered in clinical protocols for selected patients with severe multiple sclerosis [13], rheumatoid arthritis [14], systemic lupus erythromatous [15], systemic sclerosis [16], im‐ mune cytopenias and Crohn's disease [17]. Auto-HSCT for other autoimmune disorders is being considered on a developmental basis. Steroid dependency with Cushing threshold and skeletal damage could be an indication.

## **3. Allogeneic hematopoietic stem cell transplantation**

in first complete remission (CR1) subsequently suggested reduced relapse rates after auto-HSCT [1]. Auto-HSCT is also widely used to consolidate first remission in AML. The novel molecular and cytogenetic stratification methods may allow the identification of AML enti‐ ties which could benefit from autografting. The overall survival of patients receiving auto-HSCT in ALL in first remission is around 40%. The high-dose therapy followed by auto-

The results of a large European study showed that auto-HSCT can be recommended in pa‐ tients with good-risk cytogenetic characteristics of myelodysplastic syndrome [2]. Auto-HSCT can be recommended as post-remission therapy to reduce the risk of relapse. The

In myeloproliferative disorders auto-HSCT can induce responses in patients with primary

In chronic lymphocytic leukemia auto-HSCT can be considered for patients with poor-risk disease in complete or good partial remission able to withstand high-dose therapy, but it

Auto-HSCT is the standard therapy for patients with Hodgkin's lymphoma (HL) in first che‐ mosensitive relapse or second complete remission as shown by two prospective randomized clinical trials [3,4]. There is no indication for auto-HSCT in first remission, even in patients with poor prognosis at diagnosis [5,6]. Patients refractory to first-line therapy but sensitive to salvage therapy might benefit from auto-HSCT [7]. Auto-HCT might be considered as a part of a clinical protocol for patients with resistant Hodgkin's lymphoma, as an initial de‐

In many non-Hodgkin's lymphomas auto-HSCT is a standard therapy. In diffuse large B-cell lymphoma (DLBCL) auto-HSCT is a standard therapy for patients with chemosensitive re‐ lapse [9]. The role of auto-HSCT is being re-evaluated with the advance of monoclonal anti‐ bodies and use of chemo-immunotherapy as first-line treatment. Auto-HSCT remains also the standard approach for early relapsing patients with follicular lymphoma (FL) [10]. In both DLBCL and FL, auto-HSCT does not provide any clinical benefit in patients with re‐ fractory disease. Otherwise, most patients with mantle cell lymphoma are being offered an early intensification with an auto-HSCT, owing it to the inherent poor prognosis of the dis‐ ease. The retrospective analysis indicates that the results of auto-HSCT performed beyond the first remission are inferior [11]. Few studies showed an improved survival in patients with T-cell non-Hodgkin's lymphoma (NHL) who received auto-HSCT as a first line treat‐

Patients with multiple myeloma form a large group of patients being transplanted. Auto-HSCT is clearly indicated for patients <70 years of age with satisfactory general health and fitness who respond to the first-line treatment. Although new agents change the place of au‐ to-HSCT in MM, this procedure still has an established position in treatment. Best results are observed in patients achieving good response before the auto-HSCT, but some non-respond‐ ing patients also may benefit from this approach. Double auto-HSCT (or tandem auto-HSCT) has been shown to be superior to consolidation and maintenance with agents such as

HSCT can be an alternative treatment in patients in whom allo-HSCT is precluded.

myelofibrosis, but this procedure cannot be recommended out of clinical protocols.

bulking therapy to be followed by an allo-HSCT as consolidation therapy [8].

longer remission was observed in patients who undergo auto-HSCT.

140 Innovations in Stem Cell Transplantation

should be performed preferably in the context of clinical protocols.

ment, compared to those who did not.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) constitutes a standard treat‐ ment of hematological malignant and nonmalignant disorders. The possibility of finding a donor has been increased by use of unrelated donors, with similar results of transplantation when compared to results of sibling donor transplants. The use of peripheral blood stem cells, instead of bone marrow, results in faster engraftment, but also in the increased risk of chronic GvHD (Graft versus Host Disease). Reduced-intensity conditioning is used instead of high–dose myeloablative conditioning for older patients and those with comorbidities. Disease relapse is a major problem and thus it should be detected as early as possible, at the stage of the minimal residual disease or recurrent recipient chimerism and managed by im‐ munotherapy with donor lymphocyte infusions. Novel diagnostic tools and anti-microbial drugs have reduced the morbidity and mortality from infections.

Allogeneic hematopoietic stem cell transplantation connected with application of high-dose chemo- and radiotherapy was first carried out by Thomas et al. in 1957 to treat leukemia pa‐ tient in advanced stage [18]. The concept of treatment at that time was based on the previous observations conducted during the second world war, referring to destructive activity of ra‐ diation on the function of bone marrow, as well as further research conducted in the 1950's, which showed that it was possible to avoid irreversible pancytopenia thanks to the bone marrow cells transplantation in the irradiated animals. The discovery of Human Leucocyte Antigen (HLA) enabled to match appropriately the donor and the recipient, what contribut‐ ed to the significant increase in overall survival after transplantation which has been ob‐ served since 1968 [19]. The improvement of the results was undoubtedly also influenced by other factors: performing the transplantation in the optimal phase- remission of the disease, GvHD prevention, the improvement of adjunctive treatment. Nowadays more that 25.000 allo-HSCTs are being performed each year.

The main indication for allo-HSCT is acute myeloblastic leukemia (AML) and acute lympho‐ blastic leukemia (ALL). In high risk ALL and AML, when favorable prognostic genetic changes are lacking, the allo-HSCT is recommended in the first remission of the disease. The transplantation in more advanced stages of the disease leads to the higher relapse rate, as well as to the increased incidence of transplantation complications.

the matched donor depends on the frequency of occurrence of HLA-haplotypes in the whole population and the race of recipient – most donors recruited by registries worldwide belong to Caucasian race. The efforts are being made, especially in the USA, aiming to recruit high‐

Progress in Hematopoietic Stem Cell Transplantation

http://dx.doi.org/10.5772/53086

143

Phenotypic HLA-matching involving the testing of HLA-antigens by means of specific sera has been replaced by more precise molecular testing enabling the precise identification of

The question of accepting a mismatched donor for a patients, who didn't find a fully match‐ ed donor has not been finally solved. With the increasing number of observations there are recommendations concerning optimal matching not only in HLA-A, B and DR, but also in C, DQ and even DP. Many centers aim to transplant patients only from donors fully matched in 10/10 alleles of HLA-A, B, C, DR and DQ. The improved methods of typing enabling more precise molecular donor matching has improved the results of allo-HSCT from unre‐ lated donors, which are now similar to those of allo-HSCT from siblings [24]. As the allo-HSCT from an unrelated donor has to be preceded by often time-consuming search for a

The choice of the cells source depends of diagnosis and the type of conditioning treatment applied. Collection of bone marrow is preferred in nonmalignant diseases in order to avoid chronic GvHD. Transplantation of hematopoietic cells from peripheral blood is preferred when reduced intensity conditioning regimen is used, with regard to the fact that transplan‐ tation of larger number of hematopoietic cells is able to break the resistance of the recipient

The bone marrow aspirated in general anesthesia from iliac spine was for many years the main source of cells for transplantation. Except of hematopoietic cells, the bone marrow also consists of multipotential mesenchymal cells which, although are not hematopoietic cells, have a potential to differentiate in vitro and in vivo into various mesenchymal tissues, such as bone, cartilage, fat tissue, tendons and bone matrix. Mesenchymal stem cells can reduce the alloreactivity, they inhibit lymphocytes T proliferation and act immunosuppressively, what has been implemented in the form of unrelated or haploidentical mesenchymal cells

During the 1990's the cells collected in apheresis from peripheral blood after their previous stimulation with granulocytic stimulating factor (G-CSF) completely superseded the bone marrow aspiration in autologous transplantations. In the beginning of 21st century, the simi‐ lar trend occurred also in allo-HSCT. The apheresis of cells from peripheral blood usually results in collection of higher number of nucleated cells, CD34+ cells, lymphocytes CD3+ and NK cells when compared with cells aspiration from the bone marrow; it enables faster regeneration of granulocytes and platelets. It translates into the smaller risk of infections and

donor, it is important to plan the transplantation carefully in advance.

**5. The sources of hematopoietic stem cells**

and to result in the engraftment.

infusion into the treatment of acute GvHD.

smaller demand for transfusions of blood derivatives.

er number of donors of other races.

HLA allelic determinants.

Despite the introduction of tyrosine kinase inhibitors (TKIs) into the treatment of chronic myeloid leukemia (CML) over ten years ago, allo-HSCT still remains the only way of treat‐ ment capable to provide the complete recovery. The standard indication for allo-HSCT is re‐ sistance to TKIs treatment, especially in young patients. Other indications for allo-HSCT are myelodysplastic syndrome, high-risk chronic lymphocytic leukemia, selected patients with high-risk lymphoma, patients with myelofibrosis and other myeloproliferative neoplasms of unfavorable prognosis. The results of multiple myeloma treatment with the use alloHSCT are encouraging. AlloHSCT with the reduced conditioning regimen after previous auto-HSCT constitutes an interesting alternative in patients with multiple myeloma patients, who undergo single or tandem autologous transplantation [20].

Allo-HSCT is also the standard treatment in nonmalignant diseases of hematopoietic system such as severe aplastic anemia (SAA), paroxysmal nocturnal hemoglobinuria (PNH) and he‐ moglobinopathies. In some cases of inborn metabolic defects, allogeneic transplantation of donor's cells can restore the production of the deficient or lacking enzyme and eliminate the disease [21].

## **4. Hematopoietic stem cell donors**

The optimal donors are siblings possessing both haplotypes identical with the recipient. The syngeneic transplantation, i.e. from monozygotic twins, is the safest from the immunological point of view, however it is connected with the increased risk of the relapse of the disease resulting from the lack of immunological interaction between the donor cells and the recipi‐ ent cells [22].

Probability of possessing matched sibling donor is defined by the formula: 1–(0.75)*<sup>n</sup>*, were *n* indicates the number of siblings. The observed decrease in the number of newly born chil‐ dren causes problems in finding matched family donors for many patients. In rare cases with no matched sibling donor, matching donor could be found among other members of the family. In the vast majority of patients without matched sibling donor, transplantation from unrelated donor is the most frequently chosen option. The number of such transplanta‐ tions has increased considerably in the last 20 years [23]. It has been made possible thanks to dynamic development of bone marrow donors' registries, whose number of potential do‐ nors exceeded 20 million in the current year 2012. Alternatively, for those patients who are unlikely to find a matched donor, partial incompatibility could be accepted.

The most desirable model of the donors' registry organization is the development of nation‐ al ones which, for many reasons (safety of donors, clearness of procedures and financial rea‐ sons), according to WMDA's (World Marrow Donors Association) recommendations should control and supervise the recruitment of the donors within the country. The chance to find the matched donor depends on the frequency of occurrence of HLA-haplotypes in the whole population and the race of recipient – most donors recruited by registries worldwide belong to Caucasian race. The efforts are being made, especially in the USA, aiming to recruit high‐ er number of donors of other races.

Phenotypic HLA-matching involving the testing of HLA-antigens by means of specific sera has been replaced by more precise molecular testing enabling the precise identification of HLA allelic determinants.

The question of accepting a mismatched donor for a patients, who didn't find a fully match‐ ed donor has not been finally solved. With the increasing number of observations there are recommendations concerning optimal matching not only in HLA-A, B and DR, but also in C, DQ and even DP. Many centers aim to transplant patients only from donors fully matched in 10/10 alleles of HLA-A, B, C, DR and DQ. The improved methods of typing enabling more precise molecular donor matching has improved the results of allo-HSCT from unre‐ lated donors, which are now similar to those of allo-HSCT from siblings [24]. As the allo-HSCT from an unrelated donor has to be preceded by often time-consuming search for a donor, it is important to plan the transplantation carefully in advance.

## **5. The sources of hematopoietic stem cells**

changes are lacking, the allo-HSCT is recommended in the first remission of the disease. The transplantation in more advanced stages of the disease leads to the higher relapse rate, as

Despite the introduction of tyrosine kinase inhibitors (TKIs) into the treatment of chronic myeloid leukemia (CML) over ten years ago, allo-HSCT still remains the only way of treat‐ ment capable to provide the complete recovery. The standard indication for allo-HSCT is re‐ sistance to TKIs treatment, especially in young patients. Other indications for allo-HSCT are myelodysplastic syndrome, high-risk chronic lymphocytic leukemia, selected patients with high-risk lymphoma, patients with myelofibrosis and other myeloproliferative neoplasms of unfavorable prognosis. The results of multiple myeloma treatment with the use alloHSCT are encouraging. AlloHSCT with the reduced conditioning regimen after previous auto-HSCT constitutes an interesting alternative in patients with multiple myeloma patients, who

Allo-HSCT is also the standard treatment in nonmalignant diseases of hematopoietic system such as severe aplastic anemia (SAA), paroxysmal nocturnal hemoglobinuria (PNH) and he‐ moglobinopathies. In some cases of inborn metabolic defects, allogeneic transplantation of donor's cells can restore the production of the deficient or lacking enzyme and eliminate the

The optimal donors are siblings possessing both haplotypes identical with the recipient. The syngeneic transplantation, i.e. from monozygotic twins, is the safest from the immunological point of view, however it is connected with the increased risk of the relapse of the disease resulting from the lack of immunological interaction between the donor cells and the recipi‐

Probability of possessing matched sibling donor is defined by the formula: 1–(0.75)*<sup>n</sup>*, were *n* indicates the number of siblings. The observed decrease in the number of newly born chil‐ dren causes problems in finding matched family donors for many patients. In rare cases with no matched sibling donor, matching donor could be found among other members of the family. In the vast majority of patients without matched sibling donor, transplantation from unrelated donor is the most frequently chosen option. The number of such transplanta‐ tions has increased considerably in the last 20 years [23]. It has been made possible thanks to dynamic development of bone marrow donors' registries, whose number of potential do‐ nors exceeded 20 million in the current year 2012. Alternatively, for those patients who are

The most desirable model of the donors' registry organization is the development of nation‐ al ones which, for many reasons (safety of donors, clearness of procedures and financial rea‐ sons), according to WMDA's (World Marrow Donors Association) recommendations should control and supervise the recruitment of the donors within the country. The chance to find

unlikely to find a matched donor, partial incompatibility could be accepted.

well as to the increased incidence of transplantation complications.

undergo single or tandem autologous transplantation [20].

**4. Hematopoietic stem cell donors**

disease [21].

142 Innovations in Stem Cell Transplantation

ent cells [22].

The choice of the cells source depends of diagnosis and the type of conditioning treatment applied. Collection of bone marrow is preferred in nonmalignant diseases in order to avoid chronic GvHD. Transplantation of hematopoietic cells from peripheral blood is preferred when reduced intensity conditioning regimen is used, with regard to the fact that transplan‐ tation of larger number of hematopoietic cells is able to break the resistance of the recipient and to result in the engraftment.

The bone marrow aspirated in general anesthesia from iliac spine was for many years the main source of cells for transplantation. Except of hematopoietic cells, the bone marrow also consists of multipotential mesenchymal cells which, although are not hematopoietic cells, have a potential to differentiate in vitro and in vivo into various mesenchymal tissues, such as bone, cartilage, fat tissue, tendons and bone matrix. Mesenchymal stem cells can reduce the alloreactivity, they inhibit lymphocytes T proliferation and act immunosuppressively, what has been implemented in the form of unrelated or haploidentical mesenchymal cells infusion into the treatment of acute GvHD.

During the 1990's the cells collected in apheresis from peripheral blood after their previous stimulation with granulocytic stimulating factor (G-CSF) completely superseded the bone marrow aspiration in autologous transplantations. In the beginning of 21st century, the simi‐ lar trend occurred also in allo-HSCT. The apheresis of cells from peripheral blood usually results in collection of higher number of nucleated cells, CD34+ cells, lymphocytes CD3+ and NK cells when compared with cells aspiration from the bone marrow; it enables faster regeneration of granulocytes and platelets. It translates into the smaller risk of infections and smaller demand for transfusions of blood derivatives.

In the beginning allo-HSCT in the form of PBSCT (Peripheral Blood Stem Cells Transplanta‐ tion) was applied only in sibling transplantations due to the anxiety of acute GvHD occur‐ rence, however, the frequency of GvHD is similar to the one after bone marrow transplantation, despite greater number of T-lymphocytes in the transplantation material collected from peripheral blood, as it was shown in the number of studies. Thus allo-PBSCT has been successfully applied also in allo-HSCT from unrelated donors. However, the fre‐ quency of chronic GvHD is higher, and thus allo-PBSCT is applied seldom in patients with nonmalignant disease, who do not benefit from Graft versus Leukemia (GvL) effect, which is usually connected with chronic GvHD [25].

recommended as a standard in ALL. In order to avoid potential TBI consequences, such as bronchiolitis obliterans, cataract, secondary malignancy, endocrinological disorders, inhibi‐ tion of the growing process in children, TBI in AML and MDS has been replaced by busul‐ fan given at 16 mg/kg dose within 4 consecutive days before Cy [29]. The BuCy treatment has higher risk of SOS (sinusoidal obstruction syndrome), hemorrhagic cystitis and chronic GvHD. The high serum concentration of Bu (Busulfan) occurring during its oral treatment has influenced considerably its toxic complications. It is difficult to avoid it because of vari‐ ous degree of absorption from digestive tract. Thus the intravenous use of busulfan is more favorable. The reduction of SOS incidence and decrease of transplant related mortality (TRM) after intravenous use of Bu has been reported [30]. In order to further limit the toxici‐ ty, treosulfan is used instead of Bu in modern treatment programs nowadays, and addition‐ al immunosuppressive effect is obtained by parallel application of purine analoque, e.g.

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The standard preparative treatment applied in SAA comprises of Cy 200 mg/kg and antithy‐

Although the intensive conditioning treatment decreases the risk of relapse after transplan‐ tation, it does not prolong the overall survival because greater toxicity leads to increased

The concept of so-called RIC (reduced intensity conditioning) incorporates the advantage of anti-leukemic effect of donor T-lymphocytes while cytotoxic effect of conditioning regimen is decreased. The main result of RIC treatment is immunosuppressive therapy aiming to en‐ able the acceptance of the transplant by braking the immunological defence of the recipient. The anti-leukemic effect can be escalated after transplantation by means of DLI (Donor Lym‐ phocyte Infusion), whenever it is required. DLI was first used with success in CML patients, in whom the disease relapsed after conventional allo-HSCT [32]. Since then it has been used in many other diseases, including many clonal diseases of hematopoietic system, most often lymphomas and chronic lymphocytic leukemia (CLL). RIC treatment has lower toxicity when compared to conventional conditioning treatment, thus it is suitable for transplanta‐ tion in older patients and in patients with coexisting diseases in whom the application of myeloablative treatment is contraindicated. RIC treatment consists most often of purine ana‐ loque. The example of RIC treatment reduced to the minimum, after which graft occurs, is the combination of TBI dose 2Gy with fludarabine. Other exemplary RIC protocols are the combination of fludarabine with Bu at dose 8 mg/kg and ATG with Cy or with melphalan. The important element of RIC treatment is the use of immunosuppressive therapy after transplantation e.g. cyclosporine A and mycophenolate mofetil. The reduced intensity of conditioning enables the immunocompetent recipient cells to survive until the moment of transplantation, what leads to the higher risk of graft failure or incomplete graft. In some centers transplantation with RIC are performed in ambulatory, however patients often re‐

Allo-HSCT with use of RIC can be applied when autologous transplantation is ineffective. Other possibility is to apply the tandem transplantation: at first autologous one and then the allogeneic one, with use of RIC in order to reduce the TRM by separation of high-dosed cy‐

fludarabine.

mocyte globulin (ATG).

transplant related mortality [31].

quire further hospitalization due to infections or GvHD [33].

The important source of hematopoietic stem cells for allo-HSCT is a cord blood (CB), usually intended to be discarded. In many countries there are banks of frozen CB units where there are over 0,5 million units ready to be transplanted. The advantage of applying CB cells is their immediate availability and a reduced risk of GvHD, related to a relative shortage of mature T-lymphocytes in the CB. Therefore the higher level of HLA-mismatching between the donor and the the recipient is more acceptable in CB transplantation than in traditional transplantations. The unfavorable factors are a more frequent occurrence of graft failure and a slower regeneration responsible for higher risk of infections. The number of necessary nucleated cells and CD34+ cells calculated per kilogram of the recipient's body mass is lower by about one logarithm when compared to the bone marrow. A number of studies showed the importance of sufficient number of cord blood nucleated cells, for this reason it is recom‐ mended to transplant more than 2x10^7 nucleated cells per kilogram of recipient's body mass. It constitutes limitation in CB application in adults due to the small volume of cord blood and small total number of cells. Simultaneous transplantation of two CB units is suc‐ cessfully applied to solve this problem [26,27]. In vitro cells expansion to increase the num‐ ber of CB cells has not been widely used. Because of the limited, usually small number of cord blood cells, it is most often applied as the source of cells for transplantation in children.

## **6. Preparative treatment before transplantation**

The preparative treatment before transplantation (or conditioning regimen) aims to eradi‐ cate the remains of the disease and to make immunological system of recipient weaker in order to enable the acceptance of the graft by the recipient. The preparative treatment is con‐ nected with toxicities which turned out to be impossible to eliminate so far.

The choice of conditioning treatment depends of the patient's age, the main disease and co‐ existing diseases. Myeloablative conditioning regimens are characterized by strong cytotox‐ icity as well as strong immunosuppressive potential, while reduced intensity conditioning regimens differ in cytotoxic activity and immunosuppressive potential. They are chosen de‐ pending on the main disease and evaluation of the risk of graft failure.

The combination of radiotherapy (TBI- total body irradiation- at total dose of 12 Gy, deliv‐ ered in fractions) and cyclophosphamide (Cy, at total dose of 120 mg/kg administered with‐ in 2 days) has been used for over 40 years for conditioning [28]. TBI treatment is recommended as a standard in ALL. In order to avoid potential TBI consequences, such as bronchiolitis obliterans, cataract, secondary malignancy, endocrinological disorders, inhibi‐ tion of the growing process in children, TBI in AML and MDS has been replaced by busul‐ fan given at 16 mg/kg dose within 4 consecutive days before Cy [29]. The BuCy treatment has higher risk of SOS (sinusoidal obstruction syndrome), hemorrhagic cystitis and chronic GvHD. The high serum concentration of Bu (Busulfan) occurring during its oral treatment has influenced considerably its toxic complications. It is difficult to avoid it because of vari‐ ous degree of absorption from digestive tract. Thus the intravenous use of busulfan is more favorable. The reduction of SOS incidence and decrease of transplant related mortality (TRM) after intravenous use of Bu has been reported [30]. In order to further limit the toxici‐ ty, treosulfan is used instead of Bu in modern treatment programs nowadays, and addition‐ al immunosuppressive effect is obtained by parallel application of purine analoque, e.g. fludarabine.

In the beginning allo-HSCT in the form of PBSCT (Peripheral Blood Stem Cells Transplanta‐ tion) was applied only in sibling transplantations due to the anxiety of acute GvHD occur‐ rence, however, the frequency of GvHD is similar to the one after bone marrow transplantation, despite greater number of T-lymphocytes in the transplantation material collected from peripheral blood, as it was shown in the number of studies. Thus allo-PBSCT has been successfully applied also in allo-HSCT from unrelated donors. However, the fre‐ quency of chronic GvHD is higher, and thus allo-PBSCT is applied seldom in patients with nonmalignant disease, who do not benefit from Graft versus Leukemia (GvL) effect, which

The important source of hematopoietic stem cells for allo-HSCT is a cord blood (CB), usually intended to be discarded. In many countries there are banks of frozen CB units where there are over 0,5 million units ready to be transplanted. The advantage of applying CB cells is their immediate availability and a reduced risk of GvHD, related to a relative shortage of mature T-lymphocytes in the CB. Therefore the higher level of HLA-mismatching between the donor and the the recipient is more acceptable in CB transplantation than in traditional transplantations. The unfavorable factors are a more frequent occurrence of graft failure and a slower regeneration responsible for higher risk of infections. The number of necessary nucleated cells and CD34+ cells calculated per kilogram of the recipient's body mass is lower by about one logarithm when compared to the bone marrow. A number of studies showed the importance of sufficient number of cord blood nucleated cells, for this reason it is recom‐ mended to transplant more than 2x10^7 nucleated cells per kilogram of recipient's body mass. It constitutes limitation in CB application in adults due to the small volume of cord blood and small total number of cells. Simultaneous transplantation of two CB units is suc‐ cessfully applied to solve this problem [26,27]. In vitro cells expansion to increase the num‐ ber of CB cells has not been widely used. Because of the limited, usually small number of cord blood cells, it is most often applied as the source of cells for transplantation in children.

The preparative treatment before transplantation (or conditioning regimen) aims to eradi‐ cate the remains of the disease and to make immunological system of recipient weaker in order to enable the acceptance of the graft by the recipient. The preparative treatment is con‐

The choice of conditioning treatment depends of the patient's age, the main disease and co‐ existing diseases. Myeloablative conditioning regimens are characterized by strong cytotox‐ icity as well as strong immunosuppressive potential, while reduced intensity conditioning regimens differ in cytotoxic activity and immunosuppressive potential. They are chosen de‐

The combination of radiotherapy (TBI- total body irradiation- at total dose of 12 Gy, deliv‐ ered in fractions) and cyclophosphamide (Cy, at total dose of 120 mg/kg administered with‐ in 2 days) has been used for over 40 years for conditioning [28]. TBI treatment is

nected with toxicities which turned out to be impossible to eliminate so far.

pending on the main disease and evaluation of the risk of graft failure.

is usually connected with chronic GvHD [25].

144 Innovations in Stem Cell Transplantation

**6. Preparative treatment before transplantation**

The standard preparative treatment applied in SAA comprises of Cy 200 mg/kg and antithy‐ mocyte globulin (ATG).

Although the intensive conditioning treatment decreases the risk of relapse after transplan‐ tation, it does not prolong the overall survival because greater toxicity leads to increased transplant related mortality [31].

The concept of so-called RIC (reduced intensity conditioning) incorporates the advantage of anti-leukemic effect of donor T-lymphocytes while cytotoxic effect of conditioning regimen is decreased. The main result of RIC treatment is immunosuppressive therapy aiming to en‐ able the acceptance of the transplant by braking the immunological defence of the recipient. The anti-leukemic effect can be escalated after transplantation by means of DLI (Donor Lym‐ phocyte Infusion), whenever it is required. DLI was first used with success in CML patients, in whom the disease relapsed after conventional allo-HSCT [32]. Since then it has been used in many other diseases, including many clonal diseases of hematopoietic system, most often lymphomas and chronic lymphocytic leukemia (CLL). RIC treatment has lower toxicity when compared to conventional conditioning treatment, thus it is suitable for transplanta‐ tion in older patients and in patients with coexisting diseases in whom the application of myeloablative treatment is contraindicated. RIC treatment consists most often of purine ana‐ loque. The example of RIC treatment reduced to the minimum, after which graft occurs, is the combination of TBI dose 2Gy with fludarabine. Other exemplary RIC protocols are the combination of fludarabine with Bu at dose 8 mg/kg and ATG with Cy or with melphalan. The important element of RIC treatment is the use of immunosuppressive therapy after transplantation e.g. cyclosporine A and mycophenolate mofetil. The reduced intensity of conditioning enables the immunocompetent recipient cells to survive until the moment of transplantation, what leads to the higher risk of graft failure or incomplete graft. In some centers transplantation with RIC are performed in ambulatory, however patients often re‐ quire further hospitalization due to infections or GvHD [33].

Allo-HSCT with use of RIC can be applied when autologous transplantation is ineffective. Other possibility is to apply the tandem transplantation: at first autologous one and then the allogeneic one, with use of RIC in order to reduce the TRM by separation of high-dosed cy‐ totoxic treatment from immunotherapy related to allogeneic HSCT, which has been applied for the first time in patients with multiple myeloma (MM).

ever, it is connected with the higher risk of the graft failure and relapse of the disease. In cord blood transplantations, instead of methotrexate which prolonges the regeneration peri‐ od, prednisolon is used. New immunosuppressive protocols include calcineurin-inhibitors other than cyclosporine A – tacrolimus, macrolid immunosuppressant– syrolimus and my‐ cophenolan mofetil. The administration of ATG before transplantation is an important im‐ munosuppressive element used in allo-HSCT from unrelated donors. As the effective serum concentration of ATG is maintained for many weeks after infusion, it effects not only T-lym‐ phocytes of the recipient but also those of the donor [35]. The increased risk of infections is

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147

The type of GvHD prevention depends of the diagnosis, the type of conditioning treatment and the applied cell source. The GvHD prevention should be more effective in nonmalig‐

When symptoms of acute GvHD develop despite its prophylaxis, methylprednisolone at the dose of 2-5 mg per kilogram of body weight per day is used on the standard basis, usually effectively. In case of steroid resistance the risk of failure is high. The second line treatment consists of ATG, anti-IL-2 antibodies, anti-IL-2 receptor antibodies and antibodies against TNF-alpha. Photosensitizing psolarens and ultraviolet radiation in a form of extracorporal photopheresis and transplantation of mesenchymal stem cells can be also applied, but are not everywhere available. Mesenchymal stem cells have strong immunosuppressive effect, they can be obtained from the primitive connective tissue of the umbilical cord, called the Wharton's jelly, and they do not require any matching due to low levels of HLA-ABC and

The treatment of chronic GvHD consists of CsA and steroids. In patients not responding to the treatment tacrolimus, thalidomide, mycophenolan mofetil, sirolimus and irradiation of

Immunological reconstitution is of a primary importance to avoid infections after allo-HSCT. The highest risk of the infection occurs in patients with GvHD, but also in the re‐ maining patients with no GvHD it is 20 times higher than in the whole population. From 20% up to 50% of patients still require immunosuppressive treatment after 3 years from al‐ lo-HSCT, what considerably increases the risk of infectious complications in this group of

Normal endogenous Gram-negative flora from the gastrointestinal tract and exogenous catheter-related Gram-positive bacteria constitute the most frequent cause of infections in the early stage after allo-HSCT. In this stage fungal infections are also the problem, especial‐ ly other than Candida albicans, which are usually recognized with the delay. Although my‐ cological diagnosis based on PCR method is available, it has not been introduced into practice yet. Galactomannan testing and detection of specific fungal antigens in the blood are sometimes helpful. In the treatment we already administer not only conventional am‐ photericine B with considerable side effects, but also its lipid-based preparations (Abelcet,

nant disease and less intensive when lower number of cells have been transplanted.

an undesirable side effect of ATG.

lack of HLA-DR antigens.

**8.2. Infections**

patients [36].

lymphatic system with dose of 1 Gy can be applied.

## **7. Adjunctive treatment**

During the phase of pancytopenia after myeloablative conditioning patients are usually sus‐ ceptible to infections and thus they have to stay in a sterile environment, e.g. in HEPA-fil‐ tered rooms with reversed isolation. They routinely receive preventive treatment against bacteria, viruses, fungi. Moreover, the substitution treatment is applied with the use of irra‐ diated, CMV-negative red blood cells and single donor platelets concentrates. Analgetic drugs and parenteral nutrition are applied when needed. Ursodeoxycholic acid is used in order to avoid hepatic complications. G-CSF is applied to accelerate the regeneration of granulocytes, however it can delay the recovery of platelets and can increase the risk of GvHD. Erythropoietin accelerates the recovery of red blood cells system and thus it reduces the need for transfusions, but it increases the cost of the transplant procedure and it is not used on a regular basis.

## **8. Post-transplant complications**

#### **8.1. Graft versus host disease**

Acute and chronic graft versus host disease are the main complications of allo-HSCT. In pathophysiology of acute and chronic GvHD, T-lymphocytes of the donor recognize HLAmolecules of the recipient presented by the antigen presenting cells. It results in the release of interleukin-2 and activation of cytotoxic T-lymphocytes, NK-cells and macrophages. The main targets of the attack are skin, gut and liver. The most important risk factor is the HLAincompatibility between the donor and the recipient, but also minor histocompatibility anti‐ gens are responsible for the risk of GvHD, especially HY mismatch in case when the donor is female and the recipient is male [34]. Chronic GvHD occurs most often from 100 days to one year after allo-HSCT. It resembles autoimmunological diseases, e.g. systemic scleroder‐ mia and Sjoegren syndrome. Symptoms such as lichen and sclerodermic skin changes, mu‐ cositis, kserostomia, keratoconjunctivitis sicca, stricture of esophagus and vagina, cholestatic liver failure, bronchiolitis obliterans and musculitis also occur. Cachexia, immunological de‐ ficiency, additionally increasing the risk of infections especially caused by gram-plus bacte‐ ria can be also observed. The initial stage of chronic GvHD is usually more progressive when it is preceded by the acute form of the disease. It can also occurr after nonsymptomatic (quiescent) period or de-novo, without any preceding symptoms of acute GvHD. The chron‐ ic progressive GvHD has the worst prognosis.

In order to decrease the risk of GvHD a preventive immunosuppression, usually with the use of cyclosporine A (CsA) and methotrexate is applied. The removal of T-lymphocytes from the transplanted cells (T-depletion) constitutes the effective form of prevention, how‐ ever, it is connected with the higher risk of the graft failure and relapse of the disease. In cord blood transplantations, instead of methotrexate which prolonges the regeneration peri‐ od, prednisolon is used. New immunosuppressive protocols include calcineurin-inhibitors other than cyclosporine A – tacrolimus, macrolid immunosuppressant– syrolimus and my‐ cophenolan mofetil. The administration of ATG before transplantation is an important im‐ munosuppressive element used in allo-HSCT from unrelated donors. As the effective serum concentration of ATG is maintained for many weeks after infusion, it effects not only T-lym‐ phocytes of the recipient but also those of the donor [35]. The increased risk of infections is an undesirable side effect of ATG.

The type of GvHD prevention depends of the diagnosis, the type of conditioning treatment and the applied cell source. The GvHD prevention should be more effective in nonmalig‐ nant disease and less intensive when lower number of cells have been transplanted.

When symptoms of acute GvHD develop despite its prophylaxis, methylprednisolone at the dose of 2-5 mg per kilogram of body weight per day is used on the standard basis, usually effectively. In case of steroid resistance the risk of failure is high. The second line treatment consists of ATG, anti-IL-2 antibodies, anti-IL-2 receptor antibodies and antibodies against TNF-alpha. Photosensitizing psolarens and ultraviolet radiation in a form of extracorporal photopheresis and transplantation of mesenchymal stem cells can be also applied, but are not everywhere available. Mesenchymal stem cells have strong immunosuppressive effect, they can be obtained from the primitive connective tissue of the umbilical cord, called the Wharton's jelly, and they do not require any matching due to low levels of HLA-ABC and lack of HLA-DR antigens.

The treatment of chronic GvHD consists of CsA and steroids. In patients not responding to the treatment tacrolimus, thalidomide, mycophenolan mofetil, sirolimus and irradiation of lymphatic system with dose of 1 Gy can be applied.

#### **8.2. Infections**

totoxic treatment from immunotherapy related to allogeneic HSCT, which has been applied

During the phase of pancytopenia after myeloablative conditioning patients are usually sus‐ ceptible to infections and thus they have to stay in a sterile environment, e.g. in HEPA-fil‐ tered rooms with reversed isolation. They routinely receive preventive treatment against bacteria, viruses, fungi. Moreover, the substitution treatment is applied with the use of irra‐ diated, CMV-negative red blood cells and single donor platelets concentrates. Analgetic drugs and parenteral nutrition are applied when needed. Ursodeoxycholic acid is used in order to avoid hepatic complications. G-CSF is applied to accelerate the regeneration of granulocytes, however it can delay the recovery of platelets and can increase the risk of GvHD. Erythropoietin accelerates the recovery of red blood cells system and thus it reduces the need for transfusions, but it increases the cost of the transplant procedure and it is not

Acute and chronic graft versus host disease are the main complications of allo-HSCT. In pathophysiology of acute and chronic GvHD, T-lymphocytes of the donor recognize HLAmolecules of the recipient presented by the antigen presenting cells. It results in the release of interleukin-2 and activation of cytotoxic T-lymphocytes, NK-cells and macrophages. The main targets of the attack are skin, gut and liver. The most important risk factor is the HLAincompatibility between the donor and the recipient, but also minor histocompatibility anti‐ gens are responsible for the risk of GvHD, especially HY mismatch in case when the donor is female and the recipient is male [34]. Chronic GvHD occurs most often from 100 days to one year after allo-HSCT. It resembles autoimmunological diseases, e.g. systemic scleroder‐ mia and Sjoegren syndrome. Symptoms such as lichen and sclerodermic skin changes, mu‐ cositis, kserostomia, keratoconjunctivitis sicca, stricture of esophagus and vagina, cholestatic liver failure, bronchiolitis obliterans and musculitis also occur. Cachexia, immunological de‐ ficiency, additionally increasing the risk of infections especially caused by gram-plus bacte‐ ria can be also observed. The initial stage of chronic GvHD is usually more progressive when it is preceded by the acute form of the disease. It can also occurr after nonsymptomatic (quiescent) period or de-novo, without any preceding symptoms of acute GvHD. The chron‐

In order to decrease the risk of GvHD a preventive immunosuppression, usually with the use of cyclosporine A (CsA) and methotrexate is applied. The removal of T-lymphocytes from the transplanted cells (T-depletion) constitutes the effective form of prevention, how‐

for the first time in patients with multiple myeloma (MM).

**7. Adjunctive treatment**

146 Innovations in Stem Cell Transplantation

used on a regular basis.

**8.1. Graft versus host disease**

**8. Post-transplant complications**

ic progressive GvHD has the worst prognosis.

Immunological reconstitution is of a primary importance to avoid infections after allo-HSCT. The highest risk of the infection occurs in patients with GvHD, but also in the re‐ maining patients with no GvHD it is 20 times higher than in the whole population. From 20% up to 50% of patients still require immunosuppressive treatment after 3 years from al‐ lo-HSCT, what considerably increases the risk of infectious complications in this group of patients [36].

Normal endogenous Gram-negative flora from the gastrointestinal tract and exogenous catheter-related Gram-positive bacteria constitute the most frequent cause of infections in the early stage after allo-HSCT. In this stage fungal infections are also the problem, especial‐ ly other than Candida albicans, which are usually recognized with the delay. Although my‐ cological diagnosis based on PCR method is available, it has not been introduced into practice yet. Galactomannan testing and detection of specific fungal antigens in the blood are sometimes helpful. In the treatment we already administer not only conventional am‐ photericine B with considerable side effects, but also its lipid-based preparations (Abelcet, AmBisome, Amphocil) being better tolerated, but unfortunately expensive. New antifungal drugs such as echinocandins (caspofungin, anidulafungin, micafungin) and newer azole drugs (voriconazole, posaconazole) are also currently available.

prediction and analysis of the graft failure and GvHD risks. GvHD and pancytopenia can develop as the side effects of DLI. The use of T-lymphocytes in escalating doses is equally

Progress in Hematopoietic Stem Cell Transplantation

http://dx.doi.org/10.5772/53086

149

Allo-HSCT with subsequent immunotherapy can be applied in patients with metastatic sol‐ id tumors. The presence of graft versus tumor effect has been shown in kidney cancer, colon adenoma, metastatic breast, ovarian, prostate and pancreas cancers. RIC treatment has been used in these conditions in order to reduce TRM while enabling to achieve the response, which was complete in some cases [39]. The presence of fewer than 3 metastases and Kar‐ nofsky scale ≥70 constitutes beneficial prognostic factors [40]. The survival is longer in pa‐

Allo-HSCT is currently tested in animal models and in experimental clinical applications. Hematopoietic stem cells are characterized by plasticity, which means that they can form not only blood cells. Hematopoietic stem cells can be forced to transform into the cells of various tissues such as heart muscle, bone or blood vessels in suitable conditions [41]. The science dealing with plasticity of stem cells is just developing, but it arises hope for revolu‐

Allo-HSCT procedure has transformed from the experimental method of treatment of leuke‐ mia in its final stage into routine procedure applied in patients with various hematological diseases. The ability to collect and to transplant hematopoietic cells makes it possible to cure many patients with malignant and nonmalignant diseases incurable with other methods. Thanks to development of unrelated donor registries the treatment with allo-HSCT can be currently offered not only to the patients having HLA-matched sibling donor but to almost every patient in need. The observed increase of transplantations of peripheral hematopoietic stem cells results from observed faster regeneration of hematopoietic system than after bone marrow transplantation and from beneficial GvL effect in clonal diseases, although it coin‐

The patients in the older age group and those with comorbidities can be treated with allo-HSCT after preparation with RIC. Still, the main problem is the relapse of the disease, how‐ ever when it is detected early basing on chimerism analysis and minimal residual disease evaluation, it can be successfully treated with immunological intervention with DLI. Recent and current studies indicate that hematopoietic stem cells will be used for new clinical appli‐

tion in the way of thinking about transplantation and organ regeneration.

**9. New indications for transplantation of hematopoietic stem cells**

effective as high DLI dose, but it decreases the risk of GvHD [38].

tients who develop chronic GvHD after DLI.

cides with more frequent occurrence of GvHD.

cations in the near future.

**10. Conclusion**

After resolution of pancytopenia cytomegalovirus infection is a most frequent problem. Thanks to a modern diagnostic approach based on early CMV antigen detection by means of PCR methods, CMV reactivation can be detected and cured before CMV dis‐ ease is developed. The most common cause of CMV infection is latent virus reactivation in CMV-seropositive patient or CMV-transmission from a seropositive donor to a sero‐ negative recipient. Therefore the optimal situation is when the serological status of the donor and the recipient is identical. The antiviral prevention includes the substitution of blood products from CMV-seronegative donors, transfusion of immunoglobulines and administration of antiviral drugs such as gancyclovir, foscavir, cydofovir and oral val‐ gancyclovir. Polyoma- BK virus and adenovirus are common causes of dysuria, urinary tract infections and haemorrhagic cystitis in immunocompromised patients. Epstein-Barr virus can cause post-transplant lymphoproliferative disease (PTLD). The risk factors are the use of anti-thymocyte globulin and transplantation from unrelated donors. Monitor‐ ing of EBV-viremia by means of PCR methods enables to start the treatment early – to reduce the immunosuppression and to use rituximab (anti-CD20 antibody) and donor lymphocyte infusion (DLI).

#### **8.3. Relapse of the disease**

Having better adjunctive treatment and more effective GvHD prevention, the relapse of ba‐ sic disease constitutes the main cause of allo-HSCT failure. The risk of the relapse depends on the type of the disease, its stage at the moment of transplantation and the GvHD preven‐ tion applied (the more effective immunosuppression, the higher risk of the relapse). The lon‐ gest survival time is observed in patients with moderate acute or limited chronic GvHD, because of the lowest risk of the relapse.

Although the relapse after allo-HSCT can be treated by means by DLI, good prognosis refers usually to the patients with CML. In acute leukemia relapsing after allo-HSCT the tempora‐ ry response can be also achieved, but it is usually not stable.

Patients with molecular CML relapse, i.e. with reappearance of bcr/abl transcript in PCR tests, have better prognosis than those with hematological relapse. The prognosis in patients with more advanced stages of CML- relapse, acceleration phase or blastic transformation- is much worse. The relapse should by detected as soon as possible, when there is still a chance for effective immunotherapy after allo-HSCT.

The alternative to specific disease markers determination is post-transplant chimerism test‐ ing. The PCR short tandem repeats (STR) method is used. The goal of allo-HSCT is to obtain the full donor's chimerism. Detection of returning or increasing recipient's chimerism can be a sign of the relapse of the disease, similarly to the re-occurrence of minimal residual disease [37]. In such case it is recommended to use adoptive immunotherapy by reduction of immu‐ nosuppressive treatment and DLI application. The chimerism testing is important also for prediction and analysis of the graft failure and GvHD risks. GvHD and pancytopenia can develop as the side effects of DLI. The use of T-lymphocytes in escalating doses is equally effective as high DLI dose, but it decreases the risk of GvHD [38].

## **9. New indications for transplantation of hematopoietic stem cells**

Allo-HSCT with subsequent immunotherapy can be applied in patients with metastatic sol‐ id tumors. The presence of graft versus tumor effect has been shown in kidney cancer, colon adenoma, metastatic breast, ovarian, prostate and pancreas cancers. RIC treatment has been used in these conditions in order to reduce TRM while enabling to achieve the response, which was complete in some cases [39]. The presence of fewer than 3 metastases and Kar‐ nofsky scale ≥70 constitutes beneficial prognostic factors [40]. The survival is longer in pa‐ tients who develop chronic GvHD after DLI.

Allo-HSCT is currently tested in animal models and in experimental clinical applications. Hematopoietic stem cells are characterized by plasticity, which means that they can form not only blood cells. Hematopoietic stem cells can be forced to transform into the cells of various tissues such as heart muscle, bone or blood vessels in suitable conditions [41]. The science dealing with plasticity of stem cells is just developing, but it arises hope for revolu‐ tion in the way of thinking about transplantation and organ regeneration.

## **10. Conclusion**

AmBisome, Amphocil) being better tolerated, but unfortunately expensive. New antifungal drugs such as echinocandins (caspofungin, anidulafungin, micafungin) and newer azole

After resolution of pancytopenia cytomegalovirus infection is a most frequent problem. Thanks to a modern diagnostic approach based on early CMV antigen detection by means of PCR methods, CMV reactivation can be detected and cured before CMV dis‐ ease is developed. The most common cause of CMV infection is latent virus reactivation in CMV-seropositive patient or CMV-transmission from a seropositive donor to a sero‐ negative recipient. Therefore the optimal situation is when the serological status of the donor and the recipient is identical. The antiviral prevention includes the substitution of blood products from CMV-seronegative donors, transfusion of immunoglobulines and administration of antiviral drugs such as gancyclovir, foscavir, cydofovir and oral val‐ gancyclovir. Polyoma- BK virus and adenovirus are common causes of dysuria, urinary tract infections and haemorrhagic cystitis in immunocompromised patients. Epstein-Barr virus can cause post-transplant lymphoproliferative disease (PTLD). The risk factors are the use of anti-thymocyte globulin and transplantation from unrelated donors. Monitor‐ ing of EBV-viremia by means of PCR methods enables to start the treatment early – to reduce the immunosuppression and to use rituximab (anti-CD20 antibody) and donor

Having better adjunctive treatment and more effective GvHD prevention, the relapse of ba‐ sic disease constitutes the main cause of allo-HSCT failure. The risk of the relapse depends on the type of the disease, its stage at the moment of transplantation and the GvHD preven‐ tion applied (the more effective immunosuppression, the higher risk of the relapse). The lon‐ gest survival time is observed in patients with moderate acute or limited chronic GvHD,

Although the relapse after allo-HSCT can be treated by means by DLI, good prognosis refers usually to the patients with CML. In acute leukemia relapsing after allo-HSCT the tempora‐

Patients with molecular CML relapse, i.e. with reappearance of bcr/abl transcript in PCR tests, have better prognosis than those with hematological relapse. The prognosis in patients with more advanced stages of CML- relapse, acceleration phase or blastic transformation- is much worse. The relapse should by detected as soon as possible, when there is still a chance

The alternative to specific disease markers determination is post-transplant chimerism test‐ ing. The PCR short tandem repeats (STR) method is used. The goal of allo-HSCT is to obtain the full donor's chimerism. Detection of returning or increasing recipient's chimerism can be a sign of the relapse of the disease, similarly to the re-occurrence of minimal residual disease [37]. In such case it is recommended to use adoptive immunotherapy by reduction of immu‐ nosuppressive treatment and DLI application. The chimerism testing is important also for

drugs (voriconazole, posaconazole) are also currently available.

lymphocyte infusion (DLI).

148 Innovations in Stem Cell Transplantation

**8.3. Relapse of the disease**

because of the lowest risk of the relapse.

for effective immunotherapy after allo-HSCT.

ry response can be also achieved, but it is usually not stable.

Allo-HSCT procedure has transformed from the experimental method of treatment of leuke‐ mia in its final stage into routine procedure applied in patients with various hematological diseases. The ability to collect and to transplant hematopoietic cells makes it possible to cure many patients with malignant and nonmalignant diseases incurable with other methods. Thanks to development of unrelated donor registries the treatment with allo-HSCT can be currently offered not only to the patients having HLA-matched sibling donor but to almost every patient in need. The observed increase of transplantations of peripheral hematopoietic stem cells results from observed faster regeneration of hematopoietic system than after bone marrow transplantation and from beneficial GvL effect in clonal diseases, although it coin‐ cides with more frequent occurrence of GvHD.

The patients in the older age group and those with comorbidities can be treated with allo-HSCT after preparation with RIC. Still, the main problem is the relapse of the disease, how‐ ever when it is detected early basing on chimerism analysis and minimal residual disease evaluation, it can be successfully treated with immunological intervention with DLI. Recent and current studies indicate that hematopoietic stem cells will be used for new clinical appli‐ cations in the near future.

## **Author details**

Miroslaw Markiewicz\* , Malgorzata Sobczyk-Kruszelnicka, Monika Dzierzak Mietla, Anna Koclega, Patrycja Zielinska and Slawomira Kyrcz-Krzemien

\*Address all correspondence to: mir.markiewicz@wp.pl

Medical University of Silesia, Department of Hematology and Bone Marrow Transplanta‐ tion, Katowice, Poland

vanced Hodgkin's lymphoma responding to front-line therapy. J Clin Oncol. 2003;

Progress in Hematopoietic Stem Cell Transplantation

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**Author details**

Miroslaw Markiewicz\*

150 Innovations in Stem Cell Transplantation

tion, Katowice, Poland

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Medical University of Silesia, Department of Hematology and Bone Marrow Transplanta‐

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**Chapter 7**

**Current Approach to**

Hugo F. Fernandez and Lia Perez

tioning regimens used in allogeneic HCT.

**2. Indications for transplantation**

**2.1. Acute myeloid leukemia**

http://dx.doi.org/10.5772/53488

**1. Introduction**

**1.1. Aims of chapter**

Additional information is available at the end of the chapter

**Allogeneic Hematopoietic Stem Cell Transplantation**

In this Chapter we will discuss the indications for allogeneic hematopoietic stem cell trans‐ plantation (HCT). We will focus on the appropriate timing of this procedure for the different hematologic malignancies. We reviewed past approaches using myeloablative conditioning and present some of the newer reduced intensity therapies. Allogeneic transplantation is one of the first known uses of stem cells. Born from the need to rescue damaged bone marrow, it was first used in the setting of aplastic anemia and acute leukemia. Over the years, the technique has changed steadily and support for this procedure has improved immensely. Today this procedure is used to treat multiple malignant blood disorders, bone marrow failure syndromes, immune deficiency syndromes, and hemoglobinopathies. This chapter will focus on the malignant hematopathies. Another aspect of this Chapter will be to review the condi‐

Acute myeloid leukemia (AML) Is heterogeneous group of clonal disorders. The disease can present at all ages, but this disorder is most commonly seen in older patients, with a median age at presentation of 67 years. [1] AML can present in a de novo fashion or can progress from antecedent hematological disorders, including myelodysplasia and myeloproliferative neoplasms (secondary AML), or after prior exposure to chemotherapy and/or radiation

and reproduction in any medium, provided the original work is properly cited.

© 2013 Fernandez and Perez; licensee InTech. This is an open access article 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.

© 2013 The Author(s). Licensee InTech. 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,

