**1. Introduction**

146 Non-Viral Gene Therapy

You, Z., Wang, Y., Zhao, Z-G., He, Y., Xu, P., Zhao, Z., Cheng, Y., Cheng, W. & Zhang, H.

*Chinese Journal of Virology*, Vol.6, No.3, (1990), pp. 272-276, ISSN 1000-8721 Young, W., Merz, T., Ferguson-Smith, M. & Johnston, A. (1960). Chromosome Number of

Youssoufian, H. & Pyeritz, R. (2002). Mechanisms and Consequences of Somatic Mosaicism

Yu, J., Vodyanik, M., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J., Tian, S., Nie, J.,

Yu, X-J., Liang, M-F., Zhang, S-Y., Liu, Y., Li, J-D., Sun, Y-L., Zhang, L., Zhang, Q-F., Popov,

No.5858, (December 2007), pp. 1917-1920, ISSN 0036-8075

*Genetics,* Vol.52, No.7, (July 2007), pp. 607-617, ISSN 1434-5161

1999), pp. 8851-8856, ISSN 0022-538X

(2000), pp. 117-133, ISSN 0025-7931

2007), pp. 4101-4107, ISSN 0006-4971

1673, ISSN 0036-8075

1471-0056

Age. *Blood, Journal of the American Society of Hematology,* Vo.110, No.12, (December

(1990). Isolation and Identification of Two Strains of Orbivirus in Hainan Province.

the Chimpanzee, Pan troglodytes. *Science*, Vol.131, No.3414, (June 1960), pp. 1672-

in Humans. *Nature Reviews Genetics*, Vol.3, No.10, (October 2002), pp. 748-758, ISSN

Jonsdottir, G., Ruotti, V., Stewart, R., Slukvin, I. & Thomson, J. (2007). Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. *Science*, Vol.318,

V., Li, C., Qu, J., Li, Q., Zhang, Y-P., Hai, R., Wu, W., Wang, Q., Zhan, F-X., Wang, X-J., Kan, B., Wang, S-W., Wan, K-L., Jing, H-Q., Lu, J-X., Yin, W-W., Zhou, H., Guan, X-H., Liu, J-F., Bi, Z-Q., Liu, G-H., Ren, J., Wang, H., Zhao, Z., Song, J-D., He, J-R., Wan, T., Zhang, J-S., Fu, X-P., Sun, L-N., Dong, X-P., Feng, Z-J., Yang, W-Z., Hong, T., Zhang, Y., Walker, D., Wang, Y. & Li, D-X. (2011). Fever with Thrombocytopenia Associated with a Novel Bunyavirus in China. *The New England Journal of Medicine,* Vol.364, No.16, (April 2011), pp. 1523-1532, ISSN 0028-4793 Zhang, Z., Habara, Y., Nishiyama, A., Oyazato, Y., Yagi, M., Takeshima, Y. & Matsuo, M.

(2007). Identification of Seven Novel Cryptic Exons Embedded in the Dystrophin Gene and Characterization of 14 Cryptic Dystrophin Exons. *Journal of Human* 

Krauss, S. & Webster, R. (1999). Genetic Reassortment of Avian, Swine, and Human Influenza A Viruses in American Pigs. *Journal of Virology*, Vol.73, No.10, (October

(1997). Wiskott-Aldrich Syndrome/X-linked Thrombocytopenia: WASP Gene Mutations, Protein Expression, and Phenotype. *Blood, Journal of the American Society* 

Zhao, T., Zhang, Z., Rong, Z. & Xu, Y. (2011). Immunogenicity of Induced Pluripotent Stem Cells. *Nature*, Vol.474, No.7350, (May 2011), pp. 212-215, ISSN 0028-0836 Zhou, N., Senne, D., Landgraf, J., Swenson, S., Erickson, G., Rossow, K., Liu, L., Yoon, K.,

Zhu, Q., Watanabe, C., Liu, T., Hollenbaugh, D., Blaese, R., Kanner, S., Aruffo, A., Ochs, H.

*of Hematology*, Vol.90, No.7, (October 1997), pp. 2680-2689, ISSN 0006-4971 Zielenski, J. (2000). Genotype and Phenotype in Cystic Fibrosis. *Respiration*, Vol.67, No.2, The lysosomal storage disorders (LSDs) are a group of almost 50 genetic diseases, characterized by mutations and loss of activity of lysosomal enzymes or, less frequently, non-lysosomal proteins that are involved in protein maturation or lysosomal biogenesis (Meikle et al, 2004). Most LSDs have an autosomal recessive inheritance, with some exceptions as Hunter syndrome (X-linked recessive), Danon disease (X-linked dominant) and Fabry disease (X-linked with a high proportion of heterozygous affected females).

Storage of distinct undegraded or partially degraded material, usually the substrate of the defective enzyme, occurs in the lysosome. The substrate type is used to group the LSDs into general categories (table 1), including mucopolysaccharidoses (characterized by the storage of mucopolysaccharides, also called glycosaminoglycans), lipidoses (storage of lipids), glycogenoses (storage of glycogen) and oligosaccharidoses (storage of small sugar chains). Despite this categorization, many clinical similarities are observed between groups as well as within each group. Generally these diseases are multisystemic, and clinical features of many LSDs include organomegaly, central nervous system dysfunction and coarse hair and faces. Most LSDs are characterized by their progressive course with high morbidity and increased mortality, although there are significant variations between different diseases, and even among patients with the same disease (Walkley 2009). Lysosomal enzymes are ubiquitously distributed, but substrate storage is usually restricted to cells, tissues and organs with higher substrate turnover.

Recently, it has been suggested that the primary gene defect and substrate storage are triggers of a complex cascade of events that lead to many of the disease manifestations (Bellettato & Scarpa, 2010). In this context, secondary substrate storage, perturbations of Calcium homeostasis and lipid trafficking would contribute to disease pathogenesis. Other manifestations, related to the lysosome's role in vescicle trafficking, including antigen presentation, innate immunity, and signal transduction would cause inflammatory and auto-immune disturbances observed in the LSD (Parkinson-Lawrence et al., 2010). In addition, general mechanisms such as unfolded protein response, reticulum stress, oxidative stress and autophagy blockade would also play a role in the pathogenesis (Vitner et al., 2010).

The incidence and prevalence of these diseases varies from different countries and regions. For example, the overall incidence of GM1 Gangliosidosis is considered to be 1:100,000-

Non Viral Gene Transfer Approaches for Lysosomal Storage Disorders 149

sulfatase None

sulphatase ERT

sulphatase ERT

thioesterase-1 HCST

acetylgalactosaminidase None

None

therapy

None

type IIIC acetyltransferase

type IIID 252940 *GNS* N-acetylglucosamine-6-

type IVA 253000 *GALNS* N-acetylgalactosamine-6-

type VI 253200 *ARSB* N-acetylgalactosamine-4-

deficiency 272200 *SUMF1* Formylglicine-generating-

lipofuscinosis 1 256730 *PPT1* Palmitoyl protein

type IVB 253010 *GLB1* β-Galactosidase None

type VII 253220 *GUSB* β-Glucuronidase None

type IX 601492 *HYAL1* Hyaluronidase None

lipofuscinosis 2 204500 *TPP1* Tripeptydil-peptidase I GT

lipofuscinosis 3 204200 *CLN3* CLN3 protein None

lipofuscinosis 5 256731 *CLN5* CLN5 protein None

lipofuscinosis 6 601780 *CLN6* CLN6 protein None

lipofuscinosis 8 600143 *CLN8* CLN8 protein None

A/B 257200 *SMPD1* Acid sphingomyelinidase None Niemann-Pick disease C1 257220 *NPC1* NPC1 protein SSI Niemann-Pick disease C2 607625 *NPC2* NPC2 protein SSI Pompe disease 232300 *GAA* Alpha-glucosidase ERT, GT Prosaposin deficiency 176801 *PSAP* Prosaposin None Pycnodysostosis 265800 *CTSK* Cathepsin K Hormone

Sandhoff disease 268800 *HEXB* Hexosaminidase B PCT

Sialic acid storage disease 269920 *SLC17A5* Sialin None

Tay-Sachs disease 272800 *HEXA* Hexosaminidase A PCT, SSI

Table 1. List of lysosomal storage diseases with their respective OMIM accession number, gene and enzyme deficiency and treatment options (including experimental ones).

The two most widely used treatment options are Hematopoietic Stem Cell Transplantation (HSCT) and Enzyme Replacement Therapy (ERT). HSCT has proven to be

Abbreviations: GT – Gene Therapy; HSCT – Hematopoetic Stem Cell Transplantation; PCT – Pharmacological Chaperone Therapy; OMIM – Online Mendelian Inheritance in Man;

SSI – Specific Substrate Inhibition; ERT – Enzyme Replacement Therapy

UDP-N-

epimerase

acetylglucosamine-2-

Schindler disease 609241 *NAGA* Alpha-N-

Sialuria 269921 *GNE* 

enzyme

Mucopolysaccharidosis

Mucopolysaccharidosis

Mucopolysaccharidosis

Mucopolysaccharidosis

Mucopolysaccharidosis

Mucopolysaccharidosis

Multiple sulphatase

Neuronal ceroid

Neuronal ceroid

Neuronal ceroid

Neuronal ceroid

Neuronal ceroid

Neuronal ceroid

Niemann-Pick disease

1:200,000, however in some countries as Malta (1:3,700) and the South of Brazil (1:13,317) it is considerably higher (Baiotto et al, 2011). Large population studies suggest that the overall incidence of the LSDs vary from 1:5,000- 1:7,700 (Fuller et al, 2006).

The treatment options available for the LSDs were restricted to support measures until a few decades ago. Nowadays, specific treatments are available for a certain number of LSDs, even though some of them are still in the experimental phase or have limited effects. Treatment options listed in table 1 consider those already approved or under clinical trial, including compassionate use. Support measures and palliative care are not considered treatment options in this context.


1:200,000, however in some countries as Malta (1:3,700) and the South of Brazil (1:13,317) it is considerably higher (Baiotto et al, 2011). Large population studies suggest that the overall

The treatment options available for the LSDs were restricted to support measures until a few decades ago. Nowadays, specific treatments are available for a certain number of LSDs, even though some of them are still in the experimental phase or have limited effects. Treatment options listed in table 1 consider those already approved or under clinical trial, including compassionate use. Support measures and palliative care are not considered treatment

**Disease OMIM Gene Enzyme Available** 

Canavan disease 271900 *ASPA* Aspartocylase None Cystinosis 219800 *CTNS* Cystinosin Cysteamine

Fabry disease 301500 *GLA* Α-galactosidase A ERT Farber disease 228000 *ASAH1* Ceramidase HSCT Fucosidosis 230000 *FUCA1* α-L-fucosidase HSCT Galactosialidosis 256540 *CTSA* Cathepsin A None

GM1 gangliosidosis 230600 *GLB1* β-Galactosidase HSCT Krabbe disease 245200 *GALC* galactocerebrosidase HSCT

Deficiency 278000 *LIPA* Lysosomal acid lipase HSCT α- mannosidosis 248500 *MAN2B1* α -D -mannosidase HSCT β- mannosidosis 248510 *MANBA* β -D-mannosidase None

leucodystrophy 250100 *ARSA* Arylsulphatase-A HSCT

leucodystrophy 249900 *ARSA* Saposin-B HSCT Mucolipidosis type I 256550 *NEU1* Sialidase None

Mucolipidosis type IV 252650 *MCOLN1* Mucolipin 1 None

type IIIA 252900 *SGSH* Heparan-N-sulfatase None

Mucopolysaccharidosis 252930 *HGSNAT* AcetylCoa-glucosamine-N- None

type I 607014 *IDUA* α-L-iduronidase ERT, HSCT

type II 309900 *IDS* Iduronate sulfatase ERT, GT, HSCT

Mucolipidosis types II/III 252500 *GNPTAB* N-acetylglucosamine-1-

Mucolipidosis type IIIC 252605 *GNPTG*

type IIIB 252920 *NAGLU* α- N-

Gaucher disease 230800 *GBA* acid β-glucosidase ERT, GT, HSCT,

glucosaminidase HSCT

membrane protein 2 None

phosphotransferase None

acetylglucosaminidase None

N-acetylglucosamine-1 phosphotransferase γ-

subunit

**Treatments** 

(drug)

PCT, SSI

None

incidence of the LSDs vary from 1:5,000- 1:7,700 (Fuller et al, 2006).

Aspartylglucosaminuria 208400 *AGA* N-aspartyl-beta-

Danon disease 300257 *LAMP2* Lysosomal-associated

options in this context.

Lysosomal Acid Lipase

Mucopolysaccharidosis

Mucopolysaccharidosis

Mucopolysaccharidosis

Mucopolysaccharidosis

Metachromatic

Metachromatic


Abbreviations: GT – Gene Therapy; HSCT – Hematopoetic Stem Cell Transplantation; PCT – Pharmacological Chaperone Therapy; OMIM – Online Mendelian Inheritance in Man; SSI – Specific Substrate Inhibition; ERT – Enzyme Replacement Therapy

Table 1. List of lysosomal storage diseases with their respective OMIM accession number, gene and enzyme deficiency and treatment options (including experimental ones).

The two most widely used treatment options are Hematopoietic Stem Cell Transplantation (HSCT) and Enzyme Replacement Therapy (ERT). HSCT has proven to be

Non Viral Gene Transfer Approaches for Lysosomal Storage Disorders 151

The rationale for gene therapy and other enzyme-based approaches for treatment of LSDs was first introduced almost five decades ago by Christian de Duve, and can be summarized in the following sentence from his original work "In our pathogenic speculations and in our therapeutic attempts, it may be well to keep in mind that any substance which is taken up intracellularly in an endocytic process is likely to end up within lysosomes." (de Duve, 1964). His work and other pioneer studies showing cross-correction between fibroblasts from patients with Mucopolysaccharidosis type I and type II (deficient in alpha-Liduronidase and iduronate-sulphatase, respectively) established that the enzyme produced in one cell could be uptaken by a deficient cell, thus restoring its phenotype (Fratantoni et al, 1968). Later studies identified that this uptake was a saturable, receptor-mediated process, and the main actor of this process was the mannose-6-phosphate (M6P) receptor localized in the plasmatic membrane. The post-translational modification of addition of the M6P to the protein was discovered to be a signal not only to endocytosis but also for targeting nascent

These pivotal discoveries in the field of endocytosis and targeting of lysosomal enzymes provided the basis for treatments like HSCT and ERT. In the same way, LSDs may be considered good targets for gene therapy, despite their multisystemic involvement. The correction of a few cells could lead to the enzyme being secreted into the circulation and uptaken by the deficient cells, resulting in widespread correction of the biochemical defect

Since long term gene expression is desirable, most clinical and preclinical trials used viral based vectors. Initial studies on fibroblasts showed promising data using retroviruses (Anson et al, 1992). However, when tests in animal models started, it became clear that some organs as the brain would not be easily corrected, as the enzyme could not cross the bloodbrain-barrier (Elinwood et al, 2004). This is a major hurdle as most LSD shows some degree of CNS involvement. Nevertheless, CNS targeted approaches could be envisaged to overcome this obstacle. For instance, Worgall et al. (2008) showed a slowing of progression of Neuronal Ceroid Lipofuscinosis 2 in ten children treated with serotype 2 adenoassociated virus expressing *CLN2* cDNA. Another clinical trial, for Pompe disease, also used adeno-associated-based vector, but in this case serotype 1 (NCT00976352 www.clinicaltrials.gov). Other two trials performed in the late 1990s used retroviral vectors for Gaucher (Dunbar et al., 1998) and Mucopolysaccharidosis type II (NCT00004454 -

Safety issues related to immune response of the adenoviral vectors (Wilson, 2009) and the possibility of insertional mutagenesis of the retroviral vectors (Hacein-Bey-Abina S et al., 2008) led researchers to develop a series of studies in parallel using non-viral approaches to

Non-viral vectors have important safety advantages over viral approaches, including their reduced pathogenicity and capacity for insertional mutagenesis, as well as their low cost and ease of production (Fraga et al., 2008). The application of non-viral vectors to humans has, however, been held back by the poor efficiency of their delivery to cells and the transient expression of the transgenes. As new strategies are being developed for the

**2. Rationale for gene therapy in LSDs** 

hydrolases to lysosomes (Fisher et al, 1980; Sly et al., 1981).

(figure 1).

www.clinicaltrials.gov).

treat lysosomal storage disorders.

**3. Non-viral approaches** 

efficient to some of these diseases, especially if performed early enough to prevent irreversible lesions. Nevertheless, limitations such as the need of an early diagnosis, the difficulties to find a compatible donor in short time, and the high rates of morbidity and mortality associated with the procedure still limit this type of treatment (Malatack et al, 2003). Therefore, despite the many advances in this treatment over the last 30 years (Boelens et al 2010), its use has been deferred in favor of ERT whenever it is available. Enzyme replacement therapy is approved for a growing number of LSD, especially those without CNS involvement. It has proven to reduce some visceral symptoms as hepatosplenomegaly and improve respiratory function (Sifuentes et al, 2007), however difficult-to-reach organs such as the brain and the bones are still a major challenge. Innovative routes of enzyme delivery have been tested to achieve the CNS, such as intrathecal ERT (Munoz-Rojas et al, 2008; Munoz-Rojas et al, 2010).

Other treatment approaches already under clinical use or experimentation are Specific Substrate Inhibition (SSI) and Pharmacological Chaperone Therapy (PCT). SSI aims to decrease the storage by reducing substrate synthesis through an inhibitor. PCT uses small molecules able to stabilize the mutant enzyme and help it escape proteasomal degradation, thus restoring some residual enzyme activity. All such treatments have limitations (table 2), that justify the development of gene therapy approaches for these diseases.


Abbreviations: CNS – Central Nervous System

Table 2. Pros and limitations of different therapeutic approaches for lysosomal storage diseases.
