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Medicine » Medical Genetics » "Viral Gene Therapy", book edited by Ke Xu, ISBN 978-953-307-539-6, Published: July 20, 2011 under CC BY-NC-SA 3.0 license. © The Author(s).

Chapter 11

Progress and Challenges in AAV-Mediated Gene Therapy for Duchenne Muscular Dystrophy

By Shin'Ichi Takeda and Takashi Okada
DOI: 10.5772/18624

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Progress and Challenges in AAV-Mediated Gene Therapy for Duchenne Muscular Dystrophy

Takashi Okada1 and Shin’ichi Takeda

1. Introduction

Duchenne muscular dystrophy (DMD) is the most common form of childhood muscular dystrophy. DMD is an X-linked recessive disorder with an incidence of one in 3500 live male newbornsbirths (Emery, 1991). DMD causes progressive degeneration and regeneration of skeletal and cardiac muscles due to mutations in the dystrophin gene, which encodes a 427-kDa subsarcolemmal cytoskeletal protein (Hoffman et al., 1987). DMD is associated with severe, progressive muscle weakness and typically leads to death between the ages of 20 and 35 years. Due to recent advances in respiratory care, much attention is now focused on treating the cardiac conditions suffered by DMD patients.


Figure 1.

Dystrophin-glycoprotein complex.

Molecular structure of the dystrophin-glycoprotein complex and related proteins superimposed on the sarcolemma and subsarcolemmal actin network. (rRedrawn from Yoshida et al. (Yoshida et al., 2000), with modifications). cc, coiled-coil motif on dystrophin (Dys) and dystrobrevin (DB); SGC, sarcoglycan complex; ; SSPN, sarcospan; Syn, syntrophin; Cav3, caveolin-3; N and C, the N and C termini, respectively; G, G-domain of laminin; aAsterisk indicates the actin-binding site on the dystrophin rod domain; WW, WW domain.

The approximately 2.5-megabase dystrophin gene is the largest gene identified to date, and because of its size, it is susceptible to a high sporadic mutation rate. Absence of dystrophin and the dystrophin-glycoprotein complex (DGC) from the sarcolemma leads to severe muscle wasting (Figure 1). Whereas DMD is characterized by an the absence of functional protein, whereas Becker muscular dystrophy, which is commonly caused by in-frame deletions of the dystrophin gene, results in the synthesis of shows synthesis of a partially functional protein.

2. Gene-replacement strategies usingy with virus vectors

2.1. Choice of vector

Successful therapy for DMD requires the restoration of dystrophin protein in skeletal and cardiac muscles. While considerable interest has been focused on various viral vectors have been considered for tothe delivery of genes to the muscle fibers, the adeno-associated virus (AAV)-based vector is emerging as a potentiallythe favorable gene transfer vehicle with the most potential for use in DMD gene therapies.. The advantages of the AAV vector include the lack of associated disease associated with a wild-type virus, the ability to transduce non-dividing cells, and the long-term expression of the delivered transgenes (Okada et al., 2002). Serotypes 1, 6, 8 and 9 of recombinant AAV (rAAV) exhibit a potent tropism for striated muscles (Inagaki et al., 2006). Since a 5-kb genome is considered to be the upper limit for the a single AAV virion, a series of a rod-truncated micro-dystrophin genes is beneficientused in this treatment (Yuasa et al., 1998).

Due to ingenious cloning and preparation techniques, Aadenovirus vectors represent are the efficient delivery systems of episomal DNA into eukaryotic cell nuclei owing to ingenious cloning and preparation techniques (Okada et al., 1998). The utility of the adenovirus vectors has been increased by capsid modifications to that alter tropism, and by the generation of hybrid vectors to that render a capacity ofpromote chromosomal insertion (Okada et al., 2004). Also, gutted adenovirus vectors devoid of all adenoviral genes allow for the insertion of large transgenes, and portray trigger less fewer cytotoxic as well asand immunogenic effects than do those only deleted in the E1 regions (from bases 343 to 2270) and create space for the insertion of large transgenes (Hammerschmidt, 1999). Human artificial chromosomes (HACs) have the capacity to deliver a huge large gene (roughly 6-10 megabases) into host cells without integration integrating the gene into the host genome, thereby preventing the possibleility of insertional mutagenesis and genomic instability (Hoshiya et al., 2008).

A goal in clinical gene therapy pertains is tothe development of gene transfer vehicles that can integrate exogenous therapeutic genes at specific chromosomal loci, so that to prevent insertional oncogenesis is prevented. AAV can insert its genome into a specific locus, designated AAVS1, on chromosome 19 of the human genome (Kotin et al., 1992). The AAV Rep78/68 proteins and the Rep78/68-binding sequences are the trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences. A dual high-capacity adenovirus-AAV hybrid vector with full-length human dystrophin-coding sequences flanked by AAV integration-enhancing elements was experimented tested for targeted integration (Goncalves et al., 2005).

Gene correction is a process by whichwhereby sequence alterations in genes can be corrected by homologous recombination-mediated gene conversion between the recipient target locus and a donor construct encoding the correct sequence (Klug, 2005). TheAn introduction of a corrective sequence together with a site-specific nuclease to induce a double-stranded break (DSB) at sites responsible for monogenic disorders would activate gene correction. Pairs of dDesignated zinc-finger protein with tandem DNA binding sites fused to the cleavage domain of the Fok1 protein was were introduced into model systems or cell lines as pairs with tandem binding sites and produced corrections of in 10–30% of cases testedin model systems or cell lines (Porteus and Baltimore, 2003).

2.2. Modification of the dystrophin gene and promoter

Due to the large deletion in its genome, tThe gutted adenovirus vector can package 14-kb of full-length dystrophin cDNA because of the large-size deletion in its genome. Multiple proximal muscles of 7seven-day-old utrophin/dystrophin double knockout mice (dko mice), which typically show symptoms quite similar to human DMD, were effectively transduced with the gutted adenovirus vector that carriesbearing murine full-length murine dystrophin cDNA (Kawano et al., 2008). However, further improvements are needed to regulate the virus-associated host immune response still needs to be accomplished before if clinical trials can be performeutility is to be achieved.


Figure 2.

Structures of full-length and truncated dystrophin.

Helper-dependent adenovirus vector can package 14-kb of full-length dystrophin cDNA because of the large-sized deletion in its genome. A mini-dystrophin is cloned from a patient with Becker muscular dystrophy, which. It is caused by in-frame deletions resulting in a the synthesis of partially functional protein. A series of truncated micro-dystrophin cDNAs harboring only 4 four rod repeats with hinge 1, 2, and 4 (CS1); the same components, except for that the C-terminal domain is deleted C-terminal domain (delta CS1); or one rod repeat with hinge 1 and 4 (M3), are constructed to be packaged in the AAV vector.

A series of truncated dystrophin cDNAs containing rod repeats with hinge 1, 2, and 4 were constructed (Figure 2) (Yuasa et al., 1998). Although AAV vectors are too small to package the full-length dystrophin cDNA, AAV vector-mediated gene therapy with using a rod-truncated dystrophin gene provides ais promising approach (Wang et al., 2000). The structure and, particularly, the length of the rod structure, and its length in particular, is are crucial for the function of micro-dystrophin (Sakamoto et al., 2002). An AAV type 2 vector expressing micro-dystrophin (DeltaCS1) under the control of a muscle-specific MCK promoter was injected into the tibialis anterior (TA) muscles of dystrophin-deficient mdx mice (Yoshimura et al., 2004), and resulted in extensive and long-term expression of micro-dystrophin that exhibited with improved force generation.

The impact of codon usage optimization on micro-dystrophin expression and function in the mdx mouse was assessed to compare the function of two different configurations of codon-optimized micro-dystrophin genes under the control of a muscle-restrictive promoter (Spc5-12) (Foster et al., 2008). Codon optimization of micro-dystrophin significantly increased micro-dystrophin mRNA and protein levels and protein after intramuscular and systemic administration of plasmid DNA or rAAV8. By randomly assemblingy of myogenic regulatory elements into synthetic promoter recombinant libraries, several artificial promoters were isolated whose transcriptional potencies greatly exceed those of natural myogenic and viral gene promoters (Li et al., 1999).

2.3. Use of surrogate genes

An approach to usinge a surrogate gene would avoid bypass the potential immune responses associated with the delivery of exogenous dystrophin. Methods to increase expression of utrophin, a dystrophin paralog, show promise as a treatment for DMD. rAAV6 harboring a murine codon-optimized micro-utrophin transgene was intravenously administered into adult dko mice to alleviate the pathophysiological abnormalities (Odom et al., 2008). The paralogous gene efficiently behaved acted as a surrogate for dystrophin. Myostatin has beenis extensively documented as being a negative regulator of muscle growth. Systemic gene delivery of myostatin propeptide, a natural inhibitor of myostatin, enhanced body-wide skeletal muscle growth in either both normal orand mdx mice (Qiao et al., 2008). The dDelivery of various growth factors, such as insulin-like growth factor-I (IGF-I), have has been successful in promoting skeletal muscle regeneration after injury (Schertzer and Lynch, 2006).

Matrix metalloproteinases (MMPs) are key regulatory molecules in the formation, remodeling and degradation of all extracellular matrix (ECM) components in pathological processes. MMP-9 is involved predominantly in the inflammatory process during muscle degeneration (Fukushima et al., 2007). In contrast, MMP-2 is associated with ECM remodeling during muscle regeneration and fiber growth.

3. AAV-mediated transduction of of animal models

3.1. Vector production

When adenovirus helper plasmid is co-transfected into human embryonic kidney 293 cells along with a vector plasmid encoding the AAV vector as well asand an AAV packaging plasmid harboring rep-cap genes, the AAV vector is produced as efficiently as when using adenovirus infection. A large-scale transduction method to produce AAV vectors with an active gassing system makes a use of large culture vessels to processfor labor- and cost-effective transfection in a closed system. Samples containing vector particles are further purified with a two-tier CsCl gradient or dual ion-exchange chromatography to obtain highly purified vector particles.


Figure 3.

A scalable triple plasmid transfection system usingwith active gassing.

To gain acceptance as a medical treatment with a dose of over 1x1013 genome copies (g.c.)/kg body weight, AAV vectors require a scalable and economical production method. A production protocol of AAV vectors in the absence of a helper virus (Matsushita et al., 1998) is widely employed for triple plasmid transduction of human embryonic kidney 293 cells (Okada et al., 2002). The adenovirus regions that mediate AAV vector replication (namely, the VA, E2A, and E4 regions) were assembled into a helper plasmid. When this helper plasmid is co-transfected into human embryonic kidney 293 cells along with plasmids encoding the AAV vector genome as well asand rep-cap genes, the AAV vector is produced as efficiently as when using adenovirus infection (Figure 3). Importantly, contamination of most adenovirus proteins can be avoided in AAV vector stock made by this helper virus-free method. Samples containing vector particles are further purified with a two-tier CsCl gradient or dual ion-exchange chromatography to obtain highly purified vector particles (Okada et al., 2002).

Although Despite improvements in vector production, includinge the development of packaging cell lines expressing Rep/Cap, and of ways for inducible methods that induce the expression and regulation of Rep/Cap (Okada et al., 2001), maintaining such cell lines is stillremains difficult, as because the early expression of Rep proteins is toxic to cells. We developed aA scalable transfection method, with using active gassing and large culture vessels, was developed for to producing transfect the rAAV in a closed system, in a that uses large culture vessels to p rocess labor- and cost-effective infection or transfectionmanner in a closed system (Okada et al., 2005). This vector production system achieved with a yield of morehigher than 5x1013 g.c./flask was achieved by improvinged gas exchange to maintenance maintain theof physiological pH in the culture medium. Recent developments in ion-exchange chromatography also suggest that vector production using transduction culture supernatant would be compatible with current good manufacturing practice and production on an industrial scale (Okada et al., 2009). Moreover, AAV vector production in insect cells would be compatible with current good manufacturing practice production on an industrial scale (Cecchini et al., 2008).

3.2. Animal models for the gene transduction study

Dystrophin-deficient canine X-linked muscular dystrophy was found in a golden retriever with a 3’ splice-site point mutation in intron 6 (Valentine et al., 1988). The clinical and pathological characteristics of the dystrophic dogs are more similar to those of DMD patients than are those of mdx mice. We have established aA bBeagle-based model of canine X-linked muscular dystrophy, in Japan (CXMDJ),33 which is smaller and easier to handle compared tothan the golden retriever-based muscular dystrophy dog (GRMD) model, has been established in Japan, and is referred to as CXMDJ (Shimatsu et al., 2005).. The limb and temporal muscles of CXMDJ are affected by two-month-old, which is the age corresponding to the second peak of serum creatine kinase.

Interestingly, we found extensive lymphocyte-mediated immune responses to rAAV2-lacZ after direct intramuscular injection into CXMDJ dogs, despite successful delivery of the same viral construct into mouse skeletal muscle (Yuasa et al., 2007). In quite contrast to rAAV2, rAAV8-mediated transduction of canine skeletal muscles produced significantly higher transgene expression with less lymphocyte proliferation than rAAV2 (Ohshima et al., 2008).

It is increasingly important to develop strategies to treat DMD that consider the effect on cardiac involvementmuscle. The pPathology of the conduction system in CXMDJ was analyzed to seek establish the therapeutic target for DMD (Urasawa et al., 2008). Although dystrophic changes of the ventricular myocardium were not evident at the age of 1 to 13 months, Purkinje fibers showed remarkable vacuolar degeneration when dogs were as young as early as 4 four-months- of ageold. BesidesFurthermore, degeneration of Purkinje fibers was coincident with overexpression of Dp71 at the sarcolemma. The degeneration of Purkinje fibers could be associated with the distinct deep Q waves present in ECGs and the fatal arrhythmias seen in cases of dystrophin deficiency (Urasawa et al., 2008).

3.3. Immunological Issues of rAAV

Neo-antigens introduced by AAV vectors evoke significant immune reactions in DMD muscle, since increased permeability of sarcolemma allowsed a leakage of the transgene products from the dystrophin-deficient muscle fibers (Yuasa et al., 2002). rAAV2 transfer into skeletal muscles of normal dogs resulted in low and transient expression, together with intense cellular infiltrations, whereand the marked activation of cellular and humoral immune responses were remarkably activated (Yuasa et al., 2007). Furthermore, an in vitro interferon-gamma release assay showed that canine splenocytes respond to immunogens or mitogens more susceptibly strongly than do murine splenocytesones. In fact, co-administration of immunosuppressants, cyclosporine (CSP) and mycophenolate mofetil (MMF) improved rAAV2 transduction. The AAV2 capsids can induce a cellular immune response via MHC class I antigen presentation with a cross-presentation pathway, and the effects of rAAV2 has also been proposed to have an effect on human dendritic cells (DCs). have been also suggested. In contrast, other serotypes, such as rAAV8, induced less T- cell activation to a lesser degree (Ohshima et al., 2008). Immunohistochemical analysis revealed that the rAAV2-injected muscles showed higher rates of infiltration of CD4+ and CD8+ T lymphocytes in the endomysium than the rAAV8-injected muscles (Ohshima et al., 2008).

Resident antigen-presenting cells, such as dendritic cells (DCs), myoblasts, myotubes, and regenerating immature myofibers, might play a role in the immune response. Our A recent study also showed that mRNA levels of MyD88 and co-stimulating factors, such as CD80, CD86, and type I interferon, are elevated in both rAAV2- and rAAV8-transduced dog DCs in vitro (Ohshima et al., 2008). A brief course of immunosuppression with a combination of anti-thymocyte globulin (ATG), cyclosporine (CSP), and mycophenolate mofetil (MMF) was effective to in permitpermitting AAV6-mediated, long-term and robust expression of a canine micro-dystrophin in the skeletal muscle of a dog DMD model (Wang et al., 2007).

3.4. Intravascular vector administration by limb perfusion

Although recent studies suggest that vectors based on AAV are capable of body-wide transduction in rodents, translating this finding into large animals remains a challenge. Intravascular delivery can be performed as a form of limb perfusion, which might bypass the immune activation of DCs in the injected muscle (Ohshima et al., 2008). We performed limb perfusion-assisted intravenous administration of rAAV8-lacZ into the hind limb in of normal dogs and rAAV8-micro-dystrophin into the hind limb in of CXMDJ dogs (Figure 4) (Ohshima et al., 2008). Administration of rAAV8-micro-dystrophin by limb perfusion produced extensive transgene expression in the distal limb muscles of CXMDJ dogs without obvious immune responses for as long as 8 eight weeks after injection.


Figure 4.

Intravascular vector administration by limb perfusion.

(A) A blood pressure cuff is applied just above the knee of an anesthetized canine X-linked muscular dystrophy in Japan (CXMDJ dog). A 24-gauge intravenous catheter is inserted into the lateral saphenous vein, connected to a three-way stopcock, and flushed with saline. With a blood pressure cuff inflated to over 300 mmHg, saline (2.6 ml/kg) containing papaverine (0.44 mg/kg, Sigma-Aldrich, St. Louis, MO) and heparin (16 U/kg) is injected by hand over a 10 second periods. The three-way stopcock is connected to a syringe containing rAAV8 expressing micro-dystrophin (1 x 1014 vg/kg, 3.8 ml/kg). The syringe is placed in a PHD 2000 syringe pump (Harvard Apparatus, Edenbridge, UK). Five minutes after the papaverine/heparin injection, the rAAV8 is injected at a rate of 0.6 ml/sec. (B) Administration of rAAV8-micro-dystrophin by limb perfusion produces extensive transgene expression in the distal limb muscles of CXMDJ dogs without obvious immune responses at 4 four weeks after injection.

3.5. Global muscle therapies

In comparison to with fully dystrophin-deficient animals, targeted transgenic repair of skeletal muscle, but not cardiac muscle, paradoxically elicited elicits a five-fold increase in cardiac injury and dilated cardiomyopathy (Townsend et al., 2008). Because the dystrophin-deficient heart is highly sensitive to increased stress, increased activity by the repaired skeletal muscle provided provides the stimulus for heightened cardiac injury and heart remodeling. In contrast, a single intravenous injection of AAV9 vector expressing micro-dystrophin efficiently transduced transduces the entire heart in neonatal mdx mice, thereby to ameliorateing cardiomyopathy (Bostick et al., 2008).

Since a number of muscular dystrophy patients can be identified through newborn screening, neonatal transduction may lead to an effective early intervention in DMD patients. After a single intravenous injection, Rrobust skeletal muscle transduction with AAV9 vector throughout the body was observed after a single intravenous injection of AAV9 vector was observed in neonatal dogs (Yue et al., 2008). Systemic transduction was achieved in the absence of pharmacological intervention or immune suppression and lasted for at least 6 six months, whereas cardiac muscle was barely transduced in the dogs.

4. Safety and potential impact of clinical trials

The initial clinical studies lay the foundation for future studies, providing important information about vector dose, viral serotype selection, and immunogenicity in humans. The first virus-mediated gene transfer for muscle disease was carried out for limb-girdle muscular dystrophy type 2D by using rAAV1. The study, consisting of intramuscular injection of virus into a single muscle, was discharged to establish the safety of this procedure in phase I clinical trials (Rodino-Klapac et al., 2007). A The first clinical gene therapy trial for DMD was began in March 2006 (Mendell et al., 2010). This was a Phase I/IIa study in which an AAV vector was used to deliver micro-dystrophin to the biceps of boys with DMD. The trial testedstudy was conducted on six boys with DMD, each of whom was transduced with mini-dystrophin genes in a muscle of one arm in the absence of serious adverse events. Interestingly, dystrophin-specific T cells were detected after treatment, providing evidence of transgene expression. The potential for T-cell immunity to self and nonself dystrophin epitopes should be considered in designing and monitoring experimental therapies for this disease.

While low immunogenicity was considered a major strength supporting the use of rAAV in clinical trials, a number of observations have recently provided a more realistic balanced view of this procedure (Manno et al., 2006). An obvious barrier to AAV transduction is the presence of circulating neutralizing antibodies that preclude prevent the virion from binding of the virion to its cellular receptor (Scallan et al., 2006). This potential threat can be reduced by prescreening patients for AAV serotype-specific neutralizing antibodies or through the application of by performing maneuvers procedures such as plasmapheresis before gene transfer. Another challenge recently uncovered revealed is the development of a cell-mediated cytotoxic T-cell (CTL) response to AAV capsid peptides. In the human factor IX gene therapy trial in which rAAV was delivered to the liver, only short-term transgene expression was achieved and levels of therapeutic protein declined to baseline levels 10 weeks after vector infusion (Manno et al., 2006). This was accompanied by elevation of serum transaminase levels and a CTL response toward specific AAV capsid peptides. To overcome this response, transient immunosuppression may be required until AAV capsids are completely cleared. Additional findings suggest that T-cell activation requires AAV2 capsid binding to the heparan sulfate proteoglycan (HSPG) receptor, which would permitting virion shuttling into a DC pathway, as cross-presentation (Vandenberghe et al., 2006). Exposure to vVectors from other AAV clades, such as AAV8, did not lead to activatione of capsid-specific T- cells.

5. Challenges and limitations of related strategies

5.1. Design of read-through drugs

To suppress premature stop codon mutations, treatments involving aminoglycosides and other agents have been attempted. PTC124,As a novel drug capable of suppressing premature termination, a new chemical PTC124 that and selectively induces inducing ribosomal read-through of premature, but not normal, termination codons, was recently identified using nonsense-containing reporters (Welch et al., 2007). The selectivity of PTC124 for premature termination codons, its oral bioavailability and its pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.

5.2. Modification of mRNA splicing

By inducing the skipping of specific exons skipping during mRNA splicing, antisense compounds against exonic and intronic splicing regulatory sequences were shown to correct the open reading frame of the DMD gene and thus to restore truncated yet functional dystrophin expression in vitro (Takeshima et al., 1995). Intravenous infusion of an antisense phosphorothioate oligonucleotide created an in-frame dystrophin mRNA via exon skipping in a 10-year-old DMD patient possessing an out-of-frame exon 20 deletion of the dystrophin gene (Takeshima et al., 2006). Moreover, the adverse-event profile and local dystrophin-restoring effect of a single intramuscular injection of an antisense 2'-O-methyl phosphorothioate oligonucleotide, PRO051, in patients with DMD were explored (van Deutekom et al., 2007). Four patients received a dose of 0.8 mg of PRO051 in the tibialis anteriorTA muscle. Each patient showed specific skipping of exon 51 of and dystrophin in 64 to 97% of myofibers, without clinically apparent adverse eventsside effects.

The efficacy and toxicity of intravenous injection of stable morpholino phosphorodiamidate (morpholino)-induced exon skipping were tested using the CXMDJ dogs, and widespread rescue of dystrophin expression to therapeutic levels was demonstratedobserved (Yokota et al., 2009). Furthermore, a morpholino oligomer with a designed cell-penetrating peptide can efficiently target a mutated dystrophin exon in the cardiac muscles (Wu et al., 2008).

Long-term benefits can be obtained through the use of virus viral vectors expressing antisense sequences against regions within dystrophin gene. The sustained production of dystrophin at physiological levels in entire groups of muscles as well as the correction of the muscular dystrophy were achieved by treatment with exon-skipping AAV1-U7 (Goyenvalle et al., 2004).

5.3. Ex vivo gene therapy

Transplantation of genetically corrected autologous myogenic cells is a possible treatment for DMD. Freshly isolated satellite cells transduced with lentiviral vectors expressing micro-dystrophin were transplanted into the tibialis anteriorTA muscles of mdx mice, and these cells efficiently contributed to the regeneration of muscles with micro-dystrophin expression at the sarcolemma (Ikemoto et al., 2007). Mesoangioblasts are the vessel-associated stem cells and might be candidates for future stem cell therapy for DMD (Sampaolesi et al., 2006). Intra-arterial delivery of wild-type canine mesoangioblasts resulted in an the extensive recovery of dystrophin expression, normal muscle morphology and function in the GRMD. Multipotent mesenchymal stromal cells (MSCs) are less immunogenic and have the potential to differentiate and display a myogenic phenotype (Dezawa et al., 2005).

6. Future perspectives

6.1. Pharmacological Intervention

The use of a histone deacetylase (HDAC) inhibitor would likely enhance the utility of rAAV-mediated transduction strategies in the clinic (Okada et al., 2006). In contrast to the adenovirus-mediated transduction, the improved transduction with rAAV induced by the HDAC inhibitor is due to an enhanced transgene expression rather than to increased viral entry. The enhanced transduction was proposed to be related to the proposed histone-associated chromatin form of the rAAV concatemer in transduced cells. Since various HDAC inhibitors are currently being tested in clinical trials for many diseases, to utilizethe use of such agents to assistin rAAV-mediated DMD gene therapy is theoretically and practically reasonable.

6.2. Capsid modification

A DNA shuffling-based approach for developing cell type-specific vectors is an intriguing possibility to achieve altered tropism. Capsid genomes of AAV serotypes 1-9 were randomly reassembled using PCR to generate a chimeric capsid library (Li et al., 2008). A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV.

7. Conclusion

DMD remains an untreatable genetic disease that severely limits motility and life expectancy in affected children. In future, The systemic delivery of rAAV to transduce truncated dystrophin would is predicted to ameliorate the symptoms of DMD patients in the future. To translate gene transduction technologies into clinical practice, development of an effective delivery system with improved vector constructs as well as efficient immunological modulation must be required to established. A novel protocol that considersing all of these issues would help us improve the therapeutic benefits in of DMD gene therapy.


This work was supported by the Grant for Research on Nervous and Mental Disorders, Health Science Research Grants for Research on the Human Genome and Gene Therapy; and the Grant for Research on Brain Science from the Ministry of Health, Labor and Welfare of Japan. This work was also supported by Grants-in- Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). We would like to thank Dr. James M. Wilson for providing p5E18-VD2/8.


1 - B. Bostick, Y. Yue, Y. Lai, C. Long, D. Li, D. Duan, 2008 Adeno-associated virus serotype-9 microdystrophin gene therapy ameliorates electrocardiographic abnormalities in mdx mice. Hum Gene Ther, 19 8 851856 , 1043-0342
2 - S. Cecchini, A. Negrete, R. M. Kotin, 2008 Toward exascale production of recombinant adeno-associated virus for gene transfer applications. Gene Ther, 15 11 823830 , 0969-7128
3 - M. Dezawa, H. Ishikawa, Y. Itokazu, T. Yoshihara, M. Hoshino, S. Takeda, C. Ide, Y. Nabeshima, 2005 Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science, 309 5732 314317 , 0193-4511
4 - A. E. Emery, 1991 Population frequencies of inherited neuromuscular diseases--a world survey. Neuromuscul Disord, 1 1 1929 , 0960-8966
5 - H. Foster, P. S. Sharp, T. Athanasopoulos, C. Trollet, I. R. Graham, K. Foster, D. J. Wells, G. Dickson, 2008 Codon and mRNA sequence optimization of microdystrophin transgenes improves expression and physiological outcome in dystrophic mdx mice following AAV2/8 gene transfer. Mol Ther, 16 11 18251832 , 1525-0016
6 - K. Fukushima, A. Nakamura, H. Ueda, K. Yuasa, K. Yoshida, S. Takeda, S. Ikeda, 2007 Activation and localization of matrix metalloproteinase-2 and-9 in the skeletal muscle of the muscular dystrophy dog (CXMDJ). BMC Musculoskelet Disord, 8 54 1471-2474
7 - M. A. Goncalves, G. P. Van Nierop, M. R. Tijssen, P. Lefesvre, S. Knaan-Shanzer, I. Van Der Velde, D. W. Van Bekkum, D. Valerio, A. A. De Vries, 2005 Transfer of the full-length dystrophin-coding sequence into muscle cells by a dual high-capacity hybrid viral vector with site-specific integration ability. J Virol, 79 5 31463162 , 1098-5514
8 - A. Goyenvalle, A. Vulin, F. Fougerousse, F. Leturcq, J. C. Kaplan, L. Garcia, O. Danos, 2004 Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping. Science, 306 5702 17961799 , 0193-4511
9 - D. E. Hammerschmidt, 1999 Development of a gutless vector. J Lab Clin Med, 134 3 C3 0022-2143
10 - E. P. Hoffman, R. H. Brown, Jr , L. M. Kunkel, 1987 Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell, 51 6 919928 , 0092-8674
11 - H. Hoshiya, Y. Kazuki, S. Abe, M. Takiguchi, N. Kajitani, Y. Watanabe, T. Yoshino, Y. Shirayoshi, K. Higaki, G. Messina, G. Cossu, M. Oshimura, 2008 A highly Stable and Nonintegrated Human Artificial Chromosome (HAC) Containing the 2.4 Mb Entire Human Dystrophin Gene. Mol Ther, 309317 , 1525-0016
12 - M. Ikemoto, S. Fukada, A. Uezumi, S. Masuda, H. Miyoshi, H. Yamamoto, M. R. Wada, N. Masubuchi, Y. Miyagoe-Suzuki, S. Takeda, 2007 Autologous transplantation of SM/C-2.6(+) satellite cells transduced with micro-dystrophin CS1 cDNA by lentiviral vector into mdx mice. Mol Ther, 15 12 21782185 , 1525-0016
13 - K. Inagaki, S. Fuess, T. A. Storm, G. A. Gibson, C. F. Mctiernan, M. A. Kay, H. Nakai, 2006 Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol Ther, 14 1 4553 , 1525-0016
14 - R. Kawano, M. Ishizaki, Y. Maeda, Y. Uchida, E. Kimura, M. Uchino, 2008 Transduction of full-length dystrophin to multiple skeletal muscles improves motor performance and life span in utrophin/dystrophin double knockout mice. Mol Ther, 16 5 825831 , 1525-0016
15 - A. Klug, 2005 Towards therapeutic applications of engineered zinc finger proteins. FEBS Lett, 579 4 892894 , 0014-5793
16 - R. M. Kotin, R. M. Linden, K. I. Berns, 1992 Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination. Embo J, 11 13 50715078 , 0261-4189
17 - W. Li, A. Asokan, Z. Wu, T. Van Dyke, N. Diprimio, J. S. Johnson, L. Govindaswamy, M. Agbandje-Mckenna, S. Leichtle, D. E. Redmond, Jr , . T. J. Mccown, K. B. Petermann, N. E. Sharpless, R. J. Samulski, 2008 Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles. Mol Ther, 16 7 12521260 , 1525-0016
18 - X. Li, E. M. Eastman, R. J. Schwartz, R. Draghia-Akli, 1999 Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences. Nat Biotechnol, 17 3 241245 , 1087-0156
19 - C. S. Manno, G. F. Pierce, V. R. Arruda, B. Glader, M. Ragni, J. J. Rasko, M. C. Ozelo, K. Hoots, P. Blatt, B. Konkle, M. Dake, R. Kaye, M. Razavi, A. Zajko, J. Zehnder, P. K. Rustagi, H. Nakai, A. Chew, D. Leonard, J. F. Wright, R. R. Lessard, J. M. Sommer, M. Tigges, D. Sabatino, A. Luk, . H. Jiang, F. Mingozzi, L. Couto, H. C. Ertl, K. A. High, M. A. Kay, 2006 Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med, 12 3 342347 , 1078-8956
20 - T. Matsushita, S. Elliger, C. Elliger, G. Podsakoff, L. Villarreal, G. J. Kurtzman, Y. Iwaki, P. Colosi, 1998 Adeno-associated virus vectors can be efficiently produced without helper virus. Gene Ther, 5 7 938945 , 0969-7128
21 - J. R. Mendell, K. Campbell, L. Rodino-Klapac, Z. Sahenk, C. Shilling, S. Lewis, D. Bowles, S. Gray, C. Li, G. Galloway, V. Malik, B. Coley, K. R. Clark, J. Li, X. Xiao, J. Samulski, S. W. Mcphee, R. J. Samulski, C. M. Walker, 2010 Dystrophin immunity in Duchenne’s muscular dystrophy. N Engl J Med, 363 15 14291437 , 0028-4793
22 - G. L. Odom, P. Gregorevic, J. M. Allen, E. Finn, J. S. Chamberlain, 2008 Microutrophin delivery through rAAV6 increases lifespan and improves muscle function in dystrophic dystrophin/utrophin-deficient mice. Mol Ther, 16 9 15391545 , 1525-0016
23 - S. Ohshima, J. H. Shin, K. Yuasa, A. Nishiyama, J. Kira, T. Okada, S. Takeda, 2008 Transduction Efficiency and Immune Response Associated With the Administration of AAV8 Vector Into Dog Skeletal Muscle. Mol Ther, 7391 , 1525-0016
24 - T. Okada, N. J. Caplen, W. J. Ramsey, M. Onodera, K. Shimazaki, T. Nomoto, . R. Ajalli, O. Wildner, J. Morris, A. Kume, H. Hamada, R. M. Blaese, K. Ozawa, 2004 In situ generation of pseudotyped retroviral progeny by adenovirus-mediated transduction of tumor cells enhances the killing effect of HSV-tk suicide gene therapy in vitro and in vivo. J Gene Med, 6 3 288299 , 1521-2254
25 - T. Okada, H. Mizukami, M. Urabe, T. Nomoto, T. Matsushita, Y. Hanazono, A. Kume, K. Tobita, K. Ozawa, 2001 Development and characterization of an antisense-mediated prepackaging cell line for adeno-associated virus vector production. Biochem Biophys Res Commun, 288 1 6268 ., 1090-2104
26 - T. Okada, T. Nomoto, T. Yoshioka, M. Nonaka-Sarukawa, T. Ito, T. Ogura, M. Iwata-Okada, R. Uchibori, K. Shimazaki, H. Mizukami, A. Kume, K. Ozawa, 2005 Large-scale production of recombinant viruses by use of a large culture vessel with active gassing. Hum Gene Ther, 16 10 12121218 , 1043-0342
27 - T. Okada, M. Nonaka-Sarukawa, R. Uchibori, K. Kinoshita, H. Hayashita-Kinoh, Y. Nitahara-Kasahara, S. Takeda, K. Ozawa, 2009 Scalable purification of adeno-associated virus serotype 1 (AAV1) and AAV8 vectors, using dualion-exchange adsorptive membranes. Hum Gene Ther, 20 9 10131021 , 1043-0342
28 - T. Okada, J. Ramsey, J. Munir, O. Wildner, M. Blaese, 1998 Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex. Nucleic Acids Res., 26 8 19471950 , 0305-1048
29 - T. Okada, K. Shimazaki, T. Nomoto, T. Matsushita, H. Mizukami, M. Urabe, Y. Hanazono, A. Kume, K. Tobita, K. Ozawa, N. Kawai, 2002 Adeno-associated viral vector-mediated gene therapy of ischemia-induced neuronal death. Methods Enzymol, 346 378393 ., 0076-6879
30 - T. Okada, R. Uchibori, M. Iwata-Okada, M. Takahashi, T. Nomoto, M. Nonaka-Sarukawa, T. Ito, Y. Liu, H. Mizukami, A. Kume, E. Kobayashi, K. Ozawa, 2006 A histone deacetylase inhibitor enhances recombinant adeno-associated virus-mediated gene expression in tumor cells. Mol Ther, 13 4 738746 , 1525-0016
31 - M. H. Porteus, D. Baltimore, 2003 Chimeric nucleases stimulate gene targeting in human cells. Science, 300 5620 763 0193-4511
32 - C. Qiao, J. Li, J. Jiang, X. Zhu, B. Wang, J. Li, X. Xiao, 2008 Myostatin propeptide gene delivery by adeno-associated virus serotype 8 vectors enhances muscle growth and ameliorates dystrophic phenotypes in mdx mice. Hum Gene Ther, 19 3 241254 , 1043-0342
33 - L. R. Rodino-Klapac, L. G. Chicoine, B. K. Kaspar, J. R. Mendell, 2007 Gene therapy for duchenne muscular dystrophy: expectations and challenges. Arch Neurol, 64 9 12361241 , 0003-9942
34 - M. Sakamoto, K. Yuasa, M. Yoshimura, T. Yokota, T. Ikemoto, M. Suzuki, G. Dickson, Y. Miyagoe-Suzuki, S. Takeda, 2002 Micro-dystrophin cDNA ameliorates dystrophic phenotypes when introduced into mdx mice as a transgene. Biochem Biophys Res Commun, 293 4 12651272 , 1090-2104
35 - M. Sampaolesi, S. Blot, G. D’antona, N. Granger, R. Tonlorenzi, A. Innocenzi, P. Mognol, J. L. Thibaud, B. G. Galvez, I. Barthelemy, L. Perani, S. Mantero, M. Guttinger, O. Pansarasa, C. Rinaldi, Angelis. M. G. Cusella De, Y. Torrente, C. Bordignon, R. Bottinelli, G. Cossu, 2006 Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs. Nature, 444 7119 574579 , 0028-0836
36 - C. D. Scallan, H. Jiang, T. Liu, S. Patarroyo-White, J. M. Sommer, S. Zhou, L. B. Couto, G. F. Pierce, 2006 Human immunoglobulin inhibits liver transduction by AAV vectors at low AAV2 neutralizing titers in SCID mice. Blood, 107 5 18101817 , 0006-4971
37 - J. D. Schertzer, G. S. Lynch, 2006 Comparative evaluation of IGF-I gene transfer and IGF-I protein administration for enhancing skeletal muscle regeneration after injury. Gene Ther, 13 23 16571664 , 0969-7128
38 - Y. Shimatsu, M. Yoshimura, K. Yuasa, N. Urasawa, M. Tomohiro, M. Nakura, M. Tanigawa, A. Nakamura, S. Takeda, 2005 Major clinical and histopathological characteristics of canine X-linked muscular dystrophy in Japan, CXMDJ. Acta Myol, 24 2 145154 , 1128-2460
39 - Y. Takeshima, H. Nishio, H. Sakamoto, H. Nakamura, M. Matsuo, 1995 Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe. J Clin Invest, 95 2 515520 , 0021-9738
40 - Y. Takeshima, M. Yagi, H. Wada, K. Ishibashi, A. Nishiyama, M. Kakumoto, T. Sakaeda, R. Saura, K. Okumura, M. Matsuo, 2006 Intravenous infusion of an antisense oligonucleotide results in exon skipping in muscle dystrophin mRNA of Duchenne muscular dystrophy. Pediatr Res, 59 5 690694 , 0031-3998
41 - D. Townsend, S. Yasuda, S. Li, J. S. Chamberlain, J. M. Metzger, 2008 Emergent dilated cardiomyopathy caused by targeted repair of dystrophic skeletal muscle. Mol Ther, 16 5 832835 , 1525-0016
42 - N. Urasawa, M. R. Wada, N. Machida, K. Yuasa, Y. Shimatsu, Y. Wakao, S. Yuasa, T. Sano, I. Nonaka, A. Nakamura, S. Takeda, 2008 Selective vacuolar degeneration in dystrophin-deficient canine Purkinje fibers despite preservation of dystrophin-associated proteins with overexpression of Dp71. Circulation, 117 19 24372448 , 0009-7322
43 - B. A. Valentine, B. J. Cooper, A. De Lahunta, R. O’quinn, J. T. Blue, 1988 Canine X-linked muscular dystrophy. An animal model of Duchenne muscular dystrophy: clinical studies. J Neurol Sci, 88 1-3 , 6981 , 1878-5883
44 - J. C. Van Deutekom, A. A. Janson, I. B. Ginjaar, W. S. Frankhuizen, . A. Aartsma-Rus, M. Bremmer-Bout, Dunnen. J. T. Den, K. Koop, A. J. Van Der Kooi, N. M. Goemans, S. J. De Kimpe, P. F. Ekhart, E. H. Venneker, G. J. Platenburg, J. J. Verschuuren, G. J. Van Ommen, 2007 Local dystrophin restoration with antisense oligonucleotide PRO051. N Engl J Med, 357 26 26772686 , 0028-4793
45 - L. H. Vandenberghe, L. Wang, S. Somanathan, Y. Zhi, J. Figueredo, R. Calcedo, J. Sanmiguel, R. A. Desai, C. S. Chen, J. Johnston, R. L. Grant, G. Gao, J. M. Wilson, 2006 Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid. Nat Med, 12 8 967971 , 1078-8956
46 - B. Wang, J. Li, X. Xiao, 2000 Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model. Proc Natl Acad Sci U S A, 97 25 1371413719 , 0027-8424
47 - Z. Wang, C. S. Kuhr, J. M. Allen, M. Blankinship, P. Gregorevic, J. S. Chamberlain, S. J. Tapscott, R. Storb, 2007 Sustained AAV-mediated Dystrophin Expression in a Canine Model of Duchenne Muscular Dystrophy with a Brief Course of Immunosuppression. Mol Ther, 15 6 11601166 , 1525-0016
48 - E. M. Welch, E. R. Barton, J. Zhuo, Y. Tomizawa, W. J. Friesen, P. Trifillis, S. Paushkin, M. Patel, C. R. Trotta, S. Hwang, R. G. Wilde, G. Karp, J. Takasugi, G. Chen, S. Jones, H. Ren, Y. C. Moon, D. Corson, A. A. Turpoff, J. A. Campbell, M. M. Conn, A. Khan, N. G. Almstead, J. Hedrick, A. Mollin, N. Risher, M. Weetall, S. Yeh, A. A. Branstrom, J. M. Colacino, J. Babiak, W. D. Ju, S. Hirawat, V. J. Northcutt, L. L. Miller, P. Spatrick, F. He, M. Kawana, H. Feng, A. Jacobson, S. W. Peltz, H. L. Sweeney, 2007 PTC124 targets genetic disorders caused by nonsense mutations. Nature, 447 7140 8791 , 0028-0836
49 - B. Wu, H. M. Moulton, P. L. Iversen, J. Jiang, J. Li, J. Li, C. F. Spurney, A. Sali, A. D. Guerron, K. Nagaraju, T. Doran, P. Lu, X. Xiao, Q. L. Lu, 2008 Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer. Proc Natl Acad Sci U S A, 105 39 1481414819 , 0027-8424
50 - T. Yokota, Q. Lu, T. Partridge, M. Kobayashi, A. Nakamura, S. Takeda, E. P. Hoffman, 2009 Efficacy of systemic morpholino exon-skipping in Duchenne dystrophy dogs. Annals Neurol, 667676 , 0364-5134
51 - M. Yoshida, H. Hama, M. Ishikawa-Sakurai, M. Imamura, Y. Mizuno, K. Araishi, E. Wakabayashi-Takai, S. Noguchi, T. Sasaoka, E. Ozawa, 2000 Biochemical evidence for association of dystrobrevin with the sarcoglycan-sarcospan complex as a basis for understanding sarcoglycanopathy. Hum Mol Genet, 9 7 10331040 , 0964-6906
52 - M. Yoshimura, M. Sakamoto, M. Ikemoto, Y. Mochizuki, K. Yuasa, Y. Miyagoe-Suzuki, S. Takeda, 2004 AAV vector-mediated microdystrophin expression in a relatively small percentage of mdx myofibers improved the mdx phenotype. Mol Ther, 10 5 821828 , 1525-0016
53 - K. Yuasa, Y. Miyagoe, K. Yamamoto, Y. Nabeshima, G. Dickson, S. Takeda, 1998 Effective restoration of dystrophin-associated proteins in vivo by adenovirus-mediated transfer of truncated dystrophin cDNAs. FEBS Lett, 425 2 329336 , 0014-5793
54 - K. Yuasa, M. Sakamoto, Y. Miyagoe-Suzuki, A. Tanouchi, H. Yamamoto, J. Li, J. S. Chamberlain, X. Xiao, S. Takeda, 2002 Adeno-associated virus vector-mediated gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response against the transgene product. Gene Ther, 9 23 15761588 , 0969-7128
55 - K. Yuasa, M. Yoshimura, N. Urasawa, S. Ohshima, J. M. Howell, A. Nakamura, T. Hijikata, Y. Miyagoe-Suzuki, S. Takeda, 2007 Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products. Gene Ther, 14 17 12491260 , 0969-7128
56 - Y. Yue, A. Ghosh, C. Long, B. Bostick, B. F. Smith, J. N. Kornegay, D. Duan, 2008 A single intravenous injection of adeno-associated virus serotype-9 leads to whole body skeletal muscle transduction in dogs. Mol Ther, 16 12 19441952 , 1525-0016