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
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
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
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
The impact of codon usage optimization on micro-dystrophin expression and function in the
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
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
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
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
Interestingly, we found extensive lymphocyte-mediated immune responses to rAAV2-
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
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
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.
(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
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
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
Long-term benefits can be obtained through the use of virus viral vectors expressing antisense sequences against regions within
Ex vivogene 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
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.
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.