Thomas A Rando
email: rando@stanford.edu

Non-viral gene therapy for duchenne muscular dystrophy: Progress and challenges

Keywords: Muscular dystrophy, dystrophin, antisense oligonucleotide, plasmid gene therapy, gene editing

Duchenne muscular dystrophy (DMD) is one of the most common lethal, hereditary diseases of childhood. Since the identification of the genetic basis of this disorder, there has been the hope that a cure would be developed in the form of gene therapy. This has yet to be realized, but many different gene therapy approaches have seen dramatic advances in recent years. Although viralmediated gene therapy has been at the forefront of the field, several non-viral gene therapy approaches have been applied to animal and cellular models of DMD. These include plasmidmediated gene delivery, antisense-mediated exon skipping, and oligonucleotide-mediated gene editing. In the past several years, non-viral gene therapy has moved from the laboratory to the clinic. Advances in vector design, formulation, and delivery are likely to lead to even more rapid advances in the coming decade. Given the relative simplicity, safety, and cost-effectiveness of these methodologies, non-viral gene therapy continues to have great promise for future gene therapy approaches for the treatment of DMD.

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INTRODUCTION

Duchenne muscular dystrophy (DMD) is a devastating disorder of childhood, leading to loss of ambulation during the first decade of life, progressive weakness, and death usually in the twenties [START_REF] Emery | Duchenne Muscular Dystrophy[END_REF]. The molecular basis of this disease is an absence of the dystrophin protein [START_REF] Hoffman | Dystrophin: the protein product of the Duchenne muscular dystrophy locus[END_REF]. At the genetic level, dystrophin deficiency arises from mutations in the dystrophin gene, but the range of mutations is enormous both in terms of types of mutations (deletions, duplications, inversions, point mutations) and in terms of the location of the mutation along the 2.5 Mb dystrophin gene [START_REF] Worton | Duchenne muscular dystrophy: gene and gene product; mechanism of mutation in the gene[END_REF][START_REF] Amalfitano | Structure and Mutation of the Dystrophin Gene[END_REF]. Despite the advances in molecular diagnoses, accepted treatment modalities remain limited to corticosteroids and physiotherapy, and treatment is largely supportive [START_REF] Emery | Duchenne Muscular Dystrophy[END_REF][START_REF] Chamberlain | Duchenne Muscular Dystrophy: Advances in Therapeutics[END_REF].

Experimental approaches for the treatment of DMD have greatly expanded in the past decade. Gene and cell therapies continue to be at the forefront of those approaches, but pharmacologic and hybrid methods have received considerable attention [START_REF] Chamberlain | Duchenne Muscular Dystrophy: Advances in Therapeutics[END_REF]. Traditionally, gene therapy has been divided into viral and non-viral, although this distinction also can be blurred as technologies are combined. Within the category of non-viral gene therapy methodologies, three different approaches have been pursued with respect to DMD. These are the use of plasmid DNA to deliver dystrophin cDNA constructs, the use of antisense oligonucleotides (AONs) to induce exon skipping of the dystrophin pre-mRNA to convert and out-of-frame transcript to an in-frame transcript, and the use of RNA-DNA chimeric oligonucleotides (RDOs) or oligodeoxynucleotides (ODNs) for genome editing or gene correction to alter single bases in the dystrophin gene to correct point mutations or to alter mRNA splicing. Reviews of the each of these technologies have been published in manuscript and chapter form [START_REF] Rando | Oligonucleotide-mediated gene therapy for muscular dystrophies[END_REF][START_REF] Aartsma-Rus | Targeted exon skipping as a potential gene correction therapy for Duchenne muscular dystrophy[END_REF][START_REF] Wilton | RNA splicing manipulation: strategies to modify gene expression for a variety of therapeutic outcomes[END_REF][START_REF] Wolff | The Intravascular Delivery of Naked DNA for Treating Duchenne Muscular Dystrophy[END_REF][START_REF] Bertoni | Oligonucleotide-Mediated Exon Skipping and Gene Editing for Duchenne Muscular Dystrophy[END_REF]. The focus of this review is to summarize the most recent technological developments in each of these fields, to Although plasmid-mediated gene delivery, AON-induced exon skipping, and oligonucleotide-mediated genome editing have unique promises and pitfalls (Table 1 and as described below), there are also commonalities in terms of advantages and disadvantages when compared to viral-mediated gene therapy approaches. In general, given current technologies, it is considered that non-viral vectors will represent a less expensive and safer mode of therapy. The cost of production of plasmids and oligonucleotides has dramatically fallen in the past decade, whereas the generation of clinical grade viral stocks has inherently high costs. Many safety issues remain to be determined for all forms of gene therapy, but concerns over potential adverse effects of viral-mediated gene therapy have been realized in terms of morbidity and mortality in human clinical trials [START_REF] Hacein-Bey-Abina | LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1[END_REF][START_REF] Raper | Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer[END_REF]. Despite the theoretical advantages of non-viral vectors in terms of safety, caution will be warranted as these approaches also move from laboratory animals to humans.

Perhaps the most obvious and important disadvantage of non-viral vectors is one of delivery, and here delivery is meant to include everything from the distribution via the circulation of the therapeutic vector to all skeletal muscles and the heart, the process by which the vectors leave the blood to reach muscle cells, the mechanisms by which the vectors enter the cells, and finally the processes by which the vectors reach the intracellular target localization.

Although non-viral vector development can improve upon the specificity and efficiency of these phases of delivery, viruses accomplish all of these aspects of delivery without any help from the investigator. As such, important recent developments that could apply to many or all non-viral

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approaches for DMD include advances in intra-arterial and intravenous delivery protocols [START_REF] Zhang | Intraarterial delivery of naked plasmid DNA expressing full-length mouse dystrophin in the mdx mouse model of duchenne muscular dystrophy[END_REF][START_REF] Danialou | Optimization of regional intraarterial naked DNA-mediated transgene delivery to skeletal muscles in a large animal model[END_REF][START_REF] Takeshima | Intraperitoneal administration of phosphorothioate antisense oligodeoxynucleotide against splicing enhancer sequence induced exon skipping in dystrophin mRNA expressed in mdx skeletal muscle[END_REF][START_REF] Takeshima | Intravenous infusion of an antisense oligonucleotide results in exon skipping in muscle dystrophin mRNA of Duchenne muscular dystrophy[END_REF][START_REF] Alter | Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology[END_REF], adjunct treatments to promote vascular permeability to facilitate the transfer of vectors (including viral vectors) from the circulation to the tissue bed [START_REF] Gregorevic | Systemic delivery of genes to striated muscles using adeno-associated viral vectors[END_REF][START_REF] Liang | Restoration of dystrophin expression in mdx mice by intravascular injection of naked DNA containing full-length dystrophin cDNA[END_REF][START_REF] Arruda | Regional intravascular delivery of AAV-2-F.IX to skeletal muscle achieves longterm correction of hemophilia B in a large animal model[END_REF], and formulations to enhance stability and cellular uptake of non-viral vectors [START_REF] Sirsi | Poly(ethylene imine)-poly(ethylene glycol) copolymers facilitate efficient delivery of antisense oligonucleotides to nuclei of mature muscle cells of mdx mice[END_REF][START_REF] Lu | Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse[END_REF][START_REF] Williams | Induction of dystrophin expression by exon skipping in mdx mice following intramuscular injection of antisense oligonucleotides complexed with PEG-PEI copolymers[END_REF]. Although some of the most promising results have been obtained by intra-arterial injections, and even though multiple arterial injections to reach a larger subset of muscles of a patient would be a viable approach, having an intravenous deliver system would be far less invasive.

Clearly immunological challenges are faced by all gene therapy approaches. The possibility of an immune response directed against the therapeutic protein is always a concern when the individual is deficient for that protein. In the case of dystrophin, both humoral and cellular immune responses have been detected in mdx mice following delivery of full-length dystrophin cDNAs, although not following the delivery of a minidystrophin construct [START_REF] Ferrer | Immune responses to dystropin: implications for gene therapy of Duchenne muscular dystrophy[END_REF][START_REF] Braun | Immune rejection of human dystrophin following intramuscular injections of naked DNA in mdx mice[END_REF].

This suggests that the response in an individual patient might depend on the specific epitopes in the protein expressed, either from an exogenous cDNA or from modulation of the endogenous gene or transcript. Studies of plasmid-mediated gene expression indicate that the likelihood of developing immune responses against the novel protein may be ameliorated by using musclespecific promoters, and that even transient immunosuppression around the time of plasmid delivery can limit the development of an immune response [START_REF] Zhang | Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates[END_REF] The other major immunological issue is of course the possibility of an immune response against the vector itself. Although certainly safer than viral vectors in this regard, non-viral gene therapy vectors still have the potential to induce immune responses, particularly if they contain bacterial DNA sequences with unmethylated CpG motifs [START_REF] Sato | Immunostimulatory DNA sequences necessary for effective intradermal gene immunization[END_REF]. Although intramuscular injections of plasmid or double-stranded oligonucleotides bearing such sequences may result in local

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inflammatory responses [START_REF] Mcmahon | Inflammatory responses following direct injection of plasmid DNA into skeletal muscle[END_REF], and repeated injections of plasmid have been shown to result in increases in circulating levels of anti-DNA antibodies [START_REF] Mor | Do DNA vaccines induce autoimmune disease?[END_REF], there have been no reports of autoimmune diseases resulting from such treatments. Whether low levels of circulating anti-DNA antibodies generated by treatment with non-viral vectors could attenuate subsequent systemic treatments with those vectors remains to be determined.

Finally, there is one other unknown that haunts the field of gene therapy for DMD, and that is the uncertainty of the normal turnover rate for myonuclei. Estimates of myonuclear turnover rate have been determined in certain muscle groups in experimental animals [START_REF] Mcloon | Continuous myofiber remodeling in uninjured extraocular myofibers: myonuclear turnover and evidence for apoptosis[END_REF][START_REF] Schmalbruch | Dynamics of nuclei of muscle fibers and connective tissue cells in normal and denervated rat muscles[END_REF],

but there are no reliable data on the turnover of myonuclei in the large muscles of the trunk and limbs in humans. Most technologies focus on the delivery of the gene therapy vector to the differentiated myofiber, and the efficiency of satellite cell targeting is rarely examined. In the worst case scenario, myonuclear turnover rate is high and the gene therapy approach fails to target satellite cells. In that case, the therapeutic effect will necessarily be transient, as

"corrected" myonuclei are replaced by nuclei from satellite cells that do not carry the therapeutic vector. However, examples of both viral and non-viral vectors capable of transducing satellite cells after intramuscular injections have been reported [START_REF] Bertoni | Dystrophin gene repair in mdx muscle precursor cells in vitro and in vivo mediated by RNA/DNA chimeric oligonucleotides[END_REF][START_REF] Kobinger | Correction of the dystrophic phenotype by in vivo targeting of muscle progenitor cells[END_REF]. Different pseudotyped lentiviral vectors differentially transduce satellite cells and muscle fibers [START_REF] Kobinger | Correction of the dystrophic phenotype by in vivo targeting of muscle progenitor cells[END_REF], thus demonstrating that satellite cells can be specifically targeted. For non-viral technologies, such targeting would necessarily involve a carrier that is modified so as to bind specifically to muscle cells, analogous to methods that have been proposed for modifying viral vectors to target muscle [START_REF] Feero | Selection and use of ligands for receptor-mediated gene delivery to myogenic cells[END_REF]. Whether such kinds of modifications could specifically target satellite cells remains to be determined. The Solutions to these common challenges will lead to advances in all aspects of non-viral gene therapy approaches to DMD. Still, each methodology will also have its "private" hurdles as well. In the following sections, the different non-viral gene therapy approaches that are currently under active investigation for DMD are reviewed, with attention to current challenges and future directions.

NON-VIRAL GENE THERAPY TECHNOLOGIES AND EXPERIMENTAL

THERAPEUTICS FOR DMD

Plasmid-Mediated Gene Delivery

Plasmid-mediated gene therapy is based on the observation made over a decade ago that naked plasmid DNA, delivered to skeletal muscle by direct injection, is taken up in the myofiber where plasmid genes are expressed [START_REF] Wolff | Direct gene transfer into mouse muscle in vivo[END_REF]. Various parameters such as plasmid size, concentration, and promoter sequences have been tested to optimize gene delivery options [START_REF] Wolff | Conditions affecting direct gene transfer into rodent muscle in vivo[END_REF][START_REF] Manthorpe | Gene therapy by intramuscular injection of plasmid DNA: studies on firefly luciferase gene expression in mice[END_REF][START_REF] Bartlett | Long-term expression of a fluorescent reporter gene via direct injection of plasmid vector into mouse skeletal muscle: comparison of human creatine kinase and CMV promoter expression levels in vivo[END_REF][START_REF] Wells | Evaluation of plasmid DNA for in vivo gene therapy: factors affecting the number of transfected fibers[END_REF]. Advantages of plasmid gene therapy include already existing scaled-up production processes, applicability to DMD patients regardless of mutation, and simplicity. Following the initial observations that dystrophin protein could be expressed in muscles of mdx mice after simple intramuscular injections of plasmid [START_REF] Acsadi | Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs[END_REF][START_REF] Danko | Dystrophin expression improves myofiber survival in mdx muscle following intramuscular plasmid DNA injection[END_REF], much effort was devoted to improved efficacy of distribution, uptake, and expression of injected plasmids. Improvements involved adjunctive treatments, notably using electroporation and ultrasound, co-injecting enzymes such as hyaluronidase, and using non-ionic carriers for plasmid DNA [START_REF] Aihara | Gene transfer into muscle by electroporation in vivo[END_REF][START_REF] Taniyama | Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle[END_REF][START_REF] Schratzberger | Transcutaneous ultrasound augments naked DNA transfection of skeletal muscle[END_REF][START_REF] Danialou | Ultrasound increases plasmid-mediated gene transfer to dystrophic muscles without collateral damage[END_REF][START_REF] Mcmahon | Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase -increased expression with reduced muscle damage[END_REF][START_REF] Mennuni | Hyaluronidase increases electrogene transfer efficiency in skeletal muscle[END_REF][START_REF] Lemieux | A combination of poloxamers increases gene expression of plasmid DNA in skeletal muscle[END_REF]. Of these, electroporation leads to the most

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dramatic enhancement of gene expression, although there is significant muscle damage that results [START_REF] Hartikka | Electroporation-facilitated delivery of plasmid DNA in skeletal muscle: plasmid dependence of muscle damage and effect of poloxamer 188[END_REF].

Multiple intramuscular injections with electroporation would, however, be an extremely laborious and invasive method for the treatment of all muscles of a patient, not to mention the challenge of delivering plasmid to the heart and diaphragm by this method. As such, attention has focused more recently on regional or systemic delivery of plasmids. Intra-arterial delivery of plasmid in mdx mice resulted in expression of dystrophin in 1-5% of fibers in the injected limb [START_REF] Zhang | Intraarterial delivery of naked plasmid DNA expressing full-length mouse dystrophin in the mdx mouse model of duchenne muscular dystrophy[END_REF]. This approach has been used to deliver plasmids to limb muscles in non-human primates with even higher efficiencies [START_REF] Zhang | Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates[END_REF].

Given the encouraging results of plasmid-mediated gene therapy in animals combined

with technological advances and the potential of systemic delivery, this experimental therapeutic approach has moved from the laboratory to the clinic. A phase I clinical trial was initiated recently with the overall design of either a single injection or two injections of plasmid containing full-length dystrophin cDNA into single muscles of a group of 9 boys with DMD or Becker muscular dystrophy (BMD) [START_REF] Romero | Current protocol of a research phase I clinical trial of full-length dystrophin plasmid DNA in Duchenne/Becker muscular dystrophies. Part II: clinical protocol[END_REF]. The trial was designed to test for any adverse effects of low dose intramuscular plasmid injections and for immunological responses. No adverse effects were noted. In follow up biopsies three weeks after injections, dystrophin protein was detected in only 6 of the 9 boys and in only a small percentage of fibers, but no signs of an immunological response was observed [START_REF] Romero | Phase I study of dystrophin plasmid-based gene therapy in Duchenne/Becker muscular dystrophy[END_REF][START_REF] Fardeau | About a phase I gene therapy clinical trial with a full-length dystrophin gene-plasmid in Duchenne/Becker muscular dystrophy[END_REF].

As clinical trials progress, studies designed to improve the efficiency and efficacy of plasmid-mediated gene therapy continue. An unresolved issue for treatment of humans with DMD is the expected duration of plasmid gene expression. In mice, transgene expression can be detected for many months after direct intramuscular injection in normal muscle. However, there

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is clearly a continual loss of plasmid over this time [START_REF] Molnar | Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles[END_REF]. In humans, sustained expression would be necessary for years and decades. Therefore, any technological advance that would lead to plasmid persistence is likely to be an important adjunct to plasmid-mediated gene therapy for DMD. Recently, a targeted plasmid integration approach has been used to address this issue (Fig. 1). This approach is based on the finding that the co-expression of an integrase with a plasmid that contains one of the integrase recognition sequences allows for integration of the plasmid into the mammalian genome [START_REF] Thyagarajan | Site-specific genomic integration in mammalian cells mediated by phage φC31 integrase[END_REF]. This integrase technology was applied to the delivery of dystrophin-expressing plasmids to mdx muscle, and plasmid integration resulted in more sustained dystrophin expression [START_REF] Bertoni | Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration[END_REF]. An increased expression of dystrophin was seen at each time point examined, with the difference increasing with time, demonstrating the importance of integration for sustained plasmid gene expression [START_REF] Bertoni | Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration[END_REF]. The integrase-mediated enhancement of long-term gene expression in skeletal muscle was also evident when tested with plasmids expressing vascular endothelial growth factor [START_REF] Portlock | Loong-term increase in mVEGF164 in mouse hindlimb muscle mediated by phage φC31 integrase after nonviral DNA delivery[END_REF] Ideally, this technology would be truly site-specific, introducing the dystrophin plasmid into a single site in the genome, but the currently used integrases clearly integrate at multiple sites even though there are clear 'hot spots' [START_REF] Bertoni | Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration[END_REF][START_REF] Olivares | Sitespecific genomic integration produces therapeutic Factor IX levels in mice[END_REF]. Therefore, there is the theoretical risk of insertional mutagenesis, which may have resulted in the development of leukemias in three patients in a clinical trial of retroviral-mediated gene therapy for X-linked severe combined immune deficiency [START_REF] Hacein-Bey-Abina | LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1[END_REF]. However, not only does recent evidence suggest that those leukemias may have resulted from expression of the therapeutic gene as opposed to gene disruption from vector integration [START_REF] Woods | Gene therapy: therapeutic gene causing lymphoma[END_REF], but this risk is of course very low when postmitotic myonuclei are the targets as opposed to hematopoietic progenitors.

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Finally, an issue that has come to light in studies of plasmid-mediated gene therapy, even though this would apply to all gene therapy approaches, is the distribution of the vector and thus the therapeutic protein, along the length of muscle fibers in a targeted muscle. Many studies of long-term efficacy of plasmid-mediated gene delivery have been quantitated by measuring the number of muscle fibers that express the transgene over time [START_REF] Danko | Dystrophin expression improves myofiber survival in mdx muscle following intramuscular plasmid DNA injection[END_REF][START_REF] Doh | Spatial-temporal patterns of gene expression in mouse skeletal muscle after injection of lacZ plasmid DNA[END_REF]. This unfortunately underestimates protein loss; direct comparisons reveal that the total protein levels can be declining while the number of fibers that stain positively for the protein remains relatively constant since fibers with lower, but still detectable, levels of transgene expression are scored as positive [START_REF] Molnar | Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles[END_REF]. The situation is even more critical in dystrophic muscle because of the issue of segmental necrosis. Any fiber segment that does not express therapeutic levels of dystrophin is susceptible to degeneration which can then encroach upon segments with marginally therapeutic levels. Thus, any fiber in which dystrophin is not expressed at therapeutic levels along its length will eventually succumb. This was clear in studies of integrase-enhancement of plasmid gene therapy where it was found that the increased dystrophin expression and persistence by integration results in a higher number of fibers with therapeutic dystrophin levels along their entire lengths [START_REF] Bertoni | Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration[END_REF]. This finding highlights the importance of quantifying dystrophin expression not only in the muscle cross-sections but also along the longitudinal axis of the muscle.

AON-Induced Exon Skipping

AONs are composed of RNA residues or homologs and have been primarily used to block translation by binding to mRNA and leading to its degradation [START_REF] Good | Translation repression by antisense sequences[END_REF]. However, it was also noted that AONs targeted to splice sites or splicing regulatory regions of pre-mRNA could alter the splicing of that transcript, presumably by interfering with the normal splicing machinery

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11 [START_REF] Kole | Antisense effects in the cell nucleus: modification of splicing[END_REF]. The rationale for the therapeutic benefit of modifications of splicing in the case of DMD is based on the fact that most cases of DMD are caused by deletions in the dystrophin gene that lead to frame-shift mutations in the transcript [START_REF] Koenig | The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion[END_REF]. Deletions that do not cause a frame shift but simply an internally deleted protein tend to cause the milder, allelic disease, BMD. Therefore, it was hypothesized that if one could alter the splicing of a DMD gene to produce a shorter but inframe transcript that this would be a method to convert a severe DMD phenotype into a milder BMD phenotype (Fig. 2).

Proof-of-principle studies were initially published about a decade ago [START_REF] Wilton | Dystrophin gene transcripts skipping the mdx mutation[END_REF][START_REF] Dunckley | Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides[END_REF]. Progress was rapid in terms of improved vector design, target choice, and oligonucleotide chemistries, all leading to more efficient and effective exon skipping [START_REF] Mann | Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy[END_REF][START_REF] Errington | Target selection for antisense oligonucleotide induced exon skipping in the dystrophin gene[END_REF][START_REF] Aartsma-Rus | Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells[END_REF][START_REF] Aartsma-Rus | Functional analysis of 114 exon-internal AONs for targeted DMD exon skipping: indication for steric hindrance of SR protein binding sites[END_REF]. Dystrophin expression was demonstrated using AONs delivered to dystrophin-deficient muscle cells in vivo in the mdx mouse or in vitro [START_REF] Wilton | Specific removal of the nonsense mutation from the mdx dystrophin mRNA using antisense oligonucleotides[END_REF][START_REF] Van Deutekom | Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells[END_REF][START_REF] Mann | Antisenseinduced exon skipping and synthesis of dystrophin in the mdx mouse[END_REF]. Further developments toward clinical application have included methodologies, as mentioned above, to enhance the uptake of AONs in mouse muscle in vivo and the use of systemic delivery methods to begin to develop a clinically feasible delivery approach [START_REF] Alter | Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology[END_REF][START_REF] Sirsi | Poly(ethylene imine)-poly(ethylene glycol) copolymers facilitate efficient delivery of antisense oligonucleotides to nuclei of mature muscle cells of mdx mice[END_REF][START_REF] Lu | Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse[END_REF][START_REF] Williams | Induction of dystrophin expression by exon skipping in mdx mice following intramuscular injection of antisense oligonucleotides complexed with PEG-PEI copolymers[END_REF] The application of AONs to mediate exon skipping in DMD has progressed remarkably quickly from the laboratory to the clinic. Phase I clinical trials with AONs in humans are in early stages in Britain and Holland [START_REF] Wilton | Modification of pre-mRNA processing: application to dystrophin expression[END_REF]. These will involve intramuscular injections of AONs to test for safety, and it is likely that some measures of efficacy will be included in the analysis. If these phase I trials demonstrate safety, the challenge will be to scale those up for targeting large muscles, whole limbs, relatively inaccessible targets like the diaphragm and heart, and ultimately the entire body musculature. It will be at that point that the translation of systemic delivery methodologies from laboratory animals to humans will need to be tested.

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12 At the same time, the development of new generations of AONs could further enhance therapeutic efficacy. Currently, the chemistries that are being tested are 2'O-methyl phosphorothioate and morpholino-phosphorodiamidate AONs (Fig. 3). Other chemistries that have been tested include AONs containing peptide nucleic acids, locked nucleic acid, and ethylene-bridged nucleic acid backbones [START_REF] Aartsma-Rus | Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells[END_REF][START_REF] Surono | Chimeric RNA/ethylene-bridged nucleic acids promote dystrophin expression in myocytes of duchenne muscular dystrophy by inducing skipping of the nonsense mutation-encoding exon[END_REF]. Direct comparisons in terms of efficacy and toxicity in vivo will be required to determine the optimal chemistry for clinical trials.

One of the challenges of AON therapy is that it is not a 'one-size-fits-all' approach in the same way that gene delivery is. In order for an AON approach to be applicable, there needs to be an accurate molecular diagnosis, a rational plan for skipping one or more exons to produce an inframe transcript, evidence that targeting AONs to specific sequences will result in the desired transcript, and finally the design of AON vectors specifically for that particular mutation. This seemingly major hurdle of customized therapy is partially obviated by the fact there are certain deletions that are very common and that a relatively small number of deletions represent a majority of cases of DMD. Thus, a limited number of AONs will theoretically be effective in a majority of DMD patients [START_REF] Aartsma-Rus | Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients[END_REF]. In those cases where skipping of two exons would be necessary to generate an in-frame transcript, multiple AON delivery could be used as has been demonstrated [START_REF] Aartsma-Rus | Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense[END_REF]. Of course, without a defined molecular diagnosis, AON therapy is not applicable.

Finally, one of the intrinsic limitations of AON-mediated exon skipping is the fact that the therapeutic effect is transient. AONs are delivered as oligonucleotides which are unstable in the cell. In mice, detectable levels of dystrophin expression were obtained by sequential AON injections, but levels declined with a half-life of approximately 2-4 months, and this could even be an overestimate of AON stability as it may be more of a reflection of dystrophin protein

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ACCEPTED MANUSCRIPT 13 stability. Even so, protein persistence is the key determinant of the duration of therapeutic efficacy and is thus the meaningful measure. As currently configured, this would mean that AON therapy would require multiple treatments each year. While not untenable, this is clearly a serious hurdle to overcome. In mice, investigators have combined AON technology with viral expression approaches to achieve sustained expression of antisense vectors and long-term exon skipping [START_REF] Denti | Body-wide gene therapy of Duchenne muscular dystrophy in the mdx mouse model[END_REF][START_REF] De Angelis | Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Delta 48-50 DMD cells[END_REF][START_REF] Goyenvalle | Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping[END_REF]. However, this introduces the challenges of viral gene therapy, and there is no clear advantage, if one is using viral vectors, to deliver an antisense vector as opposed to delivering a dystrophin construct itself. Another possibility on the horizon is to introduce a short cDNA encoding U7-small nuclear RNA to achieve sustained exon skipping [START_REF] Brun | U7 snRNAs induce correction of mutated dystrophin pre-mRNA by exon skipping[END_REF]Thus, the technological advances that will be critical for long-term AON therapy will come in the form of oligonucleotide modification or formulation that promotes very long-term stability or sustained in vivo delivery.

Oligonucleotide-Mediated Genome Editing

The use of targeting oligonucleotides to induce single base changes in genomic DNA developed from initial studies in homologous pairing and repair activities in bacteria and simple eukaryotes [START_REF] Kotani | A role for RNA synthesis in homologous pairing events[END_REF][START_REF] Kmiec | The REC2 gene encodes the homologous pairing protein of Ustilago maydis[END_REF]. The theoretical basis for this approach is that engineered mismatches between targeting vector and genome sequence could, through endogenous DNA repair mechanisms, potentially promote targeted single base pair changes in genomic DNA [START_REF] Ye | Targeted gene correction: a new strategy for molecular medicine[END_REF][START_REF] Kren | Gene repair using chimeric RNA/DNA oligonucleotides[END_REF][START_REF] Gamper | A plausible mechanism for gene correction by chimeric oligonucleotides[END_REF][START_REF] Igoucheva | Targeted gene correction by small single-stranded oligonucleotides in mammalian cells[END_REF].

The possibility that such genome editing potential could be applied to the correction of point mutations that cause disease was compelling (Fig. 4). As such, this technology has been applied to a wide variety of models of diseases arising from point mutations in different genes [START_REF] Cole-Strauss | Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide[END_REF][START_REF] Kren | In vivo site-directed mutagenesis of the factor IX gene by chimeric RNA/DNA oligonucleotides[END_REF][START_REF] Tagalakis | Gene correction of the apolipoprotein (apo) E2 phenotype to wild-type apoE3 by in situ chimeraplasty[END_REF].

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14 Upwards of 15% of patients with DMD have point mutations as the cause of dystrophin deficiency [START_REF] Amalfitano | Structure and Mutation of the Dystrophin Gene[END_REF]. Fortuitously for this particular application, the most commonly used animal model of DMD, the mdx mouse, also has a point mutation that results in a stop codon as the cause of dystrophin deficiency. Therefore, the mdx mouse has served as an excellent model with which to test oligonucleotide-mediated dystrophin gene editing. Initial studies using RDOs and subsequent studies using ODNs demonstrated that the point mutation in the mdx dystrophin gene can indeed be corrected by targeting oligonucleotides [START_REF] Bertoni | Dystrophin gene repair in mdx muscle precursor cells in vitro and in vivo mediated by RNA/DNA chimeric oligonucleotides[END_REF][START_REF] Rando | Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides[END_REF][START_REF] Bertoni | Strand bias in oligonucleotide-mediated dystrophin gene editing[END_REF]. Correction was demonstrated at the genomic and transcript levels, and restoration of dystrophin protein expression was observed both in vivo and in vitro. In addition, RDOs were shown to be able to correct the point mutation in a canine model of DMD [START_REF] Bartlett | In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide[END_REF].

The application of this type of technology to diseases that result from point mutations is clear, but broader applications have been envisioned whereby single bases in splice sites are altered at the genomic level, thereby inducing alternate splicing to effect the same result as that achieved by AONs, namely the generation of an in-frame transcript from an out-of-frame transcript. This approach has been demonstrated in the mdx mouse by inducing skipping of the exon that contains the point mutation [START_REF] Bertoni | Restoration of dystrophin expression in mdx muscle cells by chimeraplast-mediated exon skipping[END_REF]. As described for AONs, oligonucleotide-mediated gene editing also requires individualization since the targeted base will depend on the location of the point mutation or the particular splice site to be targeted.

Other than the challenges common to all non-viral methodologies discussed in the Introduction, the major challenge for oligonucleotide-mediated gene editing is the low efficiency of this technology with current vectors that has been observed a variety of different disease applications [START_REF] Cole-Strauss | Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide[END_REF][START_REF] Kren | In vivo site-directed mutagenesis of the factor IX gene by chimeric RNA/DNA oligonucleotides[END_REF]. The was the case with the first generation RDO vectors, but has also been found with the second generation vectors, the ODNs, which yield more consistent results

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M A N U S C R I P T [START_REF] Igoucheva | Targeted gene correction by small single-stranded oligonucleotides in mammalian cells[END_REF][START_REF] Liu | In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides[END_REF]. Both RDOs and ODNs have been shown to be effective in vitro and in vivo to induce single base changes, but the efficiencies have generally been in the 1-5% range, with higher efficiencies being observed only under specific conditions [START_REF] Cole-Strauss | Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide[END_REF][START_REF] Rando | Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides[END_REF][START_REF] Bertoni | Strand bias in oligonucleotide-mediated dystrophin gene editing[END_REF][START_REF] Bertoni | Restoration of dystrophin expression in mdx muscle cells by chimeraplast-mediated exon skipping[END_REF][START_REF] Yoon | Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA•DNA oligonucleotide[END_REF][START_REF] Kren | Targeted nucleotide exchange in the alkaline phosphatase gene of HuH-7 cells mediated by a chimeric RNA/DNA oligonucleotide[END_REF][START_REF] Kren | Correction of the UDP-glucuronosyltransferase gene defect in the Gunn rat model of Crigler-Najjar syndrome type I with a chimeric oligonucleotide[END_REF][START_REF] Igoucheva | A sequence-specific gene correction by an RNA-DNA oligonucleotide in mammalian cells characterized by transfection and nuclear extract using a lacZ shuttle system[END_REF][START_REF] Alexeev | Localized in vivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide[END_REF][START_REF] Parekh-Olmedo | Targeted gene repair in mammalian cells using chimeric RNA/DNA oligonucleotides and modified single-stranded vectors[END_REF]. Therefore, current research is focusing on the enhancement of the molecular mechanisms that mediate the gene editing, and these studies include both modifications of the oligonucleotides as well as enzyme systems and cellular processes involved in the repair mechanism [START_REF] Igoucheva | Involvement of ERCC1/XPF and XPG in oligodeoxynucleotide-directed gene modification[END_REF][START_REF] Ferrara | Targeted gene repair activates Chk1 and Chk2 and stalls replication in corrected cells[END_REF][START_REF] Bertoni | Enhanced level of gene correction mediated by oligonucleotides containing CpG modification in the mdx mouse model for Duchenne muscular dystrophy[END_REF].

Improvements in genome editing efficiency is currently the most important hurdle for the consideration of a phase I clinical trial of ODNs in humans similar to those conducted or planned for plasmids and AONs.

Perhaps the greatest advantage of the gene editing approach is that it results in a permanent correction of the genetic defect in myonuclei. Therefore, the therapeutic benefit is sustained as long as the targeted myonuclei persist. The only decrement of therapeutic efficacy is that which would accompany the turnover of myonuclei and replacement by nuclei derived from satellite cells, as noted in the Introduction. However, it is possible that even this could be less of an issue for oligonucleotide-mediated gene editing than for non-viral methodologies. Although not quantitative, it was shown that targeting oligonucleotides injected into skeletal muscles of mdx mice were capable of inducing gene correction in satellite cells subsequently isolated from those muscles [START_REF] Bertoni | Dystrophin gene repair in mdx muscle precursor cells in vitro and in vivo mediated by RNA/DNA chimeric oligonucleotides[END_REF]. To the extent that ODNs could be directed both to satellite cells as well as to myofibers could, long-term permanent gene correction could be achieved. The nonsense mutation of the mdx mouse is shown as an example, but the same principles apply for deletions that generate out-of-frame transcripts in which skipping one or more exons can generate an in-frame transcript. An AON targeted to exonic splice enhancer sequences is shown.

CONCLUSIONS

AONs targeted to 5' or 3' splice sites are also effective in inducing skipping of the specific exon. a Efficiency is a difficult parameter to measure and compare because measures of efficiency vary widely. Based on in vivo type experiments in which the primary outcome is the generation of dystrophin-positive fibers, both plasmid-mediated gene delivery and AON-induced exon skipping yield much higher numbers than oligonucleotide-mediated gene editing. Of course, none of these measures are normalized to vector number or concentration in the cell. b AON-induced exon skipping results in transient dystrophin expression, plasmid-mediated gene delivery results on sustained dystrophin expression, and oligonucleotide-mediated gene editing results in permanent correction. c In general, the smaller oligonucleotides used for exon skipping and gene editing are more readily taken up into muscle fibers that are large plasmids, even when plasmids are formulated with agents to enhance uptake. d The expression of dystrophin from a plasmid is an effective treatment regardless of the nature of the endogenous gene mutation. By contrast, both AON-induced exon skipping and oligonucleotide-mediated gene editing require vectors specifically designed for the underlying mutation. However, certain AONs will be applicable to a number of different deletion mutations.

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5' 5' 3' 3'

  promising and important research directions, to enumerate specific hurdles and challenges faced by each technology, and to discuss the status of nascent clinical trials in humans.

  importance of satellite cell targeting by gene therapy vectors for sustained therapeutic efficacy may ultimately await empirical determination in clinical trials or even long-term clinical A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 7 applications since studies in animals may poorly reflect the dynamics of myonuclear turnover in humans.

  ABBREVIATIONS

Figure 1 . 29 Figure 2 .

 1292 Figure 1. Targeted plasmid integration to enhance plasmid-mediated gene delivery.

Figure 3 .

 3 Figure 3. Chemical structures of modified bases used in AON design.

Figure 4 .

 4 Figure 4. Molecular mechanism of ODN-mediated gene editing.

Figure 1 A

 1 Figure 1

Figure 2 A

 2 Figure 2

Figure 3 A-

 3 Figure 3

  Figure 4

TABLE 1

 1

		Plasmid-mediated	Antisense-induced	Oligonucleotide-mediated
		gene delivery	exon skipping	gene editing
	Efficiency of inducing	+++	+++	+
	dystrophin expression a
	Duration of effect b	+++	+	++++
	Uptake of vector after	++	++++	++++
	delivery to tissue c
	Applicability regardless of	++++	++	+
	mutation d

ACKNOWLEDGEMENTS

Supported by grants from the NIH (NIH Director's Pioneer Award) and the Muscular Dystrophy Association.

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