Dystrophin and mini-dystrophin quantification by mass spectrometry in skeletal muscle for gene therapy development in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal, degenerative muscle disorder caused by mutations in the DMD gene, leading to severe reduction or absence of the protein dystrophin. Gene therapy strategies that aim to increase expression of a functional dystrophin protein (mini-dystrophin) are under investigation. The ability to accurately quantify dystrophin/mini-dystrophin is essential in assessing the level of gene transduction. We demonstrated the validation and application of a novel peptide immunoaffinity liquid chromatography-tandem mass spectrometry (IA-LC-MS/MS) assay. Data showed that dystrophin expression in Becker muscular dystrophy and DMD tissues, normalized against the mean of non-dystrophic control tissues (n = 20), was 4-84.5% (mean 32%, n = 20) and 0.4-24.1% (mean 5%, n = 20), respectively. In a DMD rat model, biceps femoris tissue from dystrophin-deficient rats treated with AAV9.hCK.Hopti-Dys3978.spA, an adeno-associated virus vector containing a mini-dystrophin transgene, showed a dose-dependent increase in mini-dystrophin expression at 6 months post-dose, exceeding wildtype dystrophin levels at high doses. Validation data showed that inter- and intra-assay precision were ≤20% (≤25% at the lower limit of quantification [LLOQ]) and inter- and intra-run relative error was within ±20% (±25% at LLOQ). IA-LC-MS/MS accurately quantifies dystrophin/mini-dystrophin in human and preclinical species with sufficient sensitivity for immediate application in preclinical/clinical trials.

Evaluation of the dystrophin carboxy-terminal domain for micro-dystrophin gene therapy in cardiac and skeletal muscles in the DMDmdx rat model.

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the gene encoding dystrophin. Gene therapy using micro-dystrophin (MD) transgenes and recombinant adeno-associated virus (rAAV) vectors hold great promise. To overcome the limited packaging capacity of rAAV vectors, most MD do not include dystrophin carboxy-terminal (CT) domain. Yet, the CT domain is known to recruit α1- and ß1-syntrophins and α-dystrobrevin, a part of the dystrophin-associated protein complex (DAPC), which is a signaling and structural mediator of muscle cells. In this study, we explored the impact of inclusion of the dystrophin CT domain on ΔR4-23/ΔCT MD (MD1), in DMDmdx rats, which allows for relevant evaluations at muscular and cardiac levels. We showed by LC-MS/MS that MD1 expression is sufficient to restore the interactions at a physiological level of most DAPC partners in skeletal and cardiac muscles, and that inclusion of the CT domain increases the recruitment of some DAPC partners at supra-physiological levels. In parallel, we demonstrated that inclusion of the CT domain does not improve MD1 therapeutic efficacy on DMD muscle and cardiac pathologies. Our work highlights new evidences of the therapeutic potential of MD1 and strengthens the relevance of this candidate for gene therapy of DMD.

TRPC3, but not TRPC1, as a good therapeutic target for standalone or complementary treatment of DMD.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked inherited disease caused by mutations in the gene encoding dystrophin that leads to a severe and ultimately life limiting muscle-wasting condition. Recombinant adeno-associated vector (rAAV)-based gene therapy is promising, but the size of the full-length dystrophin cDNA exceeds the packaging capacity of a rAAV. Alternative or complementary strategies that could treat DMD patients are thus needed. Intracellular calcium overload due to a sarcolemma permeability to calcium (SPCa) increase is an early and critical step of the DMD pathogenesis. We assessed herein whether TRPC1 and TRPC3 calcium channels may be involved in skeletal muscle SPCa alterations and could represent therapeutic targets to treat DMD. METHODS: All experiments were conducted in the DMDmdx rat, an animal model that closely reproduces the human DMD disease. We measured the cytosolic calcium concentration ([Ca2+]c) and SPCa in EDL (Extensor Digitorum Longus) muscle fibers from age-matched WT and DMDmdx rats of 1.5 to 7 months old. TRPC1 and TRPC3 expressions were measured in the EDL muscles at both the mRNA and protein levels, by RT-qPCR, western blot and immunocytofluorescence analysis. RESULTS: As expected from the malignant hyperthermia like episodes observed in several DMDmdx rats, calcium homeostasis alterations were confirmed by measurements of early increases in [Ca2+]c and SPCa in muscle fibers. TRPC3 and TRPC1 protein levels were increased in DMDmdx rats. This was observed as soon as 1.5 months of age for TRPC3 but only at 7 months of age for TRPC1. A slight but reliable shift of the TRPC3 apparent molecular weight was observed in DMDmdx rat muscles. Intracellular localization of both channels was not altered. We thus focused our attention on TRPC3. Application of Pyr10, a specific inhibitor of TRPC3, abolished the differences between SPCa values measured in WT and DMDmdx. Finally, we showed that a rAAV-microdystrophin based treatment induced a high microdystrophin expression but only partial prevention of calcium homeostasis alterations, skeletal muscle force and TRPC3 protein increase. CONCLUSIONS: All together our results show that correcting TRPC3 channel expression and/or activity appear to be a promising approach as a single or as a rAAV-based complementary therapy to treat DMD.

Dystrophic Dmdmdx rats show early neuronal changes (increased S100ß and Tau5) at 8 months, supporting severe dystropathology in this rodent model of Duchenne muscular dystrophy.

The intrinsic necrosis of skeletal muscles in animal models of Duchenne muscular dystrophy (DMD) damages neuromuscular junctions (NMJs) with progressively altered NMJs associated with denervation and premature changes in dystrophic nerves. In the mdx mouse model of DMD, the proteins S100ß and Tau5 are significantly increased in sciatic nerves by 13 months (M) of age, far earlier (by 9 M) than in normal wildtype (WT) nerves. Since dystrophic Dmdmdx rats are reported to have a more severe dystropathology than mdx mice, we hypothesised that Dmdmdx rat nerves would show earlier neuronal changes compared with mdx nerves. We quantified levels of 8 proteins (by immunoblotting) in sciatic and radial nerves from young adult Dmdmdx rats (aged 8 M) and mdx mice (9 M), plus levels of 7 mRNAs (by qPCR) in rat nerves only. Sciatic nerves of 8 M Dmdmdx rats had more consistently increased levels of S100ß and Tau5 proteins, compared with 9 M mdx mice, supporting pronounced dystropathology in the rat model. There were no differences for mRNA levels, apart from higher gelsolin mRNA in Dmdmdx sciatic nerves. The pronounced protein changes in Dmdmdx nerves indicate a severe ongoing myonecrosis, and likely consequent myofibre denervation, for the dystrophic rat model. These data support increased neuronal proteins in dystrophic nerves as a novel pre-clinical readout of ongoing myonecrosis for DMD research. In older DMD boys, such progressive neuronal changes over many years are likely to contribute to loss of muscle function, and may complicate evaluation of late-onset clinical therapies.

Gait characterization in golden retriever muscular dystrophy dogs using linear discriminant analysis.

BACKGROUND: Accelerometric analysis of gait abnormalities in golden retriever muscular dystrophy (GRMD) dogs is of limited sensitivity, and produces highly complex data. The use of discriminant analysis may enable simpler and more sensitive evaluation of treatment benefits in this important preclinical model. METHODS: Accelerometry was performed twice monthly between the ages of 2 and 12 months on 8 healthy and 20 GRMD dogs. Seven accelerometric parameters were analysed using linear discriminant analysis (LDA). Manipulation of the dependent and independent variables produced three distinct models. The ability of each model to detect gait alterations and their pattern change with age was tested using a leave-one-out cross-validation approach. RESULTS: Selecting genotype (healthy or GRMD) as the dependent variable resulted in a model (Model 1) allowing a good discrimination between the gait phenotype of GRMD and healthy dogs. However, this model was not sufficiently representative of the disease progression. In Model 2, age in months was added as a supplementary dependent variable (GRMD_2 to GRMD_12 and Healthy_2 to Healthy_9.5), resulting in a high overall misclassification rate (83.2%). To improve accuracy, a third model (Model 3) was created in which age was also included as an explanatory variable. This resulted in an overall misclassification rate lower than 12%. Model 3 was evaluated using blinded data pertaining to 81 healthy and GRMD dogs. In all but one case, the model correctly matched gait phenotype to the actual genotype. Finally, we used Model 3 to reanalyse data from a previous study regarding the effects of immunosuppressive treatments on muscular dystrophy in GRMD dogs. Our model identified significant effect of immunosuppressive treatments on gait quality, corroborating the original findings, with the added advantages of direct statistical analysis with greater sensitivity and more comprehensible data representation. CONCLUSIONS: Gait analysis using LDA allows for improved analysis of accelerometry data by applying a decision-making analysis approach to the evaluation of preclinical treatment benefits in GRMD dogs.

Serum profiling identifies novel muscle miRNA and cardiomyopathy-related miRNA biomarkers in Golden Retriever muscular dystrophy dogs and Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy (DMD) is a fatal, X-linked neuromuscular disease that affects 1 boy in 3500 to 5000 boys. The golden retriever muscular dystrophy dog is the best clinically relevant DMD animal model. Here, we used a high-thoughput miRNA sequencing screening for identification of candidate serum miRNA biomarkers in golden retriever muscular dystrophy dogs. We confirmed the dysregulation of the previously described muscle miRNAs, miR-1, miR-133, miR-206, and miR-378, and identified a new candidate muscle miRNA, miR-95. We identified two other classes of dysregulated serum miRNAs in muscular dystrophy: miRNAs belonging to the largest known miRNA cluster that resides in the imprinting DLK1-DIO3 genomic region and miRNAs associated with cardiac disease, including miR-208a, miR-208b, and miR-499. No simple correlation was identified between serum levels of cardiac miRNAs and cardiac functional parameters in golden retriever muscular dystrophy dogs. Finally, we confirmed a dysregulation of miR-95, miR-208a, miR-208b, miR-499, and miR-539 in a small cohort of DMD patients. Given the interspecies conservation of miRNAs and preliminary data in DMD patients, these newly identified dysregulated miRNAs are strong candidate biomarkers for DMD patients.

Liver-specific transcriptional modules identified by genome-wide in silico analysis enable efficient gene therapy in mice and non-human primates.

The robustness and safety of liver-directed gene therapy can be substantially improved by enhancing expression of the therapeutic transgene in the liver. To achieve this, we developed a new approach of rational in silico vector design. This approach relies on a genome-wide bio-informatics strategy to identify cis-acting regulatory modules (CRMs) containing evolutionary conserved clusters of transcription factor binding site motifs that determine high tissue-specific gene expression. Incorporation of these CRMs into adeno-associated viral (AAV) and non-viral vectors enhanced gene expression in mice liver 10 to 100-fold, depending on the promoter used. Furthermore, these CRMs resulted in robust and sustained liver-specific expression of coagulation factor IX (FIX), validating their immediate therapeutic and translational relevance. Subsequent translational studies indicated that therapeutic FIX expression levels could be attained reaching 20-35% of normal levels after AAV-based liver-directed gene therapy in cynomolgus macaques. This study underscores the potential of rational vector design using computational approaches to improve their robustness and therefore allows for the use of lower and thus safer vector doses for gene therapy, while maximizing therapeutic efficacy.

Forelimb treatment in a large cohort of dystrophic dogs supports delivery of a recombinant AAV for exon skipping in Duchenne patients.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the dystrophin gene, without curative treatment yet available. Our study provides, for the first time, the overall safety profile and therapeutic dose of a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping to restore a functional in-frame dystrophin transcript, and injected by locoregional transvenous perfusion of the forelimb. Eighteen Golden Retriever Muscular Dystrophy (GRMD) dogs were exposed to increasing doses of GMP-manufactured vector. Treatment was well tolerated in all, and no acute nor delayed adverse effect, including systemic and immune toxicity was detected. There was a dose relationship for the amount of exon skipping with up to 80% of myofibers expressing dystrophin at the highest dose. Similarly, histological, nuclear magnetic resonance pathological indices and strength improvement responded in a dose-dependent manner. The systematic comparison of effects using different independent methods, allowed to define a minimum threshold of dystrophin expressing fibers (>33% for structural measures and >40% for strength) under which there was no clear-cut therapeutic effect. Altogether, these results support the concept of a phase 1/2 trial of locoregional delivery into upper limbs of nonambulatory DMD patients.

Dystrophin deficiency impairs cell junction formation during embryonic myogenesis from pluripotent stem cells.

Mutations in the DMD gene lead to Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder affecting young boys as they acquire motor functions. DMD is typically diagnosed at 2-4 years of age, but the absence of dystrophin has negative impacts on skeletal muscles before overt symptoms appear in patients, which poses a serious challenge in current standards of care. Here, we investigated the consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Dystrophin deficiency was linked to marked dysregulations of cell junction proteins involved in the cell state transitions characteristic of embryonic somitogenesis. Altogether, this work demonstrates that in vitro, dystrophin deficiency has deleterious effects on cell-cell communication during myogenic development, which should be considered in future therapeutic strategies for DMD.

Dystrophin deficiency impairs cell junction formation during embryonic myogenesis.

Mutations in the DMD gene lead to Duchenne muscular dystrophy, a severe X-linked neuromuscular disorder that manifests itself as young boys acquire motor functions. DMD is typically diagnosed at 2 to 4 years of age, but the absence of dystrophin negatively impacts muscle structure and function before overt symptoms appear in patients, which poses a serious challenge in the optimization of standards of care. In this report, we investigated the early consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Here, dystrophin deficiency was linked to marked dysregulations of cell junction protein families involved in the cell state transitions characteristic of embryonic somitogenesis. Altogether, this work demonstrates that in vitro, dystrophin deficiency has deleterious effects on cell-cell communication during myogenic development, which should be considered in future therapeutic strategies for DMD.

Novel chemical tyrosine functionalization of adeno-associated virus improves gene transfer efficiency in liver and retina.

Decades of biological and clinical research have led to important advances in recombinant adeno-associated viruses rAAV-based gene therapy gene therapy. However, several challenges must be overcome to fully exploit the potential of rAAV vectors. Innovative approaches to modify viral genome and capsid elements have been used to overcome issues such as unwanted immune responses and off-targeting. While often successful, genetic modification of capsids can drastically reduce vector yield and often fails to produce vectors with properties that translate across different animal species, such as rodents, non-human primates, and humans. Here, we describe a chemical bioconjugation strategy to modify tyrosine residues on AAV capsids using specific ligands, thereby circumventing the need to genetically engineer the capsid sequence. Aromatic electrophilic substitution of the phenol ring of tyrosine residues on AAV capsids improved the in vivo transduction efficiency of rAAV2 vectors in both liver and retinal targets. This tyrosine bioconjugation strategy represents an innovative technology for the engineering of rAAV vectors for human gene therapy.

Mannose-coupled AAV2: A second-generation AAV vector for increased retinal gene therapy efficiency.

Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in inflammatory reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.

PCR-based detection of gene transfer vectors: application to gene doping surveillance.

Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR.

Integration frequency and intermolecular recombination of rAAV vectors in non-human primate skeletal muscle and liver.

The comprehensive characterization of recombinant adeno-associated viral (rAAV) integration frequency and persistence for assessing rAAV vector biosafety in gene therapy is severely limited due to the predominance of episomal rAAV vector genomes maintained in vivo. Introducing rAAV insertional standards (rAIS), we show that linear amplification-mediated (LAM)-PCR and deep sequencing can be used for validated measurement of rAAV integration frequencies. Integration of rAAV2/1 or rAAV2/8, following intramuscular (IM) or regional intravenous (RI) administration of therapeutically relevant vector doses in nine adult non-human primates (NHP), occurs at low frequency between 10(-4) and 10(-5) both in NHP liver and muscle, but with no preference for specific genomic loci. High resolution mapping of inverted terminal repeat (ITR) breakpoints in concatemeric and integrated vector genomes reveals distinct vector recombination hotspots, including large deletions of up to 3 kb. Moreover, retrieval of integrated rAAV genomes indicated approximately threefold increase in liver compared to muscle. This molecular analysis of rAAV persistence in NHP provides a promising basis for a reliable genotoxic risk assessment of rAAV in clinical trials.

Muscle Pathology in Dystrophic Rats and Zebrafish Is Unresponsive to Taurine Treatment, Compared to the mdx Mouse Model for Duchenne Muscular Dystrophy.

Inflammation and oxidative stress are strongly implicated in the pathology of Duchenne muscular dystrophy (DMD), and the sulphur-containing amino acid taurine ameliorates both and decreases dystropathology in the mdx mouse model for DMD. We therefore further tested taurine as a therapy using dystrophic DMDmdx rats and dmd zebrafish models for DMD that have a more severe dystropathology. However, taurine treatment had little effect on the indices of dystropathology in both these models. While we and others have previously observed a deficiency in taurine in mdx mice, in the current study we show that the rat and zebrafish models had increased taurine content compared with wild-type, and taurine treatment did not increase muscle taurine levels. We therefore hypothesised that endogenous levels of taurine are a key determinate in potential taurine treatment efficacy. Because of this, we felt it important to measure taurine levels in DMD patient plasma samples and showed that in non-ambulant patients (but not in younger patients) there was a deficiency of taurine. These data suggest that taurine homeostasis varies greatly between species and may be influenced by age and disease progression. The potential for taurine to be an effective therapy may depend on such variables.

Intravitreal air tamponade after AAV2 subretinal injection modifies retinal EGFP distribution.

The subretinal injection protocol for the only approved retinal gene therapy (voretigene neparvovec-rzyl) includes air tamponade at the end of the procedure, but its effects on the subretinal bleb have not been described. In the present study, we evaluated the distribution of enhanced green fluorescent protein (EGFP) after subretinal injection of AAV2 in non-human primates (NHP) without (group A = 3 eyes) or with (group B = 3 eyes) air tamponade. The retinal expression of EGFP was assessed 1 month after subretinal injection with in vivo fundus photographs and fundus autofluorescence. In group A (without air), EGFP expression was limited to the area of the initial subretinal bleb. In group B (with air), EGFP was expressed in a much wider area. These data show that the buoyant force of air on the retina causes a wide subretinal diffusion of vector, away from the injection site. In the present paper, we discuss the beneficial and deleterious clinical effects of this finding. Whereas subretinal injection is likely to become more common with the coming of new gene therapies, the effects of air tamponade should be explored further to improve efficacy, reproducibility, and safety of the protocol.

Evaluation of an AAV9-mini-dystrophin gene therapy candidate in a rat model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by loss-of-function mutations in the dystrophin gene and is characterized by muscle wasting and early mortality. Adeno-associated virus-mediated gene therapy is being investigated as a treatment for DMD. In the nonclinical study documented here, we determined the effective dose of fordadistrogene movaparvovec, a clinical candidate adeno-associated virus serotype 9 vector carrying a human mini-dystrophin transgene, after single intravenous injection in a dystrophin-deficient (DMDmdx) rat model of DMD. Overall, we found that transduction efficiency, number of muscle fibers expressing the human mini-dystrophin polypeptide, improvement of the skeletal and cardiac muscle tissue architecture, correction of muscle strength and fatigability, and improvement of diastolic and systolic cardiac function were directly correlated with the amount of vector administered. The effective dose was then tested in older DMDmdx rats with a more dystrophic phenotype similar to the pathology observed in older patients with DMD. Except for a less complete rescue of muscle function in the oldest cohort, fordadistrogene movaparvovec was also found to be therapeutically effective in older DMDmdx rats, suggesting that this product may be appropriate for evaluation in patients with DMD at all stages of disease.

Systemic delivery of allogenic muscle stem cells induces long-term muscle repair and clinical efficacy in duchenne muscular dystrophy dogs.

Duchenne muscular dystrophy (DMD) is a genetic progressive muscle disease resulting from the lack of dystrophin and without effective treatment. Adult stem cell populations have given new impetus to cell-based therapy of neuromuscular diseases. One of them, muscle-derived stem cells, isolated based on delayed adhesion properties, contributes to injured muscle repair. However, these data were collected in dystrophic mice that exhibit a relatively mild tissue phenotype and clinical features of DMD patients. Here, we characterized canine delayed adherent stem cells and investigated the efficacy of their systemic delivery in the clinically relevant DMD animal model to assess potential therapeutic application in humans. Delayed adherent stem cells, named MuStem cells (muscle stem cells), were isolated from healthy dog muscle using a preplating technique. In vitro, MuStem cells displayed a large expansion capacity, an ability to proliferate in suspension, and a multilineage differentiation potential. Phenotypically, they corresponded to early myogenic progenitors and uncommitted cells. When injected in immunosuppressed dystrophic dogs, they contributed to myofiber regeneration, satellite cell replenishment, and dystrophin expression. Importantly, their systemic delivery resulted in long-term dystrophin expression, muscle damage course limitation with an increased regeneration activity and an interstitial expansion restriction, and persisting stabilization of the dog's clinical status. These results demonstrate that MuStem cells could provide an attractive therapeutic avenue for DMD patients.

AAV2/9-mediated gene transfer into murine lacrimal gland leads to a long-term targeted tear film modification.

Corneal blindness is the fourth leading cause of blindness worldwide. Since corneal epithelium is constantly renewed, non-integrative gene transfer cannot be used to treat corneal diseases. In many of these diseases, the tear film is defective. Tears are a complex biological fluid secreted by the lacrimal apparatus. Their composition is modulated according to the context. After a corneal wound, the lacrimal gland secretes reflex tears, which contain growth factors supporting the wound healing process. In various pathological contexts, the tear composition can support neither corneal homeostasis nor wound healing. Here, we propose to use the lacrimal gland as bioreactor to produce and secrete specific factors supporting corneal physiology. In this study, we use an AAV2/9-mediated gene transfer to supplement the tear film. First, we demonstrate that a single injection of AAV2/9 is sufficient to transduce all epithelial cell types of the lacrimal gland efficiently and widely. Second, we detect no adverse effect after AAV2/9-mediated nerve growth factor expression in the lacrimal gland. Only a transitory increase in tear flow is measured. Remarkably, AAV2/9 induces an important and long-lasting secretion of this growth factor in the tear film. Altogether, our findings provide a new clinically applicable approach to tackle corneal blindness.

Lack of immunotoxicity after regional intravenous (RI) delivery of rAAV to nonhuman primate skeletal muscle.

In the absence of an immune response from the host, intramuscular (IM) injection of recombinant adeno-associated virus (rAAV) results in the permanent expression of the transgene from mouse to primate models. However, recent gene transfer studies into animal models and humans indicate that the risk of transgene and/or capsid-specific immune responses occurs and depends on multiple factors. Among these factors, the route of delivery is important, although poorly addressed in large animal models. Here, we compare the IM and the drug-free regional intravenous (RI) deliveries of rAAV in nonhuman primate (NHP) skeletal muscle monitoring the host immune response toward the transgene. We show that IM is consistently associated with immunotoxicity and the destruction of the genetically modified myofibers, whereas RI allows the stable expression of the transgene. This has important implications for the design of clinical trials for gene transfer in skeletal muscle.