A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy

Background: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation. Methods: The capacity of AAV1, AAV6 or AAV8 to cross the vascular endothelial barrier carrying a micro-dystrophin cDNA was compared under identical conditions with delivery through a catheter placed in the femoral artery of the mdx mouse. Transduction efficiency was assessed by immuno-staining using an antibody (Manex1a) that recognizes the Nterminus of micro-dystrophin. The degree of physiologic correction was assessed by measuring tetanic force and protection from eccentric contraction in the extensor digitorum longus muscle (EDL). The vascular delivery paradigm found successful in the mouse was carried to the non-human primate to test its potential translation to boys with DMD. Results: Regional vascular delivery resulted in transduction by rAAV8.micro-dystrophin reaching 94.5 ± 0.9 (1 month), 91.3 ± 3.1 (2 months), and 89.6 ± 1.6% (3 months). rAAV6.micro-dystrophin treated animals demonstrated 87.7 ± 6.8 (1 month), 78.9 ± 7.4 (2 months), and 81.2 ± 6.2% (3 months) transduction. In striking contrast, rAAV1 demonstrated very low transduction efficiency [0.9 ± 0.3 (1 month), 2.1 ± 0.8 (2 months), and 2.1 ± 0.7% (3 months)] by vascular delivery. Micro-dystrophin delivered by rAAV8 and rAAV6 through the femoral artery significantly improved tetanic force and protected against eccentric contraction. Mouse studies translated to the hindlimb of cynamologous macaques using a similar vascular delivery paradigm. rAAV8 carrying eGFP in doses proportional to the mouse (5 × 1012 vg/kg in mouse vs 2 × 1012 vg/kg in monkey) demonstrated widespread gene expression [medial gastrocnemius – 63.8 ± 4.9%, lateral gastrocnemius – 66.0 ± 4.5%, EDL – 80.2 ± 3.1%, soleus – 86.4 ± 1.9%, TA – 72.2 ± 4.0%. Conclusion: These studies demonstrate regional vascular gene delivery with AAV serotype(s) in mouse and non-human primate at doses, pressures and volumes applicable for clinical trials in children with DMD

Persistent Expression of FLAG-tagged Micro dystrophin in Nonhuman Primates Following Intramuscular and Vascular Delivery

Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Nonhuman primates (NHPs) provide the ideal model to optimize vector delivery across a vascular barrier and provide accurate dose estimates for widespread transduction. To address vascular delivery and dosing in rhesus macaques, we have generated a fusion construct that encodes an eight amino-acid FLAG epitope at the C-terminus of micro-dystrophin to facilitate translational studies targeting DMD. Intramuscular (IM) injection of AAV8.MCK.micro-dys.FLAG in the tibialis anterior (TA) of macaques demonstrated robust gene expression, with muscle transduction (50–79%) persisting for up to 5 months. Success by IM injection was followed by targeted vascular delivery studies using a fluoroscopy-guided catheter threaded through the femoral artery. Three months after gene transfer, >80% of muscle fibers showed gene expression in the targeted muscle. No cellular immune response to AAV8 capsid, micro-dystrophin, or the FLAG tag was detected by interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) at any time point with either route. In summary, an epitope-tagged micro-dystrophin cassette enhances the ability to evaluate site-specific localization and distribution of gene expression in the NHP in preparation for vascular delivery clinical trials

Homologous Recombination Mediates Functional Recovery of Dysferlin Deficiency following AAV5 Gene Transfer

The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV) gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb) negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9). Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes

Impaired regeneration in calpain-3 null muscle is associated with perturbations in mTORC1 signaling and defective mitochondrial biogenesis

Abstract Background Previous studies in patients with limb-girdle muscular dystrophy type 2A (LGMD2A) have suggested that calpain-3 (CAPN3) mutations result in aberrant regeneration in muscle. Methods To gain insight into pathogenesis of aberrant muscle regeneration in LGMD2A, we used a paradigm of cardiotoxin (CTX)-induced cycles of muscle necrosis and regeneration in the CAPN3-KO mice to simulate the early features of the dystrophic process in LGMD2A. The temporal evolution of the regeneration process was followed by assessing the oxidative state, size, and the number of metabolic fiber types at 4 and 12 weeks after last CTX injection. Muscles isolated at these time points were further investigated for the key regulators of the pathways involved in various cellular processes such as protein synthesis, cellular energy status, metabolism, and cell stress to include Akt/mTORC1 signaling, mitochondrial biogenesis, and AMPK signaling. TGF-β and microRNA (miR-1, miR-206, miR-133a) regulation were also assessed. Additional studies included in vitro assays for quantifying fusion index of myoblasts from CAPN3-KO mice and development of an in vivo gene therapy paradigm for restoration of impaired regeneration using the adeno-associated virus vector carrying CAPN3 gene in the muscle. Results At 4 and 12 weeks after last CTX injection, we found impaired regeneration in CAPN3-KO muscle characterized by excessive numbers of small lobulated fibers belonging to oxidative metabolic type (slow twitch) and increased connective tissue. TGF-β transcription levels in the regenerating CAPN3-KO muscles were significantly increased along with microRNA dysregulation compared to wild type (WT), and the attenuated radial growth of muscle fibers was accompanied by perturbed Akt/mTORC1 signaling, uncoupled from protein synthesis, through activation of AMPK pathway, thought to be triggered by energy shortage in the CAPN3-KO muscle. This was associated with failure to increase mitochondria content, PGC-1α, and ATP5D transcripts in the regenerating CAPN3-KO muscles compared to WT. In vitro studies showed defective myotube fusion in CAPN3-KO myoblast cultures. Replacement of CAPN3 by gene therapy in vivo increased the fiber size and decreased the number of small oxidative fibers. Conclusion Our findings provide insights into understanding of the impaired radial growth phase of regeneration in calpainopathy

A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy-4

<p><b>Copyright information:</b></p><p>Taken from "A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy"</p><p>http://www.translational-medicine.com/content/5/1/45</p><p>Journal of Translational Medicine 2007;5():45-45.</p><p>Published online 24 Sep 2007</p><p>PMCID:PMC2082019.</p><p></p>digitorum longus (EDL) muscles (8 week time point) demonstrated significant maximum specific force improvement (mN/mm) over mdx-untreated muscles (p < 0.05) but did not reach levels of age-matched C57BL/10 wild type (WT) controls (P < 0.05). Values are presented as the means ± SEM, (B) EDL muscles were subjected to 10 cycles of isometric stimulation at 150 Hz with a 10% lengthening to induce damage during the last 100 ms of each contraction. Force values are represented as fractions of the first contraction. rAAV8.micro-dystrophin and rAAV6.micro-dystrophin treated EDL muscles exhibited significant protection from damage compared to mdx-untreated during the first two cycles (p < 0.05), while thereafter all 4 groups showed such robust injury that differences could no longer be determined. There was no significant difference between rAAV8- or rAAV6-treated and WT. (C) The ratio of force (ECC2/ECC1) after one lengthening contraction of rAAV-treated versus the initial contraction of WT is not different, but is significantly improved compared to mdx-untreated (n = 6–8 per group; P < 0.05)

Components of the dystrophin-associated protein complex are restored in mice treated with rAAV

<p><b>Copyright information:</b></p><p>Taken from "A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy"</p><p>http://www.translational-medicine.com/content/5/1/45</p><p>Journal of Translational Medicine 2007;5():45-45.</p><p>Published online 24 Sep 2007</p><p>PMCID:PMC2082019.</p><p></p>micro-dystrophin gene transfer. mice treated with rAAV8.micro-dystrophin delivered by ILP (8 week time point) were serial sectioned and stained with manex1a antibody for dystrophin, α-sarcoglycan, β-sarcoglycan, and H &E. Muscle fibers transduced with micro-dystrophin also exhibited restoration of expression of α-sarcoglycan and β-sarcoglycan (no staining in untreated mice – bottom row). Muscle fibers from serial sections exhibited identical expression localization for each protein (arrow)

Overexpression of Galgt2 in skeletal muscle prevents injury resulting from eccentric contractions in both mdx and wild-type mice

The cytotoxic T cell (CT) GalNAc transferase, or Galgt2, is a UDP-GalNAc:β1,4-N-acetylgalactosaminyltransferase that is localized to the neuromuscular synapse in adult skeletal muscle, where it creates the synaptic CT carbohydrate antigen {GalNAcβ1,4[NeuAc(orGc)α2, 3]Galβ1,4GlcNAcβ-}. Overexpression of Galgt2 in the skeletal muscles of transgenic mice inhibits the development of muscular dystrophy in mdx mice, a model for Duchenne muscular dystrophy. Here, we provide physiological evidence as to how Galgt2 may inhibit the development of muscle pathology in mdx animals. Both Galgt2 transgenic wild-type and mdx skeletal muscles showed a marked improvement in normalized isometric force during repetitive eccentric contractions relative to nontransgenic littermates, even using a paradigm where nontransgenic muscles had force reductions of 95% or more. Muscles from Galgt2 transgenic mice, however, showed a significant decrement in normalized specific force and in hindlimb and forelimb grip strength at some ages. Overexpression of Galgt2 in muscles of young adult mdx mice, where Galgt2 has no effect on muscle size, also caused a significant decrease in force drop during eccentric contractions and increased normalized specific force. A comparison of Galgt2 and microdystrophin overexpression using a therapeutically relevant intravascular gene delivery protocol showed Galgt2 was as effective as microdystrophin at preventing loss of force during eccentric contractions. These experiments provide a mechanism to explain why Galgt2 overexpression inhibits muscular dystrophy in mdx muscles. That overexpression also prevents loss of force in nondystrophic muscles suggests that Galgt2 is a therapeutic target with broad potential applications

rAAV5.DYSF delivered directly to Dysf^−/− diaphragm corrects tetanic force and resistance to fatigue.

<p>The diaphragm of 10 week old dysferlin deficient mice (129-Dysf^−/−) (n = 6 per group) was treated with 10¹¹ vg of rAAV5.DYSF via a peritoneal incision. Ten weeks post gene transfer, diaphragm muscle strips were harvested and subjected to a protocol to assess tetanic force and resistance to fatigue. (A) rAAV5.DYSF treated diaphragms demonstrated significant improvement in tetanic force (P>0.05, ANOVA) which was not different from wild-type force (129S1/SvImJ). (B) rAAV5.DYSF treated diaphragms demonstrated significant resistance to fatigue compared to untreated Dysf^−/− controls (2-way analysis of variance, P<0.001) and were not different than SVJIM wild-type controls. Force retention following ten contractions is shown.</p