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31.9 Clinical studies
1. Romero NB et al ., in the year 2004 conducted phase-I study of dystrophin plasmid-based gene therapy in Duchenne/Becker muscular dystrophy. Nine study subjects affected with DMD/BMD were registered for the trial and were injected with full length human dystrophin gene incorporated in the plasmid. The dose administered was either once with 200 or 600µg of DNA or twice, 2 weeks apart, with 600µg of DNA by injection in the radialis muscle. After 3 weeks muscle biopsy, of the region injected with the DNA, was performed and in all study subjects the vector was found in the muscle at the site of injection. Out of the nine, 6 patients were reported to express dystrophin based on the immunohistochemistry and nested RT-PCR analysis. The level of expression was found to be low with 6% complete sarcolemmal dystrophin staining and about 26% partial sarcolemmal labeling. Neither cellular, humoral anti-dystrophin response nor any side effects were observed.
Thus, based on the results obtained, it was understood that the exogenous dystrophin expression could be achieved in DMD/BMD subjects after intramuscular transfer of plasmid, without any side effects. This study directs the way for future developments in hereditary muscle dystrophy gene therapy.
2. Van Deutekom JC et al. , in the year 2007 demonstrated local dystrophin restoration using AON-PRO051. They induced specific exon skipping during messenger RNA (mRNA) splicing using antisense compounds and corrected the open reading frame (ORF) of the DMD gene and restored dystrophin expression in vitro and in animal models in vivo . They also analyzed the safety, adverse-event profile, and local dystrophin-restoring effect of a single, intramuscular dose of an AON-PRO051, in patients with this disease. Four study subjects were selected on the basis of their mutational status, muscle condition, and positive exon-skipping response to PRO051 in vitro . A dose of 0.8 mg of PRO051 was injected into the tibialis anterior muscle. After 28 days of injection muscle biopsy was performed. Safety measures were taken during the study. The composition of mRNA, and dystrophin expression were assessed. It was observed that PRO051 injection did not cause any adverse effect. Specific exon skipping occurred in each study subject in 64-97% muscle cells. Amount of dystrophin protein estimated ranged from 3 to 12% as compared to the control specimen. 17 to 35% range of quantitative ratio of dystrophin to laminin alpha2 was estimated as compared to the control specimen. This study indicates that intramuscular injection of the AON- PRO051 resulted in dystrophin synthesis in four patients with DMD who had mutations in the dystrophin gene. This study clearly shows the feasibility of further exploration of the trial with better strategies.
3. In 2012 Bowles DE et al ., conducted the first phase 1 gene therapy trial for DMD using a translational optimized AAV vector (capsid variant designated AAV2.5) derived from a rational design strategy. The vector AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. This novel vector has two benefits first it combines the improved muscle transduction capacity of AAV1 with reduced antigenic cross-reactivity against both parental serotypes and second it retains the AAV2 receptor binding capacity. A randomized double-blind placebo-controlled phase I clinical study was conducted in DMD boys. One group of study subjects were injected with AAV2.5 vector into the bicep muscle in one arm and with saline control in the other arm. Another group of study subjects were injected with AAV empty capsid instead of saline in order to distinguish an immune response to vector versus mini-dystrophin transgene. Muscle biopsy analysis revealed the presence of recombinant AAV genomes in all study subjects with up to 2.56 vector copies per diploid genome. No cellular immune response was observed against AAV2.5 capsid. This study confirms that rationally designed AAV2.5 vector was safe and well tolerated and forms the basis of designing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery).