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Relative persistence of AAV serotype 1 vector genomes in dystrophic muscle
Christina A Pacak1, Thomas Conlon, Cathryn S Mah
1Powell Gene Therapy Center, University of Florida, Gainesville, FL, USA. christina.pacak@childrens.harvard.edu
Abstract:
The purpose of this study was to assess the behavior of pseudotyped recombinant adeno-associated virus type 1 (rAAV2/1) vector genomes in dystrophic skeletal muscle. A comparison was made between a therapeutic vector and a reporter vector by injecting the hindlimb in a mouse model of Limb Girdle Muscular Dystrophy Type 2D (LGMD-2D) prior to disease onset. We hypothesized that the therapeutic vector would establish long-term persistence through prevention of myofiber turnover. In contrast, the reporter vector genome copy number would diminish over time due to disease-associated muscle degradation. One day old alpha sarcoglycan knockout mice (sgca-/-) were injected with 1 x 10(11) vector genomes of rAAV2/1-tMCK-sgca in one hindlimb and the same dose of rAAV2/1-tMCK-LacZ in the contra lateral hindlimb. Newborn mice are tolerant of the foreign transgene allowing for long-term expression of both the marker and the therapeutic gene in the null background. At 2 time-points following vector administration, hindlimb muscles were harvested and analyzed for LacZ or sarcoglycan expression. Our data demonstrate prolonged vector genome persistence in skeletal muscle from the hindlimbs injected with the therapeutic transgene as compared to hindlimbs injected with the reporter gene. We observed loss of vector genomes in skeletal muscles that were there were not protected by the benefits of therapeutic gene transfer. In comparison, the therapeutic vector expressing sarcoglycan led to reduction or elimination of myofiber loss. Mitigating the membrane instability inherent in dystrophic muscle was able to prolong the life of individual myofibers.
Insights
Gene therapy using adeno-associated virus vectors (AAV) shows potential for muscular dystrophy. Therapeutic AAV vectors persisted longer in dystrophic muscle, preventing fiber loss compared to reporter vectors.
Area of Science:
- Biomedical Engineering
- Molecular Biology
- Genetics
Background:
- Limb Girdle Muscular Dystrophy Type 2D (LGMD-2D) is characterized by progressive skeletal muscle degeneration.
- Recombinant adeno-associated virus (rAAV) vectors are being explored for gene therapy applications in muscular dystrophies.
- Understanding vector genome behavior is crucial for optimizing gene therapy efficacy.
Purpose of the Study:
- To compare the persistence of therapeutic and reporter rAAV2/1 vector genomes in dystrophic skeletal muscle.
- To investigate whether therapeutic gene transfer prevents myofiber turnover and genome loss.
- To assess the impact of sarcoglycan gene replacement on muscle integrity in a mouse model of LGMD-2D.
Main Methods:
- Injection of pseudotyped rAAV2/1 vectors (therapeutic sgca or reporter LacZ) into hindlimbs of alpha sarcoglycan knockout mice (sgca-/-) at one day of age.
- Analysis of vector genome copy number and gene expression at two time points post-administration.
- Histological assessment of myofiber loss and membrane stability.
Main Results:
- Therapeutic rAAV2/1-sgca vector genomes showed prolonged persistence in skeletal muscle compared to rAAV2/1-LacZ reporter vectors.
- Loss of vector genomes was observed in muscles treated with the reporter vector, correlating with disease-associated degradation.
- Sarcoglycan gene transfer reduced or eliminated myofiber loss, mitigating membrane instability and prolonging myofiber survival.
Conclusions:
- Therapeutic gene transfer with rAAV2/1 vectors can lead to long-term vector genome persistence in dystrophic muscle.
- Preventing myofiber degeneration through therapeutic gene delivery is key to sustained vector presence.
- rAAV-mediated sarcoglycan gene replacement offers a promising strategy for treating LGMD-2D by preserving muscle integrity.
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