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Homologous recombination mediates functional recovery of dysferlin deficiency following AAV5 gene transfer.

Grose WE, Clark KR, Griffin D, Malik V, Shontz KM, Montgomery CL, Lewis S, Brown RH, Janssen PM, Mendell JR, Rodino-Klapac LR - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
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.

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rAAV5.DYSF delivered directly to Dysf−/− diaphragm corrects tetanic force and resistance to fatigue.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.
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pone-0039233-g005: rAAV5.DYSF delivered directly to Dysf−/− diaphragm corrects tetanic force and resistance to fatigue.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.

Mentions: Functional deficiency in the diaphragm provided a substrate to test AAV5.DYSF gene transfer in dysferlin deficient mice. To deliver the vector to the diaphragm, a single incision was made from the base of the sternum to just above the pelvis in 10 week old 129-Dysf−/− mice (n = 6 per group). The diaphragm was identified and 30 µl of the vector preparation (2×1011 vg) was delivered using a 32 gauge needle prior to closing the abdominal wall. Animals were sacrificed 10 weeks post treatment and the diaphragm was isolated and subjected to maximum tetanic contractions and a muscle fatigue protocol. Treated diaphragms demonstrated a significant improvement in tetanic force (Fig. 5A, P<0.05, ANOVA) which was not different from WT force (129S1/SvImJ). Treated diaphragms also demonstrated a significant improvement in resistance to fatigue compared to saline treated controls (Fig. 5B, 2-way analysis of variance, P<0.001), and were not significantly different than WT strain controls (129S1/SvImJ).


Homologous recombination mediates functional recovery of dysferlin deficiency following AAV5 gene transfer.

Grose WE, Clark KR, Griffin D, Malik V, Shontz KM, Montgomery CL, Lewis S, Brown RH, Janssen PM, Mendell JR, Rodino-Klapac LR - PLoS ONE (2012)

rAAV5.DYSF delivered directly to Dysf−/− diaphragm corrects tetanic force and resistance to fatigue.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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376115&req=5

pone-0039233-g005: rAAV5.DYSF delivered directly to Dysf−/− diaphragm corrects tetanic force and resistance to fatigue.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.
Mentions: Functional deficiency in the diaphragm provided a substrate to test AAV5.DYSF gene transfer in dysferlin deficient mice. To deliver the vector to the diaphragm, a single incision was made from the base of the sternum to just above the pelvis in 10 week old 129-Dysf−/− mice (n = 6 per group). The diaphragm was identified and 30 µl of the vector preparation (2×1011 vg) was delivered using a 32 gauge needle prior to closing the abdominal wall. Animals were sacrificed 10 weeks post treatment and the diaphragm was isolated and subjected to maximum tetanic contractions and a muscle fatigue protocol. Treated diaphragms demonstrated a significant improvement in tetanic force (Fig. 5A, P<0.05, ANOVA) which was not different from WT force (129S1/SvImJ). Treated diaphragms also demonstrated a significant improvement in resistance to fatigue compared to saline treated controls (Fig. 5B, 2-way analysis of variance, P<0.001), and were not significantly different than WT strain controls (129S1/SvImJ).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus