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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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Recovery of membrane repair following rAAV5.DYSF injection in muscle.(A) Individual flexor digitorum brevis fibers were isolated from WT, 129-DYSF−/−, and 129-DYSFrAAV.DYSF mice and the sarcolemma was damaged in the context of a solution containing FM 1–43. Images were taken before injury (−5 s) and every 5 s for a total of 195 s. Representative initial and final images for WT, 129-DYSF−/−, and 129-DYSFrAAV.DYSF fibers are shown. (B) Fluorescence intensity from injured fibers was measured using ImageJ software, converted to change in fluorescence intensity over time, and then graphed. For clarity, only values corresponding to 15 s intervals are shown following injury. (C) The total change in fluorescence intensity over time is shown for WT, 129-DYSF−/−, and 129-DYSFrAAV.DYSF fibers at 195 s post-injury. (2-way analysis of variance, P<0.05).
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pone-0039233-g006: Recovery of membrane repair following rAAV5.DYSF injection in muscle.(A) Individual flexor digitorum brevis fibers were isolated from WT, 129-DYSF−/−, and 129-DYSFrAAV.DYSF mice and the sarcolemma was damaged in the context of a solution containing FM 1–43. Images were taken before injury (−5 s) and every 5 s for a total of 195 s. Representative initial and final images for WT, 129-DYSF−/−, and 129-DYSFrAAV.DYSF fibers are shown. (B) Fluorescence intensity from injured fibers was measured using ImageJ software, converted to change in fluorescence intensity over time, and then graphed. For clarity, only values corresponding to 15 s intervals are shown following injury. (C) The total change in fluorescence intensity over time is shown for WT, 129-DYSF−/−, and 129-DYSFrAAV.DYSF fibers at 195 s post-injury. (2-way analysis of variance, P<0.05).

Mentions: We next evaluated the ability of AAV5.DYSF treatment to restore membrane repair capability in dysferlin deficient muscle. To test this we performed a membrane wounding/resealing assay using a multi-photon laser scanning microscope on fibers isolated from the flexor digitorum brevis (FDB) muscle. We injected 2 month old 129-Dysf−/− (5 per group) with 3×1010 vg AAV5.DYSF in the (FDB) muscle. WT (129S1/SvImJ), 129-Dysf−/− and AAV5.DYSF treated 129-Dysf−/− mice were sacrificed 8 weeks post-treatment, the FDB muscle was isolated, and individual fibers were isolated following collagenase treatment. Sarcolemmal damage was induced in isolated fibers using the multiphoton laser (20% power for 5s) in the presence of FM 1–43 dye. A small area of fluorescence was detected in all fibers immediately after laser injury at the site of damage. In WT muscle fibers the sarcolemma is repaired and the amount of dye that integrates into the membrane stabilizes (Fig. 6). In contrast, dye continued to integrate into the sarcolemma of the fibers from 129-DYSF−/− muscle which resulted in significantly higher levels of fluorescence following a 3 min time course (Fig. 6). Expression of dysferlin from AAV5.DYSF-transduced fibers resulted in membrane repair that was equivalent to WT fibers further indicating that the exogenously expressed protein is fully functional (Fig. 6). Taken together, these data demonstrate that a large, potentially therapeutic cDNA can be delivered to muscle and efficiently express full-length functional dysferlin protein in muscle using AAV5.


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)

Recovery of membrane repair following rAAV5.DYSF injection in muscle.(A) Individual flexor digitorum brevis fibers were isolated from WT, 129-DYSF−/−, and 129-DYSFrAAV.DYSF mice and the sarcolemma was damaged in the context of a solution containing FM 1–43. Images were taken before injury (−5 s) and every 5 s for a total of 195 s. Representative initial and final images for WT, 129-DYSF−/−, and 129-DYSFrAAV.DYSF fibers are shown. (B) Fluorescence intensity from injured fibers was measured using ImageJ software, converted to change in fluorescence intensity over time, and then graphed. For clarity, only values corresponding to 15 s intervals are shown following injury. (C) The total change in fluorescence intensity over time is shown for WT, 129-DYSF−/−, and 129-DYSFrAAV.DYSF fibers at 195 s post-injury. (2-way analysis of variance, P<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376115&req=5

pone-0039233-g006: Recovery of membrane repair following rAAV5.DYSF injection in muscle.(A) Individual flexor digitorum brevis fibers were isolated from WT, 129-DYSF−/−, and 129-DYSFrAAV.DYSF mice and the sarcolemma was damaged in the context of a solution containing FM 1–43. Images were taken before injury (−5 s) and every 5 s for a total of 195 s. Representative initial and final images for WT, 129-DYSF−/−, and 129-DYSFrAAV.DYSF fibers are shown. (B) Fluorescence intensity from injured fibers was measured using ImageJ software, converted to change in fluorescence intensity over time, and then graphed. For clarity, only values corresponding to 15 s intervals are shown following injury. (C) The total change in fluorescence intensity over time is shown for WT, 129-DYSF−/−, and 129-DYSFrAAV.DYSF fibers at 195 s post-injury. (2-way analysis of variance, P<0.05).
Mentions: We next evaluated the ability of AAV5.DYSF treatment to restore membrane repair capability in dysferlin deficient muscle. To test this we performed a membrane wounding/resealing assay using a multi-photon laser scanning microscope on fibers isolated from the flexor digitorum brevis (FDB) muscle. We injected 2 month old 129-Dysf−/− (5 per group) with 3×1010 vg AAV5.DYSF in the (FDB) muscle. WT (129S1/SvImJ), 129-Dysf−/− and AAV5.DYSF treated 129-Dysf−/− mice were sacrificed 8 weeks post-treatment, the FDB muscle was isolated, and individual fibers were isolated following collagenase treatment. Sarcolemmal damage was induced in isolated fibers using the multiphoton laser (20% power for 5s) in the presence of FM 1–43 dye. A small area of fluorescence was detected in all fibers immediately after laser injury at the site of damage. In WT muscle fibers the sarcolemma is repaired and the amount of dye that integrates into the membrane stabilizes (Fig. 6). In contrast, dye continued to integrate into the sarcolemma of the fibers from 129-DYSF−/− muscle which resulted in significantly higher levels of fluorescence following a 3 min time course (Fig. 6). Expression of dysferlin from AAV5.DYSF-transduced fibers resulted in membrane repair that was equivalent to WT fibers further indicating that the exogenously expressed protein is fully functional (Fig. 6). Taken together, these data demonstrate that a large, potentially therapeutic cDNA can be delivered to muscle and efficiently express full-length functional dysferlin protein in muscle using AAV5.

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