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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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Analysis of genomes isolated from rAAV5.DYSF.DNA was isolated from rAAV5.DYSF vector preparation and used for Southern blot and PCR analysis. (A) Schematic of rAAV5.DYSF cassette. Strand specific hybridization probes used for Southern blot analysis are indicated by red bars. (B) Southern blot analysis of rAAV5.DYSF genomic DNA with 5′ MHCK7 probe (lane D, left side) and 3′ dysferlin probe (Lane D, right side). A 4.2 kb control vector genome was used as a standard for packaging (C in each blot). “M” denotes marker lane. (C) Electron microscopy of rAAV5 vector prep revealed virions with normal morphology.
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pone-0039233-g001: Analysis of genomes isolated from rAAV5.DYSF.DNA was isolated from rAAV5.DYSF vector preparation and used for Southern blot and PCR analysis. (A) Schematic of rAAV5.DYSF cassette. Strand specific hybridization probes used for Southern blot analysis are indicated by red bars. (B) Southern blot analysis of rAAV5.DYSF genomic DNA with 5′ MHCK7 probe (lane D, left side) and 3′ dysferlin probe (Lane D, right side). A 4.2 kb control vector genome was used as a standard for packaging (C in each blot). “M” denotes marker lane. (C) Electron microscopy of rAAV5 vector prep revealed virions with normal morphology.

Mentions: We constructed a human dysferlin cassette driven by the muscle specific MHCK7 promoter [33] (Fig. 1A). A chimeric intron was added to augment RNA processing. The cassette (7.7 kb total) was packaged into an AAV2/5 vector using standard triple transfection and purified using iodixanol gradients and ion exchange chromatography. To test whether the full-length transgene was packaged or if homologous recombination was occurring, we performed alkaline electrophoresis and southern blot analysis on vector genomes purified from the vector preparation as previously described [32]. Two probes were designed, one within the 5′ MHCK7 promoter and one located in the 3′ end of dysferlin. The findings unequivocally demonstrated that packaging did not exceed ∼5.2 kb with either probe consistent with recent reports [29], [31], [32] (Fig. 1B). AAV is known to package single strand genomes with 3′ to 5′ polarity into the pre-formed particle in an ATP dependent process starting at the 3′ ITR terminus. This is consistent with a vector “breakpoint” within this region based on the Southern blot data demonstrating packaging of up to ∼5.2 kb in length starting at a 3′ ITR termini. Despite these packaging constraints, full-length dysferlin protein was readily demonstrated in skeletal muscle consistent with a process of homologous recombination occurring within transduced myocytes to generate the full-length intact dysferlin gene (Fig. 2C) [29], [31], [32]. Using electron microscopy, AAV5 virions had normal morphology, providing further evidence that the genomes packaged did not exceed capacity (Fig. 1C).


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)

Analysis of genomes isolated from rAAV5.DYSF.DNA was isolated from rAAV5.DYSF vector preparation and used for Southern blot and PCR analysis. (A) Schematic of rAAV5.DYSF cassette. Strand specific hybridization probes used for Southern blot analysis are indicated by red bars. (B) Southern blot analysis of rAAV5.DYSF genomic DNA with 5′ MHCK7 probe (lane D, left side) and 3′ dysferlin probe (Lane D, right side). A 4.2 kb control vector genome was used as a standard for packaging (C in each blot). “M” denotes marker lane. (C) Electron microscopy of rAAV5 vector prep revealed virions with normal morphology.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376115&req=5

pone-0039233-g001: Analysis of genomes isolated from rAAV5.DYSF.DNA was isolated from rAAV5.DYSF vector preparation and used for Southern blot and PCR analysis. (A) Schematic of rAAV5.DYSF cassette. Strand specific hybridization probes used for Southern blot analysis are indicated by red bars. (B) Southern blot analysis of rAAV5.DYSF genomic DNA with 5′ MHCK7 probe (lane D, left side) and 3′ dysferlin probe (Lane D, right side). A 4.2 kb control vector genome was used as a standard for packaging (C in each blot). “M” denotes marker lane. (C) Electron microscopy of rAAV5 vector prep revealed virions with normal morphology.
Mentions: We constructed a human dysferlin cassette driven by the muscle specific MHCK7 promoter [33] (Fig. 1A). A chimeric intron was added to augment RNA processing. The cassette (7.7 kb total) was packaged into an AAV2/5 vector using standard triple transfection and purified using iodixanol gradients and ion exchange chromatography. To test whether the full-length transgene was packaged or if homologous recombination was occurring, we performed alkaline electrophoresis and southern blot analysis on vector genomes purified from the vector preparation as previously described [32]. Two probes were designed, one within the 5′ MHCK7 promoter and one located in the 3′ end of dysferlin. The findings unequivocally demonstrated that packaging did not exceed ∼5.2 kb with either probe consistent with recent reports [29], [31], [32] (Fig. 1B). AAV is known to package single strand genomes with 3′ to 5′ polarity into the pre-formed particle in an ATP dependent process starting at the 3′ ITR terminus. This is consistent with a vector “breakpoint” within this region based on the Southern blot data demonstrating packaging of up to ∼5.2 kb in length starting at a 3′ ITR termini. Despite these packaging constraints, full-length dysferlin protein was readily demonstrated in skeletal muscle consistent with a process of homologous recombination occurring within transduced myocytes to generate the full-length intact dysferlin gene (Fig. 2C) [29], [31], [32]. Using electron microscopy, AAV5 virions had normal morphology, providing further evidence that the genomes packaged did not exceed capacity (Fig. 1C).

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