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Effective delivery of large genes to the retina by dual AAV vectors.

Trapani I, Colella P, Sommella A, Iodice C, Cesi G, de Simone S, Marrocco E, Rossi S, Giunti M, Palfi A, Farrar GJ, Polishchuk R, Auricchio A - EMBO Mol Med (2013)

Bottom Line: Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans.Previous methods based on 'forced' packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns.We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B.

View Article: PubMed Central - PubMed

Affiliation: Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy.

ABSTRACT
Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on 'forced' packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.

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A–B Representative Western blot analysis of C57BL/6 (A) and Large White pig (B) retinal lysates 1 month following injection of dual AAV2/8 overlapping vectors encoding for ABCA4-3xflag (OV) or AAV2/8 vectors expressing EGFP (neg), under the control of the ubiquitous cytomegalovirus (CMV) promoter, the PR-specific Rhodopsin (RHO) and Rhodopsin kinase (RHOK) promoters, or the RPE-specific vitelliform macular dystrophy 2 (VMD2) promoter. The arrows indicate full-length proteins, the molecular weight ladder is depicted on the left, 150 μg of proteins were loaded in each lane. The number ( n) and percentage of ABCA4-positive retinas out of total retinas analyzed is depicted; α-3xflag, Western blot with anti-3xflag antibody; α-Dysferlin, Western blot with anti-Dysferlin antibody, used as loading control.
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fig03: A–B Representative Western blot analysis of C57BL/6 (A) and Large White pig (B) retinal lysates 1 month following injection of dual AAV2/8 overlapping vectors encoding for ABCA4-3xflag (OV) or AAV2/8 vectors expressing EGFP (neg), under the control of the ubiquitous cytomegalovirus (CMV) promoter, the PR-specific Rhodopsin (RHO) and Rhodopsin kinase (RHOK) promoters, or the RPE-specific vitelliform macular dystrophy 2 (VMD2) promoter. The arrows indicate full-length proteins, the molecular weight ladder is depicted on the left, 150 μg of proteins were loaded in each lane. The number ( n) and percentage of ABCA4-positive retinas out of total retinas analyzed is depicted; α-3xflag, Western blot with anti-3xflag antibody; α-Dysferlin, Western blot with anti-Dysferlin antibody, used as loading control.

Mentions: We then evaluated the best in vitro performing AAV-based systems for large gene transduction in the mouse retina. To test the dual AAV OV, which is transgene-specific, we used the therapeutic ABCA4 and MYO7A genes (Fig 3 and data not shown). We used EGFP to evaluate the AAV OZ and the dual AAV TS and hybrid AK approaches (supplementary Fig S4).


Effective delivery of large genes to the retina by dual AAV vectors.

Trapani I, Colella P, Sommella A, Iodice C, Cesi G, de Simone S, Marrocco E, Rossi S, Giunti M, Palfi A, Farrar GJ, Polishchuk R, Auricchio A - EMBO Mol Med (2013)

A–B Representative Western blot analysis of C57BL/6 (A) and Large White pig (B) retinal lysates 1 month following injection of dual AAV2/8 overlapping vectors encoding for ABCA4-3xflag (OV) or AAV2/8 vectors expressing EGFP (neg), under the control of the ubiquitous cytomegalovirus (CMV) promoter, the PR-specific Rhodopsin (RHO) and Rhodopsin kinase (RHOK) promoters, or the RPE-specific vitelliform macular dystrophy 2 (VMD2) promoter. The arrows indicate full-length proteins, the molecular weight ladder is depicted on the left, 150 μg of proteins were loaded in each lane. The number ( n) and percentage of ABCA4-positive retinas out of total retinas analyzed is depicted; α-3xflag, Western blot with anti-3xflag antibody; α-Dysferlin, Western blot with anti-Dysferlin antibody, used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927955&req=5

fig03: A–B Representative Western blot analysis of C57BL/6 (A) and Large White pig (B) retinal lysates 1 month following injection of dual AAV2/8 overlapping vectors encoding for ABCA4-3xflag (OV) or AAV2/8 vectors expressing EGFP (neg), under the control of the ubiquitous cytomegalovirus (CMV) promoter, the PR-specific Rhodopsin (RHO) and Rhodopsin kinase (RHOK) promoters, or the RPE-specific vitelliform macular dystrophy 2 (VMD2) promoter. The arrows indicate full-length proteins, the molecular weight ladder is depicted on the left, 150 μg of proteins were loaded in each lane. The number ( n) and percentage of ABCA4-positive retinas out of total retinas analyzed is depicted; α-3xflag, Western blot with anti-3xflag antibody; α-Dysferlin, Western blot with anti-Dysferlin antibody, used as loading control.
Mentions: We then evaluated the best in vitro performing AAV-based systems for large gene transduction in the mouse retina. To test the dual AAV OV, which is transgene-specific, we used the therapeutic ABCA4 and MYO7A genes (Fig 3 and data not shown). We used EGFP to evaluate the AAV OZ and the dual AAV TS and hybrid AK approaches (supplementary Fig S4).

Bottom Line: Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans.Previous methods based on 'forced' packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns.We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B.

View Article: PubMed Central - PubMed

Affiliation: Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy.

ABSTRACT
Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on 'forced' packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.

Show MeSH
Related in: MedlinePlus