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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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Representative pictures of transmission electron microscopy analysis of retinal sections from wild-type BALB/c (WT) and Abca4−/− mice injected with dual AAV trans-splicing (TS-ABCA4) and hybrid AK vectors (AK-ABCA4) or with either AAV vectors expressing EGFP or 5′-or 3′-half of the dual hybrid AK vectors (neg), as negative controls. The dotted lines indicate the edges of RPE cells. The scale bar (3.8 μm) is depicted in the figure.Quantification of the mean RPE thickness counted in at least 30 fields for each sample. The number ( n) of eyes analyzed is depicted below each bar. Abca4−/− neg includes Abca4−/− mice injected with either AAV vectors expressing EGFP ( n = 2) or 5′-( n = 3) or 3′-( n = 4) half of the dual hybrid AK vectors (neg total n = 9). The mean value is depicted above the corresponding bar. Values are represented as mean ± standard error of the mean (s.e.m.). * P ANOVA < 0.05.Recovery from light desensitization in 3 month-old Abca4−/− and BALB/c mice at 6 weeks post-injection. The relative b-wave is the ratio between the post-and the pre-desensitization b-wave amplitudes both evoked by 1 cd s/m2. The time refers to the minutes post-desensitization. The mean recovery (%) at 60 min is depicted.Data information: (A-C) WT, BALB/c eyes ( n = 4); Abca4−/− TS-ABCA4, eyes injected with dual AAV TS vectors ( n = 5); Abca4−/− AK-ABCA4, AAV hybrid AK vectors ( n = 5); Abca4−/− neg, Abca4−/− mice either not injected ( n = 2) or injected with the 5′-half of the dual AAV TS ( n = 3) or hybrid AK ( n = 2) vectors (neg total n = 7). Values are represented as mean ± standard error of the mean (s.e.m.). * P ANOVA < 0.05. More details on the statistical analysis including specific statistical values can be found in the Statistical analysis paragraph of the Materials and methods section.
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fig06: Representative pictures of transmission electron microscopy analysis of retinal sections from wild-type BALB/c (WT) and Abca4−/− mice injected with dual AAV trans-splicing (TS-ABCA4) and hybrid AK vectors (AK-ABCA4) or with either AAV vectors expressing EGFP or 5′-or 3′-half of the dual hybrid AK vectors (neg), as negative controls. The dotted lines indicate the edges of RPE cells. The scale bar (3.8 μm) is depicted in the figure.Quantification of the mean RPE thickness counted in at least 30 fields for each sample. The number ( n) of eyes analyzed is depicted below each bar. Abca4−/− neg includes Abca4−/− mice injected with either AAV vectors expressing EGFP ( n = 2) or 5′-( n = 3) or 3′-( n = 4) half of the dual hybrid AK vectors (neg total n = 9). The mean value is depicted above the corresponding bar. Values are represented as mean ± standard error of the mean (s.e.m.). * P ANOVA < 0.05.Recovery from light desensitization in 3 month-old Abca4−/− and BALB/c mice at 6 weeks post-injection. The relative b-wave is the ratio between the post-and the pre-desensitization b-wave amplitudes both evoked by 1 cd s/m2. The time refers to the minutes post-desensitization. The mean recovery (%) at 60 min is depicted.Data information: (A-C) WT, BALB/c eyes ( n = 4); Abca4−/− TS-ABCA4, eyes injected with dual AAV TS vectors ( n = 5); Abca4−/− AK-ABCA4, AAV hybrid AK vectors ( n = 5); Abca4−/− neg, Abca4−/− mice either not injected ( n = 2) or injected with the 5′-half of the dual AAV TS ( n = 3) or hybrid AK ( n = 2) vectors (neg total n = 7). Values are represented as mean ± standard error of the mean (s.e.m.). * P ANOVA < 0.05. More details on the statistical analysis including specific statistical values can be found in the Statistical analysis paragraph of the Materials and methods section.

Mentions: To evaluate the biological and therapeutic activity of the recombinant ABCA4 protein produced by dual AAV vectors, 1 month-old albino Abca4−/− mice were injected subretinally with the dual AAV TS and hybrid AK RHO-ABCA4-HA vectors (dose of each vector/eye: 1–3 × 109 GC). Three months later, eyes were harvested and immuno-electron microscopy analysis with anti-hemagglutinin (HA) antibodies of retinal sections confirmed that immunogold particles were correctly localized in PR outer segments only in animals that were injected with the combination of 5′ and 3′ dual AAV TS and hybrid AK vectors (Fig 5B). To assess the functionality of the ABCA4 protein expressed by the dual AAV vectors, we measured Abca4−/− retinal lipofuscin accumulation and recovery from light desensitization. To assess the former we performed transmission electron microscopy analysis to measure the number of RPE lipofuscin granules (Fig 5C and D) and RPE thickness (Fig 6A and B). Both were greater in the retina of Abca4−/− mice injected with control vectors (independently of the size of the control constructs, supplementary Fig S10) than in the retina of wild-type, age-matched BALB/c controls, and were significantly reduced or normalized in the eyes injected either with the therapeutic dual AAV TS or hybrid AK vectors (Figs 5C and D and 6A and B). We additionally attempted at measuring A2E, the major component of lipofuscin granules, by HPLC (Parish et al, 1998; Ben-Shabat et al, 2002; Allocca et al, 2008), however these measurements were inconsistent, even between affected and normal retinas (data not shown), thus we were not able to use these techniques in our rescue experiments. Importantly, the eyes treated with dual AAV TS and hybrid AK vectors showed improved recovery from light desensitization when compared to eyes treated with control vectors (dose of each vector/eye: 1.2 × 109 GC, Fig 6C), independently of the size of the control constructs [Student's t-test P value of dual AAV-EGFP-L ( n = 2 TS-L, n = 4 AK-L; total n = 6) versus dual AAV-EGFP ( n = 5 TS, n = 4 AK; total n = 9): 0.23].


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)

Representative pictures of transmission electron microscopy analysis of retinal sections from wild-type BALB/c (WT) and Abca4−/− mice injected with dual AAV trans-splicing (TS-ABCA4) and hybrid AK vectors (AK-ABCA4) or with either AAV vectors expressing EGFP or 5′-or 3′-half of the dual hybrid AK vectors (neg), as negative controls. The dotted lines indicate the edges of RPE cells. The scale bar (3.8 μm) is depicted in the figure.Quantification of the mean RPE thickness counted in at least 30 fields for each sample. The number ( n) of eyes analyzed is depicted below each bar. Abca4−/− neg includes Abca4−/− mice injected with either AAV vectors expressing EGFP ( n = 2) or 5′-( n = 3) or 3′-( n = 4) half of the dual hybrid AK vectors (neg total n = 9). The mean value is depicted above the corresponding bar. Values are represented as mean ± standard error of the mean (s.e.m.). * P ANOVA < 0.05.Recovery from light desensitization in 3 month-old Abca4−/− and BALB/c mice at 6 weeks post-injection. The relative b-wave is the ratio between the post-and the pre-desensitization b-wave amplitudes both evoked by 1 cd s/m2. The time refers to the minutes post-desensitization. The mean recovery (%) at 60 min is depicted.Data information: (A-C) WT, BALB/c eyes ( n = 4); Abca4−/− TS-ABCA4, eyes injected with dual AAV TS vectors ( n = 5); Abca4−/− AK-ABCA4, AAV hybrid AK vectors ( n = 5); Abca4−/− neg, Abca4−/− mice either not injected ( n = 2) or injected with the 5′-half of the dual AAV TS ( n = 3) or hybrid AK ( n = 2) vectors (neg total n = 7). Values are represented as mean ± standard error of the mean (s.e.m.). * P ANOVA < 0.05. More details on the statistical analysis including specific statistical values can be found in the Statistical analysis paragraph of the Materials and methods section.
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Related In: Results  -  Collection

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fig06: Representative pictures of transmission electron microscopy analysis of retinal sections from wild-type BALB/c (WT) and Abca4−/− mice injected with dual AAV trans-splicing (TS-ABCA4) and hybrid AK vectors (AK-ABCA4) or with either AAV vectors expressing EGFP or 5′-or 3′-half of the dual hybrid AK vectors (neg), as negative controls. The dotted lines indicate the edges of RPE cells. The scale bar (3.8 μm) is depicted in the figure.Quantification of the mean RPE thickness counted in at least 30 fields for each sample. The number ( n) of eyes analyzed is depicted below each bar. Abca4−/− neg includes Abca4−/− mice injected with either AAV vectors expressing EGFP ( n = 2) or 5′-( n = 3) or 3′-( n = 4) half of the dual hybrid AK vectors (neg total n = 9). The mean value is depicted above the corresponding bar. Values are represented as mean ± standard error of the mean (s.e.m.). * P ANOVA < 0.05.Recovery from light desensitization in 3 month-old Abca4−/− and BALB/c mice at 6 weeks post-injection. The relative b-wave is the ratio between the post-and the pre-desensitization b-wave amplitudes both evoked by 1 cd s/m2. The time refers to the minutes post-desensitization. The mean recovery (%) at 60 min is depicted.Data information: (A-C) WT, BALB/c eyes ( n = 4); Abca4−/− TS-ABCA4, eyes injected with dual AAV TS vectors ( n = 5); Abca4−/− AK-ABCA4, AAV hybrid AK vectors ( n = 5); Abca4−/− neg, Abca4−/− mice either not injected ( n = 2) or injected with the 5′-half of the dual AAV TS ( n = 3) or hybrid AK ( n = 2) vectors (neg total n = 7). Values are represented as mean ± standard error of the mean (s.e.m.). * P ANOVA < 0.05. More details on the statistical analysis including specific statistical values can be found in the Statistical analysis paragraph of the Materials and methods section.
Mentions: To evaluate the biological and therapeutic activity of the recombinant ABCA4 protein produced by dual AAV vectors, 1 month-old albino Abca4−/− mice were injected subretinally with the dual AAV TS and hybrid AK RHO-ABCA4-HA vectors (dose of each vector/eye: 1–3 × 109 GC). Three months later, eyes were harvested and immuno-electron microscopy analysis with anti-hemagglutinin (HA) antibodies of retinal sections confirmed that immunogold particles were correctly localized in PR outer segments only in animals that were injected with the combination of 5′ and 3′ dual AAV TS and hybrid AK vectors (Fig 5B). To assess the functionality of the ABCA4 protein expressed by the dual AAV vectors, we measured Abca4−/− retinal lipofuscin accumulation and recovery from light desensitization. To assess the former we performed transmission electron microscopy analysis to measure the number of RPE lipofuscin granules (Fig 5C and D) and RPE thickness (Fig 6A and B). Both were greater in the retina of Abca4−/− mice injected with control vectors (independently of the size of the control constructs, supplementary Fig S10) than in the retina of wild-type, age-matched BALB/c controls, and were significantly reduced or normalized in the eyes injected either with the therapeutic dual AAV TS or hybrid AK vectors (Figs 5C and D and 6A and B). We additionally attempted at measuring A2E, the major component of lipofuscin granules, by HPLC (Parish et al, 1998; Ben-Shabat et al, 2002; Allocca et al, 2008), however these measurements were inconsistent, even between affected and normal retinas (data not shown), thus we were not able to use these techniques in our rescue experiments. Importantly, the eyes treated with dual AAV TS and hybrid AK vectors showed improved recovery from light desensitization when compared to eyes treated with control vectors (dose of each vector/eye: 1.2 × 109 GC, Fig 6C), independently of the size of the control constructs [Student's t-test P value of dual AAV-EGFP-L ( n = 2 TS-L, n = 4 AK-L; total n = 6) versus dual AAV-EGFP ( n = 5 TS, n = 4 AK; total n = 9): 0.23].

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