Limits...
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.

Show MeSH

Related in: MedlinePlus

Semi-thin retinal sections representative of both sh1+/+ and sh1+/– eyes ( sh1+/+) injected with AAV vectors expressing EGFP and of sh1−/− eyes injected with dual AAV trans-splicing (TS-MYO7A), hybrid AK (AK-MYO7A) or the 5′-or 3′-half vectors (neg), as negative controls. The arrows point at correctly localized melanosomes, the scale bar (10 μm) is depicted in the figure.Quantification of melanosome localization in the RPE villi of sh1 mice 2–3 months following subretinal delivery of dual AAV vectors. The n of eyes analyzed is depicted below each bar. The quantification is depicted as the mean number of apical melanosomes/100 μm, the mean value is depicted above the corresponding bar. sh1−/− neg includes sh1−/− eyes injected with AAV vectors expressing either 5′-( n = 1) or 3′-( n = 2) half of the dual TS vectors, or 5′-half ( n = 2) of the dual hybrid AK vectors (neg total n = 5). Values are represented as mean ± standard error of the mean (s.e.m.). * P ANOVA < 0.05, ** P ANOVA < 0.001.Quantification of the number of rhodopsin gold particles at the PR connecting cilium of sh1 mice 2–3 months following subretinal delivery of dual AAV vectors. The n of eyes and connecting cilia analyzed is depicted below each bar. sh1−/− neg includes sh1−/− eyes injected with AAV vectors expressing either 5′-half of the dual TS vectors ( n = 3) or 5′-half ( n = 2) of the dual hybrid AK vectors (neg total n = 5). The quantification is depicted as the mean number of gold particles per length of connecting cilium, the mean value is depicted above the corresponding bar. Values are represented as mean ± standard error of the mean (s.e.m.). More details on the statistical analysis including specific statistical values can be found in the Statistical analysis paragraph of the Materials and methods section.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3927955&req=5

fig08: Semi-thin retinal sections representative of both sh1+/+ and sh1+/– eyes ( sh1+/+) injected with AAV vectors expressing EGFP and of sh1−/− eyes injected with dual AAV trans-splicing (TS-MYO7A), hybrid AK (AK-MYO7A) or the 5′-or 3′-half vectors (neg), as negative controls. The arrows point at correctly localized melanosomes, the scale bar (10 μm) is depicted in the figure.Quantification of melanosome localization in the RPE villi of sh1 mice 2–3 months following subretinal delivery of dual AAV vectors. The n of eyes analyzed is depicted below each bar. The quantification is depicted as the mean number of apical melanosomes/100 μm, the mean value is depicted above the corresponding bar. sh1−/− neg includes sh1−/− eyes injected with AAV vectors expressing either 5′-( n = 1) or 3′-( n = 2) half of the dual TS vectors, or 5′-half ( n = 2) of the dual hybrid AK vectors (neg total n = 5). Values are represented as mean ± standard error of the mean (s.e.m.). * P ANOVA < 0.05, ** P ANOVA < 0.001.Quantification of the number of rhodopsin gold particles at the PR connecting cilium of sh1 mice 2–3 months following subretinal delivery of dual AAV vectors. The n of eyes and connecting cilia analyzed is depicted below each bar. sh1−/− neg includes sh1−/− eyes injected with AAV vectors expressing either 5′-half of the dual TS vectors ( n = 3) or 5′-half ( n = 2) of the dual hybrid AK vectors (neg total n = 5). The quantification is depicted as the mean number of gold particles per length of connecting cilium, the mean value is depicted above the corresponding bar. Values are represented as mean ± standard error of the mean (s.e.m.). 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 test the ability of MYO7A expressed from dual AAV vectors to rescue the defects of the sh1−/− retina, we evaluated RPE melanosome (Fig 8A and B) and rhodopsin localization (Fig 8C) following subretinal injection of dual AAV TS and hybrid AK CBA-MYO7A vectors (dose of each vector/eye: 1.7 × 109 GC) in 1 month-old sh1−/− mice. Unlike in unaffected mice, the sh1−/− melanosomes do not enter the RPE apical microvilli (Fig 8A and B), this was significantly improved after the delivery of either dual AAV TS or hybrid AK vectors encoding MYO7A (Fig 8A and B).


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)

Semi-thin retinal sections representative of both sh1+/+ and sh1+/– eyes ( sh1+/+) injected with AAV vectors expressing EGFP and of sh1−/− eyes injected with dual AAV trans-splicing (TS-MYO7A), hybrid AK (AK-MYO7A) or the 5′-or 3′-half vectors (neg), as negative controls. The arrows point at correctly localized melanosomes, the scale bar (10 μm) is depicted in the figure.Quantification of melanosome localization in the RPE villi of sh1 mice 2–3 months following subretinal delivery of dual AAV vectors. The n of eyes analyzed is depicted below each bar. The quantification is depicted as the mean number of apical melanosomes/100 μm, the mean value is depicted above the corresponding bar. sh1−/− neg includes sh1−/− eyes injected with AAV vectors expressing either 5′-( n = 1) or 3′-( n = 2) half of the dual TS vectors, or 5′-half ( n = 2) of the dual hybrid AK vectors (neg total n = 5). Values are represented as mean ± standard error of the mean (s.e.m.). * P ANOVA < 0.05, ** P ANOVA < 0.001.Quantification of the number of rhodopsin gold particles at the PR connecting cilium of sh1 mice 2–3 months following subretinal delivery of dual AAV vectors. The n of eyes and connecting cilia analyzed is depicted below each bar. sh1−/− neg includes sh1−/− eyes injected with AAV vectors expressing either 5′-half of the dual TS vectors ( n = 3) or 5′-half ( n = 2) of the dual hybrid AK vectors (neg total n = 5). The quantification is depicted as the mean number of gold particles per length of connecting cilium, the mean value is depicted above the corresponding bar. Values are represented as mean ± standard error of the mean (s.e.m.). More details on the statistical analysis including specific statistical values can be found in the Statistical analysis paragraph of the Materials and methods section.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig08: Semi-thin retinal sections representative of both sh1+/+ and sh1+/– eyes ( sh1+/+) injected with AAV vectors expressing EGFP and of sh1−/− eyes injected with dual AAV trans-splicing (TS-MYO7A), hybrid AK (AK-MYO7A) or the 5′-or 3′-half vectors (neg), as negative controls. The arrows point at correctly localized melanosomes, the scale bar (10 μm) is depicted in the figure.Quantification of melanosome localization in the RPE villi of sh1 mice 2–3 months following subretinal delivery of dual AAV vectors. The n of eyes analyzed is depicted below each bar. The quantification is depicted as the mean number of apical melanosomes/100 μm, the mean value is depicted above the corresponding bar. sh1−/− neg includes sh1−/− eyes injected with AAV vectors expressing either 5′-( n = 1) or 3′-( n = 2) half of the dual TS vectors, or 5′-half ( n = 2) of the dual hybrid AK vectors (neg total n = 5). Values are represented as mean ± standard error of the mean (s.e.m.). * P ANOVA < 0.05, ** P ANOVA < 0.001.Quantification of the number of rhodopsin gold particles at the PR connecting cilium of sh1 mice 2–3 months following subretinal delivery of dual AAV vectors. The n of eyes and connecting cilia analyzed is depicted below each bar. sh1−/− neg includes sh1−/− eyes injected with AAV vectors expressing either 5′-half of the dual TS vectors ( n = 3) or 5′-half ( n = 2) of the dual hybrid AK vectors (neg total n = 5). The quantification is depicted as the mean number of gold particles per length of connecting cilium, the mean value is depicted above the corresponding bar. Values are represented as mean ± standard error of the mean (s.e.m.). 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 test the ability of MYO7A expressed from dual AAV vectors to rescue the defects of the sh1−/− retina, we evaluated RPE melanosome (Fig 8A and B) and rhodopsin localization (Fig 8C) following subretinal injection of dual AAV TS and hybrid AK CBA-MYO7A vectors (dose of each vector/eye: 1.7 × 109 GC) in 1 month-old sh1−/− mice. Unlike in unaffected mice, the sh1−/− melanosomes do not enter the RPE apical microvilli (Fig 8A and B), this was significantly improved after the delivery of either dual AAV TS or hybrid AK vectors encoding MYO7A (Fig 8A and B).

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