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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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Related in: MedlinePlus

Representative Western blot analysis of C57BL/6 eyecups 1 month following the injection of dual AAV trans-splicing (TS) and hybrid AK (AK) vectors encoding for MYO7A-HA under the control of the ubiquitous chicken beta-actin (CBA) promoter. The arrow indicates full-length proteins, the molecular weight ladder is depicted on the left, 100 μg of proteins were loaded in each lane. The number ( n) and percentage of MYO7A-positive eyecups out of total eyecups analyzed is depicted. AK, eyes injected with dual AAV hybrid AK vectors; TS, eyes injected with dual AAV TS vectors; neg, eyes injected with either 5′-or 3′-half of the dual AAV TS and hybrid AK vectors; α-HA, Western blot with anti-hemagglutinin (HA) antibody; α-Dysferlin, Western blot with anti-Dysferlin antibody, used as loading control.Quantification of MYO7A levels expressed from dual AAV vectors in sh1−/− eyecups relative to endogenous Myo7a expressed in littermate sh1+/– eyecups. sh1−/− eyes were injected with dual AAV TS and hybrid AK vectors encoding MYO7A under the control of the CBA promoter and analyzed 1.5 months later. sh1+/– eyes were injected with AAV vectors expressing EGFP. The number ( n) of eyes analyzed is depicted below each bar. The quantification was performed by Western blot analysis using the anti-Myo7a antibody and measurements of MYO7A and Myo7a band intensities normalized to Dysferlin. The histograms show the expression of MYO7A protein as percentage relative to sh1+/– Myo7a; the mean value is depicted above the corresponding bars. Values are represented as mean ± standard error of the mean (s.e.m.).
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fig07: Representative Western blot analysis of C57BL/6 eyecups 1 month following the injection of dual AAV trans-splicing (TS) and hybrid AK (AK) vectors encoding for MYO7A-HA under the control of the ubiquitous chicken beta-actin (CBA) promoter. The arrow indicates full-length proteins, the molecular weight ladder is depicted on the left, 100 μg of proteins were loaded in each lane. The number ( n) and percentage of MYO7A-positive eyecups out of total eyecups analyzed is depicted. AK, eyes injected with dual AAV hybrid AK vectors; TS, eyes injected with dual AAV TS vectors; neg, eyes injected with either 5′-or 3′-half of the dual AAV TS and hybrid AK vectors; α-HA, Western blot with anti-hemagglutinin (HA) antibody; α-Dysferlin, Western blot with anti-Dysferlin antibody, used as loading control.Quantification of MYO7A levels expressed from dual AAV vectors in sh1−/− eyecups relative to endogenous Myo7a expressed in littermate sh1+/– eyecups. sh1−/− eyes were injected with dual AAV TS and hybrid AK vectors encoding MYO7A under the control of the CBA promoter and analyzed 1.5 months later. sh1+/– eyes were injected with AAV vectors expressing EGFP. The number ( n) of eyes analyzed is depicted below each bar. The quantification was performed by Western blot analysis using the anti-Myo7a antibody and measurements of MYO7A and Myo7a band intensities normalized to Dysferlin. The histograms show the expression of MYO7A protein as percentage relative to sh1+/– Myo7a; the mean value is depicted above the corresponding bars. Values are represented as mean ± standard error of the mean (s.e.m.).

Mentions: We then tested the efficacy of dual AAV-mediated MYO7A gene transfer in the retina of sh1 mice, the most commonly used model of USH1B (Liu et al, 1997, 1998, 1999; Lillo et al, 2003; Gibbs et al, 2010). In sh1 mice, a deficiency in the motor Myo7a causes the mis-localization of RPE melanosomes (Liu et al, 1998), which do not enter into the RPE apical microvilli, and the accumulation of rhodopsin at the PR connecting cilium (Liu et al, 1999). MYO7A is highly expressed in the RPE and to a lesser extent in PR (Hasson et al, 1995; Liu et al, 1997), therefore we used dual AAV TS and hybrid AK vectors expressing MYO7A-HA under the transcriptional control of the ubiquitous CBA promoter. One month-old wild-type C57BL/6 mice were injected with the dual AAV vectors (dose of each vector/eye: 1.7 × 109 GC) and eyecup lysates were evaluated 1 month later using Western blot analysis with anti-HA antibodies. Results showed similarly robust and consistent levels of MYO7A expression in retinas treated with both approaches (Fig 7A). Taking advantage of our anti-Myo7a antibody able to recognize both murine and human MYO7A (although with potentially different affinity for the two orthologous proteins), we compared the levels of MYO7A achieved following delivery of dual AAV vectors to the sh1−/− eye to those expressed endogenously in the sh1+/− eye (Fig 7B). The levels of human MYO7A driven by the CBA promoter 1 month after treatment (Fig 7B; dose of each vector/eye: 1.7 × 109 GC) were 19–21% of endogenous Myo7a expressed in both RPE and PR (Fig 7B) and these remained similar at 9 months after vector delivery (MYO7A retinal levels after subretinal delivery of dual AAV-TS, quantified on Western blot of eyecup lysates as in Fig 7B: 19 ± 6% of endogenous Myo7a, n = 5). Notably, subretinal delivery of dual AAV TS and hybrid AK resulted in efficient expression of human MYO7A specifically in PR when using the RHO promoter (supplementary Fig S11). No MYO7A proteins of size different from the full-length were detected by Western blot analysis of sh1−/− eyecups treated with either dual AAV TS or hybrid AK vectors. However, two proteins smaller than the full length MYO7A (<130 KDa) are detected in vitro following infection with either the single 5′-or 3′-half of both dual AAV approaches (supplementary Fig S12 and S13).


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 Western blot analysis of C57BL/6 eyecups 1 month following the injection of dual AAV trans-splicing (TS) and hybrid AK (AK) vectors encoding for MYO7A-HA under the control of the ubiquitous chicken beta-actin (CBA) promoter. The arrow indicates full-length proteins, the molecular weight ladder is depicted on the left, 100 μg of proteins were loaded in each lane. The number ( n) and percentage of MYO7A-positive eyecups out of total eyecups analyzed is depicted. AK, eyes injected with dual AAV hybrid AK vectors; TS, eyes injected with dual AAV TS vectors; neg, eyes injected with either 5′-or 3′-half of the dual AAV TS and hybrid AK vectors; α-HA, Western blot with anti-hemagglutinin (HA) antibody; α-Dysferlin, Western blot with anti-Dysferlin antibody, used as loading control.Quantification of MYO7A levels expressed from dual AAV vectors in sh1−/− eyecups relative to endogenous Myo7a expressed in littermate sh1+/– eyecups. sh1−/− eyes were injected with dual AAV TS and hybrid AK vectors encoding MYO7A under the control of the CBA promoter and analyzed 1.5 months later. sh1+/– eyes were injected with AAV vectors expressing EGFP. The number ( n) of eyes analyzed is depicted below each bar. The quantification was performed by Western blot analysis using the anti-Myo7a antibody and measurements of MYO7A and Myo7a band intensities normalized to Dysferlin. The histograms show the expression of MYO7A protein as percentage relative to sh1+/– Myo7a; the mean value is depicted above the corresponding bars. Values are represented as mean ± standard error of the mean (s.e.m.).
© Copyright Policy - open-access
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fig07: Representative Western blot analysis of C57BL/6 eyecups 1 month following the injection of dual AAV trans-splicing (TS) and hybrid AK (AK) vectors encoding for MYO7A-HA under the control of the ubiquitous chicken beta-actin (CBA) promoter. The arrow indicates full-length proteins, the molecular weight ladder is depicted on the left, 100 μg of proteins were loaded in each lane. The number ( n) and percentage of MYO7A-positive eyecups out of total eyecups analyzed is depicted. AK, eyes injected with dual AAV hybrid AK vectors; TS, eyes injected with dual AAV TS vectors; neg, eyes injected with either 5′-or 3′-half of the dual AAV TS and hybrid AK vectors; α-HA, Western blot with anti-hemagglutinin (HA) antibody; α-Dysferlin, Western blot with anti-Dysferlin antibody, used as loading control.Quantification of MYO7A levels expressed from dual AAV vectors in sh1−/− eyecups relative to endogenous Myo7a expressed in littermate sh1+/– eyecups. sh1−/− eyes were injected with dual AAV TS and hybrid AK vectors encoding MYO7A under the control of the CBA promoter and analyzed 1.5 months later. sh1+/– eyes were injected with AAV vectors expressing EGFP. The number ( n) of eyes analyzed is depicted below each bar. The quantification was performed by Western blot analysis using the anti-Myo7a antibody and measurements of MYO7A and Myo7a band intensities normalized to Dysferlin. The histograms show the expression of MYO7A protein as percentage relative to sh1+/– Myo7a; the mean value is depicted above the corresponding bars. Values are represented as mean ± standard error of the mean (s.e.m.).
Mentions: We then tested the efficacy of dual AAV-mediated MYO7A gene transfer in the retina of sh1 mice, the most commonly used model of USH1B (Liu et al, 1997, 1998, 1999; Lillo et al, 2003; Gibbs et al, 2010). In sh1 mice, a deficiency in the motor Myo7a causes the mis-localization of RPE melanosomes (Liu et al, 1998), which do not enter into the RPE apical microvilli, and the accumulation of rhodopsin at the PR connecting cilium (Liu et al, 1999). MYO7A is highly expressed in the RPE and to a lesser extent in PR (Hasson et al, 1995; Liu et al, 1997), therefore we used dual AAV TS and hybrid AK vectors expressing MYO7A-HA under the transcriptional control of the ubiquitous CBA promoter. One month-old wild-type C57BL/6 mice were injected with the dual AAV vectors (dose of each vector/eye: 1.7 × 109 GC) and eyecup lysates were evaluated 1 month later using Western blot analysis with anti-HA antibodies. Results showed similarly robust and consistent levels of MYO7A expression in retinas treated with both approaches (Fig 7A). Taking advantage of our anti-Myo7a antibody able to recognize both murine and human MYO7A (although with potentially different affinity for the two orthologous proteins), we compared the levels of MYO7A achieved following delivery of dual AAV vectors to the sh1−/− eye to those expressed endogenously in the sh1+/− eye (Fig 7B). The levels of human MYO7A driven by the CBA promoter 1 month after treatment (Fig 7B; dose of each vector/eye: 1.7 × 109 GC) were 19–21% of endogenous Myo7a expressed in both RPE and PR (Fig 7B) and these remained similar at 9 months after vector delivery (MYO7A retinal levels after subretinal delivery of dual AAV-TS, quantified on Western blot of eyecup lysates as in Fig 7B: 19 ± 6% of endogenous Myo7a, n = 5). Notably, subretinal delivery of dual AAV TS and hybrid AK resulted in efficient expression of human MYO7A specifically in PR when using the RHO promoter (supplementary Fig S11). No MYO7A proteins of size different from the full-length were detected by Western blot analysis of sh1−/− eyecups treated with either dual AAV TS or hybrid AK vectors. However, two proteins smaller than the full length MYO7A (<130 KDa) are detected in vitro following infection with either the single 5′-or 3′-half of both dual AAV approaches (supplementary Fig S12 and S13).

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