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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 retinal lysates 1 month following the injection of dual AAV trans-splicing (TS) and hybrid AK (AK) vectors encoding for ABCA4 under the control of the PR-specific Rhodopsin promoter (RHO PROMOTER). The arrow points at full-length proteins, the molecular weight ladder is depicted on the left, 150 μg of protein were loaded in each lane. The number ( n) and percentage of ABCA4-positive retinas out of total retinas analyzed is depicted. AK, retinas injected with dual AAV hybrid AK vectors; TS, retinas injected with dual AAV TS vectors; neg, retinas injected with AAV vectors expressing EGFP, as negative controls; α-3xflag, Western blot with anti-3xflag antibody; α-Dysferlin, Western blot with anti-Dysferlin antibody, used as loading control.Representative pictures of immuno-electron microscopy analysis with anti-HA antibody of retinal sections from wild-type BALB/c (WT) and Abca4−/− mice injected with either dual AAV or with negative control vectors. The black dots represent the immuno-gold labelling of the ABCA4-HA protein. The scale bar (200 nm) is depicted in the figure.Representative pictures of transmission electron microscopy analysis showing lipofuscin granules content in the RPE of WT and Abca4−/− mice injected with either dual AAV or negative control vectors. The black arrows indicate lipofuscin granules. The scale bar (1.6 μm) is depicted in the figure.Quantification of the mean number of lipofuscin granules counted in at least 30 fields (25 μm2) for each sample. The number ( n) of eyes analyzed is depicted below each bar. 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; ** P ANOVA < 0.001. More details on the statistical analysis including specific statistical values can be found in the Statistical analysis paragraph of the Materials and methods section.Data information: (B-D) WT, BALB/c eyes; Abca4−/− neg, Abca4−/− eyes injected with either AAV vectors expressing EGFP ( n = 2) or the 5′-( n = 3) or 3′-( n = 4) half of the dual AAV hybrid AK vectors, as negative control (neg total n = 9); Abca4−/− AK-ABCA4, mice injected with dual AAV hybrid AK vectors; Abca4−/− TS-ABCA4, mice injected with dual AAV TS vectors.
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fig05: Representative Western blot analysis of C57BL/6 retinal lysates 1 month following the injection of dual AAV trans-splicing (TS) and hybrid AK (AK) vectors encoding for ABCA4 under the control of the PR-specific Rhodopsin promoter (RHO PROMOTER). The arrow points at full-length proteins, the molecular weight ladder is depicted on the left, 150 μg of protein were loaded in each lane. The number ( n) and percentage of ABCA4-positive retinas out of total retinas analyzed is depicted. AK, retinas injected with dual AAV hybrid AK vectors; TS, retinas injected with dual AAV TS vectors; neg, retinas injected with AAV vectors expressing EGFP, as negative controls; α-3xflag, Western blot with anti-3xflag antibody; α-Dysferlin, Western blot with anti-Dysferlin antibody, used as loading control.Representative pictures of immuno-electron microscopy analysis with anti-HA antibody of retinal sections from wild-type BALB/c (WT) and Abca4−/− mice injected with either dual AAV or with negative control vectors. The black dots represent the immuno-gold labelling of the ABCA4-HA protein. The scale bar (200 nm) is depicted in the figure.Representative pictures of transmission electron microscopy analysis showing lipofuscin granules content in the RPE of WT and Abca4−/− mice injected with either dual AAV or negative control vectors. The black arrows indicate lipofuscin granules. The scale bar (1.6 μm) is depicted in the figure.Quantification of the mean number of lipofuscin granules counted in at least 30 fields (25 μm2) for each sample. The number ( n) of eyes analyzed is depicted below each bar. 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; ** P ANOVA < 0.001. More details on the statistical analysis including specific statistical values can be found in the Statistical analysis paragraph of the Materials and methods section.Data information: (B-D) WT, BALB/c eyes; Abca4−/− neg, Abca4−/− eyes injected with either AAV vectors expressing EGFP ( n = 2) or the 5′-( n = 3) or 3′-( n = 4) half of the dual AAV hybrid AK vectors, as negative control (neg total n = 9); Abca4−/− AK-ABCA4, mice injected with dual AAV hybrid AK vectors; Abca4−/− TS-ABCA4, mice injected with dual AAV TS vectors.

Mentions: Although the Abca4−/− mouse model does not undergo severe PR degeneration (Wu et al, 2010a), the absence of the ABCA4-encoded all-trans retinal transporter in PR outer segments (Illing et al, 1997; Sun & Nathans, 1997) causes an accumulation of lipofuscin in PR as well as in RPE, as result of PR phagocytosis by RPE (Weng et al, 1999; Mata et al, 2001). As a consequence, both the number of lipofuscin granules in the RPE and the thickness of RPE cells are greater in Abca4−/− mice than in control mice (Allocca et al, 2008; Radu et al, 2008; Ma et al, 2011; Conley et al, 2012; Han et al, 2012). In addition, Abca4−/− mice also show delayed recovery from light desensitization (Weng et al, 1999; Maiti et al, 2006; Allocca et al, 2008; Radu et al, 2008; Han et al, 2012). Since ABCA4 is expressed specifically in PR, we generated dual AAV TS and hybrid AK vectors encoding ABCA4-3xflag under the transcriptional control of the RHO promoter. These vectors were subretinally injected in wild-type C57BL/6 mice (dose of each vector/eye: 3–5 × 109 GC) and 1 month later retinas were lysed and analyzed by Western blot with anti-3xflag antibodies (Fig 5A). Both approaches resulted in robust yet variable levels of ABCA4-3xflag expression. ABCA4-3xflag expression levels were more consistent in retinas treated with the dual AAV hybrid AK vectors (Fig 5A). No truncated and/or aberrant ABCA4 proteins were detected by Western blot analysis of C57BL/6 eyecups treated with dual AAV TS and hybrid AK vectors using anti-3xflag antibodies although two proteins (>100 KDa) smaller than the full length are produced in vitro following infection with either the single 5′-or 3′-half of both dual AAV approaches (supplementary Figs S7 and S8). In addition no evident signs of retinal toxicity were observed in Abca4−/− mice at 8 months after treatment with dual AAV TS and hybrid AK vectors by conventional histological analysis (supplementary Fig S9A).


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 retinal lysates 1 month following the injection of dual AAV trans-splicing (TS) and hybrid AK (AK) vectors encoding for ABCA4 under the control of the PR-specific Rhodopsin promoter (RHO PROMOTER). The arrow points at full-length proteins, the molecular weight ladder is depicted on the left, 150 μg of protein were loaded in each lane. The number ( n) and percentage of ABCA4-positive retinas out of total retinas analyzed is depicted. AK, retinas injected with dual AAV hybrid AK vectors; TS, retinas injected with dual AAV TS vectors; neg, retinas injected with AAV vectors expressing EGFP, as negative controls; α-3xflag, Western blot with anti-3xflag antibody; α-Dysferlin, Western blot with anti-Dysferlin antibody, used as loading control.Representative pictures of immuno-electron microscopy analysis with anti-HA antibody of retinal sections from wild-type BALB/c (WT) and Abca4−/− mice injected with either dual AAV or with negative control vectors. The black dots represent the immuno-gold labelling of the ABCA4-HA protein. The scale bar (200 nm) is depicted in the figure.Representative pictures of transmission electron microscopy analysis showing lipofuscin granules content in the RPE of WT and Abca4−/− mice injected with either dual AAV or negative control vectors. The black arrows indicate lipofuscin granules. The scale bar (1.6 μm) is depicted in the figure.Quantification of the mean number of lipofuscin granules counted in at least 30 fields (25 μm2) for each sample. The number ( n) of eyes analyzed is depicted below each bar. 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; ** P ANOVA < 0.001. More details on the statistical analysis including specific statistical values can be found in the Statistical analysis paragraph of the Materials and methods section.Data information: (B-D) WT, BALB/c eyes; Abca4−/− neg, Abca4−/− eyes injected with either AAV vectors expressing EGFP ( n = 2) or the 5′-( n = 3) or 3′-( n = 4) half of the dual AAV hybrid AK vectors, as negative control (neg total n = 9); Abca4−/− AK-ABCA4, mice injected with dual AAV hybrid AK vectors; Abca4−/− TS-ABCA4, mice injected with dual AAV TS vectors.
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Related In: Results  -  Collection

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fig05: Representative Western blot analysis of C57BL/6 retinal lysates 1 month following the injection of dual AAV trans-splicing (TS) and hybrid AK (AK) vectors encoding for ABCA4 under the control of the PR-specific Rhodopsin promoter (RHO PROMOTER). The arrow points at full-length proteins, the molecular weight ladder is depicted on the left, 150 μg of protein were loaded in each lane. The number ( n) and percentage of ABCA4-positive retinas out of total retinas analyzed is depicted. AK, retinas injected with dual AAV hybrid AK vectors; TS, retinas injected with dual AAV TS vectors; neg, retinas injected with AAV vectors expressing EGFP, as negative controls; α-3xflag, Western blot with anti-3xflag antibody; α-Dysferlin, Western blot with anti-Dysferlin antibody, used as loading control.Representative pictures of immuno-electron microscopy analysis with anti-HA antibody of retinal sections from wild-type BALB/c (WT) and Abca4−/− mice injected with either dual AAV or with negative control vectors. The black dots represent the immuno-gold labelling of the ABCA4-HA protein. The scale bar (200 nm) is depicted in the figure.Representative pictures of transmission electron microscopy analysis showing lipofuscin granules content in the RPE of WT and Abca4−/− mice injected with either dual AAV or negative control vectors. The black arrows indicate lipofuscin granules. The scale bar (1.6 μm) is depicted in the figure.Quantification of the mean number of lipofuscin granules counted in at least 30 fields (25 μm2) for each sample. The number ( n) of eyes analyzed is depicted below each bar. 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; ** P ANOVA < 0.001. More details on the statistical analysis including specific statistical values can be found in the Statistical analysis paragraph of the Materials and methods section.Data information: (B-D) WT, BALB/c eyes; Abca4−/− neg, Abca4−/− eyes injected with either AAV vectors expressing EGFP ( n = 2) or the 5′-( n = 3) or 3′-( n = 4) half of the dual AAV hybrid AK vectors, as negative control (neg total n = 9); Abca4−/− AK-ABCA4, mice injected with dual AAV hybrid AK vectors; Abca4−/− TS-ABCA4, mice injected with dual AAV TS vectors.
Mentions: Although the Abca4−/− mouse model does not undergo severe PR degeneration (Wu et al, 2010a), the absence of the ABCA4-encoded all-trans retinal transporter in PR outer segments (Illing et al, 1997; Sun & Nathans, 1997) causes an accumulation of lipofuscin in PR as well as in RPE, as result of PR phagocytosis by RPE (Weng et al, 1999; Mata et al, 2001). As a consequence, both the number of lipofuscin granules in the RPE and the thickness of RPE cells are greater in Abca4−/− mice than in control mice (Allocca et al, 2008; Radu et al, 2008; Ma et al, 2011; Conley et al, 2012; Han et al, 2012). In addition, Abca4−/− mice also show delayed recovery from light desensitization (Weng et al, 1999; Maiti et al, 2006; Allocca et al, 2008; Radu et al, 2008; Han et al, 2012). Since ABCA4 is expressed specifically in PR, we generated dual AAV TS and hybrid AK vectors encoding ABCA4-3xflag under the transcriptional control of the RHO promoter. These vectors were subretinally injected in wild-type C57BL/6 mice (dose of each vector/eye: 3–5 × 109 GC) and 1 month later retinas were lysed and analyzed by Western blot with anti-3xflag antibodies (Fig 5A). Both approaches resulted in robust yet variable levels of ABCA4-3xflag expression. ABCA4-3xflag expression levels were more consistent in retinas treated with the dual AAV hybrid AK vectors (Fig 5A). No truncated and/or aberrant ABCA4 proteins were detected by Western blot analysis of C57BL/6 eyecups treated with dual AAV TS and hybrid AK vectors using anti-3xflag antibodies although two proteins (>100 KDa) smaller than the full length are produced in vitro following infection with either the single 5′-or 3′-half of both dual AAV approaches (supplementary Figs S7 and S8). In addition no evident signs of retinal toxicity were observed in Abca4−/− mice at 8 months after treatment with dual AAV TS and hybrid AK vectors by conventional histological analysis (supplementary Fig S9A).

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