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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–F Western blot analysis of HEK293 cells infected with AAV2/2 vectors encoding for ABCA4 (A, D), MYO7A (B, E) and EGFP (C, F). The Western blot images (A-C) are representative of and the quantifications (D-F) are from n = 4 (A, C, D, F) or n = 3 (B, E) independent experiments. OZ, AAV oversize; OV, dual AAV overlapping; TS, dual AAV trans-splicing; AP, dual AAV hybrid AP; AK, dual AAV hybrid AK; TS-L, dual AAV trans-splicing EGFP with a combined genome size similar to OZ-EGFP; AK-L, dual AAV hybrid AK EGFP with a combined genome size similar to OZ-EGFP; 5′+3′, cells co-infected with 5′-and 3′-half vectors; 5′, control cells infected with the 5′-half vector; 3′, control cells infected with the 3′-half vector; α-EGFP, Western blot with anti-EGFP antibody; α-3xflag, Western blot with anti-3xflag antibody; α-Myo7a, Western blot with anti-Myo7a antibody; α-β-Tubulin, Western blot with anti-β-Tubulin antibody, used as loading control; α-Filamin A, Western blot with anti-Filamin A antibody, used as loading control. * P ANOVA < 0.05; ** P ANOVA < 0.001.A–C The arrows indicate full-length proteins, the micrograms of proteins loaded are depicted under each lane, the molecular weight ladder is depicted on the left.D–F Quantification of ABCA4 (D), MYO7A (E) and EGFP (F) protein bands. The intensity of the ABCA4, MYO7A and EGFP bands was divided by the intensity of the Filamin A (D, E) or Tubulin (F) bands. The histograms show the expression of proteins as a percentage relative to dual AAV trans-splicing (TS) vectors, the mean value is depicted above the corresponding bar. Values are represented as mean ± standard error of the mean (s.e.m.).Data information: (E) The asterisks represent significant differences with both OZ and AP. (D–F) More details on the TS and TS-L variability as well as on the statistical analysis including specific statistical values can be found in the Western blot and Statistical analysis paragraphs of the Materials and methods section, respectively.
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fig02: A–F Western blot analysis of HEK293 cells infected with AAV2/2 vectors encoding for ABCA4 (A, D), MYO7A (B, E) and EGFP (C, F). The Western blot images (A-C) are representative of and the quantifications (D-F) are from n = 4 (A, C, D, F) or n = 3 (B, E) independent experiments. OZ, AAV oversize; OV, dual AAV overlapping; TS, dual AAV trans-splicing; AP, dual AAV hybrid AP; AK, dual AAV hybrid AK; TS-L, dual AAV trans-splicing EGFP with a combined genome size similar to OZ-EGFP; AK-L, dual AAV hybrid AK EGFP with a combined genome size similar to OZ-EGFP; 5′+3′, cells co-infected with 5′-and 3′-half vectors; 5′, control cells infected with the 5′-half vector; 3′, control cells infected with the 3′-half vector; α-EGFP, Western blot with anti-EGFP antibody; α-3xflag, Western blot with anti-3xflag antibody; α-Myo7a, Western blot with anti-Myo7a antibody; α-β-Tubulin, Western blot with anti-β-Tubulin antibody, used as loading control; α-Filamin A, Western blot with anti-Filamin A antibody, used as loading control. * P ANOVA < 0.05; ** P ANOVA < 0.001.A–C The arrows indicate full-length proteins, the micrograms of proteins loaded are depicted under each lane, the molecular weight ladder is depicted on the left.D–F Quantification of ABCA4 (D), MYO7A (E) and EGFP (F) protein bands. The intensity of the ABCA4, MYO7A and EGFP bands was divided by the intensity of the Filamin A (D, E) or Tubulin (F) bands. The histograms show the expression of proteins as a percentage relative to dual AAV trans-splicing (TS) vectors, the mean value is depicted above the corresponding bar. Values are represented as mean ± standard error of the mean (s.e.m.).Data information: (E) The asterisks represent significant differences with both OZ and AP. (D–F) More details on the TS and TS-L variability as well as on the statistical analysis including specific statistical values can be found in the Western blot and Statistical analysis paragraphs of the Materials and methods section, respectively.

Mentions: We initially compared the efficiency of the various OZ, dual AAV OV, TS and hybrid AP and AK strategies for AAV-mediated large gene transduction in vitro by infecting HEK293 cells with the AAV2/2 vectors [multiplicity of infection, m.o.i.: 105 genome copies (GC)/cell of each vector] with ubiquitous promoters (CMV for ABCA4-3xflag, CBA for MYO7A-HA). Cell lysates were analyzed by Western blot with anti-3xflag (to detect ABCA4-3xflag, Fig 2A) or anti-Myo7a (Fig 2B) antibodies. All strategies resulted in the expression of proteins of the expected size. As predicted, no bands of the expected size were observed when only one of the dual AAV vectors was used for infection (Fig 2A and B). Quantification of ABCA4 and MYO7A expression (Fig 2D and E) showed that the dual AAV hybrid AP approach resulted in the lowest levels of transgene expression, while the dual AAV OV, TS and hybrid AK approaches were more efficient than the AAV OZ approach. We then confirmed this using the EGFP transgene. For this purpose we selected the best performing dual AAV strategies (TS and hybrid AK; we did not use the transgene-specific OV strategy with EGFP, which is a reporter gene) and further compared them to AAV OZ. We thus produced AAV2/2-CMV-OZ-EGFP vectors and for comparison-TS-and-AK-EGFP-L in which the combined dual AAV vector genome length is similar to that of AAV OZ (supplementary Table S1). We infected HEK293 cells with AAV2/2-CMV-EGFP vectors [multiplicity of infection, m.o.i.: 105 genome copies (GC)/cell of each vector] and performed Western blot analysis of cell lysates with anti-EGFP antibodies (Fig 2C). Similarly to what observed with the ABCA4 and MYO7A transgenes, quantification of EGFP expression (Fig 2F) showed that dual AAV TS and hybrid AK approaches are more efficient than AAV OZ.


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–F Western blot analysis of HEK293 cells infected with AAV2/2 vectors encoding for ABCA4 (A, D), MYO7A (B, E) and EGFP (C, F). The Western blot images (A-C) are representative of and the quantifications (D-F) are from n = 4 (A, C, D, F) or n = 3 (B, E) independent experiments. OZ, AAV oversize; OV, dual AAV overlapping; TS, dual AAV trans-splicing; AP, dual AAV hybrid AP; AK, dual AAV hybrid AK; TS-L, dual AAV trans-splicing EGFP with a combined genome size similar to OZ-EGFP; AK-L, dual AAV hybrid AK EGFP with a combined genome size similar to OZ-EGFP; 5′+3′, cells co-infected with 5′-and 3′-half vectors; 5′, control cells infected with the 5′-half vector; 3′, control cells infected with the 3′-half vector; α-EGFP, Western blot with anti-EGFP antibody; α-3xflag, Western blot with anti-3xflag antibody; α-Myo7a, Western blot with anti-Myo7a antibody; α-β-Tubulin, Western blot with anti-β-Tubulin antibody, used as loading control; α-Filamin A, Western blot with anti-Filamin A antibody, used as loading control. * P ANOVA < 0.05; ** P ANOVA < 0.001.A–C The arrows indicate full-length proteins, the micrograms of proteins loaded are depicted under each lane, the molecular weight ladder is depicted on the left.D–F Quantification of ABCA4 (D), MYO7A (E) and EGFP (F) protein bands. The intensity of the ABCA4, MYO7A and EGFP bands was divided by the intensity of the Filamin A (D, E) or Tubulin (F) bands. The histograms show the expression of proteins as a percentage relative to dual AAV trans-splicing (TS) vectors, the mean value is depicted above the corresponding bar. Values are represented as mean ± standard error of the mean (s.e.m.).Data information: (E) The asterisks represent significant differences with both OZ and AP. (D–F) More details on the TS and TS-L variability as well as on the statistical analysis including specific statistical values can be found in the Western blot and Statistical analysis paragraphs of the Materials and methods section, respectively.
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fig02: A–F Western blot analysis of HEK293 cells infected with AAV2/2 vectors encoding for ABCA4 (A, D), MYO7A (B, E) and EGFP (C, F). The Western blot images (A-C) are representative of and the quantifications (D-F) are from n = 4 (A, C, D, F) or n = 3 (B, E) independent experiments. OZ, AAV oversize; OV, dual AAV overlapping; TS, dual AAV trans-splicing; AP, dual AAV hybrid AP; AK, dual AAV hybrid AK; TS-L, dual AAV trans-splicing EGFP with a combined genome size similar to OZ-EGFP; AK-L, dual AAV hybrid AK EGFP with a combined genome size similar to OZ-EGFP; 5′+3′, cells co-infected with 5′-and 3′-half vectors; 5′, control cells infected with the 5′-half vector; 3′, control cells infected with the 3′-half vector; α-EGFP, Western blot with anti-EGFP antibody; α-3xflag, Western blot with anti-3xflag antibody; α-Myo7a, Western blot with anti-Myo7a antibody; α-β-Tubulin, Western blot with anti-β-Tubulin antibody, used as loading control; α-Filamin A, Western blot with anti-Filamin A antibody, used as loading control. * P ANOVA < 0.05; ** P ANOVA < 0.001.A–C The arrows indicate full-length proteins, the micrograms of proteins loaded are depicted under each lane, the molecular weight ladder is depicted on the left.D–F Quantification of ABCA4 (D), MYO7A (E) and EGFP (F) protein bands. The intensity of the ABCA4, MYO7A and EGFP bands was divided by the intensity of the Filamin A (D, E) or Tubulin (F) bands. The histograms show the expression of proteins as a percentage relative to dual AAV trans-splicing (TS) vectors, the mean value is depicted above the corresponding bar. Values are represented as mean ± standard error of the mean (s.e.m.).Data information: (E) The asterisks represent significant differences with both OZ and AP. (D–F) More details on the TS and TS-L variability as well as on the statistical analysis including specific statistical values can be found in the Western blot and Statistical analysis paragraphs of the Materials and methods section, respectively.
Mentions: We initially compared the efficiency of the various OZ, dual AAV OV, TS and hybrid AP and AK strategies for AAV-mediated large gene transduction in vitro by infecting HEK293 cells with the AAV2/2 vectors [multiplicity of infection, m.o.i.: 105 genome copies (GC)/cell of each vector] with ubiquitous promoters (CMV for ABCA4-3xflag, CBA for MYO7A-HA). Cell lysates were analyzed by Western blot with anti-3xflag (to detect ABCA4-3xflag, Fig 2A) or anti-Myo7a (Fig 2B) antibodies. All strategies resulted in the expression of proteins of the expected size. As predicted, no bands of the expected size were observed when only one of the dual AAV vectors was used for infection (Fig 2A and B). Quantification of ABCA4 and MYO7A expression (Fig 2D and E) showed that the dual AAV hybrid AP approach resulted in the lowest levels of transgene expression, while the dual AAV OV, TS and hybrid AK approaches were more efficient than the AAV OZ approach. We then confirmed this using the EGFP transgene. For this purpose we selected the best performing dual AAV strategies (TS and hybrid AK; we did not use the transgene-specific OV strategy with EGFP, which is a reporter gene) and further compared them to AAV OZ. We thus produced AAV2/2-CMV-OZ-EGFP vectors and for comparison-TS-and-AK-EGFP-L in which the combined dual AAV vector genome length is similar to that of AAV OZ (supplementary Table S1). We infected HEK293 cells with AAV2/2-CMV-EGFP vectors [multiplicity of infection, m.o.i.: 105 genome copies (GC)/cell of each vector] and performed Western blot analysis of cell lysates with anti-EGFP antibodies (Fig 2C). Similarly to what observed with the ABCA4 and MYO7A transgenes, quantification of EGFP expression (Fig 2F) showed that dual AAV TS and hybrid AK approaches are more efficient than AAV OZ.

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