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Agostina Puppo, Giulia Cesi, Donna Palmer, Pasquale Piccolo, Robin J. Parks, Philip Ng, Nicola Brunetti-Pierri, Alberto Auricchio; Adenoviral and Lentiviral Vectors for Efficient Gene Transfer to Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5578.
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© ARVO (1962-2015); The Authors (2016-present)
Photoreceptors (PR) are important targets of gene therapy as these are affected cells in inherited retinal degenerations (IRD). Several forms of IRD are due to defects in large genes which cannot be accommodated into AAV vectors, which so far have demonstrated the greatest potential in preclinical animal models and human clinical trials. PR are refractory to transduction by the most studied Adenoviral (Ad) and Lentiviral (Lv) vectors, namely Ad5 and Lv-VSVG which have the unique ability to transfer large DNA sequences. The use of Ad and Lv vectors would allow to either transfer therapeutic genes with large coding sequences or to deliver whole genomic loci including their endogenous regulatory regions. Here, we aimed at the identification of Lv and Ad vectors with high PR tropism and transduction efficiency in preparation for further testing in animal models of severe inherited PR diseases.
We collected 16 different Ad serotypes which were either isolated as naturally infectants of humans and chimpanzees or genetically capsid modified Ad5. We have also collected seven different Lv pseudotypes with heterologous envelope proteins. The vector containing either a CMV-eGFP or lacZ expression cassette were injected in adult C57/Bl6 or Cd1 mice and harvested at either four or 14 days post-injection. Transgene expression was evaluated by indirect ophthalmoscopy or by analysis of retinal sections to visualize eGFP or lacZ expression.
We identified five vectors either based on Ad serotypes or on Lv pseudotypes with PR transduction efficiency higher than Ad5 and Lv-VSVG respectively. Since the promoter used is ubiquitous, RPE, PR, and cells from the inner nuclear layer were transduced. Some vectors achieved substantial levels and extension of PR transduction of up to 40-50% of the retinal sections.
We have identified five vectors either based on Ad serotypes or on Lv pseudotypes that appear promising for efficient murine PR transduction. To better define the potential of these vectors for retinal gene therapy, we are currently performing experiments using an expression cassette including the PR-specific rhodopsin promoter. In addition, we will test these promising vectors in the pig retina, a large model retina which is highly cone-enriched.
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