June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Feasibility of multiple rAAV vector approaches for large gene delivery in the murine retina
Author Affiliations & Notes
  • Rachel Fehrman
    Ophthalmology and Visual Science, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
    Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
  • Daniel M Lipinski
    Ophthalmology and Visual Science, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Rachel Fehrman Medical College of Wisconsin, Code P (Patent); Daniel Lipinski Riflebird Therapeutics , Code O (Owner), Medical College of Wisconsin, Code P (Patent)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3847. doi:
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      Rachel Fehrman, Daniel M Lipinski; Feasibility of multiple rAAV vector approaches for large gene delivery in the murine retina. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3847.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Recombinant adeno-associated virus (rAAV) vectors are promising tools for the treatment of inherited retinal diseases and have already been used to treat several disorders, most notably LCA2. Unfortunately, rAAV vectors can only handle therapeutic cassettes of ≥4.8kb. To circumvent this limitation, multiple vector approaches have been proposed wherein the transgene cassette is split across two or more rAAV vector genomes. To be successful, this strategy relies on 1) transduction of the separate vector fragments into a single cell, and 2) correct recombination of the transgene into a complete cassette. Herein we evaluate whether the efficacy with which multiple rAAV vectors simultaneously enter and transduce individual cells within the retina represents a major bottleneck that may limit the extent of recombination and large transgene expression.

Methods : We simultaneously administered one, two, or three capsid mutant (rAAV2/2[MAX]) vectors packaging distinct fluorescent reporter genes (BFP, GFP or mCherry) via bilateral intravitreal injection (1.8μl/eye) in wild-type (C57BL/6J) mice and quantified transduction by flow cytometry following papain-dissociation of the retinae (N=6 eyes/group); a fourth group of mice was administered an equal volume of buffer as a control. Two weeks post-injection, cSLO, OCT and ERG were performed to verify gene expression, retinal thickness, and retinal function. Following final assessments, mice were euthanized and their eyes harvested for flow cytometry, qPCR, or histology.

Results : Fluorescence microscopy shows successful transduction of all three rAAV vectors. Flow cytometry suggests retinal cells are 32.26% ± 5.54 and 8.54% ± 3.42 likely to be transduced with two or three vectors, respectively, indicating decreasing transduction efficiency with multiple vector fragments. OCT and ERG both indicate an increase in toxicity relative to the number of constructs administered, with single vector injections resulting in little to no toxicity.

Conclusions : Whilst gene therapy remains a promising tool for the treatment of inherited retinal diseases, transduction efficacy appears to be a major bottleneck when two or more rAAV vectors are simultaneously utilized. Moreover, the expression of multiple constructs leads to increased toxicity that is not evident when an equivalent amount of a single vector is injected.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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