Abstract
Adeno-associated viruses (AAVs) are used extensively as a gene delivery vehicle for retinal gene therapy, yet its ability to target the anterior segment of the eye, critical to unlocking therapeutic opportunities, is less characterized. Previously, self-complimentary (sc) AAV was shown to be necessary for transduction of the cornea and trabecular meshwork (TM), limiting the size of the gene transfer cassette, likely due to a block in second strand synthesis thought to be required for functional transduction. Here, we evaluated several AAV capsids in a single stranded (ss) genome conformation for their ability to overcome the need for scAAV for targeting corneal endothelium and TM. AAV2, 8, and a recently synthetically developed AAV called Anc80L65 were evaluated in vitro and in vivo by intracameral injection in mice. Results show that although scAAV2 demonstrated superior infectivity in vitro including Human Trabecular meshwork (HTM) immortalized cell lines; Anc80L65 transduced following a single intracameral injection efficiently all components of the mouse anterior segment, including the TM, corneal stroma, and endothelial cells. These results suggest that Anc80L65 is able to overcome the requirement for scAAV genomes to enable TM and corneal targeting, expanding the potential experimental and therapeutic use of AAV gene transfer in the anterior segment of the eye.
Highlights
The adeno-associated virus (AAV) is small single-stranded DNA virus permissive in humans
With a multiplicity of infection (MOI) of 1×103 genome copies/cell (GC/cell), green fluorescent protein (GFP) was observed 1 day after infection with scAAV2 in Human embryo kidney 293 cells (HEK293) and Human Trabecular meshwork (HTM) cells, expression increased at 3 days (S1 Fig) and was detected up to 5 days after infection
We found higher GFP expression in HEK293 and HTM cells infected with scAAV2 and AAV2 (Fig 1c–1f and 1m–1p) compared to GFP expression in these cells with a MOI of 1×103 GC/ cell
Summary
The adeno-associated virus (AAV) is small single-stranded DNA virus permissive in humans. AAVs are able to transduce non-dividing cells resulting in long term transgene expression[1] and have found utility in the field of gene therapy. AAV gene therapy for a form of inherited retinal degeneration and hemophilia and multiple preclinical efforts, has identified key properties for a vector system in relation to its tissue and disease. L.H.V. is an inventor on gene therapy patents, including Anc80L65, which are licensed to various entities. Li Wang and Ru Xiao and Eva Andres-Mateos declare no potential conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials
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