Intracameral Delivery of Gene Therapy Vector scAAV2.MMP1 Results in Negligible to None Systemic Presence of Viral Genomes in a Large Animal Model.

Abstract

Purpose : We have previously shown that a single injection of a scAAV2.MMP1 viral vector lowers steroid-induced elevated IOP in sheep. With the intent of moving these results to the clinic, in this study we analyzed the viral vector genome distribution in major systemic organs and different eye tissues after administering a single therapeutic dose of the viral vector to the anterior chamber of the eye.

Methods : Ten sheep were injected in a single eye with 0.3-1X10¹¹ vg of scAAV2.MMP1 in a 100 µl volume. Tissues from major organs were harvested at necropsy from three sheep at 12, 22 and 30 days post-injection. Different eye tissues were harvested from four sheep. Tissue DNA was extracted with a DNeasy kit. Equal aliquots were used to quantify genomic DNA (gDNA) and viral genomes (vg) by TaqMan PCR. For the gDNA, samples were hybridized to a single copy human RPPH1 single-exon probe and quantified against a standard curve of RPPH1 with sheep DNA. For the vg, samples were hybridized to a human exon-spanning MMP1 cDNA probe and quantified against a standard curve of the probe with parent transgene plasmid pMG21. All runs were analyzed at CT thresholds of 0.4 (RPPH1) and 0.18 (MMP1). Data is reported as the number of double-stranded viral DNA molecules per diploid cell (vg/cell). Amount of sheep genomic DNA (1.7X10¹² MW) is calculated to be 168 ng/cell.

Results : Sheep genomic DNA concentrations were measured between 180 and 0.3 ng/µl (RPPH1 Ct values range 26 to 36). Plasmid DNA was measured between 68,000 to 6.8 copies (MMP1 Ct values range 22 to 37). In a representative sheep (22 d post injection), brain, heart, kidney, liver and lung showed <0.0003 vg/cell. Spleen showed 0.005 vg/cell. The scAAV2.MMP1 injected eye tissues from the same sheep, showed 0.02 vg/ cell (cornea), 0.03 vg/ cell (iris), 0.0002 vg/ cell (retina) and 0.4 vg/ cell (trabecular meshwork).

Conclusions : Viral genome distribution showed negligible copies in six major internal organs (0.00002-0.0005 vg/cell) and potentially re-inducible vector copies in the trabecular meshwork (0.4 vg/cell). These studies corroborate the safety of intraocular AAV vectors and reinforce the concept that this clinically safe steroid-inducible expression vector would meet requirements to address adverse ocular effects of steroid treatment in patients.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.