LaKisha K. Buie

Self-complementary AAV virus (scAAV) safe and long-term gene transfer in the trabecular meshwork of living rats and monkeys.

PURPOSE AAV vectors produce stable transgene expression and elicit low immune response in many tissues. AAVs have been the vectors of choice for gene therapy for the eye, in particular the retina. scAAVs are modified AAVs that bypass the required second-strand DNA synthesis to achieve transcription of the transgene. The goal was to investigate the ability of AAV vectors to induce long-term, safe delivery of transgenes to the trabecular meshwork of living animals. METHODS Single doses of AAV2.GFP and AAV2.RGD.GFP/Ad5.LacZ were injected intracamerally (IC) into rats (n = 28 eyes). A single dose of scAAV.GFP was IC-injected into rats (n = 72 eyes) and cynomolgus monkeys (n = 3). GFP expression was evaluated by fluorescence, immunohistochemistry, and noninvasive gonioscopy. Intraocular pressure (IOP) was measured with calibrated tonometer (rats) and Goldmann tonometer (monkeys). Differential expression of scAAV-infected human trabecular meshwork cells (HTM) was determined by microarrays. Humoral and cell-mediated immune responses were evaluated by ELISA and peripheral blood proliferation assays. RESULTS No GFP transduction was observed on the anterior segment tissues of AAV-injected rats up to 27 days after injection. In contrast, scAAV2 transduced the trabecular meshwork very efficiently, with a fast onset (4 days). Eyes remained clear and no adverse effects were observed. Transgene expression lasted >3.5 months in rats and >2.35 years in monkeys. CONCLUSIONS The scAAV viral vector provides prolonged and safe transduction in the trabecular meshwork of rats and monkeys. The stable expression and safe properties of this vector could facilitate the development of trabecular meshwork drugs for gene therapy for glaucoma.

Development of a Model of Elevated Intraocular Pressure in Rats by Gene Transfer of Bone Morphogenetic Protein 2

PURPOSE To determine whether inducing calcification in the trabecular meshwork results in elevated IOP in living rats. To use this property to create an elevated IOP animal model by gene transfer of bone morphogenetic protein 2 (BMP2). METHODS Calcification was assessed by alizarin red staining in primary human trabecular meshwork (HTM) cells and alkaline phosphatase (ALP) activity in the angle tissue. Brown Norway (BN) and Wistar rats were intracamerally injected with Ad5BMP2 (OS) and control Ad5.CMV-Null (OD). IOPs were taken twice a week and expressed as mean integral pressures. Morphology was assessed on fixed, paraffin-embedded anterior segments. Retinal ganglion cells (RGCs) were quantified on retrograde and Brn-3a-labeled flat mounts using MetaMorph software. RESULTS BMP2-treated cells displayed marked increase in calcification. Trabecular meshwork tissue showed moderate ALP activity at 13 days postinjection. Fifty-four of 55 BN and 15 of 19 Wistar rats displayed significantly elevated IOP. In a representative 29-day experiment, the integral IOP difference between treated and control eyes was 367.7 ± 83 mm Hg-days (P = 0.007). Morphological evaluation revealed a well-organized trabecular meshwork tissue, exhibiting denser matrix in the treated eyes. The Ad5BMP2-treated eye showed 34.4% ± 4.8% (P = 0.00002) loss of peripheral RGC over controls. CONCLUSIONS Gene transfer of the calcification inducer BMP2 gene to the trabecular meshwork induces elevated IOP in living rats without altering the basic structure of the tissue. This strategy generates an elevated IOP model in rats that would be useful for evaluation of glaucoma drugs targeting the outflow pathway.

Cystatin a, a potential common link for mutant myocilin causative glaucoma.

Myocilin (MYOC) is a 504 aa secreted glycoprotein induced by stress factors in the trabecular meshwork tissue of the eye, where it was discovered. Mutations in MYOC are linked to glaucoma. The glaucoma phenotype of each of the different MYOC mutation varies, but all of them cause elevated intraocular pressure (IOP). In cells, forty percent of wild-type MYOC is cleaved by calpain II, a cysteine protease. This proteolytic process is inhibited by MYOC mutants. In this study, we investigated the molecular mechanisms by which MYOC mutants cause glaucoma. We constructed adenoviral vectors with variants Q368X, R342K, D380N, K423E, and overexpressed them in human trabecular meshwork cells. We analyzed expression profiles with Affymetrix U133Plus2 GeneChips using wild-type and null viruses as controls. Analysis of trabecular meshwork relevant mechanisms showed that the unfolded protein response (UPR) was the most affected. Search for individual candidate genes revealed that genes that have been historically connected to trabecular meshwork physiology and pathology were altered by the MYOC mutants. Some of those had known MYOC associations (MMP1, PDIA4, CALR, SFPR1) while others did not (EDN1, MGP, IGF1, TAC1). Some, were top-changed in only one mutant (LOXL1, CYP1B1, FBN1), others followed a mutant group pattern. Some of the genes were new (RAB39B, STC1, CXCL12, CSTA). In particular, one selected gene, the cysteine protease inhibitor cystatin A (CSTA), was commonly induced by all mutants and not by the wild-type. Subsequent functional analysis of the selected gene showed that CSTA was able to reduce wild-type MYOC cleavage in primary trabecular meshwork cells while an inactive mutated CSTA was not. These findings provide a new molecular understanding of the mechanisms of MYOC-causative glaucoma and reveal CSTA, a serum biomarker for cancer, as a potential biomarker and drug for the treatment of MYOC-induced glaucoma.

Prevention of Nocturnal Elevation of Intraocular Pressure by Gene Transfer of Dominant-Negative RhoA in Rats

IMPORTANCE We developed a gene transfer tool for the control of nocturnal elevated intraocular pressure (IOP). OBJECTIVE To demonstrate that inhibiting the trabecular meshwork RhoA pathway by delivering a mutated, dominant-negative RhoA gene (dnRhoA) carried inside a long-expressing recombinant virus would reduce nocturnal elevated IOP in a living animal. DESIGN AND SETTING We generated an optimized recombinant viral molecule by inserting a mutated RhoA complementary DNA with a translation enhancer-promoter into a specially designed plasmid containing mutated viral terminal repeats. We then generated the virus particle, self-complementary adeno-associated virus serotype 2 carrying the mutated gene (scAAV2.dnRhoA) and assessed its function in vitro by infecting primary human trabecular meshwork cells and in vivo by injecting living rats intracamerally with therapeutic and control viruses. Three different models of 12-hour light and dark cycles were used. Viruses were injected when animals showed the circadian dark IOP elevation. The IOP measurements were conducted with a tonometer at 2 to 4 hours after onset of the nocturnal and diurnal cycles. Values at preinjection time were used as baselines. Animals were euthanized at 4 to 8 weeks after injection. EXPOSURES Intraocular injection of rodent eyes with the recombinant viral vector scAAV2.dnRhoA. MAIN OUTCOMES AND MEASURES Nocturnal elevation of IOP blocked for prolonged periods by transferred RhoA gene. RESULTS By visual inspection, human trabecular meshwork cells infected with scAAV2.dnRhoA showed diminished stress fiber formation. Living rats exhibited a circadian IOP cycle that could be reset by adjusting light conditions to facilitate light and dark nocturnal IOP studies. A single-dose injection of scAAV2.dnRhoA into the rat eyes prevented elevation of IOP during the nocturnal cycle for at least 4 weeks (mean [SE], 9.2 [0.2] mm Hg light IOP and 9.6 [0.4] mm Hg dark IOP), while control eyes showed a significantly higher IOP over baseline (9.5 [0.4] mm Hg light IOP and 13.5 [0.3] mm Hg dark IOP). CONCLUSIONS AND RELEVANCE To our knowledge, this is the first example of a gene transfer strategy that prevents nocturnal IOP elevation in living animals for prolonged periods. Inhibiting the RhoA pathway upstream of Rho kinase with a safe gene drug could provide a new enhanced treatment for long-term management of elevated nocturnal IOP.

Inducible scAAV2.GRE.MMP1 lowers IOP long-term in a large animal model for steroid-induced glaucoma gene therapy

Current treatment of glaucoma relies on administration of daily drops or eye surgery. A gene therapy approach to treat steroid-induced glaucoma would bring a resolution to millions of people worldwide who depend on glucocorticoid therapy for a myriad of inflammatory disorders. Previously, we had characterized a short-term Adh.GRE.MMP1 gene vector for the production of steroid-induced MMP1 in the trabecular meshwork and tested reduction of elevated intraocular pressure (IOP) in a sheep model. Here we conducted a trial transferring the same transgene cassette to a clinically safe vector (scAAV2), and extended the therapeutic outcome to longer periods of times. No evidence of ocular and/or systemic toxicity was observed. Viral genome distributions showed potential reinducible vector DNAs in the trabecular meshwork (0.4 v.g. per cell) and negligible copies in six major internal organs (0.00002–0.005 v.g. per cell). Histological sections confirmed successful transduction of scAAV2.GFP to the trabecular meshwork. Optimization of the sheep steroid-induced hypertensive model revealed that topical ophthalmic drug difluprednate 0.05% (durezol) induced the highest IOP elevation in the shortest time. This is the first efficacy/toxicity study of a feasible gene therapy treatment of steroid-induced hypertension using clinically accepted self-complementary adeno-associated vectors (scAAV) vectors in a large animal model.