B6 - Ophthalmic and Auditory Diseases
179: AAV-Mediated RhoA Knockdown as a Potential Gene Therapy for Glaucoma
Type: Oral Abstract Session
Presentation Details
Session Title: Ophthalmic and Auditory: Disease Focus
Glaucoma is one of the leading causes of adult blindness and affects about 80 million individuals globally. In primary open angle glaucoma (POAG), one of the high-risk factors that aggravates blindness is the accumulation of aqueous humor (AH), which results in increased intraocular pressure (IOP) and causes damage to the optic nerve. The cells of the ciliary body synthesize AH, while natural drainage happens through Schlemm’s canal and layers of the trabecular meshwork (TM). All current glaucoma treatments aim to decrease the secretion and increase the outflow of AH to reduce IOP. Rho kinase (ROCK) inhibitors are a new class of glaucoma drugs, which act on the trabecular tissue to increase AH outflow. RhoA is a ubiquitously expressed GTPase that is known to regulate actin polymerization/depolymerization. Activated GTP-bound RhoA can associate with serine/threonine kinases of the ROCK1/2 family and can assist with phosphorylation of a variety of substates, such as myosin light chain (MLC) and myosin phosphatase target subunit (MYPT-1), which contribute to increased actin polymerization, contraction of the TM, and resistance to AH outflow. Using artificial microRNAs (amiRs), we aimed to target RhoA expression. We screened eleven seed sequences targeting the coding sequence (CDS) of RhoA and its 3’-UTR in human trabecular meshwork (hTM1) cells. Three amiR candidate designs displayed reduced expression of the target gene at the protein level (60-80% reduction). Evaluation of off-target effects using small-RNA sequencing showed that endogenous microRNAs were largely unaffected. We also found that transduction of TM cells via intracameral injection by AAV2 outperformed those of AAV3b, AAV6, and AAV8; thus, the delivery of amiR-RhoA-10 by AAV2 was chosen as the final vector strategy. We demonstrate that when scAAV2-amiR-RhoA-10 is administered intracamerally to wildtype mice (7.33E8 vg/eye), mean IOP was dramatically reduced within three weeks of dosing (from 16.5 mmHg to 14.5-15.0 mmHg) and remained so for over a period of twelve weeks. Furthermore, the observed reduction was also dose dependent.In summary, our proof-of-concept study demonstrates that AAV-mediated knockdown of RhoA using amiRs can reduce intraocular pressure in mice, showing significant promise as a gene therapy for treating primary open angle glaucoma. J.X., G.G. and P.W.L.T. are corresponding authors
Plain Language Summary
One of the reasons for optical blindness in glaucoma patients is damage to the optic nerve resulting due to increased intraocular pressure (IOP). Excess discharge of the aqueous humor (AH) by the ciliary body and lack of proper drainage often leads to accumulation of AH. RhoA-dependent signaling pathways can positively influence actin polymerization, increasing contraction of the drainage channels which resists AH outflow. Artificial microRNA (amiR) scaffolds harboring RhoA-targeting sequences were designed and screened. In vitro assays showed a 60-80% decrease in RhoA expression for some of these amiR designs. The most promising amiR-RhoA design packaged with AAV2 when injected in mice intracamerally showed normalization of IOP within three weeks and was maintained for four months. In summary, our proof-of-concept study demonstrates that AAV-mediated knockdown of RhoA using amiRs can reduce intraocular pressure in mice, showing significant promise as a gene therapy for treating primary open angle glaucoma.
Tapan Sharma1,2, Meng Gao1,3, Bo Tian1,3, Haijiang Lin1,3, Jun Xie1,2,4, Guangping Gao1,2, Phillip Wl Tai1,2
1Horae Gene Therapy Center, UMass Chan Medical School, Worcester, MA,2Department of Microbiology and Physiological Systems, UMass Chan Medical School, Worcester, MA,3Department of Neurobiology, UMass Chan Medical School, Worcester, MA,4Viral Vector Core, UMass Chan Medical School, Worcester, MA"
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