Prolonged Transgene Expression with Lentiviral Vectors in the Aqueous Humor Outflow Pathway of Nonhuman Primates

Abstract

We injected lentiviral vectors into the eyes of live nonhuman primates to assess potential for glaucoma gene therapy. Anterior chambers of five cynomolgus monkeys were injected with green fluorescent protein (GFP)-encoding feline immunodeficiency viral vectors. The monkeys were monitored for in vivo transgene expression and clinical parameters. Their eyes were harvested 2-15 months postinjection for tissue analyses. All seven eyes injected with 1.0-2.0 x 10(8) transducing units (TU) showed substantial GFP fluorescence in the trabecular meshwork (TM), which was observable even by goniophotographic monitoring for up to 15 months. Only the lowest dose (0.03 x 10(8) TU) failed to result in TM fluorescence detectable in vivo, and five of the eight vector-injected eyes continued to display substantial GFP expression when enucleated eyes were examined at 2, 7, or 15 months postinjection. Some transduced cells were also detected in the iris and ciliary body. Mild, transient postinjection inflammatory responses exceeding that induced by a control saline injection were observed, but vectors did not raise intraocular pressure and were well tolerated. The results demonstrate the first lentiviral vector transduction of the nonhuman primate aqueous humor outflow pathway and support application of the system to human glaucoma gene therapy.

Open-angle glaucoma: therapeutically targeting the extracellular matrix of the conventional outflow pathway

“…Long-term expression in TM cells and in rodents has been achieved with lentivirus following a single intracameral inoculation [186,187]. The first demonstration of efficient lentiviral transduction in primates reported well tolerated, extensive viral expression with a mild inflammatory response [188]. In early AV studies, gene expression was obtained in TM cells of mice for up to 14 days following intracameral inoculation of a replication-deficient AV vector expressing a LacZ gene [189].…”

Application of Canaloplasty in Glaucoma Gene Therapy: Where Are We?

“…9 Viral vectors (FIV and scAAV) encoding genes for green fluorescent protein (GFP) show long-term (2 + years) expression in the outflow pathways of nonhuman primate eyes following transcorneal intracameral injection. 15,16 Transcorneal intracameral injection has been the method of choice for transferring transgenes/vectors into the anterior chamber (AC) of the eye. Successful transgene expression has been achieved using this route in rats, cats, and monkeys.…”

“…5,[13][14][15][16]25,26 Disadvantages of the intracameral route for transgene injection are the potential for clinically significant inflammation and possible transgene expression (a concern with high virus titers) in other anterior segment structures, such as the cornea and iris. 16 With most drug delivery methods, for example, topical drops, implants, and others, nontarget tissues are exposed to the active compound. There are a number of tissues in the anterior segment in addition to the TM/SC that would be good targets for glaucoma gene therapy.…”

“…There are a number of tissues in the anterior segment in addition to the TM/SC that would be good targets for glaucoma gene therapy. The ciliary body has potential for several applicationsthe ciliary muscle for PG-related gene transfer aimed at increasing uveoscleral outflow 13,14,16 and the ciliary processes for carbonic anhydrase inhibitor or beta-adrenergic receptor-related gene transfer aimed at inhibiting formation of aqueous humor. The question of off-target effects will be an important issue for these and other gene therapy applications moving forward.…”

“…3 In recent years, the possibility of decreasing this outflow resistance via gene therapy using TM/SC-targeted transgene delivery systems has generated considerable interest. [4][5][6][7][8][9][10][11][12][13][14][15][16] We hypothesized that a canaloplasty approach might also be used for localized transgene delivery into this region. In this article, we have briefly reviewed canaloplasty surgery and discuss its possible role in transgene delivery into the SC.…”