Gene transfer agent in development

Dec 01, 2010

Results from preclinical testing including a formal Good Laboratory Practice toxicity study, support the safety of a gene transfer agent, AAV2-sFLT01, when administered intravitreally. A phase I safety and tolerability study of this agent is currently underway.

Phase I testing is under way to evaluate intravitreal anti-vascular endothelial growth factor (VEGF) gene delivery as a treatment for exudative age-related macular degeneration (AMD), reported Dr Abraham Scaria, PhD, at the annual meeting of the Association for Research in Vision and Ophthalmology.

The novel gene transfer agent (Genzyme Corp., Framingham, Massachusetts, USA) uses adeno-associated virus serotype 2 (AAV2) as the vector to deliver the anti-VEGF molecule, sFLT01.

The anti-VEGF molecule is a hybrid protein that links the second immunoglobulin (IgG)-like domain of VEGF receptor Flt-1 to a human IgG1-Fc. It was selected as the lead candidate based on results from in vitro screening, which showed it to be a potent inhibitor of VEGF.

Dr Scaria presented favourable findings from a 12-month, formal GLP toxicity study of intravitreal injection of AAV2-sFLT01 in cynomolgus monkeys that also demonstrated long-term efficacy in preventing laser-induced CNV and persistent gene expression.

"Current treatment of neovascular AMD using anti-VEGF agents can be very effective, but generally requires monthly intravitreal injections," asserted Dr Scaria, senior scientific director, Genzyme Corp. "The results from extensive preclinical testing of AAV2-sFLT01 are encouraging in suggesting this gene therapy approach may be a viable alternative for achieving a prolonged anti-VEGF effect and obviating the need for repeated injections."

Rationale for choices

Providing additional background on the development of the investigational gene transfer agent, Dr Scaria said that AAV2 was chosen as the vector for delivering sFLT01 because of its established safety profile.

"The same viral vector is currently being used in clinical trials of gene therapy for Leber's congenital amaurosis as well as in clinical trials for the treatment of Parkinson's disease, haemophilia and cystic fibrosis," he added.

In addition, there is evidence from canine studies that AAV2-mediated gene delivery results in prolonged gene expression of at least 8 years. An earlier safety study in rats investigating biodistribution showed that the vector essentially remained in the injected eye. In an earlier mouse study, intravitreal injection with AAV2-sFLT01 resulted in persistent transduction of retinal ganglion cells with follow-up to 1 year, Dr Scaria said.

The formal Good Laboratory Practice (GLP) toxicity study in monkeys also included determination of the pattern of gene transfer and showed that it was different in the nonhuman primate eyes compared with the mice because in monkey eyes, the transduced cells were predominantly toward the front of the eye and included the transitional epithelium in the pars plana region as well as some retinal ganglion cells around the retina.

"The difference between animal species was somewhat surprising to us," Dr Scaria remarked. "However, since sFLT01 is a secreted protein, we would expect that it will reach the back of the eye where it is needed to block CNV. In fact, we found evidence of this in our efficacy testing using a laser-induced CNV model."

Safety, efficacy results

In the 12-month safety study, the cynomolgus monkeys received intravitreal injection with vehicle or one of two different doses of AAV2-sFLT01, 2E9, or 2E10 vector particles/eye. The results showed a dose-dependent increase in sFLT01 expression measured by assaying the aqueous humour. At both doses, there was persistence of expression at least for 1 year.

Comprehensive safety assessments showed no adverse events using the lower dose. In eyes injected with the 2E10 dose, no adverse effects were noted on electroretinography, tonometry, or fluorescein angiography. However, inflammation manifested as mild to moderate vitreal haze and cells were observed starting from 1 to 3 months postinjection. The inflammation was generally mild and self-resolving over 3 to 12 months.

Efficacy of AAV2-sFLT01 was investigated in a subgroup of six out of ten monkeys treated with the 2E10 dose that demonstrated the highest expression of sFLT01. CNV was induced by lasering the retina at 5 months post-injection. CNV was similar in eyes treated with AAV2 null vector compared with control eyes that received no intravitreal injection, but was suppressed in eyes treated with AAV2-sFLT01.