Apart from advancements in DNA delivery and in vectors for gene delivery into patients’ cells, zinc finger nuclease technology may prove truly transformative to gene therapy in general. Zinc finger nucleases (ZFNs) are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to the cleavage domain of a restriction endonuclease. These engineered molecules allow cellular DNA to be cut at specific points, with gene modification then occurring via the cell’s own natural repair mechanisms. They may also allow the insertion of entire genes at desired cleavage sites to replace missing or mutated genes.
Sangamo and colleagues from the University of Pennsylvania announced positive preliminary data from their Phase I trial being conducted in HIV-infected immunologic nonresponders. The patients enrolled in this study were HIV-infected individuals on highly active antiretroviral therapy with undetectable levels of virus but low T-cell counts.
The investigators used zinc fingers custom-designed to bind to specific DNA sequences in the CCR5 gene in HIV-infected patients’ T cells. The zinc finger proteins act as molecular scissors, bringing a DNA enzyme to the CCR5 gene to cut its sequence. During the repair process, a new mutation arises in the CCR5 protein, rendering it nonfunctional. Since the HIV virus uses the normal, unmodified version of CCR5 to gain access to T cells, the engineered cells became completely resistant to infection.
The data showed that a single infusion of the engineered cells was well tolerated, and the CCR5-modified cells successfully engrafted in all of the patients. The treatment also resulted in a durable improvement in total CD4+ T-cell counts in five of six patients analyzed.
The ZFN-CCR5-modified cells also exhibited normal T-cell growth kinetics and trafficking and underwent selective expansion in the gut mucosa, a major reservoir of virus in the body, suggesting, as predicted, that the cells were resistant to HIV infection.
Sangamo’s Philip Gregory, CSO and vp, research, told GEN that the goal of developing the company’s zinc finger nuclease program “has been to give investigators the ability to perform precision engineering directly on the genome itself.”
In the HIV application, he pointed out, no new DNA is being introduced. “We are really introducing a mutation ourselves just with the nucleases. We were able to do this because DNA repair mechanisms themselves are error prone. When the cells repair the break generated by the ZFN, the process occurs without error checking and mutations are introduced specifically at the site of the break. We can use this to achieve one desired outcome, knockout of the gene that’s been cleaved by the nuclease—in this case, the CCR5 gene. When the cell repairs it, it will create mutations.”
In explaining the production process for the autologous cells carrying the mutated CCR5 gene, Gregory said that the cells are collected from patients and sent to a processing facility, where they are exposed to the ZFN. The cells are expanded, and then re-infused into the patient, where they engraft, expand in the patients, and get trafficked to the normal place in the body.
“These cells are noninfectable by HIV,” Gregory said. He further explained that “just by protecting T cells, we aimed to create a reservoir of these cells that couldn’t be infected. These cells protect against loss of cells in HIV-infected patients.”
Gregory emphasized that CCR5 itself as a target “is one of the few situations in which we know the biology of the protein from the situation that exists in the natural population of patients. Patients with the Delta 32 CCR5 mutation don’t have the receptor on their cells and are completely normal but are resistant to infection.
“It turns out,” he said, “that an important feature of that mutation is that it eliminates CCR5 completely, giving rise to cells with no receptor on their surfaces.”
Sangamo says it is testing its product across a full range of HIV patients including those for whom current drug regimens are failing. “We are making good progress and are letting the data tell us where to focus our further clinical trials.”
All this progress, investigators point out, represents the culmination of years of experience, encompassing multiple disciplines from molecular biology through clinical science. And hopefully as positive clinical results continue to emerge, effective gene therapy, in whatever format, will become a real therapeutic option for intractable human diseases.