Editing Genes for a Cure

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A functional cure candidate modifies genes to create HIV-resistant cells
by Chael Needle

Gary Blick, MD, AAHIVS, Medical & Research Director of CIRCLE CARE Center and Chief Medical Officer of World Health Clinicians posing for the latter organization's HIV Equal campaign. Photo by Thomas Evans
Gary Blick, MD, AAHIVS, Medical & Research Director of CIRCLE CARE Center and Chief Medical Officer of World Health Clinicians posing for the latter organization’s HIV Equal campaign. Photo by Thomas Evans

Gary Blick, MD, AAHIVS, Medical & Research Director of CIRCLE CARE Center and Chief Medical Officer of World Health Clinicians, doesn’t throw the word “cure” around lightly.

As an HIV physician for over thirty years, he remembers when researchers were buzzing about a possible HIV vaccine in 1984 (“give it ten years”) and how the horizon of expectations kept being shifted forward (“give it ten more years”) as predicted deadlines would pass without any good news (“we’re not sure when or if”).

Now, building on years of research and the momentum created by the bone-marrow stem-cell transplant that cured the Berlin Patient (Timothy Ray Brown), attention has increasingly focused on the possibility of a functional cure, the ongoing and sustained control of viral load without antiretrovirals. A functional cure would mean that antiretrovirals, which bring with them lifelong benefits as well as lifelong challenges, such as major expenditures (the U.S. government spent $27.7 billion in research, services, and treatment of HIV in 2012 alone), kidney and bone marrow toxicities and other side effects, would no longer be needed long-term. With early but significant successes, a gene-therapy product, pioneered by Sangamo Biosciences called SB-728-T, has found its footing among the nascent field of functional-cure candidates.

Dr. Blick is excited about the results from initial clinical research on SB-728-T, but his excitement is tempered with wait-and-see realism. Sharing the latest news about this new therapy, he had a busy time as a presenter at this year’s Conference on Retroviruses and Opportunistic Infections (CROI 2014). Data from a raft of SB-728-T clinical trials led by Sangamo BioSciences, and in which Dr. Blick is an investigator, shows significant viral load reduction through an intravenous infusion of billions of a patient’s own CD4+ T-helper cells, ones that have been extracted, genetically modified, and re-infused into the body.

What’s extracted? First blood is extracted and then the individual’s own CD4+ T-helper cells are separated out to be modified. CD4+ T-helper cells, much needed for immune protection, are rendered ineffective and die off as a result of HIV infection. SB-728-T infusions have shown long-term increases in CD4+ T-helper cells, which correlates with increased CD4 central memory cells and gene-modified central memory cells.

What’s modified? Using Sangamo’s proprietary genome editing technology—zinc finger nuclease, or ZFN—the extracted cells’ DNA is modified to mimic those one percent of individuals who have been found to be immune (or largely resistant) to HIV because of a natural (and apparently harmless) genetic mutation: CCR5delta32. Having CCR5delta32 results in the expression of a shortened, or truncated, and non-functional CCR5 protein, whose co-receptors are needed for HIV to gain entry into cells. Individuals who have two copies of the mutation are virtually immune, and this is one possible mechanism that has tagged some individuals as elite controllers or long-term non-progressors, meaning some are still able to be infected by HIV but also able to indefinitely control HIV with low or undetectable HIV-1 viremia without antiretrovirals.

What happens after re-infusion? Billions of cells are reintroduced and go through a process of engraftment. Researchers used one cohort to study to what extent, and at what dose, this engraftment is primed by a short-term, one-time infusion of a chemotherapeutic agent known as Cyclophosphamide, or Cytoxan (CTX). Another cohort showed that CD4 cells can be significantly increased, viral load decreased as much as ninety-nine percent, and the ZFN-modified CD4-cell count can be increased with increasing doses of CTX therapy. Two of three subjects treated with the highest dose of CTX remain on treatment interruption for eleven and twelve weeks with stable or lowered levels of viral load.

Overall, the study showed that an individual’s T cells can be safely engineered via ZFN-based genome editing to make them HIV-resistant and safely infused, resulting in decreases in HIV viral load off antiretroviral therapy.

In another cohort discussed at CROI, one patient has shown ongoing control of viral load with nearly undetectable levels of HIV for thirty-one weeks (now thirty-five weeks as we went to press) without antiretrovirals. This patient, who looks to be on his way to a functional cure, was discovered to have one copy of the CCR5delta32 mutation and has been enrolled in a Phase II trial focused on other individuals without identical pairs of genes for the same trait (heterozygous). “We figured it would be a whole lot easier to do the gene modification if you only have one of two alleles that we needed to modify,” notes Dr. Blick. In other words, this process mimics homozygous individuals, who have two copies of the CCR5delta32 mutation. This patient at baseline had a greater percentage of T cells where both alleles were modified and fully resistant to HIV. Levels of circulating cells with biallelic modification of CCR5 may correlate with control of viral load, according to early analysis. The information will add to the bank of knowledge of how to best use this gene-modified therapy with individuals who do not carry any mutation.

Dr. Blick reports that this individual’s gene-modified T cells are also being analyzed to see if they may be hiding HIV DNA within them.

Currently the gene-modified therapy uses an adenovirus vector as a delivery system. The limitation of the adenovirus vector is that it may cause the body to produce antibodies, which ultimately can interfere with the engraftment of the associated gene-modified cells.

Thus, Sangamo is looking at other delivery systems, namely, mRNA (messenger-RNA). As RNA is a natural part of our bodies, unlike the adenovirus, explains Dr. Blick, the body will not produce any antibodies to run interference against the engraftment of the gene-modified cells and strengthen the possibility of an efficacious functional cure, as well as allow for repeated infusions, if found necessary.

The gene-modified, CCR5-disrupted cells function to not only resist infection by the most common strain of HIV but prevent its persistence.

Significantly, the modified cells have been to shown to have a longer life than the unmodified cells when exposed to HIV during a planned interruption of antiretrovirals, and, importantly, have been found to circulate where latent HIV hides out, such as gut-associated lymphoid tissue. “Lymphocytes have a normal lifespan. And T cells are lymphocytes,” reminds Dr. Blick. “So you know they’re going to have a normal lifespan, typically no greater than 120 days. If infected with HIV and not immune they’re going to die earlier than 120 days. The lifespan of the gene-modified cells—the half-life of gene-modified cells [was shown to be] something like forty-eight weeks, almost one year. We’re actually learning now that it’s going to be quite a bit longer. These gene-modified cells, for some reason, last significantly longer than if they weren’t genetically modified.”

This discovery of preferential survival has been enlightening in terms of measuring the reach of gene-modified cells. “As we’re giving 10 to 40 billion T cells, you expect to see a big bump up in the person’s CD4 cell count and then come down gradually, during treatment interruption, toward the end of treatment interruption, or if they stay on treatment interruption. But they always stayed somewhere around two to three times higher than the individual’s baseline. So, when the cells come down, where are they going?

“We know these T cells are trafficking into lymphoid tissue. These T cells are going into those reservoirs, just like the old T cells did with virus in them.” The difference here is important as antiretroviral drugs have not been shown to follow these infected cells into the reservoirs. “These gene-modified cells will not have virus in them because they’re immune and will fill up these reservoirs. We’ve demonstrated that already.”

Asked if this type of gene-modify therapy might work best in virally suppressed treatment-experienced patients, Dr. Blick responds: “At the very beginning when we started this clinical trial, about a year and a half ago, we thought a T-cell ratio, the CD4/CD8 ratio over 1 would be the parameter that would predict the best responses. We thought that an individual who was HIV-positive for less than ten years would give the best responses. We learned through the clinical trial that was not so. So now we’re treating anybody, no matter what their duration of HIV is or what their CD4/CD8 ratio is.

“But there are some exclusion criteria for these first group of patients. To get into these studies, you have to have 500 or more T cells, and you’ve got to be undetectable, because we don’t want virus replicating at the time you’re giving the gene therapy. In fact, when we give the gene therapy, we wait an additional six weeks for older T cells to die off so that the engraftment of the gene-modified T cells can take hold. And six weeks after we do the gene therapy is when they come off their medication.” As the trials are using a chemotherapeutic agent, the FDA requires other criteria to be met, as well, such as platelet counts of 200,000 and neutrophil counts of 2,500 or greater.

“We’ve been learning as we go along but so far we’ve had nothing negative to say about [the therapy],” notes Dr. Blick. Mild nausea seems to be the most severe occurrence.

Next steps in this research include the addition of a study arm to continue studying CTX dosing to achieve the best engraftment of cells (results presented were based on 1 gram/meter2; the new arm will look at 1.5); finalizing the technology for an mRNA-vector gene modification; and, once all the data from this Phase I/II study is collected about cell infusion and CTX dosing and checks out completely for safety, moving onto a Phase IIa study.

“This is really, really early data at this point,” Dr. Blick reiterates. “So, as long as everything keeps going well with this clinical trial, we have that first sign of a prolonged functional cure [in a person] now at thirty-five weeks; and now we have a second person who dropped about ninety-nine percent of his viral load, 1.9 logs. He’s still doing well. Early numbers, very few people in these Phase I clinical trials—so it’s best to say we’re cautiously optimistic.

“For me, as I’ve been doing research since 1990, understanding and uncovering all of these key findings as we go on is one of the most exciting things about doing this. We always have to give the credit to the patients for even getting involved with us and helping further the whole science of this, but just to watch this process step by step by step and learning how these things work and potentially coming up with the first functional cure for HIV is such an exciting field to be in, and such an exciting time right now.”

For more information about World Health Clinicians, log on to: www.worldhealthclinicians.org. For more information about Sangamo BioSciences, Inc., visit: www.sangamo.com.

Chael Needle wrote about a humanized BLT mouse model used in AIDS research for the March issue.