Both Heiko Jessen and Gero Hütter shared an interesting characteristic when they came up with their unique therapies that the Berlin patients received: They had little experience. Hütter had never treated an HIV patient, while Jessen was testing out the early aggressive therapy informally, with no previous experience in conducting a clinical trial. Similarly, when Sangamo gave Paula Cannon, a researcher at the University of Southern California, their CCR5 ZFNs to test, they couldn’t have been expecting much.
Cannon had no experience in gene therapy or animal models. Lack of experience had never stopped Cannon yet. She’d worked as a rock band manager and wedding dressmaker before becoming a scientist. With her charming English accent, quick wit, and catalog of A-list collaborators, Cannon was able to convince the small biotech company to let her give the CCR5 ZFNs a try. Her pitch was ambitious. She proposed using the ZFNs in hematopoietic stem cells, the stem cells that give rise to all the immune cells in the body. Those stem cells would then be engrafted in a humanized mouse model and challenged with HIV.
It was quite a proposal, considering that Cannon had never worked with stem cells or a humanized mouse model. She was a young assistant professor who had a small lab and a modest budget. Still, despite these drawbacks, Sangamo sent her, as well as other collaborators, the CCR5 ZFNs. There was little risk for the company, Cannon would either deliver the data or not. If she didn’t, then it was likely that a different collaborator would. Bringing an inexperienced graduate student, me, on, and with a tiny $50,000 grant from the California HIV/AIDS Research Program, Cannon was able to do what larger, better-funded labs could not: treat the finicky stem cells with the ZFNs, engraft them in humanized mice, and challenge with HIV. The virus put extreme pressure on the immune system. The results were phenomenal: Mice given the ZFN-modified stem cells developed a human immune system devoid of CCR5, without which the virus couldn’t enter the T cells. The mice receiving the gene therapy cleared their HIV infection. In contrast, mice that received mock-treated cells, that is, cells that received all the same manipulation but without the CCR5 ZFNs, had high levels of HIV and progressed to AIDS.
The convincing results were published in Nature Biotechnology in 2010. All Cannon needed were the right clinical collaborators to bring the technology to human trials. That’s when she was introduced to John Zaia at City of Hope hospital in Duarte, California. Together they formed what the CEO of Sangamo would call “the dream team.” The two came up with a bold plan to translate the dramatic findings from the humanized mice into humans. They postulated that the best population to test such a therapy was in HIV-positive patients who, like Timothy, had AML (acute myeloid leukemia). These were patients who needed a stem cell transplant. They couldn’t find donors who had the Δ32 mutation, so they would do the next best thing: make the stem cells look as if they came from a person naturally resistant to HIV. They would then infuse the cells back into the patient. The stem cells would travel to the bone marrow, where they would form all the cells that make up the human immune system. Like Timothy’s experience, and Carl June’s promising data, the group believed, the cells would have a survival advantage when faced with the virus. Inspired by Timothy, the group believed they could create a functional cure for HIV.
It was a bold plan and an expensive one. The safety studies needed before they could bring the new technology to a clinical trial were not trivial. Then the trial itself would be costly. This was a problem because, while the NIH funded basic research, they shied away from supporting advanced studies heading to a clinical trial. The group applied for a new kind of funding made available by CIRM (California Institute for Regenerative Medicine). Governor Arnold Schwarzenegger had formed this institute in reaction to George Bush’s freeze on federal funding for stem cell research. The data generated from Paula Cannon’s tiny $50,000 grant garnered the top score at CIRM that year. The grant, which referenced Timothy’s case, brought the funding the project needed: a whopping $14.5 million.
Cannon still finds it funny that anyone is surprised by her results. She says, “The fact that it worked, it was like the ‘Duh!’ moment. It was the most unremarkable thing. I wasn’t quite prepared for how exciting people thought the results were.”
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In 2008, Timothy Henrich was a busy second-year resident in internal medicine at Brigham and Women’s Hospital in Boston when he first heard about the Berlin patient. He immediately knew that this was where HIV research was heading. He thought the Berlin patient was “the most exciting development since antiretroviral therapy.” A young doctor interested in infectious disease, he wanted to be a part of the biggest thing to hit HIV in a decade, when the Berlin case was changing how HIV researchers saw the future, and the word cure was coming back into favor. Unfortunately, Tim was busy, his schedule packed with the heavy demands of his residency, leaving little time for research.
Two years later, Henrich was doing his fellowship in infectious disease at Brigham and Women’s Hospital when he began looking for a research project. His interest in the Berlin patient had only grown in the intervening years, and so had his need for a successful project. Henrich was in the precarious position of being a young researcher, with limited funding, desperate for a project that could bring him the publications and grants he would need to become faculty at the hospital. It’s a stressful time for any scientist starting out in their field. Funds are limited; time is precious; there are simply not enough faculty positions to go around. Under such pressure, many physician scientists tend to take the easy route, performing science that can be done quickly, to get as many publications as they can.
Henrich knew he needed a project that would get him published and bring in grants, but he didn’t want to compromise on the science. After a failed project attempt, he decided to follow in the footsteps of Gero Hütter and the Berlin patient. If scientists wanted to turn Timothy’s treatment into one accessible to HIV-positive people everywhere, they needed to understand what role each component of his treatment played in Timothy’s ultimate cure. Timothy had received chemotherapy, a conditioning regimen, and a bone marrow transplant; had graft-versus-host disease; and received donor stem cells with mutant CCR5. All signs pointed to the mutant CCR5 as the cause of Timothy’s cure. This is because Timothy went from having a Δ32 mutation in one copy of the mutant gene to having the mutation in both copies. It made logical sense that the change in Timothy’s genotype corresponded to a selective pressure exerted by the virus itself and was ultimately responsible for his ability to clear the virus. Although this made perfect sense, no one could be sure that the other factors involved in his therapy hadn’t influenced the outcome. Could the intense conditioning regimen have cleared out the virus? Or could the bone marrow transplant itself have caused the dramatic effect? This was Tim Henrich’s question.
With his advisor, Dan Kuritzkes, the director of AIDS research at Brigham and Women’s Hospital, they began to look for patients who would fit the bill. They needed to find people who had HIV but also, as a medical necessity, needed a bone marrow transplant. They wouldn’t try to find a donor who was naturally resistant to HIV, as Timothy was. Their goal wasn’t to cure HIV. Instead, they wanted to see what effect getting a bone marrow transplant had on the HIV reservoir. They hypothesized that the transplant itself, as it swapped out so many of a person’s own immune cells, would perturb the reservoir. It’s similar to Hütter’s notion of “resetting the immune system clock.” By doing so, they might also determine which cells were key to maintaining the virus reservoir.
Henrich’s study started as retrospective but, after the spectacular results, became prospective. He was working with archived samples from patients who’d already had the procedure. Quite by accident, the researchers found archived samples from two HIV-positive men with lymphoma. The men had received minimal ablation, the drugs given to clear out a person’s own cells in their bone marrow to make way for the transplant. This was in contrast to Timothy, who received an aggressive ablative conditioning regimen. Because the ablative treatment was minimal, the men were able to continue taking their antiviral drugs. Timothy’s more aggressive treatment and chemotherapy meant that he had to stop therapy. However, similar to Timothy’s experience, the donor cells homed to the patients’ bone marrow and, over time, replaced the men’s own immune cells.
What Henrich’s team found was unexpected. They had hoped to model the decay of the HIV reservoir in resting commander T cells, the cells that unwittingly hide the virus in our DNA, out of reach of current antiviral drugs. What they found, however, was no latent virus at all. The two men, who received the therapy 2.5 and 3 years ago respectively, appeared to have eradicated their viral reservoir. It was an exciting announcement at the AIDS conference in DC in July 2012. It seemed that the promises made by one of the Berlin patients had finally been fulfilled. The word cure was passed around as the story grabbed headlines. NPR ran the story under the headline TWO MORE NEARING AIDS CURE AFTER BONE MARROW TRANSPLANTS. But in actuality, the story was still more complicated.
The two men had not stopped antiviral therapy, it was unknown whether the virus would rebound. Also, while Timothy had biopsies performed in his brain, gut, and lymph nodes to search for the HIV reservoir, these two Boston patients hadn’t yet had any additional biopsies done. This is an important point, for HIV is known to hide in these T-cell-rich tissues.
Even if these hurdles were overcome, there are other reasons why this approach couldn’t be used for the majority of people living with HIV. The main drawback, as we’ve discussed, is the considerable risk involved in bone marrow transplants. As Henrich himself says, “If you don’t need a bone marrow transplant you shouldn’t get a bone marrow transplant.”
What this study makes clear is a feasible path to eradicating the virus. These researchers were able to shrink the viral reservoir, the obstacle to curing HIV. While bone marrow transplants will never be commonly used to eradicate HIV, this approach leads the way for other technologies, such as gene therapy and histone deacetylase inhibitors.
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Asier Sáez-Cirión, an assistant professor at the Pasteur Institute in Paris, was discontent with the studies on the benefits of early therapy in HIV. He wasn’t alone; many in the field have expressed frustration at the fact that we still can’t recommend early therapy and are unsure what benefits it may give, if any. To address this, Sáez-Cirión decided to go back and look at the records of 700 French HIV patients who received early therapy. With Christian’s story as inspiration, the patients in the late 1990s were given antiviral therapy during acute HIV infection. Sáez-Cirión’s is a retrospective study, requiring no new patients. The advantage to this kind of study is that you can look at large numbers of patients with little cost; the disadvantage is that you can’t modify the study since it’s already happened. Of the 700 patients, 75 of them stopped therapy after a year. Of those 75, 14 of them had not returned to therapy. These 14 became known as the VISCONTI cohort (acronym for virological and immunological studies in controllers after treatment interruption).
These 14 patients have a few unique features. They all started therapy very early: The median time to start therapy after infection was 39 days. While not as early as Christian, who received therapy within days of his infection, the cohort was treated earlier than other studies on acute HIV at the time. The VISCONTI cohort stayed on therapy between one and seven years before stopping. This, too, was different from other trials that treated for shorter periods, similar to Christian’s self-prescribed treatment interruption, which came only six months after starting therapy. Unlike Christian, the therapy the cohort received was standard and included no experimental cancer drugs. Like Christian, many of the patients experienced a short-lived spike in HIV after stopping therapy. Unlike Christian and elite controllers, the storm trooper T cells from these patients didn’t have any special ability to target HIV.
Approximately seven years after the cohort stopped therapy, researchers announced the results in 2012 at the AIDS conference in Washington, DC. The 14 patients remained off therapy. Because none of the patients had any genetic markers that could explain control of the virus, they were declared, like Timothy and Christian, functionally cured. Intriguingly, they also harbored a tiny amount of virus detectable by ultrasensitive PCR in their T cells, just like Timothy and Christian. Perhaps even more surprising was that in four of the patients, this tiny pool of virus continued to shrink, even though the men hadn’t been on therapy in years.
The pieces of the puzzle were coming together. The evidence from the VISCONTI cohort tied in perfectly with the anecdotal evidence offered from the Berlin patients as well as the toddler who was given early therapy that resulted in a functional cure. It also matched the data from the trials of histone deacetylase inhibitors. The answer wasn’t a sterilizing cure for HIV infection; not every bit of virus has to be eradicated. Instead, it’s possible to live with some HIV still hiding in the body, a tiny pool of passenger virus that was along for the ride but needed no special effort to be contained. The path to a functional cure for HIV was taking many forms, from gene therapy based on Timothy’s experience, to early therapy based on Christian’s. But it’s all leading to the same place.
• • •
David Baltimore, to put it mildly, has been interested in retroviruses for a long time. In 1975, he was awarded the Nobel Prize for discovering reverse transcriptase, work he did during his postdoctoral fellowship that revealed how retroviruses invade our DNA. Even back then, he saw the potential, reminiscing, “One of the things that struck me immediately when we found reverse transcriptase is that it was a door to gene therapy.” Those who did pursue gene therapy during those early days ran into difficulties, for the field was simply too new. But the promise was there. Researchers had figured out how retroviruses gain entry to cells and insert their genetic material into our DNA. Perhaps there was a way we could manipulate this system to insert a gene of our choosing into our DNA.
Baltimore was performing basic immunology research when once again he was intrigued by the promise of gene therapy. In the early 2000s, he partnered with Irvin Chen at UCLA. Together they tested the ability of small interfering RNAs (siRNAs) to inhibit CCR5. These short RNA molecules are able to inhibit gene expression through a mechanism known as RNA interference (RNAi). The small pieces of RNA bind to the messenger RNA (mRNA), which keeps the message, the gene information needed, from reaching the ribosome, the protein construction plant. The mRNA is like a message in a bottle, a necessary missive the cell needs to express its genes. The siRNA breaks that bottle so that the message is never delivered. Without the CCR5 message delivered, the protein can’t be expressed on the surface of the cell. This means that HIV can’t enter the cells—just as a person with a mutant CCR5 doesn’t express the protein on the surface of his T cells. Baltimore and Chen’s results, published in 2003, were promising. But the research stayed on the shelf because the next step was a human clinical trial, an expensive undertaking. “It wasn’t clear we could get support for it,” says Baltimore. Years later, he met an entrepreneur, Louis Breton, who was interested in the approach. Together they formed a small biotech named Calimmune in 2007. But they still needed funding to bring it to clinical trials. This wasn’t so easy since a gene therapy approach to treating HIV was generally considered a risky investment.
That changed when news of the Berlin patient, Timothy, broke in 2009. Suddenly, gene therapy approaches no longer seemed outlandish. This influenced not only researchers but organizations that fund researchers, such as amFAR, The Foundation for AIDS Research. In fact, when discussing the grant they awarded Baltimore in 2010, they state, “amfAR’s interest in exploring the role of gene therapy in the eradication of HIV infection stems from a February 2009 report in The New England Journal of Medicine of a patient in Berlin.” But the grant that changed the fate for Baltimore’s CCR5 siRNA approach came from CIRM, the California State–funded stem cell agency. The $20 million grant was awarded in 2010 to bring their encouraging gene therapy to clinical trials. CIRM awarded the grant, and the one for Cannon and Sangamo, based on the hopes bolstered by Timothy. The project began enrolling the first patients in March 2013. Now multiple gene therapy clinical trials, all targeting CCR5, are in motion, all based on Timothy’s cure.