Translational Research in Action
In the spring of 2011, Penn is celebrating the opening of the Smilow Center for Translational Research – a new home for Penn Medicine's emphasis on translating breakthroughs in the lab to clinical therapies for patients. The story profiled here is just one example of such research at Penn.
See more stories in this series.
Efforts to bring new treatments to patients more quickly often draw from disparate quarters and this one has it all: A miraculous recovery of an AIDS patient, a previously unrecognized ancient genetic mutation, and new hope for a deadly disease based on a molecular scissors, of sorts.
A genetic mistake that arose thousands of years ago spares rare HIV-infected individuals the ravages of AIDS. The mutation in a protein that sits on the surface of a human immune cell renders one percent of Caucasians alive today resistant to HIV infection. Researchers believe that strong natural selective pressures in the past favored individuals with this mutation.
Many labs are trying to develop approaches to cure HIV based on this rare mutation, and Penn’s School of Medicine is one of those in the midst of translating the language of ancient genetic mistakes into today’s cures.
In late 2008 an unexpected clinical development emphasized the potential for applications based on this mutation. An HIV-positive American living in Berlin, who also had leukemia, which is best treated by a bone marrow transplant, was seemingly cured of HIV. He was given marrow from a tissue-matched donor who had also been screened for the genetic mutation conferring resistance to most types of HIV. At the time of international reports on the success of his treatment, the patient had lived close to 20 months since the transplant with no detectable traces of HIV in his marrow, blood, or other tissues. He remains healthy today.
A genetic resistance to HIV Infection
The donor, born with the mutation in his CCR5 gene, did not have a working CCR5 receptor protein on the surface of his T cells. The CCR5 protein is one of the two cell-surface receptors needed for HIV to gain entry into a T cell in order to replicate. The mutation prevents HIV from attaching itself to cells. Individuals with the mutated CCR5 are seemingly not affected by the non-functional CCR5 protein, other than their resistance to HIV. (In fact, James Hoxie, MD, director of the Penn Center for AIDS Research, whose genome harbors the CCR5 mutation, was profiled in The Scientist last year about his unique genetic status.)
Much of the basic work behind understanding CCR5’s role in HIV infection came from the lab of Robert Doms, MD, PhD, chairman of Penn’s Department of Microbiology, and colleagues who in 1996 described CCR5’s relationship with HIV.
Engineering HIV-Resistant T-Cells
In research beginning in 2004, researchers at Penn’s School of Medicine and collaborators from Sangamo BioSciences in Richmond, CA, took a first step toward engineering normal T cells to mimic those that have natural HIV resistance.
Zinc fingers are proteins that normally bind to different bases in the DNA sequence to regulate the activity of genes. The zinc fingers used in this experiment were custom-designed to bind to specific DNA sequences in the CCR5 gene. The zinc finger protein acts as a molecular scissors, bringing a DNA enzyme to the CCR5 gene to cut a portion of its sequence, but due to the repair process a new mutation arises in the CCR5 protein, rendering it non-functional. Without a functional CCR5 protein on the cell's surface, HIV cannot enter, leading to resistance to HIV infection. Once the modification is made to the cell’s CCR5 gene it is permanently disrupted in that cell and even in its progeny. The door to HIV’s entry into the T cell is slammed shut, and if the cells are induced to divide, a form of spreading HIV resistance can result.
The investigators used healthy human CD4 T cells and added DNA that expresses the zinc fingers. They grew the engineered cells in tissue culture flasks and transferred them into immune-deficient mice infected with HIV.
"We followed them over time and showed that those mice that received the zinc-finger-treated cells showed less viral load than controls and also had improved CD4 counts," says Elena Perez, MD, PhD, who performed the research while a postdoctoral fellow in the lab of Carl June, MD, professor of Pathology and Laboratory Medicine at Penn Medicine. (Perez is now an assistant professor at the University of South Florida.) The data suggested that, in the presence of HIV, the zinc-finger-modified cells have a selective advantage, allowing them to evade infection and able to fight opportunistic infections and HIV itself.
Clinical Trials Giving HIV-positive Participants Their Own HIV-resistant T Cells
The next step was to adapt and scale-up the treatment for a clinical trial in humans. This was performed in Penn’s Clinical Cell and Vaccine Production Facility, directed by Bruce Levine, PhD, associate professor in the Department of Pathology and Laboratory Medicine. Data from the animal studies and from the facility supported submissions to the NIH Office of Biotechnology Activities and to the FDA, which granted permission to conduct a carefully monitored clinical trial.
Sangamo and Penn started a Phase I safety and effectiveness clinical trial in humans a little over two years ago. The CCR5 gene from T cells from HIV patients was deliberately knocked out. These modified T cells were then infused back into the patients to re-establish their immune system and decrease their viral load. The first patients enrolled in the trial at Penn under the direction of Pablo Tebas, MD, director of the AIDS Clinical Trials Unit.
The zinc-finger technology provides a simpler approach compared to a bone marrow transplant. It generates a population of cells that lack the CCR5 receptor, are resistant to HIV, and can be given back to the patient to provide a reservoir of HIV-resistant functional immune cells. More importantly, these modified cells may proliferate and provide an HIV immune response.
Almost two years to the date after enrolling their first patient, June and colleagues presented interim results of the clinical trial at the 2011 Conference on Retroviruses and Opportunistic Infections. The results were hailed in the medical press as “renewing hopes for conquering AIDS," with reports appearing in such outlets as the Associated Press, the Philadelphia Inquirer, and Nature.
Nine patients have been enrolled so far in the initial trial and the number of T cells free from HIV multiplied considerably in eight of them. The average number of these cells represented about 6 percent of the participants’ total CD4 T cell count. In the first patient they treated there were more cells persisting at 450 days after the infusion than what they infused on day 0, so the effect seems durable, say the researchers.
First Successful Example of Targeted Gene Editing in a Patient’s Own Cells
June says that the most gratifying part about this phase of the research is that the procedure is safe and workable in all patients tested to date in this small trial. “This represents a new tool that will permit us to ask if we can eventually replicate the ‘Berlin’ patient with the patient’s own cells, and not require bone marrow transplant,” he says.
Other basic work in mice – performed by a team from the Doms lab -- has shown that a similar approach can be used to edit the other HIV co-receptor, CXCR4.
June foresees a series of clinical trials to increase the levels of HIV-resistant cells in patients with the goal to approach 100 percent and obviate the need for daily HIV drugs. In a larger sense, beyond the field of HIV gene therapy, this is the first successful example of targeted gene modification in patients, with implications for developing corrective gene therapy for gene disorders of the bone marrow, such as sickle cell anemia, that are currently incurable.
No matter where the basic and clinical work on either receptor leads, the translation of gene engineering to life-changing therapy is not lost on Philadelphia resident Jay Johnson, the second patient to receive the zinc-finger T cells. “I’m ecstatic.” he told the reporters. “Maybe some day I can come off drug therapy altogether. That would be a blessing.” Recently Mr. Johnson and Dr. Tebas spoke about the trial with AP Online.
DORANZ, B. (1996). A Dual-Tropic Primary HIV-1 Isolate That Uses Fusin and the ?-Chemokine Receptors CKR-5, CKR-3, and CKR-2b as Fusion Cofactors Cell, 85 (7), 1149-1158 DOI: 10.1016/S0092-8674(00)81314-8
Perez, E., Wang, J., Miller, J., Jouvenot, Y., Kim, K., Liu, O., Wang, N., Lee, G., Bartsevich, V., Lee, Y., Guschin, D., Rupniewski, I., Waite, A., Carpenito, C., Carroll, R., S Orange, J., Urnov, F., Rebar, E., Ando, D., Gregory, P., Riley, J., Holmes, M., & June, C. (2008). Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases Nature Biotechnology, 26 (7), 808-816 DOI: 10.1038/nbt1410