CROI 2013: Gene Therapy Studies Show Potential for HIV Control without Drugs

Modified immune cells that make their own fusion inhibitors reduce levels of HIV-like virus and resisted infection in animal and test tube studies, researchers reported at the 20th Conference on Retroviruses and Opportunistic Infections (CROI 2013) last month in Atlanta.

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Gene therapy approaches that involve the genetic modification of human hematopoietic stem cells have the potential to engineer HIV control by introducing cells resistant to HIV infection.

The proof of concept for this approach to HIV control in humans comes from the case of the "Berlin Patient," who received bone marrow transplants from a donor with natural resistance to HIV infection. The donor was homozygous for the CCR5 delta 32 mutation, meaning that cells potentially vulnerable to HIV infection would never display the CCR5 receptor necessary for most types of HIV to gain entry. As a result of complete ablation (destruction) of the recipient's own stem cells by chemotherapy, his CD4 cells were replaced by cells derived from the donor's CCR5-lacking population. Over 3 years after the procedure the recipient remains HIV-free without antiretroviral treatment, and has been described as "functionally cured" by physicians.

However, the prospects for reproducing this outcome using transfer of donor cells is very low due to the shortage of potential donors who are both HLA-compatible (essential for avoidance of graft-versus-host disease) and CCR5-delta 32 homozygous. In any case, the essential elements that contributed to this functional cure are still not fully understood.

The introduction of modified genes into stem cells harvested from a person's bone marrow will be a necessarily individualized treatment, but the costs of gene therapy are likely to come down in the future, and if experimental approaches prove successful in controlling HIV without antiretroviral drugs, gene therapies may deliver a cost-effective form of treatment in the future.

The first human studies of gene therapy have sought to modify the expression of the CCR5 receptor. Sangamo Biosciences is developing a zinc finger nuclease which prevents CCR5 expression; study results have been presented at a number of conferences including ICAAC 2012 and CROI 2013showing that the procedure is safe and that it results in long-term gains in CD4 cell numbers in people also taking antiretroviral therapy.

A pair of studies presented at CROI 2013 described the use of modified immune cells that make their own peptides (short chains of amino acids) and act as fusion inhibitors, similar to the drug enfuvirtide (Fuzeon or T-20). In one experiment, these cells -- which essentially made their own anti-HIV drug -- acted as a treatment or therapeutic vaccine, curbing the viral load of a laboratory virus engineered to be far more virulent than human HIV; in the other, they prevented infection by a similar virus.

In the first study, rhesus monkey stem cells -- the progenitors of T-cells and all immune cells -- were genetically altered to express a fusion inhibitor peptide called mC46. While not able to prevent infection in monkeys challenged with a highly pathogenic monkey/human SHIV (a laboratory-manufactured virus combining parts of both the human and simian immunodeficiency virus genomes, created for research purposes), it did produce infections characterized by viral loads 2.5 and 3.15 logs lower than those seen in a control group of monkeys.

In another study, human CD4 cells had a fusion-inhibitor peptide called C34 attached to their co-receptors (CCR5 or CXCR4). These cells were able to resist infection by HIV in the test tube.

Fusion Inhibitor Cells Raise CD4 Count

In the first study, researchers from the Fred Hutchinson Cancer Research Institute in Seattle took hematopoeic stem cells (HSCs) -- bone-marrow cells that are the progenitors of all blood cells -- from 2 pigtail macaque monkeys and transformed them genetically by splicing an inserted gene sequence for mC46 into their CCR5 receptor gene.

They then injected the cells back into the monkeys. One monkey had 20% of its HSCs replaced by the C34-producing cells, and the second had over 50% replaced.

A week later, the researchers infected the modified monkeys and 2 control monkeys with a particularly lethal strain of genetically engineered human/monkey SHIV, which destroys CD4 cells fast and usually develops a steady-state viral load in the order of several million copies/mL.

In the control animals, CD4 counts declined from around 600 cells/mm³ before infection to between 10 and 50 cells/mm³ within 2 to 3 weeks. In the monkeys with mC46, the CD4 cell count dipped to about 100 cells/mm³ within 2 weeks of infection, but then rose slowly back to pre-infection levels over the next 6 months.

At the time of highest SHIV viral load and fewest CD4 cells, 90% of the CD4 cells in the mC46 monkeys had the fusion-inhibitor-generating insert in them, which is what one would expect given that SHIV decimates non-mutated CD4 cells.

What was unexpected to the scientists, though, was that after the period of peak viral load, the non-mutated CD4 cells made a partial recovery -- in one monkey to about 60% of all CD4 cells and in the other about 20%. This is promising, as it shows that one would not need to replace all or even most of the CD4 cells in the body with HIV-resistant ones in order to contain HIV infection, and that increasing numbers of non-resistant cells does not lead to a new burst of virus.

This may also mean that it would not be necessary to take the dangerous step of having to destroy a person’s immune system with whole-body radiation, as happened to "Berlin Patient" Timothy Ray Brown, in order for the new cells to repopulate the immune system.

As noted, SHIV reproduces furiously, and peak viral load in all monkeys 10 days after infection reached 1 billion copies/mL. After that, viral load in the control monkeys declined to about half a million in one animal and about 10 million in the other. In the modified monkeys, however it fell to about 100,000 in the monkey with 20% of its cells replaced by mC46 cells and down to a few hundred in the one with more than 50% of its cells replaced.

Viral load was about 320-fold (2.5 logs) lower in the first monkey and about 1400-fold (3.15 logs) lower in the second. This would transform a typical HIV viral load in an untreated human of 70,000 copies/mL to 50 copies/mL before any antiretrovirals were taken.

Although there was a partial recovery of un-mutated CD4 cells in the blood, memory cells in the lymph nodes that form the reservoir of proviral HIV DNA remained predominantly the HIV-resistant mC46 cells, which is exactly where one would want them to be.

Fusion Inhibitor Human Cells Resist Viral Infection

The second experiment was a test-tube study, using human CD4 cell cultures. Researchers from the University of Pennsylvania physically attached another fusion inhibitor peptide called C34 to CCR5 or CXCR4 co-receptors. They found that the cells could not be infected with HIV.

Interestingly, although HIV attaches preferentially to one or other of these co-receptors, they found that the action of the fusion inhibitor peptide was non-specific: whichever receptor they attached it to -- whether in cells with one or both co-receptors -- the cells became resistant to infection, so the technique could work with HIV of the R5 or X4 type. The action of C34 was specific to the co-receptors, though: attaching it to the CD4 molecule produced no effect.

The co-receptors on the cells could still do their biological job of attaching to the immune-activation molecules (MIP-1 beta and SDF-1) that they attach to in the body, which indicates less likelihood of toxicity.

The researchers now hope to produce genetically altered cells that produce the fusion inhibitor peptide as part of their co-receptors, as in the previous study, and to move into animal experiments.

4/10/13

References

P Younan, P Polacino, J Kowalski, et al. Protection of Stem Cells Results in Enhanced Virus-specific Immunity with Recovery of Unprotected CD4+ T Cells in a Primate AIDS Model. 20th Conference on Retroviruses and Opportunistic Infections (CROI 2013). Atlanta, March 3-6, 2013. Abstract 127.

G Leslie, J Wang, B Haggarty, et al. T Cells Edited to Express CCR5 or CXCR4 Fused to the C34 Peptide from gp41 Heptad Repeat-2 Exhibit Robust Protection from Diverse HIV-1 Isolates. 20th Conference on Retroviruses and Opportunistic Infections (CROI 2013). Atlanta, March 3-6, 2013. Abstract 129.