Zinc Finger Nuclease that Disables CCR5 Gene May Offer Potential New HIV Treatment Approach

By Liz Highleyman

Zinc-fingers are small DNA-binding proteins that mediate DNA-protein interactions within cells, playing an important role in regulation of DNA expression.

It may be possible to create CD4 cells that are resistant to HIV infection by using zinc finger protein nucleases to disable the gene that encodes the CCR5 co-receptor, according to research presented at the 48th International Conference on Antimicrobial Agents and Chemotherapy (ICAAC 2008) last month in Washington, DC.

CCR5 is one of 2 co-receptors HIV uses to enter cells (the other is CXCR4). CCR5 antagonist drugs such as maraviroc (Selzentry) work by preventing the virus from using this co-receptor. The new results indicate that HIV entry may also be prevented by disrupting expression of CCR5 on cell surfaces.

Michael Holmes of Sangamo BioSciences and colleagues developed zinc finger protein nucleases (ZFNs) that target the CCR5 gene and cause double strand breaks at a predetermined sites in the DNA. Natural DNA repair mechanisms can then be usurped to imperfectly mend these breaks, resulting in the permanent disruption of the gene.

ZFNs were introduced into human cells using an adenovirus vector. Cell-based assays revealed that the CCR5 ZFNs produced the expected DNA breaks, leading to efficient gene disruption (> 50%) in human CD4 cells.

ZFN-treated primary CD4 cells and transformed CD4 cell lines were specifically resistant to infection with CCR5-tropic HIV, but otherwise behaved normally.

"These data demonstrate that ZFN-treated cells can be permanently modified to prevent [CCR5]-dependent HIV infection," the researchers concluded.

In laboratory cultures and in immunodeficient mice, the modified CD4 cells survived and replicated well in the presence of HIV. Mice that received ZFN-modified cells had increased numbers of CD4 cells and a statistically significant 7-fold reduction in peripheral blood HIV viral load, compared to mice given non-modified cells. Furthermore, the ZFN-altered cells appeared to have a selective advantage relative to unmodified cells.

These results led the investigators to suggest that "these cells may be able to reconstitute immune function in patients with HIV/AIDS via maintenance of an HIV-resistant CD4 population."

Therapy using this method would involve removing some CD4 cells from a patient, modifying them in the laboratory, and returning them to the body, where they would hopefully replicate to create a reservoir of protected, HIV-resistant cells, thereby preserving immune function.

By permanently modifying cells so they cannot express CCR5, the zinc finger approach has a potential advantage over small molecule CCR5 antagonists or antibodies such as PRO 140 that must be continually administered to block the co-receptor and prevent HIV entry. The zinc finger method would only alter CCR5 expression on CD4 cells -- not other cell types -- but it is not yet clear whether disrupting CCR5 expression could have unknown detrimental effects on immune function.

"We are very excited about these data and our collaboration with Sangamo to develop an HIV/AIDS therapeutic," said Carl June, MD, of the University of Pennsylvania School of Medicine in a press release issued by the company. "The ability to prevent immune cells from becoming infected by HIV has the potential to provide long term control of both the opportunistic infections characteristic of AIDS as well as the virus itself. We look forward to bringing this program into the clinic."

Sangamo plans to test this strategy in human clinical trials and intends to file an Investigational New Drug application by the end of 2008.

Univ. of Pennsylvania, Philadelphia, PA; Sangamo BioSci., Richmond, CA.

11/18/08

Reference
EE Perez, J Wang, JC Miller, and others. Establishment of HIV Resistant CD4 T Cells using Engineered Zinc Finger Protein Nucleases. 48th International Conference on Antimicrobial Agents and Chemotherapy (ICAAC 2008). Washington, DC. October 25-28, 2008. Abstract H-4044.

Other source
Sangamo BioSciences, Inc. Sangamo BioSciences Presents Data at ICAAC Demonstrating 'In Vivo' Protection Against HIV Infection by CCR5-ZFN Therapeutic Preclinical Animal Data Demonstrates Selective Survival Advantage of ZFN-Treated Immune Cells after HIV Infection and Reduced Viral Loads. Press release. October 28, 2008.