Zinc fingers make HIV-resistant T-cells

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Post by Adrian Ogier24 Nov 2011

Gene therapy that interferes with co-receptors on the surface of T-cells can protect these cells from HIV infection, and represents a potential first step toward a ‘functional cure’ for HIV.

HIV uses two different surface co-receptors (CCR5 and CXCR4) to enter and infect CD4 T-cells. If the coreceptors are blocked or disrupted, the virus is unable to enter cells. Two presentations at the CROI conference early this year looked at using gene therapy to create cells that lack these receptor proteins and therefore are protected from infection.

Jay Lalezari from San Francisco1 used zinc finger nuclease technology to disable the gene responsible for producing the CCR5 coreceptor on T-cells.

Zinc finger nucleases (ZFNs) are proteins specially designed to break specific DNA sequences.

His work draws upon knowledge gained from ‘elite controllers’, a small proportion of HIV-positive people who have a natural genetic mutation known as ‘CCR5-delta-32 deletion’.

These individuals do not express CCR5 on their T-cells and are able to maintain undetectable or very low viral load without antiretroviral therapy.

Similarly, Timothy Brown, the ‘Berlin patient’, received two bone marrow transplants to treat leukemia from a donor with the delta-32 mutation. His own immune cells were destroyed by chemotherapy to wipe out the leukemia, and his immune system was reconstituted with cells that lacked CCR5. Timothy stopped antiretroviral therapy, and three years later researchers are unable to find any trace of HIV.

Given that bone marrow transplants are not feasible on a large scale, investigators are exploring other ways to achieve a similar outcome.

Lalezari's phase 1 study included six HIV-positive participants on antiretroviral therapy. All were men mostly in their early 50s who had been HIV positive for 20 to 30 years. They all had undetectable viral load but none had experienced optimal CD4 cell recovery, all having counts between 200 and 500.

Participants underwent a procedure known as ‘apheresis’, in which blood is drawn from their body, T-cells are filtered out, and the rest of the blood is returned. Harvested T-cells are then sent to a laboratory where they were activated and treated with the zinc finger nuclease. The nuclease causes a double-strand DNA break in the CCR5 gene and the repair process permanently disrupts the gene .

Treated cells were expanded outside the body and about 25% were successfully modified, Lalezari said. These CCR5-deleted cells were then frozen, sent back to the study clinics, and re-infused back into the original patients.

Two groups of patients received doses of ten and 20 billion cells; a third group now underway will receive 30 billion cells.

"There don’t appear to be any safety issues," Lalezari said.

The ‘apheresis’ and reinfusion process was safe and generally well tolerated.

Some participants experienced flu-like symptoms, but these were temporary. No serious side-effects or abnormal lab results were observed.

In all six participants the altered CD4 cells engrafted, or took up residence in the body, and proliferated in a manner similar to normal T-cells. Five of the six experienced significant, sustained increases in the number of CD4 cells, averaging about 200 cells, though gains varied widely across patients and over time.

Five participants also experienced normalisation of the CD4 cell to CD8 cell ratio, which is typically reversed in people with HIV.

After 90 days, up to 7% of CD4 cells showed the CCR5 deletion. Rectal tissue biopsies revealed that the altered CD4 cells were distributed to the gut lining like normal T-cells.

The observed expansion of CD4 cells was on average three-times greater than expected based on the number of infused cells, Lalezari noted.

He acknowledged, however, that the alteration procedure involved activating the cells, which may have contributed to their proliferation.

The one participant who did not respond as well to the treatment had high levels of antibodies against the adenovirus vector, which may have made the CCR5 deletion procedure less effective.

These results represent a proof of concept that further validates the Berlin patient findings, Lalezari said, but he cautioned that it is too early to talk about these results as a cure.

The next step will be to test this CCR5 removal procedure in HIV-positive people with replicating virus to see if re-infusion of altered CD4 cells reduces viral load and has a clinical benefit.

Researchers will look at treatment-naive individuals as well as some multiple treatment-experienced patients who are not responding to current therapy.

The hope is to provide a reservoir of cells that are resistant to HIV infection. If the gene therapy technique is successful, it should confer a significant survival advantage, since protected cells would continue to proliferate while susceptible cells would be infected with HIV leading to their early death (because they can’t go on to infect and replicate in other protected CD4 cells).

Since HIV can use both CCR5 and CXCR4 to enter T-cells, disruption of both co-receptors would be required to fully protect a cell from infection.

Craig Wilen from the University of Pennsylvania2 presented some of the first data on gene therapy to interfere with CXCR4 expression on CD4 cells.

His team also used zinc finger nuclease technology which in this case causes a double-strand break in the CXCR4 gene .

Laboratory cell culture studies showed that the zinc finger procedure did not negatively affect T-cell proliferation. When exposed to HIV, the modified cells with disrupted CXCR4 showed a significant survival advantage.

In mice with humanised immune systems, the altered CD4 cells were protected against infection with HIV strains using the CXCR4 coreceptor.

A protective effect was evident by 14 days after re-infusion, although the effect waned over time.

Blocking CXCR4 may prove more challenging than blocking CCR5. People with the natural CCR5-delta-32 mutation are generally healthy, with minor immune system variations giving them greater resistance or susceptibility to specific infections. The potential consequences of blocking CXCR4, however, are not fully understood.

Research to date suggests that it will be necessary to target both CCR5 and CXCR4 in T-cells, Wilen said, and his studies have showed that both can be accomplished in the same cell.

Altering mature CD4 T-cells has been shown to provide protection from HIV infection, at least in the short term. But using a similar gene therapy approach on hematopoietic stem cells, which give rise to all types of blood cells including CD4 cells, might confer longer-term and perhaps life-long protection.

While the apheresis and gene therapy approach is quite expensive initially, researchers are now exploring how the procedure might be scaled up. If cell modification only needs to be repeated infrequently – or better yet, only once ever – it might prove cost effective compared with life-long antiretroviral therapy.

References for this item are also available at www.aidsmap.com.

Adapted from an article by Keith Alcorn 1 March 2011

  1. Lalezari J et al. Successful and persistent engraftment of ZFN-M-R5-D autologous CD4 T Cells (SB- 728-T) in aviremic HIV-infected subjects on HAART. 18th Conference on Retroviruses and Opportunistic Infections, abstract 46, Boston, 2011.
  2. Wilen C et al. Creating an HIV-resistant immune system: using CXCR4 ZFN to edit the human genome. 18th Conference on Retroviruses and Opportunistic Infections, abstract 47, Boston, 2011