Immune-based treatment increases HIV-resistant CD4 cells in phase 1 study

Simon Collins, HIV i-Base

One of the first oral presentations of the conference reported results from an immune-based treatment from Sangamo Biosciences called SB-728 presented by Jay Lalezari from Quest Clinical Research in San Francisco. [1]

The intervention in people who were successfully treated with HAART but whose CD4 count remained between 200 and 500 cells/mm3 led to increases in CD4 cell counts of approximately 200 cells/mm3 that were sustained for a year.

This was a small open label phase 1 study in six people. The procedure involved blood apheresis to extract CD4 cells (mean ~43 billion), genetic modification to induce resistance to HIV infection, expansion and then reinfusion several months later with a single dose infusion of either 10 or 20 billion cells. Zinc finger nuclease (ZFN)-mediated gene editing technology was used to disrupt the naturally occuring CCR5 coreceptor. ZFNs are small peptides used to introduce a double-stranded break in DNA, that when repaired adds a five base-pair insertion including two stop codes in approximately 25% of treated hematopoietic stem cells.

In this study, the modification process affected a mean 26% of cells (14 to 36%) prior to reinfusion. The percentage of CCR5 disruption in the peripheral blood 14 days after reinfusion in five participants was 6, 3, 1, 2, and 2% and this persisted for the duration of follow up. CD4 counts increased in all participants (+35 to +1038 cells/mm3 at day 14). Mean follow up was 24 weeks and 4 weeks, in the low and high dose groups, respectively, with mean CD4 increases in these five people of 208, 86, 233, 911 and 210 cells/mm3. Although increased CD4 counts could be related to increased cellular transport, rectal tissue biopsies indicated that reinfused cells reached other tissue compartments. Infusion of both doses was safe and well tolerated with mild flu-like cytokine release-related side-effects immediately following the infusion, and garlic body odour related to one of the compounds used in the freezing process.

These results suggest that ZFN-modified gene disruption of CCR5 provides a feasible approach to generate a reservoir of HIV-resistant cells. Three new patients will receive 30 billion cells as a third cohort, two of whom have already received treatment.

A second presentation reported research into developing a similar disruption to CXCR4 virus that was able to shift tropism in a humanised mouse study suggesting that genetic manipulation may be able to be designed for people at all stages of HIV disease. [2]

The research was also the focus of two presentations in a symposium on the last day of the conference called Obstacles to a Cure. [3]

Paula Cannon from University of Southern California Los Angeles reported on early research in ZFN-treated autologous hematopoietic stem cells (HSC) to generate HIV resistant cells as a functional cure in mice.

Using an immune-depleted (NSG) mouse model allowed the researchers to transplant human cells grafted at day one and then analyse human cells after 2-3 months. At this time, 65% of the mouse bone cells are human origin, 80% of thymus cells, and 20-30% bone cells. The humanised mice were then infected with HIV and their viral infection was monitored over several months. While control mice showed expected CD4 declines, ZFN-treated mice maintained immune equilibrium. These differences were more striking when looking at tissue samples with severe cell depletion in bone marrow, thymus, spleen and gut mucosa in control mice (receiving untreated HSC) compared to ZFN-treated mice, who showed similar response to HIV uninfected and untreated control mice.

CCR5 expression in spleen and gut cells were only detected in the control HSC mice and not in the ZFN-HSC animals. Intriguingly, viral load curves were similar between the treated and untreated infected animals for the first six weeks, rising to >100,000 copies/mL. They remained at this level in the untreated mice but dropped to undetectable levels in both blood and tissues of the treated mice. Similar response were observed with other R5-tropic but not X4-tropic strains of HIV.

The group is now looking at additional gene deletions for CCR5 including developing responses resistant to X4 infection.

Scale up of this technology is starting with HIV-positive people diagnosed with AIDS-related lymphoma, including EBV-related lymphoma. Treatment included removing and treating HSC prior to chemotherapy and reinfusing after chemotherapy. Endpoints include immunologic and virological endpoints. This complements the SB-728 treated CD4 cells in phase 1 studies reported below.

Carl June and colleagues from University of Pennsylvania presented combined early in vivo results from the Lalezari study reported above and from patients enrolled in second phase 1 open label study being run by Pablo Tebas at University of Pennsilvania. This second study uses single dose infusions of between 5–10 billion cells in 12 people on stable antiretrovirals: 6 with CD4 >450 (6 enrolled, 4 infused, 2 on treatment interruptions, one with data from July 2009) and 6 with <500 cells/mm3 (4 enrolled, 2 infused). This study also includes an ‘optional’ treatment interruption one month after the infusion.

Of 14 people treated, 9 have safety data. No serious events have been reported over median of 232 days (range 99–754 post infusion); 58 events were reported (48 mild, 10 moderate); 33/58 were judged related to infusion (fever, fatigue) and resolved without complications within 48 hours. No evidence was seen of genotoxicty.

All patients have an early CD4 increase post infusion of 200–300 cells/mm3 by day 14, with three people have greater than 1000 cell increases, with most people dropping to, or stabilising to, an increase of around 200 from baseline after 3 months. The CD4:CD8 ratio normalised to >1.0 in most patients out to 90 days. However, in these small numbers there were a range of responses and not all patients responded as strongly.

All patients but one maintained a level of approximately 4% of modified cells that were maintained and stable out to 6 months. The patient with longest follow-up has maintained a stable level of modified cells for over 400 days and is evidence that memory cells with stable persistence can be modified (though there are no phenotypic data on these cells yet). In vivo expansion of the modified cells was seen in 8/9 patients (mean 3-fold increase 14-30 days post infusion, but 2 people having 20–40 fold increases) with stable increases persisting for the extent of follow-up. Trafficking to other cellar tissue was demonstrated from rectal biopsy samples showing increases gene disrupted cells at least on a comparable level to that seen in blood.

The two patients who took a 12-week treatment interruption at day 28 experienced viral rebound to around 5 logs which then dropped by 1–2 logs prior to restarting treatment but larger patients numbers are needed to evaluate any consistent pattern or treatment effect.


Currently this research is exciting. Up to 10% of people who respond virologically may fail to generate a similar or sufficient immunological response to HAART, and delayed initiation of HAART correlates to reduced likelihood of normalising CD4 counts (commonly referred to as >500 cells/mm3). IL-2-associated CD4 increases either failed to produce functional immune benefits or these were offset by the toxicity associated with IL-2 treatment.

While the publicity from this study associated the research with the case of the stem cell transplantation from a CCR5-delta32-deleted donor to a patient who has since been able to discontinue antiretroviral drugs for over three years without experiencing viral rebound, it is important to realise that these are two very different approaches.


Unless mentioned otherwise, all references are to the Programme and Abstracts of the 18th Conference on Retroviruses and Opportunistic Infections, 28 February–2 March 2011, Boston.

Webcasts are available at the following link:

  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 CROI, 27 February–2 March 2011, Boston. Oral abstract 46.
  2. Wilen C et al. Creating an HIV-resistant immune system: using CXCR4 ZFN to edit the human genome. 18th CROI, 27 February–2 March 2011, Boston. Oral abstract 47.
    Webcast Session 11, Monday 10 am-12 noon; Auditorium. HIV: Innovative Therapeutic Approaches, ART, and Drug Resistance. Access from Tuesday 28th.
  3. Session 47-Symposium Obstacles to a Cure Wednesday, 4-6 pm. Webcast available online.
  4. Cannon P et al. CCR5 knock-out in hematopoietic stem cells. Oral abstract 164.
  5. Tebas P et al. Disruption of CCR5 in zinc finger nuclease-treated CD4 T cells: phase I trials. Oral abstract 165.

Links to other websites are current at date of posting but not maintained.