Gene Therapy for HIV Functional Cure PDF Print E-mail
Written by Alain Lafeuillade   
Tuesday, 27 December 2011 10:58

Gene Therapy for HIV Functional Cure


This report is part of a series of focused summaries from the “5th International Workshop on HIV Persistence, Reservoirs & Eradication Strategies” held in St Maarten, December 6-9, 2011. Gene therapy approaches were discussed in one in-depth session this year with presentations from Paula Cannon (University of Southern California), Carl June (University of Pennsylvania) and Craig Wilen (University of Pennsylvania).



Gene Therapy for HIV Functional Cure

Paula Cannon recalled that such gene therapy approaches try to engineer an HIV-resistant immune system by CCR5 gene knockout, to suppress HIV replication without lifelong ART and the associated expense, problems of access, and side effects.

This could work:

-By reducing the pool of target cells and thereby reducing HIV replication and its consequences like inflammation, even in drug sanctuary sites,

-Restoring function to the immune system, including protection of HIV-specific T cells.

Furthermore, HIV would be used as a selecting agent.

In theory it could be necessary to protect more than T cells, including macrophages, microglia, dendritic cells…

Hematopoietic Stem Cells (HSC) are, therefore, good targets for anti-HIV gene therapy.

Two kinds of designer nucleases (ZFNs, TALENs) allow permanent disruption of the CCR5 open-reading frame. They have a cutting efficiency of 30% in cell lines but as little as 4-17% in HSPC (figure 1).

Figure 1:



There are, however, challenges before translation into the clinic:

-Using adult bone marrow stem cells

-Scale-up to patient-sized dose

-Achieve >5% CCR5 disruption, >80% viability
-retain stem cell properties

-Select first patient population (patients with lymphomas?)

-Safety, including off-target effects, for first-in-man use of ZFNs in HSC

Carl June reported the results as seen at ICAAC of a single dose of zinc finger nuclease (ZFN) CCR5 modified autologous CD4 T cells to patients.


Commenting on the Berlin patient, June cited former United States Secretary of Defense Donald Rumsfeld: “There are knowns, known unknowns, and unknown unknowns”.

He therefore raised 3 main questions about this case:

1-What was the role of host conditioning: chemotherapy, radiotherapy?

2-What was the role of donor anti-host allogeneic immune response on the residual HIV-1 reservoir?

3-What was the role of ccr5 delta 32 HSC and passenger CD4+ T cells?

Carl June pointed out that the Berlin patient was treated with ~100-fold more Δ32ccr5 deficient CD4+ T-cells than with HSC.

For an adult, a typical allogeneic bone marrow graft has ~1-2 x 1010 mononuclear cells or ~2 x 108 cells/kg, of which 1 to 2% are CD34+ cells and 10-15% are T-cells (1-2 ~107 T cells/kg). In contrast, allogeneic peripheral blood grafts using G-CSF mobilization, there is a 10 to 30-fold increase in donor T-cells, so that typically 3-5 x 108 T cells/kg are given. In each case, the number of CD34+ HSCs are typically 1 to 3 x 106 CD34+ cells/kg. The Berlin patient was treated with a matched unrelated donor (MUD) transplant, with the first dose of mobilized peripheral blood allogeneic cells containing 2.3x106 CD34+ cells and 13 months later, a 2nd MUD graft with 2.1 x 106 CD34+ cells (10). If he was ~70 kg at the time of transplantation, he was therefore given a total of 5.6 x 1010 passenger T-cells, including D32ccr5 CD4+ and CD8+ cells.

June concluded that this was similar to the dose of CCR5-deficient autologous ZFN-engineered cells that they have given in their Phase I ZFN trial (average dose 4.3x1010 total CD4+ T-cells, at a mean 25% disruption efficiency of at least one CCR5 allele; n=10 patients), which is 1.1x1010 CCR5 modified cells per patient.

This infusion increased CD4 counts that persist over time. CCR5 modified CD4 cells expanded rapidly and homed to the gut. CD4 counts rose as much as 1800 cells after transplant, then declined by 90 days to about 200-300 cells over baseline. qPCR confirmed that the expected CCR5 pentamer duplications were seen and were stable.

During a planned ART interruption in 6 patients, CCR5 modified cells persisted in all patients (figure 2).

Figure 2:



Viral load rebounds reached to nearly setpoint, and seemed to start to decline around 70 days prior to restart (figure 3).

Figure 3:



The preliminary conclusions of this trial are:

-Single IV infusion of ZFN modified CD4 T-cells at doses of 10 to 30 billion total cells (~25% modified) is well tolerated to date.

-High initial transient increase and persistent increase in CD4 count observed.

-Persistent normalization of CD4:CD8 ratios seen in most.

-Engraftment of ZFN modified CD4 T-cells occurs at high levels:

-By day 14 post infusion, 1.2 to 30% of the PB CD4 compartment are edited.

-High levels of engraftment are persistent at median of 5.2% of CD4 T-cells day 90 (by Cel-1) and indicate substantial expansion in vivo.

-CCR5-modified CD4 T cells are detected in gut mucosa, demonstrating homing and persistence in rectal mucosal tissue. Decrease in CCR5 expression in rectal mucosal cells in some patients.

-ATI in immune responders suggests antiviral effect.

Finally, Craig Wilen addressed the issue of disrupting CXCR4 by ZFN.

As ~50% of ART-experienced individuals have R5X4 or X4 HIV, CXCR4-ZFNs may protect against X4 HIV and can be combined with CCR5-ZFNs to create completely HIV-resistant CD4+ T cells.

X4-ZFN treatment of CD4+ T cells preserves cell growth and viability in the presence of HIV: X4-ZFNs did not cause any adverse growth defect after ~4 weeks in culture.

Furthermore, CXCR4 disruption confers a survival advantage in the presence of HIV in vitro.

CCR5Δ32 cells respond similarly to wtCD4s suggesting CCR5 and CXCR4 can be genetically targeted simultaneously.

CXCR4 disruption conferred protection from X4 HIV in humanized mice but this effect waned over time (figure 4).


Figure 4:



CXCR4 disruption was, however, stable over time and increased with HIV challenge. The reason why the CD4 protection waned over time could be related to viral coreceptor utilization: a single YàN mutation in the V3 loop of Env predominated in X4-ZFN but not control mice. This mutation resulted in R5X4-tropic virus.

Rhesus macaque studies are ongoing with plans to conduct adoptive therapy of HIV-resistant CD4+ T cells and CCR5-disrupted CD34+ stem cells.

Key words: HIV cure, HIV eradication, Zinc finger nucleases HIV
Last Updated on Tuesday, 27 December 2011 11:08


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