Stem Cell Cures for HIV?

UNAIDS (Joint United Nations Programme on HIV and AIDS) estimated that at the end of 2009 33.3 million people were living with HIV, and that in the same year there had been 1.8 million AIDS-related deaths and 2.6 million new infections (UN Millennium Goals report 2010). The benefits of anti-retroviral therapy (ART) have been demonstrated in some patients, however, resistance can develop, therapy is expensive (meaning that the vast majority of sufferers cannot have access) and multiple organ toxicity occurs with long-term use. Therefore, a safer and more cost effective therapy is clearly required. There have been reported cases of patients living with long-term HIV infection without progression to AIDS and in other cases patients seem to have some degree of immunity when exposed to the virus – both scenarios suggest a possible genetic influence on an individual’s response to HIV infection.

The HIV virus infects CD4⁺ T cells through either the CCR5 or CXCR4 cell surface receptors and destruction of the immune system is driven by the loss of normal CD4⁺ T cells in the peripheral blood and lymphoid tissues. The more prevalent HIV R5 virus infects through the CCR5 receptor, while the HIV X4 virus infects through CXCR4. Homozygosity for a 32-bp deletion in the CCR5 (CCR5D32) allele entails a resistance to HIV infection, while heterozygous patients show slower disease progression (Levy JA., 2007, Berger et al. 1999, de Roda Husman et al. 1997). An initial study in 2009 (Hütter et al., 2009) in the New England Journal of Medicine reported that a patient (the famous “Berlin patient”) with acute myeloid leukaemia (AML) and also HIV, who received two HLA-compatible CD34⁺ peripheral-blood stem cell transplants from a patient homozygous for CCR5D32, became negative for HIV (as determined by analyses of viral RNA and cellular proviral DNA) and entered into complete AML remission. After 2 years, the CD4⁺ T cells in this patient were in normal range, and all carried the donor’s CCR5D32 gene. However, further studies were required to show whether this was indeed a cure, as much evidence shows that HIV can be present in tissues other than the bone marrow and peripheral circulation.

Now a follow up study has provided evidence that, in this patient, this strategy may well be a cure for HIV infection. This study by Allers et al., has been pre-published in the journal Blood and endeavours to address the level of restoration of normal CD4⁺ T through the body, whether the immune system contains cells which are usually HIV susceptible target cells and the stability of the size of the HIV reservoir during the process of immune reconstitution following the stem cell transplant. Comparisons between normal healthy controls, the CCR5D32 stem cell transplant recipient and normal stem cell transplanted patients were used throughout. Transplantation of CCR5D32 cells gave rise to reconstitution of the peripheral CD4⁺ T cell compartment, including enrichment of activated/effector memory CD4⁺ T cells while donor CD4⁺ T cells were also shown to have efficiently repopulated the gut mucosa, where most of these cells normally reside, with no evidence of CCR5 expression on CD4⁺ T cells. CXCR4 expression on the transplanted cells was comparable to the controls, and ex vivo infections showed that these cells where infectable by the HIV X4 virus. CCR5-expressing macrophages were detected in the colon at 5.5 months indicating the presence of some residual host immune cells, but these later became undetectable as immune reconstitution became more complete, indicating total replacement by donor cells. Indeed, HIV RNA and DNA was not detectable in any tissue studied over the next 45 months following the stem cell transplant, HIV core-directed antibodies (p17, p24) disappeared completely and serum levels of antibodies against the HIV envelope (gp41, gp120) decreased.

Yet another high profile paper on a similar theme has been published in Nature Biotechnology from the laboratory of Paula Cannon at the Keck Scholl of Medicine at the University of South California (Holt et al.). This group utilised zinc-finger nucleases (ZFNs) to disrupt the CCR5 receptor in human CD34⁺ hematopoietic/progenitor cells (HSPCs) which would mimic the CCR5D32 mutant. ZFNs are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain therefore allowing the targeting of a desired DNA sequences. These cells were then transplanted into non-obese diabetic/severe combined immunodeficient/interleukin 2rγnull (NSG) mice, which support both human haematopoiesis and HIV-1 infection. Both control and modified HSPCs were capable of multi-lineage engraftment in NSG mice and bone marrow analysis of the mice receiving ZFN-modified HSPCs showed that 11% of the cells exhibited CCR5 disruption, with secondary engraftment leading to 12-20% CCR5 disruption in the bone marrow. The mice were then challenged with an HIV-1 virus which mimics the human disease, and tested every 2 weeks post-infection for up to 12 weeks. Control mice had a pronounced loss of CD4⁺ cells, indicative of HIV-1 infection while, encouragingly, the mice receiving the modified HSPCs maintained normal CD4⁺ levels. Further analysis suggests that the protection of CD4⁺ cells in the modified HSPC mice was a consequence of selection for the CCR5-modified, HIV-1-resistant cells derived from the transplanted modified cells.

Another interesting study has been published in Molecular Therapy describing the use of iPSC technology to generate anti-HIV iPSCs and also HIV-1 resistant macrophages (Kambal et al.). The reprogramming cocktail of factors used included a combination anti-HIV lentiviral vector containing a CCR5 short hairpin RNA (shRNA) and a human/rhesus chimeric TRIM5α gene, again to mimic the CCR5D32 effect. TRIM5α is a TRIM5 transcript variant which mediates an early block to retrovirus infection. Human haematopoietic stem cells (HSCs) were used as the target cell for reprogramming, and after iPSC generation, were re-differentiated towards the hematopoietic lineage, a strategy which perhaps mitigates the problems with lineage specific epigenetic memory of the cell of origin in iPSCs. Colony forming CD133⁺ HSCs were then purified and further differentiated towards a macrophage fate. Importantly, when challenged by the HIV-1 virus these CCR5 negative cells were strongly resistant suggesting a potential patient-specific cell replacement therapy for HIV-1 infection.

Altogether these papers give great hope to those working towards a definitive cure or preventative measures for HIV infection. However, some questions do remain unanswered and some concerns unaddressed. Although the vast majority of transmitted HIV viruses are the R5 variant which gain cellular entry via CCR5, the X4 type of HIV-1 infects through the CXCR4 receptor and these studies do not address a potential strategy for protection against this mode of infection. It could be possible for HIV-1 X4 to infect and emerge in cells which are CCR5 negative, as during disease progression HIV evolves and often will also begin to use the CXCR4 receptor. Further, loss of the CCR5 gene can lead to an increased susceptibility of certain infections, including West Nile Virus (Glass et al.). Additionally, the problem of cost and getting potential cures/protective measures to those who really need them will still exist to some extent with these new strategies. Statistics show that out of an estimated 33.3 million people living with HIV globally, 22.5 million of these live in Sub-Saharan Africa (http://www.unaids.org)) and for this reason these methods of therapy will be out of their reach in the short term. However, as opposed to ART which act to slow disease progression, these new stem cell therapies may indeed provide a more definitive cure.

References

Levy JA.
HIV and the pathogenesis of AIDS.
3rd ed. Washington, DC: ASM Press, 2007

Berger EA, Murphy PM, Farber JM.
Chemokine receptors as HIV-1 co-receptors: roles in viral entry, tropism, and disease.
Annu Rev Immunol 1999; 17:657-700

de Roda Husman AM, Koot M, Cornelissen M, et al.
Association between CCR5 genotype and the clinical course of HIV-1 infection.
Ann Intern Med 1997; 127:882-890

Long-term control of HIV by CCR5D32 /D32 stem-cell transplantation.
Hütter G, Nowak D, Mossner M, Ganepola S, Müssig A, Allers K, Schneider T, Hofmann J, Kücherer C, Blau O, Blau IW, Hofmann WK, Thiel E.
N Engl J Med. 2009 Feb 12; 360(7):692-8.

Evidence for the cure of HIV infection by CCR5D32 /D32 stem cell transplantation.
Allers K, Hütter G, Hofmann J, Loddenkemper C, Rieger K, Thiel E, Schneider T.
Blood. 2010 Dec 8.

Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo.
Holt N, Wang J, Kim K, Friedman G, Wang X, Taupin V, Crooks GM, Kohn DB, Gregory PD, Holmes MC, Cannon PM.
Nat Biotechnol. 2010 Aug; 28(8):839-47.

Generation of HIV-1 Resistant and Functional Macrophages From Hematopoietic Stem Cell-derived Induced Pluripotent Stem Cells.
Kambal A, Mitchell G, Cary W, Gruenloh W, Jung Y, Kalomoiris S, Nacey C, McGee J, Lindsey M, Fury B, Bauer G, Nolta JA, Anderson JS.
Mol Ther. 2010 Nov 30.

CCR5 deficiency increases risk of symptomatic West Nile virus infection.
Glass WG, McDermott DH, Lim JK, Lekhong S, Yu SF, Frank WA, Pape J, Cheshier RC, Murphy PM.
J Exp Med. 2006 Jan 23; 203(1):35-40.

Also See

Can HIV be cured with stem cell therapy?
Deeks SG, McCune JM.
Nat Biotechnol. 2010 Aug; 28(8):807-10