Despite major advances in anti-retroviral therapy (ART), HIV-1 infection remains an epidemic cause of morbidity and mortality. Effective ART often involves costly, multi-drug regimens that are not well tolerated by a significant percentage of patients (42
), and even successful adherence to ART does not eradicate the virus, so that a rapid rebound in HIV-1 levels can occur if therapy is discontinued (43
). Consequently, an alternate approach to controlling HIV-1 replication that is being considered is the engineering of the body’s immune cells to be resistant to infection by the virus (44
). In this regard, the CCR5 co-receptor is a particularly attractive target because of the HIV-resistant phenotype of homozygous CCR5Δ32 individuals (3
), who tolerate this genotype with minimal consequences (5
). In the present study, we identified conditions that allowed the efficient disruption of the CCR5 gene in human CD34+ HSC, and further demonstrated that such modified HSC were able to generate CCR5-negative, HIV-resistant progeny in a mouse model of human hematopoiesis and HIV-1 infection, ultimately leading to the profound suppression of HIV-1 replication in vivo
. These findings suggest that modification of a patient’s own HSC by CCR5-specific ZFNs could provide a permanent supply of HIV-resistant progeny, able to replace cells killed by HIV-1, and leading to immune reconstitution and the long-term control of virus replication in the absence of anti-retroviral drugs.
The high levels of CCR5 disruption that we achieved in human HSC were possible because of an efficient gene editing technology based on zinc finger nucleases. ZFNs can be designed to bind to a specific genomic DNA sequence and, by generating a targeted DSB, can affect the efficient, specific and permanent knockout of the targeted gene (19
). In contrast to other genetic approaches to the control of HIV-1, ZFN-mediated gene disruption is a one-time, “hit-and-run” process that requires only transient expression of the ZFNs in HSC during a brief period of ex vivo
culture. Long-term expression of a therapeutic transgene is not required and, once established, the genetic mutation is present for the life of the cell and its progeny. In this way a major shortcoming of other gene therapy technologies, the need for continued expression of a foreign transgene, is avoided. Moreover, unlike small molecule, antibody, or RNAi-based treatments (44
), ZFN treatment results in disruption of the CCR5 open-reading frame and thus has the potential to completely eliminate this important HIV-1 co-receptor from the surface of cells that are bi-allelically modified. By using an optimized nucleofection procedure, we were able to overcome the technical challenges to ZFN-induced genome editing in HSC that have previously been reported (21
) and achieve, on average, disruption at 17% of the loci.
The safety and efficacy of CCR5-targeted ZFNs are currently being evaluated in a Phase I clinical trial using T lymphocytes. Pre-clinical studies included an extensive investigation into the specificity of these ZFNs for the CCR5 locus. Off-target events were only observed at significant levels at the homologous CCR2 locus (19
). Mouse studies have not found any deleterious phenotype associated with loss of CCR2 (48
), and human genetic studies have even suggested a beneficial phenotype from the loss of this gene in HIV-infected individuals (49
). Although not specifically analyzed in our study, it is likely that the use of these same CCR5 ZFN reagents in HSC will result in similar, low levels of off-target events, the consequences of which will need to be evaluated in a larger study.
Although T lymphocytes are the primary target of HIV-1 infection, HSC may represent a more attractive cell for ZFN engineering since their modification could allow the long-term production of CCR5-negative cells in a patient. The scientific rationale for CCR5 modification of HSC is strong and is supported by the recent finding that an AIDS leukemia patient undergoing HSC transplantation from a CCR5-negative donor was effectively cured of his infection, with undetectable levels of HIV-1 and rebounds in CD4 T cell levels despite discontinuing ART (9
). As shown by our data, ZFN-modified HSCs retain full functionality and give rise to CCR5-negative cells in lineages relevant to HIV-1 pathogenesis, such as T cells and monocyte/macrophages. ZFNs delivered to purified CD34+ cell populations by nucleofection were clearly capable of modifying true SCID-repopulating stem cells, and the high levels of CCR5 editing that we observed in the progeny of engrafted HSC were also maintained following secondary transplantations. Thus, the technology appears well suited to this application.
The experimental mouse model of HIV-1 infection used in these studies revealed a strong selection for CCR5-negative progeny during acute infection with a CCR5-tropic strain of HIV-1. This suggests that homozygous CCR5-negative stem cells, even if the minority, produce sufficient numbers of CCR5-negative progeny to ultimately support immune reconstitution and inhibit HIV-1 replication. Such selection is consistent with clinical observations from genetic diseases such as ADA-SCID, X-linked SCID and Wiskott-Aldrich syndrome, where normal hematopoietic cells have a selective advantage, so that spontaneous monoclonal reversions that have occurred in a small number of patients can lead to the selective outgrowth of these cells and amelioration of symptoms (50
The observation of almost complete replacement of human T cells in the intestines of infected mice with CCR5-negative cells is consistent with this tissue harboring the majority of the body’s CD4+CCR5+ effector memory cells. A characteristic feature of HIV-1 replication in mucosal tissues is an ongoing cycle of T cell death and the accompanying recruitment of replacement T cells, whose activated state means that they are also highly permissive targets for HIV-1 infection (37
). This is especially true in the GALT, which represents a key battleground in HIV-1 infection (54
). We also noted a strong selective pressure for CCR5-negative cells at the level of the thymus. This suggests that T cells would be selected to be CCR5-negative at both a precursor stage in the thymus, as well as the effector CD4+ stage in the mucosa. Ultimately, the presence of HIV-resistant CCR5-negative cells in mucosal tissues would both protect individual cells from infection, and also help to break the cycle of immune hyperactivation that may underlie much of the pathology of AIDS (57
Does ZFN modification of HSC present a reasonable treatment option? Although ART is clearly highly effective in many patients, the associated costs and potential for side effects can be considerable when extrapolated over the lifetime of a patient. In contrast, a ZFN-engineered HSC therapy could potentially provide a one-shot treatment that would be most suited to the setting of autologous HSC transplantation. Procedures for the phenotypic isolation and processing of HSC for autologous or allogeneic transplantation are well established. The use of a patient’s own stem cells may remove the requirement for full ablation of the marrow hematopoietic compartment, and the immune suppression that is necessary for allogeneic matched donor bone marrow transplantations. Indeed, the accompanying toxicity of such regimens is one of the reasons why allogeneic stem cell transplantations from CCR5Δ32 individuals will not be a feasible treatment option for HIV patients in the absence of other conditions that would necessitate the transplant.
Of note, certain HIV-infected patient populations, such as AIDS lymphoma patients, already undergo full ablation and autologous HSC rescue as part of their therapy (58
), and thereby present an opportunity for HSC-based gene therapies (44
). In addition, the experience of autologous HSC transplantation in gene therapy for patients with genetic diseases such as ADA-SCID (59
), chronic granulomatous disease (61
) and X-linked adrenoleukodystrophy (62
), is that non-myeloablative conditioning can be used to facilitate engraftment of gene-modified autologous HSC with minimal associated toxicity. It is possible that the use of non-myeloablative regimens, together with the selective advantage conferred on CCR5-negative progeny, could prove an effective combination for HIV-1 patients receiving ZFN-treated autologous HSC.
Targeting the CCR5 gene is not expected to provide protection against viruses that use alternate co-receptors such as CXCR4. However only a handful of cases of HIV-1 infection of CCR5Δ32 homozygotes have been reported (63
). CXCR4-tropic viruses have been associated with accelerated disease progression (65
), so that selection for such strains could be an undesirable consequence of targeting CCR5. However, this outcome is not generally observed in patients treated with CCR5 inhibitors unless pre-existing CXCR4-tropic viruses were present before therapy, and resistance to the drugs can occur, instead, because of viral adaptations to the drug-bound form of the CCR5 co-receptor (66
). Interestingly, the patient receiving the CCR5Δ32 HSC transplantation harbored CXCR4-utilizing viruses before the procedure, yet he did not experience a tropism switch, and instead, successfully controlled his HIV-1 infection long-term (9
). For the present, similar to the recommendations for the use of CCR5 inhibitor drugs, it may be prudent to restrict CCR5 ZFN treatment of HSC to individuals with no detectable CXCR4-tropic viruses.
In contrast to the acute HIV-1 infection modeled in this study, HIV-1 patients usually present in a chronic phase of the disease, and will frequently have virus levels that are effectively controlled by ART. The requirement for the selective pressure of active HIV-1 replication in the success of this, or other anti-HIV gene therapies is presently unknown. It has been suggested that low level virus replication continues in certain sanctuary sites, even in well-controlled patients on ART (43
), and this could provide a low level of selection, although drug intensification trials have not provided evidence of any such ongoing HIV-1 replication (69
). It is also possible that the high levels of CCR5 disruption we achieved without selection, if extrapolated to patients, could be sufficient to provide a therapeutic effect even in the absence of a strong selective pressure. Alternatively, ZFN knockout of CCR5 in HSC could be viewed as a backup strategy, expected to play an increasingly important role in controlling HIV-1 if ART fails or is withdrawn. It may also be possible to incorporate ART treatment interruptions into an overall therapeutic strategy, as recently described for HIV-infected individuals receiving autologous HSC engineered with anti-HIV ribozymes, where gene marked progeny were found at higher levels in patients who underwent treatment interruptions (70
In summary, these data demonstrate for the first time that transient ZFN treatment of human CD34+ HSC can result in efficient levels of gene disruption, while yielding cells that remain competent to engraft and support hematopoiesis. In the presence of CCR5-tropic HIV-1, CCR5-negative progeny rapidly replaced cells depleted by the virus, leading to a polyclonal population that ultimately preserved human immune cells in multiple tissues. Our findings indicate that the modification of only a minority of human HSC was sufficient to provide the same strong anti-viral benefit as was conferred by a complete CCR5Δ32 stem cell transplantation in a patient (9
). This suggests that a partially modified autologous transplant, administered under only mildly ablative transplantation regimens, may also be effective, opening up the treatment to many more HIV-infected individuals. Finally, the identification of conditions that allow the efficient use of ZFNs in human HSC suggests the use of this technology in other diseases for which HSC modification may be curative.