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. Author manuscript; available in PMC: 2013 Feb 3.
Published in final edited form as: Cell Stem Cell. 2012 Feb 3;10(2):137–147. doi: 10.1016/j.stem.2011.12.015

Hematopoietic stem cell-based gene therapy for HIV disease

Hans-Peter Kiem 1, Keith R Jerome 1, Steven G Deeks 2, Joseph M McCune 3
PMCID: PMC3274769  NIHMSID: NIHMS346725  PMID: 22305563

Abstract

Although combination antiretroviral therapy can dramatically reduce the circulating viral load in those infected with HIV, replication-competent virus persists. To eliminate the need for indefinite treatment, there is growing interest in creating a functional HIV-resistant immune system through the use of gene-modified hematopoietic stem cells (HSC). Proof-of-concept for this approach has been provided in the instance of an HIV-infected adult transplanted with allogeneic stem cells from a donor lacking the HIV co-receptor, CCR5. Here, we review this and other strategies for HSC-based gene therapy for HIV disease.

BACKGROUND

HIV infection of CD4+ T cells leads to their death through mechanisms that are both direct (e.g., cytotoxicity) and indirect (e.g., activation-induced cell death). After years of heightened T cell turnover, the capacity of the immune system to maintain normal homeostasis is depleted and patients progress to advanced immunodeficiency. The well-described morbidity and mortality associated with untreated HIV infection is readily attributed to failed immunity, with deficits in most hematopoietic lineages causally associated with disease (McCune, 2001) Effective suppression of HIV replication using combination antiretroviral therapy essentially reverses the process of immune depletion and often results in partial restoration of immune function, improved health, and prolonged life.

Although antiretroviral therapy for HIV disease is an unquestioned success, it does have a number of limitations. First, therapy does not fully restore health. Chronic inflammation and immune dysfunction often persist indefinitely during treatment, and these factors have been associated with increased risk of non-AIDS morbidity and mortality (Deeks, 2011; Kuller et al., 2008). Second, antiretroviral therapy may not be fully suppressive. There is emerging evidence that cryptic virus replication persists within dispersed hematolymphoid organs (Yukl et al., 2010), with potentially significant effects on T cell and myeloid cell homeostasis and function. Third, combination therapy requires daily adherence to regimens that often have side effects and complex drug-drug interactions, and many individuals are unable to adhere to such regimens indefinitely. Finally, resource limitations deny the prospect of life-long therapy to many individuals who need it most. Even with the massive global investment in HIV care, access to these drugs will remain incomplete and the epidemic will continue to spread.

Given the well-recognized limitations of current therapeutic approaches, there is growing interest in developing potentially curative approaches. An ideal therapeutic cure would be one that is safe, scalable, administered for a limited period of time, and prevents infection of all susceptible cells, including cells in tissues with limited bioavailability for antiretroviral drugs. To reach this goal, it has been suggested (Baltimore, 1988; Gilboa and Smith, 1994; Yu et al., 1994) that long-lived, self-renewing, multilineage hematopoietic stem cells (HSCs) could be modified such that both they and their progeny can resist HIV infection. After introduction of these modified HSCs, the host could be repopulated with an HIV-resistant hematopoietic system, including CD4+ T cells and myeloid targets. If such a system can be created, a lifelong cure would have been achieved.

To realize the goal of HSC-based gene therapy for HIV disease, the following steps must be taken (Figure 1): HSCs must be identified and purified (and/or expanded) in numbers sufficient to provide a benefit in both adults and children; methods must be devised to efficiently and stably introduce novel gene functions into HSCs; the selected gene functions must be shown to confer HIV-resistance in progeny T cells and myeloid cells; the gene-modified cells must be introduced into the patient safely and efficiently; and clinical trials must be designed to convincingly demonstrate efficacy. This review will address each of these steps in turn and conclude with additional thoughts about the worldwide dissemination and implementation of curative therapies for HIV.

Figure 1. Intracellular immunization with gene-modified hematopoietic stem cells.

Figure 1

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Long-lived, self-renewing, multilineage hematopoietic stem cells (HSCs) could be modified such that they and their progeny resist HIV infection. The host could thereafter be repopulated with a hematopoietic system (including CD4+ T and myeloid targets for HIV) that is resistant to the replication and spread of HIV.

IDENTIFICATION AND EXPANSION OF HSCs

Characterization of HSCs

A critical obstacle confronting the identification of human HSCs was the lack of suitable assays available to test the multilineage potential of candidate cells. The gold standard method to identify a stem cell is an in vivo assay in which a particular cell or cell type can be shown to repopulate and reconstitute the entire hematopoietic system after myeloablative and otherwise lethal conditioning. Ethical concerns obviously make this impossible to test in humans. A significant advance to this field was provided by the development of mouse models allowing the engraftment and multilineage differentiation of human hematopoietic progenitor cells (Bhatia et al., 1998; Guenechea et al., 2001; Kaneshima et al., 1990; Lapidot et al., 1992; Larochelle et al., 1996; McCune et al., 1988; McCune et al., 1991; Namikawa et al., 1990). A critical limitation of this approach is the inability to test the effect of the conditioning regimens on engraftment and to evaluate the long-term generation of all lineages. Accordingly, large animal models (e.g., using monkeys and dogs) were used to study HSC biology and transplantation, and studies in the early 1990s demonstrated that marrow cells can be enriched for subpopulations that possess long-term repopulating capabilities (Berenson et al., 1988). These studies used the marker CD34, which is still used today if one wishes to perform “stem cell” enrichment or selection in patients or T cell depletion. Although there are many other markers [(e.g., functional assays to identify Rholow cells (McKenzie et al., 2007) or attention to so-called “side populations” (Goodell et al., 1997)], these methods have not advanced into routine clinical use for HSC selection or transplantation. Even if more purified populations of stem cells can be obtained with novel markers, the number of such cells that can be routinely obtained may be insufficient to support rapid engraftment and expansion in vivo.

At present, it is the general consensus that “true” self-renewing human HSCs are found within a CD34+ population and that engraftment of a suitably conditioned host with a sufficient number of such cells will result in long-term multilineage hematopoiesis. To obtain the requisite number of purified cells, sophisticated methods for clinical grade, high speed cell sorting have been developed. Once this technology is refined and approaches are developed to expand such cells ex vivo, it is likely that defined hematopoietic stem and/or progenitor cell populations can be targeted for genetic modification and used to treat non-fatal conditions such as treated HIV disease.

Expansion of HSCs

Over the past 20 years, numerous efforts have been made to expand HSCs in vitro so they will be more readily accessible for use in vivo. Initial studies relied on hematopoietic growth factors such as G-CSF, SCF, Flt3, IL-3, IL-6, and thrombopoietin (McNiece et al., 2000) but these approaches failed to result in substantial HSC expansion. Since then, a number of other factors have been studied: HOXB4 and NUP98 (Gurevich et al., 2004; Krosl et al., 2003; Sauvageau et al., 2004), Notch ligands (Delaney et al., 2010), WNT (Reya et al., 2003), pleiotrophin (Himburg et al., 2010), and prostaglandin (North et al., 2009; North et al., 2007). The use of angiopoietin-like 5 and insulin-like growth factor has also been reported to provide some benefit for HSC expansion in vitro (Zhang et al., 2008). Probably the most successful expansion reagent to be identified has been the purine derivative StemRegenin 1 (SR1), which promotes the ex vivo expansion of CD34+ cells (Boitano et al., 2010), and is reportedly responsible for a 50-fold increase in the number of CD34+ cells obtained in culture and a 17-fold increase in the number of cells able to engraft in NSG mice. The ability of these expanded HSCs to engraft in humans is unknown.

Combinations of these different pathways are likely needed to optimize the procedures for expanding multipotential HSCs (Watts et al., 2010). Ongoing studies of Notch pathway modulation using Delta-1 and SR1 are being pursued in a clinical setting, primarily in allogeneic transplantation studies. These studies are unlikely to determine true expansion of long-term repopulating cells, but rather will evaluate the contribution to short-term reconstitution. Although progress in identifying a safe and effective combination regimen for expanding HSCs has been limited, there is intense interest in this area in both industry and academia. It is expected that over the next several years an approach to readily harvest HSCs from humans and expand them ex vivo will be available.

Expansion of HSCs ex vivo may be particularly important for HIV disease. For reasons that have yet to be fully defined but that likely include HIV-mediated effects on important hematolymphoid microenvironments, the number of hematopoietic progenitor cells from HIV-infected patients to support immune reconstitution may be diminished (Jenkins et al., 1998) (McCune, 2001). Recent work by Appay and colleagues has also demonstrated that CD34+ progenitor cells obtained from the peripheral blood of such patients have reduced capacity to generate mature T cells (Sauce et al., 2011). This effect of HIV on stem cell function is comparable to that observed in advanced aging and appears to be due, at least in part, to the indirect effects of chronic inflammation.

INTRODUCTION OF DE NOVO GENE FUNCTIONS INTO HSCs

Assuming that a sufficient number of the appropriate HSC population can be obtained, it will be necessary to find ways to stably and efficiently introduce novel gene functions into such cells. Two general approaches have been taken to achieve this end: the use of integrating vector systems that permit the introduction of anti-HIV genes into the genome of HSCs and non-integrating vector systems that introduce gene-modifying enzymes to effect gene disruptions or homologous recombination.

Integrating vector systems

Much progress has been made in optimizing ex vivo transduction, and there are a number of vector systems available that allow for efficient and stable gene delivery to HSCs. The introduction of multiple hematopoietic cytokines and the RetroNectin fragment (a recombinant human fibronectin fragment) have successfully facilitated substantial flexibility in the genetic manipulation of HSCs (Hanenberg et al., 1996; Kiem et al., 1998). In addition, the identification of appropriate viral pseudotypes (e.g., GALV and RD114) has improved gene transfer efficiencies. These vectors may also allow for improved HSC maintenance (Horn et al., 2002; Kiem et al., 1998; Neff et al., 2004; von Kalle et al., 1994). Recent protocols have focused on the use of safety-modified, HIV-derived lentivirus vectors (Naldini et al., 1996; Zufferey et al., 1998; Zufferey et al., 1997), an approach that allows for the generation of high-titer vectors and efficient gene transfer to hematopoietic stem/progenitor cells. Other systems have also been evaluated, such as foamy virus vectors and different VSV-G pseudotypes (Kiem et al., 2007; Trobridge et al., 2009).

Because the vector systems can be associated with genotoxicity, and hence a risk of malignancy (Baum et al., 2011; Fischer et al., 2010), the main focus of optimization for integrating vector systems has switched to safety. Unless gene knock-outs or knock-ins are performed, integrating vectors will be required to obtain lifelong expression of the anti-HIV constructs. Given that most HIV-infected adults have a good prognosis (assuming they have access to effective therapy), there will be limited interest in any vector system that has an appreciable risk of malignant transformation. Thus, gamma-retroviral vectors are unlikely to be considered in large-scale clinical trials as these have been associated with high risk of leukemia in prior transplant protocols. Fortunately, improved vector systems have been developed and evaluated, and the currently used lentivirus and foamy virus vectors appear to have limited risk for malignant transformation (Hacein-Bey-Abina et al., 2008; Modlich et al., 2009; Zhou et al., 2010). The self-inactivating (SIN) lentivirus and foamy virus vector systems are capable of integration but have a nonfunctioning LTR in the integrated provirus and rely on a weaker promoter element for expression of the transgene (Figure 2). The removal of the strong LTR promoter reduces the potential for insertional activation of nearby genes (Zufferey et al., 1998). In addition to the SIN configuration, lentivirus and foamy virus vectors have a more favorable integration site pattern compared with gamma-retroviral vectors.

Figure 2. Basic design of a SIN configured lentivirus vector.

Figure 2

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A. A traditional retroviral vector with two functional long terminal repeats (LTRs) that contain strong enhancer and promoter elements. Integration of this vector can lead to activation of nearby proto-oncogenes and thus leukemia. B. A self-inactivating (SIN) lentivirus vector design. The 3′ LTR is modified (delta) so it does not contain any transcriptional control elements. Thus, upon transduction and integration into the genome, both 5′ and 3′ LTRs are defective and will not contain any functional promoter or enhancer elements. A weaker internal promoter (e.g., PGK) can then be inserted to drive expression of the transgene and thus decrease the risk of activating any nearby proto-oncogenes.

Non-integrating vector systems or transfection

An alternative transfection approach is to utilize viral vectors that have been modified so they are unable to integrate into the host genome (Yanez-Munoz et al., 2006). A significant advantage of non-integrating vector systems is that they circumvent the risk of insertional mutagenesis and resultant malignancy. This can be done quite efficiently using integrase-deficient lentivirus vectors or adenovirus vectors (Gabriel et al., 2011). Holt et al., 2010; Lombardo et al., 2011). As discussed below, non-integrating vector systems are especially well suited for delivery of zinc finger nucleases or other DNA-editing enzymes that can induce permanent knockout of specific genetic loci after only transient expression. However, and as with all new reagents, more safety studies are needed to confirm the lack of off-target effects and genotoxicity (Mussolino and Cathomen, 2011).

SELECTION OF GENE FUNCTIONS TO CONFER RESISTANCE TO HIV

Approaches aimed at modifying HSCs to treat HIV disease can be grouped into two main themes: targeted disruption of cellular genes involved in HIV entry, such as the CCR5 co-receptor, and the introduction of genes that interfere with HIV replication, such as fusion inhibitors or host restriction factors (Figure 3).

Figure 3. Approaches to modifying hematopoietic stem cells for HIV resistance.

Figure 3

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Both transient and permanent CCR5 suppression can be achieved using non-integrating delivery vectors. By contrast, durable expression of HIV resistance elements will likely require integrating delivery vectors. While concerns remain about the possibility of insertional mutagenesis (as observed previously with gamma-retrovirus vectors), the safety of integrating approaches has improved greatly in recent years with the development of lentivirus and foamy virus vectors. Heavier arrows depict desired outcomes; lighter arrows depict less desirable outcomes.

Targeted gene knockdown or knockout

CCR5 is a G protein-coupled chemokine receptor that also functions as a critical co-receptor during entry of “R5” strains of HIV. Given redundancies in the immune system, CCR5 is not critical for normal immune function and certain healthy individuals carry a mutation in the CCR5 gene that prevents expression of a functional protein. This CCR5-Δ32 (Δ32) mutation is present in 5–14% of individuals of Europeans but is rare in persons of African or Asian descent. HIV disease progresses more slowly in individuals with a single copy of the Δ32 mutation (de Roda Husman et al., 1997), while homozygous individuals are largely resistant to HIV infection (Liu et al., 1996; Samson et al., 1996). Thus, CCR5 has been an attractive antiviral target, and the successful development of maraviroc, an allosteric, non-competitive inhibitor of CCR5 has proven that inhibition of this receptor effectively inhibits HIV replication in a safe and durable manner (Gilliam et al., 2011).

Because of its proven clinical importance, CCR5 has been intensely studied as a target for stem cell therapies. Several approaches have been taken to inhibit functional expression of CCR5, including the introduction of siRNA (Kim et al., 2010; Shimizu et al., 2010), ribozymes (DiGiusto et al., 2010; Feng et al., 2000), trans-dominant mutant forms of CCR5 (Luis Abad et al., 2003), and single chain intracellular antibodies (Steinberger et al., 2000; Swan et al., 2006). However, all of these approaches are limited by the transient nature of the disruption. Given the ability of HSCs to repopulate the peripheral immune system, it will be important to devise approaches that instead allow for durable or permanent elimination of CCR5.

The potential efficacy of permanent CCR5 elimination was dramatically illustrated by the so-called “Berlin patient,” the only documented example of an apparent cure of HIV infection (Allers et al., 2011; Hutter et al., 2009). In this case, an HIV-positive patient with acute myeloid leukemia received an allogeneic stem cell transplantation using cells from a donor homozygous for the CCR5-Δ32 mutation. The patient efficiently engrafted with CCR5-null cells and, as of this report four years later, remains free of readily detectable circulating virus, even in the absence of antiretroviral therapy (Allers et al., 2011). Unfortunately, allogeneic stem cell transplantation has significant associated morbidities, limiting the widespread application of this approach beyond patients with AIDS-associated malignancies. Furthermore, because CCR5−/− stem cell donors are rare (particularly among non-European ethnic groups) and must still be HLA-matched with prospective HCST recipients, fewer than 1% of AIDS patients would likely be eligible for this treatment (Hutter and Thiel, 2011). Thus, there is a critical need for a strategy to create autologous CCR5−/− stem cells and bypass each of these problems.

The recent emergence of DNA editing proteins, including zinc finger nucleases (Urnov et al., 2005), TAL effector nucleases (Bogdanove and Voytas, 2011), and homing endonucleases (Stoddard, 2011), has created the possibility that any genetic locus can be specifically and permanently inactivated. This approach can be applied to CCR5 in any cell type, including a patient’s own HSCs. Initial studies used zinc finger nucleases to disrupt CCR5 in primary human T cells (Perez et al., 2008). In subsequent work, zinc fingers were used to disrupt CCR5 in human hematopoietic stem cells, which were then tested in a humanized mouse model of HIV infection (Holt et al., 2010). Disruption occurred in 17% of CCR5 alleles, resulting in HSCs with either heterozygous or homozygous disruptions. The modified HSCs were able to support multilineage hematopoiesis in the mice. After subsequent HIV challenge, the CCR5-disrupted progeny cells had a selective advantage over the unmodified cells. Compared with control animals, those receiving modified cells had lower HIV loads and higher numbers of human cells. Based on these promising results, clinical trials of zinc finger nucleases targeting CCR5 in peripheral T cells are currently underway.

A potential concern of CCR5-directed therapies is that they will drive selection of so-called “X4” viruses with tropism for CXCR4, the other chemokine co-receptor utilized by HIV for binding and entry into target cells. Although there has been interest in simultaneously disrupting CXCR4 and CCR5, CXCR4 is widely expressed on many cell types throughout the body and is critical for multiple physiologic processes, including B cell, cardiovascular, and cerebellar development. Therefore, CXCR4 may not be a suitable target for disruption in pluripotent stem cells. By contrast, CXCR4-null mice have normal T cell development (Nagasawa et al., 1996) and it may be possible to successfully knock out CXCR4 in T-lineage restricted progenitor cells. An initial study of zinc finger-mediated disruption of CXCR4 in a humanized mouse model of HIV infection showed that this approach was well tolerated and provided resistance to CXCR4-tropic virus (Wilen et al., 2011). Nevertheless, the safety of CXCR4 disruption in humans remains a major concern.

Introduction of genes that interfere with HIV replication

An alternative strategy of HSC modification for HIV therapy involves inserting new genetic elements that can inhibit critical processes such as viral entry or replication (see Figure 4 and Table 1). For instance, a gp41-derived peptide (C46) that is structurally similar to the FDA-approved fusion inhibitor enfuvirtide can effectively inhibit HIV entry. An initial clinical trial of C46-expressing, modified autologous T cells in ten patients showed the therapy was well tolerated (van Lunzen et al., 2007). The addition of C46 into hematopoietic stem cells was also evaluated in a macaque model of HIV infection (Trobridge et al., 2008); marking efficiencies were 4–7% in peripheral T cells, and the modified T cells were protected from subsequent HIV challenge. Of note, resistance to enfuvirtide occurs rapidly when it is used in the absence of a fully suppressive antiretroviral regimen, raising the possibility that C46-based HSC therapy might also be associated with the development of viral resistance. However, the genetic barrier for developing C46 resistance may be higher than for enfuvirtide resistance. Resolution of this issue will ultimately require experimental evaluation in vivo.

Figure 4. The replicative cycle of HIV.

Figure 4

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Two major strains of HIV-1 (R5 and X4) bind to target cells by concerted interactions between the envelope protein (Gp120) and CD4, and the chemokine co-receptors, CCR5 and CXCR4, respectively, leading to a fusion event with the plasma membrane that allows for entry of the virion capsid into the cytoplasm. Reverse transcription of viral genomic RNA forms a series of replicative intermediates that may ultimately integrate into the host cell genome. Transcription and generation of spliced and un-spliced forms of the viral RNA allows for movement and packaging of the diploid viral genome in the cytoplasm, a step enabled in part by HIV-1 protease. Budding and release of new viral progeny for repeated rounds of infection is then facilitated by virally-encoded release and infectivity factors. Each of these steps can be (or might be) disabled by specific drug and/or gene therapy.

Table 1.

Mechanism Drug Treatment Gene Therapy/Gene Disruption
Gp120 binding inhibitors None
Co-receptor binding inhibitors Maraviroc CCR5 KO
CXCR4 KO
Fusion inhibitors Enfuvirtide C46
Reverse transcriptase Inhibitors Abacavir
Didanosine
Emtricitabine
Lamivudine
Stavudine
Tenofovir
Zidovudine
Efavirenz
Etravirine
Nevirapine
Rilpivirine
Integrase inhibitors Raltegravir
Dolutegravir
Genome disruption Evolved recombinases, endonucleases
Gene expression inhibitors TAR decoys, anti-TAT ribozymes, siRNA, shRNA
RNA export inhibitors Trans-dominant
Rev (RevM10)
Protease inhibitors Atazanavir
Amprenavir
Darunavir
Indinavir
Lopinavir/ritonavir
Nelfinavir
Saquinavir
Tipranavir
Viral release or infectivity inhibitors TRIM5α3, APOBECs, Tetherin
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Exogenous elements can also be used to interfere with other essential viral processes. In stem cells, the key HIV regulatory protein Rev has been targeted using dominant mutant or trans-dominant forms of Rev (Bonyhadi et al., 1997; Kang et al., 2002; Podsakoff et al., 2005; Su et al., 1997). HIV Tat and its overlapping genes have also been targeted in stem cells using hammerhead ribozymes, catalytically active RNA structures that target critical gene regions (Amado et al., 2004; Mitsuyasu et al., 2009). TAR decoys have been used to inhibit viral replication in stem cells by binding and sequestering the viral transactivator TAT (Banerjea et al., 2004). Both shRNA and siRNA have been used to suppress expression of many HIV genes (Rossi et al., 2007), although their transient effects may limit their application in stem cells. Finally, the integrated viral DNA itself has been directly targeted using evolved recombinases (Sarkar et al., 2007) or homing endonucleases (Aubert et al., 2011), and introduction of genes encoding such proteins could potentially protect stem cells from viral infection and replication.

Unlike their human homologs, certain primate host restriction factors, such as TRIM5α, APOBEC 3F and 3G, and tetherin, can prevent HIV infection of cells and represent other potential candidates for gene therapy. The anti-HIV effect of the primate molecules often results from sequence variation compared to their human homologs, making immunogenicity of the transgene a potential limitation. In humanized mouse models, both a human-rhesus chimeric TRIM5α (Anderson and Akkina, 2008) and a human TRIM-cyclophilin A fusion (Neagu et al., 2009) were well tolerated and protected cells from HIV challenge, supporting the promise of this approach.

Despite these encouraging results, the addition of exogenous anti-HIV genetic elements into HSC raises concerns. Insertions of genes with their own promoters may generate risks from long-term expression or insertional activation of nearby genes (Burnett and Rossi, 2009; Li et al., 2005; Tiemann and Rossi, 2009). The expressed proteins may also be immunogenic in the recipient, limiting the expansion or lifespan of modified cells. By contrast, permanent disruption of CCR5 should be possible using non-integrating delivery methods; thus, this strategy is likely to be the safest and most appealing strategy for clinical settings. Nevertheless, in preclinical studies, combinations of CCR5 knockdowns and other pathways that interfere with HIV entry or replication should be pursued to identify the most effective approach.

Will combination therapy be needed?

Once HIV disease has reached the chronic phase, the set of viral quasispecies within an infected individual is very diverse. In addition, variants that are naturally resistant to any given antiviral agent may pre-exist in that population, which explains why potent antiretroviral drugs often have only a transient effect on HIV replication when given as monotherapy. Based on the extensive experience with standard small-molecule antiretroviral drugs, the ideal stem cell-based intervention would require the virus to develop multiple mutations to become resistant. For example, a combination of two or more antiviral genes applied simultaneously could provide a sufficient genetic barrier to prevent the emergence of resistance. Efforts are already underway to develop such combination approaches. One interesting recent approach used a combination of a tat/rev shRNA, a TAR decoy, and a CCR5-targeting ribozyme to modify CD34+ stem cells in patients undergoing transplantation for HIV-associated lymphoma (DiGiusto et al., 2010). While no clinical benefit was observed given the low frequency of modified cells in this study (<0.2% of circulating PBMC), the procedure was well tolerated and modified cells persisted for at least 24 months. Improved approaches providing a higher percentage of gene-modified cells should provide durable effects and will also allow the determination of the minimal marking efficiency required for clinical benefit and the relative benefits of various therapeutic gene combinations.

Another issue distinguishing gene therapy from standard antiretroviral therapy pertains to the possible role of partial viral suppression during the early phases of treatment. If only a partially ablative conditioning regimen is used, then by definition susceptible T cell or myeloid cell targets will be available post-transplant. In the absence of antiviral therapy, the virus would be able to replicate, albeit in a more constrained manner. Natural selection due to HIV-mediated death of susceptible cells might be expected to result in a shift from a predominantly HIV-susceptible CD4+ target population to a predominantly HIV-resistant population. However, during this process, a virus population that is resistant to the targeted gene function(s) could emerge, resulting in failure of the therapy. Therefore, it will be important to determine whether a period of antiviral suppression during engraftment will be advantageous to stem cell approaches and, if so, the optimal duration of therapy.

IN VIVO ENGRAFTMENT OF TRANSDUCED HSCs

A recent publication by Holt et al. (Holt et al., 2010) has demonstrated the feasibility of targeting the CCR5 locus in NSG-repopulating cells. The authors demonstrated successful engraftment of gene-edited CD34+ cells in the NSG mouse model and subsequent protection of these mice from HIV infection. The average frequency of disruption was 17%, with an estimated bi-allelic modification of approximately 5–7%. After irradiation with 150 cGy, nearly 40% of CD45 cells engrafted. The authors also showed engraftment in secondary recipients with comparable levels of gene-edited cells. Although these data are promising, it is important to note that these animals received gene-modified cells prior to HIV infection, which is obviously of limited relevance to what might eventually occur in humans.

The rate at which HIV-resistant cells were selected in this experiment was impressive: HIV infection in this model causes rapid CD4+ T cell depletion and complete engraftment with CCR5−/− cells was evident by week 12. Given the pace of disease progression in untreated HIV infection (on the order of years rather than weeks), it is highly unlikely that such rapid replacement will occur in humans, but a focused clinical trial using this approach will be required to determine if this is the case.

PRE-TRANSPLANT CONDITIONING REGIMENS

Although the animal studies described above clearly demonstrate the feasibility and efficacy of transplanting HIV-resistant HSCs, a major question remains: how can one achieve similar engraftment in people infected with HIV? From large animal studies, we know that HSC engraftment without conditioning is likely to be very low, especially given the limited cell numbers available for humans or large animals.

A variety of conditioning approaches have been studied in nonhuman primate models, which allows for careful analysis of the level of engraftment necessary to provide protection and the most effective conditioning regimens for achieving this level. Many conditioning regimens (e.g., the use of cyclophosphamide) (Storb et al., 1970) were first tested in nonhuman primates, as were the high-dose irradiation regimens now commonly used for transplantation or stem cell gene therapy studies (Horn et al., 2002; Trobridge et al., 2008). Other advantages of nonhuman primate studies include the ability to carefully and comprehensively analyze latent virus reservoirs by analyzing peripheral blood apheresis products, GALT biopsies, and even spinal fluid for the presence of integrated HIV, as is done for human patients, and the possibility of studying the use of immunosuppressive agents, such as anti-thymocyte globulin, similar to those used in the case of the “Berlin patient.” Finally, effective HAART treatment regimens are becoming better established in monkeys, allowing for the study of structured treatment interruptions. Thus, while the NSG mouse model will facilitate rapid and efficient evaluation of the function of gene-modified or edited cells, nonhuman primate models will be important for determining the conditions necessary to achieve sufficient engraftment for long-term protection. Over the next several years, it is hoped that effective and reasonably safe conditioning regimens will be developed using nonhuman primate models.

If HSCs can be expanded ex vivo, alternative and better-tolerated conditioning regimens could be considered or conditioning regimens might be avoided altogether. This has been nicely demonstrated in mouse models, where higher doses of marrow cells were able to successfully compete with endogenous HSCs (Quesenberry et al., 1994; Stewart et al., 1993a; Stewart et al., 1993b; Stewart et al., 1998). An alternative strategy to optimize engraftment has been proposed by Mazurier et al.(Mazurier et al., 2003), who reported improved engraftment of myeloerythroid cells when injected directly into the bone marrow as opposed to intravenous injection. A number of investigators have since pursued this approach, and studies in nonhuman primates also suggested a differential engraftment pattern (Jung et al., 2007).

Progress has also been made in developing strategies to select gene-modified cells in vivo. While this approach has been shown to be highly effective when a chemo-resistance gene is being used in combination with chemotherapy (Beard et al., 2010), other strategies that do not involve chemotherapy are being developed and may provide a nontoxic selection approach (Okazuka et al., 2011).

DESIGN OF HSC-BASED GENE THERAPY CLINICAL TRIALS IN HUMANS

Depending on the therapeutic approach, there are a number of clinical development strategies that might be pursued. At least in the near future, it is likely that all proof-of-principle studies to explore how to “make space” for exogenous HSCs will be carried out in patients with AIDS lymphoma or other diseases requiring chemotherapy as a standard of care. Exceptions to this rule might include transplantation of more mature lineage-restricted gene-modified T cells and/or myeloid cells, an approach that may not require as much, if any, preparative conditioning. These studies, together with preclinical nonhuman primate studies, will hopefully teach us more about the extent to which engraftment of modified HSCs will facilitate the expansion and protection of unmodified T and myeloid cells.

From an ethical perspective, another viable approach is to perform transplants in patients for whom standard antiretroviral drugs are not a viable option. Such patients might include those with multi-drug resistant HIV, although such patients are now rare and most have very advanced disease; they may lack either sufficient stem cells for transduction and/or the functional lymphoid infrastructure necessary to support immune reconstitution. A related group of patients includes those who choose not take antiretroviral therapy or who have exhibited an inability to adhere to any regimen, but even those patients are difficult to define and are often not ideal clinical trial participants.

The ultimate patient population, however, is likely to be healthy patients who are doing well on antiretroviral therapy and who receive a transplant while remaining on therapy. In such cases, several primary outcomes might be anticipated, including a reduction in the size of the HIV reservoir, the absence of an HIV rebound during treatment interruption, and/or reconstitution of a more effective immune system.

CONCLUDING REMARKS

There is a growing international effort aimed at developing a cure for HIV infection. A central dilemma in almost all cure strategies is balancing the risks and benefits. Although the indefinite delivery of antiretroviral therapy is not possible for many individuals, those who do access and adhere to drugs generally have a good prognosis. Because most cure strategies require that complete or near complete inhibition of HIV replication be achieved with therapy, the ideal patient population for testing cure strategies will be one that is already on maximally-suppressive antiretroviral therapy. For gene therapy and stem cell modification protocols, early work has focused on treating patients who require stem cell transplants for management of a malignancy. Moving such studies into the general population will require developing gene delivery approaches with a very low risk of malignant transformation and transplant protocols that require minimum conditioning regimens. Fortunately, there are now integrating lentivirus and foamy virus vectors with significantly improved safety features, and non-integrating vector systems or simple transfection procedures are also available. In addition, it is likely that the safety of gene delivery will continue to improve and low-dose conditioning regimens have already been used successfully in other gene therapy studies (Aiuti et al., 2009). Given these advances, it is expected that it will at least theoretically soon be possible to safely modify stem cells ex vivo, to transplant cells without ablative conditioning into healthy individuals on effective antiretroviral therapy (perhaps by using large numbers of gene-modified cells or by using an in vivo selection approach), and to then carefully interrupt therapy, perhaps leading to selective advantage and expansion of gene-modified cells. Finally, cost considerations will almost certainly emerge as a dominant factor in weighing the practicality of any curative strategy. Although gene modification of stem cells will be an expensive intervention, it may prove to be cost-effective given that decades-long administration of antiretroviral therapy would cost several hundred thousand dollars per person (Schackman et al., 2006). Thus, although there are many safety and logistical barriers to be addressed, further pursuit of HSC-based gene therapy may ultimately offer a curative strategy for HIV disease.

Acknowledgments

The authors would like to acknowledge the impact of NIH-supported Martin Delaney Collaboratory grants (including U19 AI 096111, of which HPK and KRJ are co-PIs, and U19 AI96109, of which SGD and JMM are co-PIs) in bridging efforts to complete this review. We also thank the amfAR Eradication Program for its contributions in moving the cure agenda forward. This effort was supported in part by R01 AI80326 and R01 HL84345 (to HPK), Bill and Melinda Gates Foundation Grand Challenges Explorations Phase I and II awards (to KRJ), K24 AI069994 (to SGD), and R01 AI40312 (to JMM). HPK is also supported by the José Carreras/E.Donnall Thomas Endowed Chair for Cancer Research, and JMM is a recipient of the NIH Director’s Pioneer Award Program, part of the NIH Roadmap for Medical Research, through grant DPI OD00329.

Footnotes

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