Gaining momentum: stem cell therapies for HIV cure

Abstract

Purpose of review

Durable HIV-1 remission has been reported in a person who received allogeneic stem cell transplants (SCTs) involving CCR5 Δ32/Δ32 donor cells. Much of the reduction in HIV-1 burden following allogeneic SCT with or without donor cells inherently resistant to HIV-1 infection is likely due to cytotoxic graft-versus-host effects on residual recipient immune cells. Nonetheless, there has been growing momentum to develop and implement stem cell therapies that lead to durable long-term antiretroviral therapy (ART)-free remission without the need for SCT.

Recent findings

Most current research leverages gene editing techniques to modify hematopoietic stem cells which differentiate into immune cells capable of harboring HIV-1. Approaches include targeting genes that encode HIV-1 co-receptors using Zinc Finger Nucleases (ZFN) or CRISPR-Cas-9 to render a pool of adult or progenitor cells resistant to de-novo infection. Other strategies involve harnessing multipotent mesenchymal stromal cells to foster immune environments that can more efficiently recognize and target HIV-1 while promoting tissue homeostasis.

Summary

Many of these strategies are currently in a state of infancy or adolescence; nonetheless, promising preclinical and first-in-human studies have been performed, providing further rationale to focus resources on stem cell therapies.

INTRODUCTION

HIV-1 persists in CD4⁺ T and myeloid immune cells throughout multiple anatomical compartments despite suppressive antiretroviral therapy (ART) and often leads to ongoing immune dysregulation, inflammation, and long-term morbidity [1–5]. As a result, research on various HIV-1 curative strategies is of high priority. Many HIV curative strategies are designed to be ‘functional’, with the goal of reducing overall infected cell burden and minimizing persistent inflammation and immune dysregulation [4,6,7]. Other cure strategies are designed to achieve near elimination of all infected cells in the body that harbor replication-competent virus (previously referred to as a ‘sterilizing’ cure, for a lack of a more nuanced term) [8–10]. To date, allogeneic stem cell transplant (SCT) with donor cells that harbor a homozygous CCR5 Δ32/Δ32 mutation renewing hematopoietic cells resistant to CCR5-using HIV-1 is one of the few modalities that have led to sustained HIV remission off ART [11^▪,12,13^▪▪,14]. The exciting first report of such cure in the late Timothy Ray Brown (a.k.a. The Berlin Patient) and several subsequent reported transplant-related cures [11^▪,12,13^▪▪,14] has spawned an exciting era of rapidly developing stem cell and gene therapy studies that are revolutionizing HIV cure science (Fig. 1). Whereas many of these approaches may be in a state of infancy or adolescence, they hold great promise and are reviewed here. 

FIGURE 1.

Summary of stem cell and gene therapies currently being applied in HIV cure therapies. Overlapping areas indicate a combination of two techniques. Created on BioRender (4/2/2024).

Box 1.

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ALLOGENEIC STEM CELL TRANSPLANTATION

Allogeneic hematopoietic SCT entails the infusion of healthy donor stem cells into another person following chemotherapy with or without total-body irradiation to make space for donor cell engraftment and the development of long-term donor chimerism. Donor effector lymphocytes and NK cells facilitate the sustained targeting and clearance or elimination of residual host cells, a process referred to as a graft-versus-tumor effect, which is on a spectrum with the graft-versus-host disease (GVHD) where donor cells mount an aggressive cytotoxic response to tissues outside of the hematopoietic system (e.g., skin, liver, eyes, lungs, etc.) [15–17]. Allogeneic SCT involves the use of immunosuppressant medications to maintain the balance of a beneficial graft-versus-tumor effect and sustain full donor cell chimerism while preventing GVHD [18,19]. GVH effects likely play a critical role in reducing HIV-1 burden in those who stay on ART in months to years following SCT. Even in the absence of CCR5Δ32/Δ32 mutation, cells that are capable of harboring HIV are hematopoietic in origin and recognized as foreign to the donor immune system through minor antigen mismatches in otherwise HLA-matched donor cells [20–24]. Whether there is differential targeting of HIV-1-infected cells during this process is not known, but reactivation of latent HIV in host cells observed during the peri-transplant period may facilitate the innate immune-mediated clearance of infected cells.

There have been reports of four PWH thus far that have achieved long-term ART-free remission of HIV-1 through allogeneic HSCT involving CCR5 Δ32/Δ32 donor cells [11^▪,12,13^▪▪,14]. Although receiving donor cells inherently resistant to their strains of HIV-1 played a major role in achieving durable cure, elimination of residually infected host cells was likely through the GVH effects as stated above. In fact, HIV-1 cure is theoretically possible if 100% donor chimerism persists across all tissues under the cover of ART from the reduction in infected cell burden alone even without CCR5 Δ32/Δ32 donor cells [25], although in practicality, it appears that these allogeneic SCTs only lead to many months of delayed viral rebound following ART cessation [20–23,26–28]. Although HIV-1 remnants may still be detected years following CCR5 Δ32/Δ32 SCT [29], they may not be capable of leading to de-novo infection in vivo, or lack sufficient infectable target cells to sustain ongoing replication.

Although the mechanisms by which HIV is cured are essentially the same across the four reported CCR5 Δ32/Δ32 SCT recipients, one individual underwent a different type of SCT that has a broader impact on the accessibility of SCT for HIV cure [13^▪▪]. Due to difficulties in HLA matching of allogeneic stem cells, it is not always possible to find a suitable donor that has a CCR5 Δ32/Δ32 mutation in recipients not of Northern European ancestry as less than 2% of people of Northern European descent have a CCR5 Δ32/Δ32 mutation [30–33]. The ‘New York’ patient received a haploidentical donor umbilical cord-blood SCT. Given that existing cord blood banks can be screened ahead of time for CCR5 Δ32/Δ32 mutations and a greater degree of donor-host HLA-mismatch is tolerated (lower immunogenicity of donor cells), cord blood SCT may increase the chances of identified suitable CCR5 Δ32/Δ32 donors. However, cord blood-derived stem cells may take longer to engraft after SCT, resulting in prolonged cytopenias. One way to minimize engraftment issues is a technique called haploidentical-cord (haplo-cord) SCT. In haplo-cord transplant, cord blood-derived stem cells are combined with HSPCs from a half-match or haploidentical donor which creates a more supportive early posttransplant environment with less risk of prolonged cytopenias [13^▪▪,34,35]. The haplo-cord donor cells need not carry a CCR5 Δ32/Δ32 mutation as they only act as an ‘engraftment bridge’ until the cord blood cells achieve full donor chimerism. If PWH remain on ART throughout engraftment, they may have potential for HIV cure following ART cessation.

A major caveat is that many people harbor HIV that uses CXCR4 (and potentially other coreceptors) for host-cell entry [33,36,37]. Definitive determination of HIV coreceptor usage is therefore necessary to identify potential candidates for CCR5 Δ32/Δ32 allogeneic SCT [33,37]. There has also been debate over whether there are any long-term adverse health implications of CCR5 Δ32/Δ32 allogeneic SCT irrespective of HIV infection but, thus far, it appears well tolerated and rigorous studies have not demonstrated adverse clinical outcomes [11^▪,12,13^▪▪,14,38,39].

AUTOLOGOUS STEM CELL TRANSPLANTATION

Whereas allogeneic SCT offers the potential to diminish or deplete the HIV reservoir, it carries multiple risks including, but not limited to GVHD, and higher morbidity in PWH [20,40–43]. In contrast, autologous SCT in PWH does not result in long-term reductions of the HIV reservoir but is better tolerated overall. Unlike allogeneic SCT, stem cells are harvested directly from the individual, and then replaced following transplant conditioning. Autologous SCT research has demonstrated that high-dose conditioning chemotherapy alone does not impact the HIV burden over time [44–46]. The main reason for this is likely twofold. First, HIV-infected CD4⁺ T cells can be re-infused during autologous SCT. Of note, it is also possible that CD34+ hematopoietic precursors may harbor HIV, but subsequent studies have been unable to recapitulate these data [47–57]. Second, there is a lack of GVHD effects to prevent hematopoietic cells that survive conditioning from proliferating and re-establishing the viral reservoir. However, autologous SCT may act as a basis for gene-modified stem cell therapeutic approaches as discussed below.

GENETICALLY MODIFIED STEM CELL THERAPIES FOR HIV CURE

An essential next step in the pursuit of long-term HIV-1 remission is the development and implementation of gene modification therapies. As above, autologous SCT, while having no permanent impact on HIV reservoirs, offers a potential platform for the delivery and long-term persistence of hematopoietic cells that have undergone gene modification. Achieving a full and robust repertoire of gene-modified immune cell subsets will likely require modification and infusion or even transplant of hematopoietic stem cells. Several approaches have been employed, including the use of zinc finger nucleases, CRISPR-Cas9 editing, and lentiviral vector transduction.

Zinc finger nucleases

Some of the pioneering work in gene-modified cell therapy for HIV cure involves the use of zinc finger nucleases (ZFN) to edit human genes in CD4⁺ T cells ex vivo followed by infusion into PWH to render them resistant to HIV-1 infection [58]. ZFNs can induce double-stranded DNA breaks at sequences of interest that promote nonhomologous end joining [59]. Many of these initial preclinical and in-vivo studies focused primarily on CCR5 gene modification [60–62]. Following promising preclinical work [63,64], a first-in-human study of CCR5-modified autologous CD4⁺ T cell infusions using a ZFN (SB-728-T) was well tolerated. Blood measures of HIV DNA appeared to decline across the participants, and the viral load of one individual became undetectable during the planned 12-week analytical treatment interruption [60,65]. Whether or not the participant who developed undetectable viral loads would have done so without therapy is not clear given the lack of a control group, but results were nonetheless promising [60]. The study also demonstrated that modified CD4⁺ T cells had a long half-life of 48 weeks and modified T cells declined less quickly than unmodified cells during treatment interruption [60], suggesting a selective survival advantage in the setting of viral replication. In a subsequent phase I, single-arm study using this ZFN CCR5-modified CD4⁺ T cell approach, a modest delay in time to HIV-1 rebound following ART withdrawal was observed. Three individuals experienced transient control of viremia, two of whom were heterozygous for CCR5 Δ32 [66]. There are ongoing studies of ZFN-modified CD4+ T cells involving chemotherapeutic agents with minimal impact on hematopoietic precursors, such as cyclophosphamide, to make ‘space’ for modified cells (#NCT04201782, SB-728)[62].

Whereas the initial ZFN studies involved CCR5 gene modification in differentiated CD4⁺ T cells, more recent preclinical work has focused on CCR5 modification of stem cells to achieve multilineage engraftment and proliferation by modifying hematopoietic precursors [62,67]. In a nonhuman primate study (NHP), a wide array of hematopoietic cells persisted over time and demonstrated gut-tissue homing capacity [67], suggesting that modifying HSPCs is a viable and promising future direction in HIV cure.

CRISPR-Cas9

Several preclinical studies have also shown promise using CRISPR-Cas-9 technologies to modify CCR5 thereby inhibiting viral entry and subsequent replication. CRISPR-Cas9 uses a guide RNA to locate a target of interest and introduce Cas9, which cleaves the DNA, allowing new sequences to be introduced or deleted [59]. Because of promising preclinical work [68–72], a recent study used CRISPR-Cas-9 technology in a human participant with acute lymphocytic leukemia and HIV that required SCT to edit autologous stem cells ex vivo by targeting CCR5 [61]. The overall deletion efficiency of the CD34+ cells was 17.8%, and they reached full donor chimerism around week 4. Following SCT, modified cells persisted in vivo ranging around 5–8% for the 19-month follow-up time, reducing overall HIV reservoir size around ten-fold. During ATI, however, the participant experienced relatively rapid viral rebound [61], suggesting that expansion of modified cells was not rapid enough to counteract emerging viral replication.

Like ZFNs, CRISPR-Cas9 modification initially focused on CCR5, but other recent CRISPR-Cas9 efforts involve direct modification and deletion of HIV-1 genes from infected cells [68,73,74^▪,75–81]. Efficient in-vivo delivery, while also minimizing off-target modifications, will pose clear challenges in this direct editing approach. As a result, genetic modifications of hematopoietic stem cells targeting various stages of the HIV-1 life cycle are likely going to be crucial in order to establish a long-term pool of HIV-1- resistant cells in vivo.

Retroviral vectors and multitarget gene modification strategies

The use of lentiviruses to gene modify target cells has been around for decades and has been widely applied to a variety of diseases. Success has resulted in one of the first FDA approvals for the use of lentiviral delivered gene therapies (as well as another method using CRISPR-Cas-9) for sickle cell disease [82–84] and are used to generate a variety of CAR-T cells in clinical practice [85]. Lentiviral vectors are promising for HIV-1 because they can transduce a wide variety of hematopoietic cells and can carry multiple gene modification constructs [86]. For example, studies are now incorporating a variety of techniques like short hairpin RNA (shRNA), chimeric restriction factors, and viral fusion inhibitors [87–89]. Lentiviral transduction vectors are ideal compared to other retroviral vectors because of their ability to integrate large transgenes into the human genome, provide consistent gene expression, and regulate specific gene expression therefore reducing off-target effects [86].

Several ongoing clinical studies are using autologous CD34+ hematopoietic stem cell therapies in conjunction with lentiviral-based vector modification that include multiple anti-HIV inserts [87–90]. These studies are being performed in people with hematological malignancies that require autologous SCT. One study (NCT02797470) uses a CCR5 short hairpin RNA (shRNA), chimeric human-macaque TRIM5ɑ, and HIV trans-activation response element (TAR) decoy [87,90]. Another set of studies (NCT01153646, NCT02337985, NCT00569985, and NCT01961063) use a tat/rev shRNA, TAR decoy, and a CCR5 ribozyme [88]. Lastly, multiple groups have used a dual anti-HIV lentiviral vector (coined LVsh5/C46) that contains a CCR5 shRNA and a C46 fusion inhibitor peptide (NCT01734850, NCT02378922, NCT03593187) [89,91,92]. Clinical findings from these early phase trials are eagerly awaited.

One major issue with gene-modified autologous stem cell and CAR-T cell therapies in PWH that incorporate lentiviral vectors is that there is a high degree of sequence homology with native HIV-1. As a result, novel techniques are going to be necessary to co-quantify and characterize lentiviral-transduced cells and cells harboring HIV-1 as recently reviewed [93].

LIMITATIONS OF COMBINING GENE MODIFICATION WITH AUTOLOGOUS STEM CELL TRANSPLANY

A major limiting factor in the use of gene-modified therapies is the ability of modified cells to survive in multiple anatomical compartments over time. Whereas long-term persistence of zinc-finger modified CD4⁺ T cells has been identified in tissues months or years after infusion [60,66], the overall percentage of modified cells is relatively low compared to the total CD4+ T cell mass, even during analytical treatment interruption (ATI). It is not clear if these numbers will be sufficient to prevent HIV-1 rebound after ART cessation. Combination gene-therapy approaches using modified HSPCs is promising, but the durability and tissue-distribution remains unclear and may require the use of SCT conditioning regimens. A ‘less intense’ or ‘mini’ autologous gene-modified SCT approach with a more gentle conditioning regimen may be a compromise between safety and efficacy.

MESENCHYMAL STROMAL CELL THERAPY

There has been growing interest in using mesenchymal stromal cell (MSC) transplant therapy for HIV, with an overall goal of alleviating infection-induced inflammation and immune dysregulation, leading to improved viral control. Multipotent MSC are intriguing, as they have the ability to differentiate into many cell types and have complex effects on adaptive and innate immune responses, inflammation and healing [94–96]. Small proof-of-concept human studies using MSC in PWH who are classified as immunologic nonresponders (e.g. low CD4⁺ T cell counts or percentages despite consistent ART use) have been performed, with some suggestions that MSC infusions may increase CD4⁺ T cell counts [97]. More recently, pioneering work in NHPs demonstrated enhanced gut mucosal recovery and improved tissue-based, SIV-specific immune responses following MSC infusions [98]. It is not clear if or how MSC led to these findings, but MSC infusions have been overall very well tolerated with few safety issues in a variety of studies outside HIV [95,96]. Recent evidence suggests, however, that MSC may become infected with HIV and potentially contribute to the overall HIV reservoir [94]. As such, MSC studies will need to be designed carefully taking into timing of MSC infusions related to time of infection and ART initiation, among other factors.

SAFETY, ETHICAL CONSIDERATIONS, AND CONCLUDING THOUGHTS

One of the earliest attempts to institute CRISPR technology in HIV cure in vivo was the highly controversial work that used CRISPR-Cas9 to delete CCR5 in the human embryos of twins born to an HIV-positive father and HIV-negative mother in 2018 [61,99]. This work was far outside the realms of scientific or ethical justification and the long-term health impacts of genetically modifying embryos are completely unknown. As such, this research serves as a warning of the dangers surrounding unregulated gene modification and other cellular therapies [99,100]. Fortunately, most studies are ethically and scientifically grounded and have focused primarily on proven technologies that are already in human use for other illnesses. Nonetheless, gene modification techniques may have off-target effects in stem cells that impact the human genome in ways that are not fully understood or may manifest as clinical diseases over many years. A recent increase in the reporting of T cell lymphoma in people who received CAR-T cell therapies but are very rare in the general population is now emerging [101,102^▪]. In some cases, the clonal expansion included CAR-Ts and nonmodified lymphocytes in others. Whether this will hold up as epidemiologically significant over time or in larger populations of treated individuals is not known, nor are the mechanisms driving the T cell lymphomas (e.g., role of lentiviral vectors). Further investigation will be needed to clarify these unsettling but still preliminary findings.

An additional concern is the scalability and cost of gene-modified and stem cell therapies for HIV cure. For example, both the cost of gene modification treatment for sickle cell anemia exceed $2 million [103^▪]; it remains unclear how these technologies will be delivered to tens of millions of PWH, especially those living in resource-limited settings. Regardless, stem cell therapies including immunotherapies such as MSC infusion or gene modification of hematopoietic stem cells to achieve a large pool of diverse immune subsets resistant to de-novo HIV1 infection are promising and undergoing rapid development and clinical implementation. It will be up to stakeholders at all levels to guarantee that these therapies will be scalable and accessible to the millions of PWH who may benefit.

Acknowledgements

None.

Financial support and sponsorship

This work was supported by the NIH/National Institute of Allergy and Infectious Diseases K24AI174971 and R01AI176951 (T.J.H.).

Conflicts of interest

T.J.H. receives grant support from PolyBio and Merck and Co., and T.J.H. has consulted for Roche.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

• ▪ of special interest

• ▪▪ of outstanding interest