Stem cell transplantation in the context of HIV--how can we cure HIV infection?

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Stem cell transplantation in the context of HIV – how can we cure HIV infection? Expert Rev. Clin. Immunol. 10(1), 107–116 (2014)

Gerhard Bauer* and Joseph S Anderson University of California Davis, Stem Cell Program, School of Medicine, 2921 Stockton Blvd., Sacramento, CA 95817, USA *Author for correspondence: Tel.: +1 916 703 9305 Fax: +1 916 703 9310 [email protected]

All HIV target cells are derived from hematopoietic stem cells. More than two decades ago, a hypothesis was postulated that a cure for HIV may be possible by performing a transplant with HIV-resistant hematopoietic stem cells that would allow for an HIV-resistant immune system to arise. HIV-resistant stem cells could be generated by genetically modifying them with gene therapy vectors transferring anti-HIV genes. First attempts of stem cell gene therapy for HIV were carried out in the USA in the 1990s demonstrating safety, but also little efficacy at that time. The first demonstration that the postulated hypothesis was correct was the cure of an HIV-infected individual in Berlin in 2009 who received an allogeneic bone marrow transplant from a donor who lacked the CCR5 chemokine receptor, a naturally arising mutation rendering HIV target cells resistant to infection with macrophage tropic strains of HIV. In 2013, reports were published about a possible cure of HIV-infected individuals who received allogeneic bone marrow transplants with cells not resistant to HIV. We will review these stem cell transplant procedures and discuss their utility to provide a cure for HIV infection, including efficacious future stem cell gene therapy applications. KEYWORDS: allogeneic bone marrow transplantation for HIV • anti-HIV genes • bone marrow transplantation • CCR5 receptor • functional cure for HIV • HIV • HIV cure • stem cell gene therapy for HIV

Target cells for HIV are CD4+ T cells, macrophages, monocytes, dendritic cells and even brain microglia. One very interesting aspect of all of these cell types is the fact that they are all of hematopoietic origin, which means they are derived from bone marrow stem cells. A hypothesis we have been stipulating for a long time is as follows: If all hematopoietic stem cells (HSCs), their progeny and the arising mature blood cells were engineered to be resistant to HIV, the virus would not be able to replicate and the disease could be stopped, possibly eliminated. Transplantation of HIVresistant HSCs into HIV-infected patients would accomplish this. Why would anyone consider stem cell transplants for the treatment of HIV in an era where anti-HIV drugs work so well and extend the life of HIV-infected individuals dramatically? Antiretroviral therapy (ART) is not curative and has to be administered for the life of the patient. If not taken with the highest level of compliance, drug-resistant viral mutants can develop. There are also unwanted drug side effects that can become severe. Additionally, www.expert-reviews.com

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the high cost of life-long drug treatment needs to be taken into consideration, as millions of HIV-infected individuals need to be treated worldwide. A cure or prevention of the disease would be an important public health goal. It is not the purpose of this article to review HIV prevention strategies, however, it should be briefly mentioned that preventive HIV vaccines have not been efficacious. The best efficacy in a recent trial of a prime/boost vaccine for HIV was only 32% [1]. Reported cases of HIV cures

There is a very well-documented report of a cure of HIV that confirms the hypothesis postulated earlier. In 2007, a bone marrow transplant was performed in Berlin, Germany on an HIV-infected patient in order to cure his acute myeloid leukemia. This patient, now referred to as the ‘Berlin patient’, received a transplant of HSCs from a donor who was homozygous for a CCR5 delta-32 basepair mutation which renders cells deficient of CCR5 surface expression. CCR5 is a coreceptor utilized by HIV to attach to and fuse

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with HIV susceptible cells during the first stage of the HIV life cycle. This CCR5 delta-32 basepair allele is a naturally occurring HIV-resistant genotype which is found in a small percentage of northern and central European descendants. After the transplant was performed, the patient engrafted and remained free of leukemia. In 2009, an article in the New England Journal of Medicine reported that he had also displayed no rebound in HIV viral load after being taken off ART, and had undetectable viral load for a 20-month follow-up period [2]. Several years after the transplant, the patient still remains free of an HIV viral load. Many tissue biopsies have been taken, and no virus was found. A functional cure of HIV has been postulated with this patient [3]. However, two more extremely interesting cases for possible cures of HIV have been reported very recently. In July 2013, at the 7th IAS conference on HIV pathogenesis, treatment and prevention, Henrich et al. announced that in two HIV-infected individuals who were treated with allogeneic HSC transplantations, HIV DNA could not be detected in large samples of peripheral blood cells and in gut mucosa biopsies up to 4 years post-transplantation [4,101]. What was remarkable about these two cases was the fact that the donor bone marrow cells did not come from donors who had a CCR5 delta-32 basepair mutation. It was also reported at the conference that the patients have stopped taking ART, and at the time this article is being written, they have not shown any viral rebound (~15 weeks post-ART interruption). These second cases are not in concordance with our postulated hypothesis, as the transplanted stem cells were not HIV resistant. Clearly, another mechanism, in all likelihood an immunological mechanism, must be the cause for the possible cures in these patients. Types of cures

Experts may question: can we really cure someone after an established HIV infection? There are two types of cures which should be addressed. The first type of cure is called a ‘sterilizing cure’ which is one that completely eradicates HIV from the body of a treated patient [5]. It may be possible that a patient would be transplanted with enough HIV-resistant HSCs that the vast majority of the new immune cells block further HIV infection. Over time, the HIV cellular reservoirs could not be replenished and would die off, as there would not be enough HIV-susceptible cells to re-infect. Thus, the HIV infection would eventually cease to exist in a patient. A sterilizing cure may be difficult to achieve with an integrating virus, however. For instance, the human genome harbors many silenced retroviruses, in the evolutionary path they could never be eradicated, but they were rendered harmless. Should we therefore define a cure for HIV as a functional cure only, with control of the virus by the immune system? Examples for this would be the successful control of EBV or CMV in an immunocompetent individual. These viruses persist for the rest of the life of the individual, but they do not cause any disease symptoms. Only if the 108

person becomes immunocompromised can these viruses become active and cause disease. An intact immune system eliminates newly entering viruses and newly infected cells all the time. Could this be achieved in an HIV-infected individual? A functional cure would therefore occur in an HIV-infected individual when there is a balance between a partially HIVresistant immune system and a concurrent low-level HIV infection [6]. This could be achieved by transplantation of HSCs with inserted anti-HIV genes, producing a partially HIVresistant immune system when newly arising immune cells express these anti-HIV genes. Patients would be able to stop taking ART because enough HIV-resistant cells in their immune system are capable of maintaining normal immune system functions. Even though there is still a low level of HIV replication, disease progression toward AIDS would not occur due to a subpopulation of HIV-resistant immune cells that could control viral replication and HIV target cell infection. HIV-infected individuals progress in their disease to AIDS due to a low CD4+ cell number which renders the body susceptible to opportunistic infections normally controlled by a working immune system. Therefore, if normal CD4+ cell levels are maintained, opportunistic infections can also be controlled. It has been reported that HIV-infected individuals may also suffer from impaired hematopoiesis. This may be in part due to HIV infection occurring in the bone marrow space, particularly in the bone marrow stroma or may even be due to effects of ART, contributing to marrow suppression and cytopenia [7,8]. Protection from HIV infection by an HIV-resistant immune system, including reduction of the viral load with subsequent cessation of ART may also address this problem. It is known that long-term non-progressors and elite controllers who are infected with HIV can have detectable viremia. However, due to their genetic makeup or the strain of virus they are infected with, these individuals are able to control HIV infection. They are able to maintain normal CD4+ cell numbers which allow them to not progress to AIDS, even though viral replication is still occurring in their body. Also, certain non-human primates including sooty mangabeys who are naturally infected with SIV, the simian form of HIV, are able to live with ongoing virus replication without developing AIDS-like symptoms. This is likely due to the sooty mangabeys’ genetics and evolution with the virus, which allows them to maintain normal CD4+ cell levels even while SIV is infecting cells and is replicating in them [9]. HIV only infects CD4+ cells of the immune system, and all of these cells come from a single progenitor called the HSC (FIGURE 1). Every person, young and old has these stem cells, and they are the reason why we are capable of constantly producing new immune system cells for the rest of our lives. Therefore, by utilizing HSCs as targets to insert anti-HIV genes, a sustained therapy for HIV infection and possibly a one-time treatment utilizing anti-HIV gene expressing cells could be developed. Additionally, HIV does not infect true self-renewing and selfExpert Rev. Clin. Immunol. 10(1), (2014)


Stem cells to treat HIV infection

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repopulating HSCs allowing them to be used as uninfected target cells for the insertion of anti-HIV genes [10]. A report 1 by Carter et al. raised the issue that hematopoietic progenitors may be susceptible to HIV infection and perhaps be part of the latent reservoir [11]. However, there is no strong evidence that the majority of 3 CD34+ HSCs present in an individual is 4 2 HIV infected. On the other hand, stem cell gene therapy for HIV could also be beneficial when dealing with HIVinfected progenitor cells. Anti-HIV genes inserted into an isolated pool of CD34+ cells used for transplantation could also suppress HIV output from the populaT cells Macrophages Dendritic cells tion of possibly infected progenitor cells, thus limiting the HIV reservoir. AddiFigure 1. HIV target cells derived from hematopoietic stem cells. Hematopoietic tionally, when the viral load in the bone stem cells give rise to all blood cells while maintaining, at the same time the hematomarrow space can be adequately conpoietic stem cell pool. This is accomplished by asymmetric division. After cell division, one cell of the pair undergoes lineage differentiation into blood cell progenitors and trolled, the bone marrow stroma may finally mature blood cells, while the other cell of the pair remains a stem cell (1). HIV taragain be able to support normal get cells are T cells (2), macrophages (3), dendritic cells (4). hematopoiesis [7]. HIV is a chronic infection and is mostly characterized by prolonged disease progression where the virus inhibiting HIV infection could be generated [12,13]. This stratinfects human CD4+ cells and causes immune system destruction egy, therefore, has great potential to control HIV infection in and dysregulation, both directly through cell killing and indirectly patients and may possibly one day even eradicate the virus through bystander cells. Therefore, when developing a durable from HIV-infected patients. As mentioned above, in 2007, a groundbreaking procedure anti-HIV gene therapy approach, it would not be ideal to just was performed on an HIV-infected patient, now referred to as focus on treating patients with anti-HIV gene expressing mature the Berlin patient, who had developed acute myeloid leukeimmune cells, such as T cells. This type of therapy would only mia [2,3] . To cure the patient’s leukemia, he had to undergo an have a transient effect as the gene modified T cells have a defined allogeneic bone marrow transplantation after fully ablative chehalf-life and will eventually die off. Viral reservoirs would then be motherapy. The bone marrow transplant, however, did not use able to re-infect newly arising HIV-susceptible immune cells and just any HSCs. The stem cells used where from an HLApatients would continue to be infected. Multiple anti-HIV gene matched donor and were naturally resistant to HIV infection modified T-cell infusions over the life of the patient may circumdue to a homozygous mutation in the CCR5 gene called deltavent these problems, however, this would be a costly and possibly 32 which prohibits cell surface expression of CCR5. Initially, troublesome protocol due to the many large-scale T-cell culture transduction procedures required to manufacture the cell dosages. the Berlin patient has been the only documented case of an HSCs are multipotent stem cells and reside in the bone mar- HIV-infected patient being cured of HIV over an extended row, the majority of which exist in the resting phase (G0). period of time, accomplished by utilizing HIV-resistant HSCs. Once they enter their cell cycle, they can undergo either asym- This procedure, in spite of it being expensive and involving a metric or symmetric division giving rise to daughter HSCs or potentially lethal conditioning regimen, did set the stage for blood cell progenitors. These blood cell progenitors can then the possibility of developing an actual cure for HIV. It should further differentiate into HIV-susceptible cells, including CD4+ be remarked, however, that there is currently a debate about T cells, monocytes, macrophages, dendritic cells and brain whether two other patients who received allogeneic bone marmicroglia. HSCs, which are characterized by a CD34+/CD38- row transplants without the CCR5 deletion have also been phenotype are both self-renewing and self-repopulating which cured of HIV [101]. There are indicators toward this fact, and means that they will not only continuously produce immune speculations about a graft-versus-host effect being responsible cells for the rest of the life of an individual, but they will also for this, but only long-term patient follow-up will be able to determine if there will be a rebound of productive HIV infecmake more of themselves, maintaining the HSC reservoir. If engineered to become HIV-resistant with various methods tion from non-eradicated reservoirs in these patients as cells including integrating retro- or lentivector transduction, zinc- without HIV resistance have been transplanted. One major problem with trying to apply this procedure for finger modification or long-lived episomal DNA gene expression, a prolonged and self-renewing immune system capable of everyone infected with HIV worldwide is that it would be www.expert-reviews.com

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extremely difficult to find HLA-matched donors who are homozygous for the CCR5 delta-32 allele. Although there have been efforts underway to find such matched donors, particularly using umbilical cord blood, no more transplants of this kind have been performed in HIV-infected individuals. Also, using only CCR5-negative cells would not inhibit different strains of HIV which use other co-receptors for attachment and fusion, such as CXCR4-tropic strains. HIV is also highly prone to mutations due to its error-prone reverse transcriptase and, therefore, may mutate around the CCR5 delta-32 block if allowed to reverse transcribe and replicate [14]. HIV stem cell gene therapy has great potential to mimic the outcome observed in the Berlin patient. When utilizing HIV stem cell gene therapy, patients’ own autologous HSCs can be engineered with anti-HIV genes to become HIV-resistant + [15–17]. CD34 HSCs reside in the bone marrow and can be mobilized into the bloodstream of patients after administration of granulocyte colony-stimulating factor (G-CSF) or other mobilizing agents. Once in the circulation, large quantities of HSCs can be collected by apheresis which avoids a bone marrow harvest procedure. This process has been shown to be safe and has become a routine method applied for bone marrow transplantation, even in HIV-infected individuals [102]. Once the apheresis product is collected, CD34+ HSCs can be isolated by immunomagnetic bead separation and are easily readministered via intravenous injection into patients for engraftment and long-term multi-lineage hematopoiesis. After selection, CD34+ cells are transduced, for instance, with a viral vector transferring anti-HIV genes, and cultured ex vivo, on a matrix such as fibronectin, in media supplemented with a cytokine cocktail containing stem cell factor, thrombopoietin and Flt3-ligand. This culture method allows the cells to be stimulated which enables better transduction efficiency when utilizing integrating viral vectors for genetic modification. For ex vivo expansion to be relevant in HIV stem cell gene therapy protocols, however, the HSCs would need to maintain their self-renewal and repopulating/engraftment ability. There is still limited understanding of the physiological mechanisms which regulate self-renewal of HSCs. Therefore, anti-HIV gene modification needs to be performed relatively quickly, as not to leave HSCs in culture too long and to compromise their self-renewal abilities [18]. Anti-HIV genes can then be transduced into the stem cells which thereafter can either be frozen and stored for future transplantation or directly be re-infused into patients. For HIV-infected patients who have developed AIDS-related cancers including B-cell lymphomas and leukemias, a full bone marrow ablation is required using chemotherapy in order to cure their cancer [19,20]. This patient population is ideal for testing HIV stem cell gene therapy in a clinical trial setting as it can be ‘piggy-backed’ on this procedure by inserting anti-HIV genes into the purified CD34+ HSCs which were apheresed from the patient prior to receiving chemotherapy for marrow ablation. HIV-resistant stem cells can then be re-infused into patients to reconstitute their hematopoietic system. Their 110

cancer will not only be cured, but patients will also generate an HIV-resistant immune system. For HIV-infected patients who have not developed cancer, it is envisioned that a low-dose chemotherapy ablation regimen can be used. As observed with ADA-severe combined immunodeficiency patients in recent clinical trials, only a reduced intensity conditioning regimen needs to be applied for reliable engraftment of gene modified cells to occur [21–23]. Therefore, HIV-infected patients could receive a non-lethal dose of chemotherapy to make space in the bone marrow for the anti-HIV gene modified cells. This procedure may need to be performed multiple times, however, to engraft enough HIV-resistant stem cells, but would be less toxic than full bone marrow ablation. Traditional methods of gene transfer: initially, genetic modification of CD34+ HSCs for HIV stem cell gene therapy was performed with integrating retroviral vectors (Moloney murine leukemia virus based) [24,25]. Recently, however, lentiviral vectors (HIV based) have been replacing the older retroviral vectors [26,27]. In these ex vivo gene modification procedures, great variations in the levels of HSC transduction efficiency have been seen, with many sites reporting low transduction efficiencies, also translating into low in vivo gene marking levels after re-infusion of the transduced HSCs [26]. This has been the biggest hurdle in achieving any clinical benefit in HIV stem cell gene therapy clinical trials. New methods for integrating vector-based transduction procedures are currently being developed to increase the levels of anti-HIV gene expressing cells once transplanted into patients. These could include the incorporation of a selectable marker gene which enables the transduced stem cells to be purified to a very high percentage and could mimic the transplantation procedure performed in the Berlin patient, who received a pure population of stem cells carrying the CCR5 deletion [28]. This could potentially lead to a clinical benefit in all patients receiving gene modified cells. A novel technique, not utilizing integrating vectors is the application of zinc-finger nucleases to disrupt cellular genes critical for HIV replication (such as CCR5) has recently been suggested for CD34+ HSCs. However, this method has a low efficiency in gene disruption which may not easily lead to a clinical benefit [29]. Recent work in developing CD34+ HSCspecific targeting vectors has also shown some promising results [30]. Potential for in vivo gene transfer: recent research has also involved incorporating stem cell targeting ligands including stem cell factor and thrombopoietin into the envelope of viral vectors [31]. This has allowed cell-specific targeted transduction of CD34+ HSCs and could potentially be used as an in vivo HIV stem cell gene therapy procedure instead of current transduction protocols which occur ex vivo. In summary, advantages of HIV gene therapy utilizing HSCs include the possibility of a one-time treatment, controlled and/or constitutive anti-HIV gene expression in progeny arising from HSCs, and prolonged HIV inhibition after HSC transplantation. Several HIV stem cell gene therapy clinical Expert Rev. Clin. Immunol. 10(1), (2014)


trials have been performed on HIV-infected patients with viral vectors [25,26].


Clinical trials applying stem cells to generate an HIVresistant immune system

In the 1990s, several clinical trials conducted in Los Angeles in collaboration between Children’s Hospital Los Angeles and City of Hope focused specifically on transductions of HSCs using retroviral vectors. Gene transductions were carried out by an expert team at Children’s Hospital Los Angeles, while the patients were infused and followed at City of Hope. In 1996, the first stem cell gene therapy clinical trial for HIV utilized a tat/rev ribozyme transduced into fresh mobilized peripheral blood stem cells of five HIV-infected adults who had not progressed to AIDS [103]. Both a control vector transferring only neomycin resistance and a therapeutic vector transferring the anti-HIV ribozyme plus neomycin resistance were used. This was done to see if a selective survival advantage of the antiHIV gene-transduced cells in the face of a viral load would be observed. Gene transduction efficiency into hematopoietic progenitor cells, as measured in colony-forming unit assays in vitro, was about 30–40%. However, true HSC transduction efficiency, which cannot be measured in colony assays must have been low. Gene marking in the peripheral blood, which appeared after about 4 weeks, was also very low, which was expected, as no marrow ablation was performed and the number of transduced CD34+ cells infused into the patients was small compared with the existing number of CD34+ cells in the bone marrow. There was also no difference in numbers of control gene marked cells versus anti-HIV gene marked cells. There were no adverse events associated with this clinical trial that were related to gene therapy. A groundbreaking clinical trial of stem cell gene therapy for HIV was conducted soon afterward by the same group [104]. Combination HIV drug therapy had just become state of the art at that time, however, HIV lymphoma patients were still not considered eligible for high-dose chemotherapy and autologous bone marrow transplantation since their life expectancy was considered short and insurance companies did not want to take on the cost of such an expensive procedure in this patient population. The researchers believed, however, that HIV lymphoma patients would have a very similar life expectancy as non-HIV-infected individuals with lymphoma if they could be cured of their lymphoma, as they were taking ART [20]. As these patients were in need of a full marrow ablation through high-dose chemotherapy and would need a subsequent autologous bone marrow transplantation to reconstitute their bone marrow, this was also thought to be the best patient population for stem cell gene therapy for HIV. The new clinical trial provided a chance to investigate how well anti-HIV gene-transduced CD34+ cells would engraft after complete marrow ablation, and would perhaps provide a clinical benefit, if enough engraftment could be achieved. Five HIV lymphoma patients were enrolled to be treated for their lymphoma with high-dose chemotherapy and autologous bone marrow www.expert-reviews.com

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transplantation. Peripheral blood stem cells were mobilized after complete remission was achieved by chemotherapy, the patients underwent several apheresis sessions, then their CD34+ cells were isolated from the apheresis products. Isolated CD34+ cells were then cryopreserved. Half of the apheresis products, however, remained unmanipulated and was also cryopreserved. This was done for safety reasons as no engraftment data on cultured, transduced cells in a fully ablated individual were available. The US FDA therefore required that the patients would receive the stored, unmanipulated apheresis product, which could completely reconstitute their hematopoietic system, and then in addition, the transduced CD34+ cells. The patients then underwent high-dose chemotherapy. A few days before the end of the chemotherapy procedure, the cryopreserved CD34+ cells were thawed, cultured and transduced with the same anti-HIV gene and the same control that was used in the previous clinical trial. All patients engrafted. Four out of the five patients had long-term survival and are free of relapsed lymphoma more than 10 years after the procedure. One patient died of relapse from very aggressive lymphoma. There were no adverse events related to the gene therapy approach, even in a fully marrow ablated setting. There was no insertional mutagenesis and no genotoxicity. Transduction efficiency, even after thawing of CD34+ cells, was between 30 and 40%. Initially, there was very high gene marking in the peripheral blood facilitated by complete marrow ablation and engraftment of many progenitor cells; however, gene marking diminished and disappeared by 12 months. True HSCs could not be transduced with retroviral vectors. There was also no selective survival advantage for gene-transduced peripheral blood T cells, as patients had completely suppressed viral loads since they were required to continue ART, as per FDA request. In spite of the loss of gene marking over the long run, this clinical trial paved the way for future clinical HSC transplants in HIV-infected patients [102]. It could be demonstrated that HIV lymphoma patients had practically the same survival rate as non-HIVinfected lymphoma patients [20]. A clinical trial of autologous bone marrow transplantation in HIV lymphoma patients without gene therapy followed and made this procedure standard of care. It could also be demonstrated that stem cell gene therapy for HIV in a full ablation setting was safe. In 1997, the group at Children’s Hospital Los Angeles then pioneered stem cell gene therapy clinical trials in a pediatric patient population [24,25,105]. Four children with HIV infection, aged 8–17, were enrolled. Bone marrow aspirates were obtained and CD34+ cells were isolated using immunomagnetic beads. The isolated cells were then transduced with retroviral vectors transferring the neomycin resistance gene, as a control gene and an RRE decoy anti-HIV gene. Transduction efficiencies as measured by colony-forming unit assays were between 7 and 30%. The transduced CD34+ cells were infused into the patients without marrow ablation, and as expected, very low gene marking in the peripheral blood resulted. In spite of a viral load in the patients, no selective survival advantage of the anti-HIV gene-transduced cells could be observed. There were 111


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no adverse events related to the gene transfer. It could, however be demonstrated that stem cell gene therapy for HIV was safe, and that enough bone marrow CD34+ cells could be isolated and transduced, even in a pediatric population. A re-evaluation of the clinical application of anti-HIV genes followed after these initial clinical trials. It was decided at Children’s Hospital Los Angeles to perform another pediatric study of stem cell gene therapy for HIV, using the strongest anti-HIV gene available at the time, the RevM10 gene, and to select a pediatric population that had a relatively high viral load, in spite of ART [106]. It was thought that a selective survival advantage of gene-transduced cells could perhaps be seen, in spite of the fact that the FDA, at that time, would not allow marrow ablation or the interruption of ART, due to ethical reasons. Two HIV-infected children were enrolled. Bone marrow aspirates were performed and CD34+ cells were isolated as in the previous study. The cells were transduced with a RevM10 gene and a non-translated marker gene as a control. Transduction efficiencies were 15–30%. In both subjects, approximately 1:10,000 peripheral blood mononuclear cells were marked, equally with control and anti-HIV gene at around 1–3 months post-infusion. After 3 months, vector marking started to decline and disappeared, consistent with what was seen in the previous clinical trials. Both patients continued on ART, their viral load was below 10,000 genomic copies/ml. However, one patient had poor adherence to ART and discontinued ART completely at around 11 months postinfusion. The viral load in that patient started to climb, up to 261,000 genomic copies/ml. Interestingly, at that time, peripheral blood mononuclear cells marked with RevM10, that had previously been undetectable appeared again, and were detectable up to 1:10,000 cells. The non-translated marker gene marked cells, however, did not re-appear. For safety reasons, the patient was soon put on a different ART regimen and the viral load became undetectable. At the same time, the gene marked cells also declined in tight correlation with the viral load and disappeared. The second patient, who had continued on ART had no re-appearance of gene marked cells in the peripheral blood during the same time period. In spite of this being observed in just one patient, selective survival of antiHIV gene-transduced cells in the face of an HIV viral load could be demonstrated in a human clinical trial [32]. To date, the largest randomized clinical trial of stem cell gene therapy for HIV has been performed at UCLA, which enrolled 74 patients. CD34+ cells were transduced with an anti-tat ribozyme or a placebo as a control [33,34]. CD34+ cells were isolated after mobilization and no conditioning was applied for re-infusion. It was found that the viral load did not significantly decrease in the treatment arm as compared with the placebo arm but a significant difference was found in the numbers of CD4+ cells. These were significantly higher in the treatment arm. The study therefore demonstrated that antiHIV gene-transduced HSCs could produce biologically active CD4+ cells in HIV-infected individuals that have a survival advantage, as the CD4+ cell number correlated with the viral 112

load. Additionally, this large clinical trial demonstrated the safety of this treatment, as no gene therapy-related adverse events were noted. For all the previous clinical trials, oncoretroviral vectors based on the Moloney murine leukemia virus were used. The gene therapy community thought that it would be advantageous to switch to lentiviral vectors for such trials, since better transduction into quiescent HSCs could be achieved. These vectors also offer an improved safety profile as their integration pattern does not favor transcriptional start sites of active genes. Another advantage is the fact that they do not silence gene expression over the long run. However, it took considerable time for all the regulatory steps to be accomplished to start with the first HSC gene therapy clinical trial for HIV using lentiviral vectors. At City of Hope, a new combination gene therapy vector had been developed. It encompassed a ribozyme to knock down the CCR5 receptor, an shRNA targeting the tat/rev messenger RNA for HIV and a TAR decoy. A combination anti-HIV gene strategy is based on the same principle as the combination drug regimen in ART, it significantly reduces the chance for the generation of resistant viral mutants. These three anti-HIV genes were packaged into one third-generation lentiviral vector, which was extensively tested preclinically [107]. The clinical trial enrolled four HIV lymphoma patients, as it was based on the clinical trial model that had been pioneered by the same group previously [26,108]. CD34+ cells were isolated from apheresis products, transduced with lentiviral vector and re-infused into fully marrow ablated patients, however, again in conjunction with non-manipulated cells. Transduction efficiencies were lower than those achieved with retroviral vectors previously (1% long term), and gene marking in the patients was also low (0.02–0.32%, corresponding to 200–3200 copies/106 PBMCs). However, long-term persistence of gene marking (currently up to 4 years) could be demonstrated, as gene marking did not decline or disappear. This highlights stable transduction of HSCs. Additionally, all subjects have achieved long-term remission of their lymphoma, and no gene therapy related adverse events were observed. What can be learned from these clinical gene therapy trials? The correct vector choice is very important. Lentiviral vectors are the best vectors for stem cell gene therapy, as they are able to transduce resting cells, do not get silenced over the long term and allow for sustained expression of the anti-HIV genes. Combination anti-HIV genes have to be applied to circumvent the generation of HIV escape mutants. The best anti-HIV genes to use would be those which act at the pre-entry stage, prior to reverse transcription, and also at the pre-integration stage in the HIV life cycle. This would prevent HIV from mutating during the reverse transcription stage and would block the formation of new provirus. High transduction efficiency, without the increase in copy number per cell (for safety reasons), is also an important aspect which must be followed. Gene marking in the peripheral blood is directly correlated to transduction efficiency. Marrow conditioning is necessary, or low peripheral blood cell marking will be seen, as transduced Expert Rev. Clin. Immunol. 10(1), (2014)



stem cells will be diluted out in the large number of nontransduced bone marrow stem cells. This was a lesson well learned from stem cell gene therapy clinical trials of ADA deficiency. Without marrow conditioning, gene expressing T cells in the peripheral blood arose and remained at extremely low levels leading to no clinical benefit [22]. Only clinical trials applying marrow conditioning led to the long awaited cure of ADA deficiency as enough gene expressing T cells arose and were maintained in the periphery reproducibly leading to the expected clinical benefit and cure of the disease [23]. Finally, after sufficient engraftment of transduced CD34+ cells has been reached, a selective survival advantage must be given to the anti-HIV gene expressing cells in the peripheral blood to form sufficient numbers of HIV-resistant immune cells. Taken together, all these items may lead to an immune system that could theoretically control HIV infection. Based on these assumptions, a new clinical trial has been designed at the University of California Davis Medical Center [109]. A new triple combination anti-HIV gene thirdgeneration lentiviral vector was developed encompassing an shRNA directed against the CCR5 receptor, a human/rhesus macaque TRIM5a molecule preventing HIV uncoating and a TAR decoy, preventing upregulation of HIV transcription. A high titer, clinical grade lentiviral vector was manufactured and preclinical transduction experiments resulted in transduction efficiencies into CD34+ cells up to 50%. Preclinical safety and efficacy studies in a humanized mouse model have also been performed demonstrating successful engraftment of the lentivector-transduced cells and their subsequent multi-lineage hematopoiesis. A strong selective survival advantage of antiHIV gene expressing peripheral blood cells and maintenance of normal levels of human CD4+ cells was also observed upon in vivo challenge with various strains of HIV-1. The human clinical trial designed around this vector will initially recruit AIDS-induced malignancy patients, for safety reasons. Highdose chemotherapy will be given to cure the patient’s malignancy followed by reconstitution of the bone marrow with anti-HIV gene-transduced CD34+ cells. Finally, ART will be withdrawn after stable cell engraftment has been achieved and enough peripheral blood T cells are seen. This clinical trial has gone through the first regulatory steps with approval from the National Institutes of Health Recombinant DNA Advisory Committee (RAC) and the completion of a pre-IND meeting with the FDA. Upon recommendation from the RAC, it should be considered to also expand this new clinical trial to HIV-infected patients without AIDS-induced malignancies, after the protocol has been deemed safe in the initial cohort of patients. Expert commentary

It has been demonstrated that the CCR5 receptor is vital for HIV infection. Although it is the receptor for just one tropism of HIV, CCR5 tropic strains are the predominant ones and are found especially during the initial stages of infection mostly occurring through the mucosa. CXCR4 tropic strains of HIV www.expert-reviews.com

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arise much later in the natural history of HIV infection, after prolonged and continuous infection and replication in T cells. The complete absence of the CCR5 receptor in homozygous individuals is an extremely potent protection from HIV. Even heterozygous individuals who express 50% of the receptors on the cell surface are protected from HIV infection, to a degree. The absence of the CCR5 receptor acts as an HIV entry inhibitor, prior to reverse transcription. This fact is another very important aspect, as HIV cannot produce viral mutants. The first line of defense, entry inhibition, is therefore the most important one. In the Berlin patient, it has been demonstrated how vital the CCR5 receptor is in controlling HIV. But is there another aspect to the absence of a viral load in that patient? He received intense chemotherapy that eliminated his hematopoietic system. At the same time it is possible that the cells that harbored the HIV reservoir were eliminated during the intense chemotherapy. Many HIV patients have undergone high-dose chemotherapy to treat lymphoma, however, the viral load in these patients rebounded, after successful transplantation. Nonetheless, there may be a time shortly after chemotherapy where the reservoirs are not plentiful. If HIV cannot enter new cells due to the absence of CCR5, HIV may not be able to replenish the reservoirs. Therefore, a combination of both a CCR5 inhibitor and a conditioning regimen may perhaps produce the wanted effect of curing an HIV-infected patient. Another extremely important lesson to be learned from the Berlin patient is the fact that all of the new immune cells he developed lacked the CCR5 receptor. In stem cell gene therapy experiments where only very small numbers of transduced cells were transplanted, which then produced an even smaller number of peripheral blood cells that were resistant to HIV, the anti-HIV effect may not have become visible. This was due to the majority of the cells in the body being susceptible to HIV infection thus allowing the infection to continue. There may be a threshold of protected cells that needs to be reached before a clinically measurable effect becomes observable. Is the immune system capable of controlling HIV? This is very likely the case, if the immune cells cannot be attacked and killed by HIV. But this may be a numbers game. Only if enough immune cells are available, this will truly be the case. Currently, we do not know an exact number. We only know that if all immune cells are protected from HIV, control of HIV is possible. Is the Berlin patient truly free of HIV? We also do not know the answer to this question. Integrated HIV can hide in very few long-lived cells in hidden sanctuaries. Although many biopsies have been taken and showed no presence of HIV, this does not mean that the virus is really eradicated. The current status rather indicates that outstanding control of the virus has been achieved, which in itself is an extremely remarkable accomplishment. Long-term follow-up will demonstrate if HIV really has been eradicated. Can the virus rebound? If the virus, for whatever reason, manages to survive in a sanctuary site and is able to replicate, 113


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there is a theoretical possibility that resistant mutants can arise. A huge selective pressure to mutate around the absence of the CCR5 receptor will direct this mutation toward the use of other chemokine receptors, most likely the CXCR4 receptor. How about the patients that received an allogeneic bone marrow transplantation without gene modified cells that seemed to be free of HIV and were taken off ART? The mechanism why there was no rebound of viral load cannot easily be explained based on the theories outlined above. However, the possibility of immunological control through a graft-versus-host or ‘graft-versus–HIV’ effect could be explored as a hypothesis. The conditioning regimen used for the transplant may have eliminated most HIV-infected peripheral cells and also may have significantly dropped the viral load. Allogeneic immune cells could then have potentially recognized and eliminated the small amount of remaining HIV-infected cells in the course of a graft-versus-host cell reaction mediated by cytotoxic T cells, without getting infected themselves, as they may have lacked the CD4 receptor. Additionally, as the HIV strain harbored by the patient may have been adapted to the patient’s HIV target cells, allogeneic immune cells may not have been as infectable by this strain, putting them at an advantage toward control of the infection. This hypothesis, however, is postulated with the caveat that HIV from any remaining reservoirs could also adapt to the newly introduced immune cells and infect them in the long run, producing a rebound of active HIV replication. Only cells lacking suitable receptors for HIV or are expressing antiHIV genes preventing the virus from integrating may be permanently protected from infection. Five-year view

Within the next 5 years, how can the lessons learned from the Berlin patient be applied to stem cell gene therapy and to make a cure for HIV possible? If all HSCs can be made resistant to HIV, this would directly follow in the footsteps of the transplantation with bone marrow from a donor with the CCR5 mutation. Therefore, similar results would be expected. This strategy could be pursued if transduced CD34+ could be selected or enriched in vivo. There have been attempts to do this by the insertion of a chemotherapy resistance gene into the CD34+ cells. The results of clinical trials to test this in vivo enrichment method in cancer patients were unfortunately not as promising as expected. On this note, a new pre-selective anti-HIV lentiviral vector has been developed to bridge the gap between previous HIV stem cell gene therapy trials and the Berlin patient [28]. This vector allows for the purification of transduced cells away from non-transduced cells so that a pure and enriched population of anti-HIV vector-transduced cells can be transplanted into patients. The pre-selective vector expresses an extra transgene apart from the anti-HIV genes which encodes for a cell surface protein not normally found on CD34+ HSCs. When using this vector, the initial transduced efficiencies of CD34+ cells were similar to previous experiments where only around 20–50% of 114

the cells are transduced. This mixed population of HIVresistant and HIV-susceptible cells was then further processed by CD25 immunomagnetic beads to purify the transduced cell population. Initial safety experiments demonstrated that the expression of CD25 on the surface of CD34+ cells did not have any detrimental effects in colony-forming unit assays and the phenotypic profile of in vitro derived immune cells. Strong protection from HIV-1 infection was also demonstrated when compared with non-purified cells [28]. Planned in vivo safety and efficacy studies of this pre-selective anti-HIV lentiviral vector will likely produce a therapeutic candidate superior to any previous vector used in HIV gene therapy trials. New clinical trials with this pre-selective vector are planned to be conducted within the next 5 years. Alternatively, HSCs could be developed from autologous induced pluripotent stem cells (iPSCs) that could be engineered to be resistant to HIV. Integration free methods to generate iPSCs could be used. The insertion of anti-HIV genes could be controlled, and directed into safe harbors in the human genome. The gene modified iPSCs could also be highly tested and selected, and then differentiated into HSCs with high purity. This would assure that a highly tested and efficacious product would be transplanted into a patient. Currently, this technology is still under development. The in vivo functionality of iPSC-derived HSCs is still very low and needs to be improved upon. After this problem has been solved and efficient production methods have been worked out, this method will be very promising. It is not clear, however, if this will be possible within the next 5 years. Genetically modified autologous human HSCs will have the possibility to cure HIV infection if a large percentage or a pure population of gene modified cells is transplanted into patients. This method will be superior to the transplantation of allogeneic bone marrow stem cells from donors with a CCR5 deletion since allogeneic transplantation is associated with high morbidity and mortality. To make space in the bone marrow, reduced intensity chemotherapy regimens can be developed for non-cancer patients that will also allow the treatment of older patients. Combination anti-HIV genes can be used for transduction, minimizing the chance for resistant mutants to arise. If cell culture and transduction methods are simplified, this treatment will also become affordable. In summary, it is very likely that a cure for HIV is possible. If enough funding is made available for quality research in this area and enough translational laboratories make it their priority to apply this research, a cure may become reality for many HIV-infected individuals, sooner than later. Financial & competing interests disclosure

The authors received institutional funds from University of California Davis (USA). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. Expert Rev. Clin. Immunol. 10(1), (2014)


Review

Key issues • Target cells for HIV are derived from bone marrow stem cells. • Bone marrow transplantation with HIV-resistant stem cells could potentially cure HIV infection by replacing a patient’s immune system with HIV-resistant immune cells. • Stem cell gene therapy for HIV could produce an HIV-resistant immune system, but has been plagued with low clinical efficacy. • The first cure of an HIV-infected individual was achieved by allogeneic bone marrow transplantation with hematopoietic stem cells from a donor with a CCR5 receptor deletion. • Two more cases of a potential cure of HIV infection were reported after transplantation with allogeneic bone marrow stem cells not


resistant to HIV. • A functional cure for HIV is an achievable possibility. • Possibly, more than one mechanism may be responsible for a functional cure for HIV after bone marrow stem cell transplantation. • Future stem cell gene therapy for HIV approaches may be able to mimic the outcome of the first cure in a patient who received bone marrow stem cells from a donor with a CCR5 receptor deletion.

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Expert Rev. Clin. Immunol. 10(1), (2014)

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