Berlin Patient Research Articles

Hematopoietic stem cell transplantation in the treatment of HIV infection – comparison of “Berlin patient”, “London patient” and “Dusseldorf patient”

Introduction and aim. Human Immunodeficiency Virus-1 (HIV-1) remains one of the major issues in global public health. The standard therapy for HIV-1 positive patients includes using antiretroviral therapy (ART). These medications ensure suppression of viral replication but do not lead to a cure for the patient. The aim of this study was to present hematopoietic stem cell transplantation (HSCT) as a malignant treatment method which led to cure for three HIV-1 positive patients. Material and methods. Literature available in April 2023 was searched by using the PubMed and Google Scholar databases. Articles were selected using the following words: HIV, AIDS, HSCT therapy, ART therapy. Analysis of the literature. In each case of these described HSCT, the donor of hematopoietic stem cells had a homozygous mutation in the HIV co-receptor CCR5 (CCR5Δ32/Δ32). This mutation leads to a permanent lack of the protein and prevents penetration of virus by using this receptor. After transplantation, all of these 3 patients remained virus-free despite discontinuation of ART therapy. Conclusion. More research is needed to reduce the risk of using HSCT and perhaps in the future be able to use this therapy in all HIV-infected people.

Case Study of Co-morbidities --- HIV/AIDS and Blood Cancer

HIV/AIDS is considered an uncurable disease today, and patients have to take medicine uninterrupted for their whole life to control the viral load. However, there have been five special cases possibly cured --- the Berlin patient (HIV-1 and AML), the London patient (HIV-1 and Hodgkin’s Lymphoma), the Düsseldorf patient (HIV-1 and AML), the New York patient (HIV-1 and high-risk AML monosomy 7), and the City of Hope patient (HIV-1 and AML). They were infected with HIV and blood cancer concurrently and received allogeneic hematopoietic stem cell transplant (allo-HSCT). By comparing the Düsseldorf patient and the New York patient who are in relatively similar conditions, umbilical cord blood (UCB) is very likely to be the better source for transplant based on the severity of graft versus host disease (GvHD) after transplant. A possible future treatment which is to combine the haematopoietic stem cell transplant (HSCT) and gene therapy with the help of CRISPR-Cas9, is suggested.

HIV/AIDS: Current Updates on the Disease, Treatment and Prevention.

CCR5-delta 32 homozygous stem cell transplantation for HIV-infected individuals is being treated as a milestone in the global AIDS epidemic. Since 2008, when the second Berlin patient was cured from HIV after undergoing transplantation from a donor with delta-32 mutation, scientists are aiming for a long-term cure for the wider population. In 2019, a London patient became the second person to be free of HIV and came off the antiretroviral drugs completely. CCR5 gene is now being treated as a viable target for HIV treatment. It can be used in the treatment of HIV either through administration of drugs that bind to CCR5 and stop the receptor from working or through gene therapy to alter the CCR5 gene using CRISPR/Cas9 and prevent protein production. This review article aims to identify the obstacles and the need to overcome them in order to bridge the gap between current research and future potential cures for HIV.

ABO blood group should be considered and reported when red blood cell exchange transfusion is used to treat Plasmodiumfalciparum Malaria patients.

Laboratory and epidemiologic studies have clarified how persons born with malaria-resistant red blood cells (RBCs)-like group-O, sickle-trait, and C-trait RBCs-are protected against death or severe disease due to Plasmodiumfalciparum (Pf) infection. Compared to malaria-promoting RBCs-like non-O or hemoglobin-AA RBCs-inborn RBC protection against severe Pf malaria can be profound: up to 10-fold greater. Given that "the Berlin patient" success showed patients do not have to be born with disease-resistant cells to benefit from them, why have the biologically plausible benefits of exchange transfusion (ET) of malaria-resistant RBCs not yet been evaluated? Unfortunately, a 2013 ET-for-malaria meta-analysis could not quantify the impact on mortality of ET of malaria-resistant RBCs because RBC malaria resistance variables (ABO group, hemoglobin type, enzyme levels, etc.) had not been reported in any of the ET studies used in that meta-analysis. To promote evaluation of the therapeutic impact of specific malaria-resistant RBCs, we urge clinicians to always report ABO blood group (and all other RBC malaria-resistance variables they are aware of) when they use ET to rescue Pf-infected patients. Prudent selection of donor RBCs has successfully optimized ET for sickle cell disease patients, and this precedent suggests selection of special malaria-resistant donor RBCs may optimize ET for Pf-malaria patients. Given that ET is used worldwide as a rescue adjunct, we feel it is most prudent to now assume-until proven otherwise-that considering and reporting the Pf-malaria-resistance variables of the RBCs to be transfused-at least ABO status-will help optimize ET-for-malaria.

Lessons Learned From Failures and Success Stories of HIV Breakthroughs: Are We Getting Closer to an HIV Cure?

There is a continuous search for an HIV cure as the success of ART in blocking HIV replication and the role of CD4+ T cells in HIV pathogenesis and immunity do not entirely eradicate HIV. The Berlin patient, who is virus-free, serves as the best model for a ‘sterilizing cure’ and many experts are trying to mimic this approach in other patients. Although failures were reported among Boston and Essen patients, the setbacks have provided valuable lessons to strengthen cure strategies. Following the Berlin patient, two more patients known as London and Düsseldorf patients might be the second and third person to be cured of HIV. In all the cases, the patients underwent chemotherapy regimen due to malignancy and hematopoietic stem cell transplantation (HSCT) which required matching donors for CCR5Δ32 mutation – an approach that may not always be feasible. The emergence of newer technologies, such as long-acting slow-effective release ART (LASER ART) and CRISPR/Cas9 could potentially overcome the barriers due to HIV latency and persistency and eliminate the need for CCR5Δ32 mutation donor. Appreciating the failure and success stories learned from these HIV breakthroughs would provide some insight for future HIV eradication and cure strategies.

Coreceptor-Based Hematopoietic Stem Cell Gene Therapy for HIV Disease

Combination antiretroviral therapy (cART) has significantly reduced the mortality rate and morbidity, and has increased the life expectancy of the human immunodeficiency virus (HIV) infected patients. However, the current cART is incapable of eradicating viruses from the human body, and HIV remains one of the most notorious viruses mankind has ever faced. HIV-1 enters target cells through the binding of gp120 viral protein to a CD4 receptor and then to a coreceptor, C-C chemokine receptor 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4). Individuals homozygous for a 32-bp deletion in the CCR5 allele, CCR5Δ32, are almost completely resistant to HIV-1 acquisition. Moreover, several of natural CXCR4 mutants which have been identified can reduce HIV-1 entry without impairing either ligand binding or signaling. In order to get rid of indefinite treatment for HIV patients, there is a growing interest in creating an HIV-resistant immune system through the use of CCR5 and CXCR4-modified hematopoietic stem cells (HSCs). Proof of concept for this approach has been provided in the instance of "Berlin patient" transplanted with allogeneic stem cells from a donor with homozygosity for the CCR5Δ32 deletion. Here, we review the progress of coreceptor-based HSC gene therapy for HIV disease and present new strategies.

HIV-1 remission following CCR5Δ32/Δ32 haematopoietic stem-cell transplantation.

A cure for HIV-1 remains unattainable as only one case has been reported, a decade ago1,2. The individual-who is known as the 'Berlin patient'-underwent two allogeneic haematopoietic stem-cell transplantation (HSCT) procedures using a donor with a homozygous mutation in the HIV coreceptor CCR5 (CCR5Δ32/Δ32) to treat his acute myeloid leukaemia. Total body irradiation was given with each HSCT. Notably, it is unclear which treatment or patient parameters contributed to this case of long-term HIV remission. Here we show that HIV-1 remission may be possible with a less aggressive and toxic approach. An adult infected with HIV-1 underwent allogeneic HSCT for Hodgkin's lymphoma using cells from a CCR5Δ32/Δ32 donor. He experienced mild gut graft-versus-host disease. Antiretroviral therapy was interrupted 16months after transplantation. HIV-1 remission has been maintained over a further 18months. Plasma HIV-1 RNA has been undetectable at less than one copy per millilitre along with undetectable HIV-1 DNA in peripheral CD4 T lymphocytes. Quantitative viral outgrowth assays from peripheral CD4 T lymphocytes show no reactivatable virus using a total of 24million resting CD4 T cells. CCR5-tropic, but not CXCR4-tropic, viruses were identified in HIV-1 DNA from CD4 T cells of the patient before the transplant. CD4 T cells isolated from peripheral blood after transplantation did not express CCR5 and were susceptible only to CXCR4-tropic virus ex vivo. HIV-1 Gag-specific CD4 and CD8 T cell responses were lost after transplantation, whereas cytomegalovirus-specific responses were detectable. Similarly, HIV-1-specific antibodies and avidities fell to levels comparable to those in the Berlin patient following transplantation. Although at 18months after the interruption of treatment it is premature to conclude that this patient has been cured, these data suggest that a single allogeneic HSCT with homozygous CCR5Δ32 donor cells may be sufficient to achieve HIV-1 remission with reduced intensity conditioning and no irradiation, and the findingsprovide further support forthe development of HIV-1 remission strategies based on preventing CCR5 expression.

Transient immunosuppression during short interruption of HAART: Another key to HIV cure in the “Berlin patient”?

Transient immunosuppression in lentiviral infections leads to an auto-vaccination followed by the rise of serum neutralizing activity and a significant decrease in a set-point viral load, which becomes undetectable in some cases. Arguably, in the “Berlin patient” (Hütter G, et al., N Engl J Med, 2009) an induction chemotherapy-mediated transient immunosuppression episode during short interruption of HAART might have led to at least a “functional cure” before allogeneic stem cell transplantation. Neutralization-enhancing RF antibodies (NeRFa) induced as a part of secondary immune response after transient immunosuppression may have played a key role in neutralization of infectious HIV-IgG complexes in extracellular reservoirs. Transient immunosuppression during short non-structured treatment interruption (TI-SNSTI/HAART) regimen would be promising for the achievement of HIV cure on a large scale.

CCR5 delta32 Cord & Haploidentical Grafts: Allogeneic Stem Cell Transplant for HIV+ /AML Patient: A Case Report from the Impaact P1107 Observational Study

▪Background: Although current antiretroviral therapies have effectively changed the course of HIV-1 infection, it remains an incurable illness. The C-C chemokine receptor type 5 (CCR5) is the key co-receptor for HIV entry into CD4+ T cells. Homozygous 32 deletion (delta32) in CCR5 genes leads to resistance to HIV-1 infection 1. Allogeneic stem cell transplant from a donor with CCR5 delta32/32 mutation was curative for HIV in an HIV-1-infected man (Berlin Patient) with AML 2. It has been challenging to replicate this experience. We present our experience with a single case of successful engraftment of CD34-selected, related haploidentical peripheral blood and CCR5delta32 cord blood stem cell transplant (haplo-cord) in an HIV-1-infected woman who, like the Berlin patient, developed AML and is now doing well at almost one-year post transplantation.Clinical case: A 60-year-old African-American woman was diagnosed with HIV-1- infection in June 2013 and was started on an antiretroviral treatment (ART) regimen consisting of tenofovir, emtricitabine and raltegravir. Her pre-ART viral load and CD4 counts were 1,000,000 copies/ml and 1003 cells/mm3, respectively. In Nov, 2013 she had viral load < 20 copies/ml with CD4 counts of 980 cells/mm3 and her HIV-1 infection was relatively asymptomatic. In March 2017, she was diagnosed with AML with monosomy 7. Her HIV therapy after the AML diagnosis was changed to abacavir- lamivudine -dolutegravir- combination. She achieved morphologic and cytogenetic remission after 1 cycle of standard idarubicin/cytarabine induction chemotherapy. In addition, she received 1 cycle of HiDAC consolidation and was referred for allogeneic stem cell transplant. We identified a CCR5 delta32/32 mutated cord blood unit (CBU) which was 5/8 HLA matched and contained 1.3 x 107 nucleated cells/kg and 3.2 x 104 CD34+ cells/kg.She underwent a combined CD34-selected, haploidentical peripheral blood and CCR5delta32 cord blood stem cell transplant (haplo-cord) in August 2017. Her conditioning regimen was with fludarabine/melphalan/and TBI400 and she also received ATG/MMF/tacrolimus for GVHD prophylaxis. Her neutrophils and platelets engrafted on day 10 and 16, respectively and she was discharged on day +16 post-transplant. Her post-discharge hospital course was complicated by CMV reactivation (no organ involvement) 2 months post-transplant with peak viral level of 1374 copies/ml. She did not have evidence for EBV re-activation or graft-vs-host disease. Her plasma HIV viral load remained undetectable post- transplant while remaining on abacavir/lamivudine and dolutegravir-based ART.Her day+180 bone marrow showed continued AML remission and she remains in clinical remission near one year post-transplant. CD3 chimerism showed 82% haploidentical donor and 8% CBU and 10% recipient on day +15 post-transplant (Figure 1). The chimerism composition switched to 96% CBU by day+34, and became and remained 100% CBU since day+55. CD33 chimerism showed 98% haploidentical donor and 2% cord donor on day+15 post-transplant. It was 81% haploidentical donor and 19% cord on day+55 and became 100% cord by day+100. She has continued CD4, CD8, NK and CD19 cell recovery, with normal T cell subsets currently (Figure 2). Her CD4 count dropped to 27 cells/mm3 at one-month post-transplant and currently is at 673cells/mm3. She is currently 11 months post-transplant and is back to her normal daily activitiesConclusion: Haplo-cord transplantation with CCR5 delta 32/32 CBU resulted in rapid engraftment and immune replacement with dominance of the CCR5 delta 32/32 CBU graft in an HIV-1-infected woman. Successful suppression of HIV-1-replication to clinically undetectable levels was maintained throughout the transplant period for up to one year. . Correlative viral and immunological studies are ongoing, along with effects on the latent reservoir, which was detectable pre-transplant. It is possible to identify appropriate CCR5 delta 32/32 CBU units for haplo-cord transplantation in HIV-1- infected patients with implications for HIV-1 cure. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mutations in chemokine receptors and AIDS.

Chemokines are a class of chemotactic small molecule peptides whose receptors CCR5 and CXCR4 play important role in the entry of human immunodeficiency virus (HIV-1) into immune cells. Chemokines belong to G protein-coupled receptor superfamily containing seven hydrophobic transmembrane helices, causing physiological effects such as chemotaxis, immune regulation, antiviral immunity, regulation of hematopoiesis and angiogenesis, as well as cell growth and metabolism, through certain signaling pathways. Earlier studies have shown that HIV infects the human immune cells by binding to the CD4 receptor. Soon, it was discovered that HIV-1 enters into human immune cells by binding to another receptor, chemokine receptor, which acts as co-receptor for CD4 during the invasion of HIV-1 into cells. Since complex receptor binding is important for HIV-1 invasion, antagonizing the binding has become an attractive and rational drug design goal. Early studies sought to block the interaction between virus and the receptors by chemically modifying the CCR5 and CXCR4 ligands. Although drug treatment is widely used, drug treatment cannot cure AIDS; it can only inhibit the replication of the virus, and HIV/AIDS patients need to take drugs for life. In addition, anti-AIDS drugs also produce side effects such as diseases of the cardiovascular system, nervous system, and metabolic system. In 2006, the emergence of "Berlin patient" led researchers to focus on gene therapy in chemokine receptors. In 2006 and 2007, the attending physician of "Berlin patient" cured his AIDS by transplantation of the stem cells from a donor who was homozygous for the CCR5 Δ32 mutation. This review summarizes the research progress in the mutation in chemokine receptor of HIV/AIDS.

Progress in achieving long-term HIV remission

The purpose of the present review is to describe the major barriers to HIV eradication and assess the most promising cure strategies under investigation. There are significant challenges to achieve HIV eradication. These include the establishment of persistent latently infected cells, systemic chronic immune activation, and immune dysfunction. Since the announcement of the first HIV cure involving the Berlin patient, several attempts to reproduce these results have failed. Thus, it is widely accepted that long-term HIV remission would be a more feasible approach. Optimization of ART, immune-based therapies, therapeutic vaccinations, and gene editing, amongst others, are strategies aimed at controlling HIV in the absence of ART. These new strategies alone or in combination are being developed in preclinical studies and clinical trials and will provide further insight into whether long-term HIV remission is possible. The present review discusses several mechanisms that mediate the persistence of the HIV reservoir, clinical cases that provide hope in finding a functional cure of HIV, and promising interventional strategies being tested in preclinical studies and clinical trials that attempt to reduce the HIV reservoirs and/or boost the immune responses to control HIV in the absence of ART.

Inducing CCR5Δ32/Δ32 Homozygotes in the Human Jurkat CD4+ Cell Line and Primary CD4+ Cells by CRISPR-Cas9 Genome-Editing Technology

C-C chemokine receptor type 5 (CCR5) is the main co-receptor for HIV entry into the target CD4+ cells, and homozygous CCR5Δ32/Δ32 cells are resistant to CCR5-tropic HIV infection. However, the CCR5Δ32/Δ32 homozygous donors in populations are rare. Here we developed a simple approach to induce CCR5Δ32/Δ32 homozygotes through CRISPR-Cas9 genome-editing technology. Designing a pair of single-guide RNA targeting the flank region of the CCR5Δ32 mutation locus, we applied the CRISPR-Cas9 and lentiviral packaging system to successfully convert wild-type CCR5 into CCR5Δ32/Δ32 homozygotes in the human Jurkat CD4+ cell line and primary CD4+ cells, exactly the same as the naturally occurring CCR5Δ32/Δ32 mutation. The successful rate is up to 20% in Jurkat cells but less in primary CD4+ cells. The modified CCR5Δ32/Δ32 CD4+ cells are resistant to CCR5-tropic HIV infection. Whole-genome sequencing revealed no apparent off-target sites. This approach has the promise to promote HIV/AIDS therapy from the only cured unique Berlin patient to a routine autologous cell-based therapy.

The Treatment of HIV by Vibrational Herbal Medicine

The study determines the type of virus, not as a simple but as a set of complex virus system composed of three different complex virus systems. The study also relates how the HIV virus has different energy characteristics not similar to other well known viruses. The energy characteristics of HIV indicate two positive and two negative energy groups. “Normal” viruses,like Hep C, has four negative energy groups. It was finally determined that the cause for the presence of positive and negative energy groups lies in the fact that HIV is composed of three different Major Complex Virus groups and not one monolithic Major Complex Virus like Hep C. All three complex virus systems have to be eliminated to eliminate the HIV virus. Many vibrational herbal mixtures were made over the course of the last 14 years and tried, physically and by simulation, on several patients.The results were mixed until it was discovered that the virus is actually three complex systems in one. Each of the trial mixtures worked on one or two, and not totally fully, on all three systems. The results of our study of the Berlin patient and the Mississippi baby indicate that two of the three viruses were eradicated. Although the main virus in both was the same, the second was different in each, leaving the third virus system untouched. Our latest Vibrational Herbal Mixture eradicates all three complex virus systems, as opposed to the latest chemical experimental treatment which will eliminate the main complex system but will leave the other two, untouched. This may lead to false results as a patient appears to be cured and then the virus reappearing just like the case of the Berlin patient.

Modeling and Understanding the Biology of Transplant-Mediated HIV Cure in a Non-Human Primate Model

Background : The Berlin patient is thus far the only individual considered cured of HIV. Three aspects of the Berlin Patient's treatment are thought to have contributed to his cure following allogeneic hematopoietic stem cell transplantation (HCT): 1) the myeloablative conditioning regimen, 2) transplantation with HIV-resistant cells, and 3) graft-versus-host disease (GVHD). This cure occurred in the context of HCT from an unrelated donor whose cells contained two copies of the CCR5delta32 mutation, which rendered them resistant to CCR5-tropic viruses (the vast majority of the transmitted variants). He also developed GVHD, which is thought to have contributed to his cure by inducing a graft-versus-viral-reservoir (GVVR) effect. While it is still unknown which of these factors was critical to the cure achieved in this patient, the viral rebound observed following allogeneic transplantation with wild-type cells in two Boston patients suggests that HIV-resistance factors may be key to achieving a permanent HIV cure through HCT. Our lab has previously shown that transplantation with autologous unmodified hematopoietic stem cells is not sufficient to eradicate the viral reservoir in a non-human primate (NHP) model of infection. However, the relative contributions of myeloablative conditioning, GVVR, and HIV resistant cells to the clearance of the HIV reservoir have not been rigorously determined. To dissect the impact of each of these factors on the viral reservoir, we have developed the first NHP model of allogeneic bone marrow transplantation (BMT) in Simian/Human Immunodeficiency Virus (SHIV)-infected, combined antiretroviral therapy (cART)-treated rhesus macaques.Methods : We intravenously infected 6 animals with SHIV-1157ipd3N4 and left them untreated for six months, followed by six months of cART (PMPA, FTC, Raltegravir). 3 animals served as untransplanted controls, and 3 animals received haplo-identical BMT following myeloablative total body irradiation (1020 Gy) without cART discontinuation. Donor chimerism was monitored by molecular analysis and by flow cytometry. Plasma viral RNA was measured by RT-PCR, and cell-associated DNA and RNA were quantified by qPCR in 6 tissues longitudinally, and in >20 tissues at necropsy.Results : All animals showed peak SHIV plasma viral loads (1.5e7 copies/ml) 10-11 days post-infection, subsequently reaching viral set points. 1 of 6 animal controlled viremia before cART initiation. In all other animals, plasma viral RNA became undetectable 2-3 weeks post cART initiation. We euthanized the transplant recipients at day 47, 29, and 9 post-transplant, due to infection, graft-versus host disease, and renal complications, respectively. Analysis of whole blood and gated CD4+ T cells showed acquisition of 100% donor blood chimerism at day 29, with lower T cell chimerism in 1 animal. Despite undetectable SHIV plasma viremia post-transplant, the cell-associated SHIV DNA reservoir persisted in multiple tissues, including lymphoid, hematopoietic and major organs, gut and CNS, and was even increased in certain tissues of transplanted animals compared to untransplanted controls.Conclusions : Our results indicate that the DNA reservoir persists and may even increase early after transplantion, despite control of peripheral viremia. Thus, allogeneic HCT is likely associated with an initial loss of anti-HIV immunity leading to an increase in the size of the viral reservoir. This may also explain the rebound observed in the Boston patients. Thus, we propose that reservoir eradication will require additional HIV resistance factors and/or anti-HIV strategies post-transplant to enhance 1) donor cell resistance, and 2) the targeted killing of infected cells. DisclosuresKiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Kean:Regeneron: Research Funding; Bristol Myers Squibb: Consultancy; Juno: Research Funding; Kymab Ltd: Research Funding.

Autologous Stem Cell Transplantation Disrupts Adaptive Immune Responses during Rebound Simian/Human Immunodeficiency Virus Viremia.

Primary HIV-1 infection induces a virus-specific adaptive/cytolytic immune response that impacts the plasma viral load set point and the rate of progression to AIDS. Combination antiretroviral therapy (cART) suppresses plasma viremia to undetectable levels that rebound upon cART treatment interruption. Following cART withdrawal, the memory component of the virus-specific adaptive immune response may improve viral control compared to primary infection. Here, using primary infection and treatment interruption data from macaques infected with simian/human immunodeficiency virus (SHIV), we observe a lower peak viral load but an unchanged viral set point during viral rebound. The addition of an autologous stem cell transplant before cART withdrawal alters viral dynamics: we found a higher rebound set point but similar peak viral loads compared to the primary infection. Mathematical modeling of the data that accounts for fundamental immune parameters achieves excellent fit to heterogeneous viral loads. Analysis of model output suggests that the rapid memory immune response following treatment interruption does not ultimately lead to better viral containment. Transplantation decreases the durability of the adaptive immune response following cART withdrawal and viral rebound. Our model's results highlight the impact of the endogenous adaptive immune response during primary SHIV infection. Moreover, because we capture adaptive immune memory and the impact of transplantation, this model will provide insight into further studies of cure strategies inspired by the Berlin patient.IMPORTANCE HIV patients who interrupt combination antiretroviral therapy (cART) eventually experience viral rebound, the return of viral loads to pretreatment levels. However, the "Berlin patient" remained free of HIV rebound over a decade after stopping cART. His cure is attributed to leukemia treatment that included an HIV-resistant stem cell transplant. Inspired by this case, we studied the impact of stem cell transplantation in a macaque simian/HIV (SHIV) system. Using a mechanistic mathematical model, we found that while primary infection generates an adaptive immune memory response, stem cell transplantation disrupts this learned immunity. The results have implications for HIV cure regimens based on stem cell transplantation.

Novel AgoshRNA molecules for silencing of the CCR5 co-receptor for HIV-1 infection.

Allogeneic transplantation of blood stem cells from a CCR5-Δ32 homozygous donor to an HIV-infected individual, the “Berlin patient”, led to a cure. Since then there has been a search for approaches that mimic this intervention in a gene therapy setting. RNA interference (RNAi) has evolved as a powerful tool to regulate gene expression in a sequence-specific manner and can be used to inactivate the CCR5 mRNA. Short hairpin RNA (shRNA) molecules can impair CCR5 expression, but these molecules may cause unintended side effects and they will not be processed in cells that lack Dicer, such as monocytes. Dicer-independent RNAi pathways have opened opportunities for new AgoshRNA designs that rely exclusively on Ago2 for maturation. Furthermore, AgoshRNA processing yields a single active guide RNA, thus reducing off-target effects. In this study, we tested different AgoshRNA designs against CCR5. We selected AgoshRNAs that potently downregulated CCR5 expression on human T cells and peripheral blood mononuclear cells (PBMC) and that had no apparent adverse effect on T cell development as assessed in a competitive cell growth assay. CCR5 knockdown significantly protected T cells from CCR5 tropic HIV-1 infection.

Loss of immune homeostasis dictates SHIV rebound after stem-cell transplantation.

The conditioning regimen used as part of the Berlin patient's hematopoietic cell transplant likely contributed to his eradication of HIV infection. We studied the impact of conditioning in simian-human immunodeficiency virus-infected (SHIV-infected) macaques suppressed by combination antiretroviral therapy (cART). The conditioning regimen resulted in a dramatic, but incomplete depletion of CD4+ and CD8+ T cells and CD20+ B cells, increased T cell activation and exhaustion, and a significant loss of SHIV-specific Abs. The disrupted T cell homeostasis and markers of microbial translocation positively correlated with an increased viral rebound after cART interruption. Quantitative viral outgrowth and Tat/rev-induced limiting dilution assays showed that the size of the latent SHIV reservoir did not correlate with viral rebound. These findings identify perturbations of the immune system as a mechanism for the failure of autologous transplantation to eradicate HIV. Thus, transplantation strategies may be improved by incorporating immune modulators to prevent disrupted homeostasis, and gene therapy to protect transplanted cells.

TALEN-Mediated Knockout of CCR5 Confers Protection Against Infection of Human Immunodeficiency Virus.

Transcription activator-like effector nuclease (TALEN) represents a valuable tool for genomic engineering due to its single-nucleotide precision, high nuclease activity, and low cytotoxicity. We report here systematic design and characterization of 28 novel TALENs targeting multiple regions of CCR5 gene (CCR5-TALEN) which encodes the co-receptor critical for entry of human immunodeficiency virus type I (HIV-1). By systemic characterization of these CCR5-TALENs, we have identified one (CCR5-TALEN-515) with higher nuclease activity, specificity, and lower cytotoxicity compared with zinc-finger nuclease (CCR5-ZFN) currently undergoing clinical trials. Sequence analysis of target cell line GHOST-CCR5-CXCR4 and human primary CD4 T cells showed that the double-strand breaks at the TALEN targeted sites resulted in truncated or nonfunctional CCR5 proteins thereby conferring protection against HIV-1 infection in vitro. None of the CCR5-TALENs had detectable levels of off-target nuclease activity against the homologous region in CCR2 although substantial level was identified for CCR5-ZFN in the primary CD4 T cells. Our results suggest that the CCR5-TALENs identified here are highly functional nucleases that produce protective genetic alterations to human CCR5. Application of these TALENs directly to the primary CD4 T cells and CD34 hematopoietic stem cells (HSCs) of infected individuals could help to create an immune system resistant to HIV-1 infection, recapitulating the success of "Berlin patient" and serving as an essential first step towards a "functional" cure of AIDS.

An Optimal Control Approach to Structured Treatment Interruptions for HIV Patients: A Personalized Medicine Perspective

Highly Active Antiretroviral Therapy (HAART) has changed the course of human immunodeficiency virus (HIV) treatments since its introduction. However, for many patients, long term continuous HAART is expensive and can include problems with drug toxicity and side effects, as well as increased drug resistance. Because of these reasons, some HIV infected patients will voluntarily terminate HAART. Some of these patients will also interrupt the continuous prescribed therapies for short or long periods. After discontinuing HAART, patients will usually experience a rapid increase in viral load coupled with an immediate decline in CD4+ counts. The canonical example of a patient undergoing unsupervised breaks in HAART is that of the “Berlin patient”. In this case, the patient was able to control viral load in the absence of treatment by cycling HAART on and off due to non-related infections. Due to this patient, interest in the use of structured treatment interruptions (STI) as a mechanism to regulate an HIV infection piqued. This paper describes an optimal control approach to determine STI regimen for HIV patients. The optimal STI was implemented in the context of the receding horizon control (RHC) using a mathematical model for the in-vivo dynamics of an HIV type 1 infection. Using available clinical data, we calibrate the model by estimating on a patient specific basis, a best estimable set of parameters using sensitivity analysis and subset selection. We demonstrate how customized STI protocols can be designed through the variation of control parameters on a patient specific basis.

GENE-CELL THERAPY OF HIV AND HEMATOLOGICAL MALIGNANCES BASED ON HEMATOPOIETIC STEM CELL TRANSPLANTATION AND SITE-SPECIFIC GENOME EDITING

Based on the annual UNAIDS reports the number of HIVinfected patients is continually growing since 1983. Antiretroviral Therapy (ART) allows to prolong life expectancy, but the problem of life quality and overall survival is still remaining. Nowadays, in the era of ART, one of the main cause of mortality in HIV-infected patients is malignancies. Lymphomas play one of the key roles in this group of diseases. The treatment of lymphomas includes combined regiments of chemotherapy with a curative potential. High dose chemotherapy with autologous hematopoietic stem cell transplant (auto-HSCT) is the main path of the treatment for relapsed / refractory lymphomas. In the last few years with a development of the genome editing technology auto-HSCT is becoming one of the most promising methods of HIV treatment. The case of “Berlin patient” when allogeneic HSCT from donor with mutation CCR5-delta32 lead to cure from HIV and proof of concept the efficacy of the gene therapy for HIV based on HSCT. Hematopoietic stem cell transplantation with edited autologous HSC (CCR5 knockout by site-specific genome editing tools with engineering nucleases) is a comprehensive treatment for this cohort of patients. On one hand, high dose chemotherapy with auto-HSCT cures the malignancy; on the other hand auto-HSCT works as a delivery method for the edited cells and creates an environment for the HIV eradication. This review is dedicated to HIV and oncology, methods of treatment of hematological malignancies and HIV-infection using genome editing technology based on HSCT.