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PDF file - 243KB, Lack of autoreactivity to bone marrow cells in patient F5-10.

PDF file - 148KB, Supplementary Table 1. Toxicities and response to therapy with the subsequent protocol amendments.

PDF file - 190KB, Cytokine production by multiplex assay in plasma from patients F5-12 and F5-14 to study the potential development of a cytokine storm.

PDF file - 132KB, Recall whole body rash and re-expansion of the TCR transgenic cells in peripheral blood of patient F5-13 with subsequent MART-1/DC vaccination.

Purpose: It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short 1-week manufacture protocol to determine the feasibility, safety, and antitumor efficacy of this double cell therapy.Experimental Design: A clinical trial (NCT00910650) adoptively transferring MART-1 T-cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide-pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and reinfused with (n = 10) or without (n = 3) prior cryopreservation.Results: A total of 14 patients with metastatic melanoma were enrolled and 9 of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1–specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1–specific T cells in the blood as compared with cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using noncryopreserved T cells.Conclusion: Double cell therapy with ACT of TCR-engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses. Clin Cancer Res; 20(9); 2457–65. ©2014 AACR.

PDF file - 127KB, Number and function of TCR transgenic cells infused.

PDF file - 64KB, Comparison of IL-17 production by F5-10 infused TCR transgenic cells stimulated with MART-1-pulsed K562 A2.1 cells with that of patients who did not develop pancytopenia.

PDF file - 470KB, Chest X-rays of patients F5-12 and 14 who developed SAEs of acute respiratory distress.

PDF file - 278KB, Pre- and post-treatment PET scans showing evidence of major tumor response and MART-1-specific TCR transgenic cell levels in patient F5-10.

PDF file - 82KB, TCR transgenic cell therapy manufacture protocol modifications.

PDF file - 128KB, Transduction efficiencies of infused transgenic cells.

Due to the durability and persistence of reservoirs of HIV-1-infected cells, combination antiretroviral therapy (ART) is insufficient in eradicating infection. Achieving HIV-1 cure or sustained remission without ART treatment will require the enhanced and persistent effective antiviral immune responses. Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy and show promise in treating HIV-1 infection. Persistence, trafficking, and maintenance of function remain to be a challenge in many of these approaches, which are based on peripheral T cell modification. To overcome many of these issues, we have previously demonstrated successful long-term engraftment and production of anti-HIV CAR T cells in modified hematopoietic stem cells (HSCs) in vivo. Here we report the development and in vivo testing of second generation CD4-based CARs (CD4CAR) against HIV-1 infection using a HSCs-based approach. We found that a modified, truncated CD4-based CAR (D1D2CAR) allows better CAR-T cell differentiation from gene modified HSCs, and maintains similar CTL activity as compared to the full length CD4-based CAR. In addition, D1D2CAR does not mediate HIV infection or stimulation mediated by IL-16, suggesting lower risk of off-target effects. Interestingly, stimulatory domains of 4-1BB but not CD28 allowed successful hematopoietic differentiation and improved anti-viral function of CAR T cells from CAR modified HSCs. Addition of 4-1BB to CD4 based CARs led to faster suppression of viremia during early untreated HIV-1 infection. D1D2CAR 4-1BB mice had faster viral suppression in combination with ART and better persistence of CAR T cells during ART. In summary, our data indicate that the D1D2CAR-41BB is a superior CAR, showing better HSC differentiation, viral suppression and persistence, and less deleterious functions compared to the original CD4CAR, and should continue to be pursued as a candidate for clinical study., Author summary Engineering T cells with anti-HIV chimeric antigen receptors (CAR) has emerged as a promising strategy to control HIV infection through a genetic vaccination strategy. Here we report a novel CAR-based approach targeting HIV infection using the genetic modification of blood forming hematopoietic stem cells (HSCs). This novel CAR approach uses a modified HIV receptor molecule (the primary HIV receptor CD4) as well as anti-HIV agents to modify HSCs to allow them to develop into cells that are protected from HIV infection and target HIV infected cells for the life of the individual. We found this latest generation of CARs successfully modified and allowed in vivo engraftment that resulted in the development of effective anti-HIV CAR T cells with robust memory formation and viral control. Our study highlights the identification of a next-generation CAR molecule that protected cells from infection, targeted and reduced HIV burdens, and serves as an ideal developmental candidate for further clinical studies.

Summary “Shock and kill” strategies focus on purging the latent HIV-1 reservoir by treating infected individuals with therapeutics that activate the latent virus and subsequently eliminating infected cells. We have previously reported that induction of non-canonical nuclear factor κB (NF-κB) signaling through a class of small-molecule antagonists known as Smac mimetics can reverse HIV-1 latency. Here, we describe the development of Ciapavir (SBI-0953294), a molecule specifically optimized for HIV-1 latency reversal that was found to be more efficacious as a latency-reversing agent than other Smac mimetics under clinical development for cancer. Critically, this molecule induced activation of HIV-1 reservoirs in vivo in a bone marrow, liver, thymus (BLT) humanized mouse model without mediating systemic T cell activation. This study provides proof of concept for the in vivo efficacy and safety of Ciapavir and indicates that Smac mimetics can constitute a critical component of a safe and efficacious treatment strategy to eliminate the latent HIV-1 reservoir., Graphical Abstract, Highlights Ciapavir is a potent Smac mimetic specifically optimized for HIV-1 latency reversal Smac mimetics synergize with bromodomain inhibitors to reverse HIV-1 latency Ciapavir is well tolerated and does not induce broad immune activation Systemic administration of Ciapavir mediates latency reversal in a mouse model, Pache et al. report the development of Ciapavir, a potent small-molecule Smac mimetic optimized for HIV-1 latency reversal. Ciapavir shows favorable pharmacokinetic and pharmacodynamic properties in mice and induces activation of the latent HIV-1 reservoir in vivo in a humanized mouse model in the absence of systemic immune activation.

AIDS is a pandemic disease caused by HIV that affects 37 million people worldwide. Current antiretroviral therapy slows disease progression but does not eliminate latently infected cells, which resupply active virus, thus necessitating lifelong treatment with associated compliance, cost, and chemoexposure issues. Latency-reversing agents (LRAs) activate these cells, allowing for their potential clearance, thus presenting a strategy to eradicate the infection. Protein kinase C (PKC) modulators-including prostratin, ingenol esters, bryostatin, and their analogs-are potent LRAs in various stages of development for several clinical indications. While LRAs are promising, a major challenge associated with their clinical use is sustaining therapeutically meaningful levels of the active agent while minimizing side effects. Here we describe a strategy to address this problem based on LRA prodrugs, designed for controllable release of the active LRA after a single injection. As intended, these prodrugs exhibit comparable or superior in vitro activity relative to the parent compounds. Selected compounds induced higher in vivo expression of CD69, an activation biomarker, and, by releasing free agent over time, significantly improved tolerability when compared to the parent LRAs. More generally, selected prodrugs of PKC modulators avoid the bolus toxicities of the parent drug and exhibit greater efficacy and expanded tolerability, thereby addressing a longstanding objective for many clinical applications.

Summary HIV latency prevents cure of infection with antiretroviral therapy (ART) alone. One strategy for eliminating latently infected cells involves the induction of viral protein expression via latency-reversing agents (LRAs), allowing killing of host cells by viral cytopathic effects or immune effector mechanisms. Here, we combine a barcoded HIV approach and a humanized mouse model to study the effects of a designed, synthetic protein kinase C modulating LRA on HIV rebound. We show that administration of this compound during ART results in a delay in rebound once ART is stopped. Furthermore, the rebounding virus appears composed of a smaller number of unique barcoded viruses than occurs in control-treated animals, suggesting that some reservoir cells that would have contributed virus to the rebound process are eliminated by LRA administration. These data support the use of barcoded virus to study rebound and suggest that LRAs may be useful in HIV cure efforts., Graphical Abstract, Highlights A genetically barcoded HIV swarm has been constructed and characterized This swarm forms a latent reservoir in antiretroviral (ART)-treated humanized mice Administering an HIV latency-reversing agent (LRA) during ART delays rebound LRA administration during ART also reduces barcode diversity of rebounding virus, Marsden et al. construct a barcoded HIV swarm to study HIV latency formation and rebound in humanized mice. They find that the administration of a PKC modulating HIV latency-reversing agent delays rebound and reduces the genetic diversity of rebounding virus when antiretroviral therapy is stopped, suggesting the reduction of the reservoir.

Bryostatin 1 is a marine natural product under investigation for HIV/AIDS eradication, the treatment of neurological disorders, and enhanced CAR T/NK cell immunotherapy. Despite its promising activity, bryostatin 1 is neither evolved nor optimized for the treatment of human disease. Here we report the design, synthesis, and biological evaluation of several close-in analogs of bryostatin 1. Using a function-oriented synthesis approach, we synthesize a series of bryostatin analogs designed to maintain affinity for bryostatin’s target protein kinase C (PKC) while enabling exploration of their divergent biological functions. Our late-stage diversification strategy provides efficient access to a library of bryostatin analogs, which per our design retain affinity for PKC but exhibit variable PKC translocation kinetics. We further demonstrate that select analogs potently increase cell surface expression of CD22, a promising CAR T cell target for the treatment of leukemias, highlighting the clinical potential of bryostatin analogs for enhancing targeted immunotherapies., Bryostatin 1 is a unique therapeutic lead, however its scarce natural sources have hampered its use in treatment of human disease. Here, the authors use a scalable synthesis of bryostatin 1 to make close-in analogs which potently induce increased cell surface expression holding potential for immunotherapy.

Human immunodeficiency virus (HIV) remains a significant source of morbidity and mortality worldwide. No effective vaccine is available to prevent HIV transmission, and although antiretroviral therapy can prevent disease progression, it does not cure HIV infection. Substantial effort is therefore currently directed toward basic research on HIV pathogenesis and persistence and developing methods to stop the spread of the HIV epidemic and cure those individuals already infected with HIV. Humanized mice are versatile tools for the study of HIV and its interaction with the human immune system. These models generally consist of immunodeficient mice transplanted with human cells or reconstituted with a near-complete human immune system. Here, we describe the major humanized mouse models currently in use, and some recent advances that have been made in HIV research/therapeutics using these models.

HIV infection continues to be a life-long chronic disease in spite of the success of antiretroviral therapy (ART) in controlling viral replication and preventing disease progression. However, because of the high cost of treatment, severe side effects, and inefficiency in curing the disease with ART, there is a call for alternative therapies that will provide a functional cure for HIV. Cytotoxic T lymphocytes (CTLs) are vital in the control and clearance of viral infections and therefore immune-based therapies have attempted to engineer HIV-specific CTLs that would be able to clear the infection from the body. The development of chimeric antigen receptors (CARs) provides an opportunity to engineer superior HIV-specific CTLs that will be independent of the major histocompatibility complex for target recognition. A CD4-based CAR has been previously tested in clinical trials to test the antiviral efficacy of peripheral T cells armed with this CD4-based CAR. The results from these clinical trials showed the safety and feasibility of CAR T cell therapy for HIV infection; however, minimal antiviral efficacy was seen. In this review, we will discuss the various strategies being developed to enhance the therapeutic potency of anti-HIV CARs with the goal of generating superior antiviral responses that will lead to life-long HIV immunity and clearance of the virus from the body.

Combination anti-retroviral drug therapy (ART) potently suppresses HIV-1 replication but does not result in virus eradication or a cure. A major contributing factor is the long-term persistence of a reservoir of latently infected cells. To study this reservoir, we established a humanized mouse model of HIV-1 infection and ART suppression based on an oral ART regimen. Similar to humans, HIV-1 levels in the blood of ART-treated animals were frequently suppressed below the limits of detection. However, the limited timeframe of the mouse model and the small volume of available samples makes it a challenging model with which to achieve full viral suppression and to investigate the latent reservoir. We therefore used an ex vivo latency reactivation assay that allows a semiquantitative measure of the latent reservoir that establishes in individual animals, regardless of whether they are treated with ART. Using this assay, we found that latently infected human CD4 T cells can be readily detected in mouse lymphoid tissues and that latent HIV-1 was enriched in populations expressing markers of T cell exhaustion, PD-1 and TIGIT. In addition, we were able to use the ex vivo latency reactivation assay to demonstrate that HIV-specific TALENs can reduce the fraction of reactivatable virus in the latently infected cell population that establishes in vivo, supporting the use of targeted nuclease-based approaches for an HIV-1 cure. IMPORTANCE HIV-1 can establish latent infections that are not cleared by current antiretroviral drugs or the body’s immune responses and therefore represent a major barrier to curing HIV-infected individuals. However, the lack of expression of viral antigens on latently infected cells makes them difficult to identify or study. Here, we describe a humanized mouse model that can be used to detect latent but reactivatable HIV-1 in both untreated mice and those on ART and therefore provides a simple system with which to study the latent HIV-1 reservoir and the impact of interventions aimed at reducing it.

[This corrects the article DOI: 10.1371/journal.ppat.1009404.].

Although the use of chimeric antigen receptors (CARs) based on single-chain antibodies for gene immunotherapy of cancers is increasing due to promising recent results, the earliest CAR therapeutic trials were done for HIV-1 infection in the late 1990s. This approach utilized a CAR based on human CD4 as a binding domain and was abandoned for a lack of efficacy. The growing number of HIV-1 broadly neutralizing antibodies (BNAbs) offers the opportunity to generate novel CARs that may be more active and revisit this modality for HIV-1 immunotherapy. We used sequences from seven well-defined BNAbs varying in binding sites and generated single-chain-antibody-based CARs. These CARs included 10E8, 3BNC117, PG9, PGT126, PGT128, VRC01, and X5. Each novel CAR exhibited conformationally relevant expression on the surface of transduced cells, mediated specific proliferation and killing in response to HIV-1-infected cells, and conferred potent antiviral activity (reduction of viral replication in log 10 units) to transduced CD8 + T lymphocytes. The antiviral activity of these CARs was reproducible but varied according to the strain of virus. These findings indicated that BNAbs are excellent candidates for developing novel CARs to consider for the immunotherapeutic treatment of HIV-1. IMPORTANCE While chimeric antigen receptors (CARs) using single-chain antibodies as binding domains are growing in popularity for gene immunotherapy of cancers, the earliest human trials of CARs were done for HIV-1 infection. However, those trials failed, and the approach was abandoned for HIV-1. The only tested CAR against HIV-1 was based on the use of CD4 as the binding domain. The growing availability of HIV-1 broadly neutralizing antibodies (BNAbs) affords the opportunity to revisit gene immunotherapy for HIV-1 using novel CARs based on single-chain antibodies. Here we construct and test a panel of seven novel CARs based on diverse BNAb types and show that all these CARs are functional against HIV-1.

Mutations in DMD disrupt the reading frame, prevent dystrophin translation, and cause Duchenne muscular dystrophy (DMD). Here we describe a CRISPR/Cas9 platform applicable to 60% of DMD patient mutations. We applied the platform to DMD-derived hiPSCs where successful deletion and non-homologous end joining of up to 725 kb reframed the DMD gene. This is the largest CRISPR/Cas9-mediated deletion shown to date in DMD. Use of hiPSCs allowed evaluation of dystrophin in disease-relevant cell types. Cardiomyocytes and skeletal muscle myotubes derived from reframed hiPSC clonal lines had restored dystrophin protein. The internally deleted dystrophin was functional as demonstrated by improved membrane integrity and restoration of the dystrophin glycoprotein complex in vitro and in vivo. Furthermore, miR31 was reduced upon reframing, similar to observations in Becker muscular dystrophy. This work demonstrates the feasibility of using a single CRISPR pair to correct the reading frame for the majority of DMD patients.

Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation.We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions.Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function.This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by corroborating findings of others, and providing important new information on essential RPE cell biological properties.

Invariant natural killer T (iNKT) cells are potent immune cells for targeting cancer; however, their clinical application has been greatly hindered by their extremely low numbers in cancer patients. Here, we developed a proof-of-concept for hematopoietic stem cell-engineered iNKT (HSC-iNKT) cell therapy with the potential to provide therapeutic levels of iNKT cells for a patient’s lifetime. Using a human HSC engrafted mouse model and a human iNKT TCR gene engineering approach, we demonstrated the efficient and long-term generation of HSC-iNKT cells in vivo. These HSC-iNKT cells closely resembled endogenous human iNKT cells, could deploy multiple mechanisms to attack tumor cells, and effectively suppressed tumor growth in vivo in multiple human tumor xenograft mouse models. Preclinical safety studies showed no toxicity or tumorigenicity of the intended HSC-iNKT cell therapy. Collectively, these results demonstrated the feasibility, safety, and cancer therapy potential of the proposed HSC-iNKT cell therapy and laid a foundation for future clinical development.

Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy for various forms of cancer and show promise in treating HIV-1 infection. However, significant limitations are persistence and whether peripheral T cell-based products can respond to malignant or infected cells that may reappear months or years after treatment remains unclear. Hematopoietic Stem/Progenitor Cells (HSPCs) are capable of long-term engraftment and have the potential to overcome these limitations. Here, we report the use of a protective CD4 chimeric antigen receptor (C46CD4CAR) to redirect HSPC-derived T-cells against simian/human immunodeficiency virus (SHIV) infection in pigtail macaques. CAR-containing cells persisted for more than 2 years without any measurable toxicity and were capable of multilineage engraftment. Combination antiretroviral therapy (cART) treatment followed by cART withdrawal resulted in lower viral rebound in CAR animals relative to controls, and demonstrated an immune memory-like response. We found CAR-expressing cells in multiple lymphoid tissues, decreased tissue-associated SHIV RNA levels, and substantially higher CD4/CD8 ratios in the gut as compared to controls. These results show that HSPC-derived CAR T-cells are capable of long-term engraftment and immune surveillance. This study demonstrates for the first time the safety and feasibility of HSPC-based CAR therapy in a large animal preclinical model.

The human immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte (CTL) response is critical in controlling HIV infection. Since the immune response does not eliminate HIV, it would be beneficial to develop ways to enhance the HIV-specific CTL response to allow long-term viral suppression or clearance. Here, we report the use of a protective chimeric antigen receptor (CAR) in a hematopoietic stem/progenitor cell (HSPC)-based approach to engineer HIV immunity. We determined that CAR-modified HSPCs differentiate into functional T cells as well as natural killer (NK) cells in vivo in humanized mice and these cells are resistant to HIV infection and suppress HIV replication. These results strongly suggest that stem cell-based gene therapy with a CAR may be feasible and effective in treating chronic HIV infection and other morbidities.

Many important aspects of human retroviral infections cannot be fully evaluated using only in vitro systems or unmodified animal models. An alternative approach involves the use of humanized mice, which consist of immunodeficient mice that have been transplanted with human cells and/or tissues. Certain humanized mouse models can support robust infection with human retroviruses including different strains of human immunodeficiency virus (HIV) and human T cell leukemia virus (HTLV). These models have provided wide-ranging insights into retroviral biology, including detailed information on primary infection, in vivo replication and pathogenesis, latent/persistent reservoir formation, and novel therapeutic interventions. Here we describe the humanized mouse models that are most commonly utilized to study retroviral infections, and outline some of the important discoveries that these models have produced during several decades of intensive research.

SummaryX-linked Severe Combined Immunodeficiency (SCID-X1) is a genetic disease that leaves newborns at high risk of serious infection and a predicted life span of less than 1 year in the absence of a matched bone marrow donor. The disease pathogenesis is due to mutations in the gene encoding the Interleukin-2 receptor gamma chain (IL-2Rγ), leading to a lack of functional lymphocytes. With the leukemogenic concerns of viral gene therapy there is a need to explore alternative therapeutic options. We have utilized induced pluripotent stem cell (iPSC) technology and genome editing mediated by TALENs to generate isogenic subject-specific mutant and gene-corrected iPSC lines. While the subject-derived mutant iPSCs have the capacity to generate hematopoietic precursors and myeloid cells, only wild-type and gene-corrected iPSCs can additionally generate mature NK cells and T cell precursors expressing the correctly spliced IL-2Rγ. This study highlights the potential for the development of autologous cell therapy for SCID-X1 subjects.

Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy for various forms of cancer and show promise in treating HIV-1 infection. However, significant limitations are persistence and whether peripheral T cell-based products can respond to malignant or infected cells that may reappear months or years after treatment remains unclear. Hematopoietic Stem/Progenitor Cells (HSPCs) are capable of long-term engraftment and have the potential to overcome these limitations. Here, we report the use of a protective CD4 chimeric antigen receptor (C46CD4CAR) to redirect HSPC-derived T-cells against simian/human immunodeficiency virus (SHIV) infection in pigtail macaques. CAR-containing cells persisted for more than 2 years without any measurable toxicity and were capable of multilineage engraftment. Combination antiretroviral therapy (cART) treatment followed by cART withdrawal resulted in lower viral rebound in CAR animals relative to controls, and demonstrated an immune memory-like response. We found CAR-expressing cells in multiple lymphoid tissues, decreased tissue-associated SHIV RNA levels, and substantially higher CD4/CD8 ratios in the gut as compared to controls. These results show that HSPC-derived CAR T-cells are capable of long-term engraftment and immune surveillance. This study demonstrates for the first time the safety and feasibility of HSPC-based CAR therapy in a large animal preclinical model., Author summary Hematopoietic Stem/Progenitor Cell (HSPC) based gene therapy can be used to treat many infectious and genetic diseases. Here, we used an HSPC-based approach to redirect and enhance host immunity against HIV-1. We engineered HSPCs to carry chimeric antigen receptor (CAR) genes that detect and destroy HIV-infected cells. CAR therapy has shown huge potential in the treatment of cancer, but has only been applied in peripheral blood T-cells. HSPC-based CAR therapy has several benefits over T cell gene therapy, as it allows for normal T cell development, selection, and persistence of the engineered cells for the lifetime of the patient. We used a CAR molecule that hijacks the essential interaction between the virus and the cell surface molecule CD4 to redirect HSPC-derived T-cells against infected cells. We observed >2 years of stable production of CAR-expressing cells without any adverse events, and wide distribution of these cells in lymphoid tissues and gastrointestinal tract, which are major anatomic sites for HIV replication and persistence in suppressed patients. Most importantly, HSPC-derived CAR T-cells functionally responded to infected cells. This study demonstrates for the first time the safety and feasibility of HSPC based therapy utilizing an HIV-specific CAR for suppressed HIV infection.

We report a novel approach to a new class of bioengineered, monodispersed, self-assembling vault nanoparticles consisting of a protein shell exterior with a lipophilic core interior designed for drug and probe delivery. Recombinant vaults were engineered to contain a small amphipathic α-helix derived from the nonstructural protein 5A of hepatitis C virus, thereby creating within the vault lumen a lipophilic microenvironment into which lipophilic compounds could be reversibly encapsulated. Multiple types of electron microscopy showed that attachment of this peptide resulted in larger than expected additional mass internalized within the vault lumen attributable to incorporation of host lipid membrane constituents spanning the vault waist (>35 nm). These bioengineered lipophilic vaults reversibly associate with a sample set of therapeutic compounds, including all-trans retinoic acid, amphotericin B, and bryostatin 1, incorporating hundreds to thousands of drug molecules per vault nanoparticle. Bryostatin 1 is of particular therapeutic interest because of its ability to potently induce expression of latent HIV, thus representing a preclinical lead in efforts to eradicate HIV/AIDS. Vaults loaded with bryostatin 1 released free drug, resulting in activation of HIV from provirus latency in vitro and induction of CD69 biomarker expression following intravenous injection into mice. The ability to preferentially and reversibly encapsulate lipophilic compounds into these novel bioengineered vault nanoparticles greatly advances their potential use as drug delivery systems.

Highly active antiretroviral therapy (HAART) decreases plasma viremia below the limits of detection in the majority of HIV-infected individuals, thus serving to slow disease progression. However, HAART targets only actively replicating virus and is unable to eliminate latently infected, resting CD4 + T cells. Such infected cells are potentially capable of reinitiating virus replication upon cessation of HAART, thus leading to viral rebound. Agents that would eliminate these reservoirs, when used in combination with HAART, could thus provide a strategy for the eradication of HIV. Prostratin is a preclinical candidate that induces HIV expression from latently infected CD4 + T cells, potentially leading to their elimination through a virus-induced cytopathic effect or host anti-HIV immunity. Here, we report the synthesis of a series of designed prostratin analogs and report in vitro and ex vivo studies of their activity relevant to induction of HIV expression. Members of this series are up to 100-fold more potent than the preclinical lead (prostratin) in binding to cell-free PKC, and in inducing HIV expression in a latently infected cell line and prostratin-like modulation of cell surface receptor expression in primary cells from HIV-negative donors. Significantly, selected members were also tested for HIV induction in resting CD4 + T cells isolated from infected individuals receiving HAART and were found to exhibit potent induction activity. These more potent agents and by extension related tunable analogs now accessible through the studies described herein should facilitate research and preclinical advancement of this strategy for HIV/AIDS eradication.

Interferons (IFN) are essential antiviral cytokines that establish the cellular antiviral state through upregulation of hundreds of interferon-stimulated genes (ISGs), most of which have uncharacterized functions and mechanisms. We identified Cholesterol-25-hydroxylase (Ch25h) as an antiviral ISG that can convert cholesterol to a soluble antiviral factor, 25-hydroxycholesterol (25HC). Ch25h expression or 25HC treatment in cultured cells broadly inhibits enveloped viruses including VSV, HSV, HIV, and MHV68 as well as acutely pathogenic EBOV, RVFV, RSSEV, and Nipah viruses under BSL4 conditions. As a soluble oxysterol, 25HC inhibits viral entry by blocking membrane fusion between virus and cell. In animal models, Ch25h-knockout mice were more susceptible to MHV68 lytic infection. Moreover, administration of 25HC in humanized mice suppressed HIV replication and rescued T-cell depletion. Thus, our studies demonstrate a unique mechanism by which IFN achieves its antiviral state through the production of a natural oxysterol to inhibit viral entry and implicate membrane-modifying oxysterols as potential antiviral therapeutics.

HIV remains a highly important public health and clinical issue despite many recent advances in attempting to develop a cure, which has remained elusive for most people infected with HIV. HIV disease can be controlled with pharmacologic therapies; however, these treatments are expensive, may have severe side effects, and are not curative. Consequently, an improved means to control or eliminate HIV replication is needed. Cytotoxic T lymphocytes (CTLs) play a critical role in controlling viral replication and are an important part in the ability of the immune response to eradicate most viral infections. There are considerable efforts to enhance CTL responses in HIV-infected individuals in hopes of providing the immune response with armaments to more effectively control viral replication. In this review, we discuss some of these efforts and focus on the development of a gene therapy-based approach to engineer hematopoietic stem cells with an HIV-1-specific chimeric antigen receptor, which seeks to provide an inexhaustible source of HIV-1-specific immune cells that are MHC unrestricted and superior to natural antiviral T cell responses. These efforts provide the basis for further development of T cell functional enhancement to target and treat chronic HIV infection in hopes of eradicating the virus from the body.

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (cγ(-/-))-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.