Christopher W. Peterson

Constructing schemes with prescribed cohomology in arbitrary codimension

Abstract In this paper we show how to construct varieties of arbitrary dimension in P n with prescribed intermediate cohomology modules (up to shift). If the codimension is not too small then we use Kleimans Bertini theorems to guarantee that the varieties are integral or even smooth.

CCR5-edited gene therapies for HIV cure: Closing the door to viral entry

Human immunodeficiency virus (HIV) was first reported and characterized more than three decades ago. Once thought of as a death sentence, HIV infection has become a chronically manageable disease. However, it is estimated that a staggering 0.8% of the worlds population is infected with HIV, with more than 1 million deaths reported in 2015 alone. Despite the development of effective anti-retroviral drugs, a permanent cure has only been documented in one patient to date. In 2007, an HIV-positive patient received a bone marrow transplant to treat his leukemia from an individual who was homozygous for a mutation in the CCR5 gene. This mutation, known as CCR5Δ32, prevents HIV replication by inhibiting the early stage of viral entry into cells, resulting in resistance to infection from the majority of HIV isolates. More than 10 years after his last dose of anti-retroviral therapy, the transplant recipient remains free of replication-competent virus. Multiple groups are now attempting to replicate this success through the use of other CCR5-negative donor cell sources. Additionally, developments in the use of lentiviral vectors and targeted nucleases have opened the doors of precision medicine and enabled new treatment methodologies to combat HIV infection through targeted ablation or down-regulation of CCR5 expression. Here, we review historical cases of CCR5-edited cell-based therapies, current clinical trials and future benefits and challenges associated with this technology.

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

The conditioning regimen used as part of the Berlin patients 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.

Differential impact of transplantation on peripheral and tissue-associated viral reservoirs: Implications for HIV gene therapy

Autologous transplantation and engraftment of HIV-resistant cells in sufficient numbers should recapitulate the functional cure of the Berlin Patient, with applicability to a greater number of infected individuals and with a superior safety profile. A robust preclinical model of suppressed HIV infection is critical in order to test such gene therapy-based cure strategies, both alone and in combination with other cure strategies. Here, we present a nonhuman primate (NHP) model of latent infection using simian/human immunodeficiency virus (SHIV) and combination antiretroviral therapy (cART) in pigtail macaques. We demonstrate that transplantation of CCR5 gene-edited hematopoietic stem/progenitor cells (HSPCs) persist in infected and suppressed animals, and that protected cells expand through virus-dependent positive selection. CCR5 gene-edited cells are readily detectable in tissues, namely those closely associated with viral reservoirs such as lymph nodes and gastrointestinal tract. Following autologous transplantation, tissue-associated SHIV DNA and RNA levels in suppressed animals are significantly reduced (p ≤ 0.05), relative to suppressed, untransplanted control animals. In contrast, the size of the peripheral reservoir, measured by QVOA, is variably impacted by transplantation. Our studies demonstrate that CCR5 gene editing is equally feasible in infected and uninfected animals, that edited cells persist, traffic to, and engraft in tissue reservoirs, and that this approach significantly reduces secondary lymphoid tissue viral reservoir size. Our robust NHP model of HIV gene therapy and viral persistence can be immediately applied to the investigation of combinatorial approaches that incorporate anti-HIV gene therapy, immune modulators, therapeutic vaccination, and latency reversing agents.

Long-term persistence and function of hematopoietic stem cell-derived chimeric antigen receptor T cells in a nonhuman primate model of HIV/AIDS

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.

Cell and Gene Therapy for HIV Cure

As the HIV pandemic rapidly spread worldwide in the 1980s and 1990s, a new approach to treat cancer, genetic diseases, and infectious diseases was also emerging. Cell and gene therapy strategies are connected with human pathologies at a fundamental level, by delivering DNA and RNA molecules that could correct and/or ameliorate the underlying genetic factors of any illness. The history of HIV gene therapy is especially intriguing, in that the virus that was targeted was soon co-opted to become part of the targeting strategy. Today, HIV-based lentiviral vectors, along with many other gene delivery strategies, have been used to evaluate HIV cure approaches in cell culture, small and large animal models, and in patients. Here, we trace HIV cell and gene therapy from the earliest clinical trials, using genetically unmodified cell products from the patient or from matched donors, through current state-of-the-art strategies. These include engineering HIV-specific immunity in T-cells, gene editing approaches to render all blood cells in the body HIV-resistant, and most importantly, combination therapies that draw from both of these respective offensive and defensive approaches. It is widely agreed upon that combinatorial approaches are the most promising route to functional cure/remission of HIV infection. This chapter outlines cell and gene therapy strategies that are poised to play an essential role in eradicating HIV-infected cells in vivo.

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

ABSTRACT 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 models 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.

Evidence for persistence of the SHIV reservoir early after MHC haploidentical hematopoietic stem cell transplantation

Allogeneic transplantation (allo-HCT) has led to the cure of HIV in one individual, raising the question of whether transplantation can eradicate the HIV reservoir. To test this, we here present a model of allo-HCT in SHIV-infected, cART-suppressed nonhuman primates. We infect rhesus macaques with SHIV-1157ipd3N4, suppress them with cART, then transplant them using MHC-haploidentical allogeneic donors during continuous cART. Transplant results in ~100% myeloid donor chimerism, and up to 100% T-cell chimerism. Between 9 and 47 days post-transplant, terminal analysis shows that while cell-associated SHIV DNA levels are reduced in the blood and in lymphoid organs post-transplant, the SHIV reservoir persists in multiple organs, including the brain. Sorting of donor-vs.-recipient cells reveals that this reservoir resides in recipient cells. Moreover, tetramer analysis indicates a lack of virus-specific donor immunity post-transplant during continuous cART. These results suggest that early post-transplant, allo-HCT is insufficient for recipient reservoir eradication despite high-level donor chimerism and GVHD.Allogeneic hematopoietic cell transplantation (allo-HCT) has led to the cure of HIV in one individual, but the underlying mechanisms are unclear. Here, the authors present a model of allo-HCT in SHIV-infected nonhuman primates and show that the SHIV reservoir persists in multiple tissues early after transplantation.

Efficient Enrichment of Gene-Modified Primary T Cells via CCR5-Targeted Integration of Mutant Dihydrofolate Reductase

Targeted gene therapy strategies utilizing homology-driven repair (HDR) allow for greater control over transgene integration site, copy number, and expression—significant advantages over traditional vector-mediated gene therapy with random genome integration. However, the relatively low efficiency of HDR-based strategies limits their clinical application. Here, we used HDR to knock in a mutant dihydrofolate reductase (mDHFR) selection gene at the gene-edited CCR5 locus in primary human CD4+ T cells and selected for mDHFR-modified cells in the presence of methotrexate (MTX). Cells were transfected with CCR5-megaTAL nuclease mRNA and transduced with adeno-associated virus containing an mDHFR donor template flanked by CCR5 homology arms, leading to up to 40% targeted gene insertion. Clinically relevant concentrations of MTX led to a greater than 5-fold enrichment for mDHFR-modified cells, which maintained a diverse TCR repertoire over the course of expansion and drug selection. Our results demonstrate that mDHFR/MTX-based selection can be used to enrich for gene-modified T cells ex vivo, paving the way for analogous approaches to increase the percentage of HIV-resistant, autologous CD4+ T cells infused into HIV+ patients, and/or for in vivo selection of gene-edited T cells for the treatment of cancer.

Long-Term Persistence of Anti-HIV Broadly Neutralizing Antibody-Secreting Hematopoietic Cells in Humanized Mice

Broadly neutralizing antibodies (bNAbs) are among the most promising strategies to achieve long-term control of HIV-1 in the absence of combination antiretroviral therapy. Passive administration of such antibodies in patients efficiently decreases HIV-1 viremia, but is limited by the serum half-life of the protein. Here, we investigated whether antibody-secreting hematopoietic cells could overcome this problem. We genetically modified human CD34+ hematopoietic stem and progenitor cells (HSPCs) to secrete bNAbs and transplanted them into immunodeficient mice. We found that the gene-modified cells engraft and stably secrete antibodies in the peripheral blood of the animals for the 9xa0months of the study. Antibodies were predominantly expressed by human HSPC-derived T- and B cells. Importantly, we found that secreted PGT128 was able to delay HIV-1 viremia inxa0vivo and also prevent a decline in CD4+ cells. Gene-modified cells were maintained in bone marrow and were also detected in spleen, thymus, lymph nodes, and gut-associated lymphoid tissue. These data indicate that the bNAb secretion from HSPC-derived cells in mice is functional and can affect viral infection and CD4+ cell maintenance. This study paves the way for potential applications to other diseases requiring long-lasting protein or antibody delivery.