Priti Kumar - US grants

DESCRIPTION (provided by applicant): HIV transmission from infected to uninfected cells is known to be more efficient than infection with cell-free virus. In vitro, cell-cell contact stimulates HIV replication 100-10,000 fold. The ability of HIV to utilize and manipulate cell-cell contact for the purpose of efficient transmission critically contributes to the spreading of infection throughout the body and the progression to AIDS. Cell-associated HIV is also thought to play an important role during sexual as well as mother to child transmissions. Despite its importance, very little is known about the molecular mechanisms that govern contact-dependent cell to cell spread and no antiviral therapy has been directed against this step. Here we propose a team science approach to develop antiviral therapies that specifically prevent HIV cell-to-cell transmission. We will take advantage of the first robust cell-to-cell transmission assay allowing the quantitative measurement of virus spreading from cell to cell. We will apply this assay to isolate host factors required for contact-dependent cell-to-cell transmission. Our comprehensive approach will provide the scientific community with novel candidate genes that have the potential to transform research on the prevention of HIV transmission. We will then use the humanized mouse model for HIV infection to directly test if our identified lead candidate genes are required for the establishment of HIV infection in vivo following mucosal delivery. Finally, a parallel and complementary approach we will identify small molecules that function as novel therapeutics specifically interfering with HIV transmission. Our combined genetic and chemical approach will allow the development of novel preventive therapies that reduce the spreading of AIDS. This research proposal will develop antiviral therapies that target the ability of HIV to efficiently spread from cell to cell. It will first focus on basic science by identifying host genes required for HIV transmission, test their physiological relevance in vivo and initiate a translational approach to isolate small molecule inhibitors that specifically interfere with HIV transmission.

DESCRIPTION (provided by applicant): The main obstacle to RNA-interference-based inhibitors is delivering them into primary cells that are highly recalcitrant to nucleic acid uptake. As a result suppression of target gene expression within these cells for both biological and therapeutic purposes has been a major issue. In recent years there have been several advances in siRNA-carrier design that have enabled efficient and cell-specific siRNA delivery. However, formulation of these carriers is extremely cumbersome and cost-prohibitive for preliminary laboratory testing. We propose to develop novel, cost-effective, easy-to-formulate and non-immunogenic siRNA carriers for the cell-specific delivery of siRNA in vivo. These carriers will be formulated from two components- (1) a non-immunogenic protein/peptide component that binds the immunoglobulin Fc-region with a high affinity coupled to a siRNA binding domain and (ii) an antibody component capable of recognizing a specific cell-surface receptor and inducing internalization. Simple mixing or incubation of the carrier with the antibody will yield a reagent capable of siRNA delivery to the desired cell type. Importantly, delivery to the target cell type/organ will be achieved through simple intravenous injections. We will generate siRNA carriers to each of the human immune cell subtypes and defined organs/tissues and evaluate siRNA treatment efficacy in relevant murine models of human disease. The successful completion of our research plan is expected to lead to the establishment of easily translatable delivery platforms for the preclinical evaluation of potential siRNA therapeutic candidates.

DESCRIPTION: The last five years have witnessed a hallmark achievement in the field of HIV-AIDS therapy- at least three patients have been cured of HIV after bone marrow transplant. Unfortunately, transplantation procedures tend to be very risky and restricted to those patients who are at a substantial risk for death due to associated malignancies. Direct in vivo genetic engineering of hematopoietic cells for HIV resistance would mean a big step forward in the field of HIV-AIDS gene therapy. The objective of this proposal is to develop an effective method for providing a functional cure for HIV infected individuals. The approach is based on the observations that (i) subjects lacking or heterozygous for the expression of CCR5, the viral coreceptor, can be highly resistant to HIV infection (ii) hematopoietic CD34+ stem and progenitor cells (HPCs) with a mutated version of the CCR5 gene when transplanted into a HIV patient (The Berlin patient) afforded a cure from HIV. We have developed novel peptide nucleic acids (PNA) that can form a triple helical structure specifically within the CCR5 gene. The triplex formation induces natural cellular repair-recombination pathways enabling introduction of a stop codon in the CCR5 gene when a donor DNA is supplied alongside. These pathways are error-free and can thus be used to disrupt the CCR5 gene with extremely low off-target rates and thereby expression of wild-type CCR5 protein. We propose to use biocompatible nanoparticles made from the FDA-approved polymer PLGA for encapsulating PNA and donor DNA molecules for in vivo delivery and genome editing at the CCR5 locus in hematopoietic cells. The studies in this application are directed at enabling targeted and specific delivery of newer generation PNAs and nanoparticles to human hematopoietic cells by enabling penetrance into the bone marrow following simple intravenous injection. The efficacy of the approach in prophylaxis as well as therapy will be evaluated in a new generation humanized mouse model for HIV infection. The overall goal is to establish feasibility of a new minimally invasive and innovative therapeutic paradigm for HIV-1 infection: application of triplex and nanoparticle technology for the site-directed modification of the CCR5 gene in hematopoietic cells in vivo by facile IV infusion.

? DESCRIPTION (provided by applicant): There is currently considerable emphasis on strategies for eradicating HIV from infected individuals. Although much effort is focused on reactivating integrated provirus using small molecules, a second possibility is using broadly neutralizing antibodies (bNAbs) that are highly potent and active against diverse strains and clades of HIV. Recent evidence demonstrating the ability of bNAbs in delaying viral rebound after stopping antiretroviral treatment (ART) indicate that these antibodies may be able to clear or remove cells expressing envelope glycoprotein on their surface, effectively reducing the pool size of persistently-infected cells. We propose to investigate a role for bNAbs in actively eliminating or reducing the size of the persistent viral reservoir, in novel humanized models of ART-suppressed HIV infection that support antibody-effector function. In the first of three specific aims, we will construct `gutted' or helper-dependent adenoviral (HDAd) vectors, encoding the heavy and light chains of three second-generation bNAbs, in view of the downstream intramuscular application in animal models described in Aims 2 and 3. In the second aim, these vectored bNABs will be tested in HIV-infected humanized BLT mice, in which viral loads will be suppressed to undetectable levels by ART, for measurements of the latent viral pool size by genomic DNA qPCR and viral outgrowth assays. In particular, novel humanized mouse models that have active complement and functional natural killer cells will be used to assess the role of bNAb effector function in shrinking the persistently infected reservoir. In the final aim, the vectored bNAbs will be tested for their ability to shrink viral reservoirs in humanized mice derived using peripheral blood leukocytes from ART-suppressed HIV+ patients with undetectable plasma viral loads. On the basis of the investigations to be conducted in the R21 phase of this application, we anticipate establishing a critical role for bNAb-effector activit targeting persistent viral reservoirs. The R33 phase of the application will extend these studies to the testing of tetracycline-regulable bnAbs encoded in HDAds, possible due to large carrying capacity of HdAds (~30 kb). In addition to exploring newer, more potent bNAbs, we will also test the use of the recently described eCD4-Ig, that encodes both soluble CD4-Ig and a CCR5 peptidomimetic and ARM-Hs, which are bivalent small molecules capable of binding the CD4 binding pocket of gp160 and also recruiting anti-dinitrophenol antibodies for effector activity. This approach of using HDAds encoding bNAbs may also be combined with latency reversal agents, in order to clear out infected cells. The results of these investigations should accelerate the use of bNAbs as an adjunct to ART in the treatment of HIV disease, with the potential for functional eradication due to elimination or prevention of reactivation of persistently-infected cells.

ABSTRACT/SUMMARY Antiretroviral therapy (ART) has significantly reduced the mortality of HIV disease and brought viral loads in HIV patients to below detection limits (<50 copies/ml plasma). This has been critical in controlling HIV spread and there is substantial clinical evidence that zero HIV transmissions occur from HIV-infected people with non-detectable viral loads. However, side-effects and non-adherence to the compulsory daily treatment regimen limit the long-term use of ART in infected people. A foreseeable problem with antiretroviral non- adherence is the impact on non-detectable viral loads. We hypothesize that new drugs with improved pharmacological properties, and a long-acting parenteral formulation of antiretroviral drugs that could reduce the dosing to weekly, monthly, or even longer periods of time can significantly impact patient non-adherence. In recent work, we have developed a novel class of picomolar Non Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) with enhanced pharmacological properties, drug resistance profiles, and a wide margin of safety relative to the current FDA approved NNRTIs such as efavirenz and rilpivirin. Because of these novel properties, these new NNRTIs are particularly well-suited for coupling with sustained-release drug delivery technologies. A long-acting nanoformulation of our candidate NNRTI, a naphthyl catechol phenyl ether called Compound I, maintained sustained plasma levels and antiretroviral efficacy for ?3 weeks in HIV-1-infected humanized mice, confirming potential as a late-stage preclinical candidate. Given the favorable pharmacological properties and potent synergy of Compound I with other classes of currently approved FDA drugs, our overall objective is to develop Compound I as a component of long-acting combinatorial ART (cART). We envision long-acting formulations made from different biocompatible biodegradable polymers [PLGA, poly(PDL-co-DO)] that can be delivered as an intramuscular (i.m.) injection of microparticles or a subdermal biodegradable implant to provide sustained-release of NNRTIs in combination with other ARV classes over a period of months to a year. This would be similar to the FDA-approved Lupron Depot®, an 8 micron microparticle, (marketed by AbbVie and manufactured by Takeda) that provides multi-months of sustained release of the synthetic hormone, leuprolide acetate for treatment of prostate cancer and endometriosis. The aims of this application are to develop, optimize and test efficacy of Compound I as a component of long-acting cART for pre-exposure prophylaxis (PrEP) as well as late stage therapy in HIV- infected humanized NSG mice. These approaches and techniques will directly benefit patients for the treatment of HIV-1.

PROJECT SUMMARY/ABSTRACT The Berlin patient demonstrates that cure of HIV is possible, and the likely mechanism of cure (transplantation with CCR5?32 cells) suggests that cell-intrinsic resistance represents a potential therapeutic scenario for a full or functional cure for HIV/AIDS. For several years, we have studied natural, cell-intrinsic barriers to HIV-1 gene expression in cells derived from mice and other rodents. Two of these barriers map to genes encoding host proteins Cyclin T1 (CCNT1) and Exportin-1 (XPO1, also known as CRM1) that are essential regulators of HIV-1 gene expression and latency reversal. In human cells, CCNT1 interacts with the viral Tat protein to promote HIV transcriptional elongation, while XPO1 is bound by the viral Rev protein to mediate nuclear export of intron-containing viral mRNAs and the viral RNA genome. In mouse cells, Tat-Ccnt1 and Rev-Xpo1 interactions are inefficient due to species-specific differences between mouse and human orthologues of either protein. Why these highly conserved genes evolved differences in the rodent and primate lineages is unknown, and the rise of CRISPR/Cas9 technology provides us with the unprecedented opportunity to test the hypothesis that knocking-in mouse-specific features of CCNT1 and XPO1 into human T cells will suppress HIV-1 replication in vitro and in vivo with little net impact on host cellular biology. Indeed, our project is premised on exciting preliminary data showing that modifying a single species-specific codon of human CCNT1 in a human T cell line is sufficient to abolish HIV-1 Tat activity and viral replication, with no discernable effects on cell proliferation. Herein we request support for collaborative studies between the Sherer and Kumar labs to further our studies of mouse-informed CCNT1 and XPO1 gene modifications in human T cells, and to determine the potential of targeting these genes as a resistance/treatment strategy for HIV-1.

ABSTRACT/SUMMARY Despite suppression by antiretroviral therapy (ART), virus is not eliminated in HIV patients and can rebound causing full-blown infection upon ART interruption. Thus, a strategy to eliminate the virus reservoir is urgently needed. The recently discovered gene editing technique called CRISPR has tremendous potential for eradicating HIV-1. CRISPR is comprised of a Cas9 nuclease and chimeric guide RNA (gRNA). When Cas9 and gRNA designed to target HIV sequences are present in latently-infected cells, it can result in disruption of the integrated proviral genome, permanently inactivating the virus. The biggest challenge to using the CRISPR approach for HIV elimination is the absence of an in vivo delivery system for human T cells, the major cellular of HIV-1. This is a R01 application in response to RFA-AI-18-016, ?Targeted In Vivo Delivery of Gene Therapeutics for HIV Cure?. To address the challenge of Cas9/gRNA delivery to human T cells in vivo, we propose the use of a T cell-targeting lentivirus whose tropism is guided by antibodies to human CD7, a molecule expressed at high levels on all human T cells, including resting T cells which are a major reservoir for latent HIV. To address concerns of vector integration and constitutive Cas9 expression, we have generated lentiviruses that are pre-packaged with Cas9 ribonucleoproteins with no integrating DNA components. Proof- of-concept studies in virologically-suppressed HIV-infected humanized mice demonstrate that disrupting CCR5, the coreceptor for HIV-1, with this systemic approach results in ART-free virologic remission. Importantly, as the approach does not require activation or elimination of the infected cell, it addresses the limitations of conventional `Shock and Kill' approaches that have yielded promising results in clinical settings. The proposal has three specific aims- In Specific Aim 1, we will design and test broad-spectrum gRNAs targeting HIV DNA in two independent approaches expected to mutate a segment or excise the entire length of the integrated HIV provirus. The approach will be tested in ART-suppressed humanized mice for impact on virus reservoir and rebound. A comprehensive investigation of toxicity, off-target effects and virus escape will also be undertaken. In Specific Aim 2, we will perform functional studies in patient-cell derived humanized mice to determine the effects of broad-spectrum gRNAs on latent virus quasispecies from HIV+ patients. The studies will employ HIV-1 RNA Sort-Seq, a novel methodology to quantitate the inducible replication-competent HIV reservoir. In Specific Aim 3, we incorporate strategic changes in the lentiviral vector to reduce vector-associated immunogenicity and permit a single or multiple but rapid-dosing regimen with enhanced potency. The outcome of these proof-of-principle studies is expected to establish a solid platform for future studies on an approach that could significantly contribute towards a cure for HIV-AIDS.

PROJECT SUMMARY/ABSTRACT This application is a request for continuation of support for a pre-doctoral Training Program in Virology at Yale University. The goal is to provide multidisciplinary research training to predoctoral students in the molecular biology of viruses, virus-host interactions, and pathogenesis in human viral diseases. This is expected to provide the intellectual and research foundations necessary for productive careers in virology-related disciplines in academia, industry, and government. The program offers training in virtually all aspects of modern virology including viral genetics, the molecular, cellular and structural biology of viruses, adaptive and innate immune response to viruses, virus-host interactions at the cellular and organismal levels as well as translational research towards the treatment of viral diseases. The program is co-directed by Drs. Priti Kumar and Walther Mothes, and a 5 member Advisory Committee. The program has been strengthened by the addition of new Faculty trainers and courses to cover emerging critical areas of virology. As a group, the 22 trainers have an outstanding record of research accomplishment and training and many are national or international leaders in their fields. These trainers have primary appointments in 14 different Yale departments and currently have 51 predoctoral virology trainees working in their labs. Students enter the program through the Combined Program in Biological and Biomedical Sciences (BBS). The BBS unites nearly 375 faculty in basic biological and biomedical sciences at the Yale Medical School, Science Hill on the main University Campus, and the newly developed West Campus. Admission is granted to students with outstanding academic record with particular attention to research experience. The Faculty makes extensive efforts to attract and retain trainees from diverse backgrounds, particularly under- represented minority groups. The program is supported by strong institutional commitment to graduate training. Predoctoral training leading to the Ph.D. degree involves formal course work and laboratory rotations in the first year. This provides a solid and broad conceptual foundation and experimental training in microbiology, immunology, cell biology, genetics, as well as other areas of biology. Students select a research advisor at the end of the first year and defend a research proposal in a qualifying exam at the beginning of the second year. A Thesis Committee monitors student progress throughout the thesis research process. Intensive training in the methods, logic, and responsible conduct of research are supplemented with a wide array of opportunities for teaching and scientific interactions. The median time to obtain the Ph.D. degree is 5.4 years. This application requests funding to continue support for four predoctoral trainees in each year, with each trainees supported by this grant for a maximum of two years.