Drexel University

NIH Research Projects · FY 2026 · 2016-08

Project Summary/Abstract Combination antiretroviral therapy (cART) is effective at reducing viral load and suppressing HIV-1 infection, however, there still is no cure for HIV-1 disease. This is due in part to the formation of latently infected cells harboring integrated proviruses in tissue and cell reservoirs. Clustered regularly interspaced palindromic repeats (CRISPR) gene editing has shown promise as an HIV cure strategy. All Cas enzymes in use today initiate binding by recognizing a protospacer adjacent motif (PAM) followed by the complementarity between guide RNA (gRNA) and target DNA to induce DNA cleavage. Subsequent double-strand break repair by endogenous cellular processes has been shown to result in a non-random mutational distribution dictated by protospacer and flanking sequence context. Furthermore, natural genetic variation within integrated proviral sequences has been shown to decrease the CRISPR-mediated editing efficiency which is critically dependent for efficacy of the gRNA selection process. The past funding period, we have designed a patented computational pipeline to select broad- spectrum spCas9 gRNAs that account for HIV sequence variation within and between large numbers of individuals and that have no off-target effect using predictive algorithms or functional assays. Preliminary data presented here shows gRNAs have efficacy in other tissue compartments (brain) and across subtypes. In addition to these gRNA design advances, the team also showed major advancements in delivery and effectiveness in small animal studies and non-human primate studies. In order to better harness the utility of CRISPR/Cas gene editing, this project will utilize novel high-throughput biologic assays combined with state-of- the art computational biology to expand what is known about how novel Cas enzymes edit the DNA target in vitro and test HIV-1-infected patient samples ex vivo and in vivo to optimize treatment strategy (Cas:gRNA combination) selection to account for HIV sequence variation within and across tissue compartments (periphery vs brain) and subtypes. Novel resources like the Multiple Lentiviral Expression System (MuLE) and the Mammalian Synthetic Cellular Recorder Integrating Biological Events (mSCRIBE) will be leveraged to study Cas enzymology and HIV-1 reactivation at the single-cell level. We hypothesize that, Cas:gRNA targeting will induce safe and reproducible editing outcomes that are predictablly based on the enzyme, target, and surrounding nucleotide sequence. To interrogate this hypothesis, three Specific Aims will be used: (i) develop a generic model of Cas:gRNA combination repair outcomes, (ii) identify functional impact of Cas:gRNA pairs using molecular recorders, and (iii) ex vivo and in vivo testing of combined Cas:gRNA pairs on HIV. These Aims will extend the knowledge of CRIPSR editing technologies for all fields of biology using the HIV platform. It will do this in cells important for HIV research in the periphery and the CNS (T, monocyte, microglia) and under different activation states. For HIV specifically, it will extend Cas:gRNA design into other tissues and between subtypes to develop a highly significant and innovative approach to target the HIV-1 quasispecies. This will result in a highly effective treatment strategy for using CRISPR gene-editing as a potential cure for HIV infection and disease.

NIH Research Projects · FY 2026 · 2016-04

PROJECT SUMMARY The goal of this proposal is to determine whether molecularly distinct progenitor cells give rise to local astrocyte diversity. Astrocytes are estimated to comprise up to 40% of cells in the brain and growing evidence now shows that these cells possess diverse phenotypic and molecular characteristics. Nevertheless, large gaps remain in our understanding of how such distinct characteristics arise. This study focuses on the role of the molecular signaling pathway, Sonic hedgehog (Shh), which distinguishes a population of perinatal glial progenitor cells in the forebrain. Fate mapping experiments show that these progenitors generate half the population of cortical astrocytes, suggesting that the cortex harbors astrocytes from multiple lineages. The precise role of Shh signaling in astrocyte development is not well understood. This study applies molecular genetic tools together with high resolution imaging and 3D reconstruction of single cells, together with RNASeq and bioinformatic analysis to address the role of Shh signaling in defining lineage-specific phenotypic and molecular characteristics of astrocytes. We will first identify the progenitor cell responsible for Shh-mediated astrocyte production and define unique characteristics of cells within this lineage. We will then apply loss of function strategies together with RNASeq and bioinformatic analysis to interrogate the role of Shh signaling in astrocyte development and molecular identity. Finally, we will use a cellular ablation approach to determine the functional significance of cells derived from the Shh lineage. This study will advance our understanding of astrocyte diversity in the brain and illuminate novel roles for Shh signaling in cell and brain development.

NIH Research Projects · FY 2024 · 2016-01

Project Summary At the heart of angiogenesis and biomaterial vascularization lies the inflammatory response, orchestrated primarily by macrophages, which dramatically shift phenotype over time in response to microenvironmental cues. In the normal response to injury, macrophages are initially pro-inflammatory (aka M1), and at later stages they are replaced by a mixed population referred to collectively as M2 that upregulate factors associated with resolution of the wound healing process. The extent of the diversity of this M2 population in particular is not known. At later stages of angiogenesis and biomaterial vascularization, M2 macrophages are generated 1) via transition from M1 macrophages, or 2) from direct differentiation of newly arriving monocytes. The differences between the M2 macrophages arising from each population have not been investigated. Preliminary data suggest that M1-derived M2 macrophages possess enhanced angiogenic functionality, and that biomaterials that transiently stimulate the initial M1 phase may enhance the subsequent response to M2-promoting biomaterials to achieve enhanced vascularization and healing. The overarching hypothesis of this project is that biomaterials that promote sequential M1 and M2 activation of the same population of macrophages will enhance vascularization. To test this hypothesis, this work has the following goals: 1) Determine the effects of M1 pre- polarization on the functional phenotype of M2 macrophages in crosstalk with blood vessels in vitro, using primary human macrophages, gene and protein expression profiling, and tissue-engineered models of angiogenesis. 2) Determine the effects of pro-inflammatory pre-treatment on the regenerative effects of IL4- releasing biomaterials in vivo, using biomaterials that temporally control the phenotype of host macrophages in a murine hindlimb ischemia model. 3) Determine the angiogenic effects in vivo of a biomaterial-mediated macrophage cell therapy strategy that intracellularly directs a single population of macrophages from M1 to M2. This latter strategy may result in particularly beneficial biomaterials for patients who suffer from impaired leukocyte trafficking, including patients with diabetes, autoimmune disease, or those undergoing chemotherapeutic treatment for cancer. This work will advance our understanding of how biomaterials can be designed to leverage both the inflammatory and regenerative functions of macrophages to enhance angiogenesis, which will allow us to develop new strategies to treat numerous diseases characterized by pathological angiogenesis, including heart and brain ischemia, atherosclerosis, and diabetes, among many others. In addition, this project proposes a novel approach to direct tissue revascularization by controlling the actions of both recruited and exogenously administered macrophages using biomaterials.

NIH Research Projects · FY 2026 · 2013-07

PROJECT SUMMARY/ABSTRACT Access to greater varieties, amounts, and potencies of cannabis has increased dramatically in the U.S. over the past decade as more states legalize cannabis for medical and adult use. Research on cannabis users who consume in a controlled manner, that is, in a way that does not compromise responsibilities to family and work or minimizes risk to self, is limited despite the fact that most cannabis users do not become dependent or develop a disorder. Significantly, models of controlled cannabis use have been inhibited from developing historically due to factors related to stigma and illegality, but are urgently needed as states continue to legalize cannabis for medical and adult use, which increases availability and access to potent cannabis products. Our long-term goal in this renewal of a currently funded R01 is to advance the scientific understanding of safer and controlled use of cannabis in policy environments where access to cannabis and potent products is high or increasing. Our objective here is to advance the conceptualization and assessment of controlled cannabis use across different policy environments for clinical and community-wide dissemination to fill research and practical gaps in knowledge. We will achieve this objective by sampling from two ongoing longitudinal cohorts of cannabis users -- 23-37-year-old adult users in CA (n=200) and 23-37-year-old medical users in PA (n=200) -- utilizing a measurement burst design that incorporates 21 days of ecological momentary assessment (EMA) and quantitative surveys over two time points plus qualitative interviews. Our central hypothesis is that persons who exhibit greater control over their cannabis use (via greater adherence to rituals and rules, e.g., “don’t use cannabis before work or driving” or “only use on the weekends”, defined as controlled users) will report lower cannabis use and better psychosocial functioning across the study period compared to uncontrolled users. This renewal study is guided by findings from our current studies of young adult cannabis users as well as theoretical insights into controlled use of cannabis described in Zinberg’s classic book Drug, Set, and Setting – specifically the application of rules and rituals derived from social “setting.” Our central hypothesis will be tested by pursuing three specific aims: 1) Develop and test a new scale assessing the rules of controlled cannabis use to be utilized during the subsequent EMA study; 2) Determine daily cannabis use practices and psychosocial functioning among controlled and uncontrolled users in contrasting policy environments using EMA and a baseline survey (n=400); 3) Identify longitudinal patterns of daily cannabis use practices and psychosocial functioning among controlled and uncontrolled users using EMA and surveys from two time points (n=400), and qualitative interviews (n=100). Deliverables from this project include: (1) a theoretically derived instrument assessing the rules of controlled cannabis use; (2) a fine-grained, process-level understanding of controlled use of cannabis over time via EMA, annual surveys, and qualitative interviews; (3) key factors/mechanisms identified via Aims 1- 3 that can inform the development of policies and interventions to support controlled and safer use of cannabis.

NIH Research Projects · FY 2025 · 1996-08

PROJECT SUMMARY Cyclic cGMP (cGMP) enables phototransduction in vertebrate rods and cones. The cGMP synthesis by retinal guanylyl (guanylate) cyclase (RetGC), one of the most essential processes in the photoreceptor physiology, is controlled by calcium, guanylyl cyclase activating proteins (GCAPs), and retinal degeneration 3 (RD3) protein. The abnormalities in cGMP signaling cause photoreceptor dysfunction and death. Among them, deficiency in RetGC activity and regulation leads to a variety of recessive and dominant forms of congenital blindness. The basic principles of the RetGC regulation and its fundamental importance for the photoreceptor signaling and survival have been established, and the first clinical trials for RetGC-linked blindness now have begun, evolved from the earlier molecular studies. Yet, some key molecular and cellular aspects of RetGC regulation still remain insufficiently understood, including the molecular structure and the interactions of RetGC with the regulatory proteins that define its biological function in photoreceptors. This proposal, conforming to the NEI mission to support research with respect to blinding eye diseases, visual disorders and mechanisms of visual function, is built on recent advancements in understanding of how RetGC enables the photoreceptor function: (i) identification of mutations that affect RetGC interactions with GCAPs and RD3; (ii) establishing the structure on RD3 and identification of its RetGC-binding interface; (iii) development of new mouse genetic models for studying mechanisms of signal transduction and their abnormalities caused by mutations in RetGC1, GCAP1, and RD3; (iv) establishing the complex physiological role of RD3 in photoreceptor function and survival. We here propose a diversified study designed to achieve, by integrating protein biochemistry, molecular biology, and molecular genetics, better mechanistic understanding of the regulatory processes that control cGMP synthesis in photoreceptors and underlie their function and diseases. Aim 1 seeks establishing the presently unknown structure of RetGC1, the main source of cGMP in photoreceptors, whose mutants cause severe forms of blindness. Aim 2 addresses the molecular determinant of RD3 that controls RetGC trafficking in photoreceptors using transgenic mouse models. Aim 3 will delineate the dynamics of RetGC complexes with GCAPs and RD3 that enable RetGC to function in vivo. By completing these specific aims, we expect to achieve deeper and more reliable understanding of the mechanistic interactions that define the fundamental role of RetGC in photoreceptor biology and cause physiological abnormalities in congenital retinal diseases.

NIH Research Projects · FY 2026 · 1989-07

Project Summary The emergence of eukaryotes from an exclusively prokaryotic biosphere, a momentous event that charted a divergent course for life on Earth, coincided with symbiotic adoption of mitochondria. The complexity of eukaryotes could not have existed and evolved but for the energy economy supported by mitochondrial physiology. Given the strong evidence for the monophyletic origin of mitochondria, it is probable that all extant mitochondria can trace their origin to that singular primordial event at the dawn of eukaryotes. Yet, the evolution has led to tremendous variety of mitochondria, their genomes, their regulation, and their functions, which are as diverse as the organisms they reside in We are interested in understanding mitochondrial functions in malaria parasites, pathogens that extract a tremendous toll from the health and wellbeing of vast multitudes. Over 30 years ago, our group discovered the unusual mtDNA in Plasmodium that encoded just 3 mitochondrial electron transport chain (mtETC) subunits and ribosomal RNA in fragmented and scrambled pieces. With the long-term support of this grant award, we have elucidated many aspects of mitochondrial functions in malaria parasites, showing the organelle to be the target for antimalarial drugs currently in use as well as those in development. We have shown which of the canonical mitochondrial functions are critical in blood stages and which are essential only for the insect stages of the parasite. We have also identified unique and essential features of Plasmodium mitochondrion using genetic and proteomic investigations. In recent years, our efforts have been joined by other investigators working to understand the mitochondrion in apicomplexan parasites. Together, we are at a stage when many of the mysteries surrounding this organelle in Plasmodium and Toxoplasma are being addressed with the application of powerful new approaches. For the next funding period, we are proposing to conduct studies that would bring us closer to answering some of the most critical questions remaining about mitochondrial functions in malaria parasites. These include potential dual function of Complex III, understanding the nature of mitochondrial complexes, and the unusual structural features of mitochondria in asexual and sexual stages of the parasite. As before, the work we are proposing will be carried out by an outstanding collaborative team including a consortium arrangement. Specifically, we will investigate an essential mitochondrial protein an human orthologue of which has been shown to have multiple functions in mitochondrial biogenesis and metabolism. These studies will use biochemical and metabolomic approaches in addition to genetic manipulations. In a consortium arrangement, we will use cryo-electron tomography to study in situ unusual features of mitochondrial structures in asexual and sexual stages of P. falciparum as well as consequences of genetic disruptions of a select number of mitochondrial functions. We will also explore a potential secondary function of the mtETC Complex III as a reason to maintain mtDNA even in mtETC independent parasites.