Student Award Details

Familial dilated cardiomyopathy (DCM) is a heritable disorder characterized by progressive enlargement of the heart's ventricles and impaired contraction, often leading to early-onset heart failure. A significant subset of inherited DCM cases is attributed to mutations in an 18-bp segment of the RNA binding motif protein 20 (RBM20) gene, which causes aberrant splicing of critical cardiac genes. While current treatments offer symptomatic relief, there is an unmet need for therapies that correct the underlying genetic cause. This project seeks to develop a universal CRISPR gene editing strategy to replace the entire 18-bp RBM20 pathogenic cluster with a synonymous DNA sequence. The approach utilizes prime editing (PE), focusing on the optimization of the PE3b system, which has been identified as a promising strategy for achieving high precision and low indel rates. This optimization is being conducted via a high-throughput, self-targeting lentiviral screen to identify optimal guide RNA configurations. The optimized designs identified through this screen will then be validated in isogenic induced pluripotent stem cell (iPSC) lines carrying pathogenic RBM20 variants and differentiated cardiomyocytes (CMs) to assess restoration of normal cellular phenotype. This work will inform the development of an efficient, safe PE platform to treat genetically diverse cases of RBM20-DCM.

The germline contains the genetic information that will be passed to future generations. Therefore, maintaining the germline genome is essential for fertility and species survival. One mechanism of germline genome maintenance is that of the PIWI/piRNA pathway, in which small RNAs known as piRNAs interact with a PIWI Argonaute protein to form what is known as a piRNA-Induced Silencing Complex (piRISC). Through its endonuclease activity, piRISC silences repetitive elements (i.e., transposons) to protect the genome for future generations and regulates gene expression to ensure proper germ cell development and function. Loss of PIWI function leads to infertility in at least one sex in many animals, including human males. Recent work has revealed that the small zinc-finger protein, gametocyte-specific factor 1 (GTSF1), accelerates piRISC target cleavage. Loss of GTSF1 function in mice and human males leads to infertility. Preliminary kinetic evidence suggests that GTSF1 is not required for target binding or target release. However, the piRISC catalytic states associated with GTSF1 binding have not been explored. Seven GTSF1 residues have been identified as key for target cleavage, but most of these amino acids are not conserved in the other mouse GTSF paralogs, GTSF1L and GTSF2, even though they also accelerate piRISC target cleavage. This proposal seeks to test the hypothesis that mammalian GTSF proteins stabilize a catalytically active state of piRISC via key contacts with both the piRNA-target RNA duplex and PIWI protein. Aim 1 will use single-molecule FRET to probe piRISC conformational changes in the absence and presence of GTSF proteins to determine which, if any, catalytic state is stabilized in the presence of GTSF. Aim 2 will employ a high-throughput screening method to identify all amino acid positions across GTSF paralogs which are required to interact with piRISC. This study will provide insights into how GTSF1 accelerates piRISC target cleavage and determine which GTSF residues are key for its function, providing insight into why some human GTSF1 mutations lead to infertility. The proposed research will provide training in microscopy, in vitro biochemistry, and bioinformatics to prepare the fellow for a postdoc studying epigenetic inheritance and a future career as an independent investigator.

Immunotherapies such as PD-1 and CTLA-4 inhibitors have transformed cancer treatment, yet many patients remain unresponsive, underscoring the need for new ways to engage the immune system. This project investigates NLRP10, a highly expressed but poorly characterized inflammasome sensor in skin cells. Our studies show that a fungal metabolite induces mitochondrial stress sensed by NLRP10, triggering IL-1 cytokine release, cell death via pyroptosis, and immune cell recruitment and anti-tumor activity in melanoma models. We propose that NLRP10 functions as a mitochondrial damage sensor linking epithelial stress responses to tumor immunity. This research will define how NLRP10 detects mitochondrial danger signals and regulates inflammation and tumor control, revealing a previously unrecognized immune pathway at the skin–tumor interface. Ultimately, uncovering the biology of NLRP10 will expand our understanding of how innate immunity shapes skin cancer outcomes and may open new avenues for developing therapies that reprogram the tumor microenvironment to enhance anti-cancer immunity.

Synthetic messenger RNAs (mRNAs) represent a new class of biopharmaceuticals with broad clinical utility for a range of diseases. The incorporation of chemically modified uridine nucleosides, pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ), significantly reduces synthetic mRNA immunogenicity. Linear nucleoside-modified mRNAs are short-lived because they are susceptible to cellular exonucleases, hindering their broad clinical utility for a range of diseases. Synthetic circular mRNAs (circRNAs) evade cellular exonucleases, resulting in a longer half-life, but efficient circularization methods are incompatible with chemical modifications. The most efficient method of RNA circularization utilizes self-splicing group I introns. Proper folding—and thus activation—of the typical group I intron is disrupted by Ψ-modified nucleotides. A survey of self-splicing introns, however, identified a compact group I intron from the cyanobacterium Azoarcus that effectively circularizes short (~150 nt) Ψ-modified RNA. The Azoarcus intron can circularize long (~2000 nt) unmodified mRNA but not long Ψ-modified RNAs. This proposal seeks to test the hypothesis that Ψ and m1Ψ prevent circularization by disrupting tertiary interactions required for rapid and efficient intron splicing and to develop efficient circularization techniques compatible with Ψ and m1Ψ for safe and stable RNA therapeutics. Aim 1 will determine how uridine modifications affect the structure of Azoarcus group I intron. High throughput structural probing will be used to study how uridine modifications stabilize inactive conformations of the Azoarcus group I intron. Aim 2 will employ in vitro evolution to identify Azoarcus group I intron variants optimized to circularize Ψ and m1Ψ-modified RNA. The Ψ- and m1Ψ-modified circRNA will be tested in immune cells for their ability to induce an immune response. This study will provide structural insights into how nucleoside modifications change RNA structure and function and develop a straightforward methodology to prepare nucleoside-modified circular mRNAs, expanding the potential uses of mRNA therapeutics beyond vaccines. In addition, the proposed research will provide training in high-throughput sequencing, in vitro biochemistry, cellular RNA sensing, nucleic acid chemistry, and structural biology to prepare the fellow for a career as an independent investigator developing next-generation mRNA therapeutics.

While there are many FDA-approved therapies to treat relapsing-remitting multiple Sclerosis (MS), there are far less options for treating neurodegeneration in progressive disease. Intriguingly, similar to other neurodegenerative diseases (e.g. Alzheimer’s disease and frontotemporal dementia), a hallmark feature of progressive disease in MS is the loss of synapses and gray matter atrophy. Our lab recently discovered a striking loss of synapses in the visual thalamus of MS patient tissue and MS-relevant mouse and marmoset models concomitant with visual impairment. This was particularly intriguing since prolonged visual impairment is historically attributed to demyelination of the optic nerve and is a frequent occurrence in MS patients. As complement proteins were previously shown to mediate synapse elimination by phagocytic microglia in neurodegenerative disease and genetic variants in complement proteins have recently been correlated with visual impairment in MS patients, Dr. Schafer’s lab has been exploring this pathway in synapse loss in the visual thalamus in MS. First, Dr. Schafer’s lab showed that complement proteins C1q and C3 were both increased in a mouse and non-human primate model of MS (experimental autoimmune encephalomyelitis, EAE). However, unlike in development, C1q did not localize to synapses in this context. Instead, C3 was highly synaptic in EAE to induce microglia-mediated phagocytosis and elimination of synaptic material. In contrast to C3 at synapses, Dr. Reich and Dr. Schafer identified that C1q was particularly high in microglia surrounding chronic active MS lesions and loss of C1q in microglia in the mouse EAE model attenuated the inflammatory response of microglia. Still, it is unclear how C1q is modulating inflammation, and whether C1q is working upstream of C3 to regulate synapse loss or if synapse loss is occurring through the alternative pathway, independent of C1q. Also, there are many other molecules that regulate complement proteins and it is unclear how many of these complement-related proteins contribute to MS-related disease. Therefore, the overall goal of this proposal is to gain a more comprehensive understanding of how complement proteins are regulated in demyelinating disease.

Mycobacterium tuberculosis (Mtb) poses a paradox: despite its seemingly stable genome, it is highly adaptable. Historically, Mtb was viewed as genetically stable, lacking horizontal gene transfer (HGT) and accumulating single-nucleotide variants (SNVs) at a slow rate (~1x10^-7 SNV/site/year). Yet, it adapts to diverse environments, evades immune responses, and withstands antibiotics. The genetic basis for this adaptability remained unclear until recent studies highlighted transient phase variation as a crucial factor. Our previous work identified frequent genetic variation in simple sequence repeats (SSRs) within the Mtb genome (glpK), which is linked to drug resistance and reduced drug efficacy. In addition to this, a phylogenetic analysis of over 31,000 clinical isolates identified 121 SSR sites, including glpK, with a high INDEL rate likely driven by positive selection, with 44 SSRs showing more variation than expected under neutral evolution. In this grant proposal, we plan to leverage a recently generated library of strains representing each of these clinically prevalent mutations to quantify the effect of each on the bacterium’s sensitivity to antibiotics. Understanding these mechanisms comprehensively could reveal new therapeutic strategies targeting antibiotic-resistant subpopulations, potentially improving treatment success and reducing therapy duration.

Vitiligo is characterized by loss of epidermal melanocytes and an interface dermatitis with IFN-γ–driven T cell infiltration. Although melanocyte antigens are well characterized in melanoma, the antigens that drive autoimmunity in vitiligo remain unclear, in part because melanocytes are depleted in lesional skin. I hypothesize that inflammatory and injury-induced stress remodel the repertoire of peptides presented by MHC class I on melanocytes, generating neoepitopes that prime autoreactive T cells. To test this hypothesis, I will (1) map melanocyte and immune-cell states in patient-derived samples and in a vitiligo mouse model using single-cell RNA-seq and multiplex imaging; (2) define the melanocyte MHC class I peptidome by LC–MS/MS at baseline and after IFN-γ stimulation; (3) identify expanded and public TCR clonotypes and pair TCRs with cognate peptides using reporter assays; and (4) evaluate the pathogenicity of peptide-specific T cells in vivo. These studies will identify melanocyte-derived peptides that initiate disease, delineate how inflammation reshapes antigen presentation, and nominate precise therapeutic targets for intervention in vitiligo.

Due to unpublished data, a summary is not currently available.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that primarily affects children. While many pediatric patients reach sustained remission, about 20% relapse. Relapse is thought to be due to a rare, chemotherapy-resistant subpopulation of cells, termed leukemia initiating cells (L-ICs), that enter quiescence and survive chemotherapy, after which they can exit quiescence, proliferate and re-establish disease. As such, effectively targeting L-ICs represents a potential therapeutic approach to prevent T-ALL relapse. Our preliminary data suggest that BTG2, an activator of mRNA deadenylation, plays a critical role in maintaining L-IC quiescence by destabilizing Myc and potentially other cell cycle-related mRNAs. Interestingly, BTG2 is downregulated in relapsed T-ALL patients, leading us to hypothesize that the resulting upregulation of key BTG2 target mRNAs could promote L-IC activity and thus represent potential therapeutic targets. In this project, I will comprehensively identify BTG2 target mRNAs in human T-ALL cell lines using RNA-seq and poly-A tail length sequencing (PAL-seq) methodologies, and validate candidates for their ability to promote L-IC activity. Future experiments will test whether knocking down BTG2 target mRNAs improves survival in T-ALL xenografted mice. The insight gained by these studies is likely to have significant clinical relevance for high-risk T-ALL patients that currently have few targeted treatment options.

The overall goal of this proposal is to gain a new understanding of the factors that contribute to chemoresistance in BRCA1 and BRCA2 (BRCA) mutant hereditary breast and ovarian cancers (HBOC). Currently clinical strategies rely on chemotherapies and poly (ADP-ribose) polymerase inhibitors to control malignant disease. Unfortunately, tumor chemoresistance frequently occurs which necessitates studies that uncover the critical factors leading to chemoresistance. We have recently discovered chemoresistance is linked the single-stranded (ss)DNA gap suppression in multiple models of HBOCs and patient tumors. Our work has generated a paradigm shift that ssDNA predicts sensitivity whereas gap suppression predicts resistance. To expand upon our model that ssDNA gaps are the sensitizing lesions in BRCA cancers, I propose two aims. In Aim 1, I will determine if an axis of chemoresistance in BRCA1 deficient cancers, linked to restored homologous recombination, is instead mediated by gap suppression. In Aim 2, I will determine if gap suppression in a chemoresistant BRCA2 tumor model is linked to the activation of translesion synthesis and how translesion synthesis can be overcome to resensitize these cancers. Together, these aims increase our knowledge of the basic factors that lead to chemoresistance in the clinic and will provide new insights into the vulnerabilities unique to BRCA deficient cancers. Moreover, by identifying unique factors contributing to chemoresistance, we will develop an understanding for potential druggable targets and biomarkers for future personalized chemotherapy. Existing chemotherapies are constrained by their side-effect profiles, often taxing patients' tolerance and potentially inducing secondary malignancies. Generating a new understanding of the factors contributing to chemoresistance is pertinent to improving patient outcomes.