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  • Racial and Ethnic Disparities in Use of Colorectal Cancer Screening Among Adults With Chronic Medical Conditions: BRFSS 2012-2020

    Castañeda-Avila, Maira A; Tisminetzky, Mayra; Oyinbo, Atinuke G; Lapane, Kate L (2024-02-22)
    Introduction: People with chronic conditions and people with colorectal cancer (CRC) may share common risk factors; thus, CRC screening is important for people with chronic conditions. We examined racial and ethnic differences in the use of CRC screening among people with various numbers of chronic conditions. Methods: We included data on adult respondents aged 50 to 75 years from the Behavioral Risk Factor Surveillance System in 2012 through 2020. We categorized counts of 9 conditions as 0, 1, 2, 3, and ≥4. We classified self-reported CRC screening status as up to date or not. We used Poisson models to estimate adjusted prevalence ratios (APRs) among the different counts of chronic conditions in 4 racial and ethnic groups: Hispanic adults with limited English proficiency (LEP), Hispanic adults without LEP, non-Hispanic Black adults, and non-Hispanic White adults. Results: Overall, 66.5% of respondents were up to date with CRC screening. The prevalence of being up to date increased with the number of chronic conditions. We found disparities among racial and ethnic groups. Hispanic respondents with LEP had lower rates than non-Hispanic White adults of being up to date with CRC screening across all counts of chronic conditions (APR for 0 conditions = 0.67; 95% CI, 0.64-0.71; APR for ≥4 conditions = 0.85; 95% CI, 0.79-0.91). Hispanic respondents without LEP with 0, 1, or 2 conditions were less likely than non-Hispanic White respondents to be up to date with CRC screening. We found no significant differences between non-Hispanic Black and non-Hispanic White respondents. Conclusion: We found disparities among Hispanic BRFSS respondents with LEP, who had lower rates than non-Hispanic White respondents of being up to date with CRC screening, regardless of the number of chronic conditions. Tailored interventions are needed to address these disparities and improve screening rates, particularly among Hispanic people.
  • Home Diagnostics, Viral Dynamics, and Post-acute Sequelae of SARS-COV-2

    Herbert, Carly (2024-02-09)
    Introduction: The emergence of the novel coronavirus, SARS-CoV-2, has necessitated prompt evaluations of diagnostic technologies, viral dynamics, and long-term complications of COVID-19 to inform clinical and public health strategies. Methods: Using data from the RADx Clinical Studies Core collected from October 2021-February 2022, we evaluated the longitudinal performance of reverse transcriptase polymerase chain reaction (RT-PCR) and antigen-detecting rapid diagnostic tests (Ag-RDT) by day past symptom onset and close-contact exposure and compared performance by sex, age, vaccination status, and variant (Aim 1). We further examined the association between SARS-CoV-2 viral load, BMI, and sex (Aim 2). Lastly, we conducted a follow-up survey in August 2023 regarding Long COVID. We then modeled the relationship between viral clearance of SARS-CoV-2 and Long COVID (Aim 3). Results: RT-PCR and Ag-RDT showed the highest percent positivity two days past symptom onset (RT-PCR: 91.2%; Ag-RDT: 71.1%) and six days past exposure (RT-PCR: 91.8%; Ag-RDT: 86.2%). Performance did not differ by vaccination status, variant, age category, or sex. In males, increasing BMI was associated with higher viral load in a dose-response fashion, and males had significantly lower viral load than females for BMI>29. Lastly, the risk of long COVID with 3-4 symptoms and 5+ symptoms increased by 2.90 times (95% CI: 1.09-7.74) and 4.54 times (95% CI: 1.84-11.2) per viral load slope-unit increase, respectively. Conclusion: Understanding SARS-CoV-2 viral dynamics is critical to identify effective diagnostic strategies for COVID-19, explain differences in COVID-19 outcomes across sex and BMI, and understand mechanistic contributors of Long COVID.
  • Protein and Guide RNA Engineering of a Compact Cas9 for Enhanced Precision Genome Editing

    Bamidele, Nathan (2024-02-06)
    CRISPR-Cas technologies enable robust manipulation of genetic material, and have been instrumental in advancing a wide range of fields across the life sciences. Specifically, with the ability to correct or alter faulty genes, genome editing tools promise to transform the field of genetic medicine. Current CRISPR-based editors [nucleases, base editors (BEs), and prime editors (PE)] can be programed to induce efficient mutagenesis/repair, conversion, and polymerization, respectively. Presently, nucleases - the most clinically advanced genome editors - suffer from inadequate control of genome editing outcomes. Over time, the field has focused on precision editors such as BEs and PEs that do not rely on double-strand breaks and greatly improve the safety and control of genome editing outcomes. Despite these advances, challenges such as targeting scope, accuracy and in vivo delivery represent major hurdles for the therapeutic application of next-generation editing systems such as BE and PE. In this thesis, I focus on alleviating some of the key obstacles associated with effective genome editing by improving the unique properties of a compact Cas9 orthologue (Nme2Cas9 from Neisseria meningitidis). The bulk of my thesis consists of protein engineering efforts to improve the activity and targeting scope of Nme2Cas9-derived editing systems. My later work focuses on the development of chemically stabilized guide RNAs, providing a path to facilitate in vivo delivery in a variety of formats. Overall, the advances presented in this thesis contribute to the versatility of CRISPR-based genome editing systems for a variety of therapeutic and research applications.
  • Microbiota encoded fatty-acid metabolism expands tuft cells to protect tissues homeostasis during infection in the large intestine [preprint]

    Kellogg, Tasia D; Ceglia, Simona; Mortzfeld, Benedikt M; Zeamer, Abigail L; Foley, Sage E; Ward, Doyle V; Bhattarai, Shakti K; McCormick, Beth A; Reboldi, Andrea; Bucci, Vanni (2024-01-31)
    Metabolic byproducts of the intestinal microbiota are crucial in maintaining host immune tone and shaping inter-species ecological dynamics. Among these metabolites, succinate is a driver of tuft cell (TC) differentiation and consequent type 2 immunity-dependent protection against invading parasites in the small intestine. Succinate is also a growth enhancer of the nosocomial pathogen Clostridioides difficile in the large intestine. To date, no research has shown the role of succinate in modulating TC dynamics in the large intestine, or the relevance of this immune pathway to C. difficile pathophysiology. Here we reveal the existence of a three-way circuit between commensal microbes, C. difficile and host epithelial cells which centers around succinate. Through selective microbiota depletion experiments we demonstrate higher levels of type 2 cytokines leading to expansion of TCs in the colon. We then demonstrate the causal role of the microbiome in modulating colonic TC abundance and subsequent type 2 cytokine induction using rational supplementation experiments with fecal transplants and microbial consortia of succinate-producing bacteria. We show that administration of a succinate-deficient Bacteroides thetaiotaomicron knockout (Δfrd) significantly reduces the enhanced type 2 immunity in mono-colonized mice. Finally, we demonstrate that mice prophylactically administered with the consortium of succinate-producing bacteria show reduced C. difficile-induced morbidity and mortality compared to mice administered with heat-killed bacteria or the vehicle. This effect is reduced in a partial tuft cell knockout mouse, Pou2f3+/-, and nullified in the tuft cell knockout mouse, Pou2f3-/-, confirming that the observed protection occurs via the TC pathway. Succinate is an intermediary metabolite of the production of short-chain fatty acids, and its concentration often increases during dysbiosis. The first barrier to enteric pathogens alike is the intestinal epithelial barrier, and host maintenance and strengthening of barrier integrity is vital to homeostasis. Considering our data, we propose that activation of TC by the microbiota-produced succinate in the colon is a mechanism evolved by the host to counterbalance microbiome-derived cues that facilitate invasion by intestinal pathogens.
  • Association of spatial proximity to fixed-site syringe services programs with HCV serostatus and injection equipment sharing practices among people who inject drugs in rural New England, United States

    Romo, Eric; Stopka, Thomas J; Jesdale, Bill M; Wang, Bo; Mazor, Kathleen M; Friedmann, Peter D (2024-01-28)
    Background: Hepatitis C virus (HCV) disproportionately affects rural communities, where health services are geographically dispersed. It remains unknown whether proximity to a syringe services program (SSP) is associated with HCV infection among rural people who inject drugs (PWID). Methods: Data are from a cross-sectional sample of adults who reported injecting drugs in the past 30 days recruited from rural counties in New Hampshire, Vermont, and Massachusetts (2018-2019). We calculated the road network distance between each participant's address and the nearest fixed-site SSP, categorized as ≤ 1 mile, 1-3 miles, 3-10 miles, and > 10 miles. Staff performed HCV antibody tests and a survey assessed past 30-day injection equipment sharing practices: borrowing used syringes, borrowing other used injection equipment, and backloading. Mixed effects modified Poisson regression estimated prevalence ratios (aPR) and 95% confidence intervals (95% CI). Analyses were also stratified by means of transportation. Results: Among 330 PWID, 25% lived ≤ 1 mile of the nearest SSP, 17% lived 1-3 miles of an SSP, 12% lived 3-10 miles of an SSP, and 46% lived > 10 miles from an SSP. In multivariable models, compared to PWID who lived within 1 mile of an SSP, those who lived 3 to 10 miles away had a higher prevalence of HCV seropositivity (aPR: 1.25, 95% CI 1.06-1.46), borrowing other used injection equipment (aPR: 1.23, 95% CI 1.04-1.46), and backloading (aPR: 1.48, 95% CI 1.17-1.88). Similar results were observed for PWID living > 10 miles from an SSP: aPR [HCV]: 1.19, 95% CI 1.01-1.40; aPR [borrowing other used equipment]:1.45, 95% CI 1.29-1.63; and aPR [backloading]: 1.59, 95% CI 1.13-2.24. Associations between living 1 to 3 miles of an SSP and each outcome did not reach statistical significance. When stratified by means of transportation, associations between distance to SSP and each outcome (except borrowing other used injection equipment) were only observed among PWID who traveled by other means (versus traveled by automobile). Conclusions: Among PWID in rural New England, living farther from a fixed-site SSP was associated with a higher prevalence of HCV seropositivity, borrowing other used injection equipment, and backloading, reinforcing the need to increase SSP accessibility in rural areas. Means of transportation may modify this relationship.
  • Investigating the etiologies of non-malarial febrile illness in Senegal using metagenomic sequencing

    Levine, Zoë C; Sene, Aita; Mkandawire, Winnie; Deme, Awa B; Ndiaye, Tolla; Sy, Mouhamad; Gaye, Amy; Diedhiou, Younouss; Mbaye, Amadou M; Ndiaye, Ibrahima M; et al. (2024-01-25)
    The worldwide decline in malaria incidence is revealing the extensive burden of non-malarial febrile illness (NMFI), which remains poorly understood and difficult to diagnose. To characterize NMFI in Senegal, we collected venous blood and clinical metadata in a cross-sectional study of febrile patients and healthy controls in a low malaria burden area. Using 16S and untargeted sequencing, we detected viral, bacterial, or eukaryotic pathogens in 23% (38/163) of NMFI cases. Bacteria were the most common, with relapsing fever Borrelia and spotted fever Rickettsia found in 15.5% and 3.8% of cases, respectively. Four viral pathogens were found in a total of 7 febrile cases (3.5%). Sequencing also detected undiagnosed Plasmodium, including one putative P. ovale infection. We developed a logistic regression model that can distinguish Borrelia from NMFIs with similar presentation based on symptoms and vital signs (F1 score: 0.823). These results highlight the challenge and importance of improved diagnostics, especially for Borrelia, to support diagnosis and surveillance.
  • Practice Site Heterogeneity within and between Medicaid Accountable Care Organizations

    Dyer, Zachary; Alcusky, Matthew J; Himmelstein, Jay; Ash, Arlene S.; Kerrissey, Michaela (2024-01-20)
    The existing literature has considered accountable care organizations (ACOs) as whole entities, neglecting potentially important variations in the characteristics and experiences of the individual practice sites that comprise them. In this observational cross-sectional study, our aim is to characterize the experience, capacity, and process heterogeneity at the practice site level within and between Medicaid ACOs, drawing on the Massachusetts Medicaid and Children's Health Insurance Program (MassHealth), which launched an ACO reform effort in 2018. We used a 2019 survey of a representative sample of administrators from practice sites participating in Medicaid ACOs in Massachusetts (n = 225). We quantified the clustering of responses by practice site within all 17 Medicaid ACOs in Massachusetts for measures of process change, previous experience with alternative payment models, and changes in the practices' ability to deliver high-quality care. Using multilevel logistic models, we calculated median odds ratios (MORs) and intraclass correlation coefficients (ICCs) to quantify the variation within and between ACOs for each measure. We found greater heterogeneity within the ACOs than between them for all measures, regardless of practice site and ACO characteristics (all ICCs ≤ 0.26). Our research indicates diverse experience with, and capacity for, implementing ACO initiatives across practice sites in Medicaid ACOs. Future research and program design should account for characteristics of practice sites within ACOs.
  • mRNA initiation and termination are spatially coordinated [preprint]

    Calvo-Roitberg, Ezequiel; Carroll, Christine L; Venev, Sergey V; Kim, GyeungYun; Mick, Steven T; Dekker, Job; Fiszbein, Ana; Pai, Athma A (2024-01-07)
    The expression of a precise mRNA transcriptome is crucial for establishing cell identity and function, with dozens of alternative isoforms produced for a single gene sequence. The regulation of mRNA isoform usage occurs by the coordination of co-transcriptional mRNA processing mechanisms across a gene. Decisions involved in mRNA initiation and termination underlie the largest extent of mRNA isoform diversity, but little is known about any relationships between decisions at both ends of mRNA molecules. Here, we systematically profile the joint usage of mRNA transcription start sites (TSSs) and polyadenylation sites (PASs) across tissues and species. Using both short and long read RNA-seq data, we observe that mRNAs preferentially using upstream TSSs also tend to use upstream PASs, and congruently, the usage of downstream sites is similarly paired. This observation suggests that mRNA 5' end choice may directly influence mRNA 3' ends. Our results suggest a novel "Positional Initiation-Termination Axis" (PITA), in which the usage of alternative terminal sites are coupled based on the order in which they appear in the genome. PITA isoforms are more likely to encode alternative protein domains and use conserved sites. PITA is strongly associated with the length of genomic features, such that PITA is enriched in longer genes with more area devoted to regions that regulate alternative 5' or 3' ends. Strikingly, we found that PITA genes are more likely than non-PITA genes to have multiple, overlapping chromatin structural domains related to pairing of ordinally coupled start and end sites. In turn, PITA coupling is also associated with fast RNA Polymerase II (RNAPII) trafficking across these long gene regions. Our findings indicate that a combination of spatial and kinetic mechanisms couple transcription initiation and mRNA 3' end decisions based on ordinal position to define the expression mRNA isoforms.
  • Change in Sexual and Reproductive Health Knowledge among Young Women Using the Conversational Agent "Nthabi" in Lesotho: A Clinical Trial [preprint]

    Nkabane-Nkholongo, Elizabeth; Mokgatle, Mathildah; Bickmore, Timothy; Julce, Clevanne; Thompson, David; Jack, Brian (2023-12-28)
    Background: Young women worldwide face problems like unwanted pregnancy and sexually transmitted infections. Providing sexual and reproductive health education to this population remains a priority. It is unknown if using digital health interventions to deliver health education in human resource-constrained settings is effective. Methods: We conducted a clinical trial of the Nthabi intervention to determine participant's knowledge before and after discussion of family planning, folic acid and healthy eating among young women aged 18-28 years in two rural districts of Lesotho who used the Nthabi conversational agent system on either smartphones or tablets for up to six weeks. The number of correct pre- and post-test responses were compared using generalized linear models that directly estimated the proportions and percentages of correct responses. Results: Of the 172 participants enrolled, the mean age was 22.5 years, 91% were unmarried, 69% completed high school, 23% were unemployed and 66% were students. The mean number of interactions with Nthabi was Family planning was chosen to be discussed by 82 (52.2%), of the 172 participants and of those, 49 (59.8%) completed the content on this topic, and 26 (53.1%) completed the post-test. For the 11 questions about family planning, there were 717 (76.6%) correct responses on the pre-test and 320 (89.9%) on the post-test (p = 0.0233). Folic acid was chosen to be discussed by 74 (47.1%) of 172 participants, and of those, 27 (36.5%) completed the content on this topic, and all 27 (100%) completed the post-test. For the 5 questions about folic acid use, there were 181 (45.3%) correct responses on the pre-test and 111 (71.6%) on the post-test (p < 0.0001). The number of correct responses on the post-test was positively associated with the number of sessions that the participant engaged with Nthabi. Conclusion: The Nthabi conversational agent system increased knowledge of family planning methods and folic acid use among young women in Lesotho. Digital health interventions like Nthabi offer new opportunities to deliver reproductive health information in countries that have limited human resources for health. Trial registration: ClinicalTrials.gov ID: NCT04354168.
  • Stem cells and pain

    da Silva, Matheus Deroco Veloso; Piva, Maiara; Martelossi-Cebinelli, Geovana; Stinglin Rosa Ribas, Mariana; Hoffmann Salles Bianchini, Beatriz; Heintz, Olivia K; Casagrande, Rubia; Verri, Waldiceu A (2023-12-26)
    Pain can be defined as an unpleasant sensory and emotional experience caused by either actual or potential tissue damage or even resemble that unpleasant experience. For years, science has sought to find treatment alternatives, with minimal side effects, to relieve pain. However, the currently available pharmacological options on the market show significant adverse events. Therefore, the search for a safer and highly efficient analgesic treatment has become a priority. Stem cells (SCs) are non-specialized cells with a high capacity for replication, self-renewal, and a wide range of differentiation possibilities. In this review, we provide evidence that the immune and neuromodulatory properties of SCs can be a valuable tool in the search for ideal treatment strategies for different types of pain. With the advantage of multiple administration routes and dosages, therapies based on SCs for pain relief have demonstrated meaningful results with few downsides. Nonetheless, there are still more questions than answers when it comes to the mechanisms and pathways of pain targeted by SCs. Thus, this is an evolving field that merits further investigation towards the development of SC-based analgesic therapies, and this review will approach all of these aspects.
  • Intraflagellar transport: A critical player in photoreceptor development and the pathogenesis of retinal degenerative diseases

    Gupta, Mohona; Pazour, Gregory J (2023-12-23)
    In vertebrate vision, photons are detected by highly specialized sensory cilia called outer segments. Photoreceptor outer segments form by remodeling the membrane of a primary cilium into a stack of flattened disks. Intraflagellar transport (IFT) is critical to the formation of most types of eukaryotic cilia including the outer segments. This review covers the state of knowledge of the role of IFT in the formation and maintenance of outer segments and the human diseases that result from mutations in genes encoding the IFT complex and associated motors.
  • ATXN2 is a target of N-terminal proteolysis

    Chitre, Monika; Emery, Patrick (2023-12-21)
    Spinocerebellar ataxia 2 (SCA2) is a neurodegenerative disorder caused by the expansion of the poly-glutamine (polyQ) tract of Ataxin-2 (ATXN2). Other polyQ-containing proteins such as ATXN7 and huntingtin are associated with the development of neurodegenerative diseases when their N-terminal polyQ domains are expanded. Furthermore, they undergo proteolytic processing events that produce N-terminal fragments that include the polyQ stretch, which are implicated in pathogenesis. Interestingly, N-terminal ATXN2 fragments were reported in a brain extract from a SCA2 patient, but it is currently unknown whether an expanded polyQ domain contributes to ATXN2 proteolytic susceptibility. Here, we used transient expression in HEK293 cells to determine whether ATXN2 is a target for specific N-terminal proteolysis. We found that ATXN2 proteins with either normal or expanded polyQ stretches undergo proteolytic cleavage releasing an N-terminal polyQ-containing fragment. We identified a short amino acid sequence downstream of the polyQ domain that is necessary for N-terminal cleavage of full-length ATXN2 and sufficient to induce proteolysis of a heterologous protein. However, this sequence is not required for cleavage of a short ATXN2 isoform produced from an alternative start codon located just upstream of the CAG repeats encoding the polyQ domain. Our study extends our understanding of ATXN2 posttranslational regulation by revealing that this protein can be the target of specific proteolytic cleavage events releasing polyQ-containing products that are modulated by the N-terminal domain of ATXN2. N-terminal ATXN2 proteolysis of expanded polyQ domains might contribute to SCA2 pathology, as observed in other neurodegenerative disorders caused by polyQ domain expansion.
  • Genetic ablation of Sarm1 attenuates expression and mislocalization of phosphorylated TDP-43 after mouse repetitive traumatic brain injury

    Dogan, Elif O; Bouley, James; Zhong, Jianjun; Harkins, Ashley L; Keeler, Allison M; Bosco, Daryl A; Brown, Robert H; Henninger, Nils (2023-12-20)
    Traumatic brain injury (TBI), particularly when moderate-to-severe and repetitive, is a strong environmental risk factor for several progressive neurodegenerative disorders. Mislocalization and deposition of transactive response DNA binding protein 43 (TDP-43) has been reported in both TBI and TBI-associated neurodegenerative diseases. It has been hypothesized that axonal pathology, an early event after TBI, may promote TDP-43 dysregulation and serve as a trigger for neurodegenerative processes. We sought to determine whether blocking the prodegenerative Sarm1 (sterile alpha and TIR motif containing 1) axon death pathway attenuates TDP-43 pathology after TBI. We subjected 111 male Sarm1 wild type, hemizygous, and knockout mice to moderate-to-severe repetitive TBI (rTBI) using a previously established injury paradigm. We conducted serial neurological assessments followed by histological analyses (NeuN, MBP, Iba-1, GFAP, pTDP-43, and AT8) at 1 month after rTBI. Genetic ablation of the Sarm1 gene attenuated the expression and mislocalization of phosphorylated TDP-43 (pTDP-43) and accumulation of pTau. In addition, Sarm1 knockout mice had significantly improved cortical neuronal and axonal integrity, functional deficits, and improved overall survival after rTBI. In contrast, removal of one Sarm1 allele delayed, but did not prevent, neurological deficits and neuroaxonal loss. Nevertheless, Sarm1 haploinsufficient mice showed significantly less microgliosis, pTDP-43 pathology, and pTau accumulation when compared to wild type mice. These data indicate that the Sarm1-mediated prodegenerative pathway contributes to pathogenesis in rTBI including the pathological accumulation of pTDP-43. This suggests that anti-Sarm1 therapeutics are a viable approach for preserving neurological function after moderate-to-severe rTBI.
  • U.S. nursing home leadership experiences with COVID-19 and its impact on residents and staff: A qualitative analysis

    Dubé, Catherine E; Nielsen, Natalia; McPhillips, Emily; Lee Hargraves, J; Cosenza, Carol; Jesdale, Bill; Lapane, Kate L (2023-12-19)
    Objectives: To explore experiences of U.S. (United States) nursing home leadership during the COVID-19 pandemic in their efforts to address resident loneliness and social isolation and to elicit stories about personal and professional impacts on themselves and staff. Design: Qualitative inquiry via three optional open-ended questions appended to a national self-administered survey of American nursing home leaders was employed. Textual data was analyzed using an iterative reflexive thematic approach. Setting and participants: A stratified sample frame defined by facility size (beds: 30-99, 100+) and quality ratings (1, 2-4, 5) was employed. Web survey links and paper surveys were sent to 1,676 nursing home directors of nursing between February and May 2022. Results: Open text responses were collected from 271 nursing homes. Broad themes included: 1) Addressing needs of residents & families; 2) Challenges; and 3) Personal experiences of nursing home leadership/staff. Respondents described trauma to residents, staff, and leadership. Resident loneliness was addressed using existing and newer technologies and innovative indoor and outdoor activities. Residents experienced fear, illness, loss, and sometimes death. Isolation from family and lack of touch were particularly difficult. Regulations were seen as punitive while ignoring emotional needs of residents. Staffing challenges and pressures to do more with less created additional stress. Leadership and staff made significant sacrifices resulting in physical, social, and emotional consequences. Beneficial outcomes included staff bonding, professional growth, and permanent implementation of new interventions. Conclusions and implications: New and creative interventions were successfully implemented to address social isolation and loneliness. Improved Wi-Fi and other nursing home infrastructure upgrades are needed to maintain them. Reimagining often conflicting overlapping federal, state, and local regulations, grounding them in good clinical judgement, and incentivizing performance improvement should be considered. Trauma experienced by staff needs to be addressed to deal with current and future workforce needs.
  • A programmable dual-targeting di-valent siRNA scaffold supports potent multi-gene modulation in the central nervous system [preprint]

    Belgrad, Jillian; Tang, Qi; Hildebrand, Samuel; Summers, Ashley; Sapp, Ellen; Echeverria, Dimas; O'Reilly, Dan; Luu, Eric; Bramato, Brianna; Allen, Sarah; et al. (2023-12-19)
    Di-valent short interfering RNA (siRNA) is a promising therapeutic modality that enables sequence-specific modulation of a single target gene in the central nervous system (CNS). To treat complex neurodegenerative disorders, where pathogenesis is driven by multiple genes or pathways, di-valent siRNA must be able to silence multiple target genes simultaneously. Here we present a framework for designing unimolecular "dual-targeting" di-valent siRNAs capable of co-silencing two genes in the CNS. We reconfigured di-valent siRNA - in which two identical, linked siRNAs are made concurrently - to create linear di-valent siRNA - where two siRNAs are made sequentially attached by a covalent linker. This linear configuration, synthesized using commercially available reagents, enables incorporation of two different siRNAs to silence two different targets. We demonstrate that this dual-targeting di-valent siRNA is fully functional in the CNS of mice, supporting at least two months of maximal target silencing. Dual-targeting di-valent siRNA is highly programmable, enabling simultaneous modulation of two different disease-relevant gene pairs (e.g., Huntington's disease: MSH3 and HTT; Alzheimer's disease: APOE and JAK1) with similar potency to a mixture of single-targeting di-valent siRNAs against each gene. This work potentiates CNS modulation of virtually any pair of disease-related targets using a simple unimolecular siRNA.
  • An exploration of influenza A virus entry factors using CRISPR-based gene editing

    Kyawe, Pyae Phyo (2023-12-18)
    Influenza A virus (IAV) is a respiratory pathogen with a segmented negative-sense RNA genome that is capable of causing epidemics and pandemics. During the 2021-2022 influenza season, approximately 9 million people in the U.S. were infected with influenza, resulting in an estimated 5,000 deaths. The error-prone nature of the IAV polymerase results in antigenic drift and antigenic shift which contribute to low vaccine efficacy and escape from antivirals. Furthermore, the host factors required for the complete IAV infectious cycle have not been fully identified. The aim of this dissertation is to examine the host factors that may contribute to IAV infectivity in human lung cells. My goal is to understand how changes in the expression levels of host factors can impact influenza infection by CRISPR-mediated knockout or overexpression of target genes. Utilizing CRISPR screens, several candidates, whose up- or down-regulation resulted in reduced IAV infection in the human A549 cell line were identified. I confirmed that the knockout of CMAS or overexpression of B4GALNT2 inhibited IAV infection. In addition, I tested whether overexpression of two candidates from the CRISPR activation screen – DEFB127 and ADAR1 – would inhibit IAV and non-IAV viruses. Surprisingly, overexpression of the two candidates had minimal impact on IAV in A549 cells, but overexpression of ADAR1 had a pro-viral effect on other viruses. Taken together, these data provide insight into host factors modulating IAV infection and how CRISPR-mediated gene modulation can be utilized to further understand the IAV life cycle and for development of therapeutic agents for flu.
  • Experimental and Computational Methods for Identifying Death-Regulatory Genes from Chemo-Genetic Profiles

    Honeywell, Megan E (2023-12-18)
    A common approach to understanding how drugs induce their therapeutic effect is identifying the genetic determinants of drug sensitivity. This can be achieved following systematic loss- or gain-of-function genetic perturbations with CRISPR/Cas9. Because these “chemo-genetic profiles” are generally performed in a pooled format, inference of gene function is subject to several confounding influences, including variation in growth rates between clones or variation in the degree of coordination between growth and death. To overcome these issues, we developed an analysis method called MEDUSA (Method for Evaluating Death Using a Simulation-assisted Approach). MEDUSA uses time-resolved measurements and model driven constraints to reveal the combination of growth and death rates that generated the drug-treated clonal abundance. We find that MEDUSA is uniquely effective at identifying death regulatory genes, and we apply MEDUSA to determine how DNA damage-induced lethality varies in the presence and absence of p53. We find that loss of p53 switches the mechanism of DNA damage-induced death from apoptosis to a non-apoptotic form of death called MPT-driven necrosis. We find that activation of MPT by DNA damage requires high respiration, and that cell death can be exacerbated by modulating NAD+ in p53-deficient cells. These findings demonstrate the accuracy and utility of MEDUSA, both for determining the genetic dependencies of lethality and for revealing opportunities to promote the lethality of chemotherapies in a cancer specific manner.
  • Advancing RNA-Targeting Therapeutics by Oligonucleotide Engineering: RNA Activation, Off-Target Effect, and Co-Targeting Nuclear and Cytoplasmic RNA

    Wang, Feng (2023-12-15)
    Traditional protein-targeting therapeutics by small molecules can target ~3000 proteins, representing <2% of the human genome. In comparison, ~80% of human genome is transcribed into RNAs, which are mostly targetable by oligonucleotides. Thus, RNA-targeting therapeutics enable a much bigger spectrum of drug targets. With nearly 20 FDA-approved oligonucleotide drugs and numerous ongoing clinical trials, RNA-targeting therapeutics by oligonucleotides have significantly addressed unmet medical needs. However, challenges persist in gene expression enhancement and comprehensive dual-RNA modulation. Here, I set out to address these two challenges. Firstly, I identified two ASOs designed to target intron 1 of FXN pre-mRNA, leading to a ~2-fold increase in FXN mRNA levels in multiple cell models. Despite rigorous controls such as two RNA quantification assays and normalization by multiple housekeeping genes, the FXN activation by these two ASOs wasn’t driven by direct binding to the FXN pre-mRNA or a mutual off-target transcript. Surprisingly, it’s found dependent on guanosine-rich motifs in the full PS backbone, suggesting non-base-paired off-target effects. Secondly, we developed siRNASO, an oligonucleotide scaffold integrating the functionalities of siRNA and ASO into a single molecule. siRNASOs demonstrated potent single RNA silencing and comprehensive dual-RNA modulation, including RNA silencing, splicing modulation, and RNA editing in vitro and in vivo. Notably, siRNASO exhibits excellent tolerability in the mouse CNS, suggesting its potential as a therapeutic platform for CNS disorders. Overall, the research in this thesis will serve as a valuable foundation for future research and therapeutic applications, significantly contributing to the advancement of RNA-targeting therapeutics.
  • A role for mutations in AK9 and other genes affecting ependymal cells in idiopathic normal pressure hydrocephalus

    Yang, Hong Wei; Lee, Semin; Berry, Bethany C; Yang, Dejun; Zheng, Shaokuan; Carroll, Rona S; Park, Peter J; Johnson, Mark D (2023-12-15)
    Idiopathic normal pressure hydrocephalus (iNPH) is an enigmatic neurological disorder that develops after age 60 and is characterized by gait difficulty, dementia, and incontinence. Recently, we reported that heterozygous CWH43 deletions may cause iNPH. Here, we identify mutations affecting nine additional genes (AK9, RXFP2, PRKD1, HAVCR1, OTOG, MYO7A, NOTCH1, SPG11, and MYH13) that are statistically enriched among iNPH patients. The encoded proteins are all highly expressed in choroid plexus and ependymal cells, and most have been associated with cilia. Damaging mutations in AK9, which encodes an adenylate kinase, were detected in 9.6% of iNPH patients. Mice homozygous for an iNPH-associated AK9 mutation displayed normal cilia structure and number, but decreased cilia motility and beat frequency, communicating hydrocephalus, and balance impairment. AK9+/- mice displayed normal brain development and behavior until early adulthood, but subsequently developed communicating hydrocephalus. Together, our findings suggest that heterozygous mutations that impair ventricular epithelial function may contribute to iNPH.
  • A Fluorescent Reporter Mouse for Assessment of Genome Editing with Diverse Cas Nucleases and Prime Editors

    Chen, Zexiang; Kwan, Suet-Yan; Mir, Aamir; Hazeltine, Max; Shin, Minwook; Liang, Shun-Qing; Chan, Io Long; Kelly, Karen; Ghanta, Krishna S; Gaston, Nicholas; et al. (2023-12-13)
    CRISPR-based genome-editing technologies, including nuclease editing, base editing, and prime editing, have recently revolutionized the development of therapeutics targeting disease-causing mutations. To advance the assessment and development of genome editing tools, a robust mouse model is valuable, particularly for evaluating in vivo activity and delivery strategies. In this study, we successfully generated a knock-in mouse line carrying the Traffic Light Reporter design known as TLR-multi-Cas variant 1 (TLR-MCV1). We comprehensively validated the functionality of this mouse model for both in vitro and in vivo nuclease and prime editing. The TLR-MCV1 reporter mouse represents a versatile and powerful tool for expediting the development of editing technologies and their therapeutic applications.

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