Clinical trials are a fundamental tool in evaluating the safety and efficacy of new drugs, medical devices, and health system interventions. Clinical trial visits generally involve eligibility assessment, enrollment, intervention administration, data collection, and follow-up, with many of these steps performed during face-to-face visits between participants and the investigative team. Social distancing, which emerged as one of the mainstay strategies for reducing the spread of SARS-CoV-2, has presented a challenge to the traditional model of clinical trial conduct, causing many research teams to halt all in-person contacts except for life-saving research. Nonetheless, clinical research has continued during the pandemic because study teams adapted quickly, turning to virtual visits and other similar methods to complete critical research activities. The purpose of this special communication is to document this rapid transition to virtual methodologies at Clinical and Translational Science Awards hubs and highlight important considerations for future development. Looking beyond the pandemic, we envision that a hybrid approach, which implements remote activities when feasible but also maintains in-person activities as necessary, will be adopted more widely for clinical trials. There will always be a need for in-person aspects of clinical research, but future study designs will need to incorporate remote capabilities.

OBJECTIVES/GOALS: The objective of this study was to determine whether pet dogs with multicentric lymphoma (ML), a spontaneous, immunocompetent model for human non-Hodgkin lymphoma (NHL), are exposed to higher concentrations of herbicides and volatile organic compounds (VOCs) compared to matched unaffected control dogs. METHODS/STUDY POPULATION: We are prospectively enrolling dogs with ML within a single high-risk breed, the boxer dog, along with age-matched control boxers sampled within the same season. We are measuring urinary concentrations of the herbicides glyphosate (in Roundup®) and 2,4-D, as well as stable urinary metabolites of the VOCs benzene and 1,3-butadiene. To assess the genotoxic potential of herbicide and VOC exposures, we are using reverse dosimetry to estimate plasma exposures, and exposing healthy canine PBMC’s to these concentrations of herbicides and VOCs in vitroto assess double stranded DNA damage using the Comet Chip assay. RESULTS/ANTICIPATED RESULTS: Preliminary data show significantly higher benzene exposures, measured by the stable benzene metabolite PHMA, and significantly higher 2,4-D exposures at the time of diagnosis in cases versus controls. All dogs had measurable exposures to 1,3-butadiene (measured as its stable metabolite DHBM) and glyphosate.In vitro results show significant genotoxicity thresholds of 0.1 uM for both glyphosate and 2,4-D in dog lymphoid cells. To date, these predicted plasma exposures have not been reached in vivo in boxer dogs with ML or unaffected control boxers. DISCUSSION/SIGNIFICANCE: Canine multicentric lymphoma resembles human NHL and is a potentially useful model of non-occupational chemical risk for NHL in people. The goal of this research is to identify potentially preventable non-occupational chemical risk for lymphoma and support evidence-based remediation strategies to decrease lymphoma risk in both humans and dogs.

ABSTRACT IMPACT: Here, we describe extensive sex-specific differences in the transcriptomes of pancreatic neuroendocrine tumors (PNETs). Given that the clinical course of PNETs differs by sex (female sex is associated with better survival), achieving a greater understanding of the specific molecular sexual dimorphisms is invaluable for advancing personalized treatment. OBJECTIVES/GOALS: Epidemiologic studies demonstrate that pancreatic neuroendocrine tumors (PNETs) exhibit sexual dimorphisms with regards to prognosis, disease recurrence, and complication rates. We sought to compare the transcription and DNA methylation landscapes of PNETs by sex, to elucidate molecular differences that may underlie this sex disparity. METHODS/STUDY POPULATION: RNAseq data was generated from PNETs surgically resected at our institution (9 Female; 12 Male patients). RNA was extracted with the RNeasy Mini Kit, stranded sequencing libraries were prepared with TruSeq, and paired end sequencing was done on the HiSeq 2500/4000 systems. Transcript-level quantification was performed with salmon, and DESeq2 was used for differential expression analysis. To account for significant variation due to covariates other than sex, surrogate variables were computed with the SVA package and adjusted for. The goseq package was used for gene set over representation analysis. Matched DNA methylation (DNAm) and RNAseq data was downloaded from GEO (16 F; 16 M). Raw DNAm data was processed with minfi. Differential methylation analysis was done with limma and bumphunter. Analysis was done in R. RESULTS/ANTICIPATED RESULTS: We found that 826 autosomal genes were differentially expressed (DE) by sex in PNETs (at FDR ≤0.1). Gene set over representation analysis performed on the DE genes revealed significant enrichment for several processes, including ‘ascorbate & aldarate metabolism’ and ‘positive regulation of ERK1 & ERK2 cascade’ (all FDR ≤0.1). When we compared DNAm profiles between sexes, we found 8 CpGs which were differentially methylated by sex (at FDR ≤0.1), 7 of which were proximal to genes. Methylation of one of the sex-associated CpGs, overlapping the gene TIMM8B, was found to be negatively correlated with gene expression (rho=-0.41; p-value=0.02). Interestingly, TIMM8B deletion has been previously reported in other non-pancreatic neuroendocrine tumors. There were no differentially methylated regions between sexes. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our findings demonstrate that PNETs exhibit extensive sexual dimorphisms with regards to gene expression profiles but have largely congruent methylomes by sex. These molecular differences may contribute to the variability in clinical course between men and women, and their characterization is vital for the advancement of personalized medicine.

Objectives/Goals: We previously developed and evaluated a system called MyTrials, which captures biomarker data remotely. We sought to improve that platform to support additional methods of remote biomarker capture, which is especially important in research such as substance use and co-use studies where participants provide several biological verifications. Methods/Study Population: To determine how best to adapt MyTrials, we conducted qualitative interviews with key stakeholders. Our sample of end users (i.e., participants, N=10) included 6 women (3 men and 1 nonbinary participant), 6 White participants (2 Black participants, 1 Asian participant, and 1 multiracial participant), 6 participants with education beyond high school (4 participants with high school/GED), with a mean age of 36.33 years (age range: 21–75), and with a range of substance use behavior (combustible cigarettes [N=6], e-cigarettes [N=3], cannabis use [N=5], and no substance use [N=2]). Our sample of potential customers (N=22) included 12 research investigators, 2 biospecimen lab representatives, 3 research coordinators, 3 representatives from large NIH grant initiatives (ABCT, CTN, and CTSA), and 2 REDCap engineers/managers. Results/Anticipated Results: Emerging themes included 1) suggestions for technical requirements including reliability of core functions, app usability, support access, and data security/compliance and 2) suggestions for commercialization including clarity of instructions, operational logistics, trust in the data collection process, IRB/funder acceptance, and participant retention enablers. Discussion/Significance of Impact: Key feedback themes will inform product improvement to meet the needs for decentralized clinical trials to collect biological or health data without in-person participation.

OBJECTIVES/SPECIFIC AIMS: Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is expressed on Wnt/β-catenin-dependent adult stem cell populations of the colon. Cancer stem cells are hypothesized to be the driving force behind tumor progression and metastasis, making them attractive therapeutic targets. Our aim was to analyze the clinicopathologic and prognostic significance of LGR5 expression in a cohort of colorectal cancer patients with peritoneal metastasis. METHODS/STUDY POPULATION: A total of 49 Formalin-fixed paraffin-embedded (FFPE) tissue blocks of primary or metastatic tumors and their respective normal tissues were collected from the tissue bank for time period 2009-2015. LGR5 expression was assessed at the protein level through immunohistochemical (IHC) staining of tissue microarray (TMA) constructs consisting of pairs of tumor and normal colon tissue. The correlation between LGR5 expression and clinicopathologic parameters and prognosis was assessed by statistical analysis. RESULTS/ANTICIPATED RESULTS: Of the 49 patient sample, 30(61.22%) were female vs. 19 (38.78%) males. Age range at initial diagnosis ranged from 31.7 years to 84.4 years, with a median age of 61.29 years. Duration of follow-up ranged from 1 – 9 years with a median of 5 years.LGR5 expression was higher in colorectal cancer than in normal mucosa. In univariate survival analysis overexpression of LGR5 was significantly associated with improved survival (p=0.002).Of significance, LGR5 positivity was an independent prognostic marker for better prognosis in a multivariate survival analysis adjusting for prognostic variables age, stage, gender, tumor histology and grade (HR 2.67. 95% CI 1.01-7.00, P = 0.046). DISCUSSION/SIGNIFICANCE OF IMPACT: LGR5 was significantly over expressed in colorectal cancer compared to normal tissues. LGR5 was noted to be an independent prognostic variable for an improved survival outcome in colorectal cancer patients with peritoneal metastasis, making LGR5 a potential therapeutic target in colorectal cancer patients with peritoneal metastasis.

OBJECTIVES/GOALS: This study’s goal is to determine whether intraoperative treatment of facial nerves with polyethylene glycol (PEG) fusion technology improves facial paralysis outcomes. Improved facial nerve regeneration in facial paralysis patients would lead to improved recovery time and effectiveness. METHODS/STUDY POPULATION: 30 rats were utilized; 15 underwent facial nerve regeneration without PEG fusion, and 15 with PEG fusion. Facial paralysis was initiated on the left by transection of the buccal and marginal mandibular branches of facial nerve. The buccal branch was repaired though microsuture technique. Neurorrhaphy sites of rats in the PEG group were exposed to calcium free saline, methylene blue, and polyethylene glycol. Nerve continuity was assessed post-operative in 5 animals in each group through electron microscopy. Functionality was assessed in the other 10 per group by EMG and whisker analysis after surgery, and weekly for 8 weeks. At 8 weeks, nerves and distal muscles were histologically analyzed. RESULTS/ANTICIPATED RESULTS: PEG fusion technology immediately restored axonal continuity following surgery, demonstrated by electron microscopy. Electrophysiology was also similarly restored across the site immediately, determined through intraoperative nerve stimulation, in the PEG fusion group. The nonintervention group showed dramatically reduced functional recovery than the PEG fusion group following surgery, shown by lower whisking activity and poor electrophysiology outcomes. Furthermore, the PEG fusion group showed statistically significant higher fascicle counts, myelination diameter, axonal diameter, and distal muscle fibers histologically. DISCUSSION/SIGNIFICANCE OF IMPACT: This study demonstrates that polyethylene fusion technology may improve facial reanimation outcomes. PEG is already a FDA-approved drug, and thus the pathway to translational clinical application of this work may thus be streamlined, bringing new options to patients with facial paralysis.

OBJECTIVES/GOALS: Single nucleotide polymorphisms in the transcription factor 7-like 2 (TCF7L2) gene are associated with Type 2 Diabetes (T2D) and nonalcoholic fatty liver disease (NAFLD). The metabolic function of TCF7L2 in the liver remains to be fully elucidated, but we hypothesized that TCF7L2 contributes to NAFLD through regulation of zonal metabolic pathways. METHODS/STUDY POPULATION: Using single nuclei RNA sequencing, we examined Tcf7l2 expression in periportal (PP) hepatocytes around the portal triad and pericentral (PC) hepatocytes surrounding the central vein of the liver. To visualize TCF7L2 transcriptional activity we used a TCF reporter mice, which expresses an H2B-eGFP fusion protein downstream of the conserved TCF DNA binding site. We disrupted Tcf7l2 transcriptional activity in mouse liver by breeding mice with a floxed Tcf7l2 exon 11, which encodes part of the DNA binding domain (DBD), to albumin-Cre mice (Hep-TCF7L2ΔDBD). Eight-week-old mice were fed a choline-deficient amino acid-defined high fat (CDAHFD) diet for 8 weeks. In liver samples harvested from these mice, we examined disruption to several key zonated metabolic pathways, and quantified the development of fibrosis. RESULTS/ANTICIPATED RESULTS: Single nuclei analysis revealed that Tcf7l2 mRNA was expressed primarily in parenchymal cells of the liver but was ubiquitous across the liver lobule. However, in immunofluorescence analysis of TCF reporter mice, the transcriptional activity of TCF7L2 was highly restricted to PC hepatocytes. Classic PC hepatocyte markers, including glutamine synthetase (Glul), were absent in Hep-TCF7L2ΔDBD mice. Following the CDAHFD, Hep-TCF7L2ΔDBD mice developed more severe fibrosis in histological analysis, and expressed elevated levels of genes involved in fibrogenesis, collagen synthesis and TGFβsignaling. Hep-TCF7L2ΔDBD mice also displayed hepatic cholesterol accumulation following the CDAHFD, which was likely the result of impaired pericentral bile acid synthesis. DISCUSSION/SIGNIFICANCE: Our results suggest that TCF7L2 plays an important role in the regulation of zonated metabolic pathways, which may contribute to the development of fibrosis. Ongoing analyses are exploring the mechanisms regulating the zonal transcriptional activity of TCF7L2.

OBJECTIVES/GOALS: Oligodendrocytes (OL) are glial cells of the central nervous system (CNS) responsible for the energy demanding task of generating myelin sheaths during development and remyelination after demyelinating injury. One metabolite shown to significantly increase ATP production in OL is the nitrogenous organic acid, creatine. Creatine plays an essential role in ATP buffering within tissues with highly fluctuating energy demands such as brain and muscle. Interestingly, mature OL, which are the cells capable of myelin production, are the main cells in the CNS expressing the rate-limiting enzyme for creatine synthesis, guanidinoacetate methyltransferase (Gamt). Patients with mutations in Gamt display intellectual disabilities, impaired myelination and seizures. Therefore, we hypothesize that creatine may be essential for developmental myelination and improve remyelination. METHODS/STUDY POPULATION: To investigate these hypotheses, we developed a new transgenic mouse model with LoxP sites flanking exons 2-6 of the Gamt gene where excision leads to expression of a green fluorescent tag allowing us to track the cells normally expressing Gamt. RESULTS/ANTICIPATED RESULTS: In this mouse model, we show a 95% (±0.47%, n = 3) co-localization of Gamt within mature OL during postnatal (P) day P14. Next, we show that knocking out Gamt leads to a significant reduction in OL in the major CNS white matter tract, the corpus callosum, at P14 and P21 (P14: 0.007, n = 3; P21: 0.04, n = 3). Here, we also investigate whether dietary creatine can enhance remyelination in the cuprizone model of toxic demyelination. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies highlight the important role creatine plays in developmental myelination and investigate whether creatine can provide a therapeutic value during a CNS demyelinating insult.

Objectives/Goals: This project evaluates the acceptability to patients of offering free tax preparation and joint charity-care enrollment in a clinical setting, leveraging an Internal Revenue Service-supported free income tax preparation program. The goal is to reduce administrative barriers and promote continuous access to care. Methods/Study Population: In September 2025, three members of our study team conducted in-person surveys with patients and visitors in outpatient waiting areas at Parkland Health, the county safety-net health system serving Dallas County in North Texas. The 16-item bilingual (English/Spanish) survey was developed in partnership with a large, Internal Revenue Service-supported, local VITA program, Dallas Community Tax Centers (DCTC). The survey collected sociodemographic information, household income, tax-filing practices, current charity care program enrollment status, and interest in combined VITA–charity care enrollment/renewal services. Data were entered into REDCap and analyzed descriptively in Excel. Results/Anticipated Results: Of 300 approached patients, 232 completed the survey, yielding a response rate of 77% (28% Spanish-preferring). Most respondents (86%) estimated that their household income was below $67,000, which would qualify them for VITA tax preparation, yet 82% of those eligible reported that they paid a tax preparer last year. More than half (54%) of respondents were enrolled in the charity care program for the county health system, and 87%of these adults indicated that integrated tax-and-charity care enrollment/renewal services would be helpful. Awareness and reported uptake of the Earned Income Tax Credit (EITC) was low: only 11% of respondents reported applying for EITC during the recent tax season. Interest in shifting to free on-site tax prep services was high, with 72% of respondents indicating a favorable response. Discussion/Significance of Impact: Embedding tax preparation and streamlining charity-care assistance applications in clinical environments was well received and highly acceptable. Future steps include piloting joint VITA–charity-care applications services to assess impact on operational workflows, uptake of both programs, continuity of care, and health outcomes.

OBJECTIVES/GOALS: The objective of this study was to estimate the cost differences of a telerehabilitation versus outpatient session. A secondary objective was to identify areas to improve telerehabilitation delivery efficiency. We aim to improve the translation/adoption of telerehabilitation for clinical use. METHODS/STUDY POPULATION: This study used a time-driven activity-based costing (TDABC) approach including 1) observation of rehabilitation sessions and creation of manual time stamps, 2) structured and recorded interviews with two occupational therapists familiar with outpatient therapy and two therapists familiar with telerehabilitation, 3) collection of standard wages for providers, and 4) the creation of an iterative flowchart of both an outpatient and telerehabilitation session care delivery process. This study followed the reporting guidelines to ensure a standardization for TDABC research. RESULTS/ANTICIPATED RESULTS: Overall, telerehabilitation ($225.41) was more costly than outpatient therapy ($168.29) per session for a cost difference of $57.12. Primary time drivers of this finding were initial phone calls (0 mins for OP therapists versus 35 mins for TR) and post documentation (5 mins for OP versus 30 mins for TR) demands for telerehabilitation. DISCUSSION/SIGNIFICANCE: Telerehabilitation is an emerging platform with the potential to reduce costs, improve healthcare inequities, and facilitate better patient outcomes. Improvements in documentation practices, staffing, technology, and reimbursement structuring would allow for a more successful translation.

Objectives/Goals: Nearly 42% of adults in the USA have obesity; women are disproportionately affected. Women with obesity that become pregnant are especially prone to developing health condition; thus, aid is needed to achieve appropriate gestational weight gain (GWG). GWG interventions would benefit from examining the effects of social support and stress has on GWG. Methods/Study Population: The proposed study will examine whether expecting mothers’ social support predicts their perceived stress. Also, examine whether receiving aid, in the form of weekly meal delivery, changes perceived stress. Lastly, this study will explore if social support mediates the relationship between meal delivery and perceived stress. The study will use data gathered from a pilot GWG intervention focused on providing pregnant women weekly meal delivery. Participants (N = 14) will complete 2 visits at 16–20- and 35–36-weeks gestation, while receiving weekly meal deliveries. Data from both visits will be used for the purpose of this study. Women’s social support will be measured using the Multidimensional Scale of Perceived Social Support. While stress will be measured via the Perceived Stress Scale. Results/Anticipated Results: Previous behavioral weight loss studies have found that individual who endorse experiencing social support report less perceived stress. Although the relationship has seldomly been examined in pregnant women, similar results are anticipated. Often aid, such as meal delivery, can impact perceived stress and is likely to cause a positive change. To our knowledge, this is the first study to explore whether social support mediates the changes in perceived stress after receiving weekly meal delivery. Therefore, there is no known literature to suggest anticipatory results. Discussion/Significance of Impact: Few studies have examined the association between social support and perceived stress in pregnant women. This study will examine the effects social support has on a pregnant women’s stress. The proposed study will aim to identify if meal delivery changes perceived stress. If so, the study will explore if perceived social support mediates this change.

OBJECTIVES/SPECIFIC AIMS: Histones are nuclear proteins that associate with DNA to facilitate packaging into condensed chromatin. Histones are dynamically decorated with post-translational modifications (PTMs), which regulate such processes as DNA repair, gene expression, mitosis, and meiosis. Histone 3 Family 3 (H3F3) histones (H3.3), encoded by H3F3A and H3F3B, mark active genes, maintain epigenetic memory, and maintain heterochromatin and telomeric integrity. Specific somatic mutations in H3F3A and H3F3B have been strongly associated with pediatric glia and other tumors, but no germline mutations have been reported. The goal of our study was to further understand the functional effects of germline mutations of H3F3A and H3F3B. METHODS/STUDY POPULATION: We analyzed 32 patients bearing de novo germline missense mutations in H3F3A or H3F3B with core phenotypes of progressive neurologic dysfunction and congenital anomalies, but no malignancies. Patient histones were analyzed by quantitative mass spectrometry (qMS). RESULTS/ANTICIPATED RESULTS: qMS results revealed that the mutant histone proteins are present at a concentration similar to that of wild-type H3.3. qMS analysis showed strikingly aberrant PTM patterns that suggested local dysregulation. These patterns are distinct from the dominant negative somatic mutations, which cause more global PTM dysregulation. Patient cells also demonstrated upregulation of the expression of genes related to mitosis and cell division, and had a greater proliferative capacity. DISCUSSION/SIGNIFICANCE OF IMPACT: Our data suggests that the pathogenic mechanism of germline histone mutations is distinct from that of the published cancer-associated somatic histone mutations, but may converge on control of cell proliferation. Further clarification of the pathophysiology in these patients can elucidate the roles of histones and histone PTMs in human development and non-syndromic neurodegeneration. In addition, it provides a framework for targeted therapy development for this and related progressive neurologic disorders.

OBJECTIVES/GOALS: Our long-term goal is to improve the clinical outcomes of patients with status epilepticus through increasing the level of evidence surrounding guidelines. The specific objective of our proposed work is to compare the outcomes and adverse drug events between the Neurocritical Care and American Epilepsy Society levetiracetam dose recommendations. METHODS/STUDY POPULATION: This is a retrospective, single site, cohort study comparing outcomes of hospitalized patients with status epilepticus treated with levetiracetam bolus at the University of Kansas Health System. Patients outcomes will be compared based on levetiracetam bolus dose received. The primary outcome will be seizure reoccurrence within 24 hours. Secondary outcomes include number of additional anti-epileptic drugs administered, cumulative dose of benzodiazepines administered within 24 hours of levetiracetam bolus administration and incidence of adverse drug reactions. All study data will be extracted retrospectively from the EPIC chart review following patient list generation through the HERON i2b2 database query. RESULTS/ANTICIPATED RESULTS: Aim 1 will characterize levetiracetam dosing among patients admitted to the University of Kansas Medical Center for status epilepticus during routine clinical care. We hypothesize that given the inconsistency in dosing recommendations from various professional societies and drug references, we will observe inconsistent dosing of levetiracetam among those hospitalized due to status epilepticus.

Aim 2 will evaluate effectiveness and safety across the various levetiracetam doses. We estimate that adherence to the higher weight-based dosing recommendations of the 2016 American Epilepsy Society guideline may result in improved outcomes with a similar frequency and severity of adverse drug events compared to lower/fixed-dose levetiracetam dose recommendation of the 2012 Neurocritical Care guideline. DISCUSSION/SIGNIFICANCE: Notable inconsistencies across the dosing recommendations for levetiracetam exist between the guidelines for treatment of status epilepticus and commonly used tertiary drug information databases. This variation in guidance may lead to differences in dosing and warrants further exploration to better support the management of status epilepticus.

ABSTRACT IMPACT: This work assesses clinical implementation of a surface guided imaging system to improve the accuracy radiation delivery for treatment of brain lesions using a patient CT derived head phantom. OBJECTIVES/GOALS: Advancements in radiotherapy design have made clinical demand for efficient and accurate methods to deliver stereotactic radiosurgery (SRS) for treatment of intracranial lesions. This study assesses the potential of using surface guided imaging for setup using a 3D patient specific head phantom. METHODS/STUDY POPULATION: A single isocenter, multiple metastases SRS plan was generated on a CT derived RTsafe Prime phantom made of tissue equivalent materials and a polymer gel insert. Five targets of varying diameters were treated with 8Gy of radiation using two different positioning techniques. The first gel insert was irradiated within the phantom according to internal alignment with standard orthogonal x-ray imaging while the second setup used surface guided imaging, based on external anatomy. 42 hours after irradiation, the phantom was scanned in a head coil using a 1.5T MRI. MR images were fused with the patient CT data and structure set to further evaluate calculated and measured dose distributions. RESULTS/ANTICIPATED RESULTS: Discrepancies in phantom setup according to standard orthogonal x-ray imaging compared to surface guided imaging demonstrated to be <1mm in each translational (vertical, longitudinal, and lateral) and angular (rot, roll, pitch) directions. The 3D gel inserts permitted spatial analysis to compare dose distributions of measured values to those calculated in a treatment planning system (TPS). 3D GI (Gamma Index) analysis showed good alignment in high dose regions and resulted in passing rates >94% (5%/2mm) and >87% (3%/2mm). Finally, 3 of 5 targets showed better 3D GI passing rates and less geometric offset for positioning with the surface guided imaging. DISCUSSION/SIGNIFICANCE OF FINDINGS: 3D spatial analysis of human like phantoms demonstrated that patient positioning according to external anatomy performed comparable to standard methods aligning to the internal anatomy, for a multiple met SRS treatment.

Objectives/Goals: Now in its 10th year, Research Jam, Indiana CTSI’s Patient Engagement Core, has proven to be an effective approach to patient engagement, foregoing generalized CABs for study-specific groups, personally invested in the research. Here we share our methods to benefit CTSAs seeking deeper engagement. Methods/Study Population: Research Jam is unique from patient engagement efforts in other CTSAs in almost every aspect. The composition and background of our team, the methodology to our practice and often our outcomes. This poster will use 10 years of projects, publications, and participant evaluations to explore our approach both quantitatively and qualitatively to see how it has contributed to the Indiana CTSI’s commitment to community engagement. Results/Anticipated Results: We will present quantitative data from 10 years of participant evaluations illustrating that Research Jams are received positively by those that attend, and qualitative analysis of the evaluations around themes of feeling heard, valued, and integral in research. By finding themes in evaluations across 10 years of studies, with different topics, different population types, and different investigators, we can speculate on what aspects of our approach appeal to the community, contribute to their positivity toward research and researchers, and could be reproducible in other CTSAs. Discussion/Significance of Impact: We use “blueprint” instead of “toolkit” when describing our process, as we hope others can see this not as a tool to fix a problem, but a plan to be iterated on in agreement with the community that will benefit from it. The relationships researchers need to build with communities are not cookie cutter neighborhoods, but rich, colorful and vibrant ones.

OBJECTIVES/GOALS: Re-administration of inhaled gene therapies has the potential to overcome low correction efficiencies and limiting immune responses observed in previous trials of gene therapy for Cystic Fibrosis. We therefore tested the hypothesis that pre-treatment with a B-cell depleting α ± CD20 antibody would permit vector re-administration. METHODS/STUDY POPULATION: We first selected Adenoviral (Ad) vectors to study initially due to their well-known ability to elicit potent immune responses. Mice were dosed with a depleting α ± CD20 antibody or isotype control 2 days prior to delivery of Ad-Luc. 4 weeks later, mice were euthanized to assess the development of anti-vector immune responses. Flow cytometry and single-cell RNA sequencing were used to evaluate the development of lung-resident memory cells. Serum and airway antibody responses were assessed by ELISA. After 4 weeks, mice were dosed with Ad-LacZ and euthanized 3 days later to assess efficiency of second-round gene transfer by β-galactosidase activity assay. Similar methods were used in a pilot experiment with Adeno-associated virus vector (AAV), but with euthanasia 3 weeks after secondary gene transfer. RESULTS/ANTICIPATED RESULTS: Delivery of Ad vectors leads to the development of lung-resident memory B and T-cells. The depletion of B-cells prior to first-round vector delivery attenuated airway T-cell infiltration and serum IgG production, abrogated mucosal IgG and IgA production, and completely rescued secondary gene transfer. Genetically modified mouse models suggest secreted antibodies are critical in prevention of vector redosing. AAV vectors were found to be less immunogenic than Ad vectors, with only partial reduction of second-round gene transfer. However, anti-CD20 provided no benefit for AAV redelivery. DISCUSSION/SIGNIFICANCE: Mucosal humoral immunity is critical in preventing re-administration of Adenoviral vectors. Impairment of B-cell responses by α ± CD20 treatment prior to vector delivery allows re-administration and may help overcome low efficiencies of CF gene therapy. AAV vectors may be less susceptible to neutralization by pre-existing immunity.