Alzheimer’s disease (AD) is the most common age associated neurodegenerative disease in humans, with approximately 50 million people living with the condition worldwide. AD is the leading cause of dementia, memory loss, and other symptoms related to cognitive decline. Pathologically, AD is greatly associated with the formation of amyloid-beta plaques (Aβ) forming aggregates within the brain and disrupting critical neurological functions. As current treatments for AD only serve to mitigate symptoms, there is a dire need for new therapeutic drugs that address the causes of AD and more efficiently target Aβ plaque formation. Due to its ease of culturing and short life cycle, Caenorhabditis elegans is well suited for the rapid screening of candidate compounds. In this study, we utilize a C. elegans AD disease model expressing a full-length human Aβ gene for the screening of 26 FDA-approved compounds in a dose-dependent manner. We observe that at least 6 of these compounds result in a robust delay in worm paralysis. To investigate whether these compounds delay paralysis by reducing Aβ protein aggregation, we are staining worms with X-34 – a fluorescent dye that labels amyloid structures – and subsequently comparing staining patterns in worms with and without drug treatment. In addition to recording the timing of worm paralysis, we are studying behavioral changes in drug-treated and control worms to determine the effects our identified drugs on other physiological processes. In our use of a pre-clinical C. elegans model, we can apply our findings to future clinical treatments as most C. elegans genes are also conserved in humans. Therefore, by identifying FDA-approved compounds that reduce the appearance of Aβ plaques within C. elegans, we can fast track drug discovery to better treat AD in humans.

Kidney function declines with age and is accompanied by injury to the nephron glomerulus. A critical cell for filtration in the glomerulus are podocytes, which are lost with aging and non-proliferative. However, the parietal epithelial cells (PECs) lining the Bowman’s capsule of the nephron may migrate to the glomerular tuft to replace podocytes through differentiation. The mechanisms by which PECs differentiate into podocytes is still unknown. Using a mitochondrial intervention peptide, we previously showed reduction of glomerular injury and improved PEC numbers in aged mice. Mitochondrial dysfunction increases in all organs with aging; therefore, we hypothesized that mitochondrial aging dysfunction affects PEC-to-podocyte differentiation. To determine whether mitochondrial aging dysfunction plays a role in the differentiation of podocytes from PECs, we used young (18-30 yrs.) and old (50+ yrs.) primary human cells derived from kidney PECs isolated from urine. Human urine progenitor cells (hUPCs) were sorted for makers CD133+ and CD24+. For hUPC progenitor-to-podocyte differentiation, we used ‘VRADD’ medium (100 nm retinoic acid + 100 nm Vitamin D3). Proliferation and oxygen consumption rate (OCR) were measured during differentiation. To determine the differentiation of hUPC, we looked for the loss of progenitor makers CD133+ and CD24+ by qRT-PCR. In both aged and young cells, hUPC-podocyte VRAD differentiation resulted in a significant increase of OCR that peaked at day 2 relative to control. Both proliferation and OCR normalized to cell number were significantly lower in aged vs. young cells. Cells cultured with VRADD showed decreased progenitor markers CD24 and CD133 (PROM1) as compared to controls, suggesting hUPC differentiation. Our results support an aging decline in PEC-podocyte differentiation Future work will confirm whether podocyte fate was achieved with qRT-PCR and immunohistochemistry for expression podocyte specific genes/proteins WT1, SYNPO, NPHS1, and NPHS2 and additional assays to measure mitochondrial function of hUPCs across differentiation.

 Alzheimer’s disease (AD) is the most prevalent of neurodegenerative diseases, with over 6 million Americans suffering from the illness and cases increasing each year. TREAT-AD (TaRget Enablement to Accelerate Therapy Development for AD) is an NIH-funded, multi-institutional network that identifies and addresses new targets for AD drug development. Genetic targets of interest were identified via RNA-sequencing and proteomic analysis of post-mortem tissue from participants with AD. We tested the effects of inhibiting or overexpressing selected target genes hypothesized to increase the risk of Alzheimer’s. One such target is the CD44 gene, which regulates GABA receptor activity. To efficiently manipulate genetic expression, we used clustered regularly interspaced palindromic repeats (CRISPR) technology to enhance (CRISPRa) or repress (CRISPRi) genetic transcription in neural progenitor cells, stem cells in the process of differentiating into neurons. We used a cell line engineered to harbor CRISPRa and CRISPRi machinery. CRISPRa involves a catalytically inactive Cas9 protein fused to a transcriptional activator. In CRISPRi, the inactivated Cas9 is fused to a transcriptional repressor. This, along with the quantification of molecular pathways linked to Alzheimer’s, permits a window into the conditions that lead to earlier onset of AD, and thereby conditions that might be altered by new drug treatments. Here we report that underexpression of genes related to endosomal trafficking leads to changes in protein buildup that may be related to earlier onset of AD.

Herpes Simplex Virus (HSV) causes lifelong infections due to the establishment of latency in peripheral nerves. The immune response to HSV infections involves CD8 tissue resident memory T cells (CD8TRM) to sense HSV reactivation, and the innate immune cells to mitigate viral replication and spread. Plasmacytoid dendritic cells (pDC) can produce type-1 interferons (IFN-I) and prime CD8 T cells, yet their role in human HSV infections is unclear. ChipCytometry is a highly multiplexed imaging platform useful in observing cell-cell interactions, cell distribution in a tissue, and cell phenotypes. Using Chip Cytometry, we analyzed a tissue biopsy from a genital herpes lesion with a panel of 24 markers to characterize immune cell infiltration during active HSV reactivation. We showed that CD123+/CD303+ pDCs were located near activated CD8 T cells, many of which were found in lymphoid like structures in the dermis. These lymphoid like structures formed in areas where large nerve bundles and blood vessels were present and were packed with T cells and other immune cells which expressed markers for activation, exhaustion, and proliferation. Further research into pDC functions in HSV infections and cell interactions in these lymphoid like structures may provide insight on mechanisms of immune cell surveillance and response of reactivating HSV infections.

Chronic kidney disease is the ninth leading cause of death in the United States. The current process for pathological diagnosis involves pathologists manually reviewing histochemically stained tissue slides. This analysis is also used to inform further treatment and is therefore critical to patient outcomes. In this project, we aim to improve the robustness of the diagnostic process by utilizing machine learning models to identify and classify features indicative of kidney disease on whole slide images. We manually annotate Masson’s Trichrome stained kidney tissue images from our collaborators at the University of Illinois for three functional structures (tubular cytoplasm, tubular basement membrane, glomerulus) and three indicators of damage to the kidney (fibrosis, edema, and inflammation). These annotations are used to train our VGG16 convolutional neural network model to classify patches of unmarked whole slide images into the four categories: tubular cytoplasm, fibrosis, inflammation, and glomerulus. We also address data variability that often comes from differences in the histochemical staining procedure across labs resulting in inconsistency across stains/imaging that can typically affect the generalizability of deep learning models. To address this, we are training a CycleGAN for image-to-image translation as a method of stain normalization and investigating the effect on the accuracy of our VGG16 model. Additionally, I will be training a model to identify the cortex versus medulla regions of the kidney to add to the pipeline for area-specific evaluations. Our research with integrating machine learning models within renal pathology aims to decrease the time and manual labor needed in the process and increase the accuracy of diagnoses.

Mitochondria are organelles responsible for Adenosine triphosphate (ATP) production and are central in biological aging research. Laboratory interventions that extend healthy animal lifespans also work to treat severe animal mitochondrial disease, however, how these interventions work at the molecular level is still unknown. Several longevity interventions extend lifespan and treat a model of Leigh Syndrome, a severe mitochondrial disease, in mice. Among these, rapamycin inhibits the metabolic master regulator mTOR (mechanistic target of rapamycin). This evidence suggests that mitochondrial disease and biological aging share a common cause at the cellular level. When mTOR is inhibited, SIRT3 is upregulated, an enzyme that controls mitochondrial fatty acid oxidation (FAO). We are using the mouse model of Leigh Syndrome, Ndufs4 knockout (KO) mice, to ask the following question: Does rapamycin treatment require SIRT3 activity to increase FAO to treat mitochondrial disease? Preliminary results show that SIRT3 is required for lifespan extension in Ndufs4 KO mice with rapamycin treatment. SIRT3 has also been observed regulating FAO. We are using both etomoxir, a drug that inhibits FAO, and rapamycin to answer this question by measuring lifespan. We are also measuring mitochondrial FAO directly in Ndufs4 Sirt3 double KO animals. Our results are allowing us to better understand how mitochondrial disease and normative aging are related, which will streamline targeting the biology of aging and mitochondrial dysfunction in humans.

Infection from Mycobacterium tuberculosis is the second leading infectious cause of death worldwide after COVID-19, with rates of tuberculosis infection greatest in low and middle-income countries (LMICs). Tuberculous meningitis (TBM) is one of the most severe forms of M. tuberculosis disease with over half of all cases resulting in death or neurological consequences. Recent studies in our lab have found that single-nucleotide polymorphisms (SNPs) in MUC5AC, a secretory lung mucin, are associated with increased TBM susceptibility, morbidity, and mortality. The purpose of my study is to identify the functional MUC5AC SNP. Four candidate SNPs were selected within the MUC5AC promoter region based on high linkage-disequilibrium scores across multiple global populations with a SNP in the MUC5AC promoter, rs28737416. I utilized molecular cloning techniques to combine a luciferase-expressing plasmid with isolated regions of the human MUC5AC promoter containing the SNPs of interest, and subsequently transformed this recombinant plasmid into competent cells. Next, I am performing in-vitro, site-directed mutagenesis to investigate how genotypic variation in each candidate SNP influences promoter function by measuring luciferase expression. I anticipate variants in at least one SNP of interest will reduce gene expression (measured by luciferase expression), indicating functionality. Characterization of this genetic mutation will provide insight into TBM susceptibility across populations and could inform studies of novel therapeutics to treat TBM.


 Immune cells make up the body's defense against cancer and observing their spatial distribution in a tumor can provide information about patient prognosis. However, it is difficult and time consuming to identify each immune cell in images from cancer biopsies in order to perform spatial analysis. Additionally, stained immune cells are hard to distinguish by appearance in unprocessed multispectral images due to the overlapping or "mixing" of signals coming from different channels. A computational tool could efficiently identify the immune cells in a tumor. The goal of this project is to build a digital pipeline to identify each immune cell in a multispectral image of a tumor and make it generalizable to multispectral images from any source. First, we use an unsupervised method to break up mixed multispectral images into clusters. The user selects a subset of clusters that do a good job of isolating each type of immune cell. A sample of these user-selected results are used to train a supervised machine learning model. The trained model assigns a label to each cluster to classify the entire image. Preliminary results have shown that clusters can usually be assigned to the correct label with over 50% certainty. We anticipate that the clusters will show good agreement with clinician classifications. This pipeline will allow for immune cell identification with less human involvement than pathologist annotation and without requiring spectral unmixing, a preprocessing step that typically takes hours. In the future, we will test this pipeline with varying amounts of training data coming from different sources and integrate it with spatial analysis to capture immune signatures of disease.

Joubert syndrome (JS) is a neurodevelopmental condition diagnosed by the appearance of the “molar tooth sign” on axial brain magnetic imaging (MRI). Patients display hypotonia, abnormal eye movements, and ataxia. Substantial progress has been made on identifying the genetic causes of JS, which typically displays recessive inheritance. Nonetheless, the cause cannot be identified in ~25% of our cohort of JS-affected families. The contribution of variants that impact RNA splicing remains unknown. Our goal is to evaluate the role of noncanonical splice variants in the pathogenesis of JS. Canonical splice variants impact RNA splicing by disrupting the splice site directly, whereas noncanonical splice variants may affect it through alternative mechanisms, which need to be validated by RNA analysis. We previously identified genetic causes in 520 of 679 families with JS. To identify additional causes, we used SpliceAI (SpliceAI score >0.5) to identify candidate variants that impact splicing. We extracted RNA from patient cell lines and converted it into complementary DNA (cDNA). Then we used polymerase chain reaction (PCR) to amplify the affected exons with two sets of primers flanking the relevant splice junction. We evaluated PCR product size and sequence using gel electrophoresis and Sanger sequencing. We found 74 families with ≥1 canonical splice variant. An additional 34 families have ≥1 candidate noncanonical splice variant. We confirmed the pathogenicity of two of the candidate noncanonical splice variants by demonstrating an abnormal splicing event in AHI1 and MKS1 in two patient samples. By extrapolation from our data in JS, noncanonical splice variants may contribute as much as 10% to the genetic causes of recessive conditions. A precise genetic diagnosis informs prognosis, avoids unnecessary work-up, guides monitoring for associated complications, and opens the door to gene-specific treatments.

Management of muscle-invasive urothelial carcinoma is extremely limited with Anti-FGFR3 (Erdafitinib) being the only kinase-targeting therapy FDA-approved for treating advanced bladder cancer patients. Kinases represent an important family of proteins for drug development due to their well-characterized role in tumor growth and disease progression. Until the last decade, the process of identifying essential proteins was carried out by individually depleting their production using siRNA or shRNA knockdown. To expedite the process of discovery, we’ve applied a machine-learning model that predicts the activity of 428 kinase inhibitors and identifies the most essential kinases associated with promoting viability for five bladder cancer cell lines used in this study (COCAB1, SW780, COCAB11, UMUC5, & SCABER). This technique, known as polypharmacology, leverages the use of non-specific kinase inhibitors that target multiple individual kinases. We have validated the results of this computational method by measuring the effect on cell viability for 7 kinase inhibitors representing strong, moderate, and weak predicted impacts. Using live-cell imaging we quantified relative cell growth over 72 hours and found a positive correlation between predicted and observed effects on viability for all cell lines. The regression identified PTK5 (FRK) and VEGFR2 (KDR) as a common top essential kinase across all five cell lines. Currently, we are exploring validating the essentiality of these kinases by siRNA knockdown. Complete validation of this polypharmacology method would suggest continuing the evaluation of PTK5 and VEGFR2 as candidate novel therapeutic targets to treat advanced bladder cancer patients.

Autoantibodies that bind self-antigens are a component of autoimmune diseases in which the immune system attacks the host organism rather than foreign pathogens. Interstitial Lung Disease (ILD) is an autoimmune condition that presents in conjunction with autoantibodies that bind Melanoma differentiation-associated protein 5 (MDA5). Presence of anti-MDA5 antibodies knowingly serve a predictor of clinical mortality for ILD patients. MDA5 is a cytoplasmic protein that recognizes viral RNA and induces synthesis of type 1 interferons. It is unclear whether the MDA5 autoantibodies directly associate with MDA5 to alter immune function and cause disease, or merely serve as an indication of the disease’s presence. One hypothesis is that anti-MDA5 antibodies activate MDA5 inappropriately in the absence of viral infection. Another is that the autoantibodies interrupt MDA5’s response to viral RNA, in which case ILD would result from uninhibited infection. To examine these hypotheses, the Fink lab purified anti-MDA5 antibodies from the plasma of an ILD patient. They then used electroporation to introduce MDA5 autoantibodies or Immunoglobulin G (IgG) antibodies to cultured cells with varied exposure to Polyinosinic: polycytidylic acid (Poly IC), a synthetic RNA that activates MDA5. I used mRNA from these experimental cells to synthesize cDNA and run quantitative real-time reverse transcription PCR. I measured expression of IFIT3, an interferon stimulated gene (ISG) transcribed in the MDA5 pathway, relative to the RPS18 housekeeping gene. IFIT3 was upregulated in conditions with Poly IC, particularly when incubated with anti-MDA5 antibodies. This preliminarily indicates the virally activated immune response is amplified when autoantibodies are present, suggesting that blocking the MDA5 signaling pathway could be a therapeutic treatment for ILD patients. I will be analyzing expression of other ISGs to further test the validity of early results. This project will help define the mechanism of anit-MDA5 autoimmune disease in order to identify treatment strategies.

To study genetic factors and the molecular mechanism underlying the development rate of mouse embryos, two mouse subspecies were studied. I have collected mouse embryos from crosses between C57BL6/JxCAST/EIJ (B6xCast), CastxB6 (the reciprocal cross), and intercrosses within either strain to examine the effects of maternal and paternal genomes on the developmental rate. I used mouse embryos every 24 hours at gestational stages E11.5-E14.5. Staging by the Embryonic Mouse Ontogenetic Staging System (eMOSS) is used to estimate the actual developmental stage based on limb bud morphology and compared to the gestational stage based on the estimated time of conception by observation of a vaginal plug. I also used gDNA to determine the sex of each embryo to investigate whether sexes play a role in developmental rate. I observed an interesting pattern that the mouse subspecies and the source of each parental genome affect the developmental rate of embryos. I observed embryos with at least one Cast parent, have faster development than B6xB6 starting at gestational stage E13.5. The developmental rate is independent of the sex of embryos or the litter size. Single-cell transcriptomic analysis is ongoing to determine the genes and mechanisms behind the change in developmental rate of mouse embryos. It is important because the result will contribute to human development in early stages, and help to solve problems such as preterm or underdeveloped infants.

Aging is an important problem in biomedical research. Given the increased risk of death with age, techniques to delay aging hold substantial promise for human well-being. The premise of my research is that rapamycin, a drug commonly used in transplant patients which is hypothesized to have development-slowing effects, can slow development of fruit flies. One finding from a previous study, however, is that there is enormous genetic variation within the Drosophila population (akin to the variation between dog breeds) which results in varying sensitivity to rapamycin. Earlier measurements indicated that strains of fruit flies that were sensitive to rapamycin had higher levels of histamine, a molecule that participates in metabolism, when on the drug. We set out to determine whether histamine could effectively extend sensitivity to the drug to a wider range of genotypes within Drosophila. Thus, I hypothesize that introducing histamine to the food that fruit fly strains that are genetically resistant to rapamycin are consuming will increase sensitivity. To test this hypothesis, I added solutions with different concentrations of histamine to food with or without rapamycin. I then placed eggs from a resistant strain onto the food to observe the time it takes from egg to pupa. Because I hypothesize that increasing histamine levels will make a resistant strain of Drosophila sensitive to rapamycin, the strain should become sensitive and consequently show an increase in development time compared to the conditions without histamine. Each test condition of a histamine solution and rapamycin has a corresponding control with no rapamycin. Hence, if the histamine-treated conditions show slower pupation times than the histamine-free controls, the hypothesis is validated. If validated, this work could help researchers understand ways to provide the benefits of rapamycin to individuals who might otherwise be genetically resistant to its impact in both aging and medical contexts.

X inactivation is a mechanism of dosage compensation that equalizes gene expression between males (XY) and females (XX). It is mediated by the long non-coding RNA (lncRNA) Xist (X-inactive specific transcript). However, some genes escape X chromosome inactivation(XCI) in females, and thus potentially contribute to sex differences. Escape genes are often identified by allelic expression analysis based on RNA sequences and RNA FISH (fluorescent in situ hybridization) based on imaging. In addition, RNA FISH allows the examination of escape status and the location of the transcript in individual cells. Allelic expression analysis has shown that one X-linked gene Car5b escapes XCI in mouse cells. However, it is not clear whether it escapes XCI in every cell or a portion of the cells. The goal of this project is to use RNA FISH to detect and quantify the escape status of Car5b in wild-type cells and in cells carrying a deletion of an important DNA element required for Car5b escape. In order to achieve the goal, I first prepared the fluorescent probes using the cDNA plasmids of Car5b and Xist. Next, dual RNA FISH was performed for Car5b and Xist in mouse cells to visualize the location of Car5b RNA signals whereas Xist RNA signals serve as the marker of inactivated X chromosome. Analyzing the FISH results is ongoing and we expect to visualize Car5b escape in the wild-type cells but not in the mutated cells. This method can be applied to escape studies in other X-linked genes which could enhance our understanding of X-linked gene regulation and sex difference.

My project involves making new tools for the genetic manipulation of Staphylococcus aureus. S. aureus is an important and pervasive human pathogen, however, much is still not known about its pathogenesis pathway and virulence genes. To better understand the genes and mutations that make S. aureus successful at causing disease, it is necessary to induce specific genetic changes and assess their impact on the organism’s virulence. However, currently available tools for bacterial genome engineering have yet to be optimized for S. aureus. Building on a successful recombineering system, previously developed by the Salipante lab, that is able to edit the S. aureus genome, we aim to address this need by using a dominant negative mutant protein of the mismatch repair (MMR) system to achieve suppression of DNA repair. Recombineering is the process of incorporating mutagenic DNA molecules into a host genome through the recombinase enzymes. Dominant negative mutations in the highly conserved MutL protein have been shown to disrupt the DNA base MMR pathway in several other microorganisms but have not yet been evaluated in S. aureus. My project aims to construct a temperature sensitive vector that highly expresses a ssDNA recombinase that is active in S. aureus along with a dominant negative mutL mutant able to bypass the MMR pathway. We anticipate that WT mutL should be able to repair a single base mutation since it has a functional MMR, reducing the number of successful recombinants relative to experiments performed with the multiple base pair mutations. The dominant negative mutL strains should not be able to correct a single base pair mutation, resulting in a similar number of drug-resistant transformats when recombineering with either a single base pair or many base pair mutations. This will allow us to better generate custom mutant strains and subsequently test the functions of various S. aureus genes towards a variety of clinically relevant phenotypes.

Werner Syndrome is a rare autosomal recessive disorder characterized by premature aging and heightened susceptibility to cancer. The condition is caused by a loss of function in the Werner protein (WRN), a helicase that is essential for maintaining genomic stability by assisting in DNA replication and repair. While much is known about WRN's role in the cell, the mechanisms underlying the acceleration of aging in its absence remain unclear. Recent studies have suggested that aging is associated with a reduced ability to maintain heterochromatic silencing of repetitive DNA sequences in the pericentromeric regions of the genome, also known as satellite repeats. Abnormally high expression of pericentromeric satellites can have a wide range of consequences on the cell, including DNA damage and changes in global gene regulation. Moreover, DNA damage can result in the appearance of satellite DNA fragments in the cytoplasm, leading to pro-inflammatory responses. This study seeks to determine whether WRN-deficient cells transcribe higher levels of satellite RNA and have elevated levels of satellite DNA in the cytoplasm. I focus on SAT II DNA, a class of pericentromeric satellites, using a pair of human fibroblast cell lines, with or without WRN. I isolate, purify and reverse-transcribe RNA from these cells and use qPCR to quantify the levels of SAT II RNA. Additionally, I fractionate WRN-deficient and control cells into cytoplasmic and nuclear fractions, isolate their DNA, and use qPCR to quantify the levels of SAT II DNA in the cytoplasm versus the nucleus. Preliminary results suggest that WRN absence leads to increased expression of SAT II RNA in Werner-depleted cells compared to controls, which may indicate that satellite heterochromatin is not being properly maintained. These findings may impact our understanding of aging and cancer predisposition, along with the mechanisms by which heterochromatic silencing regulates gene expression and cellular function.

Hexokinases (HK) are enzymes that catalyze the first step of glycolysis – the phosphorylation of glucose to glucose-6-phosphate. Hexokinase 3 (HK-3), one of four mammalian HK isoforms, is detected in hematopoietic cells and tissues, especially in cells of myeloid lineage. Previous studies have shown that after myeloid cells undergo CRISPR/Cas9-mediated HK-3 gene disruption and differentiation to neutrophil-like cells, loss of HK-3 leads to no effect on glycolysis, but an increased cell death phenotype during differentiation to neutrophil-like cells. This study aimed to examine if HK-3 gene knockout causes a similar cell death phenotype in monocyte/macrophages cells, which are of myeloid origin. To test directly, THP-1 cells, which are monocyte-like cells that can be differentiated to macrophage-like cells, underwent CRISPR/Cas9-mediated HK-3 gene knockout via electroporation. This is a method that uses electric pulses to make pores within the cell and nuclear membrane. PCRs and DNA sequencing comparing wild-type THP-1 cells and HK-3-null THP-1 cells were done to confirm successful knockout. The HK-3-null THP-1 cells then underwent 48-hour, 250 nM PMA-induced differentiation to macrophage-like cells and were then stained with Trypan Blue to compare viability of wild-type macrophage-like cells and HK-3-null macrophage-like cells. No apparent difference was found in cell viability between wild-type macrophage-like cells and HK-3-null macrophage-like cells during differentiation and over six days post-differentiation. Cell function was not assessed. This suggests that further work should be to: (1) confirm these results by repeating the HK-3-null in THP-1 cell line process and remeasuring cell viability after differentiation, (2) perform the HK-3-null process in other cell lines to differentiate into other cells of myeloid origin to determine if cell death phenotype appears, and (3) determine if loss of HK-3 alters viability under culture conditions mimicking low oxygen conditions, such as found in hypoxic tissue areas during bacterial infection. Understanding cell viability differences from loss of HK-3 could give insight into the potential regulatory function of HK-3 during myeloid hematopoiesis. 

CRISPR Activation (CRISPRa) is a powerful discovery-based tool to evaluate gain-of-function en masse. Applied successfully to emerging cell therapies, this technology offers tremendous promise to inform next-generation therapy design. Despite this potential, translation of CRISPRa to primary cells, including T lymphocytes, has been impeded by poor transgene expression. Based on prior reports of dCas9 genotoxicity, we suspected that CRISPRa could be exerting a toxigenic effect on CAR T cells, and thereby selecting against clones with high expression. To test this hypothesis, three separate constructs were designed with inhibited transcription and/or translation of the CRISPRa transgene. Following delivery of the constructs to donor T cells, analysis by flow cytometry revealed similar levels of cell yields and no net increase in dCas9 marker positivity across all CAR T cell subsets. Further epigenetic experiments and drug studies with anti-silencing compounds revealed that transcription of the CRISPRa transgene was severely inhibited. Collectively, these findings suggest that the CRISPRa transgene does not exert a toxigenic effect on CAR T cells; rather, low CRISPRa expression is caused by transgene silencing. Targeted efforts to mitigate silencing of the CRISPRa transgene are thus warranted to achieve adequate implementation to therapeutic cell subsets.