Bile acids are liver-synthesized molecules essential for lipid absorption and metabolic  signaling. The elimination of excess cholesterol depends on bile acid synthesis, as bile acids  are produced from cholesterol. In the classical pathway, cholesterol 7-α hydroxylase (CYP7A1)  catalyzes the first and rate-limiting step, while sterol 12-α hydroxylase (CYP8B1) is necessary  for producing cholic acid. This bile acid acts as a feedback regulator, causing downregulation of  bile acid synthesis when levels are high. Because the Tarling-Vallim laboratory is studying the  intricacies of bile acid synthesis in vitro, I aimed to disrupt CYP8B1 in human embryonic kidney  293T (HEK293T) cells using CRISPR gene editing. I designed guide RNAs targeting human  CYP8B1 and completed all steps of cloning. I confirmed the CRISPR plasmid sequences  through restriction digest and Sanger sequencing, followed by maxiprep to ensure sufficient  plasmid stock. To validate the plasmids for gene disruption, I co-transfected HEK293T cells with  the CRISPR plasmids and a construct expressing human CYP8B1. Transfection efficiency was  confirmed with additional transfection of green fluorescent protein (GFP), which I visualized  using a fluorescence microscope, and total protein was quantified by bicinchoninic acid (BCA)  assay. By Western blot analysis, CYP8B1 protein was significantly reduced or absent in  samples treated with CRISPR compared to controls, demonstrating successful disruption of  CYP8B1. Future generation of human liver cell lines disrupted for this protein will provide a  molecular platform for studying regulation of bile acid synthesis in metabolic disease models.

X-Linked Agammaglobulinemia (XLA) is a devastating genetic disorder in which the BTK gene of individuals is non-functional, producing B-cells that are unable to produce antibodies to fight infection. We employ the use of adenine base editing (ABE) to restore and rescue function in the BTK gene in autologous hematopoietic stem and progenitor cells (HSPCs). This base editing method has potential for correcting BTK mutations due to ABEs' ability to cause direct conversion of A-T to G-C base pairs while minimizing insertions or deletions of other portions of the BTK gene, resulting in a higher success rate at producing a base-perfect product compared to wildtype BTK genes, something that homology-directed repair through CRISPR-Cas9 is unable to do consistently. Given this, we hypothesize that ABE may act as a one-time treatment that can restore normal B-cell development in XLA patients. The project is currently testing the base editor on the patient mutation G763A. For summer, the main goal was to grow CD34+ cells with the mutation to test various protospacer adjacent motifs (PAMs) and guide RNA (gRNA) combinations for editing efficiency. Additionally, we similarly transduced K562 cells (a myeloid cell line) with the mutation using CRISPR-Cas9-mediated homology-directed repair with various single-stranded oligonucleotides (ssODNs). Successful implementation of the base editor demonstrated modest amounts of editing in CD34+ cells, transduction of K562 cells is to be analyzed in the future. In the previous mutation (C1684T) BTK was restored to modest levels and functionality was restored.

Glioblastoma (GBM) is the most malignant and pervasive subtype of brain tumor, and accounts for 45.2% of all primary malignant and central nervous tumors. Existing therapies including surgery, chemotherapy, and radiotherapy offer limited success and is largely attributed to GBM’s high tumor heterogeneity, capacity for immune escape, aggressive growth, and the challenge of delivering effective treatments across the blood brain barrier (BBB). Therefore, it’s imperative to consider novel drug therapies offering more promising prognoses. Epidermal growth factor receptor (EGFR) is a major oncogenic driver in glioblastoma (GBM), with over 50% of tumors harboring activating alterations. EGFR is activated by ligand-induced dimerization and regulates key processes such as proliferation, differentiation, and survival. In GBM, common EGFR alterations include wild-type (WT) amplification, the EGFRvIII mutant, and extracellular domain (ECD) point mutations.Targeting these alterations with tyrosine kinase inhibitors (TKIs) has shown success in non-small cell lung cancer (NSCLC), where drugs like erlotinib, lapatinib, and osimertinib inhibit EGFR by binding to its kinase domain. However, these agents have largely failed in GBM due to poor BBB penetration, limiting their therapeutic efficacy in the central nervous system and remains a critical challenge in developing effective EGFR-targeted therapies for GBM.In response to these challenges, the Nathanson Lab has developed KTM-101 - an EGFR TKI with high brain penetrance and specificity for EGFR alterations common to GBM making KTM-101. Our central hypothesis is that KTM-101 will more potently inhibit EGFR-driven GBM due to its optimized target specificity for GBM-specific EGFR alterations. 

Cardiovascular diseases (CVDs) are a leading cause of worldwide morbidity and mortality, with most cases attributed to atherosclerosis. Long non-coding RNAs (lncRNAs) are arbitrarily defined as non-coding transcripts that are 200 nucleotides or longer. Emerging evidence suggests that lncRNAs contribute to CVD, but its role in atherosclerosis is still emerging. Here, we identified a macrophage-regulated lncRNA, Gm32997. Our preliminary studies led us to hypothesize that Gm32997 regulates crucial immune cell functions related to lesion progression, including inflammation and metabolic signaling. Gm32997 knockout or deletion did not affect foam cell formation or proliferation in-vitro. Through gene expression analysis, we found that Gm32997 represses inflammatory responses. In the future, we will determine the role of Gm32997 in atherosclerosis development using a bone-marrow transplantation model in Ldlr-/- mice. We anticipate that this research will enrich our understanding of lncRNA biology, lipid metabolism, and offer new mechanistic insights into atherosclerosis development.

Congenital limb defects during early human development range from digit malformations to complete limb absence. Understanding early limb development is vital in the prevention of limb abnormalities. Currently, the study of limb development in humans is hindered by the inaccessibility of human embryonic limb tissue and mouse model limitations. Human embryonic stem cells (hESCs) offer a powerful in vitro platform to model embryonic processes and create three-dimensional (3D) stem cell-derived organoids resembling early limb tissues. We hypothesize that creating a 3D model that replicates embryonic limb development using hESCs will further our understanding of the pathology around limb malformations. Limbs originate from mesoderm-derived lateral plate mesoderm (LPM) encased by an ectoderm-derived apical ectodermal ridge (AER). Through integration of hESC-derived LPM and AER, our aim is to create a 3D organoid that models the cellular composition and configuration of early limb formation, including the formation of the LPM and the AER. Using RT-qPCR, we measured elevated expressions of key limb LPM markers PRRX1 and HAND1, validating our ability to model these structures. Additionally, immunofluorescent (IF) imaging of the organoids showed expression of ISL1 and HAND1, demonstrating the presence of LPM limb cell types. Next steps include using qPCR and IF imaging to assess the presence of key AER markers, further confirming the identity of our organoids. Developing a biologically accurate model of these early structures will allow us to understand the regulation of limb development further, permitting the eventual implementation of treatment and prevention plans within a clinical space.