Autophagy is a process by which cells reclaim important biomolecules by engulfing intracellular materials into autophagosomes that are transported to lysosomes for degradation. Previous data demonstrated that when cancer cells are introduced into mice with systemic autophagy inactivation, tumor size decreases. Our focus is to elucidate specific cell types important for this process. The Cre-Lox system allows for targeted inactivation of autophagy through the floxed Atg7 gene with cell type-specific promoters driving expression of Cre recombinase. When Cre recombinase expression was under the control of the promoter for the S100a4 gene (commonly referred to as Fibroblast-specific protein 1), tumor size decreased, underscoring the importance of autophagy in the tumor microenvironment. However, S100a4 is expressed in various other cell types as well as fibroblasts in the tumor microenvironment, including dendritic cells and macrophages. To narrow the autophagy loss to exclusively fibroblasts we used Col1a2, a gene encoding collagen type 1, Cre-driven recombinase. When cancer cells were introduced into mice with Col1a2 driven Atg7 inactivation, the tumors were not smaller than in control mice based on volume nor weight. The finding demonstrates that autophagy inactivation in fibroblasts alone is not sufficient to reduce tumor sizes. Therefore, different cell types in the tumor microenvironment are contributing. Loss of autophagy in these other cell types are being investigated as potential explanations for reduced tumor growth.

The cap binding complex (CBC) plays multiple regulatory roles throughout the process of gene expression, most notably in the regulation of pre-mRNA processing. The CBC is composed of two subunits and is highly conserved across eukaryotes. Though the cap binding complex is of great importance to gene expression regulation, the mechanisms for regulation of the complex itself are not well understood. Interestingly, CBP80, the gene encoding the larger subunit of the complex, contains an intronic start codon (ATG) that can be utilized to generate an intronic protein isoform. Through a series of genetics-based experiments in Saccharomyces cerevisiae, we have determined that the intronic isoform is able to partially rescue the growth defect phenotype observed in cbp80∆ cells, however the mechanism of this recovery has yet to be uncovered. The intronic protein isoform does not contain Exon 1 of CBP80, which encodes part of the protein’s nuclear localization sequence. It is unclear if the intronic protein localizes to the nucleus or elsewhere within the cell. We are seeking to uncover the localization patterns of the isoform through microscopy experiments, as this could provide more insight into the function of the protein. Together, these approaches may provide an interesting mechanism by which the cap binding complex can regulate its own activity to optimize efficiency of gene expression in response to environmental changes.

Succinic semialdehyde dehydrogenase (SSADH) is an enzyme that is responsible for gamma-aminobutyric acid (GABA) metabolism in the mammalian nervous system. SSADH is encoded by the Aldh5ɑ1 gene. SSADH deficiency, defined as the partial or total loss of the SSADH enzyme, is a rare, autosomal recessive genetic disorder that results in an increase in GABA and observed weight loss, neurological impairment, lethal seizures, and death within a month. Furthermore, preliminary data suggest that SSADH-/- murine models exhibit dysmyelination, an abnormal growth of the neuron cell, which could be related to the epileptic and seizure-related phenotypes presented in both mice and clinical patients. A potential therapy has still yet to be studied for this disorder. This project aims apply an AAV-based gene therapy for SSADH deficiency in which two known AAV serotypes, 9 and B22, are packaged with Aldh5ɑ1 cDNA for injection into deficient murine models. Preliminary results indicate that mice injected with AAV9 do not survive past the literature-determined date of around day 23 to 27, while AAVB22 treated mice indicate marked weight maintenance and survivability past this time point. Molecular assays (MBP Western blotting and IHC) currently indicate that B22 expression is comparable to that of wildtype mice expression and increased compared to untreated mutants. Although more experiments are planned at this time, the current data presented by this study indicate a potential proof-of-concept for an AAV-based gene therapy for SSADH deficiency.

Current cell therapy for skeletal muscular diseases are inefficient and expensive. For volumetric muscle injury patients, the treatments are not suitable given the situation’s time-sensitive nature. With universal stem cell (USCs) development, there would be an off-the-shelf option immediately available due to immune “cloaking,” which bypasses the immune system by overexpressing 8 genes. Before supplying USCs, we need to test for complete concealment from the immune system and retention of pluripotent abilities. In the study, we test iACT Stealth Cells for these abilities by checking their universal potential and ability to differentiate into skeletal muscle progenitor cells (SMPCs) and myofiber, in vitro and in vivo, using a humanized mouse model. In vitro, USC-derived SMPCs formed myotubes and expressed myogenic genes, confirmed by qPCR. In vivo engraftment of USC-derived SMPCs showed that the cells survived using bioluminescence luciferase assay, and immunostaining of engrafted tissues confirmed formation of human muscle fibers (expressing human dystrophin). Both experiments confirmed that USCs differentiated into SMPCs. By staining for CD45+ immune cells, we confirmed with immune cell presence that USCs maintained universal potential post-engraftment. With these results, we will investigate the state of produced myofibers and the types of recruited immune cells. The study shows USCs have universal potential and can create myofibers which can advance cell therapy for muscle diseases.

Abstract Head and Neck Cancer (HNC) refers to a variety of cancers that are derived from squamous cells lining the oral cavity, oropharynx, larynx, and other structures within the head and neck region. This research analyzed HPV-16-E7 expression in both tumor and salivary samples to determine if saliva can serve as a reliable, non-invasive biomarker for HNC. Additionally, we investigated how APG-157 treatment, a drug derived from turmeric, affects the expression levels of HPV-16. The 200mg drug was orally administered to the subjects 3 times a day. HPV-16 expression levels were studied before treatment and after 4 weeks of daily treatment. To conduct this study, we utilized qPCR for DNA integration analysis and RT-qPCR for RNA expression analysis. The results indicated that there was a strong correlation between HPV-16-E7 integration and expression, as well as within the presence of HPV-16-E7 in saliva and tumor cells. This supports that saliva can be used as a non-invasive biomarker for HNC and detection will be sufficient with qPCR. We further see that detection of HPV-16 decreases post APG-157 treatment. Significance Our study shows evidence that HPV-16 is detected in both salivary and tumor samples, which is downregulated by treatment with APG-157. This leads to our hypothesis: APG-157 can be used as a treatment for HNC.