Hemoglobinopathies, a hereditary condition involving abnormalities in the structure of hemoglobin, currently require expensive and highly sophisticated medical facilities to treat using gene therapy. The purpose of this study is to provide a highly portable and scalable approach using in vivo hematopoietic stem cell (HSC) gene therapy to potentially overcome these limitations. The main idea of our in vivo HSC gene therapy approach is to mobilize HSCs from the bone marrow, and while they circulate at high numbers in the periphery, transduce them with an intravenously injected HSC-tropic, helper-dependent adenovirus HDad5/35++ gene transfer vector system. The transduced cells return to the bone marrow where they remain long-term. This study followed one animal for 22 weeks after in vivo HSC transduction with a human-γ-globin expressing HDAd5/35++ vector using Sleeping Beauty transposase 100X for integration. Treatment with G-CSF/AMD3100 (a bone marrow stimulant) resulted in efficient HSC mobilization into the periphery blood circulation. The treatment was well tolerated and after in vivo selection, gamma-globin marking in peripheral red blood cells rose to ~90% and was stable during the duration of the study. Enrichment of gene-modified cells by in vivo selection was also reflected by an increase in mgtm and gamma-globin mRNA levels. Our data suggest that in vivo gene therapy with HDAd5/35++ is feasible and side effects can be minimized or prevented with appropriate pretreatment. This is the first proof-of-concept study that in vivo HSC gene therapy could be feasible in humans and provide the necessary portability and accessibility to reach patients in places with limited medical resources. Future studies will involve the optimization of HSC mobilization, gene transfer vectors and in vivo selection.

Chronic respiratory infections and diseases are the third leading cause of death globally. The Frevert lab studies the protein versican (Vcan), an extracellular matrix proteoglycan, whose expression is highly upregulated during lung injury and inflammation. However, it is unclear whether this upregulation is an anti-inflammatory or a pro-inflammatory response. We are testing the hypothesis that the cellular source of Vcan determines its inflammatory actions; versican from myeloid cells is anti-inflammatory but versican from stromal cells is pro-inflammatory. Vcan deletion in our cells of interest can test this hypothesis. To do this we have generated Vcan^fl/fl genetically engineered mice, which allow for conditional removal of functional versican with Cre Recombinase. Cre excises versican’s exon 4, recombining exons 3-5 and creating a premature stop codon. This produces a truncated non-functional form of versican. The goal of this research project is the development of cell-specific protocols for in vitro deletion of versican in both myeloid cells (bone marrow-derived macrophages) and stromal cells (lung explant fibroblasts). These protocols will allow for further evaluation of versican’s role in the inflammatory response when different cell types are confronted by a bacterial or viral agonist. So far, I’ve been investigating the dose response and time course for exposure of macrophages to Cre to optimize its efficiency and have been able to demonstrate by qPCR that intact versican decreases and non-functional versican increases. The inflammatory response is quantified through qPCR analysis of the fold increase of Ifn-b compared to the house-keeping gene MRPL32. Ifn-b is a cytokine released by the innate immune system in response to viral pathogens. Next, I will investigate the conditions necessary for efficient Cre deletion of Vcan in fibroblasts. These experiments allow us to investigate the effects of Vcan made by different cell types furthering our understanding Vcan’s function during injury and inflammation.

X chromosome aneuploidy refers to an atypical number of X chromosomes, differing from two X chromosomes in females, or one X and one Y chromosome in males. Unusual numbers of chromosomes arise from errors in cell division that result in too many or too few chromosomes in a cell, with X aneuploidy reported to occur in around 1 in 1000 births depending on the disorder. X chromosome aneuploidy disorders, such as Klinefelter (47, XXY), Triple X (47, XXX) and Turner (45, XO) syndromes are associated with developmental abnormalities, including cognitive and cardiovascular defects. Our goal is to generate isogenic induced Pluripotency Stem Cells (iPSCs) with different numbers of X chromosomes from patients with X aneuploidy, and subsequently differentiate them into relevant cell types to identify genes affected by aneuploidy. Our lab has previously generated isogenic XXY and XY iPSCs from patients with Klinefelter’s Syndrome by removing the extra inactive X chromosome (Xi). My project is to establish and characterize isogenic lines from mosaic XXY/XY patients, who have a mixture of cell karyotypes. Specifically, I am screening iPSC clones derived from mosaic patients for expression of XIST, a gene expressed only from the Xi, by RT-PCR to determine presence or absence of the Xi. Isogenic control XY iPSC lines derived from the same patient serve as a control to XXY cells, lessening potential for confounding variables due to differences between individuals. Next, I will screen clones with different genotypes for integration of reprogramming vectors by genomic DNA-PCR. Integration-free isogenic XXY and XY clones will be differentiated into cardiomyocytes, neural progenitor cells, and cortical organoids, cell types that are associated with the adverse effects of X aneuploidy. By doing so, we hope to gain insight into gene expression and epigenetic changes associated with X aneuploidy phenotypes in a controlled genetic environment.
 

Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of wound and hospital-acquired infections. Glycopeptides (e.g. vancomycin), lipopeptides (e.g. daptomycin), and lipoglycopeptides (e.g. dalbavancin) are three classes of cell-wall/cell-membrane targeting antimicrobials effective for treating MRSA. Since in S. aureus cell wall synthesis and lipid synthesis are metabolically closely connected, response to cell-wall/cell-membrane targeting antimicrobials is likely to be mediated by lipid synthesis. Indeed, our lab previously demonstrated that the overall lipid abundance decreased in the dalbavancin-resistant S7-D2 strain (vancomycin-intermediate), isolated by in vitro exposure of a clinical MRSA isolate S7 strain (vancomycin-susceptible) to multi-stage escalated concentrations of dalbavancin. Here we examined a lipid synthesis inhibitor, AFN-1252, to determine its potential synergistic effects with vancomycin, daptomycin or dalbavancin, using the S7 and S7-D2 strains. Broth microtiter MIC measurements and static time-kill experiments were performed to evaluate potential synergy. We found that, through the MIC measurements, the pellet sizes in the wells seemed to be reduced when S7 and S7-D2 were treated with AFN-1252 at half the MIC in the presence of vancomycin. Hence, a static time-kill at 2xMIC of AFN-1252 and/or vancomycin was performed to interrogate the growth difference. S7-D2, but not S7, showed increased killing with the combination, suggesting that killing by vancomycin could be aided by AFN-1252 in vancomycin-intermediate strains. The killing by the combination in S7-D2 also approached the level of killing by vancomycin alone in S7, suggesting mechanistically membrane lipid remodeling might have happened that counteracted the slower killing in the vancomycin-intermediate strain. Our preliminary results so far suggest that lipid synthesis inhibitors might be able to enhance the MRSA treatment effects of vancomycin. In future studies, we will evaluate the potential lipid profile changes using comprehensive lipidomics analysis, and further screen and evaluate other lipid synthesis inhibitors and their potential synergistic effects with vancomycin, daptomycin or dalbavancin.

Heart disease is a major health pandemic, and the myocardial infarction (MI), also known as a heart attack, is the leading cause of death. This is because adult cardiomyocytes (CMs) present in the heart cannot divide and proliferate, preventing the heart from regenerating itself and repairing tissue damage. From developmental studies in rodents, we know the Notch signaling pathway is crucial in mediating expansion and proliferation of CMs during development, as impairments in Notch lead to cardiac defects. Notch is inactive in adult CMs, which suggests it plays a role in CM renewal; however, this mechanism is still unknown. Thus, our goal is to investigate the role of Notch in CM proliferation and cell cycle regulation. For the purpose of this study, we are using CMs differentiated from a human embryonic stem cell line, RUES2. Differentiation was completed over a 17-day period by culturing the cells as a monolayer. On Day 0, Chiron 99021 is added to activate Wnt signaling, promoting mesoderm formation. Wnt-C59 is added at Day 2 to inhibit Wnt signaling and differentiate the cells into progenitors, and B27 supplement at Day 6 promotes full differentiation into CMs. This adherent protocol recapitulates every step of natural heart development in vitro. Purity of the cell populations, as assessed by flow cytometry staining for cardiac troponin T (cTnT), was 97.1 ± 0.9% cTnT+. We then determined the proliferative capabilities of CMs in the presence of a Notch inhibitor, DAPT. DAPT inhibits gamma-secretase, a transmembrane protein that normally proteolytically cleaves Notch during signaling, thus inactivating the Notch pathway. Treatment with DAPT significantly decreased cell proliferation by about 50%, confirming that Notch directly affects CM proliferation. The results of this study increase our knowledge of CM physiology and the mechanisms behind cell cycle withdrawal, and provide new insights into improving CM renewal.

Capacitation involves biochemical and physiological processes in sperm with motility changes (including hyperactivation) that allow sperm to bind to the zona pellucida, undergo acrosome reaction and penetrate the ovum. Bovine serum albumin (BSA), has been thought to facilitate capacitation in mouse sperm through depleting the sperm membrane of cholesterol. Other albumin preparations including human serum albumin (HSA), fatty acid free (FAFHSA), and recombinant albumin (RHSA) may affect capacitation differently. Moreover, cholesteryl ester transfer protein (CeTP) may facilitate capacitation but it should not be present in yeast-derived RHSA as opposed to human blood-derived HSA; RHSA and HSA may support capacitation at different extents. These preparations were tested in motility experiments after 3-5.75 hr (mean 4 hr) capacitating incubation. Sperm were purified by 40/80% PureSperm gradients and washed in HTF-BSA, then diluted in final albumin to 10-20M/mL. FAFHSA, BSA, HSA, and RHSA had hyperactivation percentages of 2.83%, 3%, 11.3%, and 15.75% respectively. RHSA had a motility of 71.3% on average while the others ranged from 83.6% to 88%, affecting the hyperactivation results. RHSA also caused incubated sperm to agglutinate or aggregate, but its elevated hyperactivation rates relative to HSA are insignificant. These results fit previous data suggesting that BSA and FAFHSA do not support hyperactivation (therefore, capacitation) as both are abnormally below 10% hyperactivation. Since BSA contains low CeTP, this suggests CeTP may help facilitate capacitation. Previous studies demonstrated the amount of CeTP in albumin correlated well with albumin’s ability to support acrosome reaction. The present results suggest that capacitating motility patterns may not necessarily correlate with the ability to acrosome react; this hypothesis could be tested. Since fertilization is largely misunderstood; understanding the roles of Albumin or CeTP may aid in developing infertility treatments, contraceptives and family planning for a growing human populace.