Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s Sarcoma (KS), a highly vascularized tumor made up of cells of endothelial origin. KSHV establishes a predominantly latent infection in endothelial cells in culture and in the KS tumor. A previous study has shown that KSHV induction of the Warburg effect is required for the survival of latently infected endothelial cells. The Warburg effect, a common metabolic alteration in cancer cells, refers to an increase in glycolysis and a decrease in oxidative phosphorylation. The mechanism of Warburg induction by KSHV is currently unknown. I proposed to evaluate the role of endothelial cell specific hypoxia-induced factors (HIFs) on KSHV Warburg induction since HIFs have been implicated in Warburg induction in other types of cancer. I hypothesized that HIF2α mediates KSHV Warburg induction through expression of glycolytic genes. To test this, I constructed HIF2α knock-out cells using CRISPR/Cas9 gene editing. I then used RT-qPCR to measure glycolytic gene expression during KSHV infection of wild-type and HIF2α knock-out cells. I found that the transcript levels of certain glycolytic genes remained constant in KSHV-infected HIF2α knock-out cells as compared to KSHV-infected wild-type cells, showing that HIF2α is not responsible for glycolytic gene expression during KSHV infection. I am following up on the role of the KSHV latent gene KapA on the induction of glycolytic gene expression. KapA was previously found to interact with components of the Ras pathway. As the Ras pathway activates glycolytic genes through HIF1α, I hypothesize that exogenous expression of KapA will lead to increased glycolytic gene expression through increased expression of HIF1α. I will construct an endothelial cell line that overexpresses KapA using CRISPR/Cas9 system and then use RT-qPCR to evaluate glycolytic gene expression. These results will aid in the future efforts to develop antiviral drugs by targeting cellular metabolism.

Cystic Fibrosis (CF) is a genetic disorder that is characterized by recurrent pulmonary infections and a progressive decline in lung function caused by increased inflammation. While the host immune response can help fight these infections, an overzealous inflammatory response in the lungs may be harmful. Inflammation can be induced by multimeric proteins called inflammasomes, which catalyze the maturation of pro-inflammatory cytokines and pyroptosis. Our laboratory is interested in investigating genetic variants that alter the response of the NLRP3 inflammasome in human CF patients as a means of identifying potential targets for host-directed therapies. Preliminary data from the EPIC study has identified two genetic variants in CF children that is associated with colonization by P. aeruginosa, a common bacteria in the lung that can induce deleterious inflammation. We hypothesize that genetic variants in the NLRP3 inflammasome will increase the inflammatory response to bacterial infection and will lead to worsened pulmonary outcomes in patients with CF. First, we used CRISPR/Cas9 and gene editing techniques to knock out the NLRP3 gene in human cell lines. We then inserted NLRP3 genetic variants and stimulate the NLRP3 inflammasome using Pathogen associated molecular patterns such as Nigericin and ATP. The extent of inflammation was measured using ELISA (Enzyme Linked Immunosorbent Assay), and results were compared among NLRP3 genetic variants and wild type controls. The ultimate goal of this investigation is to learn how genetic variants are associated with the clinical outcomes of CF patients. Future studies can involve harnessing host-directed therapies to target specific inflammatory pathways in order to reduce harmful inflammation in CF. Finding the relationship between the NLRP3 inflammasome and CF is critical to generating an effective treatment for CF.

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder, marked by progressive muscle degeneration due to the absence or impairment of the dystrophin protein. Our laboratory has been developing methods for gene therapy of DMD. The dystrophin gene is the largest known gene, which complicates gene therapy. We have shown that gene delivery vectors based on adeno-associated virus (AAV) can be used to deliver new genes body wide. However, AAV has a ~5 kb carrying capacity, so we have been developing miniaturized dystrophins. These smaller, highly functional dystrophins, which we named micro-dystrophin (µdys) were tested in a transgenic mdx^4cv  mouse model for DMD. Furthermore, we sought to develop a non-invasive method to evaluate treatment efficiency of µdys. In this project, the gait of mdx^4cv, wild type (WT) and transgenic mice were observed to deduce potential benefits of the µdys transgene. We hypothesized that µdys treatment will enable the gait of the transgenic mice to resemble that of WT animals as opposed to mdx^4cv. To test this, gait was assessed at three time points: 1.5, 3 and 6 months of age using the video-based Noldus CatWalk XT. Our results show that the length of a single stride of the transgenic mice hind paws increased over time compared to mdx^4cv. The normal walking pattern of mice consists of placing diagonal paws on the surface, one front and one hind from opposite sides. In the case of mdx^4cv, more paws were placed down for majority of the walk, as a compensatory mechanism for muscle weakness. The transgenic mice were found to walk with a diagonal pattern more frequently, like WT mice. Similarities in the gait of transgenic and WT mice help us conclude the µdys treatment was successfully able to mitigate the effects of DMD as seen by the gait analysis.

With advances in sequencing technology it has become feasible to sequence a patient’s genome in a clinical setting, but our ability to interpret the effect of genetic variants on health and disease remains a large challenge. Genome-wide association studies (GWAS) have helped to interpret DNA variants by correlating thousands of variants in the genome with disease. However, many of these variants fall in noncoding DNA (making it hard to predict their effects) and are often in tight linkage with neighboring single nucleotide polymorphisms (SNPs) (meaning these neighboring SNPs may also be responsible for driving the correlation with disease). Consequently, most disease-associated variants still need functional validation. There is mounting evidence that a large number of these variants may be effecting gene regulation (when, where, and to what degree a gene is expressed), and several groups have tried testing whether these SNPs effect regulatory activity with traditional reporter assays. In this study, we aimed to validate GWAS candidates linked to osteoarthritis, a joint disorder with several SNPs previously associated. We compiled a list of these variants as well as all SNPs in linkage disequilibrium, for a total of 1,605 variants. To screen this list for functional candidates, we leveraged “self-transcribing active regulatory region sequencing” (STARR-seq), a reporter assay that can test thousands of sequences for regulatory activity in a single experiment. Our screen identified two variants with statistically significant differences between the major and minor alleles. To validate these findings in the genome, we introduced the minor allele of one variant into cell lines via CRISPR/Cas9. We found a similar difference in transcription between the major and minor allele in the genome as our initial episomal screen. We are currently working with our collaborators to further validate these findings in patient samples.

Epigenetic modifications regulate chromatin structure and function, playing important roles in altering DNA transcription levels and subsequently cell fate decisions. Various next-generation sequencing (NGS) methods have been developed to detect these epigenetic changes in the genome, such as chromatin immunoprecipitation followed by sequencing (ChIP-seq). Even though ChIP-seq is extensively used to analyze DNA and histone modification levels, this method is limited to one histone marker at a time and requires significant amount of input cells, which masks the profiles of cell-to-cell variation and the complex interaction between the epigenome and gene expression. To overcome these limitations in current next-generation sequencing methods, we have been developing a multiplexed assay that could detect multiple epigenetic modifications in single cells. So far, I have developed a NGS data analysis pipeline to identify potential gene candidates that are highly differentially modified by histone markers. In the future, we hope to use these gene candidates as templates to design DNA-fluorescent in situ hybridization (DNA-FISH) probes and perform Expansion Microscopy and DNA-FISH with these probes to link histone modifications to specific gene loci at high resolution. After the assay is fully developed and validated, we plan to utilize the assay to take the epigenetic profiles of hematopoietic stem cells and study cell fate decisions in hematopoiesis.

Treatment options for kidney disease are limited to dialysis and transplantation, which are of limited efficacy and availability. Human pluripotent stem cells (hPSCs) can be used to generate immunocompatible kidney organoids, which contain the major proximal structures of the nephron, including podocytes, proximal tubules, distal tubules, and endothelial cells. They can be made on-demand to study kidney disease and regeneration. Unfortunately, these organoids lack a collecting duct system, a crucial component of the kidney. To address this need, we have developed a protocol to differentiate hPSCs into ureteric bud (UB) cells, which are the precursors of collecting duct cells. First, we identified markers for the collecting duct lineage in developing kidneys. Immunofluorescence analysis of human kidney tissue revealed co-expression of cytokeratin 8 and Dolichos biflorus agglutinin (DBA) in the collecting ducts. These markers were not expressed in the proximal tubules. Next, we investigated the ability of hPSCs to express these markers. Undifferentiated hPSCs were treated with a concentration gradient of small molecules known to induce kidney lineage differentiation, including CHIR99021 and glial cell line-derived neurotropic factor (GDNF); their effects were jointly and individually examined. Immunofluorescence was used to characterize the resulting cells, which were initially stained with DBA and Lotus tetragonolobus lectin (LTL), a proximal tubule marker. We found that CHIR99021 alone was sufficient to produce DBA positive and LTL negative cells in vitro. Notably, the DBA levels in these cells were lower than observed in kidney tissue, indicating that the structures we obtained were not yet fully mature. These studies provide the first evidence that collecting duct cells can be generated from hPSCs. Optimization of this differentiation protocol will yield kidney organoid structures that include the collecting duct system, which will make them ideal for kidney disease modeling and regenerative medicine approaches to reduce the need for kidney transplants.

Approximately 14% of the United States population is currently living with chronic kidney disease (CKD), demonstrating the importance of developing effective treatments. Presently, the mechanisms and driving forces of the many diseases that comprise the spectrum of CKD are not well understood. Current methods for acquiring kidney cells for research purposes require an invasive biopsy taken directly from the kidney. We theorized that patient kidney cells can be acquired from a significantly less invasive urine sample, be expanded, and subsequently differentiated into new kidney tissues. We developed a method for accomplishing this, which enabled the collection of cells from the urine samples of over 40 different patients located across the globe with various subtypes of CKD. Reverse transcription polymerase chain reactions (RT-PCRs) probing for the expression of XIST, a RNA gene exclusively expressed in females, were conducted on urinary cells from patients who have had kidney transplants with donors of the opposite gender. Using this assay, we show that these urinary cells originated in the donor kidney. By introducing a set of transcription factors expressed in embryonic stem cells, urinary cells were further reprogrammed to induced pluripotent stem cells (iPSCs), an undifferentiated, stem cell-like state. Subsequently, the iPSCs were differentiated into patient-specific kidney organoids, marking the first time that new kidney-like structures have been generated from a urine sample. Hollow tubules, which better mimic the architecture of the kidney, were created by incorporating these urinary cells or iPSC-derived kidney organoid cells into three-dimensional microfluidic devices wherein normal cell viability and morphology was maintained up to twenty days. Immunohistological stainings indicate similar protein expression between the cell types, which will be further investigated in the upcoming months. These studies improve our ability to regenerate kidney tissue and disease processes from patients’ own bodies, starting with a simple urine sample.

Primary cilia are antenna-like structures on the surface of diverse cell types. Cilia are important for signal transduction and processes such as cell division and development, but their precise role in the cell remains poorly understood. Defects in cilia result in a wide spectrum of genetic disorders called ciliopathies, which commonly involve polycystic kidney disease (PKD), the formation of fluid-filled sacs in the kidney which eventually result in kidney failure. To understand the role of cilia in these diseases, we generated human pluripotent stem cells (hPSCs) with defects in cilia formation for the first time. We used the CRISPR-Cas9 genome editing system to introduce mutations in two different genes that are required for cilia formation, kinesin family member 3A (KIF3A) and kinesin family member 3B (KIF3B). We isolated four independent cells lines with indel mutations in exon 3 of the KIF3A gene and two in exon 2 of KIF3B. The introduced mutations led to frameshifts and protein truncations. Immunoblot analysis confirmed that no full-length protein was produced in these mutant cells. The cells were stained with acetylated alpha-tubulin, a marker of cilia. No cilia were detected in the mutant hPSCs, while an average of 48% cilia were detected in controls of identical genetic background. Surprisingly, the absence of cilia did not alter hPSC pluripotency, self-renewal, amniotic cavity formation, or growth. Further, we differentiated these cell lines into kidney organoids. The cilia deficient kidney organoids formed cysts reminiscent of PKD, while the isogenic controls did not. In conclusion, we have generated human cells and organoids lacking an entire organelle, the primary cilium, and used these to conclusively link cilia to PKD. These cilia deficient hPSC lines can be used to study cilia function, to model various ciliopathies, and to screen for drugs that can ameliorate the disease phenotypes associated with them.

Kidneys are responsible for regulating blood glucose levels in the body through the reabsorption of sugars in the proximal tubule of the nephron. Issues with glucose absorption can lead to diabetic nephropathies where too much glucose is excreted. To study kidney glucose absorption, the lab made kidney organoids, which are miniature stem cell-derived in vitro organs that mimic the structure and function of kidneys. We used induced pluripotent stem cells (iPSCs), a type of stem cell that can be generated directly from one’s own adult stem cells. In a previous experiment, the lab found glucose absorption was present through the Sodium Glucose Co-Transporter 2 (SGLT2). The lab wanted to see if this was the only channel responsible for glucose absorption. Using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, we knocked out SGLT1 and SGLT2 separately and also knocked out Glucose Transporters (GLUT1 and GLUT2) separately in the WTC11 kidney stem cell line.  We selected a guide RNA corresponding with each glucose transporter in the cell membrane of kidney organoids.  Then RNA-guided Cas9 created the mutation by breaking the sequence for each glucose transporter in a specific site of the genome.  We verified our mutations were done correctly by sending our results to a DNA sequencing company.  We expect to find mutant lines will have less glucose absorption than our control and hope to learn which transporters are more active.  We are further using this method to create double and quadruple knockout lines with multiple glucose transporters knocked out at once.  We hope to learn which transporters are most responsible for glucose absorption, and in the future, can assess when certain transporters are used over others, which will open the doors for disease models for diabetes not only in kidneys but also in other organs.