For genetic liver diseases, the standard of care is often invasive (e.g. liver transplantation), temporary (e.g. enzyme replacement therapy), and expensive. Viral gene therapy with adeno-associated virus (AAV) is an attractive alternative as it is non-invasive, inexpensive, safe, and the therapeutic benefits can be long-term. The success of AAV gene therapy for liver diseases is critically dependent on at least two factors: (i) the ability of AAV to functionally transduce target hepatocytes; and (ii) transduction levels in affected hepatocytes must reach therapeutic levels in patients of all ages and genders. However, the field is limited by a lack of mechanistic understanding of AAV transduction that prevents the rational design of better AAV therapies, as well the differences in hepatic transduction seen between males and females. The major hypothesis addressed herein is that key host factors for AAV transduction are differentially expressed in male and female hepatocytes, and that these differences are responsible for the inconsistencies we see in treatment outcomes in the different groups.
The specific aims were designed to address these needs by: (1) determining the sexually dimorphic gene expression patterns in pediatric and adult liver; (2) identifying host factors required for all stages of AAV transduction in human hepatic cells; and (3) overlaying these two independent datasets and validating target genes which both display sexual dimorphism and are required for AAV transduction in both sexes in short-term hepatocyte culture in vitro and as well as in vivo in xenotransplanted humanized liver mice. This project aims to maximize use of innovative tools to generate fundamentally new methodologies to address key problems in hepatic gene therapy and basic liver biology. Innovative tools include applying large-scale transcriptomic profiling to a compendium of pediatric and adult human livers of each gender, pairing CRISPR/Cas9 library technology with human hepatic cells to achieve libraries of singe-gene knockouts in clinically relevant cell types, and performing genome-wide loss-of-function reverse genetic screens in human hepatic cells for viral transduction factors with clinically relevant serotypes of AAV. Candidate Summary The skills, knowledge and background of Dr. Paulk make her uniquely suited to address the research questions proposed herein. Her graduate work in the world-renowned lab of Markus Grompe in applied cell and gene therapies for metabolic liver diseases resulted in 3 first-author and 3 co-author publications. In addition, she received a prestigious NIH F31 NRSA Predoctoral Fellowship and numerous academic and travel awards during this time. During Dr. Paulk's postdoctoral training in the laboratory of Mark Kay at Stanford University, she received 3 prestigious fellowships including an NIH F32 NRSA Postdoctoral Fellowship, the American Liver Foundation Hans Popper Memorial Fellowship and the Stanford Dean's Fellowship, and has a recent publication in Nature. Training Plan Summary Three diverse training needs were identified that are needed by Dr. Paulk to achieve independence: (1) advanced computational biology and bioinformatics training is needed to be at the leading edge of biomedical research; (2) training in high-throughput screening technologies to enable faster acquisition of large datasets; and (3) training in specific career development aspects tailored to the needs of one transitioning from postdoc to independent tenure-track faculty. Through highly tailored coursework, formal didactic training and practical experience, conference attendance with a greater emphasis on networking in addition to scientific pursuits, additional focus on grantsmanship, and extensive mentoring, Dr. Paulk will become a highly competitive and independent faculty candidate. Environment Summary Stanford University is a worldwide leader in biomedical research and patient care. Stanford can provide Dr. Paulk with all the services and facilities associated with a first-class research institution. In the laboratory of Dr. Kay, Dr. Paulk will be immersed in a highly innovative lab within the Human Gene Therapy Program that Dr. Kay directs. Dr. Kay has an impressive publication record (>255 papers to date) and is a world-renowned expert in AAV biology, both viral and non-viral gene therapy, as well as small RNA biology. Dr. Kay has the ability to attract highly talented scientists for collaborations with Dr. Paulk. Dr. Kay has been involved in several high-profile clinical trials for hemophilia and can train Dr. Paulk from his insights in how to transition exciting pre-clinical findings through the clinical trial process. Both Stanford University and Dr. Kay's laboratory provide a fantastically nurturing environment, rich in ideas, support, state-of-the-art resources and collaborations.

Public Health Relevance

The proposed research and career development plan are relevant to public health because they will accurately identify and measure the differences in clinically relevant gene expression patterns in pediatric and adult livers for each gender, and will further define the human cellular factors required for all stages of viral transduction in the human liver by the gene therapy vector Adeno-associated virus.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Scientist Development Award - Research & Training (K01)
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Kidney, Urologic and Hematologic Diseases D Subcommittee (DDK)
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Saslowsky, David E
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University of California San Francisco
Schools of Medicine
San Francisco
United States
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Paulk, Nicole K; Pekrun, Katja; Charville, Gregory W et al. (2018) Bioengineered Viral Platform for Intramuscular Passive Vaccine Delivery to Human Skeletal Muscle. Mol Ther Methods Clin Dev 10:144-155