Date of Award

2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Genetics, Genomics & Informatics

Research Advisor

Yong Cheng

Committee

Daniel Savic; Kevin Freeman; Shengdar Tsai; Yan Cui

Keywords

CTCF, Gene Therapy, Genomic Safe Harbor, Insulator

Abstract

Gene therapy holds enormous potential for the treatment of genetic disorders, which are caused by mutations in DNA disrupting normal gene function. These mutations can be monogenic, affecting a single gene, or polygenic, involving multiple genes. Since, the first gene therapy treatment for SCID in 1990, there have been quite progress in development of gene therapy treatments, and currently we have 38 FDA approved gene and cell therapy treatments. Although the field is moving in positive direction, there are still concerns regarding the safety and efficacy of the treatment. One of the examples being development of leukemia after treatment with SCID gene therapy. To mitigate these risks, our study focuses on identifying novel genomic safe harbor (GSH) sites and insulator elements to ensure safe and stable transgene integration without disrupting the host transcriptome. Here we developed a knowledge-based approach using common polymorphic mobile element insertions (pMEIs) along with epigenetic and 3D chromatin information to identify new genomic safe harbor (GSH) sites. Out of 19 blood specific and 5 brain specific GSH sites, we experimentally validated 3 GSH sites from blood which showed stable transgene expression without disruption of neighboring genes in the host erythroid cells. In parallel we also developed a high-throughput screening system to identify novel insulator sequences using two expression protein marker system, cyan fluorescent protein (CFP) for validation of sequence integration and green fluorescent protein (GFP) for measurement of strength of insulator sequence. Our findings showed that CTCF and RAD21 occupancy as the major but not the sole contributor for insulator activity. We also identified the influence of sequence specific features such as downstream motif to regulate the insulator function. Furthermore, we discovered that insulators could regulate gene expression by reducing nascent transcription from enhancers, underscoring their broader role in transcriptional control. In summary, our study provides significant advancements in gene therapy, offering new methods for enhancing the safety and efficacy of transgene integration. By integration of genomic and epigenomic information, we were able to understand the critical factors governing both GSH and insulator element. Thus, these discoveries not only improve our understanding but also paves way for precise and safer gene therapy strategies.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

0000-0003-2831-2498

DOI

10.21007/etd.cghs.2024.0682

Additional Files
2025-025-Shrestha-DOA.pdf (200 kB)
Declaration of Authorship
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