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Hilton, Isaac B Szadowski, Hailey M 2025-05-29T18:34:36Z 2025-05 2025-03-04 May 2025 2025-05-29T18:34:36Z https://hdl.handle.net/1911/118385 Effective cell and gene therapies (CGT) require sustained expression of therapeutic payloads, typically achieved through genomic integration of transgenic DNA via genome editing (e.g., CRISPR\Cas9) or integrating viral vectors (e.g., retrovirus, lentivirus). Integration-based approaches risk insertional mutagenesis and oncogene activation, yielding unpredictable, irreversible consequences. Inspired by latent DNA viruses with genomes persisting as episomes, we developed an extrachromosomal genetic technology (EGT). Our EGT, featuring an engineered origin of replication and tether protein, maintained non-integrated transgene expression in proliferative cells over 12 weeks. Despite this ability, our EGT’s large size limits therapeutic applications. We focused on expanding carrying capacity by investigating accommodating delivery methods—dual integrase-deficient lentiviruses (IDLVs), high-capacity adenovirus (HC AdV), lipid nanoparticles (LNPs)—and by developing miniaturized systems with alternative tether protein DNA-binding domains, such as Tet Repressor (TetR) or Zinc Finger (ZF) proteins. Optimized EGTs will enable the production of persistent, tunable CGTs without disrupting the endogenous genome. application/pdf eng Copyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder. Cell Therapy Gene Therapy Synthetic Biology Development of Extrachromosomal Genetic Technologies for Persistent and Tunable Expression of Therapeutic Payloads Thesis Text 2027-05-01 2027-05-01 Systems, Synthetic and Physical Biology Systems/Synthetic/Phys Biology Rice University Masters Master of Science embargo