# Engineering therapeutic cellular functions using robust and highly programmable extrachromosomal genetic technologies > **NIH NIH R01** · RICE UNIVERSITY · 2024 · $484,203 ## Abstract PROJECT SUMMARY/ABSTRACT Current strategies to engineer human cells for cell-based therapeutics and biotechnologies rely upon the genomic integration of transgenic payloads. Although these approaches have catalyzed transformative medical advances, the integration of transgenic DNA permanently disrupts natural genomic sequences and can lead to unexpected and even hazardous consequences. In addition, integrated transgenic DNA is often unpredictably expressed and is prone to epigenetic silencing over time, especially within primary/therapeutically useful cells. Further, the installation and validation of integrated cargoes is inefficient and costly. These critical barriers limit the extent to which human cells can be repurposed and engineered as cell-based therapeutics and these challenges are preventing biotechnological and clinical innovations. Non-integrating, double-stranded DNA viruses have evolved sophisticated solutions to these important obstacles, and they can stably persist within human cells as circularized self-contained episomes or linear extrachromosomal elements across cellular divisions and for the lifetime of infected hosts. These viruses accomplish this remarkable persistence by tailoring their own gene expression patterns, synchronizing their genomic replication, and by reshaping endogenous transcriptional networks in host cells. In this proposal, we will harness and redirect these natural abilities towards biomedically useful outputs using clinical-grade non-integrating gene therapy vectors and cell types. Our project will establish new ways to program and apply extrachromosomal DNA within human cells. In Aim 1 of this proposal, we will optimize our recently developed genetically encoded extrachromosomal modules to further refine and enable i) site-specific and tunable localization of extrachromosomal payloads, ii) programmable episomal/extrachromosomal replication, and iii) multi-layered safety switches; across a battery of human cell types to ensure robust utility. In Aim 2, we will deploy our established extrachromosomal modules in four clinically proximal primary cell types using widely adopted viral vectors for gene and cell therapies: integrase-deficient lentiviral (IDLV), high-capacity adenoviral (HCAdV), and herpes simplex viral (HSV) vectors. In Aim 3, we will build proof-of-concept sense and respond genetic circuits within IDLV, HCAdV, and HSV viral vectors to modulate the expression of transgenic extrachromosomal and endogenous therapeutic payloads by combining these platforms with synthetic CRISPR/Cas9-based transcription factors in clinically useful primary cells. Collectively, our project will broadly empower cell engineers, synthetic biologists, and biomedical researchers with new capabilities to tunably control the expression of therapeutic payloads from a wide array of extrachromosomal vector systems and across a spectrum of clinical grade cell types without the hazards and obstacles associated with genomic integ... ## Key facts - **NIH application ID:** 10937316 - **Project number:** 1R01EB036003-01 - **Recipient organization:** RICE UNIVERSITY - **Principal Investigator:** Isaac Hilton - **Activity code:** R01 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2024 - **Award amount:** $484,203 - **Award type:** 1 - **Project period:** 2024-06-01 → 2028-02-29 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10937316 ## Citation > US National Institutes of Health, RePORTER application 10937316, Engineering therapeutic cellular functions using robust and highly programmable extrachromosomal genetic technologies (1R01EB036003-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10937316. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*