Date of Award

12-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Microbiology, Immunology and Biochemistry

Track

MIB

Research Advisor

Marko Radic

Committee

Dr. Marko Radic Dr. Elizabeth Fitzpatrick Dr. Kui Li Dr. David Nelson Dr. Giedre Krenciute

Keywords

Systemic Lupus Erythematosus (SLE), Chimeric Antigen Receptor (CAR) T cells, Circular RNA, Electroporation, Coxsackievirus B3 (CVB3)

Abstract

Systemic lupus erythematosus (SLE) is a chronic, multiorgan autoimmune disease caused by the breakdown of self-tolerance that leads to aberrant activation of B cells producing autoreactive antibodies. While traditional immunosuppressants. Glucocorticoids and biologics such as rituximab offer disease management, yet they fail to prevent flares and subsequent organ failures in severe SLE. In fact, increased doses of glucocorticoids are associated with irreversible organ damage. Newly emerged Chimeric antigen receptor (CAR) T-cell therapies have resulted in unprecedented clinical success with disease remissions and complete depletion of B cells, providing long-term disease remissions. However, autologous conventional CAR T therapies are constrained by prolonged ex vivo manufacturing timeline, need for lymphodepletion, safety risks and sustained B cell aplasia. Therefore, non-viral, in vitro-transcribed (IVT) mRNA-based CAR platforms offer a controllable transient alternative. However, their utilization in therapeutic applications is heavily challenged by the rapid CAR expression decay caused by short RNA lifespan.

To address these limitations, this dissertation established a framework for screening, characterization, and molecular engineering of transient RNA CAR platforms. First, we developed a rapid screening workflow to test surface expression of RNA CAR via Jurkat cell line and electroporation. Here, we have demonstrated that dose elevation, N1-methylpseudouridine () and codon optimization do not prolong the CAR surface expression. In fact, N1-methylpseudouridine () decayed the CAR expression faster than unmodified control RNA CARs between 4 and 24 hours. Finally, to overcome this rapid degradation, we engineered a circular RNA CAR architecture using ribozyme-mediated autocleavage coupled with endogenous RtcB ligation achieving rapid circularization within 30 minutes in cells, as quantified using digital PCR (dPCR). Cap- independent translation driven by a Coxsackievirus B3 (CVB3) internal ribosome entry site (IRES) drove efficient CAR expression, without compromising antigen specific killing capacity of T cells engineered with RNA CAR

ORCID

https://orcid.org/0000-0001-9716-1932

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