Abstract
Short synthetic nucleic acids known as oligonucleotides have become a major therapeutic class for selective, sequence-specific gene regulation. The therapeutic profile of oligonucleotides can be substantially extended by addressing a few tractable engineering problems, such as chemical modifications for targeted delivery and the design of multi- targeting scaffolds. This dissertation explores the linker-structure-activity relationship and design rules for oligonucleotide conjugate-based drug candidates.
The first study investigates the design principles for a bifunctional antisense oligonucleotide (ASO) heterodimer that upregulates the SCN1A gene in Dravet syndrome. A natural antisense transcript (NAT)-targeting ASO and a splice-switching ASO were covalently linked and evaluated for in vitro combination response. Across three linker chemistries, non-cleavable linkage eliminated gene-upregulation activity, while nuclease- cleavable and GSH-responsive linkages restored the combination response. Intracellular cleavability, linker length, and conjugation directionality were identified as critical parameters dictating productive activity.
The second study extends this principle to targeted delivery. A series of trivalent mannose- conjugated siRNAs was designed and characterized, bearing either a cleavable disulfide or a non-cleavable PEG-based linker, for CD206 receptor-mediated uptake and microglia- selective target knockdown. Though in vitro activity was validated, further evaluation in CD206-overexpressing myeloid models is required to resolve how cleavability shapes target engagement.
Together, these studies define a transferable set of design rules on linker chemistry, cleavability, and directionality for next-generation oligonucleotide conjugates, and provide chemically validated lead modalities for microglia-targeted gene silencing and single-entity gene upregulation.