Profiling Brain Biodistribution and Spatial Transcriptomics of Antisense Oligonucleotides
Graduation Year
2025
Document Type
Master's Thesis
Degree
Master of Science
Program
Biological Science
Partner Organization
BioMarin Pharmauetical
Program Director
Patti Culross, MD, MPH
First Reader
Danielle Tan
Second Reader
Manuel Lopez
Abstract
Antisense oligonucleotides (ASOs) are short, single-stranded RNA molecules that can be used to treat genetic disorders. They operate by binding to complementary mRNA nucleotide sequences, triggering mechanisms that can decrease, increase, or otherwise alter protein expression. Although ASOs are emerging as a prominent area for drug development due to this targeted approach, an understanding of their delivery and effects on tissues, especially the central nervous system (CNS) remains rather limited. In some cases, an antibody to detect ASOs has been used, but since it is not commercially available, other in situ detection methods are needed. Herein, this study aimed to build a toolbox of advanced histological techniques to detect ASOs and observe their effects in the CNS. Firstly, we employed chromogenic singleplex and immunofluorescent multiplex in situ hybridization (ISH) methods to characterize the biodistribution and cellular uptake of ASOs. However, given the limitations of traditional ISH methods whereby we can image up to 4 or 5 markers before hitting the limits of spectral overlap and effective separation of fluorescent channels, we leveraged spatial transcriptomics ability to visualize up to 1000 markers as superior alternative. This enabled us to analyze and investigate the biodistribution of ASOs in the CNS along with its impact within specific cell types. Ultimately, we have developed a sophisticated analytical spatial biological toolkit to assess ASO biodistribution with defined cell populations, measure the levels of RNA following treatment, and assess overall transcriptomic changes across almost 50 different cell types within the brain. This analysis pipeline will allow for the detailed characterization and comparison of ASOs across large-scale screening efforts, improving the likelihood of a successful ASO drug development.