Activating Protein Expression Using Antisense Oligonucleotides
Zachary J Kartje | Watts Lab | F32 Award
Haploinsufficiency in diploid organisms is characterized by a working copy and nonfunctional copy of a gene, resulting in an insufficient amount of gene product (i.e., protein). This disrupts normal cell function, and can cause a myriad of diseases. Antisense oligonucleotides (ASOs) are small, predictable, and programmable tools that can be chemically engineered to directly control the stability, processing, and translation of RNA, making them useful for dissecting mechanisms of protein production. This proposal seeks to design and apply chemically-modified ASOs to systematically investigate endogenous protein repression mechanisms and identify key factors modulating full-length protein translation, using the NF1 gene as a model. NF1 is a tumor suppressor that inhibits Ras/MAPK signaling. NF1 haploinsufficiency causes neurofibromatosis type 1, a genetic disorder characterized by uncontrolled nerve cell proliferation and other complications. Steric blocking ASOs will be used to initiate translation at the primary start site in the NF1 5’-UTR and increase protein expression. ‘Gapmer’ ASOs will be used to target and degrade NF1 antisense transcripts and determine their effect on NF1 protein expression. Following sequencing of NF1 nascent RNA to identify cryptic splice sites, steric-blocking ASOs will be designed to mask nonproductive splice sites and improve pre-mRNA splicing efficiency. Synthesized ASOs will be screened in neuroblastoma cells and subsequently tested and optimized in NF1+/- haploinsufficient neurons and Schwann cells. Functionality of activated NF1 protein will be assessed by measuring Ras/MAPK activation. This project will increase our understanding of how protein expression is regulated, and may inform strategies to correct haploinsufficiency.
