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. Effective gene activation strategies for correcting haploinsufficiency have not been identified because the mechanisms that repress protein production are unclear. 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. Previous work in wild-type cells demonstrates that steric blocking ASOs can block alternative translation start sites from ribosomes and direct splicing factors to increase protein levels. Moreover, ASO ?gapmers?, which contain a central region of DNA flanked by chemically-modified nucleotides, can degrade RNAs that negatively regulate protein expression (e.g. antisense transcripts). Yet, the efficacy of these strategies in a haploinsufficiency context has not been investigated. With guidance from Dr. Jonathan Watts (ASO synthesis and chemistry), and collaborators: Dr. Athma Pai (RNA processing and bioinformatics), Dr. Anastasia Khvorova (ASO delivery and neurobiology), and Dr. Xandra Breakefield (tumor-suppressor syndromes), 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. The NF1 locus is an excellent model for this study because it possesses two alternative translation start sites ? upstream open reading frames (uORFs) in the 5? untranslated region (UTR) of the mature mRNA; is overlapped by several antisense transcripts; and likely undergoes unproductive splicing. Steric blocking ASOs that bind NF1 5?UTR uORFs have been synthesized and promising leads identified.
Aim 1 will test the efficacy of these ASO leads to initiate translation at the primary start site and increase protein expression.
Aim 2 will design and apply ASO gapmers to target and degrade NF1 antisense transcripts and determine their effect on NF1 protein expression.
Aim 3 will isolate and sequence NF1 nascent RNA to identify cryptic splice sites. ASOs will then be designed to block these sites and improve pre-mRNA splicing efficiency. For all aims, candidate ASOs will be transfected into SH-SY5Y neuroblastoma cells (which express NF1) for bulk screening. Successful candidates will then be tested and optimized in wild-type and NF1+/- haploinsufficent 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.

Public Health Relevance

A wide range of health defects result from haploinsufficiency?a condition in which cells produce an insufficient amount of a particular protein to permit normal cell function. This proposal seeks to develop and use novel nucleic acid technology to understand how the production of protein is regulated, and identify ways to increase protein levels in cells. Findings from this study will inform future efforts to correct haploinsufficiency and treat many diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1)
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Hoodbhoy, Tanya
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University of Massachusetts Medical School Worcester
Schools of Medicine
United States
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