Boosting protein synthesis using antisense oligonucleotides to treat neurodegeneration

PROJECT SUMMARY
Brain aging and disease occur alongside the loss of essential proteins and protein functions. For example,
thousands of inherited conditions are due to genetic haploinsufficiency, in which partial or complete loss of one
normal allele is sufficient to cause disease. Likewise, advanced age involves the loss of protective signaling,
such as through neurotrophic factors, that could prevent degenerative processes. In either case, strategies that
restore these single gene targets may be therapeutic. Current protein upregulation strategies, such as direct
protein delivery or viral gene therapies, are limited in the central nervous system as they lack regulatable dosing,
may distribute poorly, and can elicit neuroimmune reactions. The goal of this proposal is to investigate mRNA
regulation as an alternative approach to safely upregulate protein expression. Protein synthesis relies on mRNA
stability and translation efficiency, which are both determined by regulatory sequences in the untranslated
regions (UTRs) of mRNA. The 3’UTR encodes numerous regulatory elements that engage RNA-binding proteins
and regulatory RNAs to control mRNA translation and stability. I predict that blocking repressors from engaging
a 3’UTR could stimulate protein synthesis of a select gene of interest. Antisense oligonucleotides (ASOs) are
short, single-stranded DNA sequences that bind complementarily to RNA with high affinity and once bound, can
sterically block trans-acting elements from engaging the transcript. ASOs are a clinically proven technology that
have entered clinical trials for many neurodegenerative conditions, but there are currently no ASOs in clinical
trials that target 3’UTRs for gene-specific upregulation. Therefore, I hypothesize that ASOs that mask repressive
regulatory sequences on a 3’UTR can be used to stimulate synthesis of proteins that prevent or protect against
neurodegeneration or age-related diseases. I demonstrated the feasibility of this approach by increasing
expression of TANK-binding kinase 1 (TBK1), a protein haploinsufficient in amyotrophic lateral sclerosis (ALS)
and frontotemporal dementia (FTD) caused by dominant TBK1 mutations. In Aim 1, I will test if our existing
TBK1-upregulating ASOs can increase TBK1 expression and prevent neurodegenerative phenotypes in pre-
clinical models of TBK1 haploinsufficiency. In Aim 2, I will evaluate if particular 3’UTR cis-elements may be
universal targets for this ASO masking strategy. Aim 1 will establish the therapeutic relevance of 3’UTR-targeting
ASOs and support advancement of TBK1-upregulating ASOs to human clinical testing. Aim 2 will greatly extend
this approach to other gene targets, including neuroprotective protein factors. Completion of this proposal will
establish 3’UTR-targeted ASOs as a generalizable strategy to stimulate protein expression to treat
neurodegeneration and other age-related diseases. This training environment combines expertise in mRNA gene
repression mechanisms with experience in ASO development for neurodegeneration and, therefore, is perfectly
suited to support my endeavors to complete these aims and advance my development as a physician scientist.

Grant Number: 5F30AG082394-02
NIH Institute/Center: NIH
Principal Investigator: Ben Boros