Spinal muscular atrophy (SMA) is a progressive inherited neuromuscular disorder and the most common genetic cause of death in infants. SMA is caused by the insufficient expression of survival motor neuron (SMN) protein. The FDA approved the first drug to treat SMA in December 2016, which corrects a splicing abnormality in the SMN2 gene, and increases SMN protein. However, therapeutic rescue by this method may be limited by the number of SMN2 copies that a person has. Because individuals with the most common and severe form of SMA have only 1 or 2 copies of SMN2, they may require other therapeutic strategies. Recently, the first long noncoding RNA (lncRNA) associated with the SMN2 gene was reported. This lncRNA, called SMN-AS1, recruits an epigenetic repressive complex, called the polycomb repressive complex 2 (PRC2), to the SMN2 promoter and represses SMN2 transcription. While knockdown of SMN-AS1 using antisense oligonucleotides (ASOs) increases SMN expression in cell culture models, behavioral rescue of SMA mice is modest when combined with the SMN2 splice-correcting drug. LncRNAs have extensive secondary structure and local domains that contribute to their function. It is currently unknown how the structure and domains of SMN-AS1 contribute to its epigenetic repression of SMN2. Elucidating and blocking the domains of SMN-AS1 that contribute to SMN2 repression using modified blocking ASOs may help advance targeting SMN-AS1 as a therapeutic for treatment of SMA. This proposal aims to discover the individual functional domains of SMN-AS1 and understand how SMN-AS1 interacts with the SMN2 promoter and PRC2 to regulate SMN expression. SA#1 will employ a simplified luciferase reporter strategy to delineate domains of SMN-AS1 required for repressing SMN2. Comparison of SMN expression after overexpression of full-length or truncated forms of SMN-AS1 will help narrow in on the critical functional regions of the transcript. Experiments in SA#2 will determine if blocking different domains using blocking ASOs targeting SMN-AS1 alters SMN RNA or protein expression in cultured mouse primary cortical neurons. Finally, SA#3, will elucidate whether the domains blocked in SA#2 are required for SMN-AS1 binding to the SMN2 promoter or PRC2 in mouse primary neurons. These experiments use advanced techniques to investigate RNA:DNA and RNA:protein interactions. Furthermore, an unbiased RNA pulldown followed by mass spectrometry will identify novel proteins that bind to SMN-AS1, opening the door to investigating other potential mechanisms of action of SMN-AS1. These experiments will give insight into how lncRNAs may regulate gene expression and will facilitate better ASO design for targeting SMN-AS1 effectively as a preclinical target for treatment of SMA.

Public Health Relevance

Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder that in most cases causes infant mortality by respiratory failure. The proposed study investigates how discrete domains of a long noncoding RNA contribute to expression of the gene disrupted in SMA. Understanding these precise interactions may allow us to develop better strategies for targeting this long noncoding RNA for treatment of SMA.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1)
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Nuckolls, Glen H
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Johns Hopkins University
Schools of Medicine
United States
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