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SINEUPs: lncRNAs with the Potential to Upregulate Haploinsufficient Autism Risk Genes

A photo of a boy looking at the one flat tire of the bicycle.
In some cases, two copies of a gene are required to support typical function — if one copy of such a gene is lost or damaged, typical function can not be supported. Suzanne Tucker

Large-scale genetic studies have now uncovered more than 200 genes1,2,3 that can be linked to neurodevelopmental disorders (NDDs) or autism. Many of those genes are implicated through gene-damaging heterozygous de novo mutations that result in haploinsufficiency of the affected gene. Identifying such genes opens the door for the development of gene-specific therapeutics like gene replacement therapy. However, gene replacement therapy comes with many challenges, one of them being the risk of overexpression. Therefore, therapeutics that could result in the upregulation of the remaining healthy allele and do not interfere with endogenous regulatory mechanisms of gene expression would be ideal.

A new SFARI-supported study led by Marta Biagioli and colleagues suggests that SINEUPs, a particular class of long-non-coding (lnc) RNAs that upregulate translation of their target mRNAs4,5, may be used to overcome haploinsufficiency of the ASD risk gene CHD8 in multiple in vitro models as well as in vivo in zebrafish. SINEUPs are modular lncRNAs, consisting of a binding domain complementary to the mRNA they are targeting and an effector domain that mediates the upregulation of the target mRNA’s translation. Due to this modular structure, SINEUPs can, in principle, be designed for almost any mRNA.

Diagram showing 3 panels. Panel 1 shows normal function: Two functional genes both produce functional mRNA resulting in six proteins each (twelve total proteins). Panel 2 shows an example of haploinsufficiency: One functional gene produces one functional mRNA resulting in six proteins total, which is is not enough to support typical function. Panel 3 shows an example of how SINEUPS can overcome haploinsufficiency: One functional gene produces one functional mRNA. A SINEUP sequence attaches to the mRNA boosting the protein production from this mRNA, resulting in ten proteins total.
Lucy Reading-Ikkanda/Simons Foundation

Biagioli and colleagues designed SINEUPs targeting the long isoform of mouse, human and zebrafish CHD8 mRNA. In neural progenitor cells derived from human induced pluripotent stem cells (hiNPC) in which CHD8 was knocked down, a SINEUP delivered via electroporation resulted in a 1.8-fold upregulation of CHD8 protein levels. The team also found that molecular phenotypes (differential gene expression and altered H3K36me3 levels) previously associated with CHD8 haploinsufficiency were partially reversed in hiNPCs treated with SINEUPs. Importantly, they also achieved 2.1-fold upregulation of CHD8 in patient-derived fibroblasts carrying a frameshift mutation (c.6307_6310del) or a stop+frameshift (c.2485dupA).

To test if SINEUPs targeting CHD8 could be effective in vivo, the scientists turned toward an established zebrafish model of Chd8 haploinsufficiency, chd8p.Glu223*6,7, that presents with macrocephaly due to increased cell proliferation. They found that injection of a SINEUP targeting the zebrafish Chd8 mRNA into one-cell-stage embryos resulted in the full rescue of both phenotypes in 4.2 dpf zebrafish larvae. Yet another phenotype, reduced number of enteric neurons, was not rescued.

This study highlights SINEUPs as a potential gene targeting therapeutic with the ability to upregulate the expression of haploinsufficient NDD genes. Other investigators supported by SFARI (Jonathan Sebat, Joe Dougherty and Joseph Buxbaum) are exploring antisense oligonucleotides and small-molecule-mediated mechanisms with the same goal.

References

  1. Zhou X. et al. Nat. Genet. 54, 1305-1319 (2022) PubMed
  2. Wang T. et al. Proc. Natl. Acad. Sci. U.S.A. 119, e2203491119 (2022) PubMed
  3. SFARI Gene
  4. Zuchelli S. et al. RNA Biol. 12, 771-779 (2015) PubMed
  5. Zuchelli S. et al. Front. Cell Neurosci. 9, 174 (2015) PubMed
  6. Bernier R. et al. Cell 158, 263-276 (2014) PubMed
  7. Sugathan A. et al. Proc. Natl. Acad. Sci. U.S.A. 111, E4468-77 (2014) PubMed

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