RNA therapeutics of neuropathogenic haploinsufficiencies

Artificial transactivation of endogenous genes ad libitum is a desirable goal for a number of basic and applied research purposes. For this purpose, we recently developed a fully synthetic ribonucleoproteic transactivator prototype. Kept together via an MS2 coat protein/RNA interface, it includes a fixed, polypeptidic transactivating domain and a variable RNA domain that recognizes the desired gene. Thanks to this device, we specifically upregulated five genes, in cell lines and primary cultures of murine pallial precursors. Even if gene upregulation was small, however it was sufficient to inhibit neuronal differentiation. Our transactivator activity was restricted to cells in which the target gene is normally transcribed. These features make our prototype a promising tool for clean rescue of gene haploinsufficiencies, since it could lead to a specific overstimulation of the spared gene allele in its physiological expression domain. On the other hand, we are interested in stimulating transcription of endogenous genes by small activating RNAs (saRNAs). These are miRNA/siRNA-like molecules, supposed to destabilize transcription-inhibiting ncRNAs or facilitate the recruitment of transcription transactivating complexes to chromatin. The expression gain they elicit is small; however it may be sufficient to modify the behaviour of cells in a robust way. They comply with endogenous gene tuning. Moreover, silent genes generally do not respond to them. As such, also saRNAs are a promising tool for therapeutic stimulation of gene transcription. We selected a number of saRNAs able to stimulate haploinsufficient genes involved in CNS morphogenesis and physiology. We reconstructed their mechanism of action. We provided proof-of-principle they can be exploited for in vivo correction of defective gene expression.