RNA-therapeutics of gene haploinsufficiencies

Defective gene expression has being recognized as the main causative agent or a key factor risk of a number of rare diseases,among which several ones affecting the central nervous system (CNS). It may be due to chromosomal microdeletions, spanning one or a few contiguous genes. Alternatively, the number of alleles may be correct, however these alleles may be poorly expressed, because of anomalous cis-active signals modulating transcription, intrinsic mRNA instability and/or poor translatability. Until now, pathologies linked to defective gene expression have been hardly tractable and, presently, no feasible-scalable approach is available for their therapy.

We are interested in developing artificial ncRNA-based devices for stimulating transcription of endogenous genes ad libitum, as key scalable tools for rationale treatment of haploinsufficiencies and other defective gene expression pathologies.

Among RNA-based devices promoting transcription of endogenous genes there are small activating RNAs (saRNAs). These are promoter-targeted, miRNA/siRNA-like molecules, supposed to (a) destabilize transcription-inhibiting ncRNAs or (b) drive transcription-promoting complexes to chromatin. The transcription gain they elicit is small, however it may be sufficient to impact the macroscopic behaviour of cells in a robust and predictable way. Moreover, silent genes generally do not respond to them. As such, saRNAs are a promising tool for clean therapeutic stimulation of gene transcritpion. We selected a number of saRNAs upregulating haploinsufficient genes implicated in CNS morphogenesis and physiology. We are studying their mechanism of action. We are working at their exploitation for in vivo correction of gene haploinsufficiency.

Conversely, CRISPR-transactivators are artificial transcription factors, which can be programmed by dedicated small guide RNAs (sgRNAs) to stimulate endogenous genes ad libitum. The expression gain they elicit may be very high and they can switch silent genes on. As such, CRISPRs were proposed for therapy of haploinsufficiencies. However, that has to be considered cautiously. In fact, genes encoding for CRISPRs would be difficult to delivery in vivo, because of their very huge size. Moreover, ectopic target gene activation triggered by broad expression of these devices could be unacceptably biohazardous. Interestingly, we recently created a novel class of artificial transactivators, the NMHV transactivators, which are RNA-programmable like CRISPRs, but differ from CRISPRs in three key aspects: (1) they are seven-fold smaller; (2) the transcription gain they achieve is more moderate; (3) they do not activate silent genes. Noticeably, a number of genes challanged by NMHVs were succesfully transactivated. As such, these transactivators could effectively vicariate saRNAs in transcriptional therapy of haploinsufficiencies. We are working at miniaturizing NMHV effectors, standardizing the NMHV platform and exploiting it for in vivo correction of gene haploinsufficiencies.