SINEUPs: a novel toolbox for RNA therapeutics

RNA molecules have emerged as a new class of promising therapeutics to expand the range of druggable targets in the genome. In addition to 'canonical' protein-coding mRNAs, the emerging richness of sense and antisense long non-coding RNAs (lncRNAs) provides a new reservoir of molecular tools for RNA-based drugs. LncRNAs are composed of modular structural domains with specific activities involving the recruitment of protein cofactors or directly interacting with nucleic acids. A single therapeutic RNA transcript can then be assembled combining domains with defined secondary structures and functions, and antisense sequences specific for the RNA/DNA target of interest. As the first representative molecules of this new pharmacology, we have identified SINEUPs, a new functional class of natural antisense lncRNAs that increase the translation of partially overlapping mRNAs. Their activity is based on the combination of two domains: an embedded mouse inverted SINEB2 element that enhances mRNA translation (effector domain) and an overlapping antisense region that provides specificity for the target sense transcript (binding domain). By genetic engineering, synthetic SINEUPs can potentially target any mRNA of interest increasing translation and therefore the endogenous level of the encoded protein. In this review, we describe the state-of-the-art knowledge of SINEUPs and discuss recent publications showing their potential application in diseases where a physiological increase of endogenous protein expression can be therapeutic

SINEUP non-coding RNA activity depends on specific N6-methyladenosine nucleotides

: SINEUPs are natural and synthetic antisense long non-coding RNAs (lncRNAs) selectively enhancing target mRNAs translation by increasing their association with polysomes. This activity requires two RNA domains: an embedded inverted SINEB2 element acting as effector domain, and an antisense region, the binding domain, conferring target selectivity. SINEUP technology presents several advantages to treat genetic (haploinsufficiencies) and complex diseases restoring the physiological activity of diseased genes and of compensatory pathways. To streamline these applications to the clinic, a better understanding of the mechanism of action is needed. Here we show that natural mouse SINEUP AS Uchl1 and synthetic human miniSINEUP-DJ-1 are N6-methyladenosine (m6A) modified by METTL3 enzyme. Then, we map m6A-modified sites along SINEUP sequence with Nanopore direct RNA sequencing and a reverse transcription assay. We report that m6A removal from SINEUP RNA causes the depletion of endogenous target mRNA from actively translating polysomes, without altering SINEUP enrichment in ribosomal subunit-associated fractions. These results prove that SINEUP activity requires an m6A-dependent step to enhance translation of target mRNAs, providing a new mechanism for m6A translation regulation and strengthening our knowledge of SINEUP-specific mode of action. Altogether these new findings pave the way to a more effective therapeutic application of this well-defined class of lncRNAs

SINEUP non-coding RNAs rescue defective frataxin expression and activity in a cellular model of Friedreich's Ataxia

Friedreich's ataxia (FRDA) is an untreatable disorder with neuro- and cardio-degenerative progression. This monogenic disease is caused by the hyper-expansion of naturally occurring GAA repeats in the first intron of the FXN gene, encoding for frataxin, a protein implicated in the biogenesis of iron-sulfur clusters. As the genetic defect interferes with FXN transcription, FRDA patients express a normal frataxin protein but at insufficient levels. Thus, current therapeutic strategies are mostly aimed to restore physiological FXN expression. We have previously described SINEUPs, natural and synthetic antisense long non-coding RNAs, which promote translation of partially overlapping mRNAs through the activity of an embedded SINEB2 domain. Here, by in vitro screening, we have identified a number of SINEUPs targeting human FXN mRNA and capable to up-regulate frataxin protein to physiological amounts acting at the post-transcriptional level. Furthermore, FXN-specific SINEUPs promote the recovery of disease-associated mitochondrial aconitase defects in FRDA-derived cells. In summary, we provide evidence that SINEUPs may be the first gene-specific therapeutic approach to activate FXN translation in FRDA and, more broadly, a novel scalable platform to develop new RNA-based therapies for haploinsufficient diseases

SINEUP lncRNAs: from molecular mechanism to therapeutic application.

The post-genomic era has brought to light a previously unknown world of transcripts with the discovery of non-coding RNAs (ncRNAs). Indeed, it became evident that only as few as 1-2% of mammalian transcriptome consists of protein-coding mRNAs. Among several families of ncRNAs, long non-coding RNAs (lncRNAs) are under intense scrutiny for their heterogenicity of forms and molecular activities. A new class of antisense lncRNAs, known as SINEUPs, were previously identified for their ability to specifically enhance the translation of their target sense mRNA. LncRNAs and mRNA were transcribed from a sense/antisense pair locus with an head-to-head divergent configuration. SINEUPs activity relies on the combination of two domains: an overlapping region, or binding domain (BD), that confers specificity, and an embedded inverted SINEB2 element, or effector domain (ED), enhancing target mRNA translation. This new class of transcripts embodies the model of lncRNAs as flexible and versatile modular scaffolds enabling interactions between RNA, DNA and proteins. Furthermore, it represents a promising new RNA therapeutics platform to increase endogenous expression of a protein of interest within a physiological range. In this work, I provided new insights on the molecular mechanism of SINEUP activity, focusing on the role of N6-methyladenosine (m6A) modification, and on a Proof-Of-Concept therapeutic application of SINEUPs to rescue haploinsufficient OPA1 gene expression in Dominant Optic Atrophy (DOA). m6A is the most common RNA modification found in mRNAs and ncRNAs, where it is post-transcriptionally installed in the cell nucleus and can exert regulatory functions in many cellular processes such as nuclear export and translation. Here, I observed that both the natural SINEUP AS Uchl1, acting in rodent cells, and the synthetic shorter miniSINEUP-DJ1, acting in human cells, are m6A-modified. Results indicate METTL3 enzyme as the main responsible for SINEUP RNA modification. I then applied Nanopore direct RNA sequencing to map m6A-modified residues and a reverse transcription assay for validation. I monitored SINEUP activity upon METTL3 knock-down and in the presence of mutations on sites of m6A deposition. Interfering with a proper m6A modification led to a dominant negative effect of SINEUPs RNA on endogenous DJ1 protein levels in both experimental conditions. Applying ribosome fractionation analysis in conditions of inhibition of proper m6A deposition, I observed an enrichment of the target DJ1 mRNA associated to 40S and 60S ribosome fractions and a concomitant depletion from polysomes. These results provide a mechanistic model for its dominant negative effect on endogenous DJ1 protein. These data also suggest the presence of an m6A-dependent step in the molecular mechanism of SINEUP activity at the ribosome and contribute to a better understanding of the role of RNA modifications in the regulation of lncRNAs function. From a therapeutic point of view, SINEUPs are proposed as a new platform for the treatment of i. haploinsufficient diseases, where the lack of a functional allele prevents healthy phenotype formation; ii. complex multifactorial diseases, where increasing a compensatory pathway could preserve or restore physiological activities. Here, I applied SINEUP technology to increase endogenous levels of OPA1 protein to treat DOA, the most common inherited optic neuropathy caused in 75% of cases by heterozygous mutations in OPA1 gene. DOA is an early-onset autosomal dominant haploinsufficient disorder, with a prevalence ranging from 1:12000 to 1:50000 births and characterized by degeneration of the retinal ganglion cells that leads to optic nerve atrophy and blindness. OPA1 is a ubiquitously expressed dynamin-related GTPase protein with crucial functions in mitochondrial homeostasis, that localizes in the Inner Mitochondrial Membrane (IMM), reaching highest expression levels in brain, retina and heart. By in vitro screening, I identified OPA1-specific miniSINEUPs able to increase selectively both human and murine OPA1 proteins in a range sufficient to restore neuronal cell functions. Currently, a major limitation to the development of SINEUPs as a RNA drug is represented by their length, that should be reduced to less than 60 nts to allow cost-effective manufacturing and efficient in vivo delivery. Recently, encouraging data have proved that the incorporation of chemically modified ribonucleotides restores IVT SINEUP RNA activity, making an important progress for its development as a drug. Here, I successfully designed and tested shorter SINEUP RNA variants that allowed us to reduce their size from 250 nts down to 50 nts. Indeed, by transfecting 2’OMeA modified ASO-SINEUP-OPA1, I was able to upregulate endogenous OPA1 protein translation of around 1.8 fold, as achieved with standard plasmid-driven expression of the same nanoSINEUP-OPA1 RNA. Most importantly, I applied previously selected mini- and nanoSINEUP to prove the functional rescue of DOA patients’ fibroblasts defects in mitochondrial morphology and activity. In summary, I was able to identify OPA1-specific SINEUPs promoting the recovery of disease-associated defects in patient-derived cellular model of DOA and I optimized SINEUP technology for its development as RNA therapeutic molecule for the treatment of haploinsufficient diseases