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CHD8 represents one of the highest confidence genetic risk factors implied in Autism Spectrum Disorders, with most mutations leading to CHD8 haploinsufficiency and the insurgence of specific phenotypes, such as macrocephaly, facial dysmorphisms, intellectual disability, and gastrointestinal complaints. While extensive studies have been conducted on the possible consequences of CHD8 suppression and protein coding RNAs dysregulation during neuronal development, the effects of transcriptional changes of long non-coding RNAs (lncRNAs) remain unclear. In this study, we focused on a peculiar class of natural antisense lncRNAs, SINEUPs, that enhance translation of a target mRNA through the activity of two RNA domains, an embedded transposable element sequence and an antisense region. By looking at dysregulated transcripts following CHD8 knock down (KD), we first identified RAB11B-AS1 as a potential SINEUP RNA for its domain configuration. Then we demonstrated that such lncRNA is able to increase endogenous RAB11B protein amounts without affecting its transcriptional levels. RAB11B has a pivotal role in vesicular trafficking, and mutations on this gene correlate with intellectual disability and microcephaly. Thus, our study discloses an additional layer of molecular regulation which is altered by CHD8 suppression. This represents the first experimental confirmation that naturally occurring SINEUP could be involved in ASD pathogenesis and underscores the importance of dysregulation of functional lncRNAs in neurodevelopment.

Use of monoclonal antibodies is emerging as a highly promising and fast-developing scenario for innovative treatment of viral, autoimmune and tumour diseases. The search for diagnostic and therapeutic antibodies currently depends on in vitro screening approaches, such as phage and yeast display technologies. Antibody production still represents a critical step for preclinical and clinical evaluations. Accordingly, improving production of monoclonal antibodies represents an opportunity, to facilitate downstream target validations. SINEUP RNAs are long non-coding transcripts, possessing the ability to enhance translation of selected mRNAs. We applied SINEUP technology to semi-stable production of monoclonal antibodies in HEK293E cells, which allows for episomal propagation of the expression vectors encoding the heavy and light chains of IgGs. Co-expression of SINEUP RNA with mRNAs encoding heavy and light chains of IgG4s was able to increase the production of different anti-CLDN1 antibodies up to three-fold. Improved production of monoclonal antibodies was achieved both in transiently transfected HEK293E cells and in cellular clones with stable expression of the SINEUP. Compared to antibody preparations obtained under standard conditions, the anti-CLDN1 IgG4s produced in the presence of the SINEUP transcript showed unaltered post-translational modifications, and retained the ability to recognize their target. We thus propose SINEUP technology as a valuable tool to enhance semi-stable antibody production in human cell lines.

Glial cell-derived neurotrophic factor (GDNF) has a potent action in promoting the survival of dopamine (DA) neurons. Several studies indicate that increasing GDNF levels may be beneficial for the treatment of Parkinson’s disease (PD) by reducing neurodegeneration of DA neurons. Despite a plethora of preclinical studies showing GDNF efficacy in PD animal models, its application in humans remains questionable for its poor efficacy and side effects due to its uncontrolled, ectopic expression. Here we took advantage of SINEUPs, a new class of antisense long non-coding RNA, that promote translation of partially overlapping sense protein-coding mRNAs with no effects on their mRNA levels. By synthesizing a SINEUP targeting Gdnf mRNA, we were able to increase endogenous GDNF protein levels by about 2-fold. Adeno-associated virus (AAV)9-mediated delivery in the striatum of wild-type (WT) mice led to an increase of endogenous GDNF protein for at least 6 months and the potentiation of the DA system’s functions while showing no side effects. Furthermore, SINEUP-GDNF was able to ameliorate motor deficits and neurodegeneration of DA neurons in a PD neurochemical mouse model. Our data indicate that SINEUP-GDNF could represent a new strategy to increase endogenous GDNF protein levels in a more physiological manner for therapeutic treatments of PD., Espinoza and colleagues demonstrate that a SINEUP non-coding RNA targeting GDNF mRNA can increase GDNF protein levels in vivo for several months after viral delivery. They also show that SINEUP-GDNF ameliorates motor deficits and neurodegeneration of dopamine neurons in a mouse model of Parkinson’s disease.

The post-genomic era has unveiled the existence of a large repertory of non-coding RNAs and repetitive elements that play a fundamental role in cellular homeostasis and dysfunction. These may represent unprecedented opportunities to modify gene expression at the right time in the correct space in vivo, providing an almost unlimited reservoir of new potential pharmacological agents. Hijacking their mode of actions, the druggable genome can be extended to regulatory RNAs and DNA elements in a scalable fashion. Here, we discuss the state-of-the-art of nucleic acid-based drugs to treat neurodegenerative diseases. Beneficial effects can be obtained by inhibiting (Yin) and increasing (Yang) gene expression, depending on the disease and the drug target. Together with the description of the current use of inhibitory RNAs (small inhibitory RNAs and antisense oligonucleotides) in animal models and clinical trials, we discuss the molecular basis and applications of new classes of activatory RNAs at transcriptional (RNAa) and translational (SINEUP) levels.

Mammalian cells are an indispensable tool for the production of recombinant proteins in contexts where function depends on post-translational modifications. Among them, Chinese Hamster Ovary (CHO) cells are the primary factories for the production of therapeutic proteins, including monoclonal antibodies (MAbs). To improve expression and stability, several methodologies have been adopted, including methods based on media formulation, selective pressure and cell- or vector engineering. This review presents current approaches aimed at improving mammalian cell factories that are based on the enhancement of translation. Among well-established techniques (codon optimization and improvement of mRNA secondary structure), we describe SINEUPs, a family of antisense long non-coding RNAs that are able to increase translation of partially overlapping protein-coding mRNAs. By exploiting their modular structure, SINEUP molecules can be designed to target virtually any mRNA of interest, and thus to increase the production of secreted proteins. Thus, synthetic SINEUPs represent a new versatile tool to improve the production of secreted proteins in biomanufacturing processes.

Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs) and unveiling their functions in a wide range of biological processes their applications as biotechnological or therapeutic tools are still at their infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the Parkinson's disease-associated gene Ubiquitin carboxyl-terminal esterase L1 (Uchl1), is able to increase UchL1 protein synthesis at post-transcriptional level. Its activity requires two RNA elements: an embedded inverted SINEB2 sequence to increase translation and the overlapping region to target its sense mRNA. This functional organization is shared with several mouse lncRNAs antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs as enhancers of target mRNA translation remains unexplored. Here we define AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named this class of RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. The overlapping region is indicated as the Binding Doman (BD) while the embedded inverted SINEB2 element is the Effector Domain (ED). By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest. SINEUPs function in an array of cell lines and can be efficiently directed toward N-terminally tagged proteins. Their biological activity is retained in a miniaturized version within the range of small RNAs length. Its modular structure was exploited to successfully design synthetic SINEUPs targeting endogenous Parkinson's disease-associated DJ-1 and proved to be active in different neuronal cell lines. In summary, SINEUPs represent the first scalable tool to increase synthesis of proteins of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.

Over the past 10 years, it has emerged that pervasive transcription in mammalian genomes has a tremendous impact on several biological functions. Most of transcribed RNAs are lncRNAs and repetitive elements. In this review, we will detail the discovery of a new functional class of natural and synthetic antisense lncRNAs that stimulate translation of sense mRNAs. These molecules have been named SINEUPs since their function requires the activity of an embedded inverted SINEB2 sequence to UP-regulate translation. Natural SINEUPs suggest that embedded Transposable Elements may represent functional domains in long non-coding RNAs. Synthetic SINEUPs may be designed by targeting the antisense sequence to the mRNA of choice representing the first scalable tool to increase protein synthesis of potentially any gene of interest. We will discuss potential applications of SINEUP technology in the field of molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.

Whenever the function of a recombinant protein depends on post-translational processing, mammalian cells become an indispensable tool for their production. This is particularly true for biologics and therapeutic monoclonal antibodies (MAbs). Despite some drawbacks, Chinese Hamster Ovary (CHO) cells are the workhorse for MAbs production in academia and industry. Several methodologies have been adopted to improve expression and stability, including methods based on selective pressure or cell engineering. We have previously identified SINEUPs as a new functional class of natural and synthetic long non-coding RNAs that through the activity of an inverted SINEB2 element are able to promote translation of partially overlapping sense coding mRNAs. Here we show that by taking advantage of their modular structure, synthetic SINEUPs can be designed to increase production of secreted proteins. Furthermore, by experimentally validating antisense to elastin (AS-eln) RNA as a natural SINEUP, we show that SINEUP-mediated control may target extracellular proteins. These results lead us to propose synthetic SINEUPs as new versatile tools to optimize production of secreted proteins in manufacturing pipelines and natural SINEUPs as new regulatory RNAs in the secretory pathways.

Comprehensive analysis of mammalian transcriptomes has surprisingly revealed that a major fraction of the RNAs produced by mammalian cells and tissues is comprised of long non-coding RNAs (lncRNAs). Such RNAs were previously disregarded as useless, but recent functional studies have revealed that they have multiple regulatory functions. A large subset of these lncRNAs are antisense to protein-coding genes; such RNAs are particularly attractive to researchers because their functions are better understood than other lncRNAs and their action can be easily modulated and engineered by modifying the antisense region. We discuss various aspects of regulation by antisense RNAs and other small nucleic acids and the challenges to bring these technologies to gene therapy. Despite several remaining issues related to delivery, RNA stability, side effects, and toxicity, the field is moving quickly towards future biotechnological and health applications. Therapies based on lncRNAs may be the key to increased cell-specificity of future gene therapies.