Heavily and Fully Modified RNAs Guide Efficient SpyCas9-Mediated Genome Editing [preprint]

RNA-based drugs depend on chemical modifications to increase potency and nuclease stability, and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. No studies have yet explored chemical modification at all positions of the crRNA guide and tracrRNA cofactor. Here, we have identified several heavily-modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2\u27-OH groups) that are functional in human cells. These designs demonstrate a significant breakthrough for Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes

Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing

RNA-based drugs depend on chemical modifications to increase potency and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. Here, we explore chemical modifications at all positions of the crRNA guide and tracrRNA cofactor. We identify several heavily modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2\u27-OH groups) that are functional in human cells. These designs will contribute to Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes

Improving gene silencing oligonucleotides by incorporation of peptide nucleic acids

The use of PNAs in therapeutics is limited by its mechanism of action. PNA (peptide nucleic acid) acts as steric blocker and therefore one copy per target of these therapeutic oligonucleotides is needed. While this mechanism is very interesting in splice-switching therapeutics it falls short of Ago2 or RNase H dependent gene silencing. Although the failure of PNA to recruit these enzymes to cleave their target could be a deal breaker, the high nuclease stability, neutral backbone and high affinity of PNAs are features that could enhance efficacy of siRNAs and antisense oligonucleotides (ASOs). First, the present work discussed the design, synthesis and properties of PNAs and LNA-modified oligonucleotides, which were used to switch off a silencing modified small non-coding RNA MicAstab. Then, usage of PNAs in tandem with highly modified siRNA is discussed. The silencing activity of siRNAs containing PNA sense strand as RNA:PNA duplex was investigated. Association of PNA and siRNA was further studied and silencing activity and biophysical properties of PNA-Peptide carrier for siRNA delivery is shown. Finally, the optimisation of DNA-PNA chimeras was investigated. The synthesis of the monomers as well as the oligomerisation of LNA-DNA-PNA is described. The biophysical properties of chimeras and their ability to efficiently knock down MALAT1 RNA in cells are shown in this thesis

Next-generation peptide nucleic acid chimeras exhibit high affinity and potent gene silencing

We present a new design of mixed-backbone antisense oligonucleotides (ASOs) containing both DNA and peptide nucleic acid (PNA). Previous generations of PNA-DNA chimeras showed low binding affinity, reducing their potential as therapeutics. The addition of a 5'-wing of locked nucleic acid as well as the combination of a modified nucleotide and a PNA monomer at the junction between PNA and DNA yielded high-affinity chimeras. The resulting ASOs demonstrated high serum stability and elicited robust RNase H-mediated cleavage of complementary RNA. These properties allowed the chimeric ASOs to demonstrate high gene silencing efficacy and potency in cells, comparable with those of LNA gapmer ASOs, via both lipid transfection and gymnosis.</p

Deep learning-based noise reduction preserves quantitative MRI biomarkers in patients with brain tumors

International audienceThe use of relaxometry and Diffusion-Tensor Imaging sequences for brain tumor assessment is limited by their long acquisition time. We aim to test the effect of a denoising algorithm based on a Deep Learning Reconstruction (DLR) technique on quantitative MRI parameters while reducing scan time. In 22 consecutive patients with brain tumors, DLR applied to fast and noisy MR sequences preserves the mean values of quantitative parameters (fractional anisotropy, mean diffusivity, T1 and T2-relaxation time) and produces maps with higher structural similarity compared to long duration sequences. This could promote wider use of these biomarkers in clinical setting

Innovative developments and emerging technologies in RNA therapeutics

RNA-based therapeutics are emerging as a powerful platform for the treatment of multiple diseases. Currently, the two main categories of nucleic acid therapeutics, antisense oligonucleotides and small interfering RNAs (siRNAs), achieve their therapeutic effect through either gene silencing, splicing modulation or microRNA binding, giving rise to versatile options to target pathogenic gene expression patterns. Moreover, ongoing research seeks to expand the scope of RNA-based drugs to include more complex nucleic acid templates, such as messenger RNA, as exemplified by the first approved mRNA-based vaccine in 2020. The increasing number of approved sequences and ongoing clinical trials has attracted considerable interest in the chemical development of oligonucleotides and nucleic acids as drugs, especially since the FDA approval of the first siRNA drug in 2018. As a result, a variety of innovative approaches is emerging, highlighting the potential of RNA as one of the most prominent therapeutic tools in the drug design and development pipeline. This review seeks to provide a comprehensive summary of current efforts in academia and industry aimed at fully realizing the potential of RNA-based therapeutics. Towards this, we introduce established and emerging RNA-based technologies, with a focus on their potential as biosensors and therapeutics. We then describe their mechanisms of action and their application in different disease contexts, along with the strengths and limitations of each strategy. Since the nucleic acid toolbox is rapidly expanding, we also introduce RNA minimal architectures, RNA/protein cleavers and viral RNA as promising modalities for new therapeutics and discuss future directions for the field.ISSN:1547-6286ISSN:1555-858

Orbitofrontal sulcal patterns in catatonia

International audienceBackground: Catatonia is a psychomotor syndrome frequently observed in disorders with neurodevelopmental impairments, including psychiatric disorders such as schizophrenia. The orbitofrontal cortex (OFC) has been repeatedly associated with catatonia. It presents with an important interindividual morphological variability, with three distinct H-shaped sulcal patterns, types I, II, and III, based on the continuity of the medial and lateral orbital sulci. Types II and III have been identified as neurodevelopmental risk factors for schizophrenia. The sulcal pattern of the OFC has never been investigated in catatonia despite the role of the OFC in the pathophysiology and the neurodevelopmental component of catatonia. Methods: In this context, we performed a retrospective analysis of the OFC sulcal pattern in carefully selected homogeneous and matched subgroups of schizophrenia patients with catatonia ( N = 58) or without catatonia ( N = 65), and healthy controls ( N = 82). Results Logistic regression analyses revealed a group effect on OFC sulcal pattern in the left ( χ 2 = 18.1; p Conclusion Because the sulcal patterns are indirect markers of early brain development, our findings support a neurodevelopmental origin of catatonia and may shed light on the pathophysiology of this syndrome