{"id":52545,"date":"2023-02-22T11:25:28","date_gmt":"2023-02-22T11:25:28","guid":{"rendered":"https:\/\/fundacionnoelia.org\/?page_id=52545"},"modified":"2024-01-12T11:08:47","modified_gmt":"2024-01-12T11:08:47","slug":"developing-two-rna-directed-approaches-to-treating-the-recurrent-glycine-variants-p-g284r-and-p-g293r-in-col6a1","status":"publish","type":"page","link":"https:\/\/fundacionnoelia.org\/en\/research-projects\/developing-two-rna-directed-approaches-to-treating-the-recurrent-glycine-variants-p-g284r-and-p-g293r-in-col6a1\/","title":{"rendered":"DEVELOPING TWO RNA DIRECTED APPROACHES TO TREATING THE RECURRENT GLYCINE VARIANTS P.G284R AND P.G293R IN COL6A1"},"content":{"rendered":"[vc_row][vc_column width=”1\/6″][\/vc_column][vc_column width=”4\/6″][vc_empty_space]
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DEVELOPING TWO RNA DIRECTED APPROACHES TO TREATING THE RECURRENT GLYCINE VARIANTS P.G284R AND P.G293R IN COL6A1<\/h2>\n[\/vc_column_text][vc_empty_space][vc_column_text]Project description:<\/strong><\/p>\n

Collagen VI-related dystrophies (COL6-RD) are a group of congenital neuromuscular disorders characterized by generalized muscle weakness, progressive contractures, and respiratory dysfunction, for which there is no effective treatment. COL6-RD are commonly caused by dominant-negative pathogenic variants in the COL6A1, COL6A2<\/em> and COL6A3<\/em> genes, such as glycine substitutions of the conserved Gly-X-Y motives. Two of the most common Gly substitutions, p.G284R and p.G293R are located in the alpha1(VI) chain, in a region that constitutes a hot-spot for dominant-negative mutations.<\/p>\n

RNA directed therapeutics offer great opportunities to silence or edit dominant-negative mutations if designed to selectively target the mutant allele.<\/p>\n

 <\/p>\n

Project 1: Chemical engineering of therapeutic siRNAs for allele-specific gene silencing of dominant-negative glycine substitutions causing collagen VI-related dystrophy<\/strong><\/p>\n

To achieve the potential of RNA directed therapeutics for dominant conditions, the design of highly active, yet allele-specific, antisense oligonucleotides or siRNAs is required. Targeting single missense substitutions proves to be challenging, as the mutant target differs from the normal copy by only one nucleotide. To increase selectivity of siRNA silencing towards a common glycine substitution (G293R) in COL6A1<\/em>, we deliberately introduced a second mismatch into the siRNA design, to destabilize hybridization to the normal allele while maintaining activity on the mutant transcript. A series of siRNAs in which the location of the second mismatch was located at various positions along the siRNA sequence were first screened in HEK293 cells with fluorescence reporters for both the normal and the mutant allele. We identified at least two siRNAs that retain high silencing activity towards the mutant allele, while preserving the expression of the normal allele. Treatment of patient-derived fibroblasts with these siRNAs efficiently rescued COL6 matrix deposition.<\/p>\n

However, for success the clinical application of these siRNAs via systemic delivery, their stabilization and effective targeting to muscle interstitial fibroblast are critical. This can be achieved through chemical modifications, stabilizing the siRNA for sustained efficacy and distribution in vivo<\/em>. Full chemical stabilization is essential for in vivo efficacy but can also significantly affect guide strand thermodynamics. Thus, the chemical modification of siRNAs changes their target recognition and discrimination properties, altering their targeting profile.<\/p>\n

In collaboration with Dr. Anastasia Khvorova at RNA Therapeutics Institute, we propose a systematic series of screens targeting two glycine substitutions in COL6A1<\/em> (the aforementioned G293R and the common G284R) using fully chemically modified siRNA in vitro<\/em> in patient-derived fibroblasts and in vivo<\/em> in the humanized COL6A1<\/em> G284R and G293R mouse models.<\/p>\n

Budget: $50,000\/year<\/p>\n

 <\/p>\n

Project 2: Use of circular RNA guides to correct a hotspot of glycine substitutions in COL6-RD<\/strong><\/p>\n

Most of glycine missense pathogenic variants are caused by a single guanosine (G) to adenosine (A) change, making them suitable for the use of an RNA editing approach using endogenous adenosine deaminases (ADARs). ADARs use double-stranded RNAs as a substrate to catalyze adenosine (A)-to-inosine (I), which mimics G during translation.<\/p>\n

In our first attempt, we used an approach aimed at correcting the mutant copy using long RNA oligonucleotides able to recruit endogenous ADARs to the target site, to convert the mutant allele to wild type. This approach resulted on non-detectable editing due to long RNAs low stability and quick degradation within the cell.<\/p>\n

Here, we propose a novel approach using circular ADAR-recruiting RNAs to recruit endogenous ADAR enzymes to change a specific A to I, as a therapeutic approach for collagen VI-related dystrophies caused by G>A changes. RNA circularization improves RNA stability and increases resistance to cellular exonucleases, improving the efficiency and durability of programmable RNA editing. Strikingly, preliminary results in HEK293T with fluorescence reporter for both<\/strong> G284R and G293R alleles showed percentages of RNA editing up to 22% and 25%, respectively, using a single<\/strong> circular ADAR-recruiting RNA. This approach again will be tested in-vivo <\/em>in the humanized mouse models, using a vectorized approach.<\/p>\n

Budget: $75,000\/year<\/p>\n

 <\/p>\n

Place of development:<\/strong>
\nNINDS National Institute of Neurological Disease and Stroke, Bethesda, Maryland USA and other places if deemed appropriate.<\/p>\n

Investigators involved:<\/strong>
\nDr. B\u00f6nnemann, Carsten G., MD.
\nDra. Veronique Bolduc, PhD
\nDra. Astrid Brull, PhD<\/p>\n

Duration of the project and period of support by Fundaci\u00f3n Noelia:<\/strong>
\n2 a\u00f1os (Abril 2023 – Abril 2025)<\/p>\n

Total inversion:<\/strong>
\n250.000 DOLARES<\/p>\n

Contributions made by Fundaci\u00f3n Noelia:<\/strong>
\nAbril 2023: 125.000 $(Proof of payment<\/a>)<\/p>\n

Contributions pending to be made by the Fundaci\u00f3n Noelia:<\/strong>
\nAbril 2024: 125.000 $[\/vc_column_text][\/vc_column][vc_column width=”1\/6″][\/vc_column][\/vc_row]\n","protected":false},"excerpt":{"rendered":"

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