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Rational design of an artificial tethered enzyme for non-templated post-transcriptional mRNA polyadenylation by the second generation of the C3P3 system.
Le Boulch, Marine; Jacquet, Eric; Nhiri, Naïma; Shmulevitz, Maya; Jaïs, Philippe H.
Afiliación
  • Le Boulch M; Eukarÿs SAS, Pépinière Genopole, 4 rue Pierre Fontaine, Genopole Entreprises Campus 3, 4 Rue Pierre Fontaine, 91000, Evry-Courcouronnes, France.
  • Jacquet E; Institut de Chimie des Substances Naturelles, CNRS UPR2301, Université Paris-Saclay, Avenue de la Terrasse, 91198, Gif-Sur-Yvette, France.
  • Nhiri N; Institut de Chimie des Substances Naturelles, CNRS UPR2301, Université Paris-Saclay, Avenue de la Terrasse, 91198, Gif-Sur-Yvette, France.
  • Shmulevitz M; Medical Microbiology and Immunology, Li Ka Shing Institute of Virology, University of Alberta, 6-142J Katz Group Centre for Pharmacy and Health Research, 114 Street NW, Edmonton, AB, T6G 2E1, Canada.
  • Jaïs PH; Eukarÿs SAS, Pépinière Genopole, 4 rue Pierre Fontaine, Genopole Entreprises Campus 3, 4 Rue Pierre Fontaine, 91000, Evry-Courcouronnes, France. philippe.jais@eukarys.com.
Sci Rep ; 14(1): 5156, 2024 03 02.
Article en En | MEDLINE | ID: mdl-38431749
ABSTRACT
We have previously introduced the first generation of C3P3, an artificial system that allows the autonomous in-vivo production of mRNA with m7GpppN-cap. While C3P3-G1 synthesized much larger amounts of capped mRNA in human cells than conventional nuclear expression systems, it produced a proportionately much smaller amount of the corresponding proteins, indicating a clear defect of mRNA translatability. A possible mechanism for this poor translatability could be the rudimentary polyadenylation of the mRNA produced by the C3P3-G1 system. We therefore sought to develop the C3P3-G2 system using an artificial enzyme to post-transcriptionally lengthen the poly(A) tail. This system is based on the mutant mouse poly(A) polymerase alpha fused at its N terminus with an N peptide from the λ virus, which binds to BoxBr sequences placed in the 3'UTR region of the mRNA of interest. The resulting system selectively brings mPAPαm7 to the target mRNA to elongate its poly(A)-tail to a length of few hundred adenosine. Such elongation of the poly(A) tail leads to an increase in protein expression levels of about 2.5-3 times in cultured human cells compared to the C3P3-G1 system. Finally, the coding sequence of the tethered mutant poly(A) polymerase can be efficiently fused to that of the C3P3-G1 enzyme via an F2A sequence, thus constituting the single-ORF C3P3-G2 enzyme. These technical developments constitute an important milestone in improving the performance of the C3P3 system, paving the way for its applications in bioproduction and non-viral human gene therapy.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ARN Polimerasas Dirigidas por ADN / Poliadenilación Límite: Animals / Humans Idioma: En Revista: Sci Rep Año: 2024 Tipo del documento: Article País de afiliación: Francia Pais de publicación: Reino Unido
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ARN Polimerasas Dirigidas por ADN / Poliadenilación Límite: Animals / Humans Idioma: En Revista: Sci Rep Año: 2024 Tipo del documento: Article País de afiliación: Francia Pais de publicación: Reino Unido