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An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants.
Mol Ther Nucleic Acids. 2016 Oct 04; 5(10):e370.MT

Abstract

In cellular models we have demonstrated that a unique U1snRNA targeting an intronic region downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1) can rescue multiple exon-skipping mutations, a relevant cause of genetic disease. Here, we explored in mice the ExSpeU1 U1fix9 toward two model Hemophilia B-causing mutations at the 5' (c.519A > G) or 3' (c.392-8T > G) splice sites of F9 exon 5. Hydrodynamic injection of wt-BALB/C mice with plasmids expressing the wt and mutant (hFIX-2G5'ss and hFIX-8G3'ss) splicing-competent human factor IX (hFIX) cassettes resulted in the expression of hFIX transcripts lacking exon 5 in liver, and in low plasma levels of inactive hFIX. Coinjection of U1fix9, but not of U1wt, restored exon inclusion of variants and in the intrinsically weak FIXwt context. This resulted in appreciable circulating hFIX levels (mean ± SD; hFIX-2G5'ss, 1.0 ± 0.5 µg/ml; hFIX-8G3'ss, 1.2 ± 0.3 µg/ml; and hFIXwt, 1.9 ± 0.6 µg/ml), leading to a striking shortening (from ~100 seconds of untreated mice to ~80 seconds) of FIX-dependent coagulation times, indicating a hFIX with normal specific activity. This is the first proof-of-concept in vivo that a unique ExSpeU1 can efficiently rescue gene expression impaired by distinct exon-skipping variants, which extends the applicability of ExSpeU1s to panels of mutations and thus cohort of patients.

Authors+Show Affiliations

Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.Internation Centre for Genetic Engineering and Biotechnology, Trieste, Italy.Internation Centre for Genetic Engineering and Biotechnology, Trieste, Italy.Haemostasis & Thrombosis Center, University of Ferrara, Ferrara, Italy.Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy. LTTA Center, University of Ferrara, Ferrara, Italy.Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy. LTTA Center, University of Ferrara, Ferrara, Italy.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

27701399

Citation

Balestra, Dario, et al. "An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants." Molecular Therapy. Nucleic Acids, vol. 5, no. 10, 2016, pp. e370.
Balestra D, Scalet D, Pagani F, et al. An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants. Molecular therapy. Nucleic acids. 2016;5(10):e370.
Balestra, D., Scalet, D., Pagani, F., Rogalska, M. E., Mari, R., Bernardi, F., & Pinotti, M. (2016). An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants. Molecular Therapy. Nucleic Acids, 5(10), e370. https://doi.org/10.1038/mtna.2016.77
Balestra D, et al. An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants. Molecular therapy. Nucleic acids. 2016 Oct 4;5(10):e370. PubMed PMID: 27701399.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants. AU - Balestra,Dario, AU - Scalet,Daniela, AU - Pagani,Franco, AU - Rogalska,Malgorzata Ewa, AU - Mari,Rosella, AU - Bernardi,Francesco, AU - Pinotti,Mirko, Y1 - 2016/10/04/ PY - 2016/02/10/received PY - 2016/07/21/accepted PY - 2016/10/5/entrez PY - 2016/10/5/pubmed PY - 2016/10/5/medline SP - e370 EP - e370 JF - Molecular therapy. Nucleic acids VL - 5 IS - 10 N2 - In cellular models we have demonstrated that a unique U1snRNA targeting an intronic region downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1) can rescue multiple exon-skipping mutations, a relevant cause of genetic disease. Here, we explored in mice the ExSpeU1 U1fix9 toward two model Hemophilia B-causing mutations at the 5' (c.519A > G) or 3' (c.392-8T > G) splice sites of F9 exon 5. Hydrodynamic injection of wt-BALB/C mice with plasmids expressing the wt and mutant (hFIX-2G5'ss and hFIX-8G3'ss) splicing-competent human factor IX (hFIX) cassettes resulted in the expression of hFIX transcripts lacking exon 5 in liver, and in low plasma levels of inactive hFIX. Coinjection of U1fix9, but not of U1wt, restored exon inclusion of variants and in the intrinsically weak FIXwt context. This resulted in appreciable circulating hFIX levels (mean ± SD; hFIX-2G5'ss, 1.0 ± 0.5 µg/ml; hFIX-8G3'ss, 1.2 ± 0.3 µg/ml; and hFIXwt, 1.9 ± 0.6 µg/ml), leading to a striking shortening (from ~100 seconds of untreated mice to ~80 seconds) of FIX-dependent coagulation times, indicating a hFIX with normal specific activity. This is the first proof-of-concept in vivo that a unique ExSpeU1 can efficiently rescue gene expression impaired by distinct exon-skipping variants, which extends the applicability of ExSpeU1s to panels of mutations and thus cohort of patients. SN - 2162-2531 UR - https://www.unboundmedicine.com/medline/citation/27701399/An_Exon_Specific_U1snRNA_Induces_a_Robust_Factor_IX_Activity_in_Mice_Expressing_Multiple_Human_FIX_Splicing_Mutants_ L2 - https://linkinghub.elsevier.com/retrieve/pii/mtna201677 DB - PRIME DP - Unbound Medicine ER -
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