Antisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation
| Autor: | Luca Spiezia, Tilman M. Hackeng, Elisabetta Castoldi, Francesca Nuzzo, Paolo Simioni, Claudia M. Radu, Marco Baralle |
|---|---|
| Přispěvatelé: | Promovendi CD, Biochemie, RS: CARIM School for Cardiovascular Diseases |
| Rok vydání: | 2013 |
| Předmět: |
RNA Splicing
Immunology Biology Transfection Biochemistry RNA Small Nuclear Chlorocebus aethiops Animals Humans RNA Antisense Messenger RNA Oligonucleotide Homozygote Intron Factor V RNA Genetic Therapy Hep G2 Cells Cell Biology Hematology Molecular biology Introns COS Cells Mutation RNA splicing Factor V Deficiency Megakaryocytes Ex vivo Small nuclear RNA Minigene |
| Zdroj: | Blood, 122(23), 3825-3831. The American Society of Hematology |
| ISSN: | 1528-0020 0006-4971 |
| DOI: | 10.1182/blood-2013-04-499657 |
| Popis: | Antisense molecules are emerging as a powerful tool to correct splicing defects. Recently, we identified a homozygous deep-intronic mutation (F5 c.1296+268A>G) activating a cryptic donor splice site in a patient with severe coagulation factor V (FV) deficiency and life-threatening bleeding episodes. Here, we assessed the ability of 2 mutation-specific antisense molecules (a morpholino oligonucleotide [MO] and an engineered U7 small nuclear RNA [snRNA]) to correct this splicing defect. COS-1 and HepG2 cells transfected with a F5 minigene construct containing the patient's mutation expressed aberrant messenger RNA (mRNA) in excess of normal mRNA. Treatment with mutation-specific antisense MO (1-5 mu M) or a construct expressing antisense U7snRNA (0.25-2 mu g) dose-dependently increased the relative amount of correctly spliced mRNA by 1 to 2 orders of magnitude, whereas control MO and U7snRNA were ineffective. Patient-derived megakaryocytes obtained by differentiation of circulating progenitor cells did not express FV, but became positive for FV at immunofluorescence staining after administration of antisense MO or U7snRNA. However, treatment adversely affected cell viability, mainly because of the transfection reagents used to deliver the antisense molecules. Our data provide in vitro and ex vivo proof of principle for the efficacy of RNA therapy in severe FV deficiency, but additional cytotoxicity studies are warranted. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|