Broad objectives and specific aims To develop a correction strategy based on Exon Specific U1snRNAs (ExSpeU1) for the therapy of coagulation factor IX (FIX) deficiency (Hemophilia B, HB) caused by exon-skipping mutations. To create ExSpeU1 derivatives with improved efficacy, to develop a straightforward strategy to create animal models for splicing mutations and demonstrate the ExSpeU1 therapeutic efficacy in vivo. Background-rationale A significant proportion of disease-causing mutations affects pre-mRNA splicing by inducing exon skipping, but only few have been targeted for therapeutic purposes. We developed ExSpeU1s targeting non-conserved intronic sequences downstream of the donor site (5′ss) and proved that a unique ExSpeU1 can correct different exon-skipping mutations. Since even small increase in FIX levels (>2%) can be estimated in a quantitative manner, and result in significant amelioration of the clinical phenotype, HB is a preferred model to investigate innovative therapeutic approaches. Research design and methods We will focus on a panel of model exon-skipping mutations occurring at 3', 5' splice sites or within the exon 5 of F9 gene, and causing HB. All mutations were corrected in vitro by the first generation of ExSpeU1s. Through mechanistic studies in cellular models we will create optimized ExSpeU1s. HB mouse models will be created by liver-restricted expression of the splicing-competent human FIX cassettes in FIX knock-out mice. Long-term FIX expression will be obtained either i) via AdenoAssociated virus (AAV) vectors serotype 8 or ii) by the Sleeping Beauty transposon technology. The most active ExSpeU1s will be delivered in HB mice through AAV5 to assess restoration of FIX levels and amelioration of the bleeding phenotype. Anticipated output To establish the in vivo efficacy of a unique ExSpeU1 for a panel of different F9 exon-skipping mutations, and extend the therapeutic potential of this RNA-based strategy.
Development of a RNA-based therapeutic approach for Hemophilia B caused by exon-skipping mutations
PINOTTI, Mirko;
2014
Abstract
Broad objectives and specific aims To develop a correction strategy based on Exon Specific U1snRNAs (ExSpeU1) for the therapy of coagulation factor IX (FIX) deficiency (Hemophilia B, HB) caused by exon-skipping mutations. To create ExSpeU1 derivatives with improved efficacy, to develop a straightforward strategy to create animal models for splicing mutations and demonstrate the ExSpeU1 therapeutic efficacy in vivo. Background-rationale A significant proportion of disease-causing mutations affects pre-mRNA splicing by inducing exon skipping, but only few have been targeted for therapeutic purposes. We developed ExSpeU1s targeting non-conserved intronic sequences downstream of the donor site (5′ss) and proved that a unique ExSpeU1 can correct different exon-skipping mutations. Since even small increase in FIX levels (>2%) can be estimated in a quantitative manner, and result in significant amelioration of the clinical phenotype, HB is a preferred model to investigate innovative therapeutic approaches. Research design and methods We will focus on a panel of model exon-skipping mutations occurring at 3', 5' splice sites or within the exon 5 of F9 gene, and causing HB. All mutations were corrected in vitro by the first generation of ExSpeU1s. Through mechanistic studies in cellular models we will create optimized ExSpeU1s. HB mouse models will be created by liver-restricted expression of the splicing-competent human FIX cassettes in FIX knock-out mice. Long-term FIX expression will be obtained either i) via AdenoAssociated virus (AAV) vectors serotype 8 or ii) by the Sleeping Beauty transposon technology. The most active ExSpeU1s will be delivered in HB mice through AAV5 to assess restoration of FIX levels and amelioration of the bleeding phenotype. Anticipated output To establish the in vivo efficacy of a unique ExSpeU1 for a panel of different F9 exon-skipping mutations, and extend the therapeutic potential of this RNA-based strategy.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
