Therapeutic RNA manipulation in liver disease

Abstract

Posttranscriptional regulation of gene expression is increasingly recognized as a model for inherited and acquired disease. Recent work has expanded understanding of the range of mechanisms that regulate several of these distinct steps, including messenger RNA (mRNA) splicing, trafficking, and/or stability. Each of these pathways is implicated in disease pathogenesis, and each represents important avenues for therapeutic intervention. This review summarizes important mechanisms controlling mRNA processing and the regulation of mRNA degradation, including the role of microRNAs and RNA binding proteins. These pathways provide important opportunities for therapeutic targeting directed at splicing and degradation in order to attenuate genetic defects in RNA metabolism. We will highlight developments in vector development and validation for therapeutic manipulation of mRNA expression with a focus on potential applications in metabolic and immunomediated liver disease.

Figure 1. Mechanisms for therapeutic mRNA targeting

(A) RNA Interference (RNAi). Cellular RNAi machinery may be coopted by a variety of mRNA targeting strategies. Genomically templated pri-miRNA is transcribed in an RNA Polymerase-II (Pol II) dependent fashion and cleaved in the nucleus to pre-miRNA and transported via Exportin 5 to the cytoplasm. Exportin 5 shuttles back to the nucleus, while the pre-miRNA undergoes further processing in the cytoplasm by Dicer. The guide strand is incorporated into the RNA-induced silencing complex (RISC) that includes proteins such as Argonaute (AGO) involved in targeting mRNA and leading to passenger strand degradation. Lentiviral or plasmid encoded short hairpin RNA (shRNA) transcripts may be generated in the nucleus using either Pol II or Pol III driven by a tissue specific promoter/enhancer (TSPE) and are similarly processed to activate RNAi. Exogenously delivered cytoplasmic dsRNA or siRNA may be processed by Dicer and incorporated into the RISC complex and generally require less processing than miRNA or shRNA. Perfect or near perfect complementarity between the guide sequence and target mRNA typically leads to target cleavage. Imperfect complementarity typically results in trafficking of the mRNA to cytoplasmic P-bodies where it is degraded or sequestered for later translation. (B). Antisense oligodeoxynucleotides (ASOs). ASOs may base pair with the target mRNA transcript at almost any location, including the 3’ untranslated region (UTR) which is shown but may also be used to target the 5’ region of the target gene and the initiation AUG codon. Depending on the target location and chemical modification of the ASO, the target mRNA may be cleaved in an RNase H dependent fashion. Alternatively, the ASO may result in steric hindrance of RNA-binding proteins, preventing translation, modifying splicing, or altering mRNA stability. (C) Ribozyme mediated trans-splicing. The guide sequence of the targeting transcript base pairs with the target mRNA upstream of the non-functional or pathologic sequence or mutational site (*). The ribozyme domain of the targeting construct catalyzes splicing of the target transcript to the replacement exons, generating a functional, non-pathogenic mRNA sequence.