Trans-splicing into highly abundant albumin transcripts for production of therapeutic proteins in vivo.

Spliceosome-mediated RNA trans-splicing has emerged as an exciting mode of RNA therapy. Here we describe a novel trans-splicing strategy, which targets highly abundant pre-mRNAs, to produce therapeutic proteins in vivo. First, we used a pre-trans-splicing molecule (PTM) that mediated trans-splicing of human apolipoprotein A-I (hapoA-I) into the highly abundant mouse albumin exon 1. Hydrodynamic tail vein injection of the hapoA-I PTM plasmid in mice followed by analysis of the chimeric transcripts and protein, confirmed accurate and efficient trans-splicing into albumin pre-mRNA and production of hapoA-I protein. The versatility of this approach was demonstrated by producing functional human papillomavirus type-16 E7 (HPV16-E7) single-chain antibody in C57BL/6 mice and functional factor VIII (FVIII) and phenotypic correction in hemophilia A mice. Altogether, these studies demonstrate that trans-splicing to highly abundant albumin transcripts can be used as a general platform to produce therapeutic proteins in vivo.

Conserved and clustered RNA recognition sequences are a critical feature of signals directing RNA localization in Xenopus oocytes.

Although it is widely regarded that the targeting of RNA molecules to subcellular destinations depends upon the recognition of cis-elements found within their 3' untranslated regions (UTR), relatively little is known about the specific features of these cis-sequences that underlie their function. Interaction between specific repeated motifs within the 3' UTR and RNA-binding proteins has been proposed as a critical step in the localization of Vg1 RNA to the vegetal pole of Xenopus oocytes. To understand the relative contributions of repeated localization element (LE) sequences, we used comparative functional analysis of Vg1 LEs from two frog species, Xenopus laevis and Xenopus borealis. We show that clusters of repeated VM1 and E2 motifs are required for efficient localization. However, groups of either site alone are not sufficient for localization. In addition, we present evidence that the X. borealis Vg1 LE is recognized by the same set of RNA-binding proteins as the X. laevis Vg1 LE and is capable of productive interactions with the X. laevis transport machinery as it is sufficient to direct vegetal localization in X. laevis oocytes. These results suggest that clustered sets of cis-acting sites within the LE direct vegetal transport through specific interactions with the localization machinery.

Dynamic conformational model for the role of ITS2 in pre-rRNA processing in yeast.

Maturation of the large subunit rRNAs includes a series of cleavages that result in removal of the internal transcribed spacer (ITS2) that separates mature 5.8S and 25/28S rRNAs. Previous work demonstrated that formation of higher order secondary structure within the assembling pre-ribosomal particle is a prerequisite for accurate and efficient pre-rRNA processing. To date, it is not clear which specific sequences or secondary structures are required for processing. Two alternative secondary structure models exist for Saccharomyces cerevisiae ITS2. Chemical and enzymatic structure probing and phylogenetic comparisons resulted in one structure (Yeh & Lee, J Mol Biol, 1990, 211:699-712) referred to here as the "hairpin model." More recently, an alternate folded structure was proposed (Joseph et al., Nucleic Acids Res, 1999, 27:4533-4540), called here the "ring model." We have used a functional genetic assay to examine the potential significance of these predicted structures in processing. Our data indicate that elements of both structural models are important in efficient processing. Mutations that prevent formation of ring-specific structures completely blocked production of mature 25S rRNA, whereas those that primarily disrupt hairpin elements resulted in reduced levels of mature product. Based on these results, we propose a dynamic conformational model for the role of ITS2 in processing: Initial formation of the ring structure may be required for essential, early events in processing complex assembly and may be followed by an induced transition to the hairpin structure that facilitates subsequent processing events. In this model, yeast ITS2 elements may provide in cis certain of the functions proposed for vertebrate U8 snoRNA acting in trans.