Title

Reduction of gene expression by U1 snRNA: A step toward somatic gene therapy

Date of Completion

January 2000

Keywords

Biology, Molecular

Degree

Ph.D.

Abstract

Gene therapy may be an alternative or adjuvant for pharmacologically resistant diseases that are caused by errors in gene expression. For example, the molecular basis of osteogenesis imperfecta (type III) is incorporation of mutant collagen peptides into the mature protein causing defective bone matrix formation. Reducing mutant gene expression at the level of mRNA may facilitate treatment where conventional means fail. Association of a ribozyme with U6 snRNA and antisense with U1 snRNA were used to overcome problems encountered with inhibition of mRNA, which include failure to co-localize target and effector, low effector levels and improper presentation of the effector. AntiCAT ribozymes, were tested in vitro to establish optimal cleavage conditions. Subsequently, for analysis of in vivo activity, the ribozymes were inserted into the body of the U6 snRNA gene, which would retain transcripts in the nucleus and approximate the ribozymes with target mRNA. In transient and stable transfection ribozymes incorporated into U6 snRNA were expressed at 10% of endogenous U6 snRNA levels. However the ribozymes were ineffective when targeted against the CAT gene. The splice donor recognition sequence of the U1 snRNA gene was altered to present antisense to several target sequences, in a variation of the mechanism observed in naturally occurring mutations resulting in decreased mRNA export. U1 snRNA is normally involved in the regulation of spliceosome formation, polyadenylation and export of mRNA. In transient co-transfections of β-gal, CAT or GFP with the appropriate U1 antitarget vector, both protein activity and mRNA level were reduced by >90%. Stable co-transfectios and sequential transfections of CAT and U1 antiCAT demonstrated ≥90% inhibition of protein activity. FACS and RNA analysis of cells sequentially transfected with GFP and U1 AntiGFP showed nuclear concentration of the U1 antitarget vector and reduction of up to 90% of GFP expression. The inhibition occurs early in processing and prevents nucleus or cytoplasm accumulation of GFP RNA. In conclusion U1snRNA antitarget vectors can selectively reduce gene output and may alone, or in combination with ribozymes, prove to be of universal application to many situations involving inhibition of dominant negative gene expression. ^