IN VIVO SSDNA EXPRESSION VECTORS FOR ALTERING GENE EXPRESSION

摘要

An expression vector for altering expression of a target nucleic acid sequence in a host cell by production of single-stranded cDNA (ssDNA) in the host cell in vivo. The expression vector is comprised of a cassette comprising a sequence of interest, an inverted tandem repeat, and a primer binding site 3' to the inverted tandem repeat, and a reverse transcriptase/RNAse H coding gene, and may be transfected into the host cell. Transcription of the cassette by the host cell produces an RNA template which is reverse transcribed with the product of the RT coding gene to produce ssDNA of a specified sequence. The ssDNA is modified to remove flanking vector sequences by taking advantage of the "stem-loop" structure of the ssDNA, which forms as a result of the inverted tandem repeat that allows the ssDNA to fold back on itself, forming a double stranded DNA stem. The double-stranded stem may contain one or more restriction endonuclease recognition sites and the loop, which remains as ssDNA, can be any desired nucleotide sequence. This design allows the double-stranded stem of the stem-loop intermediate to be cleaved by the desired corresponding restriction endonuclease(s) and the loop portion is then released as a linearized, single-stranded piece of DNA. The resulting ssDNA binds to an endogenous target nucleic acid sequence to alter the expression of that sequence for such therapeutic purposes as gene activation or inactivation using duplex or triplex binding of nucleic acids, site-directed mutagenesis, interruption of cellular function by binding to specific cellular proteins, or interfering with RNA splicing functions.