The present invention provides methods for making zinc finger proteins with high affinity for their targets, by sequentially selecting each finger of the protein.

General strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites

Methods and means are provided for reducing the phenotypic expression of a nucleic acid of interest in eucaryotic cells, particularly in plant cells, by introducing chimeric genes encoding sense and antisense RNA molecules directed towards the target nucleic acid, which are capable of forming a double stranded RNA region by base-pairing between the regions with sense and antisense nucleotide sequence or by introducing the RNA molecules themselves. Preferably, the RNA molecules comprises simultaneously both sense and antisense nucleotide sequence.

Methods and means for obtaining modified phenotypes

The present invention provides methods for modulating expression of endogenous cellular genes using recombinant zinc finger proteins.

Regulation of endogenous gene expression in cells using zinc finger proteins

The present invention relates to methods and reagents useful for modulating gene expression in various applications such as therapeutic, diagnostic, target assessment and genome discovery applications. Specifically, the present invention relates to synthetic chemically modified small nucleic acid molecules capable of mediating RNA interference (RNAi) to a target nucleic acid sequence, such as short interfering nucleic acids (siNA), short interfering RNA (siRNA), double stranded RNA (dsRNA). ), MicroRNA (miRNA), and short hairpin RNA (shRNA) molecules. Small nucleic acid molecules are useful in the treatment of any disease or condition that responds to regulation of gene expression in a cell, tissue or organism.

RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION USING CHEMICALLY MODIFIED SHORT INTERFERING NUCLEIC ACID (siNA)

The invention provides criteria and methods for selecting optimum subsequence(s) from a target for targeting by a zinc finger protein. Some of the methods of targetgene site selection seek to identify one or more target segments having a DNA motif containing one or more so-called D-able subsites having the sequence 5'NNGK3'. Other methods of the invention are directed to selection of target segments within target genes using a correspondence regime between different triplets of three bases and the three possible positions of a triplet within a nine-base site. In another aspect, the invention provides methods of designing zinc finger proteins that bind to a preselected target site. These methods can be used following the preselection of target sites according to the procedures and criteria described above. The methods of design use a database containing information about previously characterized zinc finger proteins.

Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites

Ribozymes have enormous potential as antiviral agents. We have previously reported that a hairpin ribozyme expressed under the control of the beta-actin promoter that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in the leader sequence can inhibit HIV-1 (pHXB2gpt) expression. For such a ribozyme in a retroviral vector delivery system to be useful in gene therapy for the treatment of HIV-1 infection, it must be able to inhibit the expression of multiple HIV-1 strains. We have now cloned this ribozyme into various regular expression vectors (including retroviral vectors) by using various gene expression control strategies. Here we show by transient transfection that inhibition of expression of diverse strains of HIV-1 can be achieved by this ribozyme expressed in the proper vectors. These data further support the potential of this hairpin ribozyme as a therapeutic agent for HIV-1.

Jay Rapapport

Papers

A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1

Correction: A Hairpin Ribozyme Inhibits Expression of Diverse Strains of Human Immunodeficiency Virus Type 1