DNA-BINDING PROTEINS RECOGNIZING UNIQUE SEQUENCES

DESCRIPTION (Adapted from applicant's abstract): The critical element in the development of recombinant fusion zinc-finger proteins capable of recognizing specific 18 base-pair DNA recognition sequences will be the design of a suitable peptide linker between the modular DNA binding domains. Model hybrid recombinant fusion proteins composed of the two well-characterized zinc-finger binding proteins, Zif268 and SP1 will be constructed and assayed for their relative affinity to adjacent 9 base pair nucleotide sequences. Each zinc finger monomer consists of three modular "fingers" each of which recognizes a specific base pair triplet. Several peptide linkers will be designed and tested for their ability to allow high affinity binding of both modular zinc fingers to their respective recognition sequences. It is expected that the binding affinity of the chimeric peptide for its 18-bp target sequence will be much greater than the affinities of the individual zinc-finger domains for their individual 9-bp recognition sequences. The demonstration that these hybrid DNA-binding proteins are capable of recognizing specific, adjacent 18 base pair sequences is a critical "proof of principle" step that will permit the design of other hybrid zinc-finger proteins. These proteins may have widespread application to studies of gene regulation and as potential therapeutic agents. $ = TOTAL AWARD AMTS & NOT LIMITED TO PORTION OF PROJECT RELATED TO SUBJECT OF SEARCH SUBPROJECT $ = TOTAL AWARD AMOUNT DIVIDED BY NUMBER OF SUBPROJECTS SOURCE: CRISP FORMAT F FY 97 LAST UPDATE 04-07-98 1QUERY 1536 ID SEARCH 06/01/98 PAGE 346 --PROJECT NUMBER......1 R43 GM56564-01 INVESTIGATOR NAME/ADDRESS FY 97 GREEN, ANTHONY P IRG/INTRAMURAL UNIT..ZRG3 PURESYN INC AWARD AMOUNT......... $96,600 87 GREAT VALLEY PARKWAY MALVERN, PA 19355 PERFORMING ORGANIZATION: PURESYN, INC. TITLE IMPROVED RECOMBINANT ADENOVIRUS PURIFICATION ABSTRACT: The purpose of this application is to develop commercial processes for improved purification of non-enveloped viral vectors such as adenovirus. These vectors are vehicles for vaccination or ex vivo and in vivo gene therapy. Starting from crude material, the resulting product must be of high purity and produced in high yields. In addition, these vectors must be free from contaminating cellular components such as chromosomal DNA and RNA, protein, membrane components and endotoxins. Requirements for large scale production of these vectors is creating a demand for improved purification processes. Current processes using Cesium chloride gradients are cumbersome, inefficient and difficult to scale-up. Alternative technologies based on column chromatography are often inefficient, resulting in low purity and yields and limited abilities to remove contaminants. Column fouling is a particular problem. A new approach involving an innovative technology is proposed. The technology is based on an inert, non-porous, irregularly-shaped polymer using principles of ion-pair reverse-phase chromatography. The unique properties of this resin provide for improved purity and yield resulting improved economics for vector purification. $ = TOTAL AWARD AMTS & NOT LIMITED TO PORTION OF PROJECT RELATED TO SUBJECT OF SEARCH SUBPROJECT $ = TOTAL AWARD AMOUNT DIVIDED BY NUMBER OF SUBPROJECTS SOURCE: CRISP FORMAT F FY 97 LAST UPDATE 04-07-98 1QUERY 1536 ID SEARCH 06/01/98 PAGE 347 --PROJECT NUMBER......1 R43 GM56568-01 INVESTIGATOR NAME/ADDRESS FY 97 CHOI, TED IRG/INTRAMURAL UNIT..ZRG2 SEQUANA THERAPEUTICS INC AWARD AMOUNT......... $99,743 11099 NORTH TORREY PINES RD LA JOLLA, CA 92037 PERFORMING ORGANIZATION: SEQUANA THERAPEUTICS, INC. TITLE LARGE SCALE GENE TARGETING FOR FUNCTIONAL GENOMICS ABSTRACT: We propose to develop technologies for making targeted gene inactivations in mice ten to fifty fold faster than is currently possible. Such "knockout" transgenic mice are critical for understanding gene function and disease processes at the organismal level. Instead of conventional approaches that rely on restriction enzymes to construct targeting vectors by recombinant DNA procedures, novel plasmids and methods for harnessing the recombinational machinery of bacteria to rapidly assemble targeting vectors will be developed. These vectors will be more precisely constructed and will be at least as efficient at homologous recombination than conventional targeting vectors.