Eukaryotic use of improved chimeric mutational vectors

Details Eukaryotic use of improved chimeric mutational vectors Eukaryotic use of improved chimeric mutational vectors

1999-10-28

2003-06-03

Jones, W. Gary (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S455000, C435S254110, C435S320100, C435S468000, C536S022100, C536S023100

Reexamination Certificate

active

06573046

ABSTRACT:

FIELD OF THE INVENTION
Chimeraplasty concerns the introduction of directed alterations in a specific site of the DNA of a target cell by introducing duplex oligonucleotides, which are processed by the cell's homologous recombination and error repair systems so that the sequence of the target DNA is converted to that of the oligonucleotide where they are different. The present invention concerns a chimeraplasty method that is practiced in a cell-free system.
BACKGROUND TO THE INVENTION
Chimeraplasty
Chimeraplasty in eukaryotic cells and duplex recombinagenic oligonucleotides for use therein are disclosed in U.S. Pat. No. 5,565,350, issued Oct. 15, 1996, and No. 5,731,181, issued Mar. 24, 1998 by E. B. Kmiec (collectively “Kmiec”). The recombinagenic oligonucleotides disclosed by Kmiec contained ribo-type, e.g., 2′-O-methyl-ribonucleotides, and deoxyribo-type nucleotides that were hybridized to each other and were termed Chimeric Mutational Vectors (CMV). A CMV designed to repair a mutation in the gene encoding liver/bone/kidney type alkaline phosphatase was reported in Yoon, K., et al., 1996, Proc. Natl. Acad. Sci. 93, 2071. The alkaline phosphatase gene was transiently introduced into CHO cells by a plasmid. Six hours later the CMV was introduced. The plasmid was recovered at 24 hours after introduction of the CMV and analyzed. The results showed that approximately 30% to 38% of the alkaline phosphatase genes were repaired by the CMV.
A CMV designed to correct the mutation in the human &bgr;-globin gene that causes Sickle Cell Disease and its successful use was described in Cole-Strauss, A., et al., 1996, Science 273, 1386. A CMV designed to create a mutation in a rat blood coagulation factor IX gene in the hepatocyte of a rat is disclosed in Kren et al., 1998, Nature Medicine 4, 285-290. An example of a CMV having one base of a first strand that is paired with a non-complementary base of a second strand is shown in Kren et al., June 1997, Hepatology 25, 1462.
U.S. patent application Ser. No. 08/640,517, filed May 1, 1996, by E. B. Kmiec, A. Cole-Strauss and K. Yoon, published as WO97/41141, Nov. 6, 1997, and application Ser. No. 08/906,265, filed Aug. 5, 1997, disclose methods and CMV that are useful in the treatment of genetic diseases of hematopoietic cells, e.g., Sickle Cell Disease, Thalassemia and Gaucher Disease.
An example of the use of a CMV having one base of a first strand that is paired with a non-complementary base of a second strand is shown in Kren et al., June 1997, Hepatology 25, 1462. In Kren, the strand having the different desired, sequence was the strand having 2′-O-methyl ribonucleotides, which was paired with the strand having the 3′ end and 5′ end. U.S. Pat. No. 5,565,350 described a CMV having a single segment of 2′-O-methylated RNA, which was located on the chain having the 5′ end nucleotide.
Applicants are aware of the following provisional applications that contain teaching with regard to chimeric mutational vectors: By Steer et al., Serial No. 60/045,288 filed Apr. 30, 1997; Serial No. 60/054,837 filed Aug. 5, 1997; Serial No. No. 60/064,996, filed Nov. 10, 1997; and by Steer & Roy-Chowdhury et al., Serial No. 60/074,497, filed Feb. 12, 1998, entitled “Methods of Prophylaxis and Treatment by Alteration of APO B and APO E Genes.”
Cell-Free Recombination
Various reports of homologous recombination using a cell-free extract have been published.
Hotta, Y., et al., 1985, Chromosoma 93, 140-151 report the use of an extract of yeast, mouse spermatocytes and Lilium to effect homologous recombination between two mutant pBR322 plasmids. One of the plasmids was supercoiled, the second plasmid could be linearized or supercoiled. The maximum rate of recombination was less than 1%. A similar experiment using mutant defective pSV2neo and extracts of EJ cells was reported in Kucherlapati, R. S. et al., 1985, Molecular and Cellular Biology 5, 714-720. The maximum rate of recombination was about 0.2%. Kucherlapati reported an absolute requirement that one of the mutant plasmids be linearized. In contrast Hotta, reported recombination between two circular plasmids, although the rate of recombination between circular and linear plasmids was higher.
The report of Jessberger, R., & Berg, P., 1991, Mol. & Cell. Biol. 11, 445 concerns recombination catalyzed by nuclear extracts between plasmids. It stands in contrast to both of the above in two respects. The rate of recombination reported was about 20%, in contrast to rates of less than 0.5%. In addition Jessberger observed the same rate of recombination between circularized plasmids as between a circularized and a linear plasmid.
A related experiment using human nuclear extracts was reported by Lopez, B. S., et al., 1992, Nucleic Acids Research 20, 501-506. Lopez reported recombination in a cell-free system between a linearized plasmid and an unrelated supercoiled plasmid that is not viable in the subsequent selection conditions. The linearized and supercoiled plasmid each contain a lacZ gene; which is a mutant in the linearized plasmid. The linearized plasmid is cut in the lacZ gene at a variable distance from the mutation. Homologous recombination between the site of the mutation and the cut, accordingly, results in the circularization of the plasmid that then becomes viable and the gain of lacZ function. Lopez reports no detectable homologous recombination when the cut and the mutation were 15 base pairs apart. Homologous recombination at a low level was observed when that distance was 27 base pairs. No further increase in the rate of homologous recombination was observed when the distance was made greater than 165 base pairs. Lopez et al., 1987, Nucleic Acids Research
Rad51 and Rad52 Activity in Recombination
Homologous recombination is the process whereby the genes of two chromosomes are exchanged. The rate of homologous recombination between two genetic loci is inversely proportional to their genetic linkage, tightly linked genes rarely recombine. In addition to its genetic function homologous recombination allows a somatic cell to repair DNA damaged by double strand breaks.
The first step in homologous recombination is believed to be synapse formation. A synapse is a DNA molecule in which one chain is hybridized to two other chains. Synapse formation requires an enzymatic activity and energy input from ATP hydrolysis. An artifactual assay in a cell-free system for the enzymatic activity believed to be required for synapse formation is “strand transfer.” In a typical strand transfer assay a circular single strand DNA is combined with a linear duplex to produce a “nicked” or relaxed circular duplex and a linear single strand. The Rad51 gene from yeast, mice and humans has been cloned and catalyzes strand transfer. Rad51 is believed to participate in synapse formation. Baumann, P., et al., 1996, Cell 87, 757-766; Gupta, R. C., 1997, Proc. Natl. Acad. Sci. 94, 463-468. The strand transfer activity is further enhanced by the presence of Rad52 protein and replication protein A. Baumann, P., & West, S.C., 1997, EMBO J. 16, 5198-5206; New, J. H., et al., 1998, Nature 391, 407-410; Benson, F. E., et al., 198, Nature 391, 401-404. Although RAD51 protein unlike Rec A binds to duplex DNA, Baumann & West op cit.; Benson, F. E., et al., EMBO J., 13, 5764-5771, in the presence of RAD52, its binding is directed toward single stranded DNA.
In yeast, Rad51 or Rad52 defective individuals are radiation sensitive because of an inability to repair double strand breaks. In mice, Rad51 knock out results in embryonic leathality. Tsuzuki, T., et al., Proc. Natl. Acad. Sci. 93, 6236-6240; Lin, S. D., & Hasty, P. A., Mol. Cell. Biol., 16, 7133.
Cell-Free Mismatch Repair
The intrinsic (thermodynamic) fidelity of DNA replication would lead to an unacceptably high rate of mutation without the presence of an “error correcting” mechanism. Mismatch repair is one such mechanism. In mismatch repair, duplex DNA having a base paired to a non-complementary base is processed so that one of the strands is correct

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