Chimeric mutational vectors

Details Chimeric mutational vectors Chimeric mutational vectors

1999-10-28

2001-04-03

Schwartzman, Robert A. (Department: 1635)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S024200, C435S006120, C435S069100, C435S091100, C435S320100, C435S440000, C435S455000, C435S462000, C435S463000

Reexamination Certificate

active

06211351

ABSTRACT:

1. FIELD OF THE INVENTION
The invention concerns the use of duplex oligonucleobase compounds (hereafter “duplex mutational vectors”) to specifically make alterations in the sequence of a DNA in a cell. In one embodiment the invention concerns compounds and methods of their use to make specific genetic alterations in the genome and in episomes (plasmids) of target prokaryotic cells. In a further embodiment the invention concerns methods of using bacterial cells to develop more efficient duplex mutational vectors. The structure of the duplex mutational vector (DMV) is designed so that genetic exchange between the DMV and the target gene occurs, i.e., a sequence contained in the DMV replaces the sequence of the target gene. In still further embodiments the invention concerns specific generic structures of DMV.
2. BACKGROUND OF THE INVENTION
U.S. Pat. No. 5,565,350, issued Oct. 15, 1996, and U.S. Pat. No. 5,731,181, issued Mar. 24, 1998 by E. B. Kmiec, described Chimeric Mutational Vectors (CMV), i.e., vectors having both DNA-type and RNA-type nucleobases for the introduction of genetic changes in eukaryotic cells. Such CMV were characterized by having at least 3 contiguous base pairs wherein DNA-type and RNA-type nucleobases are Watson-Crick paired with each other to form a hybrid-duplex. A CMV designed to repair a mutation in the gene encoding liver/bone/kidney type alkaline phosphatase was reported in Yoon, K., et al., March 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. Pat. No. 08/640,517, filed May 1, 1996, by E. B. Kmiec, A. Cole-Strauss and K. Yoon, published as W097/41141, Nov. 6, 1997, and U.S. Pat. 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.
The above-cited scientific publications of Yoon, Cole-Straauss and Kren describe CMV having two 2′-O-methyl RNA segments separated by an intervening DNA segment, which were located on the strand opposite the strand having the 5′ end nucleotide. 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. An oligonucleotide having complementary deoxyribonucleotides and a continuous segment of unmodified ribonucleotides on the strand opposite the strand having the 5′ end nucleotide was described in Kmiec, E. B., et al., 1994, Mol. and Cell. Biol. 14:7163-7172. The sequence of the strand was derived from the bacteriophage M13mp19,
The use of single stranded oligonucleotides to introduce specific mutations in yeast are disclosed in Yamamoto, T., et al., 1992, Genetics 131, 811-819. The oligonucleotides were between about 30 and 50 bases. Similar results were reported by Campbell, C. R., et al., 1989, The New Biologist, 1, 223-227. Duplex DNA fragments of about 160 base pairs in length have been reported to introduce specific mutations in cultured mammalian cells. Hunger-Bertling, K., et al., 1990, Molecular and Cellular Biochemistry 92, 107-116.
Applicants are aware of the following provisional applications that contain teaching with regard to uses and delivery systems of recombinagenic oligonucleotides: By Steer et al., Ser. No. 60/045,288 filed Apr. 30, 1997; Ser. No. 60/054,837 filed Aug. 5,1997; Ser. No. 60/064,996, filed Nov. 10, 1997; and by Steer & Roy-Chowdhury et al., Ser. No. 60/074,497, filed Feb. 12, 1998, entitled “Methods of Prophylaxis and Treatment by Alteration of APO B and APO E Genes.”
3. BRIEF DESCRIPTION OF THE FIGURES
FIG.
1
. An example of the conformation of a double hairpin type recombinagenic oligomer. The features are: a, first strand; b, second strand; c, first chain of the second strand; 1, 5′ most nucleobase; 2,3′ end nucleobase; 3,5′ end nucleobase; 4,3′ most nucleobase; 5, first terminal nucleobase; 6, second terminal nucleobase.
FIG.
2
. An example of the conformation of a single hairpin type recombinagenic nucleobase with an overhang. The features are as above with the addition of d, the overhang. Note that the same nucleobase is both the 5′ most nucleobase of the second strand and the 5′ end nucleobase.
4. DEFINITIONS
The invention is to be understood in accordance with the following definitions.
An oligonucleobase is a polymer of nucleobases, which polymer can hybridize by Watson-Crick base pairing to a DNA having the complementary sequence.
Nucleobases comprise a base, which is a purine, pyrimidine, or a derivative or analog thereof. Nucleobases include peptide nucleobases, the subunits of peptide nucleic acids, and morpholine nucleobases as well as nucleobases that contain a pentosefuranosyl moiety, e.g., an optionally substituted riboside or 2′-deoxyriboside. Nucleotides are pentosefuranosyl containing nucleobases that are linked by phosphodiesters. Other pentosefuranosyl containing nucleobases can be linked by substituted phosphodiesters, e.g., phosphorothioate or triesterified phosphates.
A oligonucleobase compound has a single 5′ and 3′ end nucleobase, which are the ultimate nucleobases of the polymer. Nucleobases are either deoxyribo-type or ribo-type. Ribo-type nucleobases are pentosefuranosyl containing nucleobases wherein the 2′ carbon is a methylene substituted with a hydroxyl, substituted oxygen or a halogen. Deoxyribo-type nucleobases are nucleobases other than ribo-type nucleobases and include all nucleobases that do not contain a pentosefuranosyl moiety, e.g., peptide nucleic acids.
An oligonucleobase strand generically includes regions or segments of oligonucleobase compounds that are hybridized to substantially all of the nucleobases of a complementary strand of equal length. An oligonucleobase strand has a 3′ terminal nucleobase and a 5′ terminal nucleobase. The 3′ terminal nucleobase of a strand hybridizes to the 5′ terminal nucleobase of the complementary strand. Two nucleobases of a strand are adjacent nucleobases if they are directly covalently linked or if they hybridize to nucleobases of the complementary strand that are directly covalently linked. An oligonucleobase strand may consist of linked nucleobases, wherein each nucleobase of the strand is covalently linked to the nucleobases adjacent to it. Alternatively a strand may be divided into two chains when two adjacent nucleobases are unlinked. The 5′ (or 3′) terminal nucleobase of a strand can be linked at its 5′-O (or 3′-O) to a linker, which linker is further linked to a 3′ (or 5′) terminus of a second oligonucleobase strand, which is complementary to the first strand, whereby the two strands form a single oligonucleobase compound. The linker can be an oligonucleotide, an oligonucleobase or other compound. The 5′-O and the 3′-O of a 5′ end and 3′ end nucleobase of an oligonucleobase compound can be substituted with a blocking group that protects the oligonucleobase strand. However, for example, closed circular olignucleotides do not contain 3′ or 5′ end nucleotides. Note that when an oligonucleobase compound contains a divided strand the 3′ and 5′ end nucle

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