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Compositions comprising genome segments and methods of using the same

a technology of genome segments and compositions, applied in the field of compositions and methods of treating individuals with diseases and disorders, can solve the problems of requiring additional fine and time-consuming research, being subject to mutational pressure, and unable to meet the needs of human body, etc., and achieve the effect of gene defects

Inactive Publication Date: 2007-09-27
PANAGENIC INT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009] The present invention arises from the surprising discovery that when an entire genome is administered to an individual in the form of a plurality of polynucleotide molecules that are fragments of genomic DNA, the polynucleotide molecules will circulate, be taken up by cells and translocate to the nucleus of the cells where they will recombine by homologous recombination with the genomic DNA of the cell. Accordingly, genetic defects can be corrected by administering an entire genome to an individual in the form of polynucleotide molecules that are fragments of genomic DNA that are free of the genetic defect. Individuals with diseases and disorders associated with genetic mutations can be effectively treated by the present invention.
[0015] The present invention relates to methods of preventing or reducing graft versus host disease in a recipient comprising the steps of administering to the donor an effective amount of a plurality of polynucleotide molecules that are free of vector sequences. The plurality of polynucleotide molecules collectively comprises an essentially complete genome of the recipient in polynucleotide molecules having about 100-3000 nucleotides. At least some of the plurality of polynucleotide molecules are taken up by the cells of the recipient, transported to the nucleus of the cell, and recombine with the genomic DNA of the cell by homologous recombination. The genomic DNA of the donor is replaced by genomic DNA of the recipient. Following transplantation / grafting, the level of graft versus host disease is reduced or prevented.

Problems solved by technology

But as the organism grows genomic DNA of every cell becomes subjected to mutational pressure due to environmental factors and mistakes of cellular replication and repair machinery.
When the damage is rather long and affects both DNA chains its repair becomes problematic and the damage results in mutation.
As mutations are accumulated in the cellular genome they can cause malfunction of some cells or cancerous transformation.
Because not a single mutation has been 100% proven to cause a cancer, the development of these methods requires additional fine and time-consuming research.
However the latter methods do not suggest fixing of already established or inherited mutations.

Method used

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  • Compositions comprising genome segments and methods of using the same
  • Compositions comprising genome segments and methods of using the same
  • Compositions comprising genome segments and methods of using the same

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparations of Human DNA in Cell Medium Change the Pattern of Specific Protein Expression in Human Cancer Cells in Culture

[0112] Example 1 demonstrates a capability of DNA preparations prepared from healthy donor sperm cells to influence differential expression of specific cell proteins in human tumor cell lines: breast tumor, BT474, ovarian carcinoma OVCAR5 and promielocytic leukemia HL60. The breast tumor cells are adhesive epithelial type cells forming a monolayer with multilayer cell clumps; OVCAR5 are fibroblastoid type cells forming a monolayer and HL60 cells are spherical cells that grow in suspension.

[0113] Two types of preparations were used: [0114] 1) purified total chromatin prepared from sperm cells and fragmented by nuclease digestion to oligonucleosome size (Wagner, et al. Arch. Androl. (1981) 7(3):251-7, which is incorporated herein by reference) (DNA lengths 200-3000 bp). [0115] 2) genomic DNA isolated from sperm cells (using Amersham-Pharmacia ‘genomic DNA isolat...

example 2

Introduction of Changes into Cellular Genomic DNA in Living Mice

Induction of Artificial Tolerance in Recipient Mice to a Donor Allograft

[0119] CBA and A / He are immunologically distinct mouse strains, and A / He allografts to CBA are never adopted but are always quickly rejected (Kaledin, et al. 1974 Conf. of the Res. Lab. of Exp. Biol. Models (Abstracts) Acad. Med. Sci, Moscow USSR p 5-52). The example demonstrates induction of immunological tolerance in CBA mice via injection of appropriate amounts of donor DNA preparations. The supposed mechanism of such induction is that injected donor DNA is substituting responsible alleles in CBA recipient cells to A / He specific alleles. This substitution will result in emergence of A / He antigens on CBA cells, which is probably followed by negative selection of lymphocyte clones normally responsible for the rejection.

[0120] A / He mouse strain specific hepatoma A, which is unable to grow in CBA mice mainly because of immunologic incompatibility,...

example 3

Random Human Genomic DNA Amplification in PCR Reaction

[0127] We used a random primer PCR procedure mainly according to Telenius et al (1992) supra with modifications. Telenius's 18-mer partially degenerated DOP 6-MW oligonucleotide primer was used for random DNA amplification. The typical protocol was as follows: PCR buffer with 0.2 mM MgCl2, 0.2 mM NTP, 2 μM DOP 6-MW, 40 U / ml Taq polymerase. Cycler: 95° C. 5′; 10″ 94° C., 10″ 53° C., 30″ 72° C. (the latter three steps are repeated 30 times), 10′ 72° C. Using, 1 ng of the DNA in a 50 μl reaction mixture we can get up to 10 μM DNA fragments 200-3000 bp in length. 1 liter mixtures yields 0.2 g DNA, 10 liter mixtures yields 2 g DNA. For 1 liter mixtures we need about 40,000 U Taq polymerase, 100 ml 2 mM NTP (100 mg NTP), 20 ml 100 μM DOP 6 M-W (2 μmol).

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Abstract

Methods of treating an individual who has a disease or disorder associated with one or more genetic mutations or undesirable alleles in genomic DNA of the individual, or preventing an individual from developing a disease or disorder associated with one or more genetic mutations or undesirable alleles in genomic DNA of the individual, by replacing a segment of genomic DNA that has a mutated sequence or undesirable allele with a corresponding segment of DNA that has a non-mutated sequence or desirable allele, are disclosed. Methods of inducing tolerance and preventing transplant rejection in a recipient, methods of inducing tolerance and or reducing allergies are disclosed and method of increasing fertility in a woman are disclosed. Each of the methods comprises replacing a segment of genomic DNA in an individual with a corresponding segment of DNA from another. An apparatus for doing large scale PCR preparations is disclosed. Pharmaceutical compositions that comprise a plurality of polynucleotide molecules which collectively comprise an essentially complete genome in polynucleotide molecules having about 100-3000 nucleotides are disclosed.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No. 60 / 174,155 filed Jan. 3, 2000, which is incorporated herein by reference.FIELD OF THE INVENTION [0002] The present invention relates to compositions and methods of treating individuals with diseases and disorders associated with genetic mutations. According to the present invention, DNA is administered to individuals who take it up and replace DNA with mutated sequences with DNA with non-mutated sequences or substitute existing set of alleles to a different one. The present invention also relates to compositions and methods for preventing and treating infections, allergies, transplant rejections and infertility. BACKGROUND OF THE INVENTION [0003] Every cell of an organism contains genomic DNA that keeps encoded information about all proteins of all cells of the organism and eventually about the whole organism and its development. Initially this genomic DNA is identical...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C12Q1/68C12P19/34
CPCA61K48/005A61K48/00
Inventor YAKUBOV, LEONID A.
Owner PANAGENIC INT
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