Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-03-14
2003-05-27
Reynolds, Deborah J. (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S455000, C435S462000
Reexamination Certificate
active
06569681
ABSTRACT:
BACKGROUND OF THE INVENTION
Current approaches to treating disease by administering therapeutic proteins include in vitro production of therapeutic proteins for conventional pharmaceutical delivery (e.g. intravenous, subcutaneous, or intramuscular injection) and, more recently, gene therapy.
Proteins of therapeutic interest can be produced by introducing exogenous DNA encoding the protein of therapeutic interest into appropriate cells. For example, a vector which includes exogenous DNA encoding a therapeutic protein can be introduced into cells and the encoded protein expressed. It has also been suggested that endogenous cellular genes and their expression may be modified by gene targeting. See for example, U.S. Pat. Nos. 5,272,071, 5,641,670, WO 91/06666, WO 91/06667 and WO 90/11354.
SUMMARY OF THE INVENTION
The invention is based, in part, on the use of homologous recombination between a double stranded DNA sequence and a selected target DNA, e.g., chromosomal DNA in a cell, promoted by providing an agent which enhances homologous recombination, e.g., Rad52, and an agent which inhibits non-homologous end joining, e.g., a Ku inactivating agent, in sufficiently close proximity to the DNA sequence at the targeted site. It is predicted that a higher rate of homologous recombination occurred in the presence of both Rad52 and a Ku inactivating agent than in their absence. In addition, it is predicted that gene targeting aimed at altering a targeted site in a DNA, e.g., a targeted site in the chromosomal DNA in a cell, using a selected DNA sequence as a template can be promoted by providing a Rad52 protein and a Ku inactivating agent, e.g., an anti-Ku antibody. By providing a Rad52 protein and a Ku inactivating agent in close proximity to the selected DNA sequence and the target site, a higher rate of alteration by gene targeting occurs than in the absence of a Rad52 protein and a Ku inactivating agent, e.g., an anti-Ku antibody.
Accordingly, in one aspect, the invention features, a method of promoting an alteration at a selected site in a target DNA, e.g., in the chromosomal DNA of a cell. The method includes providing, at the site: (a) a double stranded DNA sequence which includes a selected DNA sequence; (b) an agent which enhances homologous recombination, e.g., a Rad52 protein or a functional fragment thereof, or a DNA sequence which encodes Rad52 or a functional fragment thereof; and (c) an agent which inhibits non-homologous end joining, e.g., an agent which inactivates Ku, and allowing the alteration to occur. In a preferred embodiment, components (a), (b), and (c) are provided, e.g., introduced into the cell, such that, at the site of an interaction between the selected DNA sequence and the target DNA, the concentration of the agent which enhances homologous recombination and of the agent which inhibits non-homologous end joining are sufficient that an alteration of the site, e.g., homologous recombination or gene correction between the selected DNA sequence and the target DNA, occurs at a higher rate than would occur in the absence of the supplied agent which enhances homologous recombination and the agent which inhibits non-homologous end joining. The agent which inhibits non-homologous end joining is preferably provided locally. Preferably the agent which inhibits non-homologous end joining is a Ku inactivating agent such as an anti-Ku antibody.
Components (a), (b), and (c) can be introduced together, which is preferred, or separately. In addition, two of the components can be introduced together and the third can be introduced separately. For example, the DNA sequence and the agent which enhances homologous recombination, e.g., Rad52, can be introduced together or the DNA sequence and the agent which inhibits non-homologous end joining, e.g., a Ku inactivating agent, can be introduced together. In another preferred embodiment, the agent which enhances homologous recombination and the agent which inhibits non-homologous end joining can be introduced together.
Two, or preferably all, of the components can be provided as a complex. In a preferred embodiment, the method includes contacting the target DNA, e.g., by introducing into the cell, a complex which includes: (a) a double stranded DNA sequence which includes the selected DNA sequence; (b) an agent which enhances homologous recombination, e.g., a Rad52 protein or functional fragment thereof; and (c) an agent which inhibits non-homologous end joining, e.g., a Ku inactivating agent such as an anti-Ku antibody or a Ku-binding oligomer or polymer.
In a preferred embodiment, one, or more, preferably all of the components are provided by local delivery, e.g., microinjection, and are not expressed from the target genome or another nucleic acid. In a particularly preferred embodiment, the agent which inhibits non-homologous end joining, e.g., a Ku inhibiting agent, is provided by local delivery, e.g., microinjection, and is not expressed from the target genome or another nucleic acid.
In a preferred embodiment, the agent which inhibits non-homologous end joining is: an agent which inactivates hMre11, e.g., an anti-hMre11 antibody or a hMre11-binding oligomer or polymer; an agent which inactivates hRad50, e.g., an anti-hRad50 antibody or a hRad50-binding oligomer or polymer; an agent which inactivates Nbs1, e.g., an anti-Nbs1 antibody or a hNbs1-binding oligomer or polymer; an agent which inactivates human ligase 4 (hLig4), e.g., an anti-hLig4 antibody or a hLig4-binding oligomer or polymer; an agent which inactivates hXrcc4, e.g., an anti-hXrcc4 antibody or a hXrcc4-binding oligomer or polymer; an agent which inactivates a human homolog of Rap1, e.g., an antibody to a human homolog of Rap1 or an oligomer or polymer which binds a human homolog of Rap1; an agent which inactivates a human homolog of Sir2304, e.g., an antibody to a human homolog of Sir2304 or an oligomer or polymer which binds a human homolog of Sir2304; an agent which inactivates Ku, e.g., an anti-Ku antibody or a Ku-binding oligomer or polymer. Any of the agents which inhibit non-homologous end joining can be administered alone or can be administered in combination with one or more of the other agents which inhibit non-homologous end joining.
In a preferred embodiment, the DNA sequence is a linear DNA sequence. In a preferred embodiment, the linear DNA sequence can have one or more single stranded overhang(s).
In a preferred embodiment, the selected DNA sequence is flanked by a targeting sequence. The targeting sequence is homologous to the target, e.g., homologous to DNA adjacent to the site where the target DNA is to be altered or to the site where the selected DNA sequence is to be integrated. Such flanking sequence can be present at one or more, preferably both ends of the selected DNA sequence. If two flanking sequences are present, one should be homologous with a first region of the target and the other should be homologous to a second region of the target.
In a preferred embodiment, the DNA sequence has one or more protruding single stranded end, e.g., one or both of the protruding ends are 3′ ends or 5′ ends.
In a preferred embodiment, the agent which enhances homologous recombination is: a Rad52 protein or a functional fragment thereof; a Rad51 protein or a functional fragment thereof; a Rad54 protein or a functional fragment thereof; or a combination thereof.
In a preferred embodiment, the agent which enhances homologous recombination is adhered to, e.g., coated on, the DNA sequence. In a preferred embodiment, the Rad52 protein or functional fragment thereof is adhered to, e.g., coated on, the DNA sequence.
In a preferred embodiment, the Rad52 protein or fragment thereof is human Rad52 (hRad52).
In a preferred embodiment, the anti-Ku antibody is: an anti-Ku70 antibody; an anti-Ku80 antibody. In a preferred embodiment, the anti-Ku antibody is: a humanized antibody; a human antibody; an antibody fragment, e.g., a Fab, Fab′, F(ab′)
2
or F(v) fragment.
In a preferred embodiment, at least one anti-Ku antibody is covalently linked
Bertoglio Valarie
Fish & Richardson PC
Reynolds Deborah J.
Transkaryotic Therapies Inc.
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