Differential inactivation of nucleic acids by chemical modification
AssigneeHuman Gene Therapy Research Institute
InventorsSuming Wang, Charles J. Link, Jr.
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Abstract
Disclosed herein is the method for dramatically reducing cytotoxicity of viral vectors such as Herpes simplex viral while retaining gene expression. The method of the invention virtually eliminates the concern of possible recombination during virus propagation and contamination of wild-type virus and virus stock. The invention comprises use of photochemical crosslinking causing differential inactivation of viruses.
Claims21
A method of reducing cytotoxicity while retaining gene expression of Herpes Simplex Virus (HSV) vectors comprising:
- treating a plurality of said HSV vectors with an effective amount of a psoralen compound and an effective amount of ultraviolet (UV) radiation, so that random crosslink lesions are formed causing inhibition of replication while gene expression is retained.
A method of inhibiting replication of a viral vector stock while retaining gene expression, comprising:
- a) determining an effective amount of a nucleic acid damaging agent which induces random crosslink lesions, said agent comprising a crosslinking agent and an effective amount of UV radiation, which inhibits replication while retaining gene expression of a viral vector as characterized by assaying for the presence of a reporter gene product and assaying for cell lysis, and thereafter;
- b) treating said viral vector stock with said effective amount of said crosslinking agent, and;
- c) further treating said viral vector stock with said effective amount of UV radiation.
A viral vector stock with decreased cytotoxicity produced by the method of:
- treating said viral vector stock with an effective amount of a crosslinking agent, and thereafter exposing said vector to ultraviolet (UV) radiation so that random crosslink lesions are formed.
A viral vector stock with decreased cytotoxicity and retained gene expression produced by a method of random crosslink lesions which inhibit replication of Herpes Simplex Virus (HSV) vector stock while retaining gene expression, comprising:
- a) treating a plurality of HSV vectors with about 0.1 .mu.g/ml 4,5',8-trimethyl psoralen;
- b) further treating said plurality of HSV vectors with ultraviolet-A (UVA) at a wavelength between about 320 nm and 400 nm.
A method of inhibiting replication of a viral vector stock while retaining gene expression, comprising:
- a) determining an effective amount of a nucleic acid damaging agent which induces random crosslink lesions, thus inhibiting replication while retaining gene expression of a viral vector as characterized by assaying for the presence of a reporter gene product and assaying for cell lysis, and thereafter;
- b) treating said viral vector stock with said effective amount of said crosslinking agent, and;
- c) further treating said viral vector stock with said effective amount of UV radiation.
Dependencies are inferred from the claim text.