Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-06-22
2001-06-12
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C530S350000
Reexamination Certificate
active
06245543
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of molecular biology of herpes simplex viruses and vaccine technology. More specifically, the present invention relates to a means of improving gene therapy for diseases such as cancer by mutating herpes simplex virus type 1 thymidine kinases and uses thereof.
2. Description of the Related Art
The herpes simplex virus thymidine kinases (HSV-TKs)
1
are the pharmacological targets of most herpesvirus treatments (1,2), and more recently, HSV-1 TK has been utilized as a suicide gene therapeutic for cancer in combination with ganciclovir (3,4). The basis for these uses is their ability to specifically phosphorylate anti-herpesvirus nucleoside drugs such as acyclovir (ACV), ganciclovir (GCV) and 5-bromovinyldeoxyuridine (BVDU) (1,2,5). This targeting is based primarily on the differences in substrate specificity compared to the cellular TKs. The HSV-1 TK has a much broader range of substrates which include most pyrimidine nucleosides, many guanosine derivatives (e.g., ACV or GCV), and most purine and pyrimidine nucleoside triphosphates (6-9). HSV-TK also possesses a thymidylate kinase (TMPK) activity, but this activity is restricted to only deoxypyrimidine monophosphate substrates (7-9).
Proteolytic mapping studies of HSV-1 TK with the photoactive TMP analog, [
32
P]5N
3
dUMP, identified a region of the thymine base binding site inclusive in the peptide Ile
112
-Tyr
132
(10). This report, and others (7,8,11,12), concluded that the thymine base of TMP and thymidine bind in one shared site. This was subsequently confirmed in comparisons of two X-ray crystal structures of HSV-1 TK with bound thymidine or TMP (13, 14). Two initial X-ray crystal structures of HSV-1 TK have been published (13,14), one with bound thymidine or ganciclovir (13) and the other with thymidine, 5-iodo-deoxyuridine monophosphate or a complex with TMP and ADP (14). Subsequent structures have been reported with bound acyclovir, penciclovir and other nucleoside drug substrates and inhibitors (15,16). Within the pyrimidine base binding site, all structures have indicated that hydrogen bonding between Gln-125 of HSV-1 TK and the N3 and O4 atoms of the pyrimidine base was evident (13-15). In the complex with ganciclovir or acyclovir, Gln-125 was shown to form hydrogen bonds with the N1 and O6 atoms of the guanine base of GCV (13,15,16).
The prior art is deficient in lack of improved mutants of herpes simplex virus type 1 thymidine kinases useful in treating cancers in gene therapy techniques so as to maximize therapeutic efficacy and minimize untoward side effects. Increasing and/or modifying the desired substrate specificity for HSV-thymidine kinase would ameliorate these side effects. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
It has been reported that site-directed mutagenesis of (Gln-125 to Glu, Leu or Asn can modulate the substrate affinities for thymidine and ACV in the context of HSV-1 TK in antiviral drug resistance (17). To examine the role of Gln-125 in HSV-1 TK activity in the context of gene therapeutic applications, three separate site-specific mutations were made of this residue to either an Asp, Asn or Glu acid residue. These three mutants and wild-type HSV-1 TK were expressed in
E. coli,
partially purified, and then were compared for their ability to phosphorylate deoxypyrimidine and acyclic purine substrates. For each mutation, the ability to phosphorylate deoxypyrimidine substrates were greatly modified, while activity for the acyclic purines was variable. Kinetic constants for thymidine and GCV were also determined. The molecular basis for the obtained results were evaluated using Flexidock molecular modeling simulations of the different enzyme active sites. The genes for each mutant HSV-1 TK were incorporated into a retroviral plasmid for expression in two mammalian cell lines and evaluation of sensitivity to GCV killing. The potential uses of these mutants in gene therapy applications and in the design of new HSV-1 TK proteins with different activities is discussed.
In one embodiment of the present invention, there is provided a mutant herpes simplex virus type 1 thymidine kinase protein with a site-specific mutation at amino acid position 125 of wild type herpes simplex virus type 1 thymidine kinase.
In another embodiment of the present invention, there is provided a vector comprising a DNA sequence coding for the mutant herpes simplex virus type 1 thymidine kinase protein disclosed herein, a promoter and optionally an origin of replication.
In still another embodiment of the present invention, there is provided a host cell transfected with the above disclosed vector.
In still yet another embodiment of the present invention, there is provided a method of killing target cells, comprising the steps of transfecting or transducing the target cells with a gene encoding a non-human mutant herpes simplex virus type 1 thymidine kinase and then contacting the transfected or transduced cells with an effective amount of a prodrug, wherein the prodrug is a substrate for the mutant herpes simplex virus type 1 thymidine kinase to yield a toxic substance, which inhibits cellular DNA polymerases and kills the transfected or transduced target cells.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.
REFERENCES:
McKnight, Nucleic Acids Research, 8(24), 5949-5964, Dec. 1980.*
L. Stryer, “Biochemistry”, P. 16-17, Freeman & Lo; San Francisco, Apr. 1975.*
Brown et al., Nature Structural Biology 12 (10), 876-881, Nov. 1995.
Compadre Cesar
Drake Richard
Hinds Trenton
Hurlburt Barry
Rechtin Tammy
Adler Benjamin Aaron
Monshipouri Maryam
Prouty Rebecca E.
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