Designer gene therapy using an Escherichia coli purine nucleoside phosphorylase/prodrug system.
Activation of prodrugs by Escherichia coli purine nucleoside phosphorylase (PNP) provides a method for selectively killing tumor cells expressing a transfected PNP gene. This gene therapy approach requires matching a prodrug and a known enzymatic activity present only in tumor cells. The specificity of the method relies on avoiding prodrug cleavage by enzymes already present in the host cells or the intestinal flora. Using crystallographic and computer modeling methods as guides, we have redesigned E. coli PNP to cleave new prodrug substrates more efficiently than does the wild-type enzyme. In particular, the M64V PNP mutant cleaves 9-(6-deoxy-alpha-L-talofuranosyl)-6-methylpurine with a kcat/Km over 100 times greater than for native E. coli PNP. In a xenograft tumor experiment, this compound caused regression of tumors expressing the M64V PNP gene.
Bennett
Eric M
EM
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
Anand
Ruchi
R
Allan
Paula W
PW
Hassan
Abdalla E A
AE
Hong
Jeong S
JS
Levasseur
Dana N
DN
McPherson
David T
DT
Parker
William B
WB
Secrist
John A
JA
Sorscher
Eric J
EJ
Townes
Tim M
TM
Waud
William R
WR
Ealick
Steven E
SE
D000818:Animals; D001665:Binding Sites; D045744:Cell Line, Tumor; D003198:Computer Simulation; D017076:Computer-Aided Design; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D009928:Organ Specificity; D011355:Prodrugs / Q000009:adverse effects / Q000378:metabolism* / Q000627:therapeutic use*; D011487:Protein Conformation; D015202:Protein Engineering; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D011355:Prodrugs; D011683:Purine-Nucleoside Phosphorylase