Designer gene therapy using an Escherichia coli purine nucleoside phosphorylase/prodrug system.

Academic Article

Abstract

  • 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.
  • Published In

    Keywords

  • Animals, Binding Sites, Cell Line, Tumor, Computer Simulation, Computer-Aided Design, Crystallography, X-Ray, Escherichia coli, Female, Genetic Therapy, Humans, Kinetics, Mice, Models, Molecular, Molecular Structure, Mutation, Neoplasms, Organ Specificity, Prodrugs, Protein Conformation, Protein Engineering, Purine-Nucleoside Phosphorylase, Substrate Specificity
  • Author List

  • Bennett EM; Anand R; Allan PW; Hassan AEA; Hong JS; Levasseur DN; McPherson DT; Parker WB; Secrist JA; Sorscher EJ
  • Start Page

  • 1173
  • End Page

  • 1181
  • Volume

  • 10
  • Issue

  • 12