title: Intracellular delivery of purine nucleoside phosphorylase (PNP) fused to protein transduction domain corrects PNP deficiency in vitro.
abstract: Purine nucleoside phosphorylase (PNP) is an intracellular enzyme crucial for purine degradation. PNP defects result in metabolic abnormalities and fatal T cell immunodeficiency. Protein transduction domains (PTD) transfer molecules across biological membranes. We hypothesized that fusion of PTD to PNP (PTD-PNP) would be an effective method for treating PNP deficiency. We find that PTD-PNP rapidly enters PNP-deficient lymphocytes and increases intracellular enzyme activity for 96 h. Similar to endogenous PNP, PTD-PNP is predominantly distributed in the cytoplasm. PTD-PNP improve viability and correct abnormal functions of PNP-deficient T lymphocytes including their response to stimulation and IL-2 secretion. Intracellular transduction protects PTD-PNP from antibody neutralization and from elimination, which may also provide significant in vivo therapeutic advantages to PNP. In conclusion, PTD fusion is an attractive method for extended PNP intracellular enzyme replacement therapy for PNP-deficient patients as well as for the intracellular delivery of other proteins.
authors.affiliation: Infection, Immunity, Injury and Repair Program, Research Institute, Hospital for Sick Children, The University of Toronto, Toronto, Ont., Canada M5G 1X8.
authors.forename: Ana
authors.initials: A
authors.lastname: Toro
authors.forename: Melissa
authors.initials: M
authors.lastname: Paiva
authors.forename: Cameron
authors.initials: C
authors.lastname: Ackerley
authors.forename: Eyal
authors.initials: E
authors.lastname: Grunebaum
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites / Q000235:genetics; D002470:Cell Survival; D015696:Gene Products, tat / Q000235:genetics; D006801:Humans; D007376:Interleukin-2 / Q000378:metabolism; D008213:Lymphocyte Activation / Q000235:genetics; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D008661:Metabolism, Inborn Errors / Q000097:blood* / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016253:Microscopy, Immunoelectron; D011683:Purine-Nucleoside Phosphorylase / Q000172:deficiency / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D014162:Transfection / Q000379:methods
chemical_list: D000906:Antibodies; D015696:Gene Products, tat; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D011683:Purine-Nucleoside Phosphorylase