title: TAT-mediated intracellular delivery of purine nucleoside phosphorylase corrects its deficiency in mice.
abstract: Defects in purine nucleoside phosphorylase (PNP) enzyme activity result in abnormal nucleoside homeostasis, severe T cell immunodeficiency, neurological dysfunction, and early death. Protein transduction domain (PTD) can transfer molecules into cells and may help restore PNP activity in cases of PNP deficiency. However, long-term use of PTD to replace enzymes in animal models or patients has not previously been described. We fused human PNP to the HIV-TAT PTD and found that the fusion with TAT changed the retention and distribution of PNP in PNP-deficient mice. TAT induced rapid intracellular delivery of PNP into tissues, including the brain, prevented urinary excretion of PNP, and protected PNP from neutralizing antibodies, resulting in significant extension of the enzyme's biological activity in vivo. Frequent TAT-PNP injections in PNP-deficient mice corrected the metabolic disorder and immune defects with no apparent toxicity. TAT-PNP remained effective over 24 weeks of treatment, resulting in continued improvement in immune function and extended survival. Our data demonstrate that TAT changes the properties of PNP in vivo and that long-term intracellular delivery of PNP by TAT corrects PNP deficiency in mice. We provide evidence to promote further use of PTD to treat diseases that require repeated intracellular enzyme or protein delivery.
authors.affiliation: Infection, Immunity, Injury and Repair Program, Research Institute, Hospital for Sick Children and University of Toronto, Toronto, Ontario, Canada.
authors.forename: Ana
authors.initials: A
authors.lastname: Toro
authors.forename: Eyal
authors.initials: E
authors.lastname: Grunebaum
mesh_terms: D000818:Animals; D001835:Body Weight; D001921:Brain / Q000201:enzymology; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology; D002452:Cell Count; D015696:Gene Products, tat / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000201:enzymology; D008213:Lymphocyte Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010446:Peptide Fragments / Q000235:genetics*; D011683:Purine-Nucleoside Phosphorylase / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D011686:Purine-Pyrimidine Metabolism, Inborn Errors / Q000201:enzymology / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D013154:Spleen / Q000201:enzymology; D016019:Survival Analysis; D013950:Thymus Gland / Q000166:cytology / Q000201:enzymology; D014162:Transfection / Q000379:methods; D016896:Treatment Outcome; D014527:Uric Acid / Q000378:metabolism / Q000652:urine; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D015696:Gene Products, tat; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; C497137:tat peptide (47-57), Human immunodeficiency virus 1; D014527:Uric Acid; D011683:Purine-Nucleoside Phosphorylase