TAT-mediated intracellular delivery of purine nucleoside phosphorylase corrects its deficiency in mice
Ana Toro, Eyal Grunebaum
Ana Toro, Eyal Grunebaum
Published October 2, 2006
Citation Information: J Clin Invest. 2006;116(10):2717-2726. https://doi.org/10.1172/JCI25052.
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Research Article Immunology

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

Ana Toro, Eyal Grunebaum

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Figure 5

Anti-PNP antibodies neutralize TAT-PNP but do not prevent intracellular TAT-PNP transduction.

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Anti-PNP antibodies neutralize TAT-PNP but do not prevent intracellular ...
(A) Serum from PNP–/– mice treated with TAT-PNP, which contained antibodies to PNP that neutralized PNP enzyme activity, yet TAT-PNP still delivered active enzyme into PNP-deficient cells. Enzyme activity (14C-labeled inosine conversion) was measured after 20 minutes’ incubation of TAT-PNP or nonfused PNP, both at 0.1 U/ml, or PBS in the absence or presence of serum containing antibodies to TAT-PNP (diluted 1:100). Following similar incubation in the absence or presence of 2 × 105 PNP-deficient cells, activity was measured in the cell pellets. Results are representative of 3 independent experiments and are presented relative to the activity in normal control cells. **P < 0.005. (B) In spite of increasing concentrations of antibodies to TAT-PNP during treatment of PNP–/– mice with TAT-PNP, uric acid production (which is dependent on active PNP in vivo) was not affected. Twenty-four hours or later after injecting PNP–/– mice with TAT-PNP (squares), uric acid concentrations were measured in the urine and compared with uric acid from PNP–/– mice treated with nonfused PNP (triangles). Results (converted to μmol/l) are mean ± SD of urine samples collected from 6–15 mice and are compared with normal control littermates.