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 7

TAT-PNP PTD treatment for 24 weeks normalizes immune function and survival of PNP–/– mice.

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TAT-PNP PTD treatment for 24 weeks normalizes immune function and surviv...
Twenty-two PNP–/– mice were injected i.p. twice weekly with 0.5 U/g body wt of TAT-PNP for 24 weeks. Immune function and survival following treatment were not significantly different than those of normal control littermates. (A) Analysis of thymus populations was performed by flow cytometry. Single-cell suspensions from thymi were stained with PE- and FITC-conjugated anti-CD4 and anti-CD8 mAbs. The numbers of total and double-positive thymocytes in thymi of PNP–/– mice after treatment with TAT-PNP were not significantly different than in normal control littermates. (B) Function of T lymphocytes. T lymphocytes isolated from the spleen or lymph nodes were stimulated in vitro with anti-CD3. Response is expressed as stimulation indexes of the mean cpm with stimulation relative to that without stimulation. The stimulation indexes of lymphocytes isolated from PNP–/– mice after 24 weeks of TAT-PNP treatment were not significantly different from those of normal control littermates. (C) Survival of PNP–/– mice treated for 24 weeks with TAT-PNP PTD (n = 22) or a similar volume of PBS (n = 20) and of normal control littermates (n = 30) calculated by the Kaplan-Meier method. Survival of PNP–/– mice after 24 weeks of TAT-PNP treatment (77.3%) was similar to that of normal controls (93.3%) and significantly better than that of PBS-treated PNP–/– mice. #P < 0.001.