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.
View: Text | PDF
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

×

Figure 6

Treatment of PNP–/– mice with TAT-PNP prevents abnormalities in thymus and T lymphocyte number and function.

Options: View larger image (or click on image) Download as PowerPoint
Treatment of PNP–/– mice with TAT-PNP prevents abnormalities in thymus a...
Immune function of PNP–/– mice following 12 weeks of treatment with 0.5 U/g body wt of TAT-PNP or nonfused PNP was compared with that of age-adjusted healthy control littermates. (A) TAT-PNP treatment significantly increased the absolute number of thymocytes in the thymus of PNP–/– mice. (B) Analysis of single-cell suspensions from thymus stained with PE- and FITC-conjugated anti-CD4 and anti-CD8 mAbs. Isotype-matched PE- or FITC-labeled antibodies were used as negative controls. Cells expressed CD4+, CD8+, or CD4+CD8+ (double positive; DP) or showed no expression (double negative; DN). TAT-PNP treatment significantly improved the number of DP thymocytes in PNP–/– mice. (C) Analysis of the percentage of T cells in the spleen by flow cytometry. Nylon wool single-cell suspensions were stained with PE- and FITC-conjugated anti-CD3 and anti-CD19 mAbs. Isotype-matched PE- or FITC-labeled Abs were used as negative controls. Treatment with TAT-PNP significantly increased the percentage of T lymphocytes in the spleen of PNP–/– mice. (D) T lymphocyte function analysis. T lymphocytes were stimulated in vitro with anti-CD3. Proliferation is expressed as stimulation indexes (SI) of the mean cpm with stimulation divided by the mean cpm without stimulation. TAT-PNP treatment significantly increased the response of T lymphocytes in the spleen of PNP–/– mice. Results are mean ± SD of 5–10 mice. *P < 0.05.