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. 2010 May-Jun;16(5-6):216-21.
doi: 10.2119/molmed.2009.00163. Epub 2010 Feb 17.

Alpha-galactosidase A-Tat fusion enhances storage reduction in hearts and kidneys of Fabry mice

Koji Higuchi  1 , Makoto Yoshimitsu, Xin Fan, Xiaoxin Guo, Vanessa I Rasaiah, Jennifer Yen, Chuwa Tei, Toshihiro Takenaka, Jeffrey A Medin
Affiliations

Alpha-galactosidase A-Tat fusion enhances storage reduction in hearts and kidneys of Fabry mice

Koji Higuchi et al. Mol Med. 2010 May-Jun.

Abstract

The protein transduction domain from human immunodeficiency virus (HIV) Tat allows proteins to penetrate the cell membrane. Enhanced cellular uptake of therapeutic proteins could benefit a number of disorders. This is especially true for lysosomal storage disorders (LSDs) where enzyme replacement therapy (ERT) and gene therapy have been developed. We developed a novel recombinant lentiviral vector (LV) that engineers expression of alpha-galactosidase A (alpha-gal A)-Tat fusion protein for correction of Fabry disease, the second-most prevalent LSD with manifestations in the brain, kidney and heart. In vitro experiments confirmed mannose-6-phosphate independent uptake of the fusion factor. Next, concentrated therapeutic LV was injected into neonatal Fabry mice. Analysis of tissues at 26 wks demonstrated similar alpha-gal A enzyme activities but enhanced globotriaosylceramide (Gb3) reduction in hearts and kidneys compared with the alpha-gal A LV control. This strategy might advance not only gene therapy for Fabry disease and other LSDs, but also ERT, especially for cardiac Fabry disease.

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Figures

Figure 1
Figure 1
Schematic of alignment of Tat PTD domain at the carboxy-terminus of α-galactosidase A.
Figure 2
Figure 2
Plasma α-gal A activity in high-dose LV-injected Fabry mice from Experiment 1. *P < 0.001 compared with PBS-injected group. #P < 0.05 compared with LV/α-gal A-Tat-injected group.
Figure 3
Figure 3
Plasma α-gal A activity in low-dose LV-injected Fabry mice from Experiment 2.
Figure 4
Figure 4
α-Gal A and Tat mRNA expression and α-gal A enzyme activity. (A) Similar levels of α-gal A and Tat mRNA expression in transduced 3T3 cells were confirmed by RT-PCR. (B) α-Gal A activity secreted from transduced HeLa cells. (C) Uptake of secreted α-gal A and α-gal A/Tat in Fabry mouse fibroblasts with or without M6P. NT, non-transduced; RT, reverse transcriptase; sup, supernatant; M6P, mannose-6-phosphate. α-Gal A activity assays were measured in triplicates. Values are mean ± SD.
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References

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