The kinetics and tissue distribution of protein transduction in mice.
Protein transduction domains (PTDs) offer an exciting therapeutic opportunity for the treatment of many diseases. An 11-amino acid fragment of human immunodeficiency type 1 (HIV-1) TAT-protein can transduce large, biologically active proteins into mammalian cells; recent evidence has shown an in vivo PTD for the 116 kDa beta-galactosidase protein. However, there is little information on the in vivo distribution of the TAT fusion protein to define the viability of PTDs for human studies. In this study we examined the tissue kinetics and tissue distribution of the PTD-transduced TAT fusion protein in mice. Low (100 microg) or high (500 microg) doses of TAT-beta-galactosidase fusion protein were administrated to mice through four routes (portal vein, i.v., i.p., and oral). Tissues were harvested 15 min, 1h, 6h, 10h, and 24h after treatment. Distribution of beta-galactosidase in various tissues was analysed by in situ staining, enzymatic activity assay, and Western blot analysis. Beta-galactosidase enzyme activity was observed in all tissues (liver, kidney, spleen, lung, bowel, and brain). Beta-galactosidase activity peaked at 15 min in most tissues after portal vein, i.v., and i.p. administration and at 1h after oral dosing in all tissues. Beta-galactosidase activity in the liver at 15 min after portal vein injection (67 milliunits [mU]/mg) was higher than after i.v. (9.8 mU/mg), i.p. (4.4 mU/mg), and oral (0.3 mU/mg) dosing. In situ staining and Western blot results correlated closely with beta-galactosidase enzyme activity assay. The median initial half-life for activity was 2.2h, ranging from 1.2h to 3.4h (coefficient of variation=28.9%). The bioavailability of beta-galactosidase activity after an orally administered PTD was 24%. This study details the kinetics and tissue distribution of delivering of a model TAT fusion protein into the mouse via PTD. These data allow rational selection of delivery route and schedules for therapeutic PTD and will aid the use of TAT fusion protein transduction in the development of protein therapies.
Cai
Shi-Rong
SR
Department of Medicine, The Siteman Cancer Center, The Howard Hughes Medical Institute, Washington University School of Medicine, Room 1021 CSRB NT, 660 South Euclid Avenue, Campus Box 8069, St. Louis, MO 63110, USA.
Xu
Guang
G
Becker-Hapak
Michelle
M
Ma
Margaret
M
Dowdy
Steven F
SF
McLeod
Howard L
HL
D000284:Administration, Oral; D000818:Animals; D001682:Biological Availability; D016503:Drug Delivery Systems; D015696:Gene Products, tat / Q000008:administration & dosage / Q000235:genetics / Q000493:pharmacokinetics*; D006207:Half-Life; D006801:Humans; D007274:Injections, Intraperitoneal; D007275:Injections, Intravenous; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000008:administration & dosage / Q000235:genetics / Q000493:pharmacokinetics*; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000096:biosynthesis* / Q000493:pharmacokinetics*; D014018:Tissue Distribution; D014161:Transduction, Genetic; D001616:beta-Galactosidase / Q000008:administration & dosage / Q000235:genetics / Q000493:pharmacokinetics*; D054322:tat Gene Products, Human Immunodeficiency Virus
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; D001616:beta-Galactosidase