Gene therapy for long-term restoration of circulating thymulin in thymectomized mice and rats.
Thymulin is a thymic peptide possessing hypophysiotropic activity and antiinflammatory effects in the brain. We constructed a synthetic DNA sequence encoding met-FTS, a biologically active analog of thymulin, and subsequently cloned it into different expression vectors. A sequence optimized for expression of met-FTS in rodents, 5'-ATGCAGGCCAAGTCGCAGGGGGGGTCGAACTAGTAG-3', was cloned in the mammalian expression vectors pCDNA3.1(+) and phMGFP (which expresses the Monster Green Fluorescent Protein), thus obtaining pcDNA3.1-metFTS and p-metFTS-hMGFP, which express met-FTS and the fluorescent fusion protein metFTS-hMGFP, respectively. The synthetic sequence was also used to construct the adenoviral vector RAd-metFTS, which expresses met-FTS. Transfection of HEK293 and BHK cells with pcDNA3.1-metFTS (experimental groups) or pcDNA3.1 (control), led to high levels of thymulin bioactivity (>600 versus <0.1 pg/ml in experimental and control supernatants, respectively). Transfection of HEK293 and BHK cells with pmetFTS-hMGFP revealed a cytoplasmic and nuclear distribution of the fluorescent fusion protein. A single intramuscular (i.m.) injection (10(7) plaque forming units (PFU)/mouse or 10(8) PFU/rat) of RAd-metFTS in thymectomized animals (nondetectable serum thymulin) restored serum thymulin levels for at least 110 and 130 days post-injection in mice and rats, respectively. We conclude that RAd-metFTS constitutes a suitable biotechnological tool for the implementation of thymulin gene therapy in animal models of chronic brain inflammation.
Reggiani
P C
PC
Institute for Biochemical Research-Histology B, Faculty of Medicine, National University of La Plata, La Plata, Argentina.
Hereñú
C B
CB
Rimoldi
O J
OJ
Brown
O A
OA
Pléau
J-M
JM
Dardenne
M
M
Goya
R G
RG
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D006224:Cricetinae; D003593:Cytoplasm / Q000737:chemistry; D004660:Encephalitis / Q000097:blood / Q000628:therapy*; D005260:Female; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D013934:Thymectomy; D013935:Thymic Factor, Circulating / Q000032:analysis* / Q000235:genetics; D013997:Time Factors
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013935:Thymic Factor, Circulating