Gene therapy for long-term restoration of circulating thymulin in thymectomized mice and rats

Abstract

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 0 -ATGCAGGCCAAGTCGCAGGGGGGGTCGAACTAGTAG-3 0 , 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 (4600 versus o0.1 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.

Thymulin gene therapy prevents the reduction in circulating gonadotropins induced by thymulin deficiency in mice

“…This sequence was used to construct RAd-metFTS, an adenoviral vector able to achieve long-term restoration of circulating thymulin levels when intramuscularly injected in thymectomized rats and mice (25). Adenovirally delivered metFTS in the brain of immunocompetent rats showed a much longer expression than adenovirally delivered GFP and β-galactosidase (21).…”

“…Since the gene for native FTS has not been cloned, we constructed an artificial DNA sequence, optimized for expression in rat systems, which codes for a biologically active analog of FTS, metFTS (25). This sequence was used to construct RAd-metFTS, an adenoviral vector able to achieve long-term restoration of circulating thymulin levels when intramuscularly injected in thymectomized rats and mice (25).…”

“…Serum values were expressed as femtograms per milliliter of bioactive thymulin. This bioassay has been previously validated against an ELISA for thymulin (25).…”

Neuropeptides, Cytokines and Thymus Peptides as Effectors of Interactions Between Thymus and Neuroendocrine System

“…Процессы влияния других пептидов, продуцируемых различными клетками, так-же сопряжены друг с другом, и для большинства из них трудно выделить влияние пептидов только тимического происхождения. Единственный гормон, воздействия ко-торого на процессы в ЦНС являются специфичными для тимуса, -это тимулин: его уровень резко снижается или исчезает после тимэктомии [47].…”

The Thymulin-Lactotropic Axis in Rodents: Thymectomy, Immunoneutralization and Gene Transfer Studies

“…To this end we constructed a synthetic DNA sequence coding for a biologically active FTS analog called metFTS and cloned it in an adenoviral vector [17]. With this vector we assessed the effect of neonatal thymulin gene therapy on the thymus-lactotropic axis in congenitally athymic nude mice as well as the effect of thymulin gene therapy in adult female rats.…”