Long-Term Expression of the Biologically Active Growth Hormone in Genetically Modified Fibroblasts after Implantation into a Hypophysectomized Rat

Abstract

We employed the hypophysectomized rats as an animal model to explore the feasibility of using genetically engineered fibroblast cells for growth hormone gene therapy. An internal ribosome entry site (IRES)-directed bicistronic retroviral vector, PSN, which contained a porcine growth hormone (pGH) cDNA at the first cistron and a Neo(r) gene at the second cistron was used to infect primary rat embryo fibroblast (REF) cells. The infected cells (5 x 10(6) cells/rat) were injected directly into the peritoneum of syngeneic hypophysectomized rats. We demonstrate that the implanted PSN-infected REF cells could secrete biologically active pGH in vivo, leading to significant growth of the tibia at day 15 and day 57 post-implantation. We also treated the PSN-infected REF cells with collagen to form a tissue-like structure. The skin-like discs were grafted underneath the skin on the back of rats and cells were retrieved at different times. Using two criteria, semiquantitative reverse transcription-polymerase chain reaction on the pGH RNA extracted from the explants and G418 resistance conferred from the explanted cells, we demonstrate that pGH was expressed in the implanted fibroblasts up to 70 days. Despite the fact that the total pGH RNA level was reduced in the explants of long-time post-implantation, which was probably due to the reduction of transduced cells retained in the explants, the specific efficiencies of pGH RNA expression from these explants were maintained as high as the primary PSN-infected REF prior implantation. These results suggest that fibroblast cells are capable of expressing the foreign genes persistently in vivo.

Systemic action of human growth hormone following adenovirus-mediated gene transfer to rat submandibular glands

“…3 While the availability of recombinant human growth hormone (rhGH) has alleviated concerns about the coadministration of noxious contaminants with hGH purified from human cadaveric pituitaries, 4 there still exists dissatisfaction with daily rhGH injections. 3,[5][6][7][8][9] Gene transfer presents an attractive alternative for the delivery of therapeutic proteins to the bloodstream. Ideally, the gene of interest should be administered to a target tissue which is readily accessible and capable of delivering therapeutic levels of active circulating protein.…”

Different ex Vivo and Direct in Vivo DNA Administration Strategies for Growth Hormone Gene Therapy in Dwarf Animals

“…Fibroblasts are also a potentially interesting cell type for ex vivo gene delivery because of their easy accessibility, facile culture in vitro, convenient re-implantation and their potential for delivering proteins to the circulation. In 1995 a research group at the Taiwan National University employed hypophysectomized rats as an animal model to explore the feasibility of using genetically engineered fibroblasts for growth hormone gene therapy 7 . A bicistronic retroviral vector controlled by the LTR promoter, which contained a porcine growth hormone (pGH) cDNA at the first cistron and a neomycin resistance gene at the second cistron, was used to infect primary rat embryo fibroblasts.…”

New perspectives for gene therapy in endocrinology

“…A retroviral vector expressing the porcine GH cDNA was used to infect primary rat embryo ®broblasts. Transduced ®broblasts were injected into the peritoneum of syngeneic hypophysectomised rats (41). Implanted cells could secrete biologically active GH in vivo, leading to signi®cant skeletal growth up to 57 days postimplantation (41).…”