Experimental parathyroid hormone gene therapy using ØC31 integrase.
ØC31 integrase can integrate targeted plasmid DNA into preferred locations in mammalian genomes, resulting in robust, long-term expression of the integrated transgene. This system represents an effective tool that opens up promising possibilities for gene therapy. The classical treatment for hypoparathyroidism was calcium and vitamin D replacement. Recently, parathyroid hormone (PTH) replacement was reported to be a more potentially physiologic treatment option. However, PTH synthesis is technically difficult and costly. These issues may be minimized by using PTH gene therapy. We attempted to achieve site-specific genomic integration of the PTH gene into a human cell line and mice using this system. We cotransfected 293 HEK cells with PTH-attB plasmid with or without ØC31 integrase plasmid. Expression and secretion of PTH into culture supernatants and site-specific genomic integration of PTH cDNA were assessed by immunoradiometric assays and pseudo-site analysis, respectively. In in vivo experiments, we injected the PTH-attB plasmid with or without ØC31 integrase plasmid into a mouse tail vein using the hydrodynamic method. Plasma PTH concentrations were serially measured, and site-specific integration of PTH cDNA into the mouse genome was confirmed by examining hepatic genomic DNA. PTH was expressed and secreted from 293 HEK cells and mouse hepatocytes, and pseudo-site analysis confirmed the site-specific integration of PTH cDNA into the host genomes. The site-specificity and efficiency of this system are advantageous in many areas, including, potentially, gene therapy. PTH gene therapy is one candidate; however, for clinical applications, we need to regulate PTH expression and secretion in the future.
Lee
Sihoon
S
Department of Internal Medicine, Graduate School of Medicine, Gachon University of Medicine and Science, Namdong-gu, Incheon, Korea.
Hong
Soon Won
SW
Choi
Han Seok
HS
Lee
Lee Young
LY
Nam
Chunja
C
Rhee
Yumie
Y
Chung
Ung-il
UI
Lim
Sung-Kil
SK
D000818:Animals; D001287:Attachment Sites, Microbiological / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007011:Hypoparathyroidism / Q000097:blood / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D019426:Integrases / Q000235:genetics* / Q000502:physiology; D008099:Liver / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008962:Models, Theoretical; D016254:Mutagenesis, Insertional / Q000379:methods; D010281:Parathyroid Hormone / Q000032:analysis / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D016896:Treatment Outcome
D003470:Culture Media; D010281:Parathyroid Hormone; D019426:Integrases