a lentiviral gene therapy strategy for the in vitro production of feline erythropoietin慢病毒基因治疗策略的体外生产猫科动物红细胞生成素.pdfVIP

A Lentiviral Gene Therapy Strategy for the In Vitro Production of Feline Erythropoietin 1 2 3 1 Natalia Vapniarsky *, Michael Lame , Samantha McDonnel , Brian Murphy 1 Department of Pathology, Microbiology, Immunology, University of California Davis, Davis, California, United States of America, 2 Department of Molecular Biosciences, University of California Davis, Davis, California, United States of America, 3 Graduate Group of Comparative Pathology, University of California Davis, Davis, California, United States of America Abstract Nonregenerative anemia due to chronic renal failure is a common problem in domestic cats. Unfortunately, administration of recombinant human erythropoietin often only improves anemia temporarily due to antibody development. In this in vitro study, feline erythropoietin cDNA was cloned from feline renal tissue and utilized in the construction of a replication- defective lentiviral vector. The native recombinant feline erythropoietin (rfEPO) sequence was confirmed by sequencing. Upon viral vector infection of human 293H cells, Crandall Renal Feline Kidney cell line and primary feline peripheral blood mononuclear cells, bioactive rfEPO protein was produced. The presence of cellular rfEPO cDNA was confirmed by standard PCR, production of abundant rfEPO mRNA was confirmed by real-time PCR, and secretion of rfEPO protein was demonstrated by Western blot analyses, while rfEPO protein bioactivity was confirmed via an MTT proliferation bioassay. This in vitro study demonstrates the feasibility of a replication-defective lentiviral vector delivery system for the in vitro production of biologically active feline erythropoietin. Anemic cats with chronic renal failure represent