A lentiviral gene therapy strategy for the in vitro production of feline erythropoietin

PLoS One. 2012;7(9):e45099. doi: 10.1371/journal.pone.0045099. Epub 2012 Sep 18.

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 a potential in vivo application of a lentiviral gene therapy system.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Blotting, Western
  • Cats
  • Cell Line
  • DNA, Complementary / genetics
  • Erythropoietin / biosynthesis*
  • Erythropoietin / genetics
  • Genetic Therapy / methods*
  • Genetic Vectors / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Lentivirus / genetics*
  • Leukocytes, Mononuclear / metabolism
  • Leukocytes, Mononuclear / virology
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Real-Time Polymerase Chain Reaction
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics

Substances

  • DNA, Complementary
  • Recombinant Proteins
  • Erythropoietin
  • Green Fluorescent Proteins

Grant support

The work was supported by the following: Morris Animal Foundation http://www.morrisanimalfoundation.org award #D12FE803; and Winn Feline Foundation http://www.winnfelinehealth.org award #W11-035 and matched by Center of Companion Animal Health http://www.vetmed.ucdavis.edu/ccah/index.cfm. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Individuals employed or contracted by the funders played no any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.