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

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

Natalia Vapniarsky  1 , Michael Lame, Samantha McDonnel, Brian Murphy
Affiliations

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

Natalia Vapniarsky et al. PLoS One. 2012.

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. rfEPO sequence and schematic of the pLenti rfEPO vector.
The 7543 nucleotide pLenti rfEPO vector, derived from the HIV-1 based pLenti6/V5-D-TOPO (Invitrogen) is depicted schematically along with the cloned fEPO cDNA sequence. For the cDNA sequence, the start (ATG) and stop (TGA) codons are boxed while the 44th codon (GAG) is in a grey box. For the vector schematic, all of the promoter elements are light grey with an accompanying arrow. The 5′ HIV-1 LTR has a 229 nucleotide deletion in U3 replaced with the RSV enhancer/promoter (RSV prom/5′LTR). The 5′ splice donor (SD), splice acceptor (SA), packaging signal (Ψ), Rev response element (RRE), CMV promoter, feline erythopoietin cDNA (579 nucleotides), SV40 early promoter, EM7 promoter, Blasticidin resistance gene and the SV40 polyadenylation signal are depicted schematically. The 3′ HIV-1 LTR has a 52 nucleotide deletion in U3. The pUC origin and Ampicillin (bla) resistance gene/promoter are present in the vector backbone but are not depicted.
Figure 2
Figure 2. 293H Cells transfected with pGFP-EPO demonstrate abundant cytoplasmic fluorescence and GFP antigen expression.
a) In a merged image (DAPI/GFP channels), approximately 3% of 293H cells demonstrate positive green fluorescence (DAPI/GFP fluorescence microscopy, 20×magnification, scale bar = 50 µm). b) In a merged image, the transfected 293H cell cytoplasm is filled with numerous fluorescent granules (sequestration of the material into storage granules) (DAPI/GFP fluorescence microscopy, 100×magnification, scale bar = 10 µm). GFP fluorescence is not evident in the nucleus. c) GFP and GFP-EPO fusion protein-specific bands are evident in a Western blot analysis. A dark band is evident in lysates obtained from transfected cells at ∼50 kDa (GFP-EPO fusion protein, *, lane 1) and is not present in lysates from control cells (lane 2). GFP-specific degradation products that are between 25 and 37 kDa are evident in the lane derived from cells transfected with pGFP-EPO (**, lane 1) and not in the lane derived from the control cells (lane 2).
Figure 3
Figure 3. Supernatants of various cells infected with the lentiviral feEPO vector contain abundant FeEPO protein.
a) Supernatants collected from 293H cells at 6 days post infection were concentrated using centrifugal filters (lanes 7–9), or used unconcentrated (lanes 3–5). Lanes 3 and 7 represent uninfected control cells, while lanes 4 & 8 and 5 & 9 represent cells infected at an MOI of 1 and 10, respectively. Lane 1 is 10 U of commercial human EPO as a positive control; lanes 2 and 6 are blank. b) Supernatants collected from CRFK (lanes 2–7) and feline PBMC (9–14) at 3 (lanes 2–4, 9–11) and 6 (lanes 5–7, 12–14) days post infection. Lanes 2, 5, 9, & 12 represent uninfected control cells, while lanes 3, 6, 10, & 13 and 4, 7, 11, & 14 represent cells infected at an MOI of 1 and 10, respectively. Lane 1 is 10 U of commercial human EPO as a positive control; lane 8 is blank. Arrowheads: ∼34 kDa determined by molecular weight marker.
Figure 4
Figure 4. Supernatants of various cells infected with the lentiviral feEPO vector contain biologically active feEPO protein.
A) Standard curve generated by serial dilutions of commercial human EPO. MTT bioassay performed using the supernatants from B) 293H, C) CRFK, or D) feline PBMC infected (MOI 1 or 10) with the lentiviral fEPO vector (or uninfected controls) at B) 6 days or (C,D) 3 and 6 post infection. TF-1 cells treated with commercial human EPO (1 U/mL) served as positive controls for the assay, while TF-1 cells incubated in media without EPO (0 U/mL) served as negative controls. Media alone (Media) served as the reference for background absorbance. Error bars represent the standard deviation of triplicate well experiments; asterisks denote significantly different values based on one-way ANOVA with Tukey’s multiple comparison post-test (p<.05).
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Grants and funding

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.