1. Volume 19, Issue 2, Pages 302-309 (February 2011)
Lentiviral Vector Platform for Production of Bioengineered Recombinant Coagulation Factor VIII 
H Trent Spencer, Gabriela Denning, Richard E Gautney, Boro Dropulic, Andre J Roy, Lajos Baranyi, Bagirath Gangadharan, Ernest T Parker, Pete Lollar, Christopher B Doering 
Molecular Therapy 
Volume 19, Issue 2, Pages (February 2011)
DOI: /mt
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Design of ET-801i and the lentiviral production platform. (a) Design of ET-801i. The percent amino acid identities to B-domain-deleted recombinant human fVIII (h-fVIII) are 100, 90, and 88, respectively for constructs 2, 3, and 4. FVIII domain boundaries were defined using the human fVIII amino acid sequence numbering21 as follows: residues 1–372 (A1), 373–740 (A2), 1,649–1,689 (ap), 1,690–2,019 (A3), 2,020–2,172 (C1), and 2,173–2,332 (C2). (b) Schematic representation of the ET-801i transgene in the LentiMax expression vector. ET-801i was cloned into the LentiMax expression vector and recombinant lentivirus vector encoding the ET-801i transgene under the control of the elongation factor 1-α (EF-1α) promoter was generated using the LentiMax production system. cPPT, central polypurine tract; CTS, central termination sequence; LTR, long-terminal repeat; RRE, Rev-responsive element; WPRE, woodchuck hepatitis post-transcriptional regulatory element.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
ET-801i production following lentiviral transduction. (a) Serial transduction of baby hamster kidney–derived (BHK-M) cells using ET-801i lentivirus was performed at a multiplicity of infection (MOI) of 5 for transductions 1–3, MOI of 10 for transduction 4, and MOI of 20 thereafter. After each transduction, fVIII production was assessed using a one-stage coagulation assay (gray bars) and proviral copy number determined by quantitative PCR (black bars). Lenti-GFP, a LentiMax lentiviral vector expressing the green fluorescent reporter protein under the control of the elongation factor-1α (EF-1α) promoter, was used as a negative control. (b) ET-801i expression from individual BHK-M clones was studied by limiting dilution of the mixed cell population post-transduction and determination of fVIII production by one-stage coagulation assay. Error bars represent one sample standard deviation. GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Clonal analysis of BHK-M cells expressing ET-801i. A total of 26 clones were analyzed for transgene copy number and transcript levels. (a) FVIII activity versus mRNA transcript level, (b) fVIII activity versus transgene copy number, and (c) transgene copy number versus RNA transcript levels are plotted.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Pilot-scale ET-801i production run. Clone 3–10 cells were cultured in 500 cm2 flasks under serum-free conditions for 8 days. Media was collected daily (hatched bars) and fVIII activity in the bulk supernatant was measured. Gray bars represent fVIII activity as determined by one-stage coagulation assay and black circles represent the activation quotient as determined by a two-stage coagulation assay.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Analysis of purified ET-801i. (a) FVIII-containing cell culture supernatant was loaded onto an SP-Sepharose Fast Flow column and ET-801i was eluted using a linear 0.18–0.7 mol/l NaCl gradient. Fractions collected were assayed for A280, fVIII activity, AQ, and conductivity. (b) Two µg of human fVIII and ET-801i were subjected to 4–15% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions and visualized by silver staining. Where indicated, samples were treated with 100 nmol/l porcine thrombin ± PNGase F endoglycosidase for 5 minutes before analysis to demonstrate proteolytic activation and the presence of N-linked glycan modifications, respectively. BDD, B-domain-deleted; HC, heavy chain; LC, light chain; SC, single chain.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
In vivo efficacy of ET-801i. Hemophilia A mice were infused with either saline, or ET-801i at a dose of 290 units/kg. Following administration of saline or fVIII, the mice were subjected to a hemostatic challenge of 4 mm tail transection. Total blood loss was determined over a 40-minute period.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions