Defining the Optimal FVIII Transgene for Placental Cell-Based Gene Therapy to Treat Hemophilia A

Abstract

The delivery of factor VIII (FVIII) through gene and/or cellular platforms has emerged as a promising hemophilia A treatment. Herein, we investigated the suitability of human placental cells (PLCs) as delivery vehicles for FVIII and determined an optimal FVIII transgene to produce/secrete therapeutic FVIII levels from these cells. Using three PLC cell banks we demonstrated that PLCs constitutively secreted low levels of FVIII, suggesting their suitability as a transgenic FVIII production platform. Furthermore, PLCs significantly increased FVIII secretion after transduction with a lentiviral vector (LV) encoding a myeloid codon-optimized bioengineered FVIII containing high-expression elements from porcine FVIII. Importantly, transduced PLCs did not upregulate cellular stress or innate immunity molecules, demonstrating that after transduction and FVIII production/secretion, PLCs retained low immunogenicity and cell stress. When LV encoding five different bioengineered FVIII transgenes were compared for transduction efficiency, FVIII production, and secretion, data showed that PLCs transduced with LV encoding hybrid human/porcine FVIII transgenes secreted substantially higher levels of FVIII than did LV encoding B domain-deleted human FVIII. In addition, data showed that in PLCs, myeloid codon optimization is needed to increase FVIII secretion to therapeutic levels. These studies have identified an optimal combination of FVIII transgene and cell source to achieve clinically meaningful levels of secreted FVIII.

Effects of Shear Stress on Production of FVIII and vWF in a Cell-Based Therapeutic for Hemophilia A

“…The total number of cells in each channel was then used to normalize the quantification of proteins within the supernatant to 10 6 cells. Quantification of fVIII/mco-ET3 activity in the supernatants was performed as previously described ( El-Akabawy et al, 2020 ) using an activated prothrombin time (aPTT). PLC-mcoET3 under static conditions produced 3.69 ± 0.4 IU/10 6 cells/24 h of fVIII/mco-ET3 ( n = 15), but under the shear stresses of 0.05 and 0.5 dyne/cm 2 fVIII/mco-ET3 secretion significantly reduced to 2.03 ± 0.2 IU/10 6 cells/24 h ( n = 11) and 1.99 ± 0.2 IU/10 6 cells/24 h ( n = 10), respectively ( Figure 3C ) ( p < 0.05).…”

“…In addition, the gene-modified cells must possess the ability to efficiently produce and secrete FVIII, they should lodge/engraft and persist long-term within a broad range of tissues upon infusion, and they need to evade the recipient’s immune system, despite expressing a therapeutic protein that is perceived as foreign. We recently reported the development of a platform, based upon human PLC engineered with a lentiviral vector to express a mco human/porcine hybrid FVIII (ET3) molecule, PLC-mcoET3, that fulfills all of these criteria ( El-Akabawy et al, 2020 ).…”

“…Human placentas were obtained from full-term deliveries after informed consent, according to the guidelines from the Office of Human Research Protection at Wake Forest Health Sciences. Primary placental mesenchymal cells (PLC) were isolated from the placental chorionic layer after enzymatic digestion and grown in placental cell growth media (PCGM) consisting of α-minimum essential medium (α-MEM) supplemented with 17% AmnioMAX Basal Media, 15% fetal bovine serum (FBS), 2% AmnioMAX Supplement, 1% GlutaMAX, and 2.5 mg/mL gentamicin (ThermoFisher Scientific, Wilmington, DE, United States) ( El-Akabawy et al, 2020 ). PLC were transduced with an HIV-based lentiviral vector encoding a myeloid codon-optimized (mco) B domain-deleted human/porcine hybrid FVIII transgene (ET3), termed mcoET3, at a multiplicity of infection of 10 transducing units/cell ( El-Akabawy et al, 2020 ).…”

“…Primary placental mesenchymal cells (PLC) were isolated from the placental chorionic layer after enzymatic digestion and grown in placental cell growth media (PCGM) consisting of α-minimum essential medium (α-MEM) supplemented with 17% AmnioMAX Basal Media, 15% fetal bovine serum (FBS), 2% AmnioMAX Supplement, 1% GlutaMAX, and 2.5 mg/mL gentamicin (ThermoFisher Scientific, Wilmington, DE, United States) ( El-Akabawy et al, 2020 ). PLC were transduced with an HIV-based lentiviral vector encoding a myeloid codon-optimized (mco) B domain-deleted human/porcine hybrid FVIII transgene (ET3), termed mcoET3, at a multiplicity of infection of 10 transducing units/cell ( El-Akabawy et al, 2020 ). Transduced cells (PLC-mcoET3) were passaged at 70–80% confluence using TrypLE (ThermoFisher Scientific, Wilmington, DE, United States) and PLC-mcoET3 were used in these studies after they had been passaged a minimum of three times post-transduction to ensure the absence of any episomal vector.…”

“…We have previously reported that human placental cells (PLC) constitutively produce vWF ( Morsi et al, 2018 ) and FVIII ( El-Akabawy et al, 2020 ), and we developed a potential cell therapy platform by transducing human PLC with a lentiviral vector encoding a myeloid-codon-optimized (mco), bioengineered FVIII transgene that contains high-expression elements from porcine FVIII (ET3) (PLC-mcoET3) ( El-Akabawy et al, 2020 ). Moreover, PLC-mcoET3 secreted FVIII protein with functional procoagulant activity at therapeutic and clinically meaningful levels ( El-Akabawy et al, 2020 ). In addition, after prenatal administration, these cells are thought to lodge in perivascular sites, such as the liver sinusoids ( Almeida-Porada et al, 2017 ), and therefore are subjected to shear stress from fluid flow.…”