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.

Keywords: ET3; FVIII; HSQ; cell therapy; codon-optimization; gene therapy; hemophilia A; placental cells.

Graphical abstract

Figure 1

Characterization of Phenotype and FVIII Production by Placental Cell Master Banks (A) Flow cytometric characterization of cultured PLC101, PLC103, and PLC104 demonstrating the expression of characteristic mesenchymal markers (n = 3). (B) Representative image (original magnification, ×40) of immunofluorescence evaluation of FVIII expression by PLCs using an antibody specific for FVIII, showing that these cells constitutively produce FVIII protein (in red); DAPI (in blue) labels all nuclei (n = 5); the negative control consisted of slides stained in parallel, in which the primary antibody was absent. (C) Flow cytometric analysis of FVIII expression by PLCs. Upper panel: Solid-line histograms show fluorescence data for FVIII, and the dashed-line histograms depict the respective isotype controls. Lower panel: The median fluorescence intensity (MFI) ratio was obtained by dividing the MFI of FVIII by the respective MFI of the isotype (n = 3). (D) Evaluation by aPTT of FVIII activity in 24-h culture supernatants harvested from PLCs, showing the amount of functional FVIII in IU being produced by 10⁶ cells (n = 5). (E) Endogenous levels of FVIII mRNA determined by qRT-PCR, after normalization to respective GAPDH levels, in PLC101, PLC103, and PLC104 (n = 3). Experimental results are presented as the mean ± the standard error of the mean. * p < 0.05 was considered statistically significant.

Figure 2

Evaluation of FVIII Production and FVIII Secretion of Three Different PLCs/Master Cell Banks by Immunofluorescence Microscopy, aPTT, and qPCR after Transduction with mcoET3-LV (A) Representative images (original magnification, ×40) of immunofluorescence analysis of mcoET3-LV-transduced PLC101, PLC103, and PLC104, at the same MOI of 7.5, using an antibody specific for FVIII (red); DAPI (in blue) labels all nuclei (n = 5). (B) Amount of functional FVIII (IU)/10⁶ cells, evaluated by aPTT, present in 24-h culture supernatants harvested from PLCs that were plated at the same density and normalized for the number of cells present at the time of supernatant collection (n = 5). (C) mRNA levels for the mcoET3 transgene were determined by qRT-PCR in all mcoET3-LV-transduced master cell banks, using primers specific for a region of mcoET3 that differs from the endogenous FVIII sequence, and compared to the levels of endogenous FVIII in the respective non-transduced cells, using primers specific for the FVIII B domain, after normalization of each sample’s value to its respective GAPDH control (n = 3). (D) Relative fold change in endogenous FVIII mRNA after transduction with mcoET3-LV in PLC101, PLC103, and PLC104 (n = 3). Experimental results are presented as the mean ± the standard error of the mean. * p < 0.05 was considered statistically significant.

Figure 3

Phenotype of Non-transduced and mcoET3-LV-Transduced PLCs (A) Flow cytometric analysis of non-transduced and mcoET3-LV-transduced PLCs demonstrated that the mesenchymal phenotype of PLCs was maintained following transduction (n = 3). (B) Flow cytometric analysis of non-transduced and transduced PLC banks for immune receptors/markers (n = 3). (C) HLA-I and HLA-II expression on transduced and non-transduced PLCs evaluated by flow cytometry (n = 3). (D and E) Flow cytometric evaluation of Toll-like receptor molecules (D) and stress molecules (E) in transduced and non-transduced PLCs (n = 3). Experimental results are presented as the mean ± the standard error of the mean. * p < 0.05 was considered statistically significant.

Figure 4

Determination of the Optimal FVIII Transgene for Increased Synthesis and Secretion of FVIII by Transduced PLCs (A) Evaluation of FVIII:C activity in 24-h culture supernatants measured by aPTT (n = 5) after normalization for the number of cells and VCN in PLCs transduced with LVs encoding each of five different bioengineered FVIII transgenes at an identical MOI of 7.5. The transgenes used were as follows: ET3, non-codon-optimized bioengineered human/porcine hybrid FVIII; mcoET3, myeloid codon-optimized ET3; lcoET3, liver codon-optimized ET3; mcoHSQ, myeloid codon-optimized human FVIII; and lcoHSQ, liver codon-optimized human FVIII. Transduced cells were passaged three times before analysis was performed. (B) After supernatant collection, transduced PLCs were harvested and transgene-specific FVIII mRNAs were quantitated by qRT-PCR. All values were normalized to GAPDH and VCN (n = 3). (C and D) Flow cytometric analysis of BiP (C) and CHOP (D) in PLCs alone, PLCs transduced with the different FVIII transgenes, and PLCs treated with tunicamycin (positive control). The fold change in MFI of BiP (n = 3) and CHOP (n = 3) was calculated by dividing the MFI of BiP or CHOP in transduced or tunicamycin-treated PLCs by the MFI of non-transduced PLCs, after subtracting the respective isotype controls. Experimental results are presented as the mean ± the standard error of the mean. * p < 0.05 was considered statistically significant.

Figure 5

Integration Site Analysis of PLCs Transduced with the Optimal FVIII Transgene to Produce/Secrete FVIII 24 unique integration sites on chromosomes 1, 2, 3, 5, 7, 9, 10, 11, 12, 13, 14, 16, 17, 19, and 21 of transduced PLCs were identified. Their chromosomal locations are graphically depicted with green dots in the left panel. The purple text indicates the name of the gene closest to the insertion site.