Efficient and safe correction of hemophilia A by lentiviral vector-transduced BOECs in an implantable device

Abstract

Hemophilia A (HA) is a rare bleeding disorder caused by deficiency/dysfunction of the FVIII protein. As current therapies based on frequent FVIII infusions are not a definitive cure, long-term expression of FVIII in endothelial cells through lentiviral vector (LV)-mediated gene transfer holds the promise of a one-time treatment. Thus, here we sought to determine whether LV-corrected blood outgrowth endothelial cells (BOECs) implanted through a prevascularized medical device (Cell Pouch) would rescue the bleeding phenotype of HA mice. To this end, BOECs from HA patients and healthy donors were isolated, expanded, and transduced with an LV carrying FVIII driven by an endothelial-specific promoter employing GMP-like procedures. FVIII-corrected HA BOECs were either directly transplanted into the peritoneal cavity or injected into a Cell Pouch implanted subcutaneously in NSG-HA mice. In both cases, FVIII secretion was sufficient to improve the mouse bleeding phenotype. Indeed, FVIII-corrected HA BOECs reached a relatively short-term clinically relevant engraftment being detected up to 16 weeks after transplantation, and their genomic integration profile did not show enrichment for oncogenes, confirming the process safety. Overall, this is the first preclinical study showing the safety and feasibility of transplantation of GMP-like produced LV-corrected BOECs within an implantable device for the long-term treatment of HA.

Keywords: BOEC; FVIII; cell and gene therapy; endothelial cells; hemophilia A; lentiviral vector; medical device.

Graphical abstract

Figure 1

Healthy and HA BOEC isolation, LV transduction, and in vitro FVIII detection (A) Light microscope pictures of cultured healthy and HA BOECs at passage 3. (B) Representative RT-PCR analysis for the expression of endothelial markers. HUVECs and fibroblasts were used as positive and negative control, respectively. (C) RT-PCR for endothelial markers specific for blood endothelial cells (BECs). iPSC-derived ECs and fibroblasts were used as positive and negative control, respectively. (D) Representative histograms of healthy non-transduced (black line) and LV-VEC.hBDD-FVIII-transduced healthy BOECs (red line), showing endothelial marker expression and absence of hematopoietic markers. The filled-up histograms represent unstained BOECs. (E) Representative histograms of HA non-transduced (black line) and LV-VEC.hBDD-FVIII-transduced HA BOECs (red line) showing endothelial marker expression and absence of hematopoietic markers. The filled-up histograms represent unstained BOECs. (F) Matrigel assay confirming tubule formation of transduced BOECs. (G) RT-PCR, using primers specific for the exogenous F8 in non-transduced and LV-VEC.hBDD-FVIII BOECs. Unrelated transduced cells and fibroblast were used as positive and negative control, respectively. (H) FVIII intracytoplasmic staining on non-transduced (black line) or transduced healthy BOECs (red line). The filled-up histogram represents unstained BOECs. (I) FVIII intracytoplasmic staining on non-transduced (black line) or transduced HA BOECs (red line). The filled-up histogram represents unstained BOECs. (J) FVIII detection by immunofluorescence: blue, DAPI; red, anti-FVIII. Data are expressed as mean ± SD and are representative of four independent experiments.

Figure 2

Intraperitoneal implantation of BOECs with Cytodex microcarrier beads (A) Kinetics of the percentage of FVIII activity measured by aPTT assay in the plasma of transplanted NSG-HA mice. BOECs used were transduced only with LV-VEC.GFP or with both LV-VEC.hBDD-FVIII and LV-VEC.GFP. Data are expressed as mean ± SD and are representative of two independent experiments using BOECs from two healthy donors (n = 7 mice), and four independent experiments using HA BOECs from four patients (n = 23 mice). (B) Blood loss evaluation on NSG-HA mice between weeks 7 and 10 (n = 4) after cell transplantation. (C) FVIII concentration in plasma of mice transplanted with transduced or non-transduced BOECs at week 16. Data are expressed as mean ± SD (∗∗∗p < 0.0001, ∗∗p < 0.001). (D) Representative immunofluorescence on beads showing cells co-expressing GFP and CD31.

Figure 3

Large-scale expansion of HA patient-derived BOECs (A) Light microscope pictures of transduced HA BOECs pre- and post-expansion. (B) Cell size, cell density, culture time, and population doubling level during pre- and post-large-scale expansion. (C) Endothelial marker expression pre- and post-large-scale expansion expressed as stained cells versus cells with secondary isotype controls. (D) Tubulogenic assay to assess the functionality of transduced HA BOECs after pre- and post-large-scale expansion. (E) Kinetics of the percentage of FVIII activity measured by aPTT assay in plasma of transplanted NSG-HA mice. Data are expressed as mean ± SD and are representative of two independent experiments (n = 7).

Figure 4

Pathological assessment after transplantation of LV-VEC.hBDD-FVIII HA BOECs into the Cell Pouch device (A) Sernova Cell Pouches were removed at 4, 8, or 12 weeks and stained by H&E and Masson's trichrome for blinded histopathological analysis. Histology scores and representative images at 12 weeks post-transplant with 10 × 10⁶ LV-VEC.hBDD-FVIII BOECs (animal groups n = 2–3). (B) Quantification of H&E and Masson's trichrome for blinded histopathological analysis.

Figure 5

Bleeding phenotype and cell survival of LV-VEC.hBDD-FVIII HA BOECs after implantation in the Cell Pouch device (A) Bleeding assay on mice transplanted with 10 × 10⁶ or 20 × 10⁶ HA and LV-VEC.hBDD-FVIII BOECs, or left untreated (n = 3–6, mean ± SEM, ∗∗p < 0.05; ns, not significant). NSG mice were used as control for bleeding assay. (B) The transplanted Cell Pouch devices were removed from the recipient NSG-HA mice, and immunofluorescence was performed to detect cell survival within the mouse tissue by human cell staining (HLA-ABC) and blood vessel formation through staining with cross-reacting human/mouse von Willebrand factor (vWF) antibody. The images shown are representative of two transplant groups (10 × 10⁶ n = 5; 20 × 10⁶ n = 12). (C) Quantification of HLA-ABC and blood vessel formation from blinded histopathological assessment.

Figure 6

Genome wide distribution of lentiviral vector ISs (A) The pink track represents the density distribution of genes (RefSeq annotation, hg19 genome). The green tracks are the density distributions of all the ISs retrieved in the HA transduced with LV-VEC.GFP and Healthy transduced with LV-VEC.GFP groups. The blue tracks are the density distributions of all the ISs retrieved in the LV-VEC.hBDD-FVIII HA BOECs and LV-VEC.hBDD-FVIII Healthy BOECs groups. (B) Distribution of ISs of the four groups along the whole human genome and with respect to gene transcription start site (TSS).

Figure 7

Boxplot representation of clonal abundance For each sample, the abundance values for each clone are represented as dots. Clones over 10% are presented as dots labeled with the closest gene symbol (RefSeq hg19).

Figure 8

Clonal diversity comparison (A) Shannon diversity index for each transduced cell population according to cell passage and time point. (B) H index comparison between different groups.