Design and validation of a GMP stem cell manufacturing protocol for MPSII hematopoietic stem cell gene therapy

Abstract

Hematopoietic stem cell gene therapy (HSCGT) is a promising therapeutic strategy for the treatment of neurodegenerative, metabolic disorders. The approach involves the ex vivo introduction of a missing gene into patients' own stem cells via lentiviral-mediated transduction (TD). Once transplanted back into a fully conditioned patient, these genetically modified HSCs can repopulate the blood system and produce the functional protein, previously absent or non-functional in the patient, which can then cross-correct other affected cells in somatic organs and the central nervous system. We previously developed an HSCGT approach for the treatment of Mucopolysaccharidosis type II (MPSII) (Hunter syndrome), a debilitating pediatric lysosomal disorder caused by mutations in the iduronate-2-sulphatase (IDS) gene, leading to the accumulation of heparan and dermatan sulfate, which causes severe neurodegeneration, skeletal abnormalities, and cardiorespiratory disease. In HSCGT proof-of-concept studies using lentiviral IDS fused to a brain-targeting peptide ApoEII (IDS.ApoEII), we were able to normalize brain pathology and behavior of MPSII mice. Here we present an optimized and validated good manufacturing practice hematopoietic stem cell TD protocol for MPSII in preparation for first-in-man studies. Inclusion of TEs LentiBOOST and protamine sulfate significantly improved TD efficiency by at least 3-fold without causing adverse toxicity, thereby reducing vector quantity required.

Keywords: GMP cell manufacturing; MPSII; cleanroom validation studies; hCD34+ cell transduction; hematopoietic stem cell gene therapy; investigational medicinal product; lentiviral vector; mucopolysaccharidosis; transduction enhancers.

Graphical abstract

Figure 1

HSCGT overview The patient is given recombinant human granulocyte colony stimulating factor by subcutaneous injection (G-CSF; 5–16 μg/kg per day for 5–6 days) to mobilize HSPC from the bone marrow (BM) into the peripheral circulation. On the last day, plerixafor maybe given to maximize mobilization. CD34⁺ cell counts are monitored from day 3 and leukapheresis performed on days 5 and 6 provided cell count is greater than 1 × 10⁴ CD34⁺ cells/mL. Following leukapheresis, CD34⁺ cells are isolated by magnetic bead separation and a back-up of a minimum of 3 × 10⁶ CD34⁺ cells/kg cryopreserved for potential stem cell transplant rescue in the event that engraftment of the IMP fails. The main bulk of selected CD34⁺ stem cells are pre-stimulated overnight in growth media plus cytokines prior to TD with GMP LV. The following day, the transduced cells are harvested and the majority cryopreserved to make the IMP, with a subset sent for quality assurance (QA) analysis to confirm sterility, absence of mycoplasma and endotoxins, CD34⁺ cell purity and viability, normal progenitor development in the CFU assay, number of integrated VCNs, and, depending on the specific product, protein activity. The patient undergoes myeloablative conditioning before the IMP can be administered. Following transplant, the gene-corrected HSCs replenish the blood system. Differentiated progenitors can distribute throughout the body, including the brain, and produce functional protein to alleviate the disease phenotype.

Figure 2

Small scale h CD34⁺ stem cell TD optimization (A) CD34⁺ stem cells were transduced with and without TEs (±TE) at a range of vector concentrations (MOI) and then seeded in 14-day CFU assays and the number of BFU-E, CFU-GM and CFU-GEMM colonies assessed. Data are mean ± SEM. One-way ANOVA. ∗∗∗∗p < 0.0001. (B) TD efficiency was determined by picking 18 individual BFU-E and CFU-GM colonies for each condition, extracting gDNA and determining if the colonies are positive for the integrated genome by qPCR. (C) The VCN was determined in 14 day pooled CFU or LCs by qPCR. (D) IDS activity in 14-day pooled CFU or LCs. VCN vs. IDS activity in pooled CFU (E) and LCs (F).

Figure 3

Scale up of hCD34⁺ stem cell TD (A) CD34⁺ stem cell viability was evaluated by FACS post-selection, post-prestimulation and post-TD at MOI 12.5 and 25. Following 14-day CFU assay TD efficiency (B) and number and type of CFU colonies assessed (C). VCN (D) and IDS activity was assessed in liquid (E) and pooled CFU cultures (F). Data are mean ± SEM.

Figure 4

R&D pilot manufacturing run comparing culture vessel and evaluating absence of IL3 in the TD media CD34⁺ stem cells were seeded in a T75 flask containing culture media with or without IL3. Cells were transduced for 24 h before being harvested. (A) Cells taken after the final wash and from the infusion buffer were seeded in the CFU assay and BFU-E, CFU-GM and CFU-GEMM colonies counted 14 days later. (B) Total combined colony counts for each condition. (C) TD efficiency evaluated in BFU-E, CFU-GM, and combined colonies. VCN in LCs (D), in pooled CFUs from final wash (E) and infusion media (F). Data are mean ± SEM.

Figure 5

Overview of the GMP manufacturing process (1) Stem cells are isolated from the leukapheresis unit by magnetic bead separation using a CliniMACS plus or prodigy instrument. (2) The number, purity, and viability of isolated CD34⁺ cells is determined by FACS. (3) Before genetic modification, the cells undergo a pre-stimulation step in growth media + cytokines that allows more effective TD. (4) Cells are transduced with LV, which introduces correct copies of the defective gene into patient cells. (5) Following TD, cells are washed by centrifugation, cells counted, and resuspended in Cryostor at 2 × 10⁶ cell/mL prior to being cryopreserved in a controlled rate freezer (6). The IMP to be transplanted in the patient is stored in a cryobag, while smaller vials for QC analysis are stored as cryovials. (7) Before and 24 h after cryopreservation, the viability and post-thaw recovery of the product is assessed by FACS. (8) Some of the transduced cells are seeded in colony-forming unit (CFU) and LC assays. (9) Samples of the IMP and samples taken throughout the manufacturing run are assessed for sterility, mycoplasma and endotoxin. (10) Individual colonies from the CFU assay are picked and TD efficiency assessed by determining the presence of the integrated transgene by qPCR. Pooled colonies and LCs are also assessed for VCNs.

Figure 6

CFU and LC assays from Barnsley GMP runs 1 and 2 CFU colony counts from run 1 GMP product pre-cryopreservation (A), and following 6 and 12 weeks cryopreservation (B and C). (D) GMP run 1: 12-week stability and 14-day LC cell viability. CFU colony counts from run 2 GMP product pre-cryopreservation (E), and following 6 and 12 weeks cryopreservation (F and G). (H) GMP run 1 12 week stability 14 day LC cell viability. Data are mean ± SEM. One-way ANOVA. ∗∗∗p < 0.001. ∗p < 0.05.