Pre-clinical Safety and Efficacy of Lentiviral Vector-Mediated Ex Vivo Stem Cell Gene Therapy for the Treatment of Mucopolysaccharidosis IIIA

Stuart M Ellison, Aiyin Liao, Shaun Wood, Jessica Taylor, Amir Saam Youshani, Sam Rowlston, Helen Parker, Myriam Armant, Alessandra Biffi, Lucas Chan, Farzin Farzaneh, Rob Wynn, Simon A Jones, Paul Heal, H Bobby Gaspar, Brian W Bigger, Stuart M Ellison, Aiyin Liao, Shaun Wood, Jessica Taylor, Amir Saam Youshani, Sam Rowlston, Helen Parker, Myriam Armant, Alessandra Biffi, Lucas Chan, Farzin Farzaneh, Rob Wynn, Simon A Jones, Paul Heal, H Bobby Gaspar, Brian W Bigger

Abstract

Hematopoietic stem cell gene therapy is a promising therapeutic strategy for the treatment of neurological disorders, since transplanted gene-corrected cells can traffic to the brain, bypassing the blood-brain barrier, to deliver therapeutic protein to the CNS. We have developed this approach for the treatment of Mucopolysaccharidosis type IIIA (MPSIIIA), a devastating lysosomal storage disease that causes progressive cognitive decline, leading to death in early adulthood. In a previous pre-clinical proof-of-concept study, we demonstrated neurological correction of MPSIIIA utilizing hematopoietic stem cell gene therapy via a lentiviral vector encoding the SGSH gene. Prior to moving to clinical trial, we have undertaken further studies to evaluate the efficiency of gene transfer into human cells and also safety studies of biodistribution and genotoxicity. Here, we have optimized hCD34+ cell transduction with clinical grade SGSH vector to provide improved pharmacodynamics and cell viability and validated effective scale-up and cryopreservation to generate an investigational medicinal product. Utilizing a humanized NSG mouse model, we demonstrate effective engraftment and biodistribution, with no vector shedding or transmission to germline cells. SGSH vector genotoxicity assessment demonstrated low transformation potential, comparable to other lentiviral vectors in the clinic. This data establishes pre-clinical safety and efficacy of HSCGT for MPSIIIA.

Figures

Figure 1
Figure 1
GMP LV CD11b.SGSH Is Equivalent to Its Research Grade Counterpart In Vivo (A) Vector design. pCCL LV containing a human myeloid CD11b promoter driving expression of codon-optimized SGSH. (B) MPSIIIA bone marrow was lineage depleted and transduced with GMP or non-GMP CD11b.SGSH LV at an MOI of 60, then transplanted into busulfan-myeloablated MPSIIIA mice. (C) Twelve weeks post-transplantation, the donor chimerism in WBCs was determined by flow cytometry (n = 10 per group). (D) VCN was determined for each transplant batch at initial transduction following 14 days in methylcellulose culture. (E) Hematopoietic lineage development was assessed by CFU assay 12 weeks post-transplant. CFU-granulocyte, macrophage (CFU-GM); BFU-erythroid (BFU-E); CFU-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM); CFU-granulocyte (CFU-G); and CFU-macrophage (CFU-M). (F) VCN in white blood cells (WBCs), bone marrow (BM), pooled CFU colonies, and the brain was assessed 12 weeks post-transplant. (G) SGSH enzyme activity was assessed in pooled CFU colonies and BM 12 weeks post-transplant (one-way ANOVA Kruskal-Wallis test).
Figure 2
Figure 2
Characterization of Human CD34+ Cell Transduction with CD11b.GFP LV (A) Umbilical cord blood (UCB) hCD34+ cells were transduced with an increasing MOI of CD11b.GFP LV and transduction efficiency determined by counting GFP-positive colonies following colony-forming unit (CFU) assay (*p < 0.05, **p < 0.01). (B) hCD34+ cells were transduced with CD11b.GFP LV at an MOI of 100 and the transduction efficiency evaluated in BFU-E, CFU-GEMM, CFU-GM, CFU-M, and CFU-G progenitors. Representative images of CD11b.GFP LV transduced progenitors.
Figure 3
Figure 3
CD11b.SGSH LV Equivalence Testing and Dose Optimization in CD34+ Cells hCD34+ cells were transduced with GMP or non-GMP LV at an MOI of 100 and evaluated in the CFU assay. Single (TDX1) versus double (TDX2) transductions were compared. (A) Transduction efficiency determined by qPCR assessment of individual colonies and calculating the percentage of positive colonies containing integrated transgene (n = 16 BFU-E; n = 16 CFU colonies). (B) VCN determined in pooled CFU colonies by qPCR (**p < 0.005). (C) SGSH enzyme activity in pooled CFU colonies (****p < 0.0001). (D) CFU progenitor counts following TDX1 or TDX2. (E) CFU progenitor counts following TDX2. (F) Total CFU colony counts. (G) The experiment was repeated four times with different batches of CD34+ cells and the VCN range for TDX1s and TDX2s evaluated.
Figure 4
Figure 4
CD34+ Cell Transductions Scaled Up and Cryopreservation Validated for Clinical Application (A) CD34+ cell purity and viability accessed by flow cytometry at post-selection, post-prestimulation, post-transduction, and post-cryopreservation. (B) Post-thaw cell counts, viability, and total cell percentage recovery at 52 days after cryopreservation. (C) VCN of bulk liquid culture transduced cells pre- and post-cryopreservation for TDX1 and TDX2 cell groups. (D) The CFU assay was performed post-cryopreservation and the number of BFU-E and CFU colonies determined for TDX1 and TDX2 groups. (E) Evaluation of SGSH activity in transduced cells pre- and post-cryopreservation.
Figure 5
Figure 5
NSG Engraftment and Biodistribution Study (A) NSG engraftment and biodistribution study design. (B) Human leucocyte (CD45+ cells) engraftment in the hematopoietic organs of NSG mice transplanted with TDX2 or mock-transduced, 52 day cryopreserved human CD34+ cells from PBMCs. (C) Human T cell populations in the thymus of transplanted mice. (D) Stem cell (CD34+), myeloid cell (CD13+), and B cell (CD19+) populations in the BM of transplanted mice. (E) T cell (CD3+, CD4+, CD8+), stem cell (CD34+), myeloid cell (CD13+), and B cell (CD19+) populations in the spleens of transplanted mice (*p <0.05, ****p < 0.001). (F) VCN per human cell was determined in the BM, WBCs, thymus, spleen, brain, heart, liver, kidney, lungs, gastrocnemius muscle, and gonads of TDX2 transplanted mice by qPCR. VCN was also determined for transduced cells before transplant (bulk LC [liquid culture]) (G). SGSH enzyme activity in the BM of mock- and TDX2 transplanted NSG mice (****p < 0.0001).
Figure 6
Figure 6
CD11b.SGSH LV Genotoxicity (A and B) In vitro immortalization (IVIM) assay. Murine Lin− cells transduced by the indicated vectors were expanded as mass cultures for 2 weeks. An aliquot was taken for qPCR for VCN measurement. On day 15, cells were plated into 96-well plates at a density of 100 cells/well or 1,000 cells/well in 100 μL medium. Two weeks later, the wells showing abundant cell growth were counted as positive, and the frequency of replating cells was calculated based on Poisson statistics using L-Calc Software (StemCell Technologies, Vancouver, Canada). (A) WST-1 colorimetric proliferation assay to determine positive growth above baseline (red line) (n = 2). (B) Replating frequency corrected for VCN group by investigators at GOSH, UK (i) and University of Manchester (ii). Horizontal bars indicate mean values. (C) Cryptic splice site assessment. (i) 293T and (ii) CD34+ cells were transduced with LV-CD11b.SGSH at an MOI of 10. Seventy-two hours after transduction genomic DNA was isolated from cells and PCR performed to amplify the integrated transgene with primers upstream of CD11b and downstream of WPRE. (i) PCR products from LV-CD11b.SGSH transduced 293T cells (lane 1), non-transduced 293T cells (lane 2) and SGSH genome plasmid control (lane 3) were run on an electrophoresis gel. A strong band was observed at 4.1 kb indicating full-length transcript in LV-CD11b.SGSH transduced cells. No other prominent bands were detected, suggesting an absence of splice mutants. The same finding was observed in LV-CD11b.SGSH-transduced CD34+ cells (ii) (lane 4, non-template control; lane 5, SGSH plasmid control; lane 6, non-transduced control; and lane 7, LV-CD11b.SGSH transduced).

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