A scalable, GMP-compatible, autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa

Abstract

SummaryBackgroundGene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies.MethodsWe developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a combined high-efficiency reprogramming and CRISPR-based genetic correction single step to generate genome scar- free,COL7A1corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off targets, and cancer-driver variants were evaluated by targeted and whole genome sequencing.FindingsiPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT- treated mice.InterpretationDEBCT successfully overcomes previous roadblocks and represents a robust, scalable, and safe cGMP manufacturing platform for production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds.

A Spatiotemporal and Machine-Learning Platform Accelerates the Manufacturing of hPSC-derived Esophageal Mucosa

“…Classic tissue recombination experiments demonstrate that the signal interactions between epithelium and local mesenchyme are essential for tissue morphogenesis and cell fate determination 27 . We and others have demonstrated the significance of integrating supporting cellular signals into human tissue engineering 25,26,28 , reinforcing the importance of detailing the spatial architecture of developing human esophagus. To systematically map the cell types revealed in single cell atlas, we orthogonally performed unbiased Visium spatial transcriptomics (16 sections from 5 timepoints, 5 donors) and high-resolution CODEX (co-detection by indexing) proteomic profiling (48 sections/905,062 cells from 13 timepoints, 15 donors) (Figure 1C, Table S1).…”

“…While the epithelial morphogenesis has been documented superficially, the underlying stromal composition and roles in morphogenesis remain unknown. Developmental studies in skin squamous stratified epithelium and our recent work have shown that regional mesodermal morphogenic signals wire the spatialtemporal specific stratification program in surface ectoderm progenitors, and these signals are required in our hPSC-derived skin production for optimal graftability 25,26 . Therefore, to accelerate esophageal mucosa manufacturing, a holistic molecular characterization of the coordinated development of epithelium and stroma is needed.…”

Emerging Gene Therapeutics for Epidermolysis Bullosa under Development

“…Also worth noting is the fact that their own variant, LZ3 Cas9, exhibits a differential +1 insertion profile when compared to spCas9 [131]. HiFi Cas9, on the other hand, is already used by many groups in the gene editing field, including several groups working on the correction of the mutations underlying EB [134][135][136][137].…”

Innovations in the Treatment of Dystrophic Epidermolysis Bullosa (DEB): Current Landscape and Prospects

“… 27 Four gene-edited iPSCs were then differentiated into keratinocytes and fibroblasts and showed C7 restoration. 27 Recently, Neumayer et al 28 developed a method to reprogram EB patient-derived fibroblasts into iPSCs and undertake CRISPR-based correction to generate skin composite grafts. Mouse xenografts showed no tumors and exhibited stable C7 re-expression thus offering promise for future clinical translation.…”