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

Background: Gene 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. Methods: We 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, COL7A1 corrected 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. Findings: iPS 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. Interpretation: DEBCT 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.

Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa.

Genome editing with engineered site-specific endonucleases involves nonhomologous end-joining, leading to reading frame disruption. The approach is applicable to dominant negative disorders, which can be treated simply by knocking out the mutant allele, while leaving the normal allele intact. We applied this strategy to dominant dystrophic epidermolysis bullosa (DDEB), which is caused by a dominant negative mutation in the COL7A1 gene encoding type VII collagen (COL7). We performed genome editing with TALENs and CRISPR/Cas9 targeting the mutation, c.8068_8084delinsGA. We then cotransfected Cas9 and guide RNA expression vectors expressed with GFP and DsRed, respectively, into induced pluripotent stem cells (iPSCs) generated from DDEB fibroblasts. After sorting, 90% of the iPSCs were edited, and we selected four gene-edited iPSC lines for further study. These iPSCs were differentiated into keratinocytes and fibroblasts secreting COL7. RT-PCR and Western blot analyses revealed gene-edited COL7 with frameshift mutations degraded at the protein level. In addition, we confirmed that the gene-edited truncated COL7 could neither associate with normal COL7 nor undergo triple helix formation. Our data establish the feasibility of mutation site-specific genome editing in dominant negative disorders.

Pumpless microfluidic platform for drug testing on human skin equivalents.

Advances in bio-mimetic in vitro human skin models increase the efficiency of drug screening studies. In this study, we designed and developed a microfluidic platform that allows for long-term maintenance of full thickness human skin equivalents (HSE) which are comprised of both the epidermal and dermal compartments. The design is based on the physiologically relevant blood residence times in human skin tissue and allows for the establishment of an air-epidermal interface which is crucial for maturation and terminal differentiation of HSEs. The small scale of the design reduces the amount of culture medium and the number of cells required by 36 fold compared to conventional transwell cultures. Our HSE-on-a-chip platform has the capability to recirculate the medium at desired flow rates without the need for pump or external tube connections. We demonstrate that the platform can be used to maintain HSEs for three weeks with proliferating keratinocytes similar to conventional HSE cultures. Immunohistochemistry analyses show that the differentiation and localization of keratinocytes was successfully achieved, establishing all sub-layers of the epidermis after one week. Basal keratinocytes located at the epidermal-dermal interface remain in a proliferative state for three weeks. We use a transdermal transport model to show that the skin barrier function is maintained for three weeks. We also validate the capability of the HSE-on-a-chip platform to be used for drug testing purposes by examining the toxic effects of doxorubucin on skin cells and structure. Overall, the HSE-on-a-chip is a user-friendly and cost-effective in vitro platform for drug testing of candidate molecules for skin disorders.

Tetraploid cells from cytokinesis failure induce aneuploidy and spontaneous transformation of mouse ovarian surface epithelial cells.

Most ovarian cancers originate from the ovarian surface epithelium and are characterized by aneuploid karyotypes. Aneuploidy, a consequence of chromosome instability, is an early event during the development of ovarian cancers. However, how aneuploid cells are evolved from normal diploid cells in ovarian cancers remains unknown. In the present study, cytogenetic analyses of a mouse syngeneic ovarian cancer model revealed that diploid mouse ovarian surface epithelial cells (MOSECs) experienced an intermediate tetraploid cell stage, before evolving to aneuploid (mainly near-tetraploid) cells. Using long-term live-cell imaging followed by fluorescence in situ hybridization (FISH), we demonstrated that tetraploid cells originally arose from cytokinesis failure of bipolar mitosis in diploid cells, and gave rise to aneuploid cells through chromosome mis-segregation during both bipolar and multipolar mitoses. Injection of the late passage aneuploid MOSECs resulted in tumor formation in C57BL/6 mice. Therefore, we reveal a pathway for the evolution of diploid to aneuploid MOSECs and elucidate a mechanism for the development of near-tetraploid ovarian cancer cells.

A keratinocyte hypermotility/growth-arrest response involving laminin 5 and p16INK4A activated in wound healing and senescence.

Keratinocytes become migratory to heal wounds, during early neoplastic invasion, and when undergoing telomere-unrelated senescence in culture. All three settings are associated with expression of the cell cycle inhibitor p16INK4A (p16) and of the basement membrane protein laminin 5 (LN5). We have investigated cause-and-effect relationships among laminin 5, p16, hypermotility, and growth arrest. Plating primary human keratinocytes on the gamma2 precursor form of laminin 5 (LN5') immediately induced directional hypermotility at approximately 125 microm/hour, followed by p16 expression and growth arrest. Cells deficient in p16 and either p14ARF or p53 became hypermotile in response to LN5' but did not arrest growth. Plating on LN5' triggered smad nuclear translocation, and all LN5' effects were blocked by a transforming growth factor (TGF) beta receptor I (TGFbetaRI) kinase inhibitor. In contrast, plating cells on collagen I triggered a TGFbetaRI kinase-independent hypermotility unaccompanied by smad translocation or growth arrest. Plating on control surfaces with TGFbeta induced hypermotility after a 1-day lag time and growth arrest by a p16-independent mechanism. Keratinocytes serially cultured with TGFbetaRI kinase inhibitor exhibited an extended lifespan, and immortalization was facilitated following transduction to express the catalytic subunit of telomerase (TERT). These results reveal fundamental features of a keratinocyte hyper-motility/growth-arrest response that is activated in wound healing, tumor suppression, and during serial culture.

A two-stage, p16(INK4A)- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status.

With increasing frequency during serial passage in culture, primary human keratinocytes express p16(INK4A) (p16) and undergo senescence arrest. Keratinocytes engineered to express hTERT maintain long telomeres but typically are not immortalized unless, by mutation or other heritable event, they avoid or greatly reduce p16 expression. We have confirmed that keratinocytes undergo p16-related senescence during growth in culture, whether in the fibroblast feeder cell system or in the specialized K-sfm medium formulation, and that this mechanism can act as a barrier to immortalization following hTERT expression. We have characterized the p16-related arrest mechanism more precisely by interfering specifically with several regulators of cell cycle control. Epidermal, oral mucosal, corneal limbal, and conjunctival keratinocytes were transduced to express a p16-insensitive mutant cdk4 (cdk4(R24C)), to abolish p16 control, and/or a dominant negative mutant p53 (p53DD), to abolish p53 function. Expression of either cdk4(R24C) or p53DD alone had little effect on life span, but expression of both permitted cells to divide 25 to 43 population doublings (PD) beyond their normal limit. Keratinocytes from a p16(+/-) individual transduced to express p53DD alone displayed a 31-PD life span extension associated with selective growth of variants that had lost the wild-type p16 allele. Cells in which both p53 and p16 were nonfunctional divided rapidly during their extended life span but experienced telomere erosion and ultimately ceased growth with very short telomeres. Expression of hTERT in these cells immortalized them. Keratinocytes engineered to express cdk4(R24C) and hTERT but not p53DD did not exhibit an extended life span. Rare immortal variants exhibiting p53 pathway defects arose from them, however, indicating that the p53-dependent component of keratinocyte senescence is telomere independent. Mutational loss of p16 and p53 has been found to be a frequent early event in the development of squamous cell carcinoma. Our results suggest that such mutations endow keratinocytes with extended replicative potential which may serve to increase the probability of neoplastic progression.