Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen owing to mutations in the gene COL7A1 and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma. The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. We generated patient-derived COL7A1-corrected epithelial keratinocyte sheets for autologous grafting. We demonstrate the utility of sequential reprogramming and adenovirus-associated viral genome editing to generate corrected iPSC banks. iPSC-derived keratinocytes were produced with minimal heterogeneity, and these cells secreted wild-type type VII collagen, resulting in stratified epidermis in vitro in organotypic cultures and in vivo in mice. Sequencing of corrected cell lines before tissue formation revealed heterogeneity of cancer-predisposing mutations, allowing us to select COL7A1-corrected banks with minimal mutational burden for downstream epidermis production. Our results provide a clinical platform to use iPSCs in the treatment of debilitating genodermatoses, such as RDEB.

Graphical Abstract Highlights d Epigenome landscape maps reveal key transitions during epidermal lineage commitment d Network modeling identifies master regulators of lineage initiation and maturation d TFAP2C drives chromatin dynamics during initiation and primes p63-dependent maturation d Crosstalk between TFAP2C and p63 drives epigenetic transitions during differentiation

The ubiquitin-independent proteasomal degradation pathway is increasingly being recognized as important in regulation of protein turnover in eukaryotic cells. One substrate of this pathway is the pyrimidine biosynthetic enzyme thymidylate synthase (TS; EC 2.1.1.45), which catalyzes the reductive methylation of dUMP to form dTMP and is essential for DNA replication during cell growth and proliferation. Previous work from our laboratory showed that degradation of TS is ubiquitin-independent and mediated by an intrinsically disordered 27-residue region at the N-terminal end of the molecule. In the current study we show that this region, in cooperation with an ␣-helix formed by the next 15 residues, functions as a degron, i.e. it is capable of destabilizing a heterologous protein to which it is fused. Comparative analysis of the primary sequence of TS from a number of mammalian species revealed that the N-terminal domain is hypervariable among species yet is conserved with regard to its disordered nature, its high Pro content, and the occurrence of Pro at the penultimate site. Characterization of mutant proteins showed that Pro-2 protects the N terminus against N ␣ -acetylation, a post-translational process that inhibits TS degradation. However, although a free amino group at the N terminus is necessary, it is not sufficient for degradation of the polypeptide. The implications of these findings to the proteasome-targeting function of the N-terminal domain, particularly with regard to its intrinsic flexibility, are discussed.Regulated protein degradation within the cell is carried out primarily by the 26 S proteasome, a large 2-MDa complex consisting of several dozen proteins that function in recognizing and degrading its target substrates (1, 2). Typically, covalent attachment of polyubiquitin chains serves as the primary signal for target recognition by the proteasome (1, 2). However, in recent years several proteasomal substrates have been shown to be degraded without a requirement for ubiquitin modification (for a recent review, see Ref.3). Such substrates include ornithine decarboxylase (ODC) 3 (4 -6), c-Fos (7, 8), p21 Cip1 (9, 10), hepatitis virus F protein (11), and c-Jun (12), among others. Although the number of substrates identified as degraded by a ubiquitin-independent mechanism remains small, recent biochemical analyses indicate that the process may be more widespread than previously thought and contributes significantly to the regulation of protein turnover (13).Among the known substrates of the ubiquitin-independent degradation pathway, ODC has been the most studied. The degradation signal for ODC is composed of a disordered, flexible domain formed by a 37-residue region at the C terminus (4 -6). The region mediates docking of the ODC polypeptide to the proteasome and initiates its entry into the proteasomal chamber, where proteolysis proceeds in a C-to N-terminal direction (4 -6). This process is stimulated by an accessory protein termed antizyme, which binds ODC and increases the availability of i...

Definitive correction of disease causing mutations in somatic cells by homologous recombination (HR) is an attractive therapeutic approach for the treatment of genetic diseases. However, HR-based somatic gene therapy is limited by the low efficiency of gene targeting in mammalian cells and replicative senescence of primary cells ex vivo, forcing investigators to explore alternative strategies such as retro- and lentiviral gene transfer, or genome editing in induced pluripotent stem cells. Here, we report correction of mutations at the LAMA3 locus in primary keratinocytes derived from a patient affected by recessive inherited Herlitz junctional epidermolysis bullosa (H-JEB) disorder using recombinant adenoassociated virus (rAAV)-mediated HR. We identified a highly recombinogenic AAV serotype, AAV-DJ, that mediates efficient gene targeting in keratinocytes at clinically relevant frequencies with a low rate of random integration. Targeted H-JEB patient cells were selected based on restoration of adhesion phenotype, which eliminated the need for foreign sequences in repaired cells, enhancing the clinical use and safety profile of our approach. Corrected pools of primary cells assembled functional laminin-332 heterotrimer and fully reversed the blistering phenotype both in vitro and in skin grafts. The efficient targeting of the LAMA3 locus by AAV-DJ using phenotypic selection, together with the observed low frequency of off-target events, makes AAV-DJ based somatic cell targeting a promising strategy for ex vivo therapy for this severe and often lethal epithelial disorder.

Human embryonic stem cell (hESC) differentiation promises advances in regenerative medicine 1 – 3 , yet conversion into transplantable tissues remains poorly understood. Using our keratinocyte differentiation system, we employ a multi-dimensional genomics approach to interrogate the contributions of inductive morphogens retinoic acid (RA) and bone morphogenetic protein 4 (BMP4) and the epidermal master regulator p63 4 , 5 during surface ectoderm commitment. In contrast to other master regulators 6 – 9 , p63 effects major transcriptional changes only after morphogens alter chromatin accessibility, establishing an epigenetic landscape for p63 to modify. p63 distally closes chromatin accessibility and promotes accumulation of H3K27me3 modifications. Cohesin HiChIP 10 visualizations of chromosome conformation reveal that p63 and the morphogens contribute to dynamic long-range chromatin interactions, as illustrated with TFAP2C regulation 11 . Our study demonstrates the unexpected dependency of p63 on morphogenetic signaling and provides novel insights into how a master regulator can specify diverse transcriptional programs based on the chromatin landscape induced by specific morphogen exposure.