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ABSTRACTIntroductionJunctional epidermolysis bullosa (JEB) is a rare inherited genetic disorder with limited treatments beyond palliative care. A major hallmark of JEB is skin blistering caused by functional loss or complete absence of major structural proteins of the skin. Impaired wound healing in patients with JEB gives rise to chronic cutaneous ulcers that require daily care. Wound care and infection control are the current standard of care for this patient population.Areas coveredThis review covers research and clinical implementation of emerging drug, cell, and gene therapies for JEB. Current clinical trials use topical drug delivery to manipulate the inflammation and re-epithelialization phases of wound healing or promote premature stop codon readthrough to accelerate chronic wound closure. Allogeneic cell therapies for JEB have been largely unsuccessful, with autologous skin grafting emerging as a reliable method of resolving the cutaneous manifestations of JEB. Genetic correction and transplant of autologous keratinocytes have demonstrated persistent amelioration of chronic wounds in a subset of patients.Expert OpinionEmerging therapies address the cutaneous symptoms of JEB but are unable to attend to systemic manifestations of the disease. Investigations into the molecular mechanism(s) underpinning the failure of systemic allogeneic cell therapies are necessary to expand the range of effective JEB therapies.

Gene editing with CRISPR/Cas9 is revolutionizing biotechnology and medical research, yet affordable, efficient, and tailorable delivery systems are urgently needed to advance translation. Herein, a series of monodisperse amphiphilic block polymers poly[ethylene oxide-b-2-(dimethylamino) ethyl methacrylate-b-n-butyl methacrylate] (PEO-b-PDMAEMA-b-PnBMA) that housed three PEO lengths (2, 5, and 10 kDa) and a variant lacking PEO (PDMAEMA-b-PnBMA) were synthesized via controlled radical polymerization and assembled into well-defined spherical cationic micelles. The cationic micelles were complexed via electrostatic interactions with Cas9 protein/guide RNA ribonucleoproteins (RNPs) that exhibit anionic charges due to the overhanging RNA. The resulting micelleplex formulations in both phosphate-buffered saline (PBS) and water were screened via high content analysis for gene editing efficiency. The micelle variant with the 10 kDa PEO block offered the highest gene editing performance and was advanced for in-depth characterization. For the first time, quantitative static and dynamic light scattering characterization and cryogenic transmission electron microscopy images of Cas9 protein/guideRNA RNP loading into well-defined micelleplex nanoparticles are revealed, where the formulation solvent was found to play a major role in the physicochemical properties and biological performance. In PBS, the solutions containing the micelles (63 triblock polymers per micelle) were assembled with the Cas9 protein/guideRNA RNP payloads offering uniform loading of 14 RNPs per micelleplex and moderate editing efficiency; this homogeneous system offers promise for future in vivo/preclinical applications. Interestingly, when the uniform micelles were formulated with the RNP payloads in water, larger multimicelleplex nanoparticles were formed that offered double the editing efficiency of Lipofectamine 2000 (40% gene editing) due to the rapid sedimentation kinetics of the larger colloids onto adherent cells, offering promising in vitro, ex vivo, and/or cell therapy applications. This work presents the first quantitative demonstration of tailorable block polymer micelle formulations for advancing CRISPR/Cas9 RNP delivery and fundamental correlation of the solutions physics to biological performance.

Fast, efficient, and inexpensive methods for delivering functional nucleic acids to primary human cell types are needed to advance regenerative medicine and cell therapies. Plasmid-based gene editing (such as with CRISPR-Cas9) can require the delivery of plasmids that are large (∼9.5–13 kbp) in comparison to common reporter plasmids (∼5–8 kbp). To develop more efficient plasmid delivery vehicles, we investigated the effect of plasmid size on the transfection of primary human dermal fibroblasts (HDFs) and induced pluripotent stem cells (iPSCs) using a heparin-treated trehalose-containing polycation (Tr4-heparin). Transfections with 4.7 kbp to 10 kbp plasmids exhibited high rates of polyplex internalization with both plasmid sizes. However, transfection with the large plasmid was nearly eliminated in HDFs and significantly reduced in iPSCs. Molecular additives were used to probe intracellular barriers to transfection. Chloroquine treatments were used to destabilize endosomes, and dexamethasone and thymidine were used to destabilize the nuclear envelope. Destabilizing the nuclear envelope resulted in significantly increased large-plasmid-transfection, indicating that nuclear localization may be more difficult for large plasmids. To demonstrate the potential clinical utility of this formulation, HDFs and iPSCs were treated with to dexamethasone-Tr4-heparin polyplexes encoding dCas9-VP64, synthetic transcription activator, targeted to collagen type VII. These transfections enhanced collagen expression in HDFs and iPSCs by 5- and 20-fold, respectively, compared to an untransfected control and were the more effective than the Lipofectamine 2000 control. Functional plasmid transfection efficiency can be significantly improved by nuclear destabilization, which could lead to improved development of nonviral vehicles for ex vivo CRISPR-Cas9 gene editing.

Epidermolysis bullosa is classified as a genodermatosis, an inherited genetic skin disorder that results in severe, chronic skin blistering with painful and life-threatening complications. Although there is currently no cure for epidermolysis bullosa, concurrent advances in gene and stem cell therapies are converging toward combinatorial therapies that hold the promise of clinically meaningful and lifelong improvement. Recent studies using hematopoietic stem cells and mesenchymal stromal/stem cells to treat epidermolysis bullosa have demonstrated the potential for sustained, effective management of the most severe cases. Furthermore, advances in the use of gene therapy and gene-editing techniques, coupled with the development of induced pluripotent stem cells from patients with epidermolysis bullosa, allow for autologous therapies derived from a renewable population of cells that are patient-specific. Here we describe emerging treatments for epidermolysis bullosa and other genodermatoses, along with a discussion of their benefits and limitations as effective therapies.

Human induced pluripotent stem cells (h‐iPSCs) represent a potentially unlimited source for the generation of human hepatocyte‐like cells (h‐iHLCs) for the establishment of platforms to study drug‐induced hepatotoxicity, liver disease modeling, and ultimately the application of h‐iHLCs in treatment of patients with end‐stage liver disease. To understand the impact of donor‐specific factors on the generation of h‐iHLCs, the model for the direct comparison of h‐iHLCs and primary human hepatocytes (PHHs) from the same human donor is needed. This study proposes a step‐by‐step protocol for plating, expansion, and characterization of primary human hepatic non‐parenchymal cells (h‐NPCs) isolated from the human liver, the reprogramming of generated h‐NPCs into h‐iPSCs and subsequent differentiation of reprogrammed h‐iPSCs into h‐iHLCs. The ultimate goal is to compare the gene expression involved in hepatocyte metabolism between h‐iHLCs and PHHs from the same human donor thus eliminating interdonor variability. This newly developed protocol of h‐NPC culture, expansion, and reprogramming into h‐iPSCs allows: (1) utilization of a single organ source (i.e., liver) for isolation of PHHs and h‐NPCs and (2) the in‐depth study of donor factors involved in the generation of h‐iHLCs with direct comparison to PHHs from the same donor. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Plating and expansion of human hepatic NPCs in culture Basic Protocol 2: Reprogramming of h‐NPCs to h‐NPC‐derived induced pluripotent stem cells (h‐iPSCs) Basic Protocol 3: Culture, passaging, and freezing of h‐iPSCs Support Protocol 1: Confirmation of h‐NPC ability to uptake Sendai virus: GFP‐Sendai infection Support Protocol 2: Characterization of h‐NPCs amenable to transduction with Sendai particles Support Protocol 3: Characterization of h‐iPSCs: Clearance of viral reprogramming vectors Support Protocol 4: Preparation of Matrigel‐coated plates