Twin Prime Editing Mediated Exon Skipping/Reinsertion for Restored Collagen VII Expression in Recessive Dystrophic Epidermolysis Bullosa.

Gene editing nucleases, base editors, and prime editors are potential locus-specific genetic treatment strategies for recessive dystrophic epidermolysis bullosa; however, many recessive dystrophic epidermolysis bullosa COL7A1 pathogenic nucleotide variations (PNVs) are unique, making the development of personalized editing reagents challenging. A total of 270 of the ∼320 COL7A1 epidermolysis bullosa PNVs reside in exons that can be skipped, and antisense oligonucleotides and gene editing nucleases have been used to create in-frame deletions. Antisense oligonucleotides are transient, and nucleases generate deleterious double-stranded DNA breaks and uncontrolled mixtures of allele products.

Integrating Porous Silicon Nanoneedles within Medical Devices for Nucleic Acid Nanoinjection.

Porous silicon nanoneedles can interface with cells and tissues with minimal perturbation for high-throughput intracellular delivery and biosensing. Typically, nanoneedle devices are rigid, flat, and opaque, which limits their use for topical applications in the clinic. We have developed a robust, rapid, and precise substrate transfer approach to incorporate nanoneedles within diverse substrates of arbitrary composition, flexibility, curvature, transparency, and biodegradability.

Creation and characterization of novel rat model for recessive dystrophic epidermolysis bullosa: Frameshift mutation of the Col7a1 gene leads to severe blistered phenotype.

Recessive dystrophic epidermolysis bullosa is a rare genodermatosis caused by a mutation of the Col7a1 gene. The Col7a1 gene codes for collagen type VII protein, a major component of anchoring fibrils. Mutations of the Col7a1 gene can cause aberrant collagen type VII formation, causing an associated lack or absence of anchoring fibrils.

Methods for Decreasing Preweaning Mortality in a Fragile Mouse Model of Hypomorphic Collagen VII Deficiency.

Preweaning mortality is a widespread problem in laboratory mouse breeding, particularly in the case of fragile mouse models. While numerous studies explore alternative care methods to increase the survivability of common mouse strains, there remains a paucity of research into the care of mice with fragile health conditions that result from induced or natural genetic mutations. In this study, standard husbandry practices were enhanced by the addition of a softened diet, a nutritionally fortified dietary supplement, soft bedding, gentle handling techniques, decreased handling, lengthened weaning age, and dam productivity tracking.

Alloengraftment without significant toxicity or GVHD in CD45 antibody-drug conjugate-conditioned Fanconi anemia mice.

Fanconi anemia (FA) is an inherited DNA repair disorder characterized by bone marrow (BM) failure, developmental abnormalities, myelodysplasia, leukemia, and solid tumor predisposition. Allogeneic hematopoietic stem cell transplantation (allo-HSCT), a mainstay treatment, is limited by conditioning regimen-related toxicity and graft-versus-host disease (GVHD). Antibody-drug conjugates (ADCs) targeting hematopoietic stem cells (HSCs) can open marrow niches permitting donor stem cell alloengraftment.

Prevention of acute GVHD using an orthogonal IL-2/IL-2Rβ system to selectively expand regulatory T cells in vivo.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative option for patients with hematological disorders and bone marrow (BM) failure syndromes. Graft-versus-host disease (GVHD) remains a leading cause of morbidity posttransplant. Regulatory T cell (Treg) therapies are efficacious in ameliorating GVHD but limited by variable suppressive capacities and the need for a high therapeutic dose.

Accumulation of senescence observed in spinocerebellar ataxia type 7 mouse model.

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease caused by a trinucleotide CAG repeat. SCA7 predominantly causes a loss of photoreceptors in the retina and Purkinje cells of the cerebellum. Severe infantile-onset SCA7 also causes renal and cardiac irregularities.

Interrogation of RDEB Epidermal Allografts after BMT Reveals Coexpression of Collagen VII and Keratin 15 with Proinflammatory Immune Cells and Fibroblasts.

Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating genodermatosis characterized by dysfunctional collagen VII protein resulting in epithelial blistering of the skin, mucosa, and gastrointestinal tract. There is no cure for RDEB, but improvement of clinical phenotype has been achieved with bone marrow transplantation and subsequent epidermal allografting from the bone marrow transplant donor. Epidermal allografting of these patients has decreased wound surface area for up to 3 years after treatment.

Disruption of HIV-1 co-receptors CCR5 and CXCR4 in primary human T cells and hematopoietic stem and progenitor cells using base editing.

Disruption of CCR5 or CXCR4, the main human immunodeficiency virus type 1 (HIV-1) co-receptors, has been shown to protect primary human CD4 T cells from HIV-1 infection. Base editing can install targeted point mutations in cellular genomes, and can thus efficiently inactivate genes by introducing stop codons or eliminating start codons without double-stranded DNA break formation. Here, we applied base editors for individual and simultaneous disruption of both co-receptors in primary human CD4 T cells.

Future applications of 3D bioprinting: A promising technology for treating recessive dystrophic epidermolysis bullosa.

Three-dimensional (3D) bioprinting is a rapidly developing technology that has the potential to initiate a paradigm shift in the treatment of skin wounds arising from burns, ulcers and genodermatoses. Recessive dystrophic epidermolysis bullosa (RDEB), a severe form of epidermolysis bullosa, is a rare genodermatosis that results in mechanically induced blistering of epithelial tissues that leads to chronic wounds. Currently, there is no cure for RDEB, and effective treatment is limited to protection from trauma and extensive bandaging.

Mesenchymal stromal cells in wound healing applications: role of the secretome, targeted delivery and impact on recessive dystrophic epidermolysis bullosa treatment.

Mesenchymal stromal cells (MSCs) are multi-potent stromal-derived cells capable of self-renewal that possess several advantageous properties for wound healing, making them of interest to the field of dermatology. Research has focused on characterizing the unique properties of MSCs, which broadly revolve around their regenerative and more recently discovered immunomodulatory capacities. Because of ease of harvesting and expansion, differentiation potential and low immunogenicity, MSCs have been leading candidates for tissue engineering and regenerative medicine applications for wound healing, yet results from clinical studies have been variable, and promising pre-clinical work has been difficult to reproduce.

TCIRG1 Transgenic Rescue of Osteoclast Function Using Induced Pluripotent Stem Cells Derived from Patients with Infantile Malignant Autosomal Recessive Osteopetrosis.

Background: Osteoclasts are hematopoietic stem cell-derived multinucleated cells necessary for bone remodeling and resorption. TCIRG1 encodes a protein that is an adenosine triphosphate (ATP)-dependent vacuolar proton pump required for this process. Recessive loss-of-function mutations in both copies of this gene lead to impairment of osteoclast function, with increased bone density, increased skeletal mass, and early mortality.

Base Editor Correction of COL7A1 in Recessive Dystrophic Epidermolysis Bullosa Patient-Derived Fibroblasts and iPSCs.

Genome editing represents a promising strategy for the therapeutic correction of COL7A1 mutations that cause recessive dystrophic epidermolysis bullosa (RDEB). DNA cleavage followed by homology-directed repair (HDR) using an exogenous template has previously been used to correct COL7A1 mutations. HDR rates can be modest, and the double-strand DNA breaks that initiate HDR commonly result in accompanying undesired insertions and deletions (indels).

3D Printed Functional and Biological Materials on Moving Freeform Surfaces.

Conventional 3D printing technologies typically rely on open-loop, calibrate-then-print operation procedures. An alternative approach is adaptive 3D printing, which is a closed-loop method that combines real-time feedback control and direct ink writing of functional materials in order to fabricate devices on moving freeform surfaces. Here, it is demonstrated that the changes of states in the 3D printing workspace in terms of the geometries and motions of target surfaces can be perceived by an integrated robotic system aided by computer vision.

A multitask clustering approach for single-cell RNA-seq analysis in Recessive Dystrophic Epidermolysis Bullosa.

Single-cell RNA sequencing (scRNA-seq) has been widely applied to discover new cell types by detecting sub-populations in a heterogeneous group of cells. Since scRNA-seq experiments have lower read coverage/tag counts and introduce more technical biases compared to bulk RNA-seq experiments, the limited number of sampled cells combined with the experimental biases and other dataset specific variations presents a challenge to cross-dataset analysis and discovery of relevant biological variations across multiple cell populations. In this paper, we introduce a method of variance-driven multitask clustering of single-cell RNA-seq data (scVDMC) that utilizes multiple single-cell populations from biological replicates or different samples.

CRISPR/Cas9-Based Cellular Engineering for Targeted Gene Overexpression.

Gene and cellular therapies hold tremendous promise as agents for treating genetic disorders. However, the effective delivery of genes, particularly large ones, and expression at therapeutic levels can be challenging in cells of clinical relevance. To address this engineering hurdle, we sought to employ the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to insert powerful regulatory elements upstream of an endogenous gene.

Rapid generation of Col7a1 mouse model of recessive dystrophic epidermolysis bullosa and partial rescue via immunosuppressive dermal mesenchymal stem cells.

Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating and ultimately lethal blistering disease caused by mutations to the Col7a1 gene. Development of novel cell therapies for the treatment of RDEB would be fostered by having immunodeficient mouse models able to accept human cell grafts; however, immunodeficient models of many genodermatoses such as RDEB are lacking. To overcome this limitation, we combined the clustered regularly interspaced short palindromic repeats and associated nuclease (CRISPR/Cas9) system with microinjection into NOD/SCID IL2rγc (NSG) embryos to rapidly develop an immunodeficient Col7a1 mouse model of RDEB.

Rapid Induction of Cerebral Organoids From Human Induced Pluripotent Stem Cells Using a Chemically Defined Hydrogel and Defined Cell Culture Medium.

Unlabelled: Tissue organoids are a promising technology that may accelerate development of the societal and NIH mandate for precision medicine. Here we describe a robust and simple method for generating cerebral organoids (cOrgs) from human pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. By using no additional neural induction components, cOrgs appeared on the hydrogel surface within 10-14 days, and under static culture conditions, they attained sizes up to 3 mm in greatest dimension by day 28.

CRISPR/Cas9-based genetic correction for recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe disorder caused by mutations to the gene that deactivate production of a structural protein essential for skin integrity. Haematopoietic cell transplantation can ameliorate some of the symptoms; however, significant side effects from the allogeneic transplant procedure can occur and unresponsive areas of blistering persist. Therefore, we employed genome editing in patient-derived cells to create an autologous platform for multilineage engineering of therapeutic cell types.

Patient-specific naturally gene-reverted induced pluripotent stem cells in recessive dystrophic epidermolysis bullosa.

Spontaneous reversion of disease-causing mutations has been observed in some genetic disorders. In our clinical observations of severe generalized recessive dystrophic epidermolysis bullosa (RDEB), a currently incurable blistering genodermatosis caused by loss-of-function mutations in COL7A1 that results in a deficit of type VII collagen (C7), we have observed patches of healthy-appearing skin on some individuals. When biopsied, this skin revealed somatic mosaicism resulting in the self-correction of C7 deficiency.

Induced pluripotent stem cells from individuals with recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in the COL7A1 gene-encoding type VII collagen (Col7), the major component of anchoring fibrils at the dermal-epidermal junction. Individuals with RDEB develop painful blisters and mucosal erosions, and currently, there are no effective forms of therapy. Nevertheless, some advances in patient therapy are being made, and cell-based therapies with mesenchymal and hematopoietic cells have shown promise in early clinical trials.