Long-term survival after unrelated donor marrow transplantation for aplastic anaemia after optimized conditioning regimen: a retrospective multicentre cohort study.

Background: Almost all acquired severe aplastic anaemia is immune mediated and characterised by hypocellular bone marrow and ≥2 affected haematopoietic lineages. The optimal preparartive regimen for unrelated donor transplantation remains to be established. We aimed to study long-term outcomes after unrelated donor transplantation for severe aplastic anaemia with de-escalation of cyclophosphamide (Cy) dose in steps of 50 mg/kg (150, 100, 50, 0 mg/kg) in combination with total body irradiation (TBI) 2 Gy, anti-thymocyte globulin (ATG) and fludarabine.

Chondroitin sulfate proteoglycan 4 increases invasion of recessive dystrophic epidermolysis bullosa-associated cutaneous squamous cell carcinoma by modifying transforming growth factor-β signalling.

Background: Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic skin-blistering disorder that often progresses to metastatic cutaneous squamous cell carcinoma (cSCC) at chronic wound sites. Chondroitin sulfate proteoglycan 4 (CSPG4) is a cell-surface proteoglycan that is an oncoantigen in multiple malignancies, where it modulates oncogenic signalling, drives epithelial-to-mesenchymal transition (EMT) and enables cell motility.

Objectives: To evaluate CSPG4 expression and function in RDEB cSCC.

Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing.

Methods for the targeted integration of genes in mammalian genomes suffer from low programmability, low efficiencies or low specificities. Here we show that phage-assisted continuous evolution enhances prime-editing-assisted site-specific integrase gene editing (PASSIGE), which couples the programmability of prime editing with the ability of recombinases to precisely integrate large DNA cargoes exceeding 10 kilobases. Evolved and engineered Bxb1 recombinase variants (evoBxb1 and eeBxb1) mediated up to 60% donor integration (3.

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.

Selective bioelectronic sensing of pharmacologically relevant quinones using extracellular electron transfer in Lactiplantibacillus plantarum.

Redox-active small molecules containing quinone functional groups play important roles as pharmaceuticals, but can be toxic if overdosed. Despite the need for a fast and quantitative method to detect quinone and its derivatives, current sensing strategies are often slow and struggle to differentiate between structural analogs. Leveraging the discovery that microorganisms use certain quinones to perform extracellular electron transfer (EET), we investigated the use of Lactiplantibacillus plantarum as a whole-cell bioelectronic sensor to selectively sense quinone analogs.

Phage-assisted evolution and protein engineering yield compact, efficient prime editors.

Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size and improved efficiency. Using phage-assisted evolution, we improved editing efficiencies of compact reverse transcriptases by up to 22-fold and generated prime editors that are 516-810 base pairs smaller than the current-generation editor PEmax.

Engineering Human Cells Expressing CRISPR/Cas9-Synergistic Activation Mediators for Recombinant Protein Production.

Recombinant engineering for protein production commonly employs plasmid-based gene templates for introduction and expression of genes in a candidate cell system in vitro. Challenges to this approach include identifying cell types that can facilitate proper post-translational modifications and difficulty expressing large multimeric proteins. We hypothesized that integration of the CRISPR/Cas9-synergistic activator mediator (SAM) system into the human genome would be a powerful tool capable of robust gene expression and protein production.

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.

The Differing Roles of Flavins and Quinones in Extracellular Electron Transfer in Lactiplantibacillus plantarum.

Lactiplantibacillus plantarum is a lactic acid bacterium that is commonly found in the human gut and fermented food products. Despite its overwhelmingly fermentative metabolism, this microbe can perform extracellular electron transfer (EET) when provided with an exogenous quinone, 1,4-dihydroxy-2-naphthoic acid (DHNA), and riboflavin. However, the separate roles of DHNA and riboflavin in EET in have remained unclear.

Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.

Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination.

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.

Correction of Fanconi Anemia Mutations Using Digital Genome Engineering.

Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and cancer predisposition. The only curative therapy for the hematological manifestations of FA is an allogeneic hematopoietic cell transplant (HCT); however, many (>70%) patients lack a suitable human leukocyte antigen (HLA)-matched donor, often resulting in increased rates of graft-versus-host disease (GvHD) and, potentially, the exacerbation of cancer risk.

Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production.

Current hemostatic agents are obtained from pooled plasma from multiple donors requiring costly pathogen screening and processing. Recombinant DNA-based production represents an engineering solution that could improve supply, uniformity, and safety. Current approaches are typically for single gene candidate peptides and often employ non-human cells.

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.

Genes and compounds that increase type VII collagen expression as potential treatments for dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is a skin-blistering disease caused by mutations in COL7A1, which encodes type VII collagen (C7). There is no cure for DEB, but previous work has shown potential therapeutic benefit of increased production of even partially functional C7. Genome-wide screens using CRISPR-Cas9 have enabled the identification of genes involved in cancer development, drug resistance and other genetic diseases, suggesting that they could be used to identify drivers of C7 production.