Background: Disorders of fatty acid oxidation (FAOD) are estimated to account for around 1 in 10,000 live births, and with modern newborn screens, these conditions are often identified in childhood. However, not all parents will receive regular medical follow-up, and varying levels of parental health literacy can influence their reliance on online resources for information. Therefore, assessing the readability of online materials is critical to ensuring accessible and comprehensible patient education. Understanding the readability landscape informs our efforts to improve the quality of online resources and to support parents and patients in navigating the diagnosis of an FAOD. Objective: Our goal was to evaluate the readability of public facing online materials concerning the 10 most common disorders of fatty acid oxidation, with consideration given to the recommended reading levels by the National Institutes of Health (NIH) and the American Medical Association (AMA). Methods: Using Flesch-Kincaid, Coleman-Liau, and SMOG readability indices, we analyzed the top 25 internet search results for each disorder. Excluding empty or paywalled content, 232 publicly accessible materials were assessed. Results: Mean readability ranged from 11.64 to 12.85, indicating generally higher complexity than recommended. Only 15.5 % of materials met NIH's 8th grade reading level guideline, and 3.9 % met AMA's 6th grade level. Variability existed between disorders, with percentages meeting guidelines ranging from 0 % to 25 % for NIH and 0 % to 8.3 % for AMA. Conclusion: Ensuring readability of online resources for rare disorders of fatty acid oxidation is crucial, particularly given the prevalence of childhood diagnosis and varying levels of parental health literacy. Parents may rely on easily accessible but potentially complex materials found through online searches, highlighting the importance of aligning online content with recommended reading levels. Improving readability can enhance accessibility and understanding and facilitate informed decision-making and optimal care for patients.

The success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) can be limited by graft-versus-host disease (GVHD). T-cell activation is a key factor in GVHD progression. Costimulatory signals can be counterbalanced by co-inhibitory signals such as the checkpoint molecule VISTA (V-domain Ig-containing suppressor of T-cell activation)/PD-1H that restrains activation and maintains donor T-cell quiescence. A single dose of anti-VISTA mAb prevents acute GVHD lethality in multiple models. Naïve donor T-cells express moderate VISTA levels, which transiently increase in allo-HSCT recipients in association with TCR signaling, leading to heightened susceptibility to anti-VISTA mAb-mediated depletion, in contrast to donor T-cells transferred to syngeneic recipients. Anti-VISTA mAb donor T-cell depletion was compatible with rapamycin but incompatible with peri-transplant tacrolimus GVHD prophylaxis. Targeting VISTA exclusively on host cells or donor CD8+ T-cells was not protective against GVHD lethality. Instead, anti-VISTA mAb mediated deletion of alloreactive donor T-cells depended on targeting a third (non-T) cell type. Further mechanistic studies indicated that donor T-cells concurrently exposed to anti-VISTA mAb in vivo but not preincubated in vitro before adoptive T-cell transfer were eliminated via FcR-mediated phagocytosis. In a lymphoma challenge model, a graft-versus-lymphoma effect was fully retained when anti-human VISTA mAb exclusively targeted donor CD4+ T-cells and delayed but mostly retained when unseparated donor T-cells were infused. In a xenogeneic GVHD model, anti-human VISTA mAb reduced donor T-cell expansion, VISTA T-cell expression levels, and recipient lethality. Together, these data support a novel clinical translational pathway in which acute GVHD lethality can be mitigated without negating the GVL effect.

Chronic graft-versus-host disease (cGVHD) is the leading cause of morbidity and non-relapse associated mortality following allogeneic hematopoietic cell transplantation (aHSCT). Treating steroid resistant/refractory cGVHD remains challenging. Epigenetic regulators can have global transcriptional effects that control donor T-cell responses. We previously showed that inhibiting histone lysine motifs by chromatin-modifying enzymes can ameliorate murine cGVHD. Targeting donor T-cell DNA methyltransferases reduce acute GVHD. Here, we sought to investigate the DNA demethylase Tet (ten-eleven translocase) methylcytosine dioxygenases 2 (Tet2) and Tet3 in T follicular helper cell (TFH) dependent cGVHD. In a clinically relevant model of cGVHD that recapitulates pulmonary fibrosis from bronchiolitis obliterans, recipients of Tet2 deleted donor T-cells did not have improved pulmonary function tests in contrast to the markedly improved pulmonary function in Tet3 deleted donor T-cells. Tet3 deleted donor T-cells did not impair TFH-dependent germinal center (GC) formation. Unexpectedly, TET3 deficiency resulted in elevated GATA3 expression in and IL-4 production by TFH cells. TET3 deficient TFH cells supported GC B-cell immunoglobulin (Ig) class switching to nonpathogenic IgG1 but not pathogenic IgG2c allowing mice to escape cGVHD pulmonary fibrosis. Elevated GATA3 expression and disruption of IgG2c class switching was recapitulated in an in-vitro human GC culture system. These studies provide new insights into the function of Tet3 in TFH driven Ig class switching and suggest a new approach to mitigate cGVHD.

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. This alternative care plan was shown to increase the survival of a fragile recessive dystrophic epidermolysis bullosa mouse model, and some aspects could be used in developing a care plan for other fragile mouse strains.

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. With this approach, we integrated nanoneedles on medically relevant elastomers, hydrogels, plastics, medical bandages, catheter tubes, and contact lenses. The integration retains the mechanical properties and transfection efficiency of the nanoneedles. Transparent devices enable the live monitoring of cell-nanoneedle interactions. Flexible devices interface with tissues for efficient, uniform, and sustained topical delivery of nucleic acids ex vivo and in vivo. The versatility of this approach highlights the opportunity to integrate nanoneedles within existing medical devices to develop advanced platforms for topical delivery and biosensing.

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. We developed a twin prime editing strategy using the PEmax and recently evolved PE6 prime editors and dual prime editing guide RNAs flanking COL7A1 exon 5. Prime editing-mediated deletion of exon 5 with a homozygous premature stop codon was achieved in recessive dystrophic epidermolysis bullosa fibroblasts, keratinocytes, and induced pluripotent stem cells with minimal double-stranded DNA breaks, and collagen type VII protein was restored. Twin prime editing can replace the target exon with recombinase attachment sequences, and we exploited this to reinsert a normal copy of exon 5 using the Bxb1 recombinase. These findings demonstrate that twin prime editing can facilitate locus-specific, predictable, in-frame deletions and sequence replacement with few double-stranded DNA breaks as a strategy that may enable a single therapeutic agent to treat multiple recessive dystrophic epidermolysis bullosa patient cohorts.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severely debilitating disorder caused by pathogenic variants in COL7A1 and is characterized by extreme skin fragility, chronic inflammation, and fibrosis. A majority of patients with RDEB develop squamous cell carcinoma, a highly aggressive skin cancer with limited treatment options currently available. In this study, we utilized an approach leveraging whole-genome sequencing and RNA sequencing across 3 different tissues in a single patient with RDEB to gain insight into possible mechanisms of RDEB-associated squamous cell carcinoma progression and to identify potential therapeutic options. As a result, we identified PLK-1 as a possible candidate for targeted therapy and discovered microsatellite instability and accelerated aging as factors potentially contributing to the aggressive nature and early onset of RDEB squamous cell carcinoma. By integrating multitissue genomic and transcriptomic analyses in a single patient, we demonstrate the promise of bridging the gap between genomic research and clinical applications for developing tailored therapies for patients with rare genetic disorders such as RDEB.

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare genodermatosis characterized clinically by extensive inflammation, cutaneous destruction, and fibrosis that demonstrates properties similar to rapid skin aging. As tissue ages, it accumulates cellular damage and exhaustion leading to a state of senescence. Cellular senescence is an aging or disease-related phenomenon of stable exit from the cell cycle that leads to an increased inflammatory phenotype. RDEB and other EB subsets of patients need an adjunct to or alternate therapy that addresses the issues of inflammation, pain, and pruritus. Fisetin is a safe, naturally occurring compound proven to be effective at sensitizing senescent cells to cell death and ameliorating senescence-associated inflammation. In this paper, we demonstrate fisetin's ability to increase survival and reduce senescent cell burden in a hypomorphic mouse model of RDEB. ### Competing Interest Statement The authors have declared no competing interest.

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. Here, we report that single dose anti-mouse CD45-targeted ADC (CD45-ADC) facilitated stable, multilineage chimerism in 3 distinct FA mouse models representing 90% of FA complementation groups. CD45-ADC profoundly depleted host stem cell enriched Lineage(-)Sca1(+)cKit(+) cells within 48 hours. Fanca(-/-) recipients of minor-mismatched BM and single dose CD45-ADC had peripheral blood (PB) mean donor chimerism >90%; donor HSCs alloengraftment was verified in secondary recipients. In Fancc(-/-) and Fancg(-/-) recipients of fully allogeneic grafts, PB mean donor chimerism was 60% to 80% and 70% to 80%, respectively. The mean percent donor chimerism in BM and spleen mirrored PB results. CD45-ADC-conditioned mice did not have clinical toxicity. A transient <2.5-fold increase in hepatocellular enzymes and mild-to-moderate histopathological changes were seen. Under GVHD allo-HSCT conditions, wild-type and Fanca(-/-) recipients of CD45-ADC had markedly reduced GVHD lethality compared with lethal irradiation. Moreover, single dose anti-human CD45-ADC given to rhesus macaque nonhuman primates on days -6 or -10 was at least as myeloablative as lethal irradiation. These data suggest that CD45-ADC can potently promote donor alloengraftment and hematopoiesis without significant toxicity or severe GVHD, as seen with lethal irradiation, providing strong support for clinical trial considerations in highly vulnerable patients with FA.

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. This presents clinically as chronic blistering, scarring, and fibrosis, often leading to the development of cutaneous squamous cell carcinoma. Patients also experience persistent pain and pruritus. Pain management and supportive bandaging remain the primary treatment options. The pathology of recessive dystrophic epidermolysis bullosa was first described in the 1980s, and there has since been a multitude of encouraging treatment options developed. However, in vivo research has been hindered by inadequate models of the disease. The various mouse models in existence possess longevity and surface area constraints, or do not adequately model a normal human disease state. In this paper, we describe a novel rat model of recessive dystrophic epidermolysis bullosa that offers an alternative to previous murine models. An 8-base pair deletion was induced in the Col7a1 gene of Lewis rats, which was subsequently found to cause a premature stop codon downstream. Homozygous mutants presented with a fragile and chronically blistered phenotype postnatally. Further histological analysis revealed subepidermal clefting and the absence of anchoring fibrils. The generation of this novel model offers researchers an easily maintained organism that possesses a larger surface area for experimental topical and transfused therapies to be tested, which may provide great utility in the future study of this debilitating disease.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic disease resulting from inadequate type VII collagen (C7). Although recurrent skin blisters and wounds are the most apparent disease features, the impact of C7 loss is not confined to the skin and mucous membranes. RDEB is a systemic disease marred by chronic inflammation, fibrotic changes, pain, itch, and anemia, significantly impacting QOL and survival. In this narrative review, we summarize these systemic features of RDEB and promising research avenues to address them.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative option for patients with hematological disorders and bone marrow failure syndromes. Graft-versus-Host Disease (GVHD) remains a leading cause of morbidity post-transplant. Regulatory T cell (Treg) therapies are efficacious in ameliorating GVHD but limited by variable suppressive capacities and the need for a high therapeutic dose. Here, we sought to expand Treg in vivo by expressing an orthogonal IL-2Rβ receptor (oIL2Rβ) that would selectively interact with orthogonal IL-2 (oIL2) cytokine and not wildtype IL2. To test whether the orthogonal system would preferentially drive donor Treg expansion, we used a murine MHC-disparate GVHD model of lethally irradiated BALB/c mice given T-cell depleted bone marrow from C57BL/6 (B6) mice alone, or together with B6Foxp3+GFP+ Treg or oIL2Rβ transduced Treg at low cell numbers that typically do not control GVHD with WT Treg. On day 2, B6 Tcons were injected to induce GVHD. Recipients were treated with PBS or oIL2 daily for 14 days, then 3 times weekly for an additional 14 days. Mice treated with oIL2Rβ Treg and oIL2 compared to PBS had enhanced GVHD survival, in vivo selective expansion of Tregs, and greater suppression of Tcon expansion in secondary lymphoid organs, and intestines. Importantly, oIL2Rβ Treg maintained graft-versus-tumor (GVT) responses in two distinct tumor models (A20, MLL-AF9). These data demonstrate a novel approach to enhance the efficacy of Treg cell therapy in allo-HSCT using an oIL2/oIL2Rβ system that allows for selective in vivo expansion of Treg leading to GVHD protection and GVT maintenance.

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. Using cytosine base editors we observed premature stop codon introduction in up to 89% of sequenced CCR5 or CXCR4 alleles. Using adenine base editors we eliminated the start codon in CCR5 in up to 95% of primary human CD4(+) T cell and up to 88% of CD34(+) hematopoietic stem and progenitor cell target alleles. Genome-wide specificity analysis revealed low numbers of off-target mutations that were introduced by base editing, located predominantly in intergenic or intronic regions. We show that our editing strategies prevent transduction with CCR5-tropic and CXCR4-tropic viral vectors in up to 79% and 88% of human CD4(+) T cells, respectively. The engineered T cells maintained functionality and overall our results demonstrate the effectiveness of base-editing strategies for efficient and specific ablation of HIV co-receptors in clinically relevant cell types.

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. Previous reports have shown that SCA7 results in increased susceptibility to DNA damage. Since DNA damage can lead to accumulation of senescent cells, we hypothesized that SCA7 causes an accumulation of senescent cells over the course of disease. A 140-CAG repeat SCA7 mouse model was evaluated for signs of disease-specific involvement in the kidney, heart, and cerebellum, tissues that are commonly affected in the infantile form. We found evidence of significant renal abnormality that coincided with an accumulation of senescent cells in the kidneys of SCA7140Q/5Q mice, based on histology findings in addition to RT-qPCR for the cell cycle inhibitors p16Ink4a and p21Cip1 and senescence-associated ß-galactosidase (SA-ßgal) staining, respectively. The Purkinje layer in the cerebellum of SCA7140Q/5Q mice also displayed SA-ßgal+ cells. These novel findings offer evidence that senescent cells accumulate in affected tissues and may possibly contribute to SCA7's specific phenotype.

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. This study aimed to determine the phenotype of the epidermal allograft cells responsible for durable persistence of wound healing and skin integrity. We found that epidermal allografts provide basal keratinocytes coexpressing collagen VII and basal stem cell marker keratin 15. Characterization of RDEB full-thickness skin biopsies with single-cell RNA sequencing uncovered proinflammatory immune and fibroblast phenotypes potentially driven by the local environment of RDEB skin. This is further highlighted by the presence of a myofibroblast popu-lation, which has not been described in healthy control human skin. Finally, we found inflammatory fibroblasts expressing profibrotic gene POSTN , which may have implications in the development of squamous cell car-cinoma, a common, lethal complication of RDEB that lacks curative treatment. In conclusion, this study pro-vides insights into and targets for future RDEB studies and treatments.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare, incurable blistering skin disease caused by biallelic mutations in type VII collagen (C7). Advancements in treatment of RDEB have come from harnessing the immunomodulatory potential of mesenchymal stem cells (MSCs). Although human bone marrow-derived MSC (BM-MSC) trials in RDEB demonstrate improvement in clinical severity, the mechanisms of MSC migration to and persistence in injured skin and their contributions to wound healing are not completely understood. A unique subset of MSCs expressing ATP-binding cassette subfamily member 5 (ABCB5) resides in the reticular dermis and exhibits similar immunomodulatory characteristics to BM-MSCs. Our work aimed to test the hypothesis that skin-derived ABCB5+ dermal MSCs (DSCs) possess superior skin homing ability compared to BM-MSCs in immunodeficient NOD-scid IL2rgammanull (NSG) mice. Compared to BM-MSCs, peripherally injected ABCB5+ DSCs demonstrated superior homing and engraftment of wounds. Furthermore, ABCB5+ DSCs vs BM-MSCs cocultured with macrophages induced less anti-inflammatory interleukin-1 receptor antagonist (IL-1RA) production. RNA sequencing of ABCB5+ DSCs compared to BM-MSCs showed unique expression of major histocompatibility complex class II and Homeobox (Hox) genes, specifically HOXA3. Critical to inducing migration of endothelial and epithelial cells for wound repair, increased expression of HOXA3 may explain superior skin homing properties of ABCB5+ DSCs. Further discernment of the immunomodulatory mechanisms among MSC populations could have broader regenerative medicine implications beyond RDEB treatment.

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. The care of chronic wounds and burns significantly burdens the healthcare system, further illustrating the dire need for more beneficial wound care. However, in its infancy, 3D bioprinting offers therapeutic potential for wound healing and could be a breakthrough technology for the treatment of rare, incurable genodermatoses like RDEB. This viewpoint essay outlines the promise of 3D bioprinting applications for treating RDEB, including skin regeneration, a delivery system for gene-edited cells and small molecules, and disease modelling. Although the future of 3D bioprinting is encouraging, there are many technical challenges to overcome-including optimizing bioink and cell source-before this approach can be widely implemented in clinical practice.

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. Therefore, the specific mechanisms of how MSCs influence the local microenvironment in distinct wound etiologies warrant further research. Of specific interest in MSC-mediated healing is harnessing the secretome, which is composed of components known to positively influence wound healing. Molecules released by the MSC secretome can promote re-epithelialization and angiogenesis while inhibiting fibrosis and microbial invasion. This review focuses on the therapeutic interest in MSCs with regard to wound healing applications, including burns and diabetic ulcers, with specific attention to the genetic skin disease recessive dystrophic epidermolysis bullosa. This review also compares various delivery methods to support skin regeneration in the hopes of combating the poor engraftment of MSCs after delivery, which is one of the major pitfalls in clinical studies utilizing MSCs.

Background: Recessive dystrophic epidermolysis bullosa (RDEB) and junctional EB (JEB) are inherited disorders characterised by fragility and blistering of epithelial tissues leading to pain, pruritus, and adherent scarring. The severity and chronic nature of the resultant skin wounds significantly reduces quality and length of life. Current therapies primarily consist of protective bandaging and nutritional supplementation; there is no cure for these disorders. Although the skin fragility results from a lack of C7 protein (RDEB) and laminin-332 (JEB), other serious aspects of these disorders, such as inflammation that interferes with healing and aggressive squamous cell carcinoma, have not been completely elucidated. Recent research has suggested that mitochondrial function plays a significant role in skin healing. Objective: To evaluate how mitochondrial function differs in patients with RDEB and JEB. Method: The energy status of RDEB and JEB patient-derived fibroblasts was determined by Seahorse analysis and metabolite production. The energetics and overall morphology of RDEB and JEB patient-derived fibroblasts were assayed as a measure of metabolic stress. Results: EB patient-derived fibroblasts showed impaired oxidative phosphorylation with concomitant compensation by glycolysis. Morphological parameters were altered in RDEB and JEB fibroblasts compared with controls. Conclusion: This is the first study to describe changes in mitochondrial energy metabolism, metabolic profile, and mitochondrial morphology of EB patients.