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Summary: 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. Methods: Ninety-six patients with severe aplastic anaemia aged ≤65 years with adequate organ function enrolled on a trial of human leukocyte antigen (HLA)-matched or 1 HLA-locus mismatched unrelated donor marrow transplantation conducted between 02/2006 and 12/2013 in the United States (NCT00326417). Exclusion criteria were Karnofsky performance status of less than 60, clonal cytogenetic abnormalities and inherited marrow failure syndormes. The primary outcome was day-100 engraftment (achievement of absolute neutrophil recovery to at least 0.5 × 109/L without subsequent decline) and day-100 survival. The trial determined the lowest effective Cy dose as 50 mg/kg (n = 38) for day-100 engraftment and survival. Cy dose 100 mg/kg (n = 41) was also acceptable. Accrual to Cy doses 150 mg/kg (n = 15) and 0 mg/kg (n = 3) was terminated early for toxicities. The current study is an extended follow up of patients enrolled on the trial (NCT00326477) and includes 76 of 96 patients alive ≥1 year after transplantation. There were 20 deaths in the first year after transplantation (Cy 0 mg/kg [n = 2], Cy 50 mg/kg [n = 1], Cy 100 mg/kg [n = 10], Cy 150 mg/kg [n = 7]). Patients were followed prospectively from transplantation and data reported using standardized data collection forms until death, loss to follow up or last contact through November 2023. The incidence of graft failure was calculated using the cumulative incidence estimator and the probability of survival using the Kaplan–Meier estimator. Findings: The median follow up of the cohort is 8.02 (IQR) 5.16–10.12) years. With Cy 50 mg/kg, there was one graft failure and five deaths ≥1 year after transplantation. With Cy 100 mg/kg there was only one late death and no graft failure. The 8-year probabilities of survival were 85.0% (95% CI 67.3–93.5) and 75.6% (95% CI 59.4–86.1) after Cy 50 mg/kg and 100 mg/kg, respectively, P = 0.31. With Cy 0 mg/kg and 150 mg/kg, there were no graft failures or death ≥1 year after transplantation. Regardless of Cy dose 12 of 15 patients aged ≥50 years died. Interpretation: Cy 50 mg/kg or 100 mg/kg with TBI 2 Gy, ATG and fludarabine are effective conditioning regimens for unrelated donor marrow transplants for aplastic anaemia. Identification of an optimized transplantation approach for patients aged ≥50 years is needed. Funding: US National Institutes of Health.

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.

OBJECTIVES/GOALS: This study investigates whether localization of high mobility group box 1 (HMGB1) controls inflammatory signaling and DNA damage response in human keratinocytes, the cell of origin for squamous cell carcinoma (SCC). SCC is especially metastatic in chronic wounds, burns, and in patients with recessive dystrophic epidermolysis bullosa (RDEB). METHODS/STUDY POPULATION: We used CRISPR/Cas9 gene-editing to knock out HMGB1 in a keratinocyte line, p16INK4a-negative keratinocytes immortalized by ectopic hTERT expression (N/TERT-2G [46,XY]). Following gene editing, clonal keratinocyte populations were screened for knockout by PCR followed by TIDE analysis (Tracking of Indels by Decomposition) to identify indels that would result in a frameshift mutation. Total cell lysates for each clonal population were analyzed by immunoblot and immunofluorescence for HMGB1 protein. These cells will be used to assay for DNA damage sensitivity in the presence of genotoxic agents (etoposide, ultraviolet radiation, γ-irradiation). A lentiviral vector will then be used to express mutant forms of HMGB1 that localize to the nucleus (C23/45S) or cytoplasm (C106S) and DNA damage assays repeated. RESULTS/ANTICIPATED RESULTS: We have confirmed by sequencing, immunoblot, and immunofluorescence that HMGB1 is knocked out in a clonal population of N/TERT-2G human keratinocyte cells. We anticipate that cells with a complete absence of HMGB1 will have high sensitivity to DNA damaging agents, but little change in inflammatory signaling. We also expect that cells expressing mutant HMGB1 that localizes exclusively to the cytoplasm will demonstrate an increased sensitivity to DNA damage relative to wild-type controls, while mutant HMGB1 that localizes exclusively to the nucleus will be protected from DNA damage caused by exposure to genotoxic agents. DISCUSSION/SIGNIFICANCE: HMGB1 is a nuclear protein and damage associated molecular pattern that is elevated systemically in patients with RDEB, many of whom will go on to develop metastatic squamous cell carcinoma. The experiments described here investigate whether HMGB1 plays a mechanistic role in skin carcinogenesis via regulation of the DNA damage response.

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. SAMs are comprised of a “dead” Cas9 (dCas9) linked to transcriptional activators viral particle 64 (VP64), nuclear factor-kappa-B p65 subunit (p65), and heat shock factor 1 (HSF1) and are programmable to single or multiple gene targets. We integrated the components of the SAM system into human HEK293, HKB11, SK-HEP1, and HEP-g2 cells using coagulation factor X (FX) and fibrinogen (FBN) as proof of concept. We observed upregulation of mRNA in each cell type with concomitant protein expression. Our findings demonstrate the capability of human cells stably expressing SAM for user-defined singleplex and multiplex gene targeting and highlight their broad potential utility for recombinant engineering as well as transcriptional modulation across networks for basic, translational, and clinical modeling and applications.

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.

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.

Progressive immune deficiency of aging is characterized by severe thymic atrophy, contracted T cell repertoire, and poor immune function. p63 is critical for the proliferative potential of embryonic and adult stem cells, as well as thymic epithelial cells (TECs). Because p63 null mice experience rapid post-natal lethality due to epidermal and limb morphogenesis defects, studies to define a role for p63 expression in TEC biology focused on embryonic thymus development and in vitro experiments. Since post-natal thymic stromal development and function differs from that of the embryo, we assessed the impact of lineage-restricted p63 loss on pre- and post-natal murine TEC function by generating mice with a loss of p63 function targeted to TEC, termed p63TECko mice. In adult p63TECko mice, severe thymic hypoplasia was observed with a lack in a discernable segregation into medullary and cortical compartments and peripheral T cell lymphopenia. This profound thymic defect was seen in both neonatal as well as embryonic p63TECko mice. In addition to TECs, p63 also plays in important role in the development of stratified epithelium of the skin; lack of p63 results in defects in skin epidermal stratification and differentiation. Interestingly, all adult p63TECko mice lacked hair follicles despite having normal p63 expression in the skin. Together our results show a critical role of TEC p63 in thymic development and maintenance and show that p63 expression is critical for hair follicle formation.

BACKGROUND Recessive dystrophic epidermolysis bullosa (RDEB) is a rare, devastating, and life-threatening inherited skin fragility disorder that comes about due to a lack of functional type VII collagen, for which no effective therapy exists. ABCB5+ dermal mesenchymal stem cells (ABCB5+ MSCs) possess immunomodulatory, inflammation-dampening, and tissue-healing capacities. In a Col7a1–/– mouse model of RDEB, treatment with ABCB5+ MSCs markedly extended the animals’ lifespans.METHODS In this international, multicentric, single-arm, phase I/IIa clinical trial, 16 patients (aged 4–36 years) enrolled into 4 age cohorts received 3 i.v. infusions of 2 × 106 ABCB5+ MSCs/kg on days 0, 17, and 35. Patients were followed up for 12 weeks regarding efficacy and 12 months regarding safety.RESULTS At 12 weeks, statistically significant median (IQR) reductions in the Epidermolysis Bullosa Disease Activity and Scarring Index activity (EBDASI activity) score of 13.0% (2.9%–30%; P = 0.049) and the Instrument for Scoring Clinical Outcome of Research for Epidermolysis Bullosa clinician (iscorEB‑c) score of 18.2% (1.9%–39.8%; P = 0.037) were observed. Reductions in itch and pain numerical rating scale scores were greatest on day 35, amounting to 37.5% (0.0%–42.9%; P = 0.033) and 25.0% (–8.4% to 46.4%; P = 0.168), respectively. Three adverse events were considered related to the cell product: 1 mild lymphadenopathy and 2 hypersensitivity reactions. The latter 2 were serious but resolved without sequelae shortly after withdrawal of treatment.CONCLUSION This trial demonstrates good tolerability, manageable safety, and potential efficacy of i.v. ABCB5+ MSCs as a readily available disease-modifying therapy for RDEB and provides a rationale for further clinical evaluation.TRIAL REGISTRATION Clinicaltrials.gov NCT03529877; EudraCT 2018-001009-98.FUNDING The trial was sponsored by RHEACELL GmbH & Co. KG. Contributions by NYF and MHF to this work were supported by the NIH/National Eye Institute (NEI) grants RO1EY025794 and R24EY028767.

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. Successful engraftment of gene-corrected autologous hematopoietic stem cells (HSC) circumvents the need for an allogeneic HCT and has been achieved in other genetic diseases using targeted nucleases to induce site specific DSBs and the correction of mutated genes through homology-directed repair (HDR). However, this process is extremely inefficient in FA cells, as they are inherently deficient in DNA repair. Here, we demonstrate the correction of FANCA mutations in primary patient cells using ‘digital’ genome editing with the cytosine and adenine base editors (BEs). These Cas9-based tools allow for C:G > T:A or A:T > C:G base transitions without the induction of a toxic DSB or the need for a DNA donor molecule. These genetic corrections or conservative codon substitution strategies lead to phenotypic rescue as illustrated by a resistance to the alkylating crosslinking agent Mitomycin C (MMC). Further, FANCA protein expression was restored, and an intact FA pathway was demonstrated by downstream FANCD2 monoubiquitination induction. This BE digital correction strategy will enable the use of gene-corrected FA patient hematopoietic stem and progenitor cells (HSPCs) for autologous HCT, obviating the risks associated with allogeneic HCT and DSB induction during autologous HSC gene therapy.

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. We devised an approach where multiple gene products could be produced from a single population of cells. We identified gene specific Synergistic Activation Mediators (SAM) from the CRISPR/Cas9 system for targeted overexpression of coagulation factors II, VII, IX, X, and fibrinogen. The components of the CRISPR-SAM system were expressed in Human Embryonic Kidney Cells (HEK293), and single (singleplex) or multi-gene (multiplex) upregulation was assessed by quantitative RT-PCR (qRT-PCR) and protein expression by ELISA analysis. Factor II, VII, IX, and X singleplex and multiplex activation resulted in 120–4700-fold and 60–680-fold increases in gene expression, respectively. Fibrinogen sub-unit gene activation resulted in a 1700–92,000-fold increases and 80–5500-fold increases in singleplex or multiplex approaches, respectively. ELISA analysis showed a concomitant upregulation of candidate gene products. Our findings demonstrate the capability of CRISPR/Cas9 SAMs for single or multi-agent production in human cells and represent an engineering advance that augments current recombinant peptide production techniques.

Epidermolysis bullosa (EB) is a group of genetic blistering diseases characterized by mechanically fragile skin and mucocutaneous involvement. Historically, disease management has focused on supportive care. The development of new genetic, cellular, and recombinant protein therapies has shown promise, and this review summarizes a unique gene and cell therapy phenomenon termed revertant mosaicism (RM). RM is the spontaneous correction of a disease-causing mutation. It has been reported in most EB subtypes, some with relatively high frequency, and has been observed in both keratinocytes and fibroblasts. RM manifests as identifiable patches of unaffected, blister-resistant skin and can occur through a variety of molecular mechanisms, including true back mutation, intragenic crossover, mitotic gene conversion, and second-site mutation. RM cells represent a powerful autologous platform for therapy, and leveraging RM cells as a therapeutic substrate may avoid the inherent mutational risks of gene therapy/editing. However, further examination of the genomic integrity and long-term functionality of RM-derived cells, as well in vivo testing of systemic therapies with RM cells, is required to realize the full therapeutic promise of naturally occurring RM in EB.

Abstract Background X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene. 40% of X-ALD patients will convert to the deadly childhood cerebral form (ccALD) characterized by increased permeability of the brain endothelium that constitutes the blood–brain barrier (BBB). Mutation information and molecular markers investigated to date are not predictive of conversion. Prior reports have focused on toxic metabolic byproducts and reactive oxygen species as instigators of cerebral inflammation and subsequent immune cell invasion leading to BBB breakdown. This study focuses on the BBB itself and evaluates differences in brain endothelium integrity using cells from ccALD patients and wild-type (WT) controls. Methods The blood–brain barrier of ccALD patients and WT controls was modeled using directed differentiation of induced pluripotent stem cells (iPSCs) into induced brain microvascular endothelial cells (iBMECs). Immunocytochemistry and PCR confirmed characteristic expression of brain microvascular endothelial cell (BMEC) markers. Barrier properties of iBMECs were measured via trans-endothelial electrical resistance (TEER), sodium fluorescein permeability, and frayed junction analysis. Electron microscopy and RNA-seq were used to further characterize disease-specific differences. Oil-Red-O staining was used to quantify differences in lipid accumulation. To evaluate whether treatment with block copolymers of poly(ethylene oxide) and poly(propylene oxide) (PEO–PPO) could mitigate defective properties, ccALD-iBMECs were treated with PEO–PPO block copolymers and their barrier properties and lipid accumulation levels were quantified. Results iBMECs from patients with ccALD had significantly decreased TEER (2592 ± 110 Ω cm2) compared to WT controls (5001 ± 172 Ω cm2). They also accumulated lipid droplets to a greater extent than WT-iBMECs. Upon treatment with a PEO–PPO diblock copolymer during the differentiation process, an increase in TEER and a reduction in lipid accumulation were observed for the polymer treated ccALD-iBMECs compared to untreated controls. Conclusions The finding that BBB integrity is decreased in ccALD and can be rescued with block copolymers opens the door for the discovery of BBB-specific molecular markers that can indicate the onset of ccALD and has therapeutic implications for preventing the conversion to ccALD.

Pluripotent stem (PS)-cell-derived cell types hold promise for treating degenerative diseases. However, PS cell differentiation is intrinsically heterogeneous; therefore, clinical translation requires the development of practical methods for isolating progenitors from unwanted and potentially teratogenic cells. Muscle-regenerating progenitors can be derived through transient PAX7 expression. To better understand the biology, and to discover potential markers for these cells, here we investigate PAX7 genomic targets and transcriptional changes in human cells undergoing PAX7-mediated myogenic commitment. We identify CD54, integrin α9β1, and Syndecan2 (SDC2) as surface markers on PAX7-induced myogenic progenitors. We show that these markers allow for the isolation of myogenic progenitors using both fluorescent- and CGMP-compatible magnetic-based sorting technologies and that CD54+α9β1+SDC2+ cells contribute to long-term muscle regeneration in vivo. These findings represent a critical step toward enabling the translation of PS-cell-based therapies for muscle diseases.

In T cells with transgenic high-avidity T cell receptors (TCRs), endogenous and transferred TCR chains compete for surface expression and may pair inappropriately, potentially causing autoimmunity. To knock out endogenous TCR expression, we assembled 12 transcription activator-like effector nucleases (TALENs) and five guide RNAs (gRNAs) from the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system. Using TALEN mRNA, TCR knockout was successful in up to 81% of T cells. Additionally, we were able to verify targeted gene addition of a GFP gene by homology-directed repair at the TALEN target site, using a donor suitable for replacement of the reporter transgene with therapeutic TCR chains. Remarkably, analysis of TALEN and CRISPR/Cas9 specificity using integrase-defective lentiviral vector capture revealed only one off-target site for one of the gRNAs and three off-target sites for both of the TALENs, indicating a high level of specificity. Collectively, our work shows highly efficient and specific nucleases for T cell engineering.

Metabolism of oxidative stress is necessary for cellular survival. We have previously utilized the zebrafish as a model of the oxidative stress response. In this study, we found that gata1-expressing erythroid cells contributed to a significant proportion of total-body oxidative stress when animals were exposed to a strong pro-oxidant. RNA-seq of zebrafish under oxidative stress revealed the induction of tp53. Zebrafish carrying tp53 with a mutation in its DNA-binding domain were acutely sensitive to pro-oxidant exposure and displayed significant reactive oxygen species (ROS) and tp53-independent erythroid cell death resulting in an edematous phenotype. We found that a major contributing factor to ROS was increased basal mitochondrial respiratory rate without reserve. These data add to the concept that tp53, while classically a tumor suppressor and cell-cycle regulator, has additional roles in controlling cellular oxidative stress.

Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) proteins can be designed to bind specified DNA and RNA sequences and hold great promise for the accurate detection of nucleic acids for diagnostics. We integrated commercially available reagents into a CRISPR/Cas9-based lateral flow assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences with single-base specificity. This approach requires minimal equipment and represents a simplified platform for field-based deployment. We also developed a rapid, multiplex fluorescence CRISPR/Cas9 nuclease cleavage assay capable of detecting and differentiating SARS-CoV-2, influenza A and B, and respiratory syncytial virus in a single reaction. Our findings provide proof-of-principle for CRISPR/Cas9 point-of-care diagnosis as well as a scalable fluorescent platform for identifying respiratory viral pathogens with overlapping symptomology.

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. scVDMC clusters single cells in multiple scRNA-seq experiments of similar cell types and markers but varying expression patterns such that the scRNA-seq data are better integrated than typical pooled analyses which only increase the sample size. By controlling the variance among the cell clusters within each dataset and across all the datasets, scVDMC detects cell sub-populations in each individual experiment with shared cell-type markers but varying cluster centers among all the experiments. Applied to two real scRNA-seq datasets with several replicates and one large-scale droplet-based dataset on three patient samples, scVDMC more accurately detected cell populations and known cell markers than pooled clustering and other recently proposed scRNA-seq clustering methods. In the case study applied to in-house Recessive Dystrophic Epidermolysis Bullosa (RDEB) scRNA-seq data, scVDMC revealed several new cell types and unknown markers validated by flow cytometry. MATLAB/Octave code available at https://github.com/kuanglab/scVDMC.

Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

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. We achieved robust activation of the COL7A1 gene in primary human umbilical cord blood CD34+ hematopoietic stem cells and peripheral blood T-cells. CD34+ cells retained their colony forming potential and, in a second engineering step, we disrupted the T-cell receptor complex in T-cells. These cellular populations are of high translational impact due to their engraftment potential, broad circulatory properties, and favorable immune profile that supports delivery to multiple recipients. This study demonstrates the feasibility of targeted knock in of a ubiquitous chromatin opening element, promoter, and marker gene that doubles as a suicide gene for precision gene activation. This system merges the specificity of gene editing with the high level, sustained gene expression achieved with gene therapy vectors. We predict that this design concept will be highly transferrable to most genes in multiple model systems representing a facile cellular engineering platform for promoting gene expression.

Chimeric antigen receptor (CAR) T cell therapy represents the first U.S. Food and Drug Administration approved gene therapy and these engineered cells function with unprecedented efficacy in the treatment of refractory CD19 positive hematologic malignancies. CAR translation to solid tumors is also being actively investigated; however, efficacy to date has been variable due to tumor-evolved mechanisms that inhibit local immune cell activity. To bolster the potency of CAR-T cells, modulation of the immunosuppressive tumor microenvironment with immune-checkpoint blockade is a promising strategy. The impact of this approach on hematological malignancies is in its infancy, and in this review we discuss CAR-T cells and their synergy with immune-checkpoint blockade.

Jakub Tolar, Paul J OrchardDivision of Hematology, Oncology, Blood and Marrow Transplantation, Department of Pediatrics, University of Minnesota, Minneapolis, MN, USAAbstract: More than 500 patients with mucopolysaccharidosis type IH (MPS IH; Hurler syndrome) have been treated with hematopoietic cell transplantation (HCT) throughout the world since the introduction of transplantation as therapy almost 30 years ago. More recently, the availability of recombinant α-L-iduronidase (IDUA) has resulted in the widespread treatment of less severe forms of MPS I with enzyme replacement therapy (ERT). In addition, over 50 MPS IH patients have been treated with a combination of ERT and HCT. The rationale for both ERT and HCT stems from the pivotal experiments performed 4 decades ago that showed α-L-iduronidase supplied in the environment can correct the accumulation of substrate in MPS I cells. Our purpose is to address the multiple applications associated with the therapeutic delivery of IDUA: intermittent delivery of recombinant protein (ERT), continuous administration through cellular therapy (HCT), the use of other stem cells or, potentially, correction of the enzyme defect itself through gene therapy approaches. Even though gene therapy and non-hematopoietic stem cell approaches, have yet to be tested in a clinical setting, it is possible that all these approaches will in the near future be a part of a paradigm shift from unimodal to multimodal therapy for MPS I.Keywords: mucopolysaccharidosis type I, Hurler syndrome, hematopoietic cell transplantation, enzyme replacement therapy, co-modality therapy

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention.

Fanconi anemia (FA) is an inherited condition characterized by impaired DNA repair, physical anomalies, bone marrow failure, and increased incidence of malignancy. Gene editing holds great potential to precisely correct the underlying genetic cause such that gene expression remains under the endogenous control mechanisms. This has been accomplished to date only in transformed cells or their reprogrammed induced pluripotent stem cell counterparts; however, it has not yet been reported in primary patient cells. Here we show the ability to correct a mutation in Fanconi anemia D1 (FANCD1) primary patient fibroblasts. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was employed to target and correct a FANCD1 gene deletion. Homologous recombination using an oligonucleotide donor was achieved and a pure population of modified cells was obtained by using inhibitors of poly adenosine diphosphate-ribose polymerase (poly ADP-ribose polymerase). FANCD1 function was restored and we did not observe any promiscuous cutting of the CRISPR/Cas9 at off target sites. This consideration is crucial in the context of the pre-malignant FA phenotype. Altogether we show the ability to correct a patient mutation in primary FANCD1 cells in a precise manner. These proof of principle studies support expanded application of gene editing for FA.

Obliterative bronchiolitis (OB) is a significant cause of morbidity and mortality after lung transplant and hematopoietic cell transplant. Mesenchymal stromal cells (MSCs) have been shown to possess immunomodulatory properties in chronic inflammatory disease.Administration of MSCs was evaluated for the ability to ameliorate OB in mice using our established allogeneic bone marrow transplant (BMT) model.Mice were lethally conditioned and received allogeneic bone marrow without (BM) or with spleen cells (BMS), as a source of OB-causing T-cells. Cell therapy was started at 2 weeks post-transplant, or delayed to 4 weeks when mice developed airway injury, defined as increased airway resistance measured by pulmonary function test (PFT). BM-derived MSC or control cells [mouse pulmonary vein endothelial cells (PVECs) or lung fibroblasts (LFs)] were administered. Route of administration [intratracheally (IT) and IV] and frequency (every 1, 2 or 3 weeks) were compared. Mice were evaluated at 3 months post-BMT.No ectopic tissue formation was identified in any mice. When compared to BMS mice receiving control cells or no cells, those receiving MSCs showed improved resistance, compliance and inspiratory capacity. Interim PFT analysis showed no difference in route of administration. Improvements in PFTs were found regardless of dose frequency; but once per week worked best even when administration began late. Mice given MSC also had decreased peribronchiolar inflammation, lower levels of hydroxyproline (collagen) and higher frequencies of macrophages staining for the alternatively activated macrophage (AAM) marker CD206.These results warrant study of MSCs as a potential management option for OB in lung transplant and BMT recipients.

The ability of cells to detect changes in the microenvironment is important in cell signaling and responsiveness to environmental fluctuations. Our interest is in understanding how human bone marrow stromal-derived cells (MSC) and their relatives, vascular smooth muscle cells (VSMC), interact with their environment through novel receptors. We found, through a proteomics screen, that MSC express the bitter taste receptor, TAS2R46, a protein more typically localized to the taste bud. Expression was also confirmed in VSMCs. A prototypical bitter compound that binds to the bitter taste receptor class, denatonium, increased intracellular calcium release and decreased cAMP levels as well as increased the extracellular release of ATP in human MSC. Denatonium also bound and activated rodent VSMC with a change in morphology upon compound exposure. Finally, rodents given denatonium in vivo had a significant drop in blood pressure indicating a vasodilator response. This is the first description of chemosensory detection by MSC and VSMCs via a taste receptor. These data open a new avenue of research into discovering novel compounds that operate through taste receptors expressed by cells in the marrow and vascular microenvironments.

BackgroundX-linked adrenoleukodystrophy (ALD) is a metabolic, peroxisomal disease that results from a mutation in the ABCD1 gene. The most severe course of ALD progression is the cerebral inflammatory and demyelinating form of the disease, cALD. To date there is very little information on the cytokine mediators in the cerebral spinal fluid (CSF) of these boys.Methodology/principal findingsMeasurement of 23 different cytokines was performed on CSF and serum of boys with cerebral ALD and patients without ALD. Significant elevations in CSF IL-8 (29.3±2.2 vs 12.8±1.1 pg/ml, p = 0.0001), IL-1ra (166±30 vs 8.6±6.5 pg/ml, p = 0.005), MCP-1 (610±47 vs 328±34 pg/ml, p = 0.002), and MIP-1b (14.2±1.3 vs 2.0±1.4 pg/ml, pConclusions/significanceIL-8, IL-1ra, MCP-1, MIP-1b and CSF total protein were significantly elevated in patients with cALD; IL-8, MCP-1b, and CSF total protein levels correlated with disease severity determined by MRI. This is the largest report of CSF cytokine levels in cALD to date, and identification of these key cytokines will provide further insight into disease progression and perhaps lead to improved targeted therapies.

X-linked adrenoleukodystrophy results from mutations in the ABCD1 gene disrupting the metabolism of very-long-chain fatty acids. The most serious form of ALD, cerebral adrenoleukodystrophy (cALD), causes neuroinflammation and demyelination. Neuroimaging in cALD shows inflammatory changes and indicates blood-brain-barrier (BBB) disruption. We hypothesize that disruption may occur through the degradation of the extracellular matrix defining the BBB by matrix metalloproteinases (MMPs). MMPs have not been evaluated in the setting of cALD.We used a multiplex assay to correlate the concentration of MMPs in cerebrospinal fluid and plasma to the severity of brain inflammation as determined by the ALD MRI (Loes) score and the neurologic function score. There were significant elevations of MMP2, MMP9, MMP10, TIMP1, and total protein in the CSF of boys with cALD compared to controls. Levels of MMP10, TIMP1, and total protein in CSF showed significant correlation [p

BACKGROUND:The zebrafish is an increasingly popular model for studying many aspects of biology. Recently, ztert, the zebrafish homolog of the mammalian telomerase gene has been cloned and sequenced. In contrast to humans, it has been shown that the zebrafish maintains telomerase activity for much of its adult life and has remarkable regenerative capacity. To date, there has been no longitudinal study to assess whether this retention of telomerase activity equates to the retention of chromosome telomere length through adulthood. METHODOLOGY/PRINCIPAL FINDINGS:We have systematically analyzed individual organs of zebrafish with regard to both telomere length and telomerase activity at various time points in its adult life. Heart, gills, kidney, spleen, liver, and intestine were evaluated at 3 months, 6 months, 9 months, and 2 years of age by Southern blot analysis. We found that telomeres do not appreciably shorten throughout the lifespan of the zebrafish in any organ. In addition, there was little difference in telomere lengths between organs. Even when cells were under the highest pressure to divide after fin-clipping experiments, telomere length was unaffected. All aged (2 year old) tissues examined also expressed active amounts of telomerase activity as assessed by TRAP assay. CONCLUSIONS/SIGNIFICANCE:In contrast to several other species including humans, the retention of lifelong telomerase and telomeres, as we have reported here, would be necessary in the zebrafish to maintain its tremendous regenerative capacity. The ongoing study of the zebrafish's ability to maintain telomerase activity may be helpful in unraveling the complexity involved in the maintenance (or lack thereof) of telomeres in other species such the mouse or human.

Bone marrow mesenchymal stem cells are multipotent cells found lining the bone marrow cavity supporting the growth and differentiation of hematologic progenitors. There is growing evidence that these cells can, under the right circumstances, enter the peripheral circulation. We show that granulocyte colony-stimulating factor mobilized peripheral blood contains cells which form colonies and have a similar fibroblastic morphology (termed CFU-F) to bone marrow mesenchymal stem cells. These cells were found at a very low incidence (0.0002%). Mobilized peripheral blood CFU-F were successfully differentiated into osteogenic and adipogenic lineages. FACS analysis showed that the cells had a similar profile to bone marrow mesenchymal stem cells. Importantly, mobilized peripheral blood CFU-F had limited expansion potential and became senescent 20–25 days after isolation. Mobilized peripheral blood CFU-F also did not have any telomerase activity and displayed significant telomere shortening. The rarity of CFU-F in mobilized peripheral blood and the subsequent pressure to divide in cell culture probably contribute to early cellular senescence. Their potential for use in transplant or gene therapy is, therefore, limited.