Correction of recessive dystrophic epidermolysis bullosa by homology-directed repair-mediated genome editing.

Genome-editing technologies that enable the introduction of precise changes in DNA sequences have the potential to lead to a new class of treatments for genetic diseases. Epidermolysis bullosa (EB) is a group of rare genetic disorders characterized by extreme skin fragility. The recessive dystrophic subtype of EB (RDEB), which has one of the most severe phenotypes, is caused by mutations in COL7A1. In this study, we report a gene-editing approach for ex vivo homology-directed repair (HDR)-based gene correction that uses the CRISPR-Cas9 system delivered as a ribonucleoprotein (RNP) complex in combination with donor DNA templates delivered by adeno-associated viral vectors (AAVs). We demonstrate sufficient mutation correction frequencies to achieve therapeutic benefit in primary RDEB keratinocytes containing different COL7A1 mutations as well as efficient HDR-mediated COL7A1 modification in healthy cord blood-derived CD34+ cells and mesenchymal stem cells (MSCs). These results are a proof of concept for HDR-mediated gene correction in different cell types with therapeutic potential for RDEB.

The importance of immunity in the development of reliable animal models for psoriasis and atopic dermatitis.

Psoriasis (PS) and atopic dermatitis (AD) are common inflammatory skin diseases characterized by an imbalance in specific T-cell subsets, resulting in a specific cytokine profile in patients. Obtaining models closely resembling both pathologies along with a relevant clinical impact is crucial for the development of new therapies because of the high prevalence of these diseases. Single-gene mouse models developed until now do not fully reflect the complexity of these disorders, in part not only because of inherent differences between mice and humans but also because of the multifactorial nature of these pathologies. The skin-humanized mouse model developed by our group, based on a tissue engineering approach, has been used to test therapeutic strategies, although this methodology is still technically challenging and not widely available. The skin-humanized mouse models for PS and AD reproduce human skin phenotypes, providing valuable tools for drug development and testing in the preclinical setting. The tissue engineering approach allows the development of personalized medicine, covering the broad genotypic spectrum of these pathologies. This review highlights the main differences between available murine models focusing on the tissue-specific immunity of PS and AD. We discuss their contribution to unravel the complex pathophysiology of these diseases and to translate this knowledge into more accurate therapies.

Persistent hypogammaglobulinemia in pediatric solid organ transplant recipients.

INTRODUCTION: Hypogammaglobulinemia has not been well studied in pediatric solid organ transplant (SOT) recipients. We evaluated plasma immunoglobulin (Ig) and lymphocyte phenotypes among 31 pediatric heart and kidney recipients for two years post-transplant and from 10 non-transplanted children. METHODS: Plasma IgM, IgG, and IgA were quantified by immunoturbidimetric assays, IgG subclasses were quantified by bead-based multiplex immunoassay, and lymphocyte phenotypes were assessed by flow cytometry. RESULTS: Median age at transplant for SOT recipients was similar to that of the control cohort (15 vs. 12.5 years, respectively; P = .61). Mean plasma IgG and IgM levels for SOT recipients fell significantly below the control cohort means by 1 month post-transplant (P < .001 for both) and remained lower than control levels at 12-18 months post-transplant. Heart recipients had lower frequencies of a CD4+ naïve T lymphocytes relative to kidney recipients. CONCLUSIONS: Hypogammaglobulinemia was prevalent and persistent among pediatric SOT recipients and may be secondary to immunosuppressive medications, as well as loss of thymus tissue and CD45RA+   CD4+ T cells in heart recipients. Limitations of our study include but are not limited to small sample size from a single center, lack of samples for all participants at every time point, and lack of peripheral blood mononuclear cell samples for the non-transplanted cohort.

Malaria and acute kidney injury.

Malaria is a parasitic infection transmitted by mosquitos, resulting in significant morbidity and mortality. It affects 212 million worldwide, causing death in up to 303,000 children annually. In the USA, up to 1700 people are affected yearly. Although the prevalence in developed countries is less than in developing countries, travelers from low transmission areas, and those from endemic areas who later return, are very susceptible to malaria and its complications. Severe malaria can cause significant multiorgan dysfunction including acute kidney injury (AKI). The pathogenesis is not clearly understood but proposed mechanisms include acute tubular necrosis (ATN) due to impediments in renal microcirculation, infection-triggered proinflammatory reactions within the kidney, and metabolic disturbances. Providers must consider malarial infection in cases of AKI in someone with a travel history, as early recognition and treatment are crucial to improving outcomes. This article will review malaria-induced AKI in order to provide a better understanding of this infection's effect on the kidneys.

An uncommon case of arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome and review of the renal involvement: Questions.

Arthrogryposis, renal dysfunction, and cholestasis syndrome is a rare autosomal recessive disorder caused by mutations in the VPS33B and VIPAR genes. Most cases are fatal within the first year of life. Here we describe one of the two oldest patients with arthrogryposis, renal dysfunction, and cholestasis syndrome. This is a 12-year-old Hispanic female, from a non-consanguineous parents, diagnosed with an incomplete phenotype of arthrogryposis, renal dysfunction, and cholestasis syndrome with arthrogryposis and renal tubular dysfunction but without cholestasis. At 11 years of age, she was found to have impaired renal function, nephrotic-range proteinuria, Fanconi syndrome, and distal renal tubular acidosis. She also had hypercalciuria, nephrogenic diabetes insipidus, and small kidneys by renal ultrasound. Genetic analysis using whole exome sequencing showed a mutation and a partial deletion in the VPS33B gene. Further studies showed that the mother has a partial deletion in the VPS33B gene. Her medication regimen includes potassium citrate and enalapril.

Tacrolimus variability is associated with de novo donor-specific antibody development in pediatric renal transplant recipients.

BACKGROUND: Donor-specific antibody (DSA) is a risk factor for antibody-mediated rejection and shortened graft survival. We investigated the role of intrapatient variability in tacrolimus trough levels on graft outcomes (i.e., de novo DSA, rejection, graft loss) in pediatric renal transplant recipients. METHODS: This was a single-center retrospective study which included 38 pediatric renal transplant recipients. Intrapatient tacrolimus variability was defined using the coefficient of variation (CV; SD/Mean × 100) for all levels obtained after 3 months post-transplant. CV cut-points of 30%, 40%, and 50% were used in the analyses. RESULTS: The median CV 43.1% (35.0%, 58.6%). Out of 38 patients, 19 (50%) developed de novo DSA. In the logistic regression model, after adjusting for age, rejection history, maintenance immunosuppression, and CV, for every 10% increase in tacrolimus variability, the odds of developing de novo DSA increased by 53% (p = 0.048, CI 1.0005, 1.11). Age at transplant was also an independent risk factor for DSA development; every 1 year increase in age was associated with a 31% increase in the odds of developing DSA (p = 0.03, CI 1.03, 1.67). At a CV cut-point ≥ 30%, higher tacrolimus variability was associated with an increased incidence of allograft rejection (0% vs 42%, < 30 and ≥ 30% respectively, p = 0.07). As there were few graft loss events (n = 4) in our study population, an association could not be determined between tacrolimus variability and graft loss. CONCLUSION: Tacrolimus variability and age at transplant were identified as independent risk factors for de novo DSA development. There was an association between tacrolimus variability and rejection in pediatric renal transplant recipients. Adding the assessment of tacrolimus variability to current monitoring methods may be an important step towards improving graft outcomes.

Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing.

Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations in COL7A1 causing recessive dystrophic epidermolysis bullosa are amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeted deletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA (sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved very efficient targeted deletion of COL7A1 exon 80 in recessive dystrophic epidermolysis bullosa (RDEB) patient keratinocytes carrying a highly prevalent frameshift mutation. This ex vivo non-viral approach rendered a large proportion of corrected cells producing a functional collagen VII variant. The effective targeting of the epidermal stem cell population enabled long-term regeneration of a properly adhesive skin upon grafting onto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sites did not reveal any unintended nuclease activity. Our strategy could potentially be extended to a large number of COL7A1 mutation-bearing exons within the long collagenous domain of this gene, opening the way to precision medicine for RDEB.

Humanization of Tumor Stroma by Tissue Engineering as a Tool to Improve Squamous Cell Carcinoma Xenograft.

The role of stroma is fundamental in the development and behavior of epithelial tumors. In this regard, limited growth of squamous cell carcinomas (SCC) or cell-lines derived from them has been achieved in immunodeficient mice. Moreover, lack of faithful recapitulation of the original human neoplasia complexity is often observed in xenografted tumors. Here, we used tissue engineering techniques to recreate a humanized tumor stroma for SCCs grafted in host mice, by combining CAF (cancer associated fibroblasts)-like cells with a biocompatible scaffold. The stroma was either co-injected with epithelial cell lines derived from aggressive SCC or implanted 15 days before the injection of the tumoral cells, to allow its vascularization and maturation. None of the mice injected with the cell lines without stroma were able to develop a SCC. In contrast, tumors were able to grow when SCC cells were injected into previously established humanized stroma. Histologically, all of the regenerated tumors were moderately differentiated SCC with a well-developed stroma, resembling that found in the original human neoplasm. Persistence of human stromal cells was also confirmed by immunohistochemistry. In summary, we provide a proof of concept that humanized tumor stroma, generated by tissue engineering, can facilitate the development of epithelial tumors in immunodeficient mice.

Ofatumumab in post-transplantation recurrence of focal segmental glomerulosclerosis in a child.

FSGS is a potentially devastating form of nephrotic syndrome. Treatment of SRNS can be difficult, especially post-transplantation. The current therapy of post-transplant SRNS includes plasmapheresis, ACE-I, CNI, and monoclonal antibodies (rituximab). Patients who are refractory to these interventions have limited therapeutic alternatives. We present a case of a patient with SRNS secondary to FSGS. He did not respond to immunosuppressive medications prior to transplant, progressed to ESRD, and was started on chronic hemodialysis. He received a DDKT which was complicated by post-transplant FSGS recurrence. A course of plasmapheresis, rituximab, and CNI were administered with some response. Ofatumumab was then given to the patient. As a result, the patient achieved partial remission. Ofatumumab may be a safe and effective option for post-transplant recurrence of FSGS.

CRISPR/Cas9-Mediated In Situ Correction of LAMB3 Gene in Keratinocytes Derived from a Junctional Epidermolysis Bullosa Patient.

Deficiency of basement membrane heterotrimeric laminin 332 component, coded by LAMA3, LAMB3, and LAMC2 genes, causes junctional epidermolysis bullosa (JEB), a severe skin adhesion defect. Herein, we report the first application of CRISPR/Cas9-mediated homology direct repair (HDR) to in situ restore LAMB3 expression in JEB keratinocytes in vitro and in immunodeficient mice transplanted with genetically corrected skin equivalents. We packaged an adenovector carrying Cas9/guide RNA (gRNA) tailored to the intron 2 of LAMB3 gene and an integration defective lentiviral vector bearing a promoterless quasi-complete LAMB3 cDNA downstream a splice acceptor site and flanked by homology arms. Upon genuine HDR, we exploited the in vitro adhesion advantage of laminin 332 production to positively select LAMB3-expressing keratinocytes. HDR and restored laminin 332 expression were evaluated at single-cell level. Notably, monoallelic-targeted integration of LAMB3 cDNA was sufficient to in vitro recapitulate the adhesive property, the colony formation typical of normal keratinocytes, as well as their cell growth. Grafting of genetically corrected skin equivalents onto immunodeficient mice showed a completely restored dermal-epidermal junction. This study provides evidence for efficient CRISPR/Cas9-mediated in situ restoration of LAMB3 expression, paving the way for ex vivo clinical application of this strategy to laminin 332 deficiency.

COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa.

Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future.

Topical enzyme-replacement therapy restores transglutaminase 1 activity and corrects architecture of transglutaminase-1-deficient skin grafts.

Transglutaminase-1 (TG1)-deficient autosomal-recessive congenital ichthyosis (ARCI) is a rare and severe genetic skin disease caused by mutations in TGM1. It is characterized by collodion babies at birth, dramatically increased transepidermal water loss (TEWL), and lifelong pronounced scaling. The disease has a tremendous burden, including the problem of stigmatization. Currently, no therapy targeting the molecular cause is available, and the therapeutic situation is deplorable. In this study, we developed the basis for a causative therapy aiming at the delivery of the enzyme to the inner site of the keratinocytes' plasma membrane. We prepared sterically stabilized liposomes with encapsulated recombinant human TG1 (rhTG1) and equipped with a highly cationic lipopeptide vector to mediate cellular uptake. The liposomes overcame the problems of insufficient cutaneous delivery and membrane penetration and provided excellent availability and activity of rhTG1 in primary keratinocytes. To demonstrate the general feasibility of this therapeutic approach in a humanized context, we used a skin-humanized mouse model. Treatment with rhTG1 liposomes resulted in considerable improvement of the ichthyosis phenotype and in normalization of the regenerated ARCI skin: in situ monitoring showed a restoration of TG1 activity, and cholesterol clefts vanished ultrastructurally. Measurement of TEWL revealed a restoration of epidermal barrier function. We regard this aspect as a major advance over available nonspecific approaches making use of, for example, retinoid creams. We conclude that this topical approach is a promising strategy for restoring epidermal integrity and barrier function and provides a causal cure for individuals with TG1 deficiency.

Pigmentation and melanocyte supply to the epidermis depend on type XVII collagen.

Genetic deficiency of type XVII collagen (C17), laminin-332 or type VII collagen causes epidermolysis bullosa (EB). Spontaneous correction of the deficiency, also known as revertant mosaicism, is caused by a second somatic mutation that restores protein expression resulting in clinically healthy (revertant) patches surrounded by fragile (mutant) skin. Interestingly, in some patients, patches of revertant skin show hyperpigmentation. To study the possible role of affected proteins in pigmentation and melanocyte distribution, we investigated clinical documentation and skin biopsy specimens of 13 revertant EB patients having correcting mutations in the COL17A1, LAMB3 or COL7A1 genes. Analysis revealed that lack of C17 led to decreased melanin intensity and melanocyte density in the epidermis when compared with the revertant patches. Reversions of LAMB3 and COL7A1 in keratinocytes did not influence clinical pigmentation or density of melanocytes. We conclude that in human skin, melanocyte supply to the epidermis depends on C17 expression in keratinocytes.

Targeted silencing of DEFB4 in a bioengineered skin-humanized mouse model for psoriasis: development of siRNA SECosome-based novel therapies.

Psoriasis is a complex inflammatory skin disease that presents a wide variety of clinical manifestations. Human ß defensin-2 (hBD-2) is highly up-regulated in psoriatic lesions and has been defined as a biomarker for disease activity. We explored the potential benefits of targeting hBD-2 by topical application of DEFB4-siRNA-containing SECosomes in a bioengineered skin-humanized mouse model for psoriasis. A significant improvement in the psoriatic phenotype was observed by histological examination, with a normalization of the skin architecture and a reduction in the number and size of blood vessels in the dermal compartment. Treatment leads to the recovery of transglutaminase activity, filaggrin expression and stratum corneum appearance to the levels similar to those found in normal regenerated human skin. The availability of a reliable skin-humanized mouse model for psoriasis in conjunction with the use of the SECosome technology may provide a valuable preclinical tool for identifying potential therapeutic targets for this disease.