Base editing corrects the common Salla disease SLC17A5 c.115C>T variant.

Free sialic acid storage disorders (FSASDs) result from pathogenic variations in the SLC17A5 gene, which encodes the lysosomal transmembrane protein sialin. Loss or deficiency of sialin impairs FSA transport out of the lysosome, leading to cellular dysfunction and neurological impairment, with the most severe form of FSASD resulting in death during early childhood. There are currently no therapies for FSASDs. Here, we evaluated the efficacy of CRISPR-Cas9-mediated homology directed repair (HDR) and adenine base editing (ABE) targeting the founder variant, SLC17A5 c.115C>T (p.Arg39Cys) in human dermal fibroblasts. We observed minimal correction of the pathogenic variant in HDR samples with a high frequency of undesired insertions/deletions (indels) and significant levels of correction for ABE-treated samples with no detectable indels, supporting previous work showing that CRISPR-Cas9-mediated ABE outperforms HDR. Furthermore, ABE treatment of either homozygous or compound heterozygous SLC17A5 c.115C>T human dermal fibroblasts demonstrated significant FSA reduction, supporting amelioration of disease pathology. Translation of this ABE strategy to mouse embryonic fibroblasts harboring the Slc17a5 c.115C>T variant in homozygosity recapitulated these results. Our study demonstrates the feasibility of base editing as a therapeutic approach for the FSASD variant SLC17A5 c.115C>T and highlights the usefulness of base editing in monogenic diseases where transmembrane protein function is impaired.

Generation of two induced pluripotent stem cell lines (CHOCi002-A and CHOCi003-A) from Pompe disease patients with compound heterozygous mutations in the GAA gene.

Pompe disease is an autosomal recessive lysosomal storage disease caused by pathogenic variants in GAA, which encodes an enzyme integral to glycogen catabolism, acid α-glucosidase. Disease-relevant cell lines are necessary to evaluate the efficacy of genotype-specific therapies. Dermal fibroblasts from two patients presenting clinically with Pompe disease were reprogrammed to induced pluripotent stem cells using the Sendai viral method. One patient is compound heterozygous for the c.258dupC (p.N87QfsX9) frameshift mutation and the c.2227C>T (p.Q743X) nonsense mutation. The other patient harbors the c.-32-13T>G splice variant and the c.1826dupA (p.Y609X) frameshift mutation in compound heterozygosity.

CRISPR-mediated generation and characterization of a Gaa homozygous c.1935C>A (p.D645E) Pompe disease knock-in mouse model recapitulating human infantile onset-Pompe disease.

Pompe disease, an autosomal recessive disorder caused by deficient lysosomal acid α-glucosidase (GAA), is characterized by accumulation of intra-lysosomal glycogen in skeletal and oftentimes cardiac muscle. The c.1935C>A (p.Asp645Glu) variant, the most frequent GAA pathogenic mutation in people of Southern Han Chinese ancestry, causes infantile-onset Pompe disease (IOPD), presenting neonatally with severe hypertrophic cardiomyopathy, profound muscle hypotonia, respiratory failure, and infantile mortality. We applied CRISPR-Cas9 homology-directed repair (HDR) using a novel dual sgRNA approach flanking the target site to generate a Gaaem1935C>A knock-in mouse model and a myoblast cell line carrying the Gaa c.1935C>A mutation. Herein we describe the molecular, biochemical, histological, physiological, and behavioral characterization of 3-month-old homozygous Gaaem1935C>A mice. Homozygous Gaaem1935C>A knock-in mice exhibited normal Gaa mRNA expression levels relative to wild-type mice, had near-abolished GAA enzymatic activity, markedly increased tissue glycogen storage, and concomitantly impaired autophagy. Three-month-old mice demonstrated skeletal muscle weakness and hypertrophic cardiomyopathy but no premature mortality. The Gaaem1935C>A knock-in mouse model recapitulates multiple salient aspects of human IOPD caused by the GAA c.1935C>A pathogenic variant. It is an ideal model to assess innovative therapies to treat IOPD, including personalized therapeutic strategies that correct pathogenic variants, restore GAA activity and produce functional phenotypes.

A double-blinded placebo-controlled evaluation of adipose-derived mesenchymal stem cells in treatment of canine atopic dermatitis.

Mesenchymal stem cells (MSCs) have emerged as a new therapy for various immune-mediated inflammatory diseases. In this study we perform the first double-blinded, placebo-controlled evaluation of the efficacy of adipose-derived allogenic canine MSCs for the treatment of canine atopic dermatitis (cAD). Enrolled canine patients were randomly divided into placebo (PBS saline), low-dose (5 × 105 cells/kg), and high-dose (5 × 106 cells/kg) treatment groups. Each patient received three subcutaneous MSCs treatments or PBS saline at four-week intervals with injections at five sites. Patients were monitored by physical exams, pruritus visual analog scales (PVAS) signed by the primary caretaker, canine atopic dermatitis extent and severity index-4 (CADESI-4) scores by two veterinarians, and complete blood count and serum chemistry analysis along with laboratory analysis for potential biomarkers. Patients were kept off any immune-modulating drugs during the study period, and oral antibiotics and topicals were used for managing pruritus and secondary infections. The PVAS scores and the serum miR-483 levels were significantly lower in the high dose group compared to the placebo group at day90 post first-treatment. The CADESI-4 scores of the high dose group also showed downward trends. No severe adverse effects were observed in any patient in this study. The high dose MSC treatment is efficacious in alleviating the clinical signs of cAD until 30 days after the last subcutaneous administration of MSCs, and miRNA-483 may be a reliable prognostic biomarker for cAD. The MSCs efficacy and potential biomarkers should be further explored by a larger scale clinical trial.

Phosphodiesterase 4D, miR-203 and selected cytokines in the peripheral blood are associated with canine atopic dermatitis.

Canine Atopic Dermatitis (AD) is a common complex and multifactorial disease involving immune dysregulation, genetic predisposition, skin barrier defects, environmental factors and allergic sensitization. To date, diagnosis of canine AD relies on a combination of patient history, clinical examination, allergy testing and response to diet trials/therapies with no reliable biomarkers available to distinguish AD from other diseases with similar clinical presentations. A handful of studies to identify potential biomarkers in the peripheral blood of AD dogs and healthy controls have been performed with some showing inconsistent and contradictory results. In this study, we, for the first time, report statistically significant increases in expression of phosphodiesterase 4D (PDE4D) gene in peripheral blood mononuclear cells (PBMCs) and miR-203 in plasma from AD dogs compared to healthy controls. In addition, we report a statistically non-significant change of the CD4+/CD8+ ratio, a dramatic decrease of three gene markers (PIAS1, RORA and SH2B1) as well as a panel of differential expression of cytokines in AD dogs in comparison to the healthy controls. Our study provides important insight into the complexities of canine AD, and further studies to verify the specificity of these findings for canine AD at a larger-scale are warranted.

Recent Development of Wnt Signaling Pathway Inhibitors for Cancer Therapeutics.

PURPOSE OF REVIEW: Review current understanding of both canonical and non-canonical Wnt signaling in cancer and provide updated knowledge in current clinical trials of Wnt signaling drugs. RECENT FINDINGS: Important roles of both canonical and non-canonical Wnt signaling in cancer have been increasingly recognized. Recent clinical trials of several Wnt-signaling drugs have showed promising outcomes. In addition, some drugs that were originally approved for the treatment of other diseases have been recently found to block Wnt signaling, highlighting their potential to treat Wnt-dependent cancer. Dysfunction of Wnt signaling is implicated in cancer, and targeting Wnt signaling represents a useful approach to treat cancer. Current clinical trials of Wnt signaling drugs have showed promising outcomes, and repurposing the previously approved drugs for other diseases to treat Wnt-dependent cancer requires further studies.

Starvation but not locomotion enhances heart robustness in Drosophila.

Insects and vertebrates have multiple major physiological systems, each species having a circulatory system, a metabolic system, and a respiratory system that enable locomotion and survival in stressful environments, among other functions. Broadening our understanding of the physiology of Drosophila melanogaster requires the parsing of interrelationships among such major component physiological systems. By combining electrical pacing and flight exhaustion assays with manipulative conditioning, we have started to unpack the interrelationships between cardiac function, locomotor performance, and other functional characters such as starvation and desiccation resistance. Manipulative sequences incorporating these four physiological characters were applied to five D. melanogaster lab populations that share a common origin from the wild and a common history of experimental evolution. While exposure to starvation or desiccation significantly reduced flight duration, exhaustion due to flight only affected subsequent desiccation resistance. A strong association was found between flight duration and desiccation resistance, providing additional support for the hypothesis that these traits depend on glycogen and water content. However, there was negligible impact on rate of cardiac arrests from exhaustion by flight or exposure to desiccant. Brief periods of starvation significantly lowered the rate of cardiac arrest. These results provide suggestive support for the adverse impact of lipids on Drosophila heart robustness, a parallel result to those of many comparable studies in human cardiology. Overall, this study underscores clear distinctions among the connections between specific physiological responses to stress and specific types of physiological performance.