Complete genome sequence of the probiotic Bifidobacterium adolescentis strain iVS-1.

Bifidobacterium adolescentis iVS-1 is a human-isolated strain known to possess several probiotic properties. Here, its genome was completely sequenced to examine genes associated with lactose metabolism and other potentially beneficial traits, such as the production of folate and gamma-aminobutyric acid (GABA).

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.

Kombucha tea as an anti-hyperglycemic agent in humans with diabetes - a randomized controlled pilot investigation.

Introduction: Kombucha is a popular fermented tea that has attracted considerable attention due, in part, to its suggested health benefits. Previous results from animal models led us to hypothesize kombucha may reduce blood sugar levels in humans with diabetes. The objective of this pilot clinical study was to evaluate kombucha for its anti-hyperglycemic activities in adults with diabetes mellitus type II. Methods: The study was organized as a prospective randomized double-blinded crossover study at a single-center urban hospital system. Participants (n = 12) were instructed to consume either a kombucha product or a placebo control (each 240 mL) for 4 weeks. After an 8-week washout period, participants consumed the alternate product. Fasting blood glucose levels were self-determined at baseline and at 1 and 4 weeks during each treatment period. Secondary health outcomes, including overall health, insulin requirement, gut health, skin health, mental health, and vulvovaginal health were measured by questionnaire at the same time points. The kombucha microbiota was assessed by selective culturing and 16S rRNA gene (bacteria) and ITS (fungi) sequencing. Fermentation end products were assessed by HPLC. Statistical significance of changes in fasting blood glucose was determined using paired, two-tailed student's t-tests. Results: Kombucha lowered average fasting blood glucose levels at 4 weeks compared to baseline (164 vs. 116 mg/dL, p = 0.035), whereas the placebo did not (162 vs. 141 mg/dL, p = 0.078). The kombucha microbiota, as assessed by cultural enumeration, was mainly comprised of lactic acid bacteria, acetic acid bacteria, and yeast, with each group present at about 106 colony forming units (CFU)/mL. Likewise, 16S rRNA gene sequencing confirmed that lactic acid and acetic acid bacteria were the most abundant bacteria, and ITS sequencing showed Dekkera was the most abundant yeast. The primary fermentation end products were lactic and acetic acids, both less than 1%. Ethanol was present at 1.5%. Discussion: Although this pilot study was limited by a small sample size, kombucha was associated with reduced blood glucose levels in humans with diabetes. Larger follow-up studies are warranted. Clinical trial registration: ClinicalTrials.gov, identifier NCT04107207.

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.

Prebiotics enhance persistence of fermented-food associated bacteria in in vitro cultivated fecal microbial communities.

It is well established that the gastrointestinal (GI) microbiota plays a major role in human health. Dietary interventions, and consumption of fermented foods that contain live microbes, in particular, are among the approaches being investigated to modulate the GI microbiota and improve health. However, the persistence of fermented food-associated bacteria (FAB) within the GI tract is typically limited by host factors that limit colonization and competition with autochthonous microbes. In this research, we examined if the addition of prebiotics, dietary substrates that are selectively metabolized by microbes to improve health, would enhance the persistence of FAB. We evaluated the persistence of bacteria from three live microbe-containing fermented foods-kefir, sausage, and sauerkraut-in fecal microbial communities from four healthy adults. Fecal communities were propagated in vitro and were inoculated with fermented food-associated microbes from kefir, sausage, or sauerkraut at ~107 CFU/mL. Communities were diluted 1:100 every 24 h into fresh gut simulation medium to simulate microbial community turnover in the GI tract. We measured the persistence of Lactobacillaceae from fermented foods by quantitative PCR (qPCR) and the persistence of other FAB through 16S rRNA gene sequencing. FAB were unable to persist in vitro, reaching undetectable levels within 96 h. Addition of prebiotics, including xylooligosaccharides and a mixture of fructooligosaccharides and galactooligosaccharides enhanced the persistence of some species of FAB, but the level of persistence varied by fecal donor, fermented food, and prebiotic tested. Addition of prebiotics also increased the relative abundance of Bifidobacterium species, which most likely originated from the fecal microbiota. Collectively, our results support previous in vivo studies demonstrating the transient nature of FAB in the GI tract and indicate that consumption of prebiotics may enhance their persistence.

Cell and Gene Therapies for Mucopolysaccharidoses: Base Editing and Therapeutic Delivery to the CNS.

Although individually uncommon, rare diseases collectively account for a considerable proportion of disease impact worldwide. A group of rare genetic diseases called the mucopolysaccharidoses (MPSs) are characterized by accumulation of partially degraded glycosaminoglycans cellularly. MPS results in varied systemic symptoms and in some forms of the disease, neurodegeneration. Lack of treatment options for MPS with neurological involvement necessitates new avenues of therapeutic investigation. Cell and gene therapies provide putative alternatives and when coupled with genome editing technologies may provide long term or curative treatment. Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing technology and, more recently, advances in genome editing research, have allowed for the addition of base editors to the repertoire of CRISPR-based editing tools. The latest versions of base editors are highly efficient on-targeting deoxyribonucleic acid (DNA) editors. Here, we describe a number of putative guide ribonucleic acid (RNA) designs for precision correction of known causative mutations for 10 of the MPSs. In this review, we discuss advances in base editing technologies and current techniques for delivery of cell and gene therapies to the site of global degeneration in patients with severe neurological forms of MPS, the central nervous system, including ultrasound-mediated blood-brain barrier disruption.

A Prospective Treatment Option for Lysosomal Storage Diseases: CRISPR/Cas9 Gene Editing Technology for Mutation Correction in Induced Pluripotent Stem Cells.

Ease of design, relatively low cost and a multitude of gene-altering capabilities have all led to the adoption of the sophisticated and yet simple gene editing system: clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). The CRISPR/Cas9 system holds promise for the correction of deleterious mutations by taking advantage of the homology directed repair pathway and by supplying a correction template to the affected patient's cells. Currently, this technique is being applied in vitro in human-induced pluripotent stem cells (iPSCs) to correct a variety of severe genetic diseases, but has not as of yet been used in iPSCs derived from patients affected with a lysosomal storage disease (LSD). If adopted into clinical practice, corrected iPSCs derived from cells that originate from the patient themselves could be used for therapeutic amelioration of LSD symptoms without the risks associated with allogeneic stem cell transplantation. CRISPR/Cas9 editing in a patient's cells would overcome the costly, lifelong process associated with currently available treatment methods, including enzyme replacement and substrate reduction therapies. In this review, the overall utility of the CRISPR/Cas9 gene editing technique for treatment of genetic diseases, the potential for the treatment of LSDs and methods currently employed to increase the efficiency of this re-engineered biological system will be discussed.