In vivo CRISPR-Cas9-mediated DNA chop identifies a cochlear outer hair cell-specific enhancer.

Cochlear outer hair cells (OHCs) are essential for hearing. A short, OHC-specific enhancer is necessary but not yet available for gene therapeutic applications in OHC damage. Such damage is a major cause of deafness. Prestin is a motor protein exclusively expressed in OHCs. We hypothesized that the cis-regulatory DNA fragment deletion of Slc26a5 would affect its expression. We tested this hypothesis by conducting CRISPR/Cas9-mediated large DNA fragment deletion of mouse Slc26a5 intron regions. First, starting from a ~13 kbp fragment, step-by-step, we narrowed down the sequence to a 1.4 kbp segment. By deleting either a 13 kbp or 1.4 kbp fragment, we observed delayed Prestin expression. Second, we showed that 1.4 kbp was an OHC-specific enhancer because enhanced green fluorescent protein (EGFP) was highly and specifically expressed in OHCs in a transgenic mouse where EGFP was driven by the 1.4 kbp segment. More importantly, specific EGFP was also driven by its homologous 398 bp fragment in human Slc26a5. This suggests that the enhancer is likely to be evolutionarily conserved across different species.

Decreased SLC26A3 expression and function in intestinal epithelial cells in response to Cryptosporidium parvum infection.

The protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a diarrheal disease worldwide. Infection in immunocompetent hosts typically results in acute, self-limiting, or recurrent diarrhea. However, in immunocompromised individuals infection can cause fulminant diarrhea, extraintestinal manifestations, and death. To date, the mechanisms underlying CP-induced diarrheal pathogenesis are poorly understood. Diarrheal diseases most commonly involve increased secretion and/or decreased absorption of fluid and electrolytes. We and others have previously shown impaired chloride absorption in infectious diarrhea due to dysregulation of SLC26A3 [downregulated in adenoma (DRA)], the human intestinal apical membrane Cl-/HCO3- exchanger protein. However, there are no studies on the effects of CP infection on DRA activity. Therefore, we examined the expression and function of DRA in intestinal epithelial cells in response to CP infection in vitro and in vivo. CP infection (0.5 × 106 oocysts/well in 24-well plates, 24 h) of Caco-2 cell monolayers significantly decreased Cl-/HCO3- exchange activity (measured as DIDS-sensitive 125I uptake) as well as DRA mRNA and protein levels. Substantial downregulation of DRA mRNA and protein was also observed following CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mucosa of C57BL/6 mice for 24 h. However, at 48 h after infection in vivo, the effects on DRA mRNA and protein were attenuated and at 5 days after infection DRA returned to normal levels. Our results suggest that impaired chloride absorption due to downregulation of DRA could be one of the contributing factors to CP-induced acute, self-limiting diarrhea in immunocompetent hosts.

Synthesis and evaluation of tetrahydropyrazolopyridine inhibitors of anion exchange protein SLC26A4 (pendrin).

Pendrin is a transmembrane chloride/anion antiporter that is strongly upregulated in the airways in rhinoviral infection, asthma, cystic fibrosis and chronic rhinosinusitis. Based on its role in the regulation of airway surface liquid depth, pendrin inhibitors have potential indications for treatment of inflammatory airways diseases. Here, a completely regioselective route to tetrahydro-pyrazolopyridine pendrin inhibitors based on 1,3-diketone and substituted hydrazine condensation was been developed. Structure-activity relationships at the tetrahydropyridyl nitrogen were investigated using a focused library, establishing the privileged nature of N-phenyl ureas and improving inhibitor potency by greater than 2-fold.

Effective PEI-mediated delivery of CRISPR-Cas9 complex for targeted gene therapy.

The-state-of-art CRISPR/Cas9 is one of the most powerful among the approaches being developed to rescue fundamental causes of gene-based inheritable diseases. Several strategies for delivering such genome editing materials have been developed, but the safety, efficacy over time, cost of production, and gene size limitations are still under debate and must be addressed to further improve applications. In this study, we evaluated branched forms of the polyethylenimine (PEI) - branched PEI 25 kDa (BPEI-25K) - and found that it could efficiently deliver CRISPR/Cas9 plasmids. Plasmid DNA expressing both guide RNA and Cas9 to target the Slc26a4 locus was successfully delivered into Neuro2a cells and meditated genome editing within the targeted locus. Our results demonstrated that BPEI-25K is a promising non-viral vector to deliver the CRISPR/Cas9 system in vitro to mediate targeted gene therapy, and these findings contribute to an understanding of CRISPR/Cas9 delivery that may enable development of successful in vivo techniques.

SLC26A6-selective inhibitor identified in a small-molecule screen blocks fluid absorption in small intestine.

SLC26A6 (also known as putative anion transporter 1 [PAT1]) is a Cl-/HCO3- exchanger expressed at the luminal membrane of enterocytes where it facilitates intestinal Cl- and fluid absorption. Here, high-throughput screening of 50,000 synthetic small molecules in cells expressing PAT1 and a halide-sensing fluorescent protein identified several classes of inhibitors. The most potent compound, the pyrazolo-pyrido-pyrimidinone PAT1inh-B01, fully inhibited PAT1-mediated anion exchange (IC50 ~350 nM), without inhibition of the related intestinal transporter SLC26A3 (also known as DRA). In closed midjejunal loops in mice, PAT1inh-B01 inhibited fluid absorption by 50%, which increased to >90% when coadministered with DRA inhibitor DRAinh-A270. In ileal loops, PAT1inh-B01 blocked fluid absorption by >80%, whereas DRAinh-A270 was without effect. In colonic loops, PAT1inh-B01 was without effect, whereas DRAinh-A270 completely blocked fluid absorption. In a loperamide constipation model, coadministration of PAT1inh-B01 with DRAinh-A270 increased stool output compared with DRAinh-A270 alone. These results provide functional evidence for complementary and region-specific roles of PAT1 and DRA in intestinal fluid absorption, with PAT1 as the predominant anion exchanger in mouse ileum. We believe that PAT1inh-B01 is a novel tool to study intestinal ion and fluid transport and perhaps a drug candidate for small intestinal hyposecretory disorders such as cystic fibrosis-related meconium ileus and distal intestinal obstruction syndrome.

Inhibition of Pendrin by a small molecule reduces Lipopolysaccharide-induced acute Lung Injury.

Rationale: Pendrin is encoded by SLC26A4 and its mutation leads to congenital hearing loss. Additionally, pendrin is up-regulated in inflammatory airway diseases such as chronic obstructive pulmonary disease, allergic rhinitis, and asthma. In this study, the effects of a novel pendrin inhibitor, YS-01, were investigated in an LPS-induced acute lung injury (ALI) mice model, and the mechanism underlying the effect of YS-01 was examined. Methods: Lipopolysaccharide (LPS, 10 mg/kg) was intranasally instilled in wild type (WT) and pendrin-null mice. YS-01 (10 mg/kg) was administered intra-peritoneally before or after LPS inhalation. Lung injury parameters were assessed in the lung tissue and bronchoalveolar lavage fluid (BALF). Pendrin levels in the BALF of 41 patients with acute respiratory distress syndrome (ARDS) due to pneumonia and 25 control (solitary pulmonary nodule) patients were also measured. Results: LPS instillation induced lung injury in WT mice but not in pendrin-null mice. Pendrin expression was increased by LPS stimulation both in vitro and in vivo. YS-01 treatment dramatically attenuated lung injury and reduced BALF cell counts and protein concentration after LPS instillation in WT mice. Proinflammatory cytokines and NF-κB activation were suppressed by YS-01 treatment in LPS-induced ALI mice. In BALF of patients whose ARDS was caused by pneumonia, pendrin expression was up-regulated compared to that in controls (mean, 24.86 vs. 6.83 ng/mL, P < 0.001). Conclusions: A novel pendrin inhibitor, YS-01, suppressed lung injury in LPS-induced ALI mice and our data provide a new strategy for the treatment of inflammatory airway diseases including sepsis-induced ALI.

4,8-Dimethylcoumarin Inhibitors of Intestinal Anion Exchanger slc26a3 (Downregulated in Adenoma) for Anti-Absorptive Therapy of Constipation.

The chloride/bicarbonate exchanger SLC26A3 (downregulated in adenoma) is expressed mainly in colonic epithelium, where it dehydrates the stool by facilitating the final step of chloride and fluid absorption. SLC26A3 inhibition has predicted efficacy in various types of constipation including that associated with cystic fibrosis. We previously identified, by high-throughput screening, 4,8-dimethylcoumarin inhibitors of murine slc26a3 with IC50 down to ∼150 nM. Here, we synthesized a focused library of forty-three 4,8-dimethylcoumarin analogues. Structure-activity studies revealed the requirement of 4,8-dimethylcoumarin-3-acetic acid for activity. The most potent inhibitors were produced by replacements at C7, including 3-iodo- (4az) and 3-trifluoromethyl- (4be), with IC50 of 40 and 25 nM, respectively. Pharmacokinetics in mice showed predicted therapeutic concentrations of 4az for >72 h following a single 10 mg/kg oral dose. 4az at 10 mg/kg fully normalized stool water content in a loperamide-induced mouse model of constipation. The favorable inhibition potency, selectivity within the SLC26 family, and pharmacological properties of 4az support its further preclinical development.

SLC26A3 inhibitor identified in small molecule screen blocks colonic fluid absorption and reduces constipation.

SLC26A3 (downregulated in adenoma; DRA) is a Cl-/anion exchanger expressed in the luminal membrane of intestinal epithelial cells, where it facilitates electroneutral NaCl absorption. SLC26A3 loss of function in humans or mice causes chloride-losing diarrhea. Here, we identified slc26a3 inhibitors in a screen of 50,000 synthetic small molecules done in Fischer rat thyroid (FRT) cells coexpressing slc26a3 and a genetically encoded halide sensor. Structure-activity relationship studies were done on the most potent inhibitor classes identified in the screen: 4,8-dimethylcoumarins and acetamide-thioimidazoles. The dimethylcoumarin DRAinh-A250 fully and reversibly inhibited slc26a3-mediated Cl- exchange with HCO3-, I-, and thiocyanate (SCN-), with an IC50 of ~0.2 µM. DRAinh-A250 did not inhibit the homologous anion exchangers slc26a4 (pendrin) or slc26a6 (PAT-1), nor did it alter activity of other related proteins or intestinal ion channels. In mice, intraluminal DRAinh-A250 blocked fluid absorption in closed colonic loops but not in jejunal loops, while the NHE3 (SLC9A3) inhibitor tenapanor blocked absorption only in the jejunum. Oral DRAinh-A250 and tenapanor comparably reduced signs of constipation in loperamide-treated mice, with additive effects found on coadministration. DRAinh-A250 was also effective in loperamide-treated cystic fibrosis mice. These studies support a major role of slc26a3 in colonic fluid absorption and suggest the therapeutic utility of SLC26A3 inhibition in constipation.