Gene Augmentation and Readthrough Rescue Channelopathy in an iPSC-RPE Model of Congenital Blindness.

Pathogenic variants of the KCNJ13 gene are known to cause Leber congenital amaurosis (LCA16), an inherited pediatric blindness. KCNJ13 encodes the Kir7.1 subunit that acts as a tetrameric, inwardly rectifying potassium ion channel in the retinal pigment epithelium (RPE) to maintain ionic homeostasis and allow photoreceptors to encode visual information. We sought to determine whether genetic approaches might be effective in treating blindness arising from pathogenic variants in KCNJ13. We derived human induced pluripotent stem cell (hiPSC)-RPE cells from an individual carrying a homozygous c.158G>A (p.Trp53∗) pathogenic variant of KCNJ13. We performed biochemical and electrophysiology assays to confirm Kir7.1 function. We tested both small-molecule readthrough drug and gene-therapy approaches for this "disease-in-a-dish" approach. We found that the LCA16 hiPSC-RPE cells had normal morphology but did not express a functional Kir7.1 channel and were unable to demonstrate normal physiology. After readthrough drug treatment, the LCA16 hiPSC cells were hyperpolarized by 30 mV, and the Kir7.1 current was restored. Similarly, we rescued Kir7.1 channel function after lentiviral gene delivery to the hiPSC-RPE cells. In both approaches, Kir7.1 was expressed normally, and there was restoration of membrane potential and the Kir7.1 current. Loss-of-function variants of Kir7.1 are one cause of LCA. Using either readthrough therapy or gene augmentation, we rescued Kir7.1 channel function in iPSC-RPE cells derived from an affected individual. This supports the development of precision-medicine approaches for the treatment of clinical LCA16.

Multiple Dictyostelid Species Destroy Biofilms of Klebsiella oxytoca and Other Gram Negative Species.

Dictyostelids are free-living phagocytes that feed on bacteria in diverse habitats. When bacterial prey is in short supply or depleted, they undergo multicellular development culminating in the formation of dormant spores. In this work, we tested isolates representing four dictyostelid species from two genera (Dictyostelium and Polysphondylium) for the potential to feed on biofilms preformed on glass and polycarbonate surfaces. The abilities of dictyostelids were monitored for three hallmarks of activity: 1) spore germination on biofilms, 2) predation on biofilm enmeshed bacteria by phagocytic cells and 3) characteristic stages of multicellular development (streaming and fructification). We found that all dictyostelid isolates tested could feed on biofilm enmeshed bacteria produced by human and plant pathogens: Klebsiella oxytoca, Pseudomonas aeruginosa, Pseudomonas syringae, Erwinia amylovora 1189 (biofilm former) and E. amylovora 1189 Δams (biofilm deficient mutant). However, when dictyostelids were fed planktonic E. amylovora Δams the bacterial cells exhibited an increased susceptibility to predation by one of the two dictyostelid strains they were tested against. Taken together, the qualitative and quantitative data presented here suggest that dictyostelids have preferences in bacterial prey which affects their efficiency of feeding on bacterial biofilms.

VSX2 and ASCL1 Are Indicators of Neurogenic Competence in Human Retinal Progenitor Cultures.

Three dimensional (3D) culture techniques are frequently used for CNS tissue modeling and organoid production, including generation of retina-like tissues. A proposed advantage of these 3D systems is their potential to more closely approximate in vivo cellular microenvironments, which could translate into improved manufacture and/or maintenance of neuronal populations. Visual System Homeobox 2 (VSX2) labels all multipotent retinal progenitor cells (RPCs) and is known to play important roles in retinal development. In contrast, the proneural transcription factor Acheate scute-like 1 (ASCL1) is expressed transiently in a subset of RPCs, but is required for the production of most retinal neurons. Therefore, we asked whether the presence of VSX2 and ASCL1 could gauge neurogenic potential in 3D retinal cultures derived from human prenatal tissue or ES cells (hESCs). Short term prenatal 3D retinal cultures displayed multiple characteristics of human RPCs (hRPCs) found in situ, including robust expression of VSX2. Upon initiation of hRPC differentiation, there was a small increase in co-labeling of VSX2+ cells with ASCL1, along with a modest increase in the number of PKCα+ neurons. However, 3D prenatal retinal cultures lost expression of VSX2 and ASCL1 over time while concurrently becoming refractory to neuronal differentiation. Conversely, 3D optic vesicles derived from hESCs (hESC-OVs) maintained a robust VSX2+ hRPC population that could spontaneously co-express ASCL1 and generate photoreceptors and other retinal neurons for an extended period of time. These results show that VSX2 and ASCL1 can serve as markers for neurogenic potential in cultured hRPCs. Furthermore, unlike hESC-OVs, maintenance of 3D structure does not independently convey an advantage in the culture of prenatal hRPCs, further illustrating differences in the survival and differentiation requirements of hRPCs extracted from native tissue vs. those generated entirely in vitro.