Human iPSC Modeling Reveals Mutation-Specific Responses to Gene Therapy in a Genotypically Diverse Dominant Maculopathy.

Dominantly inherited disorders are not typically considered to be therapeutic candidates for gene augmentation. Here, we utilized induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in an iPSC-RPE model of recessive bestrophinopathy as well as in two models of dominant Best disease caused by different mutations in regions encoding ion-binding domains. A third dominant Best disease iPSC-RPE model did not respond to gene augmentation, but showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then subjected all three dominant Best disease iPSC-RPE models to gene editing, which produced premature stop codons specifically within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of retinal pigment epithelium (RPE) transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some individuals with dominant Best disease and that non-responders are candidates for alternate approaches such as gene editing. However, testing gene editing strategies for on-target efficiency and off-target events using personalized iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected individuals.

Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines.

Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. Although nearly all such methods yield at least partial segments of retinal structure with a mature appearance, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to understand better the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation.

Micromolded honeycomb scaffold design to support the generation of a bilayered RPE and photoreceptor cell construct.

Age-related macular degeneration (AMD) causes blindness due to loss of retinal pigment epithelium (RPE) and photoreceptors (PRs), which comprise the two outermost layers of the retina. Given the small size of the macula and the importance of direct contact between RPE and PRs, the use of scaffolds for targeted reconstruction of the outer retina in later stage AMD and other macular dystrophies is particularly attractive. We developed microfabricated, honeycomb-patterned, biodegradable poly(glycerol sebacate) (PGS) scaffolds to deliver organized, adjacent layers of RPE and PRs to the subretinal space. Furthermore, an optimized process was developed to photocure PGS, shortening scaffold production time from days to minutes. The resulting scaffolds robustly supported the seeding of human pluripotent stem cell-derived RPE and PRs, either separately or as a dual cell-layered construct. These advanced, economical, and versatile scaffolds can accelerate retinal cell transplantation efforts and benefit patients with AMD and other retinal degenerative diseases.

Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy.

Clinical genome editing is emerging for rare disease treatment, but one of the major limitations is the targeting of CRISPR editors' delivery. We delivered base editors to the retinal pigmented epithelium (RPE) in the mouse eye using silica nanocapsules (SNCs) as a treatment for retinal degeneration. Leber congenital amaurosis type 16 (LCA16) is a rare pediatric blindness caused by point mutations in the KCNJ13 gene, a loss of function inwardly rectifying potassium channel (Kir7.1) in the RPE. SNCs carrying adenine base editor 8e (ABE8e) mRNA and sgRNA precisely and efficiently corrected the KCNJ13W53X/W53X mutation. Editing in both patient fibroblasts (47%) and human induced pluripotent stem cell-derived RPE (LCA16-iPSC-RPE) (17%) showed minimal off-target editing. We detected functional Kir7.1 channels in the edited LCA16-iPSC-RPE. In the LCA16 mouse model (Kcnj13W53X/+ΔR), RPE cells targeted SNC delivery of ABE8e mRNA preserved normal vision, measured by full-field electroretinogram (ERG). Moreover, multifocal ERG confirmed the topographic measure of electrical activity primarily originating from the edited retinal area at the injection site. Preserved retina structure after treatment was established by optical coherence tomography (OCT). This preclinical validation of targeted ion channel functional rescue, a challenge for pharmacological and genomic interventions, reinforced the effectiveness of nonviral genome-editing therapy for rare inherited disorders.

Investigating cone photoreceptor development using patient-derived NRL null retinal organoids.

Photoreceptor loss is a leading cause of blindness, but mechanisms underlying photoreceptor degeneration are not well understood. Treatment strategies would benefit from improved understanding of gene-expression patterns directing photoreceptor development, as many genes are implicated in both development and degeneration. Neural retina leucine zipper (NRL) is critical for rod photoreceptor genesis and degeneration, with NRL mutations known to cause enhanced S-cone syndrome and retinitis pigmentosa. While murine Nrl loss has been characterized, studies of human NRL can identify important insights for human retinal development and disease. We utilized iPSC organoid models of retinal development to molecularly define developmental alterations in a human model of NRL loss. Consistent with the function of NRL in rod fate specification, human retinal organoids lacking NRL develop S-opsin dominant photoreceptor populations. We report generation of two distinct S-opsin expressing populations in NRL null retinal organoids and identify MEF2C as a candidate regulator of cone development.

Generation of a rod-specific NRL reporter line in human pluripotent stem cells.

Reporter lines generated in human pluripotent stem cells can be highly useful for the analysis of specific cell types and lineages in live cultures. We created the first human rod reporter line using CRISPR/Cas9 genome editing to replace one allele of the Neural Retina Leucine zipper (NRL) gene with an eGFP transgene in the WA09 human embryonic stem cell (hESC) line. After confirming successful targeting, three-dimensional optic vesicle structures were produced to examine reporter specificity and to track rod differentiation in culture. The NRL+/eGFP hESC line robustly and exclusively labeled the entirety of rods throughout differentiation, eventually revealing highly mature structural features. This line provides a valuable tool for studying human rod development and disease and testing therapeutic strategies for retinitis pigmentosa.

The surgical management of metastatic spinal tumors based on an Epidural Spinal Cord Compression (ESCC) scale.

BACKGROUND CONTEXT: There have been no previous studies looking at the outcome of surgical decompression (+/-stabilization) for various grades of epidural spinal cord compression (ESCC) due to spinal metastases. PURPOSE: The aim of this study was to determine the outcome of surgical treatment in patients with ESCC using the Bilsky six-point scale. STUDY DESIGN/SETTING: This was a retrospective cohort review of prospectively collected data. PATIENT SAMPLE: A consecutive series of 101 patients managed over the period of 3 years for ESCC due to spinal metastases in a tertiary spine surgery referral unit were included. METHODS: Data on age, gender, revised Tokuhashi score, preoperative Frankel grade, tumor histology, magnetic resonance imaging scan-based Bilsky cord compression grade, postoperative Frankel grade at last follow-up, complications, and survivorship were collected. OUTCOME MEASURES: Frankel grading system for function was used to evaluate the patient's preoperative and postoperative neurologic status. Patient survival and postoperative complications were also collected. RESULTS: Average patient age was 64.7 years (13-88 years): 62 males and 39 females. Mean follow-up was 7.3 months (3-23.3 months). Most primary tumors were in prostate, breast, renal, lung, and the blood dyscrasias. Within the lower grade of compression (Group 1; Bilsky Grades 0,1a, 1b, and 1c; n=40), 29 patients (72.5%) had no improvement in Frankel grade, seven patients (17.5%) improved, whereas four patients (10%) deteriorated neurologically after surgery. Within the higher compression grade (Group 2; Bilsky Grades 2 and 3; n=61), 37 patients (60%) did not experience neurologic change, 20 (33%) improved, whereas neurology worsened in four patients (7%). When compared with Group 2 patients, Group 1 patients had better preoperative Frankel scores but a greater number of patients in Group 2 improved their Frankel scores significantly postoperatively. The mean revised Tokuhashi score for Groups 1 and 2 was 10 and 9.1, respectively (p=.1). The complication rate for Groups 1 and 2 was 25% and 42.6%, respectively (p=.052). Survival analysis showed no difference between the groups (Group 1: median 376 days [12-1052]; Group 2: median 326 days [12-979]; p=.62). CONCLUSIONS: Surgery can achieve improvements in neurology even in higher grades of cord compression. There is a trend toward more complications and worse survival with spinal surgery in patients with higher grades of compression.

Fast food and obesity: a spatial analysis in a large United Kingdom population of children aged 13-15.

BACKGROUND: The childhood obesity epidemic is a current public health priority in many countries, and the consumption of fast food has been associated with obesity. PURPOSE: This study aims to assess the relationship between fast-food consumption and obesity as well as the relationship between fast-food outlet access and consumption in a cohort of United Kingdom teenagers. METHODS: A weighted accessibility score of the number of fast-food outlets within a 1-km network buffer of the participant's residence at age 13 years was calculated. Geographically weighted regression was used to assess the relationships between fast-food consumption at age 13 years and weight status at ages 13 and 15 years, and separately between fast-food accessibility and consumption. Data were collected from 2004 to 2008. RESULTS: The consumption of fast food was associated with a higher BMI SD score (ß=0.08, 95% CI=0.03, 0.14); higher body fat percentage (ß=2.06, 95% CI=1.33, 2.79); and increased odds of being obese (OR=1.23, 95% CI=1.02, 1.49). All these relationships were stationary and did not vary over space in the study area. The relationship between the accessibility of outlets and consumption did vary over space, with some areas (more rural areas) showing that increased accessibility was associated with consumption, whereas in some urban areas increased accessibility was associated with lack of consumption. CONCLUSIONS: There is continued need for nutritional education regarding fast food, but public health interventions that place restrictions on the location of fast-food outlets may not uniformly decrease consumption.

The geography of Fast Food outlets: a review.

The availability of food high in fat, salt and sugar through Fast Food (FF) or takeaway outlets, is implicated in the causal pathway for the obesity epidemic. This review aims to summarise this body of research and highlight areas for future work. Thirty three studies were found that had assessed the geography of these outlets. Fourteen studies showed a positive association between availability of FF outlets and increasing deprivation. Another 13 studies also included overweight or obesity data and showed conflicting results between obesity/overweight and FF outlet availability. There is some evidence that FF availability is associated with lower fruit and vegetable intake. There is potential for land use policies to have an influence on the location of new FF outlets. Further research should incorporate good quality data on FF consumption, weight and physical activity.