Molecular and functional correction of a deep intronic splicing mutation in CFTR by CRISPR-Cas9 gene editing.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. The 10th most common mutation, c.3178-2477C>T (3849+10kb C>T), involves a cryptic, intronic splice site. This mutation was corrected in CF primary cells homozygous for this mutation by delivering pairs of guide RNAs (gRNAs) with Cas9 protein in ribonucleoprotein (RNP) complexes that introduce double-strand breaks to flanking sites to excise the 3849+10kb C>T mutation, followed by DNA repair by the non-homologous end-joining pathway, which functions in all cells of the airway epithelium. RNP complexes were delivered to CF basal epithelial cell by a non-viral, receptor-targeted nanocomplex comprising a formulation of targeting peptides and lipids. Canonical CFTR mRNA splicing was, thus, restored leading to the restoration of CFTR protein expression with concomitant restoration of electrophysiological function in airway epithelial air-liquid interface cultures. Off-target editing was not detected by Sanger sequencing of in silico-selected genomic sites with the highest sequence similarities to the gRNAs, although more sensitive unbiased whole genome sequencing methods would be required for possible translational developments. This approach could potentially be used to correct aberrant splicing signals in several other CF mutations and other genetic disorders where deep-intronic mutations are pathogenic.

Comprehensive characterization of fetal and mature retinal cell identity to assess the fidelity of retinal organoids.

Characterizing cell identity in complex tissues such as the human retina is essential for studying its development and disease. While retinal organoids derived from pluripotent stem cells have been widely used to model development and disease of the human retina, there is a lack of studies that have systematically evaluated the molecular and cellular fidelity of the organoids derived from various culture protocols in recapitulating their in vivo counterpart. To this end, we performed an extensive meta-atlas characterization of cellular identities of the human eye, covering a wide range of developmental stages. The resulting map uncovered previously unknown biomarkers of major retinal cell types and those associated with cell-type-specific maturation. Using our retinal-cell-identity map from the fetal and adult tissues, we systematically assessed the fidelity of the retinal organoids in mimicking the human eye, enabling us to comprehensively benchmark the current protocols for retinal organoid generation.

Bioelectric Potential in Next-Generation Organoids: Electrical Stimulation to Enhance 3D Structures of the Central Nervous System.

Pluripotent stem cell-derived organoid models of the central nervous system represent one of the most exciting areas in in vitro tissue engineering. Classically, organoids of the brain, retina and spinal cord have been generated via recapitulation of in vivo developmental cues, including biochemical and biomechanical. However, a lesser studied cue, bioelectricity, has been shown to regulate central nervous system development and function. In particular, electrical stimulation of neural cells has generated some important phenotypes relating to development and differentiation. Emerging techniques in bioengineering and biomaterials utilise electrical stimulation using conductive polymers. However, state-of-the-art pluripotent stem cell technology has not yet merged with this exciting area of bioelectricity. Here, we discuss recent findings in the field of bioelectricity relating to the central nervous system, possible mechanisms, and how electrical stimulation may be utilised as a novel technique to engineer "next-generation" organoids.

Differentiation of brain and retinal organoids from confluent cultures of pluripotent stem cells connected by nerve-like axonal projections of optic origin.

Advances in the study of neurological conditions have been possible because of pluripotent stem cell technologies and organoids. Studies have described the generation of neural ectoderm-derived retinal and brain structures from pluripotent stem cells. However, the field is still troubled by technical challenges, including high culture costs and variability. Here, we describe a simple and economical protocol that reproducibly gives rise to the neural retina and cortical brain regions from confluent cultures of stem cells. The spontaneously generated cortical organoids are transcriptionally comparable with organoids generated by other methods. Furthermore, these organoids showed spontaneous functional network activity and proteomic analysis confirmed organoids maturity. The generation of retinal and brain organoids in close proximity enabled their mutual isolation. Suspension culture of this complex organoid system demonstrated the formation of nerve-like structures connecting retinal and brain organoids, which might facilitate the investigation of neurological diseases of the eye and brain.

Antioxidant and lipid supplementation improve the development of photoreceptor outer segments in pluripotent stem cell-derived retinal organoids.

The generation of retinal organoids from human pluripotent stem cells (hPSC) is now a well-established process that in part recapitulates retinal development. However, hPSC-derived photoreceptors that exhibit well-organized outer segment structures have yet to be observed. To facilitate improved inherited retinal disease modeling, we determined conditions that would support outer segment development in maturing hPSC-derived photoreceptors. We established that the use of antioxidants and BSA-bound fatty acids promotes the formation of membranous outer segment-like structures. Using new protocols for hPSC-derived retinal organoid culture, we demonstrated improved outer segment formation for both rod and cone photoreceptors, including organized stacked discs. Using these enhanced conditions to generate iPSC-derived retinal organoids from patients with X-linked retinitis pigmentosa, we established robust cellular phenotypes that could be ameliorated following adeno-associated viral vector-mediated gene augmentation. These findings should aid both disease modeling and the development of therapeutic approaches for the treatment of photoreceptor disorders.

Restoration of visual function in advanced disease after transplantation of purified human pluripotent stem cell-derived cone photoreceptors.

Age-related macular degeneration and other macular diseases result in the loss of light-sensing cone photoreceptors, causing irreversible sight impairment. Photoreceptor replacement may restore vision by transplanting healthy cells, which must form new synaptic connections with the recipient retina. Despite recent advances, convincing evidence of functional connectivity arising from transplanted human cone photoreceptors in advanced retinal degeneration is lacking. Here, we show restoration of visual function after transplantation of purified human pluripotent stem cell-derived cones into a mouse model of advanced degeneration. Transplanted human cones elaborate nascent outer segments and make putative synapses with recipient murine bipolar cells (BCs), which themselves undergo significant remodeling. Electrophysiological and behavioral assessments demonstrate restoration of surprisingly complex light-evoked retinal ganglion cell responses and improved light-evoked behaviors in treated animals. Stringent controls exclude alternative explanations, including material transfer and neuroprotection. These data provide crucial validation for photoreceptor replacement therapy and for the potential to rescue cone-mediated vision.

RPGR isoform imbalance causes ciliary defects due to exon ORF15 mutations in X-linked retinitis pigmentosa (XLRP).

Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause severe retinal ciliopathy, X-linked retinitis pigmentosa. Although two major alternatively spliced isoforms, RPGRex1-19 and RPGRORF15, are expressed, the relative importance of these isoforms in disease pathogenesis is unclear. Here, we analyzed fibroblast samples from eight patients and found that all of them form longer cilia than normal controls, albeit to different degrees. Although all mutant RPGRORF15 messenger RNAs (mRNAs) are unstable, their steady-state levels were similar or higher than those in the control cells, suggesting there may be increased transcription. Three of the fibroblasts that had higher levels of mutant RPGRORF15 mRNA also exhibited significantly higher levels of RPGRex1-19 mRNA. Four samples with unaltered RPGRex1-19 levels carried mutations in RPGRORF15 that resulted in this isoform being relatively less stable. Thus, in all cases, the RPGRex1-19/RPGRORF15 isoform ratio was increased, and this was highly correlative to the cilia extension defect. Moreover, overexpression of RPGRex1-19 (mimicking the increase in RPGRex1-19 to RPGRORF15 isoform ratio) or RPGRORF15 (mimicking reduction of the ratio) resulted in significantly longer or shorter cilia, respectively. Notably, the cilia length defect appears to be attributable to both the loss of the wild-type RPGRORF15 protein and to the higher levels of the RPGRex1-19 isoform, indicating that the observed defect is due to the altered isoform ratios. These results suggest that maintaining the optimal RPGRex1-9 to RPGRORF15 ratio is critical for cilia growth and that designing strategies that focus on the best ways to restore the RPGRex1-19/RPGRORF15 ratio may lead to better therapeutic outcomes.

Retinal organoids: a window into human retinal development.

Retinal development and maturation are orchestrated by a series of interacting signalling networks that drive the morphogenetic transformation of the anterior developing brain. Studies in model organisms continue to elucidate these complex series of events. However, the human retina shows many differences from that of other organisms and the investigation of human eye development now benefits from stem cell-derived organoids. Retinal differentiation methods have progressed from simple 2D adherent cultures to self-organising micro-physiological systems. As models of development, these have collectively offered new insights into the previously unexplored early development of the human retina and informed our knowledge of the key cell fate decisions that govern the specification of light-sensitive photoreceptors. Although the developmental trajectories of other retinal cell types remain more elusive, the collation of omics datasets, combined with advanced culture methodology, will enable modelling of the intricate process of human retinogenesis and retinal disease in vitro.