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B - Gene Targeting and Gene Correction -> B2 - Gene Targeting and Gene Correction – In Vitro Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

1016: Single-Cell Transcriptional Analysis of Human In Vitro Platforms to Study Genome Editing Effects in Retinal Tissues

Type: Poster Session

Poster Board Number: 1016
Presentation Details
Session Title: Thursday Poster Session
Start Time: 5/18/2023 12:00
End Time: 5/18/2023 14:00

Human stem-cell-derived retinal organoids offer powerful platforms for studying molecular programs associated with normal and disrupted eye function as well as for rapid assessment of potential targets of genome editing. The efficiency of genome editing in traditional 3D organoid systems is limited because of the low rate of transduction with non-AAV based delivery platforms. We observe that this transduction efficiency could be enhanced by removing the extracellular barriers for viral vector delivery via organoid dissociation and replating the cells into a 2D culture for transduction. However, it is unclear whether 2D platforms faithfully capture the cell type composition and gene expression programs of 3D organoids. To compare the transcriptome states of cells in 2D dissociated cultures and 3D organoids, we performed single cell RNA sequencing (scRNA-seq) of the two different platforms by taking samples from six different time points corresponding to different stages of retinal cell fate specification. To enable integrative analysis of this complex multi-sample dataset, we developed and applied a novel multi-task matrix factorization method named Tree-guided non-negative Matrix Factorization (TMF), which performs sample-specific dimensionality reduction while incorporating the relatedness of the samples. TMF identified cell clusters that were enriched in different retinal cell types. Both 2D and 3D platforms show the presence of a diverse set of cell types including Müller glia, photoreceptors, and horizontal cells. We identified a total of 22.4% of cells from 2D cultures as photoreceptors compared to 51.2% of cells from the 3D organoid cultures, while 2D cultures had a higher proportion of RPE/progenitor cell types compared to 3D cultures. Photoreceptors identified in either platform show expression of known cone and rod marker genes, which validate the identity of these cell types. The photoreceptor cells maintain functionality, as they maintain visual cycle function after delivery of the genome editor. Additionally, we compared transcriptional profiles of transduced cells with that of untransduced cells. In total, 42% of 2D cells were transduced, of which 8% comprised photoreceptors. Overall, this work shows that 2D platforms retain a sizable photoreceptor population which can be efficiently transduced, making them an effective platform for examining molecular signatures of genome-editing in a higher throughput manner.

Junha Shin, Katherine P. Mueller, Elizabeth Capowski, Saptarshi Pyne, Sunnie G. McCalla, Shivani Saxena, Kayvan Samimi, Emma Nelson, Maria A. Fernandez, Melissa Skala, David Gamm, Krishanu Saha, Sushmita Roy

University of Wisconsin-Madison, Madison, WI
  K. Saha: 1; Commercial Interest i.e. Company X; Andson Biotechnology. 1; What was received? i.e. Honorarium; ownership interest. 1; For what role? i.e. Speaker; Board member. 2; Commercial Interest i.e. Company X; Notch Therapeutics. 2; What was received? i.e. Honorarium; ownership interest. 2; For what role? i.e. Speaker; Board member.

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