Biology subjects

Frankfort, B. J.

Publications and source records attributed to Frankfort, B. J..

4 recordsLinked to original sources

bioRxiv2026

Ultrasound-enhanced retinal delivery of engineered viral vectors

The goal of this study was to examine Focused Ultrasound Blood-Retina Barrier Opening (FUS-BRBO) as a method to deliver gene therapy to the eye. We compared AAV.FUS.3, an engineered variant optimized for FUS-enhanced delivery to the central nervous system, and AAV9 for their utility as retinal gene vectors. To achieve this, mouse eyes were insonated by 4.667 MHz FUS in a custom-designed nosecone and vectors administered intravenously. We found 2.1-fold improvement of transduction of AAV.FUS.3 over AAV9 in the insonated area and 19.5-fold improvement over the uninsonated retina. Within that area, the ganglion cell layer showed the highest levels of transduction at 22% for AAV.FUS.3. We observed no gross tissue damage, change in retinal layer thickness, or increase in inflammation markers within the retina. FUS-BRBO has the potential to non-surgically deliver AAV-based vectors to spatially-defined regions of the retina. AAV.FUS.3 showed significantly improved transduction over AAV9 at the same dose. Further research to improve efficiency of delivery to the outer retina could expand the utility of FUS-BRBO.

bioengineering↗

bioRxiv2024

Sample multiplexing for retinal single-cell RNA-sequencing

Rare cell populations can be challenging to characterize using microfluidic single-cell RNA sequencing (scRNA-seq) platforms. Typically, the population of interest must be enriched and pooled from multiple biological specimens for efficient collection. However, these practices preclude the resolution of sample origin together with phenotypic data and are problematic in experiments in which biological or technical variation is expected to be high (e.g., disease models, genetic perturbation screens, or human samples). One solution is sample multiplexing whereby each sample is tagged with a unique sequence barcode that is resolved bioinformatically. We have established a scRNA-seq sample multiplexing pipeline for mouse retinal ganglion cells using cholesterol-modified-oligos and utilized the enhanced precision to investigate cell type distribution and transcriptomic variance across retinal samples. As single cell transcriptomics are becoming more widely used to research development and disease, sample multiplexing represents a useful method to enhance the precision of scRNA-seq analysis.

genomics↗

bioRxiv2024

Comprehensive single-cell atlas of the mouse retina

Single-cell RNA sequencing (scRNA-seq) has advanced our understanding of cellular heterogeneity at the single-cell resolution by classifying and characterizing cell types in multiple tissues and species. While several mouse retinal scRNA-seq reference datasets have been published, each dataset either has a relatively small number of cells or is focused on specific cell classes, and thus is suboptimal for assessing gene expression patterns across all retina types at the same time. To establish a unified and comprehensive reference for the mouse retina, we first generated the largest retinal scRNA-seq dataset to date, comprising approximately 190,000 single cells from C57BL/6J mouse whole retinas. This dataset was generated through the targeted enrichment of rare population cells via antibody-based magnetic cell sorting. By integrating this new dataset with public datasets, we conducted an integrated analysis to construct the Mouse Retina Cell Atlas (MRCA) for wild-type mice, which encompasses over 330,000 single cells. The MRCA characterizes 12 major classes and 138 cell types. It captured consensus cell type characterization from public datasets and identified additional new cell types. To facilitate the public use of the MRCA, we have deposited it in CELLxGENE, UCSC Cell Browser, and the Broad Single Cell Portal for visualization and gene expression exploration. The comprehensive MRCA serves as an easy-to-use, one-stop data resource for the mouse retina communities.

genomics↗

bioRxiv2020

Optimized culture of retinal ganglion cells and amacrine cells from adult mice

Cell culture is widely utilized to study the cellular and molecular biology of different neuronal cell populations. Current techniques to study enriched neurons in vitro are primarily limited to embryonic/neonatal animals and induced pluripotent stem cells (iPSC). Although the use of these cultures is valuable, the accessibility of purified primary adult neuronal cultures would allow for improved assessment of certain neurological diseases and pathways at the cellular level. Using a modified 7-step immunopanning technique to isolate for retinal ganglion cells (RGCs) and amacrine cells (ACs) from adult mouse retinas, we have successfully developed a model of neuronal culture that maintains for at least one week. Isolations of Thy1.2+ cells are enriched for RGCs, with the isolation cell yield being congruent to the theoretical yield of RGCs in a mouse retina. ACs of two different populations (CD15+ and CD57+) can also be isolated. The populations of these three adult neurons in culture are healthy, with neurite outgrowths in some cases greater than 500{micro}m in length. Optimization of culture conditions for RGCs and CD15+ cells revealed that neuronal survival and the likelihood of neurite outgrowth respond inversely to different culture media. Serially diluted concentrations of puromycin decreased cultured adult RGCs in a dose-dependent manner, demonstrating the potential usefulness of these adult neuronal cultures in screening assays. This novel culture system can be used to model adult neurons in vivo. Studies can now be expanded in conjunction with other methodologies to study the neurobiology of function, aging, and diseases.

neuroscience↗