Exploiting Single-Cell Tools in Gene and Cell Therapy

Abstract

Single-cell molecular tools have been developed at an incredible pace over the last five years as sequencing costs continue to drop and numerous molecular assays have been coupled to sequencing readouts. This rapid period of technological development has facilitated the delineation of individual molecular characteristics including the genome, transcriptome, epigenome, and proteome of individual cells, leading to an unprecedented resolution of the molecular networks governing complex biological systems. The immense power of single-cell molecular screens has been particularly highlighted through work in systems where cellular heterogeneity is a key feature, such as stem cell biology, immunology, and tumor cell biology. Single-cell-omics technologies have already contributed to the identification of novel disease biomarkers, cellular subsets, therapeutic targets and diagnostics, many of which would have been undetectable by bulk sequencing approaches. More recently, efforts to integrate single-cell multi-omics with single cell functional output and/or physical location have been challenging but have led to substantial advances. Perhaps most excitingly, there are emerging opportunities to reach beyond the description of static cellular states with recent advances in modulation of cells through CRISPR technology, in particular with the development of base editors which greatly raises the prospect of cell and gene therapies. In this review, we provide a brief overview of emerging single-cell technologies and discuss current developments in integrating single-cell molecular screens and performing single-cell multi-omics for clinical applications. We also discuss how single-cell molecular assays can be usefully combined with functional data to unpick the mechanism of cellular decision-making. Finally, we reflect upon the introduction of spatial transcriptomics and proteomics, its complementary role with single-cell RNA sequencing (scRNA-seq) and potential application in cellular and gene therapy.

Tumor vessel co-option: The past & the future

“…Lastly, to gather a comprehensive picture of the VCO process, one should seek to investigate the epigenome of co-opted cell types, to probe for DNA regulatory elements that may be changed in these cell types and provide an additional level of insights into the acquisition of co-opted phenotypes. Techniques, such as single-cell Assay for Transposase-Accessible Chromatin (ATAC) sequencing ( 161 ), or Methylome sequencing ( 162 – 164 ) are suitable for such endeavors.…”

Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

“…However, there is a lack of methods to probe the heterogeneity in secretory functions and link these to specific gene expression networks. Methods to sort therapeutic cell populations based on functional potency and uncover the single-cell level gene expression driving this potency can be transformative for the next generation of cell therapies 2,5 .…”

High efficiency sorting and outgrowth for single-cell cloning of mammalian cell lines

“…Single-cell cloning (SCC) is an important step in generating monoclonal cell lines. It has numerous applications, such as therapeutic protein production, drug screening, gene therapy, and it can aid in the understanding of cell function and disease mechanisms (Soitu et al 2019 ; Bode et al 2021 ). SCC applications require colonies that originate from a single cell to avoid discrepancies in product quality, inaccurate reproducibility of results, recombinant proteins instability, and poor outgrowth (Castan et al 2018 ).…”