Your One-stop Solution For
Gene And Cell Engineering Research
Genetically engineered immune cells have gained attention as they have become an increasingly effective approach against some cancer types. Viral transduction of defined T cell receptors (TCRs), chimeric antigen receptors (CARs), or other synthetic receptors can be leveraged for generation of antigen-specific T cells ex vivo. Recent studies have highlighted the advantages that lie in targeted integrations of antigen receptor genes into defined genomic loci, accomplished with programmable nucleases such as CRISPR-Cas9 in company with viral or non-viral knockin templates.
CART cell therapy has now been approved for hematological malignancies, but has not achieved effectiveness against solid tumors. In addition to engineering antigen specificity for accuracy in targeting tumors, cells must main functionality in the battle against immunosuppressive tumor microenvironments. Microenvironment challenges may include Tregs, myeloid-derived suppressor cells, dendritic cells, and stromal cells that can complicate T cell action by depriving them of necessary co-stimulatory signals or induction of inhibitory signaling.
One strategy to combat immunosuppression involves engineering cells that lack inhibitory checkpoint pathways. Antibodies to PD-1 and CTLA4 have demonstrated clinical success in re-activation of functionality in endogenous T cells in a subset of patients and tumor types. CRISPR-Cas9 has also shown to increase functionality of T cells when used for genetic ablation of checkpoint targets, including PD-1, in adoptive T cell therapies. When used in combination with single-cell RNA sequencing, coupling of pooled CRISPR gene knockouts provide a potent approach for analysis on gene perturbations to cell conditions. Next-generation T cell therapies also engineered with transgenes are now being designed to assist immunosuppression in the microenvironment.
The authors of this study developed a robust platform for analysis of functional effects of pools of knockins constructed, designed to target the same locus in parallel to yield the most effective anti-tumor activity approach. The design incorporated CRISPR targeting for introduction of candidate immunotherapeutic knockin construct libraries inserted into a specific genome position in human T cells. T cells were allowed to compete against each other in vitro and in vivo to assess enhancement of T cell function. The 36-member library of barcoded knockin templates included both published and dominant-negative receptors, synthetic “switch” receptors with engineered intracellular domains, heterologous transcription factors, metabolic regulators, and receptors.
Distinct members of the libraries that promoted T cells fitness were identified when analyzed using high-throughput pooled screening of targeted cells when exposed to resting, stimulated, and immunosuppressive in vitro conditions.
Pooled knockins paired with single-cell sequencing of template barcodes and transcriptomes revealed constructs that promoted in vivo tumor accumulation and presented high-dimensional cellular phenotypes induced by both ex vivo and in vivo tumor microenvironments. In immunodeficient mice bearing human melanoma cells, direct competition among adoptively transferred human T cells targeted using pool of constructs demonstrated a subset of constructs that also promoted in vivo accumulation of tumor infiltrating lymphocytes (TILs). Chimeric receptor TGF-bR2-41BB was also shown to enhance cell fitness in in vitro and promoted gene expression of key effector cytokines in vivo.
Gene gain-of-function effects were notably observed in targeted non-viral pooled knockin screens. Pooled non-viral HDR templates permitted targeted integration at a defined site and resulted in consistent transgene expression levels and low rates of barcode mis-assignment.
Pooled, non-viral knockin screens enhanced CRISPR technologies when applied for scalable gain-of-function screening. Particularly, CRISPR-activation increased expression of endogenous genes. With CRISPR-mediated saturating mutagenesis, gain-of-function screens can be created in cell lines at targeted genomic sites.
The authors’ integrated pooled knockin platform demonstrated TGF-βR2-41BB as an effector that increased T cell fitness in vivo and coincided with promotion of key effector cytokines including IFNɣ. Knockin of large genetic construct enabled re-wiring of synthetic receptors to signal T cells when exposed to TGF-b in the tumor microenvironment, thus presenting an enhanced clearance of an in vivo solid tumor model.
Limitations against quantitative tracking of targeted genomic integrations of pooled knockin constructs were overcome with the technology of barcoding and tracking on-target integrates of HDR templates in a pooled library. Barcoding of targeted knockin sequences also permitted for single-cell transcriptome readouts in a pool of modified cells for assessment of knockin construct effects on cell state, allowing for screening of constructs that may affect cell abundance at different in vitro and in vivo selective pressures.
Barcodes were sequenced using cDNA or genomic DNA, suggesting a future pooled phenotypic screen may be possible for assessment of coding and non-coding regions of the genome. In future studies, knockin construct size and the quantity of members in each library can be scaled to both improve knockin efficiency and reduce costs associated with DNA synthesis.
Roth, T. L., Li, P. J., Blaeschke, F., Nies, J. F., Apathy, R., Mowery, C., Yu, R., Nguyen, M. L. T., Lee, Y., Truong, A., Hiatt, J., Wu, D., Nguyen, D. N., Goodman, D., Bluestone, J. A., Ye, C. J., Roybal, K., Shifrut, E., & Marson, A. (2020). Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies. Cell, 181(3). https://doi.org/10.1016/j.cell.2020.03.039