Biology subjects

Apathy, R.

Publications and source records attributed to Apathy, R..

4 recordsLinked to original sources

bioRxiv2022

Modular Pooled Discovery of Synthetic Knockin Sequences to Program Durable Cell Therapies

Chronic stimulation can cause T cell dysfunction and limit efficacy of cellular immunotherapies. CRISPR screens have nominated gene targets for engineered T cells, but improved methods are required to compare large numbers of synthetic knockin sequences to reprogram cell functions. Here, we developed Modular Pooled Knockin Screening (ModPoKI), an adaptable platform for modular construction of DNA knockin libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors. Over 20 ModPoKI screens across human TCR and CAR T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct to enhance long-term T cell fitness and anti-cancer function in vitro and in vivo. ModPoKIs modularity allowed us to generate a [~]10,000-member library of TF combinations. Non-viral knockin of a combined BATF-TFAP4 polycistronic construct further enhanced function in vivo. ModPoKI facilitates discovery of complex gene constructs to program cellular functions. HighlightsO_LIModular pooled knockins of hundreds of TF and surface receptor constructs combined with different antigen receptors C_LIO_LIChronic stimulation screens discover programs to improve T cell persistence C_LIO_LICombinatorial knockin screens with [~]10,000 transcription factor combinations C_LIO_LIBATF-TFAP4 dual knockin construct improves CAR T cell function in vitro and in vivo C_LI

immunology↗

bioRxiv2021

Robust T cell activation requires an eIF3-driven burst in T cell receptor translation

Activation of T cells requires a rapid surge in cellular protein synthesis. However, the role of translation initiation in the early induction of specific genes remains unclear. Here we show human translation initiation factor eIF3 interacts with select immune system related mRNAs including those encoding the T cell receptor (TCR) subunits TCRA and TCRB. Binding of eIF3 to the TCRA and TCRB mRNA 3-untranslated regions (3-UTRs) depends on CD28 coreceptor signaling and regulates a burst in TCR translation required for robust T cell activation. Use of the TCRA or TCRB 3-UTRs to control expression of an anti-CD19 chimeric antigen receptor (CAR) improves the ability of CAR-T cells to kill tumor cells in vitro. These results identify a new mechanism of eIF3-mediated translation control that can aid T cell engineering for immunotherapy applications.

molecular biology↗

bioRxiv2019

Rapid discovery of synthetic DNA sequences to rewrite endogenous T cell circuits

Genetically-engineered immune cell therapies have been in development for decades1-3 and recently have proven effective to treat some types of cancer4. CRISPR-based genome editing methods, enabling more flexible and targeted sequence integrations than viral transduction, have the potential to extend the clinical utility of cell therapies5,6. Realization of this potential depends on improved knowledge of how coding and non-coding sites throughout the genome can be modified efficiently and on improved methods to discover novel synthetic DNA sequences that can be introduced at targeted sites to enhance critical immune cell functions. Here, we developed improved guidelines for non-viral genome targeting in human T cells and a pooled discovery platform to identify synthetic genome modifications that enhance therapeutically-relevant cell functions. We demonstrated the breadth of targetable genomic loci by performing large knock-ins at 91 different genomic sites in primary human T cells, and established the power of flexible genome targeting by generating cells with Genetically Engineered Endogenous Proteins (GEEPs) that seamlessly integrate synthetic and endogenous genetic elements to alter signaling input, output, or regulatory control of genes encoding key immune receptors. Motivated by success in introducing synthetic circuits into endogenous sites, we then developed a platform to facilitate discovery of novel multi-gene sequences that reprogram both T cell specificity and function. We knocked in barcoded pools of large DNA sequences encoding polycistronic gene programs. High-throughput pooled screening of targeted knock-ins to the endogenous T cell receptor (TCR) locus revealed a transcriptional regulator and novel protein chimeras that combined with a new TCR specificity to enhance T cell responses in the presence of suppressive conditions in vitro and in vivo. Overall, these pre-clinical studies provide flexible tools to discover complex synthetic gene programs that can be written into targeted genome sites to generate more effective therapeutic cells.

immunology↗

bioRxiv2019

A Cas9 nanoparticle system with truncated Cas9 target sequences on DNA repair templates enhances genome targeting in diverse human immune cell types

Virus-modified T cells are approved for cancer immunotherapy, but more versatile and precise genome modifications are needed for a wider range of adoptive cellular therapies1-4. We recently developed a non-viral CRISPR-Cas9 system for genomic site-specific integration of large DNA sequences in primary human T cells5. Here, we report two key improvements for efficiency and viability in an expanded variety of clinically-relevant primary cell types. We discovered that addition of truncated Cas9 target sequences (tCTS) at the ends of the homology directed repair (HDR) templates can interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template and enhance targeting efficiency. Further, stabilizing the Cas9 RNPs into nanoparticles with poly(glutamic acid) improved editing, reduced toxicity, and enabled lyophilized storage without loss of activity. Combining the tCTS HDR template modifications with polymer-stabilized nanoparticles increased gene targeting efficiency and viable cell yield across multiple genomic loci in diverse cell types. This system is an inexpensive, user-friendly delivery platform for non-viral genome reprogramming that we successfully applied in regulatory T cells (Tregs), {gamma}{delta}-T cells, B cells, NK cells, and primary and iPS-derived6 hematopoietic stem progenitor cells (HSPCs).

bioengineering↗