Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors

Abstract

Chimeric antigen receptor engineered T cell (CAR-T) immunotherapy has shownefficacy against a subset of hematological malignancies 1,2 , yet its autologous nature and ineffectiveness against epithelial and solid cancers limit widespread application. To overcome these limitations, targeted nucleases have been used to disrupt checkpoint inhibitors and genes involved in alloreactivity 3-6 . However, the production of allogeneic, "off-the-shelf" T cells with enhanced function requires multiplex genome editing strategies that risk off-target effects, chromosomal rearrangements, and genotoxicity due to simultaneous double-strand break (DSB) induction at multiple loci 7-10 . Moreover, it has been well documented that DSBs are toxic lesions that can drive genetic instability 11,12 .Alternatively, CRISPR/Cas9 base editors afford programmable enzymatic nucleotide conversion at targeted loci without induction of DSBs 13,14 . We reasoned this technology could be used to knockout gene function in human T cells while minimizing safety concerns associated with current nuclease platforms. Through systematic reagent and dose optimization, we demonstrate highly efficient multiplex base editing and consequent protein knockout in primary human T cells at loci relevant to the generation of allogeneic CAR-T cells including the T cell receptor ɑ constant (TRAC) locus, β-2 microglobulin (B2M), and programmed cell death 1 (PDCD1). Multiplex base edited T cells equipped with a CD19CAR killed target cells more efficiently; and importantly, both DSB induction and translocation frequency were greatly reduced compared to cells engineered with Cas9 nuclease. Collectively, our results establish a novel multiplex gene editing platform to enhance both the safety and efficacy of engineered T cell-based immunotherapies.Base editing has been previously used to induce premature stop (pmSTOP) codons for gene knockout in mice and in mammalian cells [15][16][17][18] . However, we reasoned that splice site disruption could have several advantages over induction of pmSTOP codons (Supplemental Data 1). For instance, stop codon readthrough has been shown to occur at frequencies up to 31% in some genes, and can be promoted under conditions of cellular stress 19,20 . Splice site editing mitigates this concern as it alters gene processing at the RNA level 21 , which is less likely to be bypassed at the translational level. Additionally, current base editors do not produce strict C to T edits, with even the most recent base editors producing up to 25% non-target editing (C to G/A) 22 .

Orthotopic T-Cell Receptor Replacement—An “Enabler” for TCR-Based Therapies

“…In their first in-human phase I clinical trial with CRISPR/Cas9-engineered TCR-transduced human T cells, Stadtmauer et al reported translocation frequencies of about 0.01% to 5% [ 47 ], which is in line with previous reports on translocation frequencies of Transcription activator-like effector nuclease (TALEN)- [ 84 , 85 ] and CRISPR-edited [ 86 ] T cells. Translocation frequencies are different for individual gRNA combinations [ 47 , 86 ] and can, therefore, be reduced through informed selection of specific gRNAs. In addition, Cas9 variants such as base editors that circumvent double-strand breaks were shown to decrease translocation events significantly [ 86 ].…”

“…Translocation frequencies are different for individual gRNA combinations [ 47 , 86 ] and can, therefore, be reduced through informed selection of specific gRNAs. In addition, Cas9 variants such as base editors that circumvent double-strand breaks were shown to decrease translocation events significantly [ 86 ].…”

Continuous evolution of base editors with expanded target compatibility and improved activity

“… 1 The use of programmable nucleases to generate double-stranded DNA breaks (DSBs) followed by homology-directed repair can introduce a wide variety of modifications but is inefficient in non-dividing cells, and is typically accompanied by an excess of unwanted insertions and deletions (indels), translocations, or other chromosomal rearrangements. 2 Base editing directly modifies target DNA bases in living cells and has become widely used to correct or install point mutations in organisms ranging from bacteria to human embryos. 3 Base editors use a catalytically impaired Cas9 to open a single-stranded DNA loop at a specified genomic site ( Fig.…”

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

“…Genetically modified T cells have demonstrated clinical efficacy in some therapeutic applications 27 , and there is an increasing body of evidence suggesting that the therapeutic potential of adoptive T cell therapies may be significantly enhanced by disruption of multiple genes in the same cell to achieve desirable cellular phenotypes. 28,29 Approaches using nucleases to introduce indel mutations in target genes, thereby disrupting their expression in T cells 30,31 , are effective but simultaneous creation of multiple DSBs in a target cell can result in genomic rearrangements and toxicities with variable frequencies 32,33 . Because ABEs function by making single nucleotide genomic changes without creating DSBs, multiplexed base editing with ABE8 is an attractive approach for creating genetically modified T cells.…”