Advancing to

Non-Viral T Cell T Cell-Based Therapy Workflows: From Manufacturing to the Clinic

Immunotherapies
T Cell Activation Cancer cell
and Expansion death
Leukapheresis- PFN
White blood cells GzmB
are separated from Anti-CD3/
CD28
peripheral blood T Cell Engineering CAR-T TCR T
Cell Cell

CAR-T cells have radically changed cancer treatment. CAR

Viral Non-Viral
HDR
Template IFNγ
gene TNFα
T cells engineered to express novel Chimeric Antigen RNP
Complex
Receptors (CARs), can be armed with unique
tumor-killing properties. Conventionally, viral vectors
(e.g., lentivirus) have been used to deliver sequence T Cell Separation-
Magnetic nanobeads coated with
Viral vector Electroporation
T Cell Expansion-
payloads for CAR-T cell manufacturing. However, the antibodies are used to isolate T cells
CAR-T cells TCR T cells
Generate millions of
modified T cells and
random genome integration of virally introduced CARs MHC independent
tumor antigen recognition
MHC dependent
tumor antigen recognition
infusion into patient
that has received
presents oncogenic risks. CAR-T Cell TCR T Cell
lymphodepletion
chemotherapy

CRISPR/Cas9 systems are now being applied to precisely

modify T cell receptors (TCR) and re-write antigen
specificity. Initial CRISPR-based T cell engineering has
used adeno-associated virus (AAV) vectors to deliver DNA Conventional Untargeted Viral Viral Targeted CAR-scFv Non-Viral Targeted
payloads for targeted insertion via homology-directed CAR-T Cell Engineering T Cell Engineering TCR-T Cell Engineering
repair (HDR) into the TCR gene (i.e.,TRAC locus). Yet,
despite their high editing efficiency, a limited vector Self-inactivating-deletion in 3' LTR
prevents emergence of Transfection of Packaging
Promoter
Transfection of Packaging Cell line
(e.g., HEK293 cells expressing Recombinant TCR
anti-NY-ESO
E1a/E1b helper factors)
capacity and high manufacturing costs have restricted replication-competent recombinants Cell line ITR CAR-scFv ITR
cancer antigen
Knock-out
Endogenous
TCR
Targeting the TRAC locus for
insertion of recombinant TCR or
(e.g., HEK293 cells) β

viral engineering workflows.

α
Promoter γ
ε CAR constructs improves the
α

5' LTR CAR 3' LTR Viral δ

ε
δ
β
potency and reduces the
Replication REP CAP
ε
γ
exhaustion of engineered T cells
and Capsid ε

Currently, non-viral CRISPR/Cas9 editing provides an gag pol

Helper-Viral
Replication
E4 E2a VA
Viral
attractive alternative. In non-viral systems, Packaging
rev In contrast to TCR T cells, a CAR-scFv
ribonucleoprotein complexes (RNPs) of Cas9 protein and Different vectors for gag-pol, rev and
env sequences in third-generation
targeted to the TRAC locus imparts MHC
indpendent recognition of cell surface AAV Viral Insertion
Precise
NY-ESO Insertion
HDR
Lentiviral vectors enable producing γ ε ssDNA Recombinant
guide RNA targeting the TRAC locus are
ε δ
Viral env tumor antigens, similar to conventional Particles
Envelope replication-deficient virus particles or TCR (e.g., anti
CAR-T cells TRAC dsDNA NY-ESO)
co-electroporated into T-cells with a synthetic DNA
Transduction
payload encoding a CAR-scFv or recombinant TCR of Activated Targeted CAR
(scFv) gene
Transduction T Cell Ribonucleoprotein
(RNP) Complex
Un-targeted CAR
sequence. CAR-T Cells
gene insertion
T Cells Retroviral
Particles
CAR-scFv
TCR T cells
insertion RNP Complex
Targeting TRAC
locus
Electroporation
Non-viral editing approaches are continually evolving to
improve accuracy and efficiency. Most recently, synthetic
long single-stranded DNA (long ssDNA) payloads have
been safely and efficiently delivered to simplify cGMP T Benefits of Genome Editing with Benefits of Long ssDNA HDR
cell therapy manufacturing workflows.
Ribonucleoprotein (RNP) Complex Templates for T Cell Engineering
References RNP complex stability supports high editing
Improved Efficiency
RNP
▪ Eyquem, J. et al. (2017). Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature Publishing
Group. https://doi.org/10.1038/nature21405 functionality
▪ Irving, M. et al. (2021). Choosing the Right Tool for Genetic Engineering: Clinical Lessons from Chimeric Antigen Receptor-T
Cells. Human Gene Therapy. https://www.liebertpub.com/doi/10.1089/hum.2021.173
▪ Transient nuclease activity decreases
Reduced Off-Targets
Ivica, N. A., & Young, C. M. (2021). Tracking the car-t revolution: Analysis of clinical trials of car-t and tcr-t therapies for the
treatment of cancer (1997–2020). Healthcare (Switzerland). https://doi.org/10.3390/HEALTHCARE9081062/S1
▪ Kalidasan, V. et al. (2021). A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing unspecific editing and toxicity CTS
cells as a model system. Scientific Reports. https://doi.org/10.1038/s41598-021-98657-7
▪ Mansilla-Soto, J. et al. (2022). HLA-independent T cell receptors for targeting tumors with low antigen density. Nature Medicine.
https://doi.org/10.1038/s41591-021-01621-1 RNPs eliminate risk of genome dsDNA VS. ssDNA Under GMP-suitable conditions
▪
▪
Milone, M. C., & O’Doherty, U. (2018). Clinical use of lentiviral vectors. Leukemia. https://doi.org/10.1038/s41375-018-0106-0
Moretti, A. et al. (2022). The Past, Present, and Future of Non-Viral CAR T Cells. Frontiers in Immunology.
No DNA Interference insertional events
ssDNA HDR templates designed
with Cas9 target sequences (CTS)
https://doi.org/10.3389/FIMMU.2022.867013/BIBTEX
▪ Morgan, R. A., & Boyerinas, B. (2016). Genetic Modification of T Cells. Biomedicines. support high knock-in efficiencies of
46-62%, yielding millions of
https://doi.org/10.3390/BIOMEDICINES4020009
Transient CRISPR machinery limits risk of
▪ Nguyen, D. N. et al. (2020). Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing
No Immunogenicity non-virally modified T cells
▪
efficiency. Nature Biotechnology. doi:10.1038/s41587-019-0325-6.
Roth, T. L. et al. (2018). Reprogramming human T cell function and specificity with non-viral genome targeting. Nature. adverse immunological reactions
doi:10.1038/s41586-018-0326-5.
▪ Shy, B. R. et al. (2022). High-yield genome engineering in primary cells using a hybrid ssDNA repair template and
RNPs are immediately available for editing Best for long Lower cytotoxicity
Rapid Gene Editing
small-molecule cocktails. Nature. https://doi.org/10.1038/s41587-022-01418-8
▪ Wu, L. et al. (2020). Signaling from T cell receptors (TCRs) and chimeric antigen receptors (CARs) on T cells. Cellular & Molecular Increased insertion efficiency
Immunology. https://doi.org/10.1038/s41423-020-0470-3 without the lag of transcription and translation insertions
(5-10 kb) Improved insertion accuracy
▪ Zhao, Q. et al. (2021). Engineered TCR-T Cell Immunotherapy in Anticancer Precision Medicine: Pros and Cons. Frontiers.
https://doi.org/10.3389/fimmu.2021.658753