Evidence map›Paper›PMID 37365642›Full record

ArticleMolecular cancer2023

Homology-independent targeted insertion (HITI) enables guided CAR knock-in and efficient clinical scale CAR-T cell manufacturing.

Hyatt Balke-Want, Vimal Keerthi, Nikolaos Gkitsas, Andrew G Mancini, Gavin L Kurgan, Carley Fowler, Peng Xu, Xikun Liu, Kyle Asano, Sunny Patel and 7 more

Open access · goldAbstract read
PubMed Full text at PMC Publisher
In one paragraph

Article in Molecular cancer, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 34 papers.

0numbers the graph read from it
0cells of the map it votes in
34citing papers in PubMed
9.2field-weighted citation impact, top 2% of its field
1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

The trial behind it

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

34 citing papers in PubMed, 40 citations in OpenAlex.

  1. Review
  2. Article
  3. Review
  4. Article
  5. Review
  6. Engineering T cell therapies for lung cancer.Molecular therapy. Oncology · 2026
    Article
  7. Article
  8. Ultra-large targeted DNA integrations in primary human cells.bioRxiv : the preprint server for biology · 2026
    Article
  9. Article
  10. Article
  11. Validation and analysis of 12,000 AI-driven CAR-T designs in thebioRxiv : the preprint server for biology · 2026
    Article
  12. Can we cure autoimmunity?Current opinion in immunology · 2026
    Review
  13. Article
  14. Article
  15. Article
  16. Review
  17. Review
  18. Article
  19. Article
  20. High-dimensional temporal mapping of CAR T cells reveals phenotypic and functional remodeling during manufacturing.Molecular therapy : the journal of the American Society of Gene Therapy · 2025
    Article
4 · The record

Corrections and comments

PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.

5 · Who and what money

Authors and funding

17 authors at 3 institutions in 1 country.

Hyatt Balke-WantStanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
Vimal KeerthiStanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
Nikolaos GkitsasStanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
Andrew G ManciniMaxCyte, Inc, Rockville, MD, USA.
Gavin L KurganIntegrated DNA Technologies, Inc, Coralville, IA, 52241, USA.
Carley FowlerStanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
Peng XuStanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
Xikun LiuStanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
Kyle AsanoStanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
Sunny PatelStanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
Christopher J FisherStanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
Annie K BrownStanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
Ramya H TunuguntlaStanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
Shabnum PatelStanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
Elena SotilloStanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
Crystal L MackallStanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA. cmackall@stanford.edu.
Steven A FeldmanStanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA. feldmans@stanford.edu.
Stanford University · USIntegrated DNA Technologies (United States) · USMaxCyte (United States) · US

Funding

No grant is acknowledged in the PubMed record.

6 · The paper itself

Abstract

backgroundChimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA).

methodsWe applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies in our system. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach.

resultsHere, we show that site directed CAR integration utilizing nanoplasmid DNA delivered via HITI provides high cell yields and highly functional cells. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5 × 10

conclusionsOur work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies.

Indexed as

DNAT-LymphocytesHumansImmunotherapy, AdoptiveRecombinational DNA RepairDNACRISPR/Cas9Genomic safetyGMPHomology-independent targeted insertion (HITI)Non-viral CAR-T cellTargeted insertion

Identifiers

PMID37365642
PMCPMC10291796
OpenAlexW4382137051

What OpenQuestion holds

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LicenceCC BY
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Registered trials

None linked

Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.