Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV

Meisam Naeimi Kararoudi, Shibi Likhite, Ezgi Elmas, Kenta Yamamoto, Maura Schwartz, Kinnari Sorathia, Marcelo de Souza Fernandes Pereira, Yasemin Sezgin, Raymond D. Devine, Justin M. Lyberger, Gregory K. Behbehani, Nitin Chakravarti, Branden S. Moriarity, Kathrin Meyer, Dean A. Lee

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-specific gene insertion in NK cells using CRISPR (Cas9/RNP) and AAVs. We compared AAV vectors designed to mediate gene insertion by different DNA repair mechanisms, homology arm lengths, and virus concentrations. We then validated the method for site-directed gene insertion of CD33-specific CARs into primary human NK cells. CAR transduction was efficient, its expression remained stable after expansion, and it improved efficacy against AML targets.

Original languageEnglish (US)
Article number100236
JournalCell Reports Methods
Volume2
Issue number6
DOIs
StatePublished - Jun 20 2022

Bibliographical note

Funding Information:
This work was supported by funding from the CancerFree KIDS Pediatric Cancer Research Alliance (M.N.K.), the Hyundai Hope on Wheels Foundation (D.A.L.), and the NIH-NCI (U54-CA232561, D.A.L.). We thank the Institute for Genomic Medicine at Nationwide Children's Hospital for their assistance with nucleic acid sequencing. Schematics were created with https://biorender.com. M.N.K. and D.A.L. conceived of the project; S.L. K.Y. B.S.M. K.M. G.K.B. M.d.S.F.P. and D.A.L. designed the experiments; M.N.K. S.L. K.Y. N.C. E.E. Y.S. M.S. R.D.D. J.M.L. and K.S. performed the experiments; M.N.K. K.Y. B.S.M. G.K.B. and D.A.L. analyzed and interpreted the data; and M.N.K. K.Y. B.S.M. M.S. and D.A.L. prepared the manuscript. D.A.L. reports stock from Courier Therapeutics, personal fees and stock options from Caribou Biosciences, and membership on the NIH Novel and Exception Therapies and Research Advisory Committee (NExTRAC) unrelated to the submitted work. D.A.L. also reports membership on the scientific advisory board and consulting, licensing, and royalty fees from Kiadis Pharma related to patents US62/825,007; US63/105,722; US62/928,524; PCT-US2019/032,670; PCT-US2018/020187; WO2019/222,503-A1; PCT-US2020/018,384; US62/805,394; US62/987,935; US62/900,245; and US62/815,625. M.N.K. reports consulting, licensing, and royalty fees from Kiadis Pharma related to patents US62/825,007, WO2019/222,503A1, US63/105,722, PCT-US2020/02545, US63/018,108, US62/928,524, and US62/987,935. B.S.M. is founder of and has sponsored research with Catamaran Bio and patent WO2017/214,569A1. K.M. and S.L. report licensing and royalty fees from Kiadis Pharma related to patent PCT-US2020/025,454. N.C. reports licensing and royalty fees from Kiadis Pharma related to patent PCT-US2018/020,187.

Funding Information:
This work was supported by funding from the CancerFree KIDS Pediatric Cancer Research Alliance (M.N.K.), the Hyundai Hope on Wheels Foundation (D.A.L.), and the NIH -NCI ( U54-CA232561 , D.A.L.). We thank the Institute for Genomic Medicine at Nationwide Children’s Hospital for their assistance with nucleic acid sequencing. Schematics were created with https://biorender.com .

Publisher Copyright:
© 2022 The Author(s)

Keywords

  • AAV6
  • AML
  • Acute Myeloid Leukemia
  • CAR
  • CD33CAR-NK
  • CRISPR
  • CRISPaint
  • Cas9/RNP
  • Cas9/ribonucleoprotein
  • HDR
  • NK
  • adeno-associated virus 6
  • chimeric antigen receptor
  • clustered regularly interspaced short palindromic repeats
  • gene editing
  • homology directed repair
  • natural killer cell

PubMed: MeSH publication types

  • Journal Article

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