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E - Disease Models and Clinical Applications -> Cancer – Immunotherapy, Cancer Vaccines

1502: Development of a Highly Efficient Modular and Multiplexed CRISPR Editing System for Multimodal Tracking of CAR-T Cells

Type: Poster Session

Poster Board Number: 1502
Presentation Details
Session Title: Friday Poster Session
Start Time: 5/19/2023 12:00
End Time: 5/19/2023 14:00

Intro: Translationally relevant imaging technologies that allow one to monitor the fate of chimeric antigen receptor (CAR) T cells could enable better predictions of patient response and/or side effects, as well as help evaluate CAR-T designs. Our aim was to develop a highly efficient CRISPR system for editing human T cells with multiple transgenes at loci known to improve both safety and efficacy of CAR-T cells, whilst also making them “visible” with relevant imaging modalities. T cells were edited at the TRAC locus to knock-in a CAR and simultaneously knock-out the endogenous T cell receptor (TCR). Human organic anion transporting polypeptide 1B3 (OATP1B3) for magnetic resonance imaging (MRI), human sodium iodide symporter (NIS) for positron emission tomography (PET) or Akaluc for bioluminescence imaging (BLI) were co-engineered into our CRISPR donor vectors to allow for multi-modal in vivo imaging of edited CAR-T cells. Methods: Human T cells were nucleofected with HiFi spCas9 and gRNAs targeting TRAC or AAVS1 loci. Adeno-associated viral (AAV6) vectors were designed for CD19- or HER2-CAR and reporter gene integration. Editing efficiency was determined by flow cytometry and integration by PCR. CD19+ (NALM6) or HER2+ (SKOV3) cancer cells expressing BLI reporter genes were engineered for CAR-T kill assays and preclinical mouse models. Uptake of the PET tracer [18F]tetrafluoroborate was evaluated in cells expressing NIS. Uptake of the contrast agent Gd-EOB-DTPA into OATP1B3-expressing cells was determined using 3T MRI. BLI was performed on an IVIS imaging system. Results: Flow cytometry showed >75% editing efficiency when targeting CAR AAVs to the TRAC locus. CD19CAR+/TCR- T cells significantly delayed cancer progression and extended survival in NALM6 and SKOV3 mouse models (Fig. 1). Reporter gene function was confirmed in CAR-OATP1B3 cells with MRI, in CAR-NIS cells with a gamma counter and CAR-Akaluc T cells with BLI. Multiplexing with CAR-OATP1B3/NIS at the TRAC locus and NIS/OATP1B3-LNGFR at the AAVS1 locus together resulted in duel edited cells. Single and dual edited CAR-T cells effectively killed CD19+ and HER2+ cells. Correct integration at TRAC and AAVS1 loci was confirmed by junction PCR analysis. In preclinical mouse models, BLI showed homing of CAR-Akaluc T cells to subcutaneous cancers (Fig. 2 A, B). As few as 1 x106 CAR-OATP1B3 cells pre-incubated with Gd-EOB-DTPA could be detected in subcutaneous intratumoral injections with 3T MRI (Fig. 2C). Conclusion: Our work describes a CRISPR-Cas system for highly efficient, modular editing of T cells with a CAR and preclinical and/or clinically relevant human reporter genes. These advanced CRISPR tools should have broad utility for co-editing primary cells with therapeutic genes and reporter genes to make trackable therapeutic cells with improved efficacy and safety profiles.

John J. Kelly1, Rafael E. Sanchez-Pupo1, Nourhan Shalaby1, Ying Xia1, Francisco M. Martinez1, Sean W. McRae1, Qi Qi2, Matthew S. Fox2, Justin W. Hicks2, Jonathan D. Thiessen2, Timothy J. Scholl1, John A. Ronald1

1Robarts Research Inst., University of Western Ontario, London, ON, Canada,2Lawson Health Research Institute, London, ON, Canada
 J.J. Kelly: None.

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