title: Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells.
abstract: Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19+hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease state or difficulty with manufacturing suitable numbers of CAR T cells. Alternatively, T cells from a healthy donor can be used to produce an allogeneic CAR T therapy, provided the cells are rendered incapable of eliciting graft versus host disease (GvHD). One approach to the production of these cells is gene editing to eliminate expression of the endogenous T cell receptor (TCR). Here we report a streamlined strategy for generating allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. We demonstrate that anti-CD19 CAR T cells produced in this manner do not express the endogenous TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19+tumors in an in vivo mouse model.
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA. Electronic address: dan.macleod@precisionbiosciences.com.
authors.forename: Daniel T
authors.initials: DT
authors.lastname: MacLeod
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Jeyaraj
authors.initials: J
authors.lastname: Antony
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Martin
authors.affiliation: Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Ophthalmology, University of North Carolina, Chapel Hill, NC 27599; USA.
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Moser
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Armin
authors.initials: A
authors.lastname: Hekele
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Keith J
authors.initials: KJ
authors.lastname: Wetzel
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Audrey E
authors.initials: AE
authors.lastname: Brown
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Triggiano
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Jo Ann
authors.initials: JA
authors.lastname: Hux
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Christina D
authors.initials: CD
authors.lastname: Pham
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Victor V
authors.initials: VV
authors.lastname: Bartsevich
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Caitlin A
authors.initials: CA
authors.lastname: Turner
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Janel
authors.initials: J
authors.lastname: Lape
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Samantha
authors.initials: S
authors.lastname: Kirkland
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Clayton W
authors.initials: CW
authors.lastname: Beard
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Jeff
authors.initials: J
authors.lastname: Smith
authors.affiliation: Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Ophthalmology, University of North Carolina, Chapel Hill, NC 27599; USA.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Hirsch
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Nicholson
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA.
authors.forename: Derek
authors.initials: D
authors.lastname: Jantz
authors.affiliation: Precision BioSciences, Durham, NC 27701, USA. Electronic address: bruce.mccreedy@precisionbiosciences.com.
authors.forename: Bruce
authors.initials: B
authors.lastname: McCreedy
mesh_terms: D000483:Alleles; D000818:Animals; D018941:Antigens, CD19 / Q000235:genetics*; D061249:Batch Cell Culture Techniques; D060846:Cell Engineering; D000229:Dependovirus / Q000235:genetics; D004195:Disease Models, Animal; D000072669:Gene Editing; D015870:Gene Expression; D055786:Gene Knockout Techniques; D023061:Gene Order; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016219:Immunotherapy, Adoptive; D008223:Lymphoma / Q000235:genetics / Q000276:immunology / Q000628:therapy; D051379:Mice; D009369:Neoplasms; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism*; D014161:Transduction, Genetic
chemical_list: D018941:Antigens, CD19; C000605956:CD19-specific chimeric antigen receptor; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta
keywords: chimeric antigen receptor; gene editing; homology-directed repair