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Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Confirmation of Targeted Insertion of the CAR Transgene by Digital Droplet PCR(A) Diagram showing digital PCR strategy. Two primer pairs and probes are used: one to detect the CAR transgene inserted in TRAC and another to detect FXN and serve as a reference standard for genomic DNA. (B) Activated CD3+ T cells were mock-electroporated, electroporated with TRC1-2 nuclease mRNA, or mock-electroporated and transduced with 25,000 vg/cell AAV:TRAC:CAR. Digital PCR was used to quantify targeted integration of the CAR transgene in TRAC 11 days post-electroporation/transduction. (C) Activated CD3+ T cells were electroporated with TRC1-2 mRNA and transduced with 50,000 vg/cell AAV:TRAC:CAR. CD3+ and CD3− groups were magnetically separated on day 8 post-transduction. Cells were stained for CD3 expression and CAR expression in the pre-separation samples and CD3 expression post-separation to confirm purity. Digital PCR was used to quantify targeted integration of the CAR transgene in TRAC in pre-separation, CD3+, and CD3− populations.
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fig4: Confirmation of Targeted Insertion of the CAR Transgene by Digital Droplet PCR(A) Diagram showing digital PCR strategy. Two primer pairs and probes are used: one to detect the CAR transgene inserted in TRAC and another to detect FXN and serve as a reference standard for genomic DNA. (B) Activated CD3+ T cells were mock-electroporated, electroporated with TRC1-2 nuclease mRNA, or mock-electroporated and transduced with 25,000 vg/cell AAV:TRAC:CAR. Digital PCR was used to quantify targeted integration of the CAR transgene in TRAC 11 days post-electroporation/transduction. (C) Activated CD3+ T cells were electroporated with TRC1-2 mRNA and transduced with 50,000 vg/cell AAV:TRAC:CAR. CD3+ and CD3− groups were magnetically separated on day 8 post-transduction. Cells were stained for CD3 expression and CAR expression in the pre-separation samples and CD3 expression post-separation to confirm purity. Digital PCR was used to quantify targeted integration of the CAR transgene in TRAC in pre-separation, CD3+, and CD3− populations.

Mentions: To further quantify the efficiency of CAR transgene integration into the TRAC locus, we developed a digital droplet PCR (ddPCR) assay (Figure 4A). In this assay, two primer sets are used, each in conjunction with a labeled TaqMan probe: the first set spans a portion of the 3′ end of the CAR transgene and the 3′ homology arm flanking the CAR, and the second set amplifies an unrelated gene sequence (in the FXN gene) and serves as a reference sequence to control for template number. Consistent with the conventional PCR results shown above, cells that were mock-electroporated or that were not transduced with AAV:TRAC:CAR showed no sign of gene integration in this assay (Figure 4B). However, ddPCR measured 38% targeted gene integration in DNA from cells that were both electroporated with mRNA encoding TRC1-2 and transduced with the AAV:TRAC:CAR vector (Figure 4C). When this same population of cells was analyzed by flow cytometry, total CAR expression was just over 37%, suggesting that detectable CAR expression was due to insertion of the CAR expression cassette into the TRC1-2 recognition sequence (Figure 4C). Of the 37% of CAR+ cells detected by flow cytometry, a small percentage was also CD3+. We hypothesized that this was due to targeted integration of the CAR expression cassette into the unpaired TRAC allele (which does not contribute to forming a functional TCRαβ pair), which allowed expression of both the CAR and a functional TCR. To test this hypothesis, the cells were separated into CD3− and CD3+ fractions and assayed individually by ddPCR. Indeed, the total CD3+ cell population was 13.5% CAR+ by flow cytometry, and 19% of the TRAC alleles in this CD3+ population were found to harbor an integrated CAR transgene by ddPCR. Importantly, the total CD3− population was 48.6% CAR+ by flow cytometry, and 44% of alleles in this therapeutically relevant CD3− population harbored an integrated CAR transgene by ddPCR. Taken together, the flow cytometry and ddPCR results confirm highly efficient targeted insertion of an anti-CD19 CAR into the TRAC locus, most frequently with concomitant knockout of the endogenous TCR.


Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells
Confirmation of Targeted Insertion of the CAR Transgene by Digital Droplet PCR(A) Diagram showing digital PCR strategy. Two primer pairs and probes are used: one to detect the CAR transgene inserted in TRAC and another to detect FXN and serve as a reference standard for genomic DNA. (B) Activated CD3+ T cells were mock-electroporated, electroporated with TRC1-2 nuclease mRNA, or mock-electroporated and transduced with 25,000 vg/cell AAV:TRAC:CAR. Digital PCR was used to quantify targeted integration of the CAR transgene in TRAC 11 days post-electroporation/transduction. (C) Activated CD3+ T cells were electroporated with TRC1-2 mRNA and transduced with 50,000 vg/cell AAV:TRAC:CAR. CD3+ and CD3− groups were magnetically separated on day 8 post-transduction. Cells were stained for CD3 expression and CAR expression in the pre-separation samples and CD3 expression post-separation to confirm purity. Digital PCR was used to quantify targeted integration of the CAR transgene in TRAC in pre-separation, CD3+, and CD3− populations.
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Related In: Results  -  Collection

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fig4: Confirmation of Targeted Insertion of the CAR Transgene by Digital Droplet PCR(A) Diagram showing digital PCR strategy. Two primer pairs and probes are used: one to detect the CAR transgene inserted in TRAC and another to detect FXN and serve as a reference standard for genomic DNA. (B) Activated CD3+ T cells were mock-electroporated, electroporated with TRC1-2 nuclease mRNA, or mock-electroporated and transduced with 25,000 vg/cell AAV:TRAC:CAR. Digital PCR was used to quantify targeted integration of the CAR transgene in TRAC 11 days post-electroporation/transduction. (C) Activated CD3+ T cells were electroporated with TRC1-2 mRNA and transduced with 50,000 vg/cell AAV:TRAC:CAR. CD3+ and CD3− groups were magnetically separated on day 8 post-transduction. Cells were stained for CD3 expression and CAR expression in the pre-separation samples and CD3 expression post-separation to confirm purity. Digital PCR was used to quantify targeted integration of the CAR transgene in TRAC in pre-separation, CD3+, and CD3− populations.
Mentions: To further quantify the efficiency of CAR transgene integration into the TRAC locus, we developed a digital droplet PCR (ddPCR) assay (Figure 4A). In this assay, two primer sets are used, each in conjunction with a labeled TaqMan probe: the first set spans a portion of the 3′ end of the CAR transgene and the 3′ homology arm flanking the CAR, and the second set amplifies an unrelated gene sequence (in the FXN gene) and serves as a reference sequence to control for template number. Consistent with the conventional PCR results shown above, cells that were mock-electroporated or that were not transduced with AAV:TRAC:CAR showed no sign of gene integration in this assay (Figure 4B). However, ddPCR measured 38% targeted gene integration in DNA from cells that were both electroporated with mRNA encoding TRC1-2 and transduced with the AAV:TRAC:CAR vector (Figure 4C). When this same population of cells was analyzed by flow cytometry, total CAR expression was just over 37%, suggesting that detectable CAR expression was due to insertion of the CAR expression cassette into the TRC1-2 recognition sequence (Figure 4C). Of the 37% of CAR+ cells detected by flow cytometry, a small percentage was also CD3+. We hypothesized that this was due to targeted integration of the CAR expression cassette into the unpaired TRAC allele (which does not contribute to forming a functional TCRαβ pair), which allowed expression of both the CAR and a functional TCR. To test this hypothesis, the cells were separated into CD3− and CD3+ fractions and assayed individually by ddPCR. Indeed, the total CD3+ cell population was 13.5% CAR+ by flow cytometry, and 19% of the TRAC alleles in this CD3+ population were found to harbor an integrated CAR transgene by ddPCR. Importantly, the total CD3− population was 48.6% CAR+ by flow cytometry, and 44% of alleles in this therapeutically relevant CD3− population harbored an integrated CAR transgene by ddPCR. Taken together, the flow cytometry and ddPCR results confirm highly efficient targeted insertion of an anti-CD19 CAR into the TRAC locus, most frequently with concomitant knockout of the endogenous TCR.

View Article: PubMed Central - PubMed

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.

No MeSH data available.