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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.


Related in: MedlinePlus

Combining TRC1-2 and an AAV Donor Template Results in Highly Efficient HDR-Mediated Insertion of an Anti-CD19 CAR into the TRAC Locus and Simultaneous Disruption of TCR Expression(A) Diagram of the AAV vector used to transduce cells. AAV:TRAC:CAR contains a CD19 CAR transgene with expression driven by the JeT promoter and flanked by homology arms on the 5′ (L TRAC HA) and 3′ (R TRAC HA) sides to enable targeted integration. (B) Diagram of the PCR used to confirm CAR integration by amplification with one primer located within the CAR and one primer in TRAC outside of the homology arms at both the 5′ and 3′ ends to generate 1,872-bp and 1107-bp products, respectively. (C and D) T cells were mock-electroporated or electroporated with TRC1-2 mRNA and then immediately transduced with the indicated amounts of AAV:TRAC:CAR. (C) PCR was used to confirm the presence of the CAR transgene integrated in the TRAC locus on day 3 post-electroporation and transduction as outlined in (B). (D) CAR and CD3 expression were evaluated by flow cytometry on days 3 and 8 post-electroporation and transduction.
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fig3: Combining TRC1-2 and an AAV Donor Template Results in Highly Efficient HDR-Mediated Insertion of an Anti-CD19 CAR into the TRAC Locus and Simultaneous Disruption of TCR Expression(A) Diagram of the AAV vector used to transduce cells. AAV:TRAC:CAR contains a CD19 CAR transgene with expression driven by the JeT promoter and flanked by homology arms on the 5′ (L TRAC HA) and 3′ (R TRAC HA) sides to enable targeted integration. (B) Diagram of the PCR used to confirm CAR integration by amplification with one primer located within the CAR and one primer in TRAC outside of the homology arms at both the 5′ and 3′ ends to generate 1,872-bp and 1107-bp products, respectively. (C and D) T cells were mock-electroporated or electroporated with TRC1-2 mRNA and then immediately transduced with the indicated amounts of AAV:TRAC:CAR. (C) PCR was used to confirm the presence of the CAR transgene integrated in the TRAC locus on day 3 post-electroporation and transduction as outlined in (B). (D) CAR and CD3 expression were evaluated by flow cytometry on days 3 and 8 post-electroporation and transduction.

Mentions: Next, we sought to combine the disruption of the endogenous TRAC gene with simultaneous homology-directed insertion of an anti-CD19 CAR transgene. We generated an AAV6 vector called AAV:TRAC:CAR, comprising an anti-CD19-BB-zeta CAR32 expression cassette flanked by TRAC homology arms (Figure 3A). We transduced mock-electroporated and TRC1-2-electroporated T cells at multiple vector doses and evaluated the cells by PCR and flow cytometry. To confirm site-specific integration, we designed PCR primers to amplify products spanning the homology arms on the 5′ and 3′ sides of the CAR transgene so that PCR products could only be generated by properly targeted events (Figure 3B). No PCR products were amplified from mock-electroporated cells, consistent with earlier observations that targeted gene integration in the absence of a DNA break is exceedingly rare, or in TRC1-2-electoporated mock-transduced cells (Figure 3C). In contrast, PCR products were amplified from all cells electroporated with TRC1-2 mRNA and transduced with AAV:TRAC:CAR, confirming targeted integration into the TRAC locus in these cells. Analysis of CD3 and CAR expression by flow cytometry showed a high frequency of CAR+ cells in the CD3− population (Figure 3D). At all MOIs, the frequency of CD3− cells increased between days 3 and 8 post-transduction, reflecting turnover of the TCR complex. In this experiment, integration efficiency approached 40% of total cells expressing the CAR but not CD3. Furthermore, under these conditions, >50% of the CD3− cells expressed the CAR. These data indicate that the TRC1-2 nuclease can efficiently target the insertion of a CAR transgene into the TRAC locus.


Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells
Combining TRC1-2 and an AAV Donor Template Results in Highly Efficient HDR-Mediated Insertion of an Anti-CD19 CAR into the TRAC Locus and Simultaneous Disruption of TCR Expression(A) Diagram of the AAV vector used to transduce cells. AAV:TRAC:CAR contains a CD19 CAR transgene with expression driven by the JeT promoter and flanked by homology arms on the 5′ (L TRAC HA) and 3′ (R TRAC HA) sides to enable targeted integration. (B) Diagram of the PCR used to confirm CAR integration by amplification with one primer located within the CAR and one primer in TRAC outside of the homology arms at both the 5′ and 3′ ends to generate 1,872-bp and 1107-bp products, respectively. (C and D) T cells were mock-electroporated or electroporated with TRC1-2 mRNA and then immediately transduced with the indicated amounts of AAV:TRAC:CAR. (C) PCR was used to confirm the presence of the CAR transgene integrated in the TRAC locus on day 3 post-electroporation and transduction as outlined in (B). (D) CAR and CD3 expression were evaluated by flow cytometry on days 3 and 8 post-electroporation and transduction.
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Related In: Results  -  Collection

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fig3: Combining TRC1-2 and an AAV Donor Template Results in Highly Efficient HDR-Mediated Insertion of an Anti-CD19 CAR into the TRAC Locus and Simultaneous Disruption of TCR Expression(A) Diagram of the AAV vector used to transduce cells. AAV:TRAC:CAR contains a CD19 CAR transgene with expression driven by the JeT promoter and flanked by homology arms on the 5′ (L TRAC HA) and 3′ (R TRAC HA) sides to enable targeted integration. (B) Diagram of the PCR used to confirm CAR integration by amplification with one primer located within the CAR and one primer in TRAC outside of the homology arms at both the 5′ and 3′ ends to generate 1,872-bp and 1107-bp products, respectively. (C and D) T cells were mock-electroporated or electroporated with TRC1-2 mRNA and then immediately transduced with the indicated amounts of AAV:TRAC:CAR. (C) PCR was used to confirm the presence of the CAR transgene integrated in the TRAC locus on day 3 post-electroporation and transduction as outlined in (B). (D) CAR and CD3 expression were evaluated by flow cytometry on days 3 and 8 post-electroporation and transduction.
Mentions: Next, we sought to combine the disruption of the endogenous TRAC gene with simultaneous homology-directed insertion of an anti-CD19 CAR transgene. We generated an AAV6 vector called AAV:TRAC:CAR, comprising an anti-CD19-BB-zeta CAR32 expression cassette flanked by TRAC homology arms (Figure 3A). We transduced mock-electroporated and TRC1-2-electroporated T cells at multiple vector doses and evaluated the cells by PCR and flow cytometry. To confirm site-specific integration, we designed PCR primers to amplify products spanning the homology arms on the 5′ and 3′ sides of the CAR transgene so that PCR products could only be generated by properly targeted events (Figure 3B). No PCR products were amplified from mock-electroporated cells, consistent with earlier observations that targeted gene integration in the absence of a DNA break is exceedingly rare, or in TRC1-2-electoporated mock-transduced cells (Figure 3C). In contrast, PCR products were amplified from all cells electroporated with TRC1-2 mRNA and transduced with AAV:TRAC:CAR, confirming targeted integration into the TRAC locus in these cells. Analysis of CD3 and CAR expression by flow cytometry showed a high frequency of CAR+ cells in the CD3− population (Figure 3D). At all MOIs, the frequency of CD3− cells increased between days 3 and 8 post-transduction, reflecting turnover of the TCR complex. In this experiment, integration efficiency approached 40% of total cells expressing the CAR but not CD3. Furthermore, under these conditions, >50% of the CD3− cells expressed the CAR. These data indicate that the TRC1-2 nuclease can efficiently target the insertion of a CAR transgene into the TRAC locus.

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.


Related in: MedlinePlus