Alexander Astrakhan

Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template

Therapeutic coding sequences can be targeted to the CCR5 locus of primary human T cells with high efficiency by using megaTAL nuclease and an AAV donor template. Delete and replace Newer gene-editing methods hold promise for correcting human disease but so far have been hampered by low efficiencies when used in primary cells. To address this issue, Sather et al. have devised a more effective way to both disrupt and replace the CCR5 locus in human T cells, a procedure that has already been shown to improve HIV clearance. Serotype 6 of an adeno-associated viral vector worked particularly well for delivery of megaTAL nucleases and homologous donor templates to primary human T cells, achieving efficient gene-editing rates and little toxicity. The megaTALs generate homology-directed repair (rather than previous efforts, which induce nonhomologous end-joining repair) and so was used for both deletion and accurate replacement of the CCR5 locus. The authors demonstrate that chimeric antigen receptors and an HIV fusion inhibitor inserted into the CCR5 locus ameliorate HIV infection in mice and show that their approach also works in CD34+ hematopoietic precursor cells. Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4+ T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)–mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP–modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34+ cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties.

Homology-Directed Recombination for Enhanced Engineering of Chimeric Antigen Receptor T Cells

Gene editing by homology-directed recombination (HDR) can be used to couple delivery of a therapeutic gene cassette with targeted genomic modifications to generate engineered human T cells with clinically useful profiles. Here, we explore the functionality of therapeutic cassettes delivered by these means and test the flexibility of this approach to clinically relevant alleles. Because CCR5-negative T cells are resistant to HIV-1 infection, CCR5-negative anti-CD19 chimeric antigen receptor (CAR) T cells could be used to treat patients with HIV-associated B cell malignancies. We show that targeted delivery of an anti-CD19 CAR cassette to the CCR5 locus using a recombinant AAV homology template and an engineered megaTAL nuclease results in T cells that are functionally equivalent, in both in vitro and in vivo tumor models, to CAR T cells generated by random integration using lentiviral delivery. With the goal of developing off-the-shelf CAR T cell therapies, we next targeted CARs to the T cell receptor alpha constant (TRAC) locus by HDR, producing TCR-negative anti-CD19 CAR and anti-B cell maturation antigen (BCMA) CAR T cells. These novel cell products exhibited in vitro cytolytic activity against both tumor cell lines and primary cell targets. Our combined results indicate that high-efficiency HDR delivery of therapeutic genes may provide a flexible and robust method that can extend the clinical utility of cell therapeutics.

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance

A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patients immune system and provide protection from HIV infection.

323. Efficient Generation of CART Cells by Homology Directed Transgene Integration into the TCR-Alpha Locus

Genetically engineered cancer targeting CAR-T cells have generated promising results in a series of B cell derived cancers. Development of these therapies for solid tumor applications has been much more difficult and will likely require extensive T cell engineering to improve both efficacy and safety. Advanced gene editing approaches were developed to enable simultaneous disruption of a target gene combined with introduction of exogenous transgenes at the disrupted locus. A gene specific megaTAL nuclease was used to generate double stranded DNA breaks followed by transduction with adeno-associated virus (AAV) encoding new genetic information flanked by regions of homology proximal to the nuclease breakpoint. Highly efficient introduction of a CD19-specific CAR transgene into the T cell receptor-alpha constant (TRAC) locus was demonstrated using this approach. T cells treated with the TRAC megaTAL and corresponding AAV encoding a CD19 CAR and TRAC homology arms generated greater than 50% of CD19-CAR positive T cells that no longer expressed the T cell receptor complex. In vitro assays confirmed that TRAC-targeted CD19-CAR T cells were comparable to CD19 CAR-T cells generated by lentiviral transduction in their cytotoxicity and cytokine responses against CD19+ Nalm-6 cells. These findings demonstrate the potential of megaTAL driven homology directed T cell genome engineering to obviate the need for traditional integrating viral vectors and generate a defined and potentially more potent T cell product by combining gene disruption with targeted transgene integration.

Efficient Enrichment of Gene-Modified Primary T Cells via CCR5-Targeted Integration of Mutant Dihydrofolate Reductase

Targeted gene therapy strategies utilizing homology-driven repair (HDR) allow for greater control over transgene integration site, copy number, and expression—significant advantages over traditional vector-mediated gene therapy with random genome integration. However, the relatively low efficiency of HDR-based strategies limits their clinical application. Here, we used HDR to knock in a mutant dihydrofolate reductase (mDHFR) selection gene at the gene-edited CCR5 locus in primary human CD4+ T cells and selected for mDHFR-modified cells in the presence of methotrexate (MTX). Cells were transfected with CCR5-megaTAL nuclease mRNA and transduced with adeno-associated virus containing an mDHFR donor template flanked by CCR5 homology arms, leading to up to 40% targeted gene insertion. Clinically relevant concentrations of MTX led to a greater than 5-fold enrichment for mDHFR-modified cells, which maintained a diverse TCR repertoire over the course of expansion and drug selection. Our results demonstrate that mDHFR/MTX-based selection can be used to enrich for gene-modified T cells ex vivo, paving the way for analogous approaches to increase the percentage of HIV-resistant, autologous CD4+ T cells infused into HIV+ patients, and/or for in vivo selection of gene-edited T cells for the treatment of cancer.

Abstract 1708: Effective and reversible control of anti-tumor activityin vivowith a drug-regulated CAR T cell platform (DARIC)

Redirecting T cells against tumors by introducing antigen-specific chimeric antigen receptors (CAR) has shown promising clinical results as a potential treatment strategy for certain cancers. However, traditional CARs are constitutively active, resulting in the persistent loss of all target cells (including off -tumor, on-target activity against normal tissues that express the target antigen) and enhanced potential of excessive T cell activation to drive cytokine release syndrome. While “off switches” based on suicide cassettes or other depleting cell approaches are in development, such systems by definition result in the elimination of the therapeutic cells. Here we have developed a novel drug-regulated CAR-based antigen targeting approach termed Dimerizing Agent Regulated Immune-receptor Complex (DARIC) that aims to: i) minimize the long-term toxicity of CAR T treatment; ii) allow the targeting of previously inaccessible antigens; and iii) be amenable to multiplex antigen targeting. The DARIC platform separates the antigen recognition and signaling functions of a CAR into two distinct polypeptides that are further engineered to contain the FKPB12 and FRB small-molecule regulated dimerization domains. In the absence of the dimerizing drug (e.g. rapamycin or the non-immunosuppressive rapalog AP21967) the DARIC system lacks signaling activity. However, the addition of dimerizing agent drives the interaction of the two DARIC subunits, fully restoring CAR function. Using CD19 as a model system, we show that treatment of CD19-DARIC+ T cells with rapamycin or AP21967 results in equivalent cytotoxicity, cytokine production and proliferation compared to a standard CD19-targeting CAR. Importantly, CD19-DARIC T cells were activated by picomolar levels of rapamycin and exhibited a higher antigen sensitivity than standard CD19-CAR T cells in vitro. In an aggressive CD19+Nalm-6 xenograft tumor mouse model, CD19-DARIC T cells did not exhibit anti-tumor activity in the absence of dimerizing agent. However, CD19-DARIC treated mice that received either low-dose rapamycin or AP21967 showed an equivalent level of tumor control compared to standard CD19-CAR treated animals. This activity was dependent on the presence of the dimerizing drug, as cessation of drug treatment resulted in the loss of CD19-DARIC T cell activity and the expansion of Nalm-6 tumors cells in the DARIC T cell treated mice, consistent with the ability to switch off CD19-DARIC T cells in vivo by withdrawing drug. Taken together, these results highlight the potential of the DARIC platform to facilitate the regulation of CAR T cell function both in vitro and in vivo. Citation Format: Wai-Hang Leung, Michael Certo, Holly Horton, Joel Gay, Tracy VandenBerg, Jordan Jarjour, Alexander Astrakhan. Effective and reversible control of anti-tumor activity in vivo with a drug-regulated CAR T cell platform (DARIC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1708. doi:10.1158/1538-7445.AM2017-1708

Abstract 3756: Efficient generation of CAR T cells by site-specific gene addition into the TRAC locus

Genetically engineered T cells represent a promising new approach to the treatment of cancer. Positive results in recent clinical trials exploiting T cells engineered to express chimeric antigen receptors (CARs) have highlighted the potential for these cell-based therapies in patients with B cell malignancies. To extend these successes to a broader range of tumor types may require additional T cell engineering beyond simple CAR addition, such as gene knockout and/or coupling deletion of a target gene with site-specific addition of a CAR transgene. To this end, we have developed a genome editing strategy for the simultaneous elimination of an endogenous target gene with site-specific addition of a CAR via homology-directed repair (HDR). To demonstrate the utility of this approach, we used a previously characterized megaTAL (an engineered nuclease created by the fusion of an engineered meganuclease and a transcription activator-like (TAL) -DNA binding domain) specific for the T cell receptor alpha constant region gene (TRAC). Delivery of this megaTAL to primary human T cells via mRNA electroporation results in efficient and specific disruption of the TRAC locus (Boissel et al, 2013). To achieve simultaneous target gene disruption with site-specific CAR transgene insertion, we designed an adeno-associated virus (AAV) vector for DNA template delivery encoding the CAR and flanked by regions of DNA homologous to the genome immediately surrounding the megaTAL cleavage site. Co-delivery of the megaTAL and AAV encoding a CD19-CAR with TRAC homology arms resulted in >50% CAR+TRAC- cells. In vitro assays of cytotoxicity and cytokine responses against CD19+ Nalm-6 cells confirmed that TRAC-targeted CD19-CAR T cells were comparable to CD19-CAR T cells generated by lentiviral transduction. Interestingly, a similar level of CAR T cell function was observed even though TRAC-targeted CD19-CAR T cells expressed lower amounts of the CAR, as determined by flow cytometry. Similar CAR integration efficiency and functional efficacy was observed using a TRAC-targeting AAV vector containing a distinct B cell maturation antigen (BCMA)-specific CAR. These findings demonstrate megaTAL-mediated targeted gene addition as a feasible, efficient, and potentially safer approach for generation of gene-edited CAR T cell product. Moreover, the ability to combine the disruption of a target gene with the site-specific integration of the CAR eliminates the need for randomly integrating viral vectors while satisfying the potential need for more complex genome-edited T cell products. Citation Format: Baeckseung Lee, Wai-Hang Leung, Mark Pogson, Jordan Jarjour, Alexander Astrakhan. Efficient generation of CAR T cells by site-specific gene addition into the TRAC locus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3756. doi:10.1158/1538-7445.AM2017-3756

277. Small Molecule-Regulated Antigen Recognition System for Inducible T Cell Targeting of Cancer Cells

Redirecting T cells against tumors by introducing chimeric antigen receptors (CAR) has demonstrated promising clinical results in certain cancers. However, the constitutive activity of these receptors limits which antigens can be targeted with this approach and may result in persistent elimination of healthy antigen-expressing cells. Successful CAR-T targeting of CD19+ tumors, for example, produces chronic B cell aplasia and necessitates life-long intravenous immunoglobulin (IVIG) treatment. Daric is an alternative antigen targeting approach that aims to: i) minimize the long-term toxicity of CAR-T treatment; ii) allows targeting of previously inaccessible antigens; and iii) is amenable to multiplex antigen targeting and other advanced targeting designs and strategies. The Daric system separates the antigen recognition and signaling functions of a CAR into two distinct polypeptides that contain the FKPB12 and FRB dimerization domains but lack signaling activity in the absence of a dimerization agent. Addition of the FKBP12-FRB bridging drug rapamycin or a non-immunosuppressive rapamycin analogue AP21967 heterodimerizes the signaling and antigen recognition components and restores signaling competency for antigen-dependent T cell activation. Importantly, the FKB12 and FRB dimerization partners are located extracellularly, minimizing interference with endogenously expressed signaling components and eliminating the requirement for rapamycin/AP21967 cell penetrance. A range of extracellular linkers and transmembrane domains were used to design a variety of CD19-targeting Daric constructs that exhibit minimal basal activity and robust drug-dependent antigen-specific cytolytic activity and cytokine production. The CD19-specific Daric T cells exhibited comparable levels of cytokine release, proliferation and cytolytic activity compared to a CD19-targeting CAR T cells. In vitro, Daric T cells were activated at sub-nanomolar to nanomolar concentrations of rapamycin or AP21967. No differences in cellular phenotype, expansion or functional responsiveness of Daric T cells compared to CAR T cells were observed. As expected, rapamycin was immunosuppressive to CAR T cell functionality, however Daric T cells continued to produce high level of cytokines even in the presence of rapamycin. These results highlight the potential of the Daric system to target highly expressed antigens and minimize the off-tumor on-target toxicity associated with traditional CAR designs.

398. Site-Specific Introduction of Chimeric Antigen Receptors to Primary Human T Cells

In current clinical trials, delivery of an anti-cancer chimeric antigen receptor (CAR) to patient T cells is accomplished using randomly integrating retroviruses. Designer nucleases and AAV donor templates allow an alternative delivery method that introduces the CAR gene cassette at a target locus via homologous recombination, with concomitant disruption of the endogenous gene. Here we show high-efficiency introduction of anti-CD19 and anti-BCMA CAR expression cassettes in primary human T cells at two clinically relevant loci: CCR5 and TCRa. These gene-targeted CAR+ cells (tCARs) exhibit equivalent activity in vitro against CD19+ and BCMA+ cell lines compared to lentiviral-delivered CARs, and effectively clear tumor in a murine xenograft model. Advantages over lentiviral delivery include utilization of a defined integration site for the CAR construct linked with endogenous gene disruption. The use of specific nucleases and AAV for CAR delivery expands the possibilities for a precisely engineered cell therapy product in the clinic. For example, a CCR5-tCAR may be clinically useful in HIV+ patients, where traditional delivery methods leave therapeutic cells vulnerable to HIV infection and elimination. Moreover, a TCRa-tCAR could allow for off-the-shelf CAR therapy, potentially removing limitations to current approaches. Finally, the approach described here is broadly applicable for targeted delivery of alternative therapeutic cassettes at translationally relevant sites across the human genome.

44. Novel Combination of megaTAL Nuclease-Driven Genome Engineering with a Drug Selection Cassette Increases Efficiency of HIV Gene Therapy

Human Immunodeficiency Virus (HIV) infection remains a substantial health problem worldwide. The C-C chemokine receptor 5 (CCR5) serves as a co-receptor for HIV entry into CD4+ T cells and represent an alternative therapeutic target. Early clinical trials using CCR5-targeting zinc finger nucleases demonstrated transient control of HIV infection in the course of antiretroviral treatment interruption (Tebas, NEJM, 2014). Our current work improves on these advances by combining high level of CCR5 gene disruption with preferential selection of gene modified cells. The CCR5-targeting megaTAL combines a LAGLIDADG homing endonuclease scaffold with an eleven repeat transcription activator-like (TAL) effector array to achieve site specific DNA cleavage. This nuclease produces highly efficient CCR5 targeting in primary human CD4+ T cells in vitro (70-90% disruption). To test the protective effects of megaTAL treatment, primary human CD4+ T cells treated with CCR5-megaTAL were transplanted into NOD/SCID/γc-null (NSG) ‘humanized’ mice and challenged with HIV-1. We observed a 100-fold increase of megaTAL-treated cells compared to untreated controls during an active in vivo infection demonstrating the functionality of this approach. Based on the decline of CCR5 modified cells in the clinical trials to date, we hypothesized that we could improve maintenance of HIV resistant cells by expanding them either ex vivo or in vivo. We propose that coupling megaTAL nuclease treatment with drug selection will help us achieve therapeutically relevant levels of HIV-protected cells by enabling efficient selection only of CCR5-modified T-cells. The mutant human dihydrofolate reductase (mDHFR) chemoselection system has been used to render cells resistant to lymphotoxic concentrations of the drug methotrexate (MTX). We tested our experimental approach by transducing cells with lentiviral vector encoding a mDHFR cassette followed by chemoselection in MTX at 0.02uM. This approach resulted in a six fold enrichment of gene modified primary CD4+ T cells ex vivo. Previously we have shown that combining megaTAL treatment with adeno-associated virus (AAV) transduction produces very high rates of homology-driven repair (HDR) in primary human T cells. Hence, we combined megaTAL/AAV treatment to integrate the mutant DHFR into the CCR5 locus, producing a population of MTX-resistant CD4+ cells that also lack CCR5. Primary human CD4+ T cells were transfected with CCR5-megaTAL mRNA and transduced with AAV6 containing a mutant DHFR donor template flanked by 0.6kb CCR5 homology arms. They demonstrated a greater than five-fold enrichment in MTX compared to untreated controls ex vivo. Next, we have transplanted NSG mice with 1×106 gene modified cells/mouse to assess the therapeutic potential of our approach. Mice that engraft effectively will be treated with daily injections of 0, 0.5 and 2 mg/kg of MTX to monitor preferential selection and enrichment of our target cell population. Subsequent studies will assess the long term control of viremia in these mice following HIV challenge. In conclusion, the CCR5-megaTAL nuclease platform produces very high levels of gene-modified CD4+ T-cells and protects these cells from subsequent HIV infection in vivo. Furthermore, combining targeted integration and chemical selection results in the specific selection of gene modified primary human T cells. To our knowledge we are the first group to report MTX-mediated chemoselection and expansion of CD4+ T cells following targeted integration at the CCR5 locus.