Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.
Adoptive immunotherapy using chimeric antigen receptor–modified T cells (CAR-T) has made substantial contributions to the treatment of certain B cell malignancies. Such treatment modalities could potentially obviate the need for long-term antiretroviral drug therapy in HIV/AIDS. Here, we report the development of HIV-1–based lentiviral vectors that encode CARs targeting multiple highly conserved sites on the HIV-1 envelope glycoprotein using a two-molecule CAR architecture, termed duoCAR. We show that transduction with lentiviral vectors encoding multispecific anti-HIV duoCARs confer primary T cells with the capacity to potently reduce cellular HIV infection by up to 99% in vitro and >97% in vivo. T cells are the targets of HIV infection, but the transduced T cells are protected from genetically diverse HIV-1 strains. The CAR-T cells also potently eliminated PBMCs infected with broadly neutralizing antibody-resistant HIV strains, including VRC01/3BNC117-resistant HIV-1. Furthermore, multispecific anti-HIV duoCAR-T cells demonstrated long-term control of HIV infection in vivo and prevented the loss of CD4+T cells during HIV infection using a humanized NSG mouse model of intrasplenic HIV infection. These data suggest that multispecific anti-HIV duoCAR-T cells could be an effective approach for the treatment of patients with HIV-1 infection.
A transgene, flanked by zinc finger nuclease (ZFN) cleavage sites, was deleted from a stably transformed plant by crossing it with a second plant expressing a corresponding ZFN gene. A target construct, containing a GUS reporter gene flanked by ZFN cleavage sites, a GFP reporter gene and a PAT selectable marker gene, was transformed into tobacco. Basta-resistant plants were regenerated and screened for GUS and GFP expression. A second construct, containing a ZFN gene driven by the constitutive CsVMV promoter and an HPT selectable marker gene, was also transformed into tobacco. Selected T(0) plants were grown to maturity and allowed to self-pollinate. Homozygous target plants, which expressed GUS and GFP, were crossed with homozygous ZFN plants, which expressed the ZFN gene. Numerous GUS-negative plants were observed among the hybrids with one particular cross displaying approximately 35% GUS-negative plants. Evidence for complete deletion of a 4.3 kb sequence comprising the GUS gene was obtained and sequence confirmed. Co-segregation in F(2) progenies of 'truncated' and 'intact' target sequences with expected reporter gene phenotypes were observed. Since ZFNs can be designed to bind and cleave a wide range of DNA sequences, these results constitute a general strategy for creating targeted gene deletions.
Chimeric antigen receptor T cells (CAR-T cell) targeting CD19 are effective against several subtypes of CD19-expressing hematologic malignancies. Centralized manufacturing has allowed rapid expansion of this cellular therapy, but it may be associated with treatment delays due to the required logistics. We hypothesized that point of care manufacturing of CART cells on the automated CliniMACS Prodigy R device allows reproducible and fast delivery of cells for the treatment of patients with non-Hodgkin lymphoma. Here we describe cell manufacturing results and characterize the phenotype and effector function of CART cells used in a phase I/II study. We utilized a lentiviral vector delivering a second-generation CD19 CAR construct with 4-1BB costimulatory domain and TNFRSF19 transmembrane domain. Our data highlight the successful generation of CART cells at numbers sufficient for all patients treated, a shortened duration of production from 12 to 8 days followed by fresh infusion into patients, and the detection of CART cells in patient circulation up to 1-year post-infusion.
2510 Background: Anti-CD19 CAR-T cell therapy is a breakthrough treatment (tx) for patients (pts) with relapsed/refractory (R/R) B-cell non-Hodgkin lymphoma (NHL). Despite impressive outcomes, non-response and relapse with CD19 negative disease remain challenges. Through dual B-cell antigen targeting of CD20 and CD19, with a first-in-human bispecific lentiviral CAR-T cell (LV20.19CAR), we aim to improve response rates while limiting CD19 negative relapse. Methods: Pts were treated on a Phase 1 dose escalation + expansion trial (NCT03019055) to demonstrate safety of a 41BB/CD3z LV20.19CAR T cell for adults with R/R B-cell NHL. Safety was assessed by incidence of dose limiting toxicities (DLTs) within 28 days post-infusion. Starting dose was 2.5 x 10^5 cells/kg with a target dose of 2.5 x 10^6 cells/kg. All pts received fludarabine+cyclophosphamide for lymphodepletion. Results: 11 pts have completed tx to date. 9 pts in dose escalation and 2 pts in expansion phase. Median age was 54 years (46-67) and histology included DLBCL = 5 pts, MCL = 4 pts, and CLL = 2 pts. In dose escalation, 3 pts were treated at 2.5 x 10^5 cells/kg, 3 pts at 7.5 x 10^5 cells/kg, and 3 pts at 2.5 x 10^6 cells/kg with no DLTs. As a result, 2.5 x 10^6 cells/kg was selected for expansion. In terms of safety, 6 pts developed Grade 1-2 cytokine release syndrome (CRS) and 3 pts had Grade 1-2 neurotoxicity (NTX). No patient had grade 3-4 CRS or NTX and none required ICU level care. 4 pts required 1-2 doses of tocilizumab for CRS. The day 28 overall response rate (ORR) for all pts was 82% (6/11 = complete response (CR) and 3/11 = partial response). All CR pts remain in remission, the longest > 1 year. All progressing pts underwent repeat biopsy, and all retained either CD19 or CD20 positivity. Additional pts are being enrolled in the expansion phase and updated data will be presented. Conclusions: Phase 1 results from the LV20.19 CAR T clinical trial demonstrate that infusion of 2.5 x 10^6 cells/kg is safe for further investigation with no DLTs among treated pts. Down-regulation of target antigens was not identified as a mechanism of resistance in progressing pts. With limited toxicity and encouraging ORR, dual targeted LV20.19CAR T cells merits further investigation. Clinical trial information: NCT03019055.
Transformation in grass species has traditionally relied on immature embryos and has therefore been limited to a few major Poaceae crops. Other transformation explants, including leaf tissue, have been explored but with low success rates, which is one of the major factors hindering the broad application of genome editing for crop improvement. Recently, leaf transformation using morphogenic genes Wuschel2 (Wus2) and Babyboom (Bbm) has been successfully used for Cas9-mediated mutagenesis, but complex genome editing applications, requiring large numbers of regenerated plants to be screened, remain elusive. Here we demonstrate that enhanced Wus2/Bbm expression substantially improves leaf transformation in maize and sorghum, allowing the recovery of plants with Cas9-mediated gene dropouts and targeted gene insertion. Moreover, using a maize-optimized Wus2/Bbm construct, embryogenic callus and regenerated plantlets were successfully produced in eight species spanning four grass subfamilies, suggesting that this may lead to a universal family-wide method for transformation and genome editing across the Poaceae.
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