Tumor-stromal interactions are a determining factor in cancer progression. In vivo, the interaction interface is associated with spatially-resolved distributions of cancer and stromal phenotypes. Here, we establish a micropatterned... more
Tumor-stromal interactions are a determining factor in cancer progression. In vivo, the interaction interface is associated with spatially-resolved distributions of cancer and stromal phenotypes. Here, we establish a micropatterned tumor-stromal assay (μTSA) with laser capture microdissection to control the location of co-cultured cells and analyze bulk and interfacial tumor-stromal signaling in driving cancer progression. μTSA reveals a spatial distribution of phenotypes in concordance with human estrogen receptor-positive (ER+) breast cancer samples, and Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms
BackgroundAutologous CAR-T therapies have demonstrated remarkable efficacy in treating some hematologic cancers. However, generating bespoke cell therapies creates manufacturing challenges, inconsistent products, high cost of goods, and... more
BackgroundAutologous CAR-T therapies have demonstrated remarkable efficacy in treating some hematologic cancers. However, generating bespoke cell therapies creates manufacturing challenges, inconsistent products, high cost of goods, and delays in treatment that are often incompatible with effective clinical management of patients. Strategies to create universally-compatible allogeneic CAR-T therapies have been developed as a solution to these challenges. Allogeneic CAR-Ts require mitigation of graft-versus-host-disease (GvHD), host rejection of CAR-Ts, and elimination of fratricide in instances where the target (e.g. CD7) is expressed on both malignant cells and healthy T-cells. Many allogeneic CAR-T approaches utilize DNA double strand break (DSB)-inducing nucleases to overcome these barriers. However, simultaneous induction of multiple DSBs results in unpredictable outcomes such as large-scale genomic rearrangements, megabase-scale deletions, and reduced cell proliferation. Here w...
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The foundational adenine base editors (e.g. ABE7.10) enable programmable C•G to T•A point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of... more
The foundational adenine base editors (e.g. ABE7.10) enable programmable C•G to T•A point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG PAM sites, ABE8s result in ∼1.5x higher editing at protospacer positions A5-A7 and ∼3.2x higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a ∼4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the γ-globin genes HBG1 and HBG2, with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification which is maintained when multiplexed across three loci. Delivered as mRNA, ABE8s induce no significant levels of sgRNA-independent off-target adenine deamination in genomic DNA and very low levels of adenine deaminat...
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BackgroundT-cell lymphomas and leukemias are a class of diseases lacking durable effective therapies, where median survival for patients suffering from relapsed/refractory disease is often measured in months. Translation of B-cell... more
BackgroundT-cell lymphomas and leukemias are a class of diseases lacking durable effective therapies, where median survival for patients suffering from relapsed/refractory disease is often measured in months. Translation of B-cell targeting CAR-T therapeutic success to T-cell malignancies comes with significant challenges. Notably, the shared expression of target antigens on malignant T-cells and in the T-cell product itself results in CAR-T activation and fratricide during manufacturing. To overcome the challenges associated with creating CD5-targeting CAR-Ts, we developed a process to simultaneously base edit five target genes, including CD5 and PD1, to produce potency-enhanced allogeneic anti-CD5 CAR T-cells for use as an off-the-shelf treatment for T-cell malignancies.MethodsAnti-CD5 CAR-Ts were produced in a GMP-compatible process using T-cells isolated from healthy human donors. T-cells were modified using base editing technology to simultaneously knock-out five target genes i...
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Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended... more
Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC7...
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Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for... more
Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these e...