The Kaiser Permanente (KP) Research Program on Genes, Environment and Health (RPGEH), in collaboration with the University of California-San Francisco, undertook genome-wide genotyping of .100,000 subjects that constitute the Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort. The project, which generated .70 billion genotypes, represents the first large-scale use of the Affymetrix Axiom Genotyping Solution. Because genotyping took place over a short 14-month period, creating a near-real-time analysis pipeline for experimental assay quality control and final optimized analyses was critical. Because of the multi-ethnic nature of the cohort, four different ethnic-specific arrays were employed to enhance genome-wide coverage. All assays were performed on DNA extracted from saliva samples. To improve sample call rates and significantly increase genotype concordance, we partitioned the cohort into disjoint packages of plates with similar assay contexts. Using strict QC criteria, the overall genotyping success rate was 103,067 of 109,837 samples assayed (93.8%), with a range of 92.1-95.4% for the four different arrays. Similarly, the SNP genotyping success rate ranged from 98.1 to 99.4% across the four arrays, the variation depending mostly on how many SNPs were included as single copy vs. double copy on a particular array. The high quality and large scale of genotype data created on this cohort, in conjunction with comprehensive longitudinal data from the KP electronic health records of participants, will enable a broad range of highly powered genome-wide association studies on a diversity of traits and conditions. KEYWORDS genome-wide genotyping; GERA cohort; Affymetrix Axiom; saliva DNA; quality control T HE Genetic Epidemiology Research on Adult Health and Aging (GERA) resource is a cohort of .100,000 subjects who are participants in the Kaiser Permanente Medical Care Plan, Northern California Region (KPNC), Research Program on Genes, Environment and Health (RPGEH) (detailed description of the cohort and study design can be found in dbGaP, Study Accession: phs000674.v1.p1). Genome-wide genotyping was targeted for this cohort to enable large-scale genome-wide association studies by linkage to comprehensive longitudinal clinical data derived from extensive KPNC electronic health record databases. The cohort is multi-ethnic, with 20% minority representation (African American, East Asian, and Latino or mixed), and the remaining 80% nonHispanic white. For this project, four ethnic-specific arrays were designed based on the Affymetrix Axiom Genotyping System (Hoffmann et al. 2011a,b). The genotyping assay experiment took place over a 14-month period and to our knowledge, is the single largest genotyping experiment to date, producing .70 billion genotypes. The magnitude of the experiment, in conjunction with the long duration and simultaneous high throughput, required new protocols for assuring quality control (QC) during the assays and new genotyping strategies in postassay data analysis.Samp...

Our study provides the first in vivo and in patient evidence supporting the role of CHD1 in DSB repair and in response to DNA damaging therapy. We uncover mechanistic insights that CHD1 modulates the choice between HR and NHEJ DSB repair and suggest that CHD1 loss may contribute to the genomic instability seen in this subset of PCas.

Targeting the PI3K pathway is a promising strategy for treating prostate cancers with PTEN-loss. However, current anti-PI3K therapies fail to show long lasting effects. We find that not only the PI3Kα- and PI3kβ-isoforms, but also PI3Kδ, are associated with the epithelial-mesenchymal transition (EMT), a critical process distinguishing indolent from aggressive prostate cancer. This suggests that cotargeting PI3Kα/β/δ could preempt the rebound activation of the parallel pathways induced by α- or β-isoform-selective inhibitor and prevent EMT. Indeed, BAY1082439, a new selective PI3Kα/β/δ inhibitor, is highly effective in inhibiting null prostate cancer growth and preventing EMT in the mutant metastatic model. The anti-PI3Kδ property of BAY1082439 further blocks B-cell infiltration and lymphotoxin release, which are tumor microenvironment factors that promote castration-resistant growth. Together, our data suggest a new approach for the treatment of prostate cancer by targeting both tumor cells and tumor microenvironment with PI3Kα/β/δ inhibitor. .

<div>Abstract<p>Targeting the PI3K pathway is a promising strategy for treating prostate cancers with PTEN-loss. However, current anti-PI3K therapies fail to show long lasting <i>in vivo</i> effects. We find that not only the PI3Kα- and PI3kβ-isoforms, but also PI3Kδ, are associated with the epithelial–mesenchymal transition (EMT), a critical process distinguishing indolent from aggressive prostate cancer. This suggests that cotargeting PI3Kα/β/δ could preempt the rebound activation of the parallel pathways induced by α- or β-isoform–selective inhibitor and prevent EMT. Indeed, BAY1082439, a new selective PI3Kα/β/δ inhibitor, is highly effective <i>in vivo</i> in inhibiting <i>Pten-</i>null prostate cancer growth and preventing EMT in the mutant <i>Pten/Kras</i> metastatic model. The anti-PI3Kδ property of BAY1082439 further blocks B-cell infiltration and lymphotoxin release, which are tumor microenvironment factors that promote castration-resistant growth. Together, our data suggest a new approach for the treatment of prostate cancer by targeting both tumor cells and tumor microenvironment with PI3Kα/β/δ inhibitor. <i>Mol Cancer Ther; 17(10); 2091–9. ©2018 AACR</i>.</p></div>

PACT Pharma has developed a robust single-step, targeted, non-viral method for the manufacturing of personalized adoptive cells therapies for the treatment of solid cancers (NCT03970382). For this, we insert and express a neoepitope-specific T cell receptor (neoTCR) from the endogenous locus while simultaneously abolishing the expression of endogenous TCR. This results in neoTCR-specific T cells in which neoTCR expression is naturally regulated and not impeded by competition for CD3 by the endogenous TCR. We furthermore designed a system to additionally express CD8 coreceptor along with the neoTCR on the same RNA transcript from the native TCR promoter. Both methods, currently in clinical testing, allow for the timely generation of several billion T cells expressing neoTCRs using our GMP cell manufacturing procedure. Nevertheless, the suppressive nature of some tumor microenvironments may require cells engineered with further modifications for long lasting therapeutic outcomes in certain patients. Here we describe how the flexibility of a non-viral system allows for additional complex modifications in a single step. First, we demonstrate an additional gene knock-out in the TGFBR2 gene with simultaneous TCR replacement, making the engineered cells resistant to the immunosuppressive effects of TGF-β while still maintaining neoepitope-specific recognition of the tumor. Second, we demonstrate knock-down in addition to TCR replacement. For some targets, reduction of gene expression may be favored over complete knock-out. Accordingly, we have designed an shRNA expression construct that can efficiently knock-down transcript levels of a given gene in cells expressing the neoTCR without the generation of additional double-stranded breaks, allowing for enhanced T cell function in the absence of additional genomic breaks. Third, we demonstrate a non-viral knock-in strategy to express transgenes with transcriptional regulation independent from the neoTCR. This strategy generates a product from which two genes, the neoTCR plus an additional transgene regulated by its own promoter, can be precision genome engineered into the same cell using homology directed templates that far exceed the size limitations of AAV. This technology can be successfully applied for the expression of intracellular, secreted, and membrane-bound proteins as well as proteins expressed only during the activated T cell state. In conclusion, our single-step non-viral precision genome engineering technology is highly versatile with the ability to knock-out, knock-down, knock-in, and precisely regulate additional genes in a single step. These modifications have the potential to expand the applicability of T cell drug products and are broadly applicable to a variety of other cellular therapies and research models. Citation Format: William Lu, James Byers, Charles W. Tran, Michal Mass, Tanu Shenoy, Shirley Sun, Kyle Jacoby, Stefanie Mandl. Non-viral gene editing enables multiplex single-step precision genome engineering for adoptive cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2827.