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Background Chimeric antigen receptor (CAR)-based T cell therapy and other forms of adoptive cell therapies (ACTs) have shown remarkable success in the treatment of hematologic malignancies; however, reports of clinical activity in solid tumors are limited to date. One key therapeutic challenge presented by solid tumors is the immunosuppressive tumor microenvironment (TME). Adding to the complexity, it is becoming increasingly clear that TMEs are heterogeneous (broadly classified as ‘inflamed,’ ‘immune excluded’ and ‘immune dessert’), utilizing different mechanisms of immunosuppression. Instrumental to overcoming the barriers presented by solid tumors will be the development of T cells with immune- enhancing edits that improve penetration, potency and persistence, while also preventing exhaustion in hostile TMEs. T cells with these properties may help in the development of ACTs in solid tumors. Methods CRISPR/Cas9-based functional genetic screens in T cells can enable prioritization of known targets and uncover novel targets to improve the design of genetically reprogrammed cell therapies, in an unbiased fashion. Most CRISPR screens to date have been performed in vitro with tumor cells due to the complexity of setting up CRISPR screens in primary T cells, particularly for in vivo target discovery. Here, we describe the development and careful optimization of an in vivo mouse CRISPR-screening platform to identify knock-out targets in primary T cells, with the goal of increasing T cell abundance and persistence in tumors with different TMEs. Using a mouse retroviral system to express single-guide RNA (sgRNA) libraries in T cells from Cas9 transgenic mice, we performed in vivo screens in syngeneic, fully immune-competent mouse tumor models. Results We identified both known and potential novel regulators of T cell activation and persistence. Importantly, we have discovered knock-out targets that accumulate in multiple, distinct TMEs and other targets that are TME-specific. The use of sub-genomic- focused libraries allowed us to rapidly screen in multiple tumor model systems and reproducibly identify hits across individual mice. Conclusions We have developed a fully optimized an in vivo genetic screen, which could be a rich source for target discovery, and can enable identification of functional regulators of T cells for rapid incorporation into CRISPR-engineered T cell therapies for different solid TMEs.

Summary: The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform. : Finn et al. describe the development of a transient, biodegradable LNP-based CRISPR/Cas9 delivery system that achieves >97% knockdown of serum TTR levels following a single administration. Editing levels were stable for 12 months, despite the transient nature of the delivery system and the editing components. Keywords: CRISPR, Cas9, genome editing, LNP, lipid nanoparticle, TTR, CRISPR/Cas9, liver delivery, gene therapy, sgRNA

Introduction. Despite the success of autologous chimeric antigen receptor (CAR)-T cells, barriers to a more widespread use of this potentially curative therapy include manufacturing failures and the high cost of individualized production. There is a strong desire for an immediately available cell therapy option; however, development of "off-the-shelf" T cells is challenging. Alloreactive T cells from unrelated donors can cause graft versus host disease (GvHD) for which researchers have successfully used nucleases to reduce expression of the endogenous T cell receptor (TCR) in the allogeneic product. The recognition of allogeneic cells by the host is a complex issue that has not been fully solved to date. Some approaches utilize prolonged immune suppression to avoid immune rejection and increase persistence. Although showing responses in the clinic, this approach carries the risk of infections and the durability of the adoptive T cells is uncertain. Other strategies include deletion of the B2M gene to remove HLA class I molecules and avoid recognition by host CD8 T cells. However, loss of HLA class I sends a "missing-self" signal to natural killer (NK) cells, which readily eliminate B2Mnull T cells. To overcome this, researchers are exploring insertion of the non-polymorphic HLA-E gene, which can provide partial but not full protection from NK cell-mediated lysis. Because activated T cells upregulate HLA class II, rejection by alloreactive CD4 T cells should also be addressed. Methods. Here, we developed an immunologically stealth "off-the-shelf" T cell strategy by leveraging our CRISPR/Cas9 platform and proprietary sequential editing process. To solve the issue of rejection by alloreactive CD4 and CD8 T cells, we knocked out (KO) select HLA class I and class II expression with a sequential editing process. Additionally, we utilize potent TCR-α and -β constant chain (TRAC, TRBC) gRNAs that achieve >99% KO of the endogenous TCR, addressing the risk of GvHD. An AAV-mediated insertion of a CAR or TCR into the TRAC locus is used in parallel with the TRAC KO step to redirect the T cells to tumor targets of interest. Alloreactivity by CD4 and CD8 T cells, NK killing, GvHD induction and T cell function was assessed in vitro and/or in vivo. Results. By knocking out select HLA class I and class II proteins, we were able to avoid host CD4- and CD8-T cell-mediated recognition. Edited T cells were protected from host NK cells, both in vitro and in an in vivo model engrafted with functional human NK cells. TRAC edited donor T cells did not induce GvHD in an immune compromised mouse model over the 90-day evaluation period. Using our proprietary T cell engineering process, we successfully generated allogeneic T cells with sequential KOs and insertion of a tumor-specific TCR or CAR with high yield. Importantly, these allogeneic T cells had comparable functional activity to their autologous T cell counterparts in in vitro assays (tumor cell killing and cytokine release) as well as in vivo tumor models. With a relatively small bank of donors, we can provide an "off-the-shelf" CAR or TCR-T cell solution for a large proportion of the population. Conclusions. We have successfully developed a differentiated "off-the-shelf" approach, which is expected to be safe and cost-effective. It is designed to provide long-term persistence without the need for an immune suppressive regimen. This promising strategy is being applied to our T cell immuno-oncology and autoimmune research candidates. Disclosures Zhang: Intellia Therapeutics: Current Employment. Goel: Intellia Therapeutics: Current Employment. Prodeus: Intellia Therapeutics: Current Employment. Jetley: Intellia Therapeutics: Current Employment. Tan: Intellia Therapeutics: Current Employment. Averill: Intellia Therapeutics: Current Employment. Ranade: Intellia Therapeutics: Current Employment. Balwani: Intellia Therapeutics: Current Employment. Dutta: Intellia Therapeutics: Current Employment. Sharma: Intellia Therapeutics: Current Employment. Venkatesan: Intellia Therapeutics: Current Employment. Liu: Intellia Therapeutics: Current Employment. Roy: Intellia Therapeutics: Current Employment. O′Connell: Intellia Therapeutics: Current Employment. Arredouani: Intellia Therapeutics: Current Employment. Keenan: Intellia Therapeutics: Current Employment. Lescarbeau: Intellia Therapeutics: Current Employment. Schultes: Intellia Therapeutics: Current Employment.

Recently Schaefer et al. (1) reported the presence of more than a thousand genomic differences between mice that had been edited with S. pyogenes Cas9 at the zygote stage, and a control mouse of the same strain. Given the overlap of genomic differences between the two edited mice that were not found in the control mouse, the authors concluded that these differences arose from a Cas9-dependent activity. We feel that this conclusion is inappropriate, for three key reasons: 1) there was incomplete analysis of the genetic variation; 2) there was no consideration that the variation is naturally arising in these animals; and 3) the inferred behavior of Cas9 lies outside of its understood mechanism of action. Attribution to Cas9 activity should require a burden of proof, that we believe has not been met.

Background Faced with an increasing number of choices for biologic therapies, rheumatologists have a critical need for better tools to inform rheumatoid arthritis (RA) disease management. The ability to identify patients who are unlikely to respond to first-line biologic anti-TNF therapies prior to their treatment would allow these patients to seek alternative therapies, providing faster relief and avoiding complications of disease. Methods We identified a gene expression classifier to predict, pre-treatment, which RA patients are unlikely to respond to the anti-TNF infliximab. The classifier was trained and independently evaluated using four published whole blood gene expression data sets, in which RA patients (n = 116 = 44 + 15 + 30 + 27) were treated with infliximab, and their response assessed 14–16 months post treatment according to the European League Against Rheumatism (EULAR) response criteria. For each patient, prior knowledge was used to group gene expression measurements into disease-relevant biological signaling mechanisms that were used as the input features for regularized logistic regression. Results The classifier produced a substantial enrichment of non-responders (59 %, given by the cross validated test precision) compared to the full population (27 % non-responders), while identifying nearly a third of non-responders. Given this classifier performance, treatment of predicted non-responders with alternative biologics would decrease their chance of non-response by between a third and a half, substantially improving their odds of effective treatment and stemming further disease progression. The classifier consisted of 18 signaling mechanisms, which together indicated that higher inflammatory signaling mediated by TNF and other cytokines was present pre-treatment in the blood of patients who responded to infliximab treatment. In contrast, non-responders were classified by relatively higher levels of specific metabolic activities in the blood prior to treatment. Conclusions We were able to successfully produce a classifier to identify a population of RA patients significantly enriched in anti-TNF non-responders across four different patient cohorts. Additional prospective studies are needed to validate and refine the classifier for clinical use. Electronic supplementary material The online version of this article (doi:10.1186/s12920-015-0100-6) contains supplementary material, which is available to authorized users.

Prostate cancer most commonly presents as initially castration dependent, however in a minority of patients the disease will progress to a state of castration resistance. Here, approaches for correlating alterations in the phosphoproteome with androgen independent cell survival in the LNCaP, PC3, and MDa-PCa-2b cell lines are discussed. The performance of the regression techniques multiple linear, ridge, principal component, and partial least squares regression is compared. The predictive performance of these algorithms over randomized data sets and using the Akaike Information Criterion is explored, and principal component and partial least squares regression are found to outperform other regression approaches. The effect of altering the number of features versus observations on the R(2) value and predictive performance is also examined using the partial least squares regression model. Utilizing these approaches "drivers" of castration resistant disease can be identified whose modulation alters phenotypic outcomes. These data provide an empirical comparison of the various considerations when statistically analyzing phosphorylation data with the aim of correlating with phenotypic outcomes.

Background Interferons (IFNs) have long been implicated in the pathogenesis of systemic lupus erythematosus (SLE). However, the specific consequences of IFN activity have not been well-defined. In this study, the biology associated with an IFN activity signature was assessed in SLE blood neutrophil and PBMC fractions. Objectives We assessed the alterations in the transcriptome of isolated SLE blood PBMCs and neutrophils associated with IFN dysregulation (IFN signature high vs. low), and used the data to describe activation differences between the cell types. Methods RNA was collected from isolated blood PBMC and neutrophil fractions from a cohort of 46 SLE patients and 23 healthy donors. Study patients fulfilled both ACR and SLICC criteria for SLE and represented a clinical population with SLEDAI scores 0–12 (median 2). In addition, 63% were treated with prednisone, an immunosuppressant, or both. Patients were grouped by high or low IFN activity by assessing 21 IFN-inducible genes in PBMCs, and changes in gene expression were determined by RNA sequencing. Gene expression differences were analyzed further to determine the most likely upstream mechanistic explanations for the data in each comparison. The significance of these mechanisms is based on the evaluation of two metrics: 1) supporting gene change enrichment using a hypergeometric distribution, and 2) demonstrating directional consistency as assessed by a binomial distribution. Differential mechanisms between high and low IFN groups were examined in the context of those with activity assumed to be significantly different for SLE patients vs. healthy donors. Results This analysis identified mechanisms active in high vs. low IFN neutrophils (table). High IFN neutrophils exhibited unique mechanisms, including IFNG, mTOR, and CCL5-consistent signaling. TGFB1 and MAPK1 activation were distinct in low IFN neutrophils. Most mechanisms activated in PBMCs were common between the IFN groups, and were activated to similar degrees. Conclusions In this study of an academic cohort with active SLE and low SLEDAI scores, IFN signature correlated with biologic differences that predominate in neutrophils. The work permits better understanding of the impact of IFN signaling in SLE, by demonstrating different effects in neutrophil vs. PBMC fractions. Acknowledgements Research funded by MedImmune LLC Disclosure of Interest W. White Shareholder of: AstraZeneca, Employee of: MedImmune, D. Drubin Grant/research support from: MedImmune, X. Guo Shareholder of: AstraZeneca, Employee of: MedImmune, L. Yang Shareholder of: AstraZeneca, Employee of: MedImmune, R. Zeng Shareholder of: AstraZeneca, Employee of: MedImmune, Y. Wu: None declared, M. Roberts Employee of: MedImmune, R. Lescarbeau Grant/research support from: MedImmune, A. Van Hooser Grant/research support from: MedImmune, M. Macoritto Grant/research support from: MedImmune, M. Petri Grant/research support from: MedImmune

The spread of prostate cancer cells to the bone marrow microenvironment and castration resistant growth are key steps in disease progression and significant sources of morbidity. However, the biological significance of mesenchymal stem cells (MSCs) and bone marrow derived extracellular matrix (BM-ECM) in this process is not fully understood. We therefore established an in vitro engineered bone marrow tissue model that incorporates hMSCs and BM-ECM to facilitate mechanistic studies of prostate cancer cell survival in androgen-depleted media in response to paracrine factors and BM-ECM. hMSC-derived paracrine factors increased LNCaP cell survival, which was in part attributed to IGFR and IL6 signaling. In addition, BM-ECM increased LNCaP and MDA-PCa-2b cell survival in androgen-depleted conditions, and induced chemoresistance and morphological changes in LNCaPs. To determine the effect of BM-ECM on cell signaling, the phosphorylation status of 46 kinases was examined. Increases in the phosphorylation of MAPK pathway-related proteins as well as sustained Akt phosphorylation were observed in BM-ECM cultures when compared to cultures grown on plasma-treated polystyrene. Blocking MEK1/2 or the PI3K pathway led to a significant reduction in LNCaP survival when cultured on BM-ECM in androgen-depleted conditions. The clinical relevance of these observations was determined by analyzing Erk phosphorylation in human bone metastatic prostate cancer versus non-metastatic prostate cancer, and increased phosphorylation was seen in the metastatic samples. Here we describe an engineered bone marrow model that mimics many features observed in patients and provides a platform for mechanistic in vitro studies.

Prostate cancer progression is characterized by a striking affinity to spread to bone, where it forms metastases predominately of an osteoblastic phenotype, resulting in a significant source of morbidity. Currently, animal models for studying the development of these osteoblastic metastases are limited due to a lack of spontaneous osteoblastic metastasis development. In order to recapitulate osteoblastic metastasis development, an in vitro, three dimensional culture system was created utilizing silk fibroin scaffolds. LNCaP cells were seeded to silk scaffolds with human bone marrow derived mesenchymal stem cells (hMSCs) and cultured for 6 weeks. Examination after co-culture revealed through histological analysis that hMSCs underwent osteogenic differentiation in response to LNCaP paracrine signals. Paracrine interactions between hMSCs and LNCaPs were studied and IL-6, known to promote androgen independent growth, was found to be transiently expressed by hMSCs over the first two weeks of co-culture. In addition to secreted soluble signaling factors, the effect of extracellular matrix signals on the LNCaP cells was examined. LNCaP cells grown in a decellularized matrix, derived from hMSCs having undergone osteogenic differentiation, showed a 12 fold survival advantage in androgen depleted conditions over controls. This data indicates the ability of the co-culture system to stimulate differentiation of hMSCs towards an osteogenic linage and release soluble and extracellular matrix cues which stimulate androgen independent disease progression. The in vitro co-culture system presents a scalable and high throughput method for studying the molecular mechanisms underlying the osteoblastic component of prostate cancer bone metastasis. Citation Information: Cancer Res 2009;69(23 Suppl):C9.

Background Although recent progress has been made in treating castration resistant prostate cancer, the interplay of signaling pathways which enable castration resistant growth is incompletely understood. A data driven, multivariate approach, was used in this study to predict prostate cancer cell survival based on the phosphorylation levels of key proteins in PC3, LNCaP, and MDA-PCa-2b cell lines in response to EGF, IGF1, IL6, TNFα, dihydrotestosterone, and docetaxel treatment. Methods The prostate cancer cell lines were treated with ligands or inhibitors, cell lyates were collected, and the amount of phosphoprotein quantified using 384 well ELISA assays. In separate experiments, relative cell viability was determined using an MTT assay. Normalized data was imported into Matlab where regression analysis was performed. Results Based on a linear model developed using partial least squares regression, p-Erk1/2 was found to correlate with castration resistant survival along with p-RPS6, and this model was determined to have a leave-one-out cross validated R2 value of 0.61. The effect of androgen on the phosphoproteome was examined, and increases in PI3K related phosphoproteins (p-Akt, p-RPS6, and p-GSK3) were observed which accounted for the majority of the significant increase in androgen-mediated cell survival. Simultaneous inhibition of the PI3K pathway and treatment with androgen resulted in a non-significant increase in survival. Given the strong effect of PI3K related signaling in enabling castration resistant survival, the specific effect of mTor versus complete inhibition was examined using targeted inhibitors. It was determine that mTor inhibition accounts for 52% of the effect of complete PI3K inhibition on cell survival. The differences in signaling between the cell lines were explored it was observed that MDA-PCa-2b exhibited far less activation of p-Erk in response to varying treatments, explaining one of the reasons for the lack of castration resistance. Conclusion In this work, regression analysis to the phosphoproteome was used to illustrate the sources of castration resistance between the cell lines including reduced p-Erk signaling in MDA-PCa-2b and variations in p-JNK across the cell lines, as well as studying the signaling pathways which androgen acts through, and determining the response to treatment with targeted inhibitors.