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Supplementary Figure 2 from Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Supplementary Figure 4 from Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Supplementary Figure 3 from Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Clinical trials have proven oncolytic virotherapy to be safe but not effective. We have shown that oncolytic viruses (OV) injected into intracranial gliomas established in rodents are rapidly cleared, and this is associated with up-regulation of markers (CD68 and CD163) of cells of monocytic lineage (monocytes/microglia/macrophages). However, it is unclear whether these cells directly impede intratumoral persistence of OV through phagocytosis and whether they infiltrate the tumor from the blood or the brain parenchyma. To investigate this, we depleted phagocytes with clodronate liposomes (CL) in vivo through systemic delivery and ex vivo in brain slice models with gliomas. Interestingly, systemic CL depleted over 80% of peripheral CD163+ macrophages in animal spleen and peripheral blood, thereby decreasing intratumoral infiltration of these cells, but CD68+ cells were unchanged. Intratumoral viral titers increased 5-fold. In contrast, ex vivo CL depleted only CD68+ cells from brain slices, and intratumoral viral titers increased 10-fold. These data indicate that phagocytosis by both peripheral CD163+ and brain-resident CD68+ cells infiltrating tumor directly affects viral clearance from tumor. Thus, improved therapeutic efficacy may require modulation of these innate immune cells. In support of this new therapeutic paradigm, we observed intratumoral up-regulation of CD68+ and CD163+ cells following treatment with OV in a patient with glioblastoma. [Cancer Res 2007;67(19):9398–406]

Supplementary Figure 1B from Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Supplementary Figure 5 from Depletion of Peripheral Macrophages and Brain Microglia Increases Brain Tumor Titers of Oncolytic Viruses

Protein secretion is an essential cellular process that permits cell growth, movement and communication. Traffic of proteins within the eukaryotic secretory pathway is mediated by transport intermediates that bud from one compartment, populated with appropriate cargo proteins, and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein-protein interactions that drive capture into transport vesicles. Human PCSK9 is a major determinant of cholesterol metabolism, whose secretion is mediated by a specific cargo adaptor of the ER export machinery, SEC24A. We map a series of protein-protein interactions between PCSK9, its ER export receptor, SURF4, and SEC24A, that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-PBA, a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins.

Agonist bias occurs when different ligands produce distinct signalling outputs when acting at the same receptor. However, its physiological relevance is not always clear. Using primary human cells and gene editing techniques, we demonstrate endogenous agonist bias with physiological consequences for the calcitonin receptor-like receptor, CLR. By switching the receptor-activity modifying protein (RAMP) associated with CLR we can “re-route” the physiological pathways activated by endogenous agonists calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2). AM2 promotes calcium-mediated nitric oxide signalling whereas CGRP and AM show pro-proliferative effects in cardiovascular cells, thus providing a rationale for the expression of the three peptides. CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and their importance in endogenous GPCR signalling., Clark et al. explore the ability of ligands to activate the calcitonin-like receptor (CLR) in primary endothelial cells, and the influence of co-expressed receptor-activity modifying proteins (RAMPs). Their study reveals that GPCR agonist bias occurs naturally in human cells and plays a fundamental role in providing unique functions to endogenous agonists.

Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1β rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest., Author summary Nerve connections that are severed in spinal cord injury do not heal, which can lead to permanent paralysis. Lack of repair may in part be due to prolonged inflammation of the injury site. In contrast, zebrafish show excellent repair of nerve connections after spinal injury and this is associated with controlling inflammation. Due to recent advances in genetic technology (CRISPR/Cas9) we can now determine the function of genes that influence regeneration in the living zebrafish in a matter of days. Here we devise a very rapid screening method for the function of inflammation-related genes in zebrafish larvae after spinal cord injury. We find a number of genes that are necessary for repair of nerve connections and control of the inflammation after injury. This provides important leads to improve our understanding of the role of inflammation in spinal cord injury. Moreover, our fast and robust screening method can be adopted by other researchers to screen for gene functions in a whole animal, which was previously not easily possible.

Methods to measure cellular target engagement are increasingly being used in early drug discovery. The Cellular Thermal Shift Assay (CETSA) is one such method. CETSA can investigate target engagement by measuring changes in protein thermal stability upon compound binding within the intracellular environment. It can be performed in high-throughput, microplate-based formats to enable broader application to early drug discovery campaigns, though high-throughput forms of CETSA have only been reported for a limited number of targets. CETSA offers the advantage of investigating the target of interest in its physiological environment and native state, but it is not clear yet how well this technology correlates to more established and conventional cellular and biochemical approaches widely used in drug discovery. We report two novel high-throughput CETSA (CETSA HT) assays for B-Raf and PARP1, demonstrating the application of this technology to additional targets. By performing comparative analyses with other assays, we show that CETSA HT correlates well with other screening technologies and can be applied throughout various stages of hit identification and lead optimization. Our results support the use of CETSA HT as a broadly applicable and valuable methodology to help drive drug discovery campaigns to molecules that engage the intended target in cells.

Modified messenger RNAs (mRNAs) hold great potential as therapeutics by using the body's own processes for protein production. However, a key challenge is efficient delivery of therapeutic mRNA to the cell cytosol and productive protein translation. Lipid nanoparticles (LNPs) are the most clinically advanced system for nucleic acid delivery; however, a relatively narrow therapeutic index makes them unsuitable for many therapeutic applications. A key obstacle to the development of more potent LNPs is a limited mechanistic understanding of the interaction of LNPs with cells. To address this gap, we performed an arrayed CRISPR screen to identify novel pathways important for the functional delivery of MC3 lipid-based LNP encapsulated mRNA (LNP-mRNA). Here, we have developed and validated a robust, high-throughput screening-friendly phenotypic assay to identify novel targets that modulate productive LNP-mRNA delivery. We screened the druggable genome (7795 genes) and validated 44 genes that either increased (37 genes) or inhibited (14 genes) the productive delivery of LNP-mRNA. Many of these genes clustered into families involved with host cell transcription, protein ubiquitination, and intracellular trafficking. We show that both UDP-glucose ceramide glucosyltransferase and V-type proton ATPase can significantly modulate the productive delivery of LNP-mRNA, increasing and decreasing, respectively, with both genetic perturbation and by small-molecule inhibition. Taken together, these findings shed new light into the molecular machinery regulating the delivery of LNPs into cells and improve our mechanistic understanding of the cellular processes modulating the interaction of LNPs with cells.

Acute CRISPR/Cas9 targeting offers the opportunity for scalable phenotypic genetic screening in zebrafish. However, the unpredictable efficiency of CRISPR gRNA (CrRNA) activity is a limiting factor. Here we describe how to resolve this by prescreening CrRNAs for high activity in vivo, using a simple standardised assay based on restriction fragment length polymorphism analysis (RFLP). We targeted 350 genomic sites with synthetic RNA Oligo guide RNAs (sCrRNAs) in zebrafish embryos and found that almost half exhibited > 90% efficiency in our RFLP assay. Having the ability to preselect highly active sCrRNAs (haCRs), we carried out a focussed phenotypic screen of 30 macrophage-related genes in spinal cord regeneration and found 10 genes whose disruption impaired axonal regeneration. Four (tgfb1a, tgfb3, tnfa, sparc) out of 5 stable mutants subsequently analysed retained the acute haCR phenotype, validating the efficiency of this approach. Mechanistically, lack of tgfb1a leads to a prolonged immune response after injury, which inhibits regeneration. Our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, and can be applied to study any biological function of interest.HIGHLIGHTS- Synthetic CRISPR gRNAs are highly active- in vivo pre-screening allows rapid assessment of CRISPR gRNA activity- Phenotypic CRISPR screen reveals crucial genes for spinal cord regeneration- tgfb1a promotes spinal regeneration by controlling inflammation

As gene therapy is experiencing a second wave of interest, rAAV has rapidly emerged as one of the most attractive viral transfer tools, thanks to its favorable safety profile and long-term transgene expression. The recent approval of rAAV molecules for the treatment of congenital amaurosis and spinal muscular atrophy has been paving the road for the development of several rAAV-based therapeutics targeting a continuously increasing range of indications. To support this rapid growth, the field is in high demand for robust and scalable and economically viable manufacturing processes. This review will provide an overview of the most common platforms currently adopted at the manufacturing level for rAAV production, focusing on the challenges and bottlenecks experienced in the upstream process.

10-20% of lung adenocarcinoma patients harbour activating mutations in EGFR. Although treatment with the EGFR kinase inhibitor osimertinib has improved overall survival in such patients, almost all patients ultimately develop drug resistance. In many cases the molecular resistance mechanisms remain unknown. A systematic identification of the involved genes and pathways is critical to overcome osimertinib resistance. To define the resistance landscape of EGFR kinase inhibition we performed genome-wide gain and loss of function CRISPR screens in EGFR mutant lung cancer cell lines treated with osimertinib. Resistance hits were enriched for genes in previously identified resistance pathways including PI3K (PTEN, TSC2), MAPK (NF1, MET), cell death (BCL2L11, BAX), the mediator complex (MED24, MED19) and ubiquitination (KCTD5, LZTR1). A secondary screen of 63 resistance genes that combined high content microscopy with CRISPR gene knockouts demonstrated that 21% (13/63) of genes were associated with increased nuclear localisation of YAP1/WWTR1, indicating transcriptional activation of the Hippo pathway. According to our screening data, many resistance hits mapped onto the Hippo signaling axis - upstream regulators (NF2, AMOTL2), core signaling genes (LATS1, LATS2), main effectors (WWTR1, YAP1), transcriptional co-effectors (TEAD3, FOSL1, VGLL4) and the SWI/SNF complex (ARID2, SMARCA4, SMARCB1, PBRM1). Hippo signaling is mediated through YAP1 and WWTR1 which bind to TEAD transcription factors and activate transcriptional programs affecting cell proliferation and apoptosis. We confirmed using CRISPR that knockout (NF2) or overexpression (YAP1, WWTR1) of key Hippo genes in the EGFR mutant lung cancer cell lines PC-9, HCC827 and HCC4006 resulted in up to 60-fold increased resistance to osimertinib and elevated activity of a TEAD reporter system, indicating activation of Hippo transcriptional programs. To maintain osimertinib resistance, expression of both YAP1 and WWTR1 was necessary, suggesting non-redundant roles for both Hippo main effectors in mediating osimertinib resistance. The combination of osimertinib and a TEAD inhibitor (MYF-01-37) reversed the resistance phenotype in NF2 KO cell line models in long-term proliferation assays and also substantially repressed the emergence of drug-tolerant persister cells in PC-9, HCC827 and HCC4006 cell lines following osimertinib treatment. These cells exhibited an enhanced apoptotic response when treated with combination of osimertinib and TEAD inhibitor. Consequently, we propose Hippo signaling as an important target mechanism for the prevention of resistance to osimertinib. Citation Format: Matthias Pfeifer, Jonathan S. Brammeld, Stacey Price, Matthew Martin, Hannah Thorpe, Aurelie Bornot, Erica Banks, Nin Guan, Shanade Dunn, Maria Luisa Guerriero, Daniel O'Neill, James Pilling, Davide Gianni, James Brownell, Paul Smith, Ultan McDermott. Gain and loss of function genome-wide CRISPR screens identify Hippo signaling as an important driver of resistance in EGFR mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1100.

ObjectiveTo test the hypothesis that dimethyl fumarate (DMF, Tecfidera) elicits different biological changes from DMF combined with monoethyl fumarate (MEF) (Fumaderm, a psoriasis therapy), we investigated DMF and MEF in rodents and cynomolgus monkeys. Possible translatability of findings was explored with lymphocyte counts from a retrospective cohort of patients with MS.MethodsIn rodents, we evaluated pharmacokinetic and pharmacodynamic effects induced by DMF and MEF monotherapies or in combination (DMF/MEF). Clinical implications were investigated in a retrospective, observational analysis of patients with MS treated with DMF/MEF (n = 36).ResultsIn rodents and cynomolgus monkeys, monomethyl fumarate (MMF, the primary metabolite of DMF) exhibited higher brain penetration, whereas MEF was preferentially partitioned into the kidney. In mice, transcriptional profiling for DMF and MEF alone identified both common and distinct pharmacodynamic responses, with almost no overlap between DMF- and MEF-induced differentially expressed gene profiles in immune tissues. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated oxidative stress response pathway was exclusively regulated by DMF, whereas apoptosis pathways were activated by MEF. DMF/MEF treatment demonstrated that DMF and MEF functionally interact to modify DMF- and MEF-specific responses in unpredictable ways. In patients with MS, DMF/MEF treatment led to early and pronounced suppression of lymphocytes, predominantly CD8+ T cells. In a multivariate regression analysis, the absolute lymphocyte count (ALC) was associated with age at therapy start, baseline ALC, and DMF/MEF dosage but not with previous immunosuppressive medication and sex. Furthermore, the ALC increased in a small cohort of patients with MS (n = 6/7) after switching from DMF/MEF to DMF monotherapy.ConclusionsFumaric acid esters exhibit different biodistribution and may elicit different biological responses; furthermore, pharmacodynamic effects of combinations differ unpredictably from monotherapy. The strong potential to induce lymphopenia in patients with MS may be a result of activation of apoptosis pathways by MEF compared with DMF.

Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukaemia (AML), bromodomain and extra terminal protein (BET) inhibitors are being explored as a promising therapeutic avenue in numerous cancers. While clinical trials have reported single-agent activity in advanced haematological malignancies, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukaemia, here we perform a chromatin-focused RNAi screen in a sensitive MLL-AF9;Nras(G12D)-driven AML mouse model, and investigate dynamic transcriptional profiles in sensitive and resistant mouse and human leukaemias. Our screen shows that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET inhibitor resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodelling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukaemias regardless of their sensitivity, resistant leukaemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signalling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic chromatin immunoprecipitation sequencing and self-transcribing active regulatory region sequencing of enhancer profiles reveal that BET-resistant states are characterized by remodelled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signalling as a driver and candidate biomarker of primary and acquired BET resistance in leukaemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.

Androgen Receptor (AR) is a key driver in prostate cancer. Direct targeting of AR has valuable therapeutic potential. However, the lack of disease relevant cellular methodologies capable of discriminating between inhibitors that directly bind AR and those that instead act on AR co-regulators has made identification of novel antagonists challenging. The Cellular Thermal Shift Assay (CETSA) is a technology enabling confirmation of direct target engagement with label-free, endogenous protein in living cells. We report the development of the first high-throughput CETSA assay (CETSA HT) to identify direct AR binders in a prostate cancer cell line endogenously expressing AR. Using this approach, we screened a pharmacology library containing both compounds reported to directly engage AR, and compounds expected to target AR co-regulators. Our results show that CETSA HT exclusively identifies direct AR binders, differentiating them from co-regulator inhibitors where other cellular assays measuring functional responses cannot. Using this CETSA HT approach we can derive apparent binding affinities for a range of AR antagonists, which represent an intracellular measure of antagonist-receptor Ki performed for the first time in a label-free, disease-relevant context. These results highlight the potential of CETSA HT to improve the success rates for novel therapeutic interventions directly targeting AR.

Background Recently, tangles and plaque-like aggregates have been identified in certain cases of dilated cardiomyopathy (DCM), traditionally labeled idiopathic (iDCM), where there is no specific diagnostic test or targeted therapy. This suggests a potential underlying cause for some of the iDCM cases. Objectives This study sought to identify the make-up of myocardial aggregates to understand the molecular mechanisms of these cases of DCM; this strategy has been central to understanding Alzheimer’s disease. Methods Aggregates were extracted from human iDCM samples with high congophilic reactivity (an indication of plaque presence), and the findings were validated in a larger cohort of samples. We tested the expression, distribution, and activity of cofilin in human tissue and generated a cardiac-specific knockout mouse model to investigate the functional impact of the human findings. We also modeled cofilin inactivity in vitro by using pharmacological and genetic gain- and loss-of-function approaches. Results Aggregates in human myocardium were enriched for cofilin-2, an actin-depolymerizing protein known to participate in neurodegenerative diseases and nemaline myopathy. Cofilin-2 was predominantly phosphorylated, rendering it inactive. Cardiac-specific haploinsufficiency of cofilin-2 in mice recapitulated the human disease’s morphological, functional, and structural phenotype. Pharmacological stimulation of cofilin-2 phosphorylation and genetic overexpression of the phosphomimetic protein promoted the accumulation of “stress-like” fibers and severely impaired cardiomyocyte contractility. Conclusions Our study provides the first biochemical characterization of prefibrillar myocardial aggregates in humans and the first report to link cofilin-2 to cardiomyopathy. The findings suggest a common pathogenetic mechanism connecting certain iDCMs and other chronic degenerative diseases, laying the groundwork for new therapeutic strategies.

The triple-angiokinase inhibitor nintedanib is an orally available, potent, and selective inhibitor of tumor angiogenesis by blocking the tyrosine kinase activities of vascular endothelial growth factor receptor (VEGFR) 1–3, platelet-derived growth factor receptor (PDGFR)-α and -β, and fibroblast growth factor receptor (FGFR) 1–3. Nintedanib has received regulatory approval as second-line treatment of adenocarcinoma non–small cell lung cancer (NSCLC), in combination with docetaxel. In addition, nintedanib has been approved for the treatment of idiopathic lung fibrosis. Here we report the results from a broad kinase screen that identified additional kinases as targets for nintedanib in the low nanomolar range. Several of these kinases are known to be mutated or overexpressed and are involved in tumor development (discoidin domain receptor family, member 1 and 2, tropomyosin receptor kinase A (TRKA) and C, rearranged during transfection proto-oncogene [RET proto oncogene]), as well as in fibrotic diseases (e.g., DDRs). In tumor cell lines displaying molecular alterations in potential nintedanib targets, the inhibitor demonstrates direct antiproliferative effects: in the NSCLC cell line NCI-H1703 carrying a PDGFRα amplification (ampl.); the gastric cancer cell line KatoIII and the breast cancer cell line MFM223, both driven by a FGFR2 amplification; AN3CA (endometrial carcinoma) bearing a mutated FGFR2; the acute myeloid leukemia cell lines MOLM-13 and MV-4-11-B with FLT3 mutations; and the NSCLC adenocarcinoma LC-2/ad harboring a CCDC6-RET fusion. Potent kinase inhibition does not, however, strictly translate into antiproliferative activity, as demonstrated in the TRKA-dependent cell lines CUTO-3 and KM-12. Importantly, nintedanib treatment of NCI-H1703 tumor xenografts triggered effective tumor shrinkage, indicating a direct effect on the tumor cells in addition to the antiangiogenic effect on the tumor stroma. These findings will be instructive in guiding future genome-based clinical trials of nintedanib.

Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disorder characterized by extracellular deposition of amyloid fibrils composed by mutated transthyretin (TTR) mainly in the peripheral nervous system. At present, liver transplantation is still the standard treatment to halt the progression of clinical symptoms in FAP, but new therapeutic strategies are emerging, including the use of TTR stabilizers. Here we propose to establish a new gene therapy approach using adeno-associated virus (AAV) vectors to deliver the trans-suppressor TTR T119M variant to the liver of transgenic TTR V30M mice at different ages. This TTR variant is known for its ability to stabilize the tetrameric protein. Analysis of the gastrointestinal tract of AAV-treated animals revealed a significant reduction in deposition of TTR non-fibrillar aggregates in as much as 34% in stomach and 30% in colon, as well as decreased levels of biomarkers associated with TTR deposition, namely the endoplasmic reticulum stress marker BiP and the extracellular matrix protein MMP-9. Moreover, we showed with different studies that our approach leads to an increase in tetrameric and more stable forms of TTR, in favor of destabilized monomers. Altogether our data suggest the possibility to use this gene therapy approach in a prophylactic manner to prevent FAP pathology.

Enhancing remyelination is a key therapeutic strategy for demyelinating diseases such as multiple sclerosis. To achieve this goal, a central challenge is being able to quantitatively and longitudinally track functional remyelination, especially with translatable biomarkers that can be performed in both preclinical models and in the clinic. We developed the methodology to stably measure multi-modal sensory evoked potentials from the skull surface over the course of months in individual mice and applied it to a genetic mouse model of oligodendrocyte ablation and demyelination. We found that auditory and somatosensory evoked potential latencies reliably increased over time during the early phase of the model and recovered spontaneously and almost completely during a later phase. Histological examination supported the interpretation that the evoked potential latency changes dynamically reflect changes in CNS myelination. Specifically, we found reduction of myelination in corresponding brain regions at the time that sensory evoked potentials were maximally impacted. Importantly, we also found that myelination levels recovered when evoked potential latencies recovered. Other changes known to associate with demyelination were also observed at the time of delayed evoked potentials, including the emergence of white matter vacuoles and increased markers for activated microglia and macrophages; these changes also fully reversed by the time that evoked potentials recovered. Our results support the hypothesis that skull-surface recorded evoked potential latencies can dynamically track CNS myelination changes. The methods developed here allow for longitudinally tracking functional myelination changes in vivo in preclinical rodent models with a quantitative biomarker that can also be applied clinically and will facilitate translational development of CNS remyelinating therapies.

Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery.

Oncolytic herpes simplex virus (HSV) vectors have been used in early phase human clinical trials as a therapy for recurrent malignant glioblastoma. This treatment proved safe but limited improvements in patient survival were observed. The potency of these vectors might be enhanced by targeting vector infectivity to tumor cells. Glioma tumors often express a mutant form (vIII) of the epidermal growth factor receptor (EGFR) resulting in the presence of a novel epitope on the cell surface. This epitope is specifically recognized by a single chain antibody designated MR1-1. HSV-1 infection involves initial binding to heparan sulfate (HS) on the cell surface mediated primarily by the viral envelope, glycoprotein C (gC). Here we joined the MR1-1 single chain antibody (scFv) to the gC sequence deleted for the HS binding domain (HSBD) as a means of targeting viral attachment to EGFRvIII on glial tumor cells. Virions bearing MR1-1-modified-gC had 5-fold increased infectivity for EGFRvIII-bearing human glioma U87 cells compared to mutant receptor-deficient cells. Further, MR1-1/EGFRvIII mediated infection was more efficient for EGFRvIII-positive cells than was wild-type virus for either positive or negative cells. Sustained infection of EGFRvIII+ glioma cells by MR1-1-modified-gC bearing oncolytic virus, as compared to wild-type gC oncolytic virus, was also shown in subcutaneous tumors in vivo using firefly luciferase as a reporter of infection. These data demonstrate that HSV tropism can be manipulated so that virions recognize a cell specific binding site with increased infectivity for the target cell. The retargeting of HSV infection to tumor cells should enhance vector specificity, tumor cell killing and vector safety.

Background— Heart failure is a debilitating condition resulting in severe disability and death. In a subset of cases, clustered as idiopathic dilated cardiomyopathy (iDCM), the origin of heart failure is unknown. In the brain of patients with dementia, proteinaceous aggregates and abnormal oligomeric assemblies of β-amyloid impair cell function and lead to cell death. Methods and Results— We have similarly characterized fibrillar and oligomeric assemblies in the hearts of iDCM patients, pointing to abnormal protein aggregation as a determinant of iDCM. We also showed that oligomers alter myocyte Ca 2+ homeostasis. Additionally, we have identified 2 new sequence variants in the presenilin-1 ( PSEN1 ) gene promoter leading to reduced gene and protein expression. We also show that presenilin-1 coimmunoprecipitates with SERCA2a. Conclusions— On the basis of these findings, we propose that 2 mechanisms may link protein aggregation and cardiac function: oligomer-induced changes on Ca 2+ handling and a direct effect of PSEN1 sequence variants on excitation-contraction coupling protein function.

The microtubule-associated protein tau (MAPT) and alpha-synuclein (SNCA) genes play central roles in neurodegenerative disorders. Mutations in each gene cause familial disease, whereas common genetic variation at both loci contributes to susceptibility to sporadic neurodegenerative disease. Here, we demonstrate exquisite gene regulation of the human MAPT and SNCA transgene loci and functional complementation in neuronal cell cultures and organotypic brain slices using the herpes simplex virus type 1 (HSV-1) amplicon-based infectious bacterial artificial chromosome (iBAC) vector to express complete loci >100 kb. Cell cultures transduced by iBAC vectors carrying a 143 kb MAPT or 135 kb SNCA locus expressed the human loci similar to the endogenous gene. We focused on analysis of the iBAC-MAPT vector carrying the complete MAPT locus. On transduction into neuronal cultures, multiple MAPT transcripts were expressed from iBAC-MAPT under strict developmental and cell type-specific control. In primary neurons from Mapt(-/-) mice, the iBAC-MAPT vector expressed the human tau protein, as detected by enzyme-linked immunosorbent assay and immunocytochemistry, and restored sensitivity of Mapt(-/-) neurons to Abeta peptide treatment in dissociated neuronal cultures and in organotypic slice cultures. The faithful retention of gene expression and phenotype complementation by the system provides a novel method to analyze neurological disease genes.

Glioblastoma multiforme (GBM) is a serious form of brain cancer for which there is currently no effective treatment. Alternative strategies such as adeno-associated virus (AAV) vector mediated-genetic modification of brain tumor cells with genes encoding anti-tumor proteins have shown promising results in preclinical models of GBM, although the transduction efficiency of these tumors is often low. As higher transduction efficiency of tumor cells should lead to enhanced therapeutic efficacy, a means to rapidly engineer AAV vectors with improved transduction efficiency for individual tumors is an attractive strategy. Here we tested the possibility of identifying high-efficiency AAV vectors for human U87 glioma cells by selection in culture of a newly constructed chimeric AAV capsid library generated by DNA shuffling of six different AAV cap genes (AAV1, AAV2, AAV5, AAVrh.8, AAV9, and AAVrh.10). After seven rounds of selection, we obtained a chimeric AAV capsid that transduces U87 cells at high efficiency (97% at a dose of 104 genome copies/cell), and at low doses it was 1.45–1.6-fold better than AAV2, which proved to be the most efficient parental capsid. Interestingly, the new AAV capsid displayed robust gene delivery properties to all glioma cells tested (including primary glioma cells) with relative fluorescence indices ranging from 1- to 14-fold higher than AAV2. The selected vector should be useful for in vitro glioma research when efficient transduction of several cell lines is required, and provides proof-of-concept that an AAV library can be used to generate AAV vectors with enhanced transduction efficiency of glioma cells.

Clinical trials have proven oncolytic virotherapy to be safe but not effective. We have shown that oncolytic viruses (OV) injected into intracranial gliomas established in rodents are rapidly cleared, and this is associated with up-regulation of markers (CD68 and CD163) of cells of monocytic lineage (monocytes/microglia/macrophages). However, it is unclear whether these cells directly impede intratumoral persistence of OV through phagocytosis and whether they infiltrate the tumor from the blood or the brain parenchyma. To investigate this, we depleted phagocytes with clodronate liposomes (CL) in vivo through systemic delivery and ex vivo in brain slice models with gliomas. Interestingly, systemic CL depleted over 80% of peripheral CD163+ macrophages in animal spleen and peripheral blood, thereby decreasing intratumoral infiltration of these cells, but CD68+ cells were unchanged. Intratumoral viral titers increased 5-fold. In contrast, ex vivo CL depleted only CD68+ cells from brain slices, and intratumoral viral titers increased 10-fold. These data indicate that phagocytosis by both peripheral CD163+ and brain-resident CD68+ cells infiltrating tumor directly affects viral clearance from tumor. Thus, improved therapeutic efficacy may require modulation of these innate immune cells. In support of this new therapeutic paradigm, we observed intratumoral up-regulation of CD68+ and CD163+ cells following treatment with OV in a patient with glioblastoma. [Cancer Res 2007;67(19):9398–406]

Clinical trials are testing oncolytic viruses (OVs) as therapies for cancer. We have shown that animals that have brain tumors and are treated with a herpes simplex virus (HSV)-derived OV live significantly longer when cyclophosphamide (CPA) is preadministered. Here, we explore the mechanisms behind this finding. In a syngeneic rat glioma model, intratumoral HSV administration is associated with rapid increase of natural killer cells, microglia/macrophages (CD68+and CD163+), and IFN-γ. Pretreatment with CPA enhances HSV replication and oncolysis and reduces an HSV-mediated increase in CD68+and CD163+cells and intratumoral IFN-γ. Molecular imaging shows CPA pretreatment to inhibit HSV-induced infiltration of tumor-associated phagocytic cells. Our results reveal molecular and cellular mechanisms that inhibit intratumoral spread of HSV and suggest a therapeutic path for improving the efficacy of virotherapy as a treatment for cancer.

X-ray powder diffraction data for the trigonal form of disilver trihydrogen paraperiodate, Ag2H3IO6, are reported. The cell parameters are a=5.941 89(3), c=12.7253(1) Å, V=389.089(6) Å3, and ρ=5.65 g cm−3 for Z=3. A full-profile Rietveld refinement confirms the monophasic nature of the sample and the presence of two-dimensional corrugated layers of the A2BO6 type linked by hydrogen bonds. Short unsupported Ag–Ag contacts (about 3.02 Å) of the argentophilic type are also shown.

Oncolytic adenoviruses exhibiting tumor-selective replication are promising anticancer agents. Insertion and expression of a transgene encoding tissue inhibitor of metalloproteinase-3 (TIMP-3), which has been reported to inhibit angiogenesis and tumor cell infiltration and induce apoptosis, may improve the antitumor activity of these agents. To assess the effects of TIMP-3 gene transfer to glioma cells, a replication-defective adenovirus encoding TIMP-3 (Ad.TIMP-3) was employed. Ad.TIMP-3 infection of a panel of glioma cell cultures decreased the proliferative capacity of these cells and induced morphologic changes characteristic for apoptosis. Next, a conditionally replicating adenovirus encoding TIMP-3 was constructed by inserting the TIMP-3 expression cassette into the E3 region of the adenoviral backbone containing a 24-bp deletion in E1A. This novel oncolytic adenovirus, AdΔ24TIMP-3, showed enhanced oncolytic activity on a panel of primary cell cultures and two glioma cell lines compared with the control oncolytic virus AdΔ24Luc. In vivo inhibition of matrix metalloproteinase (MMP) activity by AdΔ24TIMP-3 was shown in s.c. glioma xenografts. The functional activity of TIMP-3 was imaged noninvasively using a near-IR fluorescent MMP-2-activated probe. Tumoral MMP-2 activity was significantly reduced by 58% in the AdΔ24TIMP-3-treated tumors 24 hours after infection. A study into the therapeutic effects of combined oncolytic and antiproteolytic therapy was done in both a s.c. and an intracranial model for malignant glioma. Treatment of s.c. (U-87MG) or intracranial (U-87δEGFR) tumors with AdΔ24TIMP-3 and AdΔ24Luc both significantly inhibited tumor growth and prolonged survival compared with PBS-treated controls. However, expression of TIMP-3 in the context of AdΔ24 did not significantly affect the antitumor efficacy of this oncolytic agent. ©2005 American Association for Cancer Research. Chemicals / CAS: tissue inhibitor of metalloproteinase 3, 145809-21-8, 164781-40-2; Matrix Metalloproteinase 2, EC 3.4.24.24; Tissue Inhibitor of Metalloproteinase-3

The beta-amyloid peptide (Abeta) present in the senile plaques of Alzheimer's disease derives from the cleavage of a membrane protein, named APP, driven by two enzymes, known as beta- and gamma-secretases. The mechanisms regulating this cleavage are not understood. We have developed an experimental system to identify possible extracellular signals able to trigger the cleavage of an APP-Gal4 fusion protein, which is detected by measuring the expression of the CAT gene transcribed under the control of the Gal4 transcription factor, which is released from the membrane upon the cleavage of APP-Gal4. By using this assay, we purified a protein contained in the C6 cell-conditioned medium, which activates the cleavage of APP-Gal4 and which we demonstrated to be PDGF-BB. The APP-Gal4 processing induced by PDGF is dependent on the gamma-secretase activity, being abolished by an inhibitor of this enzyme, and is the consequence of the activation of a pathway downstream of the PDGF-receptor, which includes the non-receptor tyrosine kinase Src and the small G-protein Rac1. These findings are confirmed by the observation that a constitutively active form of Src increases Abeta generation and that, in cells stably expressing APP, the generation of A is strongly decreased by the Src tyrosine kinase inhibitor PP2.

Background Recently, tangles and plaque-like aggregates have been identified in certain cases of dilated cardiomyopathy (DCM). This suggests a potential underlying cause for the one-third of cases, traditionally labeled idiopathic (iDCM), where there is no specific diagnostic test or targeted therapy. Objectives This study sought to identify the make-up of myocardial aggregates to understand the molecular mechanisms of these cases of DCM; this strategy has been central to understanding Alzheimer's disease. Methods Aggregates were extracted from human iDCM samples with high congophilic reactivity (an indication of plaque presence), and the findings were validated in a larger cohort of samples. We tested the expression, distribution, and activity of cofilin in human tissue and generated a cardiac-specific knockout mouse model to investigate the functional impact of the human findings. We also modeled cofilin inactivity in vitro by using pharmacological and genetic gain- and loss-of-function approaches. Results Aggregates in human myocardium were enriched for cofilin-2, an actin-depolymerizing protein known to participate in neurodegenerative diseases and nemaline myopathy. Cofilin-2 was predominantly phosphorylated, rendering it inactive. Cardiac-specific haploinsufficiency of cofilin-2 in mice recapitulated the human disease's morphological, functional, and structural phenotype. Pharmacological stimulation of cofilin-2 phosphorylation and genetic overexpression of the phosphomimetic protein promoted the accumulation of "stress-like" fibers and severely impaired cardiomyocyte contractility. Conclusions Our study provides the first biochemical characterization of prefibrillar myocardial aggregates in humans and the first report to link cofilin-2 to cardiomyopathy. The findings suggest a common pathogenetic mechanism connecting certain iDCMs and other chronic degenerative diseases, laying the groundwork for new therapeutic strategies. © 2015 American College of Cardiology Foundation.

The multigenic family of mammalian Fe65s encodes three highly similar proteins with the same modular organisation: a WW domain and two phosphotyrosine-binding domains. The PTB2 domain of these proteins binds to the cytosolic domains of the Alzheimer's β-amyloid precursor protein APP and related proteins APLP1 and APLP2, generating a highly redundant system that is hard to dissect by reverse genetics. By searching potential Fe65-like genes in the nematode Caenorhabditis elegans, we identified a single gene, feh-1 (Fe65 homolog-1), encoding a protein with a high sequence similarity to mammalian Fe65s. FEH-1 is also functionally related to mammalian orthologues;in fact its PTB2 domain binds to APL-1, the product of the C. elegansorthologue of APP. Staining with specific antibodies show that the neuromuscular structures of the pharynx are the sites in which FEH-1 is present at highest levels. Expression studies with reporters indicate that the feh-1 gene is also expressed by a subset of the worm neurons. We generated and isolated a deletion allele of feh-1, and the corresponding homozygous mutants arrest as late embryos or as L1 larvae,demonstrating for the first time an essential role for a Fe65-like gene in vivo. The pharynx of homozygous larvae does not contract and the worms cannot feed. Analysis of pharyngeal pumping in heterozygous worms and in feh-1 RNA-interfered worms indicates that dosage of feh-1function affects the rate of pharyngeal contraction in C. elegans. Interference with apl-1 double-stranded RNA showed a similar effect on pharyngeal pumping, suggesting that FEH-1 and APL-1 are involved in the same pathway. The non-redundant system of the nematode will prove useful for studying the basic biology of the Fe65-APP interaction and the molecular events regulated by this evolutionarily conserved system of interacting proteins.

The following was incorrectly omitted from the Acknowledgments: "We would like to thank Danielle Morse for the production of AAV-GFP vectors and Dr. Bakhos Tannous Ph.D, Director of Vector Development and Production Core, MGH, Charlestown, MA". In addition, the name of the third author was spelled incorrectly. The correct name is: Xuan Zhang. The correct citation is: Prabhakar S, Goto J, Zhang X, Sena-Esteves M, Bronson R, et al. (2013) Stochastic Model of Tsc1 Lesions in Mouse Brain. PLoS ONE 8(5): e64224. doi:10.1371/journal.pone.0064224

Immunoglobulin light chain (LC) amyloidosis (AL) results from overproduction of circulating amyloidogenic LC proteins and subsequent amyloid fibril deposition in organs. Mortality in AL amyloidosis patients is highly associated with a rapidly progressive AL cardiomyopathy, marked by profound impairment of diastolic and systolic cardiac function and significant early mortality. While myocardial fibril deposition contributes to the severe diastolic dysfunction seen in AL cardiomyopathy patients, the degree of fibril deposition has not been found to correlate with prognosis. Previously, we and others showed a direct cardiotoxic effect of amyloidogenic LC proteins (AL-LC), which may contribute to the pathophysiology and mortality observed in AL cardiomyopathy patients. However, the mechanisms underlying AL-LC related cardiotoxicity remain unknown. Mammalian stanniocalcin1 (STC1) is associated with a number of cellular processes including oxidative stress and cell death. Herein, we find that STC1 expression is elevated in cardiac tissue from AL cardiomyopathy patients, and is induced in isolated cardiomyocytes in response to AL-LC, but not non-amyloidogenic LC. STC1 overexpression in vitro recapitulates the pathophysiology of AL-LC mediated cardiotoxicity, with increased ROS production, contractile dysfunction and cell death. Overexpression of STC1 in vivo results in significant cardiac dysfunction and cell death. Genetic silencing of STC1 prevents AL-LC induced cardiotoxicity in cardiomyocytes and protects against AL-LC induced cell death and early mortality in zebrafish. The cardiotoxic effects of STC1 appears to be mediated via mitochondrial dysfunction as indicated by loss of mitochondrial membrane potential, ROS production and increased mitochondrial calcium levels. Collectively, this work identifies STC1 as a critical determinant of AL-LC cardiotoxicity.

Schwannomas are tumors formed by proliferation of dedifferentiated Schwann cells. Patients with neurofibromatosis 2 (NF2) and schwannomatosis develop multiple schwannomas in peripheral and cranial nerves. Although benign, these tumors can cause extreme pain and compromise sensory/motor functions, including hearing and vision. At present, surgical resection is the main treatment modality, but it can be problematic because of tumor inaccessibility and risk of nerve damage. We have explored gene therapy for schwannomas, using a model in which immortalized human NF2 schwannoma cells expressing a fluorescent protein and luciferase are implanted in the sciatic nerve of nude mice. Direct injection of an adeno-associated virus (AAV) serotype 1 vector encoding caspase-1 (ICE) under the Schwann-cell specific promoter, P0, leads to regression of these tumors with essentially no vector-mediated neuropathology, and no changes in sensory or motor function. In a related NF2 xenograft model designed to cause measurable pain behavior, the same gene therapy leads to tumor regression and concordant resolution of tumor-associated pain. This AAV1-P0-ICE vector holds promise for clinical treatment of schwannomas by direct intratumoral injection to achieve reduction in tumor size and normalization of neuronal function.

Background— Ventricular arrhythmias are life-threatening complications of heart failure and myocardial ischemia. Increased diastolic Ca 2+ overload occurring in ischemia leads to afterdepolarizations and aftercontractions that are responsible for cellular electric instability. We inquired whether sarcoplasmic reticulum Ca 2+ ATPase pump (SERCA2a) overexpression could reduce ischemic ventricular arrhythmias by modulating Ca 2+ overload. Methods and Results— SERCA2a overexpression in pig hearts was achieved by intracoronary gene delivery of adenovirus in the 3 main coronary arteries. Homogeneous distribution of the gene was obtained through the left ventricle. After gene delivery, the left anterior descending coronary artery was occluded for 30 minutes to induce myocardial ischemia followed by reperfusion. We compared this model with a model of permanent coronary artery occlusion. Twenty-four–hour ECG Holter recordings showed that SERCA2a overexpression significantly reduced the number of episodes of ventricular tachycardia after reperfusion, whereas no significant difference was found in the occurrence of sustained or nonsustained ventricular tachycardia and ventricular fibrillation in pigs undergoing permanent occlusion. Conclusions— We show that Ca 2+ cycling modulation using SERCA2a overexpression reduces ventricular arrhythmias after ischemia-reperfusion. Strategies that modulate postischemic Ca 2+ overload may have clinical promise for the treatment of ventricular arrhythmias.

Commercially-available WCMP slurries perform differently under diverse modifications: pH variations, dilutions, ageing, etc. In order to understand the physico-chemical basics of the mechanism and selectivity of these slurries, we have performed a number of measurements, including determination of effective CMP removal rates on blank and patterned wafers. The results of the performances of the different modifications have been compared, and a few optimized conditions and recipes derived. Our results surprisingly show that pH has a much greater influence on the removal rate of tungsten and silicon dioxide than the particles content, and that the way a certain pH is reached (or maintained) significantly affects the performances of the prepared slurries.We propose that nitric acid can be used to dilute selective slurries in order to keep high removal rates. Indeed, diluting slurries with an acidic solution of the same pH maintains particle charges far from zero, and keeps oxidizing conditions stable. However, the effects of the acidic dilution of the slurries include a slight reduction of removal rate and a decrease in defect density