Disease-relevant Functions Research Articles

Empowering gene delivery with protein engineering platforms.

The repertoire of therapeutic proteins has been substantially augmented by molecular engineering approaches, which have seen remarkable advancement in recent years. In particular, advances in directed evolution technologies have empowered the development of custom-designed proteins with novel and disease-relevant functions. Whereas engineered proteins have typically been administered through systemic injection of the purified molecule, exciting progress in gene delivery affords the opportunity to elicit sustained production of the engineered proteins by targeted cells in the host organism. Combining developments at the leading edge of protein engineering and gene delivery has catapulted a new wave of molecular and cellular therapy approaches, which harbor great promise for personalized and precision medicine. This mini-review outlines currently used display platforms for protein evolution and describes recent examples of how the resulting engineered proteins have been incorporated into DNA- and cell-based therapeutic platforms, both in vitro and in vivo. Collectively, the strategies detailed herein provide a framework for synthesizing molecular engineering workflows with gene therapy systems for a breadth of applications in research and medicine.

NIMG-86. REVEALING THE BIOLOGY BEHIND MRI SIGNATURES IN GLIOBLASTOMA

Abstract The brain is notoriously difficult to access because of its location and protective mechanisms, both internal and external. Imaging-based approaches, such as multiparametric magnetic resonance imaging (mpMRI), provide a method to bypass these obstacles. mpMRI can capture a wide range of data from tumors embedded in brain tissue through various imaging features. However, the biological function captured by each imaging feature remains largely unknown, particularly within the aggressive disease glioblastoma multiforme (GBM). We integrated our GBM SYstems Genetics Network AnaLysis (gbmSYGNAL) gene regulatory network (Plaisier, et al. 2016), based on the multi-omic patient data from The Cancer Genome Atlas (TCGA), with mpMRI-localized patient tumor biopsies. The gbmSYGNAL network was reconstructed within the new image-localized biopsies, and associations revealed a strong connection between the spoiled gradient recalled echo + contrast (SPGR+C) feature and the immune hallmarks of cancer. The SPGR+C method generates an image by employing a contrast agent that bypasses the blood brain barrier (BBB) through leaky blood vessels that have weakened from the force of the rapidly growing GBM tumor. Furthermore, a significant association between SPGR+C signal and the presence of both infiltrating macrophages and T cells indicated that peripheral immune cells may also cross the BBB at these leaky sites. Separately, the mean diffusivity (MD) & T2 features were linked to the sustained angiogenesis hallmark. These features displayed high association with angiogenic genes, such as FLT1, KDR, and PDGFRB, and correlated with the presence of histologically identified vessels. Detection of these disease-relevant functions via mpMRI features has powerful clinical applications, providing a non-invasive approach to GBM diagnosis, prognosis, and treatment direction.

Single-Cell Analysis of the Normal Mouse Aorta Reveals Functionally Distinct Endothelial Cell Populations.

The cells that form the arterial wall contribute to multiple vascular diseases. The extent of cellular heterogeneity within these populations has not been fully characterized. Recent advances in single-cell RNA-sequencing make it possible to identify and characterize cellular subpopulations. We validate a method for generating a droplet-based single-cell atlas of gene expression in a normal blood vessel. Enzymatic dissociation of 4 whole mouse aortas was followed by single-cell sequencing of >10 000 cells. Clustering analysis of gene expression from aortic cells identified 10 populations of cells representing each of the main arterial cell types: fibroblasts, vascular smooth muscle cells, endothelial cells (ECs), and immune cells, including monocytes, macrophages, and lymphocytes. The most significant cellular heterogeneity was seen in the 3 distinct EC populations. Gene set enrichment analysis of these EC subpopulations identified a lymphatic EC cluster and 2 other populations more specialized in lipoprotein handling, angiogenesis, and extracellular matrix production. These subpopulations persist and exhibit similar changes in gene expression in response to a Western diet. Immunofluorescence for Vcam1 and Cd36 demonstrates regional heterogeneity in EC populations throughout the aorta. We present a comprehensive single-cell atlas of all cells in the aorta. By integrating expression from >1900 genes per cell, we are better able to characterize cellular heterogeneity compared with conventional approaches. Gene expression signatures identify cell subpopulations with vascular disease-relevant functions.

Abstract 394: Elucidating tissue-specific effects of the 8q24 multicancer risk locus via CRISPR/Cas9 scarless genome editing

Abstract In the past decade, genome-wide association studies have identified a large number of genomic variants, specifically single-nucleotide polymorphisms (SNPs), associated with increased cancer risk. The molecular mechanisms underlying the vast majority of these associations remain largely unknown. Precise cellular models containing the variant of interest could be a crucial tool for establishing disease-relevant function in order to translate these discoveries into clinical application. CRISPR/Cas9 gene editing has provided a convenient and flexible method for creating isogenic cell lines via the homology-directed repair (HDR) pathway; however, methods for efficient generation and subsequent isolation of precisely edited cells have proven both expensive and time consuming. In response to this need, we have combined CRISPR/Cas9 HDR gene editing with an innovative high-throughput genotyping pipeline utilizing KASP (Kompetitive Allele-Specific PCR) technology to create scarless isogenic cell models of cancer risk variants in ~1 month without selectable markers or specialized methods such as digital droplet PCR or NGS. Utilizing this technology, we have been able to create cell lines differing by only a single base to model risk-associated SNPs located in the 8q24 risk locus in multiple cell types. The 8q24 risk locus is associated with increased risk for colorectal, breast, prostate, and, more recently, thyroid cancer. In this study, we use engineered isogenic cell lines modeling functional risk SNP rs6983267 in each of these cancer types to identify tissue-specific mechanisms of this multicancer risk locus. Rs6983267 is located within a known c-Myc enhancer and has been shown to exert risk-allele specific increase in enhancer activity. Oncogenic c-Myc is known to play a significant role in the pathogenesis of many cancers. However, how or if this mechanism varies between the different cancers this locus has been associated with remains uncertain. From preliminary studies, we do observe cell type variation in the regulatory effect of rs6983267. Not only does there seem to be a varying effect of the risk allele on c-Myc expression across the different cell types, but also risk allele enhancement of binding of the TCF7L2 transcription factor to the c-Myc enhancer rs6983267 is located within. Interestingly, rs6983267's regulatory effect seems most significant between homozygous reference cells and heterozygous cells within the same tissue type. We plan to delve further into these differences using RNA-seq and 4C-seq to assess whole transcriptomic and chromosomal interaction changes with changes in risk status. This study is one of first to compare risk mechanisms across different cancer tissue types; using precisely edited cellular models, we are able to detect cell-type specific variations in functional risk SNP effects on cancer development. Citation Format: Nicole B. Coggins, Henriette O'Geen, David J. Segal, Luis G. Carvajal-Carmona. Elucidating tissue-specific effects of the 8q24 multicancer risk locus via CRISPR/Cas9 scarless genome editing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 394.

Purification of Soluble Recombinant Human Tau Protein from Bacteria Using Double-tag Affinity Purification.

Dysfunction of the microtubule-associated protein Tau (encoded by the MAPT gene) has been implicated in more than twenty neurodegenerative diseases, including Alzheimer's. As such, the physiological and disease-relevant functions of Tau have garnered great interest in the research community. One barrier hampering investigations into the functions of Tau and the generation of pharmacological agents targeting Tau has been the difficulty of obtaining soluble Tau protein in purified form. Here, we describe a protocol that uses dual affinity tag purification to selectively purify soluble recombinant Tau protein from bacteria that is functionally active for downstream applications including immunization, microtubule binding assays, and protein-protein interaction studies.

A METHYLATION-TO-EXPRESSION FEATURE MODEL FOR GENERATING ACCURATE PROGNOSTIC RISK SCORES AND IDENTIFYING DISEASE TARGETS IN CLEAR CELL KIDNEY CANCER.

Many researchers now have available multiple high-dimensional molecular and clinical datasets when studying a disease. As we enter this multi-omic era of data analysis, new approaches that combine different levels of data (e.g. at the genomic and epigenomic levels) are required to fully capitalize on this opportunity. In this work, we outline a new approach to multi-omic data integration, which combines molecular and clinical predictors as part of a single analysis to create a prognostic risk score for clear cell renal cell carcinoma. The approach integrates data in multiple ways and yet creates models that are relatively straightforward to interpret and with a high level of performance. Furthermore, the proposed process of data integration captures relationships in the data that represent highly disease-relevant functions.

Chemokine receptor expression of T cells in the cerebrospinal fluid of relapsing multiple sclerosis

Multiple Sclerosis (MS) is an autoimmune-mediated chronic demyelinating disease of the central nervous system. The role of CD4+ T helper T cells in the pathogenesis of this disease is established. CD4+ T cells comprise various Th subsets, including Th1 and Th17 cells. The contributution of each T cell subset to MS pathology is not fully elucidated. As each Th subset have characteristic chemokine receptor expression patterns, we compared the frequency of various inflammatory chemokine receptor positive Th cells in peripheral blood with those from cerebrospinal fluid (CSF) for each MS patient. CCR2+CCR5+CD4+ T cells were selectively enriched in the CSF of MS patients but not in other neurological diseases. Peripheral CCR2+CCR5+CD4+ T cells produced both IFN-γ and IL-17, expressed Matrix Metalloproteinase-9 (MMP9) and Osteopontin, and transmigrated in an in vitro blood brain barrier model more efficiently than other Th cells, suggesting a BBB-invading mechanism and an important role in the relapse of MS. This study illustrates the potential of method in which examining the combination of chemokine receptors could reveal a Th subset with disease-relevant function.

A Platelet-Inspired Paradigm for Nanomedicine Targeted to Multiple Diseases

Platelets are megakaryocyte-derived anucleated cells found in the blood. They are mainly responsible for rendering hemostasis or clotting to prevent bleeding complications. Decreased platelet numbers or deficiencies in platelet functions can lead to various acute or chronic bleeding conditions and hemorrhage. On the other hand, dysregulated hyperactivity of the clotting process can lead to thrombosis and vascular occlusion. There is significant evidence that beyond hemostasis and thrombosis, platelets play crucial mechanistic roles in other disease scenarios such as inflammation, immune response and cancer metastasis by mediating several cell-cell and cell-matrix interactions, as well as aiding the disease microenvironment via secretion of multiple soluble factors. Therefore, elucidating these mechanistic functions of platelets can provide unique avenues for developing platelet-inspired nanomedicine strategies targeted to these diseases. To this end, the current review provides detailed mechanistic insight into platelets' disease-relevant functions and discusses how these mechanisms can be utilized to engineer targeted nanomedicine systems.

MicroRNAs act complementarily to regulate disease-related mRNA modules in human diseases

MicroRNAs (miRNAs) play a key role in regulating mRNA expression, and individual miRNAs have been proposed as diagnostic and therapeutic candidates. The identification of such candidates is complicated by the involvement of multiple miRNAs and mRNAs as well as unknown disease topology of the miRNAs. Here, we investigated if disease-associated miRNAs regulate modules of disease-associated mRNAs, if those miRNAs act complementarily or synergistically, and if single or combinations of miRNAs can be targeted to alter module functions. We first analyzed publicly available miRNA and mRNA expression data for five different diseases. Integrated target prediction and network-based analysis showed that the miRNAs regulated modules of disease-relevant genes. Most of the miRNAs acted complementarily to regulate multiple mRNAs. To functionally test these findings, we repeated the analysis using our own miRNA and mRNA expression data from CD4+ T cells from patients with seasonal allergic rhinitis. This is a good model of complex diseases because of its well-defined phenotype and pathogenesis. Combined computational and functional studies confirmed that miRNAs mainly acted complementarily and that a combination of two complementary miRNAs, miR-223 and miR-139-3p, could be targeted to alter disease-relevant module functions, namely, the release of type 2 helper T-cell (Th2) cytokines. Taken together, our findings indicate that miRNAs act complementarily to regulate modules of disease-related mRNAs and can be targeted to alter disease-relevant functions.

RNAi–Based Functional Profiling of Loci from Blood Lipid Genome-Wide Association Studies Identifies Genes with Cholesterol-Regulatory Function

Genome-wide association studies (GWAS) are powerful tools to unravel genomic loci associated with common traits and complex human disease. However, GWAS only rarely reveal information on the exact genetic elements and pathogenic events underlying an association. In order to extract functional information from genomic data, strategies for systematic follow-up studies on a phenotypic level are required. Here we address these limitations by applying RNA interference (RNAi) to analyze 133 candidate genes within 56 loci identified by GWAS as associated with blood lipid levels, coronary artery disease, and/or myocardial infarction for a function in regulating cholesterol levels in cells. Knockdown of a surprisingly high number (41%) of trait-associated genes affected low-density lipoprotein (LDL) internalization and/or cellular levels of free cholesterol. Our data further show that individual GWAS loci may contain more than one gene with cholesterol-regulatory functions. Using a set of secondary assays we demonstrate for a number of genes without previously known lipid-regulatory roles (e.g. CXCL12, FAM174A, PAFAH1B1, SEZ6L, TBL2, WDR12) that knockdown correlates with altered LDL–receptor levels and/or that overexpression as GFP–tagged fusion proteins inversely modifies cellular cholesterol levels. By providing strong evidence for disease-relevant functions of lipid trait-associated genes, our study demonstrates that quantitative, cell-based RNAi is a scalable strategy for a systematic, unbiased detection of functional effectors within GWAS loci.

Active Metalloproteases of the A Disintegrin And Metalloprotease (ADAM) Family: Biological Function and Structure

The "a disintegrin and metalloproteases" (ADAMs) are membrane-anchored metzincins of the adamalysin subfamily. This review gives an overview over the biological function and structure of ADAMs focusing on members of the family that display proteolytic activity. ADAMs are involved in a range of human diseases such as cancer metastasis, inflammatory disorders, neurological disease or asthma. It is, however, often difficult to assign a definitive role to a specific member of the ADAM family in a given disease mechanism due to overlapping activities and redundancy in function, as shown in various knock-out studies. The review discusses the structural domains that are not directly linked to protease activity followed by a more detailed overview over the role of the metalloprotease domain. Different family members are critically reviewed with respect to their role in biological processes with particular emphasis on disease-relevant functions.