Ian Møller-Nielsen, Department of Biomedicine
A. B. Rovsing, Department of Biomedicine; F. Nedergaard, Department of Biomedicine;
Jacob Giehm Mikkelsen, Department of Biomedicine
Due to their involvement in autoimmune disease and their ability to function as protein factories, primary B cells are interesting targets for genome editing. However, primary B cells are difficult to culture and hard to transduce using standard vesicular stomatitis G protein (VSV-G)-pseudotyped lentiviral vectors.
To study genome editing in primary B cells, we have engineered murine MS-5 cells expressing human CD40 ligand (CD40L). Using such MS-5(CD40L) cells as feeder cells, primary B cells proliferate and are prevented from undergoing apoptosis. Furthermore, lentiviral vectors pseudotyped with measles virus (MV) glycoproteins were shown to enable efficient gene transfer to primary B cells.
Based on these findings, we set out to explore genome editing in primary B cells using engineered lentivirus-derived nanoparticles (LVNPs). LVNPs represent a novel platform for delivery of active CRISPR-Cas9/sgRNA ribonucleoprotein (RNP) complexes without
delivery of a viral genome or a transgene. Using VSV-G-pseudotyped LVNPs, we demonstrated effective gene editing in human and murine cell lines. Notably, by
pseudotyping LVNPs with MV glycoproteins, targeted gene knock out in HEK293T cells was observed. Also, we generated a MV pseudotype variant, ‘MV-SLAM’, which specifically mediates virus uptake in cells expressing signaling lymphocyte activation molecule (SLAM). Using MV-SLAM-pseudotyped LVNPs, gene editing was achieved specifically in engineered SLAM-expressing HEK293T cells. Finally, targeted ex vivo gene knockout in primary B cells was achieved by treating B cells with MV-pseudotyped LVNPs, leading to markedly higher levels of editing than with VSV-G-pseudotyped LVNPs.
Keywords: Genetic engineering, Cell biology, Other
Enrichment of cells that carry CRISPR-mediated transgene integrations by transient CRISPRa of a silent reporter gene
Nanna Steengaard Mikkelsen, Department of Biomedicine
R. O. Bak, Department of Biomedicine Aarhus University, Aarhus Institute of Advanced Studies (AIAS) Aarhus University
The CRISPR/Cas system has revolutionized the field of genome editing by its ability to simply and precisely edit the genomes of living organisms. Double-strand DNA breaks at specific target sites mediated by the CRISPR/Cas system can be repaired through the cellular repair mechanism homology-directed repair (HDR), which can be harnessed to introduce sequences from an exogenous homologous template. Exploiting the HDR mechanism for site-specific integration of a specific sequence has wide applications in both research and clinical gene and cellular therapies for example by integrating a
therapeutic cDNA to correct disease-causing mutations in stem cells or by producing gene modified CAR T cells.
However, this approach is challenged by the relatively low HDR frequencies that can be obtained in some cell lines and primary cells, thereby impairing potential gene and cellular therapies. Cells that have undergone HDR cannot directly be distinguished and sorted from cells that have not undergone HDR unless a reporter gene has been
incorporated in the HDR template. However, permanent expression of a reporter gene is not desired in most therapeutic contexts.
This project seeks to develop a novel enrichment strategy based on transient CRISPR activation of an otherwise silent and clinically relevant reporter gene co-integrated along a gene of interest. Such an enrichment strategy may be widely applicable in both research and gene and cellular therapies, where this strategy has the potential to enrich for
correctly gene modified cells and thereby improve gene and cellular therapies.
Keywords: Genetic engineering, Genetic engineering, Other
Multisite repeatability of hyperpolarized 13C MRI
Nikolaj Bøgh, Department of Clinical Medicine, MR Research Center
Nikolaj Bøgh, Department of Clinical Medicine; Jeremy W Gordon, University of California San Francisco; Esben SS Hansen, Department of Clinical Medicine; Robert A Bok, University of California San Francisco; Jakob U Blicher, Department of Clinical Medicine; Jasmine Y Hu, University of California San Francisco; Peder E Z Larson, University of California San Francisco; Daniel B Vigneron, University of California San Francisco; Christoffer Laustsen, Department of Clinical Medicine
Magnetic resonance imaging (MRI) with hyperpolarized [1-13C]pyruvate is an emerging technology for in vivo imaging of the balance of glycolytic to oxidative metabolism. It uniquely positioned to evaluate metabolic changes in the face of cancerous disease and its treatment, but the comparability between sites and protocols is unknown. In this study, we aimed to evaluate the agreement of hyperpolarized MRI between two sites across protocols.
We included three healthy volunteers, who underwent repeated imaging of the brain with various protocols. In total, we performed 13 scans. The common metrics of pyruvate to lactate conversion were calculated, including the model-based kPL and the model-free lactate-to-pyruvate ratio. Agreement was evaluated with intraclass correlation coefficients (ICCs), where 0 is no agreement and 1 is perfect.
The mean model-based pyruvate-to-lactate conversion (kPL) across all examinations was 0.02 ± 0.006 s-1. One of the employed protocols displayed a
systematically ~40% lower kPL due to a longer echo time. Upon adjustment for this, the ICC was 0.71 (P = 0.025) between sites and 0.83 between examinations (P < 0.001). The ICCs were considerably lower for the model-free lactate-to-pyruvate ratio, as this fails to consider differences in flip-angles between protocols.
In conclusion, MRI with hyperpolarized [1-13C]pyruvate shows good agreement between sites and examinations. Some differences in protocols can and should be adjusted for when comparing data retrospectively, while care should be taken to use identical protocols when conducting prospective multi-site studies. Collectively, our results inform and support larger multicenter studies.
Keywords: Medical technology and diagnostic techniques, Clinical neuroscience, Molecular metabolism and endocrinology
Nucleic acids detection by degradation of quenched fluorescent probes by CRISPR ‘collateral damage’
Alexander Rafael Lavilla Labial, Department of Biomedicine
J. Haldrup, Department of Biomedicine; J. G. Mikkelsen, Department of Biomedicine
The ability to rapidly and effectively detect nucleic acids with single-base specificity and high sensitivity may be of high use to clinical diagnosis and monitoring. Characterization of CRISPR/Cas12 and CRISPR/Cas13 has revealed that these proteins upon recognition of their target sequences are further activated and cleave nearby non-target DNAs and RNAs, respectively. Here we explore two nucleic acids detection platforms dubbed HOLMES and SHERLOCK to detect specific target sequences. By using PCR, or LAMP, amplification of target DNA and subsequently detection by Cas12 or Cas13, we aim to achieve fluorescent readouts in less than an hour. This readout can be quantified using classical plate readers providing an easy-to-use method for rapid quantification of nucleic acids carrying the exact target sequence.
One can envision the use of this system to detect recurrent biomarkers in cancer patients.
By analyzing the blood plasma containing circulating tumor DNA (ctDNA) originating from necrotic/apoptotic tumor cells, it should be possible to detect the biomarker with high specificity and sensitivity, providing rapid molecular profiling of tumor DNA isolated from cancer patients.
Keywords: Medical technology and diagnostic techniques, Oncology, Other
A novel method for simultaneous detection of five kynurenine metabolites as blood biomarkers for post-concussion syndrome
Peter Preben Eggertsen, Department of Clinical Medicine, Hammel Neurorehabilitation Centre and University Research Clinic, Aarhus University
RKJ. Olsen, Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University; JF. Nielsen, Hammel Neurorehabilitation Centre and University Research Clinic;
J. Palmfeldt, Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University
Introduction
At least 10.000 suffer from a concussion each year in Denmark, and 10-15% develop persisting symptoms such as headache, fatigue, and depression. This is defined as post- concussion syndrome (PCS), and the pathophysiology is unknown.
The kynurenine pathway describes the degradation of tryptophan, and its metabolites are involved in psychiatric disorders and may be important in PCS as well.
However, the metabolites are chemically diverse and difficult to detect simultaneously.
The aim of the study is to develop a method of measuring kynurenine metabolites in serum.
Methods
Metabolites from the kynurenine pathway (tryptophan, kynurenine, kynurenic acid,
quinolinic acid, and 3-hydroxykynurenine) including their isotope-labelled standards, were measured in aqueous solution and in serum from four healthy persons using high-
performance liquid chromatography-tandem mass spectrometry (LC-MS). Serum was prepared by precipitating proteins with either methanol, acetonitrile, acetone, or
trichloroacetic acid. Several LC-MS parameters, such as columns and mobile phases, were tested.
Results
All metabolites were detectable in aqueous solution, and mass spectrometry signal intensities correlated to the concentrations linearly (R2≈0.999).
In serum, the detection of quinolinic acid was complicated by significant matrix effects which were dependent on the method of protein precipitation and LC-MS parameters.
Limit of detection, linearity in serum, and lower limit of quantification will be presented at the PhD Day.
Perspectives
The method shows potential and will be used to measure serum concentrations of the metabolites in PCS patients and a healthy control group.
Keywords: Medical technology and diagnostic techniques, Basic neuroscience, Laboratory science
Development of noninvasive prenatal testing for monogenic disorders using circulating fetal cells in maternal blood
Line Dahl Jeppesen, Department of Clinical Medicine, Center for Fetal Diagnostics
I. Vogel, Center for Fetal Diagnostics, Department of Clinical Medicine, Aarhus University &
Department of Clinical Genetics, Aarhus University Hospital; L. Hatt, ARCEDI Biotech; D.L.
Lildballe, Department of Molecular Medicine (MoMA), Aarhus University Hospital.
Prenatal diagnosis using invasive tissue sampling involves a risk of miscarriage (<0.2 %) and is associated with severe discomfort for pregnant woman. Therefore, a long-sought goal in prenatal care is a safe and accurate noninvasive prenatal test (NIPT). NIPT for aneuploidy and copy number variants can be done using circulating trophoblast cells isolated from maternal blood samples. This study aimed to develop novel strategies for cell-based NIPT using circulating trophoblasts to test for cystic fibrosis (CF), repeat disorders and recessive disorders. Pregnant women (GA 10-14) that opted for prenatal diagnostics for disorders of interest were included. Circulating trophoblasts were enriched and isolated.
Nine pregnancies were tested for CF using multiplex-PCR and fragment analysis to test for 50 disease-causing variants. The cell-based NIPT showed 4 normal, 2 carriers of
Phe508del, 1 carrier of Arg334Trp and two inconclusive results. A repeat expansion in the DMPK gene, causing the development of autosomal dominant myotonic dystrophy, was detected using repeat-primed PCR and haplotyping in two pregnancies. In the first pregnancy, cell-based NIPT detected a repeat expansion >150 CTG-repeats in the fetus.
In the second case, two normal alleles were detected. In a pregnancy at risk of autosomal recessive diastrophic dysplasia, Sanger sequencing of two regions in SLC26A2 detected two pathogenic variants. The results of cell-based NIPT were in concordance with those of invasive prenatal diagnostics. In conclusion, cell-based NIPT can be developed to test for various monogenic disorders in the fetus as a risk-free alternative to invasive tissue
sampling.
Keywords: Medical technology and diagnostic techniques, Gynecology and obstetrics, Other