Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective. Keywords: CRISPR; FRGN; ex vivo; genome editing; hepatocyte transplantation; liver-humanized mouse; primary hepatocytes; urea cycle disorder

Endogenous thrombospondin-1 and proteases in the regulation of lymphocyte adhesion and motility

The human immune system, which protects the body from invading pathogens, largely depends on the proper function of lymphocytes, which are highly motile and constantly recirculate the blood and lymph. Adhesive and motile capability is often amplified or uncontrolled during chronic inflammatory conditions such as autoimmune diseases. This thesis comprises four studies of T lymphocyte motility and adhesion aiming to elucidate the regulative role of endogenous secretion of enzymes and the matricellular protein thrombospondin-1 (TSP-1). We initially investigated the expression of matrix metalloproteinases (MMPs) in seven leukemia T cell lines and found a strict correlation between secretion of MMP-9, its natural inhibitor tissue inhibitor of MMPs (TIMP-1) and ability to infiltrate a threedimensional (3D) gel of extracellular (ECM) components. However, cell migration to two-dimensional (2D) ECM-components was not correlated to MMP-9/TIMP-1 expression. The role for MMP-9 and TIMP-1 in motility was unclear since an inhibitor of MMP-9 activity rather enhanced infiltrative capacity over 24h. We conclude that MMP-9 and TIMP-1 play a role for spontaneous T lymphocyte motility in 3D-matrices, which is a functional property separated from ability to migrate to 2D ECMcomponents. In our following papers (Paper II-IV), we found that T cell contact with collagen type I or beta1- and beta2-integrin ligands induced cell surface expression of TSP-1 and the TSP-1 receptor low density lipoprotein receptor-related protein (LRP)/CD91. Interaction of TSP-1 with its cell surface receptors calreticulin (CRT), LRP and integrin associated protein (IAP)/CD47 was promoting motility of T cells in 3D collagen through CD47. T cell surface TSP-1 also induced polarized spreading on fibronectin and ICAM-1 via binding to and signaling through CD47. T cell lines without endogenous expression of TSP-1 showed no spontaneous infiltration of collagen type I but became motile in the presence of exogenous TSP-1. We further found constitutive cell surface association of functional granzyme B on activated T cells, which continuously cleaved TSP-1, reduced infiltration of collagen type I and maintained non-polarized spreading on fibronectin and ICAM-1. This process depended on internalization of TSP-1 fragments via LRP. In summary, we have included TSP-1 and its receptors CRT, LRP and CD47 in a model for regulation of T cell motility and adhesion, stating that TSP-1 drives infiltrative capacity and polarized spreading of T cells through receptor communication in cis within the same plasma membrane, generated by cross-linking of its receptors CRT, LRP and CD47. Cleavage of TSP-1 by granzyme B and possibly other enzymes, followed by internalization of fragments via LRP, reduces motility and results in nonpolarized spreading

Perioperative Myocardial Damage Cardiac Outcome in Patients-at-Risk undergoing Non-Cardiac Surgery

Despite increasingly sophisticated perioperative management, cardiovascular complications continue to be major challenges for the clinician. As a growing number of elderly patients with known coronary artery disease (CAD) or with risk factors for CAD are undergoing noncardiac surgery, cardiovascular complications will remain a significant clinical problem in the future. The overall objective of this thesis was to study the incidence of myocardial damage and perioperative adverse cardiac events, to determine predictors of poor outcome and to assess the effect of a medical intervention in patients at risk undergoing non-cardiac surgery. The studies in this thesis were conducted on a total of 952 patients undergoing non-cardiac surgery. Studies I and IV were multicenter studies; whereas the patients included in studies II and III underwent non-cardiac surgery at Linkoping University Hospital, Sweden. The correlation between postoperative myocardial damage and short- and long-term outcome were studied in 546 patients, aged 70 years or older undergoing non-cardiac surgery of at least 30 minutes duration. This study showed a close correlation between postoperative myocardial damage and poor short- as well as long-term outcome. Elevated Troponin T was a stron

Gene expression.

<p>Gene expression in preadipocytes (denoted Undiff) and differentiated human preadipocytes in 2D aligned and random fibers Analyzed data is ΔCt values from technical duplicates. A) PCA analysis of the variation between samples. B) A heatmap was created from mean expression levels in three donors, and the expression profiles were analyzed with hierarchical clustering (UPGMA method with Euclidian method for distance measure).</p

Lipolysis in differentiated and mature adipocytes.

<p>Lipolysis was measured as glycerol release and normalized between 0 and 100% to determine EC50 values for human preadipocytes differentiated in 2D (black, solid line), n = 18 differentiated at three separate occasions, human preadipocytes differentiated in aligned (green line), n = 18 differentiated at three separate occasions and human preadipocytes differentiated in random fibers (magenta line), n = 18 differentiated at three separate occasions, mature adipocytes (black dashed line) measured as mean of triplicates isolated from 5 different subjects. A) All four data sets normalized to min 0 and max 100% and fitted to the same slope, no significant differences between the EC50s. B) All four data sets normalized to min 0 and max 100% and fitted to individual, three parameter slopes. C) Fully differentiated human preadipocytes (N = 3, n = 6) or human mature adipocytes (N = 7) were lysed levels of HSL was measured with western blot as arbitrary units normalized to loading control actin. P-value <0.05 is indicated with *, bars or lines without a star were not found to be significantly different from any other bar in the grap.</p

Proliferation of human adipose derived stem cells.

<p>P-values are indicated with * when p<0.05 and *** when p<0.001. 2D (black), aligned (green) and random (magenta). A) Proliferation was measured by nuclear count at the indicated time points during the differentiation, in wells seeded with 2D (56250 c/cm²), aligned (112500 c/cm²) and random (112500 c/cm²), n = 10. B) Number of cells was measured as in 1A. C) Live-dead images.14-days differentiated cells were stained live cells (calcein AM) indicated with green and dead cells (Ethidium homodimer) indicated with red thenimaged.</p

Lipid accumulation during adipogenic differentiation of human preadipocytes in 2D and fiber matrixes.

<p>P-values are indicated with ** when p<0.01 and *** when p<0.001, bars without a star were not found to be significantly different from any other bar in the grap. Mature adipocytes (open bars), 2D (black), aligned (green) and random (magenta). A) Upper: 14 days differentiated cells were stained with oil-red-o and imaged in a bright field microscope. Lower: Lipid accumulation was measured as absorbance of eluted oil-o-red at 490 nm at the differentiation day 2 and 14 and normalized to the cell number counted using Cellavista after staining with Hoechst nuclear dye, n = 10. B) Upper: 14 days differentiated cells unstained imaged with CARS (red signals) and MPEF (green signals) microscopy (Order of the images: 2D, aligned, random; scalebar: 30 µm). Lower: Size distribution of lipid droplets in the adipocytes. Presented is the distribution between 6 and 14 µm in 1 µm bins. 30 cells were evaluated per condition. The total droplet number evaluated in each condition was set to 100%. C) Approximation of the droplet size distribution with a Lorentizian function from the data set in 2B. Each data set was normalized to the total number of counted lipid droplets per well in each condition. D) Levels of perilipin in fully differentiated human preadipocytes (N = 3, n = 6) and primary, mature adipocytes (N = 7) measured as a.u. with western blot and normalized to actin content and to a standard as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113620#s2" target="_blank">methods</a> section.</p

Fitting parameters for the Lorentizian distribution in Figure 3C.

<p>Fitting parameters for the Lorentizian distribution in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113620#pone-0113620-g003" target="_blank">Figure 3C</a>.</p

Human adipose derived stem cells growing in PCL fiber matrices.

<p>A) Normalized intensities of the CARS (2845 cm⁻¹) signal of the fibers (violet, solid) and the MPEF signal (495–530 nm) (black, dashed lines, one line for each cell) of the cells in the matrices in the z dimension for the aligned (left) and random (right) matrices. 5 cells were investigated for each condition, for the random matrix two fibers were shown to visualize the different fiber profiles in this matrix. B) 3D reconstructions of the MPEF signals of the cells on/in the fiber matrices for aligned (left) and random (right) matrices. Nuclei are indicated with circles and fibers with arrowheads (scalebar: 30 µm).</p