Murine cDNA Research Articles

A Mechanistic Role for RECK in the Regulation of Hepatocellular Inflammation

RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a membrane‐anchored glycoprotein, modifies the extracellular matrix involved in various cancers, including hepatocellular carcinoma (HCC). Its role in regulating inflammatory and fibrogenic processes has also been postulated. Both inflammation and fibrosis contribute to progression of nonalcoholic steatohepatitis (NASH) to HCC. Here, we tested the hypothesis that inducing or sustaining RECK expression will inhibit proinflammatory amphiregulin and epidermal growth factor receptor (EGFR) signaling, progression of NASH and the development of HCC using an in vitro cell culture model employing isolated adult mouse primary hepatocytes. Ectopic RECK overexpression (gain‐of‐function) was achieved by the adenoviral (Adv) transduction of murine RECK cDNA. Adv.GFP served as a control. RECK expression was silenced (loss‐of‐function) by transducing RECK‐specific siRNA using lipofectamine. Nonspecific siRNA served as a control. Hepatocytes were exposed to TNFα in the presence or absence of inhibitors against ADAM 10 (A Disintegrin and Metalloproteinase Domain‐Containing Protein 10) and/or ADAM17 – two well‐established pro‐inflammatory sheddases. Results show that RECK overexpression inhibited TNFα‐induced ADAM10/17 activity, and amphiregulin (an EGFR ligand) and EGFR expression. In the presence of ADAM10/17 inhibitors, no further reduction was observed in amphiregulin and EGFR activity, suggesting a maximal inhibitory effect of RECK on EGFR activity. In contrast, silencing RECK significantly increased amphiregulin secretion, and this effect was reversed by inhibition of ADAM10/17, indicating RECK signals via regulation of these sheddases. Thus, RECK not only regulates sheddase activity of ADAM10 and 17, but also downstream amphiregulin and EGFR signaling under pro‐inflammatory stimuli. Because increased and sustained EGFR activity contributes to progression of NASH to HCC in preclinical models, our results indicate that inducing RECK has the potential to inhibit hepatocellular inflammation in the setting of NASH and HCC.

A lung tropic AAV vector improves survival in a mouse model of surfactant B deficiency

Surfactant protein B (SP-B) deficiency is an autosomal recessive disorder that impairs surfactant homeostasis and manifests as lethal respiratory distress. A compelling argument exists for gene therapy to treat this disease, as de novo protein synthesis of SP-B in alveolar type 2 epithelial cells is required for proper surfactant production. Here we report a rationally designed adeno-associated virus (AAV) 6 capsid that demonstrates efficiency in lung epithelial cell transduction based on imaging and flow cytometry analysis. Intratracheal administration of this vector delivering murine or human proSFTPB cDNA into SP-B deficient mice restores surfactant homeostasis, prevents lung injury, and improves lung physiology. Untreated SP-B deficient mice develop fatal respiratory distress within two days. Gene therapy results in an improvement in median survival to greater than 200 days. This vector also transduces human lung tissue, demonstrating its potential for clinical translation against this lethal disease.

Absence of p.R50X Pygm read-through in McArdle disease cellular models.

ABSTRACTMcArdle disease is an autosomal recessive disorder caused by the absence of muscle glycogen phosphorylase, which leads to blocked muscle glycogen breakdown. We used three different cellular models to evaluate the efficiency of different read-through agents (including amlexanox, Ataluren, RTC13 and G418) in McArdle disease. The first model consisted of HeLa cells transfected with two different GFP-PYGM constructs presenting the Pygm p.R50X mutation (GFP-PYGM p.R50X and PYGM Ex1-GFP p.R50X). The second cellular model was based on the creation of HEK293T cell lines stably expressing the PYGM Ex1-GFP p.R50X construct. As these plasmids encode murine Pygm cDNA without any intron sequence, their transfection in cells would allow for analysis of the efficacy of read-through agents with no concomitant nonsense-mediated decay interference. The third model consisted of skeletal muscle cultures derived from the McArdle mouse model (knock-in for the p.R50X mutation in the Pygm gene). We found no evidence of read-through at detectable levels in any of the models evaluated. We performed a literature search and compared the premature termination codon context sequences with reported positive and negative read-through induction, identifying a potential role for nucleotide positions −9, −8, −3, −2, +13 and +14 (the first nucleotide of the stop codon is assigned as +1). The Pygm p.R50X mutation presents TGA as a stop codon, G nucleotides at positions −1 and −9, and a C nucleotide at −3, which potentially generate a good context for read-through induction, counteracted by the presence of C at −2 and its absence at +4.

219 Invitro culture environment influences the ability to generate porcine primordial germ cell-like from induced pluripotent stem cells

The ability to generate primordial germ cell-like (PGCLs) from induced pluripotent stem cells (iPSCs) in swine could greatly contribute to regenerative medicine. Herein, we aimed to generate porcine PGCLs (ipPGCLs) from iPSCs derived from different culture systems. Porcine (p)iPSCs from fibroblasts of stillborn animals (n=3) were transduced with lentiviral vectors containing murine OCT4, SOX2, c-MYC, and KLF4 cDNAs and maintained in iPSC medium on mouse embryonic fibroblasts (MEFs). The cells were divided into three groups: (1) supplemented with 10ngmL−1 basic fibroblast growth factor (bFGF) and murine leukemia inhibitory factor (LIF), (2) only bFGF, or (3) only LIF. The piPSC colonies were generated and characterised for pluripotency. To induce piPSCs into ipPGCLs, three or more cell lines from each culture condition (after passage 20) were differentiated into epiblast stem cell-like cells (EpiLCs) by culture with 20ngmL−1 Activin A, 12ngmL−1 bFGF, and 1% knockout serum replacement (KSR) for 2 days. Then, cells were further induced to differentiate by nonadherent culture and supplementation with 500ngmL−1 bone morphogenetic protein (BMP)4, 500ngmL−1 BMP8a, LIF, 100ngmL−1 stem cell factor (SCF), and 50ngmL−1 epidermal growth factor for 4 days. The ipPGCLs were characterised by cell morphology and detection of germ cell markers by immunofluorescence and gene expression. Statistical analysis was determined by one-way ANOVA (Prism Software). Co-location quantification was determined using the plugin Colocalization Threshold in Image J software (National Institutes of Health). On average, the efficiency rate of iPSC generation was 71% for the iPSCs-bFGF group, 17% for the LIF group, and 85% for the bFGF+LIF group. All iPSCs colonies were positive for alkaline phosphatase and OCT4, SOX2, NANOG, TRA1-60, TRA1-81, SSEA1, and SSEA4 by immunofluorescence. Embryoid body assay revealed that the piPSCs were able to differentiate into three germ layers. The culture condition did not influence the expression of OCT4, NANOG, and KLF4 based on qRT-PCR, however; SOX2 was upregulated in the LIF group (P<0.05). The ipPGCLs generated showed a round morphology. Analysis of endogenous pluripotent genes OCT4, SOX2, and NANOG throughout differentiation (fibroblasts, iPSCs, EpiLCs, and PGCLs) revealed a mild upregulation in ipPGCLs, while OCT4 was slightly downregulated in ipPGCLs from iPSCs-LIF group. PRDM14 and STELLA were not observed in ipPGCLs, although BLIMP1 was present; DAZL and VASA were mildly upregulated. The STELLA, VASA, OCT4, and SOX2 proteins were detected in ipPGCLs, and DAZL was detected only in ipPGCLs from the iPSCs-FGF group. Protein co-localization analysis showed that ipPGCLs from the iPSCs-FGF group were 100% OCT4+STELLA-positive, 55% positive for DAZL+SOX2, and 66% positive for VASA+NANOG; for the LIF group: 99.3% were OCT4+STELLA positive, DAZL was not detected, 95.2% were positive for SOX2 and 85.6% for VASA+NANOG. In the bFGF+LIF group, 95.8% were positive for OCT4+STELLA, DAZL and SOX2 were not observed, and 70% were positive for VASA+NANOG. Exogenous reprogramming factors were still expressed and did not differ between groups. These results indicate that, under our conditions, the iPSCs-FGF group may represent the best culture condition for induction into ipPGCLs. Financial support for this study was provided by FAPESP (2015/25564-0 and 2015/26818-5).

218 Urine samples as a noninvasive source of induced pluripotent cells in the swine model

Noninvasive collection of cells used for generation of induced pluripotent stem cells (iPSC) generation would simplify its use for regenerative and reproductive purposes in veterinary medicine, although it is still unpublished in species other than humans. This study aimed to derive urine progenitor cells (UPCs) invitro from urine samples collected from swine, and then to reprogram them into iPSCs. For that, urine samples were collected from three females, and cells were isolated and cultured from each following the human UPCs protocol (Steichen et al. 2017 Curr. Protoc. Hum. Genet. 21, 7.1-21.7.22; https://doi.org/10.1002/cphg.26). Approximately 200mL of urine samples were collected in sterile flasks and centrifuged at 300×g; the pellet was washed in Dulbecco's PBS, resuspended, and cultured in 45% Dulbecco's modified Eagle's medium (DMEM)-high glucose, 5% fetal bovine serum (FBS), 50% Renal Epithelial Cell Growth basal medium (REBM) supplemented with 1% glutamine, 1% nonessential amino acids (NEAA), penicillin/streptomycin (P/S), and REGM supplements hEGF, insulin, hydrocortisone, GA-1000, FBS, transferrin, triiodothyronine, epinephrine (Lonza), and 10ngmL−1 basic fibroblast growth factor (bFGF). The UPCs first colonies were observed approximately 1 week after and resembled epithelial-like cells. At passage 2, one cell line was transduced with murine OCT4, SOX2, KLF4, and C-MYC cDNAs (OSKM) using a lentiviral vector. After 5 days, cells were plated onto mouse embryonic fibroblasts and cultured in knockout DMEM/F12, 20% knockout serum replacement, NEAA, L-glutamine, 2-mercaptoethanol, and P/S supplemented with 10ngmL−1 bFGF. Efficiency of reprogramming was 8.45%, measured by analysing the number of typical iPSC colonies relative to the transduced cells plated, after ~12 days. Three clonal lines (C1, C4, and C6) were maintained invitro and characterised regarding pluripotency markers for at least 30 passages. All three lines were positive for alkaline phosphatase activity in passages 15 and also 22. Immunocytochemistry analysis revealed that C6 (passage 22) was positive for the pluripotency genes OCT4 (1:100, SC), SOX2 (1:500, AB), SSEA1 (1:50, SC), TRA1- 81 (1:50, Millipore), and NANOG (1:100, AB), whereas C1 and C4 (passages 23 and 22, respectively) were positive only for OCT4, SOX2, and SSEA1. The expression of exogenous and endogenous pluripotency factors (OCT4, SOX2, and NANOG) was evaluated by qRT-PCR, comparing the three clonal lines at passages 16/17 and 21/22 and comparing different passages (10, 11, 14, 17, and 22) on C1 line. No statistical difference was observed between cell lines when compared in different passages, perhaps because of the great variation between lines. However, analysis of C1 line over time showed that pluripotency genes increased and exogenous vector expression decreased during early passages (±10 passages); however, after passage 17, OCT4 and NANOG decreased whereas SOX2 and exogenous vector expression increased. In conclusion, it was possible to reprogram UPCs into iPSCs and maintain them in culture for at least 30 passages; however, iPSCS were dependent on exogenous factors. These results represent the partial reprogramming of UPCs to iPSCs in animals for the first time, enabling the generation of invitro disease models using a noninvasive method. Financial support for this study was received from FAPESP (2019/02811-2, 2015/26818-5), CNPq (433133/2018-0), and CAPES.

Affinity Purification of NF1 Protein–Protein Interactors Identifies Keratins and Neurofibromin Itself as Binding Partners

Neurofibromatosis Type 1 (NF1) is caused by pathogenic variants in the NF1 gene encoding neurofibromin. Definition of NF1 protein–protein interactions (PPIs) has been difficult and lacks replication, making it challenging to define binding partners that modulate its function. We created a novel tandem affinity purification (TAP) tag cloned in frame to the 3’ end of the full-length murine Nf1 cDNA (mNf1). We show that this cDNA is functional and expresses neurofibromin, His-Tag, and can correct p-ERK/ERK ratios in NF1 null HEK293 cells. We used this affinity tag to purify binding partners with Strep-Tactin®XT beads and subsequently, identified them via mass spectrometry (MS). We found the tagged mNf1 can affinity purify human neurofibromin and vice versa, indicating that neurofibromin oligomerizes. We identify 21 additional proteins with high confidence of interaction with neurofibromin. After Metacore network analysis of these 21 proteins, eight appear within the same network, primarily keratins regulated by estrogen receptors. Previously, we have shown that neurofibromin levels negatively regulate keratin expression. Here, we show through pharmacological inhibition that this is independent of Ras signaling, as the inhibitors, selumetinib and rapamycin, do not alter keratin expression. Further characterization of neurofibromin oligomerization and binding partners could aid in discovering new neurofibromin functions outside of Ras regulation, leading to novel drug targets.

Abstract 870: Cardiac Gene Therapy With Relaxin Receptor 1 Overexpression Protects Against Acute Myocardial Infarction and Associated Adverse Remodeling

Background: Relaxin signaling confers cardioprotection against acute myocardial infarction (MI) and mitigates adverse cardiac remodeling. We investigated the role of relaxin receptor (RXFP1) overexpression via cardiotropic AAV9 vectors in attenuating MI-associated adverse remodeling. Methods and Results: Murine RXFP1 cDNA with CMV promoter was introduced into AAV cis plasmids for generation of AAV9-RXFP1 viral vectors. 1.0 x 10 11 viral genomes in 100μL saline were administered i.v. to 8-week-old CD1 male mice. Four weeks later, overexpression was confirmed via qPCR of RXFP1 mRNA in whole cardiac tissue lysates ( Fig A ) and primary cardiomyocytes ( Fig B ) versus control. Overexpressing (AAV9-RXFP1) mice were subjected to MI (30 minutes of coronary artery ligation followed by reperfusion for 24h or 7d). Immunohistochemical staining of cardiac sections 7d post-MI shows increased RXFP1 expression in AAV9-RXFP1 mice, as shown in Fig C . Infarct size was reduced at 24h post-MI (TTC staining, Fig D ) and fractional shortening (M mode echo) was preserved at 24h and 7d post-MI in AAV9-RXFP1 mice ( Fig E ); endocardial strain rate assessment (speckle tracking analysis) showed preserved function in the affected ventricular segments of AAV9-RXFP1 mice 7d post-MI ( Fig F ). Picrosirius staining showed reduced LV fibrosis at 7d post-MI in AAV9-RXFP1 mice ( Fig G ). Conclusion: Administration of AAV9-RXFP1 successfully upregulates RXFP1 in cardiac tissue and primary cardiomyocytes, reduces infarct size and fibrosis, and preserves cardiac function post MI. Targeting cardiac RXFP1 via gene therapy could be a novel strategy to prevent MI-related adverse remodeling.

Tumor cell IDO1 increases intratumoral immunosuppressive Tregs independent of enzyme activity

Abstract Objective Indoleamine 2, 3 dioxygenase 1 (IDO1) is a rate limiting enzyme that converts tryptophan (Trp) into kynurenine (Kyn). IDO1 knockdown in murine glioblastoma (GBM) cells suppresses intratumoral regulatory T cell (Treg) recruitment and increases survival, but unexpectedly, has no effect on intratumoral Trp levels. Moreover, while GBM cell IDO1 overexpression increases intratumoral Treg levels, treatment with a pharmacological IDO1 enzyme inhibitor fails to reverse this effect. These novel data led us to question whether GBM cell IDO1 recruits Tregs independent of enzyme activity, in vivo. Methods Ido1−/− mouse GBM cells reconstituted with wild-type or enzyme-null (H350A) murine Ido1 cDNA, and IDO1−/− human GBM cells were created. HPLC for Trp/Kyn levels, cell proliferation, cell viability, and splenic macrophage-GBM cell co-cultures, were studied in vitro. Syngeneic or humanized mice intracranially-engrafted with modified mouse or human GBM cells, respectively, were studied for the effect(s) on Treg accumulation and/or survival. Results As compared with IDO1 WT GBM cells, IDO1 H350A GBM cells show a significant decrease of Trp metabolism without affecting cell proliferation and viability. As compared with vector control GBM cells, co-culture for both IDO1 WT and H350A GBM cells induces greater numbers of mature macrophages. Independent of enzyme activity, GBM cell IDO1 expression increases intratumoral Tregs and decreases animal survival. Conclusions We created novel GBM models for elucidating the mechanism of metabolism-independent, IDO1-mediated intratumoral Treg accumulation. Given the majority of cancers with IDO1 expression, we suspect that our work may be generalizable to many types of malignancies.

Transient Scleraxis Overexpression Combined with Cyclic Strain Enhances Ligament Cell Differentiation.

Efforts to generate tissue-engineered anterior cruciate ligament replacements are limited by a lack of methods to derive mature ligament cells. Viral overexpression of the tendon/ligament marker scleraxis (Scx) can drive cell differentiation; however, the use of viral vectors hampers translation to clinical use. In this study, C3H10T1/2 cells were transiently transfected with expression vectors containing the full-length murine Scx cDNA and cultured in three-dimensional collagen hydrogels under static or cyclic strain for up to 14 days. β-galactosidase (LacZ) transfected cells served as controls. Cell morphology and gene expression for ligament-related genes, in addition to contraction (hydrogel width), mechanical properties, and glycosaminoglycan (GAG) and DNA content of hydrogels, were quantified and compared over time, between Scx and LacZ groups, and between static and cyclically strained constructs. Increased Scx expression was maintained for the entire 14-day study in both static and cyclically strained constructs. In static culture, overexpression of Scx resulted in greater cell elongation and construct contraction compared to LacZ controls. There were no differences in gene expression, DNA, or GAG content between Scx and LacZ constructs cultured under static conditions and no differences in DNA content between Scx and LacZ constructs. When exposed to cyclic strain, Scx-overexpressing cells maintained the elongated phenotype exhibited in static constructs, increased GAG production compared to static culture, and increased expression of the ligament-related genes collagen type I, decorin, and tenascin-C compared to strained LacZ controls. Cyclically strained constructs containing Scx-overexpressing cells had increased maximum load and stiffness compared to LacZ controls. The maintenance of increased Scx expression throughout the 14 day study and subsequent increases in ligament marker gene expression and mechanical properties with cyclic, but not static strain, suggest that transient transfection may be a viable alternative to viral transduction of Scx for ligament engineering studies and support a synergistic effect of Scx and mechanical strain on driving early ligament cell differentiation.

Conditional disruption of hepatic carbamoyl phosphate synthetase 1 in mice results in hyperammonemia without orotic aciduria and can be corrected by liver-directed gene therapy

Carbamoyl phosphate synthetase 1 (CPS1) is a urea cycle enzyme that forms carbamoyl phosphate from bicarbonate, ammonia and ATP. Bi-allelic mutations of the CPS1 gene result in a urea cycle disorder presenting with hyperammonemia, often with reduced citrulline, and without orotic aciduria. CPS1 deficiency is particularly challenging to treat and lack of early recognition typically results in early neonatal death. Therapeutic interventions have limited efficacy and most patients develop long-term neurologic sequelae. Using transgenic techniques, we generated a conditional Cps1 knockout mouse. By loxP/Cre recombinase technology, deletion of the Cps1 locus was achieved in adult transgenic animals using a Cre recombinase-expressing adeno-associated viral vector. Within four weeks from vector injection, all animals developed hyperammonemia without orotic aciduria and died. Minimal CPS1 protein was detectable in livers. To investigate the efficacy of gene therapy for CPS deficiency following knock-down of hepatic endogenous CPS1 expression, we injected these mice with a helper-dependent adenoviral vector (HDAd) expressing the large murine CPS1 cDNA under control of the phosphoenolpyruvate carboxykinase promoter. Liver-directed HDAd-mediated gene therapy resulted in survival, normalization of plasma ammonia and glutamine, and 13% of normal Cps1 expression. A gender difference in survival suggests that female mice may require higher hepatic CPS1 expression. We conclude that this conditional murine model recapitulates the clinical and biochemical phenotype detected in human patients with CPS1 deficiency and will be useful to investigate ammonia-mediated neurotoxicity and for the development of cell- and gene-based therapeutic approaches.

Neurofibromin (NF1) genetic variant structure-function analyses using a full-length mouse cDNA.

Neurofibromatosis type 1 (NF1) is caused by pathogenic variants or mutations in the NF1 gene that encodes neurofibromin. We describe here a new approach to determining the functional consequences of NF1 genetic variants. We established a heterologous cell culture expression system using a full-length mouse Nf1 cDNA (mNf1) and human cell lines. We demonstrate that the full-length murine cDNA produces a>250kDa neurofibromin protein that is capable of modulating Ras signaling. We created mutant cDNAs representing NF1 patient variants with different clinically relevant phenotypes, and assessed their ability to produce mature neurofibromin and restore Nf1 activity in NF1-/- cells. These cDNAs represent variants in multiple protein domains and various types of clinically relevant predicted variants. This approach will help advance research on neurofibromin structure and function, determine pathogenicity for missense variants, and allow for the development of activity assays and variant-directed therapeutics.

Overlap of SEC23A and SEC23B Function Suggests a Novel Therapeutic Approach for Congenital Dyserythropoietic Anemia Type II

Congenital Dyserythropoietic Anemia type II (CDAII) is an autosomal recessive disease characterized by anemia and increased bone marrow (BM) bi/multi-nucleated erythroblasts. CDAII results from loss of function mutations in SEC23B encoding a core component of coat complex protein II (COPII) vesicles, which transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Despite the identification of the genetic cause of CDAII, the pathophysiology of the disease remains unknown.Morpholino-induced SEC23B deficiency in zebrafish (ZF) has been previously reported to result in an erythroid phenotype mimicking CDAII (Shwartz et al, Nature genetics 2009), suggesting conservation of the underlying CDAII mechanism from fish to humans. Thus, we were puzzled to observe the absence of anemia or other CDAII characteristics in mice with erythroid specific (EpoR -Cre) and pan-hematopoietic (Vav1 -Cre) SEC23B deficiency (Khoriaty et al, MBC and Khoriaty et al, Sci Rep).To re-examine the ZF phenotype, we injected the morpholino targeting Sec23b into one-cell stage ZF embryos demonstrating no increase in circulating bi-nucleated erythroid cells, in contrast to the previous report. Given the variable knock-down that can result from morpholinos, we next generated ZF heterozygous for a 53 bp deletion (Sec23b+/-) using CRISPR/Cas9 genome editing. Intercrosses between Sec23b+/- ZF demonstrated lethality of Sec23b-/- ZF between days 17-21. However, the percentage of circulating bi-nucleated erythrocytes observed at day 16 was indistinguishable between Sec23b-/- ZF and wildtype (WT) clutch mate controls.Mammals and fish express two paralogs for SEC23, SEC23A and SEC23B, encoding highly related (~85%) proteins. To investigate the different functions of SEC23A and SEC23B, we defined the SEC23A and SEC23B interactomes using “BioID” (proximity dependent biotinylation) in HEK293 cells expressing BirA*-tagged SEC23A, SEC23B, or GFP control. Surprisingly, SEC23A and SEC23B exhibit indistinguishable interactomes. We also demonstrated that both mouse and human SEC23 paralogs can complement SEC23 deficiency in yeast. Similarly, rescue of the Sec23b-/- lethal phenotype in ZF by a Sec23a transgene demonstrated at least partial functional overlap of SEC23A/SEC23B function in vertebrates.To extend these observations to mammals, we genetically engineered the murine Sec23a cDNA into the endogenous mouse genomic locus of Sec23b . We demonstrated that SEC23B-deficient mice (previously shown to die perinatally from pancreatic degeneration) are rescued by SEC23A, exhibiting normal survival and pancreas histology, with no abnormalities apparent on detailed hematologic and anatomic examination.The expression of SEC23A and SEC23B mRNAs in human and mouse BMs were examined by qRT-PCR. SEC23B is the predominantly expressed paralog in human BM, with greater levels of SEC23A and reduced SEC23B in mouse BM. We therefore hypothesized that mice with erythroid deficiency of SEC23A alone or combined SEC23A/SEC23B deficiency might exhibit an erythroid defect. We first generated mice with erythroid-specific SEC23A deficiency, with the latter mice exhibiting no anemia or other CDAII characteristic. In contrast, mice with combined erythroid SEC23A and SEC23B deficiency die at ~E12.5, exhibiting reduced size and appear white in color compared to their WT litter mate controls, consistent with requirement of SEC23 in the erythroid compartment.Taken together, these data suggest complete (or near complete) overlap in function between SEC23A and SEC23B, and suggest that therapies that increase the expression of either SEC23 paralog might prove effective in treating CDAII. This paradigm might also apply to other disorders due to mutations in paralogous genes. Finally, our findings also suggest that a switch in paralog expression could account for other disparate disease phenotypes observed between animal models and humans. DisclosuresNo relevant conflicts of interest to declare.

Modular organization of a hypocretin gene minimal promoter.

Orexins or hypocretins are neurotransmitters produced by a small population of neurons in the lateral hypothalamus. This family of peptides modulates sleep-wake cycle, arousal and feeding behaviors; however, the mechanisms regulating their expression remain to be fully elucidated. There is an interest in defining the key molecular elements in orexin regulation, as these may serve to identify targets for generating novel therapies for sleep disorders, obesity and addiction. Our previous studies showed that the expression of orexin was decreased in mice carrying null-mutations of the transcription factor early B-cell factor 2 (ebf2) and that the promoter region of the prepro-orexin (Hcrt) gene contained two putative ebf-binding sites, termed olf-1 sites. In the present study, a minimal promoter region of the murine Hcrt gene was identified, which was able to drive the expression of a luciferase reporter gene in the human 293 cell line. Deletion of the olf1-site proximal to the transcription start site of the Hcrt gene increased reporter gene expression, whereas deletion of the distal olf1-like site decreased its expression. The lentiviral transduction of murine transcription factor ebf2 cDNA into 293 cells increased the gene expression driven by this minimal Hcrt-gene promoter and an electrophoretic mobility shift assays demonstrated that the distal olf1-like sequence was a binding site for ebf2.

Abnormal von Willebrand factor secretion, factor VIII stabilization and thrombus dynamics in type 2N von Willebrand disease mice

Background von Willebrand factor (VWF) and factor VIII (FVIII) circulate as a non-covalent complex, with VWF serving as the carrier for FVIII. VWF indirectly influences secondary hemostasis by stabilizing FVIII and transporting it to the site of primary hemostasis. Type 2N von Willebrand disease involves impaired binding of VWF to FVIII, resulting in decreased plasma levels of FVIII. Objectives In these studies, we characterize the impact of three type 2N VWD variants (R763A, R854Q, R816W) on VWF secretion, FVIII stabilization and thrombus formation in a murine model. Methods Type 2N VWD mice were generated by hydrodynamic injections of mutant murine VWF cDNAs and the influence of these variants on VWF secretion and FVIII binding was evaluated. In vivo hemostasis and the dynamics of thrombus formation and embolization were assessed using a murine tail vein transection hemostasis model and an intravital thrombosis model in the cremaster arterioles. Results Type 2N VWD variants were associated with decreased VWF secretion using cell and animal-based models. FVIII-binding to type 2N variants was impaired in vitro and was variably stabilized in vivo by expressed or infused 2N variant VWF protein. Both transgenic type 2N VWD and FVIII knockout (KO) mice demonstrated impaired thrombus formation associated with decreased thrombus stability. Conclusions The type 2N VWD phenotype can be recapitulated in a murine model and is associated with both quantitative and qualitative VWF deficiencies and impaired thrombus formation. Patients with type 2N VWD may have normal primary hemostasis formation but decreased thrombus stability related to ineffective secondary hemostasis.

Discovery and characterization of two novel CB1 receptor splice variants with modified N-termini in mouse.

Numerous studies have been carried out in the mouse model, investigating the role of the cannabinoid receptor type 1 (CB1). However, mouse CB1 (mCB1) receptor differs from human CB1 (hCB1) receptor in 13 amino acid residues. Two splice variants, hCB1a and hCB1b, diverging in their amino-termini, have been reported to be unique for hCB1 and, via different signaling properties, contribute to CB1 receptor physiology and pathophysiology. We hypothesized that splice variants also exist for the mCB1 receptor and have different signaling properties. On murine hippocampal cDNA, we identified two novel mCB1 receptor splice variants generated by splicing of introns with 117bp and 186bp in the N-terminal domain, corresponding to deletions of 39 or 62 amino acids, respectively. The mRNAs for the splice variants mCB1a and mCB1b are expressed at low levels in different brain regions. Western blot analysis of protein extracts from stably transfected HEK293 cells indicates a strongly reduced glycosylation because of the absence of two glycosylation sites in mCB1b. On-cell western analysis in these stable lines revealed increased internalization of mCB1a and mCB1b upon stimulation with the agonist WIN55,212-2 as compared to mCB1. Results also point toward an increased affinity to SR141716 for mCB1a, as well as slightly enhanced inhibition of neurotransmission compared to mCB1. In mCB1b, agonist-induced MAPK phosphorylation was decreased compared to mCB1 and mCB1a. Identification of mouse CB1 receptor splice variants may help to explain differences found between human and mouse endocannabinoid systems and improve the understanding of CB1 receptor signaling and trafficking in different species.

Low-Dose Gene Therapy for Murine PKU Using Episomal Naked DNA Vectors Expressing PAH from Its Endogenous Liver Promoter.

Limited duration of transgene expression, insertional mutagenesis, and size limitations for transgene cassettes pose challenges and risk factors for many gene therapy vectors. Here, we report on physiological expression of liver phenylalanine hydroxylase (PAH) by delivery of naked DNA/minicircle (MC)-based vectors for correction of homozygous enu2 mice, a model of human phenylketonuria (PKU). Because MC vectors lack a defined size limit, we constructed a MC vector expressing a codon-optimized murine Pah cDNA that includes a truncated intron and is under the transcriptional control of a 3.6-kb native Pah promoter/enhancer sequence. This vector, delivered via hydrodynamic injection, yielded therapeutic liver PAH activity and sustained correction of blood phenylalanine comparable to viral or synthetic liver promoters. Therapeutic efficacy was seen with vector copy numbers of <1 vector genome per diploid hepatocyte genome and was achieved at a vector dose that was significantly lowered. Partial hepatectomy and subsequent liver regeneration was associated with >95% loss of vector genomes and PAH activity in liver, demonstrating that MC vectors had not integrated into the liver genome. In conclusion, MC vectors, which do not have a defined size-limitation, offer a favorable safety profile for hepatic gene therapy due to their non-integration in combination with native promoters.

Expression of a recombinant full-length LRP1B receptor in human non-small cell lung cancer cells confirms the postulated growth-suppressing function of this large LDL receptor family member.

Low-density lipoprotein (LDL) receptor-related protein 1B (LRP1B), a member of the LDL receptor family, is frequently inactivated in multiple malignancies including lung cancer. LRP1B is therefore considered as a putative tumor suppressor. Due to its large size (4599 amino acids), until now only minireceptors or receptor fragments have been successfully cloned. To assess the effect of LRP1B on the proliferation of non-small cell lung cancer cells, we constructed and expressed a transfection vector containing the 13.800 bp full-length murine Lrp1b cDNA using a PCR-based cloning strategy. Expression of LRP1B was analyzed by quantitative RT-PCR (qRT-PCR) using primers specific for human LRP1B or mouse Lrp1b. Effective expression of the full length receptor was demonstrated by the appearance of a single 600 kDa band on Western Blots of HEK 293 cells. Overexpression of Lrp1b in non-small cell lung cancer cells with low or absent endogenous LRP1B expression significantly reduced cellular proliferation compared to empty vector-transfected control cells. Conversely, in Calu-1 cells, which express higher endogenous levels of the receptor, siRNA-mediated LRP1B knockdown significantly enhanced cellular proliferation. Taken together, these findings demonstrate that, consistent with the postulated tumor suppressor function, overexpression of full-length Lrp1b leads to impaired cellular proliferation, while LRP1B knockdown has the opposite effect. The recombinant Lrp1b construct represents a valuable tool to unravel the largely unknown physiological role of LRP1B and its potential functions in cancer pathogenesis.

434. Adoptive Treg Cell Therapy Using Factor VIII-Specific CAR Regulatory T Cells Regulates Anti-Factor VIII Immune Responses in Hemophilia A Mice

The immune response to factor VIII (FVIII; F8 in constructs) protein limits the effectiveness of treatments for hemophilia A (HemA) patients. Our previous studies demonstrated that regulatory T cells (Tregs) play a pivotal role in modulation of anti-FVIII immune responses. In particular, adoptive transfer of Tregs isolated from FVIII-primed HemA/Foxp3 mice attenuated anti-FVIII immune responses induced by gene transfer of FVIII plasmid in HemA mice. For developing adoptive Treg therapy, we successfully expanded FVIII-sensitized polyclonal Tregs using an FVIII-specific expansion protocol in vitro and showed that these cells had increased FVIII-specific suppressive activity compared with nonspecific Tregs. However, FVIII-specific Tregs in the polyclonal population are still in very small numbers. In this study, we explored the strategy to generate FVIII-specific Tregs using the chimeric antigen receptor (CAR) approach. Lentiviral vector (LV) incorporating a high-binding anti-FVIII antibody-derived variable region (scFv) linked to signaling and costimulatory moieties of immune receptors (third generation CAR) and fused with a murine Foxp3 cDNA (F8CAR-Foxp3-LV) was prepared and used to transduce murine CD4+T cells. Flow cytometry analysis confirmed extracellular scFv and intracellular Foxp3 expression in transduced cells (F8CAR-Tregs). In vitro suppressive assay showed that transduced CD4+T cells had significantly higher FVIII-specific suppressive activity than untransduced cells towards FVIII-specific CD4+ effector T cells (Teffs). In addition, 1×106 transduced cells and untransduced cells were adoptively transferred into HemA mice. One day after cell transfer, the treated mice were challenged with FVIII plasmid injected hydrodynamically. The anti-FVIII antibody titers are evaluated overtime. It is expected that F8CAR-Foxp3-LV transduced cells will prevent or decrease the production of anti-FVIII antibodies. We have also prepared a LV incorporating only the F8CAR region (F8CAR-LV). CD4+CD25− and CD4+CD25+ cells isolated from HemA mice were transduced with F8CAR-LV to generate FVIII-specific Teffs and Tregs, respectively. In vitro FVIII-specific suppressive assays using CD4+ Teffs from FVIII-primed HemA mice or F8CAR-LV transduced Teffs as responder cells are performed to compare the suppressive function of F8CAR-LV transduced CD4+CD25+ cells and F8CAR-Foxp3-LV transduced CD4+ cells. CD4+CD25+ cells isolated from FVIII-primed HemA mice and untransduced CD4+CD25+ and CD4+ cells from HemA mice are used as control cells. These experiments compare the extent of suppression towards specific F8CAR Teffs and polyclonal FVIII-specific Teffs as well as the potency of suppressive function between two differently engineered F8CAR Tregs. Finally, adoptive transfer experiments into HemA mice using the transduced and control cell populations are performed to evaluate their in vivo function to protect the HemA mice from anti-FVIII antibody production. We anticipate that compared with nonspecific and polyclonally expanded Tregs, FVIII-specific CAR Tregs will exert superior suppressive activity towards anti-FVIII immune responses without triggering systemic immune suppression.

355. Minicircles Show Improved Hepatic Expression of Their Transgene from a Natural Endogenous Promoter and Are Lost Upon Partial Hepatectomy Due to the Episomal Nature of the Vector

We have previously showed the successful treatment of phenylketonuria (PKU) in the PKU mouse model (C57Bl/6-Pahenu2) by using non-viral naked DNA minicircle (MC) vectors, which are devoid of any viral or bacterial sequences, upon liver-directed phenylalanine hydroxylase (Pah) gene transfer via hydrodynamic vein injection (Viecelli et al., Hepatology 2014). Here we improved the efficacy of transgene expression and investigated the fate of MC vectors in mouse liver. We found that the therapeutic doses of MCs could be significantly lowered by using a codon-optimized murine Pah cDNA in combination with a truncated 5’-intron. Moreover, when using the natural or endogenous 3.6 kb murine Pah-promoter to drive the Pah transgene, vector doses could again be lowered compared to two other “minimal” liver-specific promoters, a synthetic hybrid enhancer/promoter (P3) or the classical CBA (modified cytomegalovirus enhancer/chicken β-actin) promoter. Following 70% partial hepatectomy, MC-vector-treated PKU mice showed normal liver regeneration and blood L-Phe concentration increased to pretreatment levels, indicating that treated PKU mice had lost therapeutic MC vectors during liver regeneration. These results corroborate previous observations that MC-DNA do not integrate - or only at a very low frequency that cannot be detected by our assays - and thus express their transgene as episomal vectors. In conclusion, MC-vectors which do not have a defined size-limitation, offer a favorable safety profile due to their non-integrating behavior in combination with a (large) natural or endogenous promoter, and at the same time have the potential for long-term gene-therapy of liver defects.

766. Genetic Correction of Ifnγr1 Deficiency in Hematopoietic Cells Repairs the Cellular Phenotype of Mendelian Susceptibility to Mycobacterial Disease

Mendelian Susceptibility to Mycobacterial Disease (MSMD) is a rare primary immunodeficiency characterized by recurrent severe infections with otherwise only weakly virulent mycobacteria. MSMD is associated with mutations in different genes, all leading to an impaired activation of macrophages by T-cells and a defective innate immune response. Specifically mutations in the interferon-gamma (IFNγ)-receptor-1 or-2 (IFNγR1 /2) genes result in a life-threatening disease phenotype with most patients dying in early childhood. We here introduce a novel gene therapy approach for IFNγR1 deficiency. Thus, we have designed a 3rd generation SIN lentiviral vector expressing the murine cDNA of Ifnγr1 from a spleen focus forming virus (SFFV) promoter element coupled by an IRES to GFP (Lv.SFFV.Ifnyr1.iGFP). Transduction of hematopoietic stem/progenitor cells from Ifnγrl−/− mice showed expression of Ifnγr1 by FACS and qRT-PCR and no abnormalities in clonogenic growth when compared to WT control cells. Moreover, differentiation of transduced cells towards macrophages (MΦ) by M-CSF was normal as determined by morphology on cytospins and surface marker expression of CD11b, CD200R, CD115, CD45 and F4/80. When subjecting corrected MΦ to stimulation with IFNγ, corrected cells were able to build a functional IFNγR1-Ifnγ-IFNγR2 and internalize IFNγ as efficiently as WT cells, as suggested by the clearance of IFNγ from the medium within 24h of stimulation. Consequently, Lv. SFFV. Ifnγr1.iGFP corrected MΦ revealed restored up-regulation of HLA-DR and CD86 (B7.2) comparable to WT-MΦ. IFNγ dependent T-cell activation was evaluated using T cell receptor- transgenic T cells recognizing ovalbumin (OVA). Both WT and corrected MΦ were unable to activate T-cells in the presence of IFNγ and OVA, whereas MΦ from Ifnγr1−/− mice induced significantly stronger proliferation of T cells. This observation was accompanied by the induction of indoleamine 2,3-dioxygenase (IDO) in WT and corrected MΦ, suggesting that IDO enables MΦ to deplete tryptophan from the medium and interferes with T-cell proliferation. Finally, Ifnγrl downstream signaling showed restored phosphorylation of STAT1 in corrected MΦ, consistent with induction of iNos and Irf1 upon stimulation with IFNγ. Moreover, correction of Ifnγrl−/− MΦ led to a significantly improved anti-mycobacterial activity as measured by comparable killing of Mycobacterium Avium in corrected and WT MΦ. Thus, we here introduce a novel gene therapy approach for MSMD in the context of Ifnγr1 deficiency.