Dissection of pleiotropic effects of variants in and adjacent to F8 exon 19 and rescue of mRNA splicing and protein function.

The pathogenic significance of nucleotide variants commonly relies on nucleotide position within the gene, with exonic changes generally attributed to quantitative or qualitative alteration of protein biosynthesis, secretion, activity, or clearance. However, these changes may exert pleiotropic effects on both protein biology and mRNA splicing due to the overlapping of the amino acid and splicing codes, thus shaping the disease phenotypes. Here, we focused on hemophilia A, in which the definition of F8 variants' causative role and association to bleeding phenotypes is crucial for proper classification, genetic counseling, and management of affected individuals. We extensively characterized a large panel of hemophilia A-causing variants (n = 30) within F8 exon 19 by combining and comparing in silico and recombinant expression analyses. We identified exonic variants with pleiotropic effects and dissected the altered protein features of all missense changes. Importantly, results from multiple prediction algorithms provided qualitative results, while recombinant assays allowed us to correctly infer the likely phenotype severity for 90% of variants. Molecular characterization of pathogenic variants was also instrumental for the development of tailored correction approaches to rescue splicing affecting variants or missense changes impairing protein folding. A single engineered U1snRNA rescued mRNA splicing of nine different variants and the use of a chaperone-like drug resulted in improved factor VIII protein secretion for four missense variants. Overall, dissection of the molecular mechanisms of a large panel of HA variants allowed precise classification of HA-affected individuals and favored the development of personalized therapeutic approaches.

Human myofibroblasts increase the arrhythmogenic potential of human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have the potential to remuscularize infarcted hearts but their arrhythmogenicity remains an obstacle to safe transplantation. Myofibroblasts are the predominant cell-type in the infarcted myocardium but their impact on transplanted hiPSC-CMs remains poorly defined. Here, we investigate the effect of myofibroblasts on hiPSC-CMs electrophysiology and Ca2+ handling using optical mapping of advanced human cell coculture systems mimicking cell-cell interaction modalities. Human myofibroblasts altered the electrophysiology and Ca2+ handling of hiPSC-CMs and downregulated mRNAs encoding voltage channels (KV4.3, KV11.1 and Kir6.2) and SERCA2a calcium pump. Interleukin-6 was elevated in the presence of myofibroblasts and direct stimulation of hiPSC-CMs with exogenous interleukin-6 recapitulated the paracrine effects of myofibroblasts. Blocking interleukin-6 reduced the effects of myofibroblasts only in the absence of physical contact between cell-types. Myofibroblast-specific connexin43 knockdown reduced functional changes in contact cocultures only when combined with interleukin-6 blockade. This provides the first in-depth investigation into how human myofibroblasts modulate hiPSC-CMs function, identifying interleukin-6 and connexin43 as paracrine- and contact-mediators respectively, and highlighting their potential as targets for reducing arrhythmic risk in cardiac cell therapy.

Adenovirus serotype 5 hexon mediates liver gene transfer.

Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.

Peri-operative thrombophilia in patients undergoing liver resection for colorectal metastases.

BACKGROUND: Routine chemical venous thromboembolism (VTE) prophylaxis for liver surgery remains controversial, and often delayed post-operatively due to perceived bleeding risk. This study asked whether patients undergoing hepatectomy for colorectal metastases (CRM) were at risk from VTE pre-operatively, and the impact of hepatectomy on that risk. METHODS: Single-centre prospective observational cohort study of patients undergoing open hepatectomy for CRM, comparing pre-, peri- and post-operative haemostatic variables. RESULTS: Of 336 hepatectomies performed October 2017-December 2019, 60 resections in 57 patients were recruited. There were 28 (46.7%) major resections, with median (interquartile range [IQR]) blood loss 150.0 (76.3-263.7) mls, no blood transfusions, post-operative VTE events or deaths. Patients were prothrombotic pre-operatively (high median factor VIIIC and increased thrombin generation velocity index), an effect exacerbated post-hepatectomy. Major hepatectomies had a significantly greater median drop in Protein C, rise in Factor VIIIC and von Willebrand Factor, versus minor resections (p = 0.001, 0.005, 0.001 respectively). Patients with parenchymal transection times greater than median (40 min), had significantly increased median (IQR) PMBC-TFmRNA expression [1.65(0.93-2.70)2ddCt], versus quicker transections [0.99(0.69-1.28)2ddCt, p = 0.020]. CONCLUSIONS: Patients with CRM are prothrombotic pre-operatively, an effect exacerbated by hepatectomy, particularly longer, complex resections, suggesting chemical thromboprophylaxis be considered early in the patient pathway.

The EAHAD blood coagulation factor VII variant database.

Hereditary blood coagulation factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder resulting from variants in the gene encoding FVII (F7). Integration of genetic variation with functional consequences on protein function is essential for the interpretation of the pathogenicity of novel variants. Here, we describe the integration of previous locus-specific databases for F7 into a single curated database with enhanced features. The database provides access to in silico analyses that may be useful in the prediction of variant pathogenicity as well as cross-species sequence alignments, structural information, and functional and clinical severity described for each variant, where appropriate. The variant data is shared with the F7 Leiden Open Variation Database. The updated database now includes 221 unique variants, representing gene variants identified in 728 individuals. Single nucleotide variants are the most common type (88%) with missense representing 74% of these variants. A number of variants are found with relatively high minor allele frequencies that are not pathogenic but contribute significantly to the likely pathogenicity of coinherited variants due to their effect on FVII plasma levels. This comprehensive collection of curated information significantly aids the assessment of pathogenicity.

The European Association for Haemophilia and Allied Disorders (EAHAD) Coagulation Factor Variant Databases: Important resources for haemostasis clinicians and researchers.

INTRODUCTION: Advances in genomic sequencing have facilitated the sequencing of genes associated with disorders of haemostasis. The identification of variants within genes and access to curated data incorporating structural, functional, evolutionary as well as phenotypic data has become increasingly important in order to ascribe pathogenicity. AIM: The European Association for Haemophilia and Allied Disorders (EAHAD) Coagulation Factor Variant Database Project aims to provide a single port of entry to a web-accessible resource for variants in genes involved in clinical bleeding disorders. RESULTS: New databases have evolved from previously developed single gene variant coagulation database projects, incorporating new data, new analysis tools and a new common database architecture with new interfaces and filters. These new databases currently present information about the genotype, phenotype (laboratory and clinical) and structural and functional effects of variants described in the genes of factor (F) VII (F7), FVIII (F8), FIX (F9) and von Willebrand factor (VWF). CONCLUSION: The project has improved the quality and quantity of information available to the haemostasis research and clinical communities, thereby enabling accurate classification of disease severity in order to make assessments of likely pathogenicity.

Clustered F8 missense mutations cause hemophilia A by combined alteration of splicing and protein biosynthesis and activity.

Dissection of pleiotropic effects of missense mutations, rarely investigated in inherited diseases, is fundamental to understanding genotype-phenotype relationships. Missense mutations might impair mRNA processing in addition to protein properties. As a model for hemophilia A, we investigated the highly prevalent F8 c.6046c>t/p.R2016W (exon 19) mutation. In expression studies exploiting lentiviral vectors, we demonstrated that the amino acid change impairs both Factor VIII (FVIII) secretion (antigen 11.0±0.4% of wild-type) and activity (6.0±2.9%). Investigations in patients' ectopic F8 mRNA and with minigenes showed that the corresponding nucleotide change also decreases correct splicing to 70±5%, which is predicted to lower further FVIII activity (4.2±2%), consistently with patients' levels (<1-5%). Masking the mutated exon 19 region by antisense U7snRNA supported the presence of a splicing regulatory element, potentially affected by several missense mutations causing hemophilia A. Among these, the c.6037g>a (p.G2013R) reduced exon inclusion to 41±3% and the c.6053a>g (p.E2018G) to 28±2%, similarly to a variant affecting the 5' splice site (c.6113a>g, p.N2038S, 26±2%), which displayed normal protein features upon recombinant expression. The p.G2013R reduced both antigen (7.0±0.9%) and activity (8.4±0.8%), while the p.E2018G produced a dysfunctional molecule (antigen: 69.0±18.1%; activity: 19.4±2.3%). In conclusion, differentially altered mRNA and protein patterns produce a gradient of residual activity, and clarify genotype-phenotype relationships. Data detail pathogenic mechanisms that, only in combination, account for moderate/severe disease forms, which in turn determine the mutation profile. Taken together we provide a clear example of interplay between mRNA and protein mechanisms of disease that operate in shaping many other inherited disorders.

Manipulating adenovirus hexon hypervariable loops dictates immune neutralisation and coagulation factor X-dependent cell interaction in vitro and in vivo.

Adenoviruses are common pathogens, mostly targeting ocular, gastrointestinal and respiratory cells, but in some cases infection disseminates, presenting in severe clinical outcomes. Upon dissemination and contact with blood, coagulation factor X (FX) interacts directly with the adenovirus type 5 (Ad5) hexon. FX can act as a bridge to bind heparan sulphate proteoglycans, leading to substantial Ad5 hepatocyte uptake. FX "coating" also protects the virus from host IgM and complement-mediated neutralisation. However, the contribution of FX in determining Ad liver transduction whilst simultaneously shielding the virus from immune attack remains unclear. In this study, we demonstrate that the FX protection mechanism is not conserved amongst Ad types, and identify the hexon hypervariable regions (HVR) of Ad5 as the capsid proteins targeted by this host defense pathway. Using genetic and pharmacological approaches, we manipulate Ad5 HVR interactions to interrogate the interplay between viral cell transduction and immune neutralisation. We show that FX and inhibitory serum components can co-compete and virus neutralisation is influenced by both the location and extent of modifications to the Ad5 HVRs. We engineered Ad5-derived HVRs into the rare, native non FX-binding Ad26 to create Ad26.HVR5C. This enabled the virus to interact with FX at high affinity, as quantified by surface plasmon resonance, FX-mediated cell binding and transduction assays. Concomitantly, Ad26.HVR5C was also sensitised to immune attack in the absence of FX, a direct consequence of the engineered HVRs from Ad5. In both immune competent and deficient animals, Ad26.HVR5C hepatic gene transfer was mediated by FX following intravenous delivery. This study gives mechanistic insight into the pivotal role of the Ad5 HVRs in conferring sensitivity to virus neutralisation by IgM and classical complement-mediated attack. Furthermore, through this gain-of-function approach we demonstrate the dual functionality of FX in protecting Ad26.HVR5C against innate immune factors whilst determining liver targeting.

The role of the tissue factor pathway in haemostasis and beyond.

PURPOSE OF REVIEW: The role of tissue factor (TF) in the initiation of the blood coagulation network leading to generation of a fibrin clot has been well defined over the past 50 years. Although much is known about this sequence of events and its regulation, many important questions remain unresolved. More recently, a complex role for TF in cellular processes independent of fibrin generation has emerged. This review summarizes some of the advances in this field. RECENT FINDINGS: TF is the cellular receptor and cofactor for factor VII/VIIa; however, controversy still surrounds expression of TF within the vasculature, the role of circulating microvesicle pools of TF and mechanisms of 'encryption' of TF activity. However, there have been significant advances in the role of TF-initiated cell signalling. Lastly, an alternatively spliced TF transcript has been identified and some insights into its role in cancer cell metastasis/proliferation have been elucidated. SUMMARY: Understanding of TF structure function has increased substantially; however, multiple controversies still surround some aspects of its regulation. TF has emerged as a pivotal player in orchestrating not only fibrin generation but wound repair. Derangement of these repair processes contributes significantly to the pathophysiology of a number of disease processes.

Expression of human tissue factor pathway inhibitor on vascular smooth muscle cells inhibits secretion of macrophage migration inhibitory factor and attenuates atherosclerosis in ApoE-/- mice.

BACKGROUND: Tissue factor (TF) and coagulation proteases are involved in promoting atherosclerosis, but the molecular and cellular bases for their involvement are unknown. METHODS AND RESULTS: We generated a new strain (ApX4) of apolipoprotein E-deficient mice expressing a membrane-tethered human tissue factor pathway inhibitor fusion protein on smooth muscle actin-positive cells, including vascular smooth muscle cells (SMCs). ApX4 mice developed little atherosclerosis on either a normal chow or high-fat diet. Lipid levels were similar to those in parental ApoE(-/-) mice, and there was no detectable difference in systemic (circulating) tissue factor pathway inhibitor levels or activity. The small lipid-rich lesions that developed had markedly reduced leukocyte infiltrates, and in contrast to ApoE(-/-) mice, SMCs did not express macrophage migratory inhibitory factor (MIF), including at sites distant from atheromatous lesions. Low levels of circulating MIF in ApX4 mice normalized to levels seen in ApoE(-/-) mice after injection of an inhibitory anti-human tissue factor pathway inhibitor antibody, which also led to MIF expression by tissue factor-positive medial SMCs. MIF production by SMCs in ApoE(-/-) mice in vitro and in vivo was shown to be dependent on tissue factor and protease-activated receptor signaling, which were inhibited in ApX4 mice. CONCLUSIONS: Our data indicate that tissue factor plays a hitherto unreported role in the generation of MIF by SMCs in atherosclerosis-prone ApoE(-/-) mice, inhibition of which significantly prevents the development of atherosclerosis, through inhibition of leukocyte recruitment. These data significantly enhance our understanding of the pathophysiology of this important pathology and suggest new potential translational strategies to prevent atheroma formation.

Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant.

Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAV-expression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon-optimized hFVIII cDNA in which a 226 amino acid (aa) B-domain spacer replaced the entire B domain and a hybrid liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared with non-codon-optimized variants. A further twofold improvement in potency was achieved by replacing the 226-aa N6 spacer with a novel 17-aa peptide (V3) in which 6 glycosylation triplets from the B domain were juxtaposed. The resulting 5.2-kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732 ± 162% of normal) in HA knock-out mice following administration of 2 × 10(12) vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15 ± 4% of normal was observed at this dose in a nonhuman primate. hFVIII expression above 100% was observed in 3 macaques that received a higher dose of either this vector or the N6 variant. These animals developed neutralizing anti-FVIII antibodies that were abrogated with transient immunosuppression. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy.

Hyperactive piggyBac transposons for sustained and robust liver-targeted gene therapy.

The development of robust nonviral vectors could facilitate clinical gene therapy applications and may overcome some of the immune complications of viral vectors. Nevertheless, most nonviral gene deliver approaches typically yield only transient and/or low gene expression. To address these caveats, we have explored piggyBac transposons to correct hemophilia B by liver-directed factor IX (FIX) gene therapy in hemophilic mice. To achieve this, we combined the use of: (i) a hyperactive codon-optimized piggyBac transposase, (ii) a computationally enhanced liver-specific promoter, (iii) a hyperfunctional codon-optimized FIX transgene (FIX R338L Padua), and (iv) a modification of the transposon terminal repeats. This combination strategy resulted in a robust 400-fold improvement in vector performance in hepatocytes, yielding stable supraphysiologic human FIX activity (>1 year). Liver-specific expression resulted in the induction of FIX-specific immune tolerance. Remarkably, only very low transposon/transposase doses were required to cure the bleeding diathesis. Similarly, PB transposons could be used to express supraphysiologic factor VIII levels using low transposon/transposase doses. PB transposition did not induce tumors in a sensitive hepatocellular carcinoma-prone mouse model. These results underscore the potency and relative safety of the latest generation PB transposons, which constitutes a versatile platform for stable and robust secretion of therapeutic proteins.

AAV-mediated gene transfer in the perinatal period results in expression of FVII at levels that protect against fatal spontaneous hemorrhage.

We explored adeno-associated viral vector (AAV)-mediated gene transfer in the perinatal period in animal models of severe congenital factor VII (FVII) deficiency, a disease associated with early postnatal life-threatening hemorrhage. In young adult mice with plasma FVII < 1% of normal, a single tail vein administration of AAV (1 × 10(13) vector genomes [vg]/kg) resulted in expression of murine FVII at 266% ± 34% of normal for ≥ 67 days, which mediated protection against fatal hemorrhage and significantly improved survival. Codon optimization of human FVII (hFVIIcoop) improved AAV transgene expression by 37-fold compared with the wild-type hFVII cDNA. In adult macaques, a single peripheral vein injection of 2 × 10(11) vg/kg of the hFVIIcoop AAV vector resulted in therapeutic levels of hFVII expression that were equivalent in males (10.7% ± 3.1%) and females (12.3% ± 0.8%). In utero delivery of this vector in the third trimester to fetal monkeys conferred expression of hFVII at birth of 20.4% ± 3.7%, with a gradual decline to > 1% by 7 weeks. Re-administration of an alternative serotype at 12 months postnatal age increased hFVII levels to 165% ± 6.2% of normal, which remained at therapeutic levels for a further 28 weeks without toxicity. Thus, perinatal AAV-mediated gene transfer shows promise for disorders with onset of pathology early after birth.

Neointimal hyperplasia after endoluminal injury in mice is dependent on tissue factor- and angiopoietin-2 dependent interferon gamma production by fibrocytes and macrophages.

Background: The intimal hyperplasia (IH) and vascular remodelling that follows endovascular injury, for instance after post-angioplasty re-stenosis, results in downstream ischaemia and progressive end organ damage. Interferon gamma (IFNγ) is known to play a critical role in this process. In mouse models we have previously shown that fibrocytes expressing tissue factor (TF) are recruited early to the site of injury. Through thrombin generation and protease activated receptor-1 (PAR-1) activation, fibrocytes secrete angiopoietin-2, stimulate neointimal cell proliferation, inhibit apoptosis and induce CXCL-12 production, all of which contribute to the progressive IH that then develops. In this study we investigated the relationship between TF, angiopoietin-2 and IFNγ. Methods and results: IH developing in carotid arteries of wild-type mice 4 weeks after endoluminal injury contained a significant proportion of IFNγ+ fibrocytes and macrophages, which we show, using a previously defined adoptive transfer model, were derived from circulating CD34+ cells. IH did not develop after injury in IFNγ-deficient mice, except after transplantation of WT bone marrow or adoptive transfer of WT CD34+ cells. In vitro, CD34+ cells isolated from post-injury mice did not express IFNγ, but this was induced when provided with FVIIa and FX, and enhanced when prothrombin was also provided: In both cases IFNγ secretion was TF-dependent and mediated mainly through protease activated PAR-1. IFNγ was predominantly expressed by fibrocytes. In vivo, all IFNγ+ neointimal cells in WT mice co-expressed angiopoietin-2, as did the small numbers of neointimal cells recruited in IFNγ-/- mice. Adoptively transferred WT CD34+ cells treated with either an anti-TIE-2 antibody, or with siRNA against angiopoetin-2 inhibited the expression of IFNγ and the development of IH. Conclusion: TF-dependent angiopoietin-2 production by newly recruited fibrocytes, and to a lesser extent macrophages, switches on IFNγ expression, and this is necessary for the IH to develop. These novel findings enhance our understanding of the pathophysiology of IH and expose potential targets for therapeutic intervention.

Codon optimization of human factor VIII cDNAs leads to high-level expression.

Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

Inhibition of thrombin receptor signaling on α-smooth muscle actin(+) CD34(+) progenitors leads to repair after murine immune vascular injury.

OBJECTIVE: The goal of this study was to use mice expressing human tissue factor pathway inhibitor (TFPI) on α-smooth muscle actin (α-SMA)(+) cells as recipients of allogeneic aortas to gain insights into the cellular mechanisms of intimal hyperplasia (IH). METHODS AND RESULTS: BALB/c aortas (H-2(d)) transplanted into α-TFPI-transgenic (Tg) mice (H-2(b)) regenerated a quiescent endothelium in contrast to progressive IH seen in C57BL/6 wild-type (WT) mice even though both developed aggressive anti-H-2(d) alloresponses, indicating similar vascular injuries. Adoptively transferred Tg CD34(+) (but not CD34(-)) cells inhibited IH in WT recipients, indicating the phenotype of α-TFPI-Tg mice was due to these cells. Compared with syngeneic controls, endogenous CD34(+) cells were mobilized in significant numbers after allogeneic transplantation, the majority showing sustained expression of tissue factor and protease-activated receptor-1 (PAR-1). In WT, most were CD45(+) myeloid progenitors coexpressing CD31, vascular endothelial growth factor receptor-2 and E-selectin; 10% of these cells coexpressed α-SMA and were recruited to the neointima. In contrast, the α-SMA(+) human TFPI(+) CD34(+) cells recruited in Tg recipients were from a CD45(-) lineage. WT CD34(+) cells incubated with a PAR-1 antagonist or taken from PAR-1-deficient mice inhibited IH as Tg cells did. CONCLUSIONS: Specific inhibition of thrombin generation or PAR-1 signaling on α-SMA(+) CD34(+) cells inhibits IH and promotes regenerative repair despite ongoing immune-mediated damage.

Bleeding due to disruption of a cargo-specific ER-to-Golgi transport complex.

Mutations in LMAN1 (also called ERGIC-53) result in combined deficiency of factor V and factor VIII (F5F8D), an autosomal recessive bleeding disorder characterized by coordinate reduction of both clotting proteins. LMAN1 is a mannose-binding type 1 transmembrane protein localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC; refs. 2,3), suggesting that F5F8D could result from a defect in secretion of factor V and factor VIII (ref. 4). Correctly folded proteins destined for secretion are packaged in the ER into COPII-coated vesicles, which subsequently fuse to form the ERGIC. Secretion of certain abundant proteins suggests a default pathway requiring no export signals (bulk flow; refs. 6,7). An alternative mechanism involves selective packaging of secreted proteins with the help of specific cargo receptors. The latter model would be consistent with mutations in LMAN1 causing a selective block to export of factor V and factor VIII. But approximately 30% of individuals with F5F8D have normal levels of LMAN1, suggesting that mutations in another gene may also be associated with F5F8D. Here we show that inactivating mutations in MCFD2 cause F5F8D with a phenotype indistinguishable from that caused by mutations in LMAN1. MCFD2 is localized to the ERGIC through a direct, calcium-dependent interaction with LMAN1. These findings suggest that the MCFD2-LMAN1 complex forms a specific cargo receptor for the ER-to-Golgi transport of selected proteins.

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor.

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users' operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

A cluster of basic amino acids in the factor X serine protease mediates surface attachment of adenovirus/FX complexes.

Hepatocyte transduction following intravenous administration of adenovirus 5 (Ad5) is mediated by interaction between coagulation factor X (FX) and the hexon. The FX serine protease (SP) domain tethers the Ad5/FX complex to hepatocytes through binding heparan sulfate proteoglycans (HSPGs). Here, we identify the critical HSPG-interacting residues of FX. We generated an FX mutant by modifying seven residues in the SP domain. Surface plasmon resonance demonstrated that mutations did not affect binding to Ad5. FX-mediated, HSPG-associated cell binding and transduction were abolished. A cluster of basic amino acids in the SP domain therefore mediates surface interaction of the Ad/FX complex.

Biodistribution and retargeting of FX-binding ablated adenovirus serotype 5 vectors.

A major limitation for adenoviral transduction in vivo is the profound liver tropism of adenovirus type 5 (Ad5). Recently, we demonstrated that coagulation factor X (FX) binds to Ad5-hexon protein at high affinity to mediate hepatocyte transduction after intravascular delivery. We developed novel genetically FX-binding ablated Ad5 vectors with lower liver transduction. Here, we demonstrate that FX-binding ablated Ad5 predominantly localize to the liver and spleen 1 hour after injection; however, they had highly reduced liver transduction in both control and macrophage-depleted mice compared with Ad5. At high doses in macrophage-depleted mice, FX-binding ablated vectors transduced the spleen more efficiently than Ad5. Immunohistochemical studies demonstrated transgene colocalization with CD11c(+), ER-TR7(+), and MAdCAM-1(+) cells in the splenic marginal zone. Systemic inflammatory profiles were broadly similar between FX-binding ablated Ad5 and Ad5 at low and intermediate doses, although higher levels of several inflammatory proteins were observed at the highest dose of FX-binding ablated Ad5. Subsequently, we generated a FX-binding ablated virus containing a high affinity Ad35 fiber that mediated a significant improvement in lung/liver ratio in macrophage-depleted CD46(+) mice compared with controls. Therefore, this study documents the biodistribution and reports the retargeting capacity of FX binding-ablated Ad5 vectors in vitro and in vivo.