Factor VIII Expression Research Articles

Potential Long-Term Treatment of Hemophilia A by Neonatal Co-Transplantation of Cord Blood Derived Endothelial Colony-Forming Cells and Placental Mesenchymal Stromal Cells

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the Factor VIII (FVIII) gene leading to deficient blood coagulation. As a monogenic disorder, HA is an ideal target for cellbased gene therapy, but successful treatment has been hampered by insufficient engraftment of potential therapeutic cells. In this study, we sought to determine whether co-transplantation of endothelial colonyforming cells (ECFCs) and placenta-derived mesenchymal stromal cells (PMSCs) can achieve long-term engraftment and FVIII expression. ECFCs and PMSCs were transduced with a B domain deleted factor VIII (BDD-FVIII) expressing lentiviral vector and luciferase, green fluorescent protein or Td-Tomato containing lentiviral tracking vectors. They were transplanted intramuscularly into neonatal or adult immunodeficient mice. In vivo bioluminescence imaging showed that the ECFC only and the cotransplantation groups but not the PMSCs only group achieved long-term engraftment for at least 26 weeks, and the co-transplantation group showed a higher engraftment than the ECFC only group at 16 and 20 weeks of age. In addition, cell transplantation at the neonatal age achieved higher engraftment than at the adult age. Immunohistochemical analyses further showed that the engrafted ECFCs expressed FVIII, maintained endothelial phenotype and generated functional vasculature. Next, cotransplantation of ECFCs and PMSCs into F8 knock-out HA mice reduced the blood loss volume from 562.13±19.84µl to 155.78±44.93µl in a tail-clip assay. This work demonstrated that co-transplantation of ECFCs with PMSCs at the neonatal age is a potential strategy to achieve stable, long-term engraftment, and thus holds great promise for cell-based treatment of HA. Funding Statement: This work was in part supported by UC Davis Department of Surgery departmental fund, and the UC Davis Medical Center Interdepartmental Seed Grant. Declaration of Interests: The authors declare: None. Ethics Approval Statement: All animal procedures were approved by The University of California, Davis (UCD) institutional animal care and use committee (IACUC). All facilities used during the study period were accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

Gene Therapy with BMN 270 Results in Therapeutic Levels of FVIII in Mice and Primates and Normalization of Bleeding in Hemophilic Mice.

Hemophilia A is an X-linked bleeding disorder caused by mutations in the gene encoding the factor VIII (FVIII) coagulation protein. Bleeding episodes in patients are reduced by prophylactic therapy or treated acutely using recombinant or plasma-derived FVIII. We have made an adeno-associated virus 5 vector containing a B domain-deleted (BDD) FVIII gene (BMN 270) with a liver-specific promoter. BMN 270 injected into hemophilic mice resulted in a dose-dependent expression of BDD FVIII protein and a corresponding correction of bleeding time and blood loss. At the highest dose tested, complete correction was achieved. Similar corrections in bleeding were observed at approximately the same plasma levels of FVIII protein produced either endogenously by BMN 270 or following exogenous administration of recombinant BDD FVIII. No evidence of liver dysfunction or hepatocyte endoplasmic reticulum stress was observed. Comparable doses in primates produced similar levels of circulating FVIII. These preclinical data support evaluation of BMN 270 in hemophilia A patients.

Invitro expression of mutant factor VII proteins and characterization of their clinical significance.

FactorVII (FVII) serves an essential role in the initiation of blood coagulation. Mutations in conserved residues within its serine protease domain may lead to dysregulated coagulation activity. The objective of the present study was to elucidate the impact of altering two conserved residues, H348R andS282R, on the functional properties of the FVII protein. The mutation‑harboring fragments were derived from genomic DNA of a FVII deficient patient. The fragments were integrated into a pcDNA vector containing FVII cDNA of HepG2 cells. The wild-type and mutated FVII constructs were transfected into CHO‑K1 cells as a mammalian cell model. The coagulation activity, antigen levels and intracellular localization of the recombinant proteins were studied in association with their pathological importance. Results indicated that FVII activity was not detectable in conditioned media of the cells transfected with the mutated constructs. The H348R mutation reduced the expression of intracellular and secreted forms of the FVII protein. Following S282R transfection, intracellular FVII expression showed no significant variation; however, extracellular protein was reduced. The pattern of intracellular localization of mutated FVII remained unaltered in comparison to the wild-type protein. Inconclusion, the present study suggested that missense mutations within the serine protease domain of FVII affect extracellular levels in addition to the coagulation activity of FVII. These results may contribute to further understanding of the molecular pathogenesis of FVII deficiency and the development of pharmaceutical candidates with improved therapeutic properties.

A Retrospective Study of the Cytokine Profile Changes in Mice with FVIII Inhibitor Development After Adeno-Associated Virus-Mediated Gene Therapy in a Hemophilia A Mouse Model.

The development of inhibitory autoantibodies to the infused clotting factor VIII (FVIII) is a major complication for severe hemophilia A management. Novel therapy options for hemophilia have significantly progressed in the last decade, and a gene therapy cure for hemophilia is becoming a reality. However, mechanistic studies of FVIII autoantibodies (FVIII inhibitors) have lagged behind and remain a challenge for both protein replacement and gene therapy. FVIII inhibitor formation is assumed to be a classical T cell-dependent immune response in which cytokines/chemokines play an important role. The study of cytokine profile changes during FVIII inhibitor development may be helpful to understand the mechanism of inhibitor development and to explore potential novel approaches that will minimize the risk. After FVIII-/- mice were treated with intravenous administration of an adeno-associated virus 8 vector encoding human FVIII, FVIII expression peaked at week 2 (W2), and FVIII inhibitor was thoroughly developed at week 8 (W8). W8 plasma that showed positive FVIII inhibitor, and W2 samples with negative FVIII inhibitor (anti-FVIII[+]), were subjected to multiplex cytokines measurement. W8 and W2 samples were both negative for FVIII inhibitor (anti-FVIII[-]) as the control. In comparison to mice in the anti-FVIII(-) group, mice in the anti-FVIII(+) group exhibited significantly elevated pro-inflammatory cytokines of interleukin (IL)-1, IL-6, IL-12p40, monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1, MIP-2, and tumor necrosis factor alpha (TNF-α), especially at higher titers. The anti-inflammatory cytokine of transforming growth factor beta (TGF-β) was decreased at W2 in both groups. Multivariate analysis of the risk factors for FVIII inhibitor development showed peak FVIII activity at W2. IL-6 and TNF-α at W8 were positively correlated with inhibitor formation, and negatively correlated with the age starting gene therapy. Collectively, the elevated monocyte derived pro-inflammatory cytokines/chemokines, together with the decreased anti-inflammatory cytokine of TGF-β at an early time point, may contribute to the persistent inflammatory environment in favor of an immune response toward FVIII inhibitor development.

Hemophilia Gene Therapy: Ready for Prime Time?

Hemophilia A and B are congenital, X-linked bleeding disorders caused by mutations in the genes encoding for the blood clotting factor VIII (FVIII) or factor IX (FIX), respectively. Since the beginning of gene therapy, hemophilia has been considered an attractive disease target that served as a trailblazer for the field at large. Different technologies have been explored to efficiently and safely deliver the therapeutic FVIII and FIX genes into the patients' cells. Currently, the most promising vectors for hemophilia gene therapy are adeno-associated viral vectors (AAVs) and lentiviral vectors. More recently, gene editing approaches based on designer nucleases or CRISPR/Cas, have also been considered to minimize risks associated with random vector integration and insertional mutagenesis though off-target issues would have to be carefully and comprehensively assessed. In the past two decades, several phase 1 hemophilia gene therapy clinical trials have been initiated with varying success. In particular, the early gene therapy clinical trials in hemophilia B patients based on AAV showed either transient or subtherapeutic clotting factor expression levels. This could be ascribed, at least in part, to suboptimal vector design and/or inadvertent immune consequences triggering hepatic inflammation. Hence, there was an unmet need to further increase vector safety and efficacy in future trials, preferably by using lower vector doses. It is particularly encouraging that sustained therapeutic FVIII and FIX expression levels have recently been attained after gene therapy in patients with severe hemophilia paving the way towards pivotal trials and commercialization. Nevertheless, transient liver toxicity still occurs and the use of transient immunosuppression was still required to curtail inadvertent immune responses, especially at high vector doses. To further boost clotting factor expression levels, codon-usage optimized synthetic FVIII or FIX transgenes have been employed. Alternatively, we and others have shown that the incorporation of hyperactive gain-of-function R338L mutation in the FIX gene substantially increased the overall efficacy. It is inevitable that the continued improvements in vector engineering and new insights in the vector-patient interactions will further benefit the development of a safe and effective cure for hemophilia A and B.

A Novel Platform for Immune Tolerance Induction in Hemophilia A Mice

Hemophilia A (HA) is an X-linked bleeding disease caused by factor VIII (FVIII) deficiency. We previously demonstrated that FVIII is produced specifically in liver sinusoid endothelial cells (LSECs) and to some degree in myeloid cells, and thus, in the present work, we seek to restrict the expression of FVIII transgene to these cells using cell-specific promoters. With this approach, we aim to limit immune response in a mouse model by lentiviral vector (LV)-mediated gene therapy encoding FVIII. To increase the target specificity of FVIII expression, we included miRNA target sequences (miRTs) (i.e., miRT-142.3p, miRT-126, and miRT-122) to silence expression in hematopoietic cells, endothelial cells, and hepatocytes, respectively. Notably, we report, for the first time, therapeutic levels of FVIII transgene expression at its natural site of production, which occurred without the formation of neutralizing antibodies (inhibitors). Moreover, inhibitors were eradicated in FVIII pre-immune mice through a regulatory Tcell-dependent mechanism. In conclusion, targeting FVIII expression to LSECs and myeloid cells by using LVs with cell-specific promoter minimized off-target expression and immune responses. Therefore, at least for some transgenes, expression at the physiologic site of synthesis can enhance efficacy and safety, resulting in long-term correction of genetic diseases such as HA.

Synergistic Inhibitory Effect of Traditional Chinese Medicine Astragaloside IV and Curcumin on Tumor Growth and Angiogenesis in an Orthotopic Nude-Mouse Model of Human Hepatocellular Carcinoma.

The aim of the present study was to investigate the efficacy of the traditional Chinese medicine (TCM), astragaloside IV (AS-IV) and curcumin on tumor growth and angiogenesis in an orthotopic nude-mouse model of human hepatocellular carcinoma (HCC). We have previously shown the usefulness of orthotopic models of human cancer for evaluation of the efficacy of TCM. Nude mice with orthotopic HepG2 HCC were treated with vehicle control (0.01 ml/g normal saline), cisplatinum (2 mg/kg), AS-IV (20 mg/kg), curcumin (100 mg/kg) or AS-IV plus curcumin (20 mg/kg + 100 mg/kg). Tumor inhibition in each group was evaluated by tumor weight at autopsy. The effect of AS-IV and curcumin on tumor angiogenesis was assessed by CD34 staining and expression of fibroblast growth factor-2 (FGF2), matrix metalloproteinase 2 (MMP2), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), thrombosis-related factor tissue factor (TF) and coagulation factor VII (FVII), as well as microRNAs miR-122 and miR-221. AS-IV and curcumin alone and in combination significantly reduced mean tumor weight compared to vehicle control (p<0.05). Tumor microvessel count was reduced by AS-IV and curcumin alone. Expression of FGF2, MMP2, VEGF, HGF, TF and FVII was reduced by AS-IV and curcumin alone. AS-IV and curcumin alone up-regulated expression of miR-122 and down-regulated that of miR-221. The combination of AS-IV and curcumin demonstrated significant synergistic effects on microvessel count as well as on expression of angiogenic and thrombosis-related factors and microRNAs. The present study indicates future clinical potential of combination therapy with AS-IV and curcumin for HCC.

Patterns of expression of factor VIII and von Willebrand factor by endothelial cell subsets in vivo

AbstractCirculating factor VIII (FVIII) is derived from liver and from extrahepatic sources probably of endothelial origin, but the vascular sites of FVIII production remain unclear. Among organs profiled, only liver and lymph nodes (LNs) show abundant expression of F8 messenger RNA (mRNA). Transcriptomic profiling of subsets of stromal cells, including endothelial cells (ECs) from mouse LNs and other tissues, showed that F8 mRNA is expressed by lymphatic ECs (LECs) but not by capillary ECs (capECs), fibroblastic reticular cells, or hematopoietic cells. Among blood ECs profiled, F8 expression was seen only in fenestrated ECs (liver sinusoidal and renal glomerular ECs) and some high endothelial venules. In contrast, von Willebrand factor mRNA was expressed in capECs but not in LECs; it was coexpressed with F8 mRNA in postcapillary high endothelial venules. Purified LECs and liver sinusoidal ECs but not capECs from LNs secrete active FVIII in culture, and human and mouse lymph contained substantial FVIII:C activity. Our results revealed localized vascular expression of FVIII and von Willebrand factor and identified LECs as a major cellular source of FVIII in extrahepatic tissues.

5. Development of Enhancing Intraosseous Delivery Efficiency of LV-Factor VIII Variants in Platelets of Hemophilia A Mice

Introduction: Our previous studies demonstrated that human factor VIII (FVIII) specifically expressed in megakaryocytes and then stored in platelets of Hemophilia A (HemA) mice can partially correct their phenotype over 5 months in mice with or without pre-existing inhibitors. This was achieved by intraosseous (IO) delivery of lentiviral vectors (LVs) carrying a transgene encoding human FVIII variant (BDDhFVIII/N6; abbreviated as F8) driven by a megakaryocyte-specific promoter (Gp1bα) without preconditioning as required in ex vivo gene therapy. Methods: In this study, we aimed at enhancing transgene expression by two strategies. One was to enhance LV transduction efficiency by suppressing the innate and adaptive immune responses against LVs and LV-transduced cells using pharmacological agents. The other was to improve FVIII gene expression by incorporating a new human FVIII variant, F8X10K12 (a 10-amino acid change in the A1 domain and a 12-amino acid change in the light chain; a kind gift from Dr. Weidong Xiao). Results: First, the immune competent C57BL6 mice were pretreated with both dexamethasone (Dex) (IP, 5 mg/kg at -24h, -4h, 4h and 24h) and anti-CD8α monoclonal antibody (mAb) (IP, 4 mg/kg on day -1, 4, 11, 16 and 21), or Dex only. IO infusion of GFP-LVs (1.1×108 i.f.u./mouse) driven by a ubiquitous MND promoter was performed on day 0. On day 7, Dex only and combination drugs + LVs treated mice (n=3) produced higher numbers of GFP+ total bone marrow cells (17.7±3.5% and 11.8±2.1% vs 6.9±3.1%, P=0.0001 and 0.005) and GFP+Lineage-Sca1+cKit+ HSCs (55.5±3.1% and 48.3±6.1% vs 44.4±17.2%, P=0.1 and 0.31) compared with LV-only treated mice (n=3). Most importantly, in the long term, higher numbers of GFP+ cells (2.4±0.4% vs 0.5±0.1%, P<0.001) in the total bone marrow and GFP+HSCs (10.7±3.3% vs 2.6±0.6%, P<0.001) were observed in combination drugs + LVs treated mice (n=3) compared with LV-only treated mice on day 160 after LV infusion (n=3), which was confirmed by higher LV copy number in bone marrow cells of drugs + LVs treated mice. Second, we tested the FVIII expression levels from two human FVIII variants in HemA mice by hydrodynamic injection of plasmids driven by a human elongation factor-1 promoter (pEF1α-F8X10K12 or pEF1α-F8, 50 µg/mouse, n=8), respectively. Compared with F8, F8X10K12 produced a 25-fold increase (147±27% vs 3,734±477%) in the clotting activity determined by an aPTT assay on day 4 post injection. Then two LVs containing F8X10K12 or F8 transgene driven by EF1α promoter (E-F8X10K12-LV or E-F8-LV) were constructed and used to transduce 293T cells, respectively. Flow cytometry data showed that E-F8X10K12-LV produced a significant increase of hFVIII+293T cells (77.8% vs 15%) and MFI (795 vs 541) compared to E-F8-LV at the same doses. These results indicated that F8X10K12 may further enhance FVIII gene expression in platelets for more effective therapy. LVs containing F8X10K12 or F8 transgene driven by Gp1bα promoter (G-F8X10K12-LV or G-F8-LV) were subsequently generated and were intraosseously delivered into HemA mice to test the FVIII efficiency in platelets by ELISA, thrombin generation assay and tail clipping. Conclusion: We found that administration of Dex that efficiently inhibited initial innate immune responses to LVs in vivo combined with anti-CD8α mAb that depleted subsequent cytotoxic CD8+ T cells improved the transduction efficiency of LVs and persistence of transduced cells, leading to over 10% GFP+HSCs in treated mice up to 160 days. In addition, a new FVIII variant, F8X10K12, can significantly enhance FVIII expression in mice following hydrodynamic injection of plasmids and in LV-transduced cells. Taken together, IO infusion of G-F8X10K12-LV into HemA mice pretreated with Dex and antiCD8α mAb can be used to further enhance and prolong transgene efficiency in platelets for effective correction of hemophilia A.

52. Therapeutic Factor VIII Expression After AAV Delivery in Non-Human Primates

Adeno-associated viral (AAV) vector delivery of factor VIII (FVIII) has been challenging due to its intrinsic properties that result in inefficient expression compared to similarly sized proteins. Early studies of AAV delivery in hemophilia A mice and dogs suggested that the therapeutic vector dose for FVIII will be higher than for factor IX. However, higher vector loads may induce stronger immune responses against capsid antigens, as evidenced in the clinical studies of AAV delivery for hemophilia B. The use of codon-optimization and novel FVIII variants with enhanced biological properties may provide strategies to increase FVIII expression or secretion to support clinical studies for hemophilia A. One published study has reported clinically relevant levels of hFVIII following AAV-hFVIII delivery in non-human primates (NHPs). This study utilized a hFVIII variant that included a 17 amino acid synthetic sequence within the 14 amino acid B-domain region that increased hFVIII expression compared to the parental B-domain deleted FVIII-SQ transgene (McIntosh, 2013). While this and other variants may increase expression after AAV delivery, the use of non-native FVIII sequences may also increase the risk of development of neutralizing antibodies to potential neoantigens. In order to generate an AAV-hFVIII vector capable of expressing therapeutic levels of FVIII at a clinically relevant vector dose without introduction of any neoantigens, 28 hFVIII-SQ sequences were generated and introduced into our optimized expression cassette containing a modified transthyretin (TTRm) promoter. The constructs were initially screened by hydrodynamic delivery of plasmid DNA which identified 11 candidates that expressed FVIII 2-7 fold higher than our first generation codon optimized construct, CO3. AAV vectors (n=9) were generated using a novel AAV capsid, Spark100, with the best performing FVIII constructs. Hem A/CD4 KO mice were administered the vectors alongside CO3 (4×10e12vg/kg). At 8 weeks post vector administration, 2/9 expressed hFVIII similar to CO3, 5/9 were 4-8 fold higher than CO3 while 2/9 (SPK-8003 and SPK-8005) were >10 fold more potent than CO3. SPK-8005 was then evaluated in a dose escalation study in cynomologus macaques (n=3/group) treated with 3 doses: 2×10e12, 5×10e12 and 1×10e13 vg/kg and compared to vehicle controls (n=2). At 2 weeks post AAV administration, average hFVIII levels in the low, mid and high dose cohorts were 12.7 ± 2.1, 22.6 ± 0.8 and 54.1 ± 15.6 percent of normal, respectively. By 3-4 weeks, hFVIII expression started to decline in most of the animals concomitant with generation of antibodies against human FVIII. Of note, this is an expected and well-described observation that occurs in immune competent animal models due to differences between human and endogenous FVIII protein sequences. The 2 macaques that did not develop anti-hFVIII antibodies had sustained FVIII expression through the last time point evaluated. Finally, no vector-related toxicity events were observed. In summary, extensive codon-optimization identified novel AAV-hFVIII constructs capable of achieving therapeutic FVIII levels in macaques at clinically relevant doses. To our knowledge, the hFVIII levels observed in this study are the highest reported in a large animal model after treatment with an AAV vector expressing an unmodified FVIII-SQ protein. These safety and efficacy results in NHPs support the use of SPK-8005 hepatic gene transfer for the potential treatment of hemophilia A.

728. Tissue-Directed Transgene Engineering for AAV and Lentivector Gene Therapy Approaches

Clinical gene therapy frequently is encumbered by low transgene product biosynthesis at predictably safe vector doses. It has been hypothesized that the presence of rare codons may regulate transgene product expression through depletion of the available cognate tRNA pool. Codon optimization is the prominent strategy utilized to overcome this hypothesized limitation and involves replacing rare, presumably translation rate-limiting, codons with the most frequent ones. Typical algorithms attempt to match the codon usage frequency of the transgene coding sequence to that of target organism's total mRNA pool, which has been shown to approximate the overall available tRNA concentrations. Upon closer examination, it appears that both codon frequency and tRNA content vary between tissue types. Therefore, we hypothesize that codon-optimization can be improved by tailoring to the codon-frequency of the most highly expressed genes present in target cell types. Our tissue-directed codon optimization algorithm utilizes novel codon usage indices generated from target cell gene expression data. As proof of concept, we developed tissue-codon optimized variants of coagulation factor VIII (FVIII) to be utilized in lenti- and adeno-associated viral (LV and AAV, respectively) vectors. These two vectors are the leading platforms for clinical gene therapy of hemophilia A. LV is utilized to target autologous hematopoietic cell types ex vivo while AAV is delivered intravenously to genetically-modify hepatocytes. However to date, development-stage LV and AAV gene therapy products for hemophilia A have been characterized by low-level FVIII transgene product biosynthesis. Initially, we designed human hepatocyte-, monocyte- and standard overall human-optimized FVIII constructs (LCO, MCO and HCO, respectively) to be compared to wild-type FVIII. Upon initial examination, expression of LCO-FVIII was shown to be 3-fold greater than either MCO- or wild-type FVIII from the human hepatoma cell line, HepG2, following transient transfection. In contrast, LCO-FVIII and MCO-FVIII expression was diminished 12 and 4-fold, respectively, compared to wild-type in the ‘neutral’ human embryonic kidney 293T cell line. Furthermore, following hydrodynamic injection of naked plasmid DNA into hemophilia A mice, LCO-FVIII exhibited a sustained 10-fold increase in FVIII expression relative to the HCO-FVIII comparator. In attempt to generate a lead candidate for clinical translation, we utilized several of our most promising vector components to construct a liver-optimized AAV8 vector consisting of LCO-ET3, our previously described high-expression bioengineered FVIII variant, transcriptionally driven by a novel 146bp liver-directed promoter and adjacent MVM intron. Following intravenous delivery into hemophilia A mice, vector doses of 1e12 and 1e11 vector genomes per kg achieved sustained, predictable curative plasma FVIII levels of 200% and 20% of normal human levels, respectively. These initial results support the utility of our novel approach of clinical tissue-directed transgene optimization.

Efficient generation of FVII gene knockout mice using CRISPR/Cas9 nuclease and truncated guided RNAs.

We investigated the effects of 5′-end truncated CRISPR RNA-guided Cas9 nuclease (tru-RGN, 17/18 nucleotides) on genome editing capability in NIH/3T3 cells, and its efficiencies on generating Factor VII (FVII) gene-knockout (KO) mice. In cultured cells, RGNs on-target editing activity had been varied when gRNAs was truncated, higher at Site Two (tF7–2 vs. F7–2, 49.5 vs. 30.1%) while lower in other two sites (Site One, tF7–1 vs.F7–1, 12.1 vs. 23.6%; Site Three, tF7–3 vs.F7–3, 7.7 vs 10.9%) (P < 0.05). Out of 15 predicated off–target sites, tru-RGNs showed significantly decreased frequencies at 5 sites. By microinjecting tru-RGN RNAs into zygotes, FVII KO mice were generated with higher efficiency at Site Two (80.1 vs. 35.8%) and Site One (55.0 vs 3.7%) (P < 0.05), but not at Site three (39.4 vs 27.8%) (P > 0.05) when compared with standard RGN controls. Knockout FVII mice demonstrated a delayed prothrombin time and decreased plasma FVII expression. Our study first demonstrates that truncated gRNAs to 18 complementary nucleotides and Cas9 nucleases, can effectively generate FVII gene KO mice with a significantly higher efficiency in a site-dependent manner. In addition, the off-target frequency was much lower in KO mice than in cell lines via RGN expression vector-mediated genome editing.

Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium

ABSTRACTSignal peptides play an important role in directing and efficiently transporting secretory proteins to their proper locations in the endoplasmic reticulum of mammalian cells. The aim of this study was to enhance the expression of recombinant coagulation factor VII (rFVII) in CHO cells by optimizing the signal peptides and type of fed-batch culture medium used. Five sub-clones (O2, I3, H3, G2 and M3) with different signal peptide were selected by western blot (WB) analysis and used for suspension culture. We compared rFVII expression levels of 5 sub-clones and found that the highest rFVII expression level was obtained with the IgK signal peptide instead of Ori, the native signal peptide of rFVII. The high protein expression of rFVII with signal peptide IgK was mirrored by a high transcription level during suspension culture. After analyzing culture and feed media, the combination of M4 and F4 media yielded the highest rFVII expression of 20 mg/L during a 10-day suspension culture. After analyzing cell density and cell cycle, CHO cells feeding by F4 had a similar percentage of cells in G0/G1 and a higher cell density compared to F2 and F3. This may be the reason for high rFVII expression in M4+F4. In summary, rFVII expression was successfully enhanced by optimizing the signal peptide and fed-batch medium used in CHO suspension culture. Our data may be used to improve the production of other therapeutic proteins in fed-batch culture.

Overexpression of factor VIII after AAV delivery is transiently associated with cellular stress in hemophilia A mice

Factor VIII (FVIII) is a large glycoprotein that is challenging to express both in vitro and in vivo. Several studies suggest that high levels of FVIII expression can lead to cellular stress. After gene transfer, transgene expression is restricted to a subset of cells and the increased FVIII load per cell may impact activation of the unfolded protein response. We sought to determine whether increased FVIII expression in mice after adeno-associated viral liver gene transfer would affect the unfolded protein response and/or immune response to the transgene. The FVIII gene was delivered as B-domain deleted single chain or two chain (light and heavy chains) at a range of doses in hemophilia A mice. A correlation between FVIII expression and anti-FVIII antibody titers was observed. Analysis of key components of the unfolded protein response, binding immunoglobulin protein (BiP), and C/EBP homologous protein (CHOP), showed transient unfolded protein response activation in the single chain treated group expressing >200% of FVIII but not after two chain delivery. These studies suggest that supraphysiological single chain FVIII expression may increase the likelihood of a cellular stress response but does not alter liver function. These data are in agreement with the observed long-term expression of FVIII at therapeutic levels after adeno-associated viral delivery in hemophilia A dogs without evidence of cellular toxicity.

Neither Factor VIII Nor the Putative F8B Protein Is Detectable in Human Peripheral Blood Mononuclear Cells

Factor VIII (FVIII) is encoded by 26 exons comprising the F8 gene. The F8 mRNA transcript encodes the full-length FVIII protein, which consists of 2332 amino acids. A CpG island within intron 22 of F8 includes an initiation site for a second, shorter transcript termed F8B. F8B contains a short exon spliced in-frame to F8 exons 23-26, and this mRNA transcript is expressed in multiple human tissues. The putative protein encoded by this gene corresponds to 8 amino acids encoded by the exon within F8 intron 22, followed by the FVIII C2 domain sequence. Earlier studies have unambiguously demonstrated FVIII expression in human endothelial cells, including liver and lung. Animal model studies have indicated FVIII is expressed, possibly exclusively, in endothelial cells. Other recent reports have presented evidence for FVIII and F8B protein expression in human peripheral blood mononuclear cells (PBMCs). In this study, we utilized several methods to test the hypothesis that FVIII and/or the putative F8B protein is expressed in human PBMCs.Frozen PBMCs from healthy human subjects were thawed and then washed 5-10X to ensure removal of plasma-derived FVIII. Samples containing 50 million PBMCs were lysed in buffer containing protease inhibitors, centrifuged, and the supernatants analyzed by immunoprecipitation (IP) followed by SDS-PAGE and Western blots. The IP experiments utilized both polyclonal anti-FVIII antibodies and cocktails of monoclonal antibodies (mAbs) specific for different FVIII domains, and Western blots used several combinations of primary and secondary antibodies. Negative controls consisted of BHK cell lysates, and positive controls were purified FVIII and lysates of BHK cells transduced with a plasmid encoding FVIII. Western blots carried out with sensitive chemiluminescence detection identified the expected bands in the positive control lanes. Bands at expected positions for FVIII or F8B proteins (~240kDa, 75 kDa, 25-30 kDa) visualized by Coomassie-blue staining of lysates analyzed by SDS-PAGE were cut from the gels, subjected to trypsin digestion and then analyzed by mass spectrometry (LC-MS). FVIII-derived peptides were detected only in the positive control samples.Intracellular staining (ICS) of permeabilized PBMCs was carried out using mAbs specific for the FVIII A1, A2, C1, and C2 domains and a mAb specific for the FVIII light chain. The mAbs were covalently labeled with Alexa-Fluor 488 to avoid possible nonspecific binding of labeled secondary antibodies. PBMCs from (1) normal control subjects; (2) a severe hemophilia A subject with an intron-22 inversion mutation and (3) a severe hemophilia A subject with a deletion of F8 exons 3-6 were analyzed. All 5 anti-FVIII mAbs detected FVIII protein in FVIII-expressing BHK cells, while none detected FVIII in BHK cells or in any of the human PBMC samples. Our results are consistent with several recent studies reporting proteomics analysis of human PBMCs, which have identified no FVIII peptides or proteins. We conclude that if FVIII or F8B proteins are expressed in human PBMCs, their concentration is below the detection limits of these assays. DisclosuresNo relevant conflicts of interest to declare.

Effective Therapy for Murine Hemophilia A by Enhancing Factor VIII Expression in Platelets

Introduction: Platelets may comprise an ideal vehicle for delivering factor VIII (FVIII) in hemophilia A (HemA) as FVIII stored in platelet α-granules is protected from neutralization by inhibitory antibodies and, during bleeding, activated platelets locally excrete their contents to promote clot formation. In our previous study, it was demonstrated that intraosseous (IO) infusion of lentiviral vectors (LVs) carrying a transgene encoding human factor VIII variant (BDDhFVIII/N6; abbreviated as F8) driven by a megakaryocyte-specific promoter (Gp1bα) successfully transduced hematopoietic stem cells (HSCs) in HemA mice without the requirement of preconditioning as in ex vivo gene therapy. FVIII expressed and then stored in platelets can partially correct the HemA phenotype over 5 months in mice with or without pre-existing inhibitors.Methods: In this study, we aimed at enhancing transgene expression by two strategies. One was to improve the efficiency of human FVIII cDNA by testing a new human FVIII variant (a kind gift from Dr. Weidong Xiao), F8X10K12 (a 10-amino acid change in the A1 domain and a 12-amino acid change in the light chain). The other was to enhance LV transduction efficiency by suppressing the innate and adaptive immune responses against LVs and LV-transduced cells using pharmacological agents.Results:We first tested the FVIII expression levels from two human FVIII variants by hydrodynamic injection of plasmids driven by a human elongation factor-1 promoter (pEF1α-F8X10K12 or pEF1α-F8, 50 μg/mouse, n=8), respectively. Compared with F8, F8X10K12 produced a 25-fold increase (147±27% vs 3,734±477%) in the clotting activity determined by an aPTT assay on day 4 post injection. Based on this result, two LVs containing F8X10K12 or F8 transgene driven by EF1α promoter (E-F8X10K12-LV or E-F8-LV) were constructed and used to transduce 293T cells, respectively. Flow cytometry data showed that E-F8X10K12-LV produced a significant increase of hFVIII+ 293T cells (77.8% vs 15%) and MFI (795 vs 541) compared to E-F8-LV at the same doses (Figure 1). These results indicated that F8X10K12 may further enhance FVIII gene expression for more effective therapy. Two LVs containing F8X10K12 or F8 transgene driven by Gp1bα promoter (G-F8X10K12-LV or G-F8-LV) were subsequently generated. Secondly, the immune competent C57BL6 mice were pretreated with both dexamethasone (IP, 5 mg/kg at -24h, -4h, 4h and 24h) and anti-CD8α monoclonal antibody (mAb) (IP, 4 mg/kg on day -1, 4, 11, 16 and 21). IO infusion of GFP-LVs (1.1×108 i.f.u./mouse) driven by a ubiquitous MND promoter was performed on day 0 (Figure 2a). On day 7, drugs + LVs treated mice (n=3) produced higher numbers of GFP+ total bone marrow cells (11.8±2.1% vs 6.9±3.1%, P =0.005) and GFP+ Lineage- Sca1+ cKit+ HSCs (48.3±6.1% vs 44.4±17.2%, P =0.31) compared with LV-only treated mice (n=3) (Figure 2b). Most importantly, in the long term, higher numbers of GFP+ cells (2.4±0.4% vs 0.5±0.1%, P <0.001) in the total bone marrow and GFP+ HSCs (10.7±3.3% vs 2.6±0.6%, P <0.001) were observed in drugs + LVs treated mice (n=3) compared with LV-only treated mice on day 160 after LV infusion (n=3) (Figure 2c).Conclusion: We found that a new FVIII variant, F8X10K12, can significantly enhance FVIII expression in mice following hydrodynamic injection of plasmids and in LV-transduced cells. In addition, administration of dexamethasone that efficiently inhibited initial innate immune responses to LVs in vivo combined with anti-CD8α mAb that depleted subsequent cytotoxic CD8+ T cells improved the transduction efficiency of LVs and persistence of transduced cells, leading to over 10% GFP+ HSCs in treated mice up to 160 days. Taken together, IO infusion of G-F8X10K12-LV into HemA mice pretreated with dexamethasone and antiCD8α mAb can be used to further enhance and prolong transgene expression levels in platelets for effective correction of hemophilia A. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Liver Tropism and Factor VIII Transgene Expression after Intrauterine Chorionic Villus Stem Cell Transplantation

INTRODUCTION: Gestation is characterized by rapid mitosis and histogenesis in an immune naïve environment. Growth and development of vital organs are occurring continuously at different time points throughout pregnancy under the regulation of paracrine signals, transcription and growth factors. Appropriate timing to target the developing organ of interest during in utero stem cell transplantation (IUT) may result in high level engraftment in that organ as well as recognition of foreign cells as self. Hemophilia A, is an X-linked recessive bleeding disorder caused by a mutation of the gene that encodes Factor VIII (FVIII) which is primarily produced by the liver. We hypothesize that introduction of stem cells in utero during hepatogenesis in mice (e9.5-15 days) will result in localization and persistence of stem cells in the liver.METHODOLOGY: We reported isolation and characterization of early preterm chorionic villus derived mesenchymal stem cells (PMSCs) at GA 9-13 weeks. PMSCs (n =5) were transduced with a lentiviral vector encoding B-domain deleted FVIII, GFP and luciferase reporter genes (pCCLc-MNDU3-eGFP/LUC-PGK-F8-WPRE) at an MOI (Multiplicity of Infection) of 3. Chromogenic assay was performed on cell supernatant of transduced PMSCs 72 hours after transduction. Prior to transplantation, PMSCs were plated at different densities ranging from 250,000 cells to 2 x10^6 cells and subjected to bioluminescence imaging. Intraperitoneal transplantation of PBS (control group) or with 1 x10^6 PMSCs (experimental group) was conducted in fetal mice at e12-14 days using a 33 gauge non-coring needle. Bioluminescence analysis was performed 24 hours after IUT on dams, and 12-15 days after transplantation. Mice were euthanized and liver tissue was harvested from all animals. Expression of FVIII was analyzed by RT-PCR from both groups.RESULTS: Bioluminescence imaging revealed focal density and transgene expression for transduced cells in vitro, in vivo at 24 hours and in postnatal pups 12-14 days after transplantation. Postmortem bioluminescence analysis of IUT transplanted mice showed positive bioluminescence in the livers of pups injected with transduced PMSCs. RT-PCR analysis of the liver samples using human specific FVIII primers revealed FVIII expression in liver tissues in PMSC treated mice while negative for PBS treated mice.CONCLUSION: These results demonstrate the functional survival and localization of genetically modified PMSCs in the liver after intrauterine transplantation for up to two weeks. This study provide better understanding of the cell fate of stem cells after IUT and suggest that appropriate timing of stem cell transplantation in utero may lead to immunological tolerance. DisclosuresPowell:CSL Behring: Employment.

Novel Gene Therapy Strategy for Hemophilia a By Hydrodynamic Gene Delivery Combined with Non-Viral Piggybac Transposon Vector in Canine Model

Currently, hemophilia A patients are treated with plasma-derived or recombinant factor VIII (FVIII) concentrates. These treatments have substantially improved the management of bleedings in hemophilia and more recently long-acting recombinant FVIII concentrates are available in clinic, but hemophilia A patients still require the repeated intravenous injections frequently 2-3 times per week. Therefore, gene and cell-based therapy is an attractive strategy for the treatment of the disease because continuous expression of FVIII levels as low as 1-5 % of normal have been shown to ameliorate the bleeding phenotype as well as improve the quality of life. Although several attempts have been made for hemophilia A gene therapy, systemic delivery of viral vectors into liver cells in vivo, however, resulted in a transient and low-level expression of FVIII, because the large size of FVIII cDNA prohibited the efficient delivery of conventional viral vectors. Moreover, there remain concerns over the safety of these approaches such as adverse immunological reactions or virus-mediated cytotoxicity. Hydrodynamic gene delivery (HGD) is considered to be an alternative candidate to viral vectors. Previously, we tested the HGD in hemophilia A mouse model with the combination of non-viral piggyBac transposon vectors which can transfer the full-length FVIII transgene. As a result, we confirmed the sustained FVIII expression for over 300 days without any immune responses (Matsui H, et.al. PLos One 9(8) e104597, 2014).While HGD system is effective and safe in small animal such as mouse (20-25g body weight), the major challenge is in applying this functioning procedure in the patients with hemophilia A. For this purpose, the current study focus on an assessment of the safety of liver-target HGD in dogs as the first step toward hydrodynamic gene therapy in clinic. Hemophilia A carrier dog was provided from Dr. Lillicrap group at Queen's University and we have already developed hemophilia A dog colony in our institution. We used both normal and hemophilia A dogs in this study. In brief, under general anesthesia using isoflurane, beagle dog (10-12Kg body weight) was placed on the table of fluoroscopy machine. A 18G peripheral catheter was directly inserted into the saphenous vein followed by insertion of a 0.038 inch guide wire, a long sheath (5Fr) and an injection balloon catheter. The balloon catheter was placed and inflated just below the branch of hepatic vein using the image-guided interventional radiology technique. Liver-targeted HGD was performed into 4 liver lobes, the right lateral, right medial, left medial, and left lateral lobe, separately using piggyBac transposon vector expressing either GFP or FVIII. An injection of saline containing plasmid DNA were performed at the speed of 10mL/second sequentially into 4 liver lobes through the hepatic veins with 20 minutes interval between each injections.Various physiological parameters including heart rate, blood pressure, oxygen saturation were continuously monitored and recorded before, during and after each injection. For the assessment of tissue damage, blood samples were collected at the various time points. For the assessment of GFP expression, the liver samples were collected under echo-guided liver biopsy both 5 and 120 days after HGD. Although, transient 5-10 fold increase in hepatobiliary enzymes, including AST, ALT, were observed, there is no significant impact on physiological parameters, such as systemic circulation, respiration and cardiac function.By histological examination of liver samples, approximately 70-80% of hepatocytes in all 4 lobes were positive with GFP both 5 and 120 days after HGD. These results suggest that HGD system combined with interventional radiology technique is capable for the large animal with safe, efficient and less invasive. Now, we are conducting FVIII gene transfer by HGD in canine hemophilia A model. DisclosuresNo relevant conflicts of interest to declare.

Immune Tolerance Developed in Platelet-Targeted FVIII Gene Therapy in Hemophilia Mice Is CD4+ T Cell-Mediated

Immune response to factor VIII (FVIII) is not only a severe complication in protein replacement therapy, but also a major concern in gene therapy of hemophilia A. Our previous studies have demonstrated that platelet-targeted FVIII (2bF8) gene therapy together with in vivo drug-selection of transduced cells can not only rescue the bleeding diathesis but also induce anti-FVIII specific immune tolerance in FVIIInull mice. In the current study, we investigated 1) whether our non-selectable lentiviral vector (LV) for the induction of platelet-FVIII expression is sufficient to induce immune tolerance and 2) which cell compartment is tolerized after platelet gene therapy. Platelet-specific FVIII expression was introduced by 2bF8LV-transduction of hematopoietic stem cells followed by syngeneic transplantation into FVIIInull mice preconditioned with 660 cGy total body irradiation (TBI) or Busulfan (Bu) plus ATG (anti-thymocyte globulin). After bone marrow transplantation and reconstitution, animals were analyzed by PCR, qPCR, FVIII:C assay, and tail clipping test to confirm the success of 2bF8 gene therapy. Sixteen weeks after transplantation, animals were challenged with recombinant human FVIII (rhF8) via retro-orbital venous administration at a dose of 50 U/kg weekly for 4 weeks. The titers of anti-FVIII inhibitory antibodies (inhibitors) were determined by Bethesda assay. The CFSE-labeled CD4 T cell proliferation assay and ELISPOT-based memory B cell maturation assay were used to determine which cell compartment is tolerized to FVIII after 2bF8 gene therapy. The level of platelet-FVIII expression was 1.44 ± 0.39 mU/108 platelets (n = 6) in the 660 cGy group, which is not significantly different from the level obtained from the Bu+ATG group [3.04 ± 1.19 mU/108 platelets (n = 6)]. Even after rhF8 challenge, no antibodies were detected in 2bF8LV-transduced recipients in either group. In contrast, all animals in the control group that did not undergo gene therapy developed various levels of inhibitors (204±97 BU/ml, n=7). The frequency of regulatory T cells in both 660 cGy TBI (11.01±0.52%) and Bu+ATG (11.02±0.68%) groups were significantly higher than the control group (8.05±0.57%). T cell proliferation assay demonstrated that CD4+ T cells from 2bF8 LV-transduced recipients that had been challenged with rhF8 did not proliferate when restimulated with rhF8 in vitro. The daughter CD4+ T cells in the group with 10 U/ml of rhF8 were 5.84 ± 2.49% (n = 6), which was not significantly different from the control group without no rhF8 stimulation (0 U/ml) (5.33 ± 1.72%). CD4+ T cells from primed FVIIInull mice did proliferate after rhF8 restimulation. The proliferated daughter cell was 13.12 ± 6.76% (n = 7) in the group with rhF8 (10 U/ml) re-stimulation, which is significantly higher than the group without rhF8 co-culture (4.99 ± 1.16%). Since FVIII-specific memory B cell maturation is CD4+ T cell dependent, we isolated CD4+ T and memory B cells from 2bF8LV-transduced or FVIIInull mice after rhF8 immunization and co-cultured with rhF8 followed by ELISPOT assay to examine the antibody secreting cells. No spots were detected when memory B cells from rhF8-primed FVIIInull mice were co-cultured with CD4+ T cells from 2bF8LV-transduced recipients. In contrast, when memory B cells from either rhF8 immunized 2bF8LV-transduced or untreated FVIIInull mice were cultured with CD4+ T cells from rhF8-primed FVIIInull mice, there were 142 and 205 anti-FVIII antibody secreting cells, respectively, detected per 106 cells seeded. These results indicate that CD4+ T cells from 2bF8LV-transduced mice are tolerized to rhF8 stimulation. In conclusion, 2bF8 lentiviral gene transfer without in vivo selection of genetically manipulated cells can introduce FVIII-specific immune tolerance in hemophilia A mice and this immune tolerance is CD4+ T cell-mediated. DisclosuresBaumgartner:Novo Nordisk: Research Funding. Shi:BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETTED GENE EXPRESSION..

Factor VII promotes hepatocellular carcinoma progression through ERK-TSC signaling.

We previously demonstrated PAR2 starts upstreamed with tissue factor (TF) and factor VII (FVII), inhibited autophagy via mTOR signaling in HCC. However, the mechanism underlying for merging functions of PAR2 with the coagulation system in HCC progression remained unclear. The present study aimed to investigate the role of TF, FVII and PAR2 in tumor progression of HCC. The expressions of TF, FVII and PAR2 from HCC specimens were evaluated by immunohistochemical stains and western blotting. We found that the expression of FVII, but not TF and PAR2, directly related to the vascular invasion and the clinical staging. Importantly, a lower level of FVII expression was significantly associated with the longer disease-free survival. The addition of FVII but not TF induced the expression of PAR2 and phosphorylation of ERK1/2, whereas knockdown of FVII decreased PAR2 expression and ERK1/2 phosphorylation in HCC cell lines. Furthermore, levels of phosphor-TSC2 (Ser664) were increased after treatment with FVII and PAR2 agonist whereas these were significantly abolished in the presence of a potent and specific MEK/ERK inhibitor U0126. Moreover, mTOR knockdown highly reduced Hep3B migration, which could be reverted by FVII but not TF and PAR2. These results indicated that FVII/PAR2 signaling through MEK/ERK and TSC2 axis for mTOR activation has potent effects on the migration of HCC cells. In addition, FVII/PAR2 signaling elicits an mTOR-independent signaling, which promotes hepatoma cell migration in consistent with the clinical observations. Our study indicates that levels of FVII, but not TF, are associated with tumor migration and invasiveness in HCC, and provides clues that evaluation of FVII expression in HCC may be useful as a prognostic indicator in patients with HCC and may form an alternative target for further therapy.