Targeted genome engineering based on CRISPR/Cas9 system to enhance FVIII expression in vitro.

BACKGROUND: Hemophilia A is caused by a deficiency of coagulation factor VIII in the body due to a defect in the F8 gene. The emergence of CRISPR/Cas9 gene editing technology will make it possible to alter the expression of the F8 gene in hemophiliacs, while achieving a potential cure for the disease. METHODS: Initially, we identified high-activity variants of FVIII and constructed donor plasmids using enzymatic digestion and ligation techniques. Subsequently, the donor plasmids were co-transfected with sgRNA-Cas9 protein into mouse Neuro-2a cells, followed by flow cytometry-based cell sorting and puromycin selection. Finally, BDD-hF8 targeted to knock-in the mROSA26 genomic locus was identified and validated for FVIII expression. RESULTS: We identified the p18T-BDD-F8-V3 variant with high FVIII activity and detected the strongest pX458-mROSA26-int1-sgRNA1 targeted cleavage ability and no cleavage events were found at potential off-target sites. Targeted knock-in of BDD-hF8 cDNA at the mROSA26 locus was achieved based on both HDR/NHEJ gene repair approaches, and high level and stable FVIII expression was obtained, successfully realizing gene editing in vitro. CONCLUSIONS: Knock-in of exogenous genes based on the CRISPR/Cas9 system targeting genomic loci is promising for the research and treatment of a variety of single-gene diseases.

Modified expi293 cell culture system using piggyBac transposon enables efficient production of human FVIII.

Human blood coagulation factor VIII (hFVIII) is used in hemostatic and prophylactic treatment of patients with hemophilia A. Biotechnological innovations have enabled purification of the culture medium of rodent or human cells harboring the hFVIII expression cassette. However, cell lines express hFVIII protein derived from an exogenous expression vector at a lower level than most other proteins. Here, we describe hFVIII production using piggyBac transposon and the human-derived expi293F cell line. Use of a drug selection protocol, rather than transient expression protocol, allowed cells harboring hFVIII expression cassettes to efficiently produce hFVIII. In heterogeneous drug-selected cells, the production level was maintained even after multiple passages. The specific activity of the produced hFVIII was comparable to that of the commercial product and hFVIII derived from baby hamster kidney cells. We also applied codon optimization to the hFVIII open reading frame sequences in the transgene, which increased production of full-length hFVIII, but decreased production of B-domain-deleted human FVIII (BDD-hFVIII). Low transcriptional abundance of the hF8 transgene was observed in cells harboring codon-optimized BDD-hFVIII expression cassettes, which might partially contribute to decreased hFVIII production. The mechanism underlying these distinct outcomes may offer clues to highly efficient hFVIII protein production.

Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A.

Hemophilia A (HA) is a bleeding disorder caused by deficiency of the coagulation factor VIII (F8). F8 replacement is standard of care, whereas gene therapy (F8 gene) for HA is an attractive investigational approach. However, the large size of the F8 gene and the immunogenicity of the product present challenges in development of the F8 gene therapy. To resolve these problems, we synthesized a shortened F8 gene (F8-BDD) and cloned it into a lentiviral vector (LV). The F8-BDD produced mainly short cleaved inactive products in LV-transduced cells. To improve F8 functionality, we designed two novel F8-BDD genes, one with an insertion of eight specific N-glycosylation sites (F8-N8) and another which restored all N-glycosylation sites (F8-299) in the B domain. Although the overall protein expression was reduced, high coagulation activity (>100-fold) was detected in the supernatants of LV-F8-N8- and LV-F8-299-transduced cells. Protein analysis of F8 and the procoagulation cofactor, von Willebrand Factor, showed enhanced interaction after restoration of B domain glycosylation using F8-299. HA mouse hematopoietic stem cell transplantation studies illustrated that the bleeding phenotype was corrected after LV-F8-N8 or -299 gene transfer into the hematopoietic stem cells. Importantly, the F8-299 modification markedly reduced immunogenicity of the F8 protein in these HA mice. In conclusion, the modified F8-299 gene could be efficiently packaged into LV and, although with reduced expression, produced highly stable and functional F8 protein that corrected the bleeding phenotype without inhibitory immunogenicity. We anticipate that these results will be beneficial in the development of gene therapies against HA.

Genetic analysis of non-severe hemophilia A phenotype with A discrepancy between one-stage and chromogenic factor VIII activity assays.

INTRODUCTION: The diagnosis of hemophilia A (HA) is based on the measurement of factor VIII activity (VIII:C). About one-third of non-severe HA patients show a discrepancy of VIII:C measured by one-stage (VIII:C 1st) and chromogenic (VIII:C chr) assays. Different mutations in the F8 gene may cause the discrepancy in results of the FVIII activity assay. The aim of this study was to investigate F8 gene mutations in patients with assay discrepancies and to evaluate their impact on the results of VIII:C assays. METHODS: Mutation analysis was performed on 41 individuals with a discrepancy in VIII:C 1st and FVIII: C chr assays by direct sequencing. In addition, the effect of the variants on FVIII macromolecule structure was investigated by in silico and bioinformatics tools. RESULTS: Genetic analysis disclosed 22 different variants, of which 19 were identified for the first time to be involved in the phenotype of VIII:C discrepancy. Most of the variants related to the higher VIII:C 1st were found in A1, A2, A3 domains. The variant related to VIII:C chr > VIII:C 1st was located in the thrombin cleavage site. In silico analysis showed the effect of variants on FVIII macromolecule stability, which may be the possible mechanism causing the discrepancy. CONCLUSION: Our data shed light on the impact of genetic defects on VIII:C assay and provided evidence that the consideration of these mutations may open a new window to the proper diagnosis and treatment monitoring of non-severe HA patients.

Characterization and visualization of murine coagulation factor VIII-producing cells in vivo.

Coagulation factors are produced from hepatocytes, whereas production of coagulation factor VIII (FVIII) from primary tissues and cell species is still controversial. Here, we tried to characterize primary FVIII-producing organ and cell species using genetically engineered mice, in which enhanced green fluorescent protein (EGFP) was expressed instead of the F8 gene. EGFP-positive FVIII-producing cells existed only in thin sinusoidal layer of the liver and characterized as CD31high, CD146high, and lymphatic vascular endothelial hyaluronan receptor 1 (Lyve1)+. EGFP-positive cells can be clearly distinguished from lymphatic endothelial cells in the expression profile of the podoplanin- and C-type lectin-like receptor-2 (CLEC-2)+. In embryogenesis, EGFP-positive cells began to emerge at E14.5 and subsequently increased according to liver maturation. Furthermore, plasma FVIII could be abolished by crossing F8 conditional deficient mice with Lyve1-Cre mice. In conclusion, in mice, FVIII is only produced from endothelial cells exhibiting CD31high, CD146high, Lyve1+, CLEC-2+, and podoplanin- in liver sinusoidal endothelial cells.

Immunohistochemical Studies of Age-Related Changes in Cell Proliferation and Angiogenesis during the Healing of Acetic Acid-Induced Gastric Ulcers in Rats.

Cell proliferation and angiogenesis are of utmost importance for healing to take place. The KI67 and EGFR proteins are markers of cell proliferation, while CD31 and factor VIII are markers of angiogenesis. To elucidate the mechanism responsible for delayed healing of the gastric injury in old age, we analyzed the expression of these markers in rats of different months during the healing of an acetic acid-induced gastric ulcer. Male Wistar rats (aged 3, 6, 12, and 18 months) divided into four groups, according to their ages, formed the experimental animals. Stomach tissue samples were collected on days 3, 7, 14, and 21 after induction for assessment of ulcer healing. The area of gastric mucosa healed was inversely proportional to age. The expression of markers of proliferation (KI67 and EGFR) and angiogenesis (factor VIII and CD31) decreased significantly (p < 0.05) in older rats when compared with younger ones (3 months > six months > 12 months > 18 months) on days 7, 14, and 21 after induction of gastric ulcer. This study revealed that the slower gastric ulcer healing rate in older rats might be due to reduced epithelial cell proliferation and angiogenic activities.

Perfusion process combining low temperature and valeric acid for enhanced recombinant factor VIII production.

Perfusion operation mode remains the preferred platform for production of labile biopharmaceuticals (e.g., blood factors) and is also being increasingly adopted for production of stable products (e.g., monoclonal antibodies). Regardless of the product, process development typically aims at maximizing production capacity. In this work, we investigated the impact of perfusion cultivation conditions on process productivity for production of human factor VIII (FVIII). Recombinant CHO cells were cultivated in bioreactors coupled to inclined settlers and the effects of reducing the temperature to 31°C with or without valeric acid (VA) supplementation were evaluated. Increases in cell specific productivity (qp ) up to 2.4-fold (FVIII concentration) and up to 3.0-fold (FVIII biological activity) were obtained at 31°C with VA compared to the control at 37°C. Biological activity is the most important quality attribute for FVIII and was positively affected by mild hypothermia in combination with the chemical inducer. The low temperature conditions resulted in enhanced product transcript levels, suggesting that the higher qp is related to the increased mRNA levels. Furthermore, a high-producer subclone was evaluated under the perfusion conditions optimized for the parental clone (31°C with VA), yielding increases in qp of 6-fold and 15-fold compared to the parental clone cultivated under the same condition and at 37°C, respectively. The proposed perfusion strategy enables increased product formation without increasing production costs, being potentially applicable to perfusion production of other CHO-derived biopharmaceuticals. To the best of our knowledge, this is the first report showing the benefits of perfusion combining mild hypothermia with VA supplementation.

Universal Correction of Blood Coagulation Factor VIII in Patient-Derived Induced Pluripotent Stem Cells Using CRISPR/Cas9.

Hemophilia A (HA) is caused by genetic mutations in the blood coagulation factor VIII (FVIII) gene. Genome-editing approaches can be used to target the mutated site itself in patient-derived induced pluripotent stem cells (iPSCs). However, these approaches can be hampered by difficulty in preparing thousands of editing platforms for each corresponding variant found in HA patients. Here, we report a universal approach to correct the various mutations in HA patient iPSCs by the targeted insertion of the FVIII gene into the human H11 site via CRISPR/Cas9. We derived corrected clones from two types of patient iPSCs with frequencies of up to 64% and 66%, respectively, without detectable unwanted off-target mutations. Moreover, we demonstrated that endothelial cells differentiated from the corrected iPSCs successfully secreted functional protein. This strategy may provide a universal therapeutic method for correcting all genetic variants found in HA patients.

New technologies in gene therapy for inducing immune tolerance in hemophilia A.

INTRODUCTION: Conventional hemophilia treatment is based on repeated infusion of the missing clotting factor. This therapy is lifelong, expensive and can result in the formation of neutralizing antibodies, thus causing failure of the treatment and requiring higher doses of the replacement drug. Areas covered: Gene and cell therapies offer the advantage of providing a definitive and long-lasting correction of the mutated gene, promoting its physiological expression and preventing neutralizing antibody development. This review focuses on the most recent approaches that have been shown to prevent and even eradicate immune response toward the replaced factor. Expert commentary: Despite the encouraging data demonstrated by ongoing clinical trials and pre-clinical studies, more extensive investigations are necessary to establish the long-term safety and efficacy of gene therapy treatments in maintaining immune tolerance.

Perspectives on progressive strategies and recent trends in the production of recombinant human factor VIII.

Factor VIII (fVIII), a glycoprotein cofactor, plays a crucial role in the intrinsic blood coagulation pathway. As one of the most essential blood clotting factors known today, fVIII is the largest and most complex commercialized therapeutic protein used in the treatment of hemophilia A, an X-linked recessive disorder. Two lyophilized fVIII concentrates (viz., plasma fractionated and recombinant) are in use to treat hemorrhagic episodes in patients suffering with hemophilia A. Recombinant fVIII (rfVIII) products that are devoid of human and animal protein expressed in mammalian cells can be used as an alternative to plasma derived (pd) products fractionated from human blood. Although effective, the expression of rfVIII in heterologous mammalian expression systems at an industrial scale is complicated due to complex fVIII structure and non-human pattern of post-translational modifications, particularly glycosylation. Chinese hamster ovary (CHO) is the most commonly used host cell line for the production of various biotherapeutics. Product safety and adaptability to grow in suspension is the most desirable feature that makes CHO, a suitable host for rfVIII production. Even though the therapeutic and commercial application of rfVIII protein from CHO has increased extensively, further studies are required at cellular to bioprocess level to overcome the challenges in production, purification and processing. Efficient strategies are required to attain better products pertaining to the glycosylation path, productivity, stability, etc., to bring down the cost of expensive therapeutics like rfVIII that obviates these biotherapeutics affordable to common man. This review summarizes the various approaches and developments that have been in practice in fVIII production.

Expression and assembly of largest foreign protein in chloroplasts: oral delivery of human FVIII made in lettuce chloroplasts robustly suppresses inhibitor formation in haemophilia A mice.

Inhibitor formation is a serious complication of factor VIII (FVIII) replacement therapy for the X-linked bleeding disorder haemophilia A and occurs in 20%-30% of patients. No prophylactic tolerance protocol currently exists. Although we reported oral tolerance induction using FVIII domains expressed in tobacco chloroplasts, significant challenges in clinical advancement include expression of the full-length CTB-FVIII sequence to cover the entire patient population, regardless of individual CD4+ T-cell epitope responses. Codon optimization of FVIII heavy chain (HC) and light chain (LC) increased expression 15- to 42-fold higher than the native human genes. Homoplasmic lettuce lines expressed CTB fusion proteins of FVIII-HC (99.3 kDa), LC (91.8 kDa), C2 (31 kDa) or single chain (SC, 178.2 kDa) up to 3622, 263, 3321 and 852 µg/g in lyophilized plant cells, when grown in a cGMP hydroponic facility (Fraunhofer). CTB-FVIII-SC is the largest foreign protein expressed in chloroplasts; despite a large pentamer size (891 kDa), assembly, folding and disulphide bonds were maintained upon lyophilization and long-term storage as revealed by GM1-ganglioside receptor binding assays. Repeated oral gavages (twice/week for 2 months) of CTB-FVIII-HC/CTB-FVIII-LC reduced inhibitor titres ~10-fold (average 44 BU/mL to 4.7 BU/mL) in haemophilia A mice. Most importantly, increase in the frequency of circulating LAP-expressing CD4+ CD25+ FoxP3+ Treg in tolerized mice could be used as an important cellular biomarker in human clinical trials for plant-based oral tolerance induction. In conclusion, this study reports the first clinical candidate for oral tolerance induction that is urgently needed to protect haemophilia A patients receiving FVIII injections.

Hemophilia gene therapy comes of age.

Concurrent with the development of recombinant factor replacement products, the characterization of the F9 and F8 genes over 3 decades ago allowed for the development of recombinant factor products and made the hemophilias a target disease for gene transfer. The progress of hemophilia gene therapy has been announced in 3 American Society of Hematology scientific plenary sessions, including the first "cure" in a large animal model of hemophilia B in 1998, first in human sustained vector-derived factor IX activity in 2011, and our clinical trial results reporting sustained vector-derived factor IX activity well into the mild or normal range in 2016. This progression to clinically meaningful success combined with numerous ongoing recombinant adeno-associated virus (rAAV)-mediated hemophilia gene transfer clinical trials suggest that the goal of gene therapy to alter the paradigm of hemophilia care may soon be realized. Although several novel therapeutics have recently emerged for hemophilia, gene therapy is unique in its potential for a one-time disease-altering, or even curative, treatment. This review will focus on the prior progress and current clinical trial investigation of rAAV-mediated gene transfer for hemophilia A and B.

Immune tolerance induced by platelet-targeted factor VIII gene therapy in hemophilia A mice is CD4 T cell mediated.

Essentials The immune response is a significant concern in gene therapy. Platelet-targeted gene therapy can restore hemostasis and induce immune tolerance. CD4 T cell compartment is tolerized after platelet gene therapy. Preconditioning regimen affects immune tolerance induction in platelet gene therapy. SUMMARY: Background Immune responses are a major concern in gene therapy. Our previous studies demonstrated that platelet-targeted factor VIII (FVIII) (2bF8) gene therapy together with in vivo drug selection of transduced cells can rescue the bleeding diathesis and induce immune tolerance in FVIIInull mice. Objective To investigate whether non-selectable 2bF8 lentiviral vector (LV) for the induction of platelet-FVIII expression is sufficient to induce immune tolerance and how immune tolerance is induced after 2bF8LV gene therapy. Methods Platelet-FVIII expression was introduced by 2bF8LV transduction and transplantation. FVIII assays and tail bleeding tests were used to confirm the success of platelet gene therapy. Animals were challenged with rhF8 to explore if immune tolerance was induced after gene therapy. Treg cell analysis, T-cell proliferation assay and memory B-cell-mediated ELISPOT assay were used to investigate the potential mechanisms of immune tolerance. Results We showed that platelet-FVIII expression was sustained and the bleeding diathesis was restored in FVIIInull mice after 2bF8LV gene therapy. None of the transduced recipients developed anti-FVIII inhibitory antibodies in the groups preconditioned with 660 cGy irradiation or busulfan plus ATG treatment even after rhF8 challenge. Treg cells significantly increased in 2bF8LV-transduced recipients and the immune tolerance developed was transferable. CD4+ T cells from treated animals failed to proliferate in response to rhF8 re-stimulation, but memory B cells could differentiate into antibody secreting cells in 2bF8LV-transduced recipients. Conclusion 2bF8LV gene transfer without in vivo selection of manipulated cells can introduce immune tolerance in hemophilia A mice and this immune tolerance is CD4+ T cell mediated.

A common polymorphism decreases LRP1 mRNA stability and is associated with increased plasma factor VIII levels.

The low-density lipoprotein receptor-related protein 1 (LRP1) gene is associated with increased levels of plasma factor VIII (FVIII). We aimed to explore eight functional genetic LRP1 variants for their potential roles in regulating FVIII levels and acute ischemic stroke (AIS). This genetic association study enrolled 192 patients with AIS and 134 controls. There were no significant differences in the genetic frequency of the eight functional single-nucleotide polymorphisms (SNPs) between the control and AIS groups. However, while analyzing the association between the eight SNPs and plasma FVIII levels, subjects with T/T genotype of rs1800137 (vs. CC+CT) were found to be associated with higher FVIII levels (23.5IU/dL; 95% confidence interval, 7.4-39.5IU/dL; P=0.0044) after adjusting for age, gender, estimated glomerular filtration rate, O blood type, inflammatory state, and body mass index. An analysis of the mRNA stability and abundance was designed and performed using minigene system transfected into HepG2 cells to assess the possible differences in mRNA stabilities between rs1800137 CC (rs1800137C) and TT (rs1800137T) genotypes. Site-directed mutagenesis revealed that rs1800137T accounts for the observed decrease in mRNA stability. The SNP rs1800137, located in exon 8, has been identified as an exon-splicing enhancer in silico. However, alternative splicing of LRP1 without inclusion of exon 8 was not identified. In transfected HepG2 cells, cycloheximide slowed down the degradation of the rs1800137T-containing minigene. These results demonstrate that synonymous SNP rs1800137 can lead to increased plasma FVIII levels due to decreased mRNA stability via translation-dependent mRNA degradation associated with codon optimality.

Production of recombinant coagulation factors: Are humans the best host cells?

The main treatment option for Hemophilia A/B patients involves the administration of recombinant coagulation factors on-demand or in a prophylactic approach. Despite the safety and efficacy of this replacement therapy, the development of antibodies against the coagulation factor infused, which neutralize the procoagulant activity, is a severe complication. The production of recombinant coagulation factors in human cell lines is an efficient approach to avoid such complication. Human cell lines can produce recombinant proteins with post translation modifications more similar to their natural counterpart, reducing potential immunogenic reactions. This review provides a brief overview of the most important characteristics of recombinant FVIII and FIX products available on the market and the improvements that have recently been achieved by the production using human cell lines.

Stable high-level expression of factor VIII in Chinese hamster ovary cells in improved elongation factor-1 alpha-based system.

BACKGROUND: Recombinant factor VIII (FVIII), used for haemophilia A therapy, is one of the most challenging among the therapeutic proteins produced in heterologous expression systems. Deletion variant of FVIII, in which the entire domain B is replaced by a short linker peptide, was approved for medical use. Efficacy and safety of this FVIII deletion variant are similar to full-length FVIII preparations while the level of production in CHO cells is substantially higher. Typical levels of productivity for CHO cell lines producing deletion variant FVIII-BDD SQ, described elsewhere, are 0.5-2 IU/ml, corresponding to the concentration of FVIII of about 0.2 µg/ml. Using standard vectors based on the cytomegalovirus promoter (CMV) and the dihydrofolate reductase cDNA we have previously obtained the cell line secreting 0.5 IU/ml of FVIII-BDD, which roughly corresponds to the previously published data. RESULTS: An expression system based on CHO genomic sequences including CHO-EEF1A promoter and Epstein-Barr virus terminal repeat fragment allowed us to achieve 80-fold increase in the production level as compared with the conventional expression system based on the CMV promoter. Immediately after the primary selection FVIII -producing cells secreted 5-10 IU/ml of FVIII-BDD, and after multi-stage methotrexate-driven amplification a stable clonal line 11A4H was selected, secreting 39 IU/ml of FVIII-BDD in the simple batch culturing conditions, which considerably exceeds known indicators for industrial producers of this protein. In contrast to other FVIII-BDD producing lines 11A4H accumulates low proportion of the secreted FVIII on the membrane. Its productivity may be further increased approximately two-fold by adding sodium butyrate and butylated hydroxyanisol to the culture medium. A five-stage purification process for the factor VIII was employed. It allowed isolation of the intact FVIII-BDD as was confirmed by mass spectrometry. Purified FVIII-BDD has a specific activity of 11,000 IU/mg, similar to known recombinant FVIII drugs. CONCLUSIONS: The recombinant FVIII-BDD was produced in CHO cells without addition of any animal-derived materials, purified and characterized. Novel genetic constructions for the expression of heterologous proteins combined with optimized cultivation method allowed to obtain the secretion level of biologically active recombinant FVIII increased by almost ten times as compared with the previously published analogues.

Characteristics of Minimally Oversized Adeno-Associated Virus Vectors Encoding Human Factor VIII Generated Using Producer Cell Lines and Triple Transfection.

Several ongoing clinical studies are evaluating recombinant adeno-associated virus (rAAV) vectors as gene delivery vehicles for a variety of diseases. However, the production of vectors with genomes >4.7 kb is challenging, with vector preparations frequently containing truncated genomes. To determine whether the generation of oversized rAAVs can be improved using a producer cell-line (PCL) process, HeLaS3-cell lines harboring either a 5.1 or 5.4 kb rAAV vector genome encoding codon-optimized cDNA for human B-domain deleted Factor VIII (FVIII) were isolated. High-producing "masterwells" (MWs), defined as producing >50,000 vg/cell, were identified for each oversized vector. These MWs provided stable vector production for >20 passages. The quality and potency of the AAVrh8R/FVIII-5.1 and AAVrh8R/FVIII-5.4 vectors generated by the PCL method were then compared to those prepared via transient transfection (TXN). Southern and dot blot analyses demonstrated that both production methods resulted in packaging of heterogeneously sized genomes. However, the PCL-derived rAAV vector preparations contained some genomes >4.7 kb, whereas the majority of genomes generated by the TXN method were ≤4.7 kb. The PCL process reduced packaging of non-vector DNA for both the AAVrh8R/FVIII-5.1 and the AAVrh8R/FVIII-5.4 kb vector preparations. Furthermore, more DNA-containing viral particles were obtained for the AAVrh8R/FVIII-5.1 vector. In a mouse model of hemophilia A, animals administered a PCL-derived rAAV vector exhibited twofold higher plasma FVIII activity and increased levels of vector genomes in the liver than mice treated with vector produced via TXN did. Hence, the quality of oversized vectors prepared using the PCL method is greater than that of vectors generated using the TXN process, and importantly this improvement translates to enhanced performance in vivo.

Evaluation of the virus clearance capacity and robustness of the manufacturing process for the recombinant factor VIII protein, turoctocog alfa.

Turoctocog alfa is a B-domain-truncated recombinant factor VIII protein produced in a Chinese hamster ovary (CHO) cell line. The aim of this study was to evaluate the virus clearance capacity and robustness of the turoctocog alfa purification process. Virus clearance evaluation studies were conducted utilising a scaled-down version of the manufacturing process. Total virus clearance was evaluated using the ecotropic murine leukaemia virus (eMuLV) as a model for non-infectious retrovirus-like particles (RVLPs) and certain enveloped viruses. Other viruses utilised included: infectious bovine rhinotracheitis (IBRV), minute virus of mice (MVM), bovine enterovirus (BEV) and Reo-3 virus (Reo-3). Robust clearance of all model viruses was demonstrated with either new or reused resins. Overall, virus reduction factors were: >18.0 log10 (eMuLV); 11.0 log10 (MVM); >11.8 log10 (Reo-3; >5.0 log10 using nanofiltration); >15.3 log10 (BEV) and >12.7 log10 (IBRV). Taken together, these values demonstrate that the purification process for turoctocog alfa effectively removes a range of enveloped and non-enveloped viruses of different physicochemical properties and sizes.

BAY 81-8973, a full-length recombinant factor VIII: Human heat shock protein 70 improves the manufacturing process without affecting clinical safety.

BAY 81-8973 is a full-length, unmodified recombinant human factor VIII (FVIII) approved for the treatment of hemophilia A. BAY 81-8973 has the same amino acid sequence as the currently marketed sucrose-formulated recombinant FVIII (rFVIII-FS) product and is produced using additional advanced manufacturing technologies. One of the key manufacturing advances for BAY 81-8973 is introduction of the gene for human heat shock protein 70 (HSP70) into the rFVIII-FS cell line. HSP70 facilitates proper folding of proteins, enhances cell survival by inhibiting apoptosis, and potentially impacts rFVIII glycosylation. HSP70 expression in the BAY 81-8973 cell line along with other manufacturing advances resulted in a higher-producing cell line and improvements in the pharmacokinetics of the final product as determined in clinical studies. HSP70 protein is not detected in the harvest or in the final BAY 81-8973 product. However, because this is a new process, clinical trial safety assessments included monitoring for anti-HSP70 antibodies. Most patients, across all age groups, had low levels of anti-HSP70 antibodies before exposure to the investigational product. During BAY 81-8973 treatment, 5% of patients had sporadic increases in anti-HSP70 antibody levels above a predefined threshold (cutoff value, 239 ng/mL). No clinical symptoms related to anti-HSP70 antibody development occurred. In conclusion, addition of HSP70 to the BAY 81-8973 cell line is an innovative technology for manufacturing rFVIII aimed at improving protein folding and expression. Improved pharmacokinetics and no effect on safety of BAY 81-8973 were observed in clinical trials in patients with hemophilia A.

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

UNLABELLED: Circulating 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.