AAV mediated gene therapy for haemophilia B: From the early attempts to modern trials.

Early gene therapy clinical trials for the treatment of Haemophilia B have been instrumental to our global understanding of gene therapy and have significantly contributed to the rapid expansion of the field. The use of adeno-associated viruses (AAVs) as vectors for gene transfer has successfully led to therapeutic expression of coagulation factor IX (FIX) in severe haemophilia B patients. Expression of FIX has remained stable following a single administration of vector for up to 8 years at levels that are clinically relevant to reduce the incidence of spontaneous bleeds and have permitted a significant change in the disease management with reduction or elimination of the need for coagulation factor concentrates. These trials have also shed light on several concerns around AAV-mediated gene transfer such as the high prevalence of pre-existing immunity against the vector capsid as well as the elevation of liver transaminases that is associated with a loss of FIX transgene expression in some patients. However, this field is advancing very rapidly with the development of increasingly more efficient strategies to overcome some of these obstacles and importantly raise the possibility of a functional cure, which has been long sought after. This review overviews the evolution of gene therapy for haemophilia B over the last two decades.

Extravascular factor IX pool fed by prophylaxis is a true hemostatic barrier against bleeding.

BACKGROUND: Factor (F)IX can bind to type IV collagen in the endothelial basement membrane and diffuse into extravascular spaces. Previous studies in rodents have reported a large biodistribution of FIX. OBJECTIVES: The aim of the study was to evaluate the potential hemostatic activity of extravascular FIX and its role in protecting against joint bleeds. METHODS: The capacity of 4 different FIX molecules (plasma-derived and recombinant) to bind type I and type IV collagen was studied here. FIX molecules were also administered intravenously at doses of 50 to 3000 IU/kg in FIX knockout mice. RESULTS: A specific FIX signal was detected in immunohistochemistry in the liver as well as in muscles and knee joints with recombinant FIX molecules injected at 1000 and 3000 IU/kg but not at the usual clinical doses of 50 to 100 IU/kg, while plasma-derived FIX generated a FIX signal at all doses, including 50 IU/kg. Such a signal was also detected after five 100 IU/kg daily infusions of recombinant FIX, suggesting that FIX can accumulate in the extravascular space during prophylaxis. The extravascular procoagulant activity of FIX, assessed in saphenous vein bleeding assays, was significantly higher in hemophilia B mice after these 5 days of prophylaxis compared to a single infusion of 100 IU/kg of FIX and assessment of FIX activity 7 days later. CONCLUSION: Taken together, these results show that in individuals with severe hemophilia B receiving regular prophylaxis with FIX, extravascular accumulation of FIX over time may have a significant impact on the coagulation capacity and protection toward bleeding.

Protein S antibody as an adjunct therapy for hemophilia B.

ABSTRACT: Hemophilia B (HB) is caused by an inherited deficiency of plasma coagulation factor IX (FIX). Approximately 60% of pediatric patients with HB possess a severe form of FIX deficiency (<1% FIX activity). Treatment typically requires replacement therapy through the administration of FIX. However, exogenous FIX has a limited functional half-life, and the natural anticoagulant protein S (PS) inhibits activated FIX (FIXa). PS ultimately limits thrombin formation, which limits plasma coagulation. This regulation of FIXa activity by PS led us to test whether inhibiting PS would extend the functional half-life of FIX and thereby prolong FIX-based HB therapy. We assayed clotting times and thrombin generation to measure the efficacy of a PS antibody for increasing FIX activity in commercially obtained plasma and plasma from pediatric patients with HB. We included 11 pediatric patients who lacked additional comorbidities and coagulopathies. In vivo, we assessed thrombus formation in HB mice in the presence of the FIXa ± PS antibody. We found an accelerated rate of clotting in the presence of PS antibody. Similarly, the peak thrombin formed was significantly greater in the presence of the PS antibody, even in plasma from patients with severe HB. Furthermore, HB mice injected with PS antibody and FIX had a 4.5-fold higher accumulation of fibrin at the thrombus induction site compared with mice injected with FIX alone. Our findings imply that a PS antibody would be a valuable adjunct to increase the effectiveness of FIX replacement therapy in pediatric patients who have mild, moderate, and severe HB.

Humanization and functional characterization of enhanced coagulation factor IX variants identified through ancestral sequence reconstruction.

BACKGROUND: Laboratory resurrection of ancient coagulation factor (F) IX variants generated through ancestral sequence reconstruction led to the discovery of a FIX variant, designated An96, which possesses enhanced specific activity independent of and additive to that provided by human p.Arg384Lys, referred to as FIX-Padua. OBJECTIVES: The goal of the current study was to identify the amino acid substitution(s) responsible for the enhanced activity of An96 and create a humanized An96 FIX transgene for gene therapy application. METHODS: Reductionist screening approaches, including domain swapping and scanning residue substitution, were used and guided by one-stage FIX activity assays. In vitro characterization of top candidates included recombinant high-purity preparation, specific activity determination, and enzyme kinetic analysis. Final candidates were packaged into adeno-associated viral (AAV) vectors and delivered to hemophilia B mice. RESULTS: Five of 42 total amino acid substitutions in An96 appear sufficient to retain the enhanced activity of An96 in an otherwise human FIX variant. Additional substitution of the Padua variant further increased the specific activity 5-fold. This candidate, designated ET9, demonstrated 51-fold greater specific activity than hFIX. AAV2/8-ET9 treated hemophilia B mice produced plasma FIX activities equivalent to those observed previously for AAV2/8-An96-Padua, which were 10-fold higher than AAV2/8-hFIX-Padua. CONCLUSION: Starting from computationally inferred ancient FIX sequences, novel amino acid substitutions conferring activity enhancement were identified and translated into an AAV-FIX gene therapy cassette demonstrating high potency. This ancestral sequence reconstruction discovery and sequence mapping refinement approach represents a promising platform for broader protein drug and gene therapy candidate optimization.

Augmented Degradation of Factors VIII and IX in the Intermittent Movement State.

The most common clinical presentation of hemophilia A and hemophilia B is bleeding in large joints and striated muscles. It is unclear why bleeding has a predilection to affect joints and muscles. As muscles and joints are involved in intermittent movement, we explored whether this phenomenon could be associated with an impact on factor VIII and IX levels. Purified proteins and a mouse model were assessed using coagulation assays, Western blot analysis and immuno-staining. Movement caused an increase in thrombin activity and a decrease in factor VIII and factor IX activity. The decrease in factor VIII activity was more significant in the presence of thrombin and during movement. Under movement condition, sodium ions appeared to enhance the activity of thrombin that resulted in decreased factor VIII activity. Unlike factor VIII, the reduction in factor IX levels in the movement condition was thrombin-independent. High factor VIII levels were found to protect factor IX from degradation and vice versa. In mice that were in movement, factor VIII and IX levels decreased in the microcirculation of the muscle tissue compared with other tissues and to the muscle tissue at rest. Movement had no effect on von Willebrand factor levels. Movement induces reduction in factor VIII and IX levels. It enables an increase in the binding of sodium ions to thrombin leading to enhanced thrombin activity and augmented degradation of factor VIII. These data suggest a potential mechanism underlying the tendency of hemophilia patients to bleed in muscles and joints.

Gene Therapy Approaches for the Treatment of Hemophilia B.

In contrast to the standard enzyme-replacement therapy, administered from once per 7-14 days to 2-3 times a week in patients with severe hemophilia B, as a result of a single injection, gene therapy can restore F9 gene expression and maintain it for a prolonged time. In clinical research, the approach of delivering a functional copy of a gene using adeno-associated viral (AAV) vectors is widely used. The scientific community is actively researching possible modifications to improve delivery efficiency and expression. In preclinical studies, the possibility of genome editing using CRISPR/Cas9 technology for the treatment of hemophilia B is also being actively studied.

Cost-effectiveness analysis of recombinant factor IX Fc fusion protein compared with recombinant factor IX for the treatment of moderate-severe to severe hemophilia B in China.

OBJECTIVE: To evaluate the lifetime cost-effectiveness of recombinant factor IX Fc fusion protein (rFIXFc) and recombinant factor IX (rFIX) for the treatment of hemophilia B (HB) in China. METHODS: We developed a decision-analytic Markov model including three health states: alive, requiring surgery, and dead. This model estimated the lifetime cost and quality-adjusted life-years (QALYs) of prophylaxis in childhood, followed by on-demand treatment in adulthood for moderate-severe to severe HB patients from China's healthcare system perspective. Efficacy data derived from pivotal clinical trials, clinical guideline recommendations, and expert consultation were applied to two scenarios (full dose and low dose). One-way sensitivity analysis and probabilistic sensitivity analysis (PSA) were performed to assess the robustness of the model. OUTCOMES: Lifetime cost, QALYs, and the incremental cost-effectiveness ratio were calculated, and the results were compared with willingness-to-pay (WTP) thresholds of one to three times the gross domestic product per capita of China in 2021 ($12,551-$37,653). RESULTS: RFIXFc was associated with lower cost and more QALYs than rFIX in both scenarios, which suggested that it is a dominant strategy (more effective and cheaper) for moderate-severe to severe HB in China. In the full-dose scenario, rFIXFc saved more money and yielded more QALYs than in the low-dose scenario (low doses are the typical clinical reality in China). PSA demonstrated that rFIXFc had an over 90% probability of being cost-effective with full-dose and low-dose treatment at WTP thresholds of $12,551-$37,653. CONCLUSIONS: Compared with rFIX, rFIXFc appears to be a cost-effective option for the lifetime management of moderate-severe to severe HB patients in China.

Pathogenesis and treatment of osteoporosis in patients with hemophilia.

INTRODUCTION: Hemophilia is a rare X-linked recessive inherited bleeding disorder caused by mutations of the genes encoding coagulation factor VIII (FVIII) or IX (FIX). Patients with hemophilia (PWH) often have a high risk of osteoporosis and fractures that is usually ignored. Herein, we review the underlying mechanisms of osteoporosis and the increased risk of fractures and their treatment in patients with FVIII or FIX deficiency. METHODS: The PubMed, Web of Science, Embase, and Cochrane Library databases were searched to identify original research articles, meta-analyses, and scientific reviews on the mechanisms or treatment of osteoporosis in PWH. RESULTS: The pathogenic mechanisms of osteoporosis in PWH are multifactorial and remain unclear. The available evidence shows that FVIII and FIX deficiency may directly affect bone metabolism by interfering with the RANK/RANKL/OPG pathway. Other potential mechanisms of osteoporosis in PWH include thrombin deficiency and the unloading and immobilization of bone, which will affect osteoblast and osteoclast activity by changing the cytokine profiles. The treatment of osteoporosis in PWH includes antiresorptive, anabolic, and dual-action drugs; weight-bearing exercise; fall prevention; and prophylactic coagulation factor replacement therapy. However, clinical studies of the efficacy of anti-osteoporotic agents in osteoporosis of PWH are urgently needed. CONCLUSION: This review summarizes recent progress in research on the pathogenesis of osteoporosis in PWH and provides insights into potential treatment for osteoporosis in PWH.

A novel mouse whole blood thrombin generation assay sensitive to FXI- and FIX-mediated amplification of coagulation.

Thrombin generation (TG) assays serve as a valuable tool to study the amplifying roles of intrinsic pathway factors in human coagulation and provide functional insights into the increased bleeding observed in individuals deficient in factors (F) XI, IX, or VIII. Mice are used extensively in hemostasis research owing to the availability of coagulation factor-deficient mice. However, phenotypic differences between mouse and human TG have become apparent. In this study, we describe a novel, calibrated mouse whole blood (WB) TG assay used to assess the amplifying roles of intrinsic pathway factors in mouse coagulation. WB- and plasma-TG was triggered with either silica or tissue factor (TF) in samples from wild-type mice and mice deficient for FXII, FXI, or FIX. Expectedly, silica-triggered WB-TG and platelet-poor plasma (PPP)-TG were significantly reduced by deficiencies for FXII, FXI, or FIX. FXII deficiency had no effect on WB-TG or PPP-TG when triggered with TF. However, FXI deficiency resulted in significantly reduced WB-TG triggered by low concentrations of TF but had no effect on TF-triggered PPP-TG. FIX deficiency profoundly reduced WB-TG when triggered by low or high concentrations of TF whereas TG in PPP or platelet-rich plasma was only moderately reduced under these conditions. In conclusion, we have developed a novel mouse WB-TG assay with enhanced sensitivity to FXI- and FIX-dependent amplification of coagulation compared with an established plasma-TG assay. The enhanced sensitivity of WB-TG to FXI and FIX-dependent amplification of coagulation suggests an important role of blood cells in this process.

Recent advances in the production of recombinant factor IX: bioprocessing and cell engineering.

Appropriate treatment of Hemophilia B is vital for patients' quality of life. Historically, the treatment used was the administration of coagulation Factor IX derived from human plasma. Advancements in recombinant technologies allowed Factor IX to be produced recombinantly. Successful recombinant production has triggered a gradual shift from the plasma derived origins of Factor IX, as it provides extended half-life and expanded production capacity. However, the complex post-translational modifications of Factor IX have made recombinant production at scale difficult. Considerable research has therefore been invested into understanding and optimizing the recombinant production of Factor IX. Here, we review the evolution of recombinant Factor IX production, focusing on recent developments in bioprocessing and cell engineering to control its post-translational modifications in its expression from Chinese Hamster Ovary (CHO) cells.

Markers of Hereditary Thrombophilia with Unclear Significance.

Thrombophilia leads to an increased risk of venous thromboembolism. Widely accepted risk factors for thrombophilia comprise deficiencies of protein C, protein S, and antithrombin, as well as the factor V "Leiden" mutation, the prothrombin G20210A mutation, dysfibrinogenemia, and, albeit less conclusive, increased levels of factor VIII. Besides these established markers of thrombophilia, risk factors of unclear significance have been described in the literature. These inherited risk factors include deficiencies or loss-of-activity of the activity of ADAMTS13, heparin cofactor II, plasminogen, tissue factor pathway inhibitor (TFPI), thrombomodulin, protein Z (PZ), as well as PZ-dependent protease inhibitor. On the other hand, thrombophilia has been linked to the gain-of-activity, or elevated levels, of α2-antiplasmin, angiotensin-converting enzyme, coagulation factors IX (FIX) and XI (FXI), fibrinogen, homocysteine, lipoprotein(a), plasminogen activator inhibitor-1 (PAI-1), and thrombin-activatable fibrinolysis inhibitor (TAFI). With respect to the molecular interactions that may influence the thrombotic risk, more complex mechanisms have been described for endothelial protein C receptor (EPCR) and factor XIII (FXIII) Val34Leu. With focus on the risk for venous thrombosis, the present review aims to give an overview on the current knowledge on the significance of the aforementioned markers for thrombophilia screening. According to the current knowledge, there appears to be weak evidence for a potential impact of EPCR, FIX, FXI, FXIII Val34Leu, fibrinogen, homocysteine, PAI-1, PZ, TAFI, and TFPI on the thrombotic risk.

High expression of serine and arginine-rich splicing factor 9 (SRSF9) is associated with hepatocellular carcinoma progression and a poor prognosis.

BACKGROUND: Serine and arginine-rich splicing factor 9 (SRSF9) has been linked to the occurrence and progression of various cancers; however, its effects and mechanism of action hepatocellular carcinoma (HCC) have not been reported. In this study, we used a bioinformatics approach and in vitro assays to evaluate the expression of SRSF9 in HCC, its prognostic value, and its underlying regulatory mechanisms, including analyses of related pathways and the role of methylation. METHODS: Transcriptomic and DNA methylation data for 357 HCC cases and 50 paratumor tissues in The Cancer Genome Atlas database were obtained. Additionally, protein expression data for cell lines and tissue samples were obtained from the Human Protein Atlas. The CMap databased was used to predict candidate drugs targeting SRSF9. Various cell lines were used for in vitro validation. RESULTS: SRSF9 expression was significantly elevated in HCC and was negatively regulated by its methylation site cg06116271. The low expression of SRSF9 and hypermethylation of cg06116271 were both associated with a longer overall survival time. A correlation analysis revealed ten genes that were co-expressed with SRSF9; levels of CDK4, RAN, DENR, RNF34, and ANAPC5 were positively correlated and levels of RBP4, APOC1, MASP2, HP, and HPX were negatively correlated with SRSF9 expression. The knockdown of SRSF9 in vitro inhibited the proliferation and migration of HCC cells and significantly reduced the expression of proteins in the Wnt signaling pathway (DVL2 and ß-catenin) and cell cycle pathway (Cyclin D and Cyclin E). A CMap analysis identified two drugs, camptothecin and apigenin, able to target and inhibit the expression of SRSF9. CONCLUSIONS: This study expands our understanding of the molecular biological functions of SRSF9 and cg06116271 and provides candidate diagnostic and therapeutic targets for HCC.

Intergrated Transcriptomic and Proteomic Analysis Revealed the Differential Responses to Novel Duck Reovirus Infection in the Bursa of Fabricius of Cairna moschata.

The bursa of Fabricius is an immunologically organ against the invasion of duck reovirus (DRV), which is a fatal bird virus belonging to the Reoviridae family. However, responses of the bursa of Fabricius of Cairna moschata to novel DRV (NDRV) infection are largely unknown. Transcriptomes and proteomes of the samples from control and two NDRV strain (HN10 and JDm10) with different virulence were analyzed. Differentially expressed genes and differential accumulated proteins were enriched in the serine protease system and innate immune response clusters. Most of the immune-related genes were up-regulated under both JDm10/HN10 infections. However, the immune-related proteins were only accumulated under HN10 infection. For the serine protease system, coagulation factor IX, three chains of fibrinogen, and complements C8, C5, and C2s were significantly up-regulated by the HN10 infection, suggesting that the serine protease-mediated immune system might be involved in the resistance to NDRV infection. For the innate and adaptive immune system, RIG-I, MDA5, MAPK20, and IRF3 were significantly up-regulated, indicating their important roles against invaded virus. TLR-3 and IKBKB were only up-regulated in the liver cells, MAPK20 was only up-regulated in the bursa of Fabricius cells, and IRAK2 was only up-regulated in the spleen samples. Coagulation factor IX was increased in the bursa of Fabricius, not in the liver and spleen samples. The data provides a detailed resource for studying the proteins participating in the resistances of the bursa of Fabricius of duck to NDRV infections.

Nanobodies against factor XI apple 3 domain inhibit binding of factor IX and reveal a novel binding site for high molecular weight kininogen.

BACKGROUND: Factor XI (FXI) is a promising target for novel anticoagulants because it shows a strong relation to thromboembolic diseases, while fulfilling a mostly supportive role in hemostasis. Anticoagulants targeting FXI could therefore reduce the risk for thrombosis, without increasing the chance of bleeding side effects. OBJECTIVES: To generate nanobodies that can interfere with FXIa mediated activation of factor IX (FIX). METHODS: Nanobodies were selected for binding to the apple 3 domain of FXI and their effects on FXI and coagulation were measured in purified protein systems as well as in plasma-based coagulation assays. Additionally, the binding epitope of selected nanobodies was assessed by hydrogen-deuterium exchange mass spectrometry. RESULTS: We have identified five nanobodies that inhibit FIX activation by FXI by competing with the FIX binding site on FXI. Interestingly, a sixth nanobody was found to target a different binding epitope in the apple 3 domain, resulting in competition with the FXI-high molecular weight kininogen (HK) interaction. CONCLUSIONS: We have characterized a nanobody targeting the FXI apple 3 domain that elucidates the binding orientation of HK on FXI. Moreover, we have produced five nanobodies that can inhibit the FXI-FIX interaction.

Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology.

BACKGROUND: Hemophilia B is a rare inherited genetic bleeding disorder caused by a deficiency or lack of coagulation factor IX, the gene for which (F9) is located on the X chromosome. Hemophilia B is currently incurable and the standard treatment is coagulation factor replacement therapy. Although gene therapy has the potential to cure hemophilia, significant barriers are still needed to be overcome, e.g., off-target effects and immunoreactivity, so new approaches must be explored. Nonsense mutations account for 8% of all the hemophilia B mutation types and can result in the development of coagulation factor inhibitors. In this study, CRISPR/Cas9 technology was used to construct a mouse embryonic stem cell model with a hemophilia B nonsense mutation (F9 c.223C > T) in humans to investigate the pathogenesis and treatment of nonsense mutations in hemophilia B. METHODS: First, a donor plasmid with a mutation (F9 c.223 C > T) and sgRNAs were constructed. Second, both the donor plasmid and the px330-sgRNA were electroporated into mouse embryonic stem cell, and the mutant cells were then screened using puromycin and red fluorescence. Third, the mutant cell lines were tested for pluripotency and the ability to differentiate into three layers. Finally, the effect of mutation on gene function was studied in the differentiation system. RESULTS: The mutant vector and effective sgRNA were constructed, and the mutant cell line was screened. This mutant cell line exhibited pluripotency and the ability to differentiate into three layers. This point mutation affects F9 expression at both the RNA and protein levels in the differentiation system. CONCLUSIONS: The mutant cell line obtained in the current study had a single-base mutation rather than a base deletion or insertion in the exon, which is more similar to clinical cases. In addition, the mutant has the characteristics of mouse embryonic stem cells, and this point mutation affects F9 gene transcription and translation, which can be used as a disease model for studying the pathogenesis and treatment of hemophilia at the stem cell level.

Long-term correction of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration.

CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications. However, it is still infeasible because homologous recombination (HR) is inefficient, especially for non-dividing cells. To overcome the challenge, we report that a homology-independent targeted integration (HITI) strategy is used for permanent integration of high-specificity-activity Factor IX variant (F9 Padua, R338L) at the albumin (Alb) locus in a novel hemophilia B (HB) rat model. The knock-in efficiency reaches 3.66%, as determined by droplet digital PCR (ddPCR). The clotting time is reduced to a normal level four weeks after treatment, and the circulating factor IX (FIX) level is gradually increased up to 52% of the normal level over nine months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through primer-extension-mediated sequencing (PEM-seq), no significant off-target effect is detected. This study not only provides a novel model for HB but also identifies a promising therapeutic approach for rare inherited diseases.

Effect of prepropeptide replacement on γ-carboxylation and activity of recombinant coagulation factor IX.

Based on observations indicating that the γ-carboxylase enzyme has a lower affinity for the protein C (PC) propeptide and that the γ-carboxylase region in the PC propeptide has a higher net charge, expression of recombinant chimeric factor IX (FIX) equipped with the PC propeptide was studied. The prepropeptide of FIX was replaced with that of PC by SOEing PCR and after cloning, recombinant pMT-prepro PC/FIX was transfected into insect Drosophila S2 cells. The expression and activity of expressed FIX were analyzed employing antigen and activity analyses 72 h of post-induction with copper. Higher secretion (1.2 fold) and activity (1.6 fold) levels were observed for chimeric prepro- PC/FIX in relation to wild-type FIX. Furthermore, after barium citrate precipitation, the evaluation of fully γ-carboxylated FIX indicated that more than 51% of the total FIX produced with the PC prepropeptide was fully γ-carboxylated, representing a substantial improvement (twofold) over a system employing the native FIX propeptide in which 25% of the protein is fully γ-carboxylated. The data illustrated that the expression of FIX using the PC propeptide led to much higher fully γ-carboxylated material, which is preferred to FIX constructs tolerating the sequence for the native FIX propeptide expressed in heterologous S2 systems.

GGCX mutants that impair hemostasis reveal the importance of processivity and full carboxylation to VKD protein function.

γ-Glutamyl carboxylase (GGCX) generates multiple carboxylated Glus (Glas) in vitamin K-dependent (VKD) proteins that are required for their functions. GGCX is processive, remaining bound to VKD proteins throughout multiple Glu carboxylations, and this study reveals the essentiality of processivity to VKD protein function. GGCX mutants (V255M and S300F) whose combined heterozygosity in a patient causes defective clotting and calcification were studied using a novel assay that mimics in vivo carboxylation. Complexes between variant carboxylases and VKD proteins important to hemostasis (factor IX [FIX]) or calcification (matrix Gla protein [MGP]) were reacted in the presence of a challenge VKD protein that could potentially interfere with carboxylation of the VKD protein in the complex. The VKD protein in the complex with wild-type carboxylase was carboxylated before challenge protein carboxylation occurred and became fully carboxylated. In contrast, the V255M mutant carboxylated both forms at the same time and did not completely carboxylate FIX in the complex. S300F carboxylation was poor with both FIX and MGP. Additional studies analyzed FIX- and MGP-derived peptides containing the Gla domain linked to sequences that mediate carboxylase binding. The total amount of carboxylated peptide generated by the V255M mutant was higher than that of wild-type GGCX; however, the individual peptides were partially carboxylated. Analysis of the V255M mutant in FIX HEK293 cells lacking endogenous GGCX revealed poor FIX clotting activity. This study shows that disrupted processivity causes disease and explains the defect in the patient. Kinetic analyses also suggest that disrupted processivity may occur in wild-type carboxylase under some conditions (eg, warfarin therapy or vitamin K deficiency).

Structural, functional, and immunogenicity implications of F9 gene recoding.

Hemophilia B is a blood clotting disorder caused by deficient activity of coagulation factor IX (FIX). Multiple recombinant FIX proteins are currently approved to treat hemophilia B, and several gene therapy products are currently being developed. Codon optimization is a frequently used technique in the pharmaceutical industry to improve recombinant protein expression by recoding a coding sequence using multiple synonymous codon substitutions. The underlying assumption of this gene recoding is that synonymous substitutions do not alter protein characteristics because the primary sequence of the protein remains unchanged. However, a critical body of evidence shows that synonymous variants can affect cotranslational folding and protein function. Gene recoding could potentially alter the structure, function, and in vivo immunogenicity of recoded therapeutic proteins. Here, we evaluated multiple recoded variants of F9 designed to further explore the effects of codon usage bias on protein properties. The detailed evaluation of these constructs showed altered conformations, and assessment of translation kinetics by ribosome profiling revealed differences in local translation kinetics. Assessment of wild-type and recoded constructs using a major histocompatibility complex (MHC)-associated peptide proteomics assay showed distinct presentation of FIX-derived peptides bound to MHC class II molecules, suggesting that despite identical amino acid sequence, recoded proteins could exhibit different immunogenicity risks. Posttranslational modification analysis indicated that overexpression from gene recoding results in suboptimal posttranslational processing. Overall, our results highlight potential functional and immunogenicity concerns associated with gene-recoded F9 products. These findings have general applicability and implications for other gene-recoded recombinant proteins.

Calumenin knockdown, by intronic artificial microRNA, to improve expression efficiency of the recombinant human coagulation factor IX.

OBJECTIVES: To improve the expression efficiency of recombinant hFIX, by enhancing its γ-carboxylation, which is inhibited by Calumenin (CALU), we used intronic artificial microRNAs (amiRNAs) for the CALU downregulation. METHODS: Two human CALU (hCALU)-specific amiRNAs were designed, validated and inserted within a truncated form of the hFIX intron 1, in either 3'- or 5'-untranslated regions of the hFIX cDNA, in an expression vector. After transfections of a human cell line with the recombinant constructs, processing of the miRNAs confirmed by RT-PCR, using stem-loop primers. The hFIX and hCALU expression assessments were done based on RT-PCR results. The Gamma(γ)-carboxylation of the expressed hFIX was examined by a barium citrate precipitation method, followed by Enzyme-Linked Immunosorbent Assay. RESULTS: Efficient CALU down regulations, with more than 30-fold decrease, occurred in the cells carrying either of the two examined the 3'-located amiRNAs. The CALU downregulation in the same cells doubled the FIX γ-carboxylation, although the transcription of the FIX decreased significantly. On the other hand, while the expression of the amiRNAs from the 5'-located intron had no decreasing effect on the expression level of CALU, the level of hFIX transcription in these cells increased almost twofold compared to the construct without amiRNA. CONCLUSION: The CALU downregulation, consistent with efficient hFIX γ-carboxylation, occurred in the cells carrying either of the two amiRNAs containing constructs, although it was affected by the locations of the amiRNA carrying introns, suggesting a possible need to optimize the conditions for the amiRNAs expression.