Factor V Research Articles

Decoding the complexity of on-target integration: characterizing DNA insertions at the CRISPR-Cas9 targeted locus using nanopore sequencing

BackgroundCRISPR-Cas9 technology has advanced in vivo gene therapy for disorders like hemophilia A, notably through the successful targeted incorporation of the F8 gene into the Alb locus in hepatocytes, effectively curing this disorder in mice. However, thoroughly evaluating the safety and specificity of this therapy is essential. Our study introduces a novel methodology to analyze complex insertion sequences at the on-target edited locus, utilizing barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, magnetic bead-based long amplicon enrichment, and nanopore sequencing.ResultsWe identified the expected F8 insertions and various fragment combinations resulting from the in vivo linearization of the double-cut plasmid donor. Notably, our research is the first to document insertions exceeding ten kbp. We also found that a small proportion of these insertions were derived from sources other than donor plasmids, including Cas9-sgRNA plasmids, genomic DNA fragments, and LINE-1 elements.ConclusionsOur study presents a robust method for analyzing the complexity of on-target editing, particularly for in vivo long insertions, where donor template integration can be challenging. This work offers a new tool for quality control in gene editing outcomes and underscores the importance of detailed characterization of edited genomic sequences. Our findings have significant implications for enhancing the safety and effectiveness of CRISPR-Cas9 gene therapy in treating various disorders, including hemophilia A.

Factor VIII as a Novel Biomarker for Diagnosis, Prognosis, and Therapy Prediction in Human Cancer and Other Disorders.

Coagulation factor VIII (FVIII) is an essential cofactor in the coagulation cascade, encoded by the F8 gene on the long arm of chromosome X (Xq28). FVIII is normally circulated in complex with Von Willebrand factor (VWF) and has relevant emerging extracoagulative functions. Dysregulation of FVIII is associated with tumor progression, and could be used as a novel biomarker for tumor screening and monitoring. In breast cancer, bladder cancer, colorectal carcinoma, esophageal carcinoma, hepatocellular carcinoma and lung cancer, F8 is regarded as an oncogene. In coronary heart disease, hemophilia A and liver disease, F8 dysregulation has been recognized as a potential biomarker for disease diagnosis and prognosis. However, the basis of these differential expression levels remains to be understood. In this review, which is a mixture of literature review and bioinformatics analysis we described the biological functions and characteristics of FVIII, and also its expression level in non-malignant disorders and various cancers.

De Novo Noninversion Variants Implicated in Sporadic Hemophilia A: A Variant Origin and Timing Study.

Sporadic hemophilia A (HA) enables the persistence of HA in the population. F8 gene inversion originates mainly in male germ cells during meiosis. To date, no studies have shown the origin and timing of HA sporadic noninversion variants (NIVs); herein, we assume that HA-sporadic NIVs are generated as a de novo variant. Of the 125 registered families with HA, 22 were eligible for inclusion. We conducted a linkage analysis using F8 gene markers and amplification refractory mutation system-quantitative polymerase chain reaction to confirm the origin of the sporadic NIVs (~0% mutant cells) or the presence of a mosaic variant, which requires further confirmation of the origin in the parent. Nine mothers, four maternal grandmothers, and six maternal grandfathers were confirmed to be the origin of sporadic NIVs, which most likely occurred in the zygote within the first few cell divisions and in single sperm cells, respectively. Three mothers had mosaic variants, which most likely occurred early in postzygotic embryogenesis. All maternal grandparents were free from sporadic NIV. In conclusion, F8 NIVs in sporadic HA were found to be caused primarily by de novo variants. Our studies are essential for understanding the genetic pathogenesis of HA and improving current genetic counseling.

Pleiotropic effects of different exonic nucleotide changes at the same position contribute to hemophilia B phenotypic variation

BackgroundThe disease-causing effects of genetic variations often depend on their location within a gene. Exonic changes generally lead to alterations in protein production, secretion, activity, or clearance. However, owing to the overlap between proteins and splicing codes, missense variants can also affect messenger RNA splicing, thus adding a layer of complexity and influencing disease phenotypes. ObjectivesTo extensively characterize a panel of 13 exonic variants in the F9 gene occurring at 6 different factor IX positions and associated with varying severities of hemophilia B (HB). MethodsComputational predictions, splicing analysis, and recombinant factor IX assays were exploited to characterize F9 variants. ResultsWe demonstrated that 5 (38%) of 13 selected F9 exonic variants have pleiotropic effects. Although bioinformatic approaches accurately classified effects, extensive experimental assays were required to elucidate and deepen the molecular mechanisms underlying the pleiotropic effects. Importantly, their characterization was instrumental in developing tailored RNA therapeutics based on engineered U7 small nuclear RNA to mask cryptic splice sites and compensatory U1 small nuclear RNA to enhance exon definition. ConclusionOverall, albeit a multitool bioinformatic approach suggested the molecular effects of multiple HB variants, the deep investigation of molecular mechanisms revealed insights into the HB phenotype–genotype relationship, enabling accurate classification of HB variants. Importantly, knowledge of molecular mechanisms allowed the development of tailored RNA therapeutics, which can also be translated to other genetic diseases.

Frequency and Association of Polymorphisms in F2, F7, and PROS1 Coagulation Genes with Disease Severity in Coronavirus Disease 2019.

Abnormal transcriptomic profiles of coagulation genes have been linked to coagulopathies in patients with coronavirus disease 2019 (COVID-19). The objective of the present study was to explore the frequency of genotypes and potential association of polymorphisms in genes encoding coagulation factors with the disease severity in COVID-19 patients.The patients were clinically categorized into four groups of COVID-19 disease severity (asymptomatic, mild, moderate, and severe). Three variants of genes, involving the coagulation genes rs3136516 (F2 gene), rs6042 (F7 gene), and rs6123 (PROS1 gene), were studied. Polymorphisms were genotyped by Sanger DNA sequencing.Most of the subjects had moderate COVID-19 infection (n = 53, 62.4%), followed by mild (n = 16, 18.8%), and severe infections (n = 15, 17.6%). The frequency of the rs3136516 AG genotype was considerably higher in non-ICU patients compared to ICU patients (51.3% vs 34.1%, OR 3.167, 95% CI 1.094-9.170, P = .031). Furthermore, the dominant genetic model (AA + AG vs GG) was significantly associated with a decreased probability of admission to the ICU in COVID-19 patients (OR 0.340, 95% CI 0.127 - 0.905, and P = .028). No other variants of the coagulation genes studied were found to be associated with the severity of COVID-19 disease, admission to the ICU, and mortality (P > .05).The rs3136516 AG genotype could predispose COVID-19 patients to increased disease severity and therefore admission to the ICU, while the dominant genetic model (AA + AG vs GG) of rs3136516 exerts a protective role.

АССОЦИАЦИЯ ПОЛИМОРФИЗМА ГЕНОВ СИСТЕМЫ ГЕМОСТАЗА С КЛИНИКО-ЛАБОРАТОРНЫМИ ПОКАЗАТЕЛЯМИ У ПАЦИЕНТОВ С ИНФАРКТОМ МИОКАРДА

The article presents the analysis of the association between polymorphic variants of the FGA, FGG, F2, F5, F11, F13, PAI-1, and GP6 genes and clinical, instrumental, and laboratory data in patients with myocardial infarction from the Republic of Belarus. It has been demonstrated that in patients with myocardial infarction, there exist statistically significant correlations between polymorphic variants of the genes F11 (rs2289252 and rs2036914), F13 (rs5985), F5 (rs6025), PAI-1 (rs1799889), FGG (rs2066865), and a variety of laboratory values and functional indicators of the myocardium. A comprehensive analysis of the genetic component (DNA polymorphism) and the outcomes of dynamic follow-up of the patient in hospital enable clinicians to accurately assess potential hazards of the occurrence and unfavorable progression of a number of diseases including myocardial infarction.

Molecular identification of the most frequent pathotypes of Escherichia coli in calves with diarrhoea in the Cajamarca region of Peru

Background: Colibacillosis caused by Escherichia coli causes significant economic losses in the livestock sector worldwide and is one of the calves' leading causes of diarrhoea. Aim: This study aimed to identify the most frequent E. coli molecularly pathotypes in calves with diarrhoea in six provinces of the Cajamarca region in the northern highlands of Peru. Methods: Twenty-eight herds of dairy cattle under a semi-intensive rearing system were evaluated; 95 samples were isolated from calves with diarrhoea up to the first month of life, 62 males and 33 females, during the rainy season. Results: The presence of virulence genes of E. coli strains was more prevalent in males; the astA (89.47%), st (83.15%) and f5 (57.89%) genes were more expressed, and the lt (17.89%) and stx2 (1.05%) genes were less expressed. The eae gene (21.05%) was more present in females. Conclusion: When E. coli strains express virulence genes astA, st and f5 and their atypical double, triple and quadruple combination between different observed pathotypes, they give rise to the formation of several pathotypes by the horizontal transfer of virulence genes, which can cause colibacillosis processes in more virulent calves, which is one of the most important causes of diarrhoea in calves in the region of Cajamarca, compromising the sanitary viability in the herds.

Patient with congenital factor VII deficiency undergoing brain tumor neurosurgery successfully treated with recombinant factor VIIa and fresh frozen plasma: A case report and literature review

Rationale: Congenital factor VII deficiency is the most common among rare bleeding disorders, characterized by spontaneous or traumatic bleeding. The clinical manifestation is heterogeneous, ranging from asymptomatic phenotype to life-threatening hemorrhages. Intracranial hemorrhage is a common complication of brain tumor neurosurgery, which significantly challenges the perioperative management of patients with hemostatic defects. Patient concerns: This report presented a 55-year-old man with congenital factor VII deficiency, who had no history of hemorrhage or family history. He underwent a craniotomy for the treatment of papillary craniopharyngioma. Diagnoses: The patient was diagnosed as papillary craniopharyngioma, factor VII deficiency, and atrial fibrillation. Interventions: To prevent bleeding, a total of 8 doses of recombinant activated factor VII and 1 dose of fresh frozen plasma were administered as the perioperative replacement therapy. This scheme was guided by a pharmacodynamic evaluation, laboratory tests, and imaging examinations. Outcomes: No excessive surgical bleeding was observed during the 22-day treatment. The patient was found to have compound heterozygous mutations, Ala304Thr (c.910G > A) and IVS5-2A > G (c.572-2A > G), in the F7 gene. Lessons: This is the first reported case in which surgical hemorrhage secondary to brain tumor resection was successfully controlled in the presence of congenital factor VII deficiency. Perioperative coagulation state, hemostasis, and thrombosis events should be closely observed, and the interval and dosage of recombinant factor VIIa should be adjusted accordingly.

Complete F9 Gene Deletion, Duplication, and Triplication Rearrangements: Implications for Factor IX Expression and Clinical Phenotypes.

Factor IX (FIX) plays a critical role in blood coagulation. Complete deletion of F9 results in severe hemophilia B, whereas the clinical implications of complete F9 duplication and triplication remain understudied. To investigate the rearrangement mechanisms underlying complete F9 deletion (cases 1 and 2), duplication (cases 3 and 4), and triplication (case 5), and to explore their association with FIX expression levels and clinical impacts. Plasma FIX levels were detected using antigen and activity assays. CNVplex technology, optical genome mapping, and long-distance polymerase chain reaction were employed to characterize the breakpoints of the chromosomal rearrangements. Cases 1 and 2 exhibited FIX activities below 1%. Case 3 displayed FIX activities within the reference range. However, cases 4 and 5 showed a significant increase in FIX activities. Alu-mediated nonallelic homologous recombination was identified as the cause of F9 deletion in case 1; FoSTeS/MMBIR (Fork Stalling and Template Switching/microhomology-mediated break-induced replication) contributed to both F9 deletion and tandem duplication observed in cases 2 and 3; BIR/MMBIR (break-induced replication/microhomology-mediated break-induced replication) mediated by the same pair of low-copy repeats results in similar duplication-triplication/inversion-duplication (DUP-TRP/INV-DUP) rearrangements in cases 4 and 5, leading to complete F9 duplication and triplication, respectively. Large deletions involving the F9 gene exhibit no apparent pattern, and the extra-hematologic clinical phenotypes require careful analysis of other genes within the deletion. The impact of complete F9 duplication and triplication on FIX expression might depend on the integrity of the F9 upstream sequence and the specific rearrangement mechanisms. Notably, DUP-TRP/INV-DUP rearrangements significantly elevate FIX activity and are closely associated with thrombotic phenotypes.

Association evaluations of oral anticoagulants with dementia risk based on genomic and real-world data

BackgroundSeveral observational studies have suggested that oral anticoagulants (OACs) might reduce the risk of dementia in the elderly, but the evidence is inconclusive. And the consistency of this relationship across different OAC classes and dementia subtypes is still uncertain. MethodsTo comprehensively evaluate this association, we applied Mendelian randomization (MR) combined with pharmacovigilance analysis. MR was used to assess the associations between genetic proxies for three target genes of OACs (VKORC1, F2, and F10) and dementia, including Alzheimer's disease (AD) and vascular dementia (VaD). This genetic analysis was supplemented with real-world pharmacovigilance data, employing disproportionality analysis for more reliable causal inference. ResultsIncreased expression of the VKORC1 gene was strongly associated with increased risk of dementia, especially for AD (OR = 1.28, 95% CI = 1.14–1.43; p value < 0.001). Based on pharmacovigilance data, vitamin K antagonists (VKAs, inhibitors targeting VKORC1) exhibited a protective effect against dementia risk (ROR = 0.43, 95% CI = 0.28–0.67). Additional sensitivity analyses, including different MR models and cohorts, supported these results. Conversely, no strong causal associations of genetically proxied F2 and F10 target genes with dementia and its subtypes were found. ConclusionsThis study reveals that the inhibition of genetically proxied VKORC1 expression or VKAs exposure is associated with a reduced risk of Alzheimer's dementia. However, there is little evidence to support similar associations with direct oral anticoagulants (F2 inhibitors and F10 inhibitors). Further research is warranted to clinically validate our findings.

Leiden mutation (rs6025) in a severe COVID-19 pneumonia patient with Down syndrome: a clinical case

COVID-19 was first reported in December 2019 in Wuhan (Hubei Province, China). Later, the pandemic of this disease took the world by storm, challenging the medical community. Its clinical manifestations vary from asymptomatic to a severe course that requires hospitalization and intensive therapy with oxygen support. The mortality rate in patients with a severe course of COVID-19 can exceed 50% The majority of fatal cases of COVID-19 were associated with thrombotic events, despite the prophylactic use of anticoagulant therapy. Numerous theoretical overviews and research articles indicate the need for genetic testing in patients with COVID-19 to determine the genetic profile of proteins involved in thrombophilia. According to the researchers, the Leiden mutation (G1691A, rs6025) of the FV gene is one of the promising candidates for testing. The aim of the work was to demonstrate the clinical features of the severe course of COVID-19 in the presence of the Leiden mutation. 58 patients with COVID-19 of the intensive care unit were genotyped. The Leiden mutation in the heterozygous state was found only in one patient, who had Down syndrome. The Leiden mutation was detected with a frequency of 1.72% in the investigated group. The described clinical case clearly showed that individuals with Down syndrome, associated with hereditary thrombophilia are at risk of undesirable clinical consequences in the treatment of COVID-19. The condition of the patient with the Leiden mutation was severe when admitted to the hospital. The score according to the sequential organ failure assessment scale was 2 points. Bilateral multisegmental pneumonia was detected on the X-ray. On the second day after admission, due to the development of acute respiratory distress syndrome and multiple organ failure, the patient was transferred to the intensive care unit, where he received oxygen therapy through a facial mask. Medical treatment was carried out according to the protocol: non-steroidal anti-inflammatory drugs, antibacterial therapy, anticoagulants, sympatho¬mimetics, and glucocorticosteroids. Despite the medical measures taken, progression of respiratory failure, renal failure, and portal hypertension was noted. On the 11th day, the patient developed asystole. Resuscitation measures were unsuccessful. Thus, the described case of a severe course of COVID-19 in a carrier of a heterozygous variant of the Leiden mutation with Down syndrome confirms the recommendations regarding the need for genetic testing for thrombophilia in high-risk groups and the appointment of personalized measures to prevent complications.

Variant spectrum of F8 and F9 in hemophilia patients from southern China and 26 novel variants.

Hemophilia, an X-linked recessive disorder, is characterized by spontaneous or trauma-induced prolonged bleeding. It is classified as hemophilia A when caused by variants in the F8 gene, and hemophilia B when caused by F9 variants. Few studies have described hemophilia variants in the Chinese population. This study aimed to investigate the clinical and genetic profiles of 193 hemophilia patients from southern China. Utilizing Sanger sequencing, multiplex ligation-dependent probe amplification, gap detection, long-range PCR, and multiplex PCR, we identified both F8 and F9 gene variants. Pregnant women with a history of hemophilia A offspring underwent amniocentesis or villus sampling for the variant detection. Variants in F8 and F9 were pinpointed in 183 patients, with 26 being novel discoveries. Notably, genetic testing was absent in the initial evaluation of 133 out of 161 patients, leading to a protracted average definitive diagnosis timeline of 2years. Remarkably, two hemophilia A cases with anticipated severe phenotypes due to protein-truncating variants presented with only moderate or mild clinical manifestations. Among the 40 fetuses tested, 34 were males, with 17 exhibiting hemizygous variants in the F8 gene. Our results contribute to the broader understanding of F8 and F9 variant spectrum and highlight the underuse of genetic analyses in southern China.

Genotype-Phenotype Relationship among 785 Unrelated White Women with Inherited Congenital Factor VII Deficiency: A Three-Center Database Study.

Congenital factor VII (FVII) deficiency, a rare bleeding disorder resulting from mutations in the F7 gene with autosomal recessive inheritance, exhibits clinical heterogeneity that lacks a strong correlation with FVII:C levels. The objective of this study was to discern genetic defects and assess their associations with the clinical phenotype in a substantial cohort comprising 785 white women exhibiting FVII:C levels below the age-dependent cut-off percentage. Individuals with verified inherited factor VII deficiency underwent i) genotyping using the Sanger method and multiplex ligation-dependent probe amplification (MLPA) to identify F7 mutations, including common polymorphic variants. Additionally, they were ii) categorized based on clinical bleeding scores (BS). Thrombophilic variants and blood groups were also determined in the study participants. The probands in this study encompassed both asymptomatic individuals (referred for a laboratory investigation due to recurrent prolonged prothrombin time; n = 221) and patients who manifested mild, moderate, or severe bleeding episodes (n = 564). The spectrum of bleeding symptoms included epistaxis, gum bleeding, gastrointestinal bleeding, hematuria, postoperative bleeding, and gynecologic hemorrhage. The median ISTH bleeding score (BS) recorded within a two-year period prior to the work-up was 2 (0-17). Notably, this score was significantly higher in symptomatic women compared to their asymptomatic counterparts (3 versus 0; p < 0.001). The corresponding PBAC score before hormonal treatment stood at 225 (5-1200), exhibiting a positive correlation with the ISTH BS (rho = 0.38; p = 0.001). Blood group O was more prevalent in symptomatic women compared to asymptomatic individuals (58 versus 42%; p = 0.01). Among the 329 women (42%), known and novel mutations in the F7 gene, encompassing coding regions, exon/intron boundaries, and the promoter region, were identified, while common polymorphisms were detected in 647 subjects (95%). Logistic regression analysis, adjusted for clinical and laboratory data (including blood group, FVII activity, the presence of F7 gene mutations and/or polymorphisms, thrombophilia status, and additional factor deficiencies) revealed that older age at referral (increase per year) (odds/95% CI: 1.02/1.007-1.03), the presence of blood group O (odds/95% CI: 1.9/1.2-3.3), and the coexistence of further bleeding defects (odds/95% CI: 1.8/1.03-3.1) partially account for the differences in the clinical bleeding phenotype associated with FVII deficiency. The clinical phenotype in individuals with FVII deficiency is impacted by factors such as age, blood group, and the concurrent presence of other bleeding defects.

Analysis of a Chinese pedigree affected with Hereditary coagulation factor Ⅻ deficiency due to compound heterozygous variants of F12 gene

To analyze the clinical phenotypes and genetic variants of a Chinese pedigree affected with Hereditary coagulation factor Ⅻ (FⅫ) deficiency. A pedigree presented at the First Affiliated Hospital of Air Force Medical University on December 24,2021 was selected as the study subject. Activated partial thromboplastin time (APTT) and coagulation factor Ⅻ activity (FⅫ:C) were determine by a clotting method, and FⅫ antigen was detected with an ELISA assay. Following the extraction of genomic DNA, all exons and flanking regions of the F12 gene were subjected to Sanger sequencing. Clustalx-2.1-win, PROVEAN and Swiss-PDB Viewer software was used to analyze the conservation of amino acids at the variant sites, impact of of the variants on the amino acid substitutions and the protein structure. The APTT of the proband has prolonged to 70.2 s. Her FⅫ:C and FⅫ:Ag have decreased to 12% and 13%, respectively. DNA sequencing revealed that the proband has harbored c.346G>A (p.Gly97Ser) and c.1583C>A (p.Ser509Tyr) heterozygous compound missense variants in exons 5 and 13 of the F12 gene, respectively. Her father and sister were heterozygous carriers for the c.346G>A (p.Gly97Ser) variant, whilst her mother and brother were heterozygous for the c.1583C>A (p.Ser509Tyr) variant. The c.346G>A (p.Gly97Ser) and c.1583C>A (p.Ser509Tyr) compound heterozygous variants of the F12 gene probably underlay the pathogenesis of hereditary coagulation FⅫ deficiency in this pedigree.

Homozygous missense variant F12 (Gly506Asp) associated with severe factor XII deficiency: a case report

BackgroundFactor XII deficiency can be related to either homozygous or compound heterozygous pathogenic variants in the F12 gene. The disease is commonly known as Hageman trait and is inherited in both autosomal recessive or dominant patterns. Clinically, factor XII deficiency is not associated with bleeding but conversely has been linked to thrombotic events, recurrent pregnancy loss, and hereditary angioedema. Molecular data of F12 deficiency are scarce and have revealed varying results between cases. However, most of the reported variants are missense mutations, gross deletions, or small insertion. Factor XII deficiency has been reported in the Saudi population in several studies, either as isolated case reports or included within the studies of rare bleeding factors deficiency. However, molecular data are lacking as no case report of genetic studies related to factor XII deficiency has been published in our local population, to the best of our knowledge.Case reportHerein we describe a homozygous missense variant involving exon 12 within F12 gene (5:176,830,269 G>A; p.Gly506Asp) in a 36-year-old Saudi multiparous female referred from the surgical clinic with significantly high activated partial thromboplastin time during preoperative assessment for sleeve gastrectomy. The patient had no history of bleeding episodes during the previous deliveries nor any tooth extractions. She had single event of spontaneous abortion during the 15th week of gestation without any bleeding complication. There was no history of thrombosis or skin manifestations, and she was not taking any medicines. There was no family history of bleeding or thrombosis. Family history revealed consanguinity as the parents are first-degree cousins. Physical examination was unremarkable. Upon investigation, the prolonged activated partial thromboplastin time was fully corrected by a 1:1 mixing study with normal pool plasma while lupus anticoagulant tests were negative. Factor assays and von Willebrand factor tests are all within normal ranges except for factor XII, which was severely deficient. A homozygous missense variant involving exon 12 within F12 gene (5:176,830,269 G>A; p.Gly506Asp) was identified.ConclusionF12 (5:176,830,269 G>A; p.Gly506Asp) variant is likely to be a pathogenic variant among homozygous factor XII-deficient patients. Genetic counseling and management of the patients and families should be based on clinical evaluation.

To Test or Not to Test: Routine Thrombophilia Diagnostic Screening of Women with Reproductive Failures.

Recurrent reproductive failure is a global health issue affecting a significant number of women. Thrombophilias have been implicated as a possible cause. Inherited thrombophilias include a single nucleotide variant on factor V Leiden and prothrombin. The aim of this study was to evaluate the association between the following single nucleotide variants: factor V Leiden (c.1601G>A), the prothrombin gene (c.*97G>A) and the reproductive failure in the Polish population. The study was conducted in a group of 545 patients with recurrent pregnancy loss, RPL (≥2 miscarriages), and in a group of 641 patients with infertility. The distribution of genotypes for the selected variants were determined by RFLP-PCR and by the real-time PCR method. A variant of the F5 gene was found in 5.14% of patients with RPL and in 6.08% of infertile women. A variant of the F2 gene was identified in 0.73% of patients with RPL and in 2.03% of women with infertility. The frequency in the study groups did not differ from that in the general population. No association between the studied variants of the F5 gene or the F2 gene and the predisposition to reproductive wastage was found. Recommendations for routine thrombophilia testing in women with recurrent miscarriages should be revisited. The decision regarding testing should be made individually depending on additional factors indicating an increased risk of venous thromboembolism.

RAAV8-PC Gene Therapy for Severe Protein C Deficiency Using a Novel Murine Model

Introduction Protein C (PC) is a vitamin K-dependent protein, synthesized in the liver. The PROC gene is located on chromosome 2 at position 2q13-14 and encodes 9 exons. Severe protein C deficiency (SPCD) is a rare devastating autosomal recessive disease that presents at birth with purpura fulminans (PF) and is associated with high rates of morbidity and mortality. As current management options are limited and burdensome, there is a large unmet need for new treatments including curative therapies. Because small increases in PC may significantly ameliorate the clinical presentation of SPCD and potentially prevent PF, we hypothesize that SPCD could be treated by PC gene therapy (GT). Aim To establish a murine model enabling the survival of SPCD mice and to evaluate PC expression after recombinant adeno-associated viral vector- PC (rAAV8-PC) gene therapy. Methods The study was conducted with C57Bl/6 PC heterozygous mice (a kind gift of Prof. Weiler, [Wisconsin]), severe hemophilia A (HA) mice and wild type C57Bl/6 mice. Genotyping was performed by PCR using appropriate primers for murine PROC and F8 genes. rAAV8-PC was produced by transfecting HEK293 cells with PX-680 as rAAV helper, AAV8 and PC plasmid with an alpha-1 antitrypsin promotor. Extraction and purification were performed using a commercial kit. Viral concentration was determined by a designated commercial qPCR assay. rAAV8 - PC (10 12 /kg Vg of AAV8-PC) was injected via the tail vein to 5-8 weeks old mice. Blood samples were collected from the facial vein into 1.5 ml tubes containing 0.109M sodium citrate at blood: anticoagulant ratio of 1:1 (vol/vol). PC plasma levels were measured prior and following rAAV8-PC infusion using a commercial ELISA assay for PC antigen. Thrombin generation was measured in plasma samples using a modification of the calibrated automated thrombinoscope assay. Results As expected, breeding of heterozygous PC mice yielded only 3/200 homozygous SPCD mice (PC levels= 0), that survived for less than 24 hours. Thus, GT for heterozygous PC mice was considered; however, PC plasma levels in heterozygous PC mice were not substantially different from those in WT mice (median levels in heterozygous PC and WT mice: 216 ng/ml vs. 190 ng/ml, respectively), which precluded this option. Hence, with the aim of increasing the survival rate and duration of homozygous SPCD mice, we decided to rebalance the severe thrombotic disorder of SPCD, by induction of a genetically engineered combination with the severe coagulopathy of Hemophilia. We established an alternative SPCD murine model by breeding severe HA mice with heterozygous PC mice. This approach yielded homozygous SPCD mice with severe HA (PROC-/F8-), exhibiting a significantly prolonged survival compared to homozygous SPCD mice with normal F8 levels (over 90% survived over 6 weeks). Genotyping and ELISA of PC levels confirmed PC homozygosity (null mutation) as well as severe HA. Nineteen PROC-/F8- mice were injected with rAAV8-PC into the tail vein and followed for at least 12 weeks. Nine mice died during blood sampling at different time points and were excluded from further analysis. Individual responses to PC- GT were noted among the remaining 10 mice, reflected by non-uniform PC expression measured by ELISA (initial PC increase, time to peak and PC peak plasma levels). An increase in PC level was noted at week 2-5 post vector infusion, reaching PC peak level between week 2-10 post GT. Table 1 summarizes the individual patterns of 10 PROC-/F8-mice that underwent GT. The functional activity of PC in the murine plasma following GT was supported by thrombin generation assays. Analyses performed in PROC-/F8-mice before and after GT demonstrated endogenous thrombin potential (ETP) and peak hight decrease following GT, as compared to untreated PROC-/F8-mice. Conclusion We present here a novel murine model of combined PC and factor VIII deficiency that supports the survival of SPCD mice and enables PC gene therapy studies. Our results provide the first proof of concept that infusion of rAAV8-PC results in increased PC plasma levels in mice, and may serve as a basis for future studies investigating GT for SPCD. The concept of modulating a severe genetic prothrombotic disorder, which is nearly incompatible with life, by genetic combination with severe coagulopathy may offer a valuable tool enabling investigation and potential development of treatment modalities for the prothrombotic disorder.

Liver Sinusoidal Endothelial Cells but Not Hepatocytes Express a Fluorescent Reporter Under the Endogenous F8 Promoter in a New Hemophilia a Mouse Model

Hemophilia A (HA) is an inherited bleeding disorder caused by mutations in the F8 gene leading to a deficiency in coagulation factor VIII (FVIII). Treatment of patients with HA has centered around replacement of FVIII protein. Gene therapy provides the possibility of permanently altering the HA phenotype. Gene therapy for HA induces FVIII expression in hepatocytes, despite the well supported belief that FVIII is not made within hepatocytes but rather in liver sinusoidal endothelial cells (LSECs). Ectopic expression of FVIII in hepatocytes has been implicated in mild toxicities and FVIII decline observed in adeno-associated viral (AAV) vector-based gene therapy, the causes of which remain unknown. Extrahepatic production of FVIII, e.g. in lymphoid tissues, has been controversial. A better understanding of where FVIII is produced is needed to improve gene therapy for HA and inform development of other therapeutic interventions. We developed in collaboration with Jackson Laboratory a new strain of hemophilic mice on the C57BL/6J background expressing the fluorescent protein mScarlet under the endogenous F8 promoter. Using the CRISPR/Cas9 gene editing system, we inserted a transgene encoding mScarlet into exon 1 of the mouse F8 gene through homology-directed repair. We selected mScarlet for its relative brightness and stability, to maximize the chances of detecting its expression despite weak F8 promoter activity. The donor DNA consisted of two homology arms flanking the mScarlet-encoding transgene followed by the bovine growth hormone polyadenylation [poly(A)] signal. Insertion of the transgene at the first F8 codon in exon 1 and inclusion of poly(A) ensured knock-out of F8. Male animals showed a bleeding phenotype consistent with other HA mouse strains, such as C57BL/6J HA and B6/129 HA. We performed retro-orbital bleeding and tested mouse plasma samples using the activated partial thromboplastin time (aPTT) assay. Animals expressing mScarlet (n=4) and C57BL/6J HA mice (n=3) had similarly prolonged aPTT (mean 57 seconds) compared to non-hemophilic C57BL/6J mice (n=5, 41 seconds). Production of mScarlet was first evaluated in the hepatocytes and LSECs of mScarlet mice (n=3) using flow cytometry. The livers were perfused in situ via the portal vein with a perfusion and collagenase medium. The livers were then processed into single cell suspensions, and hepatocytes and LSECs were separated by centrifugation. An anti-CD31 antibody was used to identify LSECs. Hepatocytes were identified by exclusion of CD45.2 + cells. CD31 +cells from mScarlet mice but not from control C57BL/6J mice expressed mScarlet. Neither mouse strain had detectable mScarlet in hepatocytes. Flow cytometry was then used to evaluate mScarlet in the splenic endothelial cells (ECs) and leukocytes. Using in situtranscardiac perfusion, mScarlet or control C57BL/6J mice received a perfusion and collagenase medium. Following processing into single cell suspensions, ECs were isolated from other splenic cells by magnetic separation using CD31 microbeads. Anti-CD31 and anti-CD45.2 antibodies were used to identify ECs and splenic leukocytes, respectively. Neither cell type from mScarlet or control mice had detectable mScarlet. We next evaluated mScarlet expression via fluorescent microscopy of liver and spleen sections. Before sectioning, tissues were fixed using in situ transcardiac perfusion fixation. Since mScarlet fluorescence was not detectable in tissue sections, we employed an anti-mScarlet antibody. Anti-mScarlet binding was detectable in liver sections from mScarlet but not control C57BL/6J mice. The staining pattern was consistent with the sinusoidal arrangement of hepatic capillaries running between rows of hepatocytes toward central venules. We found the same staining pattern using an anti-CD31 antibody, supporting that the mScarlet-expressing cells were LSECs but not hepatocytes. No anti-mScarlet binding was detected in the spleens from either mScarlet or C57BL/6J mice. We propose that FVIII is produced in LSECs but not in hepatocytes, splenic endothelial cells, splenic or hepatic leukocytes. Our novel mouse model expressing a well-defined and readily detectable reporter protein overcomes the limitations of immunohistochemistry using anti-FVIII antibodies, which complicated previous studies on the cellular origins of FVIII. Further investigation of extrahepatic FVIII production is ongoing.

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

BackgroundHemophilia 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. MethodsInitially, 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. ResultsWe 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. ConclusionsKnock-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.

Four-Year Follow-up of the Alta Study, a Phase 1/2 Study of Giroctocogene Fitelparvovec (PF-07055480/SB-525) Gene Therapy in Adults with Severe Hemophilia Α

Introduction: Hemophilia A is an X-linked ( F8 gene) recessive disorder of hemostasis that results in insufficient factor VIII (FVIII) activity. Adeno-associated virus (AAV)-based gene therapy enables delivery of a modified F8 cDNA, allowing synthesis of functional endogenous FVIII, which prevents bleeding events. We present updated results with nearly 4 years of follow-up on an ongoing gene therapy study in participants with severe hemophilia A (FVIII activity &amp;lt;1%). Methods:The phase 1/2 Alta study (NCT03061201) is a dose-ranging study of giroctocogene fitelparvovec (PF-07055480, previously called SB-525), a recombinant AAV serotype 6 vector encoding a modified B-domain-deleted F8 coding sequence. Four ascending doses of giroctocogene fitelparvovec (9e11, 2e12, 1e13, and 3e13 vg/kg) were infused into adults aged ≥18 years with severe hemophilia A across 4 cohorts (n=2 each). The high-dose (3e13 vg/kg) cohort was expanded to 5 participants. Key endpoints included safety, circulating FVIII activity, use of FVIII replacement therapy, and frequency of bleeding events. Results: Eleven male participants were enrolled in the study (mean [SD] age, 30.3 [7.8] years; White, 81.8%). As of the cutoff date (May 19, 2023), participants had been followed for 153 to 290 weeks. Two participants left the study after Week 156. Of the remaining, 1 participant had not yet completed 4 years (208 weeks). The most common treatment-related adverse events (AEs) reported in the high-dose cohort (n=5) were elevated liver enzymes and infusion-related reactions: increased alanine aminotransferase (ALT; n=3 [60.0%]), increased aspartate aminotransferase (AST; n=2 [40.0%]), pyrexia (n=3 [60.0%]), and tachycardia (n=2 [40.0%]). Treatment-related serious AEs were reported in 1 participant in the high-dose cohort who experienced hypotension and fever, with onset ≈6 h after infusion; the events fully resolved with treatment. AEs (all causality) of ALT increases requiring ≥7 days of corticosteroids were observed in 4 of 5 participants in the high-dose cohort. ALT elevations were managed with tapering courses of corticosteroids (median duration: 56 days; range: 7-135 days), with maintenance of efficacious levels of FVIII activity. Participants in the high-dose cohort have not required steroids since Week 65, have had ALT values in the normal range (follow-up: 156-208 weeks) and normal findings via liver MRI (follow-up: 104-208 weeks). No participant developed a confirmed inhibitor to FVIII. No thrombotic events or liver masses have been detected. Of the 5 participants in the high-dose cohort, 2 had data available through Week 208 and FVIII activity was maintained in the mild to normal range ( Table), consistent with Week 156 results. Of those without Week 208 data, 2 had data through Week 182. One participant maintained FVIII activity in the mild range (14.1% and 24.1% of normal, measured with a chromogenic and 1-stage assay, respectively); the other had FVIII activity of 3.1% and 7.2%. The remaining participant left the study after Week 156, with FVIII activity maintained in the mild range (11.8% and 22.9%). In the high-dose cohort, the mean annualized total bleeding rate [(number of all bleeding episodes starting 3 weeks after study drug infusion) / (observation period in years)] was 0 for the first year post infusion and 1.2 (SD 2.58) throughout the total duration of follow-up. In this cohort, the participant with the lowest FVIII activity level experienced a total of 22 bleeds, with 21 necessitating treatment (8 traumatic; 7 spontaneous; 6 unknown). The other 4 participants had no or very minimal bleeds, including 1 who experienced a bleed in a target joint. No participants in the high-dose cohort have resumed prophylaxis. Conclusion:A single infusion of giroctocogene fitelparvovec gene therapy in participants with severe hemophilia A remains generally well tolerated over a period of nearly 4 years post infusion, with associated increases in FVIII levels in the moderate to normal range, without sustained AEs and with no AEs associated with increased liver function tests since Week 59. The ongoing phase 3 study (NCT04370054) in a larger cohort will provide more long-term data on the safety and durability of giroctocogene fitelparvovec in participants with moderately severe to severe hemophilia A.