A Dedifferentiated Sinusoidal Endothelium Impacts Liver-Directed Gene Transfer in Hemophilia-a Mice

Abstract Hemophilia A is an X-linked recessive bleeding disorder that affects 1 in 5000 males and is caused by procoagulant factor VIII deficiency. Affected people are at danger of spontaneous bleeding into organs, which can be fatal and lead to persistent damage. Current therapy includes intravenous infusion of FVIII protein concentrate which carries the danger of transmitting blood-borne diseases. As a result of recent advancements in liver-directed gene transfer, gene therapy based innovative strategy for treating hemophilia has emerged. In patients with severe hemophilia B, intravenous infusion of an adeno-associated viral (AAV) vector encoding factor IX (FIX) under the control of a liver-directed promoter resulted in expression of FIX for a considerable period of time. In hemophilia-A patients, gene treatment utilizing AAV vectors has demonstrated to be less effective than Hemophilia B due to the size of the F8 coding sequence and the decreased release of FVIII protein. Among other concerns high immunogenicity of FVIII with 25-30% of hemophilia A patients forming inhibitors and overexpression of FVIII in hepatocytes triggering a cellular stress response are significantly challenging. A phase 1 clinical trial is now being conducted to examine the AAV8 induced liver directed gene expression strategy to circumvent these challenges. The Factor VIII null mouse has been effective in understanding the disease pathogenesis as well as the development of liver directed novel gene therapy techniques to treat hemophilia. FVIII is predominantly produced in the liver. Thus, liver directed adenoviral and retroviral vectors have been studied by several groups to understand the gene delivery method in hemophilia. A few of these studies have shown limited effectiveness in hemophilia animal models. Although the coagulation anomaly seen in hemophilia murine model was completely repaired immediately after liver directed adenovirus-mediated treatment, the effect was transient. Additionally, adeno associated virus (AAV8)-FVIII overexpression has been associated with increased cellular stress. In this study we evaluated the stability and efficacy of liver driven gene transfer mechanism in FVIII null mouse using recombinant AAV8 vector. Recombinant AAV8 vector delivered through the systemic circulation successfully transduces to target tissues via passing through the permeable barrier of sinusoidal endothelial cell. The bidirectional passage through sinusoidal endothelial cell is mainly supported by the presence of discontinuous fenestrated endothelium. Remarkably, we found that liver directed gene transfer was significantly delayed in FVIII null mice. Using quantitative liver intravital imaging we found that upon AAV8-GFP administration liver sinusoidal endothelial cells shows increased apoptosis. Moreover, structural analysis of the liver sinusoidal endothelial cells using intravital and electron micrograph imaging showed significant structural functional difference in liver sinusoidal endothelial cells of FVIII KO mouse. Work is currently underway to understand how absence of FVIII can affect the LSECs. In conclusion, detailed molecular characterization of LSEC-mediated liver directed gene transfer in a hemophilia mouse model is critical for understanding the efficacy and stability of gene-based hemophilia treatment. Disclosures Sundd: Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring Inc: Research Funding.

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960. Lentiviral Modification of Liver Sinusoidal Endothelial Cells (LSEC) for Cell and Gene Therapy in Hemophilia A

Molecular Therapy ◽

10.1016/s1525-0016(16)45166-x ◽

2007 ◽

Vol 15 ◽

pp. S366

Keyword(s):

Gene Therapy ◽

Endothelial Cells ◽

Hemophilia A ◽

Liver Sinusoidal Endothelial Cells ◽

Sinusoidal Endothelial Cells ◽

Cell And Gene Therapy

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Molecular Profiling of Liver Sinusoidal Endothelial Cells in Comparison to Hepatocytes: Reflection on Which Cell Type Should Be the Target for Gene Therapy

Hämostaseologie ◽

10.1055/a-1282-2286 ◽

2020 ◽

Vol 40 (S 01) ◽

pp. S26-S31

Author(s):

Osman El-Maarri ◽

Muhammad Ahmer Jamil ◽

Johannes Oldenburg

Keyword(s):

Gene Therapy ◽

Endothelial Cells ◽

Hemophilia A ◽

Molecular Profile ◽

Liver Sinusoidal Endothelial Cells ◽

Sinusoidal Endothelial Cells ◽

Genome Wide ◽

Human Factor Viii ◽

Underlying Mechanisms ◽

Methylation Profiling

AbstractHuman factor VIII (FVIII), which deficiency leads to hemophilia A, is largely synthesized and secreted by the liver sinusoidal endothelial cells (LSECs). However, the characteristics of these cells that secrete FVIII are not well known. We have previously reported that based on genome-wide expression and CpG methylation profiling, LSECs have a distinct molecular profile that distinguishes them from other endothelial cells. Hepatocytes are targeted by gene therapy protocols to treat hemophilia A. However, the hepatocyte is not the natural site for FVIII synthesis and current gene therapy protocols are eliciting immune responses that require immune suppression with corticosteroid therapy in a fairly high proportion of patients over a significant period of time. Cellular stress because of ectopic FVIII expression and codon optimization are discussed as potential underlying mechanisms. Here, we highlight the molecular differences between LSECs and hepatocytes.

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Liver Sinusoidal Endothelial Cells as Possible Vehicles for Gene Therapy: A Comparison Between Plasmid-Based and Lentiviral Gene Transfer Techniques

Endothelium ◽

10.1080/10623320802174464 ◽

2008 ◽

Vol 15 (4) ◽

pp. 165-173 ◽

Cited By ~ 5

Author(s):

Jesus Paez-Cortez ◽

Ramon Montano ◽

John Iacomini ◽

Jose Cardier

Keyword(s):

Gene Therapy ◽

Endothelial Cells ◽

Gene Transfer ◽

Liver Sinusoidal Endothelial Cells ◽

Sinusoidal Endothelial Cells ◽

Gene Transfer Techniques

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FcγRIIB on liver sinusoidal endothelial cells is essential for antibody-induced GPVI ectodomain shedding in mice

Blood ◽

10.1182/blood-2016-05-714378 ◽

2016 ◽

Vol 128 (6) ◽

pp. 862-865 ◽

Cited By ~ 5

Author(s):

David Stegner ◽

Michael Popp ◽

Viola Lorenz ◽

Jacqueline K. Wax ◽

J. Engelbert Gessner ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Sinusoidal Endothelial Cell ◽

Liver Sinusoidal Endothelial Cell ◽

Ectodomain Shedding ◽

Liver Sinusoidal Endothelial Cells ◽

Sinusoidal Endothelial Cells ◽

Key Points

Key Points Antibody-induced shedding of platelet GPVI in vivo and the associated transient thrombocytopenia depend on liver sinusoidal endothelial cell-expressed FcγRIIB.

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First-in-human Gene Therapy Study of AAVhu37 Capsid Vector Technology in Severe Hemophilia A

Blood ◽

10.1182/blood-2019-125764 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4630-4630

Author(s):

Steven Pipe ◽

Michael Becka ◽

Elke Detering ◽

Konstantina Vanevski ◽

Toshko Lissitchkov

Keyword(s):

Gene Therapy ◽

Gene Transfer ◽

Research Funding ◽

Hemophilia A ◽

Severe Hemophilia ◽

Care Providers ◽

Dose Cohort ◽

Fviii Activity ◽

Equity Ownership ◽

Hemostatic Efficacy

Background: Gene therapy for hemophilia A has the potential to reduce the treatment burden for care-providers and patients, by eliminating the need for regular factor VIII (FVIII) prophylaxis through the long-term expression of endogenous FVIII at levels sufficient to provide bleed protection. BAY 2599023 (AAVhu37FVIII) is a non-replicating adeno-associated virus (AAV) vector, based on the AAV serotype hu37, which contains a single-stranded DNA genome encoding a B-domain deleted FVIII, under the control of a liver-specific promotor/enhancer combination optimized for transgenic expression. The AAVhu37 capsid is a member of the hepatotropic Clade E family and has been selected based on nonclinical studies demonstrating efficient liver-directed FVIII gene transfer, favorable biodistribution as well as durable FVIII expression. BAY 2599023 is the first clinical-stage AAV gene therapy vector based on the AAVhu37 serotype. This analysis reports safety and FVIII activity following a single intravenous infusion of BAY 2599023 in the first-dose cohort of a phase I/II open-label, first time in human dose-finding study (NCT03588299) of previously treated, severe hemophilia A patients. Patients/Methods: Two participants were enrolled sequentially; each received a single infusion of AAVhu37 (0.5 x 1013 GC/kg). Patients were males ≥18 years with no history of FVIII inhibitor development, no detectable immunity to the AAVhu37 capsid, and >150 exposure days to FVIII products. Primary endpoints were adverse events (AEs), serious AEs and AEs/SAEs of special interest (S/AESI); the secondary endpoint was change in FVIII activity from baseline. Informed patient consent, and ethics committee approval at each local site, were obtained. Results: Following more than 15 weeks of safety observation, no SAEs, AEs related to study drug, nor S/AESI were reported. Liver enzymes (alanine aminotransferase and aspartate aminotransferase) remained <1.5 of baseline. Corticosteroids were not used in either patient. Clear evidence of FVIII expression was observed in both patients with stable values of ~5% and ~17% in the first and second patient, respectively. An early read-out also indicated hemostatic efficacy in both; the first patient had successfully halted prophylaxis for 6 weeks, while the second one, treated on-demand with 99 bleeds recorded in the 12 months prior to gene transfer, has been bleed free for over 5.5 months to date. Conclusions: BAY 2599023 was previously shown in non-clinical studies to have a good safety profile, with the potential to achieve endogenous expression of FVIII at therapeutic levels, over an extended period of time. In this first-in-human clinical study with BAY 2599023, two patients have been treated with BAY 2599023 at the starting dose of 0.5 x 1013 GC/kg and no safety concerns have been reported to date. Measurable expression of endogenous FVIII and an early read-out of hemostatic efficacy have been demonstrated in both patients. Overall, data generated from this first dose cohort demonstrate that successful translation from pre-clinical to clinical development and proof-of-mechanism for BAY 2599023 was achieved. Disclosures Pipe: HEMA Biologics: Consultancy; Shire: Consultancy; Roche/Genentech: Consultancy; Sanofi: Consultancy; Freeline: Consultancy; Apcintex: Consultancy; Novo Nordisk: Consultancy; Catalyst Bioscience: Consultancy; CSL Behring: Consultancy; Bayer: Consultancy; uniQure: Consultancy; BioMarin: Consultancy; Pfizer: Consultancy; Spark Therapeutics: Consultancy. Becka:Bayer: Employment. Detering:Bayer: Employment. Vanevski:Bayer: Employment. Lissitchkov:Octapharma: Equity Ownership, Research Funding; Bayer: Consultancy, Equity Ownership, Honoraria, Other: Principal investigator for clinical trials, Research Funding; Sobi: Consultancy, Equity Ownership, Honoraria; Sanofi: Equity Ownership, Research Funding; Roche: Consultancy, Equity Ownership, Honoraria, Speakers Bureau; Shire: Consultancy, Equity Ownership, Honoraria, Speakers Bureau. OffLabel Disclosure: Gene therapy for haemophilia treatment.

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