Ultrasound-mediated gene delivery specifically targets liver sinusoidal endothelial cells for sustained FVIII expression in hemophilia A mice.

The ability to target the native production site of factor VIII (FVIII)-liver sinusoidal endothelial cells (LSECs)-can improve the outcome of hemophilia A (HA) gene therapy. By testing a matrix of ultrasound-mediated gene delivery (UMGD) parameters for delivering a GFP plasmid into the livers of HA mice, we were able to define specific conditions for targeted gene delivery to different cell types in the liver. Subsequently, two conditions were selected for experiments to treat HA mice via UMGD of an endothelial-specific human FVIII plasmid: low energy (LE; 50 W/cm2, 150 µs pulse duration) to predominantly target endothelial cells or high energy (HE; 110 W/cm2, 150 µs pulse duration) to predominantly target hepatocytes. Both groups of UMGD-treated mice achieved persistent FVIII activity levels of ∼10% over 84 days post treatment; however, half of the HE-treated mice developed low-titer inhibitors while none of the LE mice did. Plasma transaminase levels and histological liver examinations revealed minimal transient liver damage that was lower in the LE group than in the HE group. These results indicate that UMGD can safely target LSECs with a lower-energy condition to achieve persistent FVIII gene expression, demonstrating that this novel technology is highly promising for therapeutic correction of HA.

Blunting specific T-dependent antibody responses with engineered "decoy" B cells.

Antibody inhibitors pose an ongoing challenge to the treatment of subjects with inherited protein deficiency disorders, limiting the efficacy of both protein replacement therapy and corrective gene therapy. Beyond their central role as producers of serum antibody, B cells also exhibit many unique properties that could be exploited in cell therapy applications, notably including antigen-specific recognition and the linked capacity for antigen presentation. Here we employed CRISPR-Cas9 to demonstrate that ex vivo antigen-primed Blimp1-knockout "decoy" B cells, incapable of differentiation into plasma cells, participated in and downregulated host antigen-specific humoral responses after adoptive transfer. Following ex vivo antigen pulse, adoptively transferred high-affinity antigen-specific decoy B cells were diverted into germinal centers en masse, thereby reducing participation by endogenous antigen-specific B cells in T-dependent humoral responses and suppressing both cognate and linked antigen-specific immunoglobulin (Ig)G following immunization with conjugated antigen. This effect was dose-dependent and, importantly, did not impact concurrent unrelated antibody responses. We demonstrated the therapeutic potential of this approach by treating factor VIII (FVIII)-knockout mice with antigen-pulsed decoy B cells prior to immunization with an FVIII conjugate protein, thereby blunting the production of serum FVIII-specific IgG by an order of magnitude as well as reducing the proportion of animals exhibiting functional FVIII inhibition by 6-fold.

Current animal models of hemophilia: the state of the art.

Hemophilia is the most well-known hereditary bleeding disorder, with an incidence of one in every 5000 to 30,000 males worldwide. The disease is treated by infusion of protein products on demand and as prophylaxis. Although these therapies have been very successful, some challenging and unresolved tasks remain, such as reducing bleeding rates, presence of target joints and/or established joint damage, eliminating the development of inhibitors, and increasing the success rate of immune-tolerance induction (ITI). Many preclinical trials are carried out on animal models for hemophilia generated by the hemophilia research community, which in turn enable prospective clinical trials aiming to tackle these challenges. Suitable animal models are needed for greater advances in treating hemophilia, such as the development of better models for evaluation of the efficacy and safety of long-acting products, more powerful gene therapy vectors than are currently available, and successful ITI strategies. Mice, dogs, and pigs are the most commonly used animal models for hemophilia. With the advent of the nuclease method for genome editing, namely the CRISPR/Cas9 system, it is now possible to create animal models for hemophilia other than mice in a short period of time. This review presents currently available animal models for hemophilia, and discusses the importance of animal models for the development of better treatment options for hemophilia.

Recapitulating the immune system of hemophilia A patients with inhibitors using immunodeficient mice.

BACKGROUND AND AIM: Treating hemophilia A patients who develop inhibitors remains a clinical challenge. A mouse model of hemophilia A can be used to test the efficacy of strategies for inhibitor suppression, but the differences in the immune systems of mice and humans limit its utility. To address this shortcoming, we established a humanized NOD/SCID-IL2rγnull hemophilia A (hu-NSG-HA) mouse model with a severely deficient mouse immune system presenting a patient's adapted immune cells. METHODS AND RESULTS: Through intrasplenic injection with patient inhibitor-positive peripheral blood mononuclear cells (PBMCs), utilizing an adeno-associated viral delivery system expressing human BLyS, and regular FVIII challenge, human C19+ B cells were expanded in vivo to secrete anti-FVIII antibodies. Both the inhibitor and the human anti-FVIII IgG, including the predominant subclasses (IgG1 and IgG4) present in the majority of inhibitor patients, were detected in the mouse model. We further segregated and expanded the different clones of human anti-FVIII-secreting cells through subsequent transplantation of splenocytes derived from hu-NSG-HA mice into another NSG-HA mouse. By transplanting a patient's PBMCs into the NSG-HA mouse model, we demonstrated the success of reintroducing a strong anti-FVIII immune response for a short period in mice with the immune systems of inhibitor-positive patients. CONCLUSION: Our results demonstrate a potential tool for directly obtaining functional human-derived antigen-specific antibodies and antibody-secreting cells, which may have therapeutic value for testing patient-specific immune responses to treatment options to assist in clinical decisions.

Influence of N-glycosylation in the A and C domains on the immunogenicity of factor VIII.

The most significant complication in hemophilia A treatment is the formation of inhibitors against factor VIII (FVIII) protein. Glycans and glycan-binding proteins are central to a properly functioning immune system. This study focuses on whether glycosylation of FVIII plays an important role in induction and regulation of anti-FVIII immune responses. We investigated the potential roles of 4 N-glycosylation sites, including N41 and N239 in the A1 domain, N1810 in the A3 domain, and N2118 in the C1 domain of FVIII, in moderating its immunogenicity. Glycomics analysis of plasma-derived FVIII revealed that sites N41, N239, and N1810 contain mostly sialylated complex glycoforms, while high mannose glycans dominate at site N2118. A missense variant that substitutes asparagine (N) to glutamine (Q) was introduced to eliminate glycosylation on each of these sites. Following gene transfer of plasmids encoding B domain deleted FVIII (BDD-FVIII) and each of these 4 FVIII variants, it was found that specific activity of FVIII in plasma remained similar among all treatment groups. Slightly increased or comparable immune responses in N41Q, N239Q, and N1810Q FVIII variant plasmid-treated mice and significantly decreased immune responses in N2118Q FVIII plasmid-treated mice were observed when compared with BDD-FVIII plasmid-treated mice. The reduction of inhibitor response by N2118Q FVIII variant was also demonstrated in AAV-mediated gene transfer experiments. Furthermore, a specific glycopeptide epitope surrounding the N2118 glycosylation site was identified and characterized to activate T cells in an FVIII-specific proliferation assay. These results indicate that N-glycosylation of FVIII can have significant impact on its immunogenicity.