Evaluating clinically translatable conditioning for platelet gene therapy in murine hemophilia A with inhibitors.

Background: Platelet gene therapy is effective in hemophilia A (HA) mice even with inhibitors. Fludarabine (Flu), along with busulfan (Bu) or melphalan (Mel), preconditioning has been shown to be highly effective for hematopoietic stem cell transplantation in the clinic.

Objectives: To evaluate the efficacy of Bu-Flu and Mel-Flu preconditioning in platelet gene therapy of HA with inhibitors.

Lipid nanoparticles and siRNA targeting plasminogen provide lasting inhibition of fibrinolysis in mouse and dog models of hemophilia A.

Antifibrinolytic drugs are used extensively for on-demand treatment of severe acute bleeding. Controlling fibrinolysis may also be an effective strategy to prevent or lessen chronic recurring bleeding in bleeding disorders such as hemophilia A (HA), but current antifibrinolytics have unfavorable pharmacokinetic profiles. Here, we developed a long-lasting antifibrinolytic using small interfering RNA (siRNA) targeting plasminogen packaged in clinically used lipid nanoparticles (LNPs) and tested it to determine whether reducing plasmin activity in animal models of HA could decrease bleeding frequency and severity.

Platelet-targeted gene therapy induces immune tolerance in hemophilia and beyond.

Blood platelets have unique storage and delivery capabilities. Platelets play fundamental roles in hemostasis, inflammatory reactions, and immune responses. Beyond their functions, platelets have been used as a target for gene therapy.

Pre-existing anti-factor VIII immunity alters therapeutic platelet-targeted factor VIII engraftment following busulfan conditioning through cytotoxic CD8 T cells.

Background: We previously demonstrated that busulfan preconditioning enabled sustained therapeutic platelet-derived factor VIII (FVIII) expression in naïve FVIII mice transplanted with 2bF8-transduced Sca-1 cells. However, in mice with pre-existing inhibitors, platelet-FVIII expression was lost.

Objective: In this study, we aimed to describe the mechanism of this platelet-FVIII loss.

Targeting transmembrane-domain-less MOG expression to platelets prevents disease development in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system with no cure yet. Here, we report genetic engineering of hematopoietic stem cells (HSCs) to express myelin oligodendrocyte glycoprotein (MOG), specifically in platelets, as a means of intervention to induce immune tolerance in experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. The platelet-specific αIIb promoter was used to drive either a full-length or truncated MOG expression cassette.

A novel mouse model of type 2N VWD was developed by CRISPR/Cas9 gene editing and recapitulates human type 2N VWD.

Type 2N von Willebrand disease is caused by mutations in the factor VIII (FVIII) binding site of von Willebrand factor (VWF), resulting in dysfunctional VWF with defective binding capacity for FVIII. We developed a novel type 2N mouse model using CRISPR/Cas9 technology. In homozygous VWF2N/2N mice, plasma VWF levels were normal (1167 ± 257 mU/mL), but the VWF was completely incapable of binding FVIII, resulting in 53 ± 23 mU/mL of plasma FVIII levels that were similar to those in VWF-deficient (VWF-/-) mice.

Thromboelastometry assessment of hemostatic properties in various murine models with coagulopathy and the effect of factor VIII therapeutics.

Background: Rotational thromboelastometry (ROTEM) has been commonly used to assess the viscoelastic properties of the blood clotting process in the clinic for patients with a hemostatic or prothrombotic disorder.

Objective: To evaluate the capability of ROTEM in assessing hemostatic properties in whole blood from various mouse models with genetic bleeding or clotting disease and the effect of factor VIII (FVIII) therapeutics in FVIII mice.

Methods: Mice with a genetic deficiency in either a coagulation factor or a platelet glycoprotein were used in this study.

Platelet-targeted hyperfunctional FIX gene therapy for hemophilia B mice even with preexisting anti-FIX immunity.

Gene therapy may lead to a cure for hemophilia B (HB) if it is successful. Data from clinical trials using adeno-associated virus (AAV)-mediated liver-targeted FIX gene therapy are very encouraging. However, this protocol can be applied only to adults who do not have liver disease or anti-AAV antibodies, which occur in 30% to 50% of individuals.

Platelet gene therapy induces robust immune tolerance even in a primed model via peripheral clonal deletion of antigen-specific T cells.

While platelet-specific gene therapy is effective in inducing immune tolerance to a targeted protein, how the reactivity of pre-existing immunity affects the efficacy, and whether CD8 T cells were involved in tolerization, is unclear. In this study, ovalbumin (OVA) was used as a surrogate protein. Platelet-OVA expression was introduced by 2bOVA lentivirus transduction of Sca-1 cells from either wild-type (WT)/CD45.

In vivo enrichment of genetically manipulated platelets for murine hemophilia B gene therapy.

Our previous studies have demonstrated that platelet-targeted factor IX (FIX) gene therapy can introduce sustained platelet-FIX expression in hemophilia B (FIX ) mice. In this study, we aimed to enhance platelet-FIX expression in FIX mice with O -methylguanine-DNA-methyltransferase (MGMT)-mediated in vivo drug selection of transduced cells under nonmyeloablative preconditioning. We constructed a novel lentiviral vector (2bF9/MGMT lentivirus vector), which harbors dual genes, the FIX gene driven by the αIIb promoter (2bF9) and the MGMT P140K gene under the murine stem cell virus promoter.

Induction of activated T follicular helper cells is critical for anti-FVIII inhibitor development in hemophilia A mice.

The development of neutralizing anti-FVIII antibodies (inhibitors) is a major complication of FVIII protein replacement therapy in patients with hemophilia A (HA). Although multiple lines of evidence indicate that the immune response against FVIII is CD4 T-cell-dependent and many FVIII-derived CD4 epitopes have already been discovered, the role of T follicular helper (TFH) cells in FVIII inhibitor development is unknown. TFH cells, a newly identified subset of CD4 T cells, are characterized by expression of the B-cell follicle-homing receptor CXCR5 and PD-1.

Nongenotoxic antibody-drug conjugate conditioning enables safe and effective platelet gene therapy of hemophilia A mice.

Gene therapy offers the potential to cure hemophilia A (HA). We have shown that hematopoietic stem cell (HSC)-based platelet-specific factor VIII (FVIII) (2bF8) gene therapy can produce therapeutic protein and induce antigen-specific immune tolerance in HA mice, even in the presence of inhibitory antibodies. For HSC-based gene therapy, traditional preconditioning using cytotoxic chemotherapy or total body irradiation (TBI) has been required.

The impact of GPIbα on platelet-targeted FVIII gene therapy in hemophilia A mice with pre-existing anti-FVIII immunity.

Essentials Platelet-specific FVIII gene therapy is effective in hemophilia A mice even with inhibitors. The impact of platelet adherence via VWF/GPIbα binding on platelet gene therapy was investigated. GPIbα does not significantly affect platelet gene therapy of hemophilia A with inhibitors.

Platelet Gene Therapy Promotes Targeted Peripheral Tolerance by Clonal Deletion and Induction of Antigen-Specific Regulatory T Cells.

Delivery of gene therapy as well as of biologic therapeutics is often hampered by the immune response of the subject receiving the therapy. We have reported that effective gene therapy for hemophilia utilizing platelets as a delivery vehicle engenders profound tolerance to the therapeutic product. In this study, we investigated whether this strategy can be applied to induce immune tolerance to a non-coagulant protein and explored the fundamental mechanism of immune tolerance induced by platelet-targeted gene delivery.

LAMA2 gene mutation update: Toward a more comprehensive picture of the laminin-α2 variome and its related phenotypes.

Congenital muscular dystrophy type 1A (MDC1A) is one of the main subtypes of early-onset muscle disease, caused by disease-associated variants in the laminin-α2 (LAMA2) gene. MDC1A usually presents as a severe neonatal hypotonia and failure to thrive. Muscle weakness compromises normal motor development, leading to the inability to sit unsupported or to walk independently.

The impact of von Willebrand factor on factor VIII memory immune responses.

Immune tolerance induction (ITI) with aggressive infusion of factor VIII (FVIII) is the current strategy used to eradicate FVIII inhibitors and restore normal FVIII pharmacokinetics in inhibitor patients. Whether the use of FVIII products containing von Willebrand factor (VWF) will affect the efficacy of ITI is still controversial. In this study, we explored the impact of VWF on FVIII memory immune responses in hemophilia A (HA) mice.

TGF-β1 along with other platelet contents augments Treg cells to suppress anti-FVIII immune responses in hemophilia A mice.

Platelets are a rich source of many cytokines and chemokines including transforming growth factor β 1 (TGF-β1). TGF-β1 is required to convert conventional CD4 T (Tconv) cells into induced regulatory T (iTreg) cells that express the transcription factor Foxp3. Whether platelet contents will affect Treg cell properties has not been explored.

The immunogenicity of platelet-derived FVIII in hemophilia A mice with or without preexisting anti-FVIII immunity.

Evidence shows that factor VIII (FVIII) ectopically expressed in platelets (2bF8) is therapeutic in FVIII(null) mice even with anti-FVIII inhibitory antibodies (inhibitors). If current efforts to generate platelets in vitro succeed, genetically manipulated platelets containing FVIII may be used therapeutically in hemophilia A patients with inhibitors. One important concern is the immunogenicity of platelet-derived FVIII.

Platelet gene therapy corrects the hemophilic phenotype in immunocompromised hemophilia A mice transplanted with genetically manipulated human cord blood stem cells.

Our previous studies have demonstrated that platelet FVIII (2bF8) gene therapy can improve hemostasis in hemophilia A mice, even in the presence of inhibitory antibodies, but none of our studies has targeted human cells. Here, we evaluated the feasibility for lentivirus (LV)-mediated human platelet gene therapy of hemophilia A. Human platelet FVIII expression was introduced by 2bF8LV-mediated transduction of human cord blood (hCB) CD34(+) cells followed by xenotransplantation into immunocompromised NSG mice or NSG mice in an FVIII(null) background (NSGF8KO).

Platelet gene therapy by lentiviral gene delivery to hematopoietic stem cells restores hemostasis and induces humoral immune tolerance in FIX(null) mice.

Here, we developed a clinically translatable platelet gene therapy approach for hemophilia B. Platelet-targeted FIX (2bF9) expression was introduced by transplantation of hematopoietic stem cells (HSCs) transduced with 2bF9 lentivirus (LV). Sustained therapeutic levels of platelet-FIX expression were obtained in FIX(null) mice that received 2bF9 LV-transduced HSCs.

Correction of murine Bernard-Soulier syndrome by lentivirus-mediated gene therapy.

Bernard-Soulier syndrome (BSS) is an inherited bleeding disorder caused by a defect in the platelet glycoprotein (GP) Ib-IX-V complex. The main treatment for BSS is platelet transfusion but it is often limited to severe bleeding episodes or surgical interventions due to the risk of alloimmunization. We have previously reported successful expression of human GPIbα (hGPIbα) in human megakaryocytes using a lentiviral vector (LV) encoding human GP1BA under control of the platelet-specific integrin αIIb promoter (2bIbα).