Phenotypic Correction Of Murine Hemophilia Research Articles

Sustained Phenotypic Correction of Murine Hemophilia A with Pre-Existing Anti-FVIII Immunity Using Lentivirus-Mediated Platelet-Specific FVIII Gene Transfer.

Abstract 29The development of inhibitory antibodies to exogenous factor VIII (FVIII) is considered a severe and important complication of FVIII infusion in hemophilia A patients. Gene therapy of hemophilia A with inhibitors is especially challenging because functional FVIII activity may be inactivated by circulating inhibitory antibodies if transgene protein is constitutively secreted into the blood circulation. Our previous studies have demonstrated that syngeneic transplantation of hematopoietic stem cells from 2bF8 transgenic mice that express platelet-specific FVIII can efficiently restore hemostasis to hemophilic mice with pre-existing inhibitory antibodies. In the current study, we assessed whether lentivirus-mediated 2bF8 gene transfer could efficiently introduce 2bF8 transgene expression and ameliorate the hemorrhagic phenotype in hemophilic mice with pre-existing immunity. To mimic the clinical situation of an autologous transplant in an inhibitor patient, both donor and recipient FVIIInull mice were immunized with recombinant human B-domain deleted FVIII to induce inhibitory antibody development. Platelet-derived FVIII expression in FVIIInull mice was introduced by 2bF8 lentiviral-mediated bone marrow (BM) transduction and syngeneic transplantation. Following BM reconstitution, mice were analyzed by PCR, quantitative real-time PCR, platelet lysate FVIII activity assay, and inhibitor assay. Phenotypic correction was assessed by tail clip survival test and electrolytic-induced thrombus formation. Expression of the 2bF8 product was detected in all recipients that received 2bF8 lentivirus transduced BM cells, indicating viable engraftment of BM genetically modified with the 2bF8 lentivirus transfer vector. Functional platelet-FVIII activity levels in the transduced mice with pre-existing immunity ranged from 0.36 to 6.18 mU/108 platelets (mean 1.56 ± 1.76 mU/108 platelets, n = 10), which was not significantly different from the levels obtained from a parallel non-inhibitor model (1.46 ± 0.87 mU/108 platelets, n = 4). Real-time PCR demonstrated that there was an average of 0.17 ± 0.05 LV DNA copies per cell in peripheral white blood cells from transduced mice. FVIII inhibiter titer gradually declined with the time, indicating that transduced platelet FVIII is well protected from exposure to the immune system, avoiding activation of a memory response. The tail clip survival test showed that 90% of mice survived tail clip challenge. The electrolytic injury model demonstrated that hemostasis was improved in recipients that received 2bF8 lentivirus-transduced BM cells. Furthermore, BM transferred from the primary transplant recipients into immunized FVIIInull secondary recipients demonstrated sustained platelet-FVIII expression, resulting in the correction of the hemophilia A phenotype with pre-existing immunity. This shows that gene transfer has occurred within long-term repopulating hematopoietic stem cells even in the presence of inhibitory antibodies. These results demonstrate that lentivirus-mediated bone marrow transduction/transplantation can provide sustained improvement of hemostasis in hemophilic mice with pre-existing immunity, indicating that this approach may be a promising strategy for gene therapy of hemophilia A with inhibitory antibodies in humans. Disclosures: Montgomery:GTI Diagnostics: Consultancy; Baxter: Consultancy; AstraZeneca: Consultancy; Bayer: Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

The therapeutic effect of bone marrow–derived liver cells in the phenotypic correction of murine hemophilia A

AbstractThe transdifferentiation of bone marrow cells (BMCs) into hepatocytes has created enormous interest in applying this process to the development of cellular medicine for degenerative and genetic diseases. Because the liver is the primary site of factor VIII (FVIII) synthesis, we hypothesized that the partial replacement of mutated liver cells by healthy cells in hemophilia A mice could manage the severity of the bleeding disorder. We perturbed the host liver with acetaminophen to facilitate the engraftment and hepatic differentiation of lineage-depleted enhanced green fluorescent protein-expressing BMCs. Immunohistochemistry experiments with the liver tissue showed that the donor-derived cells expressed the markers of both hepatocytes (albumin and cytokeratin-18) and endothelial cells (von Willebrand factor). The results of fluorescent in situ hybridization and immunocytochemistry experiments suggested that differentiation was direct in this model. The BMC-recipient mice expressed FVIII protein and survived in a tail clip challenge experiment. Furthermore, a coagulation assay confirmed that the plasma FVIII activity was maintained at 20.4% (± 3.6%) of normal pooled plasma activity for more than a year without forming its inhibitor. Overall, this report demonstrated that BMCs rescued the bleeding phenotype in hemophilia A mice, suggesting a potential therapy for this and other related disorders.

Phenotypic correction of murine hemophilia A using an iPS cell-based therapy

Hemophilia A is caused by mutations within the Factor VIII (FVIII) gene that lead to depleted protein production and inefficient blood clotting. Several attempts at gene therapy have failed for various reasons-including immune rejection. The recent generation of induced pluripotent stem (iPS) cells from somatic cells by the ectopic expression of 3 transcription factors, Oct4, Sox2, and Klf4, provides a means of circumventing the immune rejection barrier. To date, iPS cells appear to be indistinguishable from ES cells and thus provide tremendous therapeutic potential. Here we prepared murine iPS cells from tail-tip fibroblasts and differentiated them to both endothelial cells and endothelial progenitor cells by using the embryoid body differentiation method. These iPS cells express major ES cell markers such as Oct4, Nanog, SSEA-1, alkaline phosphatase, and SALL4. Endothelial/endothelial progenitor cells derived from iPS cells expressed cell-specific markers such as CD31, CD34, and Flk1 and secreted FVIII protein. These iPS-derived cells were injected directly into the liver of irradiated hemophilia A mice. At various times after transplantation (7-90 days) hemophilia A mice and their control mice counterparts were challenged by a tail-clip bleeding assay. Nontransplanted hemophilia A mice died within a few hours, whereas transplanted mice survived for more than 3 months. Plasma FVIII levels increased in transplanted hemophilia A mice during this period to 8% to 12% of wild type and corrected the hemophilia A phenotype. Our studies provide additional evidence that iPS cell therapy may be able to treat human monogenetic disorders in the future.

127. Gene Delivery of a High Activity Analog of FVIIa Results in Phenotypic Correction of Murine Hemophilia at Substantially Lower Vector-Doses Than FVIIa

127. Gene Delivery of a High Activity Analog of FVIIa Results in Phenotypic Correction of Murine Hemophilia at Substantially Lower Vector-Doses Than FVIIa

406. Long-Term Phenotypic Correction of Murine Hemophilia A and Immunological Differences of Bioengineered FVIII Variants Delivered by Helper-Dependent Adenoviral Vectors

406. Long-Term Phenotypic Correction of Murine Hemophilia A and Immunological Differences of Bioengineered FVIII Variants Delivered by Helper-Dependent Adenoviral Vectors

84. Assessing Hemostatic Efficacy of Continuous Expression of Factor VIIa after AAV-Mediated, Liver-Directed Gene Transfer in Hemophilia A and B Mice

We have shown that portal vein infusion of an AAV vector expressing an engineered secreted activated murine factor VIIa (mFVIIa) results in long-term phenotypic correction of murine hemophilia B (HB) mice, as judged by shortening of the prothrombin time (PT), activated partial thromboplastin time (aPTT), and the tail-clip bleeding time (J Clin Invest. 2004; 113: 1025). We sought to compare endpoints for hemostasis in hemophilia A and B mice infused with AAV-mFVIIa, analyzing VIIa levels, the tail-clip assay, the FeCl3-carotid artery model, and real-time imaging of clot formation utilizing the cremasteric muscle arteriole. Twenty-two (N=22) Hemophilia A (HA) and forty-eight (N=48) HB mice were injected with AAV-mFVIIa via the portal vein at doses ranging from 1.0|[times]|10^8 to 4.8|[times]|10^12 vg/mouse. Based on a PT assay we observed a dose response with circulating VIIa levels ranging from 11 ng/ml for the untreated and the lowest dose to 1000 ng/ml for the higher doses. In HB mice (N=15) injected at 1.2|[times]|10^12 vg/mouse, the mean PT shortened from 23 to 17 sec, and the aPTT from 86 to 43 seconds, 8 weeks post vector injection. Measurement of the blood loss post tail-clip was documented as a statistically significant reduction in blood loss in HB AAV-mFVIIa-treated mice (N=5) relative to the untreated HB mice (N=10) * [P-value 25ug/kg), animals developed complete, albeit transient, occlusion, but neither FVIIa protein (>90ug/kg) nor AAV-mFVIIa mice exhibited formation of an occlusive thrombus in HB mice. Similar results have been obtained in hemophilia A mice. Together these data suggest that 1) AAV-mFVIIa is functional in supporting hemostasis as judged by in vitro coagulation assays and by some but not all in vivo hemostasis challenges; and 2) results in these hemostasis assays may vary as a function of the diameter of the vessel that is injured. Which of these endpoints most faithfully reflects hemostasis in humans is not yet clear, but the major morbidity of hemophilia is from joint bleeds which likely begin as bleeding in the microcirculation. Our studies provide clear evidence of improvement of in vivo hemostasis and support continuous expression of FVIIa as an alternative strategy for the treatment of hemophilia.

In vivo dose threshold effect of adenovirus-mediated factor VIII gene therapy in hemophiliac mice.

While much is known about adenovirus biology from its development as a therapeutic gene delivery vehicle, an important question remains regarding the appropriate in vivo vector dose. We describe here an in vivo dose threshold effect with an adenoviral vector expressing human Factor VIII (FVIII) in hemophiliac mice. Upon administration of vector doses between 6 x 10(10) and 2 x 10(10) vector particles per mouse, FVIII was expressed linearly, whereas a dose of 1 x 10(10) vector particles per mouse did not result in detectable levels of FVIII activity. In contrast, in vitro transduction studies demonstrated linear transgene expression over 2 to 3 log units. To further define this dose threshold effect, a vector-mixing study was performed. Mice were injected with a total vector dose of 6 x 10(10) particles containing admixtures of FVIII vector plus a control vector lacking a transgene (null vector). With the admixture, FVIII activity was detected in mice that received 1 3 1010 particles of the FVIII vector, indicating that maintenance of the total viral input at 6 x 10(10) particles per mouse circumvented the threshold dose effect. This threshold dose effect could not be attributed to dose-dependent differences in liver toxicity nor to dose-dependent induction of cellular and humoral immune responses. Southern blot analysis of livers revealed that mice receiving the vector admixture contained FVIII DNA, accounting for the observed FVIII expression, whereas mice receiving 1 x 10(10) particles of FVIII vector had barely detectable FVIII DNA. These results suggest that the threshold effect is an in vivo phenomenon that will have important implications in defining the therapeutic window of adenoviral vectors for clinical applications.

Sustained phenotypic correction of murine hemophilia A by in vivo gene therapy: Connelly S, Andrews JL, Gallo AM, et al. Blood 91:3273–3281, 1998

Sustained phenotypic correction of murine hemophilia A by in vivo gene therapy: Connelly S, Andrews JL, Gallo AM, et al. Blood 91:3273–3281, 1998

Sustained Phenotypic Correction of Murine Hemophilia A by In Vivo Gene Therapy

Hemophilia A is caused by a deficiency of blood coagulation factor VIII (FVIII) and has been widely discussed as a candidate for gene therapy. While the natural canine model of hemophilia A has been valuable for the development of FVIII pharmaceutical products, the use of hemophiliac dogs for gene therapy studies has several limitations such as expense and the long canine generation time. The recent creation of two strains of FVIII-deficient mice provides the first small animal model of hemophilia A. Treatment of hemophiliac mice of both genotypes with potent, human FVIII-encoding adenoviral vectors resulted in expression of biologically active human FVIII at levels, which declined, but remained above the human therapeutic range for over 9 months. The duration of expression and FVIII plasma levels achieved were similar in both hemophiliac mouse strains. Treated mice readily survived tail clipping with minimal blood loss, thus showing phenotypic correction of murine hemophilia A by in vivo gene therapy.

Sustained Phenotypic Correction of Murine Hemophilia A by In Vivo Gene Therapy

AbstractHemophilia A is caused by a deficiency of blood coagulation factor VIII (FVIII) and has been widely discussed as a candidate for gene therapy. While the natural canine model of hemophilia A has been valuable for the development of FVIII pharmaceutical products, the use of hemophiliac dogs for gene therapy studies has several limitations such as expense and the long canine generation time. The recent creation of two strains of FVIII-deficient mice provides the first small animal model of hemophilia A. Treatment of hemophiliac mice of both genotypes with potent, human FVIII-encoding adenoviral vectors resulted in expression of biologically active human FVIII at levels, which declined, but remained above the human therapeutic range for over 9 months. The duration of expression and FVIII plasma levels achieved were similar in both hemophiliac mouse strains. Treated mice readily survived tail clipping with minimal blood loss, thus showing phenotypic correction of murine hemophilia A by in vivo gene therapy.