Correction Of Hemophilia Research Articles

Correction of hemophilia as a proof of concept for treatment of monogenic diseases by fetal spleen transplantation

Previous clinical attempts to correct genetic deficiencies such as hemophilia or Gaucher disease by transplantation of allogeneic spleen were associated with aggressive graft versus host disease, mediated by mature T cells derived from the donor spleen. We show that a fetal pig spleen harvested at the embryonic day 42 stage, before the appearance of T cells, exhibited optimal growth potential upon transplantation into SCID mice, and the growing tissue expressed factor VIII. Transplantation of embryonic day 42 spleen tissue into hemophilic SCID mice led to complete alleviation of hemophilia within 2-3 months after transplant, as demonstrated by tail bleeding and by assays for factor VIII blood levels. These results provide a proof of principle to the concept that transplantation of a fetal spleen, obtained from a developmental stage before the appearance of T cells, could provide a novel treatment modality for genetic deficiencies of an enzyme or a factor that can be replaced by the growing spleen tissue.

Factor VIII ectopically targeted to platelets is therapeutic in hemophilia A with high-titer inhibitory antibodies

Inhibitory immune response to exogenously infused factor VIII (FVIII) is a major complication in the treatment of hemophilia A. Generation of such inhibitors has the potential to disrupt gene therapy for hemophilia A. We explore what we believe to be a novel approach to overcome this shortcoming. Human B-domain-deleted FVIII (hBDDFVIII) was expressed under the control of the platelet-specific alphaIIb promoter in platelets of hemophilic (FVIIInull) mice to create 2bF8trans mice. The FVIII transgene product was stored in platelets and released at the site of platelet activation. In spite of the lack of FVIII in the plasma of 2bF8trans mice, the bleeding phenotype of FVIIInull mice was corrected. More importantly, the bleeding phenotype was corrected in the presence of high inhibitory antibody titers introduced into the mice by infusion or by spleen cell transfer from recombinant hBDDFVIII-immunized mice. Our results demonstrate that this approach to the targeted expression of FVIII in platelets has the potential to correct hemophilia A, even in the presence of inhibitory immune responses to infused FVIII.

1086. T Cell Responses to Canine Factor IX and AAV Capsid Antigens in Hemophilia B Dogs after Intravascular Gene Delivery to Skeletal Muscle

Previous studies have shown successful correction of hemophilia B in dogs by muscle-directed gene delivery of canine F.IX (cF.IX) using an adeno-associated virus (AAV) serotype 2. Intravascular delivery of the cF.IX gene to skeletal muscle allowed long-term expression of cF.IX of 4-20%, thereby correcting the disease phenotype. However, concerns over the risk of immune responses against the transgene and viral vector antigens have focused recent work on further characterizing the immunogenicity of both AAV capsid and F.IX in a gene therapy setting.

167. Polyethyleneimines as Vehicles for Oral Gene Delivery

Oral gene therapy aims to treat immune-, gastrointestinal- or systemic diseases by ingestion of genetic materials. Compared to the other routes of administration, the oral route is the most convenient, but also the most challenging for gene delivery. Previous studies have shown that oral delivery of chitosan nanoparticles complexed with 0.6 mg DNA encoding FVIII resulted in phenotypic correction of Hemophilia A. The treated animals, unlike control group survived a tail-clipping test.

Erratum to “Sustained Phenotypic Correction of Hemophilia A Mice Following Oncoretroviral-Mediated Expression of a Bioengineered Human Factor VIII Gene in Long-Term Hematopoietic Repopulating Cells”

Erratum to “Sustained Phenotypic Correction of Hemophilia A Mice Following Oncoretroviral-Mediated Expression of a Bioengineered Human Factor VIII Gene in Long-Term Hematopoietic Repopulating Cells”

603. Correction of Hemophilia B Phenotype by Novel Method of Regional Intravenous Delivery of AAV Vector to Skeletal Muscle of Hemophilia B Dogs

Top of pageAbstract Intramuscular (IM) injection of AAV-F.IX in humans was safe and resulted in gene transfer and expression. However, at the doses administered, it did not result in sustained circulating levels >1% of normal. Although access to the skeletal muscle can be easily obtained by direct IM injection. However, achievement of target doses in humans proved impractical because of the large number of injections required. We sought to exploit techniques that allowed widespread transduction of muscle fibers. The major obstacle to vector distribution to large areas of skeletal muscle is the vascular endothelial barrier. However, the rich network of capillaries can be chemically and/or mechanically modified to ensure vascular leakage of vectors. In hemophilia B dogs, we showed (Blood, 2005) that infusion of AAV2-canineF.IX through femoral artery in the presence of histamine to increase capillary permeability, results in long-term expression of robust therapeutic F.IX levels (4-14% normal). Here we report the application of a novel approach, afferent transvenular extravasation (ATVE), in which the vector is injected through a superficial saphenous vein in the distal part of the limb under elevated hydrostatic pressure. Briefly, a tourniquet was placed at the level of the proximal thigh. AAV2-F.IX was diluted in 20 ml of PBS/kg and infused rapidly under the pressure of 300 mmHg. The vector-solution dwells for 15 min before tourniquet release. Hemophilia B dogs treated with AAV2 at doses of 1|[times]|1012vg/kg (n=2) expressed F.IX levels of 2% (100-125ng/ml). Increasing the vector dose to 3|[times]|1012vg/kg (n=3) resulted in circulating F.IX plateau levels of 6%-23% (300-1160 ng/ml). F.IX is biologically active as judged by excellent correlation between clotting times/antigen levels and near normalization of the whole blood clotting time. A sixth dog received an AAV dose of 8.5|[times]|1012vg/kg; this dose seems to be excessive since after F.IX levels increased to 713 ng/ml (day 21) we documented a slow decrease to <100ng/ml due to low-level of a non-neutralizing antibody to F.IX. Muscle enzymes levels increased transiently in 2 of 6 dogs up to day 2 post-injection. Endothelial cell damage markers such as vWF and IL-6 assayed at several time points following vector delivery did not differ from baseline levels. Thus, ATV provides an efficient, non-invasive, and safe strategy for widespread delivery of vector to skeletal muscle in the absence of vasoactive drugs. The doses required to achieve therapeutic F.IX levels (6-23%) are similar to doses already prove to be safe in earlier clinical trials. Furthermore, ATVE abolishes the dose advantage in gene transfer of liver-directed over muscle-directed gene transfer for hemophilia B. These results establish an experimental basis for clinical studies of this delivery method in humans with hemophilia B.

36. Permanent Phenotypic Correction of Haemophilia B in Immunocompetent Mice by Prenatal Gene Therapy

Protein replacement treatments often trigger unwanted immune responses in the host. Induction of immune tolerance would be a desirable goal to mitigate such events, and would eliminate the need for life-long immunosuppressive therapy in affected individuals. Hemophilia B is an X-linked bleeding disorder caused by blood coagulation factor IX (FIX) deficiency. Some hemophiliacs develop neutralizing antibodies to FIX (inhibitors) that render the infused FIX ineffective. In this study we evaluated whether oral delivery of hFIX-DNA nanoparticles can downregulate the immune response against hFIX in immunized mice. A group of C57BL/6 mice was immunized with recombinant hFIX in Freund's adjuvant followed by three boost at day 7, 14, and 21. As expected, all mice developed high titer of anti-hFIX antibodies as measured by day 28. At this time, half the mice were treated with a single oral dose of plasmid DNA nanoparticles containing hFIX cDNA under the control of the b -actin promoter. The other half was left untreated, and served as control. By ELISA, we detected circulating levels of hFIX in the treated mice by day 3 after the oral treatment. In contrast, control mice had undetectable levels of circulating hFIX. Importantly, the titer of inhibitors against hFIX was measured by a Bethesda assay. Control mice had inhibitors, on average, of 40 BU/ml on day 21 after the oral administration of DNA. In contrast, the level of inhibitors became undetectable in the treated group by day 21. The cellular immune response was also assessed. Stimulation of splenocytes from orally treated mice with hFIX resulted in increased secretion of transforming growth factor-b, but no detectable interferon-g production. In contrast, stimulation of splenocytes from immunized control mice with hFIX showed an increase in the production of interferon-g, and no secretion of transforming growth factor-b. Additionally, a hFIX-specific cytotoxic T-lymphocyte (CTL) response was detected in the control, but not in the orally treated mice. Thus, our results suggest that the oral delivery of DNA nanoparticles downregulated the pre-existing immune responses against human FIX. This downregulation may result from the induction of a shift from a proinflammatory T helper cell type 1 to an anti-inflammatory T helper cell type 2 immune response. In conclusion, we have shown that pre-existing immune responses against hFIX can be overcome. This strategy may facilitate the delivery of proteins in a pre-sensitized host. Similar approaches may have potential applications for the induction of immune tolerance in autoimmune diseases. This strategy may also have implications for the prevention of unwanted immune responses elicited by the expression of novel transgenes.

930. In Vivo Functional Correction of Hemophilia a Mice Via a Novel Spliceosome-Mediated RNA Trans-Splicing Repair Mechanism

930. In Vivo Functional Correction of Hemophilia a Mice Via a Novel Spliceosome-Mediated RNA Trans-Splicing Repair Mechanism

257. Dual Vectors Expressing Murine Factor VIII Result in Sustained Correction of Hemophilia a Mice

257. Dual Vectors Expressing Murine Factor VIII Result in Sustained Correction of Hemophilia a Mice

480. Sustained Correction of Hemophilia B Phenotype Following Intravascular Delivery of AAV Vector to Skeletal Muscle

480. Sustained Correction of Hemophilia B Phenotype Following Intravascular Delivery of AAV Vector to Skeletal Muscle

Neonatal correction of hemophilia B after in utero gene therapy

Neonatal correction of hemophilia B after in utero gene therapy

Generation of Retroviral Packaging and Producer Cell Lines for Large-Scale Vector Production and Clinical Application: Improved Safety and High Titer

For many applications, human clinical therapies using retroviral vectors still require many technological improvements in key areas of vector design and production. These improvements include higher unprocessed manufacturing titers, complement-resistant vectors, and minimized potential to generate replication-competent retrovirus (RCR). To address these issues, we have developed a panel of human packaging cell lines (PCLs) with reduced homology between retroviral vector and packaging components. These reduced-homology PCLs allowed for the use of a novel high multiplicity of transduction ("high m.o. t.") method to introduce multiple copies of provector within vector-producing cell lines (VPCLs), resulting in high-titer vector without the generation of RCR. In a distinct approach to increase vector yields, we integrated manufacturing parameters into screening strategies and clone selection for large-scale vector production. Collectively, these improvements have resulted in the development of diverse VPCLs with unprocessed titers exceeding 2 x 10(7) CFU/ml. Using this technology, human Factor VIII VPCLs yielding titers as high as 2 x 10(8) CFU/ml unprocessed supernatant were generated. These cell lines produce complement-resistant vector particles (N. J. DePolo et al., J. Virol. 73: 6708-6714, 1999) and provide the basis for an ongoing Factor VIII gene therapy clinical trial.

Long-term expression of human coagulation factor VIII and correction of hemophilia A after in vivo retroviral gene transfer in factor VIII-deficient mice.

Hemophilia A is caused by a deficiency in coagulation factor VIII (FVIII) and predisposes to spontaneous bleeding that can be life-threatening or lead to chronic disabilities. It is well suited for gene therapy because a moderate increase in plasma FVIII concentration has therapeutic effects. Improved retroviral vectors expressing high levels of human FVIII were pseudotyped with the vesicular stomatitis virus G glycoprotein, were concentrated to high-titers (10(9)-10(10) colony-forming units/ml), and were injected intravenously into newborn, FVIII-deficient mice. High-levels (>/=200 milliunits/ml) of functional human FVIII production could be detected in 6 of the 13 animals, 4 of which expressed physiologic or higher levels (500-12,500 milliunits/ml). Five of the six expressers produced FVIII and survived an otherwise lethal tail-clipping, demonstrating phenotypic correction of the bleeding disorder. FVIII expression was sustained for >14 months. Gene transfer occurred into liver, spleen, and lungs with predominant FVIII mRNA expression in the liver. Six of the seven animals with transient or no detectable human FVIII developed FVIII inhibitors (7-350 Bethesda units/ml). These findings indicate that a genetic disease can be corrected by in vivo gene therapy using retroviral vectors.

Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors.

Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.

Elucidation of N-linked oligosaccharide structures of recombinant human factor VIII using fluorophore-assisted carbohydrate electrophoresis.

Characterization of the carbohydrate moiety is a critical measure of manufacturing process consistency of recombinant human Factor VIII (rFVIII) in Chinese-hamster ovary (CHO) cells. FVIII, a large (300 kDa) glycoprotein, is employed therapeutically for the correction of haemophilia A. While N-linked and O-linked oligosaccharides are found in this protein, the current study focuses on the N-linked oligosaccharides. The N-linked oligosaccharides from rFVIII were released using either peptide N-glycosidase F or endoglycosidase H, derivatized with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonate, and analysed by fluorophore-assisted carbohydrate electrophoresis (FACE). The electrophoretically resolved oligosaccharide bands were isolated and individual bands subjected to digestion with defined pools of exoglycosidases and re-electrophoresed on FACE sequencing gels. The resulting gel patterns were interpreted, based on band mobility shifts, to obtain the sequence structure of the oligosaccharides. A total of eight acidic and 12 neutral structures were identified, and the majority of the oligosaccharides (approximately 92%) were found to be sialylated. All of the major oligosaccharide structures found in CHO-cell-derived rFVIII have also been reported to be present in plasma-derived FVIII. Among the most abundant are disialylated, biantennary, core-fucosylated (approximately 40%), followed by trisialylated, triantennary, core-fucosylated and monosialo, biantennary, core-fucosylated structures (each approximately 18%). The Gal alpha 1-3Gal structures reported to be present in baby-hamster-kidney-cell-derived rFVIII were not found in the CHO-cell-derived protein. The glycosylation patterns were consistent in six random lots of rFVIII [coefficient of variation (%) 3-14] based on percentage lane luminance data of bands that represent approximately 98% of all asparagine-linked oligosaccharides.

Factor IX activity derived from purified prothrombin

<h2>Abstract</h2> Factor IX-like activity could be generated from highly purified prothrombin in the presence of phospholipids and CaCl₂ when BaCO₃-adsorbed Factor XI deficient plasma and Factor XIa were present, when BaSO₄-adsorbed hemophilia B plasma was present or when only Factor XIa was present. Factor IX-like activity was observed to increase when prothrombin, Factor XIa, phospholipids and CaCl₂ were incubated while prothrombin activity was observed to disappear. A 3-hour incubation sample was shown to correct only hemophilia B plasma; it did not correct hemophilia A nor Factor X deficient plasmas.