A factor IX-deficient mouse model for hemophilia B gene therapy

Wang, Lili; Zoppé, Monica; Hackeng, Tilman M.; Griffin, John H.; Lee, Kuo‐Fen; Verma, Inder M.

doi:10.1073/pnas.94.21.11563

Cited by 161 publications

(127 citation statements)

References 26 publications

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“…23 Peak hFIX:C levels, as determined by a one-stage clotting assay, were 8 Ϯ 0.7 and 27 Ϯ 6 IU/mL in the low-and high-dose cohorts, respectively ( Figure 3B). These levels were significantly above background (untreated HB hemophiliac mice hFIX:C level ϭ 0.03 U/mL) and significantly higher than the normal human FIX:C value, which is defined as 1 IU/mL.…”

Section: Scaav Vectors Mediate Substantially Higher Transduction Of Tmentioning

confidence: 99%

“…The HB mouse strain, based on 129/sv mice with disruption of the FIX gene, was obtained from Inder Verma (Salk Institute, La Jolla, CA). 23 Tail-vein administration of rAAV vector particles was performed in 7-to 10-week-old male mice as described before. 13 A two-thirds partial hepatectomy was performed at 16 weeks after tail-vein administration of 1 ϫ 10 10 scAAV2/8-LP1-hFIXco as previously described.…”

Section: Animal Studiesmentioning

confidence: 99%

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Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver

Nathwani

Gray

et al. 2006

Blood

365

381

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Transduction with recombinant adenoassociated virus (AAV) vectors is limited by the need to convert its single-stranded (ss) genome to transcriptionally active double-stranded (ds) forms. For AAVmediated hemophilia B (HB) gene therapy, we have overcome this obstacle by constructing a liver-restricted mini-human factor IX (hFIX) expression cassette that can be packaged as complementary dimers within individual AAV particles. Molecular analysis of murine liver transduced with these self-complementary (sc) vectors demonstrated rapid formation of active ds-linear genomes that persisted stably as concatamers or monomeric circles. This unique property resulted in a 20-fold improvement in hFIX expression in mice over comparable ssAAV vectors. Administration of only 1 ؋ 10 10 scAAV particles led to expression of hFIX at supraphysiologic levels (8I U/mL) and correction of the bleeding diathesis in FIX knock-out mice. Of importance, therapeutic levels of hFIX (3%-30% of normal) were achieved in nonhuman primates using a significantly lower dose of scAAV than required with ssAAV. Furthermore, AAV5-pseudotyped scAAV vectors mediated successful transduction in macaques with pre-existing immunity to AAV8. Hence, this novel vector represents an important advance for hemophilia B gene therapy. IntroductionThe liver is an important target for gene therapy of a variety of genetic disorders, one of which is hemophilia B (HB), a lifethreatening bleeding disorder that arises from mutations in the blood coagulation factor IX (FIX) gene. By maintaining plasma FIX levels above 1% of normal (Ͼ 0.05 g/mL), the incidence of spontaneous hemorrhage is dramatically reduced and so the therapeutic end point for HB gene therapy is modest. 1 Currently, adeno-associated virus (AAV) vectors are the most promising for HB gene therapy and have been the focus of 2 recent clinical trials. 2 Efficient transduction with AAV is, however, limited by the need to convert its single-stranded (ss) genome into transcriptionally active double-stranded (ds) forms in target cells because of its dependence on host-cell-mediated DNA synthesis of the leading strand 3 or annealing of complementary genomes derived from separate virions. 4 Coinfection with adenovirus 5 or priming the target tissues with genotoxic agents 6,7 can enhance ds transgene formation, but the clinical use of these approaches is limited by potential toxicity. Rapid uncoating of the viral genome, as recently described with AAV8 vectors, allows efficient annealing of the ssAAV provirus to form double-stranded genomes. This unique biologic property is responsible for the 10-to 100-fold higher transduction of the liver with rAAV8 when compared with AAV2 vectors in murine models. [8][9][10] Even so, almost 10 13 AAV8 vector particles are required to achieve 100% hepatocyte transduction in mice, a level that is required for successful gene therapy of some metabolic disorders of the liver. 11 This high vector dose is problematic because it may (1) Supported by The Assisi Foundation of Memphis; the Ame...

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Section: Scaav Vectors Mediate Substantially Higher Transduction Of Tmentioning

confidence: 99%

Section: Animal Studiesmentioning

confidence: 99%

Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver

Nathwani

Gray

et al. 2006

Blood

365

381

View full text Add to dashboard Cite

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“…129/Sv hemophilia B mice with a disrupted FIX gene were obtained from Professor Inder Verma. 40 All animal work was carried out in accordance with UK Home office regulations and was compliant with the local ethical review committee. Mice were irradiated (2 Â 500 cGy in a divided dose, 4 h apart) and i.v.…”

Section: Animalsmentioning

confidence: 99%

Permanent partial phenotypic correction and tolerance in a mouse model of hemophilia B by stem cell gene delivery of human factor IX

Bigger

Siapati

Mistry³

et al. 2005

Gene Ther

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Immune responses against an introduced transgenic protein are a potential risk in many gene replacement strategies to treat genetic disease. We have developed a gene delivery approach for hemophilia B based on lentiviral expression of human factor IX in purified hematopoietic stem cells. In both normal C57Bl/6J and hemophilic 129/Sv recipient mice, we observed the production of therapeutic levels of human factor IX, persisting for at least a year with tolerance to human factor IX antigen. Secondary and tertiary recipients also demonstrate long-term production of therapeutic levels of human factor IX and tolerance, even at very low levels of donor chimerism. Furthermore, in hemophilic mice, partial functional correction of treated mice and phenotypic rescue is achieved. These data show the potential of a stem cell approach to gene delivery to tolerize recipients to a secreted foreign transgenic protein and, with appropriate modification, may be of use in developing treatments for other genetic disorders.

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“…8,9 In addition, precise regulation of transgene expression is not required, because levels of 1% to 2% may be therapeutic and levels up to 100% are still within the normal range. The existence of small and large animal models of this disease [10][11][12][13][14][15][16] facilitates analysis of efficacy before clinical studies are initiated, and measurement of clinical therapeutic end points (circulating levels of F.IX) is straightforward.…”

Section: Introductionmentioning

confidence: 99%

AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B

Manno

Chew

Hutchison

et al. 2003

Blood

695

561

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Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (F.IX). Previously, we established an experimental basis for gene transfer as a method of treating the disease in mice and hemophilic dogs through intramuscular injection of a recombinant adeno-associated viral (rAAV) vector expressing F.IX. In this study we investigated the safety of this approach in patients with hemophilia B. In an open-label dose-escalation study, adult men with severe hemophilia B (F.IX < 1%) due to a missense mutation were injected at multiple intramuscular sites with an rAAV vector. At doses ranging from 2 ؋ 10 11 vector genomes (vg)/kg to 1.8 ؋ 10 12 vg/ kg, there was no evidence of local or systemic toxicity up to 40 months after injection. Muscle biopsies of injection sites performed 2 to 10 months after vector administration confirmed gene transfer as evidenced by Southern blot and transgene expression as evidenced by immunohistochemical staining. Preexisting high-titer antibodies to AAV did not prevent gene transfer or expression. Despite strong evidence for gene transfer and expression, circulating levels of F.IX were in all cases less than 2% and most were less than 1%. Although more extensive transduction of muscle fibers will be required to develop a therapy that reliably raises circulating levels to more than 1% in all subjects, these results of the first parenteral administration of rAAV demonstrate that administration of AAV vector by the intramuscular route is safe at the doses tested and effects gene transfer and expression in humans in a manner similar to that seen in animals. (Blood.

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A factor IX-deficient mouse model for hemophilia B gene therapy

Cited by 161 publications

References 26 publications

Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver

Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver

Permanent partial phenotypic correction and tolerance in a mouse model of hemophilia B by stem cell gene delivery of human factor IX

AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B

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