Targeted Inactivation of the Coagulation Factor IX Gene Causes Hemophilia B in Mice

Blood ◽  
1998 ◽  
Vol 92 (1) ◽  
pp. 168-174 ◽  
Author(s):  
Ramendra Krishna Kundu ◽  
Frank Sangiorgi ◽  
Lang-Ying Wu ◽  
Kotoku Kurachi ◽  
W. French Anderson ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Ix ◽  
Hemophilia B ◽  
Current Therapy ◽  
Male Mice ◽  
Female Mice ◽  
Carrier Female ◽  
Hemizygous Male ◽  
The Mean ◽  
Therapy Strategies

Hemophilia B is a leading target for gene therapy because current therapy is not optimal. Hence, a murine model of factor IX (F. IX) deficiency was generated to develop gene therapy strategies for hemophilia B. A targeting vector was created by replacing a 3.2-kb segment of the gene encompassing the catalytic domain with a phosphoglycerokinase promoter-driven neomycin resistant (neor) gene cassette. The transfected embryonic stem cell clones generated chimeric male mice, and germ line transmission of the inactivated F. IX gene was observed in their offsprings. Southern analysis confirmed the mutant genotype in hemizygous male and carrier female mice. F. IX transcripts were not detected in liver RNA isolated from hemizygous mice, and lower levels of F. IX mRNA were noted in carrier female mice when compared with those of normal litter mates. As expected, the mean F. IX coagulant titer of affected male mice was 2.8 U/dL (n = 10), while the mean F. IX titer of carrier female mice was 35 U/dL (n = 14), compared with 69 U/dL (n = 9) for the normal female mice and 92 U/dL (n = 22) for normal male and female litter mates. Further, the tail bleeding time of hemizygous mice was markedly prolonged (>3 hours) compared with those of normal and carrier female litter mates (15 to 20 minutes). Seven of 19 affected male mice died of exsanguination after tail snipping, and two affected mice died of umbilical cord bleeding. Currently, there are 10 affected mice surviving at 4 months of age. Aside from the factor IX defect, the carrier female and hemizygous male mice had no liver pathology by histologic examination, were fertile, and transmitted the F. IX gene mutation in the expected Mendelian frequency. Taken together, we have generated a F. IX knockout mouse for evaluation of novel gene therapy strategies for hemophilia B.

Extravascular Administration of Factor IX: Potential for Replacement Therapy of Canine and Human Hemophilia B

1997 ◽  
Vol 77 (05) ◽  
pp. 0944-0948 ◽  
Author(s):  
Darla Liles ◽  
Charles N Landen ◽  
Dougald M Monroe ◽  
Celeste M Lindley ◽  
Marjorie s Read ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Vitamin K ◽  
Absorption Rate ◽  
Venous Access ◽  
Factor Ix ◽  
Hemophilia B ◽  
Current Therapy ◽  
Home Therapy ◽  
Routes Of Administration ◽  
Plasma Compartment

SummaryCurrent therapy for hemophilia B requires large intravenous doses of factor IX (F.IX) given in the clinic or at home. Although home therapy is possible for many patients, it is often complicated by factors such as the lack of good venous access. Very little is known about extravascular routes for administering proteins like F.IX (57 kD) or other vitamin K-dependent procoagulant factors into the circulation. Questions about the absorption rate from extravascular administration as well as plasma recovery and bioavailability have arisen recently with the growing availibility of highly purified procoagulant proteins and increased interest in gene therapy of hemophilia B. Therefore, a group of studies were undertaken to determine the absorption rate, plasma recovery, and bioavailability of high purity, human plasma-derived F.IX concentrates administered via extravascular routes in hemophilia B dogs and in one human hemophilia B subject. Five hemophilia B dogs were given human F.IX via either a subcutaneous (SC), intramuscular (IM), intra- peritoneal (IP) or intravenous (IV) route. In a subsequent study, a single SC administration of human F.IX was compared to an identical IV dose of F.IX in the human hemophilia B subject. All extravascular routes of F.IX administration in both the canine and human gave lower levels of circulating plasma F.IX than the IV route, however all routes resulted in measurable F.IX activity. Of the extravascular routes, the IM injection in the canine resulted in a bioavailibility of 82.8%, while the SC injection resulted in a bioavailability of 63.5%. F.IX reached the plasma compartment by all extravascular routes used, confirming that F.IX can be absorbed extravascularly. The duration of measurable F.IX activity following extravascular administration is prolonged beyond that typically seen with IV administration. These data show that significant levels of F.IX may be obtained via SC injection in canine and ‘ human hemophilia B subjects and further highlight the potential of extravascular routes of administration for future experimental and clinical uses of F.IX and other procoagulant proteins.

Efficient Induction of Tolerance to FIX by Direct Intramuscular Delivery of AAV1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3287-3287
Author(s):  
Ellen F. Cohn ◽  
Meagan E. Kelly ◽  
Jiacai Zhuo ◽  
Hengjun Chao
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Immune Responses ◽  
Intramuscular Injection ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Current Therapy ◽  
Efficient Induction ◽  
Induction Of Tolerance

Abstract Hemophilia B is an X-linked recessive genetic disease resulting from deficiency in coagulation factor IX (FIX). The current therapy for hemophilia B is life-long replacement of FIX through recombinant FIX or purified blood products in response to bleeding events. However, this replacement therapy is non-prophylactic, costly, and can be complicated by formation of inhibitory anti-FIX antibodies in up to 5% of patients. While somatic gene therapy is expected to provide a final cure for hemophilia B, it may also cause high incidence of FIX antibodies formation and other adverse immune responses following gene delivery. Direct intramuscular injection of adeno-associated virus (AAV) is a safe and promising procedure for hemophilia B gene therapy. This treatment, however, elicits anti-FIX antibodies in immune competent animal models. We have previously reported that intramuscular injection of AAV1 expressed high levels of canine FIX and induced FIX tolerance in a mouse model of hemophilia B, but AAV2 elicited anti-FIX antibodies. Here, we report efficient induction of human FIX (hFIX) tolerance in naive as well as FIX-pre-immunized animals by direct intramuscular injection of AAV1 vectors. Following injection of 1×1011 of AAV1 expressing hFIX per mouse in hemostatically-normal and FIX knock out mice, we detected close to 1000ng/ml of hFIX antigen by ELISA 8 weeks post AAV injection (n=5). No significant level of anti-FIX antibodies could be detected in these mice, by either ELISA or modified Bethesda inhibitor assay. In addition, subsequent challenge with recombinant hFIX in complete Freund’s adjuvant did not cause anti-FIX antibodies to be produced and the level of hFIX in the blood remained constant. However, anti-FIX antibodies, but not hFIX antigen, were measured in the mice injected with the same dose of AAV2 (n=7). Subsequent injection of AAV1 vector into the skeletal muscle of these AAV2-injected mice resulted in the disappearance of anti-FIX antibodies and emergence of FIX antigen at similar levels to AAV1-injected naive mice in the circulation of these mice. In addition, direct intramuscular injection of AAV1 also induced FIX tolerance in mice that developed anti-FIX antibodies after exposure to recombinant FIX proteins (n=6). Similar experiments in mice with different genetic and MHC backgrounds have also demonstrated efficient induction of tolerance to FIX, implying that AAV1-hFIX can induce tolerance regardless of MHC haplotype. We hypothesize that the immediate expression of high levels of FIX from the non-pathogenic AAV1 induces FIX tolerance. To elucidate the mechanism of different immune responses to FIX following intramuscular injection of AAV1 and AAV2, we are examining variations in antigen presentation, interaction between antigen presenting cells and antigen-specific T cells, and fate of antigen-specific T cells following intramuscular injection of AAV1 and AAV2 vectors. In summary, our results demonstrate efficient induction of FIX following direct intramuscular injection of AAV1 vectors. Investigations to elucidate the underlying mechanism are ongoing in our lab.

scholarly journals AMT-060 Gene Therapy in Adults with Severe or Moderate-Severe Hemophilia B Confirm Stable FIX Expression and Durable Reductions in Bleeding and Factor IX Consumption for up to 5 Years

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 26-26
Author(s):  
Frank W.G. Leebeek ◽  
Karina Meijer ◽  
Michiel Coppens ◽  
Peter Kampmann ◽  
Robert Klamroth ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Board Of Directors ◽  
Research Funding ◽  
Factor Ix ◽  
Hemophilia B ◽  
First Year ◽  
Severe Hemophilia ◽  
Advisory Committees ◽  
The Mean

Background: Gene therapy aims to provide long-term therapeutic benefit from a single administration. AMT-060 is an adeno-associated virus serotype 5 (AAV5) vector with a codon-optimized wildtype human factor IX (FIX) gene and liver-specific promoter. AMT-060 is being evaluated in an ongoing study of 10 participants with severe/moderate-severe hemophilia B (Phase 1/2 study, NCT02396342) over 5 years. Aim: To describe efficacy and safety outcomes from an analysis at up to 5-years post-AMT-060. Methods: Adult males with FIX activity ≤2% and a severe bleeding phenotype received a single intravenous infusion of AMT-060 (5x1012 gc/kg, Cohort 1, n=5) or (2×1013 gc/kg, Cohort 2, n=5). Assessments included FIX activity, FIX replacement use, annualized bleeding rate (ABR), treatment-related adverse events (TRAE), immunological and inflammatory biomarkers up to 5 years (Cohort 1) and 4.5 years (Cohort 2). Results: As of November 2019, for Cohort 1 the mean FIX activity (at 4.0 years) was 5.1% as compared to 4.4% in the first year, 6.8% in the second year, 7.3% in the third year and 7.0% in the fourth year. Mean FIX activity for Cohort 2 was 7.5% as compared to 7.1% in the first year, 8.4% in the second year 7.9% in the third year, and 7.4% in the fourth year. Eight of 9 participants using prophylaxis at baseline were able to discontinue use. During the last 12, and 6 months of observation respectively, the mean annualized bleed rate (ABR) was 3.3. for Cohort 1 and 0.0 for Cohort 2. These represent, respectively, a reduction in mean ABR to the year prior to treatment of 77% and 100% for Cohort 1 and Cohort 2. During this same period the consumption of FIX replacement therapy declined 90% and 100% relative to pre-treatment, respectively for Cohort 1 and Cohort 2. No participants developed FIX inhibitors or signs of sustained AAV5 capsid-specific T-cell activation. As previously reported, TRAE were mainly reported in the first 3.5 months after treatment, including three participants who experienced transient mild elevations in alanine aminotransferase. One additional TRAE (joint swelling post-exercise) was observed during the last 12 months of observation post-treatment. Updated data, up to 5-years of observation, will be presented for the first time. Conclusions: Long-term stable endogenous FIX activity and reductions in ABR and FIX replacement use were sustained over multiple years following a single treatment with AMT-060. There were no additional safety concerns with longer term follow-up. This data supports the ongoing Phase 3 study of the enhanced construct etranacogene dezaparvovec (AMT-061), which encodes the highly active Padua FIX variant. Disclosures Meijer: Bayer: Research Funding; Sanquin: Research Funding; Pfizer: Research Funding; Bayer: Speakers Bureau; Sanquin: Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; BMS: Speakers Bureau; Aspen: Speakers Bureau; Uniqure: Consultancy. Kampmann:Uniqure: Speakers Bureau; Shire Pharmaceuticals: Speakers Bureau. Klamroth:CSL Behring: Research Funding, Speakers Bureau; Novo Nordisk: Consultancy, Research Funding, Speakers Bureau; Octapharma: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Takeda/Shire: Consultancy, Research Funding, Speakers Bureau; Sobi: Consultancy, Speakers Bureau; Biotest: Speakers Bureau; Grifols: Speakers Bureau; Biomarin: Consultancy, Research Funding, Speakers Bureau; Bayer: Consultancy, Research Funding, Speakers Bureau. Castaman:Novo Nordisk: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding; Ablynx: Honoraria; Alexion: Honoraria; Bayer: Honoraria; CSL Behring: Honoraria, Research Funding; Kedrion: Speakers Bureau; Sobi: Honoraria, Research Funding, Speakers Bureau; Uniqure: Honoraria, Membership on an entity's Board of Directors or advisory committees; Werfen: Speakers Bureau; Baxalta/Shire: Honoraria. Bönig:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Healthineers: Current equity holder in publicly-traded company; Sandor-Hexal: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphor: Research Funding; Miltenyi: Honoraria, Research Funding; Erydel: Research Funding; Chugai: Honoraria, Research Funding; Bayer: Research Funding; Terumo BCT: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kiadis: Honoraria; Uniqure: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Stage: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Fresenius: Honoraria; medac: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Sawyer:uniQure: Current Employment. Miesbach:UniQure: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BioMarin Pharmaceutical Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: AMT-060 = AAV5 vector gene therapy in subjects with moderate to severe hemophilia B

scholarly journals Sustained Expression of Therapeutic Level of Factor IX in Hemophilia B Dogs by AAV-Mediated Gene Therapy in Liver

2000 ◽  
Vol 1 (2) ◽  
pp. 154-158 ◽  
Author(s):  
Lili Wang ◽  
Timothy C. Nichols ◽  
Marjorie S. Read ◽  
Dwight A. Bellinger ◽  
Inder M. Verma
Keyword(s):  
Gene Therapy ◽  
Factor Ix ◽  
Hemophilia B ◽  
Therapeutic Level

scholarly journals Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5095-5103 ◽  
Author(s):  
G Hortelano ◽  
A Al-Hendy ◽  
FA Ofosu ◽  
PL Chang
Keyword(s):  
Gene Therapy ◽  
Human Factor ◽  
Ex Vivo ◽  
Cost Effective ◽  
Factor Ix ◽  
Hemophilia B ◽  
Therapy Strategy ◽  
Human Factor Ix

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.

scholarly journals Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant

2017 ◽  
Vol 377 (23) ◽  
pp. 2215-2227 ◽  
Author(s):  
Lindsey A. George ◽  
Spencer K. Sullivan ◽  
Adam Giermasz ◽  
John E.J. Rasko ◽  
Benjamin J. Samelson-Jones ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Activity Factor

Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy

Blood ◽  
2002 ◽  
Vol 99 (8) ◽  
pp. 2670-2676 ◽  
Author(s):  
Jane D. Mount ◽  
Roland W. Herzog ◽  
D. Michael Tillson ◽  
Susan A. Goodman ◽  
Nancy Robinson ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Large Animal Model ◽  
Null Mutation ◽  
Large Animal ◽  
Adeno Associated Virus ◽  
Increased Risk

Abstract Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (FIX). Using adeno-associated virus (AAV)–mediated, liver-directed gene therapy, we achieved long-term (> 17 months) substantial correction of canine hemophilia B in 3 of 4 animals, including 2 dogs with an FIX null mutation. This was accomplished with a comparatively low dose of 1 × 1012 vector genomes/kg. Canine FIX (cFIX) levels rose to 5% to 12% of normal, high enough to result in nearly complete phenotypic correction of the disease. Activated clotting times and whole blood clotting times were normalized, activated partial thromboplastin times were substantially reduced, and anti-cFIX was not detected. The fourth animal, also a null mutation dog, showed transient expression (4 weeks), but subsequently developed neutralizing anti-cFIX (inhibitor). Previous work in the canine null mutation model has invariably resulted in inhibitor formation following treatment by either gene or protein replacement therapies. This study demonstrates that hepatic AAV gene transfer can result in sustained therapeutic expression in a large animal model characterized by increased risk of a neutralizing anti-FIX response.

Neonatal or hepatocyte growth factor–potentiated adult gene therapy with a retroviral vector results in therapeutic levels of canine factor IX for hemophilia B

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 3924-3932 ◽  
Author(s):  
Lingfei Xu ◽  
Cuihua Gao ◽  
Mark S. Sands ◽  
Shi-Rong Cai ◽  
Timothy C. Nichols ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Growth Factor ◽  
Gene Transfer ◽  
Hepatocyte Growth Factor ◽  
Retroviral Vector ◽  
Factor Ix ◽  
Hemophilia B ◽  
Hepatocyte Growth

AbstractHemophilia B is a bleeding disorder resulting from factor IX (FIX) deficiency that might be treated with gene therapy. Neonatal delivery would correct the disease sooner than would transfer into adults, and could reduce immunological responses. Neonatal mice were injected intravenously with a Moloney murine leukemia virus–based retroviral vector (RV) expressing canine FIX (cFIX). They achieved 150% to 280% of normal cFIX antigen levels in plasma (100% is 5 μg/mL), which was functional in vitro and in vivo. Three newborn hemophilia B dogs that were injected intravenously with RV achieved 12% to 36% of normal cFIX antigen levels, which improved coagulation tests. Only one mild bleed has occurred during 14 total months of evaluation. This is the first demonstration of prolonged expression after neonatal gene therapy for hemophilia B in mice or dogs. Most animals failed to make antibodies to cFIX, demonstrating that neonatal gene transfer may induce tolerance. Although hepatocytes from newborns replicate, those from adults do not. Adult mice therefore received hepatocyte growth factor to induce hepatocyte replication prior to intravenous injection of RV. This resulted in expression of 35% of normal cFIX antigen levels for 11 months, although all mice produced anti-cFIX antibodies. This is the first demonstration that high levels of FIX activity can be achieved with an RV in adults without a partial hepatectomy to induce hepatocyte replication. We conclude that RV-mediated hepatic gene therapy is effective for treating hemophilia B in mice and dogs, although the immune system may complicate gene transfer in adults.

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