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.

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.

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.

scholarly journals Role of the vector genome and underlying factor IX mutation in immune responses to AAV gene therapy for hemophilia B

2014 ◽  
Vol 12 (1) ◽  
pp. 25 ◽  
Author(s):  
Geoffrey L Rogers ◽  
Ashley T Martino ◽  
Irene Zolotukhin ◽  
Hildegund CJ Ertl ◽  
Roland W Herzog
Keyword(s):  
Gene Therapy ◽  
Immune Responses ◽  
Factor Ix ◽  
Hemophilia B ◽  
Underlying Factor ◽  
Vector Genome

Improved muscle-derived expression of human coagulation factor IX from a skeletal actin/CMV hybrid enhancer/promoter

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2536-2542
Author(s):  
J. Nathan Hagstrom ◽  
Linda B. Couto ◽  
Ciaran Scallan ◽  
Melissa Burton ◽  
Mark L. McCleland ◽  
...  
Keyword(s):  
Intramuscular Injection ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Biologically Active ◽  
Expression Cassette ◽  
Hemophilia B ◽  
C2c12 Myotubes ◽  
Coagulation Factor Ix ◽  
Cmv Enhancer ◽  
Actin Promoter

Hemophilia B is caused by the absence of functional coagulation factor IX (F.IX) and represents an important model for treatment of genetic diseases by gene therapy. Recent studies have shown that intramuscular injection of an adeno-associated viral (AAV) vector into mice and hemophilia B dogs results in vector dose–dependent, long-term expression of biologically active F.IX at therapeutic levels. In this study, we demonstrate that levels of expression of approximately 300 ng/mL (6% of normal human F.IX levels) can be reached by intramuscular injection of mice using a 2- to 4-fold lower vector dose (1 × 1011 vector genomes/mouse, injected into 4 intramuscular sites) than previously described. This was accomplished through the use of an improved expression cassette that uses the cytomegalovirus (CMV) immediate early enhancer/promoter in combination with a 1.2-kilobase portion of human skeletal actin promoter. These results correlated with enhanced levels of F.IX transcript and secreted F.IX protein in transduced murine C2C12 myotubes. Systemic F.IX expression from constructs containing the CMV enhancer/promoter alone was 120 to 200 ng/mL in mice injected with 1 × 1011vector genomes. Muscle-specific promoters performed poorly for F.IX transgene expression in vitro and in vivo. However, the incorporation of a sequence from the -skeletal actin promoter containing at least 1 muscle-specific enhancer and 1 enhancer-like element further improved muscle-derived expression of F.IX from a CMV enhancer/promoter-driven expression cassette over previously published results. These findings will allow the design of a clinical protocol for therapeutic levels of F.IX expression with lower vector doses, thus enhancing efficacy and safety of the protocol.

scholarly journals Impact of the Underlying Mutation and the Route of Vector Administration on Immune Responses to Factor IX in Gene Therapy for Hemophilia B

2009 ◽  
Vol 17 (10) ◽  
pp. 1733-1742 ◽  
Author(s):  
Ou Cao ◽  
Brad E Hoffman ◽  
Babak Moghimi ◽  
Sushrusha Nayak ◽  
Mario Cooper ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Immune Responses ◽  
Factor Ix ◽  
Hemophilia B

Self-Complementary AAV Vectors Delivered Intramuscularly Do Not Break Tolerance In a Partial Knockout Model Of Hemophilia B

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4209-4209
Author(s):  
Geoffrey L. Rogers ◽  
Roland W Herzog
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Immune Responses ◽  
Molecular Form ◽  
Factor Ix ◽  
Hemophilia B ◽  
Gag Protein ◽  
Human Factor Ix ◽  
Muscle Gene ◽  
Knockout Model

Abstract Self-complementary adeno-associated viral (scAAV) vectors have become a popular tool for AAV gene therapies due to their increased transgene expression relative to single-stranded (ss)AAV vectors, at least for models in which the transgene can fit within these restrictive constructs. The most recent clinical trial for hemophilia B used a scAAV8 vector. However, recent reports by our lab and others have suggested that these vectors are also more immunogenic than their single-stranded brethren. We previously showed that, during hepatic gene transfer, the innate immune response is greater with a scAAV vector. Moreover, Wu et al. demonstrated an enhanced T and B cell response to a secreted form of HIV’s gag protein when it was delivered via scAAV. While this was beneficial for their vaccine model, it could potentially be problematic for therapeutic gene transfer applications. To investigate whether this phenomenon could deleteriously impact gene therapy for hemophilia B, we used a partial knockout model of hemophilia B. This mouse model has the murine factor IX gene knocked out, and then a truncated form of human factor IX (hF.IX) is inserted. Specifically, the mice we used have a late stop codon (LS) at amino acid 338; while hF.IX can be detected in the liver, this mutated protein is not released into circulation (crm-). During muscle gene transfer with AAV2, LS mice have an interesting response: while they form antibodies to hF.IX, there is no CD8+ T cell infiltrate detected in the transduced tissue. We then set out to determine whether this partial tolerance could be disrupted using a scAAV vector. Using AAV1, which is a more relevant serotype for muscle gene therapy, we injected single-stranded and self-complementary vectors (1011 vg/mouse) and measured the resulting immune responses. Surprisingly, unlike with AAV2, the LS mice completely tolerated AAV1-mediated intramuscular delivery of hF.IX. They did not make detectable anti-hF.IX IgG1, and their plasma had no Bethesda activity through 4 weeks post-injection. In contrast, hF.IX null mice (HBKO) made 10708±3869 ng anti-hF.IX IgG1 and developed inhibitory activity of 22.5±26 Bethesda units 4 weeks after delivery of a scAAV vector. Consequently, LS mice had circulating hF.IX levels of 148.9±18.64 (ssAAV1) and 60.12±8.71 ng/mL (scAAV1), while hF.IX was undetectable in the plasma of HBKO mice at this time point. The CD8 response was similarly lacking in LS mice, with splenic responses to hF.IX detected by ELISPOT reduced by ∼2-10 fold relative to HBKO mice. Infiltrating CD8 cells were detected in the muscle of HBKO but not LS mice. In conclusion, our data suggest that the underlying mutation within the patient is a more important risk factor for immune responses to the transgene than the molecular form of the AAV genome. Disclosures: Herzog: Genzyme: AAV technology, AAV technology Patents & Royalties.

Adeno-Associated Virus-Mediated Gene Transfer of Factor IX for Treatment of Hemophilia B by Gene Therapy

1999 ◽  
Vol 82 (08) ◽  
pp. 540-546 ◽  
Author(s):  
Roland Herzog ◽  
Katherine High
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Biologically Active ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Severe Hemophilia ◽  
Dog Model ◽  
Life Threatening

IntroductionPatients with severe hemophilia have circulating blood coagulation factor VIII (hemophilia A) or factor IX (hemophilia B) levels below 1% of normal due to a genetic defect in the respective X-linked gene. The resulting bleeding disorder is characterized by spontaneous joint bleeds or, in a more life-threatening situation, into critical closed spaces, such as the intracranial or retroperitoneal space. Current treatment for hemophilia is based on intravenous infusions of clotting factor concentrates. These can be episode-based in response to bleeds (which does not prevent ongoing tissue damage nor the risk of a life-threatening bleed) or prophylactic (an expensive and not always practical alternative). The goal of a gene-based therapy is to introduce a functional clotting factor gene into a patient in order to provide a continuous supply of factor levels above 1%.1,2 Clinical endpoints for the efficacy of potential gene therapy trials for hemophilia are, therefore, well-defined and unequivocal.The relatively small size of the factor IX coding sequence (1.4 kb) and the fact that a number of cell types other than hepatocytes (which normally synthesize factor IX) are capable of producing biologically-active factor IX have contributed to the development of hemophilia B into an important model for the treatment of genetic diseases by gene therapy. The factor IX gene can be incorporated into a variety of vector systems. Various target tissues can be chosen for gene transfer as long as the secreted factor IX reaches the circulation and tight regulation of transgene expression is not required.3 Possibly most important in research on gene therapy for coagulation factor deficiencies, and genetic disorders in general, is the availability of a large animal model with severe disease. In this case, it is the well-characterized hemophilia B dogs maintained at the University of North Carolina at Chapel Hill. The animals contain a point-mutation in the portion of the factor IX gene encoding the catalytic domain. This mutation results in an absence of circulating factor IX antigen and, consequently, severe hemophilia B that closely mimics the human disease.4 Gene therapy strategies for hemophilia B have typically established a method of gene transfer, resulting in expression of factor IX in mice, and subsequently, attempted scale-up to the dog model. These investigations have established experiments in the hemophilic dog model as a critical step for the assessment of the efficacy of gene therapy protocols showing initial promise in mice. For example, reimplantation of primary myoblasts that had been transduced ex vivo with a retrovirus was successful in mice, but not in the canine model.5 Adenoviral gene transfer, characterized by varying success in mice, depending on the strain and dose used, has persistently resulted in high, but transient expression following intravenous infusion into dogs.6,7 Cellular immune responses and hepatotoxicity have limited the expression of factor IX from adenoviral vectors to just a few weeks. Repeat administration of the vector was complicated by the induction of neutralizing antibodies to viral particles in injected animals following the first administration. Retroviral gene transfer to hepatocytes was successful in long-term expression of factor IX in hemophilia B dogs but required a partial hepatectomy prior to infusion of the vector through the portal vein. The resulting expression levels were no higher than 0.1% of normal human factor IX levels.8

scholarly journals BAX 335 hemophilia B gene therapy clinical trial results - potential impact of CpG sequences on gene expression

Blood ◽  
2020 ◽  
Author(s):  
Barbara A Konkle ◽  
Christopher Walsh ◽  
Miguel A Escobar ◽  
Neil C Josephson ◽  
Guy Young ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Immune Responses ◽  
Phase 1 ◽  
Factor Ix ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Open Label ◽  
Serious Adverse Events ◽  
Dose Escalation Study ◽  
Cpg Oligodeoxynucleotides

Gene therapy has the potential to maintain therapeutic blood clotting factor IX (FIX) levels in patients with hemophilia B by delivering a functional human F9 gene into liver cells. This phase 1/2, open-label dose-escalation study investigated BAX 335 (AskBio009, AAV8.sc-TTR-FIXR338Lopt), an adeno-associated virus (AAV) serotype 8-based FIX Padua gene therapy, in patients with hemophilia B. This report focuses on 12-month interim analyses of safety, pharmacokinetics, effects on FIX activity, and immune responses for dosed participants. Eight adult males (\aged 20-69 years; range FIX activity, 0.5%-2.0%) received 1 of 3 BAX 335 IV doses: 2.0 × 1011; 1.0 × 1012; or 3.0 × 1012 vector genomes/kg. Three (37.5%) participants had 4 serious adverse events (SAEs), all considered unrelated to BAX 335. No SAE led to death. No clinical thrombosis, inhibitors, or other FIX Padua-directed immunity were reported. FIX expression was measurable in 7 of 8 participants; peak FIX activity displayed dose dependence (32.0%-58.5% in cohort 3). One participant achieved sustained therapeutic FIX activity of ~20%, without bleeding or replacement therapy, for 4 years; in others, FIX activity was not sustained beyond 5-11 weeks. In contrast to some previous studies, corticosteroid treatment did not stabilize FIX activity loss. We hypothesize that the loss of transgene expression could have been caused by stimulation of innate immune responses including CpG oligodeoxynucleotides introduced into the BAX 335 coding sequence by codon optimization. Registered at www.clinicaltrials.gov as NCT01687608.

scholarly journals Coagulation Factor IX for Hemophilia B Therapy

Acta Naturae ◽  
2012 ◽  
Vol 4 (2) ◽  
pp. 62-73
Author(s):  
N. A. Orlova ◽  
S. V. Kovnir ◽  
I. I. Vorobiev ◽  
A. G. Gabibov
Keyword(s):  
Gene Therapy ◽  
Recombinant Protein ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Coagulation Cascade ◽  
Transgenic Organisms ◽  
Current State ◽  
Coagulation Factor Ix ◽  
Functional Factor

Factor IX is a zymogen enzyme of the blood coagulation cascade. Inherited absence or deficit of the IX functional factor causes bleeding disorder hemophilia B, which requires constant protein replacement therapy. Reviewed herein are the current state in the manufacturing of FIX, improved variants of the recombinant protein for therapy, transgenic organisms for obtaining FIX, and the advances in the gene therapy of hemophilia B.

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