Coagulation factor IX gene transfer to non-human primates using engineered AAV3 capsid and hepatic optimized expression cassette

We have developed a new helper-dependent (HD) adenoviral vector FTC that contains 3 cis-acting sequences as stuffer DNA: a human fragment of alphoid repeat DNA, matrix-attachment regions (MARs), and the hepatocyte control region enhancer. To determine the most robust human coagulation factor IX (hFIX) expression cassette in an adenovirus, we first tested different hFIX expression sequences with or without flanking MARs in first-generation adenoviral vectors. After intravenous infusion of the vector, serum levels of up to 100 000 ng/mL hFIX (normal level, 5000 ng/mL) were obtained at nontoxic doses. In order to make a direct comparison, a first-generation and a gene-deleted vector with identical hFIX expression cassettes were constructed. Both first-generation and HD adenovirus–treated animals demonstrated a threshold effect in a dose-response study. With the administration of 2 × 109transducing particles of either vector, supraphysiological serum levels of hFIX were obtained, with the highest expression (41 000 ng/mL) occurring during the first 2 months after injection. The serum factor IX concentrations, while remaining in the therapeutic range, slowly declined by 95% over a period of 1 year. At this dose, interleukin-6 and tumor necrosis factor–α serum concentrations were elevated in animals that received the first-generation but not the HD vector. This study compares the properties of a gene-deleted and first-generation adenovirus with equivalent expression cassettes and suggests that thecis-DNA elements contained in the vector and expression cassette have important effects on gene expression in vivo.

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

Blood ◽

10.1182/blood.v95.8.2536.008k26_2536_2542 ◽

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.

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Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector

Nature Medicine ◽

10.1038/4743 ◽

1999 ◽

Vol 5 (1) ◽

pp. 56-63 ◽

Cited By ~ 413

Author(s):

Roland W. Herzog ◽

Edmund Y. Yang ◽

Linda B. Couto ◽

J. Nathan Hagstrom ◽

Dan Elwell ◽

...

Keyword(s):

Gene Transfer ◽

Blood Coagulation ◽

Viral Vector ◽

Coagulation Factor ◽

Factor Ix ◽

Hemophilia B ◽

Coagulation Factor Ix

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Orphan designation: Recombinant adeno-associated viral vector serotype S3 containing codon-optimised expression cassette encoding human coagulation factor IX variant, Treatment of haemophilia B

Case Medical Research ◽

10.31525/cmr-8794f1 ◽

2019 ◽

Author(s):

Keyword(s):

Viral Vector ◽

Coagulation Factor ◽

Factor Ix ◽

Expression Cassette ◽

Haemophilia B ◽

Orphan Designation ◽

Coagulation Factor Ix

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Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.94.11.5804 ◽

1997 ◽

Vol 94 (11) ◽

pp. 5804-5809 ◽

Cited By ~ 351

Author(s):

R. W. Herzog ◽

J. N. Hagstrom ◽

S.-H. Kung ◽

S. J. Tai ◽

J. M. Wilson ◽

...

Keyword(s):

Gene Transfer ◽

Blood Coagulation ◽

Human Blood ◽

Intramuscular Injection ◽

Coagulation Factor ◽

Factor Ix ◽

Stable Gene ◽

Adeno Associated Virus ◽

Coagulation Factor Ix ◽

Human Blood Coagulation

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

Blood ◽

10.1182/blood.v95.8.2536 ◽

2000 ◽

Vol 95 (8) ◽

pp. 2536-2542 ◽

Cited By ~ 55

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

Abstract 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.

Download Full-text