Preparation of a recombinant adeno-associated viral vector with a mutation of human factor IX in large scale and its expressionin vitro andin vivo

2001 ◽  
Vol 46 (16) ◽  
pp. 1367-1371
Author(s):  
Huazhong Lu ◽  
Li Chen ◽  
Xuefeng Wang ◽  
Daru Lu ◽  
Xinfang Qiu ◽  
...  
Keyword(s):  
Large Scale ◽  
Human Factor ◽  
Viral Vector ◽  
Factor Ix ◽  
Human Factor Ix

Adeno-Associated Viral Vector-Mediated Gene Transfer of Human Blood Coagulation Factor IX Into Mouse Liver

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4600-4607 ◽  
Author(s):  
Hiroyuki Nakai ◽  
Roland W. Herzog ◽  
J. Nathan Hagstrom ◽  
Johannes Walter ◽  
Szu-Hao Kung ◽  
...  
Keyword(s):  
Portal Vein ◽  
Plasma Levels ◽  
Mouse Liver ◽  
Human Factor ◽  
Viral Vector ◽  
High Titer ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Immunofluorescence Staining ◽  
Human Factor Ix

Abstract Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (1012 to 1013 particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1α promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 × 1010 particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1α-F.IX of 2.7 × 1011particles resulted in plasma levels of 700 to 3,200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1α-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.

Adeno-Associated Viral Vector-Mediated Gene Transfer of Human Blood Coagulation Factor IX Into Mouse Liver

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4600-4607 ◽  
Author(s):  
Hiroyuki Nakai ◽  
Roland W. Herzog ◽  
J. Nathan Hagstrom ◽  
Johannes Walter ◽  
Szu-Hao Kung ◽  
...  
Keyword(s):  
Portal Vein ◽  
Plasma Levels ◽  
Mouse Liver ◽  
Human Factor ◽  
Viral Vector ◽  
High Titer ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Immunofluorescence Staining ◽  
Human Factor Ix

Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (1012 to 1013 particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1α promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 × 1010 particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1α-F.IX of 2.7 × 1011particles resulted in plasma levels of 700 to 3,200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1α-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.

scholarly journals Expression and characterization of human factor IX. Factor IXthr-397 and factor IXval-397

1991 ◽  
Vol 266 (23) ◽  
pp. 15213-15220
Author(s):  
N. Hamaguchi ◽  
P.S. Charifson ◽  
L.G. Pedersen ◽  
G.D. Brayer ◽  
K.J. Smith ◽  
...  
Keyword(s):  
Human Factor ◽  
Factor Ix ◽  
Human Factor Ix

scholarly journals Cooperative activation of human factor IX by the human extrinsic pathway of blood coagulation

1991 ◽  
Vol 266 (17) ◽  
pp. 11317-11327 ◽  
Author(s):  
J.H. Lawson ◽  
K.G. Mann
Keyword(s):  
Blood Coagulation ◽  
Human Factor ◽  
Factor Ix ◽  
Extrinsic Pathway ◽  
Human Factor Ix ◽  
Cooperative Activation

scholarly journals Tyrosine 69 of the first epidermal growth factor-like domain of human factor IX is essential for clotting activity.

1993 ◽  
Vol 268 (24) ◽  
pp. 17727-17733
Author(s):  
P.E. Hughes ◽  
G. Morgan ◽  
E.K. Rooney ◽  
G.G. Brownlee ◽  
P. Handford
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Human Factor ◽  
Factor Ix ◽  
Human Factor Ix ◽  
Epidermal Growth

scholarly journals The binding of human factor IX to endothelial cells is mediated by residues 3-11.

1992 ◽  
Vol 267 (29) ◽  
pp. 20529-20531
Author(s):  
W.F. Cheung ◽  
N Hamaguchi ◽  
K.J. Smith ◽  
D.W. Stafford
Keyword(s):  
Endothelial Cells ◽  
Human Factor ◽  
Factor Ix ◽  
Human Factor Ix

scholarly journals The three-dimensional structure of the first EGF-like module of human factor IX: Comparison with EGF and TGF-α

1992 ◽  
Vol 1 (1) ◽  
pp. 81-90 ◽  
Author(s):  
M. Baron ◽  
D.G. Norman ◽  
T.S. Harvey ◽  
I.D. Campbell ◽  
P.A. Handford ◽  
...  
Keyword(s):  
Human Factor ◽  
Three Dimensional ◽  
Factor Ix ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Human Factor Ix

scholarly journals Electroimmunoassay of Factor IX in the Detection of Haemophilia B Carriers

1977 ◽  
Author(s):  
F. Panicucci ◽  
U. Baicchi ◽  
A. Sagripanti ◽  
E. Pinori
Keyword(s):  
Genetic Variants ◽  
Human Factor ◽  
Factor Ix ◽  
Agarose Gel ◽  
Haemophilia B ◽  
Method Factor ◽  
Human Factor Ix ◽  
Monospecific Antiserum ◽  
Normal Women ◽  
Almost All

Parallel determinations of factor IX activity and factor IX antigen were carried out on 28 haemophilia B carriers and on 20 normal women. Factor IX activity was measured by a one stage method. Factor IX antigen was quantified by electroimmuno assay in agarose gel containing heterologous monospecific antiserum against human factor IX.The activity was observed to be at the same level as the antigen in normal women. Discrepancy was not found between the antigen and the activity in almost all of carriers: only in the mothers of haemophiliacs Bor B+ or BM the factor IX antigen resulted greater than that activity. Our results show that electroimmuno-assay may be used to study carriers of haemophilia B genetic variants and confirm that in the majority of cases they can probably be diagnosed on the basis of factor IX activity alone.

scholarly journals Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 438-445
Author(s):  
TD Palmer ◽  
AR Thompson ◽  
AD Miller
Keyword(s):  
Human Factor ◽  
Normal Skin ◽  
Human Fibroblasts ◽  
Retroviral Vectors ◽  
Factor Ix ◽  
Skin Fibroblasts ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Human Factor Ix

Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

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