scholarly journals Liver Transduction with Recombinant Adeno-Associated Virus Is Primarily Restricted by Capsid Serotype Not Vector Genotype

2006 ◽  
Vol 80 (1) ◽  
pp. 426-439 ◽  
Author(s):  
Dirk Grimm ◽  
Kusum Pandey ◽  
Hiroyuki Nakai ◽  
Theresa A. Storm ◽  
Mark A. Kay
Keyword(s):  
Current Model ◽  
Factor Ix ◽  
In Vivo Studies ◽  
Adeno Associated Virus ◽  
Copy Numbers ◽  
Human Factor Ix ◽  
Dna Copy Numbers ◽  
Vector Dna

ABSTRACT We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a gfp gene or the human factor IX (hfIX) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of gfp vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six hfIX variants into AAV-8 and infused mice via the portal vein with doses of 5 × 1010 to 1.8 × 1012 particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in ∼80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV.

scholarly journals Novel Caprine Adeno-Associated Virus (AAV) Capsid (AAV-Go.1) Is Closely Related to the Primate AAV-5 and Has Unique Tropism and Neutralization Properties

2005 ◽  
Vol 79 (24) ◽  
pp. 15238-15245 ◽  
Author(s):  
Alejandra E. Arbetman ◽  
Michael Lochrie ◽  
Shangzhen Zhou ◽  
Jennifer Wellman ◽  
Ciaran Scallan ◽  
...  
Keyword(s):  
Protein Expression ◽  
Neutralization Assay ◽  
Biological Properties ◽  
Scid Mice ◽  
Factor Ix ◽  
In Vivo Model ◽  
Adeno Associated Virus ◽  
Human Factor Ix

ABSTRACT Preexisting humoral immunity to adeno-associated virus (AAV) vectors may limit their clinical utility in gene delivery. We describe a novel caprine AAV (AAV-Go.1) capsid with unique biological properties. AAV-Go.1 capsid was cloned from goat-derived adenovirus preparations. Surprisingly, AAV-Go.1 capsid was 94% identical to the human AAV-5, with differences predicted to be largely on the surface and on or under the spike-like protrusions. In an in vitro neutralization assay using human immunoglobulin G (IgG) (intravenous immune globulin [IVIG]), AAV-Go.1 had higher resistance than AAV-5 (100-fold) and resistance similar to that of AAV-4 or AAV-8. In an in vivo model, SCID mice were pretreated with IVIG to generate normal human IgG plasma levels prior to the administration of AAV human factor IX vectors. Protein expression after intramuscular administration of AAV-Go.1 was unaffected in IVIG-pretreated mice, while it was reduced 5- and 10-fold after administration of AAV-1 and AAV-8, respectively. In contrast, protein expression after intravenous administration of AAV-Go.1 was reduced 7.1-fold, similar to the 3.8-fold reduction observed after AAV-8administration in IVIG-pretreated mice, and protein expression was essentially extinguished after AAV-2 administration in mice pretreated with much less IVIG (15-fold). AAV-Go.1 vectors also demonstrated a marked tropism for lung when administered intravenously in SCID mice. The pulmonary tropism and high neutralization resistance to human preexisting antibodies suggest novel therapeutic uses for AAV-Go.1 vectors, including targeting diseases such as cystic fibrosis. Nonprimate sources of AAVs may be useful to identify additional capsids with distinct tropisms and high resistance to neutralization by human preexisting antibodies.

Adenovirus-mediated regulatable Expression of human Factor IX in vitro and in vivo

2003 ◽  
pp. 72-80
Author(s):  
M. A. Srour ◽  
H. Fechner ◽  
X. Wang ◽  
U. Siemetzki ◽  
T. Albert ◽  
...  
Keyword(s):  
Human Factor ◽  
Factor Ix ◽  
Human Factor Ix

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.

355 COMPARISON OF Tn5 AND SLEEPING BEAUTY SYSTEMS IN BOVINE EMBRYOS AND IN OVINE OFFSPRING

2015 ◽  
Vol 27 (1) ◽  
pp. 265
Author(s):  
R. J. Bevacqua ◽  
R. Fernandéz Martín ◽  
A. Gibbons ◽  
D. Teixeira ◽  
N. G. Canel ◽  
...  
Keyword(s):  
Sleeping Beauty ◽  
Factor Ix ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Fluorescent Reporter ◽  
Human Factor Ix ◽  
Recombinant Human Factor Ix ◽  
Beta Lactoglobulin

Current techniques for the production of transgenic domestic animals remain inefficient. Only recently, DNA transposons resulted in improved efficiencies for mouse and pig transgenesis. In this work, we evaluated Tn5 and Sleeping Beauty systems for transgenesis in bovine and ovine species. First, both transposon systems were assessed in vitro in bovine embryos employing transposons carrying fluorescent reporter genes. In vitro-produced bovine zygotes were microinjected with either 1) a complex of Tn5:egfp transposon (20 ng μL–1) (protein: transgene with mosaic ends recognised by Tn5, in Mg+2 free medium), or 2) two plasmids carrying Sleeping Beauty 100X (pSB100X, 5 ng μL–1) and pT2/Venus transposon (10 ng μL–1). In vitro results for Tn5 transgenesis in bovine showed that blastocysts, Day 4 egfp embryos and egfp blastocysts rates for the group injected with Tn5:egfp did not differ from the group injected with the egfp transposon alone (73/145, 50%; 86/145, 59%; and 65/145, 45% v. 65/129, 50%; 87/129, 67%; and 57/129, 44%, respectively). For SB transgenesis, blastocysts, D4 Venus embryos, and Venus blastocysts rates did not differ between co-injection of pSB100X and pT2/Venus or injection with pT2/Venus alone (46/99, 46.5%; 64/99, 64.6%; and 33/99, 33.3% v. 41/83, 49.4%; 52/83, 62.7%; and 26/83, 31.3%, respectively). However, Venus intensity in blastocysts was markedly higher for the group co-injected with pSB100X and pT2/Venus respective to pT2/Venus alone. Both systems were assessed in vivo for the production of transgenic lambs employing a functional transposon (hrFIX, recombinant human factor IX driven by a Beta-lactoglobulin promoter). Laparoscopic artificial insemination of donor sheep was performed, and presumptive zygotes were flushed from the oviducts. The microinjections were done identically as described for the bovine embryos. A total of 24 presumptive zygotes were recovered and injected with the Tn5:hrFIX complex. Then, 21 zygotes were transferred to 5 synchronized ewes; one pregnancy of siblings was obtained, and one animal was born. Genomic DNA from skin, placenta, and blood was genotyped by PCR, but the hrFIX gene could not be detected. For the SB approach, 64 presumptive zygotes were recovered from 4 superovulated ewes, microinjected with the SB plasmids, and 21 of them were transferred to 7 oestrous synchronized recipients. The remaining zygotes were cultured in vitro and blastocysts (n = 7) were vitrified. Currently, 3 donor ewes are pregnant, one with siblings (4 total fetuses). Deliveries are expected by the end of August of this year. Our results indicate that both Tn5 and SB systems are capable of resulting in the production of transgene expressing embryos, and the presence of the transposases does not affect embryo viability. However, phenotyping of blastocyst stages does not seem to be predictive for stable transgene integration. The in vivo results will help to better address the suitability of Tn5 and SB approaches for the production of transgenic sheep.

Incorporation of the factor IX Padua mutation into FIX-Triple improves clotting activity in vitro and in vivo

2013 ◽  
Vol 110 (08) ◽  
pp. 244-256 ◽  
Author(s):  
Chung-Yang Kao ◽  
Shu-Jhu Yang ◽  
Mi-Hua Tao ◽  
Yung-Ming Jeng ◽  
I-Shing Yu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Replacement Therapy ◽  
Therapeutic Potential ◽  
Factor Ix ◽  
Adeno Associated Virus ◽  
Viral Dose ◽  
Better Than

SummaryUsing gain-of-function factor IX (FIX) for replacement therapy for haemophilia B (HB) is an attractive strategy. We previously reported a high-activity FIX, FIX-Triple (FIX-V86A/E277A/R338A) as a good substitute for FIX-WT (wild-type) in protein replacement therapy, gene therapy, and cell therapy. Here we generated a new recombinant FIXTripleL (FIX-V86A/E277A/R338L) by replacing the alanine at residue 338 of FIX-Triple with leucine as in FIX-Padua (FIX-R338L). Purified FIX-TripleL exhibited 22-fold higher specific clotting activity and 15-fold increased binding affinity to activated FVIII compared to FIXWT. FIX-TripleL increased the therapeutic potential of FIX-Triple by nearly 100% as demonstrated with calibrated automated thrombogram and thromboelastography. FIX-TripleL demonstrated a normal clearance rate in HB mice. The clotting activity of FIX-TripleL was consistently 2- to 3-fold higher in these mice than that of FIX-Triple or FIXR338L. Gene delivery of adeno-associated virus (AAV) in HB mice showed that FIX-TripleL had 15-fold higher specific clotting activity than FIX-WT, and this activity was significantly better than FIX-Triple (10-fold) or FIX-R338L (6-fold). At a lower viral dose, FIX-TripleL improved FIX activity from sub-therapeutic to therapeutic levels. Under physiological conditions, no signs of adverse thrombotic events were observed in long-term AAV-FIX-treated C57Bl/6 mice. Hepatocellular adenomas were observed in the high- but not the medium- or the lowdose AAV-treated mice expressing FIX-WT or FIX-Triple, indicating the advantages of using hyperfunctional FIX variants to reduce viral doses while maintaining therapeutic clotting activity. Thus, incorporation of the FIX Padua mutation significantly improves the clotting function of FIX-Triple so as to optimise protein replacement therapy and gene therapy.

scholarly journals A Limited Number of Transducible Hepatocytes Restricts a Wide-Range Linear Vector Dose Response in Recombinant Adeno-Associated Virus-Mediated Liver Transduction

2002 ◽  
Vol 76 (22) ◽  
pp. 11343-11349 ◽  
Author(s):  
Hiroyuki Nakai ◽  
Clare E. Thomas ◽  
Theresa A. Storm ◽  
Sally Fuess ◽  
Sharon Powell ◽  
...  
Keyword(s):  
Dose Response ◽  
Transgene Expression ◽  
Linear Increase ◽  
Factor Ix ◽  
Adeno Associated Virus ◽  
Linear Dose ◽  
Human Factor Ix ◽  
Wide Range ◽  
High Doses

ABSTRACT Recombinant adeno-associated virus (rAAV) vectors are promising vehicles for achieving stable liver transduction in vivo. However, the mechanisms of liver transduction are not fully understood, and furthermore, the relationships between rAAV dose and levels of transgene expression, total number of hepatocytes transduced, and proportion of integrated vector genomes have not been well established. To begin to elucidate the liver transduction dose response with rAAV vectors, we injected mice with two different human factor IX or Escherichia coli lacZ-expressing AAV serotype 2-based vectors at doses ranging between 4.0 × 108 and 1.1 × 1013 vector genomes (vg)/mouse, in three- to sixfold increments. A 2-log-range linear dose-response curve of transgene expression was obtained from 3.7 × 109 to 3.0 × 1011 vg/mouse. Vector doses above 3.0 × 1011 vg/mouse resulted in disproportionately smaller increases in both the number of transduced hepatocytes and levels of transgene expression, followed by saturation at doses above 1.8 × 1012 vg/mouse. In contrast, a linear increase in the number of vector genomes per hepatocyte was observed up to 1.8 × 1012 vg/mouse concomitantly with enhanced vector genome concatemerization, while the proportion of integrated vector genomes was independent of the vector dose. Thus, the mechanisms that restrict a wide-range linear dose response at high doses likely involve decreased functionality of vector genomes and restriction of transduction to fewer than 10% of total hepatocytes. Such information may be useful to determine appropriate vector doses for in vivo administration and provides further insights into the mechanisms of rAAV transduction in the liver.

scholarly journals Pharmacokinetics of Human Factor IX in a dog with Severe Hemophilia B.

1977 ◽  
Author(s):  
P.A. Gentry ◽  
A.R. Thompson ◽  
A.W. Forrey
Keyword(s):  
Human Factor ◽  
Numerical Procedure ◽  
Factor Ix ◽  
Hemophilia B ◽  
High Yield ◽  
Severe Hemophilia ◽  
Activity Data ◽  
Human Factor Ix

In preparing a factor IX concentrate with a high yield and low hepatitis and thromboembolic risks, we have tested this material for survival in an in vivo system, the hemophiliac dog. By following the disappearance of radiolabeled, isolated factor IX in addition to the classic clotting assays, data on protein survival and more accurate kinetic parameters were obtained.Crude factor IX concentrate was prepared by batchwise adsorption-elution with DEAE-Sephadex using cryoprecipitate-poor human plasma. Isolated human factor IX was radiolabeled with 125I by chloramine-T without in vitro loss of clotting activity (Thompson, J Clin Invest, in press, 1977). A preparation containing both crude and isolated factor IX was then subjected to filtration (0.22 μm) and lyophilization; clotting and radioactivity were not altered by these steps.Following infusion of the combined preparation into a dog with severe hemophilia B (0% baseline factor IX) 10 post infusion samples were taken over 96 h for determination of radioactivity and factor IX clotting activity. These data were then analyzed by fitting to a two exponential expression using a Marquart non-linear least squares numerical procedure for a two compartment open model. The central volume was 14.5% of the animal’s body weight; the total volume of distribution was 28% with a t 1/2 distribution of 114 min. The t 1/2 elimination was 20 h; the slower phase of elimination (β, or that affected by redistribution) had a t 1/2 of 40 h. Factor IX clotting activity from the crude concentrate closely paralleled radioactivity from the isolated factor IX throughout the 96 h; t 1/2 β was slightly longer from the clotting activity data.

scholarly journals Heterogeneity of Factor IX in Therapeutic Concentrates

1977 ◽  
Author(s):  
D. Ménaché ◽  
D. Aronson
Keyword(s):  
A Priori ◽  
Active Material ◽  
Factor Ix ◽  
Biologically Active ◽  
Human Factor Ix ◽  
At Iii ◽  
Unit Factor ◽  
Activated Complex

In vivo and in vitro experience shows that factor IX concentrates are partially activated. A rabbit antibody to human factor IX was used to investigate the factor IX antigenic content and electrophoretic mobility of commercial products as well as experimental “activated products”.“Rocket” Immunoelectrophoresis of all concentrates showed a 1.5–3 fold increased antigenic content/unit factor IX clotting activity when compared to plasma. Two dimensional crossed Immunoelectrophoresis of standard factor IX preparation produced a single sharp peak whether electrophoresed in Ca or EDTA containing buffer. “Activated” preparations produced a dome shaped precipitin arc. The addition of plasma to factor IX concentrates yielded a marked shoulder only when the electrophoresis was done in EDTA. This effect could not be reproduced by the addition of antithrombin III (AT-III). The addition of plasma to the activated IX (IXa) revealed an even more pronounced heterogeneity whether in Ca or EDTA. The addition of AT-III produced a second precipitin peak when activated IX was electrophoresed in the presence of Ca++.These results indicate that at least three forms of factor IX exist in factor IX preparations. The absence of detectable AT-III reacting material in the regular IX preparations is a priori evidence of the absence of major amounts of IXa, whereas the presence of AT-III reacting material in the “activated” complex is evidence of biologically active material.

A Distinction between the Role of Precursor and Activated Forms of Clotting Factors in the Genesis of Stasis Thrombi

1967 ◽  
Vol 18 (01/02) ◽  
pp. 012-023 ◽  
Author(s):  
S Wessler ◽  
E. T Yin ◽  
L. W Gaston ◽  
Iona Nicol
Keyword(s):  
Human Serum ◽  
Factor Ix ◽  
In Vivo Studies ◽  
Factor Vii ◽  
Factor X ◽  
Clotting Factors ◽  
Insight Into

Summary1. In vivo studies in rabbits have shown that the liver diminishes the thrombo-genicity of infused human serum.2. In vitro rabbit liver perfusions with human serum have demonstrated the loss of serum thrombogenicity within 5 min after the onset of the perfusion. Associated with this loss of thrombotic capacity is a marked decrease in the activation product (AP) and labile factor IX (PPA) activity in the infused serum.3. The liver appears to have the capacity to discriminate between circulating activated clotting activities such as AP and PPA and inactive procoagulants such as stable or genuine factor IX, factor VII and factor X. The latter are not cleared from the circulation by the liver.4. These studies provide some insight into the mechanism whereby circulating activated clotting activities and retarded blood flow predispose to thrombosis.

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