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 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.

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.

Targeting lentiviral vector expression to hepatocytes limits transgene-specific immune response and establishes long-term expression of human antihemophilic factor IX in mice

Blood ◽  
2004 ◽  
Vol 103 (10) ◽  
pp. 3700-3709 ◽  
Author(s):  
Antonia Follenzi ◽  
Manuela Battaglia ◽  
Angelo Lombardo ◽  
Andrea Annoni ◽  
Maria Grazia Roncarolo ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Transfer ◽  
Immune Responses ◽  
Transgene Expression ◽  
Factor Ix ◽  
Stable Gene ◽  
Human Factor Ix ◽  
Immunocompetent Mice

Abstract Stable gene replacement by in vivo administration of lentiviral vectors (LVs) has therapeutic potential for metabolic disorders and other systemic diseases. We studied the expression of intracellular and secreted proteins by LVs in immunocompetent mice. Liver, spleen, and bone marrow cells were efficiently transduced. However, transgene expression, driven by a ubiquitous promoter, was limited by transgene-specific cellular and humoral immune responses, leading to the clearance of transduced cells. After green fluorescent protein (GFP) gene transfer, the liver showed infiltration of CD8+ cytotoxic T cells, and GFP-specific CD8+ T cells were isolated from the spleen. After human factor IX (hF.IX) gene transfer, anti-hF.IX antibodies were induced. These immune responses were not detected in mice injected with heat-inactivated or genome-lacking LVs or in GFP-transgenic mice, indicating that they were specifically triggered by transgene expression in vivo. Intriguingly, selective targeting of LV expression to hepatocytes limited the immune responses to the transgenes. By this approach, high levels of hF.IX, potentially in the therapeutic range, were reached and maintained long term in immunocompetent mice, without inducing antibody formation. These results prompt further studies in relevant animal models to explore the potential of in vivo LV administration for the gene therapy of hemophilias and other liver-based diseases.

scholarly journals Proteolytic inactivation of blood coagulation factor IX by thrombin

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1302-1308 ◽  
Author(s):  
W Kisiel ◽  
KJ Smith ◽  
BA McMullen
Keyword(s):  
Human Factor ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Light Chains ◽  
Factor X ◽  
Amino Terminal ◽  
Coagulation Factor Ix ◽  
Human Factor Ix ◽  
Coagulant Activity

Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin- cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.

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

In vivo biological response to recombinant interferon-gamma during a phase I dose-response trial in patients with metastatic melanoma.

1990 ◽  
Vol 8 (6) ◽  
pp. 1070-1082 ◽  
Author(s):  
J M Kirkwood ◽  
M S Ernstoff ◽  
T Trautman ◽  
G Hebert ◽  
Y Nishida ◽  
...  
Keyword(s):  
Interferon Gamma ◽  
Dose Response ◽  
Dose Range ◽  
Cell Subset ◽  
Optimal Dosage ◽  
Antitumor Effects ◽  
Ifn Gamma ◽  
Recombinant Interferon ◽  
Wide Range

Interferon-gamma (IFN gamma), as produced by recombinant DNA technology, has shown a wide range of immunomodulatory activity in vitro and in vivo. Clinical studies have attempted to establish a dose-response relationship to define optimal dosage ranges for induction of effector cell function and host response in patients with cancer. We conducted a randomized trial to test the in vivo biologic activity of five daily dosages ranging from 3 to 3,000 micrograms/m2, administered by daily 2-hour bolus injection or by continuous infusion for 14 days. We demonstrate comparable immunobiologic effects of recombinant IFN gamma (rIFN gamma; Biogen, Inc, Cambridge, MA) administered by these two schedules at the various dosages tested, and have defined a relationship of dose to biologic response over this 3-log10 dose range. Oligo 2'5' adenylate synthetase (2'5'As) induction, natural-killer (NK) cell activity, and T-cell subset distribution (heightened T helper/suppressor ratio) showed the most consistent treatment-associated changes and the greatest immunobiologic effects at dosages of 300 to 1,000 micrograms/m2. Mononuclear cell DR and DQ antigen expression showed no consistent dose-related treatment effect. The relevance of the phenotypic, functional, and enzymologic effects observed in this trial to any clinical antitumor effects of IFN gamma in cancer therapy must now be established.

scholarly journals 860. Sustained Long_Term In Vivo Expression of Human Factor IX from Keratinocytes Transduced with HIV_Based Vectors

2006 ◽  
Vol 13 ◽  
pp. S331-S332
Author(s):  
Angeles Escarti-Nebot ◽  
Fernando Serrano ◽  
Marcela delRio ◽  
Fernando Larcher ◽  
Antonio Bernad
Keyword(s):  
Human Factor ◽  
Factor Ix ◽  
Human Factor Ix ◽  
In Vivo Expression

scholarly journals Lack of Repeat Transduction by Recombinant Adeno-Associated Virus Type 5/5 Vectors in the Mouse Airway

2007 ◽  
Vol 81 (22) ◽  
pp. 12360-12367 ◽  
Author(s):  
Stephanie G. Sumner-Jones ◽  
Deborah R. Gill ◽  
Stephen C. Hyde
Keyword(s):  
Transgene Expression ◽  
Lung Diseases ◽  
Repeated Administration ◽  
Transduction Efficiency ◽  
Viral Gene ◽  
Rapid Rise ◽  
Adeno Associated Virus ◽  
Subsequent Failure

ABSTRACT While recombinant adeno-associated virus (rAAV) vectors promote long-term transgene expression in the lungs and other organs, the goal of correcting chronic inherited lung diseases such as cystic fibrosis with this type of viral gene transfer vector is limited by the requirement of achieving stable potent transgene expression, potentially requiring vector readministration. Here we evaluated the abilities of rAAV type 5/5 (rAAV5/5) vectors based on the genome and capsid of AAV5 to efficiently transduce the lungs and nasal epithelium of mice after repeated administration. Transduction efficiency as judged by reporter gene expression was markedly reduced on a second rAAV5/5 administration and effectively abolished on a third. Varying the period between administrations from 8 to 36 weeks did not allow efficient repeated administration. A rapid rise in anti-AAV5 antibodies was noted after rAAV5/5 vector administration that was sustained for the entire period of investigation (in some cases exceeding 9 months). Furthermore, this antibody response and subsequent failure to repeatedly administer the vector were not rescued by the in vivo expression of CTLA4Ig from an rAAV5/5 vector. These results suggest that without the development of an effective and clinically acceptable immunosuppression strategy, treatments for chronic diseases that require repeated administration of rAAV5/5 vectors will be unsuccessful.

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.

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