scholarly journals Improved Hepatic Gene Transfer by Using an Adeno-Associated Virus Serotype 5 Vector

2002 ◽  
Vol 76 (20) ◽  
pp. 10497-10502 ◽  
Author(s):  
Federico Mingozzi ◽  
Jörg Schüttrumpf ◽  
Valder R. Arruda ◽  
Yuhong Liu ◽  
Yi-Lin Liu ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Transgene Expression ◽  
Coagulation Factor ◽  
Liver Parenchyma ◽  
Factor Ix ◽  
Transduction Efficiency ◽  
Stable Gene ◽  
Inherited Disorders ◽  
Virus Serotype ◽  
Serotype 5

ABSTRACT Adeno-associated viral (AAV) vectors have been shown to direct stable gene transfer and expression in hepatocytes, which makes them attractive tools for treatment of inherited disorders such as hemophilia B. While substantial levels of coagulation factor IX (F.IX) have been achieved using AAV serotype 2 vectors, use of a serotype 5 vector further improves transduction efficiency and levels of F.IX transgene expression by 3- to 10-fold. In addition, the AAV-5 vector transduces a higher proportion of hepatocytes (∼15%). The subpopulations of hepatocytes transduced with either vector widely overlap, with the AAV-5 vector transducing additional hepatocytes and showing a wider area of transgene expression throughout the liver parenchyma.

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 Induction and role of regulatory CD4+CD25+ T cells in tolerance to the transgene product following hepatic in vivo gene transfer

Blood ◽  
2007 ◽  
Vol 110 (4) ◽  
pp. 1132-1140 ◽  
Author(s):  
Ou Cao ◽  
Eric Dobrzynski ◽  
Lixin Wang ◽  
Sushrusha Nayak ◽  
Bethany Mingle ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Antibody Formation ◽  
Coagulation Factor ◽  
Gene Replacement ◽  
Factor Ix ◽  
In Vivo Gene Transfer

Abstract Gene replacement therapy is complicated by the risk of an immune response against the therapeutic transgene product, which in part is determined by the route of vector administration. Our previous studies demonstrated induction of immune tolerance to coagulation factor IX (FIX) by hepatic adeno-associated viral (AAV) gene transfer. Using a regulatory T-cell (Treg)–deficient model (Rag-2−/− mice transgenic for ovalbumin-specific T-cell receptor DO11.10), we provide first definitive evidence for induction of transgene product-specific CD4+CD25+ Tregs by in vivo gene transfer. Hepatic gene transfer–induced Tregs express FoxP3, GITR, and CTLA4, and suppress CD4+CD25− T cells. Tregs are detected as early as 2 weeks after gene transfer, and increase in frequency in thymus and secondary lymphoid organs during the following 2 months. Similarly, adoptive lymphocyte transfers from mice tolerized to human FIX by hepatic AAV gene transfer indicate induction of CD4+CD25+GITR+ that suppresses antibody formation to FIX. Moreover, in vivo depletion of CD4+CD25+ Tregs leads to antibody formation to the FIX transgene product after hepatic gene transfer, which strongly suggests that these regulatory cells are required for tolerance induction. Our study reveals a crucial role of CD4+CD25+ Tregs in preventing immune responses to the transgene product in gene transfer.

Stable Gene Transfer and Expression in Human Primary T-Cells by the Sleeping Beauty Transposon System.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5539-5539
Author(s):  
Xianzheng Zhou ◽  
Xin Huang ◽  
Andrew C. Wilber ◽  
Lei Bao ◽  
Dong Tuong ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Sleeping Beauty ◽  
Stable Gene ◽  
Cell Clones ◽  
Stable Gene Expression

Abstract The Sleeping Beauty (SB) transposon system is a non-viral DNA delivery system in which a transposase directs integration of an SB transposon into TA-dinucleotide sites in the genome. To determine whether the SB transposon system can mediate integration and long-term transgene expression in human primary T-cells, freshly isolated peripheral blood lymphocytes (PBLs) without prior activation were nucleofected with SB vectors carrying a DsRed reporter gene. Plasmids containing the SB transposase on the same (cis) (n=10) or separate molecule (trans) (n=8) as the SB transposon mediated long-term and stable reporter gene expression in human primary T-cells. We observed that delivery of SB transposase-encoding plasmid in trans effectively mediated stable gene expression in primary T-cells, exhibiting about a 3-fold increase (11% vs. 3% with 10 microgram plasmid on day 21) in potency in comparison with the cis vector (p<0.0001). In addition, a transposase mutant construct was incapable of mediating stable gene expression in human PBLs (n=6, p<0.0001), confirming that catalytic DDE domain is necessary for transposition in human primary T-cells. Immunophenotyping analysis in transposed T-cells showed that both CD4 and CD8 T-cells were transgene positive. SB-mediated high level of transgene expression in human T-cells was maintained in culture for at least 4 months without losing observable expression. Southern hybridization analysis showed a variety of transposon integrants among the 6 DsRed positive T-cell clones and no transposon sequences identifiable in the 2 DsRed negative clones. Sequencing of transposon:chromosome junctions in 5 out of 6 transposed T-cell clones confirmed that stable gene expression was due to SB-mediated transposition. In other studies, PBLs were successfully transfected using the SB transposon system and shown to stably and functionally express a fusion protein consisting of a surface receptor useful for positive T-cell selection and a “suicide” gene useful for elimination of transfected T-cells after chemotherapy. This study is the first report demonstrating that the SB transposon system can mediate stable gene transfer in human primary PBLs, which may be more advantageous for T-cell based gene therapies over widely used virus-based or conventional mammalian DNA vectors in terms of simplicity, stability, efficiency and safety.

scholarly journals Supraspinal gene transfer by intrathecal adeno-associated virus serotype 5

2014 ◽  
Vol 8 ◽  
Author(s):  
Daniel J. Schuster ◽  
Lalitha R. Belur ◽  
Maureen S. Riedl ◽  
Stephen A. Schnell ◽  
Kelly M. Podetz-Pedersen ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Adeno Associated Virus ◽  
Virus Serotype ◽  
Serotype 5

scholarly journals Immune deviation by mucosal antigen administration suppresses gene-transfer–induced inhibitor formation to factor IX

Blood ◽  
2006 ◽  
Vol 108 (2) ◽  
pp. 480-486 ◽  
Author(s):  
Ou Cao ◽  
Elina Armstrong ◽  
Alexander Schlachterman ◽  
Lixin Wang ◽  
David K. Okita ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Intranasal Administration ◽  
Viral Vector ◽  
Cell Epitope ◽  
Coagulation Factor ◽  
Antigen Expression ◽  
Gene Replacement ◽  
Factor Ix ◽  
Hemophilia B ◽  
Immune Deviation

Formation of inhibitory antibodies is a serious complication of protein or gene replacement therapy for hemophilias, congenital X-linked bleeding disorders. In hemophilia B (coagulation factor IX [F.IX] deficiency), lack of endogenous F.IX antigen expression and other genetic factors may increase the risk of antibody formation to functional F.IX. Here, we developed a protocol for reducing inhibitor formation in gene therapy by prior mucosal (intranasal) administration of a peptide representing a human F.IX-specific CD4+ T-cell epitope in hemophilia B mice. C3H/HeJ mice with a F.IX gene deletion produced inhibitory IgG to human F.IX after hepatic gene transfer with an adeno-associated viral vector. These animals subsequently lost systemic F.IX expression. In contrast, repeated intranasal administration of the specific peptide resulted in reduced inhibitor formation, sustained circulating F.IX levels, and sustained partial correction of coagulation following hepatic gene transfer. This was achieved through immune deviation to a T-helper–cell response with increased IL-10 and TGF-β production and activation of regulatory CD4+CD25+ T cells.

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