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483. AAV Vector Mediated Anti-Angiogenic Gene Therapy for Collagen-Induced Arthritis in Mice
APPLICATIONS IN DISEASE 483. AAV Vector Mediated Anti-Angiogenic Gene Therapy for Collagen-Induced Arthritis in Mice Hiroshi Takahashi,1,3 Koichi Miyake,1,2 Shinichi Yoshino,3 Takashi Shimada.1,2 1 Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan; 2Division of Gene Therapy Research, Center for Advanced Medical Technology, Nippon Medical School, Tokyo, Japan; 3Department of Joint Disease and Rheumatism, Nippon Medical School, Tokyo, Japan.
482. Secretion of Soluble TNFR:Fc Fusion Protein Following Endotracheal, Intravenous or Intramuscular Administration of an AAV-TNFR:Fc Vector Pseudotyped with Capsids of Alternate AAV Serotypes Elizabeth M. Bruckheimer,1 Linda C. Rogers,1 Ziv Sandalon,1 Mara S. Lippa,1 Martin W. Lock,1 Simon G. Godwin,1 Richard W. Peluso,1 Haim Burstein.1 1 Research, Targeted Genetics Corporation, Seattle, WA, United States. TNF-α antagonists such as anti-TNF-α monoclonal antibodies or soluble tumor necrosis factor receptors have proven to be useful therapeutics for the treatment of autoimmune inflammatory diseases including rheumatoid and psoriatic arthritis. We have recently demonstrated that local (intraarticular) or systemic (intramuscular) administration of an AAV-ratTNFR:Fc vector, encoding the rat tumor necrosis factor-α receptor: immunoglobulin (IgG1) Fc fusion gene, to rats with experimental arthritis led to suppression of disease as reflected in decreased inflammatory cell infiltration, pannus formation, cartilage and bone destruction, and mRNA expression of joint proinflammatory cytokines. The current study was aimed at evaluating the serum expression levels, in rats, of the TNFR:Fc fusion protein following either endotracheal, intramuscular or intravenous administration of an AAV-ratTNFR:Fc vector pseudotyped with capsids from AAV serotypes 1, 2 or 5. Intramuscular delivery proved to be the most efficient route of administration and resulted in very high and sustained serum levels of TNFR:Fc protein. AAV2/1 was 100- to 1000-fold more efficient than AAV2/5 and AAV2/2, while AAV2/5 was up to 5-fold more efficient than AAV2/2. Following intravenous administration, AAV2/ 1 demonstrated an over a 100-fold enhanced expression compared with AAV2/5 and AAV2/2, and AAV2/5 was up to 10-fold more efficient than AAV2/2. The serum levels of TNFR:Fc protein were below the limit of detection (< 2 ng/mL) over 9-weeks post-AAV2/ 2 administration to the lung, although anti-AAV2 neutralizing antibodies were clearly evident. AAV2/1 administration to the lung resulted in secretion of detectable TNFR:Fc protein but was less efficient than AAV2/5. These and additional results including those from readministration studies will be discussed.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
Objective. We have previously demonstrated that HIV vector mediated intra-articular expression of angiostatin efficiently inhibits progress of collagen-induced arthritis in mice. Using the same model mice, we examined the utility of AAV-vector which is more clinically applicable. Methods. Collagen-induced-arthritic model mice were generated by immunization with bovine type II collagen and Freund’s complete adjuvant. The AAV vectors containing the angiostatin expression unit (AAV-angiostatin) or the EGFP expression unit (AAV-EGFP) were injected into the right knee joints before arthritis development. As a control, phosphate buffered saline (PBS) was injected into the left joints. The incidence and severity of arthritis were histologically evaluated by scoring synovial hyperplasia, cartilage erosion and bone erosion of arthritis. Angiogenesis in knee joints was studied immunohistochemistrically by measurement of von Willebrand factor levels. Results. AAV vectors were demonstrated to be capable of efficient gene transfer into synovial cells, chondrocytes and infiltrating cells. The expression of transgene was confirmed by RNA analyses. The extent of synovial hyperplasia and other parameters of arthritis were significantly reduced in the knee joints injected with AAVangiostatin compared with the joints injected with either AAV-EGFP or PBS. Immunohistochemical analyses also demonstrated that the angiogenic index was significantly decreased in the treated joints. Conclusion. AAV-vector-mediated expression of angiostatin efficiently inhibits the progress of collagen-induced arthritis. The safety of AAV vectors was confirmed in the recent clinical protcol of Hemophillia gene therapy. Angiostatic gene therapy using AAV vector may provide a new approach to the effective treatment of rheumatoid arthritis.
484. Investigation of rAAV5 as a Vector for Gene Transfer to Mouse Lung Stephanie G. Sumner,1,3 Lee A. Davies,1,3 Anusha Varathalingam,1,3 John A. Chiorini,2 Deborah R. Gill,1,3 Stephen C. Hyde.1,3 1 Gene Medicine Research Group, NDCLS, University of Oxford, Oxford, United Kingdom; 2Gene Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, MD, United States; 3UK CF Gene Therapy Consortium, United Kingdom. Adeno-associated virus serotype 5 (AAV5) has recently emerged as a likely candidate for gene delivery to the airways. Its receptor is 2,3-linked sialic acid which is present on the apical surface of epithelia. Furthermore vectors derived from this family of non-pathogenic viruses have demonstrated a weaker immunogenicity than other types of virus (e.g. Adenovirus) reflected in particularly long persistence and, under certain conditions, AAV vectors can be readministered efficiently. For these reasons, it is an attractive candidate for gene therapy of Cystic Fibrosis. However, most of the longevity and readministration studies having been carried out with vectors based on AAV2, further characterisation of AAV5 vectors is a necessary stepping stone to its development as gene transfer agent. Recombinant AAV5 (rAAV5) vectors were investigated for S189
482. Secretion of Soluble TNFR:Fc Fusion Protein Following Endotracheal, Intravenous or Intramuscular Administration of an AAV-TNFR:Fc Vector Pseudotyped with Capsids of Alternate AAV Serotypes Elizabeth M. Bruckheimer,1 Linda C. Rogers,1 Ziv Sandalon,1 Mara S. Lippa,1 Martin W. Lock,1 Simon G. Godwin,1 Richard W. Peluso,1 Haim Burstein.1 1 Research, Targeted Genetics Corporation, Seattle, WA, United States. TNF-α antagonists such as anti-TNF-α monoclonal antibodies or soluble tumor necrosis factor receptors have proven to be useful therapeutics for the treatment of autoimmune inflammatory diseases including rheumatoid and psoriatic arthritis. We have recently demonstrated that local (intraarticular) or systemic (intramuscular) administration of an AAV-ratTNFR:Fc vector, encoding the rat tumor necrosis factor-α receptor: immunoglobulin (IgG1) Fc fusion gene, to rats with experimental arthritis led to suppression of disease as reflected in decreased inflammatory cell infiltration, pannus formation, cartilage and bone destruction, and mRNA expression of joint proinflammatory cytokines. The current study was aimed at evaluating the serum expression levels, in rats, of the TNFR:Fc fusion protein following either endotracheal, intramuscular or intravenous administration of an AAV-ratTNFR:Fc vector pseudotyped with capsids from AAV serotypes 1, 2 or 5. Intramuscular delivery proved to be the most efficient route of administration and resulted in very high and sustained serum levels of TNFR:Fc protein. AAV2/1 was 100- to 1000-fold more efficient than AAV2/5 and AAV2/2, while AAV2/5 was up to 5-fold more efficient than AAV2/2. Following intravenous administration, AAV2/ 1 demonstrated an over a 100-fold enhanced expression compared with AAV2/5 and AAV2/2, and AAV2/5 was up to 10-fold more efficient than AAV2/2. The serum levels of TNFR:Fc protein were below the limit of detection (< 2 ng/mL) over 9-weeks post-AAV2/ 2 administration to the lung, although anti-AAV2 neutralizing antibodies were clearly evident. AAV2/1 administration to the lung resulted in secretion of detectable TNFR:Fc protein but was less efficient than AAV2/5. These and additional results including those from readministration studies will be discussed.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
Objective. We have previously demonstrated that HIV vector mediated intra-articular expression of angiostatin efficiently inhibits progress of collagen-induced arthritis in mice. Using the same model mice, we examined the utility of AAV-vector which is more clinically applicable. Methods. Collagen-induced-arthritic model mice were generated by immunization with bovine type II collagen and Freund’s complete adjuvant. The AAV vectors containing the angiostatin expression unit (AAV-angiostatin) or the EGFP expression unit (AAV-EGFP) were injected into the right knee joints before arthritis development. As a control, phosphate buffered saline (PBS) was injected into the left joints. The incidence and severity of arthritis were histologically evaluated by scoring synovial hyperplasia, cartilage erosion and bone erosion of arthritis. Angiogenesis in knee joints was studied immunohistochemistrically by measurement of von Willebrand factor levels. Results. AAV vectors were demonstrated to be capable of efficient gene transfer into synovial cells, chondrocytes and infiltrating cells. The expression of transgene was confirmed by RNA analyses. The extent of synovial hyperplasia and other parameters of arthritis were significantly reduced in the knee joints injected with AAVangiostatin compared with the joints injected with either AAV-EGFP or PBS. Immunohistochemical analyses also demonstrated that the angiogenic index was significantly decreased in the treated joints. Conclusion. AAV-vector-mediated expression of angiostatin efficiently inhibits the progress of collagen-induced arthritis. The safety of AAV vectors was confirmed in the recent clinical protcol of Hemophillia gene therapy. Angiostatic gene therapy using AAV vector may provide a new approach to the effective treatment of rheumatoid arthritis.
484. Investigation of rAAV5 as a Vector for Gene Transfer to Mouse Lung Stephanie G. Sumner,1,3 Lee A. Davies,1,3 Anusha Varathalingam,1,3 John A. Chiorini,2 Deborah R. Gill,1,3 Stephen C. Hyde.1,3 1 Gene Medicine Research Group, NDCLS, University of Oxford, Oxford, United Kingdom; 2Gene Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, MD, United States; 3UK CF Gene Therapy Consortium, United Kingdom. Adeno-associated virus serotype 5 (AAV5) has recently emerged as a likely candidate for gene delivery to the airways. Its receptor is 2,3-linked sialic acid which is present on the apical surface of epithelia. Furthermore vectors derived from this family of non-pathogenic viruses have demonstrated a weaker immunogenicity than other types of virus (e.g. Adenovirus) reflected in particularly long persistence and, under certain conditions, AAV vectors can be readministered efficiently. For these reasons, it is an attractive candidate for gene therapy of Cystic Fibrosis. However, most of the longevity and readministration studies having been carried out with vectors based on AAV2, further characterisation of AAV5 vectors is a necessary stepping stone to its development as gene transfer agent. Recombinant AAV5 (rAAV5) vectors were investigated for S189