481. Biological Effect of a Tetracycline Regulated AAV2 Vector Delivering Feline Erythropoietin in Mice

APPLICATIONS IN DISEASE 479. Anti-Tumor Effect with Adeno-Associated Virus Serotype 1 Vectors Encoding Murine Endostatin Chengwen Li,1 Chaoying Yin,2 Hongyan Zhao,2 R. Jude Samulski,1,3,4 Terry Van Dyke.14 1 Gene Therapy Center; 2Department of Biochemistry & Biophysics; 3Department of Pharmacology; 4Lineberg Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States. Endostatin is a specific inhibitor of endothelial proliferation and migration and may cause apoptosis of endothelial cell. Gene transfer of endostatin represents an alternative method of delivering angiogenic polypeptide inhibitor. rAAV has been shown to confer long-term transgene expression in vivo, with serotype 1 of AAV demonstrating the best gene expression levels in muscular. Here we report that endostatin can effectively suppress growth of xenografted tumor cells LCCC after rAAV1 mediated muscular transduction. After 5x10 10 rAAV1/murine endostatin was injected into muscles about10ug/ml endostatin was detected in the sera of transduced animals. The tumor size of treated mice was reduced by half of control mice that received GFP vector injection. In vitro experiments displayed that the sera of mice with rAAV1/endostatin inhibited the proliferation and tube formation of human umbilical blood epithelial cells. When gender of mice was compared, we did not observe a difference in endostatin levels. Finally, we checked the relationship of concentration of endostatin in sera and the inhibition effect of tumor growth. It was determined that the transgene expression was dose-dependent however, the suppression effect of tumor growth did not follow a dose relationship. Although the mechanism for these observations remains to be elucidated, all of these studies demonstrated that endostatin can effectively inhibit tumor growth.

480. Double-Stranded AAV: A Promising Vector for Cancer Gene Therapy Zhong Wang,1 Tong Zhu,1 Jian Zhang,1 Hsin-I Ma,1 Juan Li,1 Xiao Xiao.1 1 Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA. The slow onset of transgene expression and lower transduction efficiency in many tumor cells limit the use of traditional singlestranded AAV as a vector for cancer gene therapy. Recently we constructed a novel double-stranded AAV (dsAAV) vector and showed it delivered much stronger and more accelerated transduction in cancer cell lines as well as in murine muscle and liver in vivo. In the present study, we further examined the potential of this dsAAV vector(serotype 2) for cancer gene therapy. We first investigated its transduction to different human and murine tumor cell lines, and found that dsAAV-CMV-EGFP vector could achieve more than 80% transduction efficiency in most of the human cell lines at 1000 particles per cell (cervical cancer HeLa, prostate cancer PC3, DU145, TSU and LNCaP; colon cancer HCT116, Follicular thyroid carcinoma FTC133, head and neck cancer 1483, osteosarcoma cell SaOS2, brain cancer U87, breast cancer MCF7 , MDA231 and BT549, hepatocarcinoma HepG2), and murine tumor cell lines( B16 melenoma, Hepa1-6 liver cancer) and 9L rat gliosarcoma cell line at a higher dose(10000 particles per cell). In a subcutaneous murine B16 tumor model, strong EGFP expressions were also detected at 2 days after intratumoral injection of 50ul dsAAV-CMV-EGFP (4x1011particles). The in vitro and in vivo data suggest that dsAAV vector can effectively deliver target gene to tumor cells. Furthermore, intrasplenic injection of dsAAV-CB-endostatin virus (2x1011 particles) induced rapid, high-level and sustained secretion of endostatin in murine serum (by 42 days postinjection, serum concentration of endostatin reached 1300 ± 257ng/ml and lasted for S188

months), demonstrating that the dsAAV vector is also an excellent alternative for indirect cancer gene therapy approaches ( such as anti-angiogenesis strategies), which produce anti-tumor proteins in normal organs and tissue. Taken together, these results indicate that dsAAV can be used as an attractive vector for cancer gene therapy.

481. Biological Effect of a Tetracycline Regulated AAV2 Vector Delivering Feline Erythropoietin in Mice Mark C. Walker,1 Sergei Zolotukhin,2 Tamara C. Mandell,1 Susan B. Washer,1 Barry J. Byrne.3 1 Applied Genetic Technologies Corporation, Alachua, FL, United States; 2Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, United States; 3Pediatrics, Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, United States. Delivery of feline erythropoietin (fEPO) cDNA by an adenoassociated (AAV) viral vectors holds promise for the treatment of hypoproliferative anemia associated with chronic renal failure (CRF) in cats. CRF exists when renal parenchymal disease/injury develops over an extended period and is characterized by polyuria, then azotemia, eventually leading to uremia. The prevalence of renal failure among cats more than 10 years of age is 7.7%, indicating a large unmet need. fEPO is a glycoprotein hormone produced in the peritubular interstitial cells of the kidney that controls the proliferation, differentiation and survival of erythroid progenitors in response to hypoxia. Treatment of anemic cats with recombinant human erythropoietin (hEPO) corrects most of the clinical signs of CRF, however, 80% of cats develop an immune response to hEPO or the human albumin to which it is bound. 70% of these cats develop anemia refractory to treatment with hEPO and 25% become transfusion dependent. Beyond species specificity, successful treatment of hypoproliferative anemia requires the expression of EPO to be tightly controlled because of its biological potency and the detrimental effects of polycythemia. The biological effects of a single intramuscular injection of an AAV2 vector containing fEPO cDNA and a bi-directional CMV promoter controlled by a tetracycline-dependent transactivator (rtTA-M2-fEPO) was investigated. Transcription was further modified by the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Mice were divided into 2 treatment groups and a control group. Treatment groups were injected with 2 x 109 IU or 2 x 108 IU into the left quadriceps muscle, respectively. Blood was collected by tail clip weekly and hematocrit values were manually calculated. fEPO levels were tightly regulated by tetracycline (Dox) which was administered in a commercial mouse diet (Dox Diet; 2g/kg). Results of the study show a rapid, profound increase in mouse hematocrit after the administration of Dox. Similarly, a rapid decline reaching baseline hematocrit values were seen soon after Dox was withdrawn. Statistical analysis with an unpaired t-test showed that all groups were significantly different at all time points except for trial day 159 (p > 0.05). These results suggest minimal fEPO expression when Dox is withdrawn. The results are illustrated below. Further studies are ongoing examining the biological response of this vector in normal and CRF cats.

Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts

Copyright © The American Society of Gene Therapy

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