[CANCER RESEARCH 64, 1781–1787, March 1, 2004] Adeno-Associated Virus 2-Mediated Antiangiogenic Cancer Gene Therapy: Long- Term Efficacy of a Vector Encoding Angiostatin and Endostatin over Vectors Encoding a Single Factor Selvarangan Ponnazhagan, 1 Gandham Mahendra, 1 Sanjay Kumar, 1 Denise R. Shaw, 2 Cecil R. Stockard, 1 William E. Grizzle, 1 and Sreelatha Meleth 2 Departments of 1 Pathology and 2 Medicine and the Comprehensive Cancer Center, The University of Alabama at Birmingham, Birmingham, Alabama ABSTRACT Angiogenesis is characteristic of solid tumor growth and a surrogate marker for metastasis in many human cancers. Inhibition of tumor an- giogenesis using antiangiogenic drugs and gene transfer approaches has suggested the potential of this form of therapy in controlling tumor growth. However, for long-term tumor-free survival by antiangiogenic therapy, the factors controlling tumor neovasculature need to be system- ically maintained at stable therapeutic levels. Here we show sustained expression of the antiangiogenic factors angiostatin and endostatin as secretory proteins by recombinant adeno-associated virus 2 (rAAV)- mediated gene transfer. Both vectors provided significant protective effi- cacy in a mouse tumor xenograft model. Stable transgene persistence and systemic levels of both angiostatin and endostatin were confirmed by in situ hybridization of the vector-injected tissues and by serum ELISA measurements, respectively. Whereas treatment with rAAV containing either endostatin or angiostatin alone resulted in moderate to significant protection, the combination of endostatin and angiostatin gene transfer from a single vector resulted in a complete protection. These data suggest that AAV-mediated long-term expression of both endostatin and angiosta- tin may have clinical utility against recurrence of cancers after primary therapies and may represent rational adjuvant therapies in combination with radiation or chemotherapy. INTRODUCTION Increasing evidence demonstrates the importance of angiogenesis in solid tumor growth and metastasis (1– 4). In the absence of neo- vasculature, tumors do not grow beyond a few millimeters and remain dormant (5, 6). Thus, novel antiangiogenic treatment strategies that can effectively control tumor growth are under intense investigation. Although many antiangiogenic factors have been implicated in the regulation of tumor growth and metastasis, the most potent have been angiostatin, endostatin, thrombospondin-1, tissue inhibitor of metallo- proteases, and soluble vascular endothelial growth factor (VEGF) receptors (7–10). Preclinical studies using purified antiangiogenic factors indicated therapeutic effects of antiangiogenic compounds in minimizing the size of established tumors (11–15). However, clinical trials with some of these factors have not demonstrated expected antitumor effects (16 –19). Administration of purified antiangiogenic factors, although capable of producing significant growth inhibition of tumor cells in animal models, may be limited by their short half-life. Hence, pro- duction of antiangiogenic factors after gene transfer may overcome these limitations. The potential of antiangiogenic gene therapy in cancer is currently being evaluated using viral and nonviral vectors (20 –23). In contrast to genetic therapies targeting tumor cells directly with genes encoding prodrug-converting enzymes or cytokines/chemokines for oncolysis, which requires high-efficiency transduction of recombinant vectors to cancer cells directly, antiangiogenic gene therapy requires vectors capable of sustained, long-term expression without vector-associated toxicity or immunity. Additionally, systemic levels of antiangiogenic factors by gene transfer may be accomplished by targeting nontumor cells, using normal tissues to provide a stable platform for transgene expression as secretory proteins. Adeno-associated virus (AAV)- based vectors are nonpathogenic and less immunogenic compared with other gene therapy vectors. The AAV genome persists stably in transduced cells and affects long-term transgene expression. Thus, AAV meets the requirements for gene transfer vectors that may be used for antiangiogenic therapy. The present study evaluated recombinant AAV (rAAV) encoding secretable forms of human angiostatin and endostatin. The results demonstrate a strong antiproliferative effect of rAAV-mediated an- giostatin or endostatin gene transfer on primary human umbilical vein endothelial cells (HUVEC) in vitro and significant protective effect against the growth of a human angiogenesis-dependent tumor xe- nograft in vivo. Furthermore, the combination of both angiostatin and endostatin long-term gene therapy from a single vector resulted in a synergistic effect over therapy with vectors encoding a single factor alone. MATERIALS AND METHODS Cells and Reagents. Human embryonic kidney cell line 293 was purchased from American Type Culture Collection and maintained in Iscove’s modified essential medium supplemented with 10% newborn calf serum. Human ovarian cancer cell line SKOV3.ip1 was a kind gift of Dr. David Curiel (The Univer- sity of Alabama at Birmingham, Birmingham, AL) and maintained as de- scribed previously (24). Primary HUVEC were a gift of Dr. Raj Singh (The University of Alabama at Birmingham, Birmingham, AL). Restriction endo- nucelases and other modifying enzymes were purchased from either New England Biolabs (Beverly, MA) or Promega Corp. (Madison, WI). Mouse monoclonal (clone 79735) and goat polyclonal antibodies to human angiostatin were obtained from R&D Systems (Minneapolis, MN), and a mouse mono- clonal antibody to human endostatin (clone EN2.1.99) was obtained from Leinco Technologies (St. Louis, MO). Secondary antibodies and colorimetric substrates were purchased from Amersham (Piscataway, NJ). Purified recom- binant human angiostatin was purchased from R&D Systems. Construction of Recombinant Plasmids, Production, and Purification of rAAV. All rAAV plasmids were constructed using pSub201 as the back bone (25). cDNA containing human angiostatin and endostatin sequences were isolated from a plasmid pBlast human Endo::Angio (Invivogen, San Diego, CA). For construction of the rAAV plasmid encoding endostatin, a region containing the human interleukin 2 secretory signal sequence was genetically fused to the endostatin coding region, amplified from the plasmid pBlast human Endo::Angio by PCR, and subcloned into an AAV plasmid containing cytomegalovirus (CMV) promoter, sequences of internal ribosome entry site (IRES), and a green fluorescent protein (GFP) gene followed by a synthetic polyadenylation signal sequence (polyA). Construction of rAAV encoding Received 6/17/03; revised 11/6/03; accepted 12/12/03. Grant support: Career Development Award of NIH-Specialized Programs of Re- search Excellence grant in ovarian cancer 5 P50-CA8359, NIH Grants R01CA90850 and R01CA98817, and United States Army Department of Defense Grants BC010494 and PC020372 (to S. Ponnazhagan). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Selvarangan Ponnazhagan, Department of Pathology, LHRB 513, 701 19th Street South, University of Alabama at Birmingham, Birmingham, AL 35294-0007. Phone: (205) 934-6731, Fax: (205) 975-9927, E-mail: sponnazh@path. uab.edu. 1781 Downloaded from http://aacrjournals.org/cancerres/article-pdf/64/5/1781/2523385/zch00504001781.pdf by guest on 24 November 2023