Recombinant adenoviruses containing the canine factor IX (FIX) cDNA were directly introduced in the hind leg muscle of mice. We show that (i) in nude mice, high expression (1-5 ,ug/ml in plasma) of FIX protein can be detected for >300 days; (ii) in contrast, expression of FIX protein was transient (7-10 days) in normal mice; (iii) CD8+ lymphocytes could be detected within 3 days in the infected muscle tissue; (iv) use of i32-microglobulin and immunoglobulin M heavy chain "knockout" mice showed that lack of sustained expression of FIX protein is due to cell-mediated and humoral immune responses; (v) normal mice, once infected with recombinant adenovirus, could not be reinfected efficiently for at least 30 days due to neutralizing viral antibodies; and, finally, (vi) using immunosuppressive drugs, some normal mice can be tolerized to produce and secrete FLIX protein for >5 months. We conclude that currently available adenoviral vectors have serious limitations for use for longterm gene therapy.Gene therapy holds a great promise for the treatment of many genetic diseases. Retroviral vectors have been extensively used to deliver genes into a wide variety of cell types but require ex vivo approaches because of their inability to infect postmitotic cells. In recent years much attention has been focused on a vector system that can deliver genes with high efficiency to a wide spectrum of nondividing cells in vivo. In particular, recombinant adenoviruses have been demonstrated to be very useful (1-5). Unfortunately, several studies with different gene products have revealed that following infection in a wide variety of target tissues, only transient expression is observed (6-12). Since the cells infected with the recombinant adenoviruses are rapidly eliminated, it is likely that the host immune system plays a major role in preventing sustained expression of the foreign genes. We have therefore undertaken a characterization of the specific immune responses and antigens involved in the lack of sustained expression by adenoviral factor IX (FIX) mediated gene therapy. in pXCJL1 were cotransfected with the pJM17 plasmid (14) into 293 cells (15), respectively, and recombinant adenoviral plaques were isolated and further purified by two rounds of plaque assays as described (16). The adenoviral vectors were propagated in 293 cells and purified by double CsCl banding as described (16). The purified virus was dialyzed against phosphate-buffered saline (PBS) and stored in aliquots with 15% glycerol at -80°C. MATERIALS AND METHODSAnimal Procedures. Adult Swiss Webster mice and nude (nu/nu) athymic mice were purchased from Harlan-SpragueDawley. 132-Microglobulin knockout, 132m(-/-), mice (17) and IgM heavy chain (,t chain) knockout, ,uMT/,uMT mice (18) were kindly provided by Leonard Shultz (The Jackson Laboratory). About 1 x 109 plaque-forming units (pfu) of purified AdMCdF9 or AdMCLacZ virus was diluted into 100 ,ul of PBS and injected into muscles of both hind legs of anesthetized adult mice at 6-10 weeks of age (5-10 ,ul ...

Tumor immune surveillance and cancer immunotherapies are thought to depend on the intratumoral infiltration of activated CD8(+) T cells. Intratumoral CD8(+) T cells are rare and lack activity. IL-10 is thought to contribute to the underlying immune suppressive microenvironment. Defying those expectations we demonstrate that IL-10 induces several essential mechanisms for effective antitumor immune surveillance: infiltration and activation of intratumoral tumor-specific cytotoxic CD8(+) T cells, expression of the Th1 cytokine interferon-γ (IFNγ) and granzymes in CD8(+) T cells, and intratumoral antigen presentation molecules. Consequently, tumor immune surveillance is weakened in mice deficient for IL-10 whereas transgenic overexpression of IL-10 protects mice from carcinogenesis. Treatment with pegylated IL-10 restores tumor-specific intratumoral CD8(+) T cell function and controls tumor growth.

Checkpoint kinase 1 (CHK1) is an essential serine/threonine kinase that responds to DNA damage and stalled DNA replication. CHK1 is essential for maintenance of replication fork viability during exposure to DNA antimetabolites. In human tumor cell lines, ablation of CHK1 function during antimetabolite exposure led to accumulation of double-strand DNA breaks and cell death. Here, we extend these observations and confirm ablation of CHK2 does not contribute to these phenotypes and may diminish them. Furthermore, concomitant suppression of cyclin-dependent kinase (CDK) activity is sufficient to completely antagonize the desired CHK1 ablation phenotypes. These mechanism-based observations prompted the development of a high-content, cell-based screen for g-H2AX induction, a surrogate marker for double-strand DNA breaks. This mechanism-based functional approach was used to optimize small molecule inhibitors of CHK1. Specifically, the assay was used to mechanistically define the optimal in-cell profile with compounds exhibiting varying degrees of CHK1, CHK2, and CDK selectivity. Using this approach, SCH 900776 was identified as a highly potent and functionally optimal CHK1 inhibitor with minimal intrinsic antagonistic properties. SCH 900776 exposure phenocopies short interfering RNA-mediated CHK1 ablation and interacts synergistically with DNA antimetabolite agents in vitro and in vivo to selectively induce dsDNA breaks and cell death in tumor cell backgrounds. Mol Cancer Ther; 10(4); 591-602. Ó2011 AACR.

Although growth factor proteins display potent tissue repair activities, difficulty in sustaining localized therapeutic concentrations limits their therapeutic activity. We reasoned that enhanced histogenesis might be achieved by combining growth factor genes with biocompatible matrices capable of immobilizing vectors at delivery sites. When delivered to subcutaneously implanted sponges, a platelet-derived growth factor B-encoding adenovirus (AdPDGF-B) formulated in a collagen matrix enhanced granulation tissue deposition 3- to 4-fold (p < or = 0.0002), whereas vectors encoding fibroblast growth factor 2 or vascular endothelial growth factor promoted primarily angiogenic responses. By day 8 posttreatment of ischemic excisional wounds, collagen-formulated AdPDGF-B enhanced granulation tissue and epithelial areas up to 13- and 6-fold (p < 0.009), respectively, and wound closure up to 2-fold (p < 0.05). At longer times, complete healing without excessive scar formation was achieved. Collagen matrices were shown to retain both vector and transgene products within delivery sites, enabling the transduction and stimulation of infiltrating repair cells. Quantitative PCR and RT-PCR demonstrated both vector DNA and transgene mRNA within wound beds as late as 28 days posttreatment. By contrast, aqueous formulations allowed vector seepage from application sites, leading to PDGF-induced hyperplasia in surrounding tissues but not wound beds. Finally, repeated applications of PDGF-BB protein were required for neotissue induction approaching equivalence to a single application of collagen-immobilized AdPDGF-B, confirming the utility of this gene transfer approach. Overall, these studies demonstrate that immobilizing matrices enable the controlled delivery and activity of tissue promoting genes for the effective regeneration of injured tissues.

Keratinocyte growth factor , (KGF) , a member of the fibroblast growth factor (FGF) family , is involved in wound healing. It also promotes the differentiation of many epithelial tissues and proliferation of epithelial cells as well as pancreatic duct cells. Additionally, many members of the highly homologous FGF family (including KGF) , influence both growth and cellular morphology in the developing embryo. We have previously observed elevated levels of KGF in our interferon-␥ transgenic mouse model of pancreatic regeneration. To understand the role of KGF in pancreatic differentiation , we generated insulin promoter-regulated KGF transgenic mice. Remarkably , we have found that ectopic KGF expression resulted in the emergence of hepatocytes within the islets of Langerhans in the pancreas. Additionally , significant intraislet duct cell proliferation in the pancreata of transgenic KGF mice was observed. The unexpected appearance of hepatocytes and proliferation of intraislet duct cells in the pancreata of these mice evidently stemmed directly from local exposure to KGF. (Am J Pathol 1999, 154:683-691)