Matrix immobilization enhances the tissue repair activity of growth factor gene therapy vectors.

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.

Uptake of adenoviral vectors via fibroblast growth factor receptors involves intracellular pathways that differ from the targeting ligand.

Target-specific delivery of adenoviral gene therapy vectors has been achieved by introducing basic fibroblast growth factor (FGF2) onto viral capsids. FGF2-retargeted vectors enter the cell through high-affinity FGF receptors while normal adenoviral receptor interactions are ablated. In addition, FGF2-mediated targeting permits a higher level of transgene expression and in vivo efficacy. We now present studies on the intracellular pathways and mechanisms of transduction by FGF2-retargeted adenovirus. FGF2 retargeting results in increased virion entry. Nuclear delivery is also increased, but to a level that is directly proportional to virion entry. In addition, after entry, the retargeted particle rapidly localizes to the nucleus in a time frame similar to that of adenovirus alone. Transgene expression is always enhanced with FGF2-mediated delivery, whether overall transduction of the population is increased, equivalent, or decreased relative to nontargeted adenoviral vectors. However, the increase in transgene expression does not correlate quantitatively with enhanced cellular entry, indicating that other factors may influence transgene expression levels. The increase in transgene expression occurs only when the FGF2-retargeting moiety is physically complexed with the adenoviral vector, indicating a requirement for a spatial link between the ligand and the virus particle. The FGF2-adenoviral complex activates the FGF receptor-mediated proliferative signaling cascade, but this signal transduction is not required for the enhanced level of gene expression observed after FGF2-mediated delivery. These findings emphasize that, in addition to altering receptor tropism, the influence of FGF2 retargeting extends to intracellular adenoviral trafficking pathways. Although the increased delivery of virions into the cell and nucleus contributes to the enhanced transgene expression observed with FGF2 retargeting, other as yet undefined cellular mechanisms also contribute to this process.

FGF2-Targeted adenovirus encoding platelet-derived growth factor-B enhances de novo tissue formation.

Gene therapy has yet to achieve reproducible clinical efficacy, due to inadequate gene delivery, inadequate gene expression, or dose-limiting toxicity. We have developed a gene therapy technology for tissue repair and regeneration that employs a structural matrix for DNA delivery. The matrix holds the DNA vector at the treatment site and provides a scaffolding for in-growth and accumulation of repair cells and efficient DNA transfection. We now report, for the first time, matrix-mediated delivery of targeted DNA vectors for soft tissue repair. A collagen matrix was used to deliver an adenoviral vector encoding platelet-derived growth factor-B (AdPDGF-B), resulting in efficient transgene expression in vitro and in vivo. Increases in the overall levels of expression and in the relative amounts of secreted PDGF-BB were achieved when AdPDGF-B was conjugated to fibroblast growth factor (FGF2) such that the virus was targeted for cellular uptake via FGF receptors. Matrix-mediated delivery of AdPDGF-B enhanced wound healing responses in vivo, and FGF2 targeting generated effects comparable to nontargeted vectors at significantly lower doses. Therefore, matrix-mediated delivery in combination with FGF2 targeting overcomes some of the safety and efficacy limitations of current gene therapy strategies and is an attractive therapeutic approach for tissue repair and regeneration.

Isolation and characterization of a thymidylate synthase-deficient human colon tumor cell line.

Following mutagenesis of the human colorectal tumor cell line HCT C with ethyl methanesulfonate, clonal sublines were isolated that survived on medium toxic to cells expressing thymidylate synthase (TS). The subline exhibiting the lowest TS activity, designated as C18, was characterized. Extracts from C18 cells were mixed with extracts from parental C cells to determine whether the TS-deficient phenotype is trans-acting. No effect was observed on the activity of TS in parental extracts. The levels of functional TS in C18 cells were analyzed by the binding of the mechanism-based inhibitor 5-fluoro-2'-deoxyuridylate (FdUMP) under conditions that allowed for the detection of 10 fmol of TS. Only a low level of FdUMP-TS complexes was detected in C18 extracts. The level of TS expression in C18 cells was similar to that in parental C cells, as indicated by immunoblot and RNA analyses. DNA sequence analysis of TS cDNA from C18 cells revealed the existence of a point mutation (C-->T) at nucleotide 647 that predicts the replacement of Ser216 by a leucine residue. That the C18 cell line was homozygous for this mutation was indicated by restriction fragment-length polymorphism analysis and by primer extension analysis. To provide additional evidence that substitution of Ser216 by a leucine residue created a defective protein, a TS-deficient bacterial strain was transformed with an expression vector containing the mutated human TS cDNA. The transformed strain exhibited thymidine auxotrophy, indicating that the mutant TS (Leu216) is nonfunctional.

Retargeted delivery of adenoviral vectors through fibroblast growth factor receptors involves unique cellular pathways.

A major goal of gene therapy is to improve target specificity by delivering vectors through alternative cellular receptors. We previously reported that adenoviral vector delivery through basic fibroblast growth factor (FGF2) receptors enhances both cellular transduction and in vivo efficacy. We now present studies addressing the cellular pathways and mechanisms underlying these events. Cellular receptors for adenoviruses are not required for transduction by FGF2-retargeted vectors. Moreover, alpha(V) integrins can antagonize FGF2 retargeting, in contrast to their obligatory role in non-retargeted vector delivery. By contrast, high-affinity FGF receptors, which are overexpressed on potential tumor targets, are required for FGF2-retargeted transduction. Low-affinity heparan sulfate proteoglycan interactions, however, are not a prerequisite, in marked contrast to their obligatory role in FGF2 mitogenic signaling. By comparing receptor expression and ligand binding with transgene expression, we also demonstrate that FGF2 retargeting enhances transduction by mechanisms other than increasing the number of targeted cells. Rather, the use of alternative targeting ligands supports the conclusion that specific receptor interactions and intracellular events serve to enhance transgene expression. Together, these studies highlight the unique delivery and transduction pathways used by FGF2-retargeted adenoviruses, and help define the basis for their enhanced in vivo efficacy.

Fibroblast growth factor 2 retargeted adenovirus has redirected cellular tropism: evidence for reduced toxicity and enhanced antitumor activity in mice.

Adenovirus (Ad) have been used as vectors to deliver genes to a wide variety of tissues. Despite achieving high expression levels in vivo, Ad vectors display normal tissue toxicity, transient expression, and antivector immune responses that limit therapeutic potential. To circumvent these problems, several retargeting strategies to abrogate native tropism and redirect Ad uptake through defined receptors have been attempted. Despite success in cell culture, in vivo results have generally not shown sufficient selectivity for target tissues. We have previously identified (C. K. Goldman et al., Cancer Res., 57: 1447-1451, 1997) the fibroblast growth factor (FGF) ligand and receptor families as conferring sufficient specificity and binding affinity to be useful for targeting DNA in vivo. In the present studies, we retargeted Ad using basic FGF (FGF2) as a targeting ligand. Cellular uptake is redirected through high-affinity FGF receptors (FGFRs) and not the more ubiquitous lower-affinity Ad receptors. Initial in vitro experiments demonstrated a 10- to 100-fold increase in gene expression in numerous FGFR positive (FGFR+) cell lines using FGF2-Ad when compared with Ad. To determine whether increased selectivity could be detected in vivo, FGF2-Ad was administered i.v. to normal mice. FGF2-Ad demonstrates markedly decreased hepatic toxicity and liver transgene expression compared with Ad treatment. Importantly, FGF2-Ad encoding the herpes simplex virus thymidine kinase (TK) gene transduces Ad-resistant FGFR+ tumor cells both ex vivo and in vivo, which results in substantially enhanced survival (180-260%) when the prodrug ganciclovir is administered. Because FGFRs are up-regulated on many types of malignant or injured cells, this broadly useful method to redirect native Ad tropism and to increase the potency of gene expression may offer significant therapeutic advantages.

Targeted delivery of DNA encoding cytotoxic proteins through high-affinity fibroblast growth factor receptors.

Nonviral DNA delivery strategies for gene therapy have generally been limited by a lack of specificity and efficacy. However, ligand-mediated endocytosis can specifically deliver DNA in vitro to cells bearing the appropriate cognate receptors. Similarly, in order to circumvent problems related to efficacy, DNA must encode proteins with high intrinsic activities. We show here that the ligand basic fibroblast growth factor (FGF2) can target FGF receptor-bearing cells with DNA encoding therapeutic proteins. Delivery of genes encoding saporin, a highly potent ribosomal inactivating protein, or the conditionally cytotoxic herpes simplex virus thymidine kinase, a protein that can kill cells by activating the prodrug ganciclovir, is demonstrated. The saporin gene was codon optimized for mammalian expression and demonstrated to express functional protein in a cell-free assay. FGF2-mediated delivery of saporin DNA or thymidine kinase DNA followed by ganciclovir treatment resulted in a 60 and 75% decrease in cell number, respectively. Specificity of gene delivery was demonstrated in competition assays with free FGF2 or with recombinant soluble FGF receptor. Alternatively, when histone H1, a ligand that binds to cell surface heparan sulfate proteoglycans ("low-affinity" FGF receptors), was used to deliver DNA encoding thymidine kinase, no ganciclovir sensitivity was observed. These findings establish the feasibility of using ligands such as FGF2 to specifically deliver genes encoding molecular chemotherapeutic agents to cells.

Toxin gene-mediated growth inhibition of lung adenocarcinoma in an animal model of pleural malignancy.

Transduction of malignant cells with toxin genes provides a novel strategy by which to promote tumor cell destruction. Whereas the capacity of the toxin gene/prodrug combination cytosine deaminase/fluorocytosine to inhibit growth of human metastatic pulmonary adenocarcinoma cell lines in vitro is established, the in vivo efficacy of this binary system has not yet been determined. For the development of toxin gene therapy for the treatment of lung adenocarcinoma metastatic to the pleural space, a reliable, disease-specific model is required. The serosa of the rat small intestine resembles the basal lamina of the pleura and provides the basis for a more convenient model than direct injection of tumor into the pleural space. Adenocarcinoma cells are inoculated into everted denuded rat intestine configured as a sac. Immunocytochemical and histological analyses show rapid cell growth with characteristics that mimic nodular metastatic intrapleural disease. In the context of this model, systemically delivered fluorocytosine significantly inhibits the growth of cytosine deaminase-expressing human lung adenocarcinoma cells. The dosing schedule required 30 days; neither addition of an enzyme inhibitor that increases the half-life of fluorocytosine nor intralumenal drug delivery is effective in shortening (to 15 days) the protocol. We conclude that CD continues to hold promise as a toxin gene for lung adenocarcinoma gene therapy, and that prolonged prodrug administration may be required for maximum efficacy.

Transduction of non-small cell lung cancer cells by adenoviral and retroviral vectors.

Gene transfer into a panel of non-small cell lung cancer (NSCLC) cells by adenoviral (Ad) and retroviral (RV) vectors was studied. Indexed to multiplicity of infection (MOI), Ad vectors transduce squamous, adenosquamous, and malignant mesothelioma cells with greater efficiency than large cells or adenocarcinoma cells. Transduction-sensitive cells bind the Ad vector with specificity for the Ad fiber knob, and internalize vector efficiently. Transduction-refractory cells bind and internalize vector by less efficient pathways. Like Ad vectors, there is heterogeneity in RV transduction efficiencies of different NSCLC subtypes. With respect to the most common cell type metastatic to the pleural space (adenocarcinoma), amphotropic retroviral vectors transduce cells of this subtype more efficiently (at a lower MOI) than Ad. RV transduction is not solely dependent on cellular replication, and both permissive and refractory cell lines express the mRNA for the amphotropic RV receptor. These observations suggest that neither Ad nor RV vectors will suffice a priori as the optimal gene transfer vehicle, and successful gene therapy of lung cancer may require tumor-specific or patient-specific vectors.

Retroviral gene transfer is inhibited by chondroitin sulfate proteoglycans/glycosaminoglycans in malignant pleural effusions.

Gene therapy may be an important adjuvant for treating cancer in the pleural space. The initial results of retroviral gene transfer to cancer cells in malignant pleural effusions revealed that transduction was markedly inhibited, and studies to characterize the inhibitory factor(s) were performed. The inhibition was contained within the soluble, rather than cellular, components of the effusions and was demonstrated with amphotropic, gibbon ape leukemia virus, and vesicular stomatitis virus-glycoprotein pseudotyped retroviral vectors. After excluding complement proteins, a series of studies identified chondroitin sulfates (CSs) as the inhibitory substances. First, treatment of the effusions with mammalian hyaluronidase or chondroitinases, but not Streptomyces hyaluronidase, abolished the inhibitory activity. Second, addition of exogenous CS glycosaminoglycans mimicked the inhibition observed with pleural effusions. Third, immunoassays and biochemical analyses of malignant pleural effusion specimens revealed CS in relevant concentrations within pleural fluid. Fourth, proteoglycans/glycosaminoglycans isolated from the effusions inhibited retroviral gene transfer. Analyses of the mechanism of inhibition indicate that the chondroitin sulfates interact with vector in solution rather than at the target cell surface. These results suggest that drainage of the malignant pleural effusion, and perhaps enzymatic pretreatment of the pleural cavity, will be necessary for efficient retroviral vector mediated gene delivery to pleural metastases.

Killing Epstein-Barr virus-positive B lymphocytes by gene therapy: comparing the efficacy of cytosine deaminase and herpes simplex virus thymidine kinase.

Epstein-Barr virus (EBV)-positive lymphomas are frequent among immunosuppressed patients. We have examined the feasibility of killing EBV-immortalized B lymphocytes by gene transfer involving the use of "suicide" genes whose expression in target cells renders them susceptible to killing by a prodrug. We examined two gene/prodrug pairs: the Escherichia coli cytosine deaminase (CD) gene with the prodrug 5-fluorocytosine (5-FC), and the herpes simplex virus thymidine kinase (HSV-TK) gene with the prodrug ganciclovir. Retroviral vectors and drug selection were used to obtain CD or HSV-TK expression in cells. Both the CD/5-FC and the HSV-TK/ganciclovir combinations yielded substantial killing of EBV-immortalized B lymphocytes in vitro, although the CD/5-FC regimen had a significantly greater therapeutic margin than the HSV-TK/ganciclovir combination. The CD/5-FC pair, but not the HSV-TK/ganciclovir pair, was shown to have a "bystander killing effect" in vitro. When only 30% of the cells expressed the suicide gene, scid mouse tumors regressed in both the CD/5-FC regimen and the HSV-TK/ganciclovir regimen, documenting an in vivo bystander effect with both regimens. However, a greater percentage of tumors completely regressed with the CD/5-FC regimen. Overall, the sum of our data indicates that the CD/5-FC combination is the more promising regimen for treatment of EBV-associated lymphomas in vivo.

Comparison of the effects of three different toxin genes and their levels of expression on cell growth and bystander effect in lung adenocarcinoma.

Transduction of malignant cells with toxin genes provides a novel means to promote tumor cell destruction. The efficacy of a toxin gene is dependent on the cell type targeted, the quantity of exogenous protein synthesized, and the mechanisms of growth inhibition and bystander killing. To develop gene therapy for targeting metastatic lung adenocarcinoma, the toxic activity of herpes simplex virus type 1-thymidine kinase, Escherichia coli cytosine deaminase, and human deoxycytidine kinase were investigated in metastatic human lung adenocarcinoma cell lines H1437 and H2122. Cells were transduced stably with retroviral vectors containing the toxin gene cDNA under the control of either a strong [cytomegalovirus (CMV) immediate early promotor and enhancer] or an intermediate strength (Moloney murine leukemia virus long terminal repeat) promotor. A comparison of toxin gene efficacy was based on the level of specific enzyme activity, the concentration of prodrug required to inhibit cell growth by 50%, and the magnitude of the bystander effect. In lung adenocarcinoma cell lines, cytosine deaminase, driven by the CMV promoter, was superior to thymidine kinase and deoxycytidine kinase in its ability to achieve high levels of specific enzyme activity, to induce growth inhibition, and to affect neighboring cell growth. Therefore, cytosine deaminase expressed from the CMV promotor seems to be the most promising toxin gene for human lung adenocarcinoma gene therapy.

A naturally occurring tyrosine to histidine replacement at residue 33 of human thymidylate synthase confers resistance to 5-fluoro-2'-deoxyuridine in mammalian and bacterial cells.

Structural changes in the macromolecular targets of pharmacological agents can result in alterations in the efficacy of these agents. In previous studies, we identified a variant structural form of thymidylate synthase (TS) that is associated with relative resistance to 5-fluoro-2'-deoxyuridine, in a human colonic tumor cell line. We now report on the use of DNA transfer techniques to examine directly the effects of each TS form on drug response. TS cDNA constructs, corresponding to the normal or variant TS mRNA, were expressed in Chinese hamster lung cells or in Escherichia coli, and response to 5-fluoro-2'-deoxyuridine was determined. We observed that expression of the variant TS, which differs from the normal form by a tyrosine to histidine substitution at residue 33, confers a 4-fold level of drug resistance in the mammalian cells, as well as in bacteria. The possible role of Tyr-33 in 5-fluoropyrimidine-mediated inhibition of TS is discussed.