RESUMO
Platelet-derived growth factor-BB (PDGF-BB) stimulates repair of healing-impaired chronic wounds such as diabetic ulcers and periodontal lesions. However, limitations in predictability of tissue regeneration occur due, in part, to transient growth factor bioavailability in vivo. Here, we report that gene delivery of PDGF-B stimulates repair of oral implant extraction socket defects. Alveolar ridge defects were created in rats and were treated at the time of titanium implant installation with a collagen matrix containing an adenoviral (Ad) vector encoding PDGF-B (5.5 x 10(8) or 5.5 x 10(9) pfu ml(-1)), Ad encoding luciferase (Ad-Luc; 5.5 x 10(9) pfu ml(-1); control) or recombinant human PDGF-BB protein (rhPDGF-BB, 0.3 mg ml(-1)). Bone repair and osseointegration were measured through backscattered scanning electron microscopy, histomorphometry, micro-computed tomography and biomechanical assessments. Furthermore, a panel of local and systemic safety assessments was performed. Results indicated that bone repair was accelerated by Ad-PDGF-B and rhPDGF-BB delivery compared with Ad-Luc, with the high dose of Ad-PDGF-B more effective than the low dose. No significant dissemination of the vector construct or alteration of systemic parameters was noted. In summary, gene delivery of Ad-PDGF-B shows regenerative and safety capabilities for bone tissue engineering and osseointegration in alveolar bone defects comparable with rhPDGF-BB protein delivery in vivo.
Assuntos
Perda do Osso Alveolar/terapia , Regeneração Óssea , Implantação Dentária Endóssea , Terapia Genética , Osseointegração , Fator de Crescimento Derivado de Plaquetas/genética , Adenoviridae/genética , Animais , Becaplermina , Humanos , Masculino , Perda da Inserção Periodontal , Proteínas Proto-Oncogênicas c-sis , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/uso terapêutico , Engenharia TecidualRESUMO
BACKGROUND: Demineralized bone matrix and recombinant human bone morphogenetic protein-2 or 7 (BMP-2 or BMP-7)-containing collagenous matrix have been shown to induce new bone formation in orthotopic and heterotopic sites. We examined the ability of subcutaneous implants of collagen combined with adenoviral vector containing the BMP-2 gene (AdBMP-2) to induce bone formation in rats. We also evaluated whether targeting the AdBMP-2 vector through an alternative receptor pathway, fibroblast growth factor (FGF), would increase the vector's potency. METHODS: In a time-course study, rat subcutaneous sites were implanted with (1) AdBMP-2 in rat-bone-derived collagen or (2) rat-bone-derived collagen alone. Samples were collected three, seven, fourteen, or thirty-five days after treatment. In a dose-response study, bone induction by AdBMP-2 in collagen (AdBMP-2/collagen) or by AdBMP-2 and FGF2 Fab' anti-adenovirus knob protein antibody in collagen (FGF2-AdBMP-2/collagen) was tested at fourteen days. Viral vector doses of 1 x 10(9) PN (viral particle number), 3 x 10(9) PN, 1 x10(10) PN, 3 x 10(10) PN, or 1 x 10(11) PN per implant were used. Equal amounts of collagen (25 mg) were used to formulate all implants. Explanted tissues were evaluated histologically to determine bone formation, specific activity of alkaline phosphatase, and calcium content. RESULTS: AdBMP-2/collagen implants induced robust bone formation. New bone was formed by the fourteenth day after implantation. In contrast, little or no bone was induced by the implant containing collagen alone. FGF2-AdBMP-2/collagen implants stimulated significantly more bone formation (p < 0.05) than did AdBMP-2/collagen implants, regardless of the dose of viral particles. CONCLUSIONS: Local delivery of AdBMP-2 in a collagen matrix rapidly induces bone formation, and targeting the virus through FGF receptors enhances the osteogenic potential of AdBMP-2.
Assuntos
Proteínas Morfogenéticas Ósseas/administração & dosagem , Implantes Experimentais , Osteogênese/efeitos dos fármacos , Proteínas Recombinantes/administração & dosagem , Fator de Crescimento Transformador beta/administração & dosagem , Adenoviridae/genética , Fosfatase Alcalina/metabolismo , Animais , Matriz Óssea , Proteína Morfogenética Óssea 2 , Células Cultivadas , Relação Dose-Resposta a Droga , Fator 2 de Crescimento de Fibroblastos/fisiologia , Vetores Genéticos , Ratos , Ratos Long-EvansRESUMO
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.
Assuntos
Colágeno/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Vetores Genéticos/administração & dosagem , Fator de Crescimento Derivado de Plaquetas/genética , Próteses e Implantes , Cicatrização , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Becaplermina , Cicatriz/induzido quimicamente , Sistemas de Liberação de Medicamentos/efeitos adversos , Sistemas de Liberação de Medicamentos/instrumentação , Orelha/patologia , Matriz Extracelular/metabolismo , Feminino , Terapia Genética/efeitos adversos , Terapia Genética/instrumentação , Terapia Genética/métodos , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Granuloma/induzido quimicamente , Humanos , Hiperplasia/induzido quimicamente , Imuno-Histoquímica , Masculino , Especificidade de Órgãos , Fator de Crescimento Derivado de Plaquetas/efeitos adversos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/uso terapêutico , Proteínas Proto-Oncogênicas c-sis/efeitos adversos , Proteínas Proto-Oncogênicas c-sis/genética , Proteínas Proto-Oncogênicas c-sis/metabolismo , Proteínas Proto-Oncogênicas c-sis/uso terapêutico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Transdução Genética , Transgenes/genéticaRESUMO
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.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Vetores Genéticos/farmacocinética , Ligantes , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/genética , Transdução de Sinais , Fatores de Tempo , Transdução Genética , Transgenes/genética , Células Tumorais CultivadasRESUMO
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.
Assuntos
Dependovirus/genética , Fator 2 de Crescimento de Fibroblastos/genética , Terapia Genética/métodos , Proteínas Proto-Oncogênicas c-sis/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Animais , Bovinos , Células Cultivadas , Colágeno/metabolismo , Ensaio de Imunoadsorção Enzimática , Fibroblastos/metabolismo , Humanos , Masculino , Álcool de Polivinil/química , Ratos , Ratos Sprague-Dawley , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Regeneração , Transfecção , Transgenes , CicatrizaçãoRESUMO
The utility of adenoviral vectors for cancer therapy is limited due to their lack of specificity for tumor cells. In order to target adenovirus to tumor, the natural tropism of the adenovirus should be ablated and replaced by a tumor-specific binding domain. To this end, a neutralizing anti-fiber antibody conjugated to an anti-EpCAM antibody was created that targets the adenovirus to the EpCAM antigen present on tumor cells. The EpCAM antigen was chosen as the target because this antigen is highly expressed on a variety of adenocarcinomas of different origin such as breast, ovary, colon and lung, whereas EpCAM expression is limited in normal tissues. In these studies, the EpCAM-targeted adenovirus was shown to infect specifically cancer cell lines of different origin expressing EpCAM such as ovary, colon and head and neck. Gene transfer was blocked by excess anti-EpCAM antibody and dramatically reduced in EpCAM negative cell lines, thus showing the specificity of the EpCAM-targeted adenovirus. Importantly, infection with targeted adenovirus was independent of CAR, which is the natural receptor for adenovirus binding, since blocking of CAR with recombinant fiber knob did not affect infection with targeted adenovirus. Apart from the cancer cell lines, the efficacy of targeted viral infection was studied in freshly isolated primary human colon cancer cells. As colon cancer predominantly metastasizes to liver, and adenovirus has a high tropism for hepatocytes, we also sought to determine if the EpCAM-targeted adenovirus showed reduced infectivity of human liver cells. The bispecific antibody could successfully mediate gene transfer to primary human colon cancer cells, whereas it almost completely abolished infection of liver cells. This work thus demonstrates that EpCAM-targeted adenoviral vectors can be specifically directed to a wide variety of adenocarcinomas. This approach may prove to be useful for selective gene therapy of cancer.
Assuntos
Adenocarcinoma/terapia , Adenoviridae/genética , Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Neoplasias do Colo/terapia , Molécula de Adesão da Célula Epitelial , Marcação de Genes/métodos , Terapia Genética/métodos , Humanos , Integrinas/metabolismo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Adenoviridae , Técnicas de Transferência de Genes , Vetores Genéticos , Receptores de Fatores de Crescimento de Fibroblastos , Expressão Gênica , Marcação de Genes , Terapia Genética , Humanos , Transdução de Sinais/genética , Células Tumorais CultivadasRESUMO
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.
Assuntos
Adenoviridae/fisiologia , Vírus Defeituosos/fisiologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunotoxinas/metabolismo , beta-Galactosidase/metabolismo , Adenoviridae/genética , Adenoviridae/imunologia , Alanina Transaminase/sangue , Animais , Antivirais/uso terapêutico , Aspartato Aminotransferases/sangue , Biomarcadores/sangue , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Vírus Defeituosos/genética , Vírus Defeituosos/imunologia , Feminino , Fator 2 de Crescimento de Fibroblastos/administração & dosagem , Fator 2 de Crescimento de Fibroblastos/imunologia , Ganciclovir/uso terapêutico , Expressão Gênica , Vetores Genéticos/imunologia , Imunotoxinas/administração & dosagem , Imunotoxinas/toxicidade , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Melanoma Experimental/metabolismo , Melanoma Experimental/virologia , Camundongos , Necrose , Especificidade de Órgãos , Receptores Virais/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/metabolismo , Simplexvirus/enzimologia , Timidina Quinase/uso terapêutico , Transfecção , Transgenes , Tropismo , beta-Galactosidase/administração & dosagem , beta-Galactosidase/toxicidadeRESUMO
Basic fibroblast growth factor (FGF2) has potent mitogenic and angiogenic activities that have been implicated in tumor development and malignant progression. The biological effects of FGF2 and other members of the FGF ligand family are mediated by 4 transmembrane tyrosine kinase receptors (FGFRs). To better understand the roles of FGFRs in cancer, the expression of FGF2 and each of the 4 FGFRs was assessed by RNase protection analysis of 60 human tumor cell lines, representing 9 tumor types. Expression of at least one FGFR isoform was detected in 90% and FGF2 mRNA in 35% of the cell lines. Our comprehensive analysis of FGF2 and FGFR expression in human tumor cell lines provides evidence that FGF signaling pathways are active in a majority of human tumor cell lines, and lends support to the development of anti-tumor strategies that target FGFRs.
Assuntos
Fator 2 de Crescimento de Fibroblastos/análise , Neoplasias/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/análise , Fator 2 de Crescimento de Fibroblastos/genética , Humanos , RNA Mensageiro/análise , Receptores de Fatores de Crescimento de Fibroblastos/genética , Ribonucleases/farmacologia , Células Tumorais CultivadasRESUMO
Adenoviral vectors have proven useful for transducing a variety of cell types. However, both adenoviral resistant cell types and vector-related toxicities (due to non-specific tropism), limit their widespread clinical utility. These limitations can be greatly reduced by targeting adenoviral vectors to alternative cell surface receptors. One ligand family, highly effective at targeting adenoviral vectors, is the family of fibroblast growth factors (FGFs). The FGFs allow a high degree of targeting specificity due to their cognate high affinity FGF receptors, which are expressed on cells undergoing repair and regeneration. Recent publications, reviewed herein, demonstrate that FGF-targeted adenoviruses result in enhanced potency and reduced toxicity, and thus substantially increase the therapeutic index in vivo. As a result, vector delivery through FGF receptors provides the targeting specificity required for successful local and systemic clinical applications of gene therapy.
Assuntos
Adenoviridae/genética , Fator 2 de Crescimento de Fibroblastos/genética , Terapia Genética/métodos , Vetores Genéticos , Animais , Fator 2 de Crescimento de Fibroblastos/metabolismo , Terapia Genética/efeitos adversos , Humanos , Ligantes , Camundongos , Ratos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismoRESUMO
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.
Assuntos
DNA/administração & dosagem , Imunotoxinas , N-Glicosil Hidrolases , Proteínas de Plantas/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Timidina Quinase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/genética , Sistema Livre de Células , Cricetinae , DNA Complementar , Dados de Sequência Molecular , Proteínas de Plantas/administração & dosagem , Proteínas Inativadoras de Ribossomos Tipo 1 , Ribossomos , Saporinas , Simplexvirus/enzimologia , Timidina Quinase/administração & dosagem , TransfecçãoRESUMO
A number of preclinical and human clinical gene therapy trials using adenoviral vectors have shown that the number of viral particles necessary to give adequate levels of gene transfer can be associated with significant vector-related toxicity. In an effort to reduce the number of adenoviral particles required for a given level of gene transfer, we sought to redirect adenoviral infection via a receptor that is highly expressed on the target cells. By using basic fibroblast growth factor (FGF2) as the targeting ligand, adenovirus-mediated gene transfer to the human ovarian cancer cell line SKOV3.ip1 was significantly enhanced, permitting the transduction of a greater number of target cells to be achieved by a given dose of virus. In a murine model of human ovarian carcinoma, an FGF2-redirected adenoviral vector carrying the gene for herpes simplex virus thymidine kinase (AdCMVHSV-TK) was shown to result in a significant prolongation of survival compared with the same number of particles of unmodified AdCMVHSV-TK. In addition, equivalent survival rates were achieved with a 10-fold lower dose of the FGF2-redirected AdCMVHSV-TK compared with the unmodified vector. To our knowledge, this is the first report demonstrating that strategies to enhance the efficiency of in vivo transduction of adenoviral vectors will be of clinical utility.
Assuntos
Adenoviridae/genética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Terapia Genética , Neoplasias Ovarianas/terapia , Simplexvirus/enzimologia , Timidina Quinase/genética , Animais , Feminino , Humanos , Camundongos , Camundongos SCID , Neoplasias Ovarianas/mortalidade , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
Over-expression of the epidermal growth factor receptor (EGFR) is a hallmark of numerous solid tumors, thus providing a means of selectively targeting therapeutic agents. Heparin-binding epidermal growth factor (HBEGF) binds to EGFRs with high affinity and to heparan sulfate proteoglycans, resulting in increased mitogenic potential compared to other EGF family members. We have investigated the feasibility of using HBEGF to selectively deliver a cytotoxic protein into EGFR-expressing tumor cells. Recombinant fusion proteins consisting of mature human HBEGF fused to the plant ribosome-inactivating protein saporin (SAP) were expressed in Escherichia coli. Purified HBEGF-SAP chimeras inhibited protein synthesis in a cell-free assay and competed with EGF for binding to receptors on intact cells. A construct with a 22-amino-acid flexible linker (L22) between the HBEGF and SAP moieties exhibited an affinity for the EGFR that was comparable to that of HBEGF. The sensitivity to HBEGF-L22-SAP was determined for a variety of human tumor cell lines, including the 60 cell lines comprising the National Cancer Institute Anticancer Drug Screen. HBEGF-L22-SAP was cytotoxic in vitro to a variety of EGFR-bearing cell lines and inhibited growth of EGFR-over-expressing human breast carcinoma cells in vivo. In contrast, the fusion protein had no effect on small-cell lung carcinoma cells, which are EGFR-deficient. Our results demonstrate that fusion proteins composed of HBEGF and SAP exhibit targeting specificity and cytotoxicity that may be of therapeutic value in treating a variety of EGFR-bearing malignancies.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Imunotoxinas/metabolismo , N-Glicosil Hidrolases , Proteínas de Plantas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Estudos de Viabilidade , Vetores Genéticos , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1 , Saporinas , Células Tumorais Cultivadas/metabolismoRESUMO
Kaposi's sarcoma (KS) is a major AIDS-related malignancy associated with significant morbidity and mortality. Current chemotherapeutic regimens are associated with a dismal prognosis. In an effort to develop a new approach to KS treatment, we devised a gene therapy-based adenovirus retargeting schema that redirects the adenovirus to fibroblast growth factor receptors endogenously present on the cell surface of KS cells. By using a bifunctional conjugate consisting of a blocking antiadenoviral knob Fab linked to basic fibroblast growth factor, FGF2, the gene transduction of KS cells was enhanced 7.7-44 fold; recombinant adenoviruses encoding either the firefly luciferase reporter gene, or the herpes simplex thymidine kinase gene, demonstrated quantitative enhancement of expression in the KS cell lines. In this regard, two KS cell lines that were previously refractory to native adenovirus transduction could be successfully transduced by the addition of the conjugate. This study thus addresses the utility of adenoviral retargeting to the FGF receptor in KS cells that are ordinarily transduction refractory to standardized approaches and allows practical development of gene therapy approaches for the treatment of human KS.
Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Adenoviridae/genética , Terapia Genética , Vetores Genéticos/uso terapêutico , Fragmentos Fab das Imunoglobulinas/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Sarcoma de Kaposi/terapia , Transfecção/métodos , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Luciferases/genética , Luciferases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/imunologia , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/virologia , Células Tumorais Cultivadas/virologiaRESUMO
Ligand-mediated targeting of DNA was validated by condensing a plasmid DNA encoding the beta-galactosidase (beta-gal) gene with a basic fibroblast growth factor (FGF2) that was first chemically conjugated to polylysine (K). The conditions that gave optimal binding of this FGF2 to DNA also generated the highest level of beta-gal expression when added to FGF2 target cells like COS-1, 3T3, baby hamster kidney (BHK), or endothelial cells. This beta-gal activity increased in a time- and dose-dependent manner and was dependent on the inclusion of FGF2 in the complex. FGF receptor specificity was demonstrated by competition of the complex with FGF2 and heparin, and by the failure of cytochrome c or histone H1 to mimic the gene-targeting effects of FGF2. The expression of beta-gal was also endosome dependent because chloroquine increased beta-gal expression 8-fold and endosome disruptive peptides increased expression of beta-gal 26-fold. Taken together these findings establish that DNA can be introduced into cells through the high affinity FGF receptor complex, and while its efficiency will require significant enhancements to achieve sustained and elevated transgene expression, the possibility that the technique could be used to deliver DNAs encoding cytotoxic molecules is discussed.
Assuntos
DNA/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Marcação de Genes , Células 3T3 , Animais , Células COS , Cricetinae , Grupo dos Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Fator 2 de Crescimento de Fibroblastos/metabolismo , Histonas/metabolismo , Camundongos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismoRESUMO
In order to produce sufficient quantities of fibroblast growth factor-saporin (rFGF-2-SAP) mitotoxin for preclinical evaluation in models of diseases such as cancer and restenosis, we have undertaken the large-scale expression, purification, and characterization of the recombinant molecule. The fusion gene encoding rFGF-2-SAP was cloned into the inducible pET 11a expression vector and transformed into Escherichia coli strain BL21 (DE3). The transformants were grown using a fed-batch fermentation until the A600 reached 85. At this stage, induction of the expression of the fusion protein led to the production of approximately 2.2 mg/liter per A600 unit. The soluble mitotoxin was purified to homogeneity from cell lysates via expanded bed adsorption chromatography followed by cation-exchange, heparin-affinity, and size-exclusion chromatography. Purified rFGF-2-SAP contained less than 0.5 EU/mg of endotoxin, as determined by gel clot analyses. The highly purified rFGF-2-SAP retained the toxin's ability to inhibit protein synthesis as measured in a cell-free system and was cytotoxic to a number of normal and neoplastic cell lines bearing FGF receptors. Binding studies establish that the fusion protein exerts its effects via the FGF high-affinity receptor.
Assuntos
Fator 2 de Crescimento de Fibroblastos/genética , Imunotoxinas , N-Glicosil Hidrolases , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusão/genética , Ligação Competitiva , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Corantes/metabolismo , Citotoxinas/farmacologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Fator 2 de Crescimento de Fibroblastos/isolamento & purificação , Expressão Gênica/genética , Espectrometria de Massas , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Plasmídeos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Inativadoras de Ribossomos Tipo 1 , Ribossomos/efeitos dos fármacos , SaporinasRESUMO
Basic fibroblast growth factor (FGF-2) and saporin were chemically conjugated using the crosslinker, N-succinimidyl-3(2-pyridyldithio)-propionate. When purified, the conjugate was found to be heterogeneous as analyzed by SDS/PAGE, size-exclusion HPLC and reverse-phase HPLC. Therefore, we sought to synthesize a molecule that would be homogeneous and thus easier to characterize and evaluate its efficacy and toxicity for pharmaceutical drug development. A homogeneous chemical conjugate was successfully synthesized by using a mutant FGF-2 with Cys96 replaced by Ser ([S96]FGF-2) and a recombinant saporin mutant containing a single Cys at the -1 position (C-SAP). The latter was expressed in Escherichia coli and isolated to 99% purity by expanded-bed adsorption chromatography followed by cation-exchange chromatography. The cysteine in C-SAP was activated by Ellman's reagent and then reacted with the only available cysteine (position 78) in [S96]FGF-2 to produce the homogeneous conjugate, designated as FGF2-C-SAP. The purified FGF2-C-SAP was more than 98% pure as judged by HPLC. In vitro biological assays indicated that FGF2-C-SAP was a potent inhibitor of protein synthesis in a cell-free system and was cytotoxic to FGF-receptor-bearing cells.
Assuntos
Fator 2 de Crescimento de Fibroblastos/química , Imunotoxinas , N-Glicosil Hidrolases , Proteínas de Plantas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Reagentes de Ligações Cruzadas/química , Cisteína/química , Primers do DNA/química , Ácido Ditionitrobenzoico/metabolismo , Eletroforese em Gel de Poliacrilamida , Fator 2 de Crescimento de Fibroblastos/isolamento & purificação , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Mutação Puntual/genética , Biossíntese de Proteínas , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Inativadoras de Ribossomos Tipo 1 , Ribossomos/metabolismo , Saporinas , Succinimidas/químicaRESUMO
The transformer gene of Drosophila is regulated by Sex-lethal-dependent 3' splice site blockage. 40 nucleotides immediately upstream of the regulated splice site are sufficient to direct sex-specific regulated splicing in transgenic animals. This entire region appears to be necessary for regulation and for efficient Sex-lethal binding. Natural splice sites containing partial homology to transformer do not show regulation. Mutations which replace the 16 nucleotides surrounding the branch point or alter single nucleotides near the splice site eliminate or reduce regulation without eliminating splicing. Mutations which reduce or eliminate regulation in vivo reduce binding to Sex-lethal in vitro, consistent with the hypothesis that these mutations bring about their effects by altering Sex-lethal binding rather than by altering binding sites for additional non-Sex-lethal factors.
Assuntos
Proteínas de Drosophila , Drosophila/genética , Hormônios de Inseto/genética , Splicing de RNA , Proteínas de Ligação a RNA , Animais , Sequência de Bases , Sítios de Ligação , Feminino , Hormônios de Inseto/metabolismo , Masculino , Dados de Sequência Molecular , Mutação , Fatores SexuaisRESUMO
Sex-specific alternative splicing of RNA from the Drosophila transformer gene involves competition between two 3' splice sites. In the absence of Sex-lethal activity (as in males), only one site functions; in the presence of Sex-lethal activity (as in females), both sites function. Information for sex-specific splice site choice is contained within the intron itself. Deletions of the splice site used in males lead to Sex-lethal-independent use of the otherwise female-specific site. The relative amounts of unspliced and spliced RNA derived from these mutant genes do not change with changes in Sex-lethal activity. Specific nucleotide changes in the non-sex-specific splice site do not affect splicing activity but eliminate Sex-lethal-induced regulation. A deletion removing material between the two splice sites does not eliminate sex-specific regulation, while a deletion of the female splice site leads to a female-specific increase in unspliced RNA. These results are consistent with a model in which female-specific factors block the function of the non-sex-specific 3' splice site.
Assuntos
Drosophila melanogaster/genética , Regulação da Expressão Gênica , Splicing de RNA , Diferenciação Sexual , Animais , Mapeamento Cromossômico , Análise Mutacional de DNA , Feminino , Íntrons , Masculino , RibonucleasesRESUMO
The transformer gene is one of a set of regulatory genes that form the hierarchy controlling all aspects of somatic sexual differentiation in Drosophila melanogaster. The gene transformer occupies an intermediate position in this hierarchy. Analysis of this gene has allowed us to determine the mechanism by which it is regulated in a sex-specific manner and to examine the way in which the regulatory hierarchy is organized. The female-specific expression of the tra gene, previously inferred from genetic observations, is based on sex-specific alternative splicing of tra pre-mRNA and is not the result of sex-specific transcriptional activation. The female-specific RNA produced by this alternative splicing is the functional mediator of tra activity. Multiple genetic, molecular, and transformation experiments show that female-specific activation of genes or gene products occurs in the order Sex lethal greater than transformer greater than transformer-2 greater than doublesex greater than or equal to intersex greater than female differentiation. The results do not distinguish the level at which transformer might regulate the downstream gene transformer-2. Neither transformer nor any of the down-stream genes feedback on, or participate in, alternative splicing of transformer RNA. The mechanism by which Sex lethal regulates transformer splicing appears to be a repression of the use of one of a pair of splice acceptor sites.