ABSTRACT
Efficient intravenous delivery is the greatest single hurdle, with most nanotherapeutics frequently found to be unstable in the harsh conditions of the bloodstream. In the case of nanotherapeutics for gene delivery, viral vectors are often avidly recognized by both the innate and the adaptive immune systems. So, most modern delivery systems have benefited from being coated with hydrophilic polymers. Self-assembling delivery systems can achieve both steric and lateral stabilization following surface coating, endowing them with much improved systemic circulation properties and better access to disseminated targets; similarly, gene delivery viral vectors can be 'stealthed' and their physical properties modulated by surface coating. Polymers that start degrading under acidic conditions are increasingly investigated as a pathway to trigger the release of drugs or genes once the carrier reaches a slightly acidic tumor environment or after the carrier has been taken up by cells, resulting in the localization of the polymer in acidic endosomes and lysosomes. Advances in the design of acid-degradable drug and gene delivery systems have been focused and discussed in this article with stress placed on HPMA-based copolymers. We designed a system that is able to "throw away" the polymer coat after successful transport of the vector into a target cell. Initial biological studies were performed and it was demonstrated that this principle is applicable for real adenoviral vectors. It was shown that the transfection ability of coated virus at pH 7.4 is 75 times lower then transfection at pH 5.4.
Subject(s)
Chemistry, Pharmaceutical , Gene Transfer Techniques , Nanomedicine/methods , Adenoviridae/genetics , Endosomes , Genetic Vectors , Humans , Hydrogen-Ion Concentration , Lysosomes , Microspheres , Polymers , TransfectionABSTRACT
A breast cancer-selective oncolytic adenovirus was engineered to express antagonists of vascular endothelial growth factor (VEGF) and Notch signaling to combine direct anticancer activity with disruption of tumor-associated angiogenesis. Replication of the parental virus, AdEHE2F, is stimulated by estrogen receptor (ER), E2F1 and hypoxia, and it mediates selective lysis of breast cancer cells in vitro and in vivo. Here, we encoded soluble Flt-1 (sFlt1) and soluble Dll4 (sDll4) under control of the E3 promoter. sFlt1 (the extra-cellular domain of VEGF receptor 1) binds VEGF-A and inhibits stimulation of VEGFR2, decreasing angiogenic stimulus. Conversely, sDll4 (the extracellular domain of Delta-like 4) antagonizes Notch signaling to prevent endothelial maturation. We hypothesized that these agents might show additive or synergistic activity. In vitro, sFlt1 inhibited endothelial cell proliferation and sprouting, whereas sDll4 increased the number of vascular branchpoints. In ER-positive ZR75.1 tumors in vivo AdEHE2F showed the potent direct virotherapy with no augmentation owing to sFlt1 or sDll4; however, in ER-negative MDA-231 tumors efficacy was enhanced by encoding sFlt1 or sDll4, with survival time extending to double that of controls. There was also a dramatic decrease in the total number of tumour blood vessels, as well as the number of perfused vessels, suggesting that improved efficacy reflects combined anti-tumour and anti-vascular effects.
Subject(s)
Breast Neoplasms/therapy , Embolization, Therapeutic/methods , Oncolytic Virotherapy/methods , Adaptor Proteins, Signal Transducing , Adenoviridae/genetics , Adenoviridae/metabolism , Adenoviridae/physiology , Animals , Breast Neoplasms/blood supply , Breast Neoplasms/pathology , Breast Neoplasms/virology , Calcium-Binding Proteins , Cell Growth Processes/physiology , Combined Modality Therapy , Female , Gene Expression , Human Umbilical Vein Endothelial Cells , Humans , Immunohistochemistry , Intercellular Signaling Peptides and Proteins/biosynthesis , Intercellular Signaling Peptides and Proteins/genetics , Mice , Mice, Nude , Neovascularization, Pathologic/therapy , Receptors, Estrogen/metabolism , Signal Transduction , Transgenes , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-1/biosynthesis , Vascular Endothelial Growth Factor Receptor-1/genetics , Xenograft Model Antitumor AssaysABSTRACT
Cervical cancer is attributable to continuous expression of the E6 and E7 oncoproteins of the high-risk human papillomaviruses. These proteins target p53 and members of the retinoblastoma cellular regulatory protein family respectively for degradation, disrupting cellular control over apoptosis, senescence and the cell cycle. Delivery of short interfering RNAs (siRNAs) targeting mRNA from the HPV16 E6/E7 open reading frame to HPV16-positive cell lines, led to an 80% reduction in full-length transcripts and 60% reduction in total (full-length and spliced) transcripts. Downregulation of E6 mRNA led to increased levels of p53 detectable by western blot and resulted in an eightfold increase in luciferase expression from a p53-responsive reporter plasmid. Downregulation of E7 mRNA reduced the levels of E7 protein and increased the levels of hypophosphorylated pRb. Cellular proliferation was reduced after siRNA delivery; the effect being greater in SiHa cells than CaSki cells and when full-length transcripts encoding E6 and spliced transcripts encoding E7 were both targeted. There was no loss of proliferation in human papillomavirus-negative cell lines. Elevation of p53 in the absence of changes to Rb led to 35% of CaSki cells undergoing apoptosis, whereas in SiHa cells restoration of both p53 and hypophosphorylated Rb had the greatest effect with 25% of cells undergoing apoptosis. The combined use of oncogene-targeting siRNA with either carboplatin, irinotecan, leptomycin B or doxorubicin led to additive toxicity, whereas, with cisplatin, led to sub-additive toxicity. In contrast, siRNA combined with paclitaxel treatment resulted in synergistic toxicity, with intronic siRNA (which mainly targets E6) more effective than exonic siRNA (which targets both E6 and E7). The growth of SiHa xenograft tumors was reduced using paclitaxel combined with intronic and exonic siRNA, compared with exonic siRNA alone, confirming the synergistic relationship between p53 restoration and paclitaxel.
Subject(s)
Human papillomavirus 16/genetics , Oncogene Proteins, Viral/genetics , Paclitaxel/pharmacology , RNA, Small Interfering/genetics , Repressor Proteins/genetics , Uterine Cervical Neoplasms/therapy , Animals , Blotting, Western , Cell Growth Processes/drug effects , Cell Growth Processes/genetics , Cell Line, Tumor , Female , Humans , Mice , Mice, Nude , Papillomavirus E7 Proteins , RNA, Messenger/genetics , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/metabolism , Transfection , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/virologyABSTRACT
Oncolytic viruses are regulated by the tumor phenotype to replicate and lyse cancer cells selectively. To identify optimal strategies for breast cancer we compared five adenoviruses with distinct regulatory mechanisms: Ad-dl922-947 (targets G1-S checkpoint); Ad-Onyx-015 and Ad-Onyx-017 (target p53/mRNA export); Ad-vKH1 (targets Wnt pathway), and AdEHE2F (targets estrogen receptor/G1-S checkpoint/hypoxic signaling). The quantity of virus required to kill 50% of breast cancer cells after 6 days (EC(50), plaque-forming units per cell) was measured. The most potent virus was Ad-dl922-947 (EC(50), 0.01-5.4 in SkBr3, MDA-231, MDA-468, MCF7, and ZR75.1 cells), followed by wild-type (Ad-WT; EC(50), 0.3-5.5) and AdEHE2F (EC(50), 1.4-3.9). Ad-vKH1 (EC(50), 7.2-72.1), Ad-Onyx-017 (EC(50), 8.4-167), and Ad-Onyx-015 (EC(50), 17.7-377) showed less activity. Most viruses showed limited cytotoxicity in normal human cells, including breast epithelium MCF10A (EC(50), >722) and fibroblasts (EC(50), >192) and only moderate cytotoxicity in normal microvascular endothelial cells (HMVECs; EC(50), 42.8-149), except Ad-dl922-947, which was active in HMVECs (EC(50), 1.6). After injection into MDA-231 xenografts, Ad-WT, AdEHE2F, and Ad-dl922-947 showed replication, assessed by hexon staining and quantitative polymerase chain reaction measurement of viral DNA, and significantly inhibited tumor growth, leading to extended survival. After intravenous injection Ad-dl922-947 showed DNA replication (233% of the injected dose was measured in liver after 3 days) whereas AdEHE2F did not. Overall, AdEHE2F showed the best combination of low toxicity in normal cells and high activity in breast cancer in vitro and in vivo, suggesting that molecular targeting using estrogen response elements, hypoxia response elements, and a dysregulated G1-S checkpoint is a promising strategy for virotherapy of breast cancer.
Subject(s)
Adenoviruses, Human/genetics , Breast Neoplasms/therapy , Oncolytic Virotherapy/methods , Oncolytic Viruses/genetics , Adenoviruses, Human/physiology , Animals , Base Sequence , Breast Neoplasms/pathology , Breast Neoplasms/virology , Cell Cycle , Cell Death , Cell Line , Cell Line, Tumor , DNA Primers/genetics , Female , Humans , Mice , Mice, SCID , Neoplasm Transplantation , Oncolytic Viruses/physiology , Transplantation, Heterologous , Virus ReplicationABSTRACT
Direct intratumoural (IT) administration of adenovirus is widely used, however little is known about the resulting distribution of virus particles. Here we have evaluated the influence of tumour size, volume of injectate and occlusion of injection sites (to prevent retrograde seepage) on particle biodistribution and transgene expression. In subcutaneous MDA-231 xenografts, IT injection of relatively large volumes (4 x 20% (vol/vol) injections) resulted in just 40% of the administered dose being retained in tumour tissue after 30 min, with 15% in the liver thought to reflect systemic 'overflow'. Occlusion of the injection sites using surgical adhesive increased retention of the vector to 80% in the tumour with no increase in liver levels. Spread of expression was enhanced using multiple injection sites, but not by using larger injectate volumes. In ZR75.1 breast carcinoma xenografts virus distribution was different, with no evidence of systemic overflow leading to hepatic transduction following IT injection. Typically, clinical doses employ up to 30% vol/vol IT injections. Depending on the tumour, this may give considerable systemic overflow and might account for the high frequency of fevers observed. Virus performance might be improved by tailoring volumes and frequency of IT injection for tumour biology or histotype.
Subject(s)
Adenoviridae/physiology , Breast Neoplasms/therapy , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Transduction, Genetic/methods , Adenoviridae/genetics , Adenoviridae Infections/pathology , Adenoviridae Infections/virology , Animals , Breast Neoplasms/pathology , Breast Neoplasms/virology , Female , Gene Expression , Humans , Injections, Intralesional , Liver/virology , Mice , Mice, Nude , Transgenes , Xenograft Model Antitumor AssaysABSTRACT
Effective gene therapy for disseminated metastatic cancer is currently impossible because of poor delivery of vector to target sites. Modification of viral vectors to target advanced cancer has long been a challenge. In this study, we aimed to redirect adenovirus tropism to infect prostate cancer cells via alpha6beta1 integrins, whose expression is upregulated during prostate cancer progression. To ablate normal mechanisms of infection and provide a framework for attachment of targeting ligands, viruses were non-genetically modified with pHPMA-ONp polymer. Addition of polymer-coated virus to prostate cells showed significantly reduced transgene expression compared with unmodified virus. To restore infectivity, an alpha6-integrin binding peptide (-SIKVAV-) derived from laminin was incorporated onto the surface of the polymer-coated viruses. Photon correlation spectroscopic analysis revealed a small increase in the mean diameter of the particles following retargeting. Addition of -SIKVAV- peptide restored virus infectivity of PC-3 cells in a ligand concentration-dependent manner that was significantly improved following removal of unincorporated polymer and peptide. Competition assays using cells preincubated with Ad5 fiber protein or free -SIKVAV- peptide confirmed that entry of retargeted viruses was mediated via the incorporated ligand. Application of retargeted viruses to a panel of human cell lines revealed varying levels of transduction efficiency. Flow cytometric analysis of cells using anti-alpha6 integrin and anti-beta1 integrin antibodies demonstrated that for prostate cells, greater transduction efficiency correlated with higher levels of expression of both integrin subunits. Furthermore with the exception of LNCaP cells, increased alpha6beta1 integrin expression correlated with advanced disease. Intravenous administration of retargeted viruses to tumor-bearing mice resulted in slower plasma clearance and greatly reduced liver tropism, and hence toxicity compared with unmodified virus, while maintaining reporter gene expression in the tumor. The data suggest that YESIKVAVS-retargeted viruses have potential for systemic delivery for the treatment of metastatic disease.
Subject(s)
Adenoviridae/genetics , Genetic Therapy/methods , Genetic Vectors , Integrin alpha6/metabolism , Laminin/chemistry , Oligopeptides/chemistry , Polymers/chemistry , Prostatic Neoplasms/therapy , Adenoviridae/chemistry , Adenoviridae/metabolism , Cell Line, Tumor , Humans , Integrin alpha6/analysis , Male , Prostatic Neoplasms/chemistry , Prostatic Neoplasms/secondary , Transduction, GeneticABSTRACT
Tumor-associated vasculature is a relatively accessible component of solid cancers that is essential for tumor survival and growth, providing a vulnerable target for cancer gene therapy administered by intravenous injection. Several features of tumor-associated vasculature are different from normal vasculature, including overexpression of receptors for angiogenic growth factors, markers of vasculogenesis, upregulation of coagulation cascades, aberrant expression of adhesion molecules and molecular consequences of hypoxia. Many of these differences provide candidate targets for tumor-selective 'transductional targeting' of genetically- or chemically modified vectors and upregulated gene expression can also enable 'transcriptional targeting', regulating tumor endothelia-selective expression of transgenes following nonspecific gene delivery. Tumor vasculature also represents an important site of therapeutic action by the secreted products of antiangiogenic gene therapies that are expressed in non-endothelial cells. In this review we assess the challenges faced and the vectors that may be suitable for gene delivery to exploit these targets. We also overview some of the strategies that have been developed to date and highlight the most promising areas of research.
Subject(s)
Endothelium, Vascular , Genetic Therapy , Neoplasms/therapy , Animals , Humans , Neovascularization, Pathologic , Transcription, GeneticABSTRACT
Use of synthetic vectors to deliver genomes of conditionally replicating lytic viruses combines the strengths of viral and non-viral approaches by enabling neutralising antibody resistant deployment of cancer virotherapy. Adenovirus is particularly suitable for this application since all proteins essential for replication can be expressed from the input DNA, although the presence of terminal protein (TP) covalently linked to the 5' termini of the input virus genomes both improves expression of transgenes encoded in the input DNA and also enhances replication. These roles of TP were distinguished in experiments where E1-deleted Ad(GFP)DNA bearing TP (Ad(GFP)DNA-TP), delivered with DOTAP, gave a two-fold greater frequency of transduction than Ad(GFP)DNA(without TP) in non-complementing A549 cells, while in 293 cells (which support replication of E1-deleted viruses) the presence of TP mediated a much greater differential transgene expression, commensurate with its ability to promote replication. Subsequent studies using AdDNA for virotherapy, therefore, included covalently linked TP. AdDNA-TP delivered to A549 cells using a synthetic polyplex vector was shown to be resistant to levels of neutralising antisera that completely ablated infection by wild-type adenovirus, enabling polyplex/Ad(wild type)DNA-TP to mediate a powerful cytopathic effect. Similarly in vivo, direct injection of a polyplex/Ad(wild type)DNA-TP into A549 tumours was neutralising antibody-resistant and enabled virus replication, whereas intact virus was neutralised by the antibody and failed to infect. The delivery of adenovirus genomes-TP using synthetic vectors should provide a strategy to bypass neutralising antibodies and facilitate clinical application of replicating adenovirus for cancer virotherapy.
Subject(s)
Adenoviridae/genetics , Antibodies, Viral/immunology , DNA, Viral/administration & dosage , Genetic Therapy/methods , Neoplasms/therapy , Vaccines, Synthetic/administration & dosage , Adenoviridae/immunology , Animals , Antigen-Antibody Reactions , DNA, Viral/immunology , Genetic Engineering , Mice , Mice, SCID , Neoplasm Transplantation , Neoplasms/immunology , Neoplasms/virology , Transduction, Genetic/methods , Transfection/methodsABSTRACT
Systemic delivery of adenoviral vectors is a major goal in cancer gene therapy, but is currently prohibited by rapid hepatic uptake of virus following intravenous injection with levels of viable virus in the murine plasma typically falling to less than 0.1% after 30 min. We have used a surface-masking technique based on multivalent copolymers of poly(N-(2-hydroxypropyl)methacrylamide) to ablate all pathways of receptor-mediated infection, combined with dose modulation to achieve partial saturation of nonspecific uptake pathways. Polymer coating gave at least 100-fold decreased hepatic transgene expression at all doses and even high doses of coated virus (pc-virus) showed no weight loss or stimulation of serum transaminases. Low doses of virus and pc-virus (10(9) viral particles (vp)/mouse) were mainly captured by the liver (assessed by quantitative PCR), although higher doses led to greater fractional persistence in the plasma (measured after 30 min). Coated virus at a dose of 6 x 10(11) vp/mouse showed nearly 50% plasma circulation, representing a 3.5-fold greater area under the concentration-time curve (0-30 min) compared to unmodified virus. Such an increase in the bioavailability of adenovirus, coupled with substantial decreases in toxicity and unwanted transgene expression is an important step towards producing systemically available tumour-targeted viruses.
Subject(s)
Adenoviridae , Genetic Vectors/genetics , Liver/virology , Plasma/virology , Polymers , Animals , Gene Expression , Gene Targeting/methods , Genetic Therapy/methods , Injections, Intravenous , Mice , Neoplasms/therapy , Phagocytosis/physiology , Receptors, Virus/genetics , Recombination, Genetic/genetics , Transduction, Genetic/methods , Transgenes/genetics , Tumor Cells, CulturedABSTRACT
BACKGROUND: Efficient neuronal gene therapy is a goal for the long-term repair and regeneration of the injured central nervous system (CNS). We investigated whether targeting cDNA to neurons with cholera toxin b chain conjugated non-viral polyplexes led to increased efficiency of non-viral gene transfer in the CNS. Here, we illustrate the potential for this strategy by demonstrating enhanced transfection of a differentiated neuronal cell type, PC12. METHODS: In vitro transfection efficiency of a cholera toxin b chain-poly(D-lysine) molecular conjugate (CTb-K(100)) was compared by fluorescence-activated cell sorting (FACS) analysis of green fluorescent protein (GFP) expression and luminometric measurement of beta-galactosidase (beta-gal) expression, to untargeted poly(D-lysine) (K(100)) in undifferentiated and NGF-differentiated PC12 cells. RESULTS: Transfection of undifferentiated PC12 cells with CTb-K(100) polyplexes resulted in a 36-fold increase in levels of pCMV-DNA(LacZ) expression and a 20-fold increase in the frequency of transduction with pCMV-DNA(GFP), compared with untargeted K(100) polyplexes. Treatment of PC12 cells with 50 ng/ml/day of NGF for 14 days led to differentiation to a neuronal phenotype. Transfection of NGF-differentiated cells with CTb-K(100) polyplexes resulted in a 133-fold increase in levels of pCMV-DNA(LacZ) expression and a 11-fold increase in the percentage of cells transduced with pCMV-DNA(GFP), compared with untargeted K(100) polyplexes. Transfection was dependent on CTb, with CTb-K(100)-mediated transfections competitively inhibited with free CTb in both PC12 phenotypes. CONCLUSIONS: Non-viral systems for gene transfer in damaged CNS show superior toxicological profiles to most viruses but are limited by inefficient and non-selective gene expression in target tissue. Cholera toxin is known to interact preferentially with neuronal cells of the central and peripheral nervous systems, mediating binding through the b subunit, CTb, and the pentasaccharide moiety of the gangliosaccharide, GM1, which is present at high levels on the neuronal cell surface. Here, we show that a molecular conjugate of the CTb subunit, covalently linked to poly(D-lysine), is able to successfully target and significantly enhance transfection of a neuronal cell type, NGF-differentiated rat PC12 pheochromocytoma cells. This observation encourages the further development of non-viral strategies for the delivery of therapeutic genes to neurons.
Subject(s)
Cholera Toxin/genetics , Gene Transfer Techniques , Neurons/physiology , Polylysine/genetics , Animals , Binding, Competitive , Cell Differentiation/drug effects , Cell Differentiation/genetics , Chemistry, Physical/methods , Cholera Toxin/metabolism , DNA, Complementary , G(M1) Ganglioside/metabolism , Gene Expression , Nerve Growth Factor/pharmacology , PC12 Cells , Rats , Transfection/methodsSubject(s)
Adenoviridae/genetics , Gene Transfer Techniques , Genetic Therapy/methods , Animals , Genetic Vectors , HumansABSTRACT
Colorectal cancer is an important public health problem worldwide. Gene therapy has therapeutic potential for patients with advanced or recurrent colorectal cancer, incurable by conventional treatments. To date, many strategies of gene therapy have been explored, including mutant gene correction, prodrug activation, immune stimulation and genetically-modified oncolytic viruses. Although the preclinical results of gene therapy for colorectal cancer have shown promise, gene therapy is still at an early stage of clinical development and has not yet shown a significant therapeutic benefit for patients. The main obstacles for introduction of gene therapy to patients are poor targeting selectivity of the vectors and inefficient gene transfer. As the science supporting tumour-selective vectors evolves, gene therapy may expand rapidly in the clinical practice of colorectal cancer treatment.
Subject(s)
Colorectal Neoplasms/therapy , Genetic Therapy , Animals , Combined Modality Therapy , Humans , Immunotherapy , Prodrugs , Viruses/geneticsABSTRACT
Inefficient nuclear delivery restricts transgene expression using polyelectrolyte DNA vectors. To increase transfer from the cytoplasm to the nucleus, we have covalently linked adenovirus hexon protein to polyethylenimine (PEI, 800 kDa). Activity of the conjugate was compared with PEI and PEI linked to albumin. Hexon-containing complexes gave 10-fold greater transgene expression in HepG2 cells than PEI/DNA or complexes containing albumin, without increasing cell uptake. Following cytoplasmic injection into Xenopus laevis oocytes, hexon-containing complexes showed reporter gene expression to be elevated by 10-fold compared with PEI/DNA. The ability of hexon to promote nuclear delivery of PEI/DNA nanoparticles was compared with that of classical nuclear localization sequences (NLS) by measuring transgene expression following intracytoplasmic microinjection of hexon-PEI/DNA complexes and NLS-albumin-PEI/DNA complexes in rat-1 fibroblasts. The resulting nuclear transfer efficiency was in the following order: hexon-PEI/DNA>NLS-albumin-PEI/DNA>PEI/DNA>DNA alone>albumin-PEI/DNA. The activities of both NLS-albumin-PEI and hexon-PEI were abolished by co-injection of wheat germ agglutinin, suggesting that both act by means of the nuclear pore complex (NPC); in contrast, excess free NLS-albumin abolished transgene expression with NLS-albumin-PEI/DNA, but only partially inhibited hexon-PEI/DNA. Nuclear transfer efficiency following cytoplasmic injection was dependent on DNA concentration for all materials, although hexon conjugates showed much better activity than NLS-albumin at low DNA doses (500-1000 plasmids/cell). Our data are consistent with hexon mediating nuclear delivery of plasmid complexes by means of the NPC, using mechanisms that are only partially dependent on the classical NLS import pathway. The hexon-mediated mechanism of nuclear import enables substantially better transgene expression, particularly when DNA concentrations in the cytoplasm are limiting.
Subject(s)
Capsid Proteins , Capsid/metabolism , Cell Nucleus/metabolism , Gene Expression , Genetic Therapy/methods , Plasmids/genetics , Polyethyleneimine/metabolism , Transgenes/genetics , Active Transport, Cell Nucleus , Animals , Capsid/genetics , Cattle , Fibroblasts , Fluorescein-5-isothiocyanate , Genetic Vectors/genetics , Humans , Microinjections , Microscopy, Electron , Nuclear Localization Signals , Oocytes/cytology , Oocytes/metabolism , Rabbits , Rats , Serum Albumin, Bovine/analysis , Serum Albumin, Bovine/genetics , Serum Albumin, Bovine/metabolism , Transfection , Tumor Cells, Cultured , Xenopus laevisABSTRACT
Entry of exogenously applied DNA into the cytoplasm and subsequent transport into the nucleus are major cellular barriers for nonviral gene delivery vectors. To overcome these barriers, we have covalently attached the cationic peptide melittin to poly(ethylenimine) (PEI). This conjugate condensed DNA into small, discrete particles (<100 nm in diameter), and the membrane lytic activity of melittin enabled efficient release of the DNA into the cytoplasm, as monitored by fluorescence microscopy and flow cytometry. Compared with PEI, the transfection activity was strongly increased within a broad range of cell lines and types tested, including different tumor cell lines but also primary hepatocytes and human umbilical vein endothelial cells. The early onset of gene expression (within 4 h, reaching maximal values after 12 h) and the high reporter gene expression achieved in slowly dividing or confluent cells suggested a further role of melittin after releasing the DNA into the cytoplasm. Intracytoplasmic microinjection of melittin-containing PEI.DNA complexes into fibroblasts produced 40% cellular frequency of reporter gene expression that was inhibitable by co-injection of wheat germ agglutinin, whereas simple PEI.DNA complexes showed only 10%. These data suggest that melittin enables release of nonviral gene transfer particles into the cytoplasm and also enhances their transport into the nucleus, possibly via the cationic cluster KRKR near the C terminus of the peptide.
Subject(s)
Gene Transfer Techniques , Genetic Vectors , Melitten/pharmacology , Active Transport, Cell Nucleus , Animals , Bromodeoxyuridine/metabolism , Cell Line , Cell Nucleus/metabolism , Cells, Cultured , Cytoplasm/metabolism , Endosomes/metabolism , Endothelium, Vascular/cytology , Erythrocytes/metabolism , Fibroblasts/metabolism , Flow Cytometry , Genes, Reporter , HeLa Cells , Humans , Luciferases/metabolism , Mice , Microscopy, Electron , Microscopy, Fluorescence , Mitosis , Photons , Plasmids/metabolism , Polyethyleneimine/chemistry , Polyethyleneimine/pharmacology , Protein Structure, Tertiary , Rats , Spectrophotometry , Time Factors , Transfection , Tumor Cells, CulturedABSTRACT
Synthetic vectors were evaluated for their ability to mediate efficient mRNA transfection. Initial results indicated that lipoplexes, but not polyplexes based on polyethylenimine (PEI, 25 and 22 kDa), poly(L-lysine) (PLL, 54 kDa) or dendrimers, mediated efficient translation of mRNA in B16-F10 cells. Significant mRNA transfection was achieved by lipoplex delivery in quiescent (passage 0) human umbilical vein endothelial cells (HUVEC), and by passage 4, 10.7% of HUVEC were transfected compared to 0.84% with DNA. Lack of expression with PEI 25 kDa/mRNA or PLL 54 kDa/mRNA in a cell-free translation assay and following cytoplasmic injection into Rat1 cells indicated that these polyplexes were too stable to release mRNA. In contrast, polyplexes formed using smaller PEI 2 kDa and PLL 3.4 kDa gave 5-fold greater expression in B16-F10 cells compared to DOTAP, but were dependent on chloroquine for transfection activity. Endosomolytic activity was incorporated by conjugating PEI 2 kDa to melittin and resulting PEI 2 kDa-melittin/mRNA polyplexes mediated high transfection levels in HeLa cells (31.1 +/- 4.1%) and HUVEC (58.5 +/- 2.9%) in the absence of chloroquine, that was potentiated to 52.2 +/- 2.7 and 71.6 +/- 1.7%, respectively, in the presence of chloroquine. These results demonstrate that mRNA polyplexes based on peptide-modified low molecular weight polycations can possess versatile properties including endosomolysis that should enable efficient non-viral mRNA transfection of quiescent and post-mitotic cells.
Subject(s)
Oligopeptides/physiology , RNA/metabolism , Transfection/methods , Amino Acid Sequence , Animals , Cell Line , Cell-Free System/metabolism , Gene Expression , Green Fluorescent Proteins , HeLa Cells , Humans , Luciferases/genetics , Luciferases/metabolism , Luminescent Proteins/genetics , Melitten/chemistry , Melitten/genetics , Microinjections , Mitosis , Molecular Sequence Data , Oligopeptides/genetics , Protein Biosynthesis , RNA/genetics , RNA, Messenger/administration & dosage , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rabbits , Reticulocytes/chemistry , Reticulocytes/metabolism , Time Factors , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
We have developed polyelectrolyte gene delivery vectors that display good extracellular stability and are activated intracellularly to permit transgene expression. The strategy comprises covalent crosslinking of primary amines in poly-L-lysine/DNA complexes with a crosslinking agent that can later be cleaved by reduction. Crosslinked complexes maintained the same size and surface charge but showed increased stability against polyelectrolyte exchange with poly-L-aspartic acid. Surface modification with polyethyleneglycol improved solubility and masked their positive surface charge. Crosslinked complexes showed 10-fold increased plasma circulation following intravenous administration to Balb/c mice. In the absence of chloroquine, the levels of transgene expression in B16F10 murine melanoma cells were similar for crosslinked and non-crosslinked complexes, however, chloroquine selectively potentiated transgene expression by the non-crosslinked complexes. Cellular uptake of the complexes was the same, irrespective of crosslinking. Following microinjection into the cytoplasm of Xenopus oocytes, or the cytoplasm or nucleus of Rat-1 fibroblasts, crosslinked complexes mediated the same transgene expression as non-crosslinked complexes, indicating crosslinked complexes are rapidly reduced and activated intracellularly. We therefore hypothesize that the lower in vitro transfection activity of crosslinked complexes in the presence of chloroquine is due to reduced transfer from endosome to cytoplasm, mainly due to increased stability against destabilization by chloroquine. The extended systemic circulation together with triggered intracellular activation makes these complexes a promising system for targeted gene delivery in vivo.
Subject(s)
Disulfides/pharmacokinetics , Gene Transfer Techniques , Genetic Vectors/pharmacokinetics , Animals , Cross-Linking Reagents , DNA/pharmacokinetics , Electrolytes , Female , Liver/metabolism , Mice , Mice, Inbred BALB C , Microinjections , Polyethylene Glycols/pharmacology , Polylysine/pharmacokinetics , Sodium Chloride/pharmacology , Solubility , Tissue Distribution , Transcription, Genetic , XenopusABSTRACT
Conjugation of folate to proteins permits receptor-mediated endocytosis via the folate receptor (FR) and delivery of the conjugate into the cytoplasm of cells. Since many cancers up-regulate the FR it has enabled the targeting of toxins to tumor cells resulting in specific cell death. However, current conjugation methods rely on chemistries that can affect certain catalytic subunits, such as the A-chain of the plant toxin gelonin. As a result many folate-targeted toxins are a compromise between receptor/ligand interaction and toxin activity. We describe the first example of folate conjugated to a protein via carbohydrate residues, using a novel SH-folate intermediate. The folate-gelonin conjugate retains over 99% of toxin activity in a cell-free translational assay compared with unmodified gelonin and is able to bind the FR at the same affinity as free folic acid (10(-10) m). Additionally, the conjugate exhibits prolonged inhibition of protein synthesis in FR positive cell lines in vitro. Folate linked to gelonin via amino conjugation exhibits the same affinity for FR as free folic acid but the toxin is 225-fold less active in a cell-free translational assay. The effect of different conjugation methods on toxin activity and the implications for folate targeting of other glycoproteins are discussed.
Subject(s)
Carrier Proteins/chemistry , Folic Acid/chemistry , Folic Acid/metabolism , Plant Proteins/chemistry , Protein Synthesis Inhibitors/chemistry , Receptors, Cell Surface , Ribosomes/metabolism , Binding, Competitive , Carrier Proteins/metabolism , Cell Line , Cell-Free System , Dose-Response Relationship, Drug , Folate Receptors, GPI-Anchored , HeLa Cells , Humans , Inhibitory Concentration 50 , Ligands , Models, Chemical , Protein Binding , Protein Biosynthesis , Ribosome Inactivating Proteins, Type 1 , Sulfides/chemistry , Time FactorsABSTRACT
Inefficient transport of DNA from the cytoplasm into the nucleus remains a limiting step in the development of non-viral gene delivery systems. This is particularly acute in non-dividing cells, where entry to the nucleus is thought to occur only through the nuclear pore complex. Active import of physiological proteins is mediated by nuclear localization sequences (NLSs) within cargo proteins such as transcription factors. Here we review current knowledge of this import machinery and consider its exploitation by mammalian viruses. Significant research effort has been directed at incorporating NLSs into synthetic gene delivery systems to take advantage of this physiological pathway. Both non-covalent and covalent methods of conjugation are evaluated, with NLS linkage to both DNA and carrier, and compared with activities of simple cationic polymers. Finally, progress in the field of DNA sequence-specific nuclear import is examined and the current state of the technology assessed.
Subject(s)
Active Transport, Cell Nucleus , Cell Nucleus/metabolism , Gene Transfer Techniques , Transgenes , Adenoviridae/physiology , DNA/genetics , DNA/metabolism , Dependovirus/physiology , Genetic Vectors , Herpesviridae/physiology , Humans , Nuclear Localization Signals/metabolismABSTRACT
Effective gene therapy for diseases of the circulation requires vectors capable of systemic delivery. The molecular weight of poly(L-lysine) (pLL) has a significant effect on the circulation of pLL/DNA complexes in mice, with pLL(211)/DNA complexes displaying up to 20 times greater levels in the blood after 30 minutes compared with pLL(20)/DNA. It is shown that pLL(20)/DNA complexes fix mouse complement C3 in vitro, independent of immunoglobulin binding; are less soluble in the blood in vivo; bind erythrocytes; are rapidly removed by the liver, where they associate predominantly with Kupffer cells; and result in a rapid increase in hepatic leukocytes expressing high levels of complement receptor 3 (CR3). The circulation properties of these complexes are also dependent on the type of DNA used, with circular plasmid DNA complexes exhibiting increased circulation compared with linear DNA. PLL(211)/DNA complexes bind erythrocytes and associate with Kupffer cells but, in contrast, do not fix mouse complement in vitro and are unaffected by the type of DNA used. In rats, both types of complexes produce hematuria and are rapidly removed from the circulation. Correlation of in vivo and in vitro results suggests that the solubility of complexes in physiological saline and species-matched complement fixation and erythrocyte lysis may correlate with systemic circulation. Analysis using human blood in vitro shows no hemolysis, but both types of complexes fix complement and bind IgG, suggesting that pLL/DNA complexes may be rapidly cleared from the human circulation.