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Leonard W Seymour

Publications and source records attributed to Leonard W Seymour.

At least 19 recordsLinked to original sources
PubMed2006

Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons.

Non-viral vectors are promising vehicles for gene therapy but delivery of plasmid DNA to post-mitotic cells is challenging as nuclear entry is particularly inefficient. We have developed and evaluated a hybrid mRNA/DNA system designed to bypass the nuclear barrier to transfection and facilitate cytoplasmic gene expression. This system, based on co-delivery of mRNA(A64) encoding for T7 RNA polymerase (T7 RNAP) with a T7-driven plasmid, produced between 10- and 2200-fold higher gene expression in primary dorsal root ganglion neuronal (DRGN) cultures isolated from Sprague-Dawley rats compared to a cytomegalovirus (CMV)-driven plasmid, and 30-fold greater expression than the enhanced T7-based autogene plasmid pR011. Cell-free assays and in vitro transfections highlighted the versatility of this system with small quantities of T7 RNAP mRNA required to mediate expression at levels that were significantly greater than with the T7-driven plasmid alone or supplemented with T7 RNAP protein. We have also characterized a number of parameters, such as mRNA structure, intracellular stability and persistence of each nucleic acid component that represent important factors in determining the transfection efficiency of this hybrid expression system. The results from this study demonstrate that co-delivery of mRNA is a promising strategy to yield increased expression with plasmid DNA, and represents an important step towards improving the capability of non-viral vectors to mediate efficient gene transfer in cell types, such as in DRGN, where the nuclear membrane is a significant barrier to transfection.

Animals↗
PubMed2006

The future of gene therapy in the UK.

Gene therapy encompasses a spectrum of therapeutic strategies, ranging from the compelling concept of using wild type copies of genes to correct the root cause of recessive genetic disorders through to using genes to mediate powerful and selective toxicity to cancer cells. Inspirational for the general public as well as the bioscience community, gene therapy has been grabbing the headlines--for good and bad reasons--regularly for the past 15 years. In this personal appraisal, Professor Len Seymour assesses the progress of gene therapy in the UK and what it might deliver in the foreseeable future.

Biotechnology↗
PubMed2006

Adenovirus type 5 interactions with human blood cells may compromise systemic delivery.

Intravenous delivery of adenovirus vectors requires that the virus is not inactivated in the bloodstream. Serum neutralizing activity is well documented, but we show here that type 5 adenovirus also interacts with human blood cells. Over 90% of a typical virus dose binds to human (but not murine) erythrocytes ex vivo, and samples from a patient administered adenovirus in a clinical trial showed that over 98% of viral DNA in the blood was cell associated. In contrast, nearly all viral genomes in the murine bloodstream are free in the plasma. Adenovirus bound to human blood cells fails to infect A549 lung carcinoma cells, although dilution to below 1.7 x 10(7) blood cells/ml relieves this inhibition. Addition of blood cells can prevent infection by adenovirus that has been prebound to A549 cells. Adenovirus also associates with human neutrophils and monocytes ex vivo, particularly in the presence of autologous plasma, giving dose-dependent transgene expression in CD14-positive monocytes. Finally, although plasma with a high neutralizing titer (defined on A549 cells) inhibits monocyte infection, weakly neutralizing plasma can actually enhance monocyte transduction. This may increase antigen presentation following intravenous injection, while blood cell binding may both decrease access of the virus to extravascular targets and inhibit infection of cells to which the virus does gain access.

Adenoviruses, Human↗
PubMed2006

Identification of oligopeptides binding to peritoneal tumors of gastric cancer.

This is a report of in vivo intraperitoneal biopanning, and we successfully identified a novel peptide to target the multiple peritoneal tumors of gastric cancer. A phage display library was injected directly into the abdominal cavity of mice bearing peritoneal tumors of human gastric cancer, and phages associated with the tumors were subsequently reclaimed from isolated samples. The tumor-associated phages were amplified and the biopanning cycle was repeated five times to enrich for high affinity tumor-selective binding peptides. Finally, a tri-peptide motif, KLP, which showed homology with laminin 5 (a ligand for alpha3beta1 integrin), was identified as a binding peptide for peritoneal tumors of gastric cancer. Phage clones displaying the sequence KLP showed 64-fold higher binding to peritoneal tumors than control phage and were preferentially distributed in tumors rather than in normal organs after intraperitoneal injection into mice. In addition, the KLP phages were more likely to bind to cancer cells in malignant ascites derived from a patient with recurrent gastric cancer. Synthesized peptide containing the motif KLP (SWKLPPS) also showed a strong binding activity to peritoneal tumors without cancer growth effect. Liposomes conjugated with SWKLPPS peptide appeared significantly more often in tumors than control liposomes after intraperitoneal injection into mice. Furthermore, modification of liposomes with SWKLPPS peptide enhanced the antitumor activity of adriamycin on gastric cancer cells. The peptide motif KLP seems a potential targeting ligand for the treatment of peritoneal metastasis of gastric cancer.

Amino Acid Motifs↗
PubMed2005

Ultrasound-enhanced transfection activity of HPMA-stabilized DNA polyplexes with prolonged plasma circulation.

Cancer gene therapy would greatly benefit from the possibility to deliver therapeutic genes via tumor-targeted systemic intravenous delivery. The main objective of this study was to determine biophysical, transfection, and pharmacokinetic properties of DNA complexes with reducible polycations that are reversibly stabilized by surface coating with multivalent HPMA copolymers. The specific goals were to evaluate compatibility of these polyplexes with extended plasma circulation, molecular targeting, and ultrasound-enhanced transfection activity. It was demonstrated that using polyplexes based on reducible polycations allows increasing transfection activity and preserving extended plasma circulation half-life observed for control polyplexes based on non-reducible polycations. In addition, the reversibly stabilized polyplexes were compatible with both molecular targeting using protein ligands as well as physical targeting using ultrasound-directed cavitation in vitro. As such, the described gene delivery vectors have the potential to permit efficient systemic delivery of therapeutic genes targeted by a local focused ultrasound treatment.

Animals↗
PubMed2005

A versatile reducible polycation-based system for efficient delivery of a broad range of nucleic acids.

Synthetic vectors based on reducible polycations consisting of histidine and polylysine residues (HIS RPCs) were evaluated for their ability to deliver nucleic acids. Initial experiments showed that RPC-based vectors with at least 70% histidine content mediated efficient levels of gene transfer without requirement for the endosomolytic agent chloroquine. Significant gene transfer was observed in a range of cell types achieving up to a 5-fold increase in the percentage of transfected cells compared to 25 kDa PEI, a gold standard synthetic vector. In contrast to 25 kDa PEI, HIS RPCs also mediated efficient transfer of other nucleic acids, including mRNA encoding green fluorescent protein in PC-3 cells and siRNA directed against the neurotrophin receptor p75(NTR) in post-mitotic cultures of rat dorsal root ganglion cell neurons. Experiments to elevate intracellular glutathione and linear profiling of cell images captured by multiphoton fluorescent microscopy highlighted that parameters such as the molecular weight and rate of cleavage of HIS RPCs were important factors in determining transfection activity. Altogether, these results demonstrate that HIS RPCs represent a novel and versatile type of vector that can be used for efficient cytoplasmic delivery of a broad range of nucleic acids. This should enable different or a combination of therapeutic strategies to be evaluated using a single type of polycation-based vector.

Animals↗
PubMed2005

Synthesis and in vitro evaluation of macromolecular antitumour derivatives based on phenylenediamine mustard.

Poly-[N-(2-hydroxyethyl)-L-glutamine] (PHEG) and poly(ethylene glycol) (PEG)-grafted PHEG conjugates of N,N-di(2-chloroethyl)-4-phenylenediamine mustard (PDM) were synthetised. A collagenase-sensitive oligopeptide spacer was selected to link the cytotoxic agent PDM onto the polymeric carrier. First, the oligopeptide-drug conjugate, L-pro-L-leu-gly-L-pro-gly-PDM, was prepared. In a second step, the low molecular weight PDM derivative and PEG-NH(2) were coupled to a N,N-disuccinimidylcarbonate activated PHEG. Dynamic laser light scattering measurements indicated the formation of aggregates. The presence of human serum albumin had no significant effect on the diameter of the conjugates. The hydrolytic stability of the conjugates was investigated in buffer solutions. The conjugates showed an improved stability compared to the parent nitrogen mustard. The enzymatic degradation studies of the polymeric conjugates were performed in the presence of collagenase type IV (Clostridiopeptidase A; EC 3.4.24.3), cathepsin B (EC 3.4.22.1), cathepsin D (EC 3.4.23.5) and tritosomes. Only the bacterial collagenase type IV was able to cleave the spacer releasing free PDM and its peptidyl derivative, gly-L-pro-gly-PDM. The in vitro cytotoxicity of the conjugates was evaluated against HT1080 fibrosarcoma cells and MDA adenocarcinoma cells. All conjugates showed low toxicity towards these cell lines.

Animals↗
PubMed2005

Barriers to Gene Delivery Using Synthetic Vectors.

Progress has been made in the development of different types of nucleic acids such as DNA and siRNA with the potential to form the basis of new treatments for genetic and acquired disorders. The lack of suitable vectors for the delivery of nucleic acids, however, represents a major hurdle to their continued development and therapeutic application. Synthetic vectors based on polycations are promising vectors for gene delivery as they are relatively safe and can be modified by the incorporation of ligands for targeting to specific cell types. However, the levels of gene expression mediated by synthetic vectors are low compared to viral vectors. The aim of this chapter is to give an overview of the main barriers that have been identified as limiting gene transfer using polycation-based synthetic vectors. The chapter is divided into two sections to focus on both extracellular and intracellular barriers. We describe novel strategies that are being used to develop increasingly sophisticated vectors in an attempt to overcome these barriers. For instance, we describe approaches to prolong the plasma circulation of polyplexes by the incorporation onto their surface of hydrophilic polymers such as polyethylene glycol (PEG) and poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). In addition, strategies to improve transfer of nucleic acids from the outside of the cell to the nucleus are described to overcome barriers such as escape from endocytic vesicles and translocation across the nuclear membrane. Furthermore, we highlight new types of vectors based on reducible polycations that are triggered by the intracellular environment to facilitate efficient cytoplasmic release of nucleic acids.

Journal Article↗
PubMed2005

Enhanced gene transfer activity of peptide-targeted gene-delivery vectors.

We have evaluated the capacity of the cell-binding heptapeptide SIGYPLP to enhance transgene expression using non-viral and viral gene delivery vectors. Targeted polyplex based vectors showed good levels of DNA uptake in freshly isolated human umbilical vein endothelial cells (HUVECs) compared to untargeted controls, whilst displaying only modest increases in reporter gene activity. The targeted polyplexes showed reduced levels of DNA uptake in cells of a none endothelial origin although they mediated higher levels of transgene expression. The enhanced efficiency of transgene expression may relate to the more rapid rate of cell division. However, since in vivo application of polyplexes is compromised by instability to serum proteins, serum-resistant polyplexes (surface modified with multivalent reactive hydrophilic polymers based on poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA)) were also evaluated for their ability to mediate transgene expression. Surface modification of polyplexes with pHPMA ablates non-specific cell entry, reducing levels of transgene expression, whilst the incorporation of the SIGYPLP peptide into the hydrophilic polymer resulted in restored transgene expression in all formulations tested. The technology of surface modification using pHPMA can also be applied in the context of viruses, masking receptor-binding epitopes and enabling the linkage of novel cell targeting ligands, enabling construction of a virus with receptor-specific infectivity. Retargeting of adenovirus based vectors using the same polymer-peptide construct enhanced levels of transgene expression in HUVECs to greater than 15 times that observed using parental (unmodified) virus, whilst restoring levels of transgene expression in non-endothelial cell lines tested. The use of constructs based on conjugates between hydrophilic polymers and small receptor-binding oligopeptides as agents for retargeting viral or non-viral vectors to cellular receptors represents a simple alternative to the use of antibodies as targeting ligands for cell specific gene delivery.

Cell Line↗
PubMed2005

Barriers to gene delivery using synthetic vectors.

Progress has been made in the development of different types of nucleic acids such as DNA and siRNA with the potential to form the basis of new treatments for genetic and acquired disorders. The lack of suitable vectors for the delivery of nucleic acids, however, represents a major hurdle to their continued development and therapeutic application. Synthetic vectors based on polycations are promising vectors for gene delivery as they are relatively safe and can be modified by the incorporation of ligands for targeting to specific cell types. However, the levels of gene expression mediated by synthetic vectors are low compared to viral vectors. The aim of this chapter is to give an overview of the main barriers that have been identified as limiting gene transfer using polycation-based synthetic vectors. The chapter is divided into two sections to focus on both extracellular and intracellular barriers. We describe novel strategies that are being used to develop increasingly sophisticated vectors in an attempt to overcome these barriers. For instance, we describe approaches to prolong the plasma circulation of polyplexes by the incorporation onto their surface of hydrophilic polymers such as polyethylene glycol (PEG) and poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). In addition, strategies to improve transfer of nucleic acids from the outside of the cell to the nucleus are described to overcome barriers such as escape from endocytic vesicles and translocation across the nuclear membrane. Furthermore, we highlight new types of vectors based on reducible polycations that are triggered by the intracellular environment to facilitate efficient cytoplasmic release of nucleic acids.

Active Transport, Cell Nucleus↗
PubMed2004

Formulation of a microparticle carrier for oral polyplex-based DNA vaccines.

Oral induction of a disseminated mucosal immune response with polyplex-based DNA vaccines requires the delivery of intact polyplexes (polyelectrolyte complexes formed by self-assembly of plasmid DNA with a cationic polymer) to subepithelial lymphoid tissue (e.g. Peyer's patches) within the gastrointestinal tract. This work describes the formulation of a microparticle polyplex carrier allowing the potential of this approach to be realised. PEGylated PEI/DNA polyplexes (DNA concentration 20 microg/ml) formed at N/P 5:0 (defined as the ratio of polycation amino groups to DNA phosphates) were stable to salt-induced aggregation and could be concentrated to a final DNA concentration of 1 mg/ml without polyplex size increase. Polyplexes containing 1:1 polyethylene glycol (PEG)/polyethylenimine (PEI) ratio (mass/mass) gave similar levels of luciferase gene expression in B16F10 cells compared to non-PEG complexes. Poly-(D,L-lactide-co-glycolide) (PLGA) microparticles containing PEGylated polyplexes (approximately 17% DNA encapsulation efficiency) were formulated using a modified double emulsion solvent evaporation method. The microencapsulation and release of intact polyplexes from the microparticle carrier was demonstrated using polyanion (heparin sulfate and poly(aspartic acid) (PAA)) displacement techniques and electron microscopy. Microparticles containing PEGylated polyplexes (24 microg beta-galactosidase DNA) were given orally to Wistar rats. Significant transgene expression (compared to background) was found in peripheral tissue (spleen) 72 h after administration. This work demonstrates the potential application of microparticle carriers for mucosal polyplex-based vaccination.

Administration, Oral↗
PubMed2004

Polymer-coated polyethylenimine/DNA complexes designed for triggered activation by intracellular reduction.

BACKGROUND: Site-specific gene delivery requires vectors that combine stability in the delivery phase with substantial biological activity within target cells. The use of biological trigger mechanisms provides one promising means to achieve this, and here we report a transfection trigger mechanism based on intracellular reduction. METHODS: Plasmid DNA was condensed with thiolated polyethylenimine (PEI-SH) and the resulting nanoparticles surface-coated using thiol-reactive poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) with 2-pyridyldisulfanyl or maleimide groups, forming reducible disulphide-linked or stable thioether-linked coatings, respectively. RESULTS: Both sets of polymer-coated complexes had similar size and were stable to a 250-fold excess of the polyanion poly(aspartic acid) (PAA). Reduction with dithiothreitol (DTT) allowed complete release of DNA from disulphide-linked coated complexes, whereas complexes with thioether-linked coating remained stable. Disulphide-linked complexes showed 40-100-fold higher transfection activity than thioether-linked ones, and activity was selectively further enhanced by boosting intracellular glutathione using glutathione monoethyl ester or decreased using buthionine sulfoximine. The chloroquine- and serum-independent transfection activity of disulphide-linked coated complexes suggests this system may provide a viable trigger mechanism to enable site-specific transfection in complex biological settings. CONCLUSIONS: Linkage of hydrophilic polymer coating to PEI/DNA complexes via reducible disulphide bonds offers a means of fulfilling the contradictory requirements for extracellular stability and intracellular activity.

DNA↗
PubMed2003

Trial and error.

Explore the source record for details and available documents.

Adenoviridae↗
PubMed2003

Vectors based on reducible polycations facilitate intracellular release of nucleic acids.

BACKGROUND: Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L-lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids. METHODS: RPCs of molecular weight (mwt) 45 and 187 kDa were prepared by oxidative polycondensation of the peptide Cys-Lys(10)-Cys and used to condense nucleic acids. The stability of RPC-based polyplexes to reduction was determined using electrophoresis, dynamic light scattering and fluorescence techniques. Transfection activity was studied in several cancer cell lines (HeLa, LNCaP, PC-3 and B16-F10) using luciferase and green fluorescent protein (GFP) genes as reporter genes in the presence of chloroquine or the cationic lipid (N-(1-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride) (DOTAP). A CMV-driven plasmid expressing the nitroreductase (ntr) gene was used to evaluate the therapeutic efficacy of RPC-based delivery vectors. RESULTS: A 187-fold higher level of gene expression indicated that intracellular delivery of DNA was more efficient using RPC/DOTAP compared with vectors based on non-reducible PLL. Analysis by flow cytometry also showed enhanced delivery of the GFP gene by RPC/DOTAP in HeLa (51.5 +/- 7.9%), LNCaP (55.2 +/- 6.7%) and PC-3 (66.1 +/- 3.7%) cells. Transfection with the ntr gene and treatment with the prodrug CB1954 resulted in significant cell killing, achieving IC(50) values similar to those previously attained with adenoviral vectors. Delivery of mRNA (20-75% of cells) was also more efficient using RPC/DOTAP than PLL/DOTAP (<5% of cells). CONCLUSIONS: These results demonstrate that lipid-mediated activation of RPC-based polyplexes is a useful strategy to enhance intracellular delivery of nucleic acids and potentiate therapeutic activity.

Gene Transfer Techniques↗
PubMed2003

Use of a phage display library to identify oligopeptides binding to the lumenal surface of polarized endothelium by ex vivo perfusion of human umbilical veins.

Human endothelial-specific targeting peptides were identified by biopanning within freshly-obtained human umbilical cords. Umbilical veins were cleaned in situ and M13 phage display libraries were passed through the cords. Tightly bound phage were recovered following isolation of endothelial cells by collagenase digestion and homogenisation, allowing production of enriched phage libraries for subsequent rounds of panning. After five rounds of biopanning, five promising sequences were selected and the binding of the corresponding phage clones was compared in perfused umbilical veins. Each of these peptides showed substantial binding, although the clone encoding the heptapeptide KPSGLTY showed the greatest, some 89-times greater than insertless phage. Binding of this phage clone was examined to cells in vitro, where it demonstrated at least five-times greater binding to isolated human umbilical vein endothelial cells than to 911, SKOV3, B16F10 and Cos7 cells. These initial peptides may prove useful targeting agents for endothelial-selective delivery, and this powerful approach should be readily applicable to biopanning in a broad range of human vessels ex vivo.

Animals↗
PubMed2003

Emerging developments in RNA therapeutics.

Exciting developments have recently emerged in the field of RNA therapeutics, with potential applications in the treatment of human diseases. The second International Conference on RNA in drug development was held to highlight several novel RNA-based technologies, including different approaches to silence gene expression, the broad range of diagnostic and therapeutic applications for aptamers, and the targeting of RNA with small molecules. Highlights of the meeting included the utilisation of RNA interference to silence genes, with applications for the treatment of both cancer and viral infections, and for systemic silencing of gene expression. Novel approaches to safer drug design using aptamers were presented, which would enable control of their therapeutic activity to be achieved with antidote oligonucleotides. Updates were also presented on the clinical and preclinical development of ribozymes and aptamers, including good progress in increasing the half-life of these molecules in serum.

Animals↗
PubMed2002

Physical properties and in vitro transfection efficiency of gene delivery vectors based on complexes of DNA with synthetic polycations.

Biophysical properties of polycation/DNA complexes designed for gene delivery were studied with respect to the conditions of their preparation, chemical structure and molecular weight of the polycations involved. The polycations used included a variety of cationic polymers and copolymers containing primary and tertiary amino or quaternary ammonium groups. It was found that the molecular weight and the size of these polyelectrolyte complexes (PECs) increase with increasing temperature and pH of the buffer. By decreasing the molecular weight of polycations used for PEC formation, the complexes become unstable towards coagulation in aqueous solution at lower pH. The self-assembly of DNA with low-molecular-weight polycations in water provides PECs with the lowest molecular weight, smallest size and the lowest density but their stability in NaCl solutions is very poor. Despite the complexity of the multistep transfection process, a direct correlation between the transfection efficiency in vitro and the stability of the complexes in NaCl solutions and coagulation in 0.15 M NaCl solution was found. DNA complexes with polycations containing primary amino groups showed the best stability in saline solutions and also the best transfection activity. PECs formed by polycations with quaternary ammonium groups were the least resistant to destruction by the added salt and provided the lowest activity in transfection assays. The highest transfection activity was found for DNA complexes formed with a statistical copolymer containing primary and tertiary amines.

Animals↗