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Biomedical subjects

Martin L Read

Publications and source records attributed to Martin L Read.

10 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↗
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

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

12th annual congress of the European Society of Gene Therapy.

The 2004 European Society of Gene Therapy (ESGT) meeting took place at Tampere Hall in Finland and highlighted advances in a variety of topics, including cancer, zinc-fingers, stem cells, small interfering RNA (siRNA), microRNA, and recent developments of non-viral and viral vectors. This meeting was attended by 513 participants from 32 countries, and included 106 oral and 224 poster presentations. One of the aims of this meeting was to take a critical look at gene therapy and the prospects for the future. Se-veral presentations reported on RNA-based technologies, such as siRNA, as potential new classes of therapeutics against a wide range of diseases and for use in expression libraries to identify functional genes involved in biological phenotypes. Critical assessments were made of other aspects of gene therapy, such as genome editing and the use of protein transduction domains (PTDs) in gene- and protein-based therapies, where many researchers have failed to reproduce initial findings reported in the literature. Safety issues related to viral vectors were also important areas of discussion, especially following details released by the UK Gene Therapy Advisory Committee of perhaps the first known case of lentiviral vector-associated oncogenesis. Finally, updates were presented on the clinical development of viral vectors in anticancer therapies with evidence of significant improvements in the mean survival of patients.

Clinical Trials as Topic↗
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↗
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

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↗
PubMedDate not supplied

Factors influencing the ability of nuclear localization sequence peptides to enhance nonviral gene delivery.

Nonviral gene delivery is limited by inefficient transfer of DNA from the cytoplasm to the nucleus. Nuclear localization sequence (NLS) peptides have been widely used to exploit intracellular transport mechanisms and promote nuclear uptake of DNA. However, the exact conditions to successfully utilize the properties of NLS peptides are still unclear. In the present study a panel of NLS peptides that bind different transport receptors were compared for their ability to enhance nonviral gene transfer. Several factors such as method of incorporating the NLS peptide, type of NLS peptide, DNA morphology, and proper characterization of NLS peptide/DNA conjugates were identified as important considerations in utilizing NLS peptides to enhance gene transfer. In particular, it was shown that a peptide derived from human T cell leukaemia virus type 1 (HTLV) was able to effectively condense DNA into discrete particles and mediate levels of transgene expression up to 32-fold greater than polylysine-based polyplexes. This is the first study to demonstrate efficient transfection mediated by an importin beta-binding peptide based on the HTLV sequence. Promising results were also achieved with a 7-fold increase in gene expression using a NLS peptide/DNA conjugate formed by site-specific linkage of an extended SV40 peptide via a peptide nucleic acid (PNA) clamp. Altogether, the results from this study should help to define the requirements for successful NLS-enhanced transfection.

Amino Acid Sequence↗