Designing gene delivery vectors for cardiovascular gene therapy

Genetic therapy in the cardiovascular system has been proposed for a variety of diseases ranging from prevention of vein graft failure to hypertension. Such diversity in pathogenesis requires the delivery of therapeutic genes to diverse cell types in vivo for varying lengths of time if efficient clinical therapies are to be developed. Data from extensive preclinical studies have been compiled and a certain areas have seen translation into large-scale clinical trials, with some encouraging reports. It is clear that progress within a number of disease areas is limited by a lack of suitable gene delivery vector systems through which to deliver therapeutic genes to the target site in an efficient, non-toxic manner. In general, currently available systems, including non-viral systems and viral vectors such as adenovirus (Ad) or adeno-associated virus (AAV), have a propensity to transduce non-vascular tissue with greater ease than vascular cells thereby limiting their application in cardiovascular disease. This problem has led to the development and testing of improved vector systems for cardiovascular gene delivery. Traditional viral and non-viral systems are being engineered to increase their efficiency of vascular cell transduction and diminish their affinity for other cell types through manipulation of vector:cell binding and the use of cell-selective promoters. It is envisaged that future use of such technology will substantially increase the efficacy of cardiovascular gene therapy.     

Intracellular signal-responsive gene carrier for cell-specific gene expression

We designed a peptide-polymer conjugate (CPCCtat) as a novel gene carrier that could control gene expression responding to the intracellular caspase-3 signal. This carrier consists of an uncharged main polymer chain and a cationic peptide side chain, which includes the substrate sequence of caspase-3 and the protein transduction domain sequence of HIV-1 Tat. In the present study, CPCCtat formed a tight complex with DNA through an electrostatic interaction, and in this state the gene expression was totally suppressed. In contrast, the complex disintegrated in the presence of caspase-3 due to cleavage of the cationic portion from CPCCtat. This event led to an activation of gene expression. Our results also indicate that the complex can be delivered into living cells due to the cell-permeable peptide side chain of CPCCtat. This intracellular signal-responsive system with CPCCtat will be useful for the cell-specific gene expression system.     

Site-specific gene targeting for gene expression in eukaryotes

Major advances in the use of site-specific recombinases to facilitate sustained gene expression via chromosomal targeting have been made during the past year. New tools for genomic manipulations using this technology include the discovery of epitopes in recombinases that confer nuclear localization, crystal structures that show the precise topology of recombinase-DNA-substrate synaptic complexes, manipulations of the DNA recognition sequences that select for integration over excision of DNA, and manipulations that make changes in gene expression inducible by drug administration. In addition, endogenous eukaryotic and mammalian DNA sequences have been discovered that can support site-specific recombinase-mediated manipulations.     

Multifunctional nanocomplexes for gene transfer and gene therapy

DNA formulated into aggregates with polycationic reagents are referred to by a variety of terms including non-viral vectors, synthetic vectors, lipoplexes, polyplexes and more recently nanoparticles. The capacity for delivery of multiple genes, genomic-sized constructs and siRNA delivery, with a diversity of possible formulations, as well as the possibilities of improved efficiency of in vivo gene deliveries, means that nanoparticles, or nanocomplexes to reflect self-assembling systems, will be investigated with increasing vigour in the coming years. This review briefly outlines the applications and challenges for nanoparticle technologies in the field of gene therapy then focuses on the development of a specific kind of formulation, receptor-targeted nanocomplex (RTN), that we have found to be particularly useful in our gene therapy research. An overriding guiding concept that has emerged in the development of synthetic nanodelivery systems is the idea to develop formulations and structures that mimic viruses, whilst retaining the safety elements of synthetic, non-viral systems. RTNs have been optimised and developed for airway epithelial transfection, leading towards gene therapy for cystic fibrosis and for vascular transfection in vein grafts used in bypass surgery. The modular design of the RTN platform further allows for the testing of specific hypotheses relating to the structure and functional role of components in the formation of stable particles and in the transfection pathway, leading to their ultimate disassembly in the nucleus.     

Targeted gene modification for gene therapy of stem cells

Ideally, gene therapy would correct the specific gene defect without adding potentially harmful extraneous DNA sequences. Such correction can be obtained with homologous recombination between input DNA sequences and identical (homologous) sequences in the genomic target gene. The development of techniques for obtaining virtually pure populations of hematopoietic stem cells should permit the use of the highly efficient nuclear microinjection methods for transfer of DNA. These techniques combined with new highly sensitive methods for detecting cells with the specified genetic modification of nonexpressed genes would make homologous recombination-mediated gene therapy feasible for hematopoietic stem cells. These advances are reviewed with particular emphasis on approaches to targeted gene modification of hematopoietic stem cells and speculation on directions for future research.     

Prospects for gene therapy

Experiments in animal models and human cells in vitro suggest that gene transfer using retroviral vectors may be useful to treat genetic diseases and to gain information that may improve treatment of other common diseases such as cancer. The approach to treatment of genetic diseases by inserting genes into bone marrow cells and experimental models, and a novel application of gene transfer technology to cancer research are discussed herein.     

Planning for gene therapy

In spite of some setbacks, gene therapy trials continue, while the UK government is putting in place plans to bring the potential power of genetics into mainstream public heathcare through its National Health Service.     

Intracellular signal-responsive artificial gene regulation for novel gene delivery

We describe two types of artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) or caspase-3. These molecular systems use newly synthesized cationic polymers, PAK and PAC. The PAK polymer includes substrate oligopeptide for PKA, ARRASLG, as receptor of PKA signal, while the PAC polymer possesses oligopeptide that is comprised of a substrate sequence of caspase-3, DEVD, and a cationic oligolysine, KKKKKK. These polymers formed stable complexes with DNA to totally suppress the gene expression. However, PKA or caspase-3 signal disintegrates the PAK-DNA or the PAC-DNA complex, respectively. This liberates the DNA and activated the gene expression. These systems are the first concept of an intracellular signal-responsive gene-regulation system using artificial polymer. We expect that these systems can be applied to the novel highly cell specific gene delivery strategy that is involved in our previously proposed new drug delivery concept, the drug delivery system based on responses to cellular signals.     

Immunoglobulin gene ''remnant'' DNA--implications for antibody gene recombination.

Many immunoglobulin (Ig)-producing cells retain the DNA that separates Ig variable (V) and constant (C) region genes in the germline. This "remnant" DNA must be moved during the recombination process that joins V and C genes via a joining (J) segment. We have analyzed remnant DNAs in several Ig-producing cell lines. The nucleotide sequences of kappa (kappa) light chain remnant DNAs indicate close relationships to V-J joining. We find fused V kappa and J kappa recognition sequences in five remnant DNAs, suggesting reciprocal relationships to the fused V kappa and J kappa segments produced by V-J joining. However, of sixteen plasmacytoma remnant DNAs analyzed, all involve only recombination with J kappa l. Thus, in most cell lines, remnant DNAs are not directly reciprocal to recombined kappa-genes. On the other hand, our analyses of some myelomas do indicate indirect relationships between remnant DNAs and kappa-genes. Our results suggest that multiple steps of DNA recombination occur during Ig-gene rearrangement. Because remnant DNA joining sites do not exhibit the flexibility that has been observed in Ig-gene V-J joining, our findings also suggest that the joining mechanism may involve endonuclease, exonuclease and ligase activities.     

Low copy number plasmid vectors for gene cloning and for monitoring gene expression

Abstract Derivatives of an IncW incompatibility plasmid with a low copy number are described which can be used for gene cloning or for analysing gene expression in conditions similar to those found in the host chromosome. Gene expression can be monitored after construction of operon or protein fusions with the lacZYA operon and measurement in Escherichia coli of the β-galactosidase activity.     

Invasion of gene duplication through masking for maladaptive gene flow

Gene duplication can increase an organism's ability to mask the effect of deleterious alleles present in the population, but this is typically a small effect when the source of the genetic variation is mutation. Migration can introduce orders of magnitude more deleterious alleles per generation and may therefore be an important force acting on the structure of genomes. Using formal analytical methods, we study the invasion of haplotypes containing two copies of the resident allele, assuming that a single-locus equilibrium is already established in a continent-island model of migration. Provided that the immigrant allele can be completely masked by multiple functional gene copies, a new duplication will deterministically spread so long as duplicate haplotypes are, on average, fitter than single-copy haplotypes. When fitness depends on the number of immigrant allele copies and their masking ability then the threshold for invasion depends on the rate of immigration and the rate of recombination between the gene copies. Results from several special cases, including formation of protein dimers and Dobzhansky-Muller incompatibilities, suggest that duplications can invade in a wide range of selection regimes. We hypothesize that duplication in response to gene flow may provide an explanation for the high levels of polymorphism in gene copy number observed in natural populations.     

Integrating heterogeneous gene expression data for gene regulatory network modelling

Gene regulatory networks (GRNs) are complex biological systems that have a large impact on protein levels, so that discovering network interactions is a major objective of systems biology. Quantitative GRN models have been inferred, to date, from time series measurements of gene expression, but at small scale, and with limited application to real data. Time series experiments are typically short (number of time points of the order of ten), whereas regulatory networks can be very large (containing hundreds of genes). This creates an under-determination problem, which negatively influences the results of any inferential algorithm. Presented here is an integrative approach to model inference, which has not been previously discussed to the authors' knowledge. Multiple heterogeneous expression time series are used to infer the same model, and results are shown to be more robust to noise and parameter perturbation. Additionally, a wavelet analysis shows that these models display limited noise over-fitting within the individual datasets.     

A novel receptor-targeted gene delivery system for cancer gene therapy

Some growth factor receptors, such as insulin like growth factor I and II receptor (IGF I R, IGF II R) and epidermal growth factor receptor (EGF R), have been proved to be over-expressed in a variety of human cancers derived from different tissue origins. Based on this molecular alteration, a polypeptide conjugate gene delivery system was designed and synthesized. It contains three essential moieties: a ligand oligopeptide (LOP) for receptor recognition, a polycationic polypeptide (PCP) such as protamine (PA) or poly-L-lysine (PL) as a backbone for DNA binding and an endosome-releasing oligopeptide (EROP) such as influenza haemagglutinin oligopeptide (HA20) for endosomolysis. These components are covalently conjugated as LOP-PCP-HA20 or in the form of a mixture of LOP-PCP and HA20-PCP. A 14 amino acid E5 was designed and synthesized as LOP for IGF I R and IGF II R, and a 16 amino acid GE7 as LOP for EGF R. Both E5 and GE7 systems could form stable complex with the plasmid DNA as E5-PCP/ DNA/PCP-HA20 and GE7-PCP/DNA/PCP-HA20. Using bacterial β-galactosidase gene (pSVβ-gal) as a reporter, the present system is able to efficiently target exogenous gene to human cancer cells of different tissue types with high efficiency bothin vitro and in implanted tumors in nude mice. It was also demonstrated that the transduced genes were highly expressed in cancer cells bothin vitro andin vivo. The present system will provide a novel effective vehicle to target therapeutic genes into cancer cells in gene therapy.     

Information-based methods for predicting gene function from systematic gene knock-downs

Background  

The rapid annotation of genes on a genome-wide scale is now possible for several organisms using high-throughput RNA interference assays to knock down the expression of a specific gene. To date, dozens of RNA interference phenotypes have been recorded for the nematode Caenorhabditis elegans. Although previous studies have demonstrated the merit of using knock-down phenotypes to predict gene function, it is unclear how the data can be used most effectively. An open question is how to optimally make use of phenotypic observations, possibly in combination with other functional genomics datasets, to identify genes that share a common role.     

Adeno-associated viral vectors for gene transfer and gene therapy.

Adeno-associated virus (AAV) is a defective, non-pathogenic human parvovirus that depends for growth on coinfection with a helper adenovirus or herpes virus. Recombinant adeno-associated viruses (rAAVs) have attracted considerable interest as vectors for gene therapy. In contrast to other gene delivery systems, rAAVs lack all viral genes and show long-term gene expression in vivo without immune response or toxicity. Over the past few years, many applications of rAAVs as therapeutic agents have demonstrated the utility of this vector system for long-lasting genetic modification and gene therapy in preclinical models of human disease. New production methods have increased rAAV vector titers and eliminated contamination by adenovirus. In addition, vectors for regulatable gene expression and vectors retargeted to different cells have been engineered. These advancements are expected to accelerate and facilitate further animal model studies, providing validation for use of rAAVs in human clinical trials.