For the scientist
EPI vector - Science for the scientist
The EPI-vector project is a Specific Targeted Research Project (STREP) that was funded (E2.1M) under the EC Framework Six Programme (FP6). The project was launched on 1st January 2005.
EPI-vector is supported under the directive of LIFE SCIENCES, GENOMICS AND BIOTECHNOLOGY FOR HEALTH in a section with the following remit:
Advanced genomics and its applications for health
Application of knowledge and technologies in the field of genomics and biotechnology for health
- Development and testing of new preventive and therapeutic tools, such as somatic gene and cell therapies and immunotherapies
The specific requirements of the EC project identifier (LSH-2003-1.2.4-7) are detailed as:
Molecular strategies to improve precision in gene transfer for therapeutic applications
Cell and tissue engineering, including stem cell therapy, have the potential to meet the challenges posed by many diseases, increased human longevity and the concomitant public challenges facing European society. The integration of different research activities in areas as diverse as genetics, fundamental and clinical research and ethics will provide standardized research materials such as stem cell banks, clinical research protocols and novel preventive and therapeutic instruments at a European level.
Background to the project
Many chronic human diseases cause great suffering as a result of inherited and sporadic genetic mutations. In many cases, gene therapy is able to provide therapeutic benefit if the gene defect can be complemented by expression of a normal gene product in affected cells. Protocols have been developed for this purpose. However, the majority of systems that are currently being evaluated in clinical trials suffer from potential deficiencies eg disrupted expression of host genes and immunological side effects - that compromise safety. EPI-vector will develop extra-chromosomal gene delivery systems for gene therapy and evaluate protocols for their safe use in pre-clinical model systems.
The genetic elements that regulate chromatin function at natural chromosomal loci and the molecular mechanisms that regulate function are known. Even so, it has proved an immense challenge to understand how these genetic elements might be configured to provide regulated, efficient and sustained gene expression from extra-chromosomal DNA. Experience of the best extra-chromosomal gene expression systems currently available will be used to develop a new generation of DNA vectors for safe and efficient therapeutic application.
The EPI vector concept assumes that the only truly safe system for human gene therapy will:
- Use gene delivery systems that incorporate only elements of human origin.
- Use extra-chromosomal vectors to eliminate the risk of disrupting expression from host genes within the target cells.
Experimental design of the EPI-vector program
This research programme combines a genomic and cell biology-based approach for the analysis of the relationship between chromatin structure, nuclear architecture and gene function. A range of molecular and cell biology techniques will be employed and these will be supplemented by advanced imaging techniques to explore the dynamic properties of gene expression systems in living cells. Post-genomic and bioinformatic strategies will be incorporated to aid vector design. Optimized vector systems will be evaluated using a range of model systems, focusing on human cells grown in culture, human and mouse stem cells and animal models for gene therapy.
State of the art in the field
Many of the gene therapy protocols that have been used to date include risk factors that in an ideal world would be eliminated. Extra-chromosomal vectors that are constructed from sequences of human origin provide the best prospect of delivering safe therapy protocols.
DNA vector systems for therapeutic application
EPI vector will define the configuration of genetic components that deliver efficient, sustained and regulated gene expression from extra-chromosomal gene delivery vectors.
Critical factors that have been incorporated into the design of prototypes include; gene structure and the use of different regulatory elements including S/MAR (scaffold/matrix attachment region), LCR (locus control region) elements and human origins of DNA replication.
The prototype - pEPI-1 developed by Hans Lipps achieves sustained and efficient gene expression because of the properties of a strong human S/MAR even though it is present at fewer than 10 copies per cell. A major challenge is to understand how the perfect vector system behaves once inside the nucleus of target cells.
The prototype vector was constructed to incorporate a fluorescent reporter gene eGFP which is expressed from an efficient viral promoter (pCMV). The S/MAR element from the human beta-interferon gene cloned down-stream of the reporter dictates the efficient long-term expression and retention of the episomal vector.
This chromosome spread shows DAPI stained chromosome from CHO cells containing a prototype episomal vector pEPI. The cell population has 5-10 copies of the plasmid/cell after continuous culture for 100 generations without selection pressure. When the pEPI sequences are located using FISH the episomes that survive hybridization appear to be associated with the host chromosomes commonly as single spots showing they are not integrated. To do this requires a detailed knowledge of normal nuclear function.
Nuclear architecture and gene expression
This laser scanning confocal microscopy section emphasises the complexity of nuclear compartmentalisation. Condensed chromatin is blue, transcription sites (Br- uridine) green and nuclear splicing speckles (SC35) red. The clusters of large green foci - three are seen - are active centres of RNA polmease I transcription within nucleoli. Note that it is common to see centomeric heterochromatin associated with the borders of nucleoli.
Chromosome structure and nuclear architecture combine to define natural levels and patterns of gene expression from endogenous genes. One challenged faced by EPI-vector is to reproduce this natural liaison when a gene is remove from the natural locus and expressed from an extra-chromosomal environment. Perhaps crucially, active sites of DNA and RNA synthesis are known to be associated with the nucleoskeleton (Figure 4). It is reasonable to assume that episomes used as vectors for expression of therapeutic genes must interact with these active centres just as chromosomal genes do.
The central image shows a resinless electron micrograph of a cell from which almost all chromatin has been removed. High voltage scanning em (inserts) show the intermediate filament networks of the cell - vimentin in the cytoskelton(csk), lamin proteins at the nuclear periperhery (lamina) and a nucleoskeleton (nsk) throughout the nucleoplasm. The nucleoskeleton provide a sub structure on which the nucleus is organised to define the spatial architecture of nuclear compartments. n= nucleus; no = nucleous. The nucleus is 10 micrometres across.
Enhancement of the field by the proposed project
The systematic approach chosen in EPI-vector will give novel and detailed insight into the interplay between genetic and epigenetic features that regulate gene expression. Particular focus will be placed on understanding the parameters that regulate gene expression from extra-chromosomal genes. Our detailed analysis of expression from an extra-chromosomal environment will allow us to explore features such as the dynamic properties of chromatin and the relationship to changes in chromatin architecture during gene expression. Such analyses will provide valuable insight into the regulation of gene function and define the critical design parameters that must be used in order to deliver efficient, safe and sustained gene therapy from episomal gene delivery systems in human cells.
EPI-vector will provide:
- Extra-chromosomal gene delivery vectors for efficient and sustained gene expression in mammalian cells.
- Extra-chromosomal gene delivery systems that are composed entirely of DNA sequences of human origin and so minimize known risk factors that compromise safety during human gene therapy.
- Development of gene therapy protocols using ex-vivo technologies to develop sustained gene expression in cells in culture, including stem cells.
- Regulated gene delivery systems for a wide range of therapeutic applications.
Further details can be downloaded as a PDF: EPI Vector Science ( PDF 334k)