Non-Viral Gene Therapy Vectors Carrying Genomic Constructs

Gene therapy is the use of genes or DNA for the treatment of diseases. For the treatment of inherited disorders, DNA carrying a functional gene is introduced into the cells of a patient to reverse the defect of the corresponding malfunctioning endogenous gene. Previous genetic characterization of the disease and cloning of the gene that causes it are necessary. In most cases, the cDNA of the therapeutic gene is cloned into a bacterial plasmid under the control of a strong heterologous promoter (often of viral origin). However, such constructs, called mini-genes, lack introns, promoters, enhancers, and long-range controlling elements that precisely control the temporal and spatial expression of the endogenous gene. For gene therapy of some diseases it is important to achieve expression of the therapeutic gene at specific levels. Expression at lower levels than normal might not be sufficient to correct the defect and at higher levels could result in undesirable effects. In other cases, tissue-specific expression may be very important. The elements responsible for controlled and tissue-specific expression of a gene usually lie within the introns and the sequences before and after the gene. Therefore, the use of genomic constructs which contain the introns and flanking DNA of the therapeutic gene is expected to be more effective than that of minigene constructs in gene therapy for certain genetic diseases where precise levels of the gene product are required (reviewed by (Perez-Luz & Diaz-Nido, 2010)). Bacterial Artificial Chromosomes (BACs), originating from the human genome project, contain genomic loci of approximately 180 kb on average and cover the entire human genome (Osoegawa et al., 2001). These sequenced BACs can accommodate most genes along with their regulatory elements and can serve as tools in gene therapy using genomic constructs. Gene therapy as a modern therapeutic tool should provide a permanent cure to the patient by long-term maintenance and expression of the administered gene. This can be achieved either by integration of the transgene into the natural chromosomes or by other mechanisms for its replication and nuclear retention. One of the most important aspects of gene therapy is the choice of the vector that will deliver and express the corrective gene in the appropriate cells. Current vectors fall into two categories: viral and non-viral. Apart from determining the method of delivery, the type of vector also determines the fate of the therapeutic gene within the cells. For instance, the vector may have the ability to remain extra-chromosomally. Non-viral artificial chromosome vectors and adeno-associated viral, adenoviral, Herpes viral and EBV vectors are all