A new third generation lentiviral gene expression system is now available. The system consists of a DNA-based recombinant DNA construct and is optimized for high-copy number replication in E. coli, effective viral transduction of a variety of cells, and integration of the vector into the host genome. By contrast, conventional transfection is limited to delivering genes to a single cell and losing them over time, especially in rapidly dividing cells. Using this approach, a genetically-modified viral vector can permanently transfer the genetic material to a host cell.
In order to produce a replication-competent lentivirus, the vector has to be modified. The recombination process requires multiple complex recombination reactions to occur in the recombinant virus. This is detected by PCR analysis. In addition, a self-inactivation mechanism is required for a third-generation vector, which results from a deletion in the long terminal repeat sequence. This eliminates the proviral enhancer sequence that would otherwise be integrated into the target cell.
Recombination-competent lentiviral vectors include an inactivating gene. A viral transcription-competent lentivirus contains two active promoters. The CMV promoter will encode the desired gene, while SV40 will encode an antibiotic-resistant sequence. Neither of these vectors contain a stop codon, indicating that they will be ineffective in causing a cellular mutagenesis.
A replication-competent virus is highly efficient, allowing rapid modulation of gene expression in different species. Furthermore, the engineered virus will not become self-inactivated, and will only retain its internal promoter. Additionally, a post-transcriptional regulatory element will be present in each vector. The goal is to obtain a third-generation lentiviral vector that will be more effective for clinical use.
A third-generation lentiviral gene expression vectors are stable and can be used to express a variety of genes. The resulting lentiviruses are designed to produce a specific protein. The adapted lentiviruses have several advantages. They are self-inactivating, which means they can be safely transferred to humans. They also require no maintenance or purification, making them ideal for human-use.
Another advantage of a third-generation lentiviral gene expression vector is its high efficiency. Unlike previous lentiviral vectors, the new system is immune-inactive, which ensures safety when using it in a clinical trial. It also has many advantages. These lentiviral particles can be made with low-cost, easily manufactured g-retroviral-expression systems.
The cloning of a new third-generation lentiviral vectors is a step forward in gene therapy. It is designed to stably integrate into chromosomes of the target cells. It also avoids the destruction of cloned viral particles by cytotoxic T cells. The newly developed lentiviral vectors are immune-competent.