Lentivirus transduction is a biological method that uses modified viruses, called lentiviruses, to introduce new genetic material into cells. This process, known as transduction, involves the virus-mediated transfer of genetic information. Lentiviruses are a type of retrovirus, unique in their ability to permanently integrate their genetic payload into the host cell’s genome, even in non-dividing cells. This makes them a valuable tool for stable, long-term gene expression in various biological applications.
The Lentiviral Vector System
Scientists use an engineered “vector system” for gene delivery, designed for safety and efficiency. This system involves multiple plasmids, each carrying specific viral components, co-transfected into producer cells like HEK293T cells. The transfer plasmid contains the gene of interest, flanked by long terminal repeat (LTR) sequences important for integration into the host cell’s DNA. It also includes regulatory elements and a promoter to drive gene expression.
Packaging plasmids provide the necessary viral proteins in trans, meaning they are supplied separately from the genetic material to be delivered. These include the gag gene for structural proteins; the pol gene for enzymes like reverse transcriptase and integrase; and the rev gene for regulating viral RNA transport. The envelope plasmid supplies the surface protein, commonly the G protein from vesicular stomatitis virus (VSV-G), which determines the range of host cells the lentivirus can infect.
Splitting these viral genes across multiple plasmids prevents the generation of replication-competent viral particles, enhancing safety. Earlier generations used fewer plasmids, but modern “third-generation” systems separate components onto up to four distinct plasmids for increased biosafety. This engineering ensures viral particles deliver their genetic payload but cannot replicate independently once inside target cells.
Mechanism of Gene Delivery
Lentiviral particles, produced in packaging cells, are introduced to target cells. The first step involves the binding of the lentiviral particle’s envelope protein, such as VSV-G, to specific receptors on the target cell surface.
Following binding, the viral particle enters the host cell through endocytosis, where the cell membrane engulfs the particle. Once inside, the viral core releases into the cytoplasm, and the viral RNA genome is uncoated. Within the cytoplasm, the viral enzyme reverse transcriptase converts the single-stranded viral RNA into a double-stranded DNA copy.
This newly synthesized viral DNA is actively transported into the nucleus. Inside the nucleus, another viral enzyme, integrase, facilitates the insertion of the viral DNA into the host cell’s chromosomal DNA. This integration allows the delivered gene to become a stable part of the host cell’s genome. The new genetic material is then replicated along with the host cell’s own DNA during cell division, ensuring stable, long-term expression in subsequent cell generations.
Applications in Research and Medicine
Lentivirus transduction is a widely used tool in scientific research and medicine, capable of efficiently delivering and stably integrating genes into both dividing and non-dividing cells. In basic research, it creates stable cell lines that consistently express a particular gene, useful for studying gene function through overexpression or by reducing gene expression using short hairpin RNAs (shRNAs). Researchers also employ lentiviral vectors to develop in vitro and in vivo disease models, enabling a better understanding of disease mechanisms and testing therapies.
In clinical medicine, lentiviruses are central to gene therapy approaches. A notable application is in Chimeric Antigen Receptor (CAR)-T cell therapy, a cancer treatment. Here, T-cells are harvested from a patient and genetically modified ex vivo using lentiviral vectors to express a CAR that specifically recognizes and targets cancer cells. These engineered CAR-T cells are then expanded in the laboratory and infused back into the patient to fight the malignancy. Approved CAR-T therapies, such as Kymriah and Yescarta, utilize lentiviral vectors for this modification.
Lentiviruses are also used in gene therapy for inherited genetic disorders, replacing a faulty gene with a functional copy. For example, Libmeldy, an approved therapy for metachromatic leukodystrophy, uses a lentiviral vector to deliver the correct gene to stem cells. The stable integration of lentiviral vectors ensures the therapeutic gene continues to be expressed long-term, providing lasting treatment outcomes for patients.
Inherent Safety Features and Protocols
Lentiviral vector systems used in laboratories and clinics incorporate engineered safeguards to minimize risks. A primary safety feature is the use of Self-Inactivating (SIN) long terminal repeats (LTRs) in the transfer plasmid. These modified LTRs contain a deletion in the U3 region of the 3′ LTR, which prevents the virus from reactivating or activating nearby cellular genes after integration. This deletion transfers to the 5′ LTR during reverse transcription, rendering the integrated provirus replication-incompetent.
Another safety measure is the “split genome” design, where viral genes for particle production (gag, pol, rev, and env) are separated onto multiple distinct plasmids. This separation reduces the probability of recombination events that could lead to a replication-competent virus. Third-generation lentiviral systems, for instance, use four plasmids, making simultaneous recombination events rare. Non-essential viral accessory genes, which contribute to virulence in wild-type viruses, are also removed from these vector systems.
Due to their ability to transduce human cells, work involving lentiviral vectors is conducted under Biosafety Level 2 (BSL-2) containment conditions. This involves specific laboratory practices, such as working in a biosafety cabinet, using personal protective equipment like lab coats and gloves, and adhering to waste decontamination protocols. These measures protect researchers and the environment.