Lentiviral vector transduction refers to the use of modified lentiviruses to perform gene editing in cell and gene therapies. Most often, lentiviral vectors are used in gene-modified cell therapies, where cells are modified ex vivo and then injected into patients to deliver the therapy. Scientists use the existing structure and mechanisms of the lentiviral particle to carry the payload - the genetic material - into the cell, which will augment the cell to change protein expression, add a construct that targets certain cancers, or another therapeutic effect.
Lentiviruses are a part of the Retroviridae family, also called retroviruses. The human immunodeficiency virus (HIV) is perhaps the most well-known lentivirus, primarily responsible for the acquired immunodeficiency syndrome (AIDS). Retroviruses are characterized by their use of viral reverse transcriptase and integrase, which allows the virus to insert genetic information into the host genome. The genetic information delivered by a retrovirus integrates into the DNA of the host cell, which makes for stable genetic engineering, and that when cells divide, the genetic change is propagated into the newly-created cell. Unlike other retroviruses, lentiviruses can transduce both non-dividing and actively dividing cell types, meaning that lentiviruses can transduce a greater variety of cell types than other viruses in their family.
While lentiviral vectors are derived from the HIV-1 virus, they are heavily modified to ensure patient safety. The critical components of a lentiviral vector are split across multiple plasmids. This separation of genetic elements renders the lentivirus incapable of self-replication, which is the primary differentiation between a lentivirus (replication competent) and a lentiviral vector (replication incompetent).
(i) Transfer plasmid
This plasmid encodes the gene of interest, which is the genetic payload the lentiviral vector will deliver to the cell. The payload includes not only the transgene, but also a promotor to drive its expression and may also include regulatory elements. Taken together, these elements comprise the sequence to be integrated into the host cell. This sequence is bordered on both sides by long terminal repeat (LTR) sequences; these facilitate integration into the host genome.
(ii) Packaging plasmids
Packaging plasmids contain core viral components and ensure the proper assembly and packaging of the lentiviral particles. Modern LVV production systems will have two packaging plasmids (GAG/POL and REV), which increases safety by requiring multiple recombination events to enable self-replication.
(iii) Envelope plasmid
The envelope plasmid contains genes for proteins that appear on the surface of the lentiviral vector particle. These surface proteins govern how the vector will transduce cells. Different envelope plasmids will create different surface proteins, which can make certain LVV particles better suited to transduce certain cell types. Most commonly, vesicular stomatitis virus envelope proteins (VSV-G) are used, binding to the LDL receptor (LDL-R) found on many cell types. Certain cell types, such as NK cells or γδ T cells, lack sufficient LDL-R, thus alternative envelopes such as baboon envelope (BaEV), which binds to ASCT1 and ASCT2, may be used to target these cell types.
Lentiviral vectors offer many advantages:
(i) The size of lentiviral vectors means they can carry larger payloads and thus can deliver more complicated gene edits.
(ii) Gene edits are integrated into the host genome, which create long-term therapeutic benefits
(iii) LVVs can transduce dividing and non-dividing cells, so there’s a wide range of possible target cells and thus enable a wide range of applications.
Lentiviral vector transduction involves the delivery of genetic material to the target cell by exploiting the natural ability of lentiviruses to insert their genetic material into dividing and non-dividing cells. The process begins with the binding of the viral envelope protein to a specific receptor on the target cell membrane. This binding triggers the entry of the viral particle into the cell, releasing the viral RNA into the cytoplasm. Once inside the cell, the viral RNA is reverse transcribed into DNA by the viral enzyme reverse transcriptase provided on one of the helper plasmids. The resulting viral DNA is then transported into the nucleus, where it integrates into the host cell genome with the help of viral integrase. This integration is a critical step that distinguishes lentiviruses from other gene delivery systems, as it allows for stable and heritable expression of the introduced genes.
(i) Know your cells – different primary cell types have different needs and will require different optimal cell density, concentration, media, or even different surface proteins on LVVs. Further, the target cell type will help determine what promoter should be used to drive expression of the transgene.
(ii) High quality lentiviral vectors – well-designed vectors of high purity and concentration are necessary for effective transduction. Not only will low quality lentiviral vectors decrease your overall expression, but they can also lead to adverse effects including proliferations arrest, improper modification of cell biomarkers, and more.
(iii) Identify your optimal MOI – MOI stands for multiplicity of infection and it represents the number of lentiviral vector particles you need per cell in order to obtain the maximal percentage of transduced cells. If your lentiviral vector is of low quality, you may need a significantly higher MOI, which will significantly increase your cost of goods sold (COGS).
Lentiviral vectors are used across many modalities within gene-modified cell therapy.
Lentiviral vector is the most common method of gene delivery in T cell therapy (predominantly CAR-T). VSV-G is today considered the standard for this application. Lentiviral vectors are used for this application due to their large payload capacity, which allows them to carry the genes for the complex constructs required for CAR-T and TCR-T therapies.
Lentiviral vectors are of interest for NK cell therapies, but the VSV-G pseudotyped LVV has a very low transduction efficiency for this cell type, which creates cost concerns. Developers have used other retroviruses and non-viral methods to get around this issue; however, the BaEV pseudotyped LVV has been demonstrated to overcome this challenge, but up until recently it hasn’t been available beyond research.
Lentiviral vectors are used to direct differentiation. These can be used to test out different cell types and investigate multiple possible therapeutic directions.
(i) Study disease mechanisms
(ii) Screen potential drug candidates
Lentiviral vectors are used with HSCs to insert a wildtype gene to overcome a defective endogenous gene, allowing the hematopoietic linage to broadly express the functional gene, creating lasting treatments for genetic blood disorders, like sickle cell anemia. Similar to NK cells, VSV-G LVVs have been historically ineffective in transducing HSCs. BaEV lentiviral vectors have shown promise with HSCs as well, and as the BaEV LVV technology progresses into the clinic it could facilitate significant advancements in this space.
Ensuring patient safety is always critical, and when considering viral vectors, one of the most important factors regarding safety is making sure the viral vector can not self-replicate, and thus cause adverse effects. To ameliorate these safety concerns, LVVs have been designed to self-inactivate. Lentiviruses are also immunogenic, this means they can trigger immune responses, potentially limiting gene therapy effectiveness. Strategies to minimize immunogenicity include modifying vector components and alternative delivery methods. Lentiviral vectors have integration site preferences, so their integration is not completely random; however, they can still integrate into regions of the genome that can be problematic - solving one genetic problem, just to cause another. This is a known issue with retroviruses, and is mitigated through careful vector and construct design, keeping vector copy number low, as well as making sure the LVV is well-characterized.
Lentiviral vector transduction is evolving, with research improving safety, efficiency, and specificity. Advances in LVVs for hard-to-transduce cells like those targeted by BaEV LVVs promise greater efficiencies, lower batch volumes, and reduced costs. Non-viral methods such as mRNA and lipid nanoparticles (LNPs) offer less immunogenic alternatives, enhancing safety. As vector design, delivery, and understanding of host-virus interactions improve, lentiviral vector transduction applications in research and clinical settings will expand.
Lentiviral vector transduction is essential in cell and gene therapy, enabling manipulation of cellular processes and treatment of cancers and genetic disorders. Despite challenges, ongoing efforts to optimize safety and efficacy highlight its transformative potential in genetic research and therapy.