CAR-T Lentivirus Packaging
BioInnovatise Viral Vector Team
Updated July 8, 2024
Genes encoding a chimeric antigen receptor (CAR) have shown tremendous success in oncology applications. J&J and Bristol-Myers Squibb have commercialized several therapies for cancer patients using lentiviral vectors.
CAR-T gene modifications using lentiviral vectors are generally preferred over other viral vectors such as AAV or adenovirus due to several key advantages including host cell integration, transduction efficiency, and genetic payload capacity (CAR encoded constructs are generally large with regulatory elements).
As a researcher or scientist, you may be aware that CAR-T therapeutics are better suited to lentiviral vectors but you may not be aware of the differences between a CAR-T and Non-CAR-T lentivirus and what the packaging process difference is. There are also downstream and upstream production differences when it comes to CAR-T lentivirus and non-CAR-T lentivirus plasmid DNA and deciding which generation of lentivirus packaging to choose. Our research team has overviewed some of the major differences and how best prepare your research utilizing lentiviral vectors.
CAR-T Lentivirus vs. Non-CAR-T Lentivirus Overview
CAR-T Lentivirus
- DNA Gene Modification: Carries the gene encoding a chimeric antigen receptor. This receptor is engineered to recognize and bind to specific proteins i.e. antigen on the surface of cancer cells.
- Target Cells: Used to transduce host T cells. Once modified, these T cells can recognize and attack cancer cells.
- Application: Involves extracting T cells from a patient, modifying them with the CAR-T lentivirus, and then reintroducing them into the patient to fight cancer.
Non-CAR-T Lentivirus
- DNA Gene Modification: Can carry a wide range of genes depending on the intended therapeutic application. This might include genes for correcting genetic disorders, introducing therapeutic proteins, or other functions.
- Target Cells: Can transduce a variety of cell types, not limited to just T cells.
- Application: Used for a variety of gene therapy, cell therapy, immunology, precision medicine, regenerative medicine, and vaccine applications, not limited to oncology.
Key Differences
- Specificity: CAR-T lentivirus is specifically engineered for T cells to target cancer, whereas non-CAR-T lentivirus can be used for broader gene therapy purposes.
- Genetic Payload: CAR-T lentivirus carries CAR genes, while non-CAR-T lentivirus carries genes relevant to the particular therapy being developed.
- Clinical Use: CAR-T lentivirus is part of a highly specialized and personalized cancer treatment protocol, while non-CAR-T lentivirus has wider applications across different medical fields.
CAR-T Lentivirus Plasmid DNA vs. Non-CAR-T Lentivirus Plasmid DNA
CAR-T Lentivirus Plasmid DNA Construct
The CAR Gene: The main feature of the plasmid DNA construct which encodes a synthetic receptor designed to target specific antigens on cancer cells.
Single-chain variable fragment: Part of the CAR that recognizes the target antigen.
Hinge and Transmembrane Domain: Connects the ScFv to the intracellular signaling domains.
Intracellular Signaling Domains: Usually includes CD3ζ (zeta) chain and one or more co-stimulatory domains (e.g., CD28 or 4-1BB) to fully activate T cells upon antigen recognition.
Promoter: A promoter that drives the expression of the CAR gene in T cells. Often a strong, ubiquitous promoter like EF-1α or PGK is used to ensure high levels of expression in T cells.
Selection Marker: A gene that allows for the selection of successfully transduced cells. This can be an antibiotic resistance gene or a popular fluorescent marker like GFP or Luciferase.
Lentiviral Elements: Elements required for packaging and integration, such as the long terminal repeats (LTRs), packaging signal (ψ), and any additional regulatory sequences.
Non-CAR-T Lentivirus Plasmid DNA Construct
Therapeutic Gene: Contains the gene of interest relevant to the specific gene therapy, immunotherapy, precision medicine, regenerative medicine, vaccine application. This could be a gene to replace a defective gene in genetic disorders, a gene encoding a therapeutic protein, or any other gene with therapeutic potential.
Promoter: A promoter suitable for the target cells and the desired level of expression. This can vary widely depending on the application and target tissue. Promoters can be tissue-specific e.g., liver-specific promoters or ubiquitous like fluorescent marker proteins.
Selection Marker: Similar to CAR-T constructs, non-CAR-T lentiviral vectors often include a selection marker for identifying successfully transduced cells.
Lentiviral Elements: Includes the necessary lentiviral elements for packaging, integration, and expression, such as LTRs and the packaging signal.
If you are interested in adding a CAR gene on your plasmid DNA construct, adding a lentiviral backbone to your plasmid DNA construct, or have any other desired modifications, our cloning team can help. Learn more about our molecular cloning services and mutagenesis service.
CAR-T Lentivirus Packaging vs. Non-CAR-T Lentivirus Packaging
Because of differences between CAR-T lentivirus plasmid DNA and non-CAR-T plasmid DNA as well as the lentiviral vector itself, there are several differences between CAR-T lentivirus packaging and non-CAR-T lentivirus packaging in order to efficiently deliver the genetic payload to a host cell.
The similarities between CAR-T lentivirus packaging and Non-CAR-T are the usage of transfection plasmids (transfer vector plasmid, packaging plasmid, and envelope plasmid), virus production, harvesting, purification, and QC assays.
The differences between CAR-T lentivirus are packaging and Non-CAR-T lentivirus packaging are not as drastic as some may believe. The three differences are limited to the transfer vector plasmid, biosafety, and QC testing.
- Transfer Vector Plasmid: As mentioned above, CAR-T lentivirus plasmid DNA contains the CAR gene and other regulatory elements to ensure the efficient expression of chimeric antigen receptors on host T cells.
- Biosaftey and Patient Safety: Given the application in oncology, additional safety measures might be incorporated for CAR-T lentivirus packaging. These could include:
- Self-inactivating LTRs: To reduce the risk of insertional mutagenesis.
- Insulators and Barrier Elements: To minimize potential activation of oncogenes.
- Suicide Genes: Optional inclusion of suicide genes to allow for the elimination of modified cells if needed.
- Rigorous testing is required to ensure the lentivirus is safe for use in humans, regardless of oncology or non-oncology applications. However oncology applications do have an increased inherent risk. Risk mitigation may include testing for replication-competent lentivirus, ensuring the correct CAR construct is present, and verifying that the vector can effectively transduce T cells and express the CAR gene.
At BioInnovatise, we preform every lentivirus packaging to match a customer’s research application by using their requested lentivirus packaging protocol.
The above diagram illustrates the production process for lentivirus packaging BioInnovatise, where our team uses a HEK293T cell line for lentivirus packaging cells.
Lentivirus Packaging Generations
CAR-T lentivirus typically achieves higher transduction efficiency and safety with third generation lentiviral systems compared to second generation systems. However this is also a commonality for all lentiviral vector applications today.
The use of third-generation vectors is standard in clinical applications of CAR-T cell therapy due to their improved safety profile, higher efficiency, and compliance with regulatory standards. Our facility can produce second and third generation lentivirus packaging upon request. If you want to learn more about 2nd generation vs 3rd generation lentivirus, read our lentivirus packaging generations resource.
Let’s get started, our team is excited to bring your CAR-T lentivirus research to life.
If you have any question about your production or need help deciding what production features best fit your research application, contact our viral vector team.
Learn about our quick turnaround lentivirus packaging services.
Want to learn more about the latest in lentivirus based research? Our colleagues at ScienceDirect and Genetic Engineering & Biotechnology News continuously collect and publish the latest information on lentivirus-based research.
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