Pseudotyping of Lentiviral Vector for Targeting CD81+ Cells
To meet the challenging requirements, Creative Biolabs has equipped a team of experienced scientists with facilities and processes designed specifically to provide the best strategy and protocols customized to lentiviral vector services for gene therapy. We have successfully accomplished a number of projects and have delivered numerous novel or validated lentiviral vectors in a range of therapeutic areas.
Introduction of CD81+
CD81, also known as TATP-1, is a member of the tetramer family that plays an important role in regulating cell development, activation, growth, and metastasis. Pilot studies indicate that CD81+ is a cell-surface protein with four hydrophobic domains and is involved in mediating many signal pathways. In general, CD81+ is regarded as a glycoprotein that can specifically bind to integrins. Furthermore, recent researchers have revealed that CD81+ can enhance the cell fusion of muscles and can improve the maintenance of muscle tubes. In addition, more recent studies suggest that CD81+ gene should be an attractive target for a wide variety of malignant tumors, such as breast cancer, brain cancer, and melanoma. The mutation in the CD81+ gene can lead to the low or no expression of other molecules, including CD62 and CD19, on the surface of T cell or B cell. Scientists have theorized that CD81+ is necessary for many signal transductions, which can be infected by a range of human viruses, for instance, hepatitis C virus. As a result, a number of viral vectors have been developed for increasing the immune response of CD81 cells. Among them, lentiviral vectors are one of the most advanced vectors and have been widely used for gene delivery.
What is Lentiviral Pseudotyping?
Lentiviral pseudotyping involves the replacement of the native envelope glycoprotein of a lentiviral vector with the envelope protein from a heterologous virus.
Figure 1. Pseudotyping of lentiviral vectors.1
- Altering Tropism: The native HIV-1 envelope restricts infection primarily to CD4+ T cells. By swapping this with an envelope protein from another virus (such as VSV, Measles, or Rabies virus), the "pseudotyped" vector acquires the binding properties of the donor virus.
- Role of Envelope Proteins: The envelope protein is the key that unlocks cell entry. It determines which cell surface receptors the virus can bind to, thereby dictating the specific tissue or cell type the vector can infect. This flexibility allows researchers to direct gene delivery to virtually any cell type.
Targeting CD81+ Cells
CD81+ is a protein member of the tetraspanin family, which plays a crucial role in organizing cell membrane domains and mediating signal transduction.
Significance of CD81+ Cells
- Hepatocytes: CD81+ is a primary receptor for the Hepatitis C Virus (HCV). Targeting CD81+ liver cells is essential for developing anti-HCV therapies and liver-directed gene editing.
- Immune Cells: CD81+ is expressed on B cells and T cells, playing a role in immune synapse formation and B-cell receptor signaling.
Therapeutic Applications
- Liver Diseases: Precision delivery of therapeutic genes to hepatocytes for metabolic disorders or viral hepatitis.
- Immunotherapy: Modulating the function of CD81+ lymphocytes for cancer immunotherapy or autoimmune disease treatment.
VSV-G Pseudotyped Lentiviral Vectors
While specific targeting is vital, the Vesicular Stomatitis Virus Glycoprotein (VSV-G) remains the gold standard for high-efficiency lentiviral pseudotyping.
What is VSV-G?
VSV-G is the envelope glycoprotein of the Vesicular Stomatitis Virus. It is the most widely used protein for pseudotyping because of its remarkable stability and ability to confer broad tropism.
Mechanism of Action
- Broad Entry: VSV-G mediates cell entry via the LDL receptor family, which is ubiquitously expressed on most mammalian cells.
- pH-Dependent Fusion: Upon endocytosis, the acidic environment of the endosome triggers a conformational change in VSV-G, facilitating fusion between the viral and endosomal membranes and releasing the viral core into the cytoplasm.
Benefits of VSV-G Pseudotyping for Gene Delivery
High Transduction Efficiency: Capable of achieving very high viral titers.
- Robustness: The VSV-G protein stabilizes the viral particle, allowing for concentration via ultracentrifugation without loss of infectivity.
- Wide Host Range: Infects a vast array of cell types, including dividing and non-dividing cells.
VSV-G in Targeting CD81+ Cells
While VSV-G is broad by nature, it serves as an excellent platform for targeting CD81+ cells:
- High-Efficiency Control: It is used to ensure maximal gene transfer into CD81+ cell lines (e.g., Huh-7) to establish baseline efficacy.
- Engineering Potential: VSV-G can be engineered (e.g., via insertional mutagenesis) to display peptides that enhance affinity for specific subsets of CD81+ cells while reducing binding to off-target receptors.
Advantages of Pseudotyping Lentiviral Vectors for CD81+ Cells
Utilizing pseudotyped vectors offers distinct advantages for research and therapy involving CD81+ cells:
- Enhanced Specificity: By using CD81-tropic envelopes or engineered ligands, off-target effects on non-liver or non-immune cells can be minimized.
- Improved Efficiency: Optimized pseudotypes overcome natural barriers to infection, ensuring high expression levels of the therapeutic gene.
- Safety Profile: Targeted entry reduces the required viral load, potentially lowering the risk of insertional mutagenesis in non-target tissues.
- Versatility: Suitable for both in vitro mechanistic studies and in vivo therapeutic delivery.
Challenges and Considerations
While powerful, targeting CD81+ cells presents specific challenges that our team is equipped to handle:
- Safety & Immune Response: Viral envelopes can be immunogenic. We optimize vector purity to minimize immune stimulation.
- Off-Target Effects: VSV-G is extremely potent but lacks specificity. For in vivo applications, we recommend surface-engineered variants to restrict entry to CD81+ tissues.
- Manufacturing Scalability: producing high-titer pseudotyped vectors (especially with complex envelopes like HCV E1/E2) can be difficult. Our optimized production protocols ensure consistent high yields.
Services At Creative Biolabs
Creative Biolabs provides a panel of services to design suitable lentiviral vectors for our worldwide customers. Up to now, we have established a variety of lentiviral vectors targeting CD81+ cells with gene-of-interest. For example, a three-generation lentiviral vector has been constructed, and the data indicate that this vector can mediate stable gene transfer into CD81+ cells in murine tumor models.
Moreover, other studies conducted by our labs have also illustrated that lentiviral vectors are perfect gene transfer systems for genetic therapy of primary human T lymphocytes. They can trigger a strong specific CD81+ T-cell response against various types of lymphohematologic disorders. Additionally, we also have a well-mature pseudotyping platform to improve the host range and change the tropism of lentiviral vectors. Glycoproteins (GPs) from different kinds of enveloped viruses, including equine infectious anemia virus (EIAV) and vesicular stomatitis virus (VSV), are commonly used for increasing the expression of targeted genes in vector-producing CD81+ cells. And we can also conduct lentiviral vectors studies in various kind of GPs derived from different viruses, such as:
Case Studies and Applications
Hepatitis C Research
Researchers used our HCV-pseudotyped particles (HCVpp) to screen for neutralizing antibodies that block viral entry by interfering with the E2-CD81 interaction.
Liver Gene Therapy
A study utilized engineered lentiviral vectors to deliver Factor VIII specifically to CD81+ hepatocytes in murine models, demonstrating restored clotting function with reduced off-target expression.
CAR-T Cell Engineering
VSV-G pseudotyped vectors were used to efficiently transduce primary T cells with Chimeric Antigen Receptors (CARs), streamlining the manufacturing of cellular immunotherapies.
Customer Reviews
Review 1: "We partnered with Creative Biolabs for pseudotyping lentiviral vectors targeting CD81+ cells. Their team was knowledgeable and helped us navigate through the complexities of vector development. The results were impressive, with high specificity and low off-target effects. Highly recommend!"
— Dr. John Smith, Senior Researcher
Review 2: "Creative Biolabs provided us with excellent support in developing pseudotyped lentiviral vectors. Their expertise in VSV-G pseudotyping has been pivotal in our research targeting liver cells. The customer service was responsive, and we're seeing great results in our experiments."
— Prof. Maria Lopez, Director of Gene Therapy Research
Frequently Asked Questions
Q: What is the difference between pseudotyping and other gene delivery methods?
A: Pseudotyping involves modifying the viral envelope protein to alter the tropism of the lentiviral vector, allowing it to specifically enter cells via distinct receptors (like CD81+). Other methods, such as lipofection or electroporation, are physical or chemical methods that often lack the specificity and stable integration capabilities of targeted viral delivery.
Q: How do you ensure the lentiviral vectors are safe for therapeutic use?
A: All our vectors undergo rigorous quality control and safety testing. This includes testing for Replication-Competent Lentivirus (RCL), sterility, endotoxin levels, and specific toxicity. For CD81+ targeting, we validate that the vector delivers its payload precisely without unintended cytotoxic effects.
Q: Can VSV-G pseudotyped vectors target all types of cells?
A: VSV-G mediates entry into a very broad range of mammalian cell types due to the ubiquitous nature of its receptor. However, for exclusive targeting of specific subsets (like only CD81+ cells in a mixed population), further surface engineering or alternative envelope proteins are required to restrict this broad tropism.
Q: How long does it take to develop a pseudotyped lentiviral vector targeting CD81+ cells?
A: The development timeline varies based on project complexity. Standard VSV-G preparations are rapid (1-2 weeks). Custom engineering for CD81 specificity typically takes several weeks to optimize the envelope design, produce the vector, and perform validation assays.
Q: Are there any potential side effects of using pseudotyped lentiviral vectors?
A: As with all gene delivery systems, risks include potential immune responses or off-target integration. However, by using self-inactivating (SIN) vectors and optimizing the envelope for specificity, we significantly minimize these risks.
Connect with Us Anytime!
Pseudotyping lentiviral vectors represents a cutting-edge approach to gene delivery, combining the stable integration of lentiviruses with the targeting precision required for modern medicine. Whether utilizing the robust VSV-G envelope for maximum efficiency or engineered variants for CD81+ specificity, this technology opens new doors for treating liver diseases and advancing immunotherapy. We are committed to providing high-quality, custom-tailored vector solutions to accelerate your research. Contact our team today to discuss your project requirements and learn how our Lentiviral Pseudotyping Services can help you target CD81+ cells with precision and efficiency.
Reference
- Gutierrez-Guerrero A, Cosset F L, Verhoeyen E. Lentiviral vector pseudotypes: precious tools to improve gene modification of hematopoietic cells for research and gene therapy. Viruses, 2020, 12(9): 1016. https://doi.org/10.3390/v12091016 (Distributed under Open Access license CC BY 4.0, without modification.)
