Pseudotyping of Lentiviral Vector for Targeting Hepatocytes
Retargeting of lentiviral vectors (LVs) entry to cell types of interest is a key factor in improving the safety and efficacy of gene transfer. Pseudotyped LVs have the ability to transduce specific cell types, such as neurons, hepatocytes, cancer cells, and the like. In particular, LVs pseudotyped with Sendai virus F protein can efficiently deliver genes to the liver, providing a new strategy for the treatment of hepatic diseases. Creative Biolabs offers a comprehensive portfolio of services designed to overcome the barriers of liver-directed gene delivery. Our platform covers the entire development cycle—from envelope design to in vivo biodistribution studies—tailored specifically for hepatocyte transduction.
Viral Vector Targeting Hepatocytes
As known, in vivo gene transfer holds great promise for the treatment of several inherited and acquired diseases. Stable expression of therapeutic genes in the liver may be beneficial for patients with various hepatic and systemic diseases, including metabolic and infectious diseases, hemophilia, hypercholesterolemia and lipid storage diseases. However, achieving efficient delivery and long-term expression of transgenes to hepatocytes without toxicity has been a challenge. Until recently, the development of novel vectors such as large-capacity adenoviral vectors, adeno-associated virus (AAV) vectors and LVs are changing this situation.
LVs are capable of efficiently integrating a sizable gene expression cassette into a target cell, providing a stable and inherited genetic modification. More importantly, it can transduce quiescent cells and provide long-term expression. These properties make it a favorable gene delivery vehicle for the treatment of inherited liver disease and systemic diseases.
Figure 1. Lentiviral particle structure and its pseudotyping.1
Hepatocyte
Hepatocyte Cytokines
Hepatocytes are not passive recipients of gene vectors; they are active participants in the liver's immune and signaling microenvironment.
Production & Signaling
Hepatocytes secrete and respond to a vast array of cytokines and growth factors, including IL-6, TGF-β, TNF-α, HGF (Hepatocyte Growth Factor), and the CXCL family. These factors regulate liver regeneration via autocrine and paracrine signaling.
Impact on Transduction
- Viral Entry: Cytokine stimulation (e.g., HGF) can modulate the expression of surface receptors, potentially creating "windows of opportunity" for viral entry.
- Intracellular Trafficking & Stability: Inflammatory cytokines (like Interferons) can trigger antiviral states that degrade incoming viral capsids or silence transgene expression. Our vector designs consider these signaling pathways to ensure sustained expression even in inflamed liver tissue.
Hepatocyte Functions
Successful gene delivery must account for the metabolic machinery of the hepatocyte.
Core Functions:
- Metabolism: Hepatocytes are the center for lipid, glucose, and bile acid metabolism, as well as Phase I/II drug detoxification via CYP450 enzymes.
- Synthesis: They produce critical plasma proteins like albumin and clotting factors.
Polarity & Endocytosis
Hepatocytes exhibit distinct apical (bile canalicular) and basolateral (sinusoidal) polarity. This polarization dictates receptor distribution. Our pseudotyping strategies specifically target basolateral receptors accessible from the bloodstream to facilitate efficient receptor-mediated endocytosis.
Primary Human Hepatocytes (PHHs)
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The Gold Standard
PHHs are the most physiologically relevant model for validating liver-targeted vectors, maintaining the metabolic competence and receptor profiles seen in vivo. -
Challenges
Working with PHHs requires specialized expertise due to their limited lifespan, donor-to-donor variability, and sensitivity to viral toxicity.
The Pseudotyping of LVs
LVs are effective gene delivery vectors suitable for delivering long-term transgene expression in a variety of cell types. They are typically surface-modified (pseudotyped) with various heterologous viral envelopes to alter their tropism, thus targeting specific cell types. The most common example is the envelope glycoprotein from the vesicular stomatitis virus (VSVG). The pseudotype of lentiviral VSV-G protein broadens the breadth of its cell tropism and can infect various cells through the G protein. In addition to VSVG pseudotyped, many other viral glycoproteins (GPs) have also been used, such as GPs from Ross River Virus (RRV) and Lyssavirus.
Applications
- Inherited Metabolic Disorders: Gene research for Hemophilia A/B, Phenylketonuria (PKU), and Wilson's disease.
- Gene Editing: Precise delivery of CRISPR/Cas9 or Base Editors for permanent correction of hepatic mutations.
- Functional Genomics: High-throughput screens to identify novel drug targets in liver metabolism.
- Ex Vivo Therapy: Modification of hepatocytes for bio-artificial liver devices or autologous transplantation.
Pseudotyping of LVs Targeting Hepatocytes at Creative Biolabs
It has demonstrated that in vivo studies using VSVG pseudotyped LVs have low transduction efficiency in hepatocytes. Therefore, GPs from other viruses are developed to pseudotype LV to target hepatocytes, especially glycoprotein of Sendai virus. The Sendai virus F envelope protein (SV-F) is capable of binding specifically to the hepatic asialoglycoprotein receptor (ASGP-R), mediating the fusion of the viral envelope with the cell membrane. Human immunodeficiency virus (HIV) and murine leukemia virus (MLV) vectors can be pseudotyped with Sendai virus fusion protein F. These vectors can transduce human hepatoma cells and primary human hepatocytes efficiently. Furthermore, several different approaches are also taken at Creative Biolabs to significantly increase the titer of these pseudotyped vectors.
We provide a comprehensive "Design-to-Data" service:
- Custom Envelope Design: In silico modeling and cloning of targeted glycoproteins.
- Optimization: Screening multiple pseudotypes to maximize the Specificity Index.
- Scalable Production: From small-scale research aliquots to large-scale preclinical batches.
VSV-G Pseudotyped Lentiviral Vectors
While we specialize in targeted vectors, VSV-G (Vesicular Stomatitis Virus G protein) remains the industry benchmark.
- Pros: Extremely high titers, high physical stability, and broad tropism (pan-cellular entry).
- Cons: Significant off-target transduction and sensitivity to complement inactivation in human serum.
- Our Stance: We use VSV-G as a control to benchmark the specificity and efficiency of our novel hepatocyte-targeted pseudotypes.
Advanced Engineering Strategies
- Envelope Mutagenesis: Point mutations to ablate native binding sites while introducing novel targeting domains.
- Tropism Detargeting: "Shielding" the virus to prevent non-specific binding to blood components or non-liver cells.
- Promoter Combination: Improving safety by using pseudotyping with liver-specific promoters to restrict transgene expression to the liver, even with off-target entry.
Lentiviral Vector Production and Quality Control
Our manufacturing platform ensures quality.
- Packaging: Proprietary helper plasmids optimized for the expression of complex or chimeric envelopes.
- Purification: Chromatography-based purification to remove impurities and concentrate vectors without damaging the fragile modified envelopes.
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Quality Control (QC):
- Infectious Titer: qPCR and flow cytometry.
- Envelope Incorporation: Western blot analysis to verify the presence and density of targeting ligands.
- Functional Assay: Validation of specific transduction in ASGPR+ cells vs. receptor-negative controls.
Advantages of Choosing Our Service
Deep Hepatic Expertise
We understand the nuances of hepatocyte biology, including polarity, cytokine responses, and receptor cycling.
Modular Platform
Ability to mix-and-match envelopes, ligands, and promoters.
High Reproducibility
Standardized SOPs ensure batch-to-batch consistency.
Safety Focused
Strategies designed to minimize immunogenicity and off-target integration.
Case Studies
Case Study 1: Enhanced Specificity via Nanobody Display Objective: Deliver a therapeutic enzyme specifically to hepatocytes while sparing splenic immune cells. Solution: Engineered a lentivirus displaying an anti-ASGPR nanobody. Result: Achieved a 5-fold increase in liver-to-spleen transduction ratio compared to VSV-G controls in a murine model.
Case Study 2: Validation in Primary Human Hepatocytes Objective: Screen a library of chimeric envelopes for human liver entry. Result: Identified two novel variants that maintained >80% viability and high transgene expression in donor-derived PHHs, overcoming the toxicity often seen with high-dose VSV-G.
Frequently Asked Questions
Q: Can lentiviral vectors efficiently transduce primary human hepatocytes (PHHs)?
A: Yes. While PHHs are harder to transduce than cell lines, our optimized pseudotypes (particularly those targeting ASGPR) show significantly improved uptake in PHHs compared to standard vectors.
Q: How do hepatocyte cytokines influence transduction efficiency?
A: Cytokines like IFN-α can induce antiviral states that limit transduction. Our vectors are designed to evade these immune sensors, and our protocols include media optimization to mitigate cytokine interference.
Q: How does this approach compare with AAV liver targeting?
A: AAV is non-integrating and episomal, meaning expression dilutes as hepatocytes divide (liver regeneration). Lentiviruses integrate into the genome, providing permanent expression for the life of the cell and its progeny, which is ideal for pediatric patients or regenerating livers.
Q: Can hepatocyte-specific promoters be combined with pseudotyping?
A: Yes, and this is a gold-standard strategy to enhance targeting specificity. Pseudotyping controls "which cell type is entered" (pre-transcriptional targeting), while tissue-specific promoters' control "in which cell the gene is expressed" (transcriptional targeting). Their combination enables dual specificity, greatly improving safety.
Connect with Us Anytime!
As a leader suppler in lentivirus production, Creative Biolabs provides high-quality lentivirus services ranging from LVs optimization (such as pseudotyping of LVs), design, as well as safety determination services for basic research. No matter what stage your research is at, our team of scientists will work with you to provide the custom service that best meets your needs. For more information, please contact us.
Reference
- Jargalsaikhan B E, Muto M, Ema M. The Era of Gene Therapy: The Advancement of Lentiviral Vectors and Their Pseudotyping. Viruses, 2025, 17(8): 1036. https://doi.org/10.3390/v17081036 (Distributed under Open Access license CC BY 4.0, without modification.)
