Lentiviruses are a subgroup of retroviruses with an RNA genome that is reverse-transcribed into DNA after infection. Their genome includes gag (structural proteins), pol (reverse transcriptase and integrase), and env (envelope glycoproteins). Lentiviruses enter host cells via membrane fusion or endocytosis, reverse-transcribe their RNA into DNA, and integrate into the host genome—preferably within transcriptionally active regions. Unlike γ-retroviruses (e.g., MLV), lentiviruses can infect non-dividing cells, making them valuable vectors for gene therapy, especially for quiescent cells such as HSCs and resting T cells. Lentiviral vector systems have evolved through three generations to improve safety.
What Are the Elements Involved in Lentiviral Vector Structure and Function?
Key Components
Accessory & Regulatory Genes
| Abbreviation | Full Name | Function |
| gag | Group-Specific Antigen | Encodes structural proteins of the virus |
| MA | Matrix | Viral matrix protein |
| CA | Capsid | Viral capsid protein |
| NC | Nucleocapsid | Viral nucleocapsid protein |
| pol | Polymerase | Encodes enzymes required for reverse transcription and integration |
| PR | Protease | Proteolytic cleavage of viral protein precursors |
| RT | Reverse Transcriptase | Converts RNA genome into DNA |
| IN | Integrase | Integrates viral DNA into host genome |
| env | Envelope | Encodes envelope glycoproteins |
| VSV-G | Vesicular Stomatitis Virus G | Envelope protein used for pseudotyping; enables broad host tropism |
| LTR | Long Terminal Repeat | Contains essential elements for transcription, reverse transcription, and integration |
| Ψ (psi) | Packaging Signal | Required for packaging of viral RNA into the capsid |
| U3 | Unique 3′ region | Promoter/enhancer region; required for transcription and reverse transcription (initiation) |
| U5 | Unique 5′ region | Required for reverse transcription (extension) |
| R | Repeat region | Found at both ends; essential during reverse transcription |
| Abbreviation | Full Name | Function |
| Vif | Viral Infectivity Factor | Enhances viral replication, counteracts host defenses |
| Vpr | Viral Protein R | Involved in nuclear import, immune evasion |
| Vpu | Viral Protein U | Enhances replication, downregulates CD4 |
| RRE | Rev Response Element | Rev binding site, facilitates nuclear export of RNA |
| Rev | Regulator of Virion | Nuclear export of unspliced and singly-spliced viral RNAs |
| Tat | Trans-Activator of Transcription | Enhances transcription of viral genes |
| polyA | poly-Adenine tail | Improves mRNA stability, export, and translation |
Advantages of Lentiviral Vectors
Clinical Applications
With the rapid development of gene therapy technologies, lentiviral vectors have found various important clinical applications due to their unique advantages. These include the correction of primary immunodeficiencies, engineered tumor-specific T-cell receptors (TCRs), and chimeric antigen receptor (CAR) T-cell therapy. Below are partial gene therapies that utilize lentiviral vectors:
| Company | Therapy | Indication | Clinical Progress & Efficacy |
| Bluebird Bio | Zynteglo | Transfusion-dependent β-thalassemia | FDA approved in 2022, patients are free from transfusion dependency, long-term follow-up shows hemoglobin near normal levels |
| Bluebird Bio | SKYSONA | Early active cerebral adrenoleukodystrophy (CALD) | FDA approved in 2022, 90.6% of patients had no severe disability within 24 months |
| Oxford Biomedica | Axo-Lenti-PD | Parkinson's disease | Ongoing clinical trials, improvement in neurological function |
| Orchard Therapeutics | Libmeldy (OTL-200) | Metachromatic leukodystrophy (MLD) | EMA approved in 2020, only approved MLD therapy, significant improvements in motor and cognitive functions |
| Orchard Therapeutics | Strimvelis | ADA-SCID | EMA approved in 2016, first ex vivo autologous gene therapy |
| Mustang Bio | MB-107/MB-207 | X-linked severe combined immunodeficiency (XSCID) | Phase 1/2 trials show significant immune reconstitution |
| AVROBIO | AVR-RD-02 | Gaucher disease | Phase 1/2 trials ongoing |
| Rocket Pharmaceuticals | RP-L201 | Leukocyte adhesion deficiency type I (LAD-I) | Clinical trials show good safety and efficacy |
| Interius BioTherapeutics | INT2104 | B-cell malignancies | Approved for Phase I clinical trial by Australian TGA in July 2024, single-dose intravenous infusion, no lymphodepletion required |
| Umoja Biopharma | UB-VV111 | Hematologic malignancies | FDA IND approval in August 2024, Phase I trial initiation, including CAR-T-treated patients |
| Kelonia Therapeutics | anti-BCMA CAR iGPS | Multiple myeloma | Preclinical data superior to in vitro culture, good targeting capabilities |
As interest in gene and cell therapies accelerates, the demand for scalable, cost-effective lentiviral vector manufacturing platforms has intensified. With advancements in drug characterization and safety analysis technologies, lentiviral vector-based gene therapies are now widely used in late-stage clinical trials and therapies approved by regulatory agencies.
Creative Biogene is dedicated to propelling engineered cell and gene therapies forward through cutting-edge solutions. Our comprehensive end-to-end lentiviral workflows deliver exceptional products, services, and specialized expertise to empower the development of transformative lentiviral gene therapies.
Creative Biogene delivers expert custom cloning services into lentiviral backbones, accommodating diverse genetic elements including ORFs, shRNA, miRNA, sgRNA, lncRNA, and circRNA—all with versatile tagging options. Every lentiviral construct undergoes rigorous verification through gold-standard Sanger sequencing, guaranteeing perfect alignment with NCBI reference sequences.
Inventory Highlights:
Creative Biogene offers a diverse portfolio of lentiviral vector systems tailored for specific research and clinical applications:
Enables long-term gene expression through host genome integration, ideal for stable cell lines and transgenic models. Features high-titer production (>1×10⁹ TU/mL), broad host compatibility via VSV-G pseudotyping, and a complete workflow from gene synthesis to QC.
Provides transient expression without genome integration risk, achieving ultra-high titers (1×10¹⁰ TU/mL) suitable for in vivo applications while meeting FDA safety guidelines for gene therapy vectors.
Specifically designed for CAR-T therapy applications with T cell-optimized vectors, serum-free processing for clinical-grade production, and comprehensive quality control measures for regulatory compliance.
Delivers stable gene knockdown for functional genomics research using algorithm-optimized shRNA design with validated silencing efficiency ≥70% and compatibility with both in vitro and in vivo applications.
Expands targeting capabilities through alternative envelope proteins, offering tissue-specific targeting options and specialized controls for experimental validation.
Integrates fluorescent or secreted reporters for real-time monitoring, pathway-specific response systems, and pre-built libraries for applications requiring visual tracking or high-throughput screening.
Creative Biogene employs an advanced third-generation packaging system to produce lentivirus in both standardized and customized formats. Our sophisticated in-house production infrastructure combines premium reagents, streamlined quality-controlled workflows, and an expert R&D team—delivering consistently reliable, high-titer lentiviral particles for cutting-edge research and therapeutic applications.
Distinguished Advantages:
How do We Support Your LV Production for Research?
With the rise of the cell therapy industry, the demand for lentivirus has been growing yearly. Lentiviral vector production for clinical use must comply with cGMP to ensure high quality, with verified identity, purity, and potency. Producing cGMP-compliant lentiviral vectors is more challenging than retroviral vectors due to the lack of stable packaging cell lines, leading to production inconsistencies and higher costs.
Creative Biogene focuses on providing comprehensive CDMO services for cell therapy drugs. We have established an advanced GMP-grade serum-free suspension virus production platform, offering high-quality lentivirus CDMO services tailored to our client's diverse needs.
Preclinical
Plasmid construction
Pseudotype selection
Small-scale LVV production
In vitro poc
Basic assays
IND Filing
GLP tox batch
Assay development
IND CMC documentation
DMF reference
Clinical (Phase I–III)
Scale-up
Process optimization
QC testing
Method validation
Batch release
Commercial
Routine GMP production
CPV
Lifecycle management
Long-term supply
BLA/NDA Submission
PPQ runs
Comparability studies
CMC dossier finalization
Regulatory response
Customization
Regulatory Compliance
End-to-End
Clinical Integration
Creative Biogene's manufacturing infrastructure features GMP-compliant dedicated cell bank establishment areas, aseptic isolators for final product filling, and optimized processes that significantly reduce production and testing costs. Both the adherent and suspension platforms are supported by comprehensive documentation and traceability systems that fully satisfy regulatory requirements for clinical and commercial applications, ensuring a seamless transition from development to market approval.
We offer two robust lentiviral production systems with FDA DMF registration and clear IP traceability:
How do We Support Your LV Production at Every Stage?
| Service Level | Typical Titer | Production scale | Production Details | Price |
| IIT Grade | 1×10⁹ TU | 2–50L |
| Inquiry |
| IND Grade | 1×10⁹ TU | 2–50L |
| Inquiry |
| Clinical Grade | 1×10⁹ TU | 2–200L |
| Inquiry |
| Test Item | Test Method | US/EU Applicable Regulations |
| Adventitious Viral Agents | Indicator Cell Culture | USP<1237>, EP 2.6.16, FDA Points to Consider |
| Replication-Competent Lentivirus | Co-cultivation Method | USP<63>, EP 5.14, FDA Guidance for Industry |
| Appearance | Visual Inspection | USP<1790>, EP 2.2.1 |
| Sterility | Membrane Filtration | USP<71>, EP 2.6.1, 21 CFR 610.12 |
| Mycoplasma | Culture/PCR Method | USP<63>, EP 2.6.7, 21 CFR 610.30 |
| pH | pH Meter | USP<791>, EP 2.2.3 |
| Osmolality | Freezing Point Depression | USP<785>, EP 2.2.35 |
| TCR Identification | Sequencing | USP<1120>, ICH Q6B |
| Host Cell Protein | ELISA | USP<1132>, EP 2.6.34, ICH Q6B |
| Physical Titer (p24) | ELISA | USP<1235>, ASTM F2888 |
| Transduction Titer | Flow Cytometry | USP<1027>, ICH Q2(R1) |
| Bacterial Endotoxin | LAL Test | USP<85>, EP 2.6.14, 21 CFR 610.9 |
| Benzonase Residue | ELISA | USP<1132>, EP 2.6.34 |
| Residual Host DNA | q-PCR Method | USP<1130>, EP 2.6.35, ICH Q6B |
| E1A Transfer Residue | Co-cultivation Method | USP<1237>, EP 5.14 |
| SV40 Transfer Residue | Co-cultivation Method | USP<1237>, EP 5.14, FDA Points to Consider |
In the field of cell and gene therapy, stable production and compliant delivery of lentiviral vectors are key to the successful implementation of therapies. Drawing on over a decade of technical expertise, Creative Biogene has developed a practical and accessible lentiviral CDMO service platform that focuses on solving real challenges for clinical-stage clients—from process development to GMP production. Contact us today to learn more or request a quote.
Q1: How to choose between ILV and IDLV?
Q2: How is viral titer determined?
Q3: My infection efficiency is low. What factors could be affecting this, and how can I improve it?
Several factors can impact lentiviral infection efficiency:
| Factor | Potential Issues | Solutions |
| Viral activity | Decreased titer due to improper handling | Thaw on ice, avoid freeze-thaw cycles, recheck titer after 6+ months |
| Target cells | Cell type susceptibility | Consider adenovirus for difficult-to-infect cells; use centrifugation for suspension cells |
| MOI | Insufficient viral particles | Conduct MOI gradient experiments to find optimal concentration |
| Infection time | Premature or delayed media change | Change media 24h post-infection; observe fluorescence at 48-72h |
| Transfection reagent | Polybrene toxicity | Test different polybrene concentrations (1-10 μg/mL) |
For persistently low efficiency, consider:
Q4: Despite successful transduction, why is my GFP fluorescence weak after lentiviral infection?
GFP protein expression typically peaks 72–120 hours post-infection in fast-dividing cells, while slower-growing cells may take longer. Fluorescence intensity is also closely linked to promoter strength, and weak promoters result in weaker GFP signals. Lentiviruses carrying gene inserts—especially long or GC-rich sequences—often show reduced downstream fluorescent protein expression compared to control viruses. Technical issues like low-pH media, aging microscope light sources, or ambient lighting can also weaken observed fluorescence.
For optimal visualization, consider increasing exposure time when imaging target gene-containing viruses compared to control viruses. This difference in fluorescence intensity is a normal phenomenon and doesn't necessarily indicate lower transduction efficiency.
Q5: I'm trying to establish stable cell lines with lentivirus. What's the best protocol for puromycin selection?
Step 1: Determine optimal puromycin concentration
Before selection, you need to find the minimum puromycin concentration that kills >90% of your untransduced cells:
1. Seed cells in a 24-well plate at 50-60% confluence
2. After 24h, add different puromycin concentrations (try a range: 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 μg/mL)
3. Observe for 48h and select the lowest concentration that kills >90% of cells
Step 2: Perform puromycin selection
1. Infect cells with your puromycin-resistant lentivirus
2. Allow 48-72h for viral expression (when infection efficiency reaches ~80%)
3. When cells reach 60-70% confluence, add the predetermined puromycin concentration
4. Maintain untransduced control cells with the same puromycin concentration
5. After ~48h (when >90% of control cells are dead), replace with fresh media without puromycin
6. For long-term maintenance, periodically re-select with puromycin
Step 3: Expand selected cells
After selection, expand cells through appropriate passages until you have sufficient numbers for:
Pro tip: For maintenance culture after initial selection, you can reduce puromycin concentration to 1/10 - 1/2 of the selection concentration to prevent transgene loss while minimizing stress on cells.
Q6: Why does my lentivirus show good RNA interference by qPCR but poor knockdown at the protein level?
This discrepancy between qPCR and Western blot results is frustrating but not uncommon. Proteins often have long half-lives and may persist for days after mRNA levels drop, with some requiring over 5–7 days for noticeable reduction. If infection efficiency is low, overall knockdown will be limited, even if the RNA interference tool is highly effective. Technical limitations in Western blotting—such as low antibody sensitivity, signal oversaturation, or high background—can obscure real knockdown results. Additionally, some genes have post-transcriptional regulatory mechanisms that maintain protein levels despite reduced mRNA.
Troubleshooting approaches:
If qPCR consistently shows good knockdown while protein levels remain unchanged, also consider the biological significance - some systems have built-in redundancy mechanisms that may be biologically relevant.
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