Helper-Dependent Adenoviral Vectors Development Service
Helper-dependent adenoviral vectors (HDAd) lack all viral genes and they can efficiently transduce a wide variety of dividing and non-dividing cells to mediate high levels of transgene expression. The absence of viral genes in HDAd results in long-term transgene expression without chronic toxicity and has a great clonal capacity. Thus, HDAd offers tremendous potential for in vivo gene therapy. Creative Biolabs has focused on the development of biotechnology for many years and has established excellent adenovirus vector construction platforms for gene therapy. We provide a variety of adenovirus vectors constructions to meet the diverse needs of our customers.
Introduction to Helper-Dependent Adenoviral Vectors
First or second-generation adenoviral vectors are effective vectors for gene transfer in vivo. However, the leakage expression of adenoviral genes from the vector backbone causes destruction of the transduced cells resulting in short-term transgene expression and chronic toxicity. To avoid this, all the viral genes were removed from the vector backbone to produce HDAds. As a result, these HDAds are able to mediate high-level, long-term transgene expression in the absence of chronic toxicity. Furthermore, the HDAd genome is maintained episomally, thus minimizing insertional mutagenesis and germline transmission.
Figure 1 Host immune responses to adenoviral vectors. (Left side) Innate immune responses.1
Helper Dependent Adenoviral Vector Long-Term Persistence
HDAdV's sustained and long-term therapeutic gene expression, stems directly from its devirulent design and the biological fate of the vector genome within transduced cells. This sustained expression is primarily due to a significant reduction in immunogenicity. The removal of all viral genes means that transduced cells no longer produce any exogenous proteins that would otherwise be presented by the major histocompatibility complex (MHC) molecules, signaling for cell destruction by the immune system. Because there are no viral antigens to trigger an immune alarm, the free HDAdV genome remains intact, continuing to transcribe therapeutic genes.
Helper-Dependent Adenoviral Vectors vs. Traditional Vectors
In the process of gene therapy development, the choice of viral vector is perhaps the most critical early decision. High-density adenovirus (HDAdV) has significant advantages compared to traditional adenovirus (first-generation adenovirus) and other widely used platforms (e.g., adeno-associated virus, lentivirus).
| Feature | Helper-Dependent Adenoviral Vector | First-Generation Adenovirus (FG AdV) | Adeno-Associated Virus (AAV) | Lentivirus (LV) |
|---|---|---|---|---|
| Genetic Capacity | Very High | Moderate | Small | Moderate |
| Integration | Non-Integrating (Episomal) | Non-Integrating (Episomal, but less stable) | Non-Integrating (Episomal) | Integrating (into host chromosome) |
| Duration of Expression | Long-Term/Sustained | Transient (Weeks) | Long-Term (Tissue-dependent) | Permanent |
| Immunogenicity | Very Low (No viral gene expression) | High (Viral gene expression) | Low (Non-pathogenic, minimal protein expression) | Low (Non-pathogenic, minimal protein expression) |
| Cell Type Tropism | Broad (Defined by serotype) | Broad (Defined by serotype) | Highly Versatile (Defined by serotype) | Dividing and non-dividing |
| Production Complexity | High (Requires co-propagation with HV) | Moderate | Moderate (Requires Helper Plasmid) | Moderate |
Innovative Directions in Helper-Dependent Adenovirus Vector (HDAdV) Development
The inherent flexibility and robustness of the HDAdV platform have led to its widespread application in numerous cutting-edge biomedical applications, driving a new wave of breakthroughs in gene therapy and vaccinology.
A. In Vivo Cell Engineering and Reprogramming
HDAdV's highly efficient transfection capabilities and transient yet persistent expression makes it ideal for in vivo cell modification.
B. Precise Targeting and Off-Target Effects
Specifically guiding the vector to target cell types (e.g., hepatocytes, nerve cells, or specific tumor cells) maximizes therapeutic efficacy and minimizes off-target toxicity.
C. Advanced Gene Vaccines
These vaccines can induce potent cellular and humoral immunity against transgenes and possess strong multi-antigen delivery capabilities.
Our Service
Helper-dependent adenovirus vectors possess numerous characteristics that make them highly attractive for gene therapy. These vectors are completely free of viral coding sequences, enabling highly efficient transduction in vivo for high-level, sustainable transgene expression with extremely low chronic toxicity. Therefore, we offer a variety of adenovirus vector construction services, including helper-dependent adenovirus vectors with advanced system or other site-specific recombinations and helper viruses.
Large-Scale Production of High-Quality Helper-Dependent Adenoviral Vectors Using Adherent Cells
The key to the commercialization of HDAdV lies in its ability to achieve large-scale production with extremely high purity and yield. The production process itself is highly complex, requiring the co-culturing of two different genomes: the HDAdV vector (containing no viral genes) and a helper virus (HV) (containing the essential viral genes, but with packaging signals conditionally deleted).
Creative Biolabs employs a robust, large-scale production system based on adherent production cell lines, typically high-performance derivatives of the E1 complementary HEK293 cell line. Adherent culture provides a mature and scalable foundation for high-titer production while maintaining batch-to-batch consistency.
The production cycle comprises the following controlled steps:
Triple transfection/initial amplification
The HDAdV plasmid is introduced into production cells.
Helper virus co-infection
Production cells already containing the HDAdV genome are co-infected with a temperature-sensitive or Cre-Lox-controlled helper virus.
Lysis and collection
Once peak yield is reached, cells are lysed and the raw vector solution is collected.
Downstream purification
Subsequent purification is crucial to ensuring product quality. Our standard approach includes multi-step chromatographic separations.
Why Choose Our Services?
Choosing Creative Biolabs for your HDAdV development means partnering with a leader in advanced gene delivery systems, ensuring your product development is built on a rigorous scientific foundation and a forward-looking regulatory perspective.
- Unparalleled expertise and capabilities - Our team comprises PhD-level virologists and process engineers with decades of experience in high-capacity vector systems.
- Zero tolerance for intellectual property risks - We rigorously screen all processes and reagents to ensure compliance with proprietary databases and intellectual property restrictions.
- Quality by Design (QbD) approach - We implement QbD principles from the initial design phase, ensuring that critical process parameters (CPP) and critical quality attributes (CQA) are controlled and optimized.
- Dedicated project management - Each client will be assigned a dedicated project manager and scientific lead to ensure seamless communication, timely reporting, and rapid problem resolution throughout the development lifecycle.
Customer Reviews
"Our large-scale, multi-gene CAR-T cell therapy is highly complex, thus requiring high-throughput HDAdV (high-throughput adenovirus vector). Creative Biolabs played a crucial role in helping us obtain stable, high-titer HDAdV. Their rigorous analytical methods, especially in achieving helper viral contamination, exceeded our regulatory expectations. They are a truly professional and scientifically strong partner."
— Dr. Anya Sharma, CSO
"Our treatment relies on long-term systemic expression in the liver. Creative Biolabs' expertise in detargeting and targeting serotype selection is critical for systemic dosing. They provided a research-grade vector that demonstrated stable, multi-year expression in our large animal models, advancing our development by months. Their commitment to intellectual property due diligence also gave us great peace of mind."
— Dr. David Zhou, Head of Translational Research
Result Delivery
Upon completion of the HDAdV development and manufacturing process, Creative Biolabs will provide a complete outcome package to facilitate your immediate research:
- Vector Product: Bulk or aliquoted final HDAdV product formulated with customer-specified buffer solutions and transported under appropriate cold chain conditions.
- Certificate of Analysis (CoA): A formal document detailing all quality control test results, including infection titer, viral particle concentration, viral particle/IU ratio, endotoxin level, sterility, mycoplasma detection, and residual host cell components.
- Document Package: A complete set of records, including batch production records, raw material certificates, standard operating procedures (SOPs) for the developed process, and complete analytical method reports.
- Givenviral Contamination Report: A detailed report confirming that residual helper virus levels are below the required detection threshold, ensuring compliance with regulatory requirements.
Frequently Asked Questions
Q: How long does HDAdV expression last in vivo?
A: In non-dividing tissues such as the liver or central nervous system, HDAdV expression can persist for several years because the vector genome exists in a stable, free nuclear form, which does not trigger immune clearance. In rapidly dividing cells, the free nuclear form is diluted over time, resulting in a shorter expression duration, but still much longer than that of first-generation vectors.
Q: How do you ensure there is no residual helper virus (HV) contamination?
A: We employ a two-layer strategy: First, we use a proprietary helper virus design that utilizes recombinases in the production cell line to remove packaging signals, significantly reducing helper virus packaging efficiency. Second, we use rigorous high-resolution chromatography (affinity chromatography and ion exchange chromatography) in downstream processing to effectively separate high-throughput adenovirus (HDAdV) and any residual helper virus. The final helper virus level is quantified by high-sensitivity and high-quality PCR detection.
Q: What is the maximum gene capacity of your high-capacity adenovirus vector?
A: The theoretical maximum capacity is approximately 36 kilobase pairs. However, the actual load may be slightly lower, depending on the specific genetic elements contained in the vector. We recommend that you consult our technical team to determine the optimal vector configuration to meet your specific load requirements.
Q: Can your high-capacity adenovirus vectors be tissue-specific targeted modified?
A: Yes, we have experience in integrating various targeting modifications into high-capacity adenovirus vectors. These methods may include genetically modifying capsid proteins, introducing targeting ligands, or adding tissue-specific regulatory elements. The feasibility of a specific targeting strategy depends on the specific application and target tissue.
Connect with Us Anytime!
Creative Biolabs has been involved in the field of gene therapy for many years and we are committed to completing your project with high quality. We have accumulated a wealth of scientific experience from our completed projects and provide you with the best adenoviral vector construction services to ensure your requirements are met. If you are interested in our services, please contact us for more details.
Reference
- Bandara R A, Chen Z R, Hu J. Potential of helper-dependent Adenoviral vectors in CRISPR-cas9-mediated lung gene therapy. Cell & Bioscience, 2021, 11(1): 145. https://doi.org/10.1186/s13578-021-00662-w (Distributed under Open Access license CC BY 4.0, without modification.)
