AAV, Lentivirus, and Adenovirus: Choosing the Right Vector for Gene Therapy and Research

As a key tool of gene delivery, viral vector can accurately transport therapeutic genes or research genes into target cells, and realize stable expression and functional regulation of genes. From overcoming hereditary diseases to analyzing gene functions, the high efficiency and specificity of virus vectors provide possibilities for many research and treatment schemes, which is an important technical support to promote the development of life sciences.

However, if the appropriate virus vector is not selected correctly, it will bring a series of serious consequences. The mismatch between vector and target cell, abnormal gene expression regulation and other problems may lead to the failure of target gene expression, making it difficult to achieve the expected results in research or treatment. Therefore, careful and scientific selection of virus vectors is the key prerequisite for the successful development of gene therapy and basic research.

This paper introduces adenovirus, lentivirus and AAV vector in gene therapy, their application choices in clinical research, and the differences in cost, safety and supervision.

Common Viral Vector and Their Characteristic

Adenovirus, lentivirus and adeno-associated virus (AAV) vector are the three main virus vectors which are widely used. Adenovirus vector has the characteristics of high titer, infectivity, division and non-splinter cell, and can carry large foreign gene fragments, but its immunogenicity is relatively strong, and it is mainly used in gene therapy, vaccine development and other fields.

Lentiviral vectors can stably integrate foreign genes into the genome of host cells, can infect mitosis and non-splinter cell, and have low immunogenicity. They are often used in cell and gene therapy, gene editing and so on, but the packaging capacity is limited.

AAV vector has the advantages of low immunogenicity, high safety, long-term stable expression of foreign genes and so on. It can infect a variety of tissues and cell types and is widely used in gene therapy, especially for the treatment of single-gene genetic diseases, but its packaging capacity is small and its production process is complicated. Generally speaking, they have their own advantages and disadvantages in functions, characteristics and applications, and are suitable for different research and treatment scenarios.

Basic characteristics of frequently used viral vectors for gene delivery (Kasala et al., 2016)

Selecting Viral Vector for Clinical Resaerch

In the fields of gene therapy, drug research and development and basic scientific research, the choice of virus vector plays a key role in the effect of experimental or clinical treatment.

Targeting Specific Tissue

AAV vector: AAV has good safety and low immunogenicity, and is excellent in targeting nerve, eye and liver tissues. In nervous system research, AAV can cross the blood-brain barrier and deliver genes to the central nervous system, which can be used for gene therapy research of neurodegenerative diseases such as Parkinson's disease and Alzheimer's Harmo's disease. In the field of ophthalmology, AAV vector has been successfully applied to the clinical trials of hereditary retinal diseases, delivering therapeutic genes accurately to improve patients' vision.

Lentiviral vectors: Lentiviral vectors can be integrated into the genome of host cells and are suitable for gene manipulation of blood system and stem cells. In the study of hematological diseases, such as leukemia and thalassemia, lentivirus can stably introduce therapeutic genes into hematopoietic stem cells, so that their differentiated offspring cells can continue to express therapeutic proteins.

Adenovirus vector: Adenovirus has strong infection ability, which is especially suitable for gene delivery in respiratory tissues and is also a common vector for rapid vaccine development. In the study of respiratory diseases, adenovirus can directly infect respiratory epithelial cells, which can be used to study the mechanism of virus infection and gene therapy for cystic fibrosis and other diseases. In terms of vaccine development, during the COVID-19 epidemic, a variety of adenovirus vector COVID-19 vaccines were developed and put into use rapidly.

Schematic representation of different viral-based vector (Henríquez et al., 2024)

Choosing Right Vector Based on Expression Time

Rapid transient expression: Adenovirus is the first choice if the experiment or treatment needs rapid gene expression and short action time. Adenovirus vector is not integrated into the genome of the host cell, but exists in the nucleus in a free form, which can quickly start the transcription and translation of foreign genes and reach a high expression level within hours to days after infection. It is often used in short-term gene function verification, rapid protein production and other experimental scenarios.

Stable and long-term expression: AAV and lentivirus are better choices for the demand of long-term stable expression of genes. Although AAV is not integrated into the genome of the host cell, it can exist as an appendage in the nucleus for a long time, achieving sustained and stable gene expression, which can last for months or even years. Lentivirus, by integrating foreign genes into the genome of the host cell, can stably transfer the genes to the offspring cells with cell division, thus achieving long-term stable expression, which is often used to construct stable expression cell lines and long-term gene therapy.

Selecting According to Gene Size

When the target gene size is about 5kb, lentivirus or adenovirus vector can be considered. The packaging capacity of lentivirus vector can reach about 8kb, which can accommodate most genes with a size of 5kb. The packaging capacity of adenovirus vector is larger, about 36kb. Although its genome needs to be modified to make room for foreign genes, it is enough to meet the delivery demand of 5kb genes.

For genes smaller than 5kb, AAV vector can be easily competent. The packaging capacity of AAV is generally about 4.7kb. Under the premise of ensuring the stability and infection efficiency of the vector, AAV can effectively deliver smaller target genes, and its characteristics of strong targeting specificity and low immunogenicity make it an ideal choice in various research and clinical applications.

Cost and Production Considerations

In the process of biomedical R&D and industrialization, the cost of carrier construction and the difficulty of virus packaging are the core factors that affect the feasibility and economic benefits of the project. AAV, adenovirus and lentivirus, as the mainstream vectors of gene therapy and biological product development, have significant differences in production cost and production efficiency, which provide diversified choices for different research directions and industrial needs.

Judging from the construction cost of the carrier, the preparation cost of AAV carrier is high. Its construction depends on three-plasmid or four-plasmid auxiliary system, which requires strict purity of packaging plasmid and auxiliary virus, and the procurement cost of high-quality raw materials remains high. Taking the production of recombinant AAV vector as an example, the raw material cost of high-purity plasmid accounts for 30%-40% of the total production cost.

The construction of lentivirus vector is also complicated, which requires four plasmid systems to transfect 293T cells. The preparation of its packaging plasmid needs a lot of amplification and purification, which requires a higher biosafety level for the vector construction laboratory, resulting in a high overall cost. In contrast, the construction of adenovirus vector depends on mature molecular biology technology system, which can be completed by using conventional tool enzymes and plasmid vectors. The raw materials are easy to obtain, the technical threshold is low, and special equipment and complex processes are not needed, which makes the cost of vector construction lower by about 50%-60% compared with AAV and lentivirus.

Overview of bench to bedside approach for personalized treatment (Panikker et al., 2022)

In terms of the difficulty of virus packaging, AAV packaging efficiency is relatively low, and the requirements for cell culture conditions, transfection parameters and environmental control are extremely strict. It is necessary to carry out precise operation in professional biosafety facilities and equip with advanced process monitoring systems, which undoubtedly increases the complexity and cost of the packaging process. Lentivirus packaging is also facing challenges. Its transfection efficiency and virus titer are affected by many factors, such as cell density and the ratio of transfection reagents. In order to ensure virus activity and safety, the biological safety risks must be strictly controlled in the packaging process, which increases the packaging difficulty and cost.

Adenovirus, by virtue of its high replication ability, can proliferate rapidly under suitable culture conditions and realize large-scale production of virus particles. Its packaging technology has been highly standardized, the operation process is standardized and stable, and the dependence on special equipment and professionals is low, which greatly reduces the packaging difficulty and labor cost.

Although the production cost of AAV vector is high, it is favored in the field of clinical gene therapy because of its excellent safety and targeted delivery ability. Its non-integrated genome and low immunogenicity make it an ideal carrier for the treatment of rare diseases and single-gene genetic diseases. In high value-added clinical application scenarios, targeted gene delivery brings remarkable curative effect and low risk, which makes its clinical value far exceed the production cost.

Lentivirus vector plays a key role in CAR-T cell therapy and other fields because of its stable integration of foreign genes. Although the production process is complex and the cost is high, it is indispensable in high-value applications such as cancer immunotherapy. Adenovirus vector, on the other hand, has become the first choice for industrial vaccine production because of its advantages of high yield, low cost and stable production process.

The chimeric antigen receptor (CAR) design (Papaioannou et al., 2023)

Comparing Security and Supervising Differences

In the field of biomedicine, the safety of gene therapy vectors is closely related to regulatory requirements. Different vectors have their own characteristics in clinical application and regulatory approval due to their different characteristics.

AAV carrier has become the first choice for the treatment of chronic diseases with strict regulatory requirements because of its non-integrated characteristics. Its genome exists in the host cell in a free state and will not be randomly inserted into the host chromosome, thus eliminating the risk of insertion mutation from the source and reducing the possibility of malignant transformation of the cell. Preclinical studies show that AAV vector has no genome integration event in long-term cultured cell lines, and its safety has been fully verified.

Although lentiviral vector can efficiently deliver genes and realize long-term expression, it will stably integrate foreign genes into the host genome, and there is a potential risk of insertion mutation. Once foreign genes are randomly integrated, normal gene coding sequence may be interrupted or proto-oncogene may be activated, which may lead to malignant transformation of cells. In applications such as CAR-T cell therapy, the safety evaluation of lentiviral vectors by regulatory authorities is very strict. Before clinical practice, enterprises need to locate foreign gene insertion sites by genome-wide sequencing, observe cell changes through long-term cell culture, evaluate risks, and formulate a sound risk management and control plan.

Adenovirus, as a common vaccine carrier, has both advantages and disadvantages in its strong immunogenicity. On the one hand, it helps to stimulate immune response and enhance the vaccine effect. On the other hand, excessive immune response can cause fever, inflammation and other adverse reactions. Therefore, controlling immunotoxicity has become the key to the development of adenovirus vector vaccine. Regulators have set strict standards for production, quality control and clinical evaluation. Enterprises need to optimize vector design, delete immunogenicity-related gene fragments, accurately control virus dosage, and find a balance between immune activation and safety.

Cell attachment and entry of human serotype 5 adenovirus (Zhang et al., 2023)

Specific Cases Comparison of Viral Vector

In the field of gene therapy and biomedical research, the choice of virus vector directly affects the success or failure of the experiment and clinical efficacy.

Zolgensma vs. Kymriah

Vector type: Zolgensma uses AAV as the vector, and Kymriah uses Lentivirus as the vector.

Treatment of diseases: Zolgensma is used to treat spinal muscular atrophy (SMA), and the functional SMN1 gene is delivered to the body through AAV. Kymriah is a CAR-T cell therapy, which is used to treat B-cell lymphoma. Lentivirus is used to modify patients' T cells so that they can recognize and attack cancer cells expressing CD19 protein.

Mechanism of action: Zolgensma introduces normal genes into patients through AAV to make up for the functional loss caused by gene defects. Kymriah is to collect the patient's own T cells, introduce the gene encoding CAR into the T cells in vitro by lentivirus vector, make them become CAR-T cells, and then transfuse them into the patient's body to specifically recognize and kill tumor cells.

COVID-19 Vaccine Platform Comparison

Technical principle: J&J COVID-19 vaccine is an adenovirus vector vaccine. The gene encoding Covid-19 spike protein is inserted into the adenovirus vector, and human cells are infected by adenovirus, so that the cells express spike protein, thus triggering an immune response. The mRNA vaccine introduces the mRNA encoding Covid-19 spike protein into human body, directly translates it to form the corresponding antigen protein, and induces the body to produce specific immune response.

Production technology: The production technology of mRNA vaccine is relatively simple, and it is easy to scale up, and mRNA can be synthesized rapidly and in large quantities by in vitro transcription. The production of J&J vaccine involves the process of adenovirus culture, transformation and gene insertion. The production process is complicated and the production cycle is relatively long.

Immune effect: mRNA vaccine has dual mechanisms of humoral immunity and T-cell immunity, and has strong immunogenicity, which can induce a high level of neutralizing antibody. J&J vaccine can also induce immune response, but it may be slightly lower than mRNA vaccine in immunogenicity and neutralizing antibody level. However, J&J vaccine only needs one dose, while mRNA vaccine usually needs two or more doses.

Storage conditions: mRNA vaccines usually need to be stored and transported at low temperature. For example, the mRNA vaccines of Pfizer-BioNTech need to be stored at around-70℃, and the mRNA vaccines of Mardner need to be stored at-20℃. The storage conditions of J&J vaccine are relatively loose, generally it can be stored at 2-8℃, which is more convenient for global distribution and use.

Vaccine platforms and their ways of producing immunogen in cells (Li et al., 2021)

Conclusion

In a word, AAV, lentivirus and adenovirus all have irreplaceable value in the field of gene therapy and scientific research, and their unique properties provide rich choices for diverse research and treatment needs. With the rapid development of gene therapy technology and the deepening of research, the understanding and application of these three virus vectors are constantly expanding and optimizing. When selecting virus vectors, researchers and clinicians should comprehensively consider many factors, such as disease types, target cell characteristics, therapeutic objectives, immune response and so on, and make the most appropriate decision.

Choosing right viral vector based on different purposes