Virus vector is a kind of artificially modified virus particles, and its essence is a molecular tool to efficiently deliver foreign genes into host cells by using the natural infection ability of virus. In nature, viruses combine with specific receptors on the surface of host cells through surface proteins to complete the processes of adsorption, invasion and genome release, and realize the replication and spread of their own genetic materials in host cells.

Based on this characteristic, researchers genetically modified the virus: removing the gene fragments related to disease in the virus genome, and retaining its infection ability and packaging function. At the same time, the expression element of the target gene was inserted to construct a recombinant virus vector.

Focusing on virus vectors, this paper expounds in detail the biological characteristics, advantages and disadvantages of lentivirus, adenovirus, AAV and retrovirus vectors. Compares their differences in key characteristics and different applications.

Viral Vector Used in Gene Therapy

In the field of gene therapy and life science research, virus vector plays a key role as an efficient gene delivery tool. Based on the biological characteristics of different viruses, many types of virus vectors have been formed after artificial modification, such as lentivirus vector, adenovirus vector, adeno-associated virus (AAV) vector, retrovirus vector and so on. These vectors have their own advantages and disadvantages in terms of safety, targeting and gene capacity, which provide diverse choices for different experimental purposes and clinical application needs.

Lentivirus Vector

It belongs to retrovirus family, which is usually modified from human immunodeficiency virus (HIV). The genome is single-stranded RNA, and the virus particles are spherical with envelope, and the envelope is inlaid with glycoprotein spikes. It can infect splinter cell and non-splinter cell, such as neurons and cardiomyocytes. The foreign gene can be stably integrated into the genome of the host cell to realize long-term stable expression; The immunogenicity is relatively low.

The gene capacity is limited, generally 6-8kb. The preparation process is relatively complicated and the production cycle is long; There is a potential risk of insertion mutation, which may activate proto-oncogene or silence tumor suppressor gene. Widely used in gene therapy, cell therapy, drug research and development and other fields, such as the treatment of genetic diseases, tumor immunotherapy has more research and application.

Third-generation HIV-1-based lentiviral vector design (Bulcha et al., 2021)

Adenovirus Vector

An envelope-free double-stranded DNA virus of adenovirus family, with icosahedral symmetrical structure, consists of protein capsid and internal double-stranded DNA genome. High transduction efficiency, can infect many types of cells; The gene capacity is large, which can reach about 36kb. Is easy to prepare and purify, and can obtain a virus vector with high titer. The immunogenicity is strong, and repeated administration may trigger an immune response.

It usually exists in free form in host cells, and the duration of gene expression is short. May cause inflammatory reaction. It is often used in gene therapy, vaccine research and development, etc. For example, there is a vaccine with adenovirus as the carrier in the new coronavirus vaccine, which can deliver the related antigen gene of the new coronavirus to the human body and trigger an immune response.

Schematic of the genome of adenovirus type 5 (Ad5) and Ad5-based vectors (Wold et al., 2013)

AAV Vector

It belongs to parvovirus family, and is a non-pathogenic single-stranded DNA virus. The virus particles have icosahedral symmetrical structure and no envelope. The genome is composed of single-stranded DNA with inverted terminal repeats (ITR) at both ends. Low immunogenicity and high safety. It has a variety of serotypes and has specific targeting to different tissues and cells.

Long-term stable expression can be achieved, and in some cases, the gene can be integrated into a specific position in the genome of the host cell. The gene capacity is small, generally less than 4.7kb. The production is difficult and the cost is high. It is widely used in gene therapy, especially in the treatment of some hereditary diseases, such as hemophilia and spinal muscular atrophy.

AAV serotype 2 vector genome conformation (Büning et al., 2019)

Retrovirus Vector

Single-stranded RNA virus of retrovirus family. In addition to lentiviruses, γ -retroviruses are also common. The virus particles are spherical and enveloped, and the genome is two identical single-stranded RNA. Splinter cell can be efficiently infected, and foreign genes can be stably integrated into the genome of the host cell, which is beneficial to long-term expression. It can only infect splinter cell, but it is inefficient for non-splinter cell infection. The gene capacity is relatively small. There is a risk of insertion mutation. It is mainly used in gene therapy and cell therapy, such as gene therapy of hematopoietic stem cells.

Stable gene expression systems of retroviral (Vargas et al., 2016)

Key Characteristic of Four Viral Vector

Different types of virus vectors have significant differences in key characteristics such as integration, gene capacity, immunogenicity and expression persistence, which determine their applicability and limitations in different application scenarios.

Conformability

Lentivirus and retrovirus vectors have unique integration advantages. They can stably integrate foreign genes into the genome of host cells by virtue of their own reverse transcription and integration mechanism. This integration feature enables foreign genes to be stably transmitted with cell division, providing reliable guarantee for long-term gene expression, and is suitable for research and treatment that needs to continuously change the genetic characteristics of cells.

Adenovirus and AAV vector mostly exist in cells in free form, and the integration probability is low under normal circumstances. However, AAV can achieve low-frequency integration under certain conditions, such as DNA damage repair of cells, which makes it flexible in gene therapy applications.

Gene Capacity

Adenovirus vector, with its large genome structure, shows strong gene carrying capacity and can accommodate about 36kb of foreign gene fragments. This advantage makes it especially suitable for carrying multiple genes or large regulatory elements, and plays a key role in experimental or therapeutic scenarios that need to deliver complex gene expression systems at the same time.

The gene capacity of lentivirus vector is about 6-8kb, which can meet the delivery needs of most full-length coding genes, and it has excellent performance in both basic gene function research and common gene therapy applications. In contrast, the capacity of AAV vector is the smallest, only about 4.7kb, so researchers often need to simplify the design of the target gene and remove unnecessary sequences to adapt to its limited carrying space.

Immunity

The adenovirus vector has strong antigenicity, and it is easy to be recognized by the immune system and trigger a significant immune response after entering the body. This strong immune response will lead to the rapid elimination of the vector, which not only affects the therapeutic effect, but also limits the feasibility of repeated administration, making it face certain challenges in clinical application.

AAV vector stands out because of its low immunogenicity, and its immune response in clinical application is weak, which greatly reduces the rejection of the vector by the immune system and creates extremely favorable conditions for long-term treatment. It is one of the most popular vectors in the field of gene therapy at present. The immunogenicity of lentivirus vector is between adenovirus vector and AAV vector, which has achieved a certain balance between immune response and gene delivery effect.

Expression Persistence

Expression persistence is closely related to the integration of vectors. Because of the stable genome integration of lentivirus and retrovirus vectors, foreign genes can be stably expressed in host cells for a long time, which has irreplaceable advantages in treating diseases that need long-term regulation of cell function or building long-term stable cell models.

Although AAV vector mostly exists in cells in free form, its low immunogenicity reduces the clearance of the vector by the immune system, and can also achieve long-term gene expression, which shows a good application prospect in many gene therapy research. However, adenovirus vector is easy to be quickly cleared by immune system, and the expression time of foreign genes is relatively short, so it is more suitable for short-term gene expression research, such as rapid verification of gene function, vaccine development and other fields, and achieves the expected effect by stimulating immune response in a short time.

Comparing the characteristic of four viral vectors

Main Clinical Applications of Viral Vector

In the research of modern medicine, virus vector plays a key role in different disease treatment and research fields by virtue of its unique advantages.

Nervous System Diseases

The structure of nervous system is complex and the ability of cell regeneration is limited, so it is difficult for traditional treatment methods to accurately act on the target area, and virus vectors bring new opportunities for the treatment and research of nervous system diseases. AAV has become the first choice for research and treatment of nervous system because of its low immunogenicity, ability to cross the blood-brain barrier and good affinity for nerve cells. In the clinical treatment of spinal muscular atrophy (SMA), normal SMN1 gene is delivered by intrathecal injection of AAV9 vector, which successfully improves the motor function of patients.

In addition, lentiviral vectors can stably integrate foreign genes into the genome of nerve cells to achieve long-term stable expression, which is suitable for studying the pathogenesis of nerve cell development and neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease), and exploring the therapeutic targets of diseases by introducing related genes or RNA interference sequences.

Hematopoietic System Diseases

Lentiviral vectors play an important role in gene therapy of hematopoietic stem cells. For hereditary blood diseases, such as β -thalassemia and sickle cell anemia, lentivirus vector can introduce normal hemoglobin gene into patients' hematopoietic stem cells, which are cultured and amplified in vitro and then reinfused into patients' bodies to realize long-term stable production of functional blood cells.

Retrovirus vector has also been used for gene therapy of hematopoietic stem cells, but its application is gradually limited because of the risk of insertion mutation, which leads to serious side effects such as leukemia in some patients. In addition, adenovirus vector can be used to stimulate hematopoietic stem cells or immune cells in vitro, and enhance the immune function of cells by delivering genes such as cytokines and costimulatory molecules, which has potential application value in immune reconstruction and immune cell therapy after hematopoietic stem cell transplantation.

Oncotherapy

Adenovirus vector is widely used in tumor treatment. On the one hand, the modified oncolytic adenovirus can selectively replicate and lyse tumor cells. On the other hand, adenovirus vectors can also be used to deliver tumor antigens, activate the immune system of the body and enhance the anti-tumor immune response.

Lentiviral vectors also play an important role in tumor immunotherapy. By introducing chimeric antigen receptor (CAR) gene into T cells and preparing CAR-T cells, lentiviral vectors can ensure the stable expression of CAR gene in T cells. These modified CAR-T cells can accurately identify tumor cell surface antigens, exert strong anti-tumor activity, and have achieved remarkable effects in the treatment of hematological tumors such as leukemia and lymphoma.

In addition, AAV vector is gradually applied to the research of gene therapy of tumor because of its low immunogenicity, for example, by delivering tumor suppressor genes or anti-angiogenesis factors to inhibit the growth and metastasis of tumor.

MV particle structure, genome organization and targeting approaches (Cattaneo et al., 2008)

Choosing Appropriate Viral Vector

Different research objectives have different requirements on the characteristics of virus vectors, so the selection should be considered in combination with specific objectives and experimental conditions.

To achieve long-term stable expression of foreign genes, lentivirus vector is the first choice, which can integrate foreign genes into the genome of host cells and perform well in the study of primary cells and stem cells. The principle of retrovirus vector is similar, but the infection spectrum is narrow, and it can only infect splinter cell, but it still has its place in the study of stable transfection of specific cells.

AAV vector has unique advantages in tissue-specific gene delivery. Its serotypes have high targeting to specific tissues and cells and low immunogenicity, which is convenient for multiple administration. Adenovirus vector is favored because it can express genes efficiently in a short time, can infect all kinds of cells and carry a large amount of genes, which is outstanding in vaccine development. However, its immunogenicity is strong and it is more suitable for short-term gene expression research or single therapy

Conclusion

In a word, a deep understanding of various viral vector types and their clinical applicability is the cornerstone of gene therapy. With the rapid development of gene therapy technology, new strategies for virus vector engineering are constantly emerging, aiming at further optimizing vector performance and improving safety and therapeutic effect. Through continuous innovation and strict clinical evaluation, virus vectors will continue to play a revolutionary role in the future medicine, bringing new hope and effective treatment methods for many diseases that cannot be cured at present.

Summary of major viral vectors (Fajrial et al., 2020)