AAV in Gene Therapy: Rationale, Mechanisms, Applications, Safety, and Efficiency Enhancement

Gene therapy has gradually moved from theoretical conception to clinical practice, which has become the hope to overcome many difficult diseases. In this complex and challenging field, how to deliver therapeutic genes to target cells accurately, efficiently and safely is always the core problem that needs to be broken through.

Adeno-Associated Virus (AAV) has emerged in many gene delivery vector because of its unique biological characteristics, including extremely low immunogenicity, extremely wide host cell range and potential advantages of long-term stable gene expression, and has quickly become the research focus and core carrier in the field of gene therapy.

In the process of AAV infecting host cells, AAV whole genome sequencing can help researchers identify possible integration sites between AAV vector and host genome. By analysing AAV integration site, the safety of AAV vector can be evaluated.

This paper focuses on the application of AAV in gene therapy, and expounds the reasons, mechanism of action, safety issues, strategies to improve treatment efficiency, and specific application examples.

The history of the development of gene therapy for neurofibromatosis type 2 (NF2) related schwannomatosis (Yuan et al., 2024)

Why Choose AAV for Gene Therapy

In the field of gene therapy, AAV is an ideal vector. It has low immunogenicity and can significantly reduce the risk of immune rejection; Its diverse serotypes can be widely targeted at various tissues; It can make the therapeutic genes stably integrated and ensure the long-term therapeutic effect. These advantages make AAV popular.

Low Immunogenicity

Immunogenicity is one of the major problems faced by gene therapy vectors. Many traditional vectors can easily trigger immune response after entering human body, and AAV has obvious low immunogenicity advantage. AAV is an envelope-free virus, its natural structure is relatively simple, and it has established a relatively mild interaction with human hosts during its evolution. A large number of preclinical studies and clinical trial data show that the immune response induced by AAV vector in vivo is much weaker than that of other viral vectors (such as adenovirus).

Wide Coverage of Therapeutic Targets

AAV has a wide host range and can infect almost all kinds of cells of many mammals, including humans. Different serotypes of AAV have unique tropism to specific tissues and cells. This wide and selective host range enables researchers to accurately select appropriate serotype AAV vectors according to different therapeutic targets and disease types, which greatly expands the application boundary of gene therapy and opens up new ways for the treatment of many refractory diseases.

Long-term Therapeutic Effect

Although AAV belongs to non-integrated virus, it has potential stable gene integration ability in some cases. It is found that under specific cell environment and conditions, foreign genes carried by AAV vector can be integrated into specific regions of the host genome at a low frequency, which is relatively stable and can realize long-term gene expression. Compared with some transient gene delivery systems, AAV, a potentially stable gene integration feature, provides more potential therapeutic strategies for chronic diseases and monogenic diseases that need long-term treatment, and is expected to fundamentally change the natural course of diseases and bring lasting therapeutic benefits to patients.

Clustering and quantification of AAV-infected retinal cells (Öztürk et al., 2021)

AAV Mechanism in Gene Therapy

AAV is an extremely critical and potential gene carrier, and the in-depth exploration of its mechanism of action has always been the core focus of scientific attention.

Virus Structure and Infection Characteristics

The process of AAV infecting host cells is a highly complex and precisely regulated biological process, which is mainly realized by receptor-mediated endocytosis. Take AAV2, which has been studied deeply, as an example. At the initial stage of infection with host cells, it mainly binds to Heparan Sulfate Proteoglycan (HSPG), which exists widely on the cell surface. As the initial receptor of AAV2, HSPG plays a vital role in the initial recognition and adhesion of virus to cells. Subsequently, AAV2 further interacted with other auxiliary receptors, and this multi-receptor synergistic mode greatly promoted the internalization process of virus particles, enabling the virus to enter the host cell smoothly.

Once it successfully enters the cell, the virus particles pass through the endosome, an intracellular transport tool, and finally reach the nucleus safely. In the nucleus, the virus particles complete the key step, that is, the single-stranded DNA genome is accurately released from the capsid, which lays a solid material foundation for the subsequent complex and delicate gene expression process.

Genome Characteristics and Gene Expression

Single-stranded DNA genome successfully released from capsid can not be directly expressed by transcription and translation, but needs to go through a key transformation step, that is, into double-stranded DNA form. Once it is successfully transformed into double-stranded DNA, the therapeutic gene will start the transcription process and generate messenger ribonucleic acid (mRNA) under the precise control of a series of key regulatory elements such as its promoter. In the cytoplasm, mRNA interacts with ribosomes, and under the catalysis of ribosomes, protein with specific therapeutic function is accurately translated according to the genetic information carried by mRNA. These protein play a role through a series of complex biological ways, and finally achieve the therapeutic effect on diseases.

Gene Integration and Long-term Expression

In the special environment of non-splinter cell, AAV genome usually exists stably in the nucleus in the form of an episome. This free form of AAV genome has unique advantages. It can continuously and stably express therapeutic genes in the nucleus, thus providing long-term and lasting therapeutic effects for gene therapy. More importantly, the free AAV genome avoids the risk of insertion mutation that may be caused by integration into the host genome, which greatly improves the safety of gene therapy.

Although AAV vector mainly plays a role in the form of free body, it still inevitably faces some challenges in the long-term gene expression process, such as the loss of free body and gene silencing, which seriously affects the long-term stability and effectiveness of gene therapy, and is also one of the key scientific problems that need to be deeply studied and solved in the field of gene therapy.

Basic strategy of the use of AAV as a vector in gene therapy (Blasiak et al., 2024)

Safety of AAV Gene Therapy

The safety of AAV gene therapy is a core issue closely concerned by scientific and clinical fields. In-depth analysis of the safety of AAV gene therapy is very important to promote its wide application from laboratory to clinic and improve the treatment prospect of patients with refractory diseases.

Inherent Safety of AAV Carrier

AAV itself is a non-pathogenic virus, which will not cause human diseases in nature. In the application of gene therapy, AAV vector constructed by genetic engineering technology removes most of its own genes, and only the key reverse terminal repeats (ITRs) are reserved for the replication, packaging and integration of the vector. This simplified design greatly reduces the possibility of adverse reactions caused by the carrier itself.

Safety of Immune Response

When AAV vector enters the human body, the first challenge is humoral immunity. Because some people are naturally infected or under other unknown circumstances, neutralizing antibodies against different serotypes of AAV may already exist in the body. These neutralizing antibodies can combine with AAV vector to prevent it from infecting target cells and reduce the effect of gene therapy. More seriously, in some cases, the antibody-carrier complex may activate the complement system, trigger a systemic immune response, and threaten the life and health of patients.

Risk of Gene Integration

Although AAV vector exists in the nucleus in most cases as a free body, which avoids the risk of random integration into the host genome and causes insertion mutation, AAV genome still has the ability to integrate into the host cell genome under certain conditions. The most common integration site of AAV is AAVS1 site on the long arm of chromosome 19. Compared with random integration, this relative site-specific integration mode reduces the risk of proto-oncogene activation or tumor suppressor gene inactivation caused by insertion mutation to some extent. However, even site-specific integration can not completely rule out the possibility of affecting the expression of surrounding genes.

Assays used to detect immunity to AAVs (Dhungel et al., 2024)

How to Improve AAV Gene Therapy Efficiency

It is of great significance to explore deeply and adopt effective strategies to improve the efficiency of AAV gene therapy for promoting the wide application of this technology in clinical treatment.

Optimizing AAV Carrier Design

AAV capsid protein plays a central role in determining vector targeting and infection efficiency. The transformation of capsid protein by rational design or directed evolution can significantly improve the efficiency of AAV gene therapy.

The structure of vector genome has a significant impact on the therapeutic efficiency. When constructing AAV vector, it is very important to select and design regulatory elements such as promoters and enhancers reasonably. Strong promoters can drive high-level expression of therapeutic genes, but may cause immune response or genotoxicity. Therefore, it is necessary to select tissue-specific or controllable promoters according to the target cell type and treatment requirements.

Improve Drug Delivery Strategy

Different routes of administration have significant effects on the efficiency of AAV gene therapy. Intravenous injection is a common way of systemic administration, which can make the carrier widely distributed in the whole body tissues, but it may lead to unnecessary distribution of the carrier in non-target tissues and reduce the effective delivery to the target tissues.

In contrast, local administration, such as intramuscular injection and intracerebral injection, can make the vector directly act on the target tissue, increase the concentration of the vector in the target tissue and enhance the efficiency of gene transduction. At the same time, it is also very important to arrange the administration time interval reasonably. Optimizing the administration time interval can avoid excessive immune response, maintain the long-term effective expression of therapeutic genes and significantly improve the efficiency of gene therapy.

Combined Therapy

The immune response of immune system to AAV vector is an important factor affecting the treatment efficiency. Combined use of immunomodulatory drugs can inhibit the immune response to some extent and improve the efficiency of AAV gene therapy. However, the use of immunomodulatory drugs needs to carefully weigh the side effects such as the increased risk of infection caused by immunosuppression. Therefore, it is necessary to study the optimal dosage, time and mode of immunomodulatory drugs to ensure the efficiency of gene therapy and the safety of patients.

Combining AAV gene therapy with other treatment methods can exert synergistic effect and improve treatment efficiency. In tumor therapy, AAV-mediated tumor suppressor gene therapy is combined with chemotherapy or radiotherapy, which opens up a new way to improve the efficiency of AAV gene therapy.

Schematic of AdAAV complex assembly and organization (Collins et al., 2024)

Application of AAV in Gene Therapy

Introducing AAV vectors encoding tumor suppressor genes (such as p53, PTEN, etc.) into tumor cells can restore the function of tumor suppressor genes that are missing or abnormal in tumor cells, inhibit the growth of tumor cells and induce their apoptosis. In the research of lung cancer treatment, using AAV to directly inject p53 gene into tumor tissue can effectively induce tumor cell apoptosis, inhibit tumor angiogenesis, and then inhibit tumor growth.

AAV can be used to deliver genes encoding immunomodulatory factors and enhance the immune response of the body to tumor cells. The gene encoding interleukin-12(IL-12) is introduced into tumor microenvironment through AAV vector, which can activate T cells and natural killer cells and enhance the body's anti-tumor immunity.

Ex vivo validation of capsid variants at D10 post-transduction (Giacomoni et al., 2024)

Conclusion

At present, AAV has achieved initial success in the field of gene therapy, and has made achievements in the treatment of single-gene genetic diseases, tumors and neurodegenerative diseases. However, it still faces difficulties in production technology, safety and targeting. In the future, multidisciplinary integration is expected to break through the bottleneck and promote AAV gene therapy to be applied to clinic more safely and efficiently.