Vector of Choice for Gene Therapy: AAV

What is the Gene Therapy?

Currently, gene therapy is one of the highly regarded fields of treatment because it directly intervenes at the root cause of diseases rather than just addressing symptoms. This therapy has shown success in preclinical trials by regulating gene expression or silencing in diseased cells. However, gene therapy still faces several limitations in clinical applications, including immunogenicity, carcinogenicity, off-target effects, and the efficacy of gene vectors. Recombinant viruses are widely employed as vectors in gene therapy, particularly playing crucial roles in cellular uptake, gene integration, and long-term expression. Nonetheless, the use of recombinant viruses also presents challenges such as immunogenicity, potential carcinogenicity, and complex manufacturing processes. To address these challenges, scientists have developed non-viral vectors with characteristics such as immunogenicity avoidance, strong adaptability, and controllable quality. However, non-viral vectors typically exhibit lower transfection efficiency, potentially leading to passive gene expression. Adeno-associated viruses (AAV), due to their lower immunogenicity and specific integration capabilities, have garnered significant attention in the field of gene therapy.

Nucleic acid drugs for gene therapy.

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What is an Adeno-Associated Virus (AAV)?

Adeno-associated virus (AAV) is currently known as the simplest non-enveloped single-stranded DNA virus, belonging to the family of Parvoviridae. It consists of a protein capsid and a 4.7kb single-stranded DNA genome. The protein capsid is composed of three subunits: VP1, VP2, and VP3. At both ends of the AAV genome are two T-shaped inverted terminal repeat (ITR) sequences. These ITRs serve as the starting point for viral DNA replication and trigger signals for virus packaging. The rep gene within the AAV genome encodes four proteins involved in virus replication: Rep78, Rep68, Rep52, and Rep40. AAV itself cannot replicate and relies on other viruses such as adenovirus, herpesvirus, or papillomavirus for replication.

Advantages of AAV-Based Gene Therapy

• High Safety and Extremely Low Immunogenicity

Wild-type AAV is a naturally defective virus, and recombinant AAV does not integrate into the genome. It has been found to be unrelated to any diseases, exhibiting extremely low immunogenicity.

• Broad Host Range for Infection

AAV can infect both dividing and non-dividing cells.

• High Tissue Tropism

AAV comprises hundreds of serotypes, allowing for specific infection of different tissues or organs.

• Strong Infection Spread and Specificity

Characterized by small size and high titer, AAV demonstrates strong diffusion ability in the body and exhibits high tissue specificity.

• Long In Vivo Expression Time

With titers reaching up to 5*10¹³ GC/mL, AAV can achieve long-term stable expression in vivo.

AAV Gene Therapy Strategies

Gene Replacement

The most common therapeutic intervention mediated by AAV is gene replacement, characterized by introducing functional copies of genes to treat monogenic diseases. This approach is primarily used for rare and untreatable diseases.

Gene Addition

Gene addition is the most widespread application of AAV gene therapy because it can be used to treat more common complex diseases, such as chronic, autoimmune, or infectious diseases. Examples of AAV used for gene addition include ongoing clinical trials for treating rheumatoid arthritis.

Gene Silencing

Different from gene replacement, which attempts to overcome functional loss-of-function mutations, gene silencing, also known as RNA interference (RNAi), can be used to silence harmful gain-of-function mutations. In this approach, AAV transgenes encode for miRNA precursors, which are processed by the target cell's own RNAi mechanism to generate miRNAs, small non-coding RNA chains capable of base-pairing with complementary sequences within mRNA to inhibit their expression. Most efforts using AAV for gene silencing are still in preclinical research because silencing poses several specific safety issues that must be addressed. One major concern is the possibility of off-target silencing. Another issue is the mechanism of RNAi saturation, in which the expression of transgenic miRNAs may overwhelm and disrupt the production of endogenous miRNAs, leading to cellular toxicity.

What is Recombinant AAV (rAAV)?

AAV can be engineered through coat modification, surface coupling and encapsulation to address the limitations of natural AAV. The engineered AAV vector, devoid of all encoding sequences for AAV genome proteins Rep or Cap genes, retains only the inverted terminal repeat (ITR) sequences at both ends as packaging signals. Recombinant adeno-associated viruses (rAAV) used as gene therapy vectors carry capsid proteins almost identical to those of wild-type AAV. However, the portion of the genome encoding viral proteins is completely replaced by therapeutic transgenes. Unlike retroviruses, rAAV does not integrate into the host genome upon cell infection, thus avoiding the risk of cancer development. Since 1984, when the first rAAV vector was shown to transduce exogenous genes into mammalian cells, rAAV has gradually been utilized as a gene delivery vector in life science research.

Advantages of Engineered AAV

One common goal of AAV engineering is to avoid neutralization and inactivation by neutralizing antibodies (nAbs) in the bloodstream after systemic administration. Modifying the binding sites of neutralizing antibodies to prevent neutralization is a promising approach. However, many of these sites are essential for AAV transduction, making it challenging to modify them without compromising their function. Additionally, surface tethering and encapsulation using polymers, lipids, and hydrogels can protect AAV capsids from nAbs, allowing them to evade detection by the immune system and antibody reactions.

Another benefit of AAV engineering is to improve targeted delivery and activation by attaching tissue-specific ligands to capsids, surface coupling, and encapsulation materials. Passive release of AAV from encapsulation materials (such as large-pore silicon particles) can prolong circulation time and delivery time. Materials that respond to external and internal stimuli (such as light, pH changes, or enzymatic degradation) allow AAV to be released or activated in target tissues or cell types, while AAV bound to magnetic particles can be directed to targets by applying a magnetic field. Engineered AAV can also be used to overcome the limited genome size and enable multimodal therapy by combining multiple therapeutic modalities. Multiple genes in the mutated pathways of diseased cells can be reprogrammed by delivering two genes, one via AAV and the other via a second nucleic acid in a surface-coupled or encapsulated AAV vector. Incorporating growth factors encoding AAV into tissue engineering scaffolds can promote tissue regeneration through gene activation and structural support. Combining administration with AAV can interfere with abnormal activities and reprogram diseased cells simultaneously, resulting in synergistic therapeutic effects.

Prospects for AAV in Gene Therapy

With the emergence of clinically approved products on the global market and a growing number of ongoing successful clinical trials, AAV is at the forefront of gene therapy, but its smaller genome and neutralizing antibodies limit its use in many diseases. Engineered AAV can address these issues while providing additional benefits including controlled and stimulus-responsive release, extended tissue targeting, and multimodal cotransport. Multimodal engineered AAV addresses the limitations of AAV and single-modality gene therapy because they not only extend the therapeutic potential, but also synergize multiple aspects of the disease itself. Engineered AAV that combines the second therapeutic modality, which is currently being explored, is an untapped area of gene therapy that could go beyond traditional single-modality gene delivery systems. While further technological advances will need to be validated by advancing additional clinical trials, multimodal engineered AAVs are already showing promise for the future of gene therapy.