Research progress and prospect of adeno-associated virus in gene therapy

Preface

Gene therapy is currently one of the most popular therapeutic areas because it targets the underlying cause of the disease, not just the symptoms

A key consideration for the success of gene therapy is the vector used to deliver therapeutic nucleic acid to express or silence genes

In order to solve these limitations, people have developed non-viral vectors that can avoid immune response, have strong adaptability, and controllable quality.

Adeno-associated virus (AAV) is gaining momentum in the field of gene therapy, and more and more clinical trials use it for various treatments

AAV is considered to have better safety due to its lower immunogenicity and site-specific integration ability

At present, people’s research is mainly focused on engineered AAV, through molecular modification of its capsid, coupling and complexing with small molecules and polymers, and packaging in various artificial and natural carriers (including vesicles, hydrogels and Polymers), used to improve safety, effectiveness and multi-modal processing

The results of clinical trials using AAV vectors also emphasize the key role of developing effective, safe, and universal vectors to achieve ideal gene therapy

The history and virology of AAV

AAV was first discovered in the 1960s and is considered to be a contaminant of adenovirus culture

AAV is a small non-enveloped parvovirus with a genome size of about 4.

AAV cannot be copied independently

AAV can infect not only actively dividing cells, but also quiescent cells, which makes it particularly valuable for many cell populations where viral and non-viral vectors are not sensitive to gene delivery, such as retinal cells and neuronal cells

Clinically approved AAV-based gene therapy drugs

In the past 20 years, the relevance of AAV vector-based treatments in clinical transformation has continued to increase, and currently accounts for 8.

The first AAV-based gene therapy drug approved for clinical use in Europe is the fat gene tiparvovec (AT), which is used to treat lipoprotein lipase deficiency (LPLD), a rare autosomal recessive disease

There are currently 17 gene therapies approved by the US FDA, including the AAV vector voretigene neparvovec rzyl (VN) developed by Spark Therapeutics under the Luxturna trademark in 2017

The second aav-based gene therapy approved by the FDA in 2019 is Onasemogene abeparvovec xioi (OA), developed by AveXis under the trade name Zolgensma

 Application of AAV therapeutic intervention strategies

The most common AAV-mediated therapeutic intervention is gene replacement, which is characterized by the introduction of functional gene copies to treat single-gene diseases

Gene addition is one of the most widely used AAV gene therapy, because it can be used to treat more common non-single gene complex diseases, such as chronic, autoimmune or infectious diseases
.
Examples of gene addition using AAV include ongoing clinical trials for the treatment of rheumatoid arthritis
.
Currently, Arthrogen is undergoing a phase 1 clinical trial (NCT02727764)
.
The AAV vector carries the IFN-β transgene.
IFN-β is an anti-inflammatory mediator that plays a role in bone and cartilage protection
.

Unlike gene replacement, which attempts to overcome loss-of-function mutations, gene silencing, also known as RNA interference (RNAi), can be used to suppress harmful gain-of-function mutations
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In this method, the AAV transgene encodes a miRNA precursor, which is processed by the target cell's own RNAi mechanism to produce miRNA.
This is a small non-coding RNA strand that can establish bases with complementary sequences in the mRNA pair.
To suppress its expression
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Most efforts to use AAV for gene silencing are still in the preclinical research stage, because silencing has several specific safety issues that must be addressed
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A major issue is the possibility of non-target silence
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Another problem is the mechanism of RNAi saturation
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In this mechanism, the expression of transgenic miRNA may overwhelm and destroy the production of endogenous miRNA, leading to cytotoxicity
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Currently, Unique has started a clinical trial (NCT049493) using AAV gene therapy to treat Huntington's disease
.

 Challenges of AAV applications

Most AAVs successfully used in preclinical and clinical research are limited to the natural capsid serotype, but the wide clinical application of these AAV serotypes has obvious limitations
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The presence of neutralizing antibodies against AAV is still an important obstacle to systemic delivery
.

These neutralizing antibodies interfere with AAV's entry into target cells, intracellular transport and unpacking in the nucleus, thereby preventing transduction
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In order to better understand the immune response caused by AAV exposure, studies have been conducted to detect the IgG subclasses produced and found that it is mainly IgG1
.

Epidemiological studies have shown that neutralizing antibodies with different seropositivity rates can be found in 30-60% of the population
.
The most common of these neutralizing antibodies is AAV2, followed by AAV1
.

Another problem with aav-mediated gene therapy is that the size limit (4.
7kbp) of the genome, including itr, leaves only ~4.
5kbp size space, so that the target index of the transferred gene is only "small transgene Fragment expression"
.

Engineering AAV

AAV can be designed through capsid modification, surface coupling, and encapsulation to solve the limitations of natural AAV
.

The common goal of AAV engineering is to avoid the inactivation of neutralizing antibodies in the blood circulation after systemic administration
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Preventing neutralization by modifying the capsid binding sites of neutralizing antibodies is a promising method
.
However, many of these sites are critical to the transduction of AAV, so modifying it without compromising its function is a challenge
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In addition, surface tethering and encapsulation using polymers, lipids, and hydrogels can protect the AAV capsid from nAbs, making it evade detection by the immune system and antibody responses
.

Another benefit of AAV engineering is to improve targeted delivery and activation by binding tissue-specific ligands to the capsid, surface coupling and encapsulating materials
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Passive release of aav from packaging materials (such as silicon particles with large pores) can extend the production cycle and delivery time
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Materials that respond to external and internal stimuli (such as light, pH changes, or enzymatic degradation) allow AAV to be released or activated in the target tissue or cell type, while the magnetic particle-bound AAV is directed toward the target by applying a magnetic field
.

Engineered AAV can also be used to overcome the limitation of genome size and combine multiple treatment modalities for multimodal treatment
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Multiple genes in the mutation pathway of disease cells can be reprogrammed by delivering two genes, one through AAV and the other through the second nucleic acid in the surface-coupled or encapsulated AAV vector; growth factors promote tissue through gene activation and structural support Regeneration; Co-administration and AAV simultaneously interfere with abnormal activities and reorganize diseased cells, thereby producing a synergistic therapeutic effect
.

prospect

With the emergence of clinically approved products in the global market and more and more successful clinical trials, AAV is at the forefront of gene therapy, but its smaller genome and neutralizing antibodies limit its application in many diseases
.

Engineered AAV can solve these problems while providing additional benefits, including controllable and stimulating response release, expanded tissue orientation, and multimodal transport
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Multi-mode engineering AAV solves the limitations of AAV and single-mode gene therapy, because they not only expand the therapeutic potential, but also can act synergistically on multiple aspects of the disease itself
.

The engineered AAV that is currently being explored in combination with the second treatment method is an undeveloped field of gene therapy, which can surpass the traditional single-mode gene delivery system
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Although further technological advancement requires more clinical trials to verify, the multi-modal engineering AAV has shown broad prospects in future gene therapy
.