Advantages And Challenges of Clinical Application of AAV Gene Therapy

Gene therapy is one of the hottest therapeutic fields at present, and multiple gene therapies have been approved by regulatory agencies in recent years. A number of pipelines that have entered late-stage clinical studies are also approaching approval for marketing. At present, most gene therapy applications still use viral vectors. These viral vectors have been modified so that they are no longer pathogenic. Widely used viral vectors include adenoviral vectors (ADV), lentiviral vectors (LV), herpes simplex virus vectors (HSV), poxvirus vectors (PV) and adeno-associated virus vectors (AAV).

Among them, AAV is the most widely used viral vector in gene therapy projects currently under research. So, what are the clinical advantages of AAV vectors? What difficulties and challenges currently exist?

AAV belongs to the family Parvoviridae and is a non-enveloped, single-stranded linear DNA virus with a size of approximately 26 nm. It was first discovered in the culture of rhesus monkey kidney cells. The AAV genome is approximately 4700 bp, including two upstream and downstream open reading frames (ORFs), located between two inverted terminal repeats (ITRs) consisting of 145 nucleotides each.

AAV has been widely used in basic research and clinical trials due to its wide host range, high safety, low immunogenicity, stable expression and stable physical properties. And AAV has become one of the most commonly used gene therapy vectors in the world.

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Clinical Application Advantages of AAV

➤ Non-pathogenic and highly safe

Compared with other viral vectors, AAV is a replication-defective virus and has no ability to replicate autonomously. Usually AAV can only complete replication if it simultaneously infects cells with, for example, an adenovirus or a herpesvirus. In the NIH's relevant ratings for biotechnology products, AAV belongs to RG1, which is the safest level, while adenovirus vectors belong to RG2 and retroviral vectors belong to RG3. AAV has not yet been found to be pathogenic. A number of clinical trials targeting conditions such as hemophilia A, hemophilia B, retinal disease and spinal muscular atrophy (SMA) have proven AAV to be a safe and effective treatment tool.

➤ Low immunogenicity

The immunogenicity of AAV is also significantly lower than that of adenovirus or lentivirus, and the immune response induced after in vivo injection is weak and short-lived. Large-dose systemic injections, especially when administered into the tail vein, may lead to the production of trace amounts of antibodies, but very few infected cells are cleared by the cellular immune system. This is thought to be related to AAV's weak infection of antigen-presenting cells (APCs). When AAV is used to locally infect muscles, brain, eyes and other tissues, its efficiency will not be greatly affected even if repeated infections occur.

➤ Long-term stable expression

In cells that are basically non-dividing, AAV can stably express foreign genes for a long time and will not interrupt other genes of the host. AAV can be expressed for more than half a year, and its expression can even be detected after 2 years in clinical trials, which is something other vectors cannot achieve.

➤ Multiple serotypes and rich targeting properties

The capsid proteins of AAV are clustered together in an icosahedral configuration, forming spine-like protrusions on the structure. These protrusions mediate interactions with targeting cell surface proteins, giving different types of AAV the ability to specifically target different tissues. AAV2 was the first AAV vector discovered from adenovirus in the 1960s. To date, 13 primary AAV serotypes and more than 100 different variants have been discovered. Each serotype has unique cellular targeting properties and can target different cellular tissues.

➤ Wide host range

AAV can infect a wide range of mammalian cells and has been successfully used for the expression of human and non-human proteins. Compared with various vectors derived from other viruses, AAV vectors have proven to be more effective when used for gene expression in immunocompetent cells. Based on the above advantages, in recent years, the types of diseases treated by AAV have developed greatly in clinical trials, from initially targeting single-gene genetic diseases to a series of genetic defect diseases such as eye diseases, arthritis, tumors, muscular dystrophy, neurological diseases, and blood diseases.

Difficulties And Challenges in Clinical Application of AAV

Although AAV vectors have many clinical application advantages, they still have certain limitations, which also bring challenges to the clinical application of AAV.

Take Glybera, the first gene therapy drug approved for marketing, as an example. It uses AAV2 vectors to deliver genes that produce functional lipoprotein lipase to patients' skeletal muscles to reduce the incidence of pancreatitis in patients, but it cannot completely cure it. The indication for Glybera is too rare (the incidence is about one in a million) and the misdiagnosis rate is high. Due to its high treatment costs and low market demand, only one patient received the drug after its launch, and it had to be withdrawn from the market in 2017, after only three years on the market.

The product SGT-001 developed by another company, Solid Biosciences, mainly targets Duchenne muscular dystrophy and initially used the AAV9 vector. However, due to serious side effects in the first patient who received the treatment, the FDA once decided to suspend the clinical trial. After actively answering all questions, it was allowed to restart the trial.

These examples show that although AAV, as a star vector in gene therapy, has advantages that other viral vectors cannot match, there are still many limitations in clinical practice. The following is a detailed introduction to the difficulties and future development directions of AAV clinical application.

➤ Improve immunogenicity and tissue targeting

Both immunogenicity and tissue targeting depend on the AAV subtype or serotype. Since only a dozen AAV serotypes have been well described to date, many serotypes remain poorly characterized. Hundreds of AAV serotypes have been distinguished based on variable regions within the AAV capsid, with altered properties that influence immunogenicity and tissue tropism. Among them, particularly important are cell surface glycans that serve as AAV receptors, which are directly affected by the AAV variable region and affect tissue tropism. Therefore, the generation of new AAV capsids has become key to the preclinical development of gene therapy.

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➤ Cross the immune barrier

The blood-brain barrier is an important barrier to protect the central nervous system, but it also blocks the delivery and absorption of drugs, making the treatment of neurological diseases difficult. Therefore, researchers need to find drug delivery strategies that effectively breach the blood-brain barrier. Viviana Gradinaru’s team at the California Institute of Technology in the United States developed a new AAV variant—AAV.CAP-B10. The vector can break through the blood-brain barrier through blood injection and target nerve cells without being enriched in the liver. It avoids possible hepatotoxic side effects and provides a safer and more effective treatment option for brain diseases.

➤ Optimize the restrictions on carryable genes

AAV is a single-stranded DNA virus with a simple genome structure and a full length of approximately 4.7kb. Excluding the inverted terminal repeats (ITR), promoter and terminator at both ends, the length of the target gene that can be delivered is approximately 4.5kb. Compared with currently popular gene therapy vectors such as adenovirus (foreign gene capacity about 37kb), retrovirus (foreign gene capacity <8kb) or lentivirus (foreign gene capacity <5kb), AAV has a smaller gene delivery capacity, which will narrow its targeting indications to only the expression of small fragments of transgenes. Therefore, researchers need to use a series of methods to modify AAV. By modifying the genetic structure of viral vectors and expanding their vector capacity, AAV vectors can better meet the needs of different diseases.

➤ Reduce production costs

The control of large-scale production costs of AAV vectors is critical to the final product pricing. According to Nature Reviews Drug Discovery, more than 200 AAV-based cell and gene therapy clinical trials are currently underway, making it one of the most commonly used viral vectors in clinical trials. The main reason for the high price of AAV gene therapy products is that many aspects of their industrial production have not been fully optimized. How to reduce costs and expand commercial production capabilities is a major problem in the commercialization of AAV gene therapy.

➤ Expanded indications

By analyzing the types of diseases covered by gene therapy for metabolic diseases in the current clinical stage and preclinical stage, the types of diseases covered in the clinical stage are mainly concentrated in rare metabolic diseases, such as GM1 gangliosidosis, adenosine deaminase deficiency, Mucopolysaccharidosis type IIIA, etc. Gene therapies under research for metabolic diseases cover more than 50 diseases in the preclinical stage. Among them, diseases such as mannosidosis and lipoprotein lipase deficiency do not yet have clinical stage gene therapy. Patients with these diseases are expected to benefit from gene therapy in the future. As research continues to deepen, the application scope of AAV gene therapy can be further expanded.

Conclusion

AAV is at the forefront of gene therapy. For gene therapy using AAV vectors, the design and development of AAV vectors will continue to be improved in the next ten years, with the development direction of precise targeting and low-dose in vivo efficacy, thereby reducing the cost of AAV gene therapy and showing broader application prospects.