How does AAV Gene Therapy Work

The Mechanism of AAV as a Gene Therapy Vector

Recombinant AAV particles enter cells through clathrin-mediated endocytosis by binding to glycosylation receptors expressed on the host cell surface. After acidification of the endosome formed by endocytosis, the conformation of the VP1/VP2 part of the viral capsid changes, causing the virus to escape from the endosome and enter the nucleus through the nuclear pore. After entering the nucleus, the single-stranded DNA is released from the capsid. At this point, single-stranded DNA cannot yet be transcribed, and they need to become double-stranded DNA. Single-stranded DNA can be synthesized using the host cell's DNA polymerase to synthesize complementary strands, or two complementary strands released from different AAV particles can anneal to form double-stranded DNA.

The latest AAV genome design can design the single-stranded DNA carried in the capsid into a self-complementary sequence. The advantage of this sequence is that it can be transcribed without the step of replicating single-stranded DNA into double-stranded DNA. Compared with the traditional single-stranded AAV genome, its gene expression is faster and the expression level is higher. One drawback of this design, however, is that the transgene capacity carried by the AAV is halved.

The double-stranded AAV genome then uses ITRs to undergo intramolecular or intermolecular genome recombination. This process allows the AAV genome to become stable episomal DNA, resulting in the genome being able to continue gene expression in cells that are no longer undergoing mitosis.

AAV Gene Therapy Strategies

Gene replacement: The goal of this strategy is to deliver genes that express normal proteins to compensate for the effects of loss-of-function mutations. It is suitable for treating recessive single-gene diseases and has achieved great success in clinical trials. Glybera and Luxturna are good examples. Glybera is based on a recombinant AAV1 platform that delivers a transgene encoding lipoprotein lipase (LPL) and is used to treat patients with LPL deficiency. Luxturna uses the AAV2 platform to deliver the normal RPE65 gene. Both treatments use local injections into the patient's muscles and eyes.

The discovery of newer AAV serotypes allows researchers to deliver transgenes throughout the body through intravenous injection of AAV vectors. These new AAV vectors bring effective therapies for the treatment of hemophilia A and B (targeting the liver), DMD (targeting muscles throughout the body), and SMA (targeting the CNS, including the spinal cord). One example is Zolgensma, a gene therapy that uses an AAV9 vector to deliver an SMA transgene encoding the survival motor neuron protein (SMN). In many diseases, simply expressing normal genes in some cells is enough to alleviate symptoms.

Gene silencing: As opposed to gene replacement, the goal of gene silencing is to treat single-gene diseases caused by gain-of-function gene mutations, such as Huntington disease. RNAi therapy is currently the preferred strategy for AAV gene silencing platforms. However, compared with the rapid progress of synthetic RNAi therapies, RNAi therapies based on AAV delivery platforms are still in the preclinical development stage. Gene silencing needs to occur in most tissues to have a meaningful therapeutic effect. In some organs (such as the brain), this is still a challenge for AAV vectors.

In addition to the RNAi strategy, using the CRISPR-Cas system to target RNA is also a gene silencing strategy. However, the obstacles that this strategy needs to overcome are the limited size of the transgene that recombinant AAV can accommodate, and the Cas protein found in bacteria may trigger an immune response, thus reducing the efficacy.

Delivery of new genes: In addition to treating single-gene diseases, AAV-mediated gene therapy has the potential to treat complex genetic diseases or infectious diseases by delivering new genes. For example, heart failure and infectious diseases represent disease areas with high unmet need, and by introducing novel genes, gene therapy can deliver growth factors, modulate signaling pathways associated with heart failure, and express antibodies that neutralize lethal viral infections. Current clinical trials are testing treatments for HIV infection that use muscle as a "biofactory" to produce therapeutic antibodies.

Gene therapy is an innovative treatment platform that has gained industry attention. As a powerful means of delivering transgenes, the research and development of AAV vector platforms is also experiencing exponential growth. Although the study of AAV belongs to the field of virology, the process of converting AAV into a gene therapy vector requires multidisciplinary joint research. Breakthroughs in molecular biology, bioinformatics, infectious diseases, structural biology, immunology, and genomics all play an indispensable role in the successful development of AAV vector platforms.

The development of AAV vector platforms still needs to overcome several challenges, including how to cost-effectively generate sufficient quantities of therapeutic AAV vectors and overcoming the immune rejection of AAV vectors and transgenic products by the human immune system. Overcoming these challenges is critical to the promotion of AAV-mediated gene therapy.

Anti-AAV Antibody ELISA Kit
AAV Antibodies and Titration ELISA