AAV Vectors:Opening a New Era of Gene Therapy

Gene therapy is a method that aims to change individual gene expression or correct abnormal genes to treat diseases. Currently, the field of gene therapy is developing rapidly. However, different types of genetic diseases have different pathogenesis, and the phenotypes reflected in patients are functional damage or loss of different tissues and organs. Therefore, how to efficiently deliver gene therapy drugs to target tissues or target organs has become the key to disease treatment. Adeno-associated virus (AAV) is widely used in gene therapy drug delivery due to its safety, efficiency and stability.

Discovery and Biological Characteristics of AAV

It has been more than 60 years since AAV was discovered. It was first discovered in the laboratory during the preparation of adenovirus (AdV) preparations. In the more than ten years since AAV was discovered, its basic biological characteristics (such as genome composition, structure and biological function, etc.) have been gradually analyzed. AAV belongs to the Parvoviridae family and is usually silent after infecting host cells. It can only replicate itself in the presence of helper viruses (such as adenovirus and herpes virus). AAV can transduce dividing cells and non-dividing cells, and express in non-dividing cells for a long time. AAV exists in many vertebrate species, including humans and non-human primates (Non-Human Primates, NHPs). There is currently no report that AAV can cause human disease. In the 1980s, scientists successfully cloned the infectiousness of AAV2 in Escherichia coli and determined its DNA sequence and the genetic elements that make up the virus.

AAV virus consists of an icosahedral protein capsid with a diameter of about 26 nm and a single-stranded DNA genome of about 4.7 kb. There are T-shaped inverted terminal repeats (ITRs) at both ends of the genome. ITRs serve as the origin of viral replication and packaging signals. The REP and CAP genes are contained between the two ITRs. The REP gene is responsible for encoding four proteins required for viral replication and regulation, and they are named after their molecular weight: Rep78, Rep68, Rep52 and Rep40; the CAP gene is responsible for encoding the VP1, VP2 and VP3 proteins that make up the capsid. The viral capsid is composed of 60 capsid protein monomers in a ratio of 1:1:10 (VP1: VP2: VP3). In addition, the embedded part also encodes an assembly activating protein (AAP) to promote the assembly of virus particles. The AAV genome exists mainly in the form of free circular dsDNA in the cell nucleus, so it can maintain stable gene expression. According to the differences in AAV serotypes, the genomes corresponding to different serotypes are artificially numbered: for example, ITR2, REP2, and CAP2 in AAV2.

rAAV vector

The early research on AAV provided the basis for engineering transformation and application of AAV as a gene delivery vector. The sequences between the ITRs at both ends of the AAV genome were replaced with elements with gene therapy effects, and transformed into a gene expression cassette plasmid (GOI plasmid), while the REP and CAP genes in its genome were cloned separately into a new eukaryotic expression vector as a capsid plasmid (Rep/Capplasmid), and co-transfected with a helper virus expression plasmid (Helperplasmid) into eukaryotic cells, and then self-assembled in eukaryotic cells to form a new recombinant adeno-associated virus (rAAV) with the target gene sequence. Its special structure and plasmid packaging construction characteristics are the basis of AAV as a viral vector.

Currently, the construction of rAAV is mainly based on the genome skeleton of AAV2, and the REP gene of AAV2 is retained. By replacing the capsid protein genes of different serotypes, rAAVs of different serotypes can be obtained. For example, rAAV2/9 means that the main skeleton of this recombinant virus is AAV2 (including ITR2 and REP2), and its capsid protein gene is CAP9 of wild-type AAV9. Due to the limitation of its own genome capacity, the gene load of rAAV vector is about 4.7 kb. rAAV vector consists of two parts: capsid and gene expression cassette. The capsid determines the transduction characteristics, and the gene expression cassette determines the gene expression characteristics. Due to the limited targeting of wild-type AAV, its application in gene therapy is also limited. Studies have shown that almost all wild-type AAVs have a high affinity for the liver. For the treatment of liver sites, low doses of rAAV vectors have obvious effects, but if the treatment target is in other locations, to achieve an effective concentration, the dose of rAAV vector must be greatly increased.

Challenges of Gene Therapy Drugs Delivered by rAAV

At present, there are many gene therapy drugs delivered by rAAV on the market worldwide, covering various types of diseases. rAAV gene therapy has entered a stage of explosive growth, and the number of gene therapy patents and papers and clinical projects have shown an increasing trend year by year. However, rAAV drugs still face some challenges: 1) Immune response to viral capsid and delivered cargo. The applicable patients of some drugs will exclude patients with pre-existing antibodies to the vector. In addition, wild AAV naturally tends to the liver, and the natural first-pass effect of the liver organ, resulting in liver toxicity and off-target effects. 2) AAV belongs to the genus Parvovirus, with limited packaging capacity. rAAV is limited by packaging capacity and cannot be administered multiple times. 3) The production process is complex and the production cost is high. The GMP-level drug production of rAAV is subject to multiple quality controls, and its dosage is extremely large. Most diseases require personalized customization for different gene mutations, which is extremely costly.

rAAV Expression Cassette Modification

By designing and modifying the GOI plasmid, rAAV can carry the target gene. The complete expression cassette of rAAV includes ITR, promoter, target gene sequence and regulatory elements. Modification of the rAAV genome expression cassette, such as using tissue or cell-specific promoters, adding enhancers and introns with regulatory functions, etc., can not only improve the expression efficiency of rAAV, but also reduce humoral and cellular immune responses. The choice of promoter is directly related to the expression efficiency of the vector in vivo. The broad-spectrum promoters of mammals include CMV, CAG, EF1α, SV40, UBC, etc. If the gene expression is to be limited to specific tissues and organs, constitutive promoters such as Syn, MHCK7, TBG, etc. can be selected to improve the safety of the treatment process. At the same time, the specific promoter needs to be selected according to the target sequence to be expressed and the purpose of gene therapy.

rAAV Capsid Modification

Due to the existence of different serotypes of AAV, there are also differences in their tissue and organ targeting, and almost all AAV serotypes are highly targeted to the liver, thereby weakening the therapeutic effect of non-liver targeted diseases. In addition, as an exogenous substance, the immune response provoked by the AAV capsid itself is also one of the bottlenecks hindering gene therapy. Early studies analyzed the three-dimensional structure of AAV through X-ray crystal diffraction and cryo-electron microscopy, and found that there are 9 common variable regions (VRⅠ-VRⅨ) on the subunits that make up the AAV capsid. These regions determine the differences between different serotypes of AAV, including receptor recognition, gene transduction efficiency, and immune response. At the same time, the analysis and application of the functions of these regions have greatly promoted the development of AAV vectors. The optimization of AAV capsid protein has become the main research direction. The core of capsid modification is to change the recognition relationship between the original capsid antigen domain and tissue and cell receptors.

Production Cost Control

In terms of production cost control, the production of rAAV can be greatly increased based on the new baculovirus production scheme and the controllable adenovirus scheme. In the future, how to use the self-replication ability of the virus in the production stage is the key to breaking through the production limit. For the narrow application range of drugs, universal treatment strategies can be developed to quantify and normalize the variables of the same type of diseases as much as possible. In addition, the export direction of clinical transformation will no longer be limited to gene silencing, substitution, addition and other methods based on rAAV, and the characteristics of its delivery vector can be fully utilized.

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