Custom AAV-gene therapy vectors production-BrainCase (Adeno-associated Virus，AAV)

What is AAV

Adeno-associated virus (AAV) belongs to the Parvoviridae family (parvoviridae), a single-stranded DNA virus, and the modified rAAV virus tool has been widely used in gene expression, gene manipulation and gene therapy. It is the simplest type of replication-defective virus found so far, and is dependent on the presence of adenovirus, HSV, and other auxiliary viruses to produce progeny.

The genome of AAV is a single-stranded DNA molecule ~4.7 kb long. The AAV replicase protein (Rep) and capsid protein (Cap) genes are flanked by inverted terminal repeat (ITR) sequences. The virus particle does not have an envelope and is an icosahedron with a diameter of ~20 nm. The rAAV genome only conserves the AAV-derived ITRs, and all viral DNA between the ITRs is replaced by the gene of interest. The AAV ITRs serve as cis-elements, while the AAV Rep, Cap, and helper genes are provided in trans for viral replication and packaging. Therefore, rAAV not only has an excellent safety profile, but also inherits many advantages of wildtype AAV, such as low immunogenicity, multiple serotypes, a wide range of infected host cells, and the ability to mediate the long-term stable expression of foreign genes in vivo.

Table 1 Comparison of several commonly used viral vector tools  

AAV Vector Production: About AAV Serptype

In the wide application in neuroscience, rAAVs are increasingly useful tools. Different AAV serotypes exhibit different tropisms and can transduce neural cells in vivo and in vitro. In studies of the structure and function of neural circuits, most AAV serotypes generally exhibit excellent neuronal tropism. These include well-known serotypes such as AAV2/2 (also commonly referred to as AAV2), 1, 8, 9, DJ, php.s, and others. Additionally, AAV5 can efficiently target astrocytes, especially when used in conjunction with astrocyte-specific promoters such as GFAP or GfaABC1D. It is worth noting that transduction of microglial cells appears much more challenging. As of now, only capsid-modified AAV6, as well as the AAVMG1.2 series of serotypes screened by the Minmin Luo laboratory at Peking University, have been shown to enhance the transduction ability of microglial cells. Host cells infected with rAAVs are unable to produce new viral particles, rAAVs cannot efficiently transmit trans-synaptically between neurons. The anterograde, retrograde, and bidirectional transport properties of AAVs are serotype- and concentration-dependent. Therefore, it is very important for researchers to select the appropriate serotype of AAV. In general, most AAV serotypes prefer to enter the cell bodies of neurons at the injection site, and subsequent cellular trafficking may result in the spread of transgenic products throughout the whole neuron. This property may be useful for labeling neuronal projections and manipulating projection targets.

Table 2 Tissue infection tropism of different serotypes of rAAV

AAV Vector Production: About Promoter

Brain Case offers a variety of promoters for selection, which, when combined with different serotypes of AAV, can enhance the specificity of expression of the gene of interest in targeted tissues and cells. Promoters are DNA sequences that control gene transcription and can activate gene expression under specific conditions, selecting and screening appropriate promoter region is a key factor in achieving virus-specific expression. Typically, gene expression is influenced by cis-acting elements and trans-acting factors transcription factors. For example, enhancers of some genes are located far upstream or even downstream of the gene, so selecting and screening appropriate promoters for virus vectors is a crucial step. By selecting the appropriate promoter and AAV serotype, it is possible to increase the targeted expression of the gene of interest in specific tissues and cells, allowing for more precise gene regulation and therapeutic applications.

Table 3 AAV Vector Production: list of promoters targeting different organs and cells(Partial) 

Gene Therapy AAV Vectors Application

Clinically, AAV programs have largely focused on degenerative retinal disorders, which are most efficiently targeted to the outer retina via subretinal injection and the inner retina and other cell types flanking the vitreous via intravitreal injection. In the only cochlear gene transfer study to date that has been performed in humans, human adenoviral vector was injected via an oval window procedure. AAV intraparenchymal injections have proven successful in numerous pre-clinical studies using mouse models of lysosomal storage diseases (LSDs), Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD). For motor neuron disorders, multilevel injections into the spinal cord parenchyma have been successful in delivering therapeutic agents in amyotrophic lateral sclerosis (ALS) mouse models. While this therapy poses significant risks and challenges in large mammals or humans, some teams have made significant progress in this approach.

Custom AAV Service Overview

Brain Case specializes in designing and constructing AAV vectors for interfering with or expressing exogenous genes, with applications including overexpression of target genes, CRISPR-mediated gene editing, shRNA-mediated gene interference, and utilizing AAV to deliver the gene backbone of your interest, thereby achieving gene regulation.

Custom AAV Vector Prodcution Service Process

Please provide the gene template, vector backbone information, indicate which AAV serotype you will use or your personalized ideal design so that we can communicate and finalize the construction plan with you. 

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Contact BrainCase for Custom AAV

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