An AAV plasmid backbone is a DNA construct that serves as the template for creating AAV vectors in the laboratory. It contains essential elements required for the replication, packaging, and propagation of AAV vectors.
Here are some key components commonly found in an AAV plasmid backbone:
- Inverted Terminal Repeats (ITRs): ITRs are essential elements found at both ends of the AAV genome. They contain the necessary cis-acting sequences for viral DNA replication, packaging, and integration. ITRs serve as the “bookends” of the AAV vector genome, allowing it to be packaged into the AAV capsid.
- Origin of Replication (Ori): This element is required for the replication of the plasmid in bacterial hosts, such as Escherichia coli (E. coli).
- Antibiotic Resistance Gene: A selectable marker, often an antibiotic resistance gene, is included to allow for the selection of bacterial colonies containing the plasmid during cloning and propagation.
Inverted Terminal Repeats (ITRs)
Wild type ITRs are 145bp palindromes, GC rich and essential for packaging the viral DNA, replication, transcription and site-specific integration. ITRs are inherently unstable due to their secondary structure, palindromic nature, and high GC content.
- Secondary Structure: ITRs have the ability to form intricate secondary structures, such as hairpin loops and stem-loop structures. These secondary structures can make the DNA prone to rearrangements, deletions, and other mutations, particularly during replication or manipulation.
- Palindromic Nature: ITRs are palindromic, meaning they have a symmetrical sequence in which the sequence on one strand is the reverse of the sequence on the complementary strand. This palindrome structure can lead to mispairing and the formation of secondary structures, which can contribute to instability.
- High GC Content: GC-rich sequences are more stable due to the stronger hydrogen bonding between guanine and cytosine bases. However, the presence of GC-rich regions can also lead to the formation of stable secondary structures, which in turn can contribute to instability and DNA rearrangements.
The structural characteristics of ITRs can lead to challenges in maintaining their stability, particularly during plasmid propagation and AAV vector production. The instability of ITRs can result in the partial or complete loss of these important elements, which is critical for proper AAV replication, packaging, and gene delivery.
Methods to prevent ITR loss:
- Propagate AAV constructs in recombination-deficient bacterial strains, such as Stbl3s.
- Frequently detection ITRs by restriction digest using enzymes(like SmaI) or ITR Sanger sequencing or whole plasmid sequencing.
- Using modified ITRs. It has been demonstrated that an 14 bp terminal deletion at each ITR and 11 bp deletion in C-domain do not affect viability and could be used in AAV production[Savy et al., 2017][Tran et al., 2020].
Resistance markers
This selectable marker is used to identify and select bacterial cells that have successfully taken up the plasmid during cloning and propagation. The two common antibiotic resistance genes you mentioned are “Amp” (ampicillin resistance) and “Kana” (kanamycin resistance). Here’s how these antibiotic resistance markers are typically used in AAV backbone plasmids:
- Ampicillin Resistance (Amp): The “Amp” gene encodes resistance to the antibiotic ampicillin. Ampicillin is a beta-lactam whose mechanism is inhibiting cell wall synthesis. When a bacterial host (e.g., Escherichia coli) is transformed with a plasmid containing the Amp gene, the transformed bacteria can grow and divide in the presence of ampicillin, while non-transformed bacteria are inhibited. This resistance allows researchers to selectively grow bacterial colonies that have successfully taken up the AAV backbone plasmid.
- Kanamycin Resistance (Kana): The “Kana” gene encodes resistance to the antibiotic kanamycin. Kanamycin is an aminoglycoside whose mechanism of action is to inhibit protein synthesis. Similarly to ampicillin, when bacteria are transformed with a plasmid containing the Kana gene, only transformed bacteria can grow in the presence of kanamycin. Kanamycin-resistant bacteria are identified and selected during cloning and propagation.
Aminoglycoside such as kanamycin and neomycin are currently preferred, since they are rarely used in clinics and have low incidence effects of ototoxicity and nephrotoxicity.
AAV Plasmid Backbone Size
The typical size of an AAV plasmid backbone is about 3 kb. This backbone contains crucial elements for various functions, including plasmid propagation, AAV replication, and packaging. However, its small size makes it susceptible to reverse packaging, which occurs when it is inadvertently enclosed within the AAV capsid during vector production.
Efforts have been made to prevent reverse genome backbone packaging, which can compromise the functionality of AAV vectors. One approach involves using an oversized plasmid backbone that exceeds the typical AAV packaging capacity. By doing so, the aim is to reduce the chances of the entire plasmid backbone being reverse-packaged into the AAV capsid.
AAVnerGene has developed two types of oversized AAV plasmid backbones: pAAVone and pAAVdual.
The pAAVone plasmids are oversized AAV plasmid backbones that go beyond the typical AAV packaging capacity. They not only contain the essential ITRs, but also include both Ad helper genes(E2A, E4orf6 and VA RNA) and AAV helper genes(Rep and Cap). This backbone is designed to produce AAV with our unique AAVone system, which only use one plasmid.
The pAAVdual plasmids are designed to contain Ad helper genes located outside the inverted terminal repeats (ITRs). This backbone is designed to produce AAV with our pAAVdual production system.
| Type of AAV Backbone | Backbone Size | Selection Marker | Ori | Ad Genes | Rep/Cap | Genome | Production System |
| pAAV | 2.9kb | Amp | pMB1/F1 | No | No | ssAAV | AAVtri |
| pscAAV | 2.9kb | Amp | pMB1/F1 | No | No | scAAV | AAVtri |
| pAAVdual | 8.7kb | Kana | pMB1/F1 | Yes | No | ssAAV | AAVdual |
| pAAVdual-sc | 8.7kb | Kana | pMB1/F1 | Yes | No | scAAV | AAVdual |
| pAAVone | 13.2 kb | Kana | pMB1/F1 | Yes | Rep78/AAV2 | ssAAV | AAVone |
| pAAVone-sc | 13.2 kb | Kana | pMB1/F1 | Yes | Rep78/AAV2 | scAAV | AAVone |