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:

• 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.
• Origin of Replication (Ori): This element is required for the replication of the plasmid in bacterial hosts, such as Escherichia coli (E. coli).
• 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.

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.

Origin of Replication (Ori)

A high-copy origin of replication ensures that the plasmid is replicated in large quantities within the bacterial cells, facilitating ample plasmid yield upon purification.

Here is a list of bacterial origins of replication commonly used in plasmids:

1. pUC Origin
   • Copy Number: Very high (500-700 copies per cell)
   • Commonly Used In: High-yield plasmid preparation, cloning, and expression vectors.
2. ColE1 Origin
   • Copy Number: High (15-20 copies per cell)
   • Commonly Used In: General cloning and subcloning vectors.
3. pBR322 Origin
   • Copy Number: Moderate (15-20 copies per cell)
   • Commonly Used In: General cloning applications.
4. p15A Origin
   • Copy Number: Moderate to low (10-12 copies per cell)
   • Commonly Used In: Cloning vectors requiring lower copy numbers, compatible with ColE1 and pUC origins.
5. F1 Origin
   • Copy Number: Moderate, depends on vector design
   • Commonly Used In: Phagemid vectors for the production of single-stranded DNA, useful in techniques like mutagenesis and phage display.
6. pSC101 Origin
   • Copy Number: Low (5-10 copies per cell)
   • Commonly Used In: Applications requiring low-copy plasmids, often used in synthetic biology and metabolic engineering.
7. RK2 Origin
   • Copy Number: Low to moderate (4-7 copies per cell)
   • Commonly Used In: Broad-host-range plasmids for Gram-negative bacteria.
8. RSF1010 Origin
   • Copy Number: Moderate (15-30 copies per cell)
   • Commonly Used In: Broad-host-range plasmids for Gram-negative bacteria, especially in genetic engineering of non-E. coli species.
9. P1 Origin
   • Copy Number: Low to moderate (1-2 copies per cell)
   • Commonly Used In: Single-copy maintenance systems, often used in BAC (bacterial artificial chromosome) vectors.
10. R6K Origin

• Copy Number: Depends on the presence of the π protein (low-copy without π protein, high-copy with π protein)
• Commonly Used In: Specialized vectors requiring controlled copy number, often used in phage display and other specialized applications.

This list includes a variety of bacterial origins of replication used in plasmid vectors, each with unique features that make them suitable for different experimental needs.

Here’s a side-by-side comparison of the listed 10 bacterial origins of replication used in plasmid vectors:

This table provides a detailed side-by-side comparison of the key features of different bacterial origins of replication used in plasmid vectors.

Inverted Terminal Repeats (ITRs)​

Wild type ITRs are 145bp palindromes (ITR145), 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 up to  15 bp terminal deletion at each ITR (ITR130)  and a further 11 bp deletion in C-domain (ITR119) do not affect viability and could be used in AAV production [Savy et al., 2017][Tran et al., 2020]. We also confirmed that both ITR130 and ITR119 are fully recovered to intact ITR145[].ASGCT 2024, poster

AAV Plasmid Backbone Size

The typical size of an AAV plasmid backbone is less than 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 ( 13 kb) 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 AAVdual system.

References

Savy etal., 2017: Impact of Inverted Terminal Repeat Integrity on rAAV8 Production Using the Baculovirus/Sf9 Cells System

Tran et al., 2020: AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity.

Liu et al., The AAVone Single-Plasmid System Efficiently Generates High-Quality AAV Vectors. ASGCT, 2024, abstract 1430.