AAV Genomic Impurities
Price and Turnaround
| Method | Purpose | Turnaround | Price | Sample Requirement |
| qPCR-SYBR | Impurities copy number | 1-2 day | $50/target/sample | Primer sequences; ~20 μl of samples |
| qPCR-TaqMan | Impurities copy number | 1-2 day | $100/target/sample | Primer/probe sequences; ~20 μl of samples |
| ddPCR | Impurities copy number | 3-5 day | $200/target/sample | Primer/probe sequences; ~20 μl of samples |
| NGS-Illumina | Sequence details | 3-4 weeks | Request | ~100 μl of sample with titer >1E+12vg/ml |
| NGS-Pacbio | Sequence details | 3-4 weeks | Request | ~100 μl of sample with titer >1E+12vg/ml |
| NGS-Nanopore | Sequence details | 2-3 weeks | Starting from $600 | ~100 μl of sample with titer >1E+12vg/ml |
DNA Impurities in AAV vectors
Besides intact GOI genomes, AAV production also yields contaminants in the final product. These nucleic acid contaminants can be delineated into 3 broad categories:
- Incomplete AAV vector genomes,
- Nucleic acids from the producer plasmids
- Nucleic acids from the host genome of the production cell line.
AAVnerGene offers both qPCR and NGS methods to analysis the DNA impurities.
Incomplete AAV Vector Genomes
- Snapback genomes, either be completely complementary or missing the complementary sequence from one of the strands;
- Incomplete single-stranded genomes containing either the 5’ or the 3’ ITR
- Genomes that contained both ITRs but were missing an internal portion of the rAAV genome
Genome integrity analysis of vectors produced by tri-plasmid systems and AAVone. (A, B) IGV display of SMRT reads representing AAV vector genomes aligned to the standard cis plasmid (A) from the pTri-plasmid (top) and the mTri-plasmid (bottom) packaging systems, and the AAVone (B) reference genomes. Both constructs carry the CMV-Egfp cassette. Reads are shown in squished displays with soft-clipped bases visible. Read matches (gray), mismatches (colored), and insertions/deletions (speckles) are shown. Alignment coverages are shown above each display.
Plasmid related impurities
Residual plasmid DNA refers to fragments of the plasmid used during the production of AAV vectors that remain in the final product. These plasmids are typically used to transfect producer cells to generate AAV particles. Residual plasmid DNA is considered an impurity because it can potentially integrate into the host genome, leading to unwanted genetic effects. The residual plasmid DNA can be categorized into three main types:
AAV Helper Genes:
- Rep: These genes are responsible for AAV replication. The Rep proteins are essential for the replication of the AAV genome within the producer cells. Residual Rep sequences in the final product can be problematic because they can interfere with the controlled replication of the AAV vector.
- Cap: These genes encode the capsid proteins that form the shell of the AAV particle. Residual Cap sequences can contribute to the formation of empty capsids or affect the purity and stability of the AAV vector
Ad Helper Elements:
- VA RNA: VA RNAs are transcribed from adenoviral helper plasmids and are involved in inhibiting the host cell’s antiviral response. Residual VA RNA sequences can interfere with the host cell’s normal functions and immune responses.
- E2A: E2A is an adenoviral gene involved in DNA replication. Residual E2A sequences can disrupt normal cellular processes and pose a risk to genomic stability.
- E4orf6: E4orf6 is another adenoviral gene involved in mRNA processing and modulation of the host cell environment. Residual E4orf6 sequences can interfere with cellular functions and potentially affect safety.
Bacterial Elements from both Ad and AAV helper plasmids:
- Ori (Origin of Replication): The origin of replication is a sequence where DNA replication begins. Residual Ori sequences can lead to unintended replication of plasmid fragments within the host cell, potentially causing genomic instability.
- Resistant Marker: Plasmids often contain antibiotic resistance genes as selectable markers. Residual sequences of these markers in the final product can confer antibiotic resistance to bacteria if transferred, posing a risk of spreading resistance genes.
Plasmid related impurities in AAVs produced with AAVone system.
Host cell DNAs
Host cell DNA impurities in the production of AAV vectors refer to the presence of unwanted DNA fragments from the producer cells used during the manufacturing process. These impurities can pose several risks, including genomic integration, immunogenicity, and potential interference with the therapeutic efficacy of the AAV vectors. Understanding and controlling these impurities are crucial for ensuring the safety and efficacy of AAV-based gene therapies. Sources of host cell DNA impurities include:
- Genomic DNA: Fragments of the host cell’s genomic DNA can be released into the production medium during cell lysis and can co-purify with the AAV vectors. These fragments can vary in size and may include genes or regulatory elements from the host genome.
- Mitochondrial DNA: Mitochondrial DNA from the producer cells can also be present as an impurity. Although typically smaller than genomic DNA, mitochondrial DNA can still pose similar risks.
Profiling of hcDNA contaminants associated with triple-plasmid systems and AAVone. The Venn diagrams (left) show mapped reads to vector genome and to human genome hg38, as well as chimeric mapped reads for the pTri-plasmid (top), mTri-plasmid (middle), and AAVone (bottom) packaging systems. The number of reads mapped exclusively to the vector genome (ITR-to-ITR, white) and hg38 (human genome) are shown. The distribution of mapped reads to individual chromosomes can also be observed in the bar graph.
Alignment of SMRT reads from DNase-resistant DNA isolated from vectors to chrM. Reads from vectors produced by the pTri-plasmid (top), mTri-plasmid (middle), and AAVone (bottom) systems are shown mapping to the mitochondrial genome. Tracks of the mitochondrial genes are shown above.