Overview of Key AAV Analytical Testing for Gene Therapy Development

In the development of AAV-based gene therapies, ensuring the quality and efficacy of the viral vectors is paramount. Several critical tests are employed to assess various aspects of AAV vectors, each addressing specific factors that impact the safety and effectiveness of the therapy. These tests include quantifying viral genome copies, evaluating genome integrity, analyzing genetic composition, measuring the proportion of empty versus full capsids, and detecting replication-competent AAV. Together, these assessments provide a comprehensive evaluation of the AAV vectors, ensuring that they meet the highest standards required for successful and safe gene therapy applications.

Understanding the quality and integrity of Adeno-Associated Virus (AAV) samples is essential for the success of gene therapy applications. Comprehensive testing is critical to ensuring the efficacy, safety, and consistency of AAV vectors. PackGene offers over 40 AAV analytical tests that cover various aspects, including titration, genome integrity, capsid characterization, purity and aggregation, contamination, and safety to help you from early research and development to GMP production.

AAV Genome Titer by ddPCR: Provides precise quantification of viral genome copies, ensuring accurate assessment of viral load, crucial for determining effective dosages and maintaining consistency in gene therapy applications. [Learn more]

AAV Genome Integrity by CE: Evaluates the structural integrity of AAV genomes, identifying fragmentation or degradation, which is vital for maintaining the stability, efficacy, and safety of the viral vectors. [Learn more]

AAV Genome Sequencing by Nanopore: Offers detailed analysis of the AAV genome, detecting mutations, insertions, and deletions. This comprehensive genomic insight ensures the fidelity and stability of the AAV vector, essential for therapeutic use. [Learn more]

AAV Empty Capsid Rate Test by AUC: Measures the proportion of empty capsids versus full ones, which is critical for determining the potency and immunogenicity of the therapy. This gold-standard method ensures the production of high-quality viral vectors. [Learn more]

Replication Competent AAV (rcAAV) Analysis: Detects and quantifies replication-competent AAV, which is crucial for ensuring the safety of gene therapies. Minimizing rcAAV contamination is necessary to prevent unwanted viral replication in patients. [Learn more]

Learn more about key AAV analytical tests

AAV Genome Titer by ddPCR: Digital Droplet PCR (ddPCR) provides precise quantification of AAV genome copies, ensuring accurate determination of viral load. This method offers high sensitivity and specificity, which is essential for consistent and reproducible results in gene therapy applications.

AAV genome titer by ddPCR and benefits over traditional qPCR

Traditionally, rAAV genome size analysis has been performed using denaturing agarose gel electrophoresis and Southern blotting, which are time-consuming and offer limited resolution. Capillary Electrophoresis (CE) has recently emerged as a superior method for this purpose.

PackGene’s AAV genome integrity test uses CE to assess the integrity of AAV genomes, identifying any fragmentation or degradation. This method provides precise and reliable measurements, ensuring the efficacy and safety of AAV-based therapies by maintaining genome integrity. The stability and performance of the viral vector are directly impacted by genome integrity, making PackGene’s CE-based testing a crucial component in producing high-quality rAAV.

Nanopore technology involves embedding nanoscale pores in a membrane structure to detect changes in electrical potential as charged biomolecules pass through the pores.

Nanopore sequencing delivers a comprehensive and detailed analysis of the AAV genome. It enables the detection of mutations, insertions, and deletions, providing a thorough understanding of the genetic composition and ensuring the fidelity of the AAV vector for therapeutic use.

PackGene’s AAV Nanopore sequencing offers deep sequencing results to evaluate AAV genome integrity. It identifies the population of mutations within the entire AAV sample, including low-frequency mutations, whether they occur in the gene of interest or the ITR region. The long reads of nanopore sequencing enable easy readthrough of complex sequences or hairpin structures, overcoming the difficulty of aligning repetitive sequences in the gene.

During the packaging process of AAV vectors, empty capsid viral particles are produced, raising safety concerns due to their potential immunogenicity and impact on potency. This has garnered significant regulatory attention, making accurate detection of empty capsid rates crucial.

PackGene’s AAV empty capsid rate test uses analytical ultracentrifugation (AUC), the gold standard for this analysis. AUC combines a centrifuge and an optical module to accurately characterize each component in a sample by analyzing sedimentation changes over time. This highly reproducible method offers unmatched resolution, distinguishing between empty, full, and partially filled viral particles. AUC precisely measures the sedimentation coefficients and relative proportions of different capsid particles in rAAV samples, detecting intermediate products, viral vector fragments, genome fragments, and viral aggregates. Recognized and trusted by industry professionals, PackGene’s AUC method ensures the highest standards of accuracy and reliability in determining AAV empty capsid rates.

Currently, literatures have not provided a feasible guideline for detecting replication-competent AAV (rcAAV). The main challenges in developing such a method include the necessity of using calibrated helper viruses and wild-type positive viruses. Typically, in a P2 laboratory setting, this testing involves using wild-type AAV (wtAAV) and samples, which, with the help of helper viruses, undergo multiple rounds of infection of target cells. Samples are then extracted for qPCR detection.

PackGene’s Replication Competent AAV (rcAAV) Analysis leverages state-of-the-art methodologies to ensure the highest standards of accuracy and reliability. Utilizing a P2 laboratory with advanced qualifications, the analysis employs uniformly calibrated helper virus libraries and wild-type AAV (wtAAV) for testing. The use of competent cell lines guarantees precise and consistent results. PackGene’s proprietary PackRich rcAAV enrichment technology sets a new benchmark in the industry, offering a detection limit as low as 10 IU. This innovative approach allows for the effective identification and quantification of rcAAV, ensuring the safety and efficacy of gene therapy vectors.

Conclusion:

PackGene’s extensive suite of over 40 AAV analytical tests provides a thorough assessment of Adeno-Associated Virus vectors, ensuring their quality, efficacy, and safety in gene therapy applications. From quantifying viral genome copies and evaluating genome integrity to analyzing genetic composition and measuring capsid proportions, these tests address critical aspects of AAV vector performance. PackGene’s cutting-edge methodologies, including ddPCR for genome titer, capillary electrophoresis for genome integrity, nanopore sequencing for detailed genetic analysis, AUC for empty capsid rates, and sophisticated techniques for detecting replication-competent AAV, collectively support the development of high-quality and safe gene therapies. These advanced testing capabilities are essential for meeting regulatory standards and ensuring the success of gene therapy treatments.

PackGene Biotech is a world-leading CRO and CDMO, excelling in AAV vectors, mRNA, plasmid DNA, and lentiviral vector solutions. Our comprehensive offerings span from vector design and construction to AAV, lentivirus, and mRNA services. With a sharp focus on early-stage drug discovery, preclinical development, and cell and gene therapy trials, we deliver cost-effective, dependable, and scalable production solutions. Leveraging our groundbreaking π-alpha 293 AAV high-yield platform, we amplify AAV production by up to 10-fold, yielding up to 1e+17vg per batch to meet diverse commercial and clinical project needs. Moreover, our tailored mRNA and LNP products and services cater to every stage of drug and vaccine development, from research to GMP production, providing a seamless, end-to-end solution.

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