Gene therapy (introducing genetic material into living cells to fix, replace, enhance, or block a faulty gene) is rapidly gaining traction as a strategy for the treatment of genetic diseases. The techniques for delivering therapeutic genetic material are generally divided into two categories: ex vivo, where patient-derived cells are genetically modified outside the body and then reintroduced; and in vivo, where genetic material is directly delivered into the patient’s body. Among the various viral vectors studied for in vivo gene therapy, adeno-associated viruses (AAVs) have emerged as the preferred choice due to their broad tissue tropism, favorable safety profile, non-pathogenic nature, and adaptable manufacturing processes. These characteristics have made them the focus of clinical trials targeting a wide range of conditions, including neurological, metabolic, cardiovascular, and hematological disorders, as well as cancers.
As the field has grown, regulatory guidelines have naturally been put in place to ensure the safety and efficacy of gene therapy products. For therapies using AAV vectors, accurate viral quantification and impurity detection are of paramount importance. Measuring AAV capsid ratios and genome titer using fully validated solutions can support regulatory compliance.
Accurate quantification of AAV genome titer (the concentration of viral particles containing the therapeutic genome) is crucial for optimizing production processes, refining purification, and ensuring therapeutic efficacy.
AAV production generates three types of viral capsids: full, partial, and empty capsids (Figure 1). Only full capsids, which carry the therapeutic gene, are functional for gene delivery, while empty capsids lack genetic material and are non-therapeutic. Partial capsids contain incomplete genetic material and are also non-functional. While the impact of partial and empty capsids on the safety and efficacy of AAV products varies, they are generally considered impurities, and elevated levels can reduce transduction efficiency and increase the risk of immunotoxicity, posing a challenge for clinical applications. Meticulous assessment and regulation of the ratio of empty to full (“empty-full”) capsids is therefore a critical quality control parameter to ensure product quality and safety.
Figure 1. The three capsid types formed during AAV production and their therapeutic effect.
Capsid titer quantification is commonly performed using optical or antibody-based methods such as analytical ultracentrifugation (AUC), transmission electron microscopy (TEM), and enzyme-linked immunosorbent assays (ELISA) (Figure 2). Among these methods, AUC is the only one capable of high-resolution capsid titer analysis but it requires substantially large volumes of concentrated purified sample, which can be especially challenging for limited sample types, and has a long turnaround time. TEM and ELISA require slightly less sample amounts than AUC but are similarly labor-intensive and time-consuming, resulting in lower-resolution capsid titer analysis. AUC can be used to determine the capsid ratio; however, its accuracy is limited as the percentage of empty, partially full, or full capsids is determined through weight. This technique therefore cannot differentiate between capsids that contain fragmented DNA or contaminant DNA that does not contain the therapeutic gene. ELISA is often used to measure capsid titer and can be used in conjunction with qPCR to determine the capsid content ratio – this is achieved by combining the capsid and genome titer measurements independently provided by ELISA and qPCR, respectively (ELISA + qPCR). This approach is, however, time-consuming and lacks precision due to the combined error propagation of the two distinct detection methods. It also requires relatively large sample amounts, making it less suitable for modern manufacturing processes.
Figure 2. Key capabilities of current quality control methods for AAV capsid determination.
The limitations of traditional methods prompted the development of more efficient, precise, and sensitive approaches for the determination of genome titer and empty-full capsid ratio. This has led to the adoption of more advanced techniques, such as droplet-based digital PCR (e.g., ddPCR), which provides absolute genome titer quantification, with high precision and reliability, even for low-template applications. This technique also uses lower sample volumes, reducing costs and preserving limited vector batches produced by resource-intensive production processes for other tests or therapeutic applications. As such, ddPCR is being increasingly recognized as a versatile and powerful technology to advance gene therapy manufacturing and quality assurance.
The development of novel assay kits compatible with ddPCR technology is also expanding the capabilities of ddPCR, enabling the consistent and reliable assessment of AAV empty-full capsid ratios, thus addressing the limitations of traditional methods. Within a single assay, this offers a highly accurate and cost-effective method for quantifying the empty-full capsid ratio in addition to genome titer. This approach involves the binding of oligo-labeled antibodies to the capsid followed by ligation, which results in the formation of an amplicon enabling both capsid and genome titer determination. The sample is then partitioned into thousands of nanodroplets, each serving as an independent PCR reaction chamber. Following droplet generation and amplification, data analysis of controls versus the sample allows the straightforward measurement of genome titer, capsid titer, and empty-full ratio in a single reaction. Additionally, by analyzing the statistical distribution of “positive droplets” containing the target sequence and “negative droplets” lacking it, ddPCR provides absolute quantification of the target nucleic acid, i.e., the therapeutic gene, without the need for standard curves. This approach enhances sensitivity, minimizes PCR bias, reduces the input of sample required, and delivers precise insights into capsid quality.
The unique capability of ddPCR to simultaneously evaluate both the genome titer and capsid titer, as well as the empty-full ratio, provides a more accurate assessment of AAV capsid quality as compared to traditional methods.
With a fast turnaround time and high sensitivity, ddPCR produces a clearer signal-to-noise ratio and efficiently processes small sample volumes, making it well-suited for high-throughput analysis. Additionally, it can be deployed across a variety of sample types, from crude lysates to purified preparations, making it a versatile technology for ensuring comprehensive quality control in AAV manufacturing.
Innovations such as ddPCR technology enable the simultaneous determination of genome titer, capsid titer, and the empty-full capsid ratio in a single assay, bypassing the need for separate testing and overcoming the labor- and time-intensive limitations of traditional methods that require high sample volume inputs. Specifically, advancements in ddPCR technology and the development of complementary assays provide consistent and reliable capsid determination to further streamline AAV characterization, supporting more efficient manufacturing workflows.
Virus-mediated gene therapies represent a promising biological therapeutic for the treatment of a broad range of genetic disorders, however, not all aspects of their manufacturing can be controlled. This uncertainty underscores the importance of accurate quantification of viral particles and contaminants to ensure the safety, efficacy, and consistency of these therapies. ddPCR stands out as a critical tool for achieving these objectives, supporting the growing field of gene replacement therapy as it transitions from an experimental treatment to mainstream clinical application.
Chelsea Pratt, PhD, is the biopharma market development manager at Bio-Rad Laboratories. Website: www.bio-rad.com.
The post Single-Assay Evaluation of AAV Capsid Quality: Novel Digital PCR Approaches Advance Gene Therapy Research and Production appeared first on GEN - Genetic Engineering and Biotechnology News.
As the field has grown, regulatory guidelines have naturally been put in place to ensure the safety and efficacy of gene therapy products. For therapies using AAV vectors, accurate viral quantification and impurity detection are of paramount importance. Measuring AAV capsid ratios and genome titer using fully validated solutions can support regulatory compliance.
AAV capsid ratios and genome titer—their impact on efficacy
Accurate quantification of AAV genome titer (the concentration of viral particles containing the therapeutic genome) is crucial for optimizing production processes, refining purification, and ensuring therapeutic efficacy.
AAV production generates three types of viral capsids: full, partial, and empty capsids (Figure 1). Only full capsids, which carry the therapeutic gene, are functional for gene delivery, while empty capsids lack genetic material and are non-therapeutic. Partial capsids contain incomplete genetic material and are also non-functional. While the impact of partial and empty capsids on the safety and efficacy of AAV products varies, they are generally considered impurities, and elevated levels can reduce transduction efficiency and increase the risk of immunotoxicity, posing a challenge for clinical applications. Meticulous assessment and regulation of the ratio of empty to full (“empty-full”) capsids is therefore a critical quality control parameter to ensure product quality and safety.
Figure 1. The three capsid types formed during AAV production and their therapeutic effect.
Capsid titer quantification is commonly performed using optical or antibody-based methods such as analytical ultracentrifugation (AUC), transmission electron microscopy (TEM), and enzyme-linked immunosorbent assays (ELISA) (Figure 2). Among these methods, AUC is the only one capable of high-resolution capsid titer analysis but it requires substantially large volumes of concentrated purified sample, which can be especially challenging for limited sample types, and has a long turnaround time. TEM and ELISA require slightly less sample amounts than AUC but are similarly labor-intensive and time-consuming, resulting in lower-resolution capsid titer analysis. AUC can be used to determine the capsid ratio; however, its accuracy is limited as the percentage of empty, partially full, or full capsids is determined through weight. This technique therefore cannot differentiate between capsids that contain fragmented DNA or contaminant DNA that does not contain the therapeutic gene. ELISA is often used to measure capsid titer and can be used in conjunction with qPCR to determine the capsid content ratio – this is achieved by combining the capsid and genome titer measurements independently provided by ELISA and qPCR, respectively (ELISA + qPCR). This approach is, however, time-consuming and lacks precision due to the combined error propagation of the two distinct detection methods. It also requires relatively large sample amounts, making it less suitable for modern manufacturing processes.
Figure 2. Key capabilities of current quality control methods for AAV capsid determination.
Overcoming the limitations of current analytical techniques
The limitations of traditional methods prompted the development of more efficient, precise, and sensitive approaches for the determination of genome titer and empty-full capsid ratio. This has led to the adoption of more advanced techniques, such as droplet-based digital PCR (e.g., ddPCR), which provides absolute genome titer quantification, with high precision and reliability, even for low-template applications. This technique also uses lower sample volumes, reducing costs and preserving limited vector batches produced by resource-intensive production processes for other tests or therapeutic applications. As such, ddPCR is being increasingly recognized as a versatile and powerful technology to advance gene therapy manufacturing and quality assurance.
The development of novel assay kits compatible with ddPCR technology is also expanding the capabilities of ddPCR, enabling the consistent and reliable assessment of AAV empty-full capsid ratios, thus addressing the limitations of traditional methods. Within a single assay, this offers a highly accurate and cost-effective method for quantifying the empty-full capsid ratio in addition to genome titer. This approach involves the binding of oligo-labeled antibodies to the capsid followed by ligation, which results in the formation of an amplicon enabling both capsid and genome titer determination. The sample is then partitioned into thousands of nanodroplets, each serving as an independent PCR reaction chamber. Following droplet generation and amplification, data analysis of controls versus the sample allows the straightforward measurement of genome titer, capsid titer, and empty-full ratio in a single reaction. Additionally, by analyzing the statistical distribution of “positive droplets” containing the target sequence and “negative droplets” lacking it, ddPCR provides absolute quantification of the target nucleic acid, i.e., the therapeutic gene, without the need for standard curves. This approach enhances sensitivity, minimizes PCR bias, reduces the input of sample required, and delivers precise insights into capsid quality.
The unique capability of ddPCR to simultaneously evaluate both the genome titer and capsid titer, as well as the empty-full ratio, provides a more accurate assessment of AAV capsid quality as compared to traditional methods.
With a fast turnaround time and high sensitivity, ddPCR produces a clearer signal-to-noise ratio and efficiently processes small sample volumes, making it well-suited for high-throughput analysis. Additionally, it can be deployed across a variety of sample types, from crude lysates to purified preparations, making it a versatile technology for ensuring comprehensive quality control in AAV manufacturing.
Conclusions
Innovations such as ddPCR technology enable the simultaneous determination of genome titer, capsid titer, and the empty-full capsid ratio in a single assay, bypassing the need for separate testing and overcoming the labor- and time-intensive limitations of traditional methods that require high sample volume inputs. Specifically, advancements in ddPCR technology and the development of complementary assays provide consistent and reliable capsid determination to further streamline AAV characterization, supporting more efficient manufacturing workflows.
Virus-mediated gene therapies represent a promising biological therapeutic for the treatment of a broad range of genetic disorders, however, not all aspects of their manufacturing can be controlled. This uncertainty underscores the importance of accurate quantification of viral particles and contaminants to ensure the safety, efficacy, and consistency of these therapies. ddPCR stands out as a critical tool for achieving these objectives, supporting the growing field of gene replacement therapy as it transitions from an experimental treatment to mainstream clinical application.
Chelsea Pratt, PhD, is the biopharma market development manager at Bio-Rad Laboratories. Website: www.bio-rad.com.
The post Single-Assay Evaluation of AAV Capsid Quality: Novel Digital PCR Approaches Advance Gene Therapy Research and Production appeared first on GEN - Genetic Engineering and Biotechnology News.