preparation of viral sample and master mix for digital droplet pcr to accurately measure the viral genome copy number

<meta charset="utf8"></head><body>פרוטוקול סטנדרטי לכימות גנום נגיפי באמצעות Digital Droplet PCR

Gene therapy is a therapeutic modality commonly used to treat genetic disorders. The design of any given gene therapy is specific to the associated pathology of the target indication, but all gene therapies involve the intracellular delivery of genetic material to target cells in order to elicit a therapeutic effect1. Gene therapy can be further classified into several categories, including gene replacement for loss-of-function mutations, gene silencing for gain-of-function abnormalities, and gene editing techniques. Irrespective of the specific strategy employed, the therapeutic nucleic acid material (referred to as a transgene) must be packaged within a vector in order to achieve targeted intracellular delivery2.
Although a variety of viral and non-viral vector systems are available for gene therapy development, adeno-associated viruses (AAVs) are frequently chosen due to the broad viral tropism and low immunogenicity associated with this group of viruses1,2. To date, seven gene therapies utilizing AAV for therapeutic gene delivery have achieved approval from either the European Medicines Agency (EMA) or the Food and Drug Administration (FDA) targeting diseases ranging from hemophilia (e.g., Roctavian) to spinal muscular atrophy (e.g., Zolgensma)3.
Production of AAV-based gene therapies stems from an understanding of the wild-type AAV itself. AAV is a small DNA virus within the Parvoviridae family comprising 13 main serotypes (AAV1-13)3. The AAV genome comprises a ~4.7 kb, single-stranded DNA molecule containing two main open reading frames (ORFs) that encode the essential viral genes necessary for genome replication, capsid assembly, and packaging (rep, cap). The viral genome is flanked at both the 5&#39; and 3&#39; ends by palindromic nucleotide sequences, referred to as inverted terminal repeats (ITRs). These ITRs form hairpin-like structures that play crucial roles in genome replication and packaging of de novo viral genomes into newly synthesized viral capsids. AAV is a helper-dependent virus and, therefore, requires the expression of ancillary genes from other viruses, such as herpes simplex virus (HSV) or adenovirus (AdV) in order to become replication-competent1.
In order to produce AAVs, a suitable cell-based expression system is employed to facilitate the expression of the viral capsid proteins and subsequent assembly into de novo viral particles, followed by encapsidation of an ITR-flanked selected transgene (also referred to as a vector genome). This process commonly utilizes a triple plasmid system, comprising (1) a plasmid harboring helper genes derived from a helper virus, (2) a plasmid encoding the essential viral elements (rep/cap), and (3) a plasmid carrying the therapeutic expression cassette (commonly referred to as a transfer plasmid)4. The unique presence of inverted terminal repeat (ITR) packaging signals flanking the therapeutic expression cassette in the transfer plasmid ensures specific packaging of the transgene, while mostly excluding the viral genes present on the other plasmids. Co-transfection of this three-plasmid system into a cell-based expression platform (typically HEK293T cells) results in the production of transduction-competent, replication-deficient viral particles suitable for use in gene therapy applications3,4.
There are a number of critical quality attributes (CQAs) associated with the production of AAV-based gene therapies that must be assessed in order to ensure the potency, purity, and safety of the intended medicinal product4. These CQAs include virus titer, capsid content, and aggregation. Virus titer itself is a combination of the number of viral particles (capsid titer) and the number of vector genomes (vector genome titer) present in any given preparation. Ideally, the ratio between these two titers should be 1:1 since each viral particle should contain one vector genome, but inefficiencies in vector genome packaging during biosynthesis result in the co-production of empty or partially filled capsids (those containing partial vector genome sequences and/or non-vector genome sequences)5. The presence of such impurities can potentially evoke unwarranted immune responses and compete for vector-binding sites, thereby increasing the risk of immunotoxicity and reducing the rate of transduction of full capsids6. Accurate quantification of AAV genomes is therefore essential for establishing virus titer and capsid content. This impacts both basic research as well as the gene therapy industry, which requires accurate dosage in order to maintain both the safety and efficacy of medicinal products.
Digital Droplet polymerase chain reaction (dd_PCR) has become closely associated with the quantification of virus titer since it can be utilized to determine the number of vector genomes present in any given preparation7. Digital PCR itself was first introduced in the 1990s8,9&#160;and dd_PCR is an enhancement to this technology that allows for high-throughput sample processing10,11. In dd_PCR, a 20 &#181;L real-time PCR reaction is partitioned into approximately 20,000 oil-encased droplets, giving up to 96 such reactions when accommodated on a standard 96-well plate. Compared with conventional quantitative PCR (qPCR), dd_PCR offers several advantages, including enhanced sensitivity, increased precision, and more direct and absolute quantification of target sequences without the need for standard curves. Moreover, the high level of partitioning in dd_PCR reduces the impact of PCR inhibitors and minimizes the potential for bias from preferential amplification of certain templates, making it an attractive option for analytical measurement of vector genome titration.

<meta charset="utf8"></head><body>מחבר זרקור: אופטימיזציה של שיטת PCR טיפתית דיגיטלית לכימות מדויק של גנום נגיפי הקשור באדנו

כימות גנומים נגיפיים הקשורים באדנו בדגימות וקטוריות מטוהרות על ידי תגובת שרשרת של פולימראז טיפתי דיגיטלי

DD PCR is a widely used technique for determining the AAV genome titer, but a consensus protocol is currently lacking. Our protocol is a validated step-by-step guide on how to prepare samples and perform DD PCR for accurate genome titration. Compared with QPCR, DD PCR offers several advantages, including increased precision, greater robustness, and a more absolute and direct quantification of target sequences without the need for standard curves.Furthermore, with a well-designed primer probe set, DD PCR allows for specific tragedy in detection, as opposed to other techniques that detect all DNA. Developing a standardized consensus protocol for vector genome quantification will improve reliability and uniformity in recombinant AAV quality control across different laboratories. This will facilitate both the research and clinical applications of recombinant AAV vectors for the wider community.

הכנת דגימה ויראלית ומיקס מאסטר ל-Digital Droplet PCR למדידה מדויקת של מספר העתק הגנום הנגיפי

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Research

JoVE Journal

Bioengineering

Preparation of Viral Sample and Master Mix for Digital Droplet PCR to Accurately Measure the Viral Genome Copy Number

Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of AAV genome copy number in vector preparations is essential for bioprocess optimization and dosage calculation in both preclinical and clinical studies of AAV-based gene therapy products. Currently, a consensus protocol for AAV viral genome titration is lacking. Here, we present a digital droplet PCR (dd_PCR) protocol for the quantification of viral genomes in purified vector samples. Samples are treated with DNase I to eliminate unpackaged contaminant DNA. DNase-treated samples are then mixed with an appropriate primer-probe set (designed according to the target AAV genome) and PCR reagents, and then loaded into a droplet generator. The prepared droplets are transferred into a PCR plate, where PCR amplification is performed and analyzed. The viral genome titer is calculated based on the concentration (copies/&#181;L), accounting for sample dilutions. A successful measurement shows a clear separation of the positive and negative droplet clouds, has at least 10,000 accepted droplets, shows a value between 10 copies/&#181;L and 10,000 copies/&#181;L, and has a coefficient of variation (CV) between repeats lower than 20%. Reliable viral genome titration will aid in the development of safe and effective AAV-based gene therapy products.

Precise quantification of adeno-associated virus (AAV) vector genome copies is critical, but a standardized protocol has yet to be established. This protocol describes a validated method for preparing purified AAV samples and conducting digital droplet polymerase chain reaction (dd_PCR) to reliably quantify the viral genome titer.

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of ...

To begin vortex the Adeno-associated virus or AAV sample and centrifuge to ensure all liquid remains at the bottom of the tube. Add 45 microliters of DNase I containing solution to a 0.2 milliliter PCR eight tube strip tube. Transfer five microliters of the AAV sample into the tube containing the DNase solution.After vortexing, centrifuge the sample briefly to ensure all liquid remains at the bottom. Using a thermocycler, incubate the samples for one hour at 37 degrees Celsius, then cool them down to four degrees Celsius. Next, prepare appropriate dilution of the DNase's treated sample in AAV dilution buffer in a fresh 0.2 milliliter PCR eight tube strip.After serial dilution, vortex the samples and centrifuge to ensure all the liquid remains at the bottom. Combine the forward and reverse primers probe ddPCR Supermix and DNase's-Free Water in the required volumes. Vortex the master mix and briefly centrifuge the tube.Then transfer 19.8 microliters of the master mix into each tube on a fresh 0.2 milliliter PCR eight tube strip.

Droplet Generation and Digital Droplet PCR for Reliable Quantification of AAV Viral Genome Titers in Vector Samples

To begin, prepare DNA's one treated AAV sample and the dd_PCR master mix. Add 2.2 microliters of the diluted sample to 19.8 microliters of dd_PCR master mix in each tube of the PCR eight tube strip and mix well. Transfer 20 microliters of the mixture into the wells contained in the middle sample row of a droplet-generating cartridge.Then transfer 60 microliters of droplet generation oil to the wells contained in the lower oil row of the droplet-generating cartridge. Place a rubber gasket over the droplet-generating cartridge, and then place it in the droplet generator. After the droplets are generated, use an eight-channel pipette to slowly transfer 42.5 microliters of solution from the droplet generation cartridge to a multi-well PCR plate.Seal the PCR plate with an aluminum foil cover using a heat sealing machine for five seconds at 180 degrees Celsius. For amplification, place the plate in a thermal cycler with a 96-deep well reaction module and close it securely and run the PCR program. Once done, load the plate into a droplet reader, input the required information into the system software, and start the reading.A clear separation between positive and negative droplets was observed in the 1D amplitude plot, suggesting a successful measurement. The second 1D amplitude plot showed droplet rain with droplets scattered between the positive and negative clouds, indicating a potential issue with measurement accuracy. The output data from an AAV run showed consistent results when one sample was measured in duplicates at two different dilutions.