<meta charset="utf8"></head><body>Standardisiertes Protokoll für die Quantifizierung des viralen Genoms mittels digitaler Tröpfchen-PCR

<ol>
	<li>Balakrishnan, B., Jayandharan, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+biology+of+adeno-associated+virus+(AAV)+vectors+used+in+gene+therapy.">Basic biology of adeno-associated virus (AAV) vectors used in gene therapy.</a> Curr Gene Ther. 14 (2), 86-100 (2014).</li><li>Srivastava, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+tissue-tropism+of+adeno-associated+viral+vectors.">In vivo tissue-tropism of adeno-associated viral vectors.</a> Curr Opin Virol. 21, 75-80 (2016).</li><li>Pupo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV+vectors:+The+Rubik's+cube+of+human+gene+therapy.">AAV vectors: The Rubik's cube of human gene therapy.</a> Mol Ther. 30 (12), 3515-3541 (2022).</li><li>Gimpel, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+methods+for+process+and+product+characterization+of+recombinant+adeno-associated+virus-based+gene+therapies.">Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies.</a> Mol Ther Methods Clin Dev. 20, 740-754 (2021).</li><li>Brimble, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventing+packaging+of+translatable+P5-associated+DNA+contaminants+in+recombinant+AAV+vector+preps.">Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps.</a> Mol Ther Methods Clin Dev. 24, 280-291 (2022).</li><li>Wright, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Product-related+impurities+in+clinical-grade+recombinant+AAV+vectors:+Characterization+and+risk+assessment.">Product-related impurities in clinical-grade recombinant AAV vectors: Characterization and risk assessment.</a> Biomedicines. 2 (1), 80-97 (2014).</li><li>Dobnik, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+quantification+and+characterization+of+adeno-associated+viral+vectors.">Accurate quantification and characterization of adeno-associated viral vectors.</a> Front Microbiol. 10, (2019).</li><li>Sidransky, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+ras+oncogene+mutations+in+the+stool+of+patients+with+curable+colorectal+tumors.">Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors.</a> Science. 256 (5053), 102-105 (1992).</li><li>Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR.">Digital PCR.</a> Proc Natl Acad Sci USA. 96 (16), 9236-9241 (1999).</li><li>Hindson, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+droplet+digital+PCR+system+for+absolute+quantitation+of+DNA+copy+number.">High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.</a> Anal Chem. 83 (22), 8604-8610 (2011).</li><li>Pinheiro, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+droplet+digital+polymerase+chain+reaction+format+for+DNA+copy+number+quantification.">Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification.</a> Anal Chem. 84 (2), 1003-1011 (2012).</li><li>Sanmiguel, J., Gao, G., Vandenberghe, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+digital+droplet-based+AAV+genome+titration.">Quantitative and digital droplet-based AAV genome titration.</a> Methods Mol Biol. 1950, 51-83 (2019).</li><li>. Planning droplet digital PCR experiments Available from: <a target="_blank" href="https://www.bio-rad.com/en-in/life-science/learning-center/introduction-to-digital-pcr/planning-ddpcr-experiments">https://www.bio-rad.com/en-in/life-science/learning-center/introduction-to-digital-pcr/planning-ddpcr-experiments</a> (2024)</li><li>Dorange, F., Le Bec, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+approaches+to+characterize+AAV+vector+production+&amp;+purification:+Advances+and+challenges.">Analytical approaches to characterize AAV vector production &amp; purification: Advances and challenges.</a> Cell Gene Ther Insights. 4 (2), 119-129 (2018).</li><li>Lock, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+determination+of+single-stranded+and+self-complementary+adeno-associated+viral+vector+genome+titers+by+droplet+digital+PCR.">Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.</a> Hum Gene Ther Methods. 25 (2), 115-125 (2014).</li><li>Prantner, A., Maar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+concentration,+characterization,+and+integrity+analysis+of+recombinant+adeno-associated+viral+vectors+using+droplet+digital+PCR.">Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR.</a> PLoS One. 18 (1), e0280242 (2023).</li><li>Dobnik, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+quantification+and+characterization+of+adeno-associated+viral+vectors.">Accurate quantification and characterization of adeno-associated viral vectors.</a> Front Microbiol. 10, 1570 (2019).</li><li>Suoranta, T., Laham-Karam, N., Yla-Herttuala, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+protocol+for+accurate+titration+of+adeno-associated+virus+vectors.">Optimized protocol for accurate titration of adeno-associated virus vectors.</a> Hum Gene Ther. 32 (19-20), 1270-1279 (2021).</li><li>Droplet digital PCR applications guide. Bulletin 6407 Available from: <a target="_blank" href="https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf">https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf</a> (2024)</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-channel pipette 10 &#181;L</td>
			<td>Eppendorf</td>
			<td>3,12,50,00,010</td>
			<td></td>
		</tr>
		<tr>
			<td>8-channel pipette 200 &#181;L</td>
			<td>Eppendorf</td>
			<td>3,12,50,00,036</td>
			<td></td>
		</tr>
		<tr>
			<td>8-channel pipette 300 &#181;L</td>
			<td>Eppendorf</td>
			<td>3,12,50,00,052</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well PCR strip</td>
			<td>Sarstedt</td>
			<td>72.991.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler with 96&#8211;Deep Well Reaction Module</td>
			<td>Bio-Rad</td>
			<td>1851197</td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR Buffer Control for Probes 9 mL (2 x 4.5 mL)</td>
			<td>Bio-Rad</td>
			<td>1863052</td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR Supermix for Probes (No dUTP) (2 x 1 mL)</td>
			<td>Bio-Rad</td>
			<td>1863023</td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR 96-Well Plates (pkg of 25)</td>
			<td>Bio-Rad</td>
			<td>12001925</td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR Droplet Reader Oil (2 x 1L)</td>
			<td>Bio-Rad</td>
			<td>1863004</td>
			<td></td>
		</tr>
		<tr>
			<td>DG8 Cartridge Holder</td>
			<td>Bio-Rad</td>
			<td>1863051</td>
			<td></td>
		</tr>
		<tr>
			<td>DG8 Cartridges for QX200/QX100 (pkg of 24)</td>
			<td>Bio-Rad</td>
			<td>1864008</td>
			<td></td>
		</tr>
		<tr>
			<td>DG8 Gaskets for QX200/QX100 (pkg of 24)</td>
			<td>Bio-Rad</td>
			<td>1863009</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I (10U/&#181;L) + buffer</td>
			<td>Roche</td>
			<td>4716728001</td>
			<td></td>
		</tr>
		<tr>
			<td>Droplet Generation Oil for Probes (10 x 7 mL)</td>
			<td>Bio-Rad</td>
			<td>1863005</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf ep Dualfilter T.I.P.S. Filter Tip, 0.1-10 &#956;L, 34 mm, Rack, PCR Clean, STERILE&#160;</td>
			<td>Eppendorf</td>
			<td>30078500</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf ep Dualfilter T.I.P.S. Filter Tip, 20-300 &#956;L, 55 mm, Rack, PCR Clean, STERILE&#160;</td>
			<td>Eppendorf</td>
			<td>30078560</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf ep Dualfilter T.I.P.S. Filter Tip, 2-100 &#956;L, 53 mm, Rack, PCR Clean, STERILE&#160;</td>
			<td>Eppendorf</td>
			<td>30078543</td>
			<td></td>
		</tr>
		<tr>
			<td>Forward lyophilized primers and respective master stocks at 100 mM&#160;</td>
			<td>IDT</td>
			<td></td>
			<td>GFP as target sequence. Forward primer: 5&#39;-GAACGGCATCAAGGTGAACT-3&#39;</td>
		</tr>
		<tr>
			<td>Lyopohilized probe and master stock at 100 &#181;M&#160;</td>
			<td>IDT</td>
			<td></td>
			<td>GFP as target sequence. PrimeTime Eco Probe: /56-FAM/CAAGATCCG/ZEN/CCACAACATCGAGGA/3IABkFQ/</td>
		</tr>
		<tr>
			<td>Magnesium Chloride (MgCl2)</td>
			<td>Chemlab Analytical</td>
			<td>CL00.1381</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>IDT</td>
			<td>11-04-02-01</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Plate Heat Seal, foil, pierceable (pkg of 100)</td>
			<td>Bio-Rad</td>
			<td>1814040</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-68 non-ionic surfactant (100x)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>24040032</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Honeywell research chemicals</td>
			<td>31248</td>
			<td></td>
		</tr>
		<tr>
			<td>QX manager software</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>Software to analyse ddPCR data</td>
		</tr>
		<tr>
			<td>QX200 Droplet Generator</td>
			<td>Bio-Rad</td>
			<td>1864002</td>
			<td></td>
		</tr>
		<tr>
			<td>QX200 Droplet Reader</td>
			<td>Bio-Rad</td>
			<td>1864003</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent reservoir</td>
			<td>VWR</td>
			<td>613-1181</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse lyophilized primers and respective master stocks at 100 mM&#160;</td>
			<td>IDT</td>
			<td></td>
			<td>GFP as target sequence. Reverse primer: 5&#39;-TGCTCAGGTAGTGGTTGTCG-3&#39;</td>
		</tr>
		<tr>
			<td>SafeSeal reaction tube, 1.5 mL</td>
			<td>Sarstedt</td>
			<td>72.706.200</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA, sheared (10 mg/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9680</td>
			<td></td>
		</tr>
		<tr>
			<td>TE buffer</td>
			<td>IDT</td>
			<td></td>
			<td>Accompanied by primers when ordering</td>
		</tr>
		<tr>
			<td>Tris hydrochloride (Tris-HCl)</td>
			<td>Roche</td>
			<td>10812846001</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of adeno-associated viral genomes in purified vector samples by digital droplet polymerase chain reaction

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>Autor im Rampenlicht: Optimierung der digitalen Tröpfchen-PCR-Methode für eine genaue Quantifizierung des adeno-assoziierten viralen Genoms

Quantifizierung von Adeno-assoziierten viralen Genomen in gereinigten Vektorproben durch digitale Tröpfchenpolymerase-Kettenreaktion

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

quantification-adeno-associated-viral-genomes-purified-vector-samples

Research

JoVE Journal

Bioengineering

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

863 Aufrufe.  KU Leuven. Die DD-PCR ist eine weit verbreitete Technik zur Bestimmung des AAV-Genomtiters, aber ein Konsensprotokoll fehlt derzeit. Unser Protokoll ist eine validierte Schritt-für-Schritt-Anleitung zur Vorbereitung von Proben und zur Durchführung der DD-PCR für eine genaue Genomtitration. Im Vergleich zur QPCR bietet die DD-PCR mehrere Vorteile, darunter eine höhere Präzision, eine höhere Robustheit und eine absolutere und direktere Quantifizierung der Zielsequen...

Serie: Autor im Rampenlicht: Optimierung der digitalen Tröpfchen-PCR-Methode für eine genaue Quantifizierung des adeno-assoziierten viralen Genoms

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.