<meta charset="utf8"></head><body>使用数字液滴 PCR 进行病毒基因组定量的标准化方案

<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

<meta charset="utf8"></head><body>作者聚焦：优化数字微滴 PCR 方法以实现准确的腺相关病毒基因组定量

通过数字液滴聚合酶链反应定量纯化载体样品中的腺相关病毒基因组

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

1.6K 次观看.  KU Leuven. DD PCR 是一种广泛使用的测定 AAV 基因组滴度的技术，但目前缺乏共识方案。我们的实验步骤是关于如何制备样品和进行 DD PCR 以实现准确基因组滴定的经过验证的分步指南。与 QPCR 相比，DD PCR 具有多种优势，包括更高的精度、更强的稳定性以及更绝对和直接的靶序列定量，而无需标准曲线。此外，DD PCR 具有精心设计的引物探针组，与其他检测?...

视频: 作者聚焦：优化数字微滴 PCR 方法以实现准确的腺相关病毒基因组定量

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

科研

生物工程

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

一种基于液滴的单链 dna 放大术和微球 pcr 扩增法

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Bulk Droplet Vitrification for Primary Hepatocyte Preservation

Vitrification is a promising ice-free alternative for classic slow-freezing (at approximately 1 &#176;C/min) cryopreservation of biological samples. Vitrification requires extremely fast cooling rates to achieve transition of water into the glass phase while avoiding injurious ice formation. Although pre-incubation with cryoprotective agents (CPA) can reduce the critical cooling rate of biological samples, high concentrations are generally needed to enable ice-free cryopreservation by vitrification. As a result, vitrification is hampered by CPA toxicity and restricted to small samples that can be cooled fast. It was recently demonstrated that these inherent limitations can be overcome by bulk droplet vitrification. Using this novel method, cells are first pre-incubated with a low intracellular CPA concentration. Leveraging rapid osmotic dehydration, the intracellular CPA is concentrated directly ahead of vitrification, without the need to fully equilibrate toxic CPA concentrations. The cellular dehydration is performed in a fluidic device and integrated with continuous high throughput generation of large sized droplets that are vitrified in liquid nitrogen. This ice-free cryopreservation method with minimal CPA toxicity is suitable for large cell quantities and results in increased hepatocyte viability and metabolic function as compared to classical slow-freezing cryopreservation. This manuscript describes the methods for successful bulk droplet vitrification in detail.

This manuscript describes an ice-free cryopreservation method for large quantities of rat hepatocytes whereby primary cells are pre-incubated with cryoprotective agents at a low concentration and vitrified in large droplets.

用于初级肝细胞保存的散装滴滴 Vit;

Vitrification is a promising ice-free alternative for classic slow-freezing (at approximately 1 &#176;C/min) cryopreservation of biological samples. ...

The Quantification of Injectability by Mechanical Testing

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more viscous than traditional aqueous injections and may be semi-solid, therefore, their injectability cannot be assumed. This protocol describes a method to objectively assess the injectability of these materials using a standard mechanical tester. The syringe plunger is compressed by the crosshead at a set rate, and the force is measured. The maximum or plateau force value can then be used for comparison between samples, or to an absolute force limit. This protocol can be used with any material, and any syringe and needle size or geometry. The results obtained may be used to make decisions about formulations, syringe and needle sizes early in the translational process. Further, the effects of altering formulations on injectability may be quantified, and the optimum time to inject temporally changing materials determined. This method is also suitable as a reproducible way to examine the effects of injection on a material, to study phenomena such as self-healing and filter pressing or study the effects of injection on cells. This protocol is faster and more directly applicable to injectability than rotational rheology, and requires minimal post processing to obtain key values for direct comparisons.

Presented here is a protocol for quantitatively evaluating the injectability of a material through a syringe-needle system using a standard mechanical testing rig.

机械测试的注入性量化

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more ...

Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus (AAV) Capsid Variants

Gene delivery vectors derived from Adeno-associated virus (AAV) are one of the most promising tools for the treatment of genetic diseases, evidenced by encouraging clinical data and the approval of several AAV gene therapies. Two major reasons for the success of AAV vectors are (i) the prior isolation of various naturally occurring viral serotypes with distinct properties, and (ii) the subsequent establishment of powerful technologies for their molecular engineering and repurposing in high throughput. Further boosting the potential of these techniques are recently implemented strategies for barcoding selected AAV capsids on the DNA and RNA level, permitting their comprehensive and parallel in vivo stratification in all major organs and cell types in a single animal. Here, we present a basic pipeline encompassing this set of complementary avenues, using AAV peptide display to represent the diverse arsenal of available capsid engineering technologies. Accordingly, we first describe the pivotal steps for the generation of an AAV peptide display library for the in vivo selection of candidates with desired properties, followed by a demonstration of how to barcode the most interesting capsid variants for secondary in vivo screening. Next, we exemplify the methodology for the creation of libraries for next-generation sequencing (NGS), including barcode amplification and adaptor ligation, before concluding with an overview of the most critical steps during NGS data analysis. As the protocols reported here are versatile and adaptable, researchers can easily harness them to enrich the optimal AAV capsid variants in their favorite disease model and for gene therapy applications.

Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus AAV Capsid Variants

AAV peptide display library generation and subsequent validation through the barcoding of candidates with novel properties for the creation of next-generation AAVs.

通过工程、条形码和筛选腺相关病毒 （AAV） 衣壳变体分离下一代基因治疗载体

Gene delivery vectors derived from Adeno-associated virus (AAV) are one of the most promising tools for the treatment of genetic diseases, evidenced by ...

通过单步肝素亲和层析纯化 AAV 载体

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs.
This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

作者聚焦：腺相关病毒载体生产和纯化的改进方法

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. ...