Name: Auteur in de schijnwerpers: Efficiënte adeno-geassocieerde virusisolatie voor preklinische toepassingen
Uploaded: 2024-02-09T14:30:00
Description: Watch this Scientific Journal Video about Author Spotlight: Efficient Adeno-Associated Virus Isolation for Pre-Clinical Applications at JoVE.com

De samenstelling van de oplossingen en buffers die in dit protocol worden gebruikt, is weergegeven in tabel 1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>Oplossing</td>
			<td colspan="2">Compositie</td>
		</tr><tr><td rowspan="4">AAV-lysisbuffer</td>
			<td colspan="2">1,2 ml 5 M NaCl-oplossing</td>
		</tr><tr><td colspan="2">2 ml van 1 M Tris-HCl pH 8,5-oplossing</td>
		</tr><tr><td colspan="2">80 uL 1 M MgCl2-...

AAV-isolatie door centrifugatie met dubbele idixanoldichtheidsgradiënt

<ol>
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Het zuiveringsprotocol met dubbele jodixanoldichtheidsgradiënt is de universele methode omdat het van toepassing is op alle AAV-mutante varianten, ongeacht hun receptorspecificiteit. Vroege methoden van AAV-zuivering waren gebaseerd op deeltjesdichtheid en omvatten isopycnische centrifugatie in CsCl en continue sucrosedichtheidsgradiëntcentrifugatie19. Later werden serotype-specifieke benaderingen ontwikkeld, die gebruik maakten van monoklonale antilichamen gebonden aan Sepharose-kolommen<sup cl...

Recombinante adeno-geassocieerde virale (rAAV) vectoren zijn veelgebruikte hulpmiddelen voor de behandeling van genetische ziekten, waaronder spinale musculaire atrofie, retinale dystrofie en hemofilie A 1,2,3. rAAV-vectoren zijn zo gemanipuleerd dat ze geen virale genen hebben die aanwezig zijn in wildtype AAV4, een klein, niet-envelop icosahedraal virus met een lineair enkelstrengs DNA-genoom van 4,7 kb. AAV werd voor het eerst ontdekt in de jaren 1960 als een verontreiniging van adenoviruspreparaten. Ondanks de kleine capside-grootte, die de grootte van het transgen dat kan worden verpakt beperkt tot maximaal 4,9 kb exclusief ITR's6, is AAV nuttig voor transgenafgifte omdat het niet-pathogeen is bij mensen, expressie van transgen mogelijk maakt in veel delende en niet-delende celtypen en beperkte immunogene effecten heeft7.
Als leden van het geslacht dependoparvovirus is de productie van rAAV's afhankelijk van de expressie van helpergenen die aanwezig zijn in adenovirus of herpes simplex-virus8. Er zijn verschillende strategieën ontwikkeld om rAAV te produceren, maar de productie in HEK293-cellen die zijn getransformeerd met de adenovirale E1A/E1B-helpergenen is de meest gevestigde methode die tegenwoordig wordt gebruikt. De algemene benadering van rAAV-productie begint met de transfectie van HEK293-cellen met drie plasmiden die het transgen bevatten in respectievelijk omgekeerde terminale herhalingen (ITR's), AAV-rep- en cap-genen en aanvullende adenovirale helpergenen. Tweeënzeventig uur na transfectie worden cellen geoogst en verwerkt om rAAV te zuiveren die het transgen bevat.
Bij de ontwikkeling van nieuwe rAAV-vectoren voor therapeutische doeleinden is een belangrijk doel het produceren van vectoren met een verhoogde transductie-efficiëntie. Een toename van de transductie-efficiëntie van doelcellen zou een verlaging van de noodzakelijke klinische dosis rAAV betekenen, waardoor de kans op nadelige immunogene effecten, variërend van antilichaam-gemedieerde neutralisatie tot acute toxiciteiten, wordt verkleind10,11. Om de transductie-efficiëntie van rAAV-vectoren te verbeteren, kunnen wijzigingen worden aangebracht in het verpakte genoom of in de capside. Haalbare methoden om de werkzaamheid van transductie af te stemmen via verpakt genoomontwerp zijn onder meer de integratie van sterke en weefselspecifieke promotors, doordachte selectie van mRNA-verwerkingselementen en optimalisatie van coderingssequenties om de translatie-efficiëntie te verbeteren12. Wijzigingen aan de capside worden aangebracht met als doel het tropisme voor menselijke doelceltypen te vergroten. Inspanningen om nieuwe rAAV-capsiden voor transgenafgifte te ontwikkelen, worden over het algemeen gekenmerkt door een focus op ofwel rationeel ontwerp van AAV-capsiden met specifieke mutaties gericht op specifieke celreceptoren, ofwel gerichte evolutie om capsiden met tropisme te identificeren voor specifieke celtypen uit combinatorische capsidebibliotheken met hoge complexiteit zonder zich te richten op één specifieke receptor (hoewel sommige groepen deze benaderingen combineren)13, 14,15. In de gerichte evolutiebenadering worden combinatorische capsidebibliotheken geconstrueerd met behulp van een bepaalde serotype-ruggengraat met gemuteerde variabele regio's aan de buitenkant van de capside16. Combinatorische capsidebibliotheken zijn vaak opgebouwd uit AAV-serotypen die niet van mensen afkomstig zijn, waardoor het risico op reeds bestaande immuniteit tijdens klinisch gebruik afneemt10. Daarom zijn zuiveringsmethoden die op elk serotype kunnen worden toegepast, ideaal om de noodzaak van serotype-specifieke optimalisatie voor de minder vaak gebruikte serotypen die als ruggengraat voor deze bibliotheken dienen, te elimineren.
Centrifugatie met de dichtheidsgradiënt van Iodixanol wordt gebruikt om hoge titers van rAAV met een hoge besmettelijkheid te zuiveren17. In dit protocol wordt rAAV geproduceerd in suspensiecelkweek om de hoeveelheid arbeid die nodig is om grote titers van AAV te produceren te verminderen. Er is ook een centrifugatiestap opgenomen om cellysaat te zuiveren om de aanwezigheid van verontreinigende eiwitten te verminderen en het risico op virusneerslag te verminderen. Dit protocol is een kosteneffectieve methode om preparaten met een hoge zuiverheid rAAV te produceren die geschikt zijn voor preklinisch gebruik.

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		</tr>
		<tr>
			<td>Conical centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>339652</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Conical centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>339650</td>
			<td>15 mL</td>
		</tr>
		<tr>
			<td>Disposable micro-pipets</td>
			<td>Fisherbrand</td>
			<td>21-164-2G</td>
			<td>Capillaries</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline without CaCl2 and MgCl2&#160; (DPBS) (10x)</td>
			<td>Sigma-Aldrich</td>
			<td>D1408</td>
			<td></td>
		</tr>
		<tr>
			<td>ECLIPSE Ts2R-FL inverted microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Expi293 Expression Medium</td>
			<td>Gibco</td>
			<td>A1435101</td>
			<td></td>
		</tr>
		<tr>
			<td>Expi293F cells</td>
			<td>Gibco</td>
			<td>A14527</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips</td>
			<td>USA Scientific</td>
			<td>1126-7810</td>
			<td>1000 &#181;L</td>
		</tr>
		<tr>
			<td>Filter tips</td>
			<td>USA Scientific</td>
			<td>1120-8810</td>
			<td>200 &#181;L</td>
		</tr>
		<tr>
			<td>Filter tips</td>
			<td>USA Scientific</td>
			<td>1120-1810</td>
			<td>20 &#181;L</td>
		</tr>
		<tr>
			<td>Filter tips</td>
			<td>USA Scientific</td>
			<td>1121-3810</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>Hypodermic needles</td>
			<td>Tyco Healthcare</td>
			<td>820112</td>
			<td>20 GA x 1-1/2 A</td>
		</tr>
		<tr>
			<td>Ice bucket with lid</td>
			<td>VWR</td>
			<td>10146-184</td>
			<td></td>
		</tr>
		<tr>
			<td>JS-5.3 rotor</td>
			<td>Beckman Coulter</td>
			<td>368690</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride solution (1 M)</td>
			<td>Millipore Sigma</td>
			<td>M1028-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal stand and clamp&#160;</td>
			<td>Fisherbrand</td>
			<td>05-769-6Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>22600028</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Needle nose pliers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optima XE-90 ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>A94471</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced-Serum Medium</td>
			<td>Gibco</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep density gradient media (iodixanol)</td>
			<td>Serumwerk</td>
			<td>AXS-1114542</td>
			<td>60% iodixanol solution</td>
		</tr>
		<tr>
			<td>P1000 Pipet</td>
			<td>Gilson</td>
			<td>F144059M</td>
			<td></td>
		</tr>
		<tr>
			<td>P2 Pipet</td>
			<td>Gilson</td>
			<td>F144054M</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 Pipet</td>
			<td>Gilson</td>
			<td>F144056M</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 Pipet</td>
			<td>Gilson</td>
			<td>F144058M</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red&#160;solution</td>
			<td>Sigma-Aldrich</td>
			<td>P0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4474</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Aid XP pipette controller</td>
			<td>Drummond Scientific</td>
			<td>4-000-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid pCapsid</td>
			<td>De novo or Addgene, etc.&#160;</td>
			<td>N/A</td>
			<td>We used pACGrh74.&#160;</td>
		</tr>
		<tr>
			<td>Plasmid pHelper</td>
			<td>Addgene</td>
			<td>112867</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid pTransgene</td>
			<td>De novo or Addgene, etc.&#160;</td>
			<td>N/A</td>
			<td>We used pdsAAV-GFP.</td>
		</tr>
		<tr>
			<td>Pluronic F-68 polyol solution (10%)</td>
			<td>Mp Biomedicals</td>
			<td>92750049</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol 8000</td>
			<td>Research Products International</td>
			<td>P48080-500.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine HCl Max (PEI-Max)</td>
			<td>Polysciences</td>
			<td>NC1038561</td>
			<td>Dilute in water to 40 &#956;M</td>
		</tr>
		<tr>
			<td>Polypropylene centrifuge tubes, sterile</td>
			<td>Corning</td>
			<td>431123</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Polypropylene centrifuge tubes, sterile</td>
			<td>Corning</td>
			<td>430776</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>Polypropylene Optiseal tubes</td>
			<td>Beckman Coulter</td>
			<td>361625</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes</td>
			<td>Alkali Scientific</td>
			<td>SP250-B</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Serological pipettes</td>
			<td>Alkali Scientific</td>
			<td>SP225-B</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>Serological pipettes</td>
			<td>Alkali Scientific</td>
			<td>SP210-B</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Serological pipettes</td>
			<td>Alkali Scientific</td>
			<td>SP205-B</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Shaker flasks</td>
			<td>Fisherbrand</td>
			<td>PBV1000</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Shaker flasks</td>
			<td>Fisherbrand</td>
			<td>PBV50-0</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Shaker flasks</td>
			<td>Fisherbrand</td>
			<td>PBV250</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>Shaker flasks</td>
			<td>Fisherbrand</td>
			<td>PBV12-5</td>
			<td>125 mL</td>
		</tr>
		<tr>
			<td>Sodium chloride solution (5 M)</td>
			<td>Fisher Scientific</td>
			<td>NC1752640</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringes</td>
			<td>Fisherbrand</td>
			<td>14-955-458</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Millipore</td>
			<td>SLGV013SL</td>
			<td>0.22 micron</td>
		</tr>
		<tr>
			<td>Tris-HCl pH 8.5 (1 M)</td>
			<td>Kd Medical</td>
			<td>RGE3363</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue solution</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube rack assembly</td>
			<td>Beckman Coulter</td>
			<td>361646</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube spacers (x4)</td>
			<td>Beckman Coulter</td>
			<td>361669</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing for peristaltic pump</td>
			<td>Fisher Scientific</td>
			<td>14190516</td>
			<td></td>
		</tr>
		<tr>
			<td>Type 70 Ti fixed-angle titanium rotor</td>
			<td>Beckman Coulter</td>
			<td>337922</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra low temperature freezer</td>
			<td></td>
			<td></td>
			<td>Set at -70 &#176;C</td>
		</tr>
		<tr>
			<td>Vivaspin 20 centrifugal concentrator</td>
			<td>Sartorius</td>
			<td>VS2041</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath&#160;</td>
			<td></td>
			<td></td>
			<td>Set at 37 &#176;C</td>
		</tr>
	</tbody>
</table>

suspension culture production and purification of adeno-associated virus by iodixanol density gradient centrifugation for in vivo applications

Dit protocol beschrijft de productie en zuivering van recombinant adeno-geassocieerd virus (rAAV) door middel van jodixanoldichtheidsgradiëntcentrifugatie, een serotype-agnostische methode om AAV te zuiveren die voor het eerst werd beschreven in 1999. rAAV-vectoren worden veel gebruikt in gentherapietoepassingen om transgenen af te leveren aan verschillende menselijke celtypen. In dit werk wordt het recombinante virus geproduceerd door transfectie van Expi293-cellen in suspensiecultuur met plasmiden die coderen voor de transgen-, vectorcapside- en adenovirale helpergenen. Iodixanol dichtheidsgradiëntcentrifugatie zuivert volledige AAV-deeltjes op basis van deeltjesdichtheid. Bovendien zijn er drie stappen opgenomen in deze nu alomtegenwoordige methodologie om de totale virusopbrengst te verhogen, het risico op neerslag als gevolg van besmettende eiwitten te verminderen en het uiteindelijke virusproduct verder te concentreren, respectievelijk: precipitatie van virale deeltjes uit celmedia met behulp van een oplossing van polyethyleenglycol (PEG) en natriumchloride, de introductie van een tweede ronde van jodixanoldichtheidsgradiëntcentrifugatie, en bufferuitwisseling via een centrifugaalfilter. Met behulp van deze methode is het mogelijk om consistent titers te bereiken in het bereik van 1012 virale deeltjes/ml met een uitzonderlijke zuiverheid voor in vivo gebruik.

Recombinant adeno-associated viral (rAAV) vectors are widely used tools for the treatment of genetic diseases, including spinal muscular atrophy, retinal dystrophy, and hemophilia A1,2,3. rAAV vectors are engineered to lack viral genes present in wild-type AAV4, a small, non-envelope icosahedral virus with a linear single-stranded 4.7 kb DNA genome. AAV was first discovered in the 1960s as a contaminant of adenovirus preparations5. Despite its small capsid size, which limits the size of the transgene that can be packaged to a maximum of 4.9 kb excluding ITRs6, AAV is useful for transgene delivery because it is non-pathogenic in humans, allows expression of transgene in many dividing and nondividing cell types, and has limited immunogenic effects7.
As members of the genus dependoparvovirus, the production of rAAVs relies on the expression of helper genes present in adenovirus or herpes simplex virus8. Several strategies to produce rAAV have been developed, but production in HEK293 cells transformed with the adenoviral E1A/E1B helper genes is the most established method used today9. The general approach of rAAV production begins with the transfection of HEK293 cells with three plasmids containing the transgene within inverted terminal repeats (ITRs), AAV rep and cap genes, and additional adenoviral helper genes, respectively. Seventy-two hours after transfection, cells are harvested and processed to purify rAAV containing the transgene.
In the development of new rAAV vectors for therapeutic purposes, a major goal is producing vectors with increased transduction efficiency. An increase in the transduction efficiency of target cells would mean a decrease in the necessary clinical dose of rAAV, thus decreasing the likelihood of adverse immunogenic effects ranging from antibody-mediated neutralization to acute toxicities10,11. To improve the transduction efficacy of rAAV vectors, alterations can be made to the packaged genome or to the capsid. Viable methods to tune transduction efficacy via packaged genome design include the incorporation of strong and tissue-specific promoters, thoughtful selection of mRNA processing elements, and coding sequence optimization to improve translation efficiency12. Alterations to the capsid are made with the goal of increasing tropism for target human cell types. Efforts towards developing new rAAV transgene delivery vector capsids are generally characterized by a focus on either rational design of AAV capsids with specific mutations targeting specific cell receptors or directed evolution to identify capsids with tropism for specific cell types from high-complexity combinatorial capsid libraries without targeting one specific receptor (although some groups combine these approaches)13,14,15. In the directed evolution approach, combinatorial capsid libraries are constructed using a particular serotype backbone with mutated variable regions on the capsid exterior16. Combinatorial capsid libraries are often constructed from AAV serotypes not originating in humans, decreasing the risk of preexisting immunity during clinical use10. Therefore, purification methods that can be applied to any serotype are ideal to eliminate the need for serotype-specific optimization for the less commonly used serotypes serving as backbones for these libraries.
Iodixanol density gradient centrifugation is utilized to purify high titers of rAAV with high infectivity17. In this protocol, rAAV is produced in suspension cell culture to decrease the amount of labor needed to produce large titers of AAV. A centrifugation step is also included to clear cell lysate to reduce the presence of contaminating proteins and decrease the risk of virus precipitation. This protocol is a cost-effective method to produce preparations of high-purity rAAV suitable for pre-clinical use.

Auteur in de schijnwerpers: Efficiënte adeno-geassocieerde virusisolatie voor preklinische toepassingen

Productie en zuivering van adeno-geassocieerd virus door centrifugatie met diodixanoldichtheidsgradiënt voor in-vivotoepassingen

We use a directed evolution approach to identify adeno-associated viral capits with higher tropism for target human cell types. Through this research, we aim to produce recombinant adeno-associated viral vectors with improved target cell transduction and clinical relevance within the field of gene therapy. Our group recently de-targeted several AAV serotypes from the liver by incorporating capsid residues original to an AAV serotype that naturally possesses decreased liver tropism.If successfully expanded to additional serotypes, this provides the groundwork to decrease necessary RAAV clinical dose by peripheral injection and reduce adverse patient outcomes. The IODIXanol purification method while being cost effective is difficult to scale up. Additionally, in downstream applications, the directed evolution approach is performed with animal models or in vitro.So capsid isolated by directed evolution may not transduce in vivo targets in humans to the same extent as in preclinical models. This protocol is affordable and accessible for smaller labs. Additionally, this method yields high purity, recombinant adeno-associated virus that can be used for downstream preclinical in vivo applications.

Deze methode kan worden gebruikt om titers van ten minste 1012 virale deeltjes per ml te verkrijgen. Een titer kan worden verkregen (figuur 3) door qPCR met behulp van de ITR-primers in aanvullende tabel 1, door ddPCR of door een andere titermethode. Suboptimale titers kunnen het gevolg zijn van het gebruik van een cap-gen dat codeert voor een capside met een slechte verpakkingsefficiëntie.
Een andere mogelijke bron van subopt...

Watch this Scientific Journal Video about Author Spotlight: Efficient Adeno-Associated Virus Isolation for Pre-Clinical Applications at JoVE.com

Adeno-geassocieerd virus wordt geproduceerd in suspensiecelkweek en gezuiverd door centrifugatie met dubbele jodixanoldichtheidsgradiënt. Er zijn stappen opgenomen om de totale virusopbrengst te verhogen, het risico op virusneerslag te verminderen en het uiteindelijke virusproduct verder te concentreren. De verwachte uiteindelijke titers bereiken 10tot 12 virale deeltjes/ml en zijn geschikt voor preklinisch in vivo gebruik.

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a ...

We gebruiken een gerichte evolutiebenadering om adeno-geassocieerde virale capits met een hoger tropisme te identificeren voor menselijke doelceltypen. Door middel van dit onderzoek willen we recombinante adeno-geassocieerde virale vectoren produceren met verbeterde doelceltransductie en klinische relevantie binnen het gebied van gentherapie. Onze groep heeft onlangs verschillende AAV-serotypen uit de lever gedeviseerd door capside-residuen op te nemen die origineel zijn voor een AAV-serotype dat van nature een verminderd levertropisme bezit.Indien dit met succes wordt uitgebreid naar aanvullende serotypen, biedt dit de basis om de noodzakelijke klinische dosis RAAV te verlagen door perifere injectie en nadelige resultaten voor de patiënt te verminderen. De IODIXanol-zuiveringsmethode is weliswaar kosteneffectief, maar moeilijk op te schalen. Bovendien wordt in downstream-toepassingen de gerichte evolutiebenadering uitgevoerd met diermodellen of in vitro.Het is dus mogelijk dat capside geïsoleerd door gerichte evolutie in vivo doelen bij mensen niet in dezelfde mate transduceert als in preklinische modellen. Dit protocol is betaalbaar en toegankelijk voor kleinere labs. Bovendien levert deze methode een zeer zuiver, recombinant adeno-geassocieerd virus op dat kan worden gebruikt voor stroomafwaartse preklinische in vivo-toepassingen.

suspension-culture-production-purification-adeno-associated-virus

Research

JoVE Journal

Medicine

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.