Name: Author Spotlight: Efficient Adeno-Associated Virus Isolation for Pre-Clinical Applications
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Sammensætningen af de opløsninger og buffere, der anvendes i denne protokol, er angivet i tabel 1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>Opløsning</td>
			<td colspan="2">Sammensætning</td>
		</tr><tr><td rowspan="4">AAV lysis buffer</td>
			<td colspan="2">1,2 ml 5 M NaCl opløsning</td>
		</tr><tr><td colspan="2">2 ml 1 M Tris-HCl pH 8,5 opløsning</td>
		</tr><tr><td colspan="2">80 ml 1 MMgCl2-opløsn...

AAV Isolation by Double Iodixanol Density Gradient Centrifugation

<ol>
	<li>Strauss, K. 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=Onasemnogene+abeparvovec+for+presymptomatic+infants+with+three+copies+of+SMN2+at+risk+for+spinal+muscular+atrophy:+the+Phase+III+SPR1NT+trial.">Onasemnogene abeparvovec for presymptomatic infants with three copies of SMN2 at risk for spinal muscular atrophy: the Phase III SPR1NT trial.</a> Nat Med. 28 (7), 1390-1397 (2022).</li><li>Fuller-Carter, P. I., Basiri, H., Harvey, A. R., Carvalho, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused+update+on+AAV-based+gene+therapy+clinical+trials+for+inherited+retinal+degeneration.">Focused update on AAV-based gene therapy clinical trials for inherited retinal degeneration.</a> BioDrugs. 34 (6), 763-781 (2020).</li><li>George, L. 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=Multiyear+factor+VIII+expression+after+AAV+gene+transfer+for+hemophilia+A.">Multiyear factor VIII expression after AAV gene transfer for hemophilia A.</a> N Engl J Med. 385 (21), 1961-1973 (2021).</li><li>Naso, M. F., Tomkowicz, B., Perry, W. L., Strohl, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-Associated+Virus+(AAV)+as+a+vector+for+gene+therapy.">Adeno-Associated Virus (AAV) as a vector for gene therapy.</a> Biodrugs. 31 (4), 317-334 (2017).</li><li>Atchison, R. W., Casto, B. C., Hammon, W. M. c. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-associated+defective+virus+particles.">Adenovirus-associated defective virus particles.</a> Science. 149 (3685), 754-756 (1965).</li><li>Wu, Z., Yang, H., Colosi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genome+size+on+AAV+vector+packaging.">Effect of genome size on AAV vector packaging.</a> Mol Ther. 18 (1), 80-86 (2010).</li><li>Samulski, R. J., Muzyczka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV-mediated+gene+therapy+for+research+and+therapeutic+purposes.">AAV-mediated gene therapy for research and therapeutic purposes.</a> Annu Rev Virol. 1 (1), 427-451 (2014).</li><li>Zolotukhin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+adeno-associated+virus+vectors.">Production of recombinant adeno-associated virus vectors.</a> Hum Gene Ther. 16 (5), 551-557 (2005).</li><li>Penaud-Budloo, M., Fran&#231;ois, A., Cl&#233;ment, N., Ayuso, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+of+recombinant+adeno-associated+virus+production.">Pharmacology of recombinant adeno-associated virus production.</a> Mol Ther - Methods Clin Dev. 8, 166-180 (2018).</li><li>Costa-Verdera, H., 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=Understanding+and+Tackling+immune+responses+to+adeno-associated+viral+vectors.">Understanding and Tackling immune responses to adeno-associated viral vectors.</a> Hum Gene Ther. 34 (17-18), 836-852 (2023).</li><li>Ertl, H. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+tools+for+production+and+purification+of+recombinant+adenoassociated+virus+vectors.">Novel tools for production and purification of recombinant adenoassociated virus vectors.</a> Hum Gene Ther. 9 (18), 2745-2760 (1998).</li><li>Zolotukhin, S., 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=Recombinant+adeno-associated+virus+purification+using+novel+methods+improves+infectious+titer+and+yield.">Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield.</a> Gene Ther. 6 (6), 973-985 (1999).</li><li>Clark, K. R., Liu, X., Mcgrath, J. P., Johnson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+purified+recombinant+adeno-associated+virus+vectors+are+biologically+active+and+free+of+detectable+helper+and+wild-type+viruses.">Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses.</a> Hum Gene Ther. 10 (6), 1031-1039 (1999).</li><li>Debelak, 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=Cation-exchange+high-performance+liquid+chromatography+of+recombinant+adeno-associated+virus+type+2.">Cation-exchange high-performance liquid chromatography of recombinant adeno-associated virus type 2.</a> J Chromatogr B Biomed Sci App. 740 (2), 195-202 (2000).</li><li>Burova, E., Ioffe, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatographic+purification+of+recombinant+adenoviral+and+adeno-associated+viral+vectors:+methods+and+implications.">Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications.</a> Gene Ther. 12 (1), S5-S17 (2005).</li><li>Adams, B., Bak, H., Tustian, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+from+the+bench+towards+a+large+scale,+industrial+platform+process+for+adeno-associated+viral+vector+purification.">Moving from the bench towards a large scale, industrial platform process for adeno-associated viral vector purification.</a> Biotechnol Bioeng. 117 (10), 3199-3211 (2020).</li><li>Grieger, J. C., Choi, V. W., Samulski, R. 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Den dobbelte iodixanol densitetsgradient oprensningsprotokol er den universelle metode, fordi den gælder for alle AAV-mutantvarianter, uanset deres receptorspecificitet. Tidlige metoder til AAV-oprensning var afhængige af partikeldensitet og omfattede isopycnic centrifugering i CsCl og kontinuerlig saccharosedensitetsgradientcentrifugering19. Senere blev serotypespecifikke tilgange udviklet, som gjorde brug af monoklonale antistoffer bundet til Sepharose kolonne20. En ny ...

Rekombinante adeno-associerede virale (rAAV) vektorer er almindeligt anvendte værktøjer til behandling af genetiske sygdomme, herunder spinal muskelatrofi, retinal dystrofi og hæmofili A 1,2,3. rAAV-vektorer er konstrueret til at mangle virale gener, der er til stede i vildtype AAV4, en lille, ikke-konvolut icosahedral virus med et lineært enkeltstrenget 4,7 kb DNA-genom. AAV blev først opdaget i 1960'erne som en forurening af adenoviruspræparater5. På trods af sin lille kapsidstørrelse, som begrænser størrelsen af transgenet, der kan pakkes til maksimalt 4,9 kb eksklusive ITR6, er AAV nyttig til transgenlevering, fordi den er ikke-patogen hos mennesker, tillader ekspression af transgen i mange delende og ikke-delende celletyper og har begrænsede immunogene virkninger7.
Som medlemmer af slægten dependoparvovirus er produktionen af rAAV'er afhængig af ekspressionen af hjælpergener, der er til stede i adenovirus eller herpes simplex virus8. Flere strategier til produktion af rAAV er blevet udviklet, men produktion i HEK293-celler transformeret med adenovirale E1A/E1B-hjælpegener er den mest etablerede metode, der anvendes i dag9. Den generelle tilgang til rAAV-produktion begynder med transfektion af HEK293-celler med tre plasmider indeholdende transgenet inden for henholdsvis inverterede terminale gentagelser (ITR'er), AAV-rep - og cap-gener og yderligere adenovirale hjælpergener. Tooghalvfjerds timer efter transfektion høstes og behandles celler for at rense rAAV indeholdende transgenet.
I udviklingen af nye rAAV-vektorer til terapeutiske formål er et vigtigt mål at producere vektorer med øget transduktionseffektivitet. En forøgelse af målcellernes transduktionseffektivitet ville betyde et fald i den nødvendige kliniske dosis rAAV, hvilket ville mindske sandsynligheden for negative immunogene virkninger, der spænder fra antistofmedieret neutralisering til akut toksicitet10,11. For at forbedre transduktionseffektiviteten af rAAV-vektorer kan der foretages ændringer i det pakkede genom eller kapsiden. Levedygtige metoder til at indstille transduktionseffektiviteten via pakket genomdesign omfatter inkorporering af stærke og vævsspecifikke promotorer, tankevækkende udvælgelse af mRNA-behandlingselementer og kodningssekvensoptimering for at forbedre oversættelseseffektiviteten12. Ændringer i kapsiden foretages med det formål at øge tropismen for målhumane celletyper. Bestræbelser på at udvikle nye rAAV-transgenleveringsvektorkapsider er generelt kendetegnet ved fokus på enten rationelt design af AAV-kapsider med specifikke mutationer rettet mod specifikke cellereceptorer eller rettet evolution for at identificere kapsider med tropisme for specifikke celletyper fra kombinatoriske capsidbiblioteker med høj kompleksitet uden at målrette mod en bestemt receptor (selvom nogle grupper kombinerer disse tilgange)13, 14,15. I den rettede evolutionstilgang konstrueres kombinatoriske capsidbiblioteker ved hjælp af en bestemt serotype-rygrad med muterede variable regioner på kapsidet ydre16. Kombinatoriske capsidbiblioteker er ofte konstrueret af AAV-serotyper, der ikke stammer fra mennesker, hvilket reducerer risikoen for allerede eksisterende immunitet under klinisk brug10. Derfor er rensningsmetoder, der kan anvendes på enhver serotype, ideelle til at eliminere behovet for serotypespecifik optimering for de mindre almindeligt anvendte serotyper, der tjener som rygrad for disse biblioteker.
Iodixanol densitetsgradientcentrifugering anvendes til at rense høje titere af rAAV med høj infektivitet17. I denne protokol produceres rAAV i suspensionscellekultur for at reducere mængden af arbejdskraft, der er nødvendig for at producere store titere af AAV. Et centrifugeringstrin er også inkluderet for at rydde cellelysat for at reducere tilstedeværelsen af forurenende proteiner og mindske risikoen for virusudfældning. Denne protokol er en omkostningseffektiv metode til fremstilling af præparater af rAAV med høj renhed, der er egnet til præklinisk brug.

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	<tbody>
		<tr>
			<td>5810 R benchtop centrifuge</td>
			<td>Eppendorf</td>
			<td>22625501</td>
			<td></td>
		</tr>
		<tr>
			<td>8-channel peristaltic pump&#160;</td>
			<td>Watson-Marlow</td>
			<td>020.3708.00A</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated cell counter&#160;</td>
			<td>NanoEntek</td>
			<td>EVE-MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-E high-speed centrifuge</td>
			<td>Beckman Coulter</td>
			<td>369001</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>Thermo Scientific</td>
			<td>88701</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety cabinet</td>
			<td>Labconco</td>
			<td>322491101</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator with shaker&#160;</td>
			<td></td>
			<td></td>
			<td>Set at 8% CO2 and 37 &#176;C</td>
		</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

Denne protokol beskriver rekombinant adeno-associeret virus (rAAV) produktion og oprensning ved iodixanol densitetsgradientcentrifugering, en serotype-agnostisk metode til rensning af AAV først beskrevet i 1999. rAAV-vektorer anvendes i vid udstrækning i genterapiapplikationer til at levere transgener til forskellige humane celletyper. I dette arbejde produceres den rekombinante virus ved transfektion af Expi293-celler i suspensionskultur med plasmider, der koder for transgen-, vektorkapsid- og adenovirale hjælpergener. Iodixanol densitetsgradientcentrifugering renser fulde AAV-partikler baseret på partikeldensitet. Derudover er tre trin inkluderet i denne nu allestedsnærværende metode for at øge det samlede virusudbytte, mindske risikoen for udfældning på grund af forurenende proteiner og yderligere koncentrere det endelige virusprodukt, henholdsvis: udfældning af virale partikler fra cellemedier ved anvendelse af en opløsning af polyethylenglycol (PEG) og natriumchlorid, indførelsen af en anden runde af iodixanol densitetsgradientcentrifugering, og bufferudskiftning via et centrifugalfilter. Ved hjælp af denne metode er det muligt konsekvent at opnå titere i området 1012 virale partikler/ml af exceptionel renhed til in vivo-brug .

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.

Author Spotlight: Efficient Adeno-Associated Virus Isolation for Pre-Clinical Applications

Suspension kultur: produktion og oprensning af adeno-associeret virus ved iodixanol densitetsgradient centrifugering til in vivo applikationer

Denne metode kan anvendes til at opnå titere på mindst 1012 virale partikler pr. ml. En titer kan opnås (figur 3) ved qPCR ved hjælp af ITR-primerne i supplerende tabel 1, ved ddPCR eller ved enhver anden titeringsmetode. Suboptimale titere kan skyldes brug af et cap-gen , der koder for et capsid med dårlig emballageeffektivitet.
En anden mulig kilde til suboptimale resultater er den dårlige transduktionseffektivitet af E...

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

Adeno-associeret virus fremstilles i suspensionscellekultur og renses ved centrifugering med dobbelt iodixanoldensitetsgradient. Trin er inkluderet for at øge det samlede virusudbytte, mindske risikoen for virusudfældning og yderligere koncentrere det endelige virusprodukt. Forventede sluttitre når op på 1012 virale partikler/ml og er egnede til præklinisk in vivo-brug .

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

Vi bruger en rettet evolutionstilgang til at identificere adeno-associerede virale kapits med højere tropisme for målhumane celletyper. Gennem denne forskning sigter vi mod at producere rekombinante adeno-associerede virale vektorer med forbedret målcelletransduktion og klinisk relevans inden for genterapi. Vores gruppe har for nylig målrettet flere AAV-serotyper fra leveren ved at inkorporere capsidrester, der er originale til en AAV-serotype, der naturligt besidder nedsat levertropisme.Hvis det lykkes at udvide til yderligere serotyper, giver dette grundlaget for at reducere den nødvendige RAAV kliniske dosis ved perifer injektion og reducere negative patientresultater. IODIXanol-rensningsmetoden, mens den er omkostningseffektiv, er vanskelig at skalere op. Derudover udføres den rettede evolutionstilgang i downstream-applikationer med dyremodeller eller in vitro.Så kapsid isoleret ved rettet evolution kan muligvis ikke transducere in vivo-mål hos mennesker i samme omfang som i prækliniske modeller. Denne protokol er overkommelig og tilgængelig for mindre laboratorier. Derudover giver denne metode rekombinant adenoassocieret virus med høj renhed, der kan bruges til downstream prækliniske in vivo-applikationer.