Name: Autor destacado: Aislamiento eficiente de virus adenoasociados para aplicaciones preclínicas
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

La composición de las soluciones y tampones utilizados en este protocolo se proporciona en la Tabla 1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>Solución</td>
			<td colspan="2">Composición</td>
		</tr><tr><td rowspan="4">Tampón de lisis AAV</td>
			<td colspan="2">1,2 ml de solución de NaCl 5 M</td>
		</tr><tr><td colspan="2">2 ml de solución de Tris-HCl 1 M pH 8,5</td>
		</tr><tr><td colspan="2">80 uL de solu...

Aislamiento de AAV por centrifugación en doble gradiente de densidad de yodixanol

<ol>
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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. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+characterization+of+adeno-associated+viral+vectors.">Production and characterization of adeno-associated viral vectors.</a> Nat Protoc. 1 (3), 1412-1428 (2006).</li><li>Florea, 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=High-efficiency+purification+of+divergent+AAV+serotypes+using+AAVX+affinity+chromatography.">High-efficiency purification of divergent AAV serotypes using AAVX affinity chromatography.</a> Mol Ther Methods Clin Dev. 28, 146-159 (2022).</li><li>Chamberlain, K., Riyad, J. M., Weber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expressing+transgenes+that+exceed+the+packaging+capacity+of+adeno-associated+virus+capsids.">Expressing transgenes that exceed the packaging capacity of adeno-associated virus capsids.</a> Hum Gene Ther Methods. 27 (1), 1-12 (2016).</li><li>Green, E. A., Hamaker, N. K., Lee, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+vector+elements+and+process+conditions+in+transient+and+stable+suspension+HEK293+platforms+using+SARS-CoV-2+receptor+binding+domain+as+a+model+protein.">Comparison of vector elements and process conditions in transient and stable suspension HEK293 platforms using SARS-CoV-2 receptor binding domain as a model protein.</a> BMC Biotechnol. 23 (1), 7 (2023).</li><li>Erbacher, P., Zou, S., Bettinger, T., Steffan, A. M., Remy, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan-based+vector/DNA+complexes+for+gene+delivery:+Biophysical+characteristics+and+transfection+ability.">Chitosan-based vector/DNA complexes for gene delivery: Biophysical characteristics and transfection ability.</a> Pharm Res. 15 (9), 1332-1339 (1998).</li><li>Vandenberghe, L. 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=Efficient+serotype-dependent+release+of+functional+vector+into+the+culture+medium+during+adeno-associated+virus+manufacturing.">Efficient serotype-dependent release of functional vector into the culture medium during adeno-associated virus manufacturing.</a> Hum Gene Ther. 21 (10), 1251-1257 (2010).</li><li>Summerford, C., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-associated+heparan+sulfate+proteoglycan+is+a+receptor+for+adeno-associated+virus+type+2+virions.">Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions.</a> J Virol. 72 (2), 1438-1445 (1998).</li><li>Wright, J. F., 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+factors+that+contribute+to+recombinant+AAV2+particle+aggregation+and+methods+to+prevent+its+occurrence+during+vector+purification+and+formulation.">Identification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation.</a> Mol Ther. 12 (1), 171-178 (2005).</li><li>Gruntman, A. 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=Stability+and+compatibility+of+recombinant+adeno-associated+virus+under+conditions+commonly+encountered+in+human+gene+therapy+trials.">Stability and compatibility of recombinant adeno-associated virus under conditions commonly encountered in human gene therapy trials.</a> Hum Gene Ther Methods. 26 (2), 71-76 (2015).</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=Rationale+and+strategies+for+the+development+of+safe+and+effective+optimized+AAV+vectors+for+human+gene+therapy.">Rationale and strategies for the development of safe and effective optimized AAV vectors for human gene therapy.</a> Mol Ther Nucleic Acids. 32, 949-959 (2023).</li><li>Mullard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FDA+approves+first+gene+therapy+for+Duchenne+muscular+dystrophy,+despite+internal+objections.">FDA approves first gene therapy for Duchenne muscular dystrophy, despite internal objections.</a> Nat Rev Drug Discov. 22 (8), 610-610 (2023).</li><li>Center for Biologics Evaluation and Research. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approved+Cellular+and+Gene+Therapy+Products.">Approved Cellular and Gene Therapy Products.</a> US Food Drug Adm. , (2023).</li><li>Kang, 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=AAV+vectors+applied+to+the+treatment+of+CNS+disorders:+Clinical+status+and+challenges.">AAV vectors applied to the treatment of CNS disorders: Clinical status and challenges.</a> J Control Release Off J Control Release Soc. 355, 458-473 (2023).</li><li>De Wolf, D., Singh, K., Chuah, M. 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El protocolo de purificación de doble gradiente de densidad de yodixanol es el método universal porque es aplicable a cualquier variante mutante de AAV, independientemente de su especificidad de receptor. Los primeros métodos de purificación de AAV se basaban en la densidad de partículas e incluían la centrifugación isopícnica en CsCl y la centrifugación continua en gradiente de densidad de sacarosa19. Posteriormente, se desarrollaron abordajes seroespecíficos que hicieron uso de anticue...

Los vectores virales adenoasociados recombinantes (rAAV) son herramientas ampliamente utilizadas para el tratamiento de enfermedades genéticas, como la atrofia muscular espinal, la distrofia retiniana y la hemofilia A 1,2,3. Los vectores rAAV están diseñados para carecer de genes virales presentes en el AAV4 de tipo salvaje, un virus icosaédrico pequeño y sin envoltura con un genoma lineal de ADN monocatenario de 4,7 kb. El AAV se descubrió por primera vez en la década de 1960 como un contaminante de las preparaciones de adenovirus5. A pesar de su pequeño tamaño de cápside, que limita el tamaño del transgén que se puede empaquetar a un máximo de 4,9 kb excluyendo los RTI6, el AAV es útil para la administración de transgenes porque no es patógeno en humanos, permite la expresión del transgén en muchos tipos de células que se dividen y no se dividen, y tiene efectos inmunogénicos limitados7.
Como miembros del género dependoparvovirus, la producción de rAAVs se basa en la expresión de genes auxiliares presentes en el adenovirus o virus del herpes simple8. Se han desarrollado varias estrategias para producir rAAV, pero la producción en células HEK293 transformadas con los genes auxiliares adenovirales E1A/E1B es el método más establecido y utilizado en la actualidad9. El enfoque general de la producción de rAAV comienza con la transfección de células HEK293 con tres plásmidos que contienen el transgén dentro de repeticiones terminales invertidas (ITR), genes AAV rep y cap , y genes auxiliares adenovirales adicionales, respectivamente. Setenta y dos horas después de la transfección, las células se cosechan y procesan para purificar el rAAV que contiene el transgén.
En el desarrollo de nuevos vectores rAAV con fines terapéuticos, uno de los principales objetivos es producir vectores con una mayor eficiencia de transducción. Un aumento en la eficiencia de transducción de las células diana significaría una disminución de la dosis clínica necesaria de rAAV, disminuyendo así la probabilidad de efectos inmunogénicos adversos que van desde la neutralización mediada por anticuerpos hasta toxicidades agudas10,11. Para mejorar la eficacia de la transducción de los vectores rAAV, se pueden realizar alteraciones en el genoma empaquetado o en la cápside. Los métodos viables para ajustar la eficacia de la transducción a través del diseño del genoma empaquetado incluyen la incorporación de promotores fuertes y específicos de tejido, la selección cuidadosa de los elementos de procesamiento del ARNm y la optimización de la secuencia de codificación para mejorar la eficiencia de la traducción12. Las alteraciones de la cápside se realizan con el objetivo de aumentar el tropismo para los tipos de células humanas diana. Los esfuerzos para desarrollar nuevas cápsidas de vectores de administración de transgenes rAAV generalmente se caracterizan por un enfoque en el diseño racional de cápsidas de AAV con mutaciones específicas dirigidas a receptores celulares específicos o la evolución dirigida para identificar cápsidas con tropismo para tipos celulares específicos a partir de bibliotecas de cápside combinatorias de alta complejidad sin dirigirse a un receptor específico (aunque algunos grupos combinan estos enfoques)13, 14,15. En el enfoque de evolución dirigida, las bibliotecas combinatorias de la cápside se construyen utilizando una columna vertebral de serotipo particular con regiones variables mutadas en el exterior de la cápside16. Las bibliotecas combinatorias de cápside a menudo se construyen a partir de serotipos de AAV que no se originan en humanos, lo que disminuye el riesgo de inmunidad preexistente durante el uso clínico10. Por lo tanto, los métodos de purificación que se pueden aplicar a cualquier serotipo son ideales para eliminar la necesidad de una optimización específica del serotipo para los serotipos menos utilizados que sirven como columna vertebral para estas bibliotecas.
La centrifugación en gradiente de densidad de yodixanol se utiliza para purificar altos títulos de rAAV con alta infectividad17. En este protocolo, el rAAV se produce en cultivo celular en suspensión para disminuir la cantidad de mano de obra necesaria para producir grandes títulos de AAV. También se incluye un paso de centrifugación para limpiar el lisado celular para reducir la presencia de proteínas contaminantes y disminuir el riesgo de precipitación de virus. Este protocolo es un método rentable para producir preparaciones de rAAV de alta pureza adecuadas para uso preclínico.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

Este protocolo describe la producción y purificación de virus adenoasociados recombinantes (rAAV) mediante centrifugación en gradiente de densidad de yodixanol, un método independiente del serotipo para purificar AAV descrito por primera vez en 1999. Los vectores rAAV se utilizan ampliamente en aplicaciones de terapia génica para administrar transgenes a varios tipos de células humanas. En este trabajo, el virus recombinante se produce por transfección de células Expi293 en cultivo en suspensión con plásmidos que codifican los genes transgénicos, de la cápside del vector y de los genes auxiliares adenovirales. La centrifugación en gradiente de densidad de yodixanol purifica las partículas AAV completas en función de la densidad de partículas. Además, se incluyen tres pasos en esta metodología ahora omnipresente para aumentar el rendimiento total del virus, disminuir el riesgo de precipitación debido a proteínas contaminantes y concentrar aún más el producto final del virus, respectivamente: precipitación de partículas virales de medios celulares utilizando una solución de polietilenglicol (PEG) y cloruro de sodio, la introducción de una segunda ronda de centrifugación en gradiente de densidad de yodixanol, y el intercambio de tampón a través de un filtro centrífugo. Con este método, es posible lograr títulos consistentes en el rango de 10a 12 partículas virales/ml de pureza excepcional para uso in vivo .

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.

Autor destacado: Aislamiento eficiente de virus adenoasociados para aplicaciones preclínicas

Producción de cultivos en suspensión y purificación de virus adenoasociados mediante centrifugación en gradiente de densidad de yodixanol para aplicaciones in vivo

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.

Este método se puede utilizar para obtener títulos de al menos 10a 12 partículas virales por ml. Se puede obtener un título (Figura 3) por qPCR utilizando los cebadores ITR proporcionados en la Tabla Suplementaria 1, por ddPCR o por cualquier otro método de titulación. Los títulos subóptimos podrían ser el resultado del uso de un gen de la cápsula que codifica una cápside con una eficiencia de envasado deficiente.
Ot...

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

El virus adenoasociado se produce en cultivo celular en suspensión y se purifica mediante centrifugación de doble gradiente de densidad de yodixanol. Se incluyen medidas para aumentar el rendimiento total del virus, disminuir el riesgo de precipitación del virus y concentrar aún más el producto final del virus. Los títulos finales esperados alcanzan las 1012 partículas virales/ml y son adecuados para el uso preclínico in vivo .

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

Utilizamos un enfoque de evolución dirigida para identificar capitis virales adenoasociados con mayor tropismo para los tipos de células humanas diana. A través de esta investigación, nuestro objetivo es producir vectores virales adenoasociados recombinantes con una mejor transducción de células diana y relevancia clínica dentro del campo de la terapia génica. Recientemente, nuestro grupo eliminó varios serotipos de AAV del hígado mediante la incorporación de residuos de cápside originales de un serotipo de AAV que posee naturalmente un tropismo hepático disminuido.Si se expande con éxito a serotipos adicionales, esto proporciona la base para disminuir la dosis clínica necesaria de RAAV mediante inyección periférica y reducir los resultados adversos para los pacientes. El método de purificación de IODIXanol, aunque es rentable, es difícil de ampliar. Además, en aplicaciones posteriores, el enfoque de evolución dirigida se realiza con modelos animales o in vitro.Por lo tanto, es posible que la cápside aislada por evolución dirigida no transduca dianas in vivo en humanos en la misma medida que en modelos preclínicos. Este protocolo es asequible y accesible para laboratorios más pequeños. Además, este método produce virus adenoasociados recombinantes de alta pureza que se pueden utilizar para aplicaciones preclínicas in vivo posteriores.

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.