Name: Yazar Spotlight: Klinik Öncesi Uygulamalar için Verimli Adeno İlişkili Virüs İzolasyonu
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

Bu protokolde kullanılan çözeltilerin ve tamponların bileşimi Tablo 1'de verilmiştir.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>Çözüm</td>
			<td colspan="2">Kompozisyon</td>
		</tr><tr><td rowspan="4">AAV lizis tamponu</td>
			<td colspan="2">1.2 mL 5 M NaCl çözeltisi</td>
		</tr><tr><td colspan="2">2 mL 1 M Tris-HCl pH 8.5 çözeltisi</td>
		</tr><tr><td colspan="2">80 uL 1 M MgCl2 çözeltisi...

Çift İodiksanol Yoğunluk Gradyan Santrifüjleme ile AAV İzolasyonu

<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. 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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. 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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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Çift iyodiksanol yoğunluk gradyan saflaştırma protokolü evrensel yöntemdir, çünkü reseptör özgüllüklerine bakılmaksızın herhangi bir AAV mutant varyantına uygulanabilir. AAV saflaştırmanın erken yöntemleri, partikül yoğunluğuna dayanıyordu ve CsCl'de izopiknik santrifüjleme ve sürekli sükroz yoğunluk gradyan santrifüjünüiçeriyordu 19. Daha sonra, Sefaroz kolonlarına20 bağlı monoklonal antikorları kullanan serotipe özgü yaklaşımlar ge...

Rekombinant adeno ilişkili viral (rAAV) vektörler, spinal müsküler atrofi, retina distrofisi ve hemofili Adahil olmak üzere genetik hastalıkların tedavisinde yaygın olarak kullanılan araçlardır 1,2,3. rAAV vektörleri, doğrusal tek sarmallı 4.7 kb DNA genomuna sahip küçük, zarfsız bir ikosahedral virüs olan vahşi tip AAV4'te bulunan viral genlerden yoksun olacak şekilde tasarlanmıştır. AAV ilk olarak 1960'larda adenovirüs preparatlarının bir kontaminantı olarak keşfedildi5. ITR'ler6 hariç maksimum 4.9 kb ile paketlenebilen transgenin boyutunu sınırlayan küçük kapsid boyutuna rağmen, AAV, insanlarda patojenik olmadığı, birçok bölünen ve bölünmeyen hücre tipinde transgen ekspresyonuna izin verdiği ve sınırlı immünojenik etkilere sahip olduğu için transgen iletimi için yararlıdır7.
Dependoparvovirus cinsinin üyeleri olarak, rAAV'lerin üretimi, adenovirüs veya herpes simpleks virüsünde bulunan yardımcı genlerin ekspresyonuna dayanır8. rAAV üretmek için çeşitli stratejiler geliştirilmiştir, ancak adenoviral E1A/E1B yardımcı genleri ile dönüştürülen HEK293 hücrelerinde üretim günümüzde kullanılan en yaygın yöntemdir9. rAAV üretiminin genel yaklaşımı, HEK293 hücrelerinin, sırasıyla ters terminal tekrarlar (ITR'ler), AAV rep ve cap genleri ve ek adenoviral yardımcı genler içinde transgeni içeren üç plazmid ile transfeksiyonu ile başlar. Transfeksiyondan yetmiş iki saat sonra, transgeni içeren rAAV'yi saflaştırmak için hücreler toplanır ve işlenir.
Terapötik amaçlar için yeni rAAV vektörlerinin geliştirilmesinde, ana hedef, artan transdüksiyon verimliliğine sahip vektörler üretmektir. Hedef hücrelerin transdüksiyon verimliliğindeki bir artış, gerekli klinik rAAV dozunda bir azalma anlamına gelir, böylece antikor aracılı nötralizasyondan akut toksisitelere kadar değişen olumsuz immünojenik etkilerin olasılığını azaltır10,11. rAAV vektörlerinin transdüksiyon etkinliğini arttırmak için, paketlenmiş genomda veya kapsidde değişiklikler yapılabilir. Paketlenmiş genom tasarımı yoluyla transdüksiyon etkinliğini ayarlamak için uygulanabilir yöntemler arasında güçlü ve dokuya özgü promotörlerin dahil edilmesi, mRNA işleme elemanlarının dikkatli seçimi ve çeviri verimliliğini artırmak için kodlama dizisi optimizasyonu yer alır12. Kapsidde yapılan değişiklikler, hedef insan hücre tipleri için tropizmi artırmak amacıyla yapılır. Yeni rAAV transgen iletim vektörü kapsidleri geliştirmeye yönelik çabalar, genellikle, belirli hücre reseptörlerini hedefleyen spesifik mutasyonlara sahip AAV kapsidlerinin rasyonel tasarımına veya belirli bir reseptörü hedeflemeden yüksek karmaşıklıktaki kombinatoryal kapsid kütüphanelerinden belirli hücre tipleri için tropizmli kapsidleri tanımlamak için yönlendirilmiş evrime odaklanarak karakterize edilir (bazı gruplar bu yaklaşımları birleştirse de)13, 14,15. Yönlendirilmiş evrim yaklaşımında, kombinatoryal kapsid kütüphaneleri, kapsid dış16 üzerinde mutasyona uğramış değişken bölgelere sahip belirli bir serotip omurgası kullanılarak oluşturulur. Kombinatoryal kapsid kütüphaneleri genellikle insanlardan kaynaklanmayan AAV serotiplerinden oluşturulur ve klinik kullanım sırasında önceden var olan bağışıklık riskini azaltır10. Bu nedenle, herhangi bir serotipe uygulanabilen saflaştırma yöntemleri, bu kütüphaneler için omurga görevi gören daha az kullanılan serotipler için serotipe özgü optimizasyon ihtiyacını ortadan kaldırmak için idealdir.
İodiksanol yoğunluk gradyan santrifüjü, yüksek enfektiviteye sahip yüksek rAAV titrelerini saflaştırmak için kullanılır17. Bu protokolde, büyük AAV titreleri üretmek için gereken emek miktarını azaltmak için süspansiyon hücre kültüründe rAAV üretilir. Kirletici proteinlerin varlığını azaltmak ve virüs çökelmesi riskini azaltmak için hücre lizatını temizlemek için bir santrifüjleme adımı da dahildir. Bu protokol, klinik öncesi kullanıma uygun yüksek saflıkta rAAV preparatları üretmek için uygun maliyetli bir yöntemdir.

<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

Bu protokol, ilk olarak 1999'da tanımlanan AAV'yi saflaştırmak için serotipten bağımsız bir yöntem olan iyodiksanol yoğunluk gradyan santrifüjleme ile rekombinant adeno-ilişkili virüs (rAAV) üretimini ve saflaştırılmasını açıklar. rAAV vektörleri, transgenleri çeşitli insan hücre tiplerine iletmek için gen terapisi uygulamalarında yaygın olarak kullanılmaktadır. Bu çalışmada, rekombinant virüs, transgen, vektör kapsid ve adenoviral yardımcı genleri kodlayan plazmitlerle süspansiyon kültüründe Expi293 hücrelerinin transfeksiyonu ile üretilir. İodiksanol yoğunluk gradyan santrifüjü, partikül yoğunluğuna dayalı olarak tam AAV partiküllerini saflaştırır. Ek olarak, toplam virüs verimini artırmak, kontamine proteinler nedeniyle çökelme riskini azaltmak ve nihai virüs ürününü daha da yoğunlaştırmak için şu anda her yerde bulunan bu metodolojiye sırasıyla üç adım dahil edilmiştir: bir polietilen glikol (PEG) ve sodyum klorür çözeltisi kullanılarak hücre ortamından viral partiküllerin çökeltilmesi, ikinci bir iyodiksanol yoğunluk gradyan santrifüj turunun tanıtılması, ve santrifüj filtre ile tampon değişimi. Bu yöntemi kullanarak, in vivo kullanım için 1012 viral partikül / mL aralığında olağanüstü saflıkta titreleri tutarlı bir şekilde elde etmek mümkündür.

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.

Yazar Spotlight: Klinik Öncesi Uygulamalar için Verimli Adeno İlişkili Virüs İzolasyonu

İn Vivo Uygulamalar için İodiksanol Yoğunluk Gradyan Santrifüjü ile Adeno İlişkili Virüsün Süspansiyon Kültürü Üretimi ve Saflaştırılması

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.

Bu yöntem, mL başına en az 1012 viral partikül titresi elde etmek için kullanılabilir. Ek Tablo 1'de verilen ITR primerleri kullanılarak, ddPCR ile veya başka herhangi bir titre yöntemi ile qPCR ile bir titre elde edilebilir (Şekil 3). Yetersiz titreler, zayıf paketleme verimliliğine sahip bir kapsidi kodlayan bir kapak geninin kullanılmasından kaynaklanabilir.
Optimal olmayan sonuçların bir başka olası kayna?...

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

Adeno ilişkili virüs, süspansiyon hücre kültüründe üretilir ve çift iyodiksanol yoğunluk gradyan santrifüjlemesi ile saflaştırılır. Toplam virüs verimini artırmak, virüs çökelme riskini azaltmak ve nihai virüs ürününü daha da konsantre etmek için adımlar dahil edilmiştir. Beklenen nihai titreler 1012 viral partikül/mL'ye ulaşır ve klinik öncesi in vivo kullanım için uygundur.

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

Hedef insan hücre tipleri için daha yüksek tropizme sahip adeno ilişkili viral kapitleri tanımlamak için yönlendirilmiş bir evrim yaklaşımı kullanıyoruz. Bu araştırma sayesinde, gen terapisi alanında gelişmiş hedef hücre transdüksiyonu ve klinik önemi olan rekombinant adeno ilişkili viral vektörler üretmeyi amaçlıyoruz. Grubumuz yakın zamanda, doğal olarak azalmış karaciğer tropizmine sahip bir AAV serotipine orijinal kapsid kalıntılarını dahil ederek karaciğerden birkaç AAV serotipini hedefledi.Ek serotiplere başarılı bir şekilde genişletilirse, bu, periferik enjeksiyon ile gerekli RAAV klinik dozunu azaltmak ve olumsuz hasta sonuçlarını azaltmak için zemin sağlar. IODIXanol saflaştırma yönteminin uygun maliyetli olmasına rağmen ölçeklendirilmesi zordur. Ek olarak, aşağı akış uygulamalarında, yönlendirilmiş evrim yaklaşımı hayvan modelleriyle veya in vitro olarak gerçekleştirilir.Bu nedenle, yönlendirilmiş evrim tarafından izole edilen kapsid, insanlarda klinik öncesi modellerde olduğu gibi in vivo hedefleri dönüştürmeyebilir. Bu protokol daha küçük laboratuvarlar için uygun fiyatlı ve erişilebilirdir. Ek olarak, bu yöntem, klinik öncesi in-vivo uygulamalar için kullanılabilen yüksek saflıkta, rekombinant adeno ilişkili virüs verir.

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.