Name: 저자 스포트라이트: 전임상 애플리케이션을 위한 효율적인 아데노 관련 바이러스 분리
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

이 프로토콜에 사용된 용액 및 버퍼의 구성은 표 1에 나와 있습니다.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>용액</td>
			<td colspan="2">구성</td>
		</tr><tr><td rowspan="4">AAV 용해 완충액</td>
			<td colspan="2">5M NaCl 용액 1.2mL</td>
		</tr><tr><td colspan="2">2M Tris-HCl pH 1 용액 8.5mL</td>
		</tr><tr><td colspan="2">80 uL의 1 M MgCl2 용액</td>
		</tr><tr><td ...

이중 요오딕사놀 밀도 구배 원심분리에 의한 AAV 분리

<ol>
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이중 요오드산올 밀도 구배 정제 프로토콜은 수용체 특이성에 관계없이 모든 AAV 돌연변이 변이체에 적용할 수 있기 때문에 보편적인 방법입니다. AAV 정제의 초기 방법은 입자 밀도에 의존하고 CsCl에서의 isopycnic 원심분리 및 연속 자당 밀도 구배 원심분리를 포함하였다19. 나중에, 혈청형 특이적 접근법이 개발되었는데, 이는 세파로스 컬럼20에 결합된 단클론 항...

재조합 아데노 관련 바이러스(rAAV) 벡터는 척수성 근위축증, 망막 이영양증 및 혈우병 A 1,2,3을 포함한 유전 질환 치료에 널리 사용되는 도구입니다. rAAV 벡터는 선형 단일 가닥 4.7kb DNA 게놈을 가진 작고 외피가 없는 이십면체 바이러스인 야생형 AAV4에 존재하는 바이러스 유전자가 없도록 조작되었습니다. AAV는 1960년대에 아데노바이러스 제제의 오염물질로 처음 발견되었다5. AAV는 ITR을 제외하고 최대 4.9 kb로 패키징할 수 있는 전이유전자의 크기를 제한하는 작은 캡시드 크기에도 불구하고6, 인간에서 비병원성이고, 많은 분열 및 비분열 세포 유형에서 전이유전자 발현을 허용하며, 면역원성 효과가 제한적이기 때문에 전이유전자 전달에 유용하다7.
레의존파보바이러스(dependoparvovirus) 속의 구성원인 rAAV의 생산은 아데노바이러스 또는 단순 헤르페스 바이러스(herpes simplex virus)에 존재하는 도우미 유전자의 발현에 의존한다8. rAAV를 생산하기 위한 몇 가지 전략이 개발되었지만, 아데노바이러스 E1A/E1B 도우미 유전자로 형질전환된 HEK293 세포에서의 생산이 오늘날 가장 확립된 방법이다9. rAAV 생산의 일반적인 접근법은 HEK293 세포를 역말단 반복(ITR) 내에서 전이유전자를 포함하는 3개의 플라스미드, AAV rep 및 cap 유전자, 추가 아데노바이러스 도우미 유전자로 transfection하는 것으로 시작됩니다. 형질주입 후 72시간 후, 세포를 수확하고 처리하여 전이유전자를 포함하는 rAAV를 정제합니다.
치료 목적을 위한 새로운 rAAV 벡터의 개발에서 주요 목표는 형질도입 효율이 향상된 벡터를 생산하는 것입니다. 표적 세포의 형질도입 효율의 증가는 rAAV의 필요한 임상적 투여량의 감소를 의미하며, 따라서 항체 매개 중화에서 급성 독성에 이르는 면역원성 부작용의 가능성을 감소시킨다10,11. rAAV 벡터의 형질도입 효능을 개선하기 위해, 패키징된 게놈 또는 캡시드를 변경할 수 있습니다. 패키징된 게놈 디자인을 통해 형질도입 효능을 조정하는 실행 가능한 방법에는 강력한 조직 특이적 promoter의 통합, mRNA 처리 요소의 신중한 선택, 번역 효율 향상을 위한 코딩 서열 최적화가 포함됩니다12. 캡시드에 대한 변경은 표적 인간 세포 유형에 대한 영양성을 증가시키는 것을 목표로 이루어집니다. 새로운 rAAV 전이유전자 전달 벡터 캡시드를 개발하기 위한 노력은 일반적으로 특정 세포 수용체를 표적으로 하는 특정 돌연변이를 가진 AAV 캡시드의 합리적인 설계에 초점을 맞추거나, 하나의 특정 수용체를 표적으로 하지 않고 고복잡성 조합 캡시드 라이브러리에서 특정 세포 유형에 대한 영양성을 가진 캡시드를 식별하기 위한 유도 진화에 초점을 맞추는 것이 특징입니다(일부 그룹은 이러한 접근 방식을 결합하지만)13, 14,15. 유도 진화 접근법에서, 조합 캡시드 라이브러리는 캡시드 외장(16) 상에 돌연변이된 가변 영역들을 갖는 특정 혈청형 골격을 사용하여 구성된다. 조합 캡시드 라이브러리는 종종 인간에서 유래하지 않은 AAV 혈청형으로 구성되며, 임상 사용 중 기존 면역의 위험을 감소시킨다10. 따라서 모든 혈청형에 적용할 수 있는 정제 방법은 이러한 라이브러리의 중추 역할을 하는 덜 일반적으로 사용되는 혈청형에 대한 혈청형 특이적 최적화의 필요성을 제거하는 데 이상적입니다.
요오딕사놀 밀도 구배 원심분리는 높은 감염성을 가진 rAAV의 높은 역가를 정제하는 데 이용된다17. 이 프로토콜에서 rAAV는 AAV의 큰 역가를 생산하는 데 필요한 노동력을 줄이기 위해 부유 세포 배양에서 생산됩니다. 오염 단백질의 존재를 줄이고 바이러스 침전의 위험을 줄이기 위해 세포 용해물을 청소하기 위한 원심분리 단계도 포함됩니다. 이 프로토콜은 전임상 사용에 적합한 고순도 rAAV 제제를 생산하는 비용 효율적인 방법입니다.

<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

이 프로토콜은 1999년에 처음 기술된 AAV를 정제하는 혈청형에 구애받지 않는 방법인 요오드릭사놀 밀도 구배 원심분리에 의한 재조합 아데노 관련 바이러스(rAAV) 생산 및 정제에 대해 설명합니다. rAAV 벡터는 다양한 인간 세포 유형에 전이유전자를 전달하기 위한 유전자 치료 응용 분야에서 널리 사용됩니다. 이 연구에서 재조합 바이러스는 전이유전자, 벡터 캡시드 및 아데노바이러스 도우미 유전자를 암호화하는 플라스미드를 사용하여 현탁 배양에서 Expi293 세포를 transfection하여 생산됩니다. 요오딕사놀 밀도 구배 원심분리는 입자 밀도를 기반으로 전체 AAV 입자를 정제합니다. 또한, 현재 유비쿼터스가 된 이 방법론에는 총 바이러스 수율을 높이고, 단백질 오염으로 인한 침전 위험을 줄이고, 최종 바이러스 생성물을 더욱 농축하기 위한 세 가지 단계가 포함되어 있습니다: 폴리에틸렌 글리콜(PEG) 및 염화나트륨 용액을 사용한 세포 배지에서 바이러스 입자 침전, 요오드릭산올 밀도 구배 원심분리의 두 번째 라운드 도입, 원심 필터를 통한 완충액 교환. 이 방법을 사용하면 in vivo 사용을 위한 탁월한 순도의 1012 바이러스 입자/mL 범위의 역가를 일관되게 달성할 수 있습니다.

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.

저자 스포트라이트: 전임상 애플리케이션을 위한 효율적인 아데노 관련 바이러스 분리

In Vivo 응용 분야를 위한 Iodixanol Density Gradient Centrifugation에 의한 아데노 관련 바이러스의 현탁 배양 생산 및 정제

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.

이 방법은 mL당 최소10,12개의 바이러스 입자의 역가를 얻는 데 사용할 수 있습니다. 역가는 보충 표 1에 제공된 ITR 프라이머를 사용하는 qPCR, ddPCR 또는 기타 적정 방법으로 얻을 수 있습니다(그림 3). 최적이 아닌 역가는 패키징 효율이 좋지 않은 캡시드를 암호화하는 캡 유전자를 사용하여 발생할 수 있습니다.
차선의 결과를 얻...

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

아데노 관련 바이러스는 현탁 세포 배양에서 생성되며 이중 요오드화놀 밀도 구배 원심분리로 정제됩니다. 총 바이러스 수율을 높이고, 바이러스 침전의 위험을 줄이고, 최종 바이러스 산물을 더욱 농축하기 위한 단계가 포함되어 있습니다. 예상되는 최종 역가는10,12 바이러스 입자/mL에 도달하며 전임상 in vivo 사용에 적합합니다.

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

우리는 표적 인간 세포 유형에 대해 더 높은 tropism을 가진 아데노 관련 바이러스 capit를 식별하기 위해 유도 진화 접근법을 사용합니다. 본 연구를 통해 유전자 치료 분야에서 표적 세포 transduction 및 임상적 관련성이 향상된 재조합 아데노 관련 바이러스 벡터를 생산하는 것을 목표로 합니다. 우리 그룹은 최근 자연적으로 감소된 간 영양성을 가진 AAV 혈청형에 원래 캡시드 잔기를 통합하여 간에서 여러 AAV 혈청형을 탈표적화했습니다.추가 혈청형으로 성공적으로 확장될 경우, 말초 주사를 통해 필요한 RAAV 임상 용량을 줄이고 부작용 환자 결과를 줄일 수 있는 토대를 마련할 수 있습니다. IODIXanol 정제 방법은 비용 효율적이지만 확장이 어렵습니다. 또한 다운스트림 응용 분야에서는 동물 모델 또는 시험관 내에서 유도 진화 접근법이 수행됩니다.따라서 유도 진화에 의해 분리된 캡시드는 전임상 모델에서와 동일한 정도로 인간에서 생체 내 표적을 transduction하지 않을 수 있습니다. 이 프로토콜은 저렴하고 소규모 실험실에서 사용할 수 있습니다. 또한 이 방법은 다운스트림 전임상 in vivo 응용 분야에 사용할 수 있는 고순도 재조합 아데노 관련 바이러스를 생성합니다.

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.