Name: 著者スポットライト:前臨床応用のための効率的なアデノ随伴ウイルス単離(英語)
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

このプロトコルで使用した溶液および緩衝液の組成を 表1に示します。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>解決</td>
			<td colspan="2">組成</td>
		</tr><tr><td rowspan="4">AAV溶解バッファー</td>
			<td colspan="2">1.2 mL の 5 M NaCl 溶液</td>
		</tr><tr><td colspan="2">2 mL の 1 M Tris-HCl pH 8.5 溶液</td>
		</tr><tr><td colspan="2">80 uL の 1 M MgCl2 溶?...

ダブルヨウ素酸濃度グラジエント遠心分離によるAAV単離

<ol>
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ダブルヨージキサノール密度勾配精製プロトコルは、受容体の特異性に関係なく、あらゆるAAV変異体バリアントに適用できるため、普遍的な方法です。AAV精製の初期の方法は粒子密度に依存しており、CsClでのアイソピクニック遠心分離と連続スクロース密度勾配遠心分離が含まれていました19。その後、セファロースカラムに結合したモノクローナル抗体を利用した血清型?...

組換えアデノ随伴ウイルス(rAAV)ベクターは、脊髄性筋萎縮症、網膜ジストロフィー、血友病A1,2,3などの遺伝性疾患の治療に広く使用されているツールです。rAAVベクターは、直鎖状の一本鎖4.7 kbのDNAゲノムを持つ小型の非エンベロープ正二十面体ウイルスである野生型AAV4に存在するウイルス遺伝子を欠くように操作されています。AAVは、1960年代にアデノウイルス製剤の汚染物質として初めて発見されました5。AAVはキャプシドサイズが小さいため、パッケージ化できる導入遺伝子のサイズはITRを除いて最大4.9 kbに制限されますが6、ヒトでは非病原性であり、多くの分裂細胞および非分裂細胞タイプで導入遺伝子の発現を可能にし、免疫原性効果が限られているため、導入遺伝子の送達に有用です7。
dependoparvovirus属のメンバーとして、rAAVの産生は、アデノウイルスまたは単純ヘルペスウイルスに存在するヘルパー遺伝子の発現に依存しています8。rAAVを産生するいくつかの戦略が開発されていますが、アデノウイルスE1A/E1Bヘルパー遺伝子で形質転換されたHEK293細胞での産生は、現在最も確立された方法です9。rAAV産生の一般的なアプローチは、HEK293細胞を、それぞれ逆末端反復配列(ITR)、AAV rep 遺伝子および cap 遺伝子、および追加のアデノウイルスヘルパー遺伝子内に導入遺伝子を含む3つのプラスミドを用いてトランスフェクションすることから始まります。トランスフェクションの72時間後に細胞を回収し、導入遺伝子を含むrAAVを精製するためにプロセシングします。
治療目的の新しいrAAVベクターの開発では、形質導入効率を高めたベクターを作製することが大きな目標となっています。標的細胞の形質導入効率の向上は、rAAVの必要臨床用量の減少を意味し、したがって、抗体媒介性中和から急性毒性に至るまでの免疫原性への悪影響の可能性を減少させるであろう10,11。rAAVベクターの形質導入効率を向上させるために、パッケージ化されたゲノムまたはカプシドに改変を加えることができます。パッケージ化されたゲノム設計によって形質導入の有効性を調整する実行可能な方法には、強力な組織特異的プロモーターの組み込み、mRNAプロセシングエレメントの慎重な選択、および翻訳効率を向上させるためのコード配列の最適化が含まれます12。カプシドの改変は、標的となるヒト細胞タイプの向性を高めることを目的として行われます。新しいrAAV導入遺伝子導入ベクターカプシドの開発に向けた取り組みは、一般に、特定の細胞受容体を標的とする特定の変異を持つAAVカプシドの合理的な設計、または1つの特定の受容体を標的とせずに、非常に複雑なコンビナトリアルカプシドライブラリから特定の細胞タイプに対して向性を持つカプシドを同定するための指向性進化のいずれかに焦点を当てることによって特徴付けられます(ただし、これらのアプローチを組み合わせるグループもあります)13。14,15。指向性進化アプローチでは、コンビナトリアルカプシドライブラリは、カプシド外装16に変異した可変領域を有する特定の血清型骨格を用いて構築される。コンビナトリアルカプシドライブラリーは、ヒトに由来しないAAV血清型から構築されることが多く、臨床使用中の既存の免疫のリスクを低減します10。したがって、あらゆる血清型に適用できる精製法は、これらのライブラリのバックボーンとして機能する、あまり一般的に使用されない血清型に対する血清型特異的な最適化の必要性を排除するのに理想的です。
ヨウ素酸塩密度勾配遠心分離は、感染力の高いrAAVの高力価を精製するために利用される17。このプロトコルでは、rAAVはAAVの大きな力価を生成するために必要な労働量を減らすために、浮遊細胞培養で産生されます。また、細胞溶解液を除去するための遠心分離ステップも含まれており、汚染タンパク質の存在を減らし、ウイルス沈殿のリスクを低減します。このプロトコルは、前臨床使用に適した高純度rAAVの調製物を製造するための費用対効果の高い方法です。

<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

このプロトコルはiodixanol密度勾配の遠心分離、1999年に最初に記述されているAAVを浄化する血清型不可知論者方法によって組換えのadeno準のウイルス(rAAV)の生産そして浄化を記述する。rAAVベクターは、さまざまなヒト細胞タイプに導入遺伝子を送達するための遺伝子治療アプリケーションで広く使用されています。本研究では、導入遺伝子、ベクターカプシド、およびアデノウイルスヘルパー遺伝子をコードするプラスミドを用いた浮遊培養中の Expi293 細胞のトランスフェクションにより、組換えウイルスを産生します。ヨウジオジキサノール密度勾配遠心分離は、粒子密度に基づいて完全なAAV粒子を精製します。さらに、この今ではユビキタスな方法論には、総ウイルス収量の増加、汚染タンパク質による沈殿のリスクの低減、および最終的なウイルス生成物のさらなる濃縮のために、それぞれ3つのステップが含まれています:ポリエチレングリコール(PEG)と塩化ナトリウムの溶液を使用した細胞培地からのウイルス粒子の沈殿、2回目のヨージキサノール密度勾配遠心分離の導入、 遠心フィルターによる緩衝液交換。この方法を用いることで、in vivoでの使用において、10〜12個のウイルス粒子/mLの並外れた純度の力価を一貫して達成することができます。

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.

著者スポットライト:前臨床応用のための効率的なアデノ随伴ウイルス単離(英語)

In vivoアプリケーションのためのヨジキサノール密度勾配遠心分離によるアデノ随伴ウイルスの浮遊培養産生と精製

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.

この方法は、mL当たり少なくとも10〜12個のウイルス粒子の力価を得るために用いることができる。力価は、補足表1に記載のITRプライマーを用いたqPCR、ddPCR、またはその他の力価法により得ることができる(図3)。最適でない力価は、パッケージング効率の悪いカプシドをコードするキャップ遺伝子を使用した場合に生じる可能性があります。</...

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

アデノ随伴ウイルスは浮遊細胞培養で産生され、二重ヨージキサノール密度勾配遠心分離によって精製されます。ウイルスの総収量を増やし、ウイルス沈殿のリスクを低減し、最終的なウイルス生成物をさらに濃縮するためのステップが含まれています。予想される最終力価は10,12 ウイルス粒子/mLに達し、前臨床 in vivo での使用に適しています。

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

私たちは、指向性進化アプローチを用いて、標的ヒト細胞タイプについて、より高い向性を持つアデノ随伴ウイルスキャップを同定します。本研究により、標的細胞の形質導入を改善し、遺伝子治療分野における臨床的関連性を有する組換えアデノ随伴ウイルスベクターの作製を目指します。私たちのグループは最近、自然に肝臓向性が低下したAAV血清型に元のカプシド残基を組み込むことにより、肝臓からいくつかのAAV血清型を標的としました。追加の血清型への拡大に成功すれば、末梢注射による必要なRAAV臨床線量を減らし、患者の有害な転帰を減らすための基礎となります。IODIXanol精製法は、費用対効果が高いものの、スケールアップが困難です。さらに、下流のアプリケーションでは、動物モデルまたはin vitroで指向性進化アプローチが実行されます。したがって、指向性進化によって単離されたカプシドは、前臨床モデルと同じ程度にヒトのin vivo標的を形質導入しない可能性があります。このプロトコルは手頃な価格で、小規模なラボでも利用しやすいものです。さらに、この分析法により、下流の前臨床in vivoアプリケーションに使用できる高純度の組換えアデノ随伴ウイルスが得られます。

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.