O'Riordan C R, Lachapelle A L, Vincent K A, Wadsworth S C
Genzyme Corporation, Framingham, MA 01701-9322, USA.
J Gene Med. 2000 Nov-Dec;2(6):444-54. doi: 10.1002/1521-2254(200011/12)2:6<444::AID-JGM132>3.0.CO;2-1.
Adeno-associated virus (AAV) is a human parvovirus currently being developed as a vector for gene therapy applications. Traditionally AAV has been purified from cell lysates using CsCl gradients; this approach however is not likely to be useful in large-scale manufacturing. Moreover gradient-purified AAV vectors tend to be contaminated with significant levels of cellular and adenoviral proteins and nucleic acid. To address the issue of purification we have developed a process scale method for the rapid and efficient purification of recombinant AAV (rAAV) from crude cellular lysates.
The preferred method for the purification of rAAVbetagal includes treatment of virally infected cell lysates with both trypsin and nuclease followed by ion exchange chromatography using ceramic hydroxyapatite and DEAE-Sepharose in combination with cellufine sulphate affinity chromatography.
Purification of rAAV particles from crude cellular lysates co-infected with adenovirus was achieved using column chromatography exclusively. Column-purified rAAV was shown to be greater than 90% pure, free of any detectable contaminating adenovirus, biologically active, and capable of directing efficient gene transfer to the lungs of both cotton rats and mice.
This study demonstrates the feasibility of using column chromatography alone for the isolation of highly purified rAAV vector. The methods described here are advancements in procedures to purify rAAV and are adaptable for commercial production of clinical-grade rAAV vector.
腺相关病毒（AAV）是一种人类细小病毒，目前正被开发用作基因治疗应用的载体。传统上，AAV是使用CsCl梯度从细胞裂解物中纯化的；然而，这种方法在大规模生产中可能并不实用。此外，梯度纯化的AAV载体往往会被大量的细胞和腺病毒蛋白及核酸污染。为了解决纯化问题，我们开发了一种工艺规模的方法，用于从粗细胞裂解物中快速高效地纯化重组AAV（rAAV）。
纯化rAAVβ半乳糖苷酶的优选方法包括用胰蛋白酶和核酸酶处理病毒感染的细胞裂解物，然后依次使用陶瓷羟基磷灰石和DEAE-琼脂糖进行离子交换色谱，并结合硫酸纤维素亲和色谱。
仅使用柱色谱法就从与腺病毒共感染的粗细胞裂解物中纯化出了rAAV颗粒。柱纯化的rAAV显示纯度大于90%，不含任何可检测到的污染腺病毒，具有生物活性，并且能够将高效基因转移到棉鼠和小鼠的肺部。
本研究证明了仅使用柱色谱法分离高度纯化的rAAV载体的可行性。这里描述的方法是纯化rAAV程序的进步，适用于临床级rAAV载体的商业化生产。
