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Efficient Production and Purification of rAAV for Gene Therapy Applications

标题

Method of Preparing Recombinant AAV Compositions

详细技术说明

None

*Abstract

Increases Production of Highly Infectious rAAV that Is Nearly Contaminant-Free

This efficient production, isolation, and purification of recombinant adeno-associated virus (rAAV) yields at least ten times more than conventional production. The global genome editing market is projected to exceed $8 billion by 2025. rAAV vectors are useful for efficient long-term gene transfer in a variety of tissues, including lung, muscle, brain, retina, and liver. However, traditional methods of rAAV production suffer from multiple-week production time, poor vector recovery, and poor rAAV quality, hindering the developments of rAAV vectors in diagnostic and gene therapy applications. Researchers at the University of Florida have developed a protocol for producing, isolating, and purifying rAAV samples that leads to high-quality rAAV products in less than 24 hours. The yield of purified rAAV is increased by at least 10-fold over traditional methods, making broader gene therapy applications of rAAV feasible.

Application

Production and purification of rAAV that increases quantity and quality while decreasing processing time

Advantages

• Isolates and purifies rAAV, increasing quality of produced rAAV
• Quickly produces isolated and purified rAAV, significantly decreasing production time from weeks to hours
• Removes unbound proteins and contaminants, increasing yield over traditional methods by at least 10-fold

Technology

Traditional methods of rAAV production suffer from extremely long processing times (multiple weeks) as well as low-quality yields. While protocols have advanced in increasing the overall quantity of rAAV yields, the quality of the produced rAAV suffers from impurities due to insufficient isolation during production. University of Florida researchers have developed a protocol for producing, isolating, and purifying rAAV samples by reducing the concentration of helper adenovirus in the rAAV samples. It also purifies the rAAV stock by contacting the rAAV sample with a matrix that includes heparin, thereby removing unbound proteins and contaminants from the sample. The general process of production, isolation, and purification involves centrifuging the rAAV sample through at least one iodixanol gradient. The resulting purified rAAV stocks have up to 1013 particles/mL, with a particles-to-infectivity ratio less than 100, and complete their production in less than 24 hours.

Related to 10411

*IP Issue Date

Nov 14, 2000

*Principal Investigation

Name: Sergei Zolotukhin

Department:

Name: Barry Byrne

Department:

Name: Nicholas Muzyczka

Department:

申请日期

May 27, 1999

申请号码

6,146,874

其他

国家/地区

美国