Efficient Production and Purification of rAAV for Gene Therapy Applications

Technology #10411

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Researchers
Sergei Zolotukhin
Nicholas Muzyczka Ph.D.
Barry J. Byrne
Managed By
April Kilburn
Assistant Director 352-392-8929
Patent Protection
US Patent 6,660,514

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

This versatile, efficient production and purification of recombinant adeno-associated virus (rAAV) yields at least ten times more than the amount of the virus recovered in conventional production. In recent years, research demonstrates rAAV vectors are useful and efficient for 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 therapeutic applications. Despite recent advancements, rAAV vector production takes several weeks to complete and often results in a loss of virus or contamination. The quality of those vector preparations is useful in some laboratory studies but the resulting rAAV are unsuitable for clinical studies requiring highly-purified vector stocks with little contaminants. 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 process 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 rAAV production that purifies and isolates 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, removing unbound proteins and contaminants from the sample. The general method of production, purification, and isolation is performed by centrifuging the rAAV sample through at least one iodixanol gradient. The produced purified rAAV stocks have up to 1013 particles/mL, with a particles-to-infectivity ratio less than 100 in processes accomplished in less than 24 hours.