Genetic Therapy, Inc.

Hitoshi Kotani

Shuyuan Zhang

Perry Newton, Iii

Perry Newman, Iii

A method of purifying retroviral vector particles which comprises culturing retroviral vector producer cells which are capable of generating retroviral vector particles, and obtaining a supernatant containing retroviral vector particles from the culture of retroviral vector producer cells. The supernatant is clarified, and then concentrated. The supernatant then is contacted with a precipitation agent, and the supernatant and precipitation agent are centrifuged. A precipitate is recovered, resuspended, and subjected to a high-speed centrifugation. Retroviral particles then are recovered. The above method provides for increased recovery of retroviral vector particles which may be employed in gene therapy.

Purification of retroviral vectors

The present invention relates to an efficient method, termed spin transduction, of transducing cells with retroviral vectors. Retroviral particles, suspended in a pharmaceutically acceptable carrier, are contacted with the cells to be transduced, and the mixture is centrifuged for a sufficient period of time to permit transduction. This method leads to a 3- to 15-fold increase in transduction efficiency, depending on the type of cells transduced.

Method of transduction of cells with retroviral vectors

A stainless steel column housing is capped with an O-ring sealed cover to form a sealed autoclavable sterile chamber. A pair of stainless steel mesh circular cylindrical concentric inner and outer columns are within the chamber. A bed of porous glass spherical beads are packed in the region between the inner and outer mesh columns. The pH and DO are monitored and controlled while culture medium is continuously supplied to and from the sterile chamber. The outer mesh column forms an outer bulk medium chamber with the housing and the inner mesh column forms an inner bulk medium chamber. A ring sparger bubbles air and CO.sub.2 mixture into the outer bulk medium chamber, the bubbles creating pressure differentials in the bulk medium chambers. These differentials cause radially bulk medium flow through the packed bead bed without pressure gradients in the bed and axial bulk medium recirculation flow in and between the inner and outer bulk medium chambers at their axial ends. The bioreactor is suitable for retroviral vector and animal cell culture production.

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Middletown, MD, United States of America

Hitoshi Kotani