Genzyme Corporation

The Children's Hospital of Philadelphia

Spark Therapeutics, Inc.

Avigen, Inc.

Guang Qu

John Fraser Wright

Katherine A High

Bernd Hauck

Lin Lu

Younghoon Oh

Jesusa Josue-almqvist

Haibo Zhang

Laura Duncan

Yuhuan Wang

Provided are compositions and methods of transducing/transfecting cells with a molecule, such as a nucleic acid (e.g., plasmid), at high efficiency. High efficiency transduced/transfected cells can, when transduced with a nucleic acid that encodes a protein or comprises a sequence that is transcribed into a transcript of interest, produce high amounts of protein and/or transcript. High efficiency transduced/transfected cells can, when transduced with plasmids comprising (i) nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding helper proteins; and (ii) a transgene that encodes a protein or is transcribed into a transcript of interest; produce high amounts of recombinant rAAV vector.

Enhancing agents for improved cell transfection and/or rAAV vector production

Methods for preparing highly purified AAV vector formulations are provided. The highly pure AAV formulations described herein are superior for clinical use.

Scalable manufacturing platform for viral vector purification and viral vectors so purified for use in gene therapy

In accordance with the present invention, a method for increasing the yield of rLV vector particles comprising a trans gene encoding a therapeutic protein or fragment thereof is disclosed. In one approach, cells are transfected with plasmids encoding the necessary components for rLV production using a calcium chloride transfection mix at pH 7.1 wherein the calcium chloride and plasmids form a complex which is added to the cells at a constant speed. The cells are then incubated for a suitable time period wherein virus particle media is collected at least twice during the incubation period and stored in a cold storage unit, thereby reducing virus inactivation.

Large commercial scale lentiviral vector production system and vectors produced thereby

In accordance with the invention, provided herein are methods for purifying recombinant adeno-associated (rAAV) vector particles.

Column-based fully scalable rAAV manufacturing process

Methods for separating AAV empty capsids from mixtures of AAV vector particles and AAV empty capsids are described. The methods use column chromatography techniques and provide for commercially viable levels of recombinant AAV virions.

Methods for producing preparations of recombinant AAV virions substantially free of empty capsids

Methods for preparing highly purified rLV vector formulations at the scale needed to meet anticipated demand for human gene therapy are provided.

Scalable manufacturing process to produce recombinant lentiviral vectors in serum-free suspension cell culture system

Compositions and methods are provided for preparation of concentrated stock solutions of AAV virions without aggregation. Formulations for AAV preparation and storage are high ionic strength solutions (e.g. &#x3bc;&#x2dc;500 mM) that are nonetheless isotonic with the intended target tissue. This combination of high ionic strength and modest osmolarity is achieved using salts of high valency, such as sodium citrate. AAV stock solutions up to 6.4&#xd7;10vg/mL are possible using the formulations of the invention, with no aggregation being observed even after ten freeze-thaw cycles. The surfactant Pluronic&#xae; F68 may be added at 0.001% to prevent losses of virions to surfaces during handling. Virion preparations can also be treated with nucleases to eliminate small nucleic acid strands on virions surfaces that exacerbate aggregation.

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Alameda, CA, United States of America

Guang Qu