The Children's Hospital of Philadelphia

Genzyme Corporation

Spark Therapeutics, Inc.

Other

Avigen, Inc.

John Fraser Wright

Guang Qu

Katherine A High

Bernd Hauck

Lin Lu

Federico Mingozzi

Olga Zelenaia

Younghoon Oh

Jesusa Josue-almqvist

Marina Sumaroka

Quang Qu

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

The present invention provides humanized viral vectors and methods of use thereof for delivery of transgenes or therapeutic nucleic acids to human subjects. Humanized viral vectors are modified from known viral vectors such as those based on AAV by coating their surface with a human protein such as human serum albumin and optionally a lipid coating or formulation, so that the foreign or non-human nature of the vector is masked. The coating is performed in a manner that reduces or prevents binding of antibodies to the vector surface, thereby reducing or preventing antibody-mediated clearance of vector, but still allowing the vector to transduce target cells and achieve therapeutic gene transfer. Such humanized vectors therefore evade pre-existing immune surveillance, reduce immune responses, and achieve therapeutic gene transfer in the presence of pre-existing antibodies to the viral vector.

Humanized viral vectors and methods of use thereof

Methods for measuring REP-1 and REP-2 activity are provided. In certain embodiments, a method includes: (a) contacting cells that do not express endogenous functional REP-1 or REP-2 protein with an adeno-associated viral (AAV) vector comprising a CHM gene encoding a REP-1 protein or CHM like gene encoding a REP-2 protein under conditions allowing cell transduction; (b) incubating transduced cells under conditions allowing expression of the encoded REP-1 or REP-2 protein; (c) lysing the transduced cells to produce an extract comprising the encoded REP-1 or REP-2 protein and Rab small GTPase (Rabs); (d) incubating said extract with a Rab substrate for a period of time and under conditions allowing prenylation of the Rab thereby forming prenylated Rab; and (e) detecting and/or quantifying the prenylated Rab, wherein the amount of prenylated Rab reflects REP-1 or REP-2 activity thereby measuring REP-1 or REP-2 activity.

Rab Escort Protein potency assay

This application relates to the fields of gene therapy and molecular biology. In accordance with the present invention, an adeno-associated virus (AAV) vector comprising an altered capsid protein is provided. More specifically, this invention provides adeno-associated viral vectors comprising protein capsid variants which accelerate vector breakdown and clearance, thereby reducing undesirable immune responses.

AAV8 vector with enhanced functional activity and methods of use thereof

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.

Growing community of inventors

Princeton, NJ, United States of America

John Fraser Wright