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

Abstract: 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.

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

Abstract: 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.

Abstract: 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.

Abstract: Disclosed herein are packaging cell lines, in which adenovirus (Ad) E1A is constitutively expressed, that also contain integrated AAV rep and cap genes. The packaging cell lines exhibit little to no expressed Rep protein until helper virus function, such as adenovirus (Ad) E4, E2A and/or VA RNA are provided by, for example, transduction of the cells with a virus, vector or plasmid, such as an Ad-AAV hybrid virus. The promoter driving expression of AAV rep gene can be positioned far enough upstream (5?) of the rep coding sequence that E1A is unable to activate the promoter, activate substantial transcription of the rep gene and in turn produce Rep protein. Introduction of helper virus function, such as E2A, E4 and/or VA RNA into these packaging cells is able to drive AAV rep gene transcription, subsequent Rep protein expression and production of rAAV vector particles.

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

Abstract: Described and provided herein are purification, production and manufacturing methods for recombinant adeno-associated viral (rAAV) vector particles. Purification, production and manufacturing methods set forth herein, for example, include at least 2 column chromatography steps. Column chromatography steps include, for example, cation exchange chromatography, anion exchange chromatography, size exclusion chromatography and/or AAV affinity chromatography alone or in combination and in any order.

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

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

Abstract: 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.

Abstract: 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.

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

Abstract: Methods and compositions for transfecting cells with plasmids are disclosed. In certain embodiments, methods and compositions are disclosed in which transfection efficiency is significantly increased by contacting the cells being transduced with polyethyleneimine (PEI) that is free of nucleic acid during the transfection process. Therapeutically useful adeno-associated viral vectors generated according to the disclosed methods and compositions are also disclosed.

Abstract: Provided are methods for producing recombinant adeno-associated virus (rAAV) vector particles at high recovery or high titer. Also provided are methods that concentrate rAAV vectors to a high concentration, for example, up to 5E+13 (5×1013) vector genomes per milliliter (Vg/ml) with little if any rAAV aggregates.

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

Abstract: 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.

Abstract: Cells and cell lines are disclosed that are able to produce therapeutic proteins, antibodies, vectors, and viral vectors such as lentiviral vectors and adeno-associated viral (AAV) vectors. The cells and/or cell lines can have mutations or deletions in either one or both of the endogenous di-hydrofolate reductase (DHFR?/?) or glutamine synthetase (GS?/?) genes such that DHFR and/or GS expression or function is substantially reduced or eliminated.

Abstract: 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.

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