公开(公告)号：US20190292561A1

公开(公告)日：2019-09-26

申请号：US15780542

申请日：2016-12-01

Applicant: SPARK THERAPEUTICS, INC.

Inventor： Guang QU ,  Lin LU ,  John Fraser WRIGHT

IPC: C12N15/86 ,  C12N5/00 ,  C12N5/073 ,  C12N5/071

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.

公开(公告)号：US20220025396A1

公开(公告)日：2022-01-27

申请号：US17052097

申请日：2019-05-07

Applicant: Spark Therapeutics, Inc.

Inventor： Guang QU ,  Denis PHICHITH ,  Jingmin ZHOU

IPC: C12N15/86

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.

公开(公告)号：US20210079422A1

公开(公告)日：2021-03-18

申请号：US16627227

申请日：2018-06-29

Applicant: Spark Therapeutics, Inc.

Inventor： Younghoon OH ,  Guang QU

IPC: C12N15/86 ,  B01D15/34 ,  B01D15/36

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.

公开(公告)号：US20190078099A1

公开(公告)日：2019-03-14

申请号：US16088693

申请日：2017-03-30

Applicant: SPARK THERAPEUTICS, INC.

Inventor： Jingmin ZHOU ,  Guang QU ,  John Fraser WRIGHT

IPC: C12N15/52 ,  C07K14/705 ,  C12N15/85 ,  C12N9/06 ,  C12N9/22 ,  C12N9/00 ,  C12N9/12 ,  C12N15/62

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.

公开(公告)号：US20200299650A1

公开(公告)日：2020-09-24

申请号：US16088743

申请日：2017-03-31

Applicant: SPARK THERAPEUTICS, INC.

Inventor： Guang QU ,  Younghoon OH ,  Lin LU ,  John Fraser WRIGHT

IPC: C12N7/00 ,  C12N1/06 ,  B01D15/36 ,  B01D15/34 ,  B01D61/14 ,  B01D15/38

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

公开(公告)号：US20200165632A1

公开(公告)日：2020-05-28

申请号：US16619898

申请日：2018-06-06

Applicant: Spark Therapeutics, Inc.

Inventor： Guang QU ,  Lin LU ,  Jesusa JOSUE-ALMQVIST ,  John Fraser WRIGHT

IPC: C12N15/87 ,  C12N5/073

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.

公开(公告)号：US20190284576A1

公开(公告)日：2019-09-19

申请号：US16319216

申请日：2017-07-21

Applicant: Spark Therapeutics, Inc.

Inventor： Guang QU ,  John Fraser WRIGHT ,  Younghoon OH ,  Yuhuan WANG ,  Haibo ZHANG ,  Laura DUNCAN

IPC: C12N15/86 ,  C07K14/755 ,  C12N9/64 ,  C12N9/48

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.