公开(公告)号：US20180135097A1

公开(公告)日：2018-05-17

申请号：US15806136

申请日：2017-11-07

Applicant: Spark Therapeutics, Inc.

Inventor： John Fraser WRIGHT ,  Marina SUMAROKA

IPC: C12Q1/48

CPC classification number: C12Q1/48 ,  C12Y205/0106 ,  G01N2333/91171

Abstract: 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.

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：US20200164008A1

公开(公告)日：2020-05-28

申请号：US16631692

申请日：2018-07-17

Applicant: SPARK THERAPEUTICS, INC.

Inventor： John Fraser WRIGHT ,  Marcus Eugene CARR, JR.

IPC: A61K35/76 ,  C12N15/86 ,  C07K14/005 ,  C07K16/08 ,  A61K35/16 ,  G01N33/68 ,  C12N9/22 ,  C12N15/11 ,  A61M1/36 ,  A61M1/34 ,  A61M1/16

Abstract: Provided are methods of treating a subject in need of treatment for a disease caused by a loss of function or activity of a protein. Also provided are methods of treating a subject in need of treatment for a disease caused by a gain of function, activity or expression, of a protein.

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.