公开(公告)号：US11912986B2

公开(公告)日：2024-02-27

申请号：US17028836

申请日：2020-09-22

Applicant: The Regents of the University of California

Inventor： Prashant Mali ,  Udit Parekh ,  Yan Wu ,  Kun Zhang

IPC: C12N15/10 ,  C12N5/071 ,  C12N15/86 ,  A61K35/44

CPC classification number: C12N15/1065 ,  A61K35/44 ,  C12N5/069 ,  C12N15/86 ,  C12N2506/45 ,  C12N2740/15043 ,  C12N2740/15052

Abstract: Understanding the complex effects of genetic perturbations on cellular state and fitness in human pluripotent stem cells (hPSCs) has been challenging using traditional pooled screening techniques which typically rely on unidimensional phenotypic readouts. Here, Applicants use barcoded open reading frame (ORF) overexpression libraries with a coupled single-cell RNA sequencing (scRNA-seq) and fitness screening approach, a technique we call SEUSS (ScalablE fUnctional Screening by Sequencing), to establish a comprehensive assaying platform. Using this system, Applicants perturbed hPSCs with a library of developmentally critical transcription factors (TFs), and assayed the impact of TF overexpression on fitness and transcriptomic cell state across multiple media conditions. Applicants further leveraged the versatility of the ORF library approach to systematically assay mutant gene libraries and also whole gene families. From the transcriptomic responses, Applicants built genetic co-perturbation networks to identify key altered gene modules. Strikingly, we found that KLF4 and SNAI2 have opposing effects on the pluripotency gene module, highlighting the power of this method to characterize the effects of genetic perturbations. From the fitness responses, Applicants identified ETV2 as a driver of reprogramming towards an endothelial-like state.

公开(公告)号：US20210163948A1

公开(公告)日：2021-06-03

申请号：US17170693

申请日：2021-02-08

Applicant: The Regents of the University of California

Inventor： Prashant Mali ,  Dhruva Katrekar

IPC: C12N15/115 ,  A61P21/00 ,  C12N9/78

Abstract: Aspects of the disclosure relate to a gene therapy approach for diseases, disorders, or conditions caused by mutation in the stop codon utilizing modified tRNA. At least 10-15% of all genetic diseases, including muscular dystrophy (e.g. Duchene muscular dystrophy), some cancers, beta thalassemia, Hurler syndrome, and cystic fibrosis, fall into this category. Not to be bound by theory, it is believed that this approach is safer than CRISPR approaches due to minimal off-target effects and the lack of genome level changes.

公开(公告)号：US20200263180A1

公开(公告)日：2020-08-20

申请号：US16490494

申请日：2018-03-02

Applicant: The Regents of the University of California

Inventor： Prashant Mali ,  Dhruva Katrekar

IPC: C12N15/115 ,  C12N9/78 ,  A61P21/00

Abstract: Aspects of the disclosure relate to a gene therapy approach for diseases, disorders, or conditions caused by mutation in the stop codon utilizing modified tRNA. At least 10-15% of all genetic diseases, including muscular dystrophy (e.g. Duchene muscular dystrophy), some cancers, beta thalassemia, Hurler syndrome, and cystic fibrosis, fall into this category. Not to be bound by theory, it is believed that this approach is safer than CRISPR approaches due to minimal off-target effects and the lack of genome level changes.

公开(公告)号：US12297241B2

公开(公告)日：2025-05-13

申请号：US17257951

申请日：2019-07-03

Applicant: The Regents of the University of California

Inventor： Kun Zhang ,  Yan Wu ,  Amir Dailamy ,  Prashant Mali ,  Daniella McDonald ,  Udit Parekh ,  Michael Hu

IPC: C07K14/47 ,  A61K31/522 ,  A61K45/06 ,  C12N5/071 ,  C12N9/22 ,  C12N15/11 ,  C12N15/86

Abstract: This disclosure relates to methods, polynucleotides, vectors, viral particles, cells, and systems or the engineering of human tissues. One aspect of the disclosure relates to using lineage-specific miRNA binding molecules to bias tissue lineage. Another aspect of the disclosure relates to using lineage-specific transcription factor overexpression to bias tissue lineage.

公开(公告)号：US11913017B2

公开(公告)日：2024-02-27

申请号：US16312907

申请日：2017-06-28

Applicant: The Regents of the University of California

Inventor： Kun Zhang ,  Prashant Mali ,  Yan Wu ,  Dongxin Zhao

IPC: G16B25/10 ,  C12N9/22 ,  C12N15/10 ,  C12N15/113 ,  C12N15/90 ,  G16B20/20 ,  G16B40/10

CPC classification number: C12N15/907 ,  C12N9/22 ,  C12N15/102 ,  C12N15/113 ,  G16B20/20 ,  G16B25/10 ,  G16B40/10 ,  C12N2310/20 ,  C12N2320/12 ,  C12N2330/31

Abstract: A guide RNA comprising: a gRNA spacer sequence at the 5′ end of the guide RNA, wherein the spacer sequence is complementary to a target gene, a scaffold sequence that binds to Cas9, and an RNA capture and sequencing domain comprising: a barcode sequence, and a primer binding sequence; nucleic acids and vectors encoding the guide RNA; cells expressing the guide RNA; and a library comprising a plurality of guide RNAs. Also disclosed are methods of introducing a genetic perturbation into a cell, methods of assessing an effect of at least one genetic perturbation on RNA expression in a cell, methods of identifying nucleic acid sequences associated with a disease state and a method of identifying candidate therapeutic agents.

公开(公告)号：US20210198673A1

公开(公告)日：2021-07-01

申请号：US17170671

申请日：2021-02-08

Applicant: The Regents of the University of California

Inventor： Prashant Mali ,  Dhruva Katrekar

IPC: C12N15/115 ,  A61P21/00 ,  C12N9/78

Abstract: Aspects of the disclosure relate to a gene therapy approach for diseases, disorders, or conditions caused by mutation in the stop codon utilizing modified tRNA. At least 10-15% of all genetic diseases, including muscular dystrophy (e.g. Duchene muscular dystrophy), some cancers, beta thalassemia, Hurler syndrome, and cystic fibrosis, fall into this category. Not to be bound by theory, it is believed that this approach is safer than CRISPR approaches due to minimal off-target effects and the lack of genome level changes.

公开(公告)号：US20210108193A1

公开(公告)日：2021-04-15

申请号：US17028836

申请日：2020-09-22

Applicant: The Regents of the University of California

Inventor： Prashant Mali ,  Udit Parekh ,  Yan Wu ,  Kun Zhang

IPC: C12N15/10 ,  C12N5/071 ,  C12N15/86 ,  A61K35/44

Abstract: Understanding the complex effects of genetic perturbations on cellular state and fitness in human pluripotent stem cells (hPSCs) has been challenging using traditional pooled screening techniques which typically rely on unidimensional phenotypic readouts. Here, Applicants use barcoded open reading frame (ORF) overexpression libraries with a coupled single-cell RNA sequencing (scRNA-seq) and fitness screening approach, a technique we call SEUSS (ScalablE fUnctional Screening by Sequencing), to establish a comprehensive assaying platform. Using this system, Applicants perturbed hPSCs with a library of developmentally critical transcription factors (TFs), and assayed the impact of TF overexpression on fitness and transcriptomic cell state across multiple media conditions. Applicants further leveraged the versatility of the ORF library approach to systematically assay mutant gene libraries and also whole gene families. From the transcriptomic responses, Applicants built genetic co-perturbation networks to identify key altered gene modules. Strikingly, we found that KLF4 and SNAI2 have opposing effects on the pluripotency gene module, highlighting the power of this method to characterize the effects of genetic perturbations. From the fitness responses, Applicants identified ETV2 as a driver of reprogramming towards an endothelial-like state.

公开(公告)号：US20200340012A1

公开(公告)日：2020-10-29

申请号：US16325679

申请日：2017-08-18

Applicant: Prashant Mali ,  Dhruva Katrekar ,  Ana Moreno Collado ,  The Regents of the University of California

Inventor： Prashant Mali ,  Dhruva Katrekar ,  Ana Moreno Collado

IPC: C12N15/86 ,  C12N15/113 ,  C12N9/22 ,  C12N7/00 ,  A61K35/76 ,  A61P25/04 ,  A61P33/06

Abstract: The present disclosure relates to a novel delivery system with unique modular CRISPR-Cas9 architecture that allows better delivery, specificity and selectivity of gene editing. It represents significant improvement over previously described split-Cas9 systems. The modular architecture is “regulatable”. Additional aspects relate to systems that can be both spatially and temporally controlled, resulting in the potential for inducible editing. Further aspects relate to a modified viral capsid allowing conjugation to homing agents.

公开(公告)号：US11608503B2

公开(公告)日：2023-03-21

申请号：US17170671

申请日：2021-02-08

Applicant: The Regents of the University of California

Inventor： Prashant Mali ,  Dhruva Katrekar

IPC: C12N15/115 ,  A61P21/00 ,  C12N9/78 ,  A61K38/00 ,  C12N15/113

Abstract: Aspects of the disclosure relate to a gene therapy approach for diseases, disorders, or conditions caused by mutation in the stop codon utilizing modified tRNA. At least 10-15% of all genetic diseases, including muscular dystrophy (e.g. Duchene muscular dystrophy), some cancers, beta thalassemia, Hurler syndrome, and cystic fibrosis, fall into this category. Not to be bound by theory, it is believed that this approach is safer than CRISPR approaches due to minimal off-target effects and the lack of genome level changes.

公开(公告)号：US20230036370A1

公开(公告)日：2023-02-02

申请号：US17781686

申请日：2020-12-01

Applicant: The Regents of the University of California

Inventor： Prashant Mali ,  Dhruva Katrekar

IPC: C12N15/113 ,  C12N9/78 ,  C12N9/22

Abstract: Disclosed herein are engineered guide RNAs, constructs for forming engineered guide RNAs, pharmaceutical compositions thereof, methods of making the engineered guide RNAs, and methods of treating or preventing a diseases and disorders of a subject by administering one or more of the engineered guide RNAs or the constructs for forming the engineered guide RNAs.