Lymphohematopoietic engineering using cas9 base editors

A base editing and lymphatic technology, applied in genetic engineering, genetically modified cells, blood/immune system cells, etc., can solve the problem of low efficiency of a single nucleotide

Pending Publication Date: 2021-01-29
RGT UNIV OF MINNESOTA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While this approach is efficient and reliable for purely disrupting single genes by NHEJ, it is very inefficient for precisely changing single nucleotides by HDR
Furthermore, induction of multiple DSBs in multiple gene editing procedures can lead to undesired genotoxicity and the formation of potentially oncogenic gross chromosomal translocations

Method used

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  • Lymphohematopoietic engineering using cas9 base editors
  • Lymphohematopoietic engineering using cas9 base editors
  • Lymphohematopoietic engineering using cas9 base editors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 - Splice site base editing

[0068] Base editing has previously been used to induce premature stop (pmSTOP) codons for gene knockout in mouse and mammalian cells 15-18 . However, we believe that splice site disruption has several advantages over induction of the pmSTOP codon ( figure 1 ). For example, stop codon readthrough has been shown to occur at frequencies as high as 31% in some genes and can be promoted under cellular stress conditions 19，20 . Splice site editing alleviates this concern because it alters gene processing at the RNA level 21 , which is unlikely to be bypassed at the translation level. Furthermore, current base editors cannot produce strict C-T edits, and even the latest base editors produce up to 25% off-target edits (C-G / A) 22 . In the case of pmSTOP, off-target editing prevents premature stop codon formation, thereby reducing the efficiency of protein knockdown and instead generating potentially undesired amino acid changes.

...

Embodiment 2

[0114] Example 2 - Base Editing in Natural Killer (NK) Cells

[0115] method

[0116] Isolation of Peripheral Blood Mononuclear Cells (PBMC): Peripheral blood was diluted 3:1 with chilled IX PBS. Diluted blood was dropped onto 15 mL of Lymphoprep (Stem Cell Technologies). Centrifuge cells at 400 x g for 25 min without brake. The buffy coat was removed and washed with cold 1X PBS and centrifuged at 400 x g for 10 min. The supernatant was removed and cells were either frozen as PBMCs or used immediately for purification of NK cells.

[0117] CD3 - CD56 + Isolation of NK cells: adjust the density of PBMCs to 5 x 10 7 cells / mL and transfer the cells to a 14 mL polystyrene round bottom tube. NK cells were isolated using Human NK Cell Enrichment Kit or Human NK Cell Isolation Kit (Stem Cell Technologies) according to the kit instructions. Enriched cells were counted and analyzed for purity (%CD56+, %CD3+) by flow cytometry.

[0118] Stimulation of CD3-CD56+ NK cells: CD3-...

Embodiment 3-CD34

[0137] Example 3 - Base editing in CD34+ hematopoietic stem-progenitor cells (HSPC)

[0138] Materials and methods

[0139]Medium: StemSpan Serum Free Expansion Media II (SFEMII) (Stem Cell Technologies catalog #09605); 100ng / ml hSCF (Peprotech); 100ng / ml hTPO (Peprotech); 100ng / ml hFlt- 3L (Peprotech); 100 ng / ml hIL-6 (Peprotech); StemRegenin1 (0.75 μM final concentration) Cayman Chemical

[0140] Freezing medium: Cryostor CS10

[0141] Cell isolation reagents: Human UCB CD34+ Enrichment Kit (Stem Cell Technologies; variations depending on source).

[0142] Other reagents: Neon kit (Thermo Fisher Scientific, various options depending on amount and desired tip size).

[0143] Thaw samples: Thaw cells in pre-warmed medium (37°C) using the same type of medium as used for cultivation. Add 1 mL of medium to a sterile 15 mL conical tube. Thaw frozen vials in a 37°C water bath until a single ice crystal remains. Immediately move the vial to a biosafety cabinet, spray with 70...

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Abstract

Provided herein are methods and systems for targeted gene disruption (knock-out, missense mutation) and targeted gene knock-in in mammalian cells using base editors and guide RNAs (gRNAs) designed totarget splice acceptor-splice donor sites. Also provided herein are universally acceptable genetically engineered cells comprising targeted disruptions in immunotherapy-related genes and comprising aCAR / TCR for therapeutic applications.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 642,151, filed March 13, 2018, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. Background technique [0003] Precise regulation of primary human cells has diverse applications in the fields of immunotherapy, autoimmunity, and enzymatic diseases. Modulating a patient's immune cells at the genetic level is an attractive therapeutic avenue due to the permanence of the treatment and the low risk of patient rejection. One approach for gene editing in immune cells uses the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system to induce double-strand breaks (DSBs) in the gene of interest, resulting in Small insertions or deletions (collectively referred to as "indels") are formed by hypervariable repair via the Non-Homologous End Joining (NHEJ) pathway. Alternatively, precise genomic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/17A61K9/00A61P31/00
CPCA61P31/00A61K35/17C12N2740/16043C12N5/0634C12N5/10C12N2501/515C12N2310/20C12N5/0636C12N15/907C12N2510/00C12N2800/80
Inventor 布兰登·莫里亚蒂博·韦伯卡拉-琳·隆特里米查林·迪尔斯米切尔·克鲁斯纳沃克·拉尔艾米丽·乔伊·波默罗伊
Owner RGT UNIV OF MINNESOTA
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