Targeted gene insertion for improved immune cells therapy

A technology of immune cells and therapy, applied in the field of sequence-specific endonuclease reagents and donor DNA vectors, which can solve the problems of reducing immune response and lifespan

Pending Publication Date: 2019-07-16
CELLECTIS SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, a major challenge is to avoid cellular exhaustion / anergy, which significantly reduces their immune response and lifespan

Method used

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  • Targeted gene insertion for improved immune cells therapy
  • Targeted gene insertion for improved immune cells therapy
  • Targeted gene insertion for improved immune cells therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0359] Example 1: AAV-driven homologous recombination at different loci in human primary T cells under the control of an endogenous promoter knocked out of an endogenous gene.

[0360] introduce

[0361] Sequence-specific endonuclease reagents such as (Cellectis, 8 rue de la Croix Jarry, 75013 PARIS) enables site-specific induction of double-strand breaks (DSBs) at desired loci in the genome. Repair of DSBs by cellular enzymes occurs mainly through two pathways: non-homologous end joining (NHEJ) and homology-mediated repair (HDR). HDR uses homologous DNA fragments (template DNA) to repair DSBs through recombination and can be used to introduce any gene sequence contained in the template DNA. As shown therein, recombinant adeno-associated virus (rAAV) with engineered nucleases such as The template DNA is delivered together to introduce site-specific DSBs.

[0362] Integrated Matrix Design

[0363] 1.1 Insertion of an apoptotic CAR at an upregulated locus knocked out of t...

Embodiment 2

[0382] Example 2: In T cells Mediated dual-targeted integration of IL-15 and CAR-encoded substrates

[0383] Material

[0384] X-vivo-15 was obtained from Lonza (cat#BE04-418Q), IL-2 was obtained from Miltenyi Biotech (cat#130-097-748), human serum AB was obtained from Seralab (cat#GEM-100-318), human T activator CD3 / CD28 was obtained from LifeTechnology (cat#11132D), QBEND10-APC was obtained from R&D Systems (cat#FAB7227A), vioblue-labeled anti-CD3, PE-labeled anti-LNGFR, APC-labeled anti- CD25 and PE-labeled anti-PD1 were obtained from Miltenyi (cat#130-094-363, 130-112-790, 130-109-021 and 130-104-892, respectively), 48-well treated plates (CytoOne, cat#CC7682- 7548), human IL-15 Quantikine ELISA Kit was obtained from R&D systems (cat#S1500), and ONE-Glo was obtained from Promega (cat#E6110). AAV6 batches containing different matrices were obtained from Virovek, PBMC cells were obtained from Allcells (cat#PB004F) and Raji-fluorescence was obtained after transduction of ...

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PUM

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Abstract

The invention pertains to the field of adaptive cell immunotherapy. It provides with the genetic insertion of exogenous coding sequence(s) that help the immune cells to direct their immune response against infected or malignant cells. These exogenous coding sequences are more particularly inserted under the transcriptional control of endogenous gene promoters that are sensitive to immune cells activation. Such method allows the production of safer immune primary cells of higher therapeutic potential.

Description

technical field [0001] The present invention relates to the field of adaptive cellular immunotherapy. It aims to enhance the function of primary immune cells against conditions that develop immune resistance, such as tumors, thereby improving the therapeutic potential of these immune cells. The methods of the invention provide for the genetic insertion of exogenous coding sequences, which assist immune cells in directing their immune response against infected or malignant cells. These exogenous coding sequences are more particularly inserted under the transcriptional control of promoters of endogenous genes that are activated by immune cells, in the tumor microenvironment, or that are life-threatening up- or down-regulation under inflammatory conditions. The invention also provides sequence-specific endonuclease reagents and donor DNA vectors (eg, AAV vectors) to effect such targeted insertions at said specific loci. The methods of the invention help to improve the therapeu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078A61K35/17C12N15/90C12N9/22C12N5/0783
CPCA61K35/17C12N5/0634C12N5/0636C12N5/0638C12N2510/00C07K2319/03C07K14/7051A61K39/0011A61K2039/5156A61K2039/5158C12N9/22C12N15/907C12N2750/14143C12N2830/008
Inventor 布莱恩·布瑟尔菲利普·杜沙托亚历山大·朱利拉特洛朗·普瓦罗朱利安·瓦尔顿
Owner CELLECTIS SA
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