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Targeted disruption of t cell and/or HLA receptors

A cell and zinc finger nuclease technology, applied in the field of targeted destruction of T cells and/or HLA receptors, can solve the problem of cells that cannot cause graft-versus-host disease

Pending Publication Date: 2020-04-10
SANGAMO BIOSCIENCES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Furthermore, knockout of the TCR will result in cells that are unable to elicit a graft-versus-host disease (GVHD) response when introduced into the patient

Method used

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  • Targeted disruption of t cell and/or HLA receptors
  • Targeted disruption of t cell and/or HLA receptors
  • Targeted disruption of t cell and/or HLA receptors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0214] Example 1: Design of TCR-specific nucleases

[0215] TCR-specific ZFNs were constructed to site-specifically introduce double-strand breaks at the TCRα (TCRA) gene. The design of ZFNs is generally described in, Urnov et al. (2005) Nature 435(7042): 646-651, Lombardo et al. (2007) Nat Biotechhol. 25(11): 1298-306, and US Patent Publication No. 2008 / 0131962; 2015 / 016495; 2014 / 0120622; and 2014 / 0301990 and US Patent No. 8,956,828. ZFN pairs target different sites in the constant region of the TCRA gene (see Figure 1). The recognition helices and target sequences of exemplary ZFN pairs are shown in Table 1 below. The target sites of the TCRA zinc finger designers are shown in the first column. Nucleotides in the target site targeted by the ZFP recognition helix are indicated in upper case; nucleotides not targeted are indicated in lower case. The linker used to connect the Fokl nuclease domain and the ZFP DNA binding domain is also shown (see, US Patent Publication No....

Embodiment 2

[0227] Example 2: In vitro nuclease activity

[0228] ZFNs described in Table 1 were used to test nuclease activity in K562 cells. To test for cleavage activity, plasmids encoding the paired human TCRA-specific ZFN pairs described above were transfected into K562 cells with either plasmid or mRNA. K562 cells were obtained from the American Type Culture Collection and grown as recommended in RPMI medium (Invitrogen) supplemented with 10% qualified fetal bovine serum (FBS, Cyclone). For transfection, the ORFs of the active nucleases listed in Table 1 were cloned into expression vectors optimized for mRNA production containing 5' and 3' UTRs and a synthetic poly-A signal. mRNA was generated using the mMessage mMachine T7 Ultra kit (Ambion) following the manufacturer's instructions. In vitro synthesis of nuclease mRNA using a pVAX-based vector containing a T7 promoter, an appropriate nuclease, and a poly-A motif, or a pGEM-based vector, or a PCR amplicon for in vitro The poly-A...

Embodiment 3

[0240] Example 3: TCRA-specific ZFN activity in T cells

[0241] The nuclease activity of TCRA-specific ZFN pairs was also tested in human T cells. ZFN-encoding mRNAs were transfected into purified T cells. Briefly, T cells were obtained from leukopheresis products and purified using Miltenyi's CliniMACS system (CD4 and CD8 dual selection). These cells were then activated using Dynabeads (ThermoFisher) according to the manufacturer's protocol. Three days after activation, cells were transfected with three doses of mRNA (60, 120, and 250 μg / mL) using a Maxcyte electroporator (Maxcyte), OC-100, 30e6 cells / mL, and a volume of 0.1 mL. On day 10 after transfection, cells were analyzed for on target TCRA modification using deep sequencing (Miseq, Illumina). Cell viability and cell growth (total cell doublings) were measured throughout 13-14 days of culture. In addition, at day 10 of culture for CD3 staining, TCR was measured on the cell surface of treated cells using standard FA...

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Abstract

Disclosed herein are methods and compositions for inactivating TCR and / or HLA genes, using engineered nucleases comprising at least one DNA binding domain and a cleavage domain or cleavage half-domainin conditions able to preserve cell viability. Polynucleotides encoding nucleases, vectors comprising polynucleotides encoding nucleases and cells comprising polynucleotides encoding nucleases and / orcells comprising nucleases are also provided.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Application No. 62 / 521,132, filed June 16, 2017, U.S. Provisional Application No. 62 / 542,052, filed August 7, 2017, and U.S. Provisional Application No. 62 / 573,956, filed October 18, 2017 rights and interests, the disclosure of which is incorporated herein by reference in its entirety. technical field [0003] This disclosure is in the field of genome modification of human cells, including lymphocytes and stem cells. Background technique [0004] Gene therapy holds great potential for a new era of human therapeutics. These approaches will enable the treatment of conditions not addressed by standard medical practice. Gene therapy can include various variations of genome editing techniques, such as disruption (inactivation) or correction of a genetic locus, and / or insertion of an expressible transgene that can be expressed through a specific exogenous promoter operatively linked...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/85C12N15/113C12N9/22C12N15/62C12N5/0783C12N15/11
CPCC12N2501/515C12N2510/00A61K48/005C12N15/907C12N2750/14143C12N5/0636C12N9/22C07K14/7051A61P37/06A61P35/00A61K2239/48A61K39/464412A61K39/4631A61K39/4611C12N15/8509C12N2015/8518A61K48/0066C07K14/70539
Inventor A·康韦S·贾殷G·K·李D·帕斯乔恩E·J·瑞巴张雷
Owner SANGAMO BIOSCIENCES INC
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