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Modified cells and methods of therapy

A technology of cells and lymphocytes, applied in the fields of V-domain immunoglobulins, sequential cell death factor 1, and hepatitis A virus cell receptor 2, which can solve the problems of lack of identifiable molecules and lack of specific binding to tumor targets

Pending Publication Date: 2018-08-31
RGT UNIV OF MINNESOTA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these successes have been largely limited to hematological malignancies, and broader application against solid tumors has been limited by the lack of identifiable molecules expressed by cells in a particular tumor and available for specific binding to tumor targets in order to mediate tumorigenesis. molecule of destruction

Method used

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  • Modified cells and methods of therapy
  • Modified cells and methods of therapy
  • Modified cells and methods of therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0509] Example 1: Determining the transfection efficiency of various nucleic acid delivery platforms

[0510] Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from LeukoPak

[0511]This article uses leukopak collected from normal peripheral blood. Dilute blood cells 3 to 1 with chilled 1X PBS. Add the diluted blood dropwise (eg, very slowly) onto 15 mL of LYMPHOPREP (stem cell Technologies) in a 50 mL Erlenmeyer flask. Cells were spun for 25 minutes at 400x G with no brake. Slowly remove the buffy coat and place it in a sterile Erlenmeyer flask. Wash the cells with chilled 1X PBS and spin the cells at 400x G for 10 minutes. The supernatant was removed, cells were resuspended in culture medium, counted and viable frozen in freezing medium (45 mL heat-inactivated FBS and 5 mL DMSO).

[0512] Isolation of CD3+ T cells

[0513] PBMCs were thawed and plated in medium (RPMI-1640 (without phenol red), 20% FBS (heat inactivated) and 1X Gluta-MAX) for 1-2 hours. Collect c...

Embodiment 2

[0531] Embodiment 2: Determine the transfection efficiency of GFP plasmid in T cells

[0532] Transfection efficiency of primary T cells nucleoffected with Amaxa using GFP plasmid. Figure 4 The structures of four plasmids prepared for this experiment are shown: Cas9 nuclease plasmid, HPRT gRNA plasmid (CRISPR gRNA targeting human HPRT gene), Amaxa EGFPmax plasmid, and HPRT targeting vector. The HPRT targeting vector has a 0.5kb targeting arm ( Figure 5 ). Sample preparation, flow cytometry, and other methods were similar to Experiment 1. Plasmids were prepared using an endotoxin-free kit (Qiagen). Different conditions (shown in Table 3), including cell numbers and plasmid combinations, were tested.

[0533] Table 3. Different conditions used in the experiments

[0534] Sample ID

#PBMC

plasmid

GFP'(ug)

Cas9'(ug)

gRNA'(ug)

target' (ug)

1

5×10^6

GFP

5

0

0

0

2

2×10^7

Cas9

0.1

20

0

0

3 ...

Embodiment 3

[0537] Example 3: Identification of gRNAs with the highest double-strand break (DSB) induction at each locus

[0538] Design and construction of guide RNA:

[0539] Guide RNAs (gRNAs) were designed to desired gene regions using the CRISPR design program (Zhang Lab, MIT 2015). Various primers (shown in Table 4) for generating gRNAs were selected based on the highest ranking value determined by the off-target position. The gRNAs were sorted by oligonucleotide pairs: 5'-CACCG-gRNA sequence-3' and 5'-AAAC-reverse complementary gRNA sequence-C-3' (sequences of oligonucleotide pairs are listed in Table 4 ).

[0540] Table 4. Primers used to generate gRNA (CACCG sequence added to sense strand and AAAC to antisense strand for cloning purposes).

[0541]

[0542]

[0543]

[0544]The gRNAs were cloned together using the Target Sequence Cloning Protocol (Zhang Lab, MIT). Briefly, using T4PNK (NEB) and 10X T4 Ligation Buffer (T4Ligation Buffer, NEB), oligonucleotide pairs we...

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Abstract

Genetically modified compositions, such as non-viral vectors and T cells, for treating cancer are disclosed. Also disclosed are the methods of making and using the genetically modified compositions intreating cancer.

Description

[0001] cross reference [0002] This application claims U.S. Provisional Application No. 62 / 199,905, filed July 31, 2015; U.S. Provisional Application No. 62 / 232,983, filed September 25, 2015; U.S. Provisional Application No., filed January 22, 2016 62 / 286,206; U.S. Provisional Application No. 62 / 295,670, filed February 16, 2016; U.S. Provisional Application No. 62 / 330,464, filed May 2, 2016; and U.S. Provisional Application No. 62 / 330,464, filed July 8, 2016 The benefit of Application No. 62 / 360,245, all of which are hereby incorporated by reference in their entirety. Background technique [0003] Despite remarkable advances in cancer therapy over the past 50 years, there are still many tumor types that are refractory to chemotherapy, radiotherapy, or biological therapy, especially at advanced stages that cannot be resolved by surgical techniques. Recently, significant advances have been made in the genetic engineering of lymphocytes for in vivo recognition of molecular tar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/00A61K35/17A61K35/28A61K38/00C07H21/04C07K14/725
CPCC07K14/70521C07K14/7158C12N15/113C12N15/87C12N2310/20C07K14/4718C07K14/7051C12N5/0636C12N2510/00C07K14/70503C12N15/907C12N9/22C12N9/96A61P35/00A61K39/4611A61K2239/38A61K39/4632A61K39/4621A61K39/46434A61K35/00A61K35/28A61K38/00C07H21/04C12N15/01C12N15/63C07K2319/81C07K14/705C07K2319/00Y02A50/30
Inventor 布兰登·莫里亚提博·韦伯斯科特·R·麦基弗大卫·拉盖斯帕达莫达希尔·乔杜里史蒂文·A·罗森堡道格拉斯·C·帕尔默尼古拉斯·P·雷斯蒂福
Owner RGT UNIV OF MINNESOTA
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