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Methods and compositions for selectively eliminating cells of interest

A composition and selective technology that can be used in drug combinations, chemical instruments and methods, biochemical equipment and methods, etc., to solve problems such as infertility

Pending Publication Date: 2017-10-13
朱坚
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, cancer patients undergoing current systemic therapies suffer from multiple side effects ranging from nausea to infertility due to the non-selectivity of therapeutic agents

Method used

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  • Methods and compositions for selectively eliminating cells of interest
  • Methods and compositions for selectively eliminating cells of interest
  • Methods and compositions for selectively eliminating cells of interest

Examples

Experimental program
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preparation example Construction

[0075] Preparation of lipid:nucleic acid complexes, including targeted lipids (e.g., immunolipid complexes), is well known to those skilled in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al. , Cancer GeneTher.2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem.5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); US Pat.

[0076] Delivery of nucleic acids using RNA or DNA virus-based systems exploits the highly evolved process of targeting viruses to specific cells in the body and delivering the viral payload to the nucleus. Viral vectors can be administered directly to a patient (in vivo), or can be used to treat cells in vitro, and the modified cells can optionally be administered to a patient (in vivo). Traditional virus-based systems can include retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors for gene transfer. ...

Embodiment 1

[0089] The following is an example of insertion of a suicide gene into a target sequence in a cell to induce cell death.

[0090] To test the functionality of the strategy disclosed in this application, HSV-TK was integrated into the AAVS1 (adenovirus-associated virus integration site 1) locus located in the PPP1R12C gene on chromosome 19 of HEK293T cells.

[0091] Preparation of Cas9 and gRNA expression constructs

[0092] Plasmid pX330 was obtained from Addgene (ID No: 48140), which contains two expression cassettes: a human codon-optimized S. pyogenes Cas9 expression cassette, and a U6 promoter-driven cassette expressing a single guide RNA. Target the encoding to two Cas9 targets, T1 and T2 (see figure 2 ), respectively cloned into the guide RNA expression cassette of the pX330 plasmid (see Ran et al. (2013) Genome engineering using the CRISPR-Cas9system, Nature Protocols8, 2281-2308).

[0093] Preparation of target plasmid containing HSV-TK gene

[0094] Use the Mu...

Embodiment 2

[0102] The following are examples of selective depletion of cells of interest. In this example, the HSV-TK gene was inserted into cells containing the prospacer sequence and the cells were induced to die.

[0103] The cells into which the GFP gene was inserted as disclosed in Example 1 also contained the prospacer 2 sequence (SEQ ID NO: 5) downstream of the GFP gene, which was used in this Example for insertion of the suicide gene. A guide RNA was designed based on the protospacer 2 sequence and inserted into the pX330 vector to generate the CRISPR plasmid pX330-p. The targeting vector was constructed as disclosed in Example 1 and contained the HSV-tk gene flanked by a 5' homology arm consisting of the GFP gene and the same 3' homology arm as used in Example 1. Therefore, if the target vector is inserted by homologous recombination, the expression of GFP will not be disturbed.

[0104] To show selective depletion of cells containing the prospacer, HEK293T cells inserted with...

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Abstract

The present disclosure provides novel compositions and methods suitable for specifically eliminating target cells (e.g., cancer cells) without affecting non-target cells (e.g., non-cancer cells). For example, CRISPR system and the compositions of the present disclosure can be employed to specifically introduce a suicidal gene into a cancer cell in the loci of a cancer-specific target sequence, which as a result of chromosomal re-arrangement or translocation in a cancer cell presents a cancer specific sequence for a guide RNA and CAS to be recognized and such sequence is absent in a non-cancer cell. Consequently, the specific introduction of the composition(s) to cancer-specific site(s) and integration of suicide gene in the target genome, which is inapplicable to normal cells for lack of the site(s), leads to selective elimination of cancer cells but not non-cancer cells, and therefore render novel therapeutic methods and compositions for cancer treatment.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Patent Application No. 62 / 091,431, filed December 12, 2014, the disclosure of which is incorporated herein by reference. field of invention [0003] The present invention generally relates to selective cell depletion. Background of the invention [0004] Cancer is the leading cause of morbidity and death worldwide. In 2012, there were approximately 14 million new cases and 8.2 million cancer-related deaths, or 14.6% of all human deaths (WHO website (November 2014) "Cancer"). The main feature of cancer is the growth of abnormal cells with the potential to invade or spread to other parts of the body. The ideal therapy for cancer is to completely eliminate all cancer cells, leaving all healthy cells intact. However, cancer patients undergoing current systemic therapies suffer from a variety of side effects ranging from nausea to infertility due to the non-selectivity o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C07K14/47C12N9/22C12N9/64C12N9/78C12N15/113C12N15/52C12N15/85C12Q1/68A61P35/00
CPCA61K48/00C07K14/4747C12N9/22C12N9/6472C12N9/78C12N15/113C12N15/52C12N15/85C12N15/111C12N2320/30C12N15/102C12N15/63A61K48/005C12N15/907C12N2750/14143C12N2310/20A61P35/00A61P43/00
Inventor 朱坚张怡
Owner 朱坚
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