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Methods and compositions for rna-guided treatment of HIV infection

A composition, HIV-1 technology, applied in retro RNA viruses, DNA/RNA fragments, gene therapy, etc., can solve problems such as inability to target and inhibit low-level viral genome expression and replication

Inactive Publication Date: 2018-11-23
EXCISION BIOTHERAPEUTICS INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But HAART cannot inhibit low-level viral genome expression and replication in tissues, and cannot target latently infected cells that serve as HIV-1 reservoirs, such as resting memory T cells, brain macrophages, microglia, and astrocytes. glial cells, gut-associated lymphocytes

Method used

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  • Methods and compositions for rna-guided treatment of HIV infection
  • Methods and compositions for rna-guided treatment of HIV infection
  • Methods and compositions for rna-guided treatment of HIV infection

Examples

Experimental program
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preparation example Construction

[0127] The methods of the invention can be expressed in terms of the preparation of a medicament. Accordingly, the present invention includes the use of the agents and compositions described herein for the manufacture of a medicament. The compounds described herein are useful in therapeutic compositions and regimens or in the manufacture of medicaments for the treatment of diseases or conditions as described herein.

[0128] Any of the compositions described herein can be administered to any part of the host's body for subsequent delivery to target cells. Compositions can be delivered to, but are not limited to, the brain, cerebrospinal fluid, joints, nasal mucosa, blood, lungs, intestines, muscle tissue, skin, or peritoneal cavity of a mammal. In terms of route of delivery, the composition can be administered by intravenous, intracranial, intraperitoneal, intramuscular, subcutaneous, intramuscular, intrarectal, intravaginal, intrathecal, intratracheal, intradermal or transde...

example 1

[0152] Example 1: Materials and methods

[0153] Plasmid preparation : Various constructs, LTR-A, B, C and D, were generated using vectors containing human Cas9 and gRNA expression cassettes, pX260 and pX330 (Addgene).

[0154] Cell Culture and Stable Cell Lines : The TZM-b1 reporter and the U1 cell line were obtained from the NIH AIDS Reagent Program, and CHME5 microglial cells are known in the art.

[0155] Immunohistochemistry and western blot : A standard method for visualizing cells using immunocytochemistry and assessing protein expression by Western blotting.

[0156] Firefly-luciferase assay : According to the manufacturer's protocol, cells were lysed 24 hours after treatment using PassiveLysisBuffer (Promega) and analyzed with the Luciferase Reporter Gene Assay kit (Luciferase Reporter Gene Assay kit) ( Promega Corporation) for the determination. Luciferase activity was normalized to the number of cells determined by a parallel MTT assay (Vybrant, Invitrogen...

example 2

[0181] Example 2: Cas9 / LTR-gRNA inhibits HIV-1 reporter virus production in CHME5 microglial cells latently infected with HIV-1

[0182] We assessed the ability of HIV-1-directed guide RNA (gRNA) to abolish LTR transcriptional activity and eliminate proviral DNA from the genome of latently infected myeloid cells in the brain (a particularly refractory target population) as the HIV-1 reservoir. Our strategy focused on targeting the U3 region of the HIV-1 LTR promoter. Through bioinformatics screens and efficiency / off-target predictions, we identified four gRNA targets (protospacers; LTRA-D) that avoid conserved transcription factor binding sites, thereby minimizing the possibility of altering host gene expression ( Figure 5 and 13 ). We inserted DNA fragments complementary to gRNAs A-D into humanized Cas9 expression vectors (A / B in pX260; C / D in pX330) and tested their individual and combined ability to alter the activity of the integrated HIV-1 genome. We first utilized t...

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Abstract

A method of inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus by treating the host cell with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and two or more different guide RNAs (gRNAs), wherein each of the at least two gRNAs is complementary to a different target nucleic acid sequence in a long terminal repeat (LTR) in the proviral DNA, and inactivating the proviral DNA. A composition for use in inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus including isolated nucleic acid sequences comprising a CRISPR-associated endonuclease and a guide RNA, wherein the guide RNA is complementary to a target sequence in ahuman immunodeficiency virus.

Description

[0001] Statement Regarding Federally Funded Research [0002] This invention was made with US Government support under Grant Nos. R01MH093271, R01NS087971, and P30MH092177 awarded by the National Institutes of Health. The US Government has certain rights in this invention. technical field [0003] The present invention relates to compositions that specifically cleave target sequences in retroviruses, such as human immunodeficiency virus (HIV). Such a composition may be administered to a subject having or at risk of contracting HIV infection, and the composition may include a nucleic acid encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-related endonuclease in combination with A guide RNA sequence complementary to the target sequence in human immunodeficiency virus. Background technique [0004] More than three decades since the discovery of HIV-1, AIDS remains a major public health problem, affecting more than 35.3 million people worldwide. AID...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P31/12A61P31/18C12N15/63
CPCA61K31/711A61P31/18C12N9/22C12N15/102C12N2740/16021C12N15/1132C12N15/70C12N15/86C12Q1/703C12N2310/20
Inventor K·哈利利托马斯·马尔科姆
Owner EXCISION BIOTHERAPEUTICS INC
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