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Methods for screening and using crispr/cas9 guidance RNA sequence from HIV provirus genome

a genome and genome technology, applied in the direction of viruses/bacteriophages, biochemistry apparatus and processes, peptides/protein ingredients, etc., can solve the problems of serious dysregulation of neuronal function and homeostasis, astrocytes' ability to maintain homeostasis, and aids remains incurabl

Active Publication Date: 2018-09-06
FLORIDA INTERNATIONAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is about identifying specific molecules (gRNAs) that can treat HIV infections. The invention involves creating cells that have a gene that expresses a protein called a CRISPR-Cas and a piece of HIV genome with a marker gene. The cells are then screened to find gRNAs that can remove or disrupt the HIV genome. The invention also provides a composition of CRISPR-Cas protein and gRNAs that can be used to treat HIV infections in humans. This approach has the potential to lead to a cure for HIV.

Problems solved by technology

However, AIDS remains an incurable, chronic infection due to the multiple HIV latent cells in a patient.
However, the neurotoxic products released from HIV infected brain cells seriously dysregulates neuronal function and homeostasis.
Moreover, in some pathological conditions, astrocytes' ability to maintain homeostasis is disrupted.
HIV and HIV proteins impair astrocytes' ability to maintain homeostasis.
However, gene therapy for HIV / AIDS has progressed very slowly.

Method used

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  • Methods for screening and using crispr/cas9 guidance RNA sequence from HIV provirus genome
  • Methods for screening and using crispr/cas9 guidance RNA sequence from HIV provirus genome
  • Methods for screening and using crispr/cas9 guidance RNA sequence from HIV provirus genome

Examples

Experimental program
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Effect test

example 1

Establishment of Stable Cas9 Expressing Astrocyte Cell Line

[0096]A genetically engineered astrocyte cell line stably expressing Cas9 was produced. An astrocyte cell line was transfected with intact or linearized Cas9 plasmid pX 459 and cell clones were selected for Cas9 plasmid integrated based on puromycin resistance. By testing the death curve of astrocyte under the puromycin resistance, the concentration of 0.31 μg / ml was chosen for selection. More positive cell clones were obtained from the linearized plasmid group compared to intact plasmid. The cell clones were expanded and genomic DNA was extracted for a PCR screen of Cas9 gene integration. Over ten cell clones provided a specific PCR product band, which was absent from the parent cell line (FIGS. 1B-1C). After the genomic DNA PCR screen, the protein extracts of positive cell clones were prepared for protein detection using SDS-PAGE and Western blot (FIG. 1C). The cell clones presented variable levels of Cas9 protein expressi...

example 2

Establishment of HIV Latent Astrocyte Cell Model with Stable Cas9 Expression

[0097]To make a robust HIV latent cell model, a dual fluorescent protein HIV-reporter plasmid-RGH was used. The dual fluorescent protein HIV-reporter plasmid-RGH is described in the Dahabieh et al. (2013) reference, which is incorporated herein by reference in its entirety.

[0098]After RGH pseudovirus infection, when infected cells are in active state, green fluorescence proteins are expressed, and cells are green under fluorescent microscopy (FIG. 2B). When RGH pseudovirus infected cells are in latent stage, only red fluorescent proteins are expressed and cells are red under fluorescent microscopy. According to conventional method, astrocytes were infected with VSVG pseudo-type RGH HIV virus (as described in the Dahabieh et al. (2013) reference) and time-course study was performed of the cell infection status (FIG. 2B). Early during the infection, for example, over first few days, green fluorescent protein e...

example 3

Screening and Testing of gRNAs Targeting HIV Provirus in Latent Astrocyte Cell Lines

[0099]An in vitro cell platform to screen gRNAs of CRISPR / Cas9 system for the removal of HIV provirus in HIV reporter Cas9 cells is provided. The specificity and gene-editing function of CRISPR / Cas9 system mainly depends on gRNAs. As an initial test, bioinformatics tools were applied to design several gRNAs targeting LTR region, pol gene region, tat gene region and nef gene region as indicated in FIG. 2A.

[0100]RGH Cas9 clones were transfected with those gRNAs either alone or in combination of two (gLTR and gpol; gLTR and gtat; gnef and gpol; gnef and gtat) to delete a DNA fragment which includes the mCherry coding region. Therefore a successful deletion should eliminate the expression of red fluorescent protein (FIG. 2A).

[0101]After transfection, those cells were first imaged with fluorescence microscopy as indicated in FIG. 3A. Reduced expression of red fluorescent protein was observed in gRNAs trea...

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Abstract

The invention pertains to compositions and methods for identifying gRNAs that are effective in treating a latent HIV infection. An embodiment of the invention provides a cell having incorporated into its genome: a gene that expresses a CRISPR-Cas protein and an HIV pseudovirus genome, wherein the HIV pseudovirus genome has a first marker gene encoding a first marker protein under the control of HIV-1 LTR promoter and a second marker gene inserted into the nef gene of the HIV pseudovirus and encoding a second marker protein under the control of a constitutive promoter. Screening methods for identifying gRNAs that can treat a latent HIV infection are also provided. Further, compositions comprising a CRISPR-Cas protein and gRNAs that can treat a latent HIV infection are provided. Furthermore, a method for treating a latent HIV infection in a subject by administering the compositions of CRISPR-Cas protein and gRNAs are described.

Description

[0001]The Sequence Listing for this application is labeled “SeqList-03Mar17-ST25.txt”, which was created on Mar. 3, 2017, and is 4 KB. The entire contents are incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0002]Human immunodeficiency virus (HIV) / acquired immunodeficiency syndrome (AIDS) is the most severe pandemic disease in modern history and remains a major threat to humans. With HIV / AIDS prevention, diagnosis and treatment, the morbidity and mortality of AIDS decreased significantly. However, AIDS remains an incurable, chronic infection due to the multiple HIV latent cells in a patient.[0003]HIV infection of human cells can be divided into active infection and latent infection. In most human cells, HIV infection is active infection; however, in rare human cells, latent infection can occur at very early stage. These very small numbers of latently infected cells are called HIV reservoirs and they are located mainly in brain, peripheral blood, and lymph...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/22C12N15/90
CPCC12N9/22A61K38/00C12N15/907C12N2740/16043
Inventor HUANG, ZAOHUANAIR, MADHAVAN
Owner FLORIDA INTERNATIONAL UNIVERSITY
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