Chemically Ligated RNAs for CRISPR/Cas9-lgRNA Complexes as Antiviral Therapeutic Agents

a technology of lgrna and lgrna, which is applied in the direction of biochemistry apparatus and processes, peptide/protein ingredients, enzyme stabilisation, etc., can solve the problems of hsv, hbv, and hsv afflicting enormous suffering, life loss, and increased hes burden, so as to optimize the effect of target cleavag

Inactive Publication Date: 2018-08-16
ZHONG MINGHONG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0046]In certain embodiments, lgRNAs are mixtures comprising lgRNA constructs with different spacers corresponding to different loci of viral genomes and / or variants of a single locus of target genomes. Useful selection methods to identify sequences having extremely low to no homology between the foreign viral genome and host cellular genome including endogenous retroviral DNA include bioinformatics screening to minimize and / or exclude off-target human transcriptome and essential untranslated genomic sites, and to optimize the efficacy of target cleavage.DETAILED DESCRIPTION OF THE INVENTION
[0048]In some embodiments, this chemical ligation strategy provides diverse chemically modified lgRNAs for optimization for better efficacy in cleaving viral genomic DNAs.
[0049]In other embodiments, this chemical ligation strategy provides diverse chemically modified lgRNAs for minimizing off-target cleavages of host genomic DNAs with or without engineering Cas9 proteins.
[0050]In some embodiments, this chemical ligation strategy provides diverse chemically modified lgRNAs for decreasing the size of Cas9-lgRNA complex by engineering Cas9 proteins, or for full functional substitution of Cas9 with smaller natural / engineered CRISPR-associated proteins thus more amenable to delivery in human cells, for efficient administrations and better dosage forms.

Problems solved by technology

Chronic viral infections such as HIV, HBV, and HSV afflict enormous suffering, life loss, and financial burdening among the infected individuals.
These infectious diseases are incurable, and contagious to variable degrees, and are prominent threats for public health, highlighting the urgent needs for curative therapies.
Both ZFNs and TALENs are composed of protein-based programmable, sequence-specific DNA-binding modules and nonspecific DNA cleavage domains, which make multiple-site targeting extremely challenging.
This is even more challenging for HEs, which have the same protein domains for both DNA binding and cleavage.
To date, the Cas9 protein and RNAs (either dualRNA (crRNA / tracrRNA) or a single guide RNA (sgRNA, ˜100 nt)) have been mostly introduced by plasmid transfection either as whole or separately, which makes chemical modifications of RNAs extremely challenging, if not impossible, and also is limited by random integration of all or part of the plasmid DNA into the host genome and by persistent and elevated expression of Cas9 in target cells that could lead to off-target effects.
However, considering the size of sgRNA (˜100 nt), large scale chemically manufacturing such large molecules is industrially challenging and costly.
Truncating sgRNAs or crRNA / tracrRNAs is limited because of the many essential binding interactions between Cas9 and sgRNA and the complicated molecular mechanisms of recognition and cleavage of target DNAs.

Method used

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  • Chemically Ligated RNAs for CRISPR/Cas9-lgRNA Complexes as Antiviral Therapeutic Agents
  • Chemically Ligated RNAs for CRISPR/Cas9-lgRNA Complexes as Antiviral Therapeutic Agents
  • Chemically Ligated RNAs for CRISPR/Cas9-lgRNA Complexes as Antiviral Therapeutic Agents

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0129]Compound 4

[0130]Compound 2 is prepared essentially according to a reported procedure in 4 steps (Santner, T. et al. Bioconjugate Chem. 2014, 25, 188-195). Compound 1 is treated with 2-azidoethanol in dimethylacetamide at 120 ° C. at the presence of BF3.OEt2, and the resulting 2′-azido nucleoside is tritylated (DMTrCl, in pyridine, RT), and attached to an amino-functionalized support.

[0131]To compound 2 (0.99 mmol) in THF / H2O (2:1) (18 mL) is then added trimethylphosphine (1.5 mL, 1.5 mmol). Reaction is shaken at room temperature for 8 h and washed thoroughly with THF / H2O (2:1). Dioxane / H2O (1:1) (20 mL) and NaHCO3 (185 mg, 2.2 mmol) are added. The reaction mixture is cooled down to 0° C., and Fmoc-OSu (415 mg, 1.23 mmol) in dioxane (2 mL) is added. The reaction is shaked for 15 min at 0° C., and then washed with water and then THF (3×20 mL) to give compound 4.

example 2

7

[0132]

[0133]6-Benzoyl-5′-O-DMTr-adenosine 5 (1.24 g, 1.84 mmole) is dissolved in THF (40 mL) and sodium hydride (60% dispersion in mineral oil, 0.184 g, 4.6 mmole) is added in portions at 0° C. The reaction mixture is warmed up to room temperature and stirred for 15 min. The reaction is then cooled to 0° C., and propargyl bromide (80% in toluene, 0.44 mL, 3.98 mmole) is added. The reaction is then stirred under reflux for 12 h. Saturated aqueous sodium bicarbonate (10 mL) is added, and volatiles are removed in vacuo. The resulting residue is dissolved in DCM, and washed with water and saturated brine. The organic layer is collected, dried over anhydrous sodium sulfate, and concentrated till dryness in vacuo. The resulting residue is purified by silica-gel chromatography (eluent: 97:3, DCM : MeOH, 0.5% pyridine) to provide compound 6.

[0134]To a solution of compound 6 (0.30 g, 0.42 mmol) in anhydrous dichloromethane (5 mL) and diisopropylethylamine (0.15 mL, 0.83 mmol), under nitroge...

example 3

[0135]

[0136]ON-11 is prepared using 2′-TBS protected RNA phosphoramidite monomers with t-butylphenoxyacetyl protection of the A, G and C nucleobases and unprotected uracil. 0.3 M Benzylthiotetrazole in acetonitrile (Link Technologies) is used as the coupling agent, t-butylphenoxyacetic anhydride as the capping agent and 0.1 M iodine as the oxidizing agent. Oligonucleotide synthesis is carried out on an Applied Biosystems 394 automated DNA / RNA synthesizer using the standard 1.0 μmole RNA phosphoramidite cycle. Compound 4 (20 mg) is packed into a twist column. All β-cyanoethyl phosphoramidite monomers are dissolved in anhydrous acetonitrile to a concentration of 0.1 M immediately prior to use. Stepwise coupling efficiencies are determined by automated trityl cation conductivity monitoring and in all cases are >96.5%.

[0137]Fmoc is then cleaved by treatment with 20% piperidine in DMF. The resulting 3′-end aminoethyl oligonucleotide is then treated with NHS ester of 6-azido caproic acid ...

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Abstract

Provided herein are chemically ligated guide RNA oligonucleotides (lgRNA) which comprise two functional RNA modules (crgRNA and tracrgRNA) joined by non-nucleotide chemical linkers (nNt-linker), their complexes with CRISPR-Cas9, preparation methods of Cas9-lgRNA complexes, and their uses for prevention and treatments of HIV infections in humans. Also disclosed are processes and methods for preparation of these compounds.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The present application is a divisional application of U.S. application Ser. No. 15 / 006,131, filed Jan 26, 2016, with title “Chemically Ligated RNAs for CRISPR / Cas9-lgRNA Complexes as Antiviral Therapeutic Agents” and naming Minghong Zhong as inventor(s), and claims the benefits of U.S. Provisional Application Ser. No. 62 / 108,064, the entire said invention being incorporated herein by reference.TECHNICAL FIELD OF THE INVENTION[0002]The present invention relates to chemically synthesized guide RNA oligonucleotides (lgRNA) comprising two functional RNA modules (crgRNA and tracrgRNA) ligated by non-nucleotide chemical linker(s) (nNt-linker), their complexes with CRISPR-Cas9, preparation methods of Cas9-lgRNA complexes, and uses as medicinal agents in treatment of chronic viral infections.BACKGROUND OF THE INVENTION[0003]Chronic viral infections such as HIV, HBV, and HSV afflict enormous suffering, life loss, and financial burdening among the...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/11
CPCC12N2310/20C12N15/111C12N2310/3519A61K38/00C12N2310/10A61K38/1709C12N2320/30A61K31/7105C12N15/1132C12N15/11C12N9/96
Inventor ZHONG, MINGHONG
Owner ZHONG MINGHONG
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