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Isolation of extracellular vesicles (EVS) from red blood cells for gene therapy

a technology of extracellular vesicles and gene therapy, applied in the field of molecular biology and genome editing, can solve the problems of ineffective methods, relapse of patients, immunogenic and toxic, etc., and achieve the effect of precise modification of genomic dna and robust

Inactive Publication Date: 2020-07-23
CITY UNIVERSITY OF HONG KONG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes a method for delivering RNA to target cells for CRISPR genome editing. The RNA is loaded into extracellular vesicles (EVs) which are then taken up by the target cells. This technique allows for precise modification of DNA using CRISPR for research and medical applications.

Problems solved by technology

Viral transduction and nanoparticles are often used for in vivo delivery of RNAs and DNAs however, these methods are usually ineffective, immunogenic and toxic.
However, the response rate is much lower in older patients as they cannot tolerate the toxicity of chemotherapy.
Moreover, almost all the patients relapse after a certain time due to drug resistance.
However, delivery of RNAs to AML cells for gene therapies has proven challenging, especially for in vivo treatments.
Common gene therapy delivery vehicles such as adeno-associated virus (AAV) and lipid nanoparticles (LNPs) are mostly ineffective or toxic in AML models.

Method used

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  • Isolation of extracellular vesicles (EVS) from red blood cells for gene therapy
  • Isolation of extracellular vesicles (EVS) from red blood cells for gene therapy
  • Isolation of extracellular vesicles (EVS) from red blood cells for gene therapy

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Materials and Methods

[0047]Blood samples were obtained by Red Cross from healthy donors in Hong Kong with informed consents. RBCs were separated from plasma and white blood cells by centrifugation and treated with 10 mM calcium ionophore (Sigma) overnight. The purification of EVs were optimized with multiple centrifugation steps including the removal of protein contamination using a 60% sucrose cushion (ultracentrifugation at 100,000×g) that yields a homogenous population of EVs with an average diameter of ˜140 nm. Each unit of RBCs, isolated from ˜300 ml blood, yield 7.1 mg EVs on average. These EVs are enriched in EV markers, ALIX and TSG101, as shown by Western blot analysis. They also contain hemoglobin A which is a major protein from RBCs.

[0048]FIG. 1a: Culture supernatants were collected from ionophore-treated human red blood cells and subjected to multiple steps of centrifugation to remove dead cells and debris. EVs were purified by ultracentrifugation with 60% sucrose cushio...

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Abstract

A method of RNA delivery using extracellular vesicles (EVs) derived from red blood cells (RBCs). The method comprises the purification and electroporation of the EVs and applying the RNA-loaded EVs to target cells. The method further comprises the treatment of cancer using the RNA-loaded EVs.

Description

SEQUENCE LISTING[0001]The Sequence Listing file entitled “sequencelisting” having a size of 6,395 bytes and a creation date of Aug. 16, 2017, that was filed with the patent application is incorporated herein by reference in its entirety.FIELD OF INVENTION[0002]The present invention relates to the field of molecular biology and genome editing, more specifically the transfer of genetic materials to recipient cells by extracellular vesicles (EVs).BACKGROUND[0003]RNA therapeutics including antisense oligo nucleotides (ASOs), small-interfering RNA (siRNAs), synthetic mRNAs and genome editing RNA-protein complexes are emerging modalities for therapies targeting the human genomes at high specificity and great flexibility. ASOs and siRNAs have been widely used as the tools for gene knockdown in biomedical research. Their ability to silence any gene of interest offers a great potential for targeting disease-prevalent genes. Various chemical modifications or conjugations can be used to keep A...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C12N15/90C12N9/22A61K35/13A61K35/14G01N33/49C12N15/88C12N5/078
CPCA61K35/14C12N2310/11C12N15/907A61K48/005A61K48/0075C12N2310/20A61K48/0041A61K48/0091C12N2310/122G01N33/491A61K35/13A61K2035/128A61K35/00A61K48/0008C12N15/88C12N5/0641C12N9/22A61P35/02A61P35/00A61K31/7088C12N15/87
Inventor LE, THI NGUYET MINHSHI, JIAHAIWAQAS, MUHAMMAD
Owner CITY UNIVERSITY OF HONG KONG