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Exosomes comprising RNA therapeutics

An exosome, therapeutic technology, applied in retro RNA viruses, DNA/RNA fragments, fusions with RNA binding domains, etc., can solve problems such as adverse immune reactions

Active Publication Date: 2020-09-04
EVOX THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Another disadvantage of the TAMEL system is that it uses phage proteins, which may elicit an adverse immune response when the produced EVs are delivered to patients

Method used

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  • Exosomes comprising RNA therapeutics
  • Exosomes comprising RNA therapeutics
  • Exosomes comprising RNA therapeutics

Examples

Experimental program
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example 1

[0074] Example 1. Bone marrow-derived MSCs were cultured in conventional tissue culture flasks, and PEI transfection was used for transient transfection to achieve the loading and expression of mRNA cargo molecules and fusion polypeptide constructs. figure 2 The loading in EVs of mRNA cargo molecules encoding NanoLuc (RTM) and p21 obtained from BM-MSC is shown. Loading was achieved using a fusion polypeptide construct containing CD63 as an exosomal polypeptide and PUF or Cas6 as an NA binding domain. The experiment also included different numbers of binding sites for NA binding domains, namely 0, 3 and 6 binding sites, which were inserted into different positions on the 3'and / or 5'sides of the coding region.

[0075] MSC-EV was purified using a sequential combination of TFF and SEC. Only the expression of the exosomal polypeptide CD63 does not cause any mRNA to be loaded into the EV ( figure 2 The right column of the middle figure). Expression of PUF-containing fusion polypept...

example 2

[0076] Example 2. Silencing of in vitro gene expression in target cells (HEK293 cells) under EV-mediated delivery of shRNA targeting huntingtin. Lentiviral transduction was used to transduce amniotic membrane cells cultured in a shaking incubator to secrete EVs containing shRNA and fusion polypeptide constructs containing PUF or Cas6 as NA binding domains, and CD63 or CD81 binds as an EV polypeptide. The EV was purified from the supernatant using ultracentrifugation. image 3 The Y-axis shows how the huntingtin protein expression level decreases as the number of binding sites in the NA cargo molecule increases. Loading anti-huntingtin shRNA with a PUF-CD63-PUF fusion polypeptide will result in approximately 80% knockout .

example 3

[0077] Example 3. After HEK EV-mediated delivery of NanoLuc (RTM) mRN, NanoLuc (RTM) is used as a reporter gene to express in target Huh7 cells. HEK293T cells were stably transduced to express various fusion polypeptide constructs to load the reporter gene NanoLuc (RTM) mRNA cargo into the EV. The NanoLuc(RTM) mRNA cargo molecule is engineered to contain 0, 3 or 6 binding sites of the NA binding domain. The NA binding domain comprises a fusion polypeptide construct, in which case the fusion polypeptide construct is PUFx2-CD63-PUFx2 (two PUF NA-binding peptides are inserted into the N and C ends of the exosomal polypeptide CD63), PUF-CD63-PUF and Cas6-CD9-Cas6 ( Figure 4 ).

[0078] After purification of HEK-derived EVs based on TFF with bead-eluted LC, the EVs are added to HEK cells at the optimal concentration. For this assay, the optimal concentration is in each well of a 6-well plate of Huh7 target cells. 10 7 EV. Figure 4 The Y axis of shows the relative light (luminescenc...

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Abstract

The present invention pertains to extracellular vesicle (EV) therapeutics, wherein the EVs comprise nucleic acid (NA)-based therapeutics such as m RNAs, circular RNAs, mi RNAs, sh RNAs, circular RNA and / or DNA molecules. The NA therapeutics are loaded into EVs using inventive engineering protein and NA engineering strategies to enhance loading into EVs and to facilitate release of the NA cargo molecules inside target cells.

Description

Technical field [0001] The present invention relates to an extracellular vesicle (EV) therapeutic agent, wherein the EV contains an endogenously loaded RNA therapeutic agent, such as mRNA, circular RNA, miRNA, shRNA and various other RNA therapeutic agents. Background technique [0002] Nucleic acid-based therapeutics are rapidly entering clinical applications. Gene therapy, mRNA-based therapy, short oligonucleotide and siRNA-based therapy are just some of the many models in the field of RNA therapy. Naked nucleic acid (usually RNA) is difficult to deliver in the body due to its fast clearance, nuclease activity, lack of organ-specific distribution, and low cell uptake efficiency. Therefore, it is usually necessary to use a specialized delivery vehicle as a means to achieve biologically active delivery. This is especially true for non-liver targets and high molecular weight RNA therapeutics (such as mRNA and gene therapy). [0003] Extracellular vesicles (such as exosomes) are us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/65A61K47/64A61K47/69A61K48/00C12N15/85C07K19/00
CPCC12N15/87C07K2319/06C07K2319/85C12N2740/16043C12N2320/32C12N2310/531C12N2310/14C12N15/113C12N15/111A61K47/6901A61K9/5068A61K48/0025A61K48/0091C07K19/00C12N15/85C12N15/88A61K31/7088A61K9/1277C07K2319/01C12N9/22
Inventor L.埃利切利D.古普塔J.黑安J.诺丁
Owner EVOX THERAPEUTICS LTD
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