Carboxylated nanodiamond-mediated CRISPR-CAS9 delivery system

Patent metadata

Field	Value
Publication number	US-12091677-B2
Application number	US-202017071667-A
Country	US
Kind code	B2
Filing date	Oct 15, 2020
Priority date	Oct 15, 2019
Publication date	Sep 17, 2024
Grant date	Sep 17, 2024

Abstract

The present invention provides a carboxylated nanodiamond-mediated CRISPR-Cas9 delivery system for gene editing comprising nanodiamond (ND) particles as the carriers of CRISPR-Cas9 components designed to introduce the mutation in a given gene for repairing a tissue damage.

First claim

What is claimed is:

1. A delivery system for gene editing comprising nanodiamond (ND) particles as carriers of CRISPR-Cas9 components including a Cas9 protein, a guide RNA (gRNA), a template DNA designed to introduce a mutation in a given gene, wherein the ND particles each has a diameter less than 5 nm, and is functionalized by carboxylation of their surface and covalently conjugated with an mCherry protein comprising a polyhistidine tag;
wherein the ND particles are a mixture of:
(i) a first ND particle linked, through the polyhistidine tag by phosphoryl imidazole, with a first linear DNA construct for expression of the Cas9 protein, and
(ii) a second ND particle linked, through the polyhistidine tag by phosphoryl imidazole, with a second linear DNA construct for expression of the gRNA/template DNA; and wherein the ND particles enter a cell and enter a cell nucleus of the cell.

2. The delivery system of claim 1 , wherein the ND particles have a diameter in a range of 3 nm or more to less than 5 nm.

3. The delivery system of claim 1 , wherein the ND particles have a diameter of about 3 nm.

4. The delivery system of claim 1 , further comprising bovine serum albumin (BSA) mixed with the ND particles.

5. The delivery system of claim 1 , wherein the given gene is RSI gene associated with X-linked retinoschisis (XLRS).

6. The delivery system of claim 1 , wherein the first linear DNA construct encodes for Cas9 endonuclease and a green fluorescent protein (GFP) reporter.

7. The delivery system of claim 5 , wherein the first linear DNA construct encodes for Cas9 endonuclease and a GFP reporter.

8. A method for treating a disease or repairing a tissue damage in a subject, comprising delivering and internalizing the mutation in a given gene into said subject through the delivery system of claim 1 .

9. The method of claim 8 , wherein the disease is X-linked retinoschisis (XLRS).

10. The method of claim 9 , wherein the given gene is RSI gene associated with X-linked retinoschisis (XLRS).

11. A method for creating an in vitro or in vivo disease model, comprising delivering and internalizing the mutation in a given gene into induced pluripotent stem cells (iPSCs) or an animal organ through the delivery system of claim 1 .

12. The method of claim 11 , wherein the disease is X-linked retinoschisis (XLRS).

13. The method of claim 12 , wherein the given gene is RS1 gene associated with X-linked retinoschisis (XLRS).

14. The method of claim 12 , wherein the animal organ is mouse retinas.

15. The method of claim 14 , wherein the RSI gene editing in the mouse retinas results in several pathological features typical for XLRS.

16. The method of claim 15 , wherein the pathological feature typical for XLRS is aberrant photoreceptor structure.

Assignees

• Taipei Veterans General Hospital
• Univ National Chiao Tung
• Univ Nat Cheng Kung

Inventors

• Chiou Shih-Hwa
• Yang Tien-Chun
• Chang Chia-Ching
• Tzeng Yon-Hua

Classifications

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• A61K9/5115

  Inorganic compounds · CPC title