NUCLEIC ACID LOADED RED BLOOD CELL EXTRACELLULAR VESICLES
A red blood cell extracellular vesicle (RBCEV) loaded with a nucleic acid cargo; method for preparing the loaded vesicle; and the therapeutic use of the vesicle thereof are disclosed. The nucleic acid cargoes may be DNA or RNA, single stranded or double stranded, as well as linear or circular.
1 . A red blood cell extracellular vesicle (RBCEV) loaded with a DNA cargo.
2 . The RBCEV of claim 1 , wherein the DNA cargo is single stranded and has a length of at least 250 bases.
3 . The RBCEV of claim 1 , wherein the DNA cargo is single stranded and has a length of at least 2000 bases or 2000-30000 bases or more.
4 . The RBCEV of claim 1 , wherein the DNA cargo is double stranded and has a length of at least 250 base pairs.
5 . The RBCEV of claim 1 , wherein the DNA cargo is double stranded and has a length of at least 2000 base pairs or 20000-17000 base pairs or more.
6 . The RBCEV of claim 1 , wherein the DNA cargo is an expression vector comprising a gene encoding a protein or peptide.
7 . The RBCEV of claim 1 , wherein the DNA cargo is circular.
8 . The RBCEV of claim 1 , wherein the DNA cargo is a minicircle or plasmid.
9 . The RBCEV of claim 1 , wherein the DNA cargo is linear.
10 . The RBCEV of claim 1 , wherein the DNA cargo is in the lumen of the RBCEV.
11 . The RBCEV of claim 1 , wherein the RBCEV is derived or obtained from human or mammalian red blood cells.
12 . The RBCEV of claim 1 , wherein the RBCEV is isolated.
13 . An isolated red blood cell extracellular vesicle (RBCEV) containing in the lumen of the RBCEV at least one DNA cargo.
14 . A composition comprising a plurality of RBCEVs according to claim 1 .
15 . The composition of claim 14 , wherein on average each RBCEV in the composition is loaded with at least 1.0 DNA cargo.
16 . The composition of claim 14 , wherein on average each RBCEV in the composition is loaded with at least 2.0 or 3.0 DNA cargoes.
17 . The composition of claim 14 , wherein on average each RBCEV in the composition is loaded with 1.0 to 4.0 DNA cargoes.
18 . The composition of claim 14 , wherein the composition is a pharmaceutical composition or medicament.
19 . The composition of claim 18 , wherein the composition or medicament further comprises a pharmaceutically acceptable carrier, diluent, excipient or stabiliser.
20 . A method of treating a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of an RBCEV according to of claim 1 .
21 . The method of claim 20 , wherein the method involves treatment of a disease in the subject by expression of a protein or peptide from a gene sequence of the DNA cargo.
22 . The method of claim 21 , wherein the treatment comprises prevention and/or amelioration of the disease.
23 . A method for loading an extracellular vesicle with a nucleic acid cargo, the method comprising:
a. providing a nucleic acid to be loaded into an extracellular vesicle;
b. contacting the nucleic acid with an extracellular vesicle in the presence of a transfection reagent under conditions sufficient for the extracellular vesicle to be loaded with the nucleic acid; and
c. optionally washing the loaded extracellular vesicle.
24 . The method of claim 23 , wherein the extracellular vesicle is a red blood cell extracellular vesicle.
25 . The method of claim 23 , wherein the method comprises repeating step b.
26 . A method for loading an extracellular vesicle with a nucleic acid cargo, the method comprising:
a. providing a nucleic acid to be loaded into an extracellular vesicle;
b. contacting the nucleic acid with transfection reagent to allow formation of nucleic acid/transfection reagent complexes;
c. incubating or contacting the nucleic acid/transfection reagent complexes with an extracellular vesicle under conditions sufficient for the extracellular vesicle to be loaded with a nucleic acid/transfection reagent complex; and
d. optionally washing the loaded extracellular vesicle.
27 . A method for loading an extracellular vesicle with a nucleic acid cargo, the method comprising:
a. providing a nucleic acid to be loaded into an extracellular vesicle;
b. contacting the nucleic acid with transfection reagent to allow formation of nucleic acid/transfection reagent complexes;
c. incubating or contacting the nucleic acid/transfection reagent complexes with an extracellular vesicle under conditions sufficient for the extracellular vesicle to be loaded with a nucleic acid/transfection reagent complex;
d. optionally washing the loaded extracellular vesicle;
e. contacting the loaded extracellular vesicle with further nucleic acid/transfection reagent complexes; and
f. incubating or contacting the further nucleic acid/transfection reagent complexes with the loaded extracellular vesicle.
28 . The method of claim 26 , wherein the extracellular vesicle is a red blood cell extracellular vesicle.
29 . The method of claim 26 , wherein the method further comprises repeating steps b-d.
30 . The method of claim 23 , wherein the transfection reagent is a Linear Polyethylenimine Hydrochloride, optionally of MW 25,000 Da or MW 40,000 Da.
31 . The method of claim 23 , wherein the method further comprises the step of removing nucleic acid cargo not contained within the lumen of the extracellular vesicle.
32 . The method of claim 31 , wherein removing nucleic acid cargo not contained within the lumen of the extracellular vesicle comprises contacting the loaded extracellular vesicle with a nuclease, preferably an RNase or DNase.
33 . The method of claim 32 , wherein the loaded extracellular vesicle is contacted with heparin prior to contact with nuclease.
34 . The method of claim 23 , wherein the nucleic acid cargo comprises nucleic acid molecules, wherein each nucleic acid molecule is single stranded and has a length of at least 250 bases, or at least 2000 bases, or 2000-30000 bases or more.
35 . The method of claim 23 , wherein the nucleic acid cargo comprises nucleic acid molecules, wherein each nucleic acid molecule is double stranded and has a length of at least 250 base pairs, or at least 2000 base pairs, or 2000-17000 base pairs or more.
36 . The method of claim 23 , wherein the nucleic acid cargo is circular.
37 . The method of claim 36 , wherein the nucleic acid cargo is a minicircle or plasmid.
38 . The method of claim 23 , wherein the nucleic acid cargo is linear.
39 . The method of claim 23 , wherein the nucleic acid cargo is DNA.
40 . The method of claim 23 , wherein the extracellular vesicle is a microvesicle.
41 . The method of claim 23 , wherein the extracellular vesicle is an exosome.
42 . An extracellular vesicle loaded with a nucleic acid cargo, prepared or obtained by the method of claim 23 .