Targeting RNAs to microvesicles
Disclosed herein is an isolated nucleic acid molecule comprising a first nucleic acid sequence 5′-ACCCTGCCGCCTGGACTCCGCCTGT-3′ (SEQ ID NO: 22), or a functional variant thereof, operably linked to a second, heterologous nucleic acid sequence. The isolated nucleic acid molecule can be DNA (in an expression vector) and RNA (mRNA, shRNA, orncRNA). Also disclosed is a microvesicle comprising the nucleic acid molecule and a microvesicle preparation comprising the microvesicle. Also disclosed is an in vitro method of producing a microvesicle preparation enriched for a specific RNA sequence by transfecting cells with the nucleic acid sequence, and isolating microvesicles generated therefrom. Methods of delivering therapeutic RNA to a subject are also disclosed.
1. An in vitro method of producing a microvesicle preparation enriched for a specific desired RNA sequence, comprising:
a) transfecting cells in vitro with a DNA molecule in expressible form, comprising two or more copies of a first nucleic acid sequence 5′-ACCCTGCCGCCTGGACTCCGCCTGT-3′ (SEQ ID NO: 22), operably linked to a second, heterologous nucleic acid sequence encoding a specific desired RNA sequence, under conditions suitable for expression of an RNA transcript; and
b) isolating microvesicles generated by the transfected cells of step a);
to thereby produce a microvesicle preparation enriched for the specific desired RNA sequence.
2. The method of claim 1 , wherein the DNA molecule is in the context of an expression vector.
3. The method of claim 1 , wherein the first nucleic acid sequence is located 3′ of the second nucleic acid sequence.
4. The method of claim 1 , wherein the first nucleic acid sequence is located 5′ of a poly adenylation site.
5. The method of claim 1 , further comprising transfecting the cells with a construct capable of expressing a pre-miR-1289 RNA under conditions suitable for expression.
6. The method of claim 1 , wherein the specific RNA sequence is selected from the group consisting of a mRNA, a shRNA, and a regulatory ncRNA.
7. The method of claim 1 , wherein transfection is by lipofection.
8. The method of claim 1 , wherein the cells are primary cells or a continuous cell line.
9. The method of claim 8 , wherein the cells are dendritic cells.
10. An in vitro method of producing a microvesicle preparation enriched for a specific RNA sequence, comprising:
a) transfecting cells in vitro with:
(i) a DNA molecule in expressible form, comprising a first nucleic acid sequence 5′-ACCCTGCCGCCTGGACTCCGCCTGT-3′ (SEQ ID NO: 22), operably linked to a second, heterologous nucleic acid sequence encoding the specific RNA sequence; and
(ii) a construct capable of expressing a pre-miR-1289 RNA;
under conditions suitable for expression; and
b) isolating microvesicles generated by the transfected cells of step a);
to thereby produce a microvesicle preparation enriched for the specific RNA sequence.
11. The method of claim 1 , wherein the first nucleic acid sequence further comprises one or more copies of 5′-ACCCTGCCGCCTGGATCAAGCCTGT-3′ (SEQ ID NO: 38).
12. An in vitro method of producing a microvesicle preparation enriched for a specific desired RNA sequence, comprising:
a) transfecting cells in vitro with a DNA molecule in expressible form, comprising a first nucleic acid sequence comprising 5′-ACCCTGCCGCCTGGACTCCGCCTGT-3′ (SEQ ID NO: 22) and 5′-ACCCTGCCGCCTGGATCAAGCCTGT-3′ (SEQ ID NO: 38), operably linked to a second, heterologous nucleic acid sequence encoding the specific desired RNA sequence, under conditions suitable for expression of an RNA transcript; and
b) isolating microvesicles generated by the transfected cells of step a);
to thereby produce a microvesicle preparation enriched for the specific desired RNA sequence.