Co-transcriptional assembly of modified RNA nanoparticles

Patent metadata

Field	Value
Publication number	US-9719084-B2
Application number	US-201314426707-A
Country	US
Kind code	B2
Filing date	Sep 6, 2013
Priority date	Sep 7, 2012
Publication date	Aug 1, 2017
Grant date	Aug 1, 2017

Abstract

A method is provided for generating RNA nanoparticles having modified nucleotides and/or having increased nuclease resistance where the RNA nanoparticles are formed cotranscriptionally by T7 RNA polymerase in the presence of manganese ions.

First claim

What is claimed is:

1. A method of producing modified RNA nanoparticles comprising:
preparing a mixture comprising one or more dsDNA templates encoding a plurality of distinct RNA transcripts, wild-type T7 RNA polymerase, a modified nucleotide, and a buffer comprising manganese ions, and
incubating the mixture thereby transcribing the dsDNA templates to form the plurality of distinct RNA transcripts comprising the modified nucleotide, and
co-transcriptionally forming modified RNA nanoparticles by allowing the modified RNA transcripts to assemble without first separately purifying the modified RNA transcripts.

2. The method of claim 1 , wherein modified RNA nanoparticles are nuclease resistant.

3. The method of claim 1 , wherein the manganese ions are at from 0.25 mM to 0.75 mM.

4. The method of claim 1 , wherein the manganese ions are at about 0.5 mM.

5. The method of claim 1 , wherein the modified RNA nanoparticles comprise between 12 and 22 distinct RNA transcripts.

6. The method of claim 1 , wherein the modified nucleotide comprises a modified nucleoside selected from the group consisting of 5-methylcytidine, 5-methyluridine, 2-thiouridine, m 1 A (1-methyladenosine); m 2 A (2-methyladenosine); Am (2′-O-methyladenosine); ms 2 m 6 A (2-methylthio-N 6 -methyladenosine); i 6 A (N 6 -isopentenyladenosine); ms 2 i6A (2-methylthio-N 6 isopentenyladenosine); io 6 A (N 6 -(cis-hydroxyisopentenyl)adenosine); ms 2 i 6 A (2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine); g 6 A (N 6 -glycinylcarbamoyladenosine); t 6 A (N 6 -threonylcarbamoyladenosine); ms 2 t 6 A (2-methylthio-N 6 -threonyl carbamoyladenosine); m 6 t 6 A (N 6 -methyl-N 6 -threonylcarbamoyladenosine); hn 6 A(N 6 -hydroxynorvalylcarbamoyladenosine); ms 2 hn 6 A (2-methylthio-N 6 -hydroxynorvalyl carbamoyladenosine); Ar(p) (2′-O-ribosyladenosine (phosphate)); I (inosine); m 1 I (1-methylinosine); m 1 Im (1,2′-O-dimethylinosine); m 3 C (3-methylcytidine); Cm (2′-O-methylcytidine); s 2 C (2-thiocytidine); ac 4 C(N4-acetylcytidine); f 5 C (5-formylcytidine); m 5 Cm (5,2′-O-dimethylcytidine); ac 4 Cm (N 4 -acetyl-2′-O-methylcytidine); k 2 C (lysidine); m1G (1-methylguanosine); m 2 G (N2-methylguanosine); m 7 G (7-methylguanosine); Gm (2′-O-methylguanosine); m 2 2 G (N 2 ,N 2 -dimethylguanosine); m 2 Gm (N 2 ,2′-O-dimethylguanosine); m 2 2 Gm (N 2 ,N 2 ,2′-O-trimethylguanosine); Gr(p) (2′-O-ribosylguanosine (phosphate)); yW (wybutosine); o 2 yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQ 0 (7-cyano-7-deazaguanosine); preQ 1 (7-aminomethyl-7-deazaguanosine); G + (archaeosine); D (dihydrouridine); m 5 Um (5,2′-O-dimethyluridine); s 4 U (4-thiouridine); m 5 s2U (5-methyl-2-thiouridine); s 2 Um (2-thio-2′-O-methyluridine); acp 3 U (3-(3-amino-3-carboxypropyl)uridine); ho 5 U (5-hydroxyuridine); mo 5 U (5-methoxyuridine); cmo 5 U (uridine 5-oxyacetic acid); mcmo 5 U (uridine 5-oxyacetic acid methyl ester); chm 5 U (5-(carboxyhydroxymethyl)uridine)); mchm 5 U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm 5 U (5-methoxycarbonylmethyluridine); mcm 5 Um (5-methoxycarbonylmethyl-2′-O-methyluridine); mcm 5 s 2 U (5-methoxycarbonylmethyl-2-thiouridine); nm 5 s2U (5-aminomethyl-2-thiouridine); mnm 5 U (5-methylaminomethyluridine); mnm 5 s 2 U (5-methylaminomethyl-2-thiouridine); mnm 5 se2U (5-methylaminomethyl-2-selenouridine); ncm 5 U (5-carbamoylmethyluridine); ncm 5 Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm 5 U (5-carboxymethylaminomethyluridine); cmnm 5 Um (5-carboxymethylaminomethyl-2′-O-methyluridine); cmnm 5 s2U (5-carboxymethylaminomethyl-2-thiouridine); m6 2 A (N 6 ,N 6 -dimethyladenosine); Im (2′-O-methylinosine); m 4 C(N4-methylcytidine); m 4 Cm (N 4 ,2′-O-dimethylcytidine); hm 5 C (5-hydroxymethylcytidine); m 3 U (3-methyluridine); cm 5 U (5-carboxymethyluridine); m 6 Am (N6,2′-O-dimethyladenosine); m 6 2 Am (N 6 ,N 6 ,O-2′-trimethyladenosine); m 2 ,7G (N 2 ,7-dimethylguanosine); m 2 ,2,7G (N 2 ,N2,7-trimethylguanosine); m 3 Um (3,2′-O-dimethyluridine); m 5 D (5-methyldihydrouridine); f 5 Cm (5-formyl-2′-O-methylcytidine); m 1 Gm (1,2′-O-dimethylguanosine); m 1 Am (1,2′-O-dimethyladenosine); τm 5 U (5-taurinomethyluridine); τm 5 s2U (5-taurinomethyl-2-thiouridine)); imG-14 (4-demethylwyosine); imG2 (isowyosine); and ac 6 A (N6-acetyladenosine).

7. The method of claim 1 , wherein the modified nucleotide is selected from the group consisting of 2′-fluoro-dUMP, 2′-fluoro-dCMP, 2′-fluoro-dGMP, and 2′-fluoro-dAMP.

8. The method of claim 1 , wherein the modified nucleotide is 2′-fluoro-dUTP.

9. The method of claim 2 , wherein the modified RNA nanoparticles having nuclease resistance have increased serum half-life compared to a corresponding RNA nanoparticles formed from wild-type RNA.

10. The method of claim 1 , further comprising RNAse H treatment.

Assignees

• The Us Secretary Dept Of Health & Human Services
• Univ California

Inventors

• Shapiro Bruce A
• Afonin Kirill A
• Kireeva Maria L
• Kashlev Mikhail
• Jaeger Luc
• Grabow Wade W

Classifications

• C12N15/111 Primary

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C12N2310/52

  branched · CPC title

• C12N2310/3519

  Fusion with another nucleic acid · CPC title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• C12N2320/32

  Special delivery means, e.g. tissue-specific · CPC title