TRANSDUCIBLE DELIVERY OF NUCLEIC ACIDS BY REVERSIBLE PHOSPHOTRIESTER CHARGE NEUTRALIZATION PROTECTING GROUPS

• US 20090093425A1
• Filed: 07/11/2007
• Published: 04/09/2009
• Est. Priority Date: 07/12/2006
• Status: Abandoned Application

• Current Assignee: Howard Hughes Medical Institute, Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Meade, Bryan R., Petersen, Scott G., Dowdy, Steven F.
• Application Number:
  US11/776,317
• Publication Number:
  US 20090093425A1
• Time in Patent Office: Days
• US Class Current: 514/44.00R
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12N 2330/30 chemically synthesised

Abstract

This disclosure relates to nucleic acid constructs modified to have a reduced net anionic charge. In some aspects the constructs comprise phosphodiester and/or phosphothioate protecting groups. The disclosure also provide methods of making and using such constructs.

Claims

1. A nucleic acid construct comprising:

a) an oligonucleotide or polynucleotide domain comprising a phosphodiester and/or phosphothioate protecting group at one or more positions that reduces the net anionic charge of the oligonucleotide or polynucleotide backbone; and

b) at least one transduction domain comprising a membrane transport function operably linked to the oligonucleotide or polynucleotide domain.

2. The nucleic acid construct of claim 1, wherein the at least one transduction domain is a cationically charged domain.

3. The nucleic acid construct of claim 1, wherein the at least one transduction domain is a protein transduction domain (PTD).

4. The nucleic acid construct of claim 1, wherein overall charge of the nucleic acid construct is neutral or positively charged relative to a non-protected oligonucleotide or polynucleotide.

5. The nucleic acid construct of claim 1, wherein the phosphodiester and/or phosphothioate protecting group has the general formula:

R—

X—

or Q-X—

wherein X is O, S or NR¹, and R¹ is H, methyl, ethyl, S-pivaloyl thioethanol, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, or substituted heterocyclic;

wherein R is selected from the group consisting of;

R²,

wherein R² is alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, or substituted heterocyclic,

wherein R³ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, halo, cyano, or nitro,

wherein A¹ and A² are each independently one to seven atom chains, or substituted one to seven atom chains,

wherein R⁴ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, halo, cyano, or nitro,

wherein A³ and A⁴ are each independently one to seven atom chains, or substituted one to seven atom chains,

wherein R⁵ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, halo, cyano, or nitro,

wherein A⁵ is a one to seven atom chain, or substituted one to seven atom chain,

wherein X¹ and X² are each independently O, S or NR⁷, and R⁷ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, or substituted heterocyclic, and

wherein R⁶ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, halo, cyano, or nitro,

wherein A⁶ and A⁶ are each independently one to seven atom chains, or substituted one to seven atom chains,

wherein X³ and X⁴ are each independently O, S or NR⁸, and R⁸ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, or substituted heterocyclic;

wherein Q is selected from the group consisting of;

Q¹,

wherein Q¹ is a basic group with a pKa greater than or equal to 10,

wherein Q² is a basic group with a pKa greater than or equal to 10,

wherein A⁸ and A⁹ are each independently one to seven atom chains, or substituted one to seven atom chains,

wherein Q³ is a basic group with a pKa greater than or equal to 10,

wherein A¹⁰ and A¹¹ are each independently one to seven atom chains, or substituted one to seven atom chains,

wherein Q⁴ is a basic group with a pKa greater than or equal to 10,

wherein A¹² is a one to seven atom chain, or substituted one to seven atom chain,

wherein X⁵ and X⁶ are each independently O, S or NR⁹, and R⁹ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, or substituted heterocyclic, and

wherein Q⁵ is a basic group with a pKa greater than or equal to 10,

wherein A¹³ and A¹⁴ are each independently one to seven atom chains, or substituted one to seven atom chains,

wherein X⁷ and X⁸ are each independently O, S or NR¹⁰, and R¹⁰ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, or substituted heterocyclic.

6. The nucleic acid construct of claim 1, wherein the phosphodiester and/or phosphothioate protecting group is selected from the group consisting of MeO—

, EtO—

, iPrO,

7. The nucleic acid construct of claim 5, wherein the phosphodiester and/or phosphothioate protecting group comprises a structure selected from the group consisting of:

8. The nucleic acid construct of claim 5, further comprising a basic substitution, guanidinium, amine, or urea group attached to the phosphodiester and/or phosphothioate protecting group via a linker to form an RNB protecting group.

9. The nucleic acid construct of claim 8, wherein the RNB protecting group comprises a structure selected from the group consisting of:

10. The nucleic acid construct of claim 3, wherein the at least one protein transduction domain is selected from the group consisting of a polypeptide comprising a herpesviral VP22 domain;

a polypeptide comprising a human immunodeficiency virus (HIV) TAT domain;

a polypeptide comprising a homeodomain of an Antennapedia protein (Antp HD) domain;

an N-terminal cationic prion protein domain;

poly-Arg; and

functional fragments thereof.

11. The nucleic acid construct of claim 1, wherein the oligonucleotide or polynucleotide domain comprises dsRNA or siRNA.

12. A pharmaceutical composition comprising the nucleic acid construct of claim 1 and a pharmaceutically acceptable carrier.

29. A method of transfecting a cell, comprising:

contacting the cell with a nucleic acid construct of claim 1 or 13.

30. The method of claim 29, wherein the contacting is in vivo.

31. The method of claim 30, wherein the nucleic acid construct comprises an antisense molecule.

32. A method of treating a disease or disorder comprising administering a nucleic acid construct of claim 1 or 13 to a subject, wherein the oligonucleotide or polynucleotide comprises a therapeutic or diagnostic molecule.

35. The nucleic acid construct of claim 10, wherein the at least one transduction domain comprises two or more transduction domains.

36. The nucleic acid of claim 1 or 35, wherein the at least one transduction domain is reversibly or irreversibly conjugated to the 5′

, 3′

, or 5′ and

3′

end of the oligonucleotide or polynucleotide domain.

13. A nucleic acid construct comprising:

an oligonucleotide or polynucleotide comprising a phosphodiester and/or phosphothioate protecting group at one or more positions that reduces the net anionic charge of the oligonucleotide or polynucleotide backbone or provides a net cationic charge of the oligonucleotide or polynucleotide backbone.

14. The nucleic acid construct of claim 13, wherein the phosphodiester and/or phosphothioate protecting group has the general formula:

R—

X—

or Q-X—

wherein X is O, S or NR¹, and R¹ is H, methyl, ethyl, S-pivaloyl thioethanol, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, or substituted heterocyclic;

wherein R is selected from the group consisting of;

R²,

wherein R² is alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, or substituted heterocyclic,

wherein R³ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, halo, cyano, or nitro,

wherein A¹ and A² are each independently one to seven atom chains, or substituted one to seven atom chains,

wherein R⁴ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, halo, cyano, or nitro,

wherein A³ and A⁴ are each independently one to seven atom chains, or substituted one to seven atom chains,

wherein R⁵ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, halo, cyano, or nitro,

wherein A⁵ is a one to seven atom chain, or substituted one to seven atom chain,

wherein X¹ and X² are each independently O, S or NR⁷, and R⁷ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, or substituted heterocyclic, and

wherein R⁶ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, halo, cyano, or nitro,

wherein A⁶ and A⁶ are each independently one to seven atom chains, or substituted one to seven atom chains,

wherein X³ and X⁴ are each independently O, S or NR⁸, and R⁸ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, or substituted heterocyclic;

wherein Q is selected from the group consisting of;

Q¹,

wherein Q¹ is a basic group with a pKa greater than or equal to 10,

wherein Q² is a basic group with a pKa greater than or equal to 10,

wherein A⁸ and A⁹ are each independently one to seven atom chains, or substituted one to seven atom chains,

wherein Q³ is a basic group with a pKa greater than or equal to 10,

wherein A¹⁰ and A¹¹ are each independently one to seven atom chains, or substituted one to seven atom chains,

wherein Q⁴ is a basic group with a pKa greater than or equal to 10,

wherein A¹² is a one to seven atom chain, or substituted one to seven atom chain,

wherein X⁵ and X⁶ are each independently O, S or NR⁹, and R⁹ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, or substituted heterocyclic, and

wherein Q⁵ is a basic group with a pKa greater than or equal to 10,

wherein A¹³ and A¹⁴ are each independently one to seven atom chains, or substituted one to seven atom chains,

wherein X⁷ and X⁸ are each independently O, S or NR¹⁰, and R¹⁰ is H, hydroxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heterocyclic, or substituted heterocyclic.

15. The nucleic acid construct of claim 13, wherein the phosphodiester and/or phosphothioate protecting group is selected from the group consisting of MeO—

, EtO—

, iPrO,

16. The nucleic acid construct of claim 14, wherein the phosphodiester and/or phosphothioate protecting group comprises a structure selected from the group consisting of:

17. The nucleic acid construct of claim 14, comprising a basic substitution, guanidinium, amine, or urea group attached to the phosphodiester and/or phosphothioate protecting group via a linker to form an RNB protecting group.

18. The nucleic acid construct of claim 17, wherein the RNB protecting group comprises a structure selected from the group consisting of:

19. The nucleic acid construct of claim 13, further comprising a targeting domain operably linked to the oligonucleotide or polynucleotide.

20. The nucleic acid construct of claim 19, wherein the targeting moiety is an antibody that binds to a cell surface protein.

21. The nucleic acid construct of claim 19, wherein the targeting domain comprises a receptor ligand for a cell surface receptor.

22. The nucleic acid construct of claim 13, further comprising at least one cationically charged polypeptide or peptide.

23. The nucleic acid construct of claim 22, wherein the cationically charged polypeptide or peptide comprises a protein transduction domain.

24. A pharmaceutical composition comprising the nucleic acid construct of claim 13, and a pharmaceutically acceptable carrier.

25. A method comprising:

linking one or more protein transduction domain to a nucleic acid construct of claim 13.

26. The method of claim 25, wherein the one or more protein transduction domains comprise 2-5 protein transduction domains.

27. The method of claim 26, wherein the one or more protein transduction domains comprise three protein transduction domains.

33. The nucleic acid construct of claim 5 or 14, wherein R¹ is a methyl, ethyl or S-pivaloyl thioethanol.

34. The nucleic acid construct of claim 33, wherein the phosphotriester and/or phosphothioate protecting group present on a nucleotide comprising a 2′

-W, wherein W is H, F, O-Me, or O-Alkyl.

28. A method of generating a nucleic acid construct comprising:

substantially purifying a protein transduction domain;

synthesizing an oligonucleotide;

charge neutralizing the anionic charge on the oligonucleotide with a phosphotriester group; and

linking the oligonucleotide to one or more protein transduction domains.