Compositions for enhancing transport of molecules into cells
Compositions and methods for enhancing delivery of molecules, e.g. biological agents, into cells are described. The composition is a conjugate of the biological agent, preferably a nucleic acid analog having a substantially uncharged backbone, covalently linked to a peptide transporter moiety as described. Conjugation of the peptide transporter to a substantially uncharged nucleic acid analog, such as a morpholino oligomer, is also shown to enhance binding of the oligomer to its target sequence and enhance antisense activity.
1. A peptide-nucleic acid analog conjugate comprising:
i) a nucleic acid analog comprising a substantially uncharged backbone and a targeting base sequence,
ii) a carrier peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-41,
iii) a linker consisting of one or more linker subunits connected by amide bonds, and optionally a piperazine moiety, and
iv) a covalent attachment between the nucleic acid analog and the carrier peptide, the covalent attachment consisting of amide bonds to the linker,
wherein each linker subunit is of the formula —C(O)—(CH 2 ) n —NH—, wherein n is an integer from 2 to 7.
2. The peptide-nucleic acid analog conjugate of claim 1 , wherein the linker comprises a β-alanine subunit, a 6-aminohexanoic acid subunit, or a combination thereof.
3. The peptide-nucleic acid analog conjugate of claim 1 , wherein the nucleic acid analog comprises a morpholino nucleic acid analog, the morpholino nucleic acid analog comprising morpholino subunits linked by phosphorus-containing linkages, the phosphorus-containing linkages linking the morpholino nitrogen of one morpholino subunit to an exocyclic carbon at the morpholino 3-position of an adjacent morpholino subunit.
4. The peptide-nucleic acid analog conjugate of claim 3 , wherein the morpholino subunits are linked by phosphorous-containing linkages in accordance with the structure:
wherein: Y 1 is O; Z is O; X is alkyl, alkoxy, thioalkoxy or alkyl amino; and Pj is a purine or pyrimidine base-pairing moiety effective to bind to a base in a polynucleotide by base-specific hydrogen bonding.
5. The peptide-nucleic acid analog conjugate of claim 4 , wherein X is dimethylamino.
6. The peptide-nucleic acid analog conjugate of claim 1 , wherein the carrier peptide is attached to the 5′ terminus or the 3′ terminus of the nucleic acid analog.
7. The peptide-nucleic acid analog conjugate of claim 6 , wherein the carrier peptide is attached to the 5′ terminus of the nucleic acid analog.
8. The peptide nucleic-acid analog conjugate of claim 7 , wherein the carrier peptide is attached to the 5′ terminus of the nucleic acid analog via a piperazinyl moiety having the following structure:
9. The peptide-nucleic acid analog conjugate of claim 1 , wherein the targeting base sequence of the nucleic acid analog is targeted to an antisense target in a polynucleotide, wherein the antisense target is a splice site in a pre-mRNA, a translation start site in an mRNA or a cis-acting element in a viral genome.
10. The peptide-nucleic acid analog conjugate of claim 1 , wherein the carrier peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-35 and 37-41.
11. The peptide-nucleic acid analog conjugate of claim 1 , wherein the carrier peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-35.