Compositions for enhancing transport of molecules into cells
First Claim
1. A method for enhancing the ability of an nucleic acid analog having a substantially uncharged backbone and a targeting base sequence to bind to a target sequence in a nucleic acid, the method comprising conjugating to the nucleic acid analog a peptide consisting of 8 to 16 subunits selected from X subunits, Y subunits, and optional Z subunits, including at least six X subunits, at least two Y subunits, and at most three Z subunits, where >
- 50% of said subunits are X subunits, and where (a) each X subunit independently represents arginine or an arginine analog, said analog being a cationic α
-amino acid comprising a side chain of the structure R1 N═
C(NH2)R2, where R1 is H or R;
R2 is R, NH2, NHR, or NR2, where R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen;
R1 and R2 may together form a ring; and
the side chain is linked to said amino acid via R1 or R2;
(b) each Y subunit independently represents a neutral amino acid —
C(O)—
(CHR)n—
NH—
, where (i) n is 2 to 7 and each R is independently H or methyl, or (ii) n is 1 and R is a neutral side chain selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, and aralkyl, wherein said neutral side chain, when selected from substituted alkyl, alkenyl, and alkynyl, includes at most one heteroatom for every four carbon atoms; and
(c) each Z subunit independently represents an amino acid selected from alanine, asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine, serine, and threonine.
2 Assignments
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Accused Products
Abstract
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.
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Citations
68 Claims
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1. A method for enhancing the ability of an nucleic acid analog having a substantially uncharged backbone and a targeting base sequence to bind to a target sequence in a nucleic acid, the method comprising
conjugating to the nucleic acid analog a peptide consisting of 8 to 16 subunits selected from X subunits, Y subunits, and optional Z subunits, including at least six X subunits, at least two Y subunits, and at most three Z subunits, where > - 50% of said subunits are X subunits, and where
(a) each X subunit independently represents arginine or an arginine analog, said analog being a cationic α
-amino acid comprising a side chain of the structure R1 N═
C(NH2)R2, where R1 is H or R;
R2 is R, NH2, NHR, or NR2, where R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen;
R1 and R2 may together form a ring; and
the side chain is linked to said amino acid via R1 or R2;
(b) each Y subunit independently represents a neutral amino acid —
C(O)—
(CHR)n—
NH—
, where (i) n is 2 to 7 and each R is independently H or methyl, or (ii) n is 1 and R is a neutral side chain selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, and aralkyl, wherein said neutral side chain, when selected from substituted alkyl, alkenyl, and alkynyl, includes at most one heteroatom for every four carbon atoms; and
(c) each Z subunit independently represents an amino acid selected from alanine, asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine, serine, and threonine. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 65, 66)
- 50% of said subunits are X subunits, and where
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2. The method of claim 2, wherein said peptide, when conjugated to an antisense oligomer having said substantially uncharged backbone, is effective to enhance the binding of the antisense oligomer to its target sequence, relative to the antisense oligomer in unconjugated form, as evidenced by:
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(i) a decrease in expression of an encoded protein, when binding of the antisense oligomer to its target sequence is effective to block a translation start codon for the encoded protein, or (ii) an increase in expression of an encoded protein, when binding of the antisense oligomer to its target sequence is effective to block an aberrant splice site in a pre-mRNA which encodes said protein when correctly spliced.
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22. A peptide-nucleic acid analog conjugate, comprising
a nucleic acid analog having a substantially uncharged backbone and a targeting base sequence, and covalently linked to the nucleic acid analog, a peptide consisting of 8 to 16 subunits selected from X subunits, Y subunits, and optional Z subunits, including at least eight X subunits, at least two Y subunits, and at most three Z subunits, wherein > - 50% of said subunits are X subunits, and where
(a) each X subunit independently represents arginine or an arginine analog, said analog being a cationic α
-amino acid subunit comprising a side chain of the structure R1N═
C(NH2)R2, where R1 is H or R;
R2 is R, NH2, NHR, or NR2, where R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen;
R1 and R2 may together form a ring; and
the side chain is linked to said amino acid subunit via R1 or R2;
(b) said at least two Y subunits include (i) two neutral α
-amino acid subunits having side chains independently selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, and aralkyl, wherein said side chain, when selected from substituted alkyl, alkenyl, and alkynyl, includes at most one heteroatom for every four carbon atoms, and wherein said subunits are contiguous or are flanking a linker moiety, or(ii) two neutral, hydrophobic amino acid subunits —
C(O)—
(CH2)n-1(CHR)—
NH—
, where n is 2 to 7 and R is H or methyl; and
(c) Z represents an amino acid subunit selected from alanine, asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine, serine, and threonine. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)
- 50% of said subunits are X subunits, and where
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45. A method for enhancing cell uptake of a pharmacological agent, the method comprising
conjugating to the agent a peptide consisting of 8 to 16 subunits selected from X subunits, Y subunits, and optional Z subunits, including at least six X subunits, at least two Y subunits, and at most three Z subunits, wherein > - 50% of said subunits are X subunits, and where
(a) each X subunit independently represents arginine or an arginine analog, said analog being a cationic α
-amino acid comprising a side chain of the structure R1N═
C(NH2)R2, where R1 is H or R;
R2 is R, NH2, NHR, or NR2, where R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen;
R1 and R2 may together form a ring; and
the side chain is linked to said amino acid via R1 or R2;
(b) each Y subunit independently represents a neutral amino acid —
C(O)—
(CHR)—
NH—
, where R is a neutral side chain selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, and aralkyl, wherein said neutral side chain, when selected from substituted alkyl, alkenyl, and alkynyl, includes at most one heteroatom for every four carbon atoms; and
(c) each Z subunit independently represents an amino acid selected from alanine, asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine, serine, and threonine. - View Dependent Claims (46, 47, 48, 49, 50, 51, 52)
- 50% of said subunits are X subunits, and where
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53. A conjugate comprising a pharmacological agent covalently linked to a peptide, wherein the peptide consists of 8 to 16 subunits selected from X subunits, Y subunits, and optional Z subunits, including at least six X subunits, at least two Y subunits, and at most three Z subunits, wherein >
- 50% of said subunits are X subunits, and where
(a) each X subunit independently represents arginine or an arginine analog, said analog being a cationic α
-amino acid comprising a side chain of the structure R1 N═
C(NH2)R2, where R1 is H or R;
R2 is R, NH2, NHR, or NR2, where R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen;
R1 and R2 may together form a ring; and
the side chain is linked to said amino acid via R1 or R2;
(b) each Y subunit independently represents a neutral amino acid —
C(O)—
(CHR)—
NH—
, where R is a neutral side chain selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, and aralkyl, wherein said neutral side chain, when selected from substituted alkyl, alkenyl, and alkynyl, includes at most one heteroatom for every *four carbon atoms; and
(c) each Z subunit independently represents an amino acid selected from alanine, asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine, serine, and threonine. - View Dependent Claims (54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64)
- 50% of said subunits are X subunits, and where
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67. An antisense composition having
(i) a plurality of subunits connected by intersubunit linkages, and supporting a sequence of bases effective to hybridize to a complementary-sequence target polynucleotide, to form a target/antisense duplex; - and
(ii) carried on at least six contiguous intersubunit linkages, a charged moiety of the structure R1N═
C(NH2)R2, where R1 is H or R;
R2 is R, NH2, NHR, or NR2, where R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen;
R1 and R2 may together form a ring; and
the side chain moiety is linked to said amino acid subunit via R1 or R2. - View Dependent Claims (68)
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Specification