US5804604A
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Tat-derived transport polypeptides and fusion proteins
Assignee: Biogen MA Inc
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Filing: 1995-05-25
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Granted: 1998-09-08
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Expires: 2015-05-25
Abstract
Abstract This invention relates to delivery of biologically active cargo molecules, such as polypeptides and nucleic acids, into the cytoplasm and nuclei of cells in vitro and in vivo. Intracellular delivery of cargo molecules according to this invention is accomplished by the use of novel transport polypeptides which comprise HIV tat protein or one or more portions thereof, and which are covalently attached to cargo molecules. The transport polypeptides in preferred embodiments of this invention are characterized by the presence of the tat basic region (amino acids 49-57), the absence of the tat cysteine-rich region (amino acids 22-36) and the absence of the tat exon 2-encoded carboxy-terminal domain (amino acids 73-86) of the naturally-occurring tat protein. By virtue of the absence of the cysteine-rich region, the preferred transport polypeptides of this invention solve the potential problems of spurious trans-activation and disulfide aggregation. The reduced size of the preferred transport polypeptides of this invention also minimizes interference with the biological activity of the cargo molecule.
Inventors
- 👤 Alan Frankel
- 👤 Carl Pabo
- 👤 James G. Barsoum
- 👤 Stephen E. Fawell
- 👤 R. Blake Pepinsky
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 06:19
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/005; A; A61; A61K; A61K47/00; A61K47/50; A61K47/51; A61K47/62; C12; C12N; C12N15/00; C12N15/09; C12N15/87; C07K2319/00; C07K2319/01; C07K2319/10; C07K2319/55; C07K2319/70; C07K2319/71; C07K2319/80; C12N2740/00; C12N2740/00011; C12N2740/10011; C12N2740/16011; C12N2740/16311; C12N2740/16322
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
61 words
An agent for delivery of a cargo moiety consisting of a cargo moiety in combination with a transport moiety, wherein the transport moiety has the following characteristics: (i) the presence of amino acids 49-57 of HIV tat protein; (ii) the absence of amino acids 22-36 of HIV tat protein; and (iii) the absence of amino acids 73-86 of HIV tat protein.
Claim 2
Dependent Claim
61 words
An agent for delivery of a cargo moiety consisting of a cargo moiety in combination with a transport moiety, wherein the transport moiety has the following characteristics: (i) the presence of amino acids 49-57 of HIV tat protein; (ii) the absence of amino acids 22-36 of HIV tat protein; and (iii) the absence of amino acids 73-86 of HIV tat protein.
Claim 3
Dependent Claim
17 words
The agent of claim 1, wherein the cargo moiety retains biological activity following transport moiety-dependent intracellular delivery.
Claim 4
Dependent Claim
29 words
The agent of claim 2, wherein the agent is a fusion protein having a carboxy-terminal that is the cargo moiety and having an amino-terminal that is the transport moiety.
Claim 5
Dependent Claim
23 words
The agent of claim 3, wherein the cargo moiety consists of amino acids 245-365 of human papillomavirus E2 protein (SEQ ID NO: 51).
Claim 6
Dependent Claim
24 words
The agent of claim 3, wherein the carboxy-terminal cargo moiety is selected from the group consisting of therapeutic molecules, prophylactic molecules and diagnostic molecules.
Claim 7
Dependent Claim
27 words
The agent of claim 3, wherein the cargo moiety is an E2 repressor consisting of amino acids 250-410 of bovine papillomavirus E2 protein (SEQ ID NO: 56).
Claim 8
Dependent Claim
54 words
The agent of claim 3, wherein the cargo moiety is selected from the group consisting of: (a) amino acids 245-365 of human papillomavirus E2 protein (SEQ ID NO: 51); and (b) amino acids 245-365 of human papillomavirus E2 protein, wherein amino acids 300 and 309 have been changed to serine (SEQ ID NO: 55).
Claim 9
Dependent Claim
12 words
The agent of claim 3, wherein the cargo moiety is an antigen.
Claim 10
Dependent Claim
144 words
A fusion protein comprising a cargo moiety linked to a target moiety, wherein the cargo moiety is a human papillomavirus E2 repressor that retains its biological activity after delivery into a target cell and wherein the transport moiety is selected from the group consisting of: (a) amino acids 47-58 of HIV tat protein (SEQ ID NO: 47); (b) amino acids 47-72 of HIV tat protein (SEQ ID NO: 48); (c) amino acids 38-72 of HIV tat protein (SEQ ID NO: 49); (d) amino acids 38-58 of HIV tat protein (SEQ ID NO: 50); (e) amino acids 37-58 of HIV tat protein (SEQ ID NO: 3); (f) amino acids 1-21 and 38-72 of HIV tat protein (SEQ ID NO: 7); (g) amino acids 47-62 of HIV tat protein (SEQ ID NO: 52); and (h) amino acids 38-62 of HIV tat protein (SEQ ID NO: 53).
Claim 11
Dependent Claim
144 words
A fusion protein comprising a cargo moiety linked to a target moiety, wherein the cargo moiety is a human papillomavirus E2 repressor that retains its biological activity after delivery into a target cell and wherein the transport moiety is selected from the group consisting of: (a) amino acids 47-58 of HIV tat protein (SEQ ID NO: 47); (b) amino acids 47-72 of HIV tat protein (SEQ ID NO: 48); (c) amino acids 38-72 of HIV tat protein (SEQ ID NO: 49); (d) amino acids 38-58 of HIV tat protein (SEQ ID NO: 50); (e) amino acids 37-58 of HIV tat protein (SEQ ID NO: 3); (f) amino acids 1-21 and 38-72 of HIV tat protein (SEQ ID NO: 7); (g) amino acids 47-62 of HIV tat protein (SEQ ID NO: 52); and (h) amino acids 38-62 of HIV tat protein (SEQ ID NO: 53).
Claim 12
Dependent Claim
16 words
The fusion protein of claim 9, wherein the transport moiety is preceded by an amino-terminal methionine.
Claim 13
Dependent Claim
51 words
An agent for delivery of a cargo moiety, the agent comprising an HIV tat protein characterized by: (i) the presence of amino acids 49-57 of HIV tat protein; (ii) the absence of amino acids 22-36 of HIV tat protein; and (iii) the absence of amino acids 73-86 of HIV tat protein.
Claim 14
Dependent Claim
51 words
An agent for delivery of a cargo moiety, the agent comprising an HIV tat protein characterized by: (i) the presence of amino acids 49-57 of HIV tat protein; (ii) the absence of amino acids 22-36 of HIV tat protein; and (iii) the absence of amino acids 73-86 of HIV tat protein.
Claim 15
Dependent Claim
72 words
An agent for delivery of a cargo moiety, the agent comprising an HIV tat protein selected from the group consisting of: (a) amino acids 47-72 of HIV tat protein (SEQ ID NO: 48); (b) amino acids 38-72 of HIV tat protein (SEQ ID NO: 49); (c) amino acids 37-58 of HIV tat protein (SEQ ID NO: 3); and (d) amino acids 1-21 and 38-72 of HIV tat protein (SEQ ID NO: 7).
Claim 16
Dependent Claim
72 words
An agent for delivery of a cargo moiety, the agent comprising an HIV tat protein selected from the group consisting of: (a) amino acids 47-72 of HIV tat protein (SEQ ID NO: 48); (b) amino acids 38-72 of HIV tat protein (SEQ ID NO: 49); (c) amino acids 37-58 of HIV tat protein (SEQ ID NO: 3); and (d) amino acids 1-21 and 38-72 of HIV tat protein (SEQ ID NO: 7).
Claim 17
Dependent Claim
45 words
A composition of matter comprising an HIV tat protein characterized by: (i) the presence of amino acids 49-57 of HIV tat protein; (ii) the absence of amino acids 22-36 of HIV tat protein; and (iii) the absence of amino acids 73-86 of HIV tat protein.
Claim 18
Dependent Claim
45 words
A composition of matter comprising an HIV tat protein characterized by: (i) the presence of amino acids 49-57 of HIV tat protein; (ii) the absence of amino acids 22-36 of HIV tat protein; and (iii) the absence of amino acids 73-86 of HIV tat protein.
Claim 19
Dependent Claim
66 words
A composition of matter comprising an HIV tat protein selected from the group consisting of: (a) amino acids 47-72 of HIV tat protein (SEQ ID NO: 48); (b) amino acids 38-72 of HIV tat protein (SEQ ID NO: 49); (c) amino acids 37-58 of HIV tat protein (SEQ ID NO: 3); and (d) amino acids 1-21 and 38-72 of HIV tat protein (SEQ ID NO: 7).
Claim 20
Dependent Claim
66 words
A composition of matter comprising an HIV tat protein selected from the group consisting of: (a) amino acids 47-72 of HIV tat protein (SEQ ID NO: 48); (b) amino acids 38-72 of HIV tat protein (SEQ ID NO: 49); (c) amino acids 37-58 of HIV tat protein (SEQ ID NO: 3); and (d) amino acids 1-21 and 38-72 of HIV tat protein (SEQ ID NO: 7).
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