United States Patent:  6,086,900

Assignee:  Board of Regents, The University of Texas Systems (Austin, TX)

Filed:  March 24, 1998

The present invention provides methods and compositions delivery of agents into the cytoplasm of cells. Particularly, it concerns the use of membrane-penetrating toxin proteins to deliver drugs to the cytoplasm of target cells.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention, to provide compositions and methods for the transfers of various molecules across biological membranes. A variety of different uses for these compositions and methods are contemplated, as described further below.

In one embodiment, there is provided a method of providing a molecule to a cell comprising (i) conjugating said molecule to a detoxified exotoxin A (ETA) at a non-terminal site; and (ii) contacting the conjugate with said cell, whereby said conjugate is delivered to the cytoplasm of said cell. The molecule may be a nucleic acid, a peptide, a peptide-nucleic acid, an antibody, a single-chain antibody or a pharmaceutical. The antibody or single-chain antibody may be ones that have catalytic function. The nucleic acid may be DNA o RNA. Where a DNA is involved, it may be placed under the control of a eukaryotic promoter. The DNA may encode a nucleic acid binding protein, a single-chain antibody, a tumor suppressor, a cytokine, an oncogene, a hormone or a toxin. The promoter may be a CMV IE, .beta.-actin, E1A, TET or ecdysone. The DNA may encode an antisense construct, for example, an antisense construct that targets an oncogene or a viral protein. The polypeptide may be an enzyme, an antibody or a nucleic acid binding protein.

Various methods of conjugation are contemplated, for example, by a covalent bond or non-covalent. The bond may be reducible. The bond may be a carbon-sulfur bond, carbon-carbon bond, carbon-oxygen bond or a carbon-nitrogen bond. The sulfur residue of said carbon-sulfur bond may be a component of said detoxified ETA.

The detoxified ETA may be produced recombinantly. The detoxified ETA may contain a sulfur residue not found in the natural toxin. The detoxified ETA may contain a deletion, insertion or substitution in domain III. The detoxified ETA may contain a deletion of the glutamate residue at position 553 of the natural toxin.

Provision of the conjugate to a cell may be performed in vitro or in vivo. Exemplary cells include a CHO cell, a CV-1 cell, a Vero cell, an embryonic stem cell, a HeLa cell, a smooth muscle cell, a fibroblast, a tumor cell, a B-lymphocyte or a T-lymphocyte.

In another embodiment, there is provided a conjugate comprising (i) a detoxified ETA; and (ii) another molecule conjugated to said detoxified ETA at a non-terminal site. The molecule may be a nucleic acid, a peptide, a peptide-nucleic acid, an antibody, a single-chain antibody or a pharmaceutical. The antibody or single-chain antibody may be ones that have catalytic function. The nucleic acid may be DNA o RNA. Where a DNA is involved, it may be placed under the control of a eukaryotic promoter. The DNA may encode a nucleic acid binding protein, a single-chain antibody, a tumor suppressor, a cytokine, an oncogene, a hormone or a toxin. The promoter may be a CMV IE, .beta.-actin, E1A, TET or ecdysone. The DNA may encode an antisense construct, for example, an antisense construct that targets an oncogene or a viral protein. The polypeptide may be an enzyme, an antibody or a nucleic acid binding protein.

In yet another embodiment, there is provided a pharmaceutical composition comprising (i) an ETA conjugate comprising (a) a detoxified ETA, (b) another molecule conjugated to said detoxified ETA at a non-terminal site; and (ii) a pharmaceutically acceptable buffer diluent or excipient. The molecule of the pharmaceutical composition may be a nucleic acid, a peptide, a peptide-nucleic acid, an antibody, a single-chain antibody or a pharmaceutical.

Claim 1 of 62 Claims

1. A method of providing a molecule to a cell comprising:

(i) conjugating said molecule to a detoxified exotoxin A (ETA) in domain III of ETA, wherein said detoxified ETA also contains a deletion, insertion or substitution in domain III of ETA; and

(ii) contacting the conjugate with said cell,

whereby said conjugate is delivered to the cytoplasm of said cell.

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