US 7,173,154 B2Grant
Transfection reagents
Issue Date:2007-02-06
•22 Claims
•4 Drawing Sheets
Abstract
Disclosed are compounds capable of facilitating transport of biologically active agents or substances into cells having the general structure:
wherein
-
- Q is selected from the group consisting of N, O and S; L is any bivalent organic radical capable of linking each Q, such as C, CH, (CH2)l, or {(CH2)i-Y—(CH2)j}k, wherein Y is selected from the group consisting of CH2, an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide, an imine, a carbonyl, and a secondary amino group and wherein Y is optionally substituted by —X1-L′-X2-Z or -Z; R1–R6, independently of one another, are selected from the group consisting of H, —(CH2)p-D-Z, an alkyl, an alkenyl, an aryl, and an alkyl or alkyl ether optionally substituted by one or more of an alcohol, an aminoalcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, a guanidyl, or a carbamoyl group, and wherein at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group; and anyone of R1, R3, R4 and/or R6 may optionally be covalently linked with each other, with Y or with L when L is C or CH to form a cyclic moiety; Z is selected from the group consisting of amine, spermiyl, carboxyspermiyl, guanidyl, spermidinyl, putricinyl, diaminoalkyl, pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, peptide, and protein; X1 and X2, independently of one another, are selected from the group consisting of NH, O, S, alkylene, and arylene; L′ is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, alkylene ether, and polyether; D is Q or a bond; A1 and A2, independently of one another, are selected from the group consisting of CH2O, CH2S, CH2NH, C(O), C{NH), C(S) and (CH2)t; X is a physiologically acceptable anion; m, n, r, s, u, v, w and y are 0 or 1, with the proviso that when both m and n are 0 at least one of r, s, u and y is other than 0; i, j, k, l, p and are integers from 0 to about 100; q is an integer from 1 to about 1000; and a is the number of positive charge divided by the valence of the anion.
Metadata
Assignee
- Invitrogen Corp.
Inventors
- Yongliang Chu
- Malek Masoud
- Gulilat Gebeyehu
Application Information
Application Number:US 10/629,522
Filing Date:2003-07-28
Priority Date:1998-11-12
Art Unit:1621
Classifications
IPC:
C07C215/14A61K31/13C12N15/63C12N15/86C12N15/87C12N15/85C12N15/88
Patent Drawings (4 sheets)
Description
[0001] This application is a continuation of co-pending U.S. patent application Ser. No. 09/438,365, filed Nov. 12, 1999, which claims priority from U.S. Provisional Patent Application No. 60/108,117, filed Nov. 12, 1998. To the extent that they are consistent herewith, the aforementioned applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to cationic lipids and compositions of cationic lipids having utility in lipid aggregates for delivery of macromolecules and other compounds into cells.
[0004] 2. Related Art
[0005] Lipid aggregates such as liposomes have been found to be useful as agents for delivery to introduce macromolecules, such as DNA, RNA, protein, and small chemical compounds such as pharmaceuticals, to cells. In particular, lipid aggregates comprising cationic lipid components have been shown to be especially effective for delivering anionic molecules to cells. In part, the effectiveness of cationic lipids is thought to result from enhanced affinity for cells, many of which bear a net negative charge. Also in part, the net positive charge on lipid aggregates comprising a cationic lipid enables the aggregate to bind polyanions, such as nucleic acids. Lipid aggregates containing DNA are known to be effective agents for efficient transfection of target cells.
[0006] The structure of various types of lipid aggregates varies, depending on composition and method of forming the aggregate. Such aggregates include liposomes, unilamellar vesicles, multilameller vesicles, micelles and the like, having particular sizes in the nanometer to micrometer range. Methods of making lipid aggregates are by now well-known in the art. The main drawback to use of conventional phospholipid containing liposomes for delivery is that the material to be delivered must be encapsulated and the liposome composition has a net negative charge which is not attracted to the negatively charged cell surface. By combining cationic lipid compounds with a phospholipid, positively charged vesicles and other types of lipid aggregates can bind DNA, which is negatively charged, can be taken up by target cells, and can transfect target cells. (Felgner, P. L. et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413–7417; Eppstein, D. et al., U.S. Pat. No. 4,897,355.)
[0007] A well-known cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). The structure of DOTMA is:
[0008]
DOTMA by itself or in 1:1 combination with dioleoylphosphatidylethanolamine (DOPE) is formulated into liposomes using standard techniques. Felgner, et al. supra demonstrated that such liposomes provided efficient delivery of nucleic acids to some types of cells. A DOTMA:DOPE (1:1) formulation is sold under the trade name LIPOFECTIN (Life Technologies, Inc., Rockville, Md.). Another commercially available cationic lipid is 1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), which differs from DOTMA only in that the oleoyl moieties are linked via ester, rather than ether bonds to the propylamine. A related group of compounds differ from DOTMA and DOTAP in that one of the methyl groups of the trimethylammonium group is replaced by a hydroxyethyl group. Compounds of this type are similar to the Rosenthal Inhibitor (RI) of phospholipase A (Rosenthal, A. F. and Geyer, R. P. (1960) J. Biol. Chem. 235:2202–2206) which has stearoyl esters linked to the propylamine core. The dioleoyl analogs of RI are commonly abbreviated as DORI-ether and DORI-ester, depending on the linkage of the fatty acid moieties to the propylamine core. The hydroxy group can be used as a site for further functionalization.
DOTMA by itself or in 1:1 combination with dioleoylphosphatidylethanolamine (DOPE) is formulated into liposomes using standard techniques. Felgner, et al. supra demonstrated that such liposomes provided efficient delivery of nucleic acids to some types of cells. A DOTMA:DOPE (1:1) formulation is sold under the trade name LIPOFECTIN (Life Technologies, Inc., Rockville, Md.). Another commercially available cationic lipid is 1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), which differs from DOTMA only in that the oleoyl moieties are linked via ester, rather than ether bonds to the propylamine. A related group of compounds differ from DOTMA and DOTAP in that one of the methyl groups of the trimethylammonium group is replaced by a hydroxyethyl group. Compounds of this type are similar to the Rosenthal Inhibitor (RI) of phospholipase A (Rosenthal, A. F. and Geyer, R. P. (1960) J. Biol. Chem. 235:2202–2206) which has stearoyl esters linked to the propylamine core. The dioleoyl analogs of RI are commonly abbreviated as DORI-ether and DORI-ester, depending on the linkage of the fatty acid moieties to the propylamine core. The hydroxy group can be used as a site for further functionalization.
[0009] The dimyristyloxy analog of RI is known as DRMIE. A 1:1 (M/M) DMRIE:cholesterol formulation is sold under the tradename DMRIE-C (Life Technologies, Inc., Rockvilee, Md.). The structure of DMRIE is:
[0010]
[0011] Another class of compounds has been disclosed by Behr et al. (1989) Proc. Natl. Acad. Sci. USA 86:6982–6986; EPO publication 0 394 111 (Oct. 24, 1990), in which carboxyspermine has been conjugated to two types of lipids. The structures of 5-carboxyspermylglycine dioctadecylamide (DOGS) is:
[0012]
The structure of dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES) is:
The structure of dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES) is:
[0013]
[0014] Both DOGS and DPPES have been used to coat plasmids, forming a lipid aggregate complex that provides efficient transfection. The compounds are claimed to be more efficient and less toxic than DOTMA for transfection of some cell lines. DOGS is available commercially as TRANSFECTAM™ (Promega, Madison, Wis.).
[0015] Another class of compounds has been also described in which carboxy spermine has been conjugated to lipids via an amide bond (Gebeyehu, G. et al., U.S. Pat. No. 5,334,761). These compounds are useful for an efficient delivery of nucleic acids into various cells and also are intermediates for making other such lipids. 2,3-di-oleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propan-aminium (DOSP A) is available as a 3:1 (w/w) formulation with DOPE under the trade name LipofectAMINE (available from Life Technologies, Inc., Rockville, Md.). The structure of DOSPA is as follows:
[0016]
[0017] Lipid compounds with a spermine head group have also been described (Haces, A., et al., U.S. Pat. No. 5,674,908). These compounds are especially useful for delivery of nucleic acids into insect cells. A 1:1.5 (M/M) formulation of tetramethyltetrapalmityl spennine (TM-TPS) to DOPE is commercially available under the tradename CellFECTIN (Life Technologies, mc., Rockville, Md.). The structure of TM-TPS is shown below:
[0018]
[0019] A cationic cholesterol derivative (DC-Chol) has been synthesized and formulated into liposomes in combination with DOPE. (Gao. X. and Huang, L. (1991) Biochim. Res. Cornrn. 179:280–285). The compound's structure is:
[0020]
Liposomes formulated with DC-Chol are said to provide more efficient transfection and lower toxicity than DOTMA-containing liposomes for some cell lines.
Liposomes formulated with DC-Chol are said to provide more efficient transfection and lower toxicity than DOTMA-containing liposomes for some cell lines.
[0021] Lipopolylysine, formed by conjugating polylysine to DOPE, has been reported to be especially effective for transfection in the presence of serum, a condition likely to be encountered in vivo (Zhou, X. et al. (1991) Biochim. Biophys. Acta 1065: 8–14).
[0022] Despite advances in the field, a need remains for a variety of improved cationic lipid compounds. In particular, no single cationic lipid to date has been found to work well with all cell types. Since different cell types differ from one another in membrane composition, it is not surprising that different compositions and types of lipid aggregates are effective for different cell types, either for their ability to contact and fuse with target cell membranes, or for aspects of the transfer process itself. At present these processes are not well understood, consequently the design of effective liposomal precursors is largely empirical. Besides content and transfer, other factors are of importance, for example, ability to form lipid aggregates suited to the intended purpose, the possibility of transfecting cells in the presence of serum, toxicity to the target cell, stability as a carrier for the compound to be delivered, and ability to function in an in vivo environment. In addition, lipid aggregates can be improved by broadening the range of substances which can be delivered to cells. The cationic lipid compounds of the present invention have improved function with respect to several of the foregoing attributes.
SUMMARY OF THE INVENTION
[0023] The present invention provides novel cationic lipids according to the general Formula (A):
[0024]
wherein
wherein
[0025] Q is selected from the group consisting of N, O and S;
[0026] L is any bivalent organic radical capable of covalently linking each Q, such as C, CH, (CH2)l or {(CH2)i-Y—(CH2)j}k, wherein Y is selected from the group consisting of CH2, an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide, an imine, a carbonyl, and a secondary amino group and wherein Y is optionally substituted by —X1-L′-X2-Z or -Z;
[0027] R1–R6, independently of one another, are selected from the group consisting of H, —{CH2)p-D-Z, an alkyl, an alkenyl, an aryl, and an alkyl or alkyl ether optionally substituted by one or more of an alcohol, an amino alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, a guanidyl, or a carbamoyl group, and wherein at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group; and anyone or more of R1, R3, R4 and R6 may optionally be covalently linked with each other, with Y or with L when L is C or CH to form a cyclic moiety;
[0028] Z is selected from the group consisting of amine, spermiyl, carboxyspermiyl, guanidyl, spermidinyl, putricinyl, diaminoalkyl, pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, peptide, and protein;
[0029] X1 and X2, independently of one another, are selected from the group consisting of NH, O, S, alkylene, and arylene;
[0030] L′ is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, alkylene ether, and polyether;
[0031] D is Q or a bond;
[0032] A1 and A2, independently of one another, are selected from the group consisting of CH2O, CH2S, CH2NH, C(O), C(NH), C(S) and (CH2)t;
[0033] X is a physiologically acceptable anion;
[0034] m, n, r, s, u, v, w and y are 0 or 1, with the proviso that when both m and n are 0 at least one of r, s, u and y is other than 0;
[0035] i, j, k, l, p and t are from a to about 100;
[0036] q is an integer from 1 to about 1000; and
[0037] a is the number of positive charge divided by the valence of the anion.
[0038] Further, the present invention provides novel cationic lipids according to the general Formula (B):
[0039]
wherein
wherein
[0040] L is (CH2)l or {(CH2)i-Y—(CH2)j}k wherein Y is selected from the group consisting of an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, and a secondary amino group;
[0041] R1–R6, independently of one another, are selected from the group consisting of H, —(CH2)p-Z, an alkyl, an alkenyl, an aryl, and an alkyl or an alkyl ether optionally substituted by one or more of an alcohol, an aminoalcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group, and at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group, preferably having from about 2 to 100, preferably 4 to 75, more preferably 6 to 64, more preferably 8 to 50, more preferably 8 to 40, more preferably 8 to 30, more preferably 6 to 30, more preferably 4 to 30, more preferably 2 to 30, and most preferably 8 to about 24 carbon atoms, and anyone or more of R1, R3, R4 and/or R6 may optionally be covalently linked with each other to form a cyclic moiety;
[0042] Z is selected from the group consisting of amine, spermiyl, carboxyspermiyl, guanidyl, spemlidinyl, putricinyl, diaminoalkyl, pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, amino acid derivative, peptide, and protein;
[0043] A1 and A2, independently of one another, are selected from the group consisting of CH2O, CH2S, CH2NH, C(O), C(NH), C(S) and (CH2)t;
[0044] X is a physiologically acceptable anion, such as the halide anions, chloride, bromide, and iodide as well as acetate, sulfate, trifluoroacetate, etc.;
[0045] m, n, v and w are 0 or 1;
[0046] i, j, k, l, p and t are integers from 1 to about 100, more preferably 1 to 50, more preferably 1 to 25, more preferably 1 to 15, more preferably 1 to 10 and most preferably 1 to about 4;
[0047] q is an integer from 1 to about 1000, preferably from 1 to about 500, more preferably from 1 to about 250, more preferably from 1 to about 100, more preferably from 1 to about 50, more preferably from 1 to about 25, more preferably from 1 to about 12, most preferably from 1 to about 6; and
[0048] a is the number of positive charges divided by the valence of the anion, wherein when m and n are 0, then a is O.
[0049] Also, the present invention provides novel cationic lipids according to the Formula (C):
[0050]
wherein
wherein
[0051] Y is selected from the group consisting of CH2, an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide, an imine, a carbonyl, and a secondary amino group and wherein Y is optionally substituted by —X1-L′-X2-Z or -Z;
[0052] R1, R3, R4 and R6, independently of one another, are selected from the group consisting of H, —{CH2)p-D-Z, an alkyl, an alkenyl, an aryl, and an alkyl or an alkyl ether optionally substituted by one or more of an alcohol, an aminoalcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, a guanidyl, or a carbamoyl group, and at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group, most preferably having from about 8 to about 24 carbon atoms, and R1, R3, R4 and R6 may optionally be covalently linked with each other or with Y, to form a cyclic moiety;
[0053] Z is selected from the group consisting of amine, spemliyl, carboxyspemliyl, guanidyl, spemlidinyl, putricinyl, diaminoalkyl, pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, peptide, and protein;
[0054] X1 and X2, independently of one another, are selected from the group consisting of NH, O, S, alkylene, and arylene;
[0055] L′ is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, alkylene ether, and polyether;
[0056] D is Q or a bond;
[0057] m and n are 0 or 1; and
[0058] i, j, k, l and p are integers from 1 to about 10.
[0059] Further, the present invention provides compounds or polycations according to the Formula (D):
[0060]
wherein
wherein
[0061] L is C, CH, (CH2)l or {(CH2)i-Y—(CH2)j}k, wherein Y is selected from the group consisting of CH2, an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide, an imine, a carbonyl, and a secondary amino group and wherein Y is optionally substituted by —X1-L′-X2-Z or -Z;
[0062] R1–R6, independently of one another, are selected from the group consisting of H, —(CH2)p-D-Z, an alkyl, an alkenyl, an aryl, and an alkyl or an alkyl ether optionally substituted by one or more of an alcohol, an aminoalcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, a guanidyl, or a carbamoyl group, and wherein at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl groups, preferably having from about 2 to about 30 carbon atoms, more preferably from 8 to 24 carbon atoms;
[0063] Z is selected from the group consisting of amine, spenniyl, carboxyspenniyl, guanidyl, spennidinyl, putricinyl, diaminoalkyl, pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, amino acid derivative, peptide, and protein;
[0064] X1 and X2, independently of one another, are selected from the group consisting of NH, O, S, alkylene and arylene;
[0065] L′ is selected from the group consisting of alkyl ene, alkenylene, alkynylene, arylene, alkylene ether, and polyether;
[0066] A1 and A2, independently of one another, are selected from the group consisting of CH2O, CH2S, CH2NH, C(O), C{NH), C(S) and (CH2)t;
[0067] m, n, r, s, u, v, w and y are 0 or 1, with the proviso that when both m and n are 0 at least one of r, s, u and y is other than 0;
[0068] i, j, k, l, p and t are integers from 0 to about 100; and
[0069] q is an integer from 1 to about 1000.
[0070] Also, the present invention provides compounds or polycations according to the Formula (E):
[0071]
wherein
wherein
[0072] L is (CH2)l or {(CH2)i-Y—(CH2)j}k, wherein Y is selected from the group consisting of CH2, an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, and a secondary amino group;
[0073] R1–R6, independently of one another, are selected from the group consisting of H, —(CH2)p-Z, an alkyl, an alkenyl, an aryl, and an alkyl or an alkyl ether optionally substituted by one or more of an alcohol, an amino alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group, and at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkenyl or aryl group, preferably having from about 2 to about 30 carbon atoms, more preferably having from about 8 to about 24 carbon atoms;
[0074] Z is selected from the group consisting of amine, spenniyl, carboxyspenniyl, guanidyl, spennidinyl, putricinyl, diaminoalkyl, pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, amino acid derivative, peptide, and protein;
[0075] A1 and A2, independently of one another, are selected from the group consisting of CH2O, CH2S, CH2NH, C(O), C(NH), C(S) and (CH2)t;
[0076] m, n, v and w are 0 or 1;
[0077] i, j, k, l, p and t are integers from 1 to about 100; and
[0078] q is an integer from 1 to about 1000.
[0079] Also, the present invention provides novel compounds falling within the scope of the above formulae.
[0080] The compounds of the invention are useful, either alone or in combination with other lipid aggregate-forming components (e.g., DOPE, DOPC or cholesterol) for formulation into liposomes or other lipid aggregates. Such aggregates are polycationic, able to form stable complexes with anionic macromolecules, such as nucleic acids. The lipid aggregate macromolecular complex interacts with cells making the polyanionic macromolecule available for absorption and uptake by the cell.
[0081] The present invention provides a lipid aggregate comprising one or more of the compounds of the present invention. Preferably, the lipid aggregate comprises at least one lipid aggregate-forming compound. Preferably, the lipid aggregate-forming compound is selected from the group consisting of DOPE, DOPC and cholesterol.
[0082] The compounds of the present invention may also be conjugated to or mixed with or used in conjunction with a variety of useful molecules and substances such as proteins, peptides, growth factors and the like to enhance cell-targeting, uptake, internalization, nuclear targeting and expression.
[0083] This invention also includes lipid aggregates comprising one or more compounds of the present invention or mixtures thereof. Such lipid aggregates may be combined with one or more aggregate-forming components and/or transfection enhancers.
[0084] The transfection methods of the present invention employing the compounds or compositions (such as those described above) of the present invention or mixtures thereof can be applied to in vitro and in vivo transfection of cells, particularly to transfection of eukaryotic cells or tissues including animal cells, human cells, insect cells, plant cells, avian cells, fish cells, mammalian cells and the like.
[0085] Accordingly, the present invention provides a method for introducing a polyanion into a cell or cells, wherein the method comprises forming a liposome from a positively charged compound according to the invention, contacting the liposome with polyanion to form a positively-charged polyanion-liposome complex and incubating the complex with a cell or cells.
[0086] The methods of this invention can be used to generate transfected cells or tissues which express useful gene products. The methods of this invention can also be used as a step in the production of transgenic animals. The methods of this invention are useful in any therapeutic method requiring introducing of nucleic acids into cells or tissues. In particular, these methods are useful in cancer treatment, in in vivo and ex vivo gene therapy, and in diagnostic methods. See, for example, U.S. Pat. No. 5,589,466 to Felgner, et al. and U.S. patent application Ser. No. 08/450,555 filed on May 25, 1995 to Jessee, et al. The transfection compounds or compositions of this invention can be employed as research reagents in any transfection of cells or tissues done for research purposes. Nucleic acids that can be transfected by the methods of this invention include DNA and RNA from any source comprising natural bases or non-natural bases, and include those encoding and capable of expressing therapeutic or otherwise useful proteins in cells or tissues, those which inhibit expression of nucleic acids in cells or tissues, those which inhibit enzymatic activity or activate enzymes, those which catalyze reactions (ribozymes), and those which function in diagnostic assays.
[0087] The compounds, compositions and methods provided herein can also be readily adapted in view of the disclosure herein to introduce biologically active macromolecules or substances other than nucleic acids, including, among others, polyamines, polyamine acids, polypeptides, proteins, biotin, and polysaccharides into cells. Other useful materials for example, therapeutic agents, diagnostic materials and research reagents, can be introduced into cells by the methods of this invention. In a preferred aspect, any nucleic acid vector may be delivered to or into a cell by the present invention.
[0088] Accordingly, the present invention provides a method for introducing a biologically active substance into a cell, wherein the method comprises forming a liposome of a compound according to the invention and a biologically active substance and incubating the liposome with a cell or cell culture.
[0089] The invention also relates to compositions comprising the compounds of the invention and one or more additional components selected from the group consisting of nucleic acids, cells, buffers, culture media, biologically active substance, neutral lipids, and transfection enhancers, preferably a nucleic acid.
[0090] This invention also includes transfection kits which include one or more of the compounds or compositions of the present invention or mixtures thereof. Particularly, the invention provides a kit comprising one or more of the compounds of the present invention and at least one additional component selected from the group consisting of a cell, cells, a cell culture media, a nucleic acid, a transfection enhancer and instructions for transfecting a cell or cells.
[0091] The invention also relates to intermediates and methods for using such intermediates for making the compounds or compositions of the invention. The invention also relates to the compositions, compounds or components obtained by the interaction of materials (intermediates, compounds, lipids etc.) used in the synthesis methods of the invention.
[0092] Other preferred embodiments of the present invention will be apparent to one of ordinary skill in the art in view of the following drawings and description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0093] FIG. 1 is a graph showing the transfection of HEK-293 cells with cationic transfection reagents.
[0094] FIG. 2 is a graph showing transfection of COS-7 cells with cationic transfection reagents.
[0095] FIG. 3 is a graph showing transfection of CHO-KI cells with cationic transfection reagents.
[0096] FIG. 4 is a graph showing transfection of He La cells with cationic transfection reagents.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0097] The present invention relates to cationic lipids and compositions of cationic lipids having utility in lipid aggregates for delivery of macromolecules and other compounds into cells. The compounds can be used alone or in combination with other compounds to prepare liposomes and other lipid aggregates suitable for transfection or delivery of compounds to target cells, either in vitro or in vivo.
[0098] The compounds of the present invention are preferably polycationic and preferably thus form highly stable complexes with various anionic macromolecules, particularly polyanions such as nucleic acids. These compounds have the property, when dispersed in water, of forming lipid aggregates which associate strongly, via their cationic portion, with polyanions. By using an excess of cationic charges relative to the anionic compound, the polyanion-lipid complexes may be adsorbed on cell membranes, thereby facilitating uptake of the desired compound by the cells.
[0099] The present invention also relates to intermediates for preparing the compound and compositions of the invention.
[0100] More specifically, the present invention relates to a cationic lipid for transfection which has a greater transfection efficiency than commercially available products in the three most common cell types used in expression research (CHO-K1, COS-7, and HEK293) making it useful for high throughput applications; and which has a simple to use protocol as defined by the fact that no additional reagents are required (e.g., such as 10 LipofectAMINE PLUS Reagent available from Life Technologies, Inc., Rockville, Md.), no removal of serum and therefore no media changes are required, and the DNA/lipid complex do not need to be removed from the cells prior to assay.
[0101] The compounds according to the present invention have the Formula (A):
[0102]
wherein
wherein
[0103] Q is selected from the group consisting of N, O and S;
[0104] L is any bivalent organic radical capable of covalently linking each Q, such as C, CH, (CH2)l or {(CH2)i-Y—(CH2)j}k, wherein Y is selected from the group consisting of CH2, an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide, an imine, a carbonyl, and a secondary amino group and wherein Y is optionally substituted by —X1-L′-X2-Z or -Z;
[0105] R1–R6, independently of one another, are selected from the group consisting of H, —(CH2)p-D-Z, an alkyl, an alkenyl, an aryl, and an alkyl or alkyl ether optionally substituted by one or more of an alcohol, an amino alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, a guanidyl, or a carbamoyl group, and wherein at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group; and R1 and R4 or R3 and R6 may optionally be covalently linked with each other, with Y or with L when L is C or CH to form a cyclic moiety;
[0106] Z is selected from the group consisting of amine, spermiyl, carboxyspermiyl, guanidyl, spermidinyl, putricinyl, diaminoalkyl, pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, peptide, and protein;
[0107] X1 and X2, independently of one another, are selected from the group consisting of NH, O, S, alkylene, and arylene;
[0108] L′ is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, alkylene ether, and polyether;
[0109] D is Q or a bond;
[0110] A1 and A2, independently of one another, are selected from the group consisting of CH2O, CH2S, CH2NH, C(O), C(NH), C(S) and (CH2)t;
[0111] X is a physiologically acceptable anion;
[0112] m, n, r, s, u, v, w and y are 0 or 1, with the proviso that when both m and n are 0 at least one of r, s, u and y is other than 0;
[0113] i, j, k, l, p and t are integers from 0 to about 100;
[0114] q is an integer from 1 to about 1000; and
[0115] a is the number of positive charge divided by the valence of the anion.
[0116] Preferably the alkyl ether optionally substituted by one or more alcohol groups comprises a carbohydrate. Preferably, the carbohydrate is selected from the group consisting of galactose, fructose, glucose, maltose, sucrose, cellobiose, lactose, mannose, glucopyranose, mannopyranose and galactopyranose.
[0117] Preferably, i, j, k, l, p and t are integers independently selected from 1 to 100, more preferably from 1 to 50, more preferably 1 to 25, more preferably 1 to 15, more preferably 1 to 10 and most preferably 1 to about 4. Preferably, l, b and c are integers from 1 to about 4, i and j are integers from about 2 to about 3 and k is an integer from 1 to about 3.
[0118] Preferably, q is an integer from 1 to about 500, more preferably from 1 to about 250, more preferably from 1 to about 100, more preferably from 1 to about 50, more preferably from 1 to about 25, more preferably from 1 to about 12, most preferably from 1 to about 6.
[0119] Preferably, at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 2 to 100, preferably 4 to 75, more preferably 6 to 64, more preferably 8 to 50, more preferably 8 to 40, more preferably 8 to 30, more preferably 6 to 30, more preferably 4 to 30, and most preferably 8 to about 24 carbon atoms.
[0120] In all aspects of the invention, most suitable R1 and R4 groups, which can be the same or different, preferably the same, are C6-30 hydrocarbon radicals derived from fatty acids or activated derivatives thereof, such as fatty acyl chlorides. Thus, typical R1 and R4 groups are C6-30 alkyl or alkenyl groups.
[0121] Preferably, R1, R2, R3, R4, R5 and R6, independently of one another, are selected from the group consisting of H, C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group, and at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms.
[0122] Preferably Q is N.
[0123] Preferably, Y is selected from the group consisting of CH2, O, S and NH.
[0124] Useful compounds falling within the scope of the above formula (A) include compounds having the following formulae:
[0125]
wherein
wherein
[0126] Q and L are as defined above;
[0127] R1, R3, R4 and R6, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0128] r, s, u and y are 0 or 1; and
[0129] R7 and R8 are independently H or a carbohydrate;
[0130]
wherein
wherein
[0131] Q is as defined above;
[0132] R1, R2, R4 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0133] Z is selected from the group consisting of spermiyl, spermidiyl, amino acid, peptidyl, diaminoalkyl, and polyamine;
[0134] m, n, r and u are 0 or 1; and
[0135] l, b and c are integers independently selected from 1 to about 4;
[0136]
wherein
wherein
[0137] Q, R1, R4, m, n, r and u are as defined above;
[0138] R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0139] Z is selected from the group consisting of spermiyl, spermidiyl, amino acid, peptidyl, diaminoalkyl, and polyamine;
[0140] R7 and R8 are independently H or a carbohydrate; and
[0141] l is an integer from 1 to about 4;
[0142]
wherein
wherein
[0143] Q is as defined above, preferably N;
[0144] at least one of R1 and R4 are straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl groups having from about 8 to about 24 carbon atoms;
[0145] R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0146] Z is selected from the group consisting of spermiyl, spermidiyl, amino acid, peptidyl, diaminoalkyl, and polyamine;
[0147] R7 and R8 are independently H or a carbohydrate, preferably H;
[0148] m and n are as defined above; and
[0149] is an integer from 1 to about 4;
[0150]
wherein
wherein
[0151] Q, R1, R4, r, u, m and n are as defined above;
[0152] R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0153] i and j are integers from about 2 to about 3; and
[0154] k is an integer from 1 to about 3;
[0155]
wherein
wherein
[0156] Q, R1, R4, r, u, m and n are as defined above;
[0157] R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0158] i and j are integers from about 2 to about 3;
[0159] k is an integer from 1 to about 3;
[0160] L1 and L2, independently from one another, are an alkylene or an alkylene ether;
[0161] Y is selected from the group consisting of CH2, O, S and NH;
[0162]
wherein
wherein
[0163] Q, R1, R4, r, u, m and n are as defined above;
[0164] R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0165] i and j are integers from about 2 to about 3;
[0166] k is an integer from 1 to about 3;
[0167] L1 and L2, independently from one another, are an alkylene or an alkylene ether; Y is selected from the group consisting of CH2, O, S and NH;
[0168]
wherein
wherein
[0169] Q, R1, R4, r, u, m and n are as defined above;
[0170] R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0171] i and j are integers from about 2 to about 3;
[0172] k is an integer from 1 to about 3;
[0173] L1 and L2, independently from one another, are an alkylene or an alkylene ether;
[0174] Y is selected from the group consisting of CH2, O, S and NH;
[0175]
wherein
wherein
[0176] Q, R1, R2, r, u, m and n are as defined above;
[0177] R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0178] i and j are integers from about 2 to about 3;
[0179] k is an integer from 1 to about 3;
[0180] L1 and L2, independently from one another, are an alkylene or an alkylene ether;
[0181] Y is selected from the group consisting of CH2, O, S and NH; and
[0182]
wherein
wherein
[0183] Q, R1, R4, r, u, m, and n are as defined above;
[0184] R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0185] i and j are integers from about 2 to about 3;
[0186] k is an integer from 1 to about 3;
[0187] L1 and L2, independently from one another, are an alkylene or an alkylene ether;
[0188] Y is selected from the group consisting of CH2, O, S and NH.
[0189] Also, compounds of the present invention have the Formula (B):
[0190]
wherein
wherein
[0191] L is (CH2)l or {(CH2)i-Y—(CH2)j}k, wherein Y is selected from the group consisting of CH2, an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, and a secondary amino group;
[0192] R1–R6, independently of one another, are selected from the group consisting of H, —(CH2)p-Z, an alkyl, an alkenyl, an aryl, and an alkyl or alkyl ether optionally substituted by one or more of an alcohol, an aminoalcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group, and at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group, and anyone or more of R1, R4, R3 and R6 may optionally be covalently linked with each other to form a cyclic moiety;
[0193] Z is selected from the group consisting of amine, spermiyl, carboxyspermiyl, guanidyl, spermidinyl, putricinyl, diaminoalkyl, pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, peptide, and protein;
[0194] A1 and A2, independently of one another, are selected from the group consisting of CH2O, CH2S, CH2NH, C(O), C(NH), C(S) and (CH2)t;
[0195] X is a physiologically acceptable anion;
[0196] m, n, v and w are 0 or 1;
[0197] i, j, k, l, p and t are integers from 1 to about 100;
[0198] q is an integer from 1 to about 1000; and
[0199] a is the number of positive charge divided by the valence of the anion, wherein when m and n are 0, then a is 0.
[0200] Preferably, R1–R6, i, j, k, l, p, t, q, b and c are as defined with reference to Formula (A).
[0201] Preferably, Y is selected from the group consisting of CH2, O, S and NH.
[0202] Useful compounds falling within the scope of the Formula (B) include compounds having the following formulae:
[0203]
wherein
wherein
[0204] R1, R3, R4 and R6, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group, and at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms; and
[0205] l, b and c are integers independently selected from 1 to about 4;
[0206]
wherein
wherein
[0207] R1, R3, R4 and R6, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group, and at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms;
[0208] R7 and R8 are independently H or a carbohydrate; and
[0209] 1 is an integer from 1 to about 4;
[0210]
wherein
wherein
[0211] R1, R2, R4 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group, and at least one of R1, R2, R3 and R5 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms;
[0212] Z is selected from the group consisting of spermiyl, spermidiyl, amino acid, peptidyl, diaminoalkyl, and polyamine;
[0213] m and n are 0 or 1; and
[0214] l, b and c are integers independently selected from 1 to about 4;
[0215]
wherein
wherein
[0216] at least one of R1 and R4 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms; and
[0217] l, b and c are integers independently selected from 1 to about 4;
[0218]
wherein
wherein
[0219] at least one of R1 and R4 are straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl groups having from about 8 to about 24 carbon atoms; R7 and R8 are independently hydrogen or a carbohydrate, preferably hydrogen; and
[0220] l is an integer from 1 to about 4;
[0221]
wherein
wherein
[0222] Z is as defined above;
at least one of R1 and R4 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms;
[0223] R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0224] R7 and R8 are independently H or a carbohydrate;
[0225] m and n are 0 or 1;
[0226] i and j are integers from about 2 to about 3; and
[0227] k is an integer from 1 to about 3;
[0228]
wherein
wherein
[0229] at least one of R1 and R4 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms;
[0230] R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0231] m and n are 0 or 1;
[0232] i and j are integers from about 2 to about 3; and
[0233] k is an integer from 1 to about 3;
[0234]
wherein
wherein
[0235] at least one of R1 and R4 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms;
[0236] R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0237] m and n are 0 or 1;
[0238] i and j are integers from about 2 to about 3; and
[0239] k is an integer from 1 to about 3;
[0240]
wherein
wherein
[0241] at least one of R1 and R4 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms;
[0242] R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0243] m and n are 0 or 1;
[0244] i and j are integers from about 2 to about 3;
[0245] k is an integer from 1 to about 3;
[0246] L1 and L2, independently from one another, are an alkylene or an alkylene ether;
[0247] Y is selected from the group consisting of CH2, O, S and NH;
[0248]
wherein
wherein
[0249] at least one of R1 and R4 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms;
[0250] R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0251] m and n are 0 or 1;
[0252] i and j are integers from about 2 to about 3;
[0253] k is an integer from 1 to about 3;
[0254] L1 and L2, independently from one another, are an alkylene or an alkylene ether;
[0255] Y is selected from the group consisting of CH2, O, S and NH;
[0256]
wherein
wherein
[0257] at least one of R1 and R4 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms;
[0258] R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0259] m and n are 0 or 1;
[0260] i and j are integers from about 2 to about 3;
[0261] k is an integer from 1 to about 3;
[0262] L1 and L2, independently from one another, are an alkylene or an alkylene ether;
[0263] Y is selected from the group consisting of CH2, O, S and NH;
[0264]
wherein
at least one of R1 and R4 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms;
R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
wherein
at least one of R1 and R4 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms;
R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0265] m and n are 0 or 1;
[0266] i and j are integers from about 2 to about 3;
[0267] k is an integer from 1 to about 3;
[0268] L1 and L2, independently from one another, are an alkylene or an alkylene ether;
[0269] Y is selected from the group consisting of CH2, O, S and NH; and
[0270]
wherein
wherein
[0271] at least one of R1 and R4 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24 carbon atoms;
[0272] R2 and R5, independently of one another, are selected from the group consisting of H and a C1–C8 alkyl, alkenyl, aryl, and alkyl optionally substituted by one or more of an alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group;
[0273] m and n are 0 or 1;
[0274] i and j are integers from about 2 to about 3;
[0275] k is an integer from 1 to about 3;
[0276] L1 and L2, independently from one another, are an alkylene or an alkylene ether;
[0277] Y is selected from the group consisting of CH2, O, S and NH.
[0278] In each of formulae (B1) through (B13) preferably R1 and R4 are each C6-30 alkyl or alkenyl, more preferably C8-24 alkyl or alkenyl, and R2 and R5 or R3 and R6 are each hydrogen or C1-8 alkyl.
[0279] Specific compounds within the scope of the invention include the following examples. R7 and R8 in the formulae are independently H or a carbohydrate, preferably H.
[0280]
[0281] Further, the compounds according to the present invention have the Formula (C):
[0282]
wherein
wherein
[0283] Y is selected from the group consisting of CH2, an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide, an imine, a carbonyl, and a secondary amino group and wherein Y is optionally substituted by —X1-L′-X2-Z or -Z;
[0284] R1, R3, R4 and R6, independently of one another, are selected from the group consisting of H, —(CH2)p-D-Z, an alkyl, an alkenyl, an aryl, and an alkyl or an alkyl ether optionally substituted by one or more of an alcohol, an aminoalcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, a guanidyl, or a carbamoyl group, and at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group, and R1, R3, R4 and R6 may optionally be covalently linked with each other or with Y, to form a cyclic moiety;
[0285] Z is selected from the group consisting of amine, spermiyl, carboxyspermiyl, guanidyl, spermidinyl, putricinyl, diaminoalkyl, pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, peptide, and protein;
[0286] X1 and X2, independently of one another, are selected from the group consisting of NH, O, S, alkylene, and arylene;
[0287] L′ is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, alkylene ether, and polyether;
[0288] D is Q or a bond;
[0289] m and n are 0 or 1; and
[0290] i, j, k, I and p are integers independently selected from 1 to about 10.
[0291] Preferably, Y is selected from the group consisting of CH2, an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, and a secondary amino group.
[0292] Preferably, R1, R3, R4 and R6, independently of one another, are selected from the group consisting of H, —(CH2)p-Z, an alkyl, an alkenyl, an aryl, and an alkyl or an alkyl ether optionally substituted by one or more of an alcohol, an aminoalcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group, and at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkenyl or aryl group, and R1, R3, R4 and R6 may be covalently linked with each other, to form a cyclic moiety.
[0293] Preferably, at least one of R1 and R4 is straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 2 to 100, preferably 4 to 75, more preferably 6 to 64, more preferably 8 to 50, more preferably 8 to 40, more preferably 8 to 30, more preferably 6 to 30, more preferably 4 to 30, and most preferably 8 to about 24 carbon atoms.
[0294] Preferably, Y is selected from the group consisting of CH2, O, S and NH.
[0295] The compounds and polycations of the present invention have the following Formula (D):
[0296]
wherein
wherein
[0297] L is C, CH, (CH2), or {(CH2)i-Y—(CH2)j}k, wherein Y is selected from the group consisting of CH2, an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide, an imine, a carbonyl, and a secondary amino group and wherein Y is optionally substituted by —X1-L′-X2-Z or -Z.
[0298] R1–R6, independently of one another, are selected from the group consisting of H, —(CH2)p-D-Z, an alkyl, an alkenyl, an aryl, and an alkyl or an alkyl ether optionally substituted by one or more of an alcohol, an amino alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, a guanidyl, or a carbamoyl group, and wherein at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group;
[0299] Z is selected from the group consisting of amine, spermiyl, carboxyspermiyl, guanidyl, spermidinyl, putricinyl, diaminoalkyl, pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, amino acid derivative, peptide, and protein;
[0300] X1 and X2, independently of one another, are selected from the group consisting of NH, O, S, alkylene and arylene;
[0301] L′ is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, alkylene ether, and polyether;
[0302] A1 and A2, independently of one another, are selected from the group consisting of CH2O, CH2S, CH2NH, C(O), C(NH), C(S) and (CH2)t;
[0303] m, n, r, s, u, v, w and y are 0 or 1, with the proviso that when both m and n are 0 at least one of r, s, u and y is other than 0;
[0304] i, j, k, l, p and t are integers from 0 to about 100; and
[0305] q is an integer from 1 to about 1000.
[0306] Also, the compounds and the polycations of the present invention have the following Formula (E):
[0307]
wherein
wherein
[0308] L is (CH2)l or {(CH2)i-Y—(CH2)j}k, wherein Y is selected from the group consisting of CH2, an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, and a secondary amino group;
[0309] R1–R6, independently of one another, are selected from the group consisting of H, —(CH2)p-Z, an alkyl, an alkenyl, an aryl, and an alkyl or an alkyl ether optionally substituted by one or more of an alcohol, an amino alcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or a carbamoyl group, and at least one of R1, R3, R4 and R6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group; Z, A1, A2, m, n, i, j, k, l, p, t and q are as defined above.
[0310] In the above formulae (D) and (E), R1–R6, Y, i, j, k, l, p, t and q are preferably as defined with reference to Formula (A).
[0311] It would be obvious for a skilled person that when Q is O or S, the number of substituents should be according their valency.
[0312] Certain of the compounds of the invention may be insufficiently soluble in physiological media to employ for delivery and transfection methods. Those of ordinary skill in the art will appreciate that there are a variety of techniques available in the art to enhance solubility of such compounds in aqueous media. Such methods are readily applicable without undue experimentation to the compounds described herein.
Definitions
[0313] Useful aryl groups are C6-100 aryl, preferably C6-75 aryl, more preferably C6-64 aryl, more preferably C6-50 aryl, more preferably C6-40 aryl, more preferably C6-30 aryl, most preferably C6-24 aryl. Typical C6-100 aryl groups include phenyl, naphthyl, phenanthryl, anthracyl, indenyl, azulenyl, biphenyl, biphenylenyl, fluorenyl, pyrenyl, aceanthrenyl, cholantrenyl, acephenanthrenyl, violantherenyl, hexaphenyl, hexacenyl, trinaphtyl and pyranthyl groups.
[0314] Useful alkyl groups are straight chain or branched C2-100 alkyl groups, preferably C4-75 alkyl, more preferably C6-64 alkyl, more preferably C8-50 alkyl, more preferably C8-40 alkyl, more preferably C8-30 alkyl, more preferably C6-30 alkyl, more preferably C4-30 alkyl, most preferably C8-24 alkyl. Typical C2-100 alkyl groups include ethyl, propyl, isopropyl, butyl, sec.-butyl, tert.-butyl, pentyl, hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, eicosyl, docosyl, tetracosyl, hexacosyl, octacosyl and triacontyl groups. Also contemplated is a trimethylene group substituted on two adjoining positions on any benzene ring of the compounds of the invention.
[0315] Useful alkenyl groups are straight chain or branched C2-100 alkenyl, preferably C4-75 alkenyl, more preferably C6-64 alkenyl, more preferably C8-5O alkenyl, more preferably C8-40 alkenyl, more preferably C8-30 alkenyl, more preferably C6-30 alkenyl, more preferably C4-30 alkenyl, most preferably C8-24 alkenyl. Typical C2-100 alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, sec.-butenyl, hexenyl, octenyl, decenyl, dodecenyl, especially 9-dodecenyl, tetradecenyl, especially 9-tetradecenyl, hexadecenyl, especially 9-hexadecenyl, octadecenyl, especially 9-octadecenyl, eicosenyl, docosenyl, tetracosenyl, hexacosenyl, octacosenyl and triacontenyl.
[0316] Useful alkynyl groups are straight chain or branched C2-100 alkynyl, preferably C4-75 alkynyl, more preferably C6-64 alkynyl, more preferably C8-50 alkynyl, more preferably C8-40 alkynyl, more preferably C8-30 alkynyl, more preferably C6-30 alkynyl, more preferably C4-30 alkynyl, most preferably C8-24 alkynyl. Typical C2-100 alkynyl groups include ethynyl, propynyl, butynyl, -butynyl, hexynyl, octynyl, decynyl, dodecynyl, tetradecynyl, hexadecynyl, octadecynyl, eicosynyl, docosynyl, tetracosynyl, hexacosynyl, octacosynyl and triacontynyl groups.
[0317] Typical alkyl ether groups include any of the above-mentioned C2-100 alkyl groups having an ether group.
[0318] An ether group is —O—.
[0319] Typical polyether groups include the —(CHR14—CH2—O)t-, wherein R14 is H or a C1-4 alkyl group and t is an integer as defined above, preferably t is 2 to 5.
[0320] For the purposes of the invention an amide group is an organic radical having —NHC(O)— as a functional group. Typical amide groups include alkyl amides, alkenyl amides, alkynyl amides, and aryl amides, wherein alkyl, alkenyl, alkynyl and aryl are as defined above.
[0321] Typically polyamide groups include organic radicals having two or more amide groups as defined above.
[0322] Typically an ester group is an organic radical having —C(O)—O— as a functional group. Typical ester groups include R14—C(O)—O—R15, wherein R14 and R15 are alkylene, alkenylene, alkynylene and arylene groups as defined above.
[0323] Typically urea groups are organic radicals having —NH—C(O)—NH— as a functional group. Typical urea groups include R14NH—C(O)—NHR14, R14NH—C(O)—NHR15, R14R15N—C(O)—NR14R15 wherein R14 and R15 are alkylene, alkenylene, alkynylene and arylene groups as defined above.
[0324] Typically thiourea groups are organic radicals having urea group as defined above wherein the oxygen in the urea group is substituted by sulfur.
[0325] Typically guanidyl groups are organic radicals having —NH—C(NH)—NH— as a functional group. Typical guanidyl groups include R14NH—C(NH)—NHR14, R14NH—C(NH)—NHR15 and R14R15N—C(NH)—NR14R15 wherein R14 and R15 are alkylene, alkenylene, alkynylene and arylene groups as defined above.
[0326] A carbamoyl group is —NH—C(O)—O—.
[0327] Typically carbonate groups include organic radicals containing a CO32− radical, i.e., —O—C(O)—O.
[0328] A phosphate group is a PO43− radical.
[0329] A sulfate group is a SO42− radical.
[0330] A sulfoxide group is —S(O)—.
[0331] An imine group is —C(N}—.
[0332] A carbonyl group is —C(O)—.
[0333] A secondary amino group is —NH—.
[0334] Typically amino alcohol groups are organic radicals having both a secondary amino group as defined above and a hydroxyl group. Typical aminoalcohols include amino ethanol, aminopropanol and aminobutanol.
[0335] The definition “D is a bond” means that when D is not Q there is a single bond between (CH2)p and Z.
[0336] Biologically Active Substance refers to any molecule or mixture or complex of molecules that exerts a biological effect in vitro and/or in vivo, including pharmaceuticals, drugs, proteins, peptides, polypeptides, hormones, vitamins, steroids, polyanions, nucleosides, nucleotides, nucleic acids (e.g. DNA or RNA), nucleotides, polynucleotides, etc.
[0337] Cationic Lipids refers to any cationic lipids which may be used for transfection, including but not limited to, DOSPA, DOTMA, DMRIE, DOTAP, DOGS and TM-TPS.
[0338] Cell refers to eukaryotic cells of any type and from any source. Types of eukaryotic cells include epithelial, fibroblastic, neuronal, hematopoietic cells and the like from primary cells, tumor cells or immortalized cell lines. Sources of such cells include any animal such as human, canine, mouse, hamster, cat, bovine, porcine, monkey, ape, sheep, fish, insect, fungus and any plant including crop plants, ornamentals and trees.
[0339] Delivery is used to denote a process by which a desired compound is transferred to a target cell such that the desired compound is ultimately located inside the target cell or in, or on, the target cell membrane. In many uses of the compounds of the invention, the desired compound is not readily taken up by the target cell and delivery via lipid aggregates is a means for getting the desired compound into the cell. In certain uses, especially under in vivo conditions, delivery to a specific target cell type is preferable and can be facilitated by compounds of the invention.
[0340] Drug refers to any therapeutic or prophylactic agent other than food which is used in the prevention, diagnosis, alleviation, treatment, or cure of disease in man or animal.
[0341] Kit refers to transfection or protein expression kits which include one or more of the compounds of the present invention or mixtures thereof. Such kits may comprise a carrying means being compartmentalized to receive in close confinement one or more container means such as vials, test tubes and the like. Each of such container means comprises components or a mixture of components needed to perform transfection. Such kits may include one or more components selected from nucleic acids (preferably one or more vectors), cells, one or more compounds of the present invention, lipid-aggregate forming compounds, transfection enhancers, biologically active substances, etc.
[0342] Lipid Aggregate is a generic term which includes liposomes of all types both unilamellar and multilameller as well as micelles and more amorphous aggregates of cationic lipids or lipids mixed with amphiphatic lipids such as phospholipids and steroids.
[0343] Lipid Aggregate-forming Compounds refers to neutral compounds or lipids such as DOPE, DOPC and cholesterol, etc.
[0344] Target Cell refers to any cell to which a desired compound is delivered, using a lipid aggregate as carrier for the desired compound.
[0345] Transfection is used herein to mean the delivery of nucleic acid, protein or other macromolecule to a target cell, such that the nucleic acid, protein or other macromolecule is expressed or has a biological function in the cell. The term “expressible nucleic acid” includes both DNA and RNA without regard to molecular weight, and the term “expression” means any manifestation of the functional presence of the nucleic acid within the cell including, without limitation, both transient expression and stable expression. Functional aspects include inhibition of expression by oligonucleotides or protein delivery.
[0346] Transfection Enhancers refers generally to molecules and substances such as proteins, peptides, growth factors and the like that enhance cell-targeting, uptake, internalization, nuclear targeting and expression. Such molecules and substances include ligands such as insulin, transferrin, fibronectin that target the cell surface; peptides that target cellular integrin receptors; and other compounds such as Plus Reagent (available from Life Technologies, Inc., Rockville, Md.). Examples of transfection enhancers may be found in U.S. Pat. No. 5,736,392 and U.S. application Ser. No. 09/039,780 filed Mar. 16, 1998.
[0347] The invention will be further clarified by the following examples, which are intended to be purely exemplary of the invention. The polycationic lipids were prepared by following the general reaction schemes described below.
EXAMPLES
Example 1
Synthesis of N1,N4-dioleoyl-diam in obutane (I)
[0348] A solution of 1,4-diaminobutane (4.28 g, 48.6 mmol) and triethylamine (20.4 ml, 146 mmol) in 10 mL of dry methylene chloride was slowly added to a solution of oleoyl chloride (30.0 g, 99.7 mmol) in 300 ml of anhydrous methylene chloride in an ice bath 25 at 0° C. The reaction mixture was stirred vigorously with a mechanical stirrer. After the addition was complete, the ice bath was removed and the mixture was stirred at room temperature for 2.5 days. TLC analysis confirmed that the reaction had gone to completion and the product had precipitated. The excess oleoyl chloride was removed by filtration. The precipitate was washed twice with 50 ml of methylene chloride. The mother liquor was concentrated and more product precipitated. This precipitate was filtered and combined with the previous precipitate. The resulting solid was vacuum dried for 4 hours. A total of 27.0 g of a white solid of the desired product, N1,N4-dioleoyl-diaminobutane, was obtained.
Synthesis of N1,N-dioleyl-diaminobutane (II)
[0349] Lithium aluminum hydride (8.62 g, 95%, 216 mmol) was carefully added to a suspension of N1,N4-dioleoyl-diaminobutane (27.0 g, 43.8 mmol) in 400 ml of anhydrous diethyl ether at 0° C. After addition, the ice bath was removed. The reaction mixture was warmed slowly to room temperature and then heated gently to reflux with an appropriate condensing device and stirred for 16 hours. The reaction mixture was then cooled and quenched carefully at 0° C. with 70 mL of a 1 N sodium hydroxide solution. Another 500 mL of diethyl ether was added and the mixture was stirred at room temperature for additional 2 hours. The top ether layer turned clear gradually and then separated. The aqueous layer was extracted three times with 100 mL of diethyl ether each. The combined ether solution was concentrated, and dried on high vacuum overnight. Total of 17.0 g of oily colorless N1,N4-dioleyl-diaminobutane was obtained.
Synthesis of N1,N4-dioleyl-N1,N4-di-[2-hydroxy-3-(N-phthalamido)propyljdiamino-butane (III)
[0350] Diisopropylethylamine (11.1 mL, 63.7 mmol) was added to a suspension of N1,N4-dioleyl-diaminobutane (15.5 g, 26.3 mmol) and N-(2,3-epoxypropyl)-phthalimide (15.6 g, 76.8 mmol) in 110 mL of dry N,N-dimethylformamide. After purging with nitrogen, the reaction mixture was sealed in a round-bottom flask and heated to around 90° C. for 24 hours. N,N-dimethylformamide and diisopropylethylamine were removed and a yellow oil was obtained. This crude material was recrystallized from ethanol. A total of 18.6 g of a white solid, N1,N4-dioleyl-N1,N4-di-[2-hydroxy-3-(N-phthalamido)propyl]-diamino-butane was obtained.
Synthesis of N1,N4-dioleyl-N1,N4-di-[2-hydroxy-3-(N-aminopropyl)]-diaminobutane (IV) (hereinafter referred to as DHDOS)
[0351] Hydrazine (4.0 mL, 80% aq., 103 mmol) was added to a suspension of N1,N4-dioleyl-N1,N4-di-[2-hydroxy-3-(N-phthalamido )propyl]-diaminobutane (17.0 g, 17.1 mmol) in 250 mL of dry ethanol at room temperature. With an appropriate condensing device, the reaction mixture was heated to a reflux, at which point the suspension turned into a clear solution. The oil bath was set to 85° C. After 45 minutes a white solid precipitated from the solution. The reaction mixture was stirred at reflux for 4 hours before being cooled to −20° C. The white solid settled down to the bottom. The top clear ethanol solution was decanted. The residue was washed twice with cold ethanol. The combined ethanol solution was concentrated and dried overnight over vacuum. 12.4 g of oily N1,N4-dioleyl-N1,N4-di-[2-hydroxy-3-(N-aminopropyl)]-diaminobutane was obtained.
[0352] The following compounds were synthesized by the above method using the corresponding diamine and a long chain acyl chloride:
[0353] N1,N4-dimyristyl-N1,N4-di-[2-hydroxy-3-(N-aminopropyl)]-diaminobutane;
[0354] N1,N4-dipalmityl-N1,N4-di-[2-hydroxy-3-(N-aminopropyl)]-diaminobutane;
[0355] N1,N4-dipalmitolyl-N1,N4-di-[2-hydroxy-3-(N-aminopropyl)]-diaminobutane;
[0356] N1,N4-distearyl-N1,N4-di-[2-hydroxy-3-(N-aminopropyl)]-diaminobutane;
[0357] N1,N4-dilauryl-N1,N4-di-[2-hydroxy-3-(N-aminopropyl)]-diaminobutane;
[0358] N1,N2-dimyristyl-N1,N2-di-[2-hydroxy-3-(N-aminopropyl)]-diaminoethane;
[0359] N1,N2-dipalmity-N1,N2-di-[2-hydroxy-3-(N-aminopropyl)]-diaminoethane;
[0360] N1,N2-dipalmitolyl-N1,N2-di-[2-hydroxy-3-(N-aminopropyl)]-diaminoethane;
[0361] N1,N2-distearyl-N1,N2-di-[2-hydroxy-3-(N-aminopropyl)]-diaminoethane;
[0362] N1,N2-dilauryl-N1,N2-di-[2-hydroxy-3-(N-aminopropyl)]-diaminoethane;
[0363] N1,N2-dioleyl-N1,N2-di-[2-hydroxy-3-(N-aminopropyl)]-diaminoethane;
[0364] N1,N8-dimyristyl-N1,N8-di-[2-hydroxy-3-(N-aminopropyl)]-Jeffamine;
[0365] N1,N8-dipalmityl-N1,N8-di-[2-hydroxy-3-(N-aminopropyl)]-Jeffamine;
[0366] N1,N8-dipalmitolyl-N1,N8-di-[2-hydroxy-3-(N-aminopropyl)]-Jeffamine;
[0367] N1,N8-distearyl-N1,N8-di-[2-hydroxy-3-(N-aminopropyl)]-Jeffamine;
[0368] N1,N8-dilauryl-N1,N8-di-[2-hydroxy-3-(N-aminopropyl)]-Jeffamine;
[0369] N1,N8-dioleyl-N1,N8-di-[2-hydroxy-3-(N-aminopropyl)]-Jeffamine;
Synthesis of N1,N4-dioleyl-N1,N4-di-[2-hydroxy-3-(N-carboxamidine)-aminopropyl]-diaminobutane (V)
[0370] 1H-pyrazole-1-carboxamidine hydrochloride (45 mg, 0.31 mmol) was added to a solution of N1,N4-dioleyl-N1,N4-di-[2-hydroxy-3-(N-aminopropyl)]-diamino-butane (115 mg, 0.156 mmol) in 1 mL of dry N,N-dimethylformamide. The salt was not very soluble in dimethylformamide (DMF). However, the mixture turned clear after diisopropylethylamine (55 μl, 0.31 mmol) was added. The mixture was stirred under nitrogen at room temperature for 18 hours. After removal of solvent, the crude material was loaded on a C-18 reverse phase flash column, and eluted with 20% H2O in MeOH to 10% H2O in MeOH. The pure fractions were collected and concentrated. An 81 mg colorless oily N1,N4-dioleyl-N1,N4-di-[2-hydroxy-3-(N-carboxamidine)aminopropyl]-diaminobutane was obtained, which was converted to its TFA and HCL salts.
Synthesis of N1,N4-dioleyl-N1,N4-di-{2-hydroxy-3-[N(NI,NII,NIII,NIV-butoxycarbonyl-spermine carboxamido)]aminopropyl}diaminobutane (VI)
[0371] Diisopropylcarbodiimide (5.32 mL, 34.0 mmol) was added drop wise to a solution of Boc-spermine acid (21.7 g, 33.5 mmol) and N-hydroxysuccinimide (NHS) (3.91 g, 34.0 mmol) in mixed solvents (100 mL of DMF and 100 mL of CH2Cl2) at room temperature. After stirring for 2.5 hours, a solution of N1,N4-dioleyl-N1,N4-di-[2-hydroxy-3-(N-aminopropyl)]diaminobutane (10 g, 13.6 mmol) in 40 mL of methylene chloride and DMF was added. The mixture was stirred for another 5 hours before quenching with 200 mL of a 2.5% sodium bicarbonate solution. An additional 300 mL of methylene chloride was added. The aqueous solution was extracted with 120 mL of methylene chloride three times. The combined organic solution was washed with water twice and dried over anhydrous magnesium sulfate. After concentration, a pale yellow oil was obtained. The crude material was purified with silica gel, eluting with 2% MeOH in CH2Cl2 to 5% MeOH in CH2Cl2. A total of 13.1 g of white solid N1,N4-dioleyl-N1,N4-di-{2-hydroxy-3-[N-(NI,NII,NIII,NIV-butoxycarbonyl-spermine carboxamido)]aminopropyl}di-aminobutane was obtained.
Synthesis of N1,N4-dioleyl-N1,N4-di-[2-hydroxy-3-(N-spermine carboxamido)-aminopropyl]-diaminobutane (VII)
[0372] 100 mL of a solution of 4.0 M hydrogen chloride in 1,4-dioxane was added to a solution of N1,N4-dioleyl-N1,N4-di-{2-hydroxy-3-[N-(NI,NII,NIII,NIV butoxycarbonylspermine carboxamido)]aminopropyl}diaminobutane (11.8 g, 5.92 mmol) in 100 mL of 1,4-dioxane at room temperature. The reaction mixture turned cloudy 10 minutes after addition of the acid. After 2.5 hours of stirring at room temperature, the excess acid and solvent was removed. The residue was dried for at least 5 hours over vacuum before being loaded on a C-18 reverse phase flash column. The column was eluted starting with 25% H2O in MeOH, then 20%, and then 17%. Pure fractions were collected and concentrated. A 3.06 g colorless solid N1,N4-dioleyl-N1,N4-di-[2-hydroxy-3-(N-spenninecarboxamido)-aminopropyl]-diaminobutane was obtained.
[0373] The following compounds were synthesized using the protocol described above starting with the requisite diamine and long chain acyl chloride:
[0374] N1,N4-dimyristyl-N1,N4-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-diaminobutane;
[0375] N1,N4-dipalmityl-N1,N4-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-diaminobutane;
[0376] N1,N4-dipalmitolyl-N1,N4-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-diaminobutane;
[0377] N1,N4-distearyl-N1,N4-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-diaminobutane;
[0378] N1,N4-dilauryl-N1,N4-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-diaminobutane;
[0379] N1,N8-dimyristyl-N1,N8-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-Jeffamine;
[0380] N1,N8-dipalmityl-N1,N8-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-Jeffamine;
[0381] N1,N8-dipalmitolyl-N1,N8-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-Jeffamine;
[0382] N1,N8-distearyl-N1,N8-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-Jeffamine;
[0383] N1,N8-dilauryl-N1,N8-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-Jeffamine;
[0384] N1,N8-dioleyl-N1,N8-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-Jeffamine;
[0385] N1,N2-dimyristyl-N1,N2-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-diaminoethane;
[0386] N1,N2-dipalmityl-N1,N2-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-diaminoethane;
[0387] N1,N2-dipalmitolyl-N1,N2-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-diaminoethane;
[0388] N1,N2-distearyl-N1,N2-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-diaminoethane;
[0389] N1,N2-dilauryl-N1,N2-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-diaminoethane;
[0390] N1,N2-dioleyl-N1,N2-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-diaminoethane;
[0391]
Example 2
Synthesis of N1,N4-dioleyl-N1,N4-di-3-cyanopropyldiaminobutane (VIII)
[0392] Acrylonitrile (0.43 mL, 6.53 mmol) was added dropwise to a solution of N1,N4-dioleyl-diaminobutane (1.8 g, 3.06 mmol) in 20 mL of ethanol at room temperature. The mixture was stirred for 30 hours. All starting materials were converted to product as confirmed by TLC analysis. The crude material was purified using flash chromatography with a silica gel (1% MeOH in CH2Cl2. A clear oil was obtained at high yield.
Synthesis of N1,N4-dioleyl-N1,N4-di-3-(aminopropyl) -diaminobutane (XI)
[0393] A solution of LAH (9.2 mL, 1 M in ether, 9.2 mmol) was slowly added to a solution of N1,N4-dioleyl-N1,N4-di-3-cyanopropyl-diaminobutane (2.12 g, 3.05 mmol) in 15 mL of anhydrous diethyl ether at 0° C. After addition, the mixture was stirred at room temperature for 20 hours. All starting material was consumed. The reaction mixture was quenched with a 1 N NaOH solution at 0° C. After stirring 2 hours at room temperature, the mixture was extracted with diethyl ether three times. The combined ether solutions were concentrated and dried over vacuum for three hours. An oily N1,N4-dioleyl-N1,N4- di-3-(aminopropyl)diaminobutane was obtained at high yield.
Synthesis of N1,N4-dioleyl-N1,N4-di-[3-(N-spermine carboxamido)-aminopropyl]-diaminobutane (XI)
[0394] The procedure for making N1,N4-dioleyl-N1,N4-di-[2-hydroxy-3-(N-spermine carboxamido)-aminopropyl]-diaminobutane described above was followed.
[0395]
Example 3
Synthesis of Cholesterol Analogs
[0396] The cholesterol analogs can be synthesized by using the scheme given below (Scheme 3). Jeffamine is alkylated with cholestryl chloride to provide the dicholestryl jeffamine analog (XII). Further alkylation with the epoxide phthalamide (XIII) and deblocking with hydrazine gives the compound of the invention (XIV).
[0397]
Example 4
Synthesis of Monoalkyl Analogs
[0398] When monoalkyl analogs are desired, the above Scheme 1 can be modified such that one of the amines in the starting material is protected before the acylation step. Thus, tritylprotected diaminobutane (XV) is acylated with alkanoyl chloride (e.g., oleoyl chloride) followed with LAH reduction to obtain compound XVIII. The amine is then alkylated with the desired phthalamide epoxide to obtain compound XVIII. Removing the phthalamide using hydrazine renders the desired amine XIX. (See Scheme 4).
[0399]
Example 5
Synthesis of Cyclic Analogs
[0400] The following scheme (Scheme 5) can be used to make the cyclic analogs. Trityl protected amino alcohol (XX) with the desired chain is alkylated using dibromoalkyl (e.g., dibromobutane). The trityl is removed from the desired dimer (XXI) and acylated using diacyl chlorides (e.g., succinyl chloride). The amide (XXIII) is then reduced with LAB and alkylated using the desired phthalamide epoxide. Removal of the phthalamide gives the desired compound of the invention.
[0401]
Example 6
Synthesis of Polymeric Analogs
[0402] Polymeric analogs of the present invention can be synthesized by using polymeric amines such as PEI as starting material or dendrimeric polyamines. For example, PEI can be acylated with alkyloyl chloride (e.g., oleoyl chloride) and the acylated PEI can then be reduced with lithium aluminum hydride to obtain compounds of the invention.
[0403] Although the above methods exemplify the synthesis of specific compounds, the reaction schemes provide a general method for preparing a variety of compounds according to the present invention. Those of ordinary skill in the art will appreciate that alternate methods and reagents other than those specifically detailed herein can be employed or readily adapted to produce compounds of the invention.
[0404] The compounds of the present invention can be used in the same manner as are prior art compounds such as DOTMA, DOTAP, DOGS, DOSPA and the like. Methods for incorporating such cationic lipids into lipid aggregates are well-known in the art. Representative methods are disclosed by Felgner, et al., supra; Eppstein et al. supra; Behr et al. supra; Bangham, A. et al. (1965) M. Mol. Biol. 23:238–252; Olson, F. et al. (1979) Biochim. Biophys. Acta 557:9–23; Szoka, F. et al. (1978) Proc. Natl. Acad. Sci. USA 75:4194–4198; Mayhew, E. et al. (1984) Biochim. Biophys. Acta 775:169–175; Kim, S. et al. (1983) Biochim. Biophys. Acta 728: 339–348; and Fukunaga, M. et al. (1984) Endocrinol. 115:757–761. Techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion as perhaps the most commonly used. See, e.g., Mayer, L. et al. (1986) Biochim. Biophys. Acta 858:161–168. Microfluidization is used when consistently small (50–200 nm) and relatively uniform aggregates are desired (Mayhew, E., supra). Aggregates ranging from about 50 nm to about 200 nm diameter are preferred; however, both larger and smaller sized aggregates are functional.
[0405] Methods of transfection and delivery of other compounds are well-known in the art. The compounds and compositions of the present invention yield lipid aggregates that can be used in the same processes used for other known transfection agents.
[0406] It will be readily apparent to those of ordinary skill in the art that a number of general parameters are important for optimal efficiency of transfection or delivery. These parameters include, for example, the cationic lipid concentration, the concentration of compound to be delivered, the number of cells transfected, the medium employed for delivery, the length of time the cells are incubated with the polyanion-lipid complex, and the relative amounts of cationic and non-cationic lipid. It may be necessary to optimize these parameters for each particular cell type. Such optimization is routine employing the guidance provided herein and knowledge generally available to the art.
[0407] It will also be apparent to those of ordinary skill in the art that alternative methods, reagents, procedures and techniques other than those specifically detailed herein can be employed or readily adapted to produce the liposomal precursors and transfection compositions of this invention. Such alternative methods, reagents, procedures and techniques are within the spirit and scope of this invention.
[0408] The use of representative compounds of the invention are further detailed by reference to the following examples. All abbreviations used herein are standard abbreviations in the art. Specific procedures not described in detail are either referenced or well-known in the art.
Example 7
[0409] This example compares transfection of HEK-293 (human embryonic kidney-derived cell line), COS-7 (SV40 transformed monkey cell line), CHO-KI (Chinese Hamster Ovary-derived cell line), and HeLa (Human cervical carcinoma-derived cell line) cells with the β-galactosidase reporter plasmid DNA pCMV. SPORT-β-gal (LifeTechnologies, Rockville, Md.) using commercially available cationic lipid transfection reagents and the compounds of the present invention.
[0410] The cells were plated the day before transfection in 24-well tissue culture plates in a total volume of 0.4 ml DMEM (Dulbecco's Modified Eagle's Medium, Life Technologies, Rockville, Md.) culture medium containing a 1% non-essential amino acid (NEAA) solution (LifeTechnologies), and 10% FBS. For the HEK-293 and COS-7 cells, tissue culture plates were pre-coated with Poly-L-Lysine to enhance cell attachment.
[0411] The next day, DNA-transfection reagent complexes were prepared as follows:
[0412] The cationic lipid reagents and DNA were diluted separately into 25 μl aliquots of serum-free DMEM, containing 1% NEAA. For LipofectAMINE PLUS, 7–14 μl of PLUS reagent was added to the DNA, mixed, and incubated for 15 minutes at room temperature. The diluted DNA was combined with the diluted lipid and incubated at room temperature for at least 15 minutes to allow the DNA and the lipid to form complexes. Following this incubation the complexes were added directly into the culture medium dropwise and mixed by rocking the culture plate back and forth. The cells were further incubated at 37° C. for a total of 24 hours to allow expression of the lacZ transgene encoded by the reporter plasmid, pCMV. SPORT-β-gal. At 24 hours post-transfection, the growth medium and transfection complexes were removed from the wells, and the cells in each well were rinsed briefly with 1 ml of D-PBS (Dulbecco's PBS, Life Technologies, Rockville, Md.). The cells in each well were lysed by the addition of 0.15 to 2.0 ml of 0.1% Tris, pH 8.0, containing 0.1 M Triton X-100. The plates were frozen at −80° C. for a minimum of 2 hours, and thawed at room temperature or 37° C. The thawed cell lysates were cleared by centrifugation and the supernatants were assayed for β-gal activity using the enzymatic substrate ONPG. The concentration of total protein in cell lysates was also determined using a Bradford assay (Bio-Rad Laboratories, Hercules Calif.). β-gal activity in transfected cell extracts was calculated against a standard curve and expressed as ng β-gal per surface area of tissue culture plate (ng/cm2) to reflect activity per transfection, or as ng β-gal per μg of total protein (ng/μg) to reflect specific activity.
[0413] HEK-293 (FIG. 1 ), COS-7 (FIG. 2 ), CHO-K1 (FIG. 3 ), and HeLa (FIG. 4 ) cells were transfected with 0.4 or 0.8 μg of pCMV. SPORT-β-gal DNA and 0.2 to 4.0 μl of transfection reagent. The transfection reagents tested were DHDOS (IV) formulated at 2 mg/ml with the neutral co-lipid, cholesterol (at a ratio of 1:15 (M/M) DHDOS to cholesterol); DHDOS formulated at 2 mg/ml with the neutral co-lipid DOPE (dioleylphosphatidyl ethanolamine) (at a ratio of 1:1 (M/M) DHDOS to DOPE); LipofectAMINE PLUS (Life Technologies, Rockville Md.); and FuGENE™-6 (Boehringer Mannheim, Germany). DHDOS formulations were tested in the range of 0.2 to 1.5 μl; LipofectAMINE PLUS and FuGENE-6 were tested in the range of 0.2 to 4.0 μl. FuGENE-6 was used according to the manufacturer's recommended protocol. DHDOS and LipofectAMINE PLUS were used according to the above protocol. The data presented in the Figures are expressed as total activity (ng/cm2) to better compare total expression from the transfected DNA. Only data with 0.8 μg of DNA is shown, since similar results were obtained with 0.4 and 0.8 μg of DNA.
Example 8
[0414] Primary, passaged, normal human fibroblasts (NHFs) were plated in 96-well plates at a density of 1.6×104 cells per well and transfected the following day. Cells in each well were transfected with 40 ng pCMV. SPORT-β-gal DNA and 0.1 or 0.2 μl lipid.
[0415] The DNA and lipid were diluted separately into 10 μl of DMEM. The DNA was either used alone or pre-mixed with PLUS, insulin, transferrin, or an integrin-targeting peptide prior to complexing with the lipid. After 15 minutes of complexing, the DNA-lipid was added to cells. Cells were assayed for p-gal activity as described above.
[0416]
| ACTIVITY (ng/βgal/cm2) | |||||
| DNA | |||||
| and | |||||
| DNA | DNA | DNA | INTEGRIN- | ||
| and | and | and | TARGETING | ||
| LIPID | DNA | PLUS* | INSULIN | TRANSFERRIN | PEPTIDE** |
| LipofectAMINE | 10.36 | 28.6 | ND | 17.4 | ND |
| Compound of | ND | 37.8 | ND | ND | 40.9 |
| Formula X | |||||
| 1:1.5 DOPE | |||||
| 1 mg/ml | |||||
| Compound of | 29.4 | 637.9 | 195.7 | 21.7 | 587.9 |
| Formula VII | |||||
| 1:1 DOPE | |||||
| 2 mg/ml | |||||
| ND = no detectable activity | |||||
| *PLUS Reagent is available from Life Technologies, Inc., Rockville, Maryland. | |||||
| **Reference: S. L. HART, et al (1998). Human Gene Therapy, 9: 575–585. | |||||
[0417] The results show that these cationic lipid formulations can deliver DNA molecules alone, but also that delivery, and ultimately gene expression, may be enhanced when the lipids are used in conjunction with peptides or proteins that bind DNA and/or act as ligands for cell surface receptors, or otherwise enhance cellular and/or nuclear uptake.
[0418] Having now fully described the present invention in some detail by way of illustration and examples for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.
[0419] All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.
Claims
What is claimed is:
1. A compound having the formula:
2. A composition comprising the compound of claim 1 solubilized in aqueous medium.
3. The composition of claim 2 further comprising a neutral lipid.
4. The composition of claim 2 further comprising DOPE, DOPC or cholesterol.
5. A kit comprising a container means which comprises a compound having the formula:
6. The kit of claim 5 wherein the compound is solubilized in aqueous medium.
7. The kit of claim 6 wherein the aqueous medium further comprises a neutral lipid.
8. The kit of claim 6 wherein the aqueous medium further comprises DOPE, DOPC or cholesterol.
9. A method for introducing a nucleic acid into a cell, the method comprising the steps of:
contacting a compound having the formula:
with the nucleic acid for at least 15 minutes to form a complex; and incubating the complex with the cell.
10. The method of claim 9 wherein the contacting step and the incubating step are conducted in physiological medium.
11. A method for introducing a nucleic acid into a cell, the method comprising the steps of contacting a composition comprising a compound having the formula
solubilized in aqueous medium with the nucleic acid for at least 15 minutes to form a complex; and incubating the complex with a cell.
12. The method of claim 11 wherein the contacting step and the incubating step are conducted in physiological medium.
13. The method of claim 11 wherein the composition comprises a neutral lipid when it contacts the nucleic acid.
14. The method of claim 11 wherein the composition comprises DOPE, DOPC, or cholesterol when it contacts the nucleic acid.
15. A lipid aggregate made by mixing a compound having the formula
with a neutral lipid.
16. The lipid aggregate of claim 15, wherein the neutral lipid is DOPE, DOPC or cholesterol.
17. The lipid aggregate of claim 15, wherein the lipid aggregate is 50 to 200 nm in diameter.
18. The lipid aggregate of claim 15, wherein the lipid aggregate is formed using extrusion, freeze-thaw, or sonication.
19. A method for making a lipid aggregate, comprising mixing a compound having the formula
with a neutral lipid to form the lipid aggregate.
20. The method of claim 19, wherein the lipid aggregate is made by mixing the compound with the neutral lipid DOPE, DOPC or cholesterol.
21. The method of claim 19, wherein the mixing is performed to obtain a lipid aggregate having a diameter of 50 to 200 nm.
22. The method of claim 19, wherein the lipid aggregate is formed using sonication, freeze-thaw, or extrusion.
Patent Citations (286)
| Patent | Date | Inventor | Cited By |
|---|---|---|---|
| US2654785(A) | 1953-10-01 | Miescher et al. | Applicant |
| US2695314(A) | 1954-11-01 | Kosmin | Applicant |
| US2867665(A) | 1959-01-01 | Dornfield | Applicant |
| US2901461(A) | 1959-08-01 | Auerbach et al. | Applicant |
| US3152188(A) | 1964-10-01 | Kirkpatrick | Applicant |
| US3324182(A) | 1967-06-01 | De Brunner et al. | Applicant |
| US3369905(A) | 1968-02-01 | Jones et al. | Applicant |
| US4143003(A) | 1979-03-01 | Haas et al. | Applicant |
| US4235871(A) | 1980-11-01 | Papahadjopoulos et al. | Applicant |
| US4501728(A) | 1985-02-01 | Geho et al. | Applicant |
| US4683195(A) | 1987-07-01 | Mullis et al. | Applicant |
| US4683202(A) | 1987-07-01 | Mullis | Applicant |
| US4737323(A) | 1988-04-01 | Martin et al. | Applicant |
| US4767699(A) | 1988-08-01 | Vary et al. | Applicant |
| US4800159(A) | 1989-01-01 | Mullis et al. | Applicant |
| US4812449(A) | 1989-03-01 | Rideout | Applicant |
| US4837028(A) | 1989-06-01 | Allen | Applicant |
| US4889818(A) | 1989-12-01 | Gelfand et al. | Applicant |
| US4889953(A) | 1989-12-01 | Inoue et al. | Applicant |
| US4897355(A) | 1990-01-01 | Eppstein et al. | Applicant |
| US4946787(A) | 1990-08-01 | Eppstein et al. | Applicant |
| US4962022(A) | 1990-10-01 | Fleming et al. | Applicant |
| US4965188(A) | 1990-10-01 | Mullis et al. | Applicant |
| US4967008(A) | 1990-10-01 | Friedli et al. | Applicant |
| US5047342(A) | 1991-09-01 | Chatterjee | Applicant |
| US5049386(A) | 1991-09-01 | Eppstein et al. | Applicant |
| US5079352(A) | 1992-01-01 | Gelfand et al. | Applicant |
| US5091576(A) | 1992-02-01 | Bergeron | Applicant |
| US5165925(A) | 1992-11-01 | Leong | Applicant |
| US5166320(A) | 1992-11-01 | Wu et al. | Applicant |
| US5171678(A) | 1992-12-01 | Behr et al. | Applicant |
| US5186923(A) | 1993-02-01 | Piwnica-Worms et al. | Applicant |
| US5187085(A) | 1993-02-01 | Lee | Applicant |
| US5198423(A) | 1993-03-01 | Taguchi et al. | Applicant |
| US5208036(A) | 1993-05-01 | Eppstein et al. | Applicant |
| US5244797(A) | 1993-09-01 | Kotewicz et al. | Applicant |
| US5264423(A) | 1993-11-01 | Cohen et al. | Applicant |
| US5264618(A) | 1993-11-01 | Felgner et al. | Applicant |
| US5266106(A) | 1993-11-01 | Winnik et al. | Applicant |
| US5270179(A) | 1993-12-01 | Chatterjee | Applicant |
| US5276019(A) | 1994-01-01 | Cohen et al. | Applicant |
| US5277897(A) | 1994-01-01 | Piwnica-Worms et al. | Applicant |
| US5279833(A) | 1994-01-01 | Rose | Applicant |
| US5283185(A) | 1994-02-01 | Epand et al. | Applicant |
| US5286717(A) | 1994-02-01 | Cohen et al. | Applicant |
| US5328470(A) | 1994-07-01 | Nabel et al. | Applicant |
| US5328984(A) | 1994-07-01 | Pastan et al. | Applicant |
| US5334761(A) | 1994-08-01 | Gebeyehu et al. | Applicant |
| US5338532(A) | 1994-08-01 | Tomalia et al. | Applicant |
| US5350672(A) | 1994-09-01 | Oberst et al. | Applicant |
| US5354844(A) | 1994-10-01 | Beug et al. | Applicant |
| US5374553(A) | 1994-12-01 | Gelfand et al. | Applicant |
| US5409818(A) | 1995-04-01 | Davey et al. | Applicant |
| US5436149(A) | 1995-07-01 | Barnes | Applicant |
| US5455166(A) | 1995-10-01 | Walker | Applicant |
| US5455335(A) | 1995-10-01 | Kahne et al. | Applicant |
| US5459127(A) | 1995-10-01 | Felgner et al. | Applicant |
| US5484702(A) | 1996-01-01 | Ludwig | Applicant |
| US5498522(A) | 1996-03-01 | Porter | Applicant |
| US5498523(A) | 1996-03-01 | Tabor et al. | Applicant |
| US5500356(A) | 1996-03-01 | Li et al. | Applicant |
| US5506212(A) | 1996-04-01 | Hoke et al. | Applicant |
| US5510239(A) | 1996-04-01 | Baracchini, Jr. et al. | Applicant |
| US5510476(A) | 1996-04-01 | Ravikumar et al. | Applicant |
| US5512438(A) | 1996-04-01 | Ecker | Applicant |
| US5512462(A) | 1996-04-01 | Cheng | Applicant |
| US5514577(A) | 1996-05-01 | Draper et al. | Applicant |
| US5514787(A) | 1996-05-01 | Atkinson | Applicant |
| US5519134(A) | 1996-05-01 | Acevedo et al. | Applicant |
| US5521291(A) | 1996-05-01 | Curiel et al. | Applicant |
| US5521302(A) | 1996-05-01 | Cook | Applicant |
| US5527524(A) | 1996-06-01 | Tomalia et al. | Applicant |
| US5532142(A) | 1996-07-01 | Johnston et al. | Applicant |
| US5541307(A) | 1996-07-01 | Cook et al. | Applicant |
| US5543507(A) | 1996-08-01 | Cook et al. | Applicant |
| US5545412(A) | 1996-08-01 | Eppstein et al. | Applicant |
| US5545540(A) | 1996-08-01 | Mian | Applicant |
| US5547932(A) | 1996-08-01 | Curiel et al. | Applicant |
| US5550289(A) | 1996-08-01 | Eppstein et al. | Applicant |
| US5554746(A) | 1996-09-01 | Ravikumar et al. | Applicant |
| US5560929(A) | 1996-10-01 | Hedstrand et al. | Applicant |
| US5574142(A) | 1996-11-01 | Meyer, Jr. et al. | Applicant |
| US5578475(A) | 1996-11-01 | Jessee | Applicant |
| US5580859(A) | 1996-12-01 | Felgner et al. | Applicant |
| US5583198(A) | 1996-12-01 | Whittaker | Applicant |
| US5587441(A) | 1996-12-01 | Frechet et al. | Applicant |
| US5587446(A) | 1996-12-01 | Frechet et al. | Applicant |
| US5589392(A) | 1996-12-01 | Short | Applicant |
| US5589466(A) | 1996-12-01 | Felgner et al. | Applicant |
| US5595096(A) | 1997-01-01 | Coffman | Applicant |
| US5595897(A) | 1997-01-01 | Midoux et al. | Applicant |
| US5596091(A) | 1997-01-01 | Switzer | Applicant |
| US5602240(A) | 1997-02-01 | De Mesmaeker et al. | Applicant |
| US5614365(A) | 1997-03-01 | Tabor et al. | Applicant |
| US5627159(A) | 1997-05-01 | Shih et al. | Applicant |
| US5631329(A) | 1997-05-01 | Yin et al. | Applicant |
| US5635487(A) | 1997-06-01 | Wolff et al. | Applicant |
| US5641662(A) | 1997-06-01 | Debs et al. | Applicant |
| US5650096(A) | 1997-07-01 | Harris et al. | Applicant |
| US5658776(A) | 1997-08-01 | Flotte et al. | Applicant |
| US5661025(A) | 1997-08-01 | Szoka, Jr. et al. | Applicant |
| US5667774(A) | 1997-09-01 | Figuly | Applicant |
| US5670347(A) | 1997-09-01 | Gopal | Applicant |
| US5674908(A) | 1997-10-01 | Haces et al. | Applicant |
| US5676954(A) | 1997-10-01 | Brigham | Applicant |
| US5681944(A) | 1997-10-01 | Crooke et al. | Applicant |
| US5691460(A) | 1997-11-01 | Duvic et al. | Applicant |
| US5693509(A) | 1997-12-01 | Cotten et al. | Applicant |
| US5693773(A) | 1997-12-01 | Kandimalla et al. | Applicant |
| US5703055(A) | 1997-12-01 | Felgner et al. | Applicant |
| US5705385(A) | 1998-01-01 | Bally et al. | Applicant |
| US5714166(A) | 1998-02-01 | Tomalia et al. | Applicant |
| US5719131(A) | 1998-02-01 | Harris et al. | Applicant |
| US5726298(A) | 1998-03-01 | Hirai et al. | Applicant |
| US5736387(A) | 1998-04-01 | Paul et al. | Applicant |
| US5736392(A) | 1998-04-01 | Hawley-Nelson et al. | Applicant |
| US5744335(A) | 1998-04-01 | Wolff et al. | Applicant |
| US5753613(A) | 1998-05-01 | Ansell et al. | Applicant |
| US5756353(A) | 1998-05-01 | Debs | Applicant |
| US5759778(A) | 1998-06-01 | Li et al. | Applicant |
| US5759805(A) | 1998-06-01 | Feldhaus et al. | Applicant |
| US5773527(A) | 1998-06-01 | Tomalia et al. | Applicant |
| US5780053(A) | 1998-07-01 | Ashley et al. | Applicant |
| US5783565(A) | 1998-07-01 | Lee et al. | Applicant |
| US5783566(A) | 1998-07-01 | Mislick | Applicant |
| US5785992(A) | 1998-07-01 | Ansell et al. | Applicant |
| US5795587(A) | 1998-08-01 | Gao et al. | Applicant |
| US5827703(A) | 1998-10-01 | Debs et al. | Applicant |
| US5830430(A) | 1998-11-01 | Unger et al. | Applicant |
| US5830878(A) | 1998-11-01 | Gorman et al. | Applicant |
| US5834439(A) | 1998-11-01 | Haces et al. | Applicant |
| US5837092(A) | 1998-11-01 | Grieves et al. | Applicant |
| US5837283(A) | 1998-11-01 | McDonald et al. | Applicant |
| US5837533(A) | 1998-11-01 | Boutin | Applicant |
| US5840710(A) | 1998-11-01 | Lee et al. | Applicant |
| US5854224(A) | 1998-12-01 | Lockett et al. | Applicant |
| US5861397(A) | 1999-01-01 | Wheeler | Applicant |
| US5866613(A) | 1999-02-01 | Bergeron | Applicant |
| US5869606(A) | 1999-02-01 | Whittaker | Applicant |
| US5871929(A) | 1999-02-01 | Barnes | Applicant |
| US5877309(A) | 1999-03-01 | McKay et al. | Applicant |
| US5885970(A) | 1999-03-01 | Bennett et al. | Applicant |
| US5886165(A) | 1999-03-01 | Kandimalla et al. | Applicant |
| US5906922(A) | 1999-05-01 | Whittaker et al. | Applicant |
| US5908635(A) | 1999-06-01 | Thierry | Applicant |
| US5908777(A) | 1999-06-01 | Lee et al. | Applicant |
| US5916807(A) | 1999-06-01 | Bennett et al. | Applicant |
| US5919772(A) | 1999-07-01 | Szyf et al. | Applicant |
| US5929226(A) | 1999-07-01 | Padmapriya et al. | Applicant |
| US5935936(A) | 1999-08-01 | Fasbender et al. | Applicant |
| US5948614(A) | 1999-09-01 | Chatterjee | Applicant |
| US5948767(A) | 1999-09-01 | Scheule et al. | Applicant |
| US5948925(A) | 1999-09-01 | Keynes et al. | Applicant |
| US5962533(A) | 1999-10-01 | Bergeron, Jr. | Applicant |
| US5976567(A) | 1999-11-01 | Wheeler et al. | Applicant |
| US5977306(A) | 1999-11-01 | Grieve et al. | Applicant |
| US5985558(A) | 1999-11-01 | Dean et al. | Applicant |
| US6013448(A) | 2000-01-01 | Braxton et al. | Applicant |
| US6020202(A) | 2000-02-01 | Jessee | Applicant |
| US6022874(A) | 2000-02-01 | Wheeler | Applicant |
| US6022950(A) | 2000-02-01 | Murphy | Applicant |
| US6030626(A) | 2000-02-01 | Kolattukudy et al. | Applicant |
| US6031086(A) | 2000-02-01 | Switzer | Applicant |
| US6051429(A) | 2000-04-01 | Hawley-Nelson et al. | Applicant |
| US6054439(A) | 2000-04-01 | Szyf et al. | Applicant |
| US6056938(A) | 2000-05-01 | Unger et al. | Applicant |
| US6074826(A) | 2000-06-01 | Hogan et al. | Applicant |
| US6075012(A) | 2000-06-01 | Gebeyehu et al. | Applicant |
| US6086913(A) | 2000-07-01 | Tam et al. | Applicant |
| US6090627(A) | 2000-07-01 | Kemp et al. | Applicant |
| US6093564(A) | 2000-07-01 | Budowsky et al. | Applicant |
| US6103492(A) | 2000-08-01 | Yu | Applicant |
| US6110662(A) | 2000-08-01 | Foung et al. | Applicant |
| US6110916(A) | 2000-08-01 | Haces et al. | Applicant |
| US6126965(A) | 2000-10-01 | Kasid et al. | Applicant |
| US6180784(B1) | 2001-01-01 | Wolff et al. | Applicant |
| US6211140(B1) | 2001-04-01 | Sivik et al. | Applicant |
| US6214804(B1) | 2001-04-01 | Felgner et al. | Applicant |
| US6251390(B1) | 2001-06-01 | Harman et al. | Applicant |
| US6287817(B1) | 2001-09-01 | Davis et al. | Applicant |
| US6335199(B1) | 2002-01-01 | Bischoff et al. | Applicant |
| US6376248(B1) | 2002-04-01 | Hawley-Nelson et al. | Applicant |
| US6387395(B1) | 2002-05-01 | Eppstein et al. | Applicant |
| US6399663(B1) | 2002-06-01 | Haces et al. | Applicant |
| US2002/0028447(A1) | 2002-03-01 | Li et al. | Applicant |
| US2002/0077305(A1) | 2002-06-01 | Jessee et al. | Applicant |
| US2002/0086849(A1) | 2002-07-01 | Gebeyehu et al. | Applicant |
| US2002/0156049(A1) | 2002-10-01 | Haces et al. | Applicant |
| US2003/0069173(A1) | 2003-04-01 | Hawley-Nelson | Applicant |
| US2005/0014962(A1) | 2005-01-01 | Gebeyehu et al. | Applicant |
| AU158967 | 1952-02-01 | Applicant | |
| AUB-26526/92 | 1993-05-01 | Applicant | |
| DE290877(A5) | 1991-06-01 | Applicant | |
| DE4411588(C1) | 1995-09-01 | Applicant | |
| DE4411594(C1) | 1995-12-01 | Applicant | |
| EP187702(B1) | 1986-07-01 | Applicant | |
| EP329822(A2) | 1989-08-01 | Applicant | |
| EP304111(B1) | 1991-10-01 | Applicant | |
| EP359347(B1) | 1992-12-01 | Applicant | |
| EP544292(A2) | 1993-06-01 | Applicant | |
| EP684315(B1) | 1995-11-01 | Applicant | |
| EP394111(B1) | 1997-06-01 | Applicant | |
| EP821059(A2) | 1998-01-01 | Applicant | |
| EP846680(A1) | 1998-06-01 | Applicant | |
| FR1567214 | 1969-05-01 | Applicant | |
| GB892413 | 1962-03-01 | Applicant | |
| WO87/02061(A1) | 1987-04-01 | Applicant | |
| WO90/09180(A1) | 1990-08-01 | Applicant | |
| WO90/09786(A1) | 1990-09-01 | Applicant | |
| WO90/11092(A1) | 1990-10-01 | Applicant | |
| WO91/04668(A1) | 1991-04-01 | Applicant | |
| WO91/04753(A1) | 1991-04-01 | Applicant | |
| WO91/08191(A1) | 1991-06-01 | Applicant | |
| WO91/15501(A1) | 1991-10-01 | Applicant | |
| WO91/16024(A1) | 1991-10-01 | Applicant | |
| WO91/17424(A1) | 1991-11-01 | Applicant | |
| WO92/06188(A2) | 1992-04-01 | Applicant | |
| WO92/06200(A1) | 1992-04-01 | Applicant | |
| WO92/13570(A2) | 1992-08-01 | Applicant | |
| WO92/20697(A1) | 1992-11-01 | Applicant | |
| WO92/21752(A1) | 1992-12-01 | Applicant | |
| WO92/22635(A1) | 1992-12-01 | Applicant | |
| WO93/03709(A1) | 1993-03-01 | Applicant | |
| WO93/05162 | 1993-03-01 | Applicant | |
| WO93/05162(A1) | 1993-03-01 | Applicant | |
| WO93/07282(A1) | 1993-04-01 | Applicant | |
| WO93/07283(A1) | 1993-04-01 | Applicant | |
| WO93/08130(A1) | 1993-04-01 | Applicant | |
| WO93/14778 | 1993-08-01 | Applicant | |
| WO93/19768(A1) | 1993-10-01 | Applicant | |
| WO94/02499(A1) | 1994-02-01 | Applicant | |
| WO94/04696(A1) | 1994-03-01 | Applicant | |
| WO94/05624(A1) | 1994-03-01 | Applicant | |
| WO94/07899(A1) | 1994-04-01 | Applicant | |
| WO94/08004(A1) | 1994-04-01 | Applicant | |
| WO94/14475(A1) | 1994-07-01 | Applicant | |
| WO94/17093(A1) | 1994-08-01 | Applicant | |
| WO94/23751(A1) | 1994-10-01 | Applicant | |
| WO94/27435 | 1994-12-01 | Applicant | |
| WO94/27435(A1) | 1994-12-01 | Applicant | |
| WO95/02397(A1) | 1995-01-01 | Applicant | |
| WO95/16028(A1) | 1995-06-01 | Applicant | |
| WO95/17373 | 1995-06-01 | Applicant | |
| WO95/20682(A1) | 1995-08-01 | Applicant | |
| WO95/21259(A1) | 1995-08-01 | Applicant | |
| WO95/24221(A1) | 1995-09-01 | Applicant | |
| WO95/31557(A1) | 1995-11-01 | Applicant | |
| WO96/01841(A1) | 1996-01-01 | Applicant | |
| WO96/05218(A1) | 1996-02-01 | Applicant | |
| WO96/08723(A1) | 1996-03-01 | Applicant | |
| WO96/10038(A1) | 1996-04-01 | Applicant | |
| WO96/10640(A1) | 1996-04-01 | Applicant | |
| WO96/15811(A1) | 1996-05-01 | Applicant | |
| WO91/07947(A1) | 1996-06-01 | Applicant | |
| WO96/22321(A1) | 1996-07-01 | Applicant | |
| WO96/22765(A1) | 1996-08-01 | Applicant | |
| WO96/29337(A1) | 1996-09-01 | Applicant | |
| WO96/31549(A1) | 1996-10-01 | Applicant | |
| WO96/32474(A1) | 1996-10-01 | Applicant | |
| WO96/35706(A1) | 1996-11-01 | Applicant | |
| WO96/40961 | 1996-12-01 | Applicant | |
| WO97/05265(A1) | 1997-02-01 | Applicant | |
| WO97/09451(A1) | 1997-03-01 | Applicant | |
| WO97/42819 | 1997-11-01 | Applicant | |
| WO98/02190 | 1998-01-01 | Applicant | |
| WO98/06736(A1) | 1998-02-01 | Applicant | |
| WO98/14439(A1) | 1998-04-01 | Applicant | |
| WO98/19709 | 1998-05-01 | Applicant | |
| WO98/32866(A1) | 1998-07-01 | Applicant | |
| WO98/29541 | 1998-09-01 | Applicant | |
| WO98/40499 | 1998-09-01 | Applicant | |
| WO98/40502 | 1998-09-01 | Applicant | |
| WO98/47912(A1) | 1998-10-01 | Applicant | |
| WO99/02190 | 1999-01-01 | Applicant | |
| WO99/05302(A1) | 1999-02-01 | Applicant | |
| WO99/11809(A1) | 1999-03-01 | Applicant | |
| WO99/24559(A2) | 1999-05-01 | Applicant | |
| WO99/29712 | 1999-06-01 | Applicant | |
| WO99/41410(A1) | 1999-08-01 | Applicant | |
| WO99/46400(A1) | 1999-09-01 | Applicant | |
| WO/12454 | 2000-03-01 | Applicant | |
| WO/58488 | 2000-10-01 | Applicant | |
| WO/58488(A2) | 2000-10-01 | Applicant | |
| WO/58488(A3) | 2001-01-01 | Applicant | |
| WO1/07548 | 2001-02-01 | Applicant | |
| WO2/34879 | 2002-05-01 | Applicant |
Non-Patent Literature (309)
- Felgner et al. (1993) Keystone Symposium on Genetic Targeted Research and Therapeurics: Antisense and Gene Therapy, Keystone, CO; J. Cell. Biochem. Suppl. 0(17 Part E), p. 206, S306.Applicant
- Gao, X. and Huang, L. (1993), “Cationic Liposomes and Polymers for Gene Transfer,” J. Liposome Res. 3(1):17-30.Applicant
- Menkel und Cie. G.m.b.M., abstract No. 68522p, “Low-foaming detergents containing bisquaternary compounds,” Chem. Abst. 72(14):116 (Apr. 1970).Applicant
- McCluskie, M, et al., abstract No. 91045k, “Direct gene transfer to the respitatpry tract of mice with pure plasmid and lipid-formulated DNA,” Chem. Abst. 130:151 (Feb. 1999).Applicant
- International Search Report for International Application No. PCT/US99/26825, mailed Feb. 22, 2000.Applicant
- Bangham, A.D. et al., “Diffusion of Univalent Ions across the Lamellae of Swollen Phospholipids,” J. Mol. Biol. 13:238-252 (1965).Applicant
- Behr, J.-P. et al., “Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-caoted DNA,” Proc. Natl. Acad. Sci. USA 86:6982-6986 (Sep. 1989).Applicant
- Felgner, P.L. et al., “Lipofection: A highly efficient, lipid-mediated DNA—transfection procedure,” Proc. Natl. Acad. Sci. USA 84:7413-7417 (Nov. 1987).Applicant
- Fukunaga, M. et al., “Liposome Entrapment Enhances the Hypocalcemic Action of Parenterally Administered Calcitonin,” Endocrinal. 115:757-761 (1984).Applicant
- Geo, X. and Muang, L., “A Novel Cationic Liposome Reagent for Efficient Transfection of Mammalian Cells,” Biochem. Biophys. Res. Comm. 179:280-285 (Aug. 1991).Applicant
- Hart, S.L. et al., “Lipid-Mediated Enhancement of Transfection by a Nonviral Integrin-Targeting Vector,” Hum. Gene Ther. 9:575-585 (Mar. 1998).Applicant
- Kim S. et al., “Preparation of Multivesicular Liposomes,” Biochim. Biophys. Acta 728:339-348.Applicant
- Mayer, L.D. et al., “Vesicles of variable sizes produced by a rapid extrusion procedure,” Biochim. Biophys. Acta 858:161-168 (1986).Applicant
- Mayhew, E. et al., “Characterization of Liposomes Prepared Using Micromulsifier,” Biochim. Biophys. Acta 775:169-174 (1984).Applicant
- Olson, F. et al., “Preparation of Liposomes of Defined Size Distribution by Extrusion Through Polycarbonate Membranes,” Biochim. Biophys. Acta 557:9-23 (1979).Applicant
- Rosenthal, A.F. and Geyer, R.P., “A Synthetic Inhibitor of Venom Lecithinase A,” J. Biol. Chem. 235:2202-2206 (Aug. 1960).Applicant
- Szoka Jr., F. and Papehadjopoulos, D., “Procedure for preparation of tipsomes with large internal aqueous space and high capture by reverse-phase evaporation,” Proc. Natl. Acad. Sci. USA 75:4194-4198 (1978).Applicant
- Zhou, X. et al., “Lipophilic polylysines mediate efficient DNA transfection in mammalian cells,” Biochim. Biophys. Acta 1065:8-14 (1991).Applicant
- Life Technologies Inc. catalogue, Spectrum, pp. 1-8 (Sep. 1999).Applicant
- Albrecht, T., et al., “Cationic lipide mediated transfer of c-abl and bcr antisense oligonucleotides to immature normal myeloid cells: uptake, biological effects and modulation of gene expression,” Ann. Hematol. 72:73-79, Springer-Verlag (1996).Applicant
- Aslanyan, V.M. et al., “Conformation and Thermal Stability of DNA in Aqueous Solutions of β-Alanine and γ-Aminobutyric Acid,” Biophysics 29:615-620, Pergamon Press, Ltd. (1984).Applicant
- Astatke, M., et al., “How E. coli DNA Polymerase I (Klenow fragment) Distinguishes between Deoxy- and Dideoxynucleotides,” J. Mol. Biol. 278:147-165, Academic Press, Ltd. (Apr. 1998).Applicant
- Barnes, W.M., “The fidelity of Tag polymerase catalyzing PCR is improved by an N-terminal deletion,” Gene 112:29-35, Elsevier Science Publishers B.V. (1992).Applicant
- Baskaran, N., et al., “Uniform Amplification of a Mixture of Deoxyribonucleic Acids with Varying GC Content,” Genome Res. 6:633-638, Cold Spring Harbor Laboratory Press (1996).Applicant
- Behr, J.P., “Gene Transfer with Synthetic Cationic Amphiphiles: Prosepects for Gene Therapy,”Bioconjug. Chem. 5:382-389, American Chemical Society (1994).Applicant
- Benoist, C., et al., “In vivo sequence requirements of the SV40 early promoter region,” Nature 290:304-310, Macmillan Journals, Ltd. (1991).Applicant
- Bonfanti, M., et al., “p21KAFI -derived Peptides Linked to an Internalization Peptide Inhibit Human Cancer Cell Growth.” Cancer Res. 57:1442-1446, American Association for Cancer Research (Apr. 1997).Applicant
- Bottger, M., et al., “Condensation of vector DNA by the chromosomal protein HD4G1 results in efficient transfection,” Biochim. Biophys. Acta 950:221-228, Elsevier Science Publishers B.V. (1988).Applicant
- Boussif, O., et al., “A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine,” Proc. Natl. Acad. Sci. USA 92:7297-7301, National Academy Press (1995).Applicant
- Brunette, E., et al., “Lipofection does not require the removal of serum,” Nucleic Acids Res. 20:1151. Oxford University Press (1992).Applicant
- Buche, A., et al., “Glycine and other amino compounds prevent chromatin precipitation at physiological ionic strength,” FEBS Lett. 247:367-370, Elsevier Science Publishers S.V. (1989).Applicant
- Buche, A., et al., “Organic Osmotic Effectors and Chromatin Scructure,” J. Biomol. Struct. & Dynam. 8:601-618, Adenine Press (1990).Applicant
- Buche, A., et al., “Effect of Organic Effectors on Chromatin Solubility, DNA Histone HI Interactions, DNA and Histone H1 Structures,” J. Biomol. Struct. & Dynam. II:95-119, Adenine Press (1993).Applicant
- Cánovas, D., et al., “Osmoprotectants in Halomonas elongata: High-Affinity Betaine Transport System and Choline-Betaine Pathway,” J. Bacteriol. 178:7221-7226, American Society for Microbiology (1996).Applicant
- Cánovas, D., et al., “Isolation and Characterization of Salt-sensitive Mutants of the Moderate Halophile Halomonas elongata and Cloning of the Ectoine Synthesis Genes,” J. Biol. Chem. 272:25794-25801. The American Society for Biochemistry and Molecular Biology, Inc. (Oct. 1997).Applicant
- Carninci, P., et al., “Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA,” Proc. Natl. Acad. Sci. USA 95:520-524, National Academy Press (Jan. 1998).Applicant
- Chambers, S.T., et al., “Dimethylthetin Can Substitute for Glycine Betaine as an Osmoprotectant Molecule for Escherichia coli,” J. Bacteriol. 169:4845-4847, American Society for Microbiology (1987).Applicant
- Chen, X., et al., “A self-initiating eukaryocic transient gene expression system based on cotransfection of bacteriophage T7 RNA polymerase and DNA vectors containing a T7 autogene,” Nucleic Acids Res. 22:2114-2120, Oxford University Press (1994).Applicant
- Cohen, J. “Naked DNA Points Way to Vaccines,” Science 259:1691-1692, American Association for the Advancement of Science (1993).Applicant
- Cotten, M., and Wagner, E., “Non-viral approaches to gene therapy,”Curr. Opin. Biotechnol. 4:705-710, Current Biology, Ltd. (1993).Applicant
- Deamer, D.W., and Uster, P.S., “Liposome Preparation: Methods and Mechanisms,” in Liposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, NY, pp. 27-51 (1983).Applicant
- Dean, N.M., and McKay, R., “Inhibition of Protein Kinase C-α expression in mice after systemic administration of phosohorothioate antisense oligodeoxynucleotides,” Proc. Natl. Acad. Sci. USA 91:11762-11766, National Academy Press (1994).Applicant
- Elliot, G., and O'Hare P., “Intercellular trafficking of VP22-GFP fusion proteins,” Gene Ther. 6:149-151, Stockton Press (Jan. 1999).Applicant
- Felgner, P.L., and Holm, M., “Cationic Liposome-Mediated Transfection,” Focus 11:21-25, Life Technologies, Inc. (1989).Applicant
- Felgner, P.L., and Rhodes, G., “Gene therapeutics,” Nature 349:351-352, Macmillan Journals, Ltd. (1991).Applicant
- Flaman, J-M., et al., “A rapid PCR fidelity assay,” Nucleic Acids Res. 22:3259-3260, Oxford University Press (1994).Applicant
- Flock, S., et al., “Osmotic Effectors and DNA Structure: Effect of Glycine on Precipitation of DNA by Multivalent Cations” J. Biomol. Struct. Dyn. 11:87-102, Adenine Press (1995).Applicant
- Flock, S. et al., “Dielectric Constant and Ionic Strength Effects on DNA Precipitation,” Biophys. J. 70:1456-1465, Biophysical Society (1996).Applicant
- Flock, S., et al., “13Na NMR Study of the Effect of Organic Osmolytes on DNA Counterion Atmosphere,” Biophys. J. 71:1519-1529, Biophysical Society (1996).Applicant
- Foecking, M.K., and Hofstetter, H., “Powerful and versatile enhancer-promoter unit for mammalian expression vectors,” Gene 45:101-105, Elsevier Science Publishers B.V. (1986).Applicant
- Frackman, S., et al., “Betaine and DMSO: Enhancing Agents for PCR,” Promega Notes 65:27-29, Promega (Feb. 1998).Applicant
- Fritz, J.D., et al., “Gene Transfer into Mammalian Cells Using Histone-Condensed Plasmid DNA,” Hum. Gene. Ther. 7:1195-1404, Mary Ann Liebart, Inc. (1996).Applicant
- Giles, R.V., “Antisense oligonucleotide technology: From EST to therapeutics,” Curr. Opin. Mol. Ther. 2:238-252, PharmaPress, Ltd. (Jun. 2000).Applicant
- Goldman, C.K., et al., “In vitro and in vivo gene delivery mediated by a synthetic polycationic amino polymer,” Nat. Biotechnol. 15:462-466, Nature America Publishing (May 1997).Applicant
- Gorman,. C., “High Efficiency Gene Transfer into Mammalian Cells,” in DNA Cloning, vol. II, Glover, D.M., ed., IRL Press, Washington, D.C., pp. 143-190.Applicant
- Gouesbet, G., et al., “Characterization of the Erwinia chrysanthemi Osmoprotectant Transporter Gene ousA,” J. Bacteriol. 178:447-455, American Society for Microbiology (1996).Applicant
- Gublar, U , and Hoffman, B.J., “A simple and very efficient method for generating cDNA libraries,” Gene 25:263-269, Elsevier Science Publishers (1983).Applicant
- Haensler, J., and Szoka Jr., F.C., “Polyamidoamine Cascade Polymers Mediate Efficient Transfection of Cells in Culture,” Bioconjug. Chem. 4:372-379, American Chemical Society (1993).Applicant
- Harrison, G.S., et al., “Optimization of Gene Transfer Using Cationic Lipids in Cell Lines and Primary Human CD4′ and CD13′ Hematopoietic Cells,” BioTechniques 19:816-823, Eaton Publishing Company (1995).Applicant
- Hazinski, T.A., et al., “Localization and Induced Expression of Fusion Genes in the Rat Lung,” Am. J. Respir. Cell Mol. Biol. 4:206-209, American Thoracic Society (1991).Applicant
- Hengen, P.N., “Optimizing multiplex and LA-PCR with betaine,” Trends Biochem. Sci. 22:225-226, Elsevier Science, Ltd. (Jun. 1997).Applicant
- Henke, W., et al., “Betaine improves the PCR amplification of GC-rich DNA sequences,” Nucl. Acids Res. 25:3957-3958, Oxford University Press (Oct. 1997).Applicant
- Heywood, S.M., “tcRNA as a naturally occurring antisense RNA in eukaryotes,” Nucleic Acids Res. 14:6771-6772, IRL Press (1986).Applicant
- Hografe, W., et al., “Novel PCR Enhancing Factor Improves Performance of Pfu DNA Polymerase,” Strategies 10:93-96, Stratagene Cloning Systems (Aug. 1997).Applicant
- Holt, C.E., et al., “Lipofection of cDNAs in the Embryonic Vertebrate Central Nervous System,” Neuron 4:203-214, Cell Press (1990).Applicant
- Hope, M.J., et al., “Generation of Multilamellar and Unilamellar Phospholipid Vesicles,” Chem. Phys. Lipids 40:89-107, Elsevier Scientific Publishers Ireland, Ltd. (1986).Applicant
- Houssier, C., et al., “Effects of Compensatory Solutes on DNA and Chromatin Structural Organization in Solution,” Comp. Biochem. Physiol. 117A:313-318, Elsevier Science, Inc. (Jul. 1997).Applicant
- Houts, G.E., et al., “Reverse Transcriptase from Avian Myeloblastosis Virus,” J. Virol. 29:517-522, American Society for Microbiology (1979).Applicant
- Iakobashvili, R., and Lapidot, A., “Low temperature cycled PCR protocol for Klenow fragment of DNA polymerase I in the presence of proline, ” Nucleic Acids Res. 27:1566-1568. Oxford University Press (Mar. 1999).Applicant
- Innis, M.A., “PCR with 7-Deaza-2′ -Deoxyguanosine Triphoshate,” in PCR Protocols: A Guide to Methods and Applications. Innis, M.A., et al., eds., Academic Press. Inc., San Diego, CA, pp. 54-59 (1990).Applicant
- Iyer, R.P., et al., “The Automated Synthesis of Sulfur-Containing Oligodeoxyribonucleotides Using 3H-1,2-Benzodithiol-3-one 1,1-Dioxide as a Sulfur-Transfer Reagent,” J. Am. Chem. Soc. 55:4691-4699, American Chemical Society (1990).Applicant
- Iyer, R.P., et al., “3H-1,2-Benzodithiole-3-one 1,1-Dioxide as an Improved Sulfurizing Reagent in the Solid-Phase Synthesis of Oligodeoxyribonucleoside Phosphorothioates,” J. Am. Soc. 112:1253-1254, American Chemical Society (1990).Applicant
- Jakob, R., and Saenger, W., “Reversed-phase ion-pair chromotographic separation of ribulose 1,5-bisphosphate from 3-phosphoglycerate and its application as a new enzyme assay for RuBP carboxylase/oxygenase,” FEBS Lett. 183:111-114, Elsevier Science Publishers B.V. (1985).Applicant
- Johnstone, A., and Thorpe, R., “Production of antibodies,” in Immunochemistry in Practice, Johnstone A., and Thorpe, R., eds., Blackwell Scientific Publications, Oxford, Great Britain, pp. 30-47, (1987).Applicant
- Joung, I., and Engler, J.A., “Mutations in Two Cysteine-Histidine-Rich Clusters in Adenovirus Type 2 DNA Polymerase Affect DNA Binding,” J. Virol. 66:5788-5796, American Society for Microbiology (1992).Applicant
- Kondakova, N.V., et al., “Effect of Low-molecular Amines on the Conformation and Stability of the Double Helix,” Mol. Biol. (Mosk.) 9:742-746, Izdatelatvo Nauka (1975).Applicant
- English Language Translation for Kondakova, N.V., et al., “Effect of Low-molecular Amines on the Conformation and Stability of the Double Helix,” Mol. Biol. (Mosk:) 9:742-746, Izdatelstvo Nauka (1975) (Document AR77).Applicant
- Kotewicz, M.L., et al., “Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity,” Nucleic Acids Res. 16:265-277, IRL Press, Ltd. (1988).Applicant
- Landre, P.A., et al., “The use of cosolvents to enhance amplification by the polymerase chain reaction,” in PCR Strategies, Innis, M.A., et al., eds., Academic Press, San Diego, CA, pp. 1-16 (1995).Applicant
- Langel, U., et al., “Cell penetrating PNA constructs,” J. Neurochem. 69 (suppl) :S260, Absract B, Lippincott-Raven Publishers(Jul. 1997).Applicant
- Lau, Q.C., et al., “Abrogation of c-Raf expression induces apoptosis in tumor cells,” Oncogene 16:1899-1902, Stockton Press (Apr. 1998).Applicant
- Lawyer, F.C., et al., High-level Expression, Purification, and Enzymatic Characterization of Full-Length Thermus aquaticus DNA Polymerase and a Truncated Form Deficient in 5′ to 3′ Exonuclease Activity, PCR Methods Appl. 2:275-287, Cold Spring Harbor Laboratory Press (1993).Applicant
- Le Rudulier, D. et al., “Molecular Biology of Osmoregulation,” Science 224:1064-1068, American Association for the Advancement of Science (1984).Applicant
- Li, C. -J., et al., “Nonprotein amino acids from seeds of Cycas circinalis and Phaseolus vulgaris,” Phytochemistry 42:443-445, Pergamon Press (1996).Applicant
- 1995-1996 GIBCO/BRL Product Catalogue and Reference Guide. pp. 19-10 and 19-12, Life Technologies, Inc. (1995).Applicant
- Liu, X., et al., “Hypoglycemia-induced c-Jun Phosphorylation Is Mediated by c-Jun N-terminal Kinase 1 and Lyn Kinase in Drug-resistant Human Breast Carcinoma MCF-7/ADR Cells,” J. Biol. Chem. 272:11690-11693, The American Society for Biochemistry and Molecular Biology, Inc. (May 1997).Applicant
- Malin, G., and Lapidot, A., “Induction of Sysnthesis of Tetrahydropyrimidine Derivatives in Streptomyces Strains and Their Effect on Escherichia coli in Response to Osmotic and Heat Stress,” J. Bacteriol. 178:385-395, American Society for Microbiology (1996).Applicant
- Maniatis, T., et al., “The Isolation of Structural Genes from Libraries of Eucaryotic DNA,” Cell 15:687-701, MIT Press (1978).Applicant
- Marquet: R. and Houssier, C.; “Thermodynamics of Cation-Induced DNA Condensation,” J. Biomol. Struct. & Dynam. 9:159-167, Adenine Press (1991).Applicant
- Mizuno, T., et al., “A unique mechanism regulating gene expression: Translational inhibition by a complementary RNA transcript (micRNA),” Proc. Natl. Acad. Sci. USA 81:1966-1970, National Academy Press (1984).Applicant
- Monia, B.P., et al., “Sequence-specific antitumor activity of a phosphorothioate oligodeoxyribonucleotide targeted to human C-raf kinase supports an antisense mechanism of action in vivo,” Proc. Natl. Acad. Sci. USA 93:15481-15484, National Academy Press (1996).Applicant
- Murphy, A.L., and Murphy, S.J., “Catch V322: the hitch-hiker's ride to gene therapy?” Gene Ther. 6:4-5, Nature Publishing Group (Jan. 1999).Applicant
- Mycelka, D.S., and Chamberlin, M.J., “Analysis and suppression of DNA polymerase pauses associated with a trinucletide consensus,” Nucleic Acids Res. 24:2774-2781, Oxford University Press (1996).Applicant
- McCluskie, M.J., et al., “Direct Gene Transfer to the Respiratory Tract of Mice with Pure Plasmid and Lipid-Formulated DNA,” Antisense Nucleic Acid Drug Dev. 8:401-414, Mary Ann Liebert, Inc. (Oct. 1998).Applicant
- Nabel, G.J., et al., “Direct gene transfer for treatment of human cancer,” Ann. NY Acad. Sci. 792:227-231, New York Academy of Sciences (1995).Applicant
- Nabel, G.J., et al., “Immune responses in human melanoma after transfer of an allogeneic class I major histocompatibility complex gene with DNA-liposome complexes,” Proc. Natl. Acad. Sci. USA 93:15388-15393. National Academy Press (1996).Applicant
- Neckers, L.M., “Cellular Internalization of Oligodeoxynucleotides,” in Antisense Research and Applications, Crooke, S.T. and Lebleu, B., eds., CRC Press, Inc., Boca Ration, FL, pp. 451-460 (1993).Applicant
- Neurath, A.R. et al., “B cell epitope mapping of human immunodeficiency virus envelope glycoproteins with long (19- to 36-residue) synthetic peptides,” J. Gen. Virol. 71:85-95, Society for General Microbiology (1990).Applicant
- Niidome, T., et al., “Binding of Cationic α-Helical Peptides to Plasmid DNA and Their Gene Transfer Abilities into Cells,” J. Biol. Chem. 272:15307-15312, The American Society for Biochemistry and Molecular Biology, Inc. (Jun. 1997).Applicant
- Okayama, H., and Berg, P., “High-Efficiency Cloning of Full-Length cDNA,” Mol. Cell. Biol. 2:161-170, American Society for Microbiology (1982).Applicant
- Panaccio, M., et al., “FoLT PCR: A Simple PCR Protocol for Amplifying DNA Directly from Whole Blood,” BioTechniques 14:238-243, Eaton Publishing Company (1993).Applicant
- Paterson, B.M., et al., “Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free translation,” Proc. Natl. Acad. Sci. USA 74:4370-4374, National Academy Press (1977).Applicant
- Polesky, A.H., et al., “Identification of Residue Critical for the Polymerase Activity of the Klenow Fragment of DNA Polymerase I from Escherichia coli,” J. Biol. Chem. 265:14579-14591, The American Society for Biochemistry and Molecular Biology, Inc. (1990).Applicant
- Prochiantz, A., “Getting Hydrophilic compounds into cells, lessons from homeopeptides,” Curr. Opin. Neurobiol. 6:629-634, Current Biology, Ltd. (1996).Applicant
- Prochiantz, A., “Peptide nucleic acid smugglers,” Nat. Biotechnol. 16:819-820, Nature America Publishing (Sep. 1998).Applicant
- Rafferty, J.A., and Fletcher, H.L., “Sequence analysis of a family of highly repeated DNA units in Stauroderus scalaris (Orthoptera)” Intl. J. Genome Res. 1:1-16, World Scientific Publishing Company (1992).Applicant
- Rajendrakumar, C.S.V., et al., “DNA helix destabilization by proline and betaine, possible role in the salinity tolerance process,” FEBS Lett. 410:201-205, Elsevier Science B.V. (Jun. 1997).Applicant
- Randall, K., et al., “Accumulation of natural and synthetic betaines by a mammalian renal cell line,” Biochem. Cell Biol. 74:283-287, National Research Council of Canada (1996).Applicant
- Randall, K., et al., “Natural and synthetic betaines counter the effects of high NaCl and urea concentrations,” Biochim. Biophys. Acta 1291:189-194, Elsevier Science B.V. (1996).Applicant
- Rees, W.A., et al., “Betaine Can Eliminate the Base Pair Composition Dependence of DNA Melsing,” Biochemistry 12:137-144, American Chemical Society (1991).Applicant
- Rothenberg, M., et al., “Oligodeoxynucleotides as Anti-Sense Inhibitors of Gene Expression: Therapeutic Implications,” J. Natl. Cancer Inst. 81:1539-1544, Oxford University Press (1989).Applicant
- Saiki, R.K., et al., “Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase,” Science 239:487-491, American Association for the Advancement of Science (1988).Applicant
- Shepard, A.R. and Rae, J.L., “Magnetic bead capture of cDNAs from double-stranded plasmid cDNA libraries,” Nucleic Acids Res. 25:3183-3185, Oxford University Press (Aug. 1997).Applicant
- Bioactive Peptides, 1993 Sigma Chemical Company Catalogue, St. Louis, MO, pp. 1028-1034, Sigma Chemical Company (1993).Applicant
- Soltis, D.A., and Skalka, A.M., “The α and β chains of avian retrovirus reverse transcriptase independently expressed in Escherichia coli: Characterization of enzymatic activities,” Proc. Natl. Acad. Sci. USA 85:3372-3376, National Academy Press (1988).Applicant
- Stopeck, A.T., et al., “Phase I Study of Direct Gene Transfer of an Allogeneic Histocompatibility Antigen, HLA-B7, in Patients With Metastatic Melanoma,” J. Clin. Oncol. 15:341-349, American Society of Clinical Oncology (Jan. 1997).Applicant
- Szoka Jr., F., and Papahadjopoulos D., “Comparative properties and methods of preparation of lipid vesicles (liposomes),” Annu. Rev. Biophys. Bioeng. 9:467-508, Annual Reviews, Inc. (1980).Applicant
- Tang, D.-c., et al., “Genetic immunization is a simple method for eliciting an immune response,” Nature 356:152-154, Nature Publishing Group (1992).Applicant
- Thompson, L., “A Shot in the Arm for Vaccine Problems,” Washington Post, p. AO3 (Jun. 7, 1993).Applicant
- Uhlmann, E., and Peyman, A., “Antisense Oligonucleotides: A New Therapeutic Principle,” Chem. Rev. 90:544-584, American Chemical Society (1990).Applicant
- Ulmer, J.B. et al., “Heterologous Protection Against Influenza by Injection of DNA Encoding a Viral Protein,” Science 259:1745-1749, American Association for the Advancement of Science (1993).Applicant
- Urdea, M.S., “Branched DNA Signal Amplification,” Bio/Technology 12:926-928, Nature Publishing Company (1994).Applicant
- van der Krol, A.R., et al., “Modulation of Eukaryotic Gene Expression by Complementary RNA or DNA Sequences,” BioTechniques 6:958-976, Eaton Publishing Company (1988).Applicant
- Varadaraj, K. and Skinner, D.M., “Denaturants or cosolvents improve the specificity of PCR amplification of a G-C-rich DNA using genetically engineered DNA polymerases,” Gene 140:1-5, Elsevier Science B.V. (1994).Applicant
- Voytik-Harbin, S.L., et al., “Identification of Extractable Growth Factors From Small Intestinal Submucosa,” J. Cell. Biochem. 67:478-491, Wiley-Liss, Inc. (Dec. 1997).Applicant
- Wagner, E., et al., “Transferrin-polycation-DNA complexes: The effect of polycations on the structure of the complex and DNA delivery to cells,” Proc. Natl. Acad. Sci. USA 88: 4255-4259, National Academy Press (1991).Applicant
- Wang, B. et al., “Gene inoculation generates immune response against human immunodeficiency virus type 1,” Proc. Natl. Acad. Sci. USA 90:4156-4160, National Academy Press (1993).Applicant
- Weissensteiner, T., and Lanchbury, J.S., “Strategy for Controlling Preferential Amplification and Avoiding False Negatives in PCR Typing,” BioTechniques 21:1102-1108, Eaton Publishing Company (1996).Applicant
- Wen, W., et al., “Identification of a Signal for Rapid Export of Proteins from the Nucleus,” Cell 82:463-473, Cell Press (1995).Applicant
- Wickstrom, E.L., et al., “Human promyelocytic leukemia HL-60 cell proliferation and c-myc protein expression are inhibited by an antisense pentadecadeoxynucleotide targeted against c-myc mRNA,” Proc. Natl. Acad. Sci. USA 85:1028-1032, National Academy Press (1988).Applicant
- Wickstrom, E., et al., “Down-Regulation of c-MYC Antigen Expression in Lymphocytes of Eu-c-myc Transgenic Mice Treated with Anti-c-myc DNA Methylophosphonates,” Cancer Res. 52:6741-6745, American Association for Cancer Research (1992).Applicant
- Woodford, K., et al., “The Use of κ' -free buffers eliminates a common cause of premature chain termination in PCR and PCR sequencing,” Nucleic Acids Res. 23:539, Oxford University Press (1995).Applicant
- Zaitsev, S.V., et al., “H1 and HMG17 extracted from calf thymus nuclei are efficient DNA carriers in gene transfer,” Gene Ther. 4:586-592, Stockton Press (Jun. 1997).Applicant
- “Voyager™—The Power of Translocation,” Expressions 6:6, Life Technologies Inc. (Feb. 1999).Applicant
- Dialog File 351, Accession No. 8899481, Derwent WPI English language abstract for WO 93/08130 A1 (Document AO12).Applicant
- Dialog File 351, Accession No. 10428036, Derwent WPI English language abstract for DE 44 11 588 C1 (Docment AN14).Applicant
- Dialog File 351, Accession No. 10524091, Derwent WPI English language abstract for DE 44 11 594 C1 (Document AP14).Applicant
- Dialog File 351, Accession No. 10655226, Derwent WPI English language abstract for WO 96/08723 A1 (Document AL15).Applicant
- Co-pending U.S. Appl. No. 09/266,935, filed Mar. 12, 1999, inventors Li et al. (Not Published).Applicant
- Co-pending U.S. Appl. No. 09/570,526, filed May 12, 2000, inventors Astatke et al., (Not Published).Applicant
- Co-pending U.S. Appl. No. 09/608,066, filed Jun. 30, 2000, inventors Astatke et al., (Not Published).Applicant
- Albarella, J.P. et al., “Monoadduct forming photochemical reagents for labeling nucleic acids for hybridization,” Chemical Abstracts 111:385-386, Abstract No. 130176h, American Chemical Society (1989).Applicant
- Aumailley, M., et al., “Cell-Attachment Properties of Collagen Type VI and Arg-Gly-Asp Dependent Binding to its α2(VI) and α3(VI) Chains,” Exp. Cell Res. 181:463-474, Academic Press, Inc. (1989).Applicant
- Barthel, F., et al., “Laboratory Methods: Gene Transfer Optimization with Lipospermine-Coated DNA,” DNA Cell Biol. 12:553-560, Mary Ann Liebert, Inc. (1993).Applicant
- Behr, J-P., “Synthetic Gene-Transfer Vectors,” Acc. Chem. Res. 26:274-278, American Chemical Society (1993).Applicant
- Bielinska, A., et al., “Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers,” Nucleic Acids Res. 24:2176-2182, Oxford University Press (1996).Applicant
- Bonifaci, N., et al., “Nuclear translocation of an exogenous fusion protein containing HIV Tat requires unfolding,” AIDS 9:995-1000, Rapid Science Publishers (1995).Applicant
- Braunlin, W.H., et al., “Equilibrium Dialysis Studies of Polyamine Binding to DNA,” Biopolymers 21:1301-1314, John Wiley & Sons, Inc. (1982).Applicant
- Budkar, V., et al., “Protein/Amphipathic Polyamine Complexes Enable Highly Efficient Transfection with Minimal Toxicity,” Biotechniques 23:139-147, Eaton Publishing Company (Jul. 1997).Applicant
- Caminati, G., et al., “Photophysical Investigation of Starburst Dendrimers and Their Interactions with Anionic and Cationic Surfactants,” J. Am. Chem. Soc. 112:8515-8522, American Chemical Society (1990).Applicant
- Carrasco, L., “Modification of Membrane Permeability Induced by Animal Viruses Early in Infection,” Virology 113:623-629, Academic Press, Inc. (1981).Applicant
- Carrasco, L., and Eateban, M., “Modification of Membrane Permeability in Vaccinia Virus-Infected Cells,” Virology 117:62-69, Academic Press, Inc. (1982).Applicant
- Ciccarone, V., et al., “Cationic liposome-mediated transfection of eukaryotic cells: High efficency nucleic acid delivery with lipofectin® lipofectace™, and lipofectamine™ reagents,” FASEB J. 7:A1131, Abstract No. 454, Federation of American Societies for Experimental Biology (1993).Applicant
- Ciccarone, V., et al., “DMRIE-C reagent for transfection of suspension cells and for RNA transfections,” Focus 17:84-87, Life Technologies, Inc. (1995).Applicant
- Cicovsky, V., et al., “Nuclear Localization of Agrobacterium VirE2 Protein in Plant Cells,” Science 256:1802-1805, American Association for the Advancement of Science (1992).Applicant
- Cotten, M., et al., “Transferrin-polycation-mediated introduction of DNA into human leukemic cells: Stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels,” Proc. Natl. Acad. Sci. USA 87:4033-4037, National Academy Press (1990).Applicant
- Cotten, M., et al., “High-efficiency receptor-mediated delivery of small and large (48 kilobase gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles,” Proc. Natl. Acad. Sci. USA 89:6094-6098, National Academy Press (1992).Applicant
- Curiel, D.T., et al., “Adenovirus enhancement of transferrin-polylysine-mediated gene delivery,” Proc. Natl. Acad. Sci. USA 88:8850-8854, National Academy Press (1991).Applicant
- Curiel, D.T., et al., “High-Efficiency Gene Transfer Mediated by Adenovirus Coupled to DNA-Polylysine Complexes,” Hum. Gene Ther. 3:147-154, Mary Ann Liebert, Inc. (1992).Applicant
- Curiel, D.T., et al., “Gene Transfer to Respiratory Epithelial Cells via the Receptor-mediated Endocytosis Pathway,” Am. J. Respir. Cell Mol. Biol. 5:247-252, American Thoracic Society (1992).Applicant
- Dattagupts, N., and Albarella, J.P., “Photochemical nucleic acid-labeling reagent having a polyalkylamine spacer,” Chemical Abstracts 114:383, Abstract No. 114:78227w, American Chemical Society (1991).Applicant
- Dayhoff, M.O., et al., “A Model of Evolutionary Change in Proteins,” in Atlas of Protein Sequence and Structure, vol. 5, Suppl. 3, Dayhoff, M.O., ed., National Biomedical Research Foundation, Washington, D.C. pp. 345-352 (1978).Applicant
- De Roberts, E.M., et al., “Intracellular migration of nuclear proteins in Xenopus oocytes,” Nature 272:254-256, Macmillan Journals, Ltd. (1978).Applicant
- Dedhar, S., et al., “A Cell Surface Receptor Complex for Collagen Type I Recognizes the Arg-Gly-Asp Sequence” J. Cell Biol. 104:585-593, The Rockefeller University Press (1987).Applicant
- Demeneix, B.A., et al., “Gene Transfer into intact vertebrate embryos,” Int. J. Dev. Biol. 35:481-484, UBC Press (1991).Applicant
- Dingwall, C., and Laskey, R.A., “Nuclear targeting sequences—a consensus?” Trends Biochem. Sci. 16:478-481, Elsevier Science Publishers, (UK) (1991).Applicant
- Dingwall, C., et al., “Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro,” Proc. Natl. Acad. Sci. USA 86:6925-6929, National Academy Press (1989).Applicant
- Düzgùnes, N., et al., “Fusion of Liposomes Containing a Novel Cationic Lipid, N-[2,3-(Dioleyloxy)propyl]-N,N,N-trimethylammonium: Induction by Multivalent Anions and Asymmetric Fusion with Acidic Phospholipid Vesicles,” Biochemistry 28:9179-9184, American Chemical Society (1989).Applicant
- Dwarki, V.J., et al., “Cationic Liposome-Mediated RNA Transfection,” Methods Enzymol. 217:644-654, Academic Press, Inc. (1993).Applicant
- Epand, R.M., et al., “Peptide Models for the Membrane Destabilizing Actions of Viral Fusion Proteins,” Biopolymers 32:309-314, John Wiley & Sons, Inc. (1992).Applicant
- Eytan, G.D., “Use of Liposomes for reconstruction of biological functions,” Biochim. Biophys. Acta 694:185-202, Elsevier Biomedical Press (1982).Applicant
- Fawell, S., et al., “Tat-mediated delivery of heterologous proteins into cells,” Proc. Natl. Acad. Sci. USA 91:664-668, National Academy Press (1994).Applicant
- Felgner, P.L., and Ringold, G.H., “Cationic liposome-mediated transfection,” Nature 337:387-388, Nature Publishing Group (1989).Applicant
- Finlay, D.R., et al., “Nuclear transport in vitro,” J. Cell Sci. Suppl. 11:225-242, The Company of Biologists Ltd. (1989).Applicant
- FitzGerald, D.J.P., et al., “Adenovirus-Induced Release of Epidermal Growth Factor and Pseudomonas Toxin into the Cytosol of KB Cells during Receptor-Mediated Endocytosis,” Cell 32:607-617, Cell Press (1983).Applicant
- Frankel, A.D., et al., “Activity of synthetic peptides from the Tac protein of human immunodeficiancy virus type 1,” Proc. Natl. Acad. Sci. USA 86:7397-7401, National Academy Press (1989).Applicant
- Friedlander, D.R., et al., “Functional Mapping of Cytotactin: Proteolytic Fragments Active in Cell-Substrate Adhesion,” J. Cell Biol. 107:2329-2340, The Rockefeller Univeristy Press (1988).Applicant
- Gao, X., and Huang, L., “Potentiation of Cationic Liposome-Mediated Gene Delivery by Polycations,” Biochemistry 35:1027-1036, American Chemical Society (1996).Applicant
- Garcia-Bustos, J., et al., “Nuclear protein localization,” Biochim. Biophys. Acta 1071:83-101, Elsevier Science Publishers B.V. (1991).Applicant
- Gardner, J.M., and Hynes, R.O., “Interaction of Fibronectin with Its Receptor on Platelets,” Cell 42:439-448, Cell Press (1985).Applicant
- Garrigues, B., and Vidaud, L., “Synthesis of spermine and spermidine selectively substituted on the secondary amine functions,” Chemical Abstracts 111:593, Abstract No. 23282t, American Chemical Society (1989).Applicant
- Goldfarb, D.S., et al., “Synthetic peptides as nuclear localization signals,” Nature 322:641-644, Nature Publishing Group (1986).Applicant
- Goldfarb, D., and Michaud, N., “Pathways for the nuclear transport of proteins and RNAs,” Trends Cell Biol. 1:20-24, Elsevier Science Publishers, Ltd (UK) (1991).Applicant
- Gould-Fogerite, S., et al., “Chimerasome-mediated gene transfer in vitro and in vivo,” Gene 84:429-438, Elsevier (1989).Applicant
- Grant, D.S., et al., “Two Different Laminin Domains Mediate the Differentiation of Human Endothelial Cells into Capillary-like Structures In Vitro,” Cell 58:933-943, Cell Press (1989).Applicant
- Hackh's Chemical Dictionary, 4th ed., Grant, J., ed., McGraw-Hill Book Company, New York, NY p. 391 (1969).Applicant
- Haensler, J., and Stoka Jr., F.C., “Polyamidoamine Cascade Polymers Mediate Efficient Transfection of Cells in Culture,” Bioconjug. Chem. 4:372-379, American Chemical Society (1993).Applicant
- Hagstrom, J.E., et al., “Complexes of non-cationic liposomes and histone H1 mediate efficient transfection of DNA without encapsulation,” Biochim. Biophys. Acta 1284:47-55, Elsevier Science B.V. (1996).Applicant
- Harman, K., et al., “N'-Methylthermospermine in leguminous seeds,” Chemical Abstracts 117:427, Abstract No. 117:86723g, American Chemical Society (1992).Applicant
- Harbottle, R.C., et al., “RGD-mediated gene delivery and expression in epithelial cells,” Gene Therapy and Molecular Medicine, Keystone Symposium, Steamboat Springs, Co., Mar. 26-Apr. 1, 1995, J. Cell Biochem. Suppl. 21A:394, Abstract No. C6-321, Wiley-Liss (1995).Applicant
- Haverstick, D.M., et al., “Inhibition of Platelet Adhesion to Fibronectin, Fibrinogen, and von Willebrand Factor Substrates by a Synthetic Tetrapeptide Derived From the Cell-Binding Domain of Fibronectin,” Blood 66:946-952, Harcourt Brace Jovanovich (1985).Applicant
- Hawley-Nelson, P., et al., “LipofectAMINE™ reagent: A new, higher efficiency polycationic liposome transfection reagent,” Focus 15:73-79, Life Technologies, Inc. (1993).Applicant
- Hetschko, M., and Gosselck, J., “Reaktionen von sulfoniumsalzen des 1,3-dithiolans und seiner 2,2-substitutionsprodukte,” Tetrahedron Lett. 17:1691-1692, Pergamon Press (1972).Applicant
- Huckett, B., et al., “Evidence for targeted gene transfer by receptor-mediated endocytosis,” Biochem. Pahramacol. 40:253-263, Pergamon Press (1990).Applicant
- Humphries, M.J., et al., “Identification of an Alternatively Spliced Site in Human Plasma Fibronectin That Mediates Cell Type-specific Adhesion,” J. Cell Biol. 103:2637-2647, The Rockefellar University Press (1986).Applicant
- Humphries, M.J., et al., “Identification of Two Distinct Regions of the Type III Connecting Segment of Human Plasma Fibronectin That Promote Cell Type-specific Adhesion,” J. Biol. Chem. 262:6886-6892, American Society for Biochemistry and Molecular Biology (1987).Applicant
- Ito, A., et al., “Synthetic cationic amphipiles for liposome-mediated DNA transfection,” Biochem. Intl. 22:235-241, Academic Press Australia (1990).Applicant
- Kalderon, D., et al., “A Short Amino Acid Sequence Able to Specify Nuclear Location,” Cell 39:499-509, Cell Press (1984).Applicant
- Kamata, H., et al., “Amphiphilic peptides enhance the efficiency of liposome-mediated DNA transfection,” Nucleic Acids Res. 22:536-537, Oxford University Press (1994).Applicant
- Kaneda, Y., et al., “The Improved Efficient Method for Introducing Macromolecules into Cells Using HVJ (Sendai Virus) Liposomes with Gangliosides,” Exp. Cell Res. 173:56-69, Academic Press, Inc. (1987).Applicant
- Kaneda, Y., et al., “Introduction and Expression of the Human Insulin Gene in Adult Rat Liver,” J. Biol. Chem. 264:12126-12129, The American Society for Biochemistry and Molecular Biology, Inc. (1989).Applicant
- Kaneda, Y., et al., “Increased Expression of DNA Cointroduced with Nuclear Protein in Adult Rat Liver,” Science 243:375-378, American Association for the Advancement of Science (1989).Applicant
- Karlsson, S., et al., “Transfer of genes into hematopoietic cells using recombinant DNA viruses,” Proc. Natl. Acad. Sci. USA 82:158-162, National Academy Press (1985).Applicant
- Kielian, M., and Helenius, A., “Entry of Alphaviruses” in The Togaviridae and Flavivitidac, Schlesinger, S., and Schlesinger, M.J., eds., Plenum Press, New York, NY, pp. 92-119 (1986).Applicant
- Kirsch, T., et al., “Cloning, High-Yield Expression in Escherichia coli, and Purification of Biologically Active HIV-1 Tat Protein,” Protein Expr. Purif. 8:75-84, Academic Press, Inc. (1996).Applicant
- Klappe, K., et al., “Parameters Affecting Fusiom between Sendai Virus and Liposomes, Role of Viral Proteins, Liposome Composition, and pH,” Biochemistry 25:8252-8260, American Chemical Society (1986).Applicant
- Knodis, Z., and Valiuliene, S., “New reactions of 1,1-diamines,” Chemical Abstracts 120:1056, Abstract No. 270276b, American Chemical Society (1994).Applicant
- Konopka, K., et al., “Enhancement of human immunodeficiency virus type 1 infection by cationic liposomes: the role of CD4, serum and liposome-cell interactions,” J. Gen. Virol. 72:2685-2696, Society for General Microbiology (1991).Applicant
- Krsaijeveld, C.A., et al., “The effect of liposomal charge on the neutralizing antibody response against inactivated encephalomyocarditis and Semliki Forest Viruses,” Clin. Exp. Immunol. 56:509-514, Blackwell Scientific Publications (1984).Applicant
- Kukowska-Latallo, J.F., et al., “Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers,” Proc. Natl. Acad. Sci. USA 93:4897-4902, National Academy (1996).Applicant
- Lanford, R.E., et al., “Comparison of Diverse Transport Signals in Synthetic Peptide-Induced Nuclear Transport,” Exp. Cell Res. 186:32-38, Academic Press, Inc. (1990).Applicant
- Lanford, R.E., et al., “Induction of Nuclear Transport with a Synthetic Peptide Homologous to the SV40 T Antigen Transport Signal,” Cell 46:575-582, Cell Press (1986).Applicant
- Lapidot, M., and Loyter, A., “Fusion-Mediated Microinjection of Liposome-Enclosed DNA into Cultured Cells with the Aid of Influenza- Virus Glycoproteins,” Exp. Cell Res. 189, Academic Press, Inc. (1990).Applicant
- Lawler, J., et al., “Cell Attachment to Thrombospondin: The Role of ARG-GLY-ASP, Calcium, and Integrin Receptors,” J. Cell. Biol. 107:2351-2361, The Rockefeller Iniversity Press (1988).Applicant
- Ledley, F.D., “Clinical Considerations in the Design of Protcols for Somatic Gene Therapy,” Hum. Gene Ther. 2:77-83, Mary Ann Liebert, Inc. (1991).Applicant
- Legendre, J-Y., et al., “Delivery of Plasmid DNA into Mammalian Cell Lines Using pH-Sensitive Liposomes: Comparison with Cationic Liposomes,” Pharm. Res. 9:1235-1242, Plenum Publishing Corporation (1992).Applicant
- Legendre, J-Y., et al., “Cyclic amphipathic peptide-DNA complexes mediate high-efficiency transfection of adherent mammalian cells,” Proc. Natl. Acad. Sci. USA 90:893-897, National Academy Press (1993).Applicant
- Life Technologies 1993-1994 Catalog and Reference Guide, pp. 9-19 to 9-21, I-66, R-48 to R-51, Life Technologies, Inc. (1993).Applicant
- Liljeström, P., and Garoff, H., “A new generation of animal cell expression vectors based on the semliki Forest virus replicon,” Biotechology(N- Y) 9:1356-1361, Nature Publishing Company (1993).Applicant
- Loeffler, J-P., and Behr, J-P., “Gene Transfer into Primary and Established Mammalian Cell Lines with Lipopolyamine-Coated DNA,” Methods Enzymol. 217:599-618, Academic Press, Inc. (1993).Applicant
- Loyter, A., et al., “Mechanisms of DNA uptake by mammalian cells Fate of exogenously added DNA monitored by the use of fluorescent dyes,” Proc. Natl. Acad. Sci. USA 79:422-426, National Academy Press (1982).Applicant
- Malone, R.W., et al., “Cationic liposome-mediated RNA transfection,” Proc. Natl. Acad. Sci. USA 86:6077-6081, National Academy Press (1989).Applicant
- Mann, D.A., and Frankel, A.D., “Endocytosis and targeting of exogenous HIV-1 Tat protein,” EMBO J. 10:1733-1739, Oxford University Press (1991).Applicant
- Marah, M., et al., “Interactions of Semliki Forest Virus Spike Glycoprotein Rosettes and Vesicles with Cultured Cells,” J. Cell Biol. 96:455-461, The Rockefeller University Press (1994).Applicant
- Mason, P.W., et al., “RGD sequence of foot-and-mouth disease virus is essential for infecting cells via the natural receptor but can be bypassed by an antibody-dependent enhancement pathway,” Proc. Natl. Acad. Sci. USA 91:1932-1936, National Academy Press (1994).Applicant
- Mazur, W., et al., “The Efficiency of Lipofectin-Mediated Gene Transfer into Porcine and Human Coronary Smooth Muscle Cells is Dramatically Improved by the Influenza Virus Hemagglutinin Antigen,” J. Am. Coll. Cardiol. Suppl. 21:186A, Abstract No. 889-31, Elsevier (1993).Applicant
- Miyanohara, A., et al., “Partial Cell-Free Assembly of VSV-G Pseudotyped Retrovirus Particles” Molecular and Cell Biology of Gene Therapy, Keystone, Colorado, meeting date Jan. 19-25, 1998, Abstract No. 007, p. 34, Keystone Symposis (Dec. 1997).Applicant
- Murata, M., et al., “Modification of the N-terminus of membrane fusion-active peptides blocks the fusion activity,” Biochem. Biophys. Res. Commun. 179:1050-1055, Academic Press, Inc. (1991).Applicant
- Neugebauer, J.M., “detergents: An Overview,” Methods Enzymol. 182:239-253, Academic Press, Inc. (1990).Applicant
- Otero, M.J., and Carrasco, L., “Proteins are cointernalized with Virion Particles during Early Infection,” Virology 160:75-80, Academic Press, Inc. (1987).Applicant
- Parente, R.A., et al., “Association of pH-Sensitive Peptide with Membrane Vesicles: Role of Amino Acid Sequence,” Biochemistry 29:8713-8719, American Chemical Society (1990).Applicant
- Parente, R.A., et al., “Mechanism of Leakage of Phospholipid Vesicle Contents Induced by the Peptide GALA,” Biochemistry 29:8720-8728, American Chemical Society (1990).Applicant
- Pastan, I.H., and Willingham, M.C., “Journey to the Center of the Cell: Role of the Receptosome,” Science 214:504-509, American Association for the Advancement of Science (1981).Applicant
- Pepinsky, R.B., et al., “Specific Inhibition of a Human Papillomavirus E2 Trans-Activator by Intracellular Delivery of Its Repressor,” DNA Cell Biol. 13:1011-1019, Mary Ann Liebert, Inc. (1994).Applicant
- Phalen, T., and Kielian, M., “Cholesterol Is Required for Infection by Semliki Forest Virus,” J. Cell Biol. 112:615-623, The Rockefeller University Press (1992).Applicant
- Pierschbacher, M.D., and Ruoslahti, E., “Call attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule,” Nature 309:30-33, Nature Publishing Group (1984).Applicant
- Pierschbacher, M.D., and Ruoslahti, E., “Influence of Stereochemistry of the Sequence, Arg-Gly-Asp-Xaa on Binding Specificity on Cell Adhesion,” J. Biol. Chem. 262:17294-17298, The American Society for Biochemistry and Molecular Biology, Inc. (1987).Applicant
- Pinnaduwage, P., et al., “Use of quaternary ammonium detergent in liposome mediated DNA transfection of mouse L-cells,” Biochim. Biophys. Acta 985:33-37, Elsevier (1989).Applicant
- Poste, G. et al., “Lipid Vesicles as Carriers for Introducing Biologically Active Materials into Cells,” Methods Cell Biol. 14:33-71, Academic Press, Inc. (1976).Applicant
- “Eukaryotic transcription and Reporter Systems”, 1993/1994 Promega Catalog, p. 251 Promega Corporation (1993).Applicant
- Remy, J-S., et al., “Targeted gene transfer into hepatoma cells with lipopolyamine-condensed DNA particles presenting galactose ligands: A stage toward artificial virus,” Proc. Natl. Acad. Sci. USA 92:1744-1748, National Academy Press (1995).Applicant
- Rihs, H-P., “The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen,” EMBO J. 10:633-639, Oxford University Press (1991).Applicant
- Rihs, H-P., and Peters R., “Nuclear transport kinetics depend on phosphorylation-site-containing sequences flanking the karyophilic signal of the Simian virus 40 T-antigen,” EMBO J. 8:1479-1484, IRL Press (1989).Applicant
- Rose, J.K., et al., “A New Cationic Liposome Reagent Mediating Nearly Quantitative Transfection of Animal Cells,” Biotechniques 10:520-525, Eaton Publishing Company (1991).Applicant
- Rosenkranz, A.A., et al., “Receptor-Mediated Endocytosis and Nuclear Transport of a Transfecting DNA Construct,” Exp. Cell Res. 199:323-329, Academic Press, Inc. (1992).Applicant
- Ruoslahti, E., and Pierschbacher, M.D., “New Perspectives in Cell Adhesion: RGD and Integrins,” Science 238:491-497, American Association for the Advancement of Science (1987).Applicant
- Sands, J.A., “Virucidal activity of cetyltrimethylammonium bromide below the critical micelle concentration,” FEMS Microbiol. Lett. 36:261-263, Elsevier.Applicant
- Schlegel, R., et al., “Inhibition of VSV Binding and Infectivity by Phosphatidylserine: Is Phosphatidylserine a VSV-Binding Site?” Cell 32:639-646, Cell Press (1985).Applicant
- Schlegel, R., and Wade, M., “Biologically Active Peptides of the Vesicular Stomatitis Virus Glycoprotein,” J. Virol. 53:319-323, American Society for Microbiology (1985).Applicant
- Scheule, R.K., “Novel Preparation of Functional Sindbis Virosomes,” Biochemistry 25:4223-4232, American Chemical Society (1986).Applicant
- Schmid, N. and Hehr, J. P., “Location of Spermine and other Polyamines on DNA As Revealed by Photoaffinity Cleavage with Polyaminobenzenediazonium Salts,” Biochemistry 30:4357-4361, American Chemical Society (1991).Applicant
- Seth, P., et al., “Pathway of Adenovirus Enty into Cells,” in Virus Attachment and Entry into Cells, Crowell, R.L., and Longberg-Holm, K., eds., American Society for Microbiology, Washington, D.C., pp. 191-195 (1986).Applicant
- Silver, P.A., “How Proteins Enter the Nucleus,” Cell 64:489-497, Cell Press (1991).Applicant
- Smull, C.E., and Ludwig, S.H., “Enhancement of the plaque-forming capacity of poliovirus ribonucleic acid with basic proteins,” J. Bacteriol. 84:1035-1040, The Williams & Wilkins Company (1962).Applicant
- Stegmann, T., et al., “Protein-mediated membrane fusion,” Annu. Rev. Biophys. Biophys. Chem. 18:187-211, Annual Reviews, Inc. (1989).Applicant
- Sugawa, H., et al., “Large Macromolecules Can Be Introduced into Cultured Mammalian Cells Using Erythrocyte Membrane Vesicles,” Exp. Cell Res. 159:410-418, Academic Press, Inc. (1985).Applicant
- Suzuki, g., et al., “Complete amino acid sequence of human vitronectin deduced from cDNA, Similarity of cell attachment sites in vitronectin and fibronectin,” EMBO J. 4:2519-2524, IRL Press, Ltd. (1985).Applicant
- Tang, M.X., et al., “In Vitro Gene Delivery by Degraded Polyamidoamine Dendrimers,” Bioconjug. Chem. 7:703-714, American Chemical Society (1996).Applicant
- Tikchonenko, T.I., et al., “Transfer of condensed viral DNA into eukaryotic cells using proteoliposomes,” Gene 63:323-330, Elsevier (1988).Applicant
- “Transfection Reagent” in Genetic Engineering News, Mary Ann Liebert, Inc., p. 12, col. 3, (Jun. 1993).Applicant
- Trubetskoy, V.S., et al., “Use of N-terminal Modified Poly(L-lysine)-Antibody Conjugate as a Carrier for Targeted Gene Delivery in Mouse Lung Endothelial Cells,” Bioconjug. Chem. 3:323-327, American Chemical Society (1992).Applicant
- Väänänen, P., and Kääriäinen, L., “Fusion and Haemolysis of Erythrocytes causeed by Three Togaviruses: Semliki Forest, Sindbis and Rubella,” J. Gen. Virol. 46:467-475, Cambridge University Press (1980).Applicant
- van Zee, K., et al., “A Hydrophobic Protein Sequence Can Override a Nuclear Localization Signal Independently of Protein Context,” Mol. Cell. Biol. 11:5137-5146, American Society for Microbiology (1991).Applicant
- Vivès, et al., “A Truncated HIV-1 Tat Protein Basic Domain Rapidly Translocates through the Plasma Membrane and Accumulates in the Cell Nucleus,” J. Biol. Chem. 272:16010-16017, The American Society for Biochemistry and Molecular Biology, Inc. (Jun. 1997).Applicant
- Wagner, E., et al., “Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes,” Proc. Natl. Acad. Sci. USA 89:6099-6103, National Academy Press (1992).Applicant
- Wagner, E., et al., “Transferrin-polycation conjugates as carriers for DNA uptake into calls,” Proc. Natl. Acad. Sci. USA 87:3410-3414, National Academy Press (1990).Applicant
- wagner, E., et al., “Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: Toward a synthetic virus-like gene-transfer vehicle,” Proc. Natl. Acad. Sci. USA 89:7934-7938, National Academy Press (1992).Applicant
- Wagner, E., et al., “DNA-Binding Transferrin Conjugates as Functional Gene-Delivery Agents: Synthesis by Linkage of Polylysine or Ethidium Homodimer to the Transferrin Carbohydrate Moiety,” Bioconjug. Chem. 2:226-231, American Chemical Society (1991).Applicant
- Walker, C., et al., “Cationic lipids direct a viral glycoprotein into the class I major histocompatibility complex antigen-presentation pathway,” Proc. Natl. Acad. Sci. USA 89:7915-7918, National Academy Press (1992).Applicant
- Wayner, E.A., et al., “Identification and Characterization of the T Lymphocyte Adhesion Receptor for An Alternative Cell Attachment Domain (CS-1) in Plasma Fibrobectin,” J. Cell Biol. 109:1321-1330, The Rockefeller University Press (1989).Applicant
- Weinstock, L.T., et al., “Synthesis of New Polyamine Derivatives for Cancer Chemotherapeutic Studies,” J. Pharm. Sci. 70:956-959, American Pharmaceutical Association (1981).Applicant
- Wickham, T.J., et al., “Targeting of adenovirus penton base to new receptors through replacement of its RGD motif with other receptor-specific peptide motifs,” Gene Ther. 2:750-756, Stockton Press (1995).Applicant
- Wilson, J.M., et al., “Hepatocyte-directed Gene Transfer in Vivo Leads to Transient Improvement of Hypercholesterolemia in Low Density Lipoprotein Receptor-deficient Rabbits,” J. Biol. Chem. 267:963-967, The American Society for Biochemistry and Molecular Biology, inc. (1992).Applicant
- Wolff, J.A., et al., “Direct Gene Transfer into Mouse Muscle in Vivo,” Science 247:1465-1468, American Association for the Advancement of Science (1990).Applicant
- Wu, G.Y., and Wu, C.H., “Receptor mediated Gene Delivery and Expression in Vivo,” J. Biol. Chem. 263:14621-14624, The American Association for Biochemistry and Molecular Biology, Inc. (1988).Applicant
- Wu, C.H., et al., “Targeting Genes: Delivery and Persistent Expression of a Foreign Gene Driven by Mammalian Regulatory Elements in Vivo,” J. Biol. Chem. 264:16985-16987, The American Society for Biochemistry and Molecular Cell Biology, Inc. (1989).Applicant
- Wu, G.Y., and Wu, C.H., “Evidence for Targeted Gene Delivery to Hep G2 Hepatoma Cells in Vitro,” Biochemistry 27:887-892, American Chemical Society (1988).Applicant
- Wu, G.Y., et al., “Receptor mediated Gene Delivery in Vivo Partial correction of genetic analbuminemia in nagase rats,” J. Biol. Chem. 266:14338-14342, The American Society for Biochemistry and Molecular Biology , Inc. (1991).Applicant
- Yagi, K., et al., “Incorporation of Histone into Liposomes Increases the Efficiency of Liposome-Mediated Gene Transfer,” J. Clin. Biochem. Nutr. 10:21-25, Institute of Applied Biochemistry (1991).Applicant
- Yoshimura, K., et al., “Adenovirus-mediated Augmentation of Cell Transfection with Unmodified Plasmid Vectors,” J. Biol. Chem. 268:2300-2303, The American Association for Biochemistry and Molecular Biology, Inc. (1993).Applicant
- Young, J.D. E., et al., “Interaction of Enveloped Viruses with Planar Bilayer Membranes: Observations on Sendal, Influenza, Vesicular Stomatitis, and Semliki Forest Viruses,” Virology 128:186-194, Academic Press, Inc. (1983).Applicant
- Zenke, M., et al., “Receptor-mediated endocytosis of transferrin-polycation conjugates: An efficient way to introduce DNA into Hematopoietic cells,” Proc. Natl. Acad. Sci. USA 87:3655-3659, National Academy Press (1990).Applicant
- Zhou, X., and Huang, L., “DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action,” Biochim. Biophys. Acta 1189:195-203, Elsevier Science B.V. (1994).Applicant
- Zhu, Z., et al., “Transformation of tobacco protoplasts with DNA entrapped in pH-sensitive liposomes,” Plant Cell, Tissue & Organ Cult. 22:135-145, Kluwez Academic Publishers (1990).Applicant
- CAPLUS, Acession No. 1972:461856, STNEasy CAPLUS English language abstract for Hetschko, M., and Govselck, J., “Reaktionen von sulfoniumsalzen des 1,3-dithiolans und seiner 2,2-substitutionsprodukte,” Tetrahedron Lett. 17:1691-1692, Pergamon Press (1972) (Document AT23).Applicant
- Dialog File 351, Accession No. 7672839, Derwent WPI English language abstract for EP 0 304 111 B1 (Document AN4).Applicant
- Dialog File 351, Accession No. 8628544, Derwent WPI English language abstract for WO 91/04668 A1 (Document AO4).Applicant
- Dialog File 351, Accession No. 8822189, Derwent WPI English language abstract for DD 290 877 A5 (Document AN5).Applicant
- Dialog File 351, Accession No. 9150067, Derwent WPI English language abstract for WO 92/13570 A2 (Document AL6).Applicant
- Dialog File 351, Accession No. 9417386, Derwent WPI English language abstract for WO 93/07282 A1 (Document AL7).Applicant
- Dialog File 351, Accession No. 9440951, Derwent WPI English language abstract for WO 93/07283 A1 (Document AM7).Applicant
- Dialog File 351, Accession No. 9483543, Derwent WPI English language abstract for EP 0 544 292 A2 (Document AO7).Applicant
- Dialog File 351, Accession No. 10073779, Derwent WPI English language abstract for WO 94/23751 A1 (Document AN8).Applicant
- Dialog File 351, Accession No. 10968033, Derwent WPI English language abstract for WO 96/31549 A1 (Document AO10).Applicant
- “Voyager , The Power of Translocation,” Expressions 6:6, Life Technologies, Inc. (Feb. 1999).Applicant
- Dialog File 351, Accession No. 8899481, Derwent WPI English language abstract for WO 93/08130 A1 (Document A012) (1993).Applicant
- Dialog File 351, Accession No. 10428036, Derwent WPI English language abstract for DE 44 11 588 C1 (Document AN14) (1995).Applicant
- Dialog File 351, Accession No. 10524091, Derwent WPI English language abstract for DE 44 11 594 C1 (Document AP14) (1995).Applicant
- Dialog File 351, Accession No. 10655226, Derwent WPI English language abstract for WO 96/08723 A1 (Document AL15) (1996).Applicant
- Copy of co-pending U.S. Appl. No. 09/266,935, filed Mar. 12, 1999, inventors Li et al. (Not Published).Applicant
- Dialog File 351, Accession No. 8626544, Derwent WPI English language abstract for WO 91/04668 A1 (Document A04) (1991).Applicant
- Dialog File 351, Accession No. 8822189, Derwent WPI English language abstract for DD 290 877 A5 (Document AN5) (1991).Applicant
- Dialog File 351, Accession No. 9150067, Derwent WPI English language abstract for WO 92/13570 A2 (Document AL6) (1992).Applicant
- Dialog File 351, Accession No. 9417386, Derwent WPI English language abstract for WO 93/07282 A1 (Document AL7) (1993).Applicant
- Dialog File 351, Accession No. 9440951, Derwent WPI English language abstract for WO 93/07283 A1 (Document AM7) (1993).Applicant
- Dialog File 351, Accession No. 9483543, Derwent WPI English language abstract for EP 0 544 292 A2 (Document AO7) (1993).Applicant
- Dialog File 351, Accession No. 10073779, Derwent WPI English abstract for WO 94/23751 A1 (Document An8) (1994).Applicant
- Dialog File 351, Accession No. 10968033, Derwent WPI English language abstract for WO 96/31549 A1 (Document AO10) (1996).Applicant
- Banerjee et al., (2001) “Design, Synthesis, and Tranfection Biology of Novel Cationic Glycolipids for Use in Liposomal Gene Delivery,” J. Med. Chem., 44:4176-4185.Applicant