Cationic amphiphilic lipids for liposomal gene transfer

Details Cationic amphiphilic lipids for liposomal gene transfer Cationic amphiphilic lipids for liposomal gene transfer

1999-03-30

2001-07-31

Badio, Barbara P. (Department: 1616)

Organic compounds -- part of the class 532-570 series

Organic compounds

Fatty compounds having an acid moiety which contains the...

C554S107000

Reexamination Certificate

active

06268516

ABSTRACT:

The invention relates to cationic lipids of the general formula I,
whereby n can be =2,3,4,6,8 and m can be =3,6,8 and where R
1
represents H, CH
3
CH
2
CH
2
OH; R
2
represents H, CH
3
, CH
2
CH
2
OH, (CH
2
)
3
N
+
(R
1
)
3
, R
3
represents a straight chain, saturated or unsaturated aliphatic group C
7
-C
21
, Z represents CH
2
, O, NH, Y represents CH
2
, O, NH and X represents Cl, Br, I, CH
3
COO, CF
3
COO;
cationic lipids of the general formula Ia,
wherein n and m can be n=2, 3, 4, 6, or 8 and m=2, 3, 6, or 8 and R
1
=H, CH
3
, or CH
2
CH
2
OH; R
2
=H, CH
3
, CH
2
CH
2
OH, or (CH
2
)
3
N
+
(R
1
)
3
, R
3
=H, CH
3
, CH
2
CH
2
OH, or (CH
2
)
3
N
+
(R
1
)
3
, R
4
represents a straight chain saturated or unsaturated aliphatic group C
7
-C
21
, Z=CH
2
, O, or NH, Y=CH
2
, O or NH and X=Cl, Br, I, CH
3
COO, or CF
3
COO;
cationic lipids of the formula II,
whereby R
1
represents an aliphatic, aromatic or heteroaliphatic &agr;-carbon atom substituent of the &agr;-amino acids glycine, alanine, valine, leucine, isoleucine, phenylalanine, trosine proline, hydroxyproline, serine, threonine, cysteine, cystine, methionine, tryptophane arginine, lysine, ornithine, histidine, and R
2
represents a straight chain saturated or unsaturated aliphatic group C
7
-C
21
, X=Cl, Br, I, CH
3
COO, CF
3
COO, Y=CH
2
, O, NH and Z=CH
2
, O, NH;
cationic lipids of the general formula III,
whereby R
1
represents H, CH
3
, (CH
2
)
3
NH
2
+
X
−
(CH
2
)
3
NH
3
+
X
−
, R
3
=H, (CH
2
)
3
NH
3
+
X
−
, and R
2
a straight chain saturated or unsaturated aliphatic group C
7
-C
21
and X=Cl, Br, I, CH
3
COO, CF
3
COO;
cationic lipids of the general formula IV,
whereby n can be 1-4 and R represents a straight chain, saturated or unsaturated aliphatic group C
7
-C
21
, Y represents CH
2
, O, NH, Z represents CH
2
, O, NH and X represents Cl, Br, I, CH
3
COO, CF
3
COO;
cationic lipids of the general formula V,
whereby n can be =2,3,4,6,8 and m can be =2,3,6,8 and R
1
represents H, CH
3
, CH
2
CH
2
OH; R
2
represents H, CH
3
, CH
2
CH
2
OH, (CH
2
)
3
N
+
(R
1
)
3
and X represents Cl, Br, I, CH
3
COO, CF
3
COO;
cationic lipids of the general formula VI,
whereby R
1
represents H, CH
3
, (CH
2
)
3
NH
2
+
X
−
(CH
2
)
3
NH
3
+
X
−
, (CH
2
)
3
NH
3
+
X
−
, R
2
represents H, (CH
2
)
3
NH
3
+
X
−
and X represents Cl, Br, I, CH
3
COO, CF
3
COO;
cationic lipids of the general formula VII,
whereby m can be =2-6 and Y represents a group N(R)
3
+
X
−
in which R represents H, CH
3
, (CH
2
)
2
OH or a group NH—C(NH
2
+
X
−
)NH
2
in which X represents Cl, Br, I, CH
3
COO, CF
3
COO;
cationic lipids of the general formula VIII,
whereby Y represents a group N(R)
3
+
X
−
in which R represents H, CH
3
, (CH
2
)
2
OH or a group NH—C(NH
2
+
X
−
)NH
2
in which X represents Cl, Br, I, CH
3
COO, CF
3
COO;
cationic lipids of the general formulas IX und X,
whereby n can be =3,4,6,8 and m can be =2,3,6,8 and R
1
represents H, CH
3
, CH
2
CH
2
OH, R
2
represents H, CH
3
, CH
2
CH
2
OH, (CH
2
)
3
N
+
(R
1
)
3
, R represents H, CH
3
, (CH
2
)
2
OH, Y represents a carbonyl group (═O (estrone)) or a hydroxy group OH (estradiol), Z represents a group N(R)
3
+
X
−
in which R represents H, CH
3
, (CH
2
)
2
OH or a group NH—C(NH
2
+
X
−
)NH
2
and whereby X represents Cl, Br, I, CH
3
COO, CF
3
COO.
Cationic lipids of the general formulas I-X are suitable reagents for liposomal gene transfer (transfection). Applications for such transfection reagents are in medicine and gene technology. The delivery of genetic material into eukaryotic cells is a fundamental method for studies of biological functions and of increasing importance for the gene therapeutic treatment of various diseases whereby tumours have to be mentioned foremost.
One differentiates thereby between biological, physical and physico-chemical methods for the transfer of DNA, RNA and proteins into target cells [Wagner J, Madry, H., Reszka, R (1995). In vivo gene transfer: focus on the kidney. Nephrol. Dial. Transplant 10:1801-1807 Zhu J, Zhang L, Hanisch U-K, Felgner P L,. Reszka R (1996). In vivo gene therapy of experimental brain tumors by continuous administration of DNA-liposome complexes. Gene Therapy 3: 472-476, Kiehntopf, M., Brach MA & Hermann F (1995). Gentherapie in der Onkologie: Perspektiven, Chancen und Risiken. Onkologie 18 (Sonderheft): 16-26]. Physical methods like electroporation and micro injection are only suitable for ex vivo and in vitro transfer. The so-called “Jet”-injection method can be used in addition also for the in vivo gene transfer (liver, skin). Physico-chemical methods like the calcium phosphate precipitation technique (cpp) or DEAE-dextrane transfection are limited to in vitro and ex vivo applications.
Retroviral gene transfer using virus producing cells, as presently being tested in a series of clinical trials (phase III) is characterised by a relatively long lasting, however, relatively low gene expression in the dividing cells. Problematic in the retroviral gene transfer are mainly the development of a specific immuno answer against the implanted, virus producing helper cells, the possible generation of replication competent viruses and the danger of activating cellular oncogenes or, possibly, the deactivation of suppressor genes as a result of the accidental localisation of gene insertion. The comprehensive, required cell biological and medicinal preparational work and the expensive safety measures lead to high expected costs in the clinical applications of retro viral gene transfer.
Vectors based on adenoviruses are also attractive gene transfer vehicles. They acchieve high transfection rates also in non dividing tissue. However, since the DNA here is not integrated into the genome, the duration of the expression of the foreign gene is limited and the repeated application in vivo is hampered by the strong and specific immuno answer of the host organism during repeated applications.
In contrast, the liposomal gene transfer has gained in recent years in importance also for applications in vivo. The gene constructs can either be encapsulated in liposomes or are associated to their membranes. Liposomal preparations are characterised by facile handling, low immuno reaction and thus the possibility for repeated applications leading to reduced risks both for applicant and “receiver” (patient). The application of immuno liposomes as transport vehicles for genetic material is still at a very early stage.
Another also quite interesting approach constitutes the use of fusogenic liposomes which carry in their interior a complex formed by DNA and nuclear protein (HMG I) [Kaneda, Iwai, K., Uchida, T(1989). Increased expression of DNA cointroduced with nuclear protein in adult rat liver Science 243, 375-378,Kaneda Y, Kato, K., Nakanishi, M., Uchida, T.(1996) Introduction of plasmid DNA and nuclear protein into cells by using erythrocyte ghosts, liposomes, and Sendai virus. Methods-Enzymol. 221:317-327].
For a number of years now cationic liposomes are applied successfully for the transfer of DNA (Felgner P L, Gadek T R, Holm M et al. (1987). Lipofection: a highly efficient, lipid-mediated DNA transfection procedure. Proc Natl Acad Sci USA 84:7413-7417, Felgner J R Kumar R, Sridhar C N et al. (1994). Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol Chem 269:2550-2561), antisense-oligomers, proteins and ribozymes. By electrostatic interaction DNA is associated with the membrane of the liposomes and transfected into the cell by a mechanism which up to now is only incompletely understood. The transfection rate in vitro is thereby dependent on the specific cell line, however comparable with the efficacy of retroviral gene transfer. Presently the firs cationic liposome

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