Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1995-06-06
2001-10-09
Marschel, Ardin H. (Department: 1631)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S440000, C435S443000, C435S455000, C435S458000
Reexamination Certificate
active
06300317
ABSTRACT:
TECHNICAL FIELD
This invention is in the field of oligonucleotide delivery and gene therapy. In particular this invention is directed to a self-assembling polynucleotide delivery system comprising components aiding in the delivery of the polynucleotide to the desired address which are associated via noncovalent interactions with the polynucleotide. The components of this system include DNA-masking components, cell recognition components, charge-neutralization and membrane-permeabilization components, and subcellular localization components.
BACKGROUND ART
Cystic Fibrosis (CF) is a fatal recessive genetic disease characterized by abnormalities in chloride transport (McPherson & Dorner, 1991). The locus of the disease has been traced to mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). J. R. Riordan et al.,
Science
(1989) 245:1066-1073; B. Kerem et al.,
Science
(1989) 245:1073-1080. Correction of the underlying gene defect by complementation or replacement of the defective CFTR is the ultimate cure for CF. Gene therapy, the in vivo delivery and expression of genes, is a fast-developing science that can be used to replace defective genes.
Gene therapy is already feasible. T. Friedmann,
Science
(1989) 244:1275-1281; M. Bluestone,
Biotechnol
(1992) 10:132-134. Systems and polymers for delivery of polynucleotides are known in the art. P. L. Felgner,
Adv Drug Delivery Rev
(1990) 5:163-187. Adenoviral vectors have been used to transfer CFTR to the cotton rat lung in vivo. M. A. Rosenfeld et al.,
Cell
(1992) 68:143-155. Although high levels of transfection in vivo have been reported with the adenoviral vectors, non-viral delivery systems have a number of advantages and should be vigorously developed. Rosenfeld et al., supra; M. A. Rosenfeld et al.,
Science
(1991) 252:431-434.
During the past decade, a number of methods have been developed to introduce functional genes into mammalian cells in vitro. These techniques are applicable to gene therapy if the target cells can be removed from the body, treated, and the transfected cells amplified and then returned to the patient. This option is not possible for CF patients. At present the best in vivo transfection efficiencies are obtained with retroviruses (Bluestone, supra) and adenoviruses (Rosenfeld et al., supra). However the efficiency is variable and a concern is that virus based gene delivery might cause viral infection or cancer. Initial human clinical trials have revealed no acute complications of retroviral vectors but the possibility of long-term complications mandate careful patient monitoring. K. Cornetta et al.,
Human Gene Ther
(1991) 2:3-14.
The risks of using viral based vectors and the conceptual advantages in using plasmid DNA constructs for gene therapy (discussed in P. L. Felgner et al.,
Nature
(1991) 349:351-352) have led to a parallel development of various physical and chemical methods for gene transfer. The most intensely studied systems involve treatment of the cells with calcium phosphate or a cationic facilitator (Felgner et al., supra). Other popular methods involve DNA injection during physical puncture of the membrane (M. R. Capecchi,
Cell
(1980) 22:479-485) or passive uptake during permeabilization or abrasion of the cellular membrane (Felgner et al., supra). Each method is intrinsically aggressive and applicable only in vitro.
This invention is in the field of direct gene delivery that does not involve the use of viral vehicles. A non-viral carrier for gene delivery must be able to surmount many barriers: it must survive in circulation, it must be able to target the cell of choice, it must be able to introduce DNA into the cytoplasm, and it must be able to transport the DNA into the nucleus.
Masking. One concern about the direct delivery of genes in vivo is the ability of the polynucleotide to survive in circulation long enough to arrive at the desired cellular destination. “Masking”, or protecting the polynucleotides is one way to address this concern.
Microparticulates (such as the erythrocyte ghost, reconstituted viral envelopes and liposomes) have been used in part as protection in gene transfer. C. Nicolau et al.,
Crit Rev Ther Drug Carr Sys
(1989) 6:239-271; R. J. Mannio et al.,
Biotechniques
(1988) 6:682-690. The most successful liposome system uses the cationic lipid reagent N-[1(-2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). P. L. Felgner et al.,
Proc Natl Acad Sci
(
USA
) (1987) 84:7413-7417. DOTMA is mixed with phosphatidylethanolamine (PE) to form the reagent Lipofectin™. The advantage of using Lipofectin™ is that the cationic liposome is simply mixed with the DNA and added to the cell. It is not necessary to encapsulate the DNA inside of the liposome with the cationic reagents. Lipofectin™ has been used to transfect reporter genes into human lung epithelial cells in culture (L. Lu et al.,
Pflugers Arch
(1989) 415:198-203), to introduce the CAT gene into rats by intratracheal route (T. A. Hazinski et al.,
Am J Respir Cell Mol Biol
(1991) 4:206-209) and to introduce the CAT gene into mice by the intratracheal and intravenous route (K. L. Brigham et al.,
Am J Med Sci
(1989) 298:278-281; A. Bout et al., “Abstracts of the 1991 Cystic Fibrosis Conference”, Abstract no. 87 (1991)). About 50% of the airway epithelial cells transiently expressed the &bgr; galactosidase reporter gene (Hazinski et al., supra) but the level of expression was not quantitated. When chloramphenicol acetyltransferase (CAT) attached to a steroid sensitive promoter was transfected into rat lung, expression could be positively regulated by dexamethasone. Hazinski et al., supra. Cytotoxicity is a problem with high concentrations of Lipofectin™.
Substitutes for DOTMA include lipopolyamine (J. Loeffler et al.,
J Neurochem
(1990) 54:1812-1815), lipophilic polylysines (X. Zhou et al.,
Biochim Biophys Acta
(1991) 1065:8-14) and a cationic cholesterol (X. Gao et al.,
Biochem Biophys Res Comm
(1991) 179:280-285). These have been used to mediate gene transfer in culture. Although there is some improvement over transfection rates observed with Lipofectin™ (about threefold), toxicity remains a problem. Studies on the mechanism responsible for transfection using the cationic lipids have been notably lacking. The past approach has been to synthesize different cationic lipids and try them in transfection assays, rather than to systematically study how the delivery systems introduce DNA into the cell. DOTMA/PE liposomes can undergo bilayer fusion with anionic liposomes (N. Duzgunes et al.,
Biochem
(1989) 28:9179-9184) which suggests that direct entry of the DNA via the plasma membrane is involved with DOTMA's mode of action. High efficiency transfection using cationic lipids systems requires the inclusion of PE, possibly because PE can form intramembrane lipid intermediates which facilitate membrane fusion. The role of PE in membrane permeabilization and fusion has been extensively studied. E.g., M.-Z. Lai et al.,
Biochem
(1985) 24:1646-1653; H. Ellens et al.,
Biochem
(1986) 25:285-294; J. Bentz et al.,
Biochem
(1987) 26:2105-2116).
Cellular Targeting. Efficient gene transfer requires targeting of the DNA to the cell of choice. Recently, procedures based upon receptor mediated endocytosis have been described for gene transfer. G. Y. Wu et al.,
J Biol Chem
(1987) 262:4429; G. Y. Wu et al.,
J Biol Chem
(1988) 263:14621-14624. A cell-specific ligand-polylysine complex is bound to nucleic acids through charge interactions. The resulting complex is taken up by the target cells. Wu et al., supra, reported efficient transfection of the human hepatoma cell line HepG2 and of rat hepatocytes in vivo using this delivery system with asialoorosomucoid as a ligand. Huckett et al.,
Biochem Pharmacol
(1990) 40:253-263, reported stable expression of an enzymatic activity in HepG2 cells following insulin-directed targeting. Finally Wagner et al.,
Proc Natl Acad Sci
(
USA
) (1990) 87:3410-3414 and (1991) 88:4255-4259 observed transfer
Haensler Jean
Szoka, Jr. Francis C.
Crosby, Heafey Roach & May
Koenig Nathan P.
Marschel Ardin H.
The Regents of the University of California
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