Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1999-10-08
2001-04-03
Schwartzman, Robert A. (Department: 1636)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C435S458000
Reexamination Certificate
active
06210708
ABSTRACT:
FIELD OF INVENTION
The present invention is in the field of gene biotechnology and gene therapy and relates to novel virosomes, i.e., positively charged liposomal vesicles containing viral glycoproteins in the membrane, for efficient transfer of genetic material to target locations, a method of manufacture and useful applications thereof. The present cationic virosomes are particularly suitable for the specific and unspecific, non-infectious delivery of genes to target cells in vitro and in vivo.
BACKGROUND OF THE INVENTION
Liposomes are widely used as carriers for drug delivery and as protective shelters for short-lived pharmaceutical substances or against (bio)chemical attack by bodily fluids. Liposomes containing reconstituted membrane proteins or parts thereof from viral envelopes are usually called “virosomes” (Sizer et al., Biochemistry 26:5106-5113, 1987). They have been applied for non-specific delivery of various drugs and DNA molecules (Vainstein et al., Methods Enzymol. 101:492-512, 1983). It turned out to be a major drawback that these virosomes fused with the cell membrane of the target cells resulting in an uncontrolled release of the transported material into the cytoplasm of the target cells where the unprotected material was readily attacked by degradative intracellular processes.
WO 92/13525, the whole contents of which shall herewith be incorporated by reference, reports that virosomes made of phospholipid bilayer membranes which are targeted with viral spike proteins from influenza virus and with cell-specific markers such as, e.g., monoclonal antibodies, very efficiently fuse with model membranes and animal cells due to a virus-like penetration mechanism by way of receptor-mediated endocytosis. While these virosomes are successfully applied to deliver chemical substances and desired drugs to target locations, they suffer from certain disadvantages with respect to stable incorporation and transfer of charged molecules such as, for instance, negatively charged nucleic acids.
Within the last few years the delivery, notably the cell-specific delivery, of genetic material incorporated in liposomes has gained more and more attention and importance, particularly with regard to applications in anti-cancer and gene therapy. Several methods are currently available for delivery of DNA or RNA to cells: Virus mediated methods, lipid mediated methods, and other methods like microinjection and electroporation. The advantages and disadvantages of current gene transfer techniques can be summarized as follows:
a) Virus mediated gene transfer: Genes can be introduced stably and efficiently into mammalian cells by means of retroviral vectors. However, the efficiency of gene transfer to non-replicating cells is very low because retroviruses infect only dividing cells. Further, general safety concerns are associated with the use of retroviral vectors relating to, for instance, the possible activation of oncogenes. Replication-defective adenovirus has become the gene transfer vector-of-choice for a majority of investigators. The adenovirus vector mediated gene delivery involves either the insertion of the desired gene into deleted adenovirus particles or the formation of a complex between the DNA to be inserted and the viral coat of adenovirus by a transferring-polylysine bridge. The drawback of this very efficient system in vivo is an undefined risk of infection or inflammation: Despite the E1 gene deletion that renders the virus defective for replication, the remaining virus genome contains numerous open reading frames encoding viral proteins (Yang et al. 1994; Proc. Natl. Acad. Sci. USA 91, 4407-4411). Expression of viral proteins by transduced cells elicits both humoral and cellular immune responses in the animal and human body and thus, may provoke inflammation and proliferation.
In the HVJ (Sendai virus) mediated method the foreign DNA is complexed with liposomes. The liposomes are then loaded with inactivated Sendai virus (hemagglutinating virus of Japan; HVJ). This method has already been used for gene transfer in vivo to various tissues. In addition, cellular uptake of antisense oligonucleotides by HVJ-liposomes was reported (Morishita et al. 1993; J. Cell. Biochem. 17E, 239). A particular disadvantage is, however, that the HVJ-liposomes tend to non-specifically bind to red blood cells.
b) Lipid mediated gene transfer: Positively charged liposomes made of cationic lipids appear to be safe, easy to use and efficient for in vitro transfer of DNA and antisense oligonucleotides. The highly negatively charged nucleic acids interact spontaneously with cationic liposomes. Already by simple mixing of the polynucleotides with preformed cationic liposomes a complete formation of DNA-liposome complexes is achieved. However, due to the lack of fusion peptides and cell-specific markers on the liposomal membrane the in vivo transfection efficiency is very low and the incubation times are long, wherefore high doses have to be administered in order to achieve a desired effect. Consequently, undesired side-effects may occur since there is evidence that large amounts of cationic lipids can exhibit toxic effects in vivo.
Small oligonucleotides are currently being tested as therapeutic agents for the treatment of cancer and as antiviral agents. Only one of the two DNA strands is transcribed to synthesize messenger RNA (mRNA). The DNA strand transcribed into RNA is called the coding strand or sense strand. The complementary, non-coding or antisense strand has the same sequence as the mRNA. When the non-coding strand is transcribed, it produces antisense RNA molecules that are able to bind to target (sense) mRNA. Once the antisense RNA is bound to the sense RNA the resulting RNA duplex molecules cannot be translated and the production of the protein is blocked. Usually, short synthetic antisense oligonucleotides of 18 to 22 bases effectively bind to the mRNA and inhibit mRNA translation. By this mechanism antisense oligonucleotides can stop the proliferation of human cancer cells. Genes that are involved in cancer exert their effect through overexpression of their normal structural proteins. Genes such as c-fos, c-myc, L-myc, N-myc, c-myb, abl, bcr-abl, c-raf, c-erb-2, K-ras may be potential targets for antisense cancer therapy. Antisense oligonucleotides are also an attractive potential alterative to conventional drugs such as, for example, antiviral agents such as, e.g., the antisense oligonucleotides of tat and gag gene of the human immunodeficiency virus (HIV).
Liposomal membranes comprising reconstituted virus envelopes as described in the literature (Stegmann et al.; EMBO J. 6:2651-2659, 1987) may be called virosomes. They usually comprise a phospholipid bilayer containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) together with viral envelope, e.g., spike proteins embedded in the membrane. The conventional methods to incorporate genetic material into PC, PE-virosomes suffer from the drawback of a rather low efficiency of nucleic acid incorporation.
SUMMARY OF THE INVENTION
The present invention therefore relates to a novel cationic virosome which due to its specific membrane composition may very efficiently be loaded with any desired genetic material comprising long and short chain DNA or RNA, oligodeoxynucleotides, ribonucleotides, peptide nucleic acids (PNA), ribozymes (RNA molecules with enzymatic activities), genes, plasmids and vectors and, thus, convincingly overcomes the drawbacks of the prior art.
The invention further relates to a method for the efficient reconstitution of hemagglutinin of influenza virus A, particularly of strain A/Singapore, into substantially unilamellar cationic lipid vesicles resulting in the formation of cationic virosomes with a mean diameter of approximately 120-180 nm and a continuous lipid bilayer, which is substantially free from the disadvantage of leakage seen with many conventional virosome preparations. The structure of the—preferably unilamellar—cationic brayer membrane is such that the hydrophilic, positively charged heads of
Gluck Reinhard
Klein Peter
Walti Ernst Rudolf
Davis Katharine F
Nika Health Products Limited
Oliff & Berridg,e PLC
Schwartzman Robert A.
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