Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – Mixing of two or more solid polymers; mixing of solid...
Reexamination Certificate
1998-07-30
2001-04-24
Michl, Paul R. (Department: 1714)
Synthetic resins or natural rubbers -- part of the class 520 ser
Synthetic resins
Mixing of two or more solid polymers; mixing of solid...
C514S04400A, C536S023100, C536S023700, C536S023720
Reexamination Certificate
active
06221959
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to compositions of poly(nucleic acid) polymers such as RNA or DNA polymers and polycations that are associated, either covalently or noncovalently, with block copolymers of alkylethers. The complexes are well suited for use as vehicles for delivering nucleic acid into cells.
BACKGROUND OF THE INVENTION
The use of antisense poly(nucleic acids) to treat genetic diseases, cell mutations (including cancer causing or enhancing mutations) and viral infections has gained widespread attention. This treatment tool is believed to operate, in one aspect, by binding to “sense” strands of mRNA encoding a protein believed to be involved in causing the disease state sought to be treated, thereby stopping or inhibiting the translation of the mRNA into the unwanted protein. In another aspect, genomic DNA is targeted for binding by the antisense polynucleotide (forming a triple helix), for instance, to inhibit transcription. See Helene,
Anti
-
Cancer Drug Design,
6:569 (1991). Once the sequence of the mRNA sought to be bound is known, an antisense molecule can be designed that binds the sense strand by the Watson-Crick base-pairing rules, forming a duplex structure analogous to the DNA double helix.
Gene Regulation: Biology of Antisense RNA and, DNA
, Erikson and lxzant, eds., Raven Press, New York, 1991; Helene,
Anti
-
Cancer Drug Design,
6:569 (1991); Crooke,
Anti
-
Cancer Drug Design,
6:609 (1991). A serious barrier to fully exploiting this technology is the problem of efficiently introducing into cells a sufficient number of antisense molecules to effectively interfere with the translation of the targeted mRNA or the function of DNA.
One method that has been employed to overcome this problem is to covalently modify the 5′ or the 3′ end of the antisense polynucleic acid molecule with hydrophobic substituents. These modified nucleic acids generally gain access to the cells interior with greater efficiency. See, for example, Kabanov et al.,
FEBS Lett.,
259:327 (1990); Boutorin et al.,
FEBS Lett.,
23:1382-1390, 1989; Shea et al,
Nucleic Acids Res.,
18:3777-3783 (1990). Additionally, the phosphate backbone of the antisense molecules has been modified to remove the negative charge (see for example, Agris et al.,
Biochemistry,
25:6268 (1986); Cazenave and Helene in
Antisense Nucleic Acids and Proteins: Fundamentals and Applications
, Mol and Van der Krol, eds., p. 47 et seq., Marcel Dekker, New York, (1991) or the purine or pyrimidine bases have been modified (see, for example,
Antisense Nucleic Acids and Proteins: Fundamentals and Applications
, Mol and Van der Krol, eds., p. 47 et seq., Marcel Dekker, New York (1991); Milligan et al. in
Gene Therapy For Neoplastic Diseases
, Huber and Laso, eds., p. 228 et seq., New York Academy of Sciences, New York (1994). Other attempts to overcome the cell penetration barrier include incorporating the antisense poly(nucleic acid) sequence into an expression vector that can be inserted into the cell in low copy number, but which, when in the cell, can direct, the cellular machinery to synthesize more substantial amounts of antisense polynucleic molecules. See, for example, Farhood et al.,
Ann. N.Y. Acad. Sci.,
716:23 (1994). This strategy includes the use of recombinant viruses that have an expression site into which the antisense sequence has been incorporated. See, e.g., Boris-Lawrie and Temin,
Ann. N. Acad. Sci.,
716:59 (1994). Others have tried to increase membrane permeability by neutralizing the negative charges on antisense molecules or other nucleic acid molecules with polycations. See, e.g. Kabanov et al.,
Soviet Scientific Reviews, Vol.
11, Part 2 (1992); 30 Kabanov et al.,
Bioconjugate Chemistry
4:448 (1993); Wu and Wu,
Biochemistry,
27:887-892 (1988); Behr et al,
Proc. Natl. Acad Sci U.S.A.
86:6982-6986 (1989). There have been problems with systemically administering poylnucleotides due to rapid clearance degradation and low bioavailability. In some cases it would be desirable to target polynucleotide molecules to a specific site in the body to specific target cells. Also, due to poor or low transport across biological barriers (such as the blood-brain barrier) the transport of polynucleotides to targets across this barrier is decreased or impossible. Additionally, the problems with low oral or rectal bioavailability dramatically hinders the administration of such polynucleotides (including oligonucleotides).
Of course, antisense polynucleic acid molecules are not the only type of polynucleic acid molecules that can usefully be made more permeable to cellular membranes. To make recombinant protein expression systems, the expression-directing nucleic acid must be transported across the membrane and into the eukaryotic or prokaryotic cell that will produce the desired protein. For gene therapy, medical workers try to incorporate, into one or more cell types of an organism, a DNA vector capable of directing the synthesis of a protein missing from the cell or useful to the cell or organism when expressed in greater amounts. The methods for introducing DNA to cause a cell to produce a new protein, ribozyme or a greater amount of a protein or ribozyme are called “transfection” methods. See, generally,
Neoplastic Diseases
, Huber and Lazo, eds., New York Academy of Science, New York (1994); Feigner,
Adv. Drug Deliv. Rev.,
5:163 (1990); McLachlin, et al.,
Progr. Nuci. Acids Res. Mol. Biol.,
38:91 (1990); Karlsson,
S. Blood,
78:2481 (1991); Einerhand and Valerio,
Curr. Top. MicrobioL immunol,
177:217-235 (1992), Makdisi et al.,
Prog. Liver Dis.,
10:1 (1992); Litzinger and Huang,
Biochim. Biophys. Acta,
11, 13:201 (1992); Morsy et al.,
J.A.M.A.,
270:2338 (1993); Dorudi et al.,
British J. Surgery,
80:566 (1993).
A number of the above-discussed methods of enhancing cell penetration by antisense nucleic acid are generally applicable methods of incorporating a variety of poly(nucleic acids) into cells. Other general methods include calcium phosphate precipitation of nucleic acid and incubation with the target cells (Graham and Van der Eb,
Virology,
52:456, 1983), co-incubation of nucleic acid, DEAE-dextran and cells (Sompayrac and Danna,
Proc. Natl. Acad. Sci.,
12:7575, 1981), electroporation of cells in the presence of nucleic acid (Pofter et al.,
Proc. Nail. Acad. Sci.,
81:7161-7165, 1984), incorporating nucleic acid into virus coats to create transfection vehicles (Gitman et al.,
Proc. Natl Acad Sci. U.S.A.,
82:7309-7313, 1985) and incubating cells with nucleic acid incorporated into liposomes (Wang and Huang,
Proc. Natl. Acad. Sci.,
84:7851-7855, 1987).
Another problem in delivering nucleic acid to a cell is the extreme sensitivity of nucleic acids, particularly ribonucleic acids, to nuclease activity. This problem has been particularly germane to efforts to use ribonucleic acids as anti-sense oligonucleotides. Accordingly, methods of protecting nucleic acid from nuclease activity are desirable.
SUMMARY OF THE INVENTION
The invention relates to compositions of poly(nucleic acid) polymers such as RNA or DNA polymers and polycations that are associated, either covalently or noncovalently, with block copolymers of alkylethers. In a preferred embodiment, the poly(nucleic acids) are complexed with a polycation. The nucleic acid is stabilized by the complex and, in the complex, has increased permeability across cell membranes. Accordingly, the complexes are well suited for use as vehicles for delivering nucleic acid into cells.
DETAILED DESCRIPTION OF THE INVENTION
The invention thus relates to compositions of poly(nucleic acid) polymers such as RNA or DNA polymers, and polycations that are associated (either covalently or noncovalently) with cationic block copolymers.
Structure of Block Copolymers
Block copolymers are most simply defined as conjugates of at least two different polymer segments (Tirrel, M. In:
Interactions of Surfactants with Polymers and Proteins
. Goddard E. D. and Ananthapadmanabhan, K. P. (eds.), CRC Press, Boca Raton, A
Alakov Valery Y.
Kabanov Alexander V.
Vinogradov Sergey V.
Mathews, Collins Shepherd & Gould P.A.
Michl Paul R.
Supratek Pharma Inc.
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