Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Particulate form
Reexamination Certificate
1999-07-13
2001-04-17
Brusca, John S. (Department: 1635)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Particulate form
C424S489000, C435S325000, C435S440000, C514S04400A
Reexamination Certificate
active
06217912
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to gene therapy and drug delivery. More particularly, the invention relates to compositions and methods for use, and making thereof, for delivering nucleic acids as gene therapy applications or other non-soluble bioactive molecules such as protein, peptides or small non-soluble drugs.
Biodegradable polymers are gaining attention as drug delivery systems. R. Langer, New Methods of Drug delivery, 249 Science 1527-1533 (1990); B. Jeong et al., Biodegradable Block Copolymers as Injectable Drug-delivery Systems, 388 Nature 860-862 (1997). Since genes are now considered pharmaceutical agents for treating many types of diseases, and gene therapy is becoming widely used as demonstrated by many clinical trials, M. A. Kay et al., Gene Therapy, 94 Proc. Nat'l Acad. Sci. USA 1744-12746 (1997); C. Bordignon et al., Gene Therapy in Peripheral Blood Lymphocytes and Bone Marrow for ADA-immunodeficient Patients, 270 Science 470-475 (1995), there is an urgent need for a safe and efficient gene carrier. Genes are very attractive candidates for therapeutic use in a variety of disease states due to their ability to produce bioactive proteins using the biosynthetic machinery provided by host cells. A major technical impediment to gene transfer is the lack of an ideal gene delivery system. There are many established protocols for transferring genes into cells, including calcium phosphate precipitation, electroporation, particle bombardment, liposomal delivery, viral-vector delivery, and receptor-mediated gene delivery. A.V.Kavanov, Self-assembling Complexes for Gene delivery, p.L.Felgner & L.W.Seymour, J.Wiley & Sons (1998); P.L.Chang, Somatic Gene Therapy, CRC Press (1995).
Transfection methods using retroviral or adenoviral vectors have been investigated. Retroviral vectors, in particular, have been used successfully for introducing exogenous genes into the genomes of actively dividing cells such that stable transformants are obtained. D. G. Miller et al., Gene Transfer by Retrovirus Vectors Occurs Only in Cells that are Actively Replicating at the Time of Infection. 10 Mol. Cell Biol. 4239-4242 (1990). Viral vector systems often, in case of complementation of defective vectors by inserting genes into ‘helper’ cell lines, generate a transducing infectious agent. In addition, it is well known that the host immune response to adenoviruses limits their use as a transfer facilitating agent to a single administration. To address this limitation, fusion peptides of the influenza virus hemagglutinin have been employed to replace adenoviruses as endosomal lytic agents, but have met with limited success. S. Gottschalk et al., A Novel DNA-Peptide Complex for Efficient Gene Transfer and Expression in Mammalian Cells, 3 Gene Ther. 448-457 (1996). However, despite their high transfection efficiency in vitro, inserting genes into the host cell's genome in vivo depends on the viral infection pathway. Application of the viral infection pathway for human gene therapy introduces serious concerns about endogenous virus recombination, oncogenic effects, and inflammatory or immunologic reactions. G Ross et al., Gene Therapy in the United States: A Five-Year Status Report. 7 Hum. Gene Ther., 1781-1790 (1996). Because of these concerns the use of viral vectors for human gene therapy has been extremely limited.
As compared to viral gene carriers, there are several advantages to the use of non-viral based gene therapies, including their relative safety and low cost of manufacture. Non-viral gene delivery systems such as cationic liposomes or synthetic gene carriers, e.g.poly-L-lysine (PLL), are being widely sought as alternatives and investigated intensively to circumvent some problems encountered in viral vectors. K. A. Mislick et al., Transfection of Folate-polylysine DNA Complexes: Evidence for Lysosomal Delivery, 6 Bioconjugate Chem. 512-515 (1995); J.O. Rädler et al., Structure of DNA-cationic Liposome Complexes: DNA Intercalation in Multilamellar Membranes in Distinct Interhelical Packing Regimes, 275 Science 810-814 (1997); J. Cheng et al., Effect of Size and Serum Proteins on Transfection Efficiency of Poly((2-dimethylamino)ethyl methacrylate)-plasmid nanoparticles, 13 Pharm. Res. 1038-1042 (1996). There are several polymeric materials currently being investigated for use as gene carriers, of which poly-L-lysine (PLL)is the most popular, but few of them are biodegradable. Biodegradable polymers, such as polylactic/glycolic acid(negatively charged), and polylactide/glycolide (neutral) have been used as gene carriers in the form of non-soluble particulates. Amarucyama et al, Nanoparticle DNA Carrier with PLL Grafted Polysallanide Copolymer and Polylactic Acid, 8 Bioconjugate, 735-739(1997). In general, polycationinic polymers are known to be toxic and the PLL backbone is barely degraded under physiological conditions. It will remain in cells and tissue which cause undesirably high toxicity. A.Segouras & R.Dunlan, Methods for Evaluation of Biocompatibility of Synthetic Polymers, 1 J.Mater.Sci in Medicine, 61-68(1990).
In view of the foregoing it will be appreciated that providing a soluble and biodegradable gene carrier, meaning that the polymer gene carrier can break down or degrade within body to non-toxic components after the genes have been delivered, that is non viral, safe and effective would be a significant advancement in the art.
BRIEF SUMMARY OF THE INVENTION
It is an object of the present invention to provide a composition and a method for delivering nucleic acids into cells.
It is also an object of the present invention to provide a biodegradable gene carrier composition and methods of use and making thereof.
It is another object of the present invention to provide a composition and a method for gene delivery that is efficient and non toxic.
It is yet another object of the invention to provide an non-toxic, soluble, biodegradable, non-viral composition and a method of use thereof, for delivering exogenous nucleic acids into a target cell.
These and other objects can be achieved by providing a novel polymer, poly[&agr;-(4-aminobutyl)-L-glycolic acid] (PAGA), containing a biodegradable ester linkage backbone with positively charged amine groups.
A composition for use as a gene delivery carrier comprises an effective amount of PAGA admixed with an effective amount of a nucleic acid comprising the gene to be delivered.
A method for making poly[&agr;-(4-aminobutyl)-L-glycolic acid] comprises the steps of:
(a) protecting the ∈-amino group of L-lysine with an amine blocking agent to result in blocked L-lysine;
(b) deaminating the &agr;-amino group of the blocked L-lysine to result in blocked (&agr;-amino-2(S)-hydroxyhexanoic acid);
(c) polymerizing the blocked (6-amino-2(S)-hydroxyhexanoic acid) to result in blocked poly[&agr;-(4-aminobutyl)-L-glycolic acid]; and
(d) deprotecting the blocked poly[&agr;-(4-aminobutyl)-L-glycolic acid] by removing the amine blocking group to result in poly[&agr;-(4-aminobutyl)-L-glycolic acid].
A method of delivering a selected nucleic acid into a selected cell comprises the steps of:
(a) mixing an effective amount of the selected nucleic acid with an effective amount of poly[&agr;-(4-aminobutyl)-L-glycolic acid] to result in a complex;
(b) contacting the selected cell with the complex under conditions suitable to maintain the viability of the cell.
The biodegradable gene carrier of this invention, PAGA, which is an analogue of PLL, can also be grafted with polyethylene glycol (PEG), which in turn may covalently bind to a targeting moiety by a similar process as disclosed in a copending U.S. patent application, Ser. No. 09/315,240 filed May 20, 1999, which is fully incorporated herein.
REFERENCES:
patent: 5942253 (1999-08-01), Gombotz et al.
Choi Young Hun
Kim Sung Wan
Park Jong Sang
Brusca John S.
Expression Genetics, Inc.
Shibuya Mark L.
Thorpe North & Western LLP
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