Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Matrices
Reexamination Certificate
1997-08-13
2001-06-26
Nguyen, Dave Trong (Department: 1633)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Matrices
C424S450000, C530S300000, C530S333000, C435S320100, C525S054100, C525S420000
Reexamination Certificate
active
06251433
ABSTRACT:
DESCRIPTION
1. Field of the Invention
This invention relates to compositions and methods for increasing the uptake of polynucleotides into cells. Specifically, the invention relates to vectors, targeting ligands, and polycationic agents. The polycationic agents are capable of (1) increasing the frequency of uptake of polynucleotides into a cell, (2) condensing polynucleotides; and (3) inhibiting serum and/or nuclease degradation of polynucleotides.
2. Background of the Invention
Polycations, such as polylysine, have been used to facilitate delivery of nucleic acids to cell interior. Both in vitro and in vivo applications have taken advantage of this property. See, for example, Gao et al., 1996,
Biochem.
35:1027-1036.
Polynucleotides, typically DNA, may be taken into a cell by a receptor-mediated endocytosis pathway, a cellular mechanism which internalizes specific macromolecules. In general, complexes designed to be delivered in this fashion contain nucleic acids encoding the gene of interest and a polycationic agent, which acts as a DNA-binding carrier and both neutralizes the charge on the nucleic acids and condenses it.
Condensation facilitates entry of nucleic acids into cell vesicle systems by simulating a macromolecular structure. For example, polylysine condenses DNA into a toroid or doughnut-like structure. (Wagner et al., 1991,
Proc. Natl. Acad. Sci.
88:4255-4259).
Polycations previously utilized for nucleic acid delivery to cell interiors include polylysine, protamines, histones, spermine, spermidine, polyornithine, polyargnine, and putrescine.
All publications mentioned herein are incorporated herein by reference for the purpose of disclosing and describing features of the invention for which the publications are cited in connection with.
SUMMARY OF THE INVENTION
An embodiment of the invention is a vector for expression of polypeptides. The vector of the instant invention comprises: (i) an Epstein Barr Virus (EBV) origin of replication; (ii) a polynucleotide encoding an EBV origin binding protein; (iii) an enhancer; (iv) a promoter; and (v) a terminator. Polynucleotides encoding a desired polypeptide, such as erythropoietin or leptin can be inserted into the vector. Also, ribozyme and antisense polynucleotides can also be inserted into the vector.
One embodiment of the invention is a composition capable of targeting a polynucleotide to a specific cell type. The composition comprises: (i) a lipoprotein; (ii) a polynucleotide binding molecule; and (iii) a polynucleotide.
Another embodiment of the invention is a method of increasing the frequency of uptake of polynucleotides into a cell by contacting a cell with a composition comprising: (i) a lipoprotein, (ii) a polynucleotide binding molecule; and (iii) a polynucleotide.
Yet another embodiment of the invention is a method of increasing the frequency of uptake of polynucleotides into a specific cell type by contacting a population of cells with a composition comprising (i) a lipoprotein, (ii) a polynucleotide binding molecule; and (iii) a polynucleotide.
One embodiment of the invention is a polycationic agent exhibiting a net positive electrical charge at physiological pH with the following formula:
where Ta and Tc are terminating groups. A preferred subset of these compounds is the set where R
2
is hydrogen. Even more preferred are polymers comprising at least one unnatural amino acid. Also preferred are polymers where R
2
and R
3
are hydrogen and R
1
is not hydrogen, also referred to as poly N-substituted glycines or “poly NSGs.”
Another embodiment is a neutral polymer exhibiting no net positive or negative electrical charge at physiological pH with the following formula:
where Ta and Tc are terminating groups. A preferred subset of these compounds is the set where R
2
is hydrogen. Even more preferred are polymers comprising at least one unnatural amino acid. Also preferred are polymers where R
2
and R
3
are hydrogen and R
1
is not hydrogen, also referred to as poly N-substituted glycines or “poly NSGs.”
The instant polycationic agents and neutral polymers are capable of neutralizing the electrical charge of nucleic acids. A subset of these compounds are capable of (1) condensing the structure of polynucleotides and/or (2) protecting polynucleotides from serum and/or nuclease degradation.
Yet another embodiment of the invention are polycationic agents and neutral polymers that (1) target binding of nucleic acids to cell surfaces, (2) trigger cell membrane destabilization; (3) exhibit endosome buffering capacity; (4) trigger endocytosis; (5) help trigger the release of polynucleotide/lipid complexes from endosomes or (6) nuclear tropism.
Another embodiment of the invention is a composition comprising a polynucleotide of interest and an effective amount of the polycationic agent to neutralize the charge of the polynucleotide. Optionally, the composition includes a ligand which directs the complex to particular cells expressing a ligand-binding partner, and/or an endosomolytic agent, which serves to cause disruption of the endosome containing the complex.
Another embodiment of the invention is a method of condensing nucleic acids by providing an effective amount of the polycationic agents or neutral polymers of the invention and contacting the agent with the desired polynucleotides.
Also an embodiment of the invention is a method of inhibiting serum and/or nuclease degradation of nucleic acids by providing an effective amount of the the polycationic agents or neutral polymers of the inventions and contacting the agent with the desired nucleic acids.
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Wagner et al., “DNA-Binding Transferrin Conjugates as Functional Gene-Delivery Agents: Synthesis by Linkage of Polylysine or Ethidium Homodimer to the Transferrin Carbohydrate Moiety”Bioconjugate Chem. 2(4):226-231, 1991.
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Lasic and Tenpleton,
Cohen Fred E.
Dubois-Stringfellow Nathalie
Dwarki Varavani
Innis Michael A.
Murphy John E.
Blackburn Robert P.
Chiron Corporation
Gorthey LeeAnn
Lentini David P.
Nguyen Dave Trong
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