Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-12-22
2001-04-17
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S456000, C435S458000
Reexamination Certificate
active
06218112
ABSTRACT:
FIELD OF THE INVENTION
This invention is generally in the field of DNA delivery systems. More specifically, this invention describes methods of optimizing the efficiency of transfecting cells with a DNA molecule bound to peptides and derivatives thereof.
All publications cited in this specification are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Transfection of cells, whether in vitro, ex vivo or in vivo, involves not only delivery of the transfecting DNA to the cell nucleus, but also expression of the delivered DNA in the cell. Some gene delivery systems involve transfection of cells using a delivery complex in which DNA is condensed with cationic polymers such as cationic lipids or polylysine (see, for example, Cotten and Wagner (1993) Curr. Opin. Biotech., 4: 705). There is a need in the art for a gene delivery system which is stable in the bloodstream, targetable to selected tissue types, and capable of efficient transport into the cytoplasm and to the nucleus of both dividing and non-dividing cells. Some transfection techniques demonstrate that the transfecting DNA may be absorbed by cells, only to accumulate unchanged in the cytoplasm and unexpressed in the nucleus (for example, see Zaubner et al., (1995) J. Biol. Chem. 270: 18997, wherein cationic liposomes carrying DNA accumulate in the cytoplasm), suggesting that there is a need in the art for improvements in current methods of cell transfection.
SUMMARY OF THE INVENTION
This invention provides methods of screening of polyvalent cationic peptides, or derivatives of peptides (also called conjugates), for the ability to optimally transfect cells with a nucleic acid molecule condensed on the cationic peptide or conjugate based on determining the equilibrium dissociation constant (also called the equilibrium constant), K
D
or the apparent dissociation constant (also called the apparent dissociation constant), k
d
, for the interaction between the peptide or conjugate and the nucleic acid molecule.
The invention is based on the recognition that the equilibrium constant for the interaction between a peptide, or derivative thereof, and a nucleic acid molecule is readily determined by measuring the change in surface plasmon resonance (SPR) signal caused by a bimolecular interaction between the nucleic acid molecule which is immobilized on a metal sensor chip, and the peptide or conjugate which is present in a solution flowing over the chip, and that the equilibrium constant, or simply the apparent dissociation constant, is a critical predictor of optimal transfection efficiency.
As used herein, the terms “optimal transfection” or “optimally transfect” refers to obtaining the largest number of transfected cells per unit amount of nucleic acid condensed on a particular peptide or conjugate of a gene delivery complex. It will be appreciated by one of skill in the art that the proportion of transfected cells will vary for each target cell and therefore the largest number of transfected cells in a given target cell population may be. for example, 25% or 50% of the targeted cells for one selected cell population, and may be 80% or even as high as 95% for another selected cell population. It will become apparent from the specification below that “transfection” is meaningful only in terms of expression of the nucleic acid in the host cell, and thus “expression” refers to the biological activity of the nucleic acid delivered to the host cell, whether that biological activity be the production of a gene product such as a protein or an RNA, or via a measurable biological effect of the nucleic acid in the host cell.
The nucleic acid to be delivered to the host cell need not be identical in length or composition to the nucleic acid immobilized on the sensor chip. It is preferred that the nucleic acid to be delivered and the chip immobilized nucleic acid possess the same overall properties as to DNA or RNA and as to strandedness (double or single-stranded). It also is preferred that the overall lengths of the delivered and immobilized nucleic acids be approximately the same; that is, if the nucleic acid to be delivered is about 50 nucleotides in length, the immobilized nucleic acid be in the range of 10-500 nucleotides in length; if the nucleic acid to be delivered is about 1.0 kb in length, the immobilized nucleic acid be in the range of 1.0 kb-10 kb in length; if the nucleic acid to be delivered is about 50 kb in length, the immobilized nucleic acid be in the range of 10 kb-100 kb in length.
The nucleic acid molecule immobilized on the sensor ship may be a DNA or an RNA, although DNA is preferred, and it may be a double-stranded or single-stranded nucleic acid molecule. The immobilized nucleic acid may be of any desired length (e.g. generally in the range of 10 nucleotides—1 kb-50 kb), and may be of any sequence composition.
In a preferred embodiment of the methods described herein, the nucleic acid molecule immobilized on the sensor chip is a double-stranded DNA which is a plasmid. Plasmid refers to a circular or linear form of double-stranded DNA which is large enough to contain a gene (that is, a sequence comprising coding as well as regulatory regions).
The invention thus encompasses a method of screening test peptides for the ability to optimally transfect cells with a nucleic acid molecule comprising detecting a change in the surface plasmon resonance of a nucleic acid immobilized on a sensor chip and exposed to a solution of a test peptide, wherein the change in surface plasmon resonance occurs upon binding of the peptide to and dissociation of the peptide from the immobilized nucleic acid, thereby to permit either calculation of the equilibrium dissociation constant K
D
or apparent dissociation constant, k
d
; and (b) selecting the peptide having an equilibrium dissociation constant, K
D
, with a value of approximately 1×10
−12
to 1×10
−6
(or apparent dissociation constant, k
d
, with a value of approximately 1×10
−6
to 1×10
−1
).
The invention also encompasses a method of screening test peptides for the ability to optimally transfect cells with a nucleic acid molecule comprising (a) detecting a change in the surface plasmon resonance of a nucleic acid immobilized on a sensor chip and exposed to (a) a solution containing a test peptide for a time sufficient to permit binding of the peptide to the immobilized nucleic acid followed by (b) a solution lacking test peptide for a time sufficient to permit dissociation of the peptide from the immobilized nucleic acid, wherein the change in surface plasmon resonance occurs upon binding of the peptide to and dissociation of the peptide from the immobilized nucleic acid, thereby to permit calculation of the equilibrium dissociation constant K
D
; and selecting a peptide having an equilibrium dissociation constant, K
D
, with a value of approximately 1×10
−12
to 1×10
−6
.
The invention also encompasses a method of screening test peptides for the ability to optimally transfect cells with a nucleic acid molecule comprising (i) detecting a change in the surface plasmon resonance of a nucleic acid immobilized on a sensor chip and exposed to (a) a solution containing a test peptide for a time sufficient to permit binding of the peptide to the immobilized nucleic acid followed by (b) a solution lacking test peptide for a time sufficient to permit dissociation of the peptide from the immobilized nucleic acid, wherein the detection detects a change in surface plasmon resonance which occurs upon dissociation of the peptide from the immobilized nucleic acid, thereby to permit calculation of the apparent dissociation constant, k
d
; and (ii) selecting a peptide having an apparent dissociation constant, k
d
, with a value of approximately 1×10
−6
to 1×10
−1
).
In preferred embodiments of the invention, the methods further comprise, prior to the detecting step, the steps of immobilizing the nucleic acid on a sensor chip; and exposing the immobilized nucleic acid molecule to a solutio
Thatcher David R.
Wilks Paula E.
Cobra Therapeutics Limited
Ketter James
Woodcock Washburn Krutz Mackiewicz & Norris LLP
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