Amphipathic nucleic acid transporter

A nucleic acid transporter to deliver a nucleic acids into cells, comprising a cationic compound having a cationic head group for binding the nucleic acid and a lipid tail for association with the membrane. A cationic compound usually is a polyamine or a short basic peptide. The lipid tail is usually selected from the group consisting of plant steroid, animal steroid, isoprenoid compound, aliphatic lipid, pore forming protein, pore forming peptides and fusogenic peptides. The cationic head and the lipid tail are linked through a carbamate linkage. When polyamine is used, it is preferably either spermidine or spermine and the nucleic acid can be any of a variety, including triplex forming oligonucleotides, antisense oligonucleotide, aptamers, ribozymes, plasmids and DNA for gene therapy. Also described is a method for treating individuals using the transporter linked to a therapeutic nucleic acid.

FIELD OF INVENTION 
The present invention generally relates to a method of transporting nucleic 
acids into cells. More specifically, it relates to linking a polyamine or 
short basic peptide to a lipid compound for transporting nucleic acids 
into cells. 
BACKGROUND OF THE INVENTION 
It has been known that certain cationic lipids, e.g., DOTMA (Felgner, Adv. 
Drug Delivery Rev. 5:163, 1990), can improve the uptake kinetics of 
nucleic acids in some types of animal cells. Unfortunately, the 
commercially available preparations of cationic lipids are frequently 
cytotoxic at concentrations necessary for achieving cellular uptake. In 
addition, these compounds are not always effective for delivering material 
to different types of cell populations. 
There are a number of commercially available cationic lipids purported to 
improve the cellular entry of nucleic acids. The prototype compound, DOTMA 
(Felgner), has been widely used for improving transfection efficiency of 
cells. This compound is the active ingredient of the commercially 
available liposomal preparation Lipofectin (Gibco-BRL). Gibco-BRL also 
markets Lipofectace and Lipofectamine for the same purpose. Boehringer 
Mannheim supplies DOTAP, and Promega markets Transfectam. Most of these 
preparations have stringent requirements for administration to cells 
(e.g., a requirement for a low serum medium, specificity of cell types, 
quality of nucleic acids) and many are cytotoxic. Gao and Huang (Biochem. 
Biophys. Res. Comm. 179:280, 1991) have described a cholesterol-containing 
cationic lipid that is used as a component of liposomes with the potential 
to enhance the cellular uptake of nucleic acids. However, the structure of 
the cationic group and the mode of usage of the compound are different 
from the compounds of the present invention. An amino-sterol, squalamine, 
isolated from shark tissues, has been shown to be a broad-spectrum 
antibiotic by Moore et al. (Proc. Natl. Acad. Sci. 90:1354, 1993). This 
compound is a cationic steroid in which an anionic bile salt intermediate 
is linked to spermidine. Moore et al. did not suggest any use of the 
compound in improving nucleic acid delivery into cells. We are not aware 
of other publications that describe compounds identical or similar to the 
ones in this disclosure. 
There is a real need in the field of nucleic acid therapeutics to develop 
nonviral uptake enhancement reagents that are nontoxic and effective in a 
variety of cell types. The numerous commercial preparations are widely 
perceived to be ineffectual or of limited value for most aspects of 
nucleic acid delivery. The present invention addresses this problem by the 
synthesis of simple, relatively nontoxic cationic compounds that improve 
the cellular and nuclear delivery of nucleic acids. 
SUMMARY OF THE INVENTION 
An object of the present invention is to provide a transporter for moving 
nucleic acids into the cell and into the nucleus. 
Another object of the present invention is to provide a transporter for 
moving triplex forming oligonucleotides into the cell and into the 
nucleus. 
A further object of the present invention is the provision of a treatment 
method using therapeutic nucleic acids linked to a transporter. 
Thus, in accomplishing the foregoing objects, there is provided in 
accordance with one aspect of the present invention a relatively 
non-toxic, amphipathic transporter for delivery of nucleic acids into 
cells, comprising a cationic compound having a cationic head group for 
binding the nucleic acid and a lipid tail for association with the 
membrane; said cationic compound being a polyamine or a short basic 
peptide; said lipid tail being selected from the group consisting of plant 
steroid, animal steroid, isoprenoid compound, aliphatic lipid, pore 
forming protein or peptide and fusogenic peptide; and wherein the cationic 
head is linked to the lipid tail through a carbamate linkage. 
In the specific embodiments of the present invention, the cationic head 
group is a polyamine and selected from the group consisting of spermidine 
or spermine and the lipid tail is cholesterol. 
In another embodiment, the transporter is linked to a therapeutic nucleic 
acid. This combined nucleic acid/transporter can be used to treat disease. 
Other and further objects, features and advantages will be apparent in the 
following description of the presently preferred embodiments of the 
invention given for the purpose of disclosure when taken in conjunction 
with the accompanying drawings.

The drawings are not necessarily to scale. Certain features of the 
invention may be exaggerated in scale or shown in schematic form in the 
interest of clarity and conciseness. 
DETAILED DESCRIPTION 
It is readily apparent to one skilled in the art that various substitutions 
and modifications may be made to the invention disclosed herein without 
departing from the scope and spirit of the invention. 
The term "synthetic oligonucleotides" as used herein is defined as 
molecules, comprising two or more deoxyribonucleotides or ribonucleotides, 
preferably more than ten. The exact size would depend on many factors, 
including the specificity and binding affinity. 
The term "TFO" or "triplex forming oligonucleotide" as used herein refers 
to the synthetic oligonucleotides which are capable of forming a triplex 
by binding to the major groove of a duplex DNA structure. 
As used herein, a "short basic peptide" includes a peptide of two or more 
amino acids containing the amino acids arginine and lysine that are 
prevalent in proteins that bind to nucleic acids. 
The term "therapeutic nucleic acid" as used herein means any nucleic acid 
which can be used for the therapy of disease in humans or animals. This 
includes triplex forming oligonucleotides, antisense oligonucleotides, 
aptamers, ribozymes, plasmids and DNA for gene therapy. Once a therapeutic 
nucleic acid has been developed, the methods described in the present 
invention can be used to facilitate the introduction of the therapeutic 
nucleic acid into the cells. In this method the therapeutic nucleic acid 
is combined with the transporter. The therapeutic nucleic acid binds to 
the cationic head group. The combined therapeutic nucleic acid/transporter 
can then be delivered to the individual (human or animal) to be treated. 
Delivery can be any of the variety of techniques known to those skilled in 
the art. Examples of some of the techniques include oral, parental and 
direct injection. 
The word "bound" when used in the context of a nucleic acid bound to the 
transporter refers to the association of the head group with the nucleic 
acids by electrostatic, hydrophobic, ionic or other non-covalent 
interaction. One skilled in the art will recognize that the specific 
binding which is most useful in the present invention will depend on the 
specific head group and nucleic acid which are combined. 
One embodiment of the present invention is a non-toxic amphipathic 
transporter for delivery of nucleic acids into cells. The transporter is 
comprised of a cationic compound having a cationic head group for binding 
nucleic acids and a lipid tail for association with a membrane. The 
cationic compound is usually selected from a group consisting of polyamine 
or a short basic peptide. The lipid tail is usually selected from the 
group consisting of plant steroid, animal steroid, isoprenoid compound, 
aliphatic lipid, pore forming protein, pore forming peptide and a 
fusogenic peptide. In the transporter, the cationic head is linked to the 
lipid tail through a carbamate linkage. 
Although the mechanism of action of the transporter is not known, it is 
expected that the most effective compounds have a cationic head group for 
binding to nucleic acids, and a lipid tail to associate with membranes. In 
addition, the components of the transporter should have minimal toxicity 
and be biodegradable. 
In one embodiment the transporter has polyamines (spermine or spermidine) 
attached to cholesterol through a carbamate linkage (FIG. 1). The 
polyamines are ubiquitous in all animal cell types, and cholesterol is a 
component of cellular membranes. 
One skilled in the art recognizes that other related amphipathic compounds 
can be used. In each case the transporter contains a cationic group 
attached to a membrane-interactive moiety. The two functional domains are 
essential for improving the transmembrane movement of the 
oligonucleotides. The cationic group can be any polyamine or short basic 
peptides. The hydrophobic group could be any naturally occurring lipid. 
For example, these can include animal or plant steroids, isoprenoid 
compounds such as farnesol, aliphatic lipids, pore forming proteins, pore 
forming peptides, and fusogenic peptides. 
The following examples are offered by way of illustration and are not 
intended to limit the invention in any manner. 
EXAMPLE 1 
Synthesis of Polyamine-Cholesterol Conjugates 
Cholesteryl chloroformate was added dropwise to a solution of spermidine or 
spermine in methylene chloride and N,N-diisopropyl ethylamine, and stirred 
at room temperature for 2 hours. The product was purified by 
chromatographic methods and characterized by standard physical and 
spectroscopic methods. The final preparation, a white solid (&gt;95% purity) 
(FIG. 1), was solubilized and used in standard oligonucleotide uptake 
assays. 
EXAMPLE 2 
Cytotoxicity of Cationic Lipids 
Cell proliferation was measured by the Cell Titer 96 Aqueous Non 
Radioactive Cell Proliferation Assay (Promega). The absorbance at 490 nm 
is directly proportional to the number of living cells. Vero cells were 
plated at an initial density of 500 cells/well. After 20 hours, cells were 
exposed to different concentrations of cationic lipid for 4 days. Data 
points in FIG. 2 are the average of replicates for each test 
concentration. 
From FIG. 2 it can be seen that the novel cationic compounds are less toxic 
than DOTMA, as assessed by this highly sensitive cell proliferation assay. 
The polyamine-cholesterol derivatives are typically used in cell 
culture-based biological assays at concentrations ranging from 2 to 10 
.mu.g/ml, at effectively nontoxic levels. 
EXAMPLE 3 
Cellular Uptake Experiments 
Vero cells were treated with 1 .mu.M .sup.35 S-labeled 
oligonucleotide-propylamine coadministered with one of the 3 cationic 
lipids, as indicated, at about a 1:2 mass ratio. For Lipofectin only, the 
extracellular medium contained no serum, as suggested by the manufacturer. 
At various time points (0-24 h), the uptake of radiolabeled 
oligonucleotides was evaluated by scintillation counting, and 
intracellular concentration was calculated based on the known specific 
activity of the radiolabeled oligonucleotides. 
In the standard assay, the 3' end-protected oligonucleotides (e.g., 
oligo-3'-propylamine; Zendegui et al., Nuc. Acid Res. 20:307, 1992) are 
premixed with the cationic compounds and added to cells. The optimal ratio 
of oligonucleotides to cationic group must be determined empirically. 
Usually, a ratio of 1:2 by mass results in enhanced cellular uptake. The 
internalized compounds can be traced using biological assays, or by 
attaching a radiolabel or a fluorophore to the nucleic acid. 
Internalization of oligonucleotide-cationic lipid complex occurs rapidly, 
within 20-40 minutes, and fluorescence microscopy experiments suggest that 
a portion of the internalized oligonucleotides enter the nucleus. Using 
radiolabeled material, the uptake-enhancement effect is estimated to be up 
to 100 fold greater than if cells are treated with oligomers alone (FIG. 
3). The improved uptake kinetics of oligonucleotides obtained by 
coadministration with polyamine-cholesterol indicates that the 
oligonucleotides can be used at a lower effective dose in biological 
experiments. Thus, the new cationic compounds improved cellular uptake by 
up to 100 fold over controls, and uptake enhancement occurred in the 
presence of serum. 
EXAMPLE 4 
Stability of Oligonueleotides 
Vero cells were incubated at 37.degree. C. with 1 .mu.M .sup.35 S-labeled 
oligonucleotide-3'-propylamine in the presence of spermidine-cholesterol 
at about a 1:2 mass ratio. At various time points, extracellular medium 
and cells were separated and the amount of total radioactivity in each 
sample was determined by scintillation counting. Intact oligonucleotides 
were then extracted from the medium and the cells by phenol-chloroform 
extraction and ethanol precipitation, and finally electrophoresed on 12% 
denaturing polyacrylamide gels. The amount of intact radiolabeled 
oligonucleotide was quantified. The figures show the amount of total 
radioactivity and intact radiolabeled oligonucleotide, plotted as a 
percentage of control oligonucleotide. 
The integrity of oligonucleotides delivered into cells via the new uptake 
enhancers has been studied in Vero cells. End-protected oligonucleotides 
(containing 3'-propylamine; Zendegui et al., 1992) are known to be more 
stable than unmodified oligonucleotides (free 3'-OH) in the extracellular 
medium. There is a slight decrease in total radioactivity in the 
extracellular medium over 24 hours, presumably due to cellular uptake. 
Approximately 15% of the starting material is still intact after 24 hours. 
Loss of intact oligonucleotides appears to be due mostly to degradation as 
well as cellular uptake. 
When Vero cells grown in medium containing 10% bovine serum were incubated 
with .sup.35 S-labeled oligomers, approximately 10% of the counts entered 
cells at the end of 24 hours (FIG. 4). Gel analysis indicated that 
approximately a third of oligonucleotides were still intact after 24 
hours. More importantly, nearly all of the internalized material was also 
intact (FIG. 4). 
These data suggest that the material inside cells is protected from 
endonuclease digestion. 
The novel transporter of the present invention is more effective and 
versatile than the available compounds for improving the uptake of 
oligonucleotides by cells. Cells actively accumulate the oligonucleotides, 
and once inside, the oligonucleotides remain intact for longer periods of 
time. Transport into the nucleus is also facilitated. The uptake enhancers 
have utility in the delivery of any nucleic acid therapeutic that needs to 
enter cells, including but not limited to triple helix-forming 
oligonucleotides, antisense oligonucleotides, aptamers, and longer pieces 
of DNA for gene therapy. 
All patents and publications mentioned in this specification are indicative 
of the levels of those skilled in the art to which the invention pertains. 
All patents and publications are herein incorporated by reference to the 
same extent as if each individual publication was specifically and 
individually indicated to be incorporated by reference. 
One skilled in the art will readily appreciate that the present invention 
is well adapted to carry out the objects and obtain the ends and 
advantages mentioned, as well as those inherent therein. The methods, 
procedures and treatments described herein are presently representative of 
preferred embodiments, are exemplary, and are not intended as limitations 
on the scope of the invention. Changes therein and other uses will occur 
to those skilled in the art which are encompassed within the spirit of the 
invention or defined by the scope of the appended claims.