U.S. patent application number 09/450315 was filed with the patent office on 2001-09-06 for intravascular delivery of non-viral nucleic acid.
Invention is credited to BUDKER, VLADIMIR G., HAGSTROM, JAMES E., MONAHAN, SEAN D., ROZEMA, DAVID B., SLATTUM, PAUL M., WOLFF, JON A..
Application Number | 20010019723 09/450315 |
Document ID | / |
Family ID | 27382671 |
Filed Date | 2001-09-06 |
United States Patent
Application |
20010019723 |
Kind Code |
A1 |
MONAHAN, SEAN D. ; et
al. |
September 6, 2001 |
INTRAVASCULAR DELIVERY OF NON-VIRAL NUCLEIC ACID
Abstract
Disclosed is a complex for providing nucleic acid expression in
a cell. A polynucleotide and a polymer are mixed together to form
the complex wherein the zeta potential of the complex is not
positive. Then the complex is delivered to the cell wherein the
nucleic acid is expressed.
Inventors: |
MONAHAN, SEAN D.; (MADISON,
WI) ; WOLFF, JON A.; (MADISON, WI) ; SLATTUM,
PAUL M.; (MADISON, WI) ; HAGSTROM, JAMES E.;
(MIDDLETOWN, WI) ; BUDKER, VLADIMIR G.; (MADISON,
WI) ; ROZEMA, DAVID B.; (MADISON, WI) |
Correspondence
Address: |
MARK K JOHNSON
P O BOX 510644
NEW BERLIN
WI
531310644
|
Family ID: |
27382671 |
Appl. No.: |
09/450315 |
Filed: |
November 29, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60121730 |
Feb 26, 1999 |
|
|
|
60146564 |
Jul 30, 1999 |
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Current U.S.
Class: |
424/450 ;
435/455; 514/44R; 536/23.1 |
Current CPC
Class: |
A61K 48/0041 20130101;
A61K 48/0083 20130101; A61K 48/0008 20130101 |
Class at
Publication: |
424/450 ; 514/44;
435/455; 536/23.1 |
International
Class: |
A61K 048/00; C07H
021/04; C12N 015/85; A61K 009/48 |
Claims
We claim:
1. A complex for providing nucleic acid expression in a cell,
comprising: a) mixing a polynucleotide and a polymer to form the
complex wherein the zeta potential of the complex is not positive;
b) delivering the complex to the cell wherein the nucleic acid is
expressed.
2. The complex of claim 1 wherein the polymer has a positive
charge.
3. The complex of claim 2 wherein the negative charge density is
greater than the positive charge density.
4. The complex of claim 1 wherein the complex is delivered to the
cell by inserting the polynucleotide into a mammalian vessel.
5. The complex of claim 4 wherein the complex is delivered to the
cell by increasing the permeability of the vessel.
6. The process of claim 5 wherein increasing the permeability of
the vessel consists of increasing pressure against vessel
walls.
7. The process of claim 6 wherein increasing the pressure consists
of increasing a volume of fluid within the vessel.
8. The process of claim 7 wherein increasing the volume consists of
inserting the polynucleotide in a solution into the vessel.
9. The process of claim 8 wherein a specific volume of the solution
is inserted within a specific time period.
10. The process of claim 9 wherein increased pressure is controlled
by altering the specific volume of the solution in relation to the
specific time period of insertion.
11. The process of claim 1 wherein the cell is selected from the
group consisting of a liver cell, spleen cell, heart cell, kidney
cell, prostate cell, skin cell, testis cell, skeletal muscle cell,
fat, bladder cell, brain cell, pancreas cell, thymus cell, and lung
cell.
12. A process for delivering the complex of claim 1 into a
parenchymal cell of a mammal, comprising: a) making the
polynucleotide-compound complex wherein the compound is selected
from the group consisting of amphipathic compounds, polymers and
non-viral vectors; b) inserting the complex into a mammalian
vessel; c) delivering the polynucleotide to the parenchymal
cell.
13. The process of claim 12 wherein further comprising increasing
permeability of the vessel.
14. The process of claim 13 wherein increasing the permeability of
the vessel consists of increasing pressure against vessel
walls.
15. The process of claim 112 wherein the parenchymal tissue is
selected from the group consisting of liver cells, spleen cells,
heart cells, kidney cells, prostate cells, skin cells, testis
cells, skeletal muscle cells, fat, bladder cells, brain cells,
pancreas cells, thymus cells, and lung cells.
Description
FEDERALLY SPONSORED RESEARCH N/A
CROSS-REFERENCE TO RELATED APPLICATIONS (Provisional Application
Ser. No.) (Filing Date) 60/121,730 Feb. 26, 1999 60/146,564 Jul. 7,
1999
FIELD OF THE INVENTION
[0001] The invention relates to compounds and methods for use in
biologic systems. More particularly, processes that transfer
nucleic acids into cells are provided. Nucleic acids in the form of
naked DNA or a nucleic acid combined with another compound are
delivered to cells.
BACKGROUND
[0002] Biotechnology includes the delivery of a genetic information
to a cell to express an exogenous nucleotide sequence, to inhibit,
eliminate, augment, or alter expression of an endogenous nucleotide
sequence, or to express a specific physiological characteristic not
naturally associated with the cell. Polynucleotides may be coded to
express a whole or partial protein, or may be anti-sense.
[0003] A basic challenge for biotechnology and thus its subpart,
gene therapy, is to develop approaches for delivering genetic
information to cells of a patient in a way that is efficient and
safe. This problem of "drug delivery," where the genetic material
is a drug, is particularly challenging. If genetic material are
appropriately delivered they can potentially enhance a patient's
health and, in some instances, lead to a cure. Therefore, a primary
focus of gene therapy is based on strategies for delivering genetic
material in the form of nucleic acids. After delivery strategies
are developed they may be sold commercially since they are then
useful for developing drugs.
[0004] Delivery of a nucleic acid means to transfer a nucleic acid
from a container outside a mammal to near or within the outer cell
membrane of a cell in the mammal. The term transfection is used
herein, in general, as a substitute for the term delivery, or, more
specifically, the transfer of a nucleic acid from directly outside
a cell membrane to within the cell membrane. The transferred (or
transfected) nucleic acid may contain an expression cassette. If
the nucleic acid is a primary RNA transcript that is processed into
messenger RNA, a ribosome translates the messenger RNA to produce a
protein within the cytoplasm. If the nucleic acid is a DNA, it
enters the nucleus where it is transcribed into a messenger RNA
that is transported into the cytoplasm where it is translated into
a protein. Therefore if a nucleic acid expresses its cognate
protein, then it must have entered a cell. A protein may
subsequently be degraded into peptides, which may be presented to
the immune system.
[0005] It was first observed that the in vivo injection of plasmid
DNA into muscle enabled the expression of foreign genes in the
muscle (Wolff, J A, Malone, R W, Williams, P, et al. Direct gene
transfer into mouse muscle in vivo. Science 1990;247:1465-1468.).
Since that report, several other studies have reported the ability
for foreign gene expression following the direct injection of DNA
into the parenchyma of other tissues. Naked DNA was expressed
following its injection into cardiac muscle (Acsadi, G., Jiao, S.,
Jani, A., Duke, D., Williams, P., Chong, W., Wolff, J. A. Direct
gene transfer and expression into rat heart in vivo. The New
Biologist 3(1), 71-81, 1991.).
SUMMARY
[0006] In one preferred embodiment, a process is described for
delivering a polynucleotide into a parenchymal cell of a mammal,
comprising making a polynucleotide such as a nucleic acid. Then,
inserting the polynucleotide into a mammalian vessel, such as a
blood vessel and increasing the permeability of the vessel.
Finally, delivering the polynucleotide to the parenchymal cell
thereby altering endogenous properties of the cell. Increasing the
permeability of the vessel consists of increasing pressure against
vessel walls. Increasing the pressure consists of increasing a
volume of fluid within the vessel. Increasing the volume consists
of inserting the polynucleotide in a solution into the vessel
wherein the solution contains a compound which complexes with the
polynucleotide. A specific volume of the solution is inserted
within a specific time period. Increased pressure is controlled by
altering the specific volume of the solution in relation to the
specific time period of insertion. The vessel may consist of a tail
vein. The parenchymal cell is a cell selected from the group
consisting of liver cells, spleen cells, heart cells, kidney cells
and lung cells.
[0007] In another embodiment, a complex for providing nucleic acid
expression in a cell is provided, comprising mixing a
polynucleotide and a polymer to form the complex wherein the zeta
potential of the complex is not positive. Then, delivering the
complex to the cell wherein the nucleic acid is expressed.
[0008] In another embodiment, a process is described for delivering
a polynucleotide complexed with a compound into a parenchymal cell
of a mammal, comprising making the polynucleotide-compound complex
wherein the compound is selected from the group consisting of
amphipathic compounds, polymers and non-viral vectors. Inserting
the polynucleotide into a mammalian vessel and increasing the
permeability of the vessel. Then, delivering the polynucleotide to
the parenchymal cell thereby altering endogenous properties of the
cell.
[0009] In yet another embodiment, a process is described for
transfecting genetic material into a mammalian cell, comprising
designing the genetic material for transfection. Inserting the
genetic material into a mammalian blood vessel. Increasing
permeability of the blood vessel and delivering the genetic
material to the parenchymal cell for the purpose of altering
endogenous properties of the cell.
DETAILED DESCRIPTION
[0010] We have found that an intravascular route of administration
allows a polynucleotide to be delivered to a parenchymal cell in a
more even distribution than direct parenchymal injections. The
efficiency of polynucleotide delivery and expression is increased
by increasing the permeability of the tissue's blood vessel.
Permeability is increased by increasing the intravascular
hydrostatic (physical) pressure, delivering the injection fluid
rapidly (injecting the injection fluid rapidly), using a large
injection volume, and increasing permeability of the vessel wall.
Expression of a foreign DNA is obtained in large number of
mammalian organs including; liver, spleen, lung, kidney and heart
by injecting the naked polynucleotide. Increased expression occurs
when polynucleotide is mixed with another compound.
[0011] In a first embodiment the compound consists of an
amphipathic compound. Amphipathic compounds have both hydrophilic
(water-soluble) and hydrophobic (water-insoluble) parts. The
amphipathic compound can be cationic or incorporated into a
liposome that is positively-charged (cationic) or non-cationic
which means neutral, or negatively-charged (anionic). In another
embodiment the compound consists of a polymer. In yet another
embodiment the compound consists of a non-viral vector. In one
embodiment, the compound does not aid the transfection process in
vitro of cells in culture but does aid the delivery process in vivo
in the whole organism. We also show that foreign gene expression
can be achieved in hepatocytes following the rapid injection of
naked plasmid DNA in a large volume of physiologic solutions.
[0012] The term intravascular refers to an intravascular route of
administration that enables a polymer, oligonucleotide, or
polynucleotide to be delivered to cells more evenly distributed
than direct injections. Intravascular herein means within an
internal tubular structure called a vessel that is connected to a
tissue or organ within the body of an animal, including mammals.
Within the cavity of the tubular structure, a bodily fluid flows to
or from the body part. Examples of bodily fluid include blood,
lymphatic fluid, or bile. Examples of vessels include arteries,
arterioles, capillaries, venules, sinusoids, veins, lymphatics, and
bile ducts. The intravascular route includes delivery through the
blood vessels such as an artery or a vein.
[0013] Afferent blood vessels of organs are defined as vessels in
which blood flows toward the organ or tissue under normal
physiologic conditions. Efferent blood vessels are defined as
vessels in which blood flows away from the organ or tissue under
normal physiologic conditions. In the heart, afferent vessels are
known as coronary arteries, while efferent vessels are referred to
as coronary veins.
[0014] The term naked nucleic acids indicates that the nucleic
acids are not associated with a transfection reagent or other
delivery vehicle that is required for the nucleic acid to be
delivered to a target cell. A transfection reagent is a compound or
compounds used in the prior art that mediates nucleic acids entry
into cells.
Parenchymal Cells
[0015] Parenchymal cells are the distinguishing cells of a gland or
organ contained in and supported by the connective tissue
framework. The parenchymal cells typically perform a function that
is unique to the particular organ. The term "parenchymal" often
excludes cells that are common to many organs and tissues such as
fibroblasts and endothelial cells within blood vessels.
[0016] In a liver organ, the parenchymal cells include hepatocytes,
Kupffer cells and the epithelial cells that line the biliary tract
and bile ductules. The major constituent of the liver parenchyma
are polyhedral hepatocytes (also known as hepatic cells) that
presents at least one side to an hepatic sinusoid and opposed sides
to a bile canaliculus. Liver cells that are not parenchymal cells
include cells within the blood vessels such as the endothelial
cells or fibroblast cells. In one preferred embodiment hepatocytes
are targeted by injecting the polynucleotide within the tail vein
of a rodent such as a mouse.
[0017] In striated muscle, the parenchymal cells include myoblasts,
satellite cells, myotubules, and myofibers. In cardiac muscle, the
parenchymal cells include the myocardium also known as cardiac
muscle fibers or cardiac muscle cells and the cells of the impulse
connecting system such as those that constitute the sinoatrial
node, atrioventricular node, and atrioventricular bundle. In one
preferred embodiment striated muscle such as skeletal muscle or
cardiac muscle is targeted by injecting the polynucleotide into the
blood vessel supplying the tissue. In skeletal muscle an artery is
the delivery vessel; in cardiac muscle, an artery or vein is
used.
[0018] Polymers
[0019] A polymer is a molecule built up by repetitive bonding
together of smaller units called monomers. In this application the
term polymer includes both oligomers which have two to about 80
monomers and polymers having more than 80 monomers. The polymer can
be linear, branched network, star, comb, or ladder types of
polymer. The polymer can be a homopolymer in which a single monomer
is used or can be copolymer in which two or more monomers are used.
Types of copolymers include alternating, random, block and
graft.
[0020] One of our several methods of nucleic acid delivery to cells
is the use of nucleic acid-polycations complexes. It was shown that
cationic proteins like histones and protamines or synthetic
polymers like polylysine, polyarginine, polyornithine, DEAE
dextran, polybrene, and polyethylenimine are effective
intracellular delivery agents while small polycations like spermine
are ineffective.
[0021] A polycation is a polymer containing a net positive charge,
for example poly-L-lysine hydrobromide. The polycation can contain
monomer units that are charge positive, charge neutral, or charge
negative, however, the net charge of the polymer must be positive.
A polycation also can mean a non-polymeric molecule that contains
two or more positive charges. A polyanion is a polymer containing a
net negative charge, for example polyglutamic acid. The polyanion
can contain monomer units that are charge negative, charge neutral,
or charge positive, however, the net charge on the polymer must be
negative. A polyanion can also mean a non-polymeric molecule that
contains two or more negative charges. The term polyion includes
polycation, polyanion, zwitterionic polymers, and neutral polymers.
The term zwitterionic refers to the product (salt) of the reaction
between an acidic group and a basic group that are part of the same
molecule. Salts are ionic compounds that dissociate into cations
and anions when dissolved in solution. Salts increase the ionic
strength of a solution, and consequently decrease interactions
between nucleic acids with other cations.
[0022] In one embodiment, polycations are mixed with
polynucleotides for intravascular delivery to a cell. Polycations
provide the advantage of allowing attachment of DNA to the target
cell surface. The polymer forms a cross-bridge between the
polyanionic nucleic acids and the polyanionic surfaces of the
cells. As a result the main mechanism of DNA translocation to the
intracellular space might be non-specific adsorptive endocytosis
which may be more effective then liquid endocytosis or
receptor-mediated endocytosis. Furthermore, polycations are a very
convenient linker for attaching specific receptors to DNA and as
result, DNA-polycation complexes can be targeted to specific cell
types.
[0023] Additionally, polycations protect DNA in complexes against
nuclease degradation. This is important for both extra- and
intracellular preservation of DNA. The endocytic step in the
intracellular uptake of DNA-polycation complexes is suggested by
results in which DNA expression is only obtained by incorporating a
mild hypertonic lysis step (either glycerol or DMSO). Gene
expression is also enabled or increased by preventing endosome
acidification with NH.sub.4CI or chloroquine. Polyethylenimine
which facilitates gene expression without additional treatments
probably disrupts endosomal function itself. Disruption of
endosomal function has also been accomplished by linking the
polycation to endosomal-disruptive agents such as fusion peptides
or adenoviruses.
[0024] Polycations also cause DNA condensation. The volume which
one DNA molecule occupies in complex with polycations is
drastically lower than the volume of a free DNA molecule. The size
of DNA/polymer complex may be important for gene delivery in vivo.
In terms of intravenous injection, DNA needs to cross the
endothelial barrier and reach the parenchymal cells of
interest.
[0025] The average diameter of liver fenestrae (holes in the
endothelial barrier) is about 100 nm, increases in pressure and/or
permeability can increase the size of the fenestrae. The fenestrae
size in other organs is usually less. The size of the DNA complexes
is also important for the cellular uptake process. DNA complexes
should be smaller than 200 nm in at least one dimension. After
binding to the target cells the DNA-polycation complex is expected
to be taken up by endocytosis.
[0026] Polymers may incorporate compounds that increase their
utility. These groups can be incorporated into monomers prior to
polymer formation or attached to the polymer after its formation.
The gene transfer enhancing signal (Signal) is defined in this
specification as a molecule that modifies the nucleic acid complex
and can direct it to a cell location (such as tissue cells) or
location in a cell (such as the nucleus) either in culture or in a
whole organism. By modifying the cellular or tissue location of the
foreign gene, the expression of the foreign gene can be
enhanced.
[0027] The gene transfer enhancing signal can be a protein,
peptide, lipid, steroid, sugar, carbohydrate, nucleic acid or
synthetic compound. The gene transfer enhancing signals enhance
cellular binding to receptors, cytoplasmic transport to the nucleus
and nuclear entry or release from endosomes or other intracellular
vesicles.
[0028] Nuclear localizing signals enhance the targeting of the gene
into proximity of the nucleus and/or its entry into the nucleus.
Such nuclear transport signals can be a protein or a peptide such
as the SV40 large T ag NLS or the nucleoplasmin NLS. These nuclear
localizing signals interact with a variety of nuclear transport
factors such as the NLS receptor (karyopherin alpha) which then
interacts with karyopherin beta. The nuclear transport proteins
themselves could also function as NLS's since they are targeted to
the nuclear pore and nucleus.
[0029] Signals that enhance release from intracellular compartments
(releasing signals) can cause DNA release from intracellular
compartments such as endosomes (early and late), lysosomes,
phagosomes, vesicle, endoplasmic reticulum, golgi apparatus, trans
golgi network (TGN), and sarcoplasmic reticulum. Release includes
movement out of an intracellular compartment into cytoplasm or into
an organelle such as the nucleus. Releasing signals include
chemicals such as chloroquine, bafilomycin or Brefeldin Al and the
ER-retaining signal (KDEL sequence), viral components such as
influenza virus hemagglutinin subunit HA-2 peptides and other types
of amphipathic peptides.
[0030] Cellular receptor signals are any signal that enhances the
association of the gene with a cell. This can be accomplished by
either increasing the binding of the gene to the cell surface
and/or its association with an intracellular compartment, for
example: ligands that enhance endocytosis by enhancing binding the
cell surface. This includes agents that target to the
asialoglycoprotein receptor by using asialoglycoproteins or
galactose residues. Other proteins such as insulin, EGF, or
transferrin can be used for targeting. Peptides that include the
RGD sequence can be used to target many cells. Chemical groups that
react with sulfhydryl or disulfide groups on cells can also be used
to target many types of cells. Folate and other vitamins can also
be used for targeting. Other targeting groups include molecules
that interact with membranes such as lipids fatty acids,
cholesterol, dansyl compounds, and amphotericin derivatives. In
addition viral proteins could be used to bind cells.
Polynucleotides
[0031] The term nucleic acid is a term of art that refers to a
string of at least two base-sugar-phosphate combinations. (A
polynucleotide is distinguished from an oligonucleotide by
containing more than 120 monomeric units.) Nucleotides are the
monomeric units of nucleic acid polymers. The term includes
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the form
of an oligonucleotide messenger RNA, anti-sense, plasmid DNA, parts
of a plasmid DNA or genetic material derived from a virus.
Anti-sense is a polynucleotide that interferes with the function of
DNA and/or RNA. The term nucleic acids-refers to a string of at
least two base-sugar-phosphate combinations. Natural nucleic acids
have a phosphate backbone, artificial nucleic acids may contain
other types of backbones, but contain the same bases. Nucleotides
are the monomeric units of nucleic acid polymers. The term includes
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). RNA may be
in the form of an tRNA (transfer RNA), snRNA (small nuclear RNA),
rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, and
ribozymes. DNA may be in form plasmid DNA, viral DNA, linear DNA,
or chromosomal DNA or derivatives of these groups. In addition
these forms of DNA and RNA may be single, double, triple, or
quadruple stranded. The term also includes PNAs (peptide nucleic
acids), phosphorothioates, and other variants of the phosphate
backbone of native nucleic acids.
[0032] A polynucleotide can be delivered to a cell to express an
exogenous nucleotide sequence, to inhibit, eliminate, augment, or
alter expression of an endogenous nucleotide sequence, or to
express a specific physiological characteristic not naturally
associated with the cell. Polynucleotides may be coded to express a
whole or partial protein, or may be anti-sense.
[0033] A delivered polynucleotide can stay within the cytoplasm or
nucleus apart from the endogenous genetic material. Alternatively,
the polymer could recombine (become a part of) the endogenous
genetic material. For example, DNA can insert into chromosomal DNA
by either homologous or non-homologous recombination.
[0034] Vectors are polynucleic molecules originating from a virus,
a plasmid, or the cell of a higher organism into which another
nucleic fragment of appropriate size can be integrated without loss
of the vectors capacity for self-replication; vectors typically
introduce foreign DNA into host cells, where it can be reproduced.
Examples are plasmids, cosmids, and yeast artificial chromosomes;
vectors are often recombinant molecules containing DNA sequences
from several sources. A vector includes a viral vector: for
example, adenovirus; DNA; adenoassociated viral vectors (AAV) which
are derived from adenoassociated viruses and are smaller than
adenoviruses; and retrovirus (any virus in the family Retroviridae
that has RNA as its nucleic acid and uses the enzyme reverse
transcriptase to copy its genome into the DNA of the host cell's
chromosome; examples include VSV G and retroviruses that contain
components of lentivirus including HIV type viruses).
[0035] A non-viral vector is defined as a vector that is not
assembled within an eukaryotic cell.
[0036] Permeability
[0037] In another preferred embodiment, the permeability of the
vessel is increased. Efficiency of polynucleotide delivery and
expression was increased by increasing the permeability of a blood
vessel within the target tissue. Permeability is defined here as
the propensity for macromolecules such as polynucleotides to move
through vessel walls and enter the extravascular space. One measure
of permeability is the rate at which macromolecules move through
the vessel wall and out of the vessel. Another measure of
permeability is the lack of force that resists the movement of
polynucleotides being delivered to leave the intravascular
space.
[0038] To obstruct, in this specification, is to block or inhibit
inflow or outflow of blood in a vessel. Rapid injection may be
combined with obstructing the outflow to increase permeability. For
example, an afferent vessel supplying an organ is rapidly injected
and the efferent vessel draining the tissue is ligated transiently.
The efferent vessel (also called the venous outflow or tract)
draining outflow from the tissue is also partially or totally
clamped for a period of time sufficient to allow delivery of a
polynucleotide. In the reverse, an efferent is injected and an
afferent vessel is occluded.
[0039] In another preferred embodiment, the intravascular pressure
of a blood vessel is increased by increasing the osmotic pressure
within the blood vessel. Typically, hypertonic solutions containing
salts such as NaCl, sugars or polyols such as mannitol are used.
Hypertonic means that the osmolarity of the injection solution is
greater than physiologic osmolarity. Isotonic means that the
osmolarity of the injection solution is the same as the
physiological osmolarity (the tonicity or osmotic pressure of the
solution is similar to that of blood). Hypertonic solutions have
increased tonicity and osmotic pressure similar to the osmotic
pressure of blood and cause cells to shrink.
[0040] In another preferred embodiment, the permeability of the
blood vessel can also be increased by a biologically-active
molecule. A biologically-active molecule is a protein or a simple
chemical such as papaverine or histamine that increases the
permeability of the vessel by causing a change in function,
activity, or shape of cells within the vessel wall such as the
endothelial or smooth muscle cells. Typically, biologically-active
molecules interact with a specific receptor or enzyme or protein
within the vascular cell to change the vessel's permeability.
Biologically-active molecules include vascular permeability factor
(VPF) which is also known as vascular endothelial growth factor
(VEGF). Another type of biologically-active molecule can also
increase permeability by changing the extracellular connective
material. For example, an enzyme could digest the extracellular
material and increase the number and size of the holes of the
connective material.
[0041] In another embodiment a non-viral vector along with a
polynucleotide is intravascularly injected in a large injection
volume. The injection volume is dependent on the size of the animal
to be injected and can be from 1.0 to 3.0 ml or greater for small
animals (i.e. tail vein injections into mice). The injection volume
for rats can be from 6 to 35 ml or greater. The injection volume
for primates can be 70 to 200 ml or greater. The injection volumes
in terms of ml/body weight can be 0.03 ml/g to 0.1 ml/g or
greater.
[0042] The injection volume can also be related to the target
tissue. For example, delivery of a non-viral vector with a
polynucleotide to a limb can be aided by injecting a volume greater
than 5 ml per rat limb or greater than 70 ml for a primate. The
injection volumes in terms of ml/limb muscle are usually within the
range of 0.6 to 1.8 ml/g of muscle but can be greater. In another
example, delivery of a polynucleotide to liver in mice can be aided
by injecting the non-viral vector--polynucleotide in an injection
volume from 0.6 to 1.8 ml/g of liver or greater. In another
preferred embodiment, delivering a polynucleotide--non-viral vector
to a limb of a primate (rhesus monkey), the complex can be in an
injection volume from 0.6 to 1.8 ml/g of limb muscle or anywhere
within this range.
[0043] In another embodiment the injection fluid is injected into a
vessel rapidly. The speed of the injection is partially dependent
on the volume to be injected, the size of the vessel to be injected
into, and the size of the animal. In one embodiment the total
injection volume (1 - 3 mls) can be injected from 15 to 5 seconds
into the vascular system of mice. In another embodiment the total
injection volume (6 - 35 mls) can be injected into the vascular
system of rats from 20 to 7 seconds. In another embodiment the
total injection volume (80 - 200 mls) can be injected into the
vascular system of monkeys from 120 seconds or less.
[0044] In another embodiment a large injection volume is used and
the rate of injection is varied. Injection rates of less than 0.012
ml per gram (animal weight) per second are used in this embodiment.
In another embodiment injection rates of less than ml per gram
(target tissue weight) per second are used for gene delivery to
target organs. In another embodiment injection rates of less than
0.06 ml per gram (target tissue weight) per second are used for
gene delivery into limb muscle and other muscles of primates.
[0045] Reporter Molecules
[0046] There are three types of reporter (marker) gene products
that are expressed from reporter genes. The reporter gene/protein
systems include:
[0047] a) Intracellular gene products such as luciferase,
.beta.-galactosidase, or chloramphenicol acetyl transferase.
Typically, they are enzymes whose enzymatic activity can be easily
measured.
[0048] b) Intracellular gene products such as .beta.-galactosidase
or green fluorescent protein which identify cells expressing the
reporter gene. On the basis of the intensity of cellular staining,
these reporter gene products also yield qualitative information
concerning the amount of foreign protein produced per cell.
[0049] c) Secreted gene products such as growth hormone, factor IX,
or alphal-antitrypsin are useful for determining the amount of a
secreted protein that a gene transfer procedure can produce. The
reporter gene product can be assayed in a small amount of
blood.
[0050] We have disclosed gene expression achieved from reporter
genes in parenchymal cells. The terms "delivery," "delivering
genetic information," "therapeutic" and "therapeutic results" are
defined in this application as representing levels of genetic
products, including reporter (marker) gene products, which indicate
a reasonable expectation of genetic expression using similar
compounds (nucleic acids), at levels considered sufficient by a
person having ordinary skill in the art of delivery and gene
therapy. For example: Hemophilia A and B are caused by deficiencies
of the X-linked clotting factors VIII and IX, respectively. Their
clinical course is greatly influenced by the percentage of normal
serum levels of factor VIII or IX: <2%, severe; 2-5%, moderate;
and 5-30% mild. This indicates that in severe patients only 2% of
the normal level can be considered therapeutic. Levels greater than
6% prevent spontaneous bleeds but not those secondary to surgery or
injury. A person having ordinary skill in the art of gene therapy
would reasonably anticipate therapeutic levels of expression of a
gene specific for a disease based upon sufficient levels of marker
gene results. In the Hemophilia example, if marker genes were
expressed to yield a protein at a level comparable in volume to 2%
of the normal level of factor VIII, it can be reasonably expected
that the gene coding for factor VIII would also be expressed at
similar levels.
EXAMPLES
Enhanced Delivery of Naked DNA
[0051] Enhancement of In Vivo Gene Expression by
M-methyl-L-arginine (L-NMMA) Following Intravascular Delivery of
Naked DNA:
[0052] Intravascular delivery of pCILuc via the iliac artery of rat
following a short pre-treatment with L-NMMA delivery enhancer. A 4
cm long abdominal midline excision was performed in 150-200 g,
adult Sprague-Dawley rats anesthesized with 80 mg/mg ketamine and
40 mg/kg xylazine. Microvessel clips were placed on external iliac,
caudal epigastric, internal iliac and deferent duct arteries and
veins to block both outflow and inflow of the blood to the leg. 3
ml of normal saline with 0.66 mM L-NMMA were injected into the
external iliac artery. After 2 min 27 g butterfly needle was
inserted into the external iliac artery and 10 ml of DNA solution
(50 ug/ml pCILuc) in normal saline was injected within 8-9 sec.
Luciferase assays was performed 2 days after injection on limb
muscle samples (quadriceps femoris).
[0053] Organ Treatment Total Luciferase (nanograms)
[0054] Muscle (quadriceps)+papaverine 9,999
[0055] Muscle (quadriceps)+0.66 mM L-NMMA 15,398
[0056] Muscle (quadriceps)+papaverine, +0.66 mM L-NNMA 24,829
[0057] 2) Enhancement of in vivo gene expression by
aurintricarboxylic Acid (ATA) delivery enhancer following
intravascular delivery of naked DNA.
[0058] Intravascular delivery of pCILuc in the absence or presence
of aurintricarboxylic acid via tail vein injection into mice. 10
micrograms of pCILuc was diluted to 2.5 ml with Ringers solution
and aurintricarboxylic acid was added to a final concentration of
0.11 mg/ml. The DNA solution was injected into the tail vein of 25
g ICR mice with an injection time of .about.7 seconds. Mice were
sacrificed 24 hours after injection and various organs were assayed
for luciferase expression.
[0059] Organ Treatment Total Relative Light Units per Organ
[0060] Liver none 55,300,000,000
[0061] Liver+ATA 109,000,000,000
[0062] Spleen none 63,200,000
[0063] Spleen+ATA 220,000,000
[0064] Lung none 100,000,000
[0065] Lung+ATA 128,000,000
[0066] Heart none 36,700,000
[0067] Heart+ATA 32,500,000
[0068] Kidney none 15,800,000
[0069] Kidney+ATA 82,400,000
[0070] DNA/Polymer Delivery
[0071] Rapid injection of pDNA/cationic polymer complexes
(containing 10 .mu.g of pCILuc; a luciferase expression vector
utilizing the human CMV promoter) in 2.5 ml of Ringers solution
(147 mM NaCl, 4 mM KCl, 1.13 mM CaCl2) into the tail vein of ICR
mice facilitated expression levels higher than comparable
injections using naked plasmid DNA (pCILuc). Maximal luciferase
expression using the tail vein approach was achieved when the DNA
solution was injected within 7 seconds. Luciferase expression was
also critically dependent on the total injection volume and high
level gene expression in mice was obtained following tail vein
injection of polynucleotide/polymer complexes of 1, 1.5, 2, 2.5,
and 3 ml total volume. There is a positive correlation between
injection volume and gene expression for total injection volumes
over 1 ml. For the highest expression efficiencies an injection
delivery rate of greater than 0.003 ml per gram (animal weight) per
second is likely required. Injection rates of 0.004, 0.006, 0.009,
0.012 ml per gram (animal weight) per second yield successively
greater gene expression levels.
1 1
[0072] The graph above illustrates high level luciferase expression
in liver following tail vein injections of naked plasmid DNA and
plasmid DNA complexed with labile disulfide containing polycations
L-cystine -1,4-bis(3-aminopropyl)piperazine copolymer (M66) and
5,5'-Dithiobis(2-nitrobenzoic acid)-Pentaethylenehexamine Copolymer
(M72). The labile polycations were complexed with DNA at a 3:1
wt:wt ratio resulting in a positively charged complex. Complexes
were injected into 25 gram ICR mice in a total volume of 2.5 ml of
ringers solution.
2 2
[0073] The graph above indicates high level luciferase expression
in spleen, lung, heart and kidney following tail vein injections of
naked plasmid DNA and plasmid DNA complexed with labile disulfide
containing polycations M66 and M72. The labile polycations were
complexed with DNA at a 3:1 wt:wt ratio resulting in a positively
charged complex. Complexes were injected into 25 gram ICR mice in a
total volume of 2.5 ml of ringers solution.
[0074] Luciferase Expression in a Variety of Tissues Following a
Single Tail Vein Injection of pCILuc/66 Complexes
[0075] DNA and polymer 66 were mixed at a 1:1.7 wt:wt ratio in
water and diluted to 2.5 ml with Ringers solution as described.
Complexes were injected into tail vein of 25 g ICR mice within 7
seconds. Mice were sacrificed 24 hours after injection and various
organs were assayed for luciferase expression.
[0076] Organ Total Relative Light Units
[0077] Prostate 637,000
[0078] Skin (abdominal wall) 194,000
[0079] Testis 589,000
[0080] Skeletal Muscle (quadriceps) 35,000
[0081] fat (peritoneal cavity) 44,700
[0082] bladder 17,000
[0083] brain 247,000
[0084] pancreas 2,520,000
[0085] Directed Intravascular Injection of pCILuc/66 Polymer
Complexes into Dorsal Vein of Penis Results in High Level Gene
Expression in the Prostate and Other Localized Tissues
[0086] Complexes were formed as described for example above and
injected rapidly into the dorsal vein of the penis (within 7
seconds). For directed delivery to the prostate with increased
hydrostatic pressure, clamps were applied to the inferior vena cava
and the anastomotic veins just prior to the injection and removed
just after the injection (within 5 - 10 seconds). Mice were
sacrificed 24 hours after injection and various organs were assayed
for luciferase expression.
[0087] Organ Total Relative Light Units Per Organ
[0088] Prostate 129,982,450
[0089] Testis 4,229,000
[0090] fat (around bladder) 730,300
[0091] bladder 618,000
[0092] Intravascular Tail Vein Injection into Rat Results in High
Level Gene Expression in a Variety of Organs
[0093] 100 micrograms of pCILuc was diluted into 30 mis Ringers
solution and injected into the tail vein of 480 gram Harlan Sprague
Dawley rat. The entire volume was delivered within 15 seconds. 24
hours after injection various organs were harvested and assayed for
luciferase expression.
[0094] Organ Total Relative Light Units Per Organ
[0095] Liver 30,200,000,000
[0096] Spleen 14,800,000
[0097] Lung 23,600,000
[0098] Heart 5,540,000
[0099] Kidney 19,700,000
[0100] Prostate 3,490,000
[0101] Skeletal Muscle (quadriceps) 7,670,000
Cleavable Polymers
[0102] A prerequisite for gene expression is that once DNA/cationic
polymer complexes have entered a cell the polynucleotide must be
able to dissociate from the cationic polymer. This may occur within
cytoplasmic vesicles (i.e. endosomes), in the cytoplasm, or the
nucleus. We have developed bulk polymers prepared from disulfide
bond containing co-monomers and cationic co-monomers to better
facilitate this process. These polymers have been shown to condense
polynucleotides, and to release the nucleotides after reduction of
the disulfide bond. These polymers can be used to effectively
complex with DNA and can also protect DNA from DNases during
intravascular delivery to the liver and other organs. After
internalization into the cells the polymers are reduced to
monomers, effectively releasing the DNA, as a result of the
stronger reducing conditions (glutathione) found in the cell.
Negatively charged polymers can be fashioned in a similar manner,
allowing the condensed nucleic acid particle (DNA+polycation) to be
"recharged" with a cleavable anionic polymer resulting in a
particle with a net negative charge that after reduction of
disulfide bonds will release the polynucleic acid. The reduction
potential of the disulfide bond in the reducible co-monomer can be
adjusted by chemically altering the disulfide bonds environment.
This will allow the construction of particles whose release
characteristics can be tailored so that the polynucleic acid is
released at the proper point in the delivery process.
Cleavable Cationic Polymers
[0103] Cationic cleavable polymers are designed such that the
reducibility of disulfide bonds, the charge density of polymer, and
the functionalization of the final polymer can all be controlled.
The disulfide co-monomer can have reactive ends chosen from, but
not limited to the following: the disulfide compounds contain
reactive groups that can undergo acylation or alkylation reactions.
Such reactive groups include isothiocyanate, isocyanate, acyl
azide, N-hydroxysuccinimide esters, succinimide esters, sulfonyl
chloride, aldehyde, epoxide, carbonate, imidoester, carboxylate,
alkylphosphate, arylhalides (e.g. difluoro-dinitrobenzene) or
succinic anhydride.
[0104] If functional group A (cationic co-monomer) is an amine then
B (disulfide containing comonomer) can be (but not restricted to)
an isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide,
sulfonyl chloride, aldehyde (including formaldehyde and
glutaraldehyde), epoxide, carbonate, imidoester, carboxylate, or
alkylphosphate, arylhalides (difluoro-dinitrobenzene) or succinic
anhyride. In other terms when function A is an amine then function
B can be acylating or alkylating agent.
[0105] If functional group A is a sulfhydryl then functional group
B can be (but not restricted to) an iodoacetyl derivative,
maleimide, vinyl sulfone, aziridine derivative, acryloyl
derivative, fluorobenzene derivatives, or disulfide derivative
(such as a pyridyl disulfide or 5-thio-2-nitrobenzoic acid {TNB}
derivatives).
[0106] If functional group A is carboxylate then functional group B
can be (but not restricted to) a diazoacetate or an amine, alcohol,
or sulfhydryl in which carbonyldiimidazole or carbodiimide is
used.
[0107] If functional group A is an hydroxyl then functional group B
can be (but not restricted to) an epoxide, oxirane, or an carboxyl
group in which carbonyldiimidazole or carbodiimide or N,
N'-disuccinimidyl carbonate, or N-hydroxysuccinimidyl chloroformate
is used.
[0108] If functional group A is an aldehyde or ketone then function
B can be (but not restricted to) an hydrazine, hydrazide
derivative, amine (to form a Schiff Base that may or may not be
reduced by reducing agents such as NaCNBH.sub.3).
[0109] The polymer is formed by simply mixing the cationic, and
disulfide-containing co-monomers under appropriate conditions for
reaction. The resulting polymer may be purified by dialysis or
size-exclusion chromatography.
[0110] The reduction potential of the disulfide bond can be
controlled in two ways. Either by altering the reduction potential
of the disulfide bond in the disulfide-containing co-monomer, or by
altering the chemical environment of the disulfide bond in the bulk
polymer through choice the of cationic co-monomer.
[0111] The reduction potential of the disulfide bond in the
co-monomer can be controlled by synthesizing new cross-linking
reagents. Dimethyl 3,3'-dithiobispropionimidate (DTBP) is a
commercially available disulfide containing crosslinker from Pierce
Chemical Co. This disulfide bond is reduced by dithiothreitol
(DTT), but is only slowly reduced, if at all by biological reducing
agents such as glutathione. More readily reducible crosslinkers
have been synthesized by Mirus. These crosslinking reagents are
based on aromatic disulfides such as 5,5'-dithiobis(2-nitrobenzoic
acid) and 2,2'-dithiosalicylic acid. The aromatic rings activate
the disulfide bond towards reduction through delocalization of the
transient negative charge on the sulfur atom during reduction. The
nitro groups further activate the compound to reduction through
electron withdrawal which also stabilizes the resulting negative
charge (FIG. 2).
[0112] Cleavable disulfide containing co-monomers: 3
[0113] The reduction potential can also be altered by proper choice
of cationic co-monomer. For example when DTBP is polymerized along
with diaminobutane the disulfide bond is reduced by DTT, but not
glutathione. When ethylenediamine is polymerized with DTBP the
disulfide bond is now reduced by glutathione. This is apparently
due to the proximity of the disulfide bond to the amidine
functionality in the bulk polymer.
[0114] The charge density of the bulk polymer can be controlled
through choice of cationic monomer, or by incorporating positive
charge into the disulfide co-monomer. For example spermine a
molecule containing 4 amino groups spaced by 3-4-3 methylene groups
could be used for the cationic monomer. Because of the spacing of
the amino groups they would all bear positive charges in the bulk
polymer with the exception of the end primary amino groups that
would be derivitized during the polymerization. Another monomer
that could be used is N,N'-bis(2-aminoethyl)-1,3-propedia- mine
(AEPD) a molecule containing 4 amino groups spaced by 2-3-2
methylene groups. In this molecule the spacing of the amines would
lead to less positive charge at physiological pH, however the
molecule would exhibit pH sensitivity, that is bear different net
positive charge, at different pH's. A molecule such as
tetraethylenepentamine could also be used as the cationic monomer,
this molecule consists of 5 amino groups each spaced by two
methylene units. This molecule would give the bulk polymer pH
sensitivity, due to the spacing of the amino groups as well as
charge density, due to the number and spacing of the amino groups.
The charge density can also be affected by incorporating positive
charge into the disulfide containing monomer, or by using imidate
groups as the reactive portions of the disulfide containing monomer
as imidates are transformed into amidines upon reaction with amine
which retain the positive charge.
[0115] The bulk polymer can be designed to allow further
functionalization of the polymer by incorporating monomers with
protected primary amino groups. These protected primary amines can
then be deprotected and used to attach other functionalities such
as nuclear localizing signals, endosome disrupting peptides,
cell-specific ligands, fluorescent marker molecules, as a site of
attachment for further crosslinking of the polymer to itself once
it has been complexed with a polynucleic acid, or as a site of
attachment for a second anionic layer when a cleavable
polymer/polynucleic acid particle is being recharged to an anionic
particle. An example of such a molecule is
3,3'-(N',N"-tert-butoxycarbony-
l)-N-(3'-trifluoroacetamidylpropane)-N-methyldipropylammonium
bromide (see experimental), this molecule would be incorporated by
removing the two BOC protecting groups, incorporating the
deprotected monomer into the bulk polymer, followed by deprotection
of the trifluoroacetamide protecting group.
Cleavable Anionic Polymers
[0116] Cleavable anionic polymers can be designed in much the same
manner as the cationic polymers. Short, multi-valent oligopeptides
of glutamic or aspartic acid can be synthesized with the carboxy
terminus capped with ethylene diamine. This oligo can the be
incorporated into a bulk polymer as a co-monomer with any of the
amine reactive disulfide containing crosslinkers mentioned
previously. A preferred crosslinker would make use of NHS esters as
the reactive group to avoid retention of positive charge as occurs
with imidates. The cleavable anionic polymers can be used to
recharge positively charged particles of condensed polynucleic
acids.
[0117] Examples of cleavable polymers: 4
[0118] The cleavable anionic polymers can have co-monomers
incorporated to allow attachment of cell-specific ligands, endosome
disrupting peptides, fluorescent marker molecules, as a site of
attachment for further crosslinking of the polymer to itself once
it has been complexed with a polynucleic acid, or as a site of
attachment for to the initial cationic layer. For example the
carboxyl groups on a portion of the anionic co-monomer could be
coupled to an aminoalcohol such as 4-hydroxybutylamine. The
resulting alcohol containing comonomer can be incorporated into the
bulk polymer at any ratio. The alcohol functionalities can then be
oxidized to aldehydes, which can be coupled to amine containing
ligands etc. in the presence of sodium cyanoborohydride via
reductive amination.
[0119] Synthesis of Activated Disulfide Containing Co-Monomers
[0120] Synthesis of 5,5'-dithiobis(2-nitrobenzoate)propionitrile:
5,5'-dithiobis(2-nitrobenzoic acid) [Ellman's reagent] (500 mg,
1.26 mmol) was dissolved in 4.0 ml dioxane. Dicylohexylcarbodiimide
(540 mg, 2.6 mmol) and 3-hydroxypropionitrile (240 .mu.L, 188 mg,
2.60 mmol) were added. The reaction mixture was stirred overnight
at room temperature. The urea precipitate was removed by
centrifugation. The dioxane was removed on rotary evaporator. The
residue was washed with saturated bicarbonate, water, and brine;
and dried over magnesium sulfate. Solvent removal yielded 696 mg
yellow/orange foam. The residue was purified using normal phase
HPLC (Alltech econosil, 250.times.22 nm), flow rate=9.0 ml/min,
mobile phase=1% ethanol in chloroform, retention time=13 min.
Removal of solvent afforded 233 mg (36.8%) product as a yellow oil.
TLC (silica: 5% methanol in chloroform; rf=0.51).
H.sup.1NMR.quadrature.8.05 (d, 4H), 7.75 (m, 4H), 4.55 (t, 4H),
2.85 (t, 4H).
[0121] Synthesis of 5,5'-dithiobis(2-nitrobenzoic acid)dimethyl
propionimidate [DTNBP]: (113.5 mg, 0.226 mmol) was dissolved in 500
.mu.L anhydrous chloroform along with anhydrous methanol (20.0
.mu.L, 0.494 mmol). The flask was stoppered with a rubber septum,
chilled to 0.degree. C. on an ice bath, and HCl gas produced by
mixing sulfuric acid and ammonium chloride was bubbled through the
solution for a period of 10 minutes. The flask was then tightly
sealed with parafilm and placed in a -20.degree. C. freezer for a
period of 48 hours. During this time a yellow oil formed. The oil
was washed thoroughly with chloroform and dried under vacuum to
yield 137 mg (95.8%) product as a yellow foam. 5
[0122] 3,3'-(N',N"-tert-butoxycarbonyl)-N-methyldipropylamine (1).
3,3'-Diamino-N-methyldipropylamine (0.800 ml, 0.721 g, 5.0 mmol)
was dissolved in 5.0 ml 2.2 N sodium hydroxide (11 mmol). To the
solution was added Boc anhydride (2.50 ml, 2.38 g, 10.9 mmol) with
magnetic stirring. The reaction mixture was allowed to stir at room
temperature overnight (approximately 18 hours). The reaction
mixture was made basic by adding additional 2.2 N NaOH until all
t-butyl carboxylic acid was in solution. The solution was then
extracted into chloroform (2.times.20 ml). The combined chloroform
extracts were washed 2.times.10 ml water and dried over magnesium
sulfate. Solvent removal yielded 1.01 g (61.7%) product as a white
solid: .sup.1H-NMR (CDCl.sub.3) .delta.5.35 (bs, 2H), 3.17 (dt,
4H), 2.37 (t, 4H), 2.15 (s, 3H), 1.65 (tt, 4H), 1.45 (s, 18H).
6
[0123]
3,3'-(N',N"-tert-butoxycarbonyl)-N-(3'-trifluoroacetamidylpropane)--
N-methyldipropylammonium bromide (13). Compound 1 (100.6 mg, 0.291
mmol) and compound 4 (76.8 mg, 0.328 mmol) were dissolved in 0.150
ml dimethylformamide. The reaction mixture was incubated at
50.degree. C. for 3 days. TLC (reverse phase; acetonitrile: 50 mM
ammonium acetate pH 4.0; 3:1) showed 1 major and 2 minor spots none
of which corresponded to starting material. Recrystalization
attempts were unsuccessful so product was precipitated from ethanol
with ether yielding 165.5 mg (98.2%) product and minor impurities
as a clear oil: .sup.1H-NMR (CDCl.sub.3) .delta.9.12 (bs,1H), 5.65
(bs, 2H), 3.50 (m, 8H), 3.20 (m, 4H), 3.15 (s, 3H), 2.20 (m, 2H),
2.00 (m, 4H), 1.45 (s, 18H).
[0124] Intravascular Injections of DNA/Polymer Complexes
[0125] Synthesis of N,N'-Bis(t-BOC)-L-cystine:
[0126] To a solution of L-cystine (1 gm,4.2 mmol, Aldrich Chemical
Company) in acetone (10 ml) and water (10 ml) was added
2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (2.5 gm, 10
mmol, Aldrich Chemical Company) and triethylamine (1.4 ml, 10 mmol,
Aldrich Chemical Company). The reaction was allowed to stir
overnight at room temperature. The water and acetone was then by
rotary evaporation resulting in a yellow solid. The diBOC compound
was then isolated by flash chromatography on silica gel eluting
with ethyl acetate 0.1% acetic acid.
[0127] Synthesis of L-cystine-1,4-bis(3-aminopropyl)piperazine
copolymer (M66):
[0128] To a solution of N,N'-Bis(t-BOC)-L-cystine (85 mg, 0.15
mmol) in ethyl acetate (20 ml) was added
N,N'-dicyclohexylcarbodiimide (108 mg, 0.5 mmol) and
N-hyroxysuccinimide (60 mg, 0.5 mmol). After 2 hr, the solution was
filtered through a cotton plug and 1,4-bis(3-aminopropyl)pip-
erazine (54 .mu.L, 0.25 mmol) was added. The reaction was allowed
to stir at room temperature for 16 h. The ethyl acetate was then
removed by rotary evaporation and the resulting solid was dissolved
in trifluoroacetic acid (9.5 ml), water (0.5 ml) and
triisopropylsilane (0.5 ml). After 2 h, the trifluoroacetic acid
was removed by rotary evaporation and the aqueous solution was
dialyzed in a 15,000 MW cutoff tubing against water (2.times.2 l)
for 24 h. The solution was then removed from dialysis tubing,
filtered through 5 .mu.M nylon syringe filter and then dried by
lyophilization to yield 30 mg of polymer.
[0129] Injection of plasmid DNA
(pCILuc)/L-cystine-1,4-bis(3-aminopropyl)p- iperazine copolymer
(M66) complexes into the iliac artery of rats.
[0130] Complex formation--500 ug pDNA (500 ul) was mixed with M66
copolymer at a 1:3 wt:wt ratio in 500 ul saline. Complexes were
then diluted in Ringers solution to total volume of 10 mls.
[0131] Injections--total volume of 10 mls was injected into the
iliac artery of Sprague-Dawley rats (Harlan, Indianapolis, Ind.) in
approximately 10 seconds.
[0132] Expression--Animals were sacrificed after 1 week and
individual muscle groups were removed and assayed for luciferase
expression.
[0133] Rat hind limb muscle groups.
[0134] 1) upper leg posterior--6.46.times.10.sup.8 total Relative
Light Units (32 ng luciferase)
[0135] 2) upper leg anterior--3.58.times.10.sup.9 total Relative
Light Units (183 ng luciferase)
[0136] 3) upper leg middle--2.63.times.10.sup.9total Relative Light
Units (134 ng luciferase)
[0137] 4) lower leg anterior--3.19.times.10.sup.9 total Relative
Light Units (163 ng luciferase)
[0138] 5) lower leg anterior--1.97.times.10.sup.9 total Relative
Light Units (101 ng luciferase)
[0139] These results indicate that high level gene expression in
all muscle groups of the leg was facilitated by intravascular
delivery of pCILuc/M66 complexes into rat iliac artery.
[0140] Synthesis of
5,5'-Dithiobis[succinimidyl(2-nitrobenzoate)
[0141] 5,5'-dithiobis(2-nitrobenzoic acid) (50.0 mg, 0.126 mmol,
Aldrich Chemical Company) and N-hyroxysuccinimide (29.0 mg, 0.252
mmol, Aldrich Chemical Company) were taken up in 1.0 ml
dichloromethane. Dicylohexylcarbodiimide (52.0 mg, 0.252 mmol) was
added and the reaction mixture was stirred overnight at room
temperature. After 16 hr, the reaction mixture was partitioned in
EtOAc/H.sub.2O. The organic layer was washed 2.times.H.sub.2O,
1.times. brine, dried (MgSO.sub.4) and concentrated under reduced
pressure. The residue was taken up in CH.sub.2Cl.sub.2, filtered,
and purified by flash column chromatography on silica gel
(130.times.30 mm, EtOAc:CH.sub.2Cl.sub.2 1:9 eluent) to afford 42
mg (56%) 5,5'-dithiobis[succinimidyl(2-nitrobenzoate)] as a white
solid. H.sup.1NMR (DMSO) .quadrature.7.81-7.77 (d, 2H), 7.57-7.26
(m, 4H), 3.69 (s, 8 H).
[0142] Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
acid)-Pentaethylenehexam- ine Copolymer (M72):
[0143] Pentaethylenehexamine (4.2 .mu.L, 0.017 mmol, Aldrich
Chemical Company) was taken up in 1.0 ml dichloromethane and HCl (1
ml, 1 M in Et.sub.2O, Aldrich Chemical Company) was added Et.sub.2O
was added and the resulting HCl salt was collected by filtration.
The salt was taken up in 1 ml DMF and
5,5'-dithiobis[succinimidyl(2-nitrobenzoate)] (10 mg, 0.017 mmol)
was added. The resulting solution was heated to
80.degree..quadrature. C. and diisopropylethylamine (12 .mu.L,
0.068 mmol, Aldrich Chemical Company) was added dropwise. After 16
hr, the solution was cooled, diluted with 3 ml H.sub.2O, and
dialyzed in 12,000 - 14,000 MW cutoff tubing against water
(2.times.2 L) for 24 hr. The solution was then removed from
dialysis tubing and dried by lyophilization to yield 5.9 mg (58%)
of 5,5'-dithiobis(2-nitrobenzoic acid)-pentaethylenehexamine
Copolymer.
[0144] Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
acid)-Tetraethylenepenta- mine Copolymer (#M57):
[0145] Tetraethylenepentamine (3.2 .mu.L, 0.017 mmol, Aldrich
Chemical Company) was taken up in 1.0 ml dichloromethane and HCl (1
ml, 1 M in Et.sub.2O, Aldrich Chemical Company) was added Et.sub.2O
was added and the resulting HCl salt was collected by filtration.
The salt was taken up in 1 ml DMF and 5,5'-dithiobis[succinimidyl
(2-nitrobenzoate)] (10 mg, 0.017 mmol) was added. The resulting
solution was heated to 80.degree. C. and diisopropylethylamine (15
.mu.L, 0.085 mmol, Aldrich Chemical Company) was added dropwise.
After 16 hr, the solution was cooled, diluted with 3 ml H.sub.2O,
and dialyzed in 12,000 - 14,000 MW cutoff tubing against water
(2.times.2 L) for 24 h. The solution was then removed from dialysis
tubing and dried by lyophilization to yield 5.8 mg (62%) of
5,5'-dithiobis(2-nitrobenzoic acid)-tetraethylenepentamine
copolymer.
[0146] Mouse Tail Vein Injections of pDNA (pCI
Luc)/5,5'-Dithiobis(2-nitro- benzoic acid)-- Tetraethylenepentamine
Copolymer Complexes:
[0147] Complexes were prepared as follows:
[0148] Complex I: pDNA (pCI Luc, 200 .mu.g) was added to 300 .mu.L
DMSO then 2.5 ml Ringers was added.
[0149] Complex II: pDNA (pCI Luc, 200 .mu.g) was added to 300 .mu.L
DMSO then 5,5'-Dithiobis(2-nitrobenzoic
acid)-Tetraethylenepentamine Copolymer (336 .mu.g) was added
followed by 2.5 ml Ringers.
[0150] High pressure (2.5 ml) tail vein injections of the complex
were performed as previously described (Zhang, G., Budker, V.,
Wolff, J. "High Levels of Foreign Gene Expression in Hepatocytes
from Tail Vein Injections of Naked Plasmid DNA", Human Gene
Therapy, July, 1999). Results reported are for liver expression,
and are the average of two mice. Luciferase expression was
determined as previously reported (Wolff, J. A., Malone, R. W.,
Williams, P., Chong, W., Acsadi, G., Jani, A., and Feigner, P. L.,
1990 "Direct gene transfer into mouse muscle in vivo," Science 247,
1465-8.) A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany)
luminometer was used.
[0151] Results: High pressure injections
[0152] Complex I: 25,200,000 Relative Light Units
[0153] Complex II: 21,000,000 Relative Light Units
[0154] Results indicate that pDNA (pCI
Luc)/5,5'-Dithiobis(2-nitrobenzoic acid) -tetraethylenepentamine
copolymer complexes are nearly equivalent to pCI Luc DNA itself in
high pressure injections. This indicates that the pDNA is being
released from the complex and is accessible for transcription.
[0155] Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
acid)-Tetraethylenepenta- mine-Tris(2-aminoethyl)amine Copolymer
(#M58):
[0156] Tetraethylenepentamine ( 2.3 .mu.L, 0.012 mmol, Aldrich
Chemical Company) and tris(2-aminoethyl)amine (0.51 .mu.L, 0.0034
mmol, Aldrich Chemical Company) were taken up in 0.5 ml methanol
and HCl (1 ml, 1 M in Et.sub.2O, Aldrich Chemical Company) was
added. Et.sub.2O was added and the resulting HCl salt was collected
by filtration. The salt was taken up in 1 ml DMF and
5,5'-dithiobis[succinimidyl (2-nitrobenzoate)] (10 mg, 0.017 mmol)
was added. The resulting solution was heated to 80.degree. C. and
diisopropylethylamine (15 .mu.L, 0.085 mmol, Aldrich Chemical
Company) was added dropwise. After 16 hr, the solution was cooled,
diluted with 3 ml H.sub.2O, and dialyzed in 12,000 - 14,000 MW
cutoff tubing against water (2.times.2 L) for 24 h. The solution
was then removed from dialysis tubing and dried by lyophilization
to yield 6.9 mg (77%) of 5,5'-dithiobis(2-nitrobenzoic
acid)-tetraethylenepentamine-tris(- 2-aminoethyl)amine
copolymer.
[0157] Mouse Tail Vein Injections of pDNA (pCI
Luc)/5,5'-Dithiobis(2-nitro- benzoic
acid)-Tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer
Complexes:
[0158] Complexes were prepared as follows:
[0159] Complex I: pDNA (pCI Luc, 200 .mu.g) was added to 300 .mu.L
DMSO then 2.5 ml Ringers was added.
[0160] Complex II: pDNA (pCI Luc, 200 .mu.g) was added to 300 .mu.L
DMSO then 5,5'-Dithiobis(2-nitrobenzoic
acid)-Tetraethylenepentamine-Tris(2-am- inoethyl)amine Copolymer
(324 .mu.g) was added followed by 2.5 ml Ringers.
[0161] High pressure (2.5 ml) tail vein injections of the complex
were performed as previously described. Results reported are for
liver expression, and are the average of two mice. Luciferase
expression was determined a previously shown.
[0162] Results: High pressure injections
[0163] Complex I: 25,200,000 Relative Light Units
[0164] Complex II: 37,200,000 Relative Light Units
[0165] Results indicate that pDNA (pCI Luc)/
5,5'-Dithiobis(2-nitrobenzoic
acid)-tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer
Complexes are more effective than pCI Luc DNA in high pressure
injections. This indicates that the pDNA is being released from the
complex and is accessible for transcription.
[0166] Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
acid)-N,N'-Bis(2-aminoet- hyl)-1,3-propanediamine Copolymer
(#M59):
[0167] N,N'-Bis(2-aminoethyl)-1,3-propanediamine (2.8 .mu.L, 0.017
mmol, Aldrich Chemical Company) was taken up in 1.0 ml
dichloromethane and HCl (1 ml, 1 M in Et.sub.2O, Aldrich Chemical
Company) was added. Et.sub.2O was added and the resulting HCl salt
was collected by filtration. The salt was taken up in 1 ml DMF and
5,5'-dithiobis[ succinimidyl(2-nitroben- zoate)] (10 mg, 0.017
mmol) was added. The resulting solution was heated to 80.degree. C.
and diisopropylethylamine (12 .mu.L, 0.068 mmol, Aldrich Chemical
Company) was added dropwise. After 16 hr, the solution was cooled,
diluted with 3 ml H.sub.2O, and dialyzed in 12,000 - 14,000 MW
cutoff tubing against water (2.times.2 L) for 24 hr. The solution
was then removed from dialysis tubing and dried by lyophilization
to yield 5.9 mg (66%) of 5,5'-dithiobis(2-nitrobenzoic acid)
-N,N'-bis(2-aminoethyl)-1,3-propanediamine Copolymer.
[0168] Mouse Tail Vein Injections of pDNA (pCI
Luc)/5,5'-Dithiobis(2-nitro- benzoic
acid)-N,N'-Bis(2-aminoethyl)-1,3-propanediamine Copolymer
Complexes:
[0169] Complexes were prepared as follows:
[0170] Complex I: pDNA (pCI Luc, 200 .mu.g) was added to 300 .mu.L
DMSO then 2.5 ml Ringers was added.
[0171] Complex II: pDNA (pCI Luc, 200 .mu.g) was added to 300 .mu.L
DMSO then 5,5'-Dithiobis(2-nitrobenzoic
acid)-N,N'-Bis(2-aminoethyl)-1,3-propa- nediamine Copolymer (474
.mu.g) was added followed by 2.5 ml Ringers.
[0172] High pressure tail vein injections of 2.5 ml of the complex
were performed as previously described. Results reported are for
liver expression, and are the average of two mice. Luciferase
expression was determined as previously shown.
[0173] Results: High pressure injections
[0174] Complex I: 25,200,000 Relative Light Units
[0175] Complex II: 341,000 Relative Light Units
[0176] Results indicate that pDNA (pCI
Luc)/5,5'-Dithiobis(2-nitrobenzoic acid)-tetraethylenepentamine
Copolymer Complexes are less effective than pCI Luc DNA in high
pressure injections. Although the complex was less effective, the
luciferase expression indicates that the pDNA is being released
from the complex and is accessible for transcription.
[0177] Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
acid)-N,N'-Bis(2-aminoet-
hyl)-1,3-propanediamine-Tris(2-aminoethyl)amine Copolymer
(#M60):
[0178] N,N'-Bis(2-aminoethyl)-1,3-propanediamine (2.0 .mu.L, 0.012
mmol, Aldrich Chemical Company) and tris(2-aminoethyl)amine (0.51
.mu.L, 0.0034 mmol, Aldrich Chemical Company) were taken up in 0.5
ml methanol and HCl (1 ml, 1 M in Et.sub.2O, Aldrich Chemical
Company) was added. Et.sub.2O was added and the resulting HCl salt
was collected by filtration. The salt was taken up in 1 ml DMF and
5,5'-dithiobis[ succinimidyl(2-nitroben- zoate)] (10 mg, 0.017
mmol) was added. The resulting solution was heated to 80.degree. C.
and diisopropylethylamine (12 .mu.L, 0.068 mmol, Aldrich Chemical
Company) was added dropwise. After 16 hr, the solution was cooled,
diluted with 3 ml H.sub.2O, and dialyzed in 12,000 - 14,000 MW
cutoff tubing against water (2.times.2 L) for 24 hr. The solution
was then removed from dialysis tubing and dried by lyophilization
to yield 6.0 mg (70%) of 5,5'-dithiobis(2-nitrobenzoic
acid)-N,N'-bis(2-aminoethyl-
)-1,3-propanediamine-tris(2-aminoethyl)amine copolymer.
[0179] Mouse Tail Vein Injections of pDNA (pCI
Luc)/5,5'-Dithiobis(2-nitro- benzoic
acid)-N,N'-Bis(2-aminoethyl)-1,3-propanediamine-Tris(2-aminoethyl)-
amine Copolymer Complexes:
[0180] Complexes were prepared as follows:
[0181] Complex I: pDNA (pCI Luc, 200 .mu.g) was added to 300 .mu.L
DMSO then 2.5 ml Ringers was added.
[0182] Complex II: pDNA (pCI Luc, 200 .mu.g) was added to 300 .mu.L
DMSO then 5,5'-Dithiobis(2-nitrobenzoic
acid)-N,N'-Bis(2-aminoethyl)-1,3-propa-
nediamine-Tris(2-aminoethyl)amine Copolymer (474 .mu.g) was added
followed by 2.5 ml Ringers.
[0183] High pressure tail vein injections of 2.5 ml of the complex
were preformed as previously described. Results reported are for
liver expression, and are the average of two mice. Luciferase
expression was determined as previously shown.
[0184] Results: High pressure injections
[0185] Complex I: 25,200,000 Relative Light Units
[0186] Complex II: 1,440,000 Relative Light Units
[0187] Results indicate that pDNA (pCI
Luc)/5,5'-Dithiobis(2-nitrobenzoic
acid)-N,N'-Bis(2-aminoethyl)-1,3-propanediamine-Tris(2-aminoethyl)amine
Copolymer Complexes are less effective than pCI Luc DNA in high
pressure injections. Although the complex was less effective, the
luciferase expression indicates that the pDNA is being released
from the complex and is accessible for transcription.
[0188] Synthesis of guanidino-L-cvstine,
1,4-bis(3-aminopropyl)piperazine copolymer (#M67):
[0189] To a solution of cystine (1 gm, 4.2 mmol) in ammonium
hydroxide (10 ml) in a screw-capped vial was added O-methylisourea
hydrogen sulfate (1.8 gm, 10 mmol). The vial was sealed and heated
to 60.degree. C. for 16 h. The solution was then cooled and the
ammonium hydroxide was removed by rotary evaporation. The solid was
then dissolved in water (20 ml), filtered through a cotton plug.
The product was then isolated by ion exchange chromatography using
Bio-Rex 70 resin and eluting with hydrochloric acid (100 mM).
[0190] Synthesis of guanidino-L-cystinel
1,4-bis(3-aminopropyl)piperazine copolymer
[0191] To a solution of guanidino-L-cystine (64 mg, 0.2 mmol) in
water (10 ml) was slowly added N,N'-dicyclohexylcarbodiimide (82
mg, 0.4 mmol) and N-hyroxysuccinimide (46 mg, 0.4 mmol) in dioxane
(5 ml). After 16 hr, the solution was filtered through a cotton
plug and 1,4-bis(3-aminopropyl)pip- erazine (40 .mu.L, 0.2 mmol)
was added. The reaction was allowed to stir at room temperature for
16 h and then the aqueous solution was dialyzed in a 15,000 MW
cutoff tubing against water (2.times.2 l) for 24 h. The solution
was then removed from dialysis tubing, filtered through 5 .mu.M
nylon syringe filter and then dried by lyophilization to yield 5 mg
of polymer.
[0192] Particle size of
pDNA-L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer and
DNA-guanidino-L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer
complexes
[0193] To a solution of pDNA (10 .mu.g/ml) in 0.5 ml 25 mM HEPES
buffer pH 7.5 was added 10 .mu.g/ml
L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer or
guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymer. The
size of the complexes between DNA and the polymers were measured.
For both polymers, the size of the particles were approximately 60
nm.
[0194] Condensation of DNA with
L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer and
decondensation of DNA upon addition of glutathione
[0195] Fluorescein labeled DNA was used for the determination of
DNA condensation in complexes with
L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer. pDNA was
modified to a level of 1 fluorescein per 100 bases using Mirus'
LabelIT Fluorescein kit. The fluorescence was determined using a
fluorescence spectrophotometer (Shimadzu RF-1501
spectrofluorometer) at an excitation wavelength of 495 nm and an
emission wavelength of 530 nm (Trubetskoy, V. S., Slattum, P. M.,
Hagstrom, J. E., Wolff, J. A., and Budker, V. G., "Quantitative
assessment of DNA condensation," Anal Biochem 267, 309-13 (1999),
incorporated herein by reference).
[0196] The intensity of the fluorescence of the fluorescein-labeled
DNA (10 .mu.g/ml) in 0.5 ml of 25 mM HEPES buffer pH 7.5 was 300
units. Upon addition of 10 .mu.g/ml of
L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer, the intensity
decreased to 100 units. To this DNA-polycation sample was added 1
mM glutathione and the intensity of the fluorescence was measured.
An increase in intensity was measured to the level observed for the
DNA sample alone. The half life of this increase in fluorescence
was 8 minutes.
[0197] The experiment indicates that DNA complexes with
physiologically-labile disulfide-containing polymers are cleavable
in the presence of the biological reductant glutathione.
[0198] Mouse Tail Vein Injection of
DNA-L-cystine-1,4-bis(3-aminopropyl)pi- perazine copolymer and
DNA-guanidino-L-cystine 1,4-bis(3-aminopropyl)piper- azine
copolymer Complexes
[0199] Plasmid delivery in the tail vein of ICR mice was performed
as previously described. To pCILuc DNA (50 .mu.g) in 2.5 ml
H.sub.2O was added either L-cystine
-1,4-bis(3-aminopropyl)piperazine copolymer, guanidino-L-cystine
1,4-bis(3-aminopropyl)piperazine copolymer, or poly-L-lysine
(34,000 MW, Sigma Chemical Company) (50 .mu.g). The samples were
then injected into the tail vein of mice using a 30 gauge, 0.5 inch
needle. One day after injection, the animal was sacrificed, and a
luciferase assay was conducted.
3 ng/ Polycation liver poly-L-lysine 6.2 L-cystine-
1,4-bis(3-aminopropyl)piperazine copolymer 439 guanidino-L-cystine
1,4-bis(3-aminopropyl)piperazine copolymer 487
[0200] The experiment indicates that DNA complexes with the
physiologically-labile disulfide-containing polymers are capable of
being broken, thereby allowing the luciferase gene to be
expressed.
[0201] Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
acid)-Pentaethylenehexam- ine Copolymer (#M69)
[0202] Pentaethylenehexamine (4.2 .mu.L, 0.017 mmol, Aldrich
Chemical Company) was taken up in 1.0 ml dichloromethane and HCl (1
ml, 1 M in Et.sub.2O, Aldrich Chemical Company) was added Et.sub.2O
was added and the resulting HCl salt was collected by filtration.
The salt was taken up in 1 ml DMF and
5,5'-dithiobis[succinimidyl(2-nitrobenzoate)] (10 mg, 0.017 mmol)
was added. The resulting solution was heated to 80.degree. C. and
diisopropylethylamine (12 .mu.L, 0.068 mmol, Aldrich Chemical
Company) was added dropwise. After 16 hr, the solution was cooled,
diluted with 3 ml H.sub.2O, and dialyzed in 12,000-14,000 MW cutoff
tubing against water (2.times.2 L) for 24 hr. The solution was then
removed from dialysis tubing and dried by lyophilization to yield
5.9 mg (58%) of 5,5'-dithiobis(2-nitrobenzoic
acid)-pentaethylenehexamine Copolymer.
[0203] Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
acid)-Pentaethylenehexam- ine-Tris(2-aminoethyl)amine Copolymer
(#M70)
[0204] Pentaethylenehexamine (2.9 .mu.L, 0.012 mmol, Aldrich
Chemical Company) and tris(2-aminoethyl)amine (0.51 .mu.L, 0.0034
mmol, Aldrich Chemical Company) were taken up in 0.5 ml methanol
and HCl (1 ml, 1 M in Et.sub.2O, Aldrich Chemical Company) was
added. Et.sub.2O was added and the resulting HCl salt was collected
by filtration. The salt was taken up in 1 ml DMF and
5,5'-dithiobis[succinimidyl(2-nitrobenzoate)] (10 mg, 0.017 mmol)
was added. The resulting solution was heated to 80.degree. C. and
diisopropylethylamine (12 .mu.L, 0.068 mmol, Aldrich Chemical
Company) was added dropwise. After 16 hr, the solution was cooled,
diluted with 3 ml H.sub.2O, and dialyzed in 12,000-14,000 MW cutoff
tubing against water (2.times.2 L) for 24 h. The solution was then
removed from dialysis tubing and dried by lyophilization to yield
6.0 mg (64%) of 5,5'-dithiobis(2-nitrobenzoic
acid)-pentaethylenehexamine-tris(2- -aminoethyl)amine
copolymer.
[0205] pH Cleavable Polymers for Intracellular Compartment
Release
[0206] A cellular transport step that has importance for gene
transfer and drug delivery is that of release from intracellular
compartments such as endosomes (early and late), lysosomes,
phagosomes, vesicle, endoplasmic reticulum, golgi apparatus, trans
golgi network (TGN), and sarcoplasmic reticulum. Release includes
movement out of an intracellular compartment into cytoplasm or into
an organelle such as the nucleus. Chemicals such as chloroquine,
bafilomycin or Brefeldin A1. Chloroquine decreases the
acidification of the endosomal and lysosomal compartments but also
affects other cellular functions. Brefeldin A, an isoprenoid fungal
metabolite, collapses reversibly the Golgi apparatus into the
endoplasmic reticulum and the early endosomal compartment into the
trans-Golgi network (TGN) to form tubules. Bafilomycin A.sub.1, a
macrolide antibiotic is a more specific inhibitor of endosomal
acidification and vacuolar type H.sup.+-ATPase than chloroquine.
The ER-retaining signal (KDEL sequence) has been proposed to
enhance delivery to the endoplasmic reticulum and prevent delivery
to lysosomes.
[0207] To increase the stability of DNA particles in serum, we have
added to positively-charged DNA-polycation particles polyanions
that form a third layer in the DNA complex and make the particle
negatively charged. To assist in the disruption of the DNA
complexes, we have synthesized polymers that are cleaved in the
acid conditions found in the endosome, pH 5-7. We also have reason
to believe that cleavage of polymers in the DNA complexes in the
endosome assists in endosome disruption and release of DNA into the
cytoplasm.
[0208] There are two ways to cleave a polyion: cleavage of the
polymer backbone resulting in smaller polyions or cleavage of the
link between the polymer backbone and the ion resulting in an ion
and an polymer. In either case, the interaction between the polyion
and DNA is broken and the number of molecules in the endosome
increases. This causes an osomotic shock to the endosomes and
disrupts the endosomes. In the second case, if the polymer backbone
is hydrophobic it may interact with the membrane of the endosome.
Either effect may disrupt the endosome and thereby assist in
release of DNA.
[0209] To construct cleavable polymers, one may attach the ions or
polyions together with bonds that are inherently labile such as
disulfide bonds, diols, diazo bonds, ester bonds, sulfone bonds,
acetals, ketals, enol ethers, enol esters, imines, imminiums, and
enamines. Another approach is construct the polymer in such a way
as to put reactive groups, i.e. electrophiles and nucleophiles, in
close proximity so that reaction between the function groups is
rapid. Examples include having carboxylic acid derivatives (acids,
esters, amides) and alcohols, thiols, carboxylic acids or amines in
the same molecule reacting together to make esters, thiol esters,
acid anhydrides or amides.
[0210] In one embodiment, ester acids and amide acids that are
labile in acidic environments (pH less than 7, greater than 4) to
form an alcohol and amine and an anhydride are use in a variety of
molecules and polymers that include peptides, lipids, and
liposomes.
[0211] In one embodiment, ketals that are labile in acidic
environments (pH less than 7, greater than 4) to form a diol and a
ketone are use in a variety of molecules and polymers that include
peptides, lipids, and liposomes.
[0212] In one embodiment, acetals that are labile in acidic
environments (pH less than 7, greater than 4) to form a diol and an
aldehyde are use in a variety of molecules and polymers that
include peptides, lipids, and liposomes.
[0213] In one embodiment, enols that are labile in acidic
environments (pH less than 7, greater than 4) to form a ketone and
an alcohol are use in a variety of molecules and polymers that
include peptides, lipids, and liposomes.
[0214] In one embodiment, iminiums that are labile in acidic
environments (pH less than 7, greater than 4) to form an amine and
an aldehyde or a ketone are use in a variety of molecules and
polymers that include peptides, lipids, and liposomes.
[0215] pH-Sensitive Cleavage of Peptides and Polypeptides
[0216] In one embodiment, peptides and polypeptides (both referred
to as peptides) are modified by an anhydride. The amine (lysine),
alcohol (serine, threonine, tyrosine), and thiol (cysteine) groups
of the peptides are modified by the an anhydride to produce an
amide, ester or thioester acid. In the acidic environment of the
internal vesicles (pH less than 6.5, greater than 4.5) (early
endosomes, late endosomes, or lysosome) the amide, ester, or
thioester is cleaved displaying the original amine, alcohol, or
thiol group and the anhydride.
[0217] A variety of endosomolytic and amphipathic peptides can be
used in this embodiment. A positively-charged
amphipathic/endosomolytic peptide is converted to a
negatively-charged peptide by reaction with the anhydrides to form
the amide acids and this compound is then complexed with a
polycation-condensed nucleic acid. After entry into the endosomes,
the amide acid is cleaved and the peptide becomes positively
charged and is no longer complexed with the polycation-condensed
nucleic acid and becomes amphipathic and endosomolytic. In one
embodiment the peptides contains tyrosines and lysines. In yet
another embodiment, the hydrophobic part of the peptide (after
cleavage of the ester acid) is at one end of the peptide and the
hydrophilic part (e.g. negatively charged after cleavage) is at
another end. The hydrophobic part could be modified with a
dimethylmaleic anhydride and the hydrophilic part could be modified
with a citranconyl anhydride. Since the dimethylmaleyl group is
cleaved more rapidly than the citrconyl group, the hydrophobic part
forms first. In another embodiment the hydrophilic part forms alpha
helixes or coil-coil structures.
[0218] pH-Sensitive Cleavage of Lipids and Liposomes
[0219] In another embodiment, the ester, amide or thioester acid is
complexed with lipids and liposomes so that in acidic environments
the lipids are modified and the liposome becomes disrupted,
fusogenic or endosomolytic. The lipid diacylglycerol is reacted
with an anhydride to form an ester acid. After acidification in an
intracellular vesicle the diacylglycerol reforms and is very lipid
bilayer disruptive and fusogenic.
[0220] Synthesis of Citraconylpolyvinylphenol
[0221] Polyvinylphenol (10 mg 30,000 MW Aldrich Chemical ) was
dissolved in 1 ml anhydrous pyridine. To this solution was added
citraconic anhydride (100 .mu.L, 1 mmol) and the solution was
allowed to react for 16 hr. The solution was then dissolved in 5 ml
of aqueous potassium carbonate (100 mM) and dialyzed three times
against 2 L water that was at pH8 with addition of potassium
carbonate. The solution was then concentrated by lyophilization to
10 mg/ml of citraconylpolyvinylphenol.
[0222] Synthesis of Citraconylpoly-L-tyrosine
[0223] Poly-L-tyrosine (10 mg, 40,000 MW Sigma Chemical ) was
dissolved in 1 ml anhydrous pyridine. To this solution was added
citraconic anhydride (100 .mu.L, 1 mmol) and the solution was
allowed to react for 16 hr. The solution was then dissolved in 5 ml
of aqueous potassium carbonate (100 mM) and dialyzed against
3.times.2 L water that was at pH8 with addition of potassium
carbonate. The solution was then concentrated by lyophilization to
10 mg/ml of citraconylpoly-L-tyrosine.
[0224] Synthesis of Citraconylpoly-L-lysine
[0225] Poly-L-lysine (10 mg 34,000 MW Sigma Chemical ) was
dissolved in 1 ml of aqueous potassium carbonate (100 mM). To this
solution was added citraconic anhydride (100 .mu.L, 1 mmol) and the
solution was allowed to react for 2 hr. The solution was then
dissolved in 5 ml of aqueous potassium carbonate (100 mM) and
dialyzed against 3.times.2 L water that was at pH8 with addition of
potassium carbonate. The solution was then concentrated by
lyophilization to 10 mg/ml of citraconylpoly-L-lysine.
[0226] Synthesis of Dimethylmaleylpoly-L-lysine
[0227] Poly-L-lysine (10 mg 34,000 MW Sigma Chemical ) was
dissolved in 1 ml of aqueous potassium carbonate (100 mM). To this
solution was added 2,3-dimethylmaleic anhydride (100 mg, 1 mmol)
and the solution was allowed to react for 2 hr. The solution was
then dissolved in 5 ml of aqueous potassium carbonate (100 mM) and
dialyzed against 3.times.2 L water that was at pH8 with addition of
potassium carbonate. The solution was then concentrated by
lyophilization to 10 mg/ml of dimethylmaleylpoly-L-lysine.
[0228] Characterization of Particles Formed with Citraconylated and
dimethylmaleylated Polymers
[0229] To a complex of DNA (20 .mu.g/ml) and poly-L-lysine (40
.mu.g/ml) in 1.5 ml was added the various citraconylpolyvinylphenol
and citraconylpoly-L-lysine (150 .mu.g/ml). The sizes of the
particles formed were measured to be 90-120 nm and the zeta
potentials of the particles were measured to be -10 to -30 mV
(Brookhaven ZetaPlus Particle Sizer).
[0230] To each sample was added acetic acid to make the pH 5. The
size of the particles was measured as a function of time. Both
citraconylpolyvinylphenol and citraconylpoly-L-lysine DNA complexes
were unstable under acid pH. The citraconylpolyvinylphenol sample
had particles >1 .mu.m in 5 minutes and citraconylpoly-L-lysine
sample had particles >1 .mu.m in 30 minutes.
[0231] Synthesis of Glutaric Dialdehyde-Poly-Glutamic Acid (8 mer)
Copolymer
[0232] H.sub.2N-EEEEEEEE-NHCH.sub.2CH.sub.2NH.sub.2 (5.5 mg, 0.0057
mmol, Genosys) was taken up in 0.4 ml H.sub.2O. Glutaric dialdehyde
(0.52 .mu.L, 0.0057 mmol, Aldrich Chemical Company) was added and
the mixture was stirred at room temperature. After 10 min the
solution was heated to 70.degree..quadrature. C. After 15 hrs, the
solution was cooled to room temperature and dialyzed against
H.sub.2O (2.times.2L, 3500 MWCO). Lyophilization afforded 4.3 mg
(73%) glutaric dialdehyde-poly-glutamic acid (8 mer) copolymer.
[0233] Synthesis of Ketal from Polyvinylphenyl Ketone and
Glycerol
[0234] Polyvinyl phenyl ketone (500 mg, 3.78 mmol, Aldrich Chemical
Company) was taken up in 20 ml dichloromethane. Glycerol (304
.mu.L, 4.16 mmol, Acros Chemical Company) was added followed by
p-toluenesulfonic acid monohydrate (108 mg, 0.57 mmol, Aldrich
Chemical Company). Dioxane (10 ml) was added and the solution was
stirred at room temperature overnight. After 16 hrs, TLC indicated
the presence of ketone. The solution was concentrated under reduced
pressure, and the residue redissolved in DMF (7 ml). The solution
was heated to 60.degree. C. for 16 hrs. Dialysis against H.sub.2O
(1.times.3L, 3500 MWCO), followed by Lyophilization resulted in 606
mg (78%) of the ketal.
[0235] Synthesis of Ketal Acid of Polyvinylphenyl Ketone and
Glycerol Ketal
[0236] The ketal from polyvinylphenyl ketone and glycerol (220 mg,
1.07 mmol) was taken up in dichloromethane (5 ml). Succinic
anhydride (161 mg, 1.6 mmol, Sigma Chemical Company) was added
followed by diisopropylethyl amine (0.37 ml, 2.1 mmol, Aldrich
Chemical Company) and the solution was heated at reflux. After 16
hrs, the solution was concentrated, dialyzed against H.sub.2O
(1.times.3L, 3500 MWCO), and lyophilized to afford 250 mg (75%) of
the ketal acid.
[0237] Particle Sizing and Acid Lability of Poly-L-Lysine/Ketal
Acid of Polyvinylphenyl Ketone and Glycerol Ketal Complexes
[0238] Particle sizing (Brookhaven Instruments Corporation,
ZetaPlus Particle Sizer, I90, 532 nm) indicated an effective
diameter of 172 nm (40 .mu.g) for the ketal acid Addition of acetic
acid to a pH of 5 followed by particle sizing indicated a increase
in particle size to 84000.
[0239] A poly-L-lysine/ketal acid (40 .mu.g, 1:3 charge ratio)
sample indicated a particle size of 142 nm. Addition of acetic acid
(5 .mu.L, 6 N) followed by mixing and particle sizing indicated an
effective diameter of 1970 nm. This solution was heated at
40.degree. C. particle sizing indicated a effective diameter of
74000 and a decrease in particle counts.
[0240] Results:
[0241] The particle sizer data indicates the loss of particles upon
the addition of acetic acid to the mixture.
[0242] Synthesis of Ketal from Polyvinyl Alcohol and
4-Acetylbutyric Acid
[0243] Polyvinylalcohol (200 mg, 4.54 mmol, 30,000-60,000 MW,
Aldrich Chemical Company) was taken up in dioxane (10 ml).
4-acetylbutyric acid (271 .mu.L, 2.27 mmol, Aldrich Chemical
Company) was added followed by p-toluenesulfonic acid monohydrate
(86 mg, 0.45 mmol, Aldrich Chemical Company). After 16 hrs, TLC
indicated the presence of ketone. The solution was concentrated
under reduced pressure, and the residue redissolved in DMF (7 ml).
The solution was heated to 60.degree. C. for 16 hrs. Dialysis
against H.sub.2O (1.times.4L, 3500 MWCO), followed by
lyophilization resulted in 145 mg (32%) of the ketal.
[0244] Particle Sizing and Acid Lability of Poly-L-Lysine/Ketal
from Polyvinyl Alcohol and 4-Acetylbutyric Acid Complexes
[0245] Particle sizing (Brookhaven Instruments Corporation,
ZetaPlus Particle Sizer, I90, 532 nm) indicated an effective
diameter of 280 nm (743 kcps) for poly-L-lysine/ketal from
polyvinyl alcohol and 4-acetylbutyric acid complexes (1:3 charge
ratio). A poly-L-lysine sample indicated no particle formation.
Similarly, a ketal from polyvinyl alcohol and 4-acetylbutyric acid
sample indicated no particle formation. Acetic acid was added to
the poly-L-lysine/ ketal from polyvinyl alcohol and 4-acetylbutyric
acid complexes to a pH of 4.5. Particle sizing indicated particles
of 100 nm, but at a minimal count rate (9.2 kcps)
[0246] Results:
[0247] The particle sizer data indicates the loss of particles upon
the addition of acetic acid to the mixture.
[0248] Synthesis of 1,4-Bis(3-aminopropyl)piperazine Glutaric
Dialdehyde Copolymer
[0249] 1,4-Bis(3-aminopropyl)piperazine (206 .mu.L, 0.998 mmol,
Aldrich Chemical Company) was taken up in 5.0 ml H.sub.2O. Glutaric
dialdehyde was (206 .mu.L, 0.998 mmol, Aldrich Chemical Company)
was added and the solution was stirred at room temperature. After
30 min, an additional portion of H.sub.2O was added (20 ml), and
the mixture neutralized with 6 N HCl to pH 7, resulting in a red
solution. Dialysis against H.sub.2O (3.times.3L, 12,000 - 14,000 MW
cutoff tubing) and lyophilization afforded 38 mg (14 %) of the
copolymer
[0250] Particle Sizing and Acid Lability of pDNA (pCI
Luc)/1,4-Bis(3-aminopropyl)piperazine Glutaric Dialdehyde Copolymer
Complexes (#M140)
[0251] To 50 .mu.g pDNA in 2 ml HEPES (25 mM, pH 7.8) was added 135
.mu.g 1,4-bis(3-aminopropyl)piperazine glutaric dialdehyde
copolymer. Particle sizing (Brookhaven Instruments Corporation,
ZetaPlus Particle Sizer, I90, 532 nm) indicated an effective
diameter of 110 nm for the complex. A 50 .mu.g pDNA in 2 ml HEPES
(25 mM, pH 7.8) sample indicated no particle formation. Similarly,
a 135 .mu.g 1,4-bis(3-aminopropyl)piperazine glutaric dialdehyde
copolymer in 2 ml HEPES (25 mM, pH 7.8) sample indicated no
particle formation.
[0252] Acetic acid was added to the pDNA (pCI
Luc)/1,4-bis(3-aminopropyl)p- iperazine glutaric dialdehyde
copolymer complexes to a pH of 4.5. Particle sizing indicated
particles of 2888 nm, and aggregation was observed.
[0253] Results:
[0254] 1,4-Bis(3-aminopropyl)piperazine-glutaric dialdehyde
copolymer condenses pDNA, forming small particles. Upon
acidification, the particle size increases, and aggregation occurs,
indicating cleavage of the polymeric immine.
[0255] Mouse Tail Vein Injections of pDNA
(pCILuc)/1,4-Bis(3-aminopropyl)p- iperazine Glutaric Dialdehyde
Copolymer Complexes
[0256] Four complexes were prepared as follows:
[0257] Complex I: pDNA (pCI Luc, 50 .mu.g) in 12.5 ml Ringers.
[0258] Complex II: pDNA (pCI Luc, 50 .mu.g) was mixed with
1,4-bis(3-aminopropyl)piperazine glutaric dialdehyde copolymer (50
.mu.g) in 1.25 ml HEPES 25 mM, pH 8. This solution was then added
to 11.25 ml Ringers.
[0259] Complex III: pDNA (pCI Luc, 50 .mu.g) was mixed with
poly-L-lysine (94.5 .mu.g, MW 42,000, Sigma Chemical Company) in
12.5 ml Ringers.
[0260] 2.5 ml tail vein injections of 2.5 ml of the complex were
preformed as previously described. Luciferase expression was
determined as previously indicated.
[0261] Results: 2.5 ml injections
[0262] Complex I: 3,692,000 Relative Light Units
[0263] Complex II: 1,047,000 Relative Light Units
[0264] Complex III: 4,379 Relative Light Units
[0265] Results indicate an increased level of pCI Luc DNA
expression in pDNA/1,4-bis(3-aminopropyl)piperazine glutaric
dialdehyde copolymer complexes over pCI Luc DNA/poly-L-lysine
complexes. These results also indicate that the pDNA is being
released from the pDNA/1,4-Bis(3-aminopro- pyl)piperazine-glutaric
dialdehyde copolymer complexes, and is accessible for
transcription.
[0266] Negatively Charged Complexes Using Non-cleavable
polymers
[0267] Many cationic polymers such as histone (H1, H2a, H2b, H3,
H4, H5), HMG proteins, poly-L-lysine, polyethylenimine, protamine,
and poly-histidine are used to compact polynucleic acids to help
facilitate gene delivery in vitro and in vivo. A key for efficient
gene delivery using prior art methods is that the non-cleavable
cationic polymers (both in vitro and in vivo) must be present in a
charge excess over the DNA so that the overall net charge of the
DNA/polycation complex is positive. Conversely, using our
intravascular delivery process having non-cleavable cationic
polymer/DNA complexes we found that gene expression is most
efficient when the overall net charge of the complexes are negative
(DNA negative charge>polycation positive charge). Tail vein
injections using cationic polymers commonly used for DNA
condensation and in vitro gene delivery revealed that high gene
expression occurred when the net charge of the complexes were
negative.
[0268] Tail vein injection of pCILuc/polycation complexes in 2.5 ml
ringers solution into 25 gram mice (ICR, Harlan) as previously
described (Zhang et al. Hum. Gen. Ther. 10:1735, 1999) Plasmid DNA
encoding the luciferase gene was complexed with various polycations
at two different concentrations. Complexes were prepared at
polycation to DNA charge ratios of 0.5:1 (low) and 5:1 (high). This
resulted in the formation of net negatively charged particles and
net positively charged particles respectively. 24 hours after tail
vein injection the livers were removed, cell extracts were
prepared, and assayed for luciferase activity. Only complexes with
a net negative overall charge displayed high gene expression
following intravascular delivery.
[0269] The net surface charge of DNA/polymer particles formed at
two different polymer to DNA ratios was determined by zeta
potential analysis. DNA/polymer complexes were formed by mixing the
components at the indicated charge: charge ratios in 25 mM HEPES,
pH 8 at a DNA concentration of 20 micrograms per ml (pCILuc).
Complexes were assayed for zeta potential on a Brookhaven ZetaPlus
dynamic light scattering particle sizer/zeta potential
analyzer.
[0270] Results:
[0271] DNA particles were formed at two different cationic polymer
to DNA ratios of 0.5:1 (charge:charge) and 5:1 (charge:charge). At
these ratios both negative (0.5:1 ratio) and positive particles
(5:1 ratio) should be theoretically obtained. Zeta potential
analysis of these particles confirmed that the two different ratios
did yield oppositely charged particles.
[0272] Cationic polymer Cationic polymer/DNA ratio Zeta Potential
(net surface charge of particle)
4 Poly-L-lysine 0.5: 1-16.77 mV Poly-L-lysine 5: 1 + 24.11 mV (n =
7) (n = 6) Polyethylenimine 0.5: 1-12.47 mV Polyethylenimine 5: 1 +
35.74 mV (n = 7) (n = 8) Histone H1 0.5: 1-9.6 mV Histone H1 5: 1 +
20.97 mV (n = 8) (n = 8)
[0273]
5 7
[0274] High Efficiency Gene Expression Following Tail Vein Delivery
of pDNA/Cationic Peptide Complexes
[0275] Plasmid DNA (pCILuc) was mixed with an amphipathic cationic
peptide at a 1:2 ratio (charge ratio) and diluted into 2.5 ml of
Ringers solution per mouse. Complexes were injected into the tail
vein of a 25 g ICR mouse (Harlan Sprague Dawley, Indianapolis,
Ind.) in 7 seconds. Animals were sacrificed after 24 hours and
livers were removed and assayed for luciferase expression.
[0276] Complex Preparation (per mouse)
[0277] Complex I: pDNA (pCI Luc, 10 .mu.g) in 2.5 ml Ringers.
[0278] Complex II: pDNA (pCI Luc, 10 .mu.g) was mixed with cationic
peptide (18 mer KLLKKLLKLWKKLLKKLK) at a 1:2 ratio. Complexes were
diluted to 2.5 ml with Ringers solution.
[0279] Tail vein injections of 2.5 ml of the complex were preformed
as previously described. Luciferase expression was determined as
previously shown.
[0280] Results: 2.5 ml injections
[0281] Complex I: 1.63.times.10.sup.10 Relative Light Units per
liver
[0282] Complex II: 2.05.times.10.sup.10 Relative Light Units per
liver
[0283] Negatively Charged Complexes Using Labile polymers
[0284] Delivery of PEI/DNA and histone Hl/DNA particles to rat
skeletal muscle via intravascular injection into an artery.
[0285] Experimental Protocol and Methods:
[0286] PEI/DNA and histone Hl/DNA particles were injected into rat
leg muscle by either a single intra-arterial injection into the
external iliac [see Budker et al. Gene Therapy, 5:272, (1998)].
Harlan Sprague Dawley (HSD SD) rats were used for the muscle
injections. All rats used were female and approximately 150 grams
and each received complexes containing 100 micrograms of plasmid
DNA encoding the luciferase gene under control of the CMV
enhancer/promoter (pCILuc) [see Zhang et al. Human Gene Therapy,
8:1763, (1997)].
[0287] Luciferase Assays:
[0288] Results of the rat injections are provided in relative light
units (RLUs) and micrograms (.mu.g) of luciferase produced. To
determine RLUs, 10 .mu.l of cell lysate were assayed using a
EG&G Berthold LB9507 luminometer and total muscle RLUs were
determined by multiplying by the appropriate dilution factor. To
determine the total amount of luciferase expressed per muscle we
used a conversion equation that was determined in an earlier study
[see Zhang et al. Human Gene Therapy, 8:1763, (1997)] [pg
luciferase=RLUs.times.5.1.times.10.sup.-5]
6 Total Total Muscle Group RLUs Luciferase DNA/PEI particles (1:0.5
charge ratio) muscle group 1 (upper leg anterior) 3.50 .times.
10.sup.9 0.180 .mu.g muscle group 2 (upper leg posterior) 3.96
.times. 10.sup.9 0.202 .mu.g muscle group 3 (upper leg medial) 7.20
.times. 10.sup.9 0.368 .mu.g muscle group 4 (lower leg posterior)
9.90 .times. 10.sup.9 0.505 .mu.g muscle group 5 (lower leg
anterior) 9.47 .times. 10.sup.8 0.048 .mu.g muscle group 6 (foot)
6.72 .times. 10.sup.6 0.0003 .mu.g Total RLU/leg = 25.51 .times.
10.sup.9 RLU (1.303 .mu.g luciferase) DNA/PEI particles (1:5 charge
ratio) muscle group 1 (upper leg anterior) 1.77 .times. 10.sup.7
0.0009 .mu.g muscle group 2 (upper leg posterior) 1.47 .times.
10.sup.7 0.0008 .mu.g muscle group 3 (upper leg medial) 5.60
.times. 10.sup.6 0.00003 .mu.g muscle group 4 (lower leg posterior)
7.46 .times. 10.sup.6 0.00004 .mu.g muscle group 5 (lower leg
anterior) 6.84 .times. 10.sup.6 0.00003 .mu.g muscle group 6 (foot)
1.55 .times. 10.sup.6 0.000008 .mu.g Total RLU/leg = 5.39 .times.
10.sup.7 RLU (0.0018 .mu.g luciferase)
[0289]
7 Total Total Muscle Group RLUs Luciferase DNA/histone H1 particles
(1:0.5 charge ratio) muscle group 1 (upper leg anterior) 3.12
.times. 10.sup.9 0.180 .mu.g muscle group 2 (upper leg posterior)
9.13 .times. 10.sup.9 0.202 .mu.g muscle group 3 (upper leg medial)
1.23 .times. 10.sup.10 0.368 .mu.g muscle group 4 (lower leg
posterior) 5.73 .times. 10.sup.9 0.505 .mu.g muscle group 5 (lower
leg anterior) 4.81 .times. 10.sup.8 0.048 .mu.g muscle group 6
(foot) 6.49 .times. 10.sup.6 0.0003 .mu.g Total RLU/leg = 3.08
.times. 10.sup.10 RLU (1.57 .mu.g luciferase) DNA/histone H1
particles (1:5 charge ratio) muscle group 1 (upper leg anterior)
1.42 .times. 10.sup.7 0.0007 .mu.g muscle group 2 (upper leg
posterior) 5.94 .times. 10.sup.6 0.0003 .mu.g muscle group 3 (upper
leg medial) 3.09 .times. 10.sup.6 0.0002 .mu.g muscle group 4
(lower leg posterior) 2.53 .times. 10.sup.6 0.0001 .mu.g muscle
group 5 (lower leg anterior) 2.85 .times. 10.sup.6 0.0001 .mu.g
muscle group 6 (foot) 1.84 .times. 10.sup.5 0.000009 .mu.g Total
RLU/leg = 2.88 .times. 10.sup.7 RLU (0.0014 .mu.g luciferase)
[0290] The foregoing is considered as illustrative only of the
principles of the invention. Furthermore, since numerous
modifications and changes will readily occur to those skilled in
the art, it is not desired to limit the invention to the exact
construction and operation shown and described. Therefore, all
suitable modifications and equivalents fall within the scope of the
invention.
* * * * *