U.S. patent application number 10/755784 was filed with the patent office on 2004-07-15 for compositions and methods for drug delivery using amphiphile binding molecules.
Invention is credited to Budker, Vladimir, Hagstrom, James E., Monahan, Sean D., Rozema, David B., Slattum, Paul M., Wolff, Jon A..
Application Number | 20040137625 10/755784 |
Document ID | / |
Family ID | 26863520 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040137625 |
Kind Code |
A1 |
Wolff, Jon A. ; et
al. |
July 15, 2004 |
Compositions and methods for drug delivery using amphiphile binding
molecules
Abstract
The present invention relates to the delivery of desired
compounds (e.g., nucleic acids) into cells using noncovalent
delivery systems which include complexing nucleic acids,
amphipathic binding agents, and amphiphiles.
Inventors: |
Wolff, Jon A.; (Madison,
WI) ; Hagstrom, James E.; (Madison, WI) ;
Monahan, Sean D.; (Madison, WI) ; Budker,
Vladimir; (Middleton, WI) ; Rozema, David B.;
(Madison, WI) ; Slattum, Paul M.; (Madison,
WI) |
Correspondence
Address: |
Mark K. Johnson
Mirus Corporation
505 S. Rosa Rd.
Madison
WI
53719
US
|
Family ID: |
26863520 |
Appl. No.: |
10/755784 |
Filed: |
January 12, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10755784 |
Jan 12, 2004 |
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09726792 |
Nov 29, 2000 |
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6740643 |
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10755784 |
Jan 12, 2004 |
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09234606 |
Jan 21, 1999 |
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60167836 |
Nov 29, 1999 |
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Current U.S.
Class: |
435/455 ;
514/44R; 514/54 |
Current CPC
Class: |
A61K 48/00 20130101;
C07H 21/00 20130101; A61K 48/0025 20130101; A61K 47/6951 20170801;
B82Y 5/00 20130101; C12N 15/87 20130101 |
Class at
Publication: |
435/455 ;
514/044; 514/054 |
International
Class: |
A61K 048/00; A61K
031/724; C12N 015/85 |
Claims
We claim:
1. A process for obtaining an expression product by delivering a
polynucleotide to a cell, comprising: a. associating an amphiphile
binding agent, an amphiphile, and a polynucleotide thereby forming
a complex; b. delivering the complex to the cell; and, c.
expressing the polynucleotide.
2. The process of claim 1 wherein the amphiphile binding agent
consists of a cyclodextrin.
3. The process of claim 1 wherein the amphiphile binding agent is
polymeric.
4. The process of claim 1 further comprising complexing the
polynucleotide with a polycation.
5. The process of claim 1 further comprising associating a
polyanion in step (a).
6. The process of claim 1 wherein the amphiphile consists of a
polymer.
7. The process of claim 1 wherein the amphiphile consists of an
interaction modifier.
8. The process of claim 1 wherein the cell is in a mammal.
9. The process of claim 1 wherein the polynucleotide consists of
DNA.
10. The process of claim 1 wherein the polynucleotide consists of a
gene.
11. A complex for delivering and expressing DNA in a mammal,
comprising: an amphiphile binding agent, an amphiphile, and DNA in
complex.
12. The complex of claim 11 wherein the amphiphile is attached to
the DNA.
13. The complex of claim 12 wherein the amphiphile is covalently
attached to DNA.
14. The complex of claim 11 wherein the amphiphile binding agent
consists of a cyclodextrin.
15. A process for obtaining an expression product in vivo,
comprising: a. forming a complex with a cyclodextrin, an amphiphile
and a polynucleotide; b. delivering the complex to a cell in a
mammal; c. expressing the polynucleotide.
16. The process of claim 15 wherein the amphiphile binding agent is
polymeric.
17. The process of claim 15 further comprising complexing the
polynucleotide with a polycation.
18. The process of claim 15 further comprising associating a
polyanion in step (a).
19. The process of claim 15 wherein the amphiphile consists of a
polymer.
20. The process of claim 15 wherein the amphiphile consists of an
interaction modifier.
Description
[0001] This application claims priority benefit of provisional
application No. 60/167,836, filed on Nov. 29.sup.th, 1999.
[0002] This application is a continuation-in-part of pending
application No. 09/234,606 filed on Jan. 21, 1999.
FIELD OF THE INVENTION
[0003] The present invention relates to the delivery of desired
compounds (e.g., drugs and nucleic acids) into cells using
noncovalent delivery systems. The present invention provides
compositions and methods for the delivery and release of a compound
of interest to a cell.
BACKGROUND
[0004] Drug Delivery
[0005] A variety of methods and routes of administration have been
developed to deliver pharmaceuticals that include small molecular
drugs and biologically active compounds such as peptides, hormones,
proteins, and enzymes to their site of action. Parenteral routes of
administration include intravascular (intravenous, intraarterial),
intramuscular, intraparenchymal, intradermal, subdermal,
subcutaneous, intratumor, intraperitoneal, and intralymphatic
injections that use a syringe and a needle or catheter. The blood
circulatory system provides systemic spread of the pharmaceutical.
Polyethylene glycol and other hydrophilic polymers have provided
protection of the pharmaceutical in the blood stream by preventing
its interaction with blood components and to increase the
circulatory time of the pharmaceutical by preventing opsonization,
phagocytosis and uptake by the reticuloendothelial system. For
example, the enzyme adenosine deaminase has been covalently
modified with polyethylene glycol to increase the circulatory time
and persistence of this enzyme in the treatment of patients with
adenosine deaminase deficiency.
[0006] The controlled release of pharmaceuticals after their
administration is under intensive development. Pharmaceuticals have
also been complexed with a variety of biologically-labile polymers
to delay their release from depots. These polymers have included
copolymers of poly(lactic/glycolic acid) (PLGA) (Jain, R. et al.
Drug Dev. Ind. Pharm. 24, 703-727 (1998), ethylvinyl
acetate/polyvinyl alcohol (Metrikin, DC and Anand, R, Curr Opin
Ophthalmol 5, 21-29, 1994) as typical examples of biodegradable and
non-degradable sustained release systems respectively.
[0007] Transdermal routes of administration have been effected by
patches and ionotophoresis. Other epithelial routes include oral,
nasal, respiratory, and vaginal routes of administration. These
routes have attracted particular interest for the delivery of
peptides, proteins, hormones, and cytokines which are typically
administered by parenteral routes using needles. For example, the
delivery of insulin via respiratory, oral, or nasal routes would be
very attractive for patients with diabetes mellitus. For oral
routes, the acidity of the stomach (pH less than 2) is avoided for
pH-sensitive compounds by concealing peptidase-sensitive
polypeptides inside pH-sensitive hydrogel matrix (copolymers of
polyethyleneglycol and polyacrylic acid). After passing low pH
compartments of gastrointestinal tract such structures swells at
higher pH releasing thus a bioactive compound (Lowman AM et al. J.
Pharm. Sci. 88, 933-937 (1999). Capsules have also been developed
that release their contents within the small intestine based upon
pH-dependent solubility of a polymer. Copolymers of polymethacrylic
acid (Eudragit S, Rohm America) are known as polymers which are
insoluble at lower pH but readily solubilized at higher pH, so they
are used as enteric coatings (Z Hu et al. J. Drug Target., 7, 223,
1999).
[0008] Biologically active molecules may be assisted by a
reversible formation of covalent bonds. Quite often, it is found
that the drug administered to a patient is not the active form of
the drug, but is what is a called a prodrug that changes into the
actual biologically active compound upon interactions with specific
enzymes inside the body. In particular, anticancer drugs are quite
toxic and are administered as prodrugs which do not become active
until they come in contact with the cancerous cell (Sezaki, I I.,
Takakura, Y., Hashida, M. Adv. Drug. Delivery Reviews 3, 193,
1989).
[0009] Recent studies have found that pH in solid tumors is 0.5 to
1 units lower than in normal tissue (Gerweck LE et al. Cancer Res.
56, 1194 (1996). Hence, the use of pH-sensitive polymers for tumor
targeting is justified. However, this approach was demonstrated
only in vitro (Berton, M, Eur. J. Pharm. Biopharm. 47, 119-23,
1999).
[0010] Liposomes were also used as drug delivery vehicles for low
molecular weight drugs and macromolecules such as amphotericin B
for systemic fungal infections and candidiasis. Inclusion of
anti-cancer drugs such as adriamycin have been developed to
increase their delivery to tumors and reduce it to other tissue
sites (e.g. heart) thereby decreasing their toxicity. pH-sensitive
polymers have been used in conjunction with liposomes for the
triggered release of an encapsulatede drug. For example,
hydrophobically-modified N-isopropylacrylamide-methacr- ylic acid
copolymer can render regular egg phosphatidyl chloline liposomes
pH-sensitive by pH-dependent interaction of grafted aliphatic
chains with lipid bilayer (O Meyer et al., FEBS Lett., 421, 61,
1998).
[0011] Gene And Nucleic Acid-Based Delivery
[0012] Gene or polynucleotide transfer is the cardinal process of
gene therapy. The gene needs to be transferred across the cell
membrane and enter the nucleus where the gene can be expressed.
Gene transfer methods currently being explored included viral
vectors and physical-chemical methods.
[0013] Viruses have evolved over millions of year to transfer their
genes into mammalian cells. Viruses can be modified to carry a
desired gene and become a "vector" for gene therapy. Using standard
recombinant techniques, the harmful or superfluous viral genes can
be removed and replaced with the desired normal gene. This was
first accomplished with mouse retroviruses. The development of
retroviral vectors were the catalyst that promoted current gene
therapy efforts. However, they cannot infect all cell types very
efficiently, especially in vivo. Other viral vectors based on
Herpes virus are being developed to enable more efficient gene
transfer into brain cells. Adenoviral and adenoassociated vectors
are being developed to infect lung and other cells.
[0014] Besides using viral vectors, it is possible to directly
transfer genes into mammalian cells. Usually, the desired gene is
placed within bacterial plasmid DNA along with a mammalian
promoter, enhancer, and other sequences that enable the gene to be
expressed in mammalian cells. Several milligrams of the plasmid DNA
containing all these sequences can be prepared and purified from
the bacterial cultures. The plasmid DNA containing the desired gene
can be incorporated into lipid vesicles (liposomes including
cationic lipids such as Lipofectin) that then transfer the plasmid
DNA into the target cell. Plasmid DNA can also be complexed with
proteins that target the plasmid DNA to specific tissues just as
certain proteins are taken up (endocytosed) by specific cells.
Also, plasmid DNA can be complexed with polymers such as polylysine
and polyethylenimine. Another plasmid-based technique involves
"shooting" the plasmid DNA on small gold beads into the cell using
a "gun". Finally, muscle cells in vivo have the unusual ability to
take up and express plasmid DNA.
[0015] Gene therapy approaches can be classified into direct and
indirect methods. Some of these gene transfer methods are most
effective when directly injected into a tissue space. Direct
methods using many of the above gene transfer techniques are being
used to target tumors, muscle, liver, lung, and brain. Other
methods are most effective when applied to cells or tissues that
have been removed from the body and the genetically-modified cells
are then transplanted back into the body. Indirect approaches in
conjunction with retroviral vectors are being developed to transfer
genes into bone marrow cells, lymphocytes, hepatocytes, myoblasts
and skin cells.
[0016] Gene Therapy And Nucleic Acid-Based Therapies
[0017] Gene therapy promises to be a revolutionary advance in the
treatment of disease. It is a fundamentally new approach for
treating disease that is different from the conventional surgical
and pharmaceutical therapies. Conceptually, gene therapy is a
relatively simple approach. If someone has a defective gene, then
gene therapy would fix the defective gene. The disease state would
be modified by manipulating genes instead of their products, i.e
proteins, enzymes, enzyme substrates and enzyme products. Although,
the initial motivation for gene therapy was the treatment of
genetic disorders, it is becoming increasingly apparent that gene
therapy will be useful for the treatment of a broad range of
acquired diseases such as cancer, infectious disorders (AIDS),
heart disease, arthritis, and neurodegenerative disorders
(Parkinson's and Alzheimer's).
[0018] Gene therapy promises to take full-advantage of the major
advances brought about by molecular biology. While, biochemistry is
mainly concerned with how the cell obtains the energy and matter
that is required for normal function, molecular biology is mainly
concerned with how the cell gets the information to perform its
functions. Molecular biology wants to discover the flow of
information in the cell. Using the metaphor of computers, the cell
is the hardware while the genes are the software. In this sense,
the purpose of gene therapy is to provide the cell with a new
program (genetic information) so as to reprogram a dysfunctional
cell to perform a normal function. The addition of a new cellular
function is provided by the insertion of a foreign gene that
expresses a foreign protein or a native protein at amounts that are
not present in the patient.
[0019] The inhibition of a cellular function is provided by
anti-sense approaches (that is acting against messenger RNA) and
that includes oligonucleotides complementary to the messenger RNA
sequence and ribozymes. Messenger RNA (mRNA) is an intermediate in
the expression of the DNA gene. The mRNA is translated into a
protein. "Anti-sense" methods use a RNA sequence or an
oligonucleotide that is made complementary to the target mRNA
sequence and therefore binds specifically to the target messenger
RNA. When this anti-sense sequence binds to the target mRNA, the
mRNA is somehow destroyed or blocked from being translated.
Ribozymes destroy a specific mRNA by a different mechanism.
Ribozymes are RNA's that contain sequence complementary to the
target messenger RNA plus a RNA sequence that acts as an enzyme to
cleave the messenger RNA, thus destroying it and preventing it from
being translated. When these anti-sense or ribozyme sequences are
introduced into a cell, they would inactivate their specific target
mRNA and reduce their disease-causing properties.
[0020] Several recessive genetic disorders are being considered for
gene therapy. One of the first uses of gene therapy in humans has
been used for the genetic deficiency of the adenosine deaminase
(ADA) gene. Other clinical gene therapy trials have been conducted
for cystic fibrosis, familial hypercholesteremia caused by a
defective LDL-receptor gene and partial ornithine transcarbomylase
deficiency. Both indirect and direct gene therapy approaches are
being developed for Duchenne muscular dystrophy. Patients with this
type of muscular dystrophy eventually die from loss of their
respiratory muscles. Direct approaches include the intramuscular
injection of naked plasmid DNA or adenoviral vectors.
[0021] A wide variety of gene therapy approaches for cancer are
under investigation in animals and in human clinical trials. One
approach is to express in lymphocytes and in the tumor cells,
cytokine genes that stimulate the immune system to destroy the
cancer cells. The cytokine genes would be transferred into the
lymphocytes by removing the lymphocytes from the body and infecting
them with a retroviral vector carrying the cytokine gene. The tumor
cells would be similarly genetically modified by this indirect
approach to express cytokines within the tumor. Direct approaches
involving the expression of cytokines in tumor cells in situ are
also being considered. Other genes besides cytokines may be able to
induce an immune response against the cancer. One approach that has
entered clinical trials is the direct injection of HLA-B7 gene
(which encodes a potent immunogen) within lipid vesicles into
malignant melanomas in order to induce a more effective immune
response against the cancer.
[0022] "Suicide" genes are genes that kill cells that express the
gene. For example, the diphtheria toxin gene directly kills cells.
The Herpes thymidine kinase (TK) gene kills cells in conjunction
with acyclovir (a drug used to treat Herpes viral infections).
Other gene therapy approaches take advantage of our knowledge of
oncogenes and suppressor tumor genes- also known as anti-oncogenes.
The loss of a functioning anti-oncogene plays a decisive role in
childhood tumors such as retinoblastoma, osteosarcoma and Wilms
tumor and may play an important role in more common tumors such as
lung, colon and breast cancer. Introduction of the normal
anti-oncogene back into these tumor cells may convert them back to
normal cells. The activation of oncogenes also plays an important
role in the development of cancers. Since these oncogenes operate
in a "dominant" fashion, treatment will require inactivation of the
abnormal oncogene. This can be done using either "anti-sense" or
ribozyme methods that selectively inactivate a specific messenger
RNA in a cell.
[0023] Gene therapy can be used as a type of vaccination to prevent
infectious diseases and cancer. When a foreign gene is transferred
into a cell and the protein is made, the foreign protein is
presented to the immune system differently from simply injecting
the foreign protein into the body. This different presentation is
more likely to cause a cell-mediated immune response which is
important for fighting latent viral infections such as human
immunodeficiency virus (HIV causes AIDS), Herpes and
cytomegalovirus. Expression of the viral gene within a cell
simulates a viral infection and induces a more effective immune
response by fooling the body that the cell is actually infected by
the virus, without the danger of an actual viral infection.
[0024] One direct approach uses the direct intramuscular injection
of naked plasmid DNA to express a viral gene in muscle cells. The
"gun" has also been shown to be effective at inducing an immune
response by expressing foreign genes in the skin. Other direct
approaches involving the use of retroviral, vaccinia or adenoviral
vectors are also being developed. An indirect approach has been
developed to remove fibroblasts from the skin, infect them with a
retroviral vector carrying a viral gene and transplant the cells
back into the body. The envelope gene from the AIDS virus (HIV) is
often used for these purposes. Many cancer cells express specific
genes that normal cells do not. Therefore, these genes specifically
expressed in cancer cells can be used for immunization against
cancer.
[0025] Besides the above immunization approaches, several other
gene therapies are being developed for treating infectious disease.
Most of these new approaches are being developed for HIV infection
and AIDS. Many of them will involve the delivery of anti-sense or
ribozyme sequences directed against the particular viral messenger
RNA. These anti-sense or ribozyme sequences will block the
expression of specific viral genes and abort the viral infection
without damaging the infected cell. Another approach somewhat
similar to the ant-sense approaches is to overexpress the target
sequences for these regulatory HIV sequences.
[0026] Gene therapy efforts would be directed at lowering the risk
factors associated with atherosclerosis. Overexpression of the LDL
receptor gene would lower blood cholesterol in patients not only
with familial hypercholesteremia but with other causes of high
cholesterol levels. The genes encoding the proteins for HDL ("the
good cholesterol") could be expressed also in various tissues. This
would raise HDL levels and prevent atherosclerosis and heart
attacks. Tissue plasminogen activator (tPA) protein is being given
to patients immediately after their myocardial infarction to digest
the blood clots and open up the blocked coronary blood vessels. The
gene for tPA could be expressed in the endothelial cells lining the
coronary blood vessels and thereby deliver the tPA locally without
providing tPA throughout the body. Another approach for coronary
vessel disease is to express a gene in the heart that produces a
protein that causes new blood vessels to grow. This would increase
collateral blood flow and prevent a myocardial infarction from
occurring.
[0027] Neurodegenerative disorders such as Parkinson's and
Alzheimer's diseases are good candidates for early attempts at gene
therapy. Arthritis could also be treated by gene therapy. Several
proteins and their genes (such as the IL-1 receptor antagonist
protein) have recently been discovered to be anti-inflammatory.
Expression of these genes in joint (synovial) fluid would decrease
the joint inflammation and treat the arthritis.
[0028] In addition, methods are being developed to directly modify
the sequence of target genes and chromosomal DNA. The delivery of a
nucleic acid or other compound that modifies the genetic
instruction (e.g., by homologous recombination) can correct a
mutated gene or mutate a functioning gene.
[0029] Polymers for Drug and Nucleic Acid Delivery
[0030] Polymers are used for drug delivery for a variety of
therapeutic purposes. Polymers have also been used in research for
the delivery of nucleic acids (polynucleotides and
oligonucleotides) to cells with an eventual goal of providing
therapeutic processes. Such processes have been termed gene therapy
or anti-sense therapy. One of the several methods of nucleic acid
delivery to the cells is the use of DNA-polycation complexes. It
has been shown that cationic proteins like histones and protamines
or synthetic polymers like polylysine, polyarginine, polyomithine,
DEAE dextran, polybrene, and polyethylenimine may be effective
intracellular delivery agents while small polycations like spermine
are ineffective. The following are some important principles
involving the mechanism by which polycations facilitate uptake of
DNA:
[0031] Polycations provide attachment of DNA to the 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 convenient linker for attaching
specific ligands to DNA and as result, DNA- polycation complexes
can be targeted to specific cell types.
[0032] Polycations protect DNA in complexes against nuclease
degradation. This is important for both extra- and intracellular
preservation of DNA. Gene expression is also enabled or increased
by preventing endosome acidification with NH.sub.4Cl 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 to the polycation endosomal-disruptive agents such as
fusion peptides or adenoviruses.
[0033] Polycations can also facilitate DNA condensation. The volume
which one DNA molecule occupies in a complex with polycations is
drastically lower than the volume of a free DNA molecule. The size
of a DNA/polymer complex is probably critical for gene delivery in
vivo. In terms of intravenous injection, DNA needs to cross the
endothelial barrier and reach the parenchymal cells of interest.
The largest endothelia fenestrae (holes in the endothelial barrier)
occur in the liver and have an average diameter of 100 nm. The
trans-epithelial pores in other organs are much smaller, for
example, muscle endothelium can be described as a structure which
has a large number of small pores with a radius of 4 nm, and a very
low number of large pores with a radius of 20-30 nm. The size of
the DNA complexes is also important for the cellular uptake
process. After binding to the cells the DNA- polycation complex
should be taken up by endocytosis. Since the endocytic vesicles
have a homogenous internal diameter of about 100 nm in hepatocytes
and are of similar size in other cell types, DNA complexes smaller
than 100 nm are preferred.
[0034] Condensation of DNA
[0035] A significant number of multivalent cations with widely
different molecular structures have been shown to induce
condensation of DNA.
[0036] Two approaches for compacting (used herein as an equivalent
to the term condensing) DNA:
[0037] 1. Multivalent cations with a charge of three or higher have
been shown to condense DNA. These include spermidine, spermine,
Co(NH.sub.3).sub.6.sup.3+,Fe.sup.3+, and natural or synthetic
polymers such as histone Hl, protamine, polylysine, and
polyethylenimine. Analysis has shown DNA condensation to be favored
when 90% or more of the charges along the sugar-phosphate backbone
are neutralized.
[0038] 2. Polymers (neutral or anionic) which can increase
repulsion between DNA and its surroundings have been shown to
compact DNA. Most significantly, spontaneous DNA self-assembly and
aggregation process have been shown to result from the confinement
of large amounts of DNA, due to excluded volume effect.
[0039] Depending upon the concentration of DNA, condensation leads
to three main types of structures:
[0040] 1) In extremely dilute solution (about 1 ug/mL or below),
long DNA molecules can undergo a monomolecular collapse and form
structures described as toroid.
[0041] 2) In very dilute solution (about 10 ug/mL) microaggregates
form with short or long molecules and remain in suspension.
Toroids, rods and small aggregates can be seen in such
solution.
[0042] 3) In dilute solution (about 1 mg/mL) large aggregates are
formed that sediment readily.
[0043] Toroids have been considered an attractive form for gene
delivery because they have the smallest size. While the size of DNA
toroids produced within single preparations has been shown to vary
considerably, toroid size is unaffected by the length of DNA being
condensed. DNA molecules from 400 bp to genomic length produce
toroids similar in size. Therefore one toroid can include from one
to several DNA molecules. The kinetics of DNA collapse by
polycations that resulted in toroids is very slow. For example DNA
condensation by Co(NH.sub.3).sub.6Cl.sub.3 needs 2 hours at room
temperature.
[0044] The mechanism of DNA condensation is not clear. The
electrostatic force between unperturbed helices arises primarily
from a counterion fluctuation mechanism requiring multivalent
cations and plays a major role in DNA condensation. The hydration
forces predominate over electrostatic forces when the DNA helices
approach closer then a few water diameters. In a case of
DNA-polymeric polycation interactions, DNA condensation is a more
complicated process than the case of low molecular weight
polycations. Different polycationic proteins can generate toroid
and rod formation with different size DNA at a ratio of positive to
negative charge of two to five. T4 DNA complexes with polyarginine
or histone can form two types of structures; an elongated structure
with a long axis length of about 350 nm (like free DNA) and dense
spherical particles. Both forms exist simultaneously in the same
solution. The reason for the co-existence of the two forms can be
explained as an uneven distribution of the polycation chains among
the DNA molecules. The uneven distribution generates two
thermodynamically favorable conformations.
[0045] The electrophoretic mobility of DNA-polycation complexes can
change from negative to positive in excess of polycation. It is
likely that large polycations do not completely align along DNA but
form polymer loops that interact with other DNA molecules. The
rapid aggregation and strong intermolecular forces between
different DNA molecules may prevent the slow adjustment between
helices needed to form tightly packed orderly particles.
[0046] As previously stated, preparation of polycation-condensed
DNA particles is of particular importance for gene therapy, more
specifically, particle delivery such as the design of non-viral
gene transfer vectors. Optimal transfection activity in vitro and
in vivo can require an excess of polycation molecules. However, the
presence of a large excess of polycations may be toxic to cells and
tissues. Moreover, the non-specific binding of cationic particles
to all cells forestalls cellular targeting. Positive charge also
has an adverse influence on biodistribution of the complexes in
vivo.
[0047] Several modifications of DNA-cation particles have been
created to circumvent the nonspecific interactions of the
DNA-cation particle and the toxicity of cationic particles.
Examples of these modifications include attachment of steric
stabilizers, e.g. polyethylene glycol, which inhibit nonspecific
interactions between the cation and biological polyanions. Another
example is recharging the DNA particle by the additions of
polyanions, which interact with the cationic particle, thereby
lowering its surface charge, i.e. recharging of the DNA particle
U.S. Pat. No. 09/328,975. Another example is cross-linking the
polymers and thereby caging the complex described in U.S. Pat. No.
08/778,657, U.S. Pat. No. 09/000,692, U.S. Pat. No. 09/070299, and
U.S. Pat. No. 09/464,871. Nucleic acid particles can be formed by
the formation of chemical bonds and template polymerization
described in U.S. Pat. No. 08/778,657, U.S. Pat. No. 09/000,692,
U.S. Pat. No. 09/070299, and U.S. Pat. No. 09/464,871.
[0048] A potential problem with these modifications is that they
may be irreversible rendering the particle unable to interact with
the cell to be transfected, and/or incapable of escaping from the
lysosome once taken into a cell, and/or incapable of entering the
nucleus once inside the cell. A method for formation of DNA
particles that is reversible under conditions found in the cell may
allow for effective delivery of DNA. The conditions that cause the
reversal of particle formation may be, but not limited to, the pH,
ionic strength, oxidative or reductive conditions or agents, or
enzymatic activity.
[0049] DNA Template Polymerization
[0050] Low molecular weight cations with valency<+3 fail to
condense DNA in aqueous solutions under normal conditions. However,
cationic molecules with the charge<+3 can be polymerized in the
presence of DNA and the resulting polymers can cause DNA to
condense into compact structures. Such an approach is known in
synthetic polymer chemistry as template polymerization. During this
process, monomers (which are initially weakly associated with the
template) are positioned along template's backbone, thereby
promoting their polymerization. Weak elecrostatic association of
the nascent polymer and the template becomes stronger with chain
growth of the polymer. Trubetskoy et al used two types of
polymerization reactions to achieve DNA condensation: step
polymerization and chain polymerization (V S Trubetskoy, V G
Budker, L J Hanson, P M Slattum, J A Wolff, L E Hagstrom. Nucleic
Acids Res. 26:4178-4185, 1998) U.S. Pat. No. 08/778,657, U.S. Pat.
No. 09/000,692, U.S. Pat. No. 97/24089, U.S. Pat. No. 09/070299,
and U.S. Pat. No. 09/464,871. Bis(2-aminoethyl)-1,3-propanediamine
(AEPD), a tetraamine with 2.5 positive charges per molecule at pH 8
was polymerized in the presence of plasmid DNA using cleavable
disulfide amino-reactive cross-linkers dithiobis (succinimidyl
propionate) and dimethyl-3,3'-dithiobispropionimidate. Both
reactions yielded DNA/polymer complexes with significant
retardation in agarose electrophoresis gels demonstrating
significant binding and DNA condensation. Treatment of the
polymerized complexes with 100 mM dithiothreitol (DTT) resulted in
the pDNA returning to its normal supercoiled position following
electrophoresis proving thus cleavage the backbone of the. The
template dependent polymerization process was also tested using a
14 mer peptide encoding the nuclear localizing signal (NLS) of SV40
T antigen (CGYGPKKKRKVGGC; SEQ ID 1) as a cationic "macromonomer".
Other studies included pegylated comonomer (PEG-AEPD) into the
reaction mixture and resulted in "worm"-like structures (as judged
by transmission electron microscopy) that have previously been
observed with DNA complexes formed from block co-polymers of
polylysine and PEG ( M A Wolfert, E H Schacht, V Toncheva, K
Ulbrich, O Nazarova, L W Seymour Human Gene Ther. 7:2123-2133,
1996). Blessing et al used bisthiol derivative of spermine and
reaction of thiol-disulfide exchange to promote chain growth. The
presence of DNA accelerated the polymerization reaction as measured
the rate of disappearance of free thiols in the reaction mixture (T
Blessing, J S Remy, J P Behr. J. Am. Chem. Soc. 120:8519-8520,
1998).
[0051] "Caging" of polycation-condensed DNA particles.
[0052] The stability of DNA nanoassemblies based on DNA
condensation is generally low in vivo because they easily engage in
polyion exchange reactions with strong polyanions. The process of
exchange consists of two stages: 1) rapid formation of a triple
DNA-polycation-polyanion complex, 2) slow substitution of one
same-charge polyion with another. At equilibrium conditions, the
whole process eventually results in formation of a new binary
complex and an excess of a third polyion. The presence of low
molecular weight salt can greatly accelerate such exchange
reactions, which often result in complete disassembly of condensed
DNA particles. Hence, it is desirable to obtain more colloidally
stable structures where DNA would stay in its condensed form in
complex with corresponding polycation independently of environment
conditions.
[0053] The complete DNA condensation upon neutralization of only
90% of the polymer's phosphates results in the presence of unpaired
positive charges in the DNA particles. If the polycation contains
such reactive groups, such as primary amines, these unpaired
positive charges may be modified. This modification allows
practically limitless possibilities of modulating colloidal
properties of DNA particles via chemical modifications of the
complex. We have demonstrated the utility of such reactions using
traditional DNA-poly-L-lysine (DNA/PLL) system reacted with the
cleavable cross-linking reagent dimethyl-3,3'-dithiobispropionim-
idate (DTBP) which reacts with primary amino groups with formation
of amidines (V S Trubetskoy, A Loomis, P M Slattum, J E Hagstrom, V
G Budker, J A Wolff. Bioconjugate Chem. 10:624-628, 1999) U.S. Pat.
No. 08/778,657, U.S. Pat. No. 09/000,692, U.S. Pat. No. 97/24089,
U.S. Pat. No. 09/070299, and U.S. Pat. No. 09/464,871. Similar
results were achieved with other polycations including
poly(allylamine) and histone Hl. The use of another bifucntional
reagent, glutaraldehyde, has been described for stabilization of
DNA complexes with cationic peptide CWK18 (R C Adam, K G Rice. J.
Pharm. Sci. 739-746, 1999).
[0054] Recharging.
[0055] The caging approach described above could lead to more
colloidally stable DNA assemblies. However, this approach may not
change the particle surface charge. Caging with bifunctional
reagents, which preserve positive charge of amino group, keeps the
particle positive. However, negative surface charge would be more
desirable for many practical applications, i.e. in vivo delivery.
The phenomenon of surface recharging is well known in colloid
chemistry and is described in great detail for lyophobic/lyophilic
systems (for example, silver halide hydrosols). Addition of polyion
to a suspension of latex particles with oppositely-charged surface
leads to the permanent absorption of this polyion on the surface
and, upon reaching appropriate stoichiometry, changing the surface
charge to opposite one. This whole process is salt dependent with
flocculation to occur upon reaching the neutralization point.
[0056] We have demonstrated that similar layering of
polyelectrolytes can be achieved on the surface of DNA/polycation
particles (V S Trubetskoy, A Loomis, J E Hagstrom, V G Budker, J A
Wolff. Nucleic Acids Res. 27:3090-3095, 1999). The principal
DNA-polycation (DNA/pC) complex used in this study was DNA/PLL (1:3
charge ratio) formed in low salt 25 mM HEPES buffer and recharged
with increasing amounts of various polyanions. The DNA particles
were characterized after addition of a third polyion component to a
DNA/polycation complex using a new DNA condensation assay (V S
Trubetskoy, P M Slattum, J E Hagstrom, J A Wolff, V G Budker. Anal.
Biochem. 267:309-313, 1999) and static light scattering. It has
been found that certain polyanions such as poly(methacrylic acid)
and poly(aspartic acid) decondensed DNA in DNA/PLL complexes.
Suprisingly, polyanions of lower charge density such as
succinylated PLL and poly(glutamic acid), even when added in
20-fold charge excess to condensing polycation (PLL) did not
decondense DNA in DNA/PLL (1:3) complexes. Further studies have
found that displacement effects are salt-dependent. In addition,
polyglutamate but not the relatively weaker polyanion succinylated
poly-L-lysine (SPLL) displaces DNA at higher sodium chloride
concentrations. Measurement of z-potential of DNA/PLL particles
during titration with SPLL revealed the change of particle surface
charge at approximately the charge equivalency point. Thus, it can
be concluded that addition of low charge density polyanion to the
cationic DNA/PLL particles results in particle surface charge
reversal while maintaining condensed DNA core intact. Finally,
DNA/polycation complexes can be both recharged and crosslinked or
caged U.S. Pat. No. 08/778,657, U.S. Pat. No. 09/000,692, U.S. Pat.
No. 97/24089, U.S. Pat. No. 09/070299, and U.S. Pat. No.
09/464,871.
[0057] The Use of pH-Sensitive Lipids, Amphipathic Compounds, and
Liposomes for Drug and Nucleic Acid Delivery
[0058] After the landmark description of DOTMA
(N-[1-(2,3-dioleyloxy)propy- l]-N,N,N-trimethylammonium chloride)
[Felgner, P L, Gadek, T R, Holm, M, et al. Lipofection: a highly
efficient, lipid-mediated DNA-transfection procedure. Proc. Natl.
Acad. Sci. USA. 1987;84:7413-7417], a plethora of cationic lipids
have been synthesized. Basically, all the cationic lipids are
amphipathic compounds that contain a hydrophobic domain, a spacer,
and positively-charged amine. The hydrophobic domains are typically
hydrocarbon chains such as fatty acids derived from oleic or
myristic acid. The hydrocarbon chains are often joined either by
ether or ester bonds to a spacer such as glycerol. Quaternary
amines often compose the cationic groups. Usually, the cationic
lipids are mixed with a fusogenic lipid such as DOPE (dioleoyl
phosphatidyl ethanolamine) to form liposomes. The mixtures are
mixed in chloroform that is then dried. Water is added to the dried
lipid film and unilamellar liposomes form during sonication.
Multilamellar cationic liposomes and cationic liposomes/DNA
complexes prepared by the reverse-phase evaporation method have
also been used for transfection. Cationic liposomes have also been
prepared by an ethanol injection technique.
[0059] Several cationic lipids contain a spermine group for binding
to DNA. DOSPA, the cationic lipid within the LipofectAMINE
formulation (Life Technologies) contains a spermine linked via a
amide bond and ethyl group to a trimethyl, quaternary amine
[Hawley-Nelson, P, Ciccarone, V and Jessee, J. Lipofectamine
reagent: A new, higher efficiency polycationic liposome
transfection reagent. Focus 1993;15:73-79]. A French group has
synthesized a series of cationic lipids such as DOGS
(dioctadecylglycinespermine) that contain spermine [Remy, J-S,
Sirlin, C, Vierling, P, et al. Gene transfer with a series of
lipophilic DNA-binding molecules. Bioconjugate Chem.
1994;5:647-654]. DNA has also been transfected by lipophilic
polylysines which contain dipalmotoylsuccinylglycerol
chemically-bonded to low molecular weight (.about.3000 MW)
polylysine [Zhou, X, Kilbanov, A and Huang, L. Lipophilic
polylysines mediate efficient DNA transfection in mammalian cells.
Biochim. Biophys. Acta 1991;1065:8-14. Zhou, X and Huang, L. DNA
transfection mediated by cationic liposomes containing
lipopolylysine: Characterization and mechanism of action. Biochim.
Biophys. Acta 1994;1 195-203].
[0060] Other studies have used adjuvants with the cationic
liposomes. Transfection efficiency into Cos cells was increased
when amphiphilic peptides derived from influenza virus
hemagglutinin were added to DOTMA/DOPE liposomes [Kamata, H,
Yagisawa, H, Takahashi, S, et al. Amphiphilic peptides enhance the
efficiency of liposome-mediated DNA transfection. Nucleic Acids
Res. 1994;22:536-537]. Cationic lipids have been combined with
galactose ligands for targeting to the hepatocyte
asialoglycoprotein receptor [Remy, J-S, Kichler, A, Mordvinov, V,
et al. Targeted gene transfer into hepatoma cells with
lipopolyamine-condensed DNA particles presenting galactose ligands:
A stage toward artificial viruses. Proc. Natl. Acad. Sci. USA
1995;92:1744-1748]. Thiol-reactive phospholipids have also been
incorporated into cationic lipid/pDNA complexes to enable cellular
binding even when the net charge of the complex is not positive
[Kichler, A, Remy, J-S, Boussif, 0, et al. Efficient gene delivery
with neutral complexes of lipospermine and thiol-reactive
phospholipids. Biochem. Biophys. Res. Comm. 1995;209:444-450].
DNA-dependent template process converted thiol-containing detergent
possessing high critical micelle concentration into dimeric
lipid-like molecule with apparently low water solubility (JP Behr
and colleagues).
[0061] Cationic liposomes may deliver DNA either directly across
the plasma membrane or via the endosome compartment. Regardless of
its exact entry point, much of the DNA within cationic liposomes
does accumulate in the endosome compartment. Several approaches
have been investigated to prevent loss of the foreign DNA in the
endosomal compartment by protecting it from hydrolytic digestion
within the endosomes or enabling its escape from endosomes into the
cytoplasm. They include the use of acidotropic (lysomotrophic),
weak amines such as chloroquine that presumably prevent DNA
degradation by inhibiting endosomal acidification [Legendre, J.
& Szoka, F. Delivery of plasmid DNA into mammalian cell lines
using pH-sensitive liposomes: Comparison with cationic liposomes.
Pharmaceut. Res. 9, 1235-1242 (1992)]. Viral fusion peptides or
whole virus have been included to disrupt endosomes or promote
fusion of liposomes with endosomes, and facilitate release of DNA
into the cytoplasm [Kamata, H., Yagisawa, H., Takahashi, S. &
Hirata, H. Amphiphilic peptides enhance the efficiency of
liposome-mediated DNA transfection. Nucleic Acids Res. 22, 536-537
(1994). Wagner, E., Curiel, D. & Cotten, M. Delivery of drugs,
proteins and genes into celis using transferrin as a ligand for
receptor-mediated endocytosis. Advanced Drug Delivery Reviews 14,
113-135 (1994)].
[0062] Knowledge of lipid phases and membrane fusion has been used
to design potentially more versatile liposomes that exploit the
endosomal acidification to promote fusion with endosomal membranes.
Such an approach is best exemplified by anionic, pH-sensitive
liposomes that have been designed to destabilize or fuse with the
endosome membrane at acidic pH [Duzgunes, N., Straubinger, R. M.,
Baldwin, P. A. & Papahadjopoulos, D. PH-sensitive liposomes.
(eds Wilschub, J. & Hoekstra, D.) p. 713-730 (Marcel Deker INC,
1991)]. All of the anionic, pH-sensitive liposomes have utilized
phosphatidylethanolamine (PE) bilayers that are stabilized at
non-acidic pH by the addition of lipids that contain a carboxylic
acid group. Liposomes containing only PE are prone to the inverted
hexagonal phase (H.sub.II). In pH-sensitive, anionic liposomes, the
carboxylic acid's negative charge increases the size of the lipid
head group at pH greater than the carboxylic acid's pK and thereby
stabilizes the phosphatidylethanolamine bilayer. At acidic pH
within endosomes, the uncharged or reduced charge species is unable
to stabilize the phosphatidylethanolamine-rich bilayer. Anionic,
pH-sensitive liposomes have delivered a variety of
membrane-impermeant compounds including DNA. However, the negative
charge of these pH-sensitive liposomes prevents them from
efficiently taking up DNA and interacting with cells; thus
decreasing their utility for transfection. We have described the
use of cationic, pH-sensitive liposomes to mediate the efficient
transfer of DNA into a variety of cells in culture U.S. Pat. No.
08/530,598, and U.S. Pat. No. 09/020,566.
[0063] The Use of pH-Sensitive Polymers for Drug and Nucleic Acid
Delivery
[0064] Polymers that pH-sensitive are have found broad application
in the area of drug delivery exploiting various physiological and
intracellular pH gradients for the purpose of controlled release of
drugs (both low molecular weight and polymeric). pH sensitivity can
be broadly defined as any change in polymer's physico-chemical
properties over certain range of pH. More narrow definition demands
significant changes in the polymer's ability to retain (release) a
bioactive substance (drug) in a physiologically tolerated pH range
(usually pH 5.5-8). pH-sensitivity presumes the presence of
ionizable groups in the polymer (polyion). All polyions can be
divided into three categories based on their ability to donate or
accept protons in aqueous solutions: polyacids, polybases and
polyampholytes. Use of pH-sensitive polyacids in drug delivery
applications usually relies on their ability to become soluble with
the pH increase (acid/salt conversion), to form complex with other
polymers over change of pH or undergo significant change in
hydrophobicity/hydrophilicity balance. Combinations of all three
above factors are also possible.
[0065] Copolymers of polymethacrylic acid (Eudragit S, Rohm
America) are known as polymers which are insoluble at lower pH but
readily solubilized at higher pH, so they are used as enteric
coatings designed to dissolve at higher intestinal pH (Z Hu et al.
J. Drug Target., 7, 223, 1999). A typical example of pH-dependent
complexation is copolymers of polyacrylate(graft)ethyleneglycol
which can be formulated into various pH-sensitive hydrogels which
exhibit pH-dependent swelling and drug release (F Madsen et al.,
Biomaterials, 20, 1701, 1999). Hydrophobically-modified
N-isopropylacrylamide-methacrylic acid copolymer can render regular
egg PC liposomes pH-sensitive by pH-dependent interaction of
grafted aliphatic chains with lipid bilayer (O Meyer et al., FEBS
Lett., 421, 61, 1998). Polymers with pH-mediated hydrophobicity
(like polyethylacrylic acid) can be used as endosomal disruptors
for cytoplasmic drug delivery (Murthy, N., Robichaud, J. R.,
Tirrell, D. A., Stayton, P. S., Hoffman, A. S. J. Controlled
Release 61, 137, 1999).
[0066] Polybases have found broad applications as agents for
nucleic acid delivery in transfection/gene therapy applications due
to the fact they are readily interact with polyacids. A typical
example is polyethylenimine (PEI). This polymer secures nucleic
acid electrostatic adsorption on the cell surface followed by
endocytosis of the whole complex. Cytoplasmic release of the
nucleic acid occurs presumably via the so called "proton sponge"
effect according to which pH-sensitivity of PEI is responsible for
endosome rupture due to osmotic swelling during its acidification
(O Boussif et al. Proc. Natl. Acad. Sci. USA 92, 7297, 1995).
Cationic acrylates possess the similar activity (for example,
poly-((2-dimethylamino)ethyl methacrylate) (P van de Wetering et
al. J. Controlled Release 64, 193, 2000). However, polybases due to
their polycationic nature pH-sensitive polybases have not found
broad in vivo application so far, due to their acute systemic
toxicity in vivo (J H Senior, Biochim. Biophys. Acta, 1070, 173,
1991). Milder polybases (for example, linear PEI) are better
tolerated and can be used systemically for in vivo gene transfer (D
Goula et al. Gene Therapy 5, 712, 1998).
[0067] Endosome Disruption
[0068] Many biologically active compounds, in particular large
and/or charged compounds, are incapable of crossing biological
membranes. In order for these compounds to enter cells they must
either be taken up by the cells via endocytosis, into endosomes, or
there must be a disruption of the cellular membrane to allow the
compound to cross. In the case of endosomal entry, the endosomal
membrane must be disrupted to allow for the entrance of the
compound in the enterior of the cell. Therefore, either entry
pathway into the cell requires a disruption of the cellular
membrane. There exist compounds termed membrane active compounds
that disrupt membranes. One can imagine that if the membrane active
agent were operative in a certain time and place it would
facilitate the transport of the biologically active compound across
the biological membrane. The control of when and where the membrane
active compound is active is crucial to effective transport. If the
membrane active compound is too active or active at the wrong time,
then no transport occurs or transport is associated with cell
rupture and thereby cell death. Nature has evolved various
strategies to allow for membrane transport of biologically active
compounds including membrane fusion and the use membrane active
compounds whose activity is modulated such that activity assists
transport without toxicity. Many lipid-based transport formulations
rely on membrane fusion and some membrane active peptides'
activities are modulated by pH. In particular, viral coat proteins
are often pH-sensitive, inactive at neutral or basic pH and active
under the acidic conditions found in the endosome.
[0069] Small Molecular Endosomolytic Agents
[0070] A cellular transport step that has attracted attention for
gene transfer is that of 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. A number of chemicals such as
chloroquine, bafilomycin or Brefeldin Al have been used to disrupt
or modify the trafficking of molecules through intracellular
pathways. 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.l, a macrolide antibiotic is a
more specific inhibitor of endosomal acidification and vacuolar
type H.sup.+-ATPase than chloroquine.
[0071] Viruses, Proteins and Peptides for Disruption of Endosomes
and Endosomal Function
[0072] Viruses such as adenovirus have been used to induce gene
release from endosomes or other intracellular compartments (D.
Curiel, Agarwal, S., Wagner, E., and Cotten, M. PNAS 88:8850,
1991). Rhinovirus has also been used for this purpose (W. Zauner et
al. J. Virology 69:1085-92, 1995). Viral components such as
influenza virus hemagglutinin subunit HA-2 analogs has also been
used to induce endosomal release (E. Wagner et al. PNAS 89:7934,
1992). Amphipathic peptides resembling the N-terminal HA-2 sequence
has been studied (K. Mechtler and E. Wagner, New J. Chem.
21:105-111, 1997). Parts of the pseudonmonas exotoxin and diptheria
toxin have also been used for drug delivery (I. Pastan and D.
FitzGerald. J. Biol. Chem. 264:15157, 1989).
[0073] A variety of synthetic amphipathic peptides have been used
to enhance transfection of genes (N. Ohmori et al. Biochem.
Biophys. Res. Commun. 235:726, 1997). The ER-retaining signal (KDEL
sequence) has been proposed to enhance delivery to the endoplasmic
reticulum and prevent delivery to lysosomes (S. Seetharam et al. J.
Biol. Chem. 266:17376, 1991).
[0074] Other Cellular and Intracellular Gradients Useful for
Delivery
[0075] Nucleic acid and gene delivery may involve the biological pH
gradient that is active within organisms as a factor in delivering
a polynucleotide to a cell. Different pathways that may be affected
by the pH gradient include cellular transport mechanisms, endosomal
disruption/breakdown, and particle disassembly (release of the
DNA). Other gradients that can be useful in gene therapy research
involve ionic gradients that are related to cells. For example,
both Na.sup.+and K.sup.+have large concentration gradients that
exist across the cell membrane. Systems containing metal-binding
groups can utilize such gradients to influence delivery of a
polynucleotide to a cell. Changes in the osmotic pressure in the
endosome also have been used to disrupt membranes and allow for
transport across membrane layer. Buffering of the endosome pH may
cause these changes in osmotic pressure. For example, the "proton
sponge" effect of PEI (O Boussif et al. Proc. Natl. Acad. Sci. USA
92, 7297, 1995) and certain polyanions (Murthy, N., Robichaud, J.
R., Tirrell, D. A., Stayton, P. S., Hoffman, A. S. Journal of
Controlled Release 1999, 61, 137) are postulated to cause an
increase in the ionic strength inside of the endosome, which causes
a increase in osmotic pressure. This pressure increase results in
membrane disruption and release of the contents of the
endosome.
[0076] In addition to pH and other ionic gradients, there exist
other difference in the chemical environment associated with
cellular activities that may be used in gene delivery. In
particular enzymatic activity both extra and intracellularly may be
used to deliver the gene of interest either by aiding in the
delivery to the cell or escape from intracellular compartments.
Proteases, found in serum, lysosome and cytoplasm, may be used to
disrupt the particle and allow its interaction with the cell
surface or cause it fracture the intracellular compartment, e.g.
endosome or lysosome, allowing the gene to be released
intracellularly.
SUMMARY OF THE INVENTION
[0077] The invention relates to noncovalent amphiphile binding
systems for use in biologic systems. More particularly,
amphiphile-binding agents and polymers of amphiphile-binding agents
are utilized in the delivery of molecules, polymers, nucleic acids
and genes to cells.
[0078] Described in a preferred embodiment is a process for
obtaining an expression product by delivering a polynucleotide to a
cell, comprising the step of associating an amphiphile binding
agent, an amphiphile, and a polynucleotide to form a complex. Then,
delivering the complex to the cell and expressing the
polynucleotide in the cell.
[0079] In another preferred embodiment, a complex is described for
delivering and expressing DNA in a mammal, comprising an amphiphile
binding agent, an amphiphile, and DNA in complex.
[0080] Another preferred embodiment is a process for obtaining an
expression product in vivo, comprising forming a complex with a
cyclodextrin, an amphiphile and a polynucleotide. Then, delivering
the complex to a cell in a mammal which expresses the
polynucleotide.
DETAILED DESCRIPTION OF THE INVENTION
[0081] The following description provides exemplary embodiments of
the systems, compositions, and methods of the present invention.
These embodiments include a variety of systems that have been
demonstrated as effective delivery systems. The invention is not
limited to these particular embodiments.
[0082] Cyclodextrin Structure and Binding Properties
[0083] Cyclodextrins are naturally occurring cyclic oligomers of
glucose in 1-4 .alpha. linkages (structure 1
[0084] Cyclodextrin composed of six glucose units (N=6) is called
.alpha.-cyclodextrin, 7 units is called .beta.-cyclodextrin, and 8
is called .gamma.-cyclodextrin. The cyclic structure is torroidal
in shape with the center of the torroid relatively nonpolar
compared to water. For this reason, cyclodextrins will bind to
nonpolar sections of amphipathic compounds, also known as
amphiphilic compounds or amphiphiles, in water. Amphiphiles are
compounds that contain both hydrophilic and hydrophobic functional
groups. Examples include lipids, acyl-glycerol, sterols,
polyethyleneglycol, and amino acids. Hydrophilic groups indicate in
qualitative terms that the chemical moiety is water-preferring.
Typically, such chemical groups are water soluble, and are hydrogen
bond donors or acceptors with water. Examples of hydrophilic groups
include compounds with the following chemical moieties;
carbohydrates, polyoxyethylene, peptides, oligonucleotides and
groups containing amines, amides, alkoxy amides, carboxylic acids,
sulfurs, or hydroxyls. Hydrophobic groups indicate in qualitative
terms that the chemical moiety is water-avoiding. Typically, such
chemical groups are not water soluble, and tend not to hydrogen
bonds. Hydrocarbons are hydrophobic groups. Amphipathic compounds
bound by cyclodextrins include hydrophobic amino acids (e.g.
leucine and phenylalanine), surfactants (e.g. sodium dodecylsulfate
and Triton X-100), and lipids (e.g.palmitic acid). The strength of
the interaction between cyclodextrin and an amphiphilic compound
depends on the size of both the hydrophobic part of the amphiphile
and the cyclodextrin. For example, a-cyclodextrin will bind linear
alkyl chains, but not branched tertiary alkyl groups, which are
bound by .beta.-cyclodextrin (Stella, V. J., Rajewski, R. A. Pharm.
Res. 1997, 14, 556. Stella, V. J., Rao, V. M., Zannov, E. A., Zia,
V. Adv. Drug Del. Rev. 1999, 36, 3.).
[0085] Nucleic Acid Delivery by Polycations and Cationic Lipids
[0086] There are many nonviral nucleic acid complexes that have
been shown to aid in delivery of DNA into cells. Nucleic acid
includes DNA (plasmid DNA, antisense, oligonucleotides) and RNA
(ribozymes, oligonucleotides, artificial messenger RNA). In
general, these nonviral complexes may be grouped into two classes:
cationic lipid complexes (lipoplexes) and cationic polymer
(polyplexes) complexes. In either case, the polyanionic DNA is
complexed with a cation. In lipoplexes, the cations are associated
noncovalently by hydrophobic lipid-lipid interactions to form a
polycation. In polymer complexes, the positive charges are attached
covalently to form a polycation. Nucleic acids are delivered to
cells for the purpose of gene therapy and antisense therapy.
[0087] Nucleic Acids Complexes Containing Cyclodextrins
[0088] As mentioned previously, cyclodextrins form complexes with
amphipathic molecules that may be positively or negatively charged.
Therefore, a polymer composed of cyclodextrins will become a
polyion, a noncovalent amphiphilic electrolyte, when associated
with a charged amphiphile. For example, association between a
polymer composed of cyclodextrins and a cationic amphiphile will
result in a polycation that may interact with DNA. In a preferred
embodiment, a cyclodextrin-containing polymers are constructed by
reacting cyclodextrin with epichlorohydrin under alkaline
conditions to produce cyclodextrin-epichlorohydrin copolymer. This
cyclodextrin-epichlorohydrin copolymer, compacts pDNA upon addition
of cations such as 1-adamantanamine or 1-dodecylamine. The complex
of cyclodextrin-epichlorohydrin copolymer and 1-adamantanamine or
1-dodecylamine is a cationic noncovalent amphiphilic
polyelectrolyte, which is capable of condensing DNA. In addition,
cationic amphiphiles that are polymers that are bound to monomeric
or polymeric amphiphile binding agents may be used to compact DNA.
Such DNA-containing complexes may be used for transfection of
cells.
[0089] Amphiphile binding agents may also be used to create anionic
noncovalent amphiphilic polyelectrolytes. Association between a
polymer composed of cyclodextrins and an anionic amphiphile will
result in a polyanion that will interact with a positively-charged
DNA-polycation complex, i.e. "recharge" the DNA complex. In a
preferred embodiment, the complex between
cyclodextrin-epichlorohydrin copolymer and 4-t-butylbenzoic acid,
to form an anionic noncovalent amphiphilic polyelectrolyte, was
added to particles of DNA and poly-L-lysine . The resulting
particles were found to transfect cells in vitro. In addition,
anionic amphiphiles that are polymers that are bound to monomeric
or polymeric amphiphile binding agents may be used to "recharge"
DNA particles. For example, succinyloleoylpoly-L-lysine is an
anionic polymeric amphiphile which complexes with the amphiphile
binding agent .beta.-cyclodextrin and interacts ("recharges") a
poly-L-lysine condensed DNA particle. The addition of the
cyclodextrin increased the transfection of the recharged particle
33 fold over recharged particle in the absence of cyclodextrin.
[0090] Not only is the cyclodextrin the basis for the DNA-polyion
interaction, but cyclodextrin-based polyions may have properties
(e.g. surface charge and stability) different from standard
polyions. In contrast to standard polyions, the polyions derived
from cyclodextrin-containing polymers and charged amphiphiles are
reversible. The existence of the polyion is dependent upon the
concentration of the cyclodextrin-containing polymer and the
charged amphiphile, such that the disruption of the polyion maybe
trigger by simple dilution of either cyclodextrin or charged
amphiphile.
[0091] Monomeric cyclodextrins may also be incorporated into
nucleic acid complexes by association with amphiphile molecules in
a DNA complex. In this case, the cyclodextrins are not the basis
for the DNA-electrolyte interactions, but may be used to change the
properties of the DNA-electrolyte complex, e.g. stability or
surface charge. The addition of cyclodextrin into a DNA particle
also adds hydrophilic, but not charged, moieties to the particle.
Hydrophilic molecules (e.g. PEG) have been shown to increase
solubility of DNA particles, decrease the surface charge and
thereby increase their stability. Cyclodextrins have the ability to
bind to other nonionic hydrophilic molecules such as PEG.
Therefore, addition of PEG to a cyclodextrin-containing DNA
particle will result in PEG-particle interactions, which may confer
the particle with added stability. Unlike other examples of PEG
stabilization of DNA particles, the interaction between DNA
particle and PEG is transient and may release under dilute,
delivery conditions. The rate at which the PEG may be released may
be altered by the number of PEG molecules incorporated, the number
of cyclodextrins, and the incorporation of PEG derivatives with
strong cyclodextrin binding regions (e.g. t-octylphenyl group of
Triton X-100). In a preferred embodiment, addition of the
PEG-derived detergent Triton X- 100 to particles of DNA and
poly-L-lysine-succinyl-.beta.-cyclodextrin resulted in particles
that were more stable than particles without addition of the Triton
X-100.
[0092] Likewise, cell targeting ligands aid in transport to a cell
but may not be necessary, and may inhibit, transport into a cell.
In all of these cases, the reversible attachment of the interaction
modifier, through a labile bond, would be beneficial.
[0093] The present invention provides for the transfer of
polynucleotides, and other biologically active compounds into cells
in culture (also known as "in vitro"). Compounds or kits for the
transfection of cells in culture is commonly sold as "transfection
reagents" or "transfection kits". The present invention also
provides for the transfer of polynucleotides, and biologically
active compounds into cells within tissues in situ and in vivo, and
delivered intravasculary (U.S. patent application Ser. No.
08/571,536), intrarterially, intravenous, orally, intraduodenaly,
via the jejunum (or ileum or colon), rectally, transdermally,
subcutaneously, intramuscularly, intraperitoneally,
intraparenterally, via direct injections into tissues such as the
liver, lung, heart, muscle, spleen, pancreas, brain (including
intraventricular), spinal cord, ganglion, lymph nodes, lymphatic
system, adipose tissues, thryoid tissue, adrenal glands, kidneys,
prostate, blood cells, bone marrow cells, cancer cells, tumors, eye
retina, via the bile duct, or via mucosal membranes such as in the
mouth, nose, throat, vagina or rectum or into ducts of the salivary
or other exocrine glands. Compounds for the transfection of cells
in vivo in a whole organism can be sold as "in vivo transfection
reagents" or "in vivo transfection kits" or as a pharmaceutical for
gene therapy.
[0094] Definitions
[0095] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below:
[0096] Amphiphile Binding Agent
[0097] Amphiphile binding agents are compounds with molecular
weight 1,300 or less that bind through a noncovalent interaction
amphiphilic compounds in water. The basis for this interaction is
contact between hydrophobic portions of the amphiphile with
hydrophobic portions of the amphiphile binding agent. In particular
.alpha., .beta. and .gamma.-cyclodextrins, and their derivatives,
are amphiphile binding agents.
[0098] Polymeric Amphiphile Binding Agent
[0099] Polyermic amphiphile binding agent is a polymer composed of
monomers that are amphiphile binding agents.
[0100] Noncovalent Amphiphilic Electrolytes
[0101] Noncovalent amphiphilic polyelectrolytes are systems
composed of amphiphile binding agents and charged amphiphiles,
which are bound by the amphiphile binding agents. The interaction
between charged amphiphile and polymer results in a complex that
has a different charge than the amphiphile binding agent alone. The
amphiphile binding agent may be uncharged, charge positive or
neutral, but upon interaction with a charged amphiphile the charge
of the complex is different than the amphiphile binding agent
alone.
[0102] Biologically Active Compound
[0103] A biologically active compound is a compound having the
potential to react with biological components. More particularly,
biologically active compounds utilized in this specification are
designed to change the natural processes associated with a living
cell. For purposes of this specification, a cellular natural
process is a process that is associated with a cell before delivery
of a biologically active compound. In this specification, the
cellular production of, or inhibition of a material, such as a
protein, caused by a human assisting a molecule to an in vivo cell
is an example of a delivered biologically active compound.
Pharmaceuticals, proteins, peptides, polypeptides, enzyme
inhibitors, hormones, cytokines, antigens, viruses,
oligonucleotides, enzymes and nucleic acids are examples of
biologically active compounds.
[0104] Peptide and Polypeptide
[0105] Peptide and polypeptide refer to a series of amino acid
residues, more than two, connected to one another by amide bonds
between the beta or alpha-amino group and carboxyl group of
contiguous amino acid residues. The amino acids may be naturally
occurring or synthetic. Polypeptide includes proteins and peptides,
modified proteins and peptides, and non-natural proteins and
peptides. Enzymes are proteins evolved by the cells of living
organisms for the specific function of catalyzing chemical
reactions. A chemical reaction is defined as the formation or
cleavage of covalent or ionic bonds. Bioactive compounds may be
used interchangeably with biologically active compound for purposes
of this application.
[0106] Cyclodextrin
[0107] A cyclic oligomer of alpha-D-glucopyranose.
[0108] Delivery of Biologically active compound
[0109] The delivery of a biologically active compound is commonly
known as "drug delivery". "Delivered" means that the biologically
active compound becomes associated with the cell or organism. The
compound can be in the circulatory system, intravessel,
extracellular, on the membrane of the cell or inside the cytoplasm,
nucleus, or other organelle of the cell.
[0110] Parenteral routes of administration include intravascular
(intravenous, intraarterial), intramuscular, intraparenchymal,
intradermal, subdermal, subcutaneous, intratumor, intraperitoneal,
intrathecal, subdural, epidural, and intralymphatic injections that
use a syringe and a needle or catheter. An intravascular route of
administration enables a polymer or polynucleotide to be delivered
to cells more evenly distributed and more efficiently expressed
than direct injections. Intravascular herein means within a tubular
structure called a vessel that is connected to a tissue or organ
within the body. Within the cavity of the tubular structure, a
bodily fluid flows to or from the body part. Examples of bodily
fluid include blood, cerebrospinal fluid (CSF), 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. An administration route involving the
mucosal membranes is meant to include nasal, bronchial, inhalation
into the lungs, or via the eyes. Other routes of administration
include intraparenchymal into tissues such as muscle
(intramuscular), liver, brain, and kidney. Transdermal routes of
administration have been effected by patches and ionotophoresis.
Other epithelial routes include oral, nasal, respiratory, and
vaginal routes of administration.
[0111] Delivery System
[0112] Delivery system is the means by which a biologically active
compound becomes delivered. That is all compounds, including the
biologically active compound itself, that are required for delivery
and all procedures required for delivery including the form (such
volume and phase (solid, liquid, or gas)) and method of
administration (such as but not limited to oral or subcutaneous
methods of delivery).
[0113] Nucleic Acid
[0114] The term "nucleic acid" is a term of art that refers to a
polymer containing at least two nucleotides. "Nucleotides" contain
a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate
group. Nucleotides are linked together through the phosphate
groups. "Bases" include purines and pyrimidines, which further
include natural compounds adenine, thymine, guanine, cytosine,
uracil, inosine, and natural analogs, and synthetic derivatives of
purines and pyrimidines, which include, but are not limited to,
modifications which place new reactive groups such as, but not
limited to, amines, alcohols, thiols, carboxylates, and
alkylhalides. Nucleotides are the monomeric units of nucleic acid
polymers. A "polynucleotide" is distinguished here from an
"oligonucleotide" by containing more than 80 monomeric units;
oligonucleotides contain from 2 to 80 nucleotides. The term nuclei
acid includes deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA). The term encompasses sequences that include any of the known
base analogs of DNA and RNA including, but not limited to,
4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,
pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil,
5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil- , dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
[0115] DNA may be in the form of anti-sense, plasmid DNA, parts of
a plasmid DNA, product of a polymerase chain reaction (PCR),
vectors (PI, PAC, BAC, YAC, artificial chromosomes), expression
cassettes, chimeric sequences, chromosomal DNA, or derivatives of
these groups. RNA may be in the form of oligonucleotide RNA, tRNA
(transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA),
MRNA (messenger RNA), anti-sense RNA, ribozymes, chimeric
sequences, or derivatives of these groups.
[0116] "Anti-sense" is a polynucleotide that interferes with the
function of DNA and/or RNA. This may result in suppression of
expression. Natural nucleic acids have a phosphate backbone,
artificial nucleic acids may contain other types of backbones and
bases. These include PNAs (peptide nucleic acids),
phosphothionates, and other variants of the phosphate backbone of
native nucleic acids. In addition, DNA and RNA may be single,
double, triple, or quadruple stranded.
[0117] The term "recombinant DNA molecule" as used herein refers to
a DNA molecule that is comprised of segments of DNA joined together
by means of molecular biological techniques. "Expression cassette"
refers to a natural or recombinantly produced polynucleotide
molecule that is capable of expressing protein(s). A DNA expression
cassette typically includes a promoter (allowing transcription
initiation), and a sequence encoding one or more proteins.
Optionally, the expression cassette may include trancriptional
enhancers, non-coding sequences, splicing signals, transcription
termination signals, and polyadenylation signals. An RNA expression
cassette typically includes a translation initiation codon
(allowing translation initiation), and a sequence encoding one or
more proteins. Optionally, the expression cassette may include
translation termination signals, a polyadenosine sequence, internal
ribosome entry sites (IRES), and non-coding sequences.
[0118] A nucleic acid can be used to modify the genomic or
extrachromosomal DNA sequences. This can be achieved by delivering
a nucleic acid that is expressed. Alternatively, the nucleic acid
can effect a change in the DNA or RNA sequence of the target cell.
This can be achieved by homologous recombination, gene conversion,
or other, yet to be described, mechanisms.
[0119] Gene
[0120] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide or precursor (e.g., --myosin heavy
chain). The polypeptide can be encoded by a full length coding
sequence or by any portion of the coding sequence so long as the
desired activity or functional properties (e.g., enzymatic
activity, ligand binding, signal transduction, etc.) of the
full-length or fragment are retained. The term also encompasses the
coding region of a structural gene and the including sequences
located adjacent to the coding region on both the 5' and 3' ends
for a distance of about 1 kb or more on either end such that the
gene corresponds to the length of the full-length mRNA. The
sequences that are located 5' of the coding region and which are
present on the mRNA are referred to as 5' non-translated sequences.
The sequences that are located 3' or downstream of the coding
region and which are present on the mRNA are referred to as 3'
non-translated sequences. The term "gene" encompasses both cDNA and
genomic forms of a gene. A genomic form or clone of a gene contains
the coding region interrupted with non-coding sequences termed
"introns" or "intervening regions" or "intervening sequences."
Introns are segments of a gene which are transcribed into nuclear
RNA (hnRNA); introns may contain regulatory elements such as
enhancers. Introns are removed or "spliced out" from the nuclear or
primary transcript; introns therefore are absent in the messenger
RNA (mRNA) transcript. The mRNA functions during translation to
specify the sequence or order of amino acids in a nascent
polypeptide.
[0121] As used herein, the terms "nucleic acid molecule encoding,"
"DNA sequence encoding," and "DNA encoding" refer to the order or
sequence of deoxyribonucleotides along a strand of deoxyribonucleic
acid. The order of these deoxyribonucleotides determines the order
of amino acids along the polypeptide (protein) chain. The DNA
sequence thus codes for the amino acid sequence.
[0122] As used herein, the terms "an oligonucleotide having a
nucleotide sequence encoding a gene" and "polynucleotide having a
nucleotide sequence encoding a gene," means a nucleic acid sequence
comprising the coding region of a gene or in other words the
nucleic acid sequence which encodes a gene product. The coding
region may be present in either a cDNA, genomic DNA or RNA form.
When present in a DNA form, the oligonucleotide or polynucleotide
may be single-stranded (i.e., the sense strand) or double-stranded.
Suitable control elements such as enhancers/promoters, splice
junctions, polyadenylation signals, etc. may be placed in close
proximity to the coding region of the gene if needed to permit
proper initiation of transcription and/or correct processing of the
primary RNA transcript. Alternatively, the coding region utilized
in the expression vectors of the present invention may contain
endogenous enhancers/promoters, splice junctions, intervening
sequences, polyadenylation signals, etc. or a combination of both
endogenous and exogenous control elements.
[0123] The term "isolated" when used in relation to a nucleic acid,
as in "an isolated oligonucleotide" or "isolated polynucleotide"
refers to a nucleic acid sequence that is identified and separated
from at least one contaminant nucleic acid with which it is
ordinarily associated in its natural source. Isolated nucleic acid
is such present in a form or setting that is different from that in
which it is found in nature. In contrast, non-isolated nucleic
acids as nucleic acids such as DNA and RNA found in the state they
exist in nature. For example, a given DNA sequence (e.g., a gene)
is found on the host cell chromosome in proximity to neighboring
genes; RNA sequences, such as a specific mRNA sequence encoding a
specific protein, are found in the cell as a mixture with numerous
other mRNAs that encode a multitude of proteins. However, isolated
nucleic acid encoding a given protein includes, by way of example,
such nucleic acid in cells ordinarily expressing the given protein
where the nucleic acid is in a chromosomal location different from
that of natural cells, or is otherwise flanked by a different
nucleic acid sequence than that found in nature. The isolated
nucleic acid, oligonucleotide, or polynucleotide may be present in
single-stranded or double-stranded form. When an isolated nucleic
acid, oligonucleotide or polynucleotide is to be utilized to
express a protein, the oligonucleotide or polynucleotide will
contain at a minimum the sense or coding strand (i.e., the
oligonucleotide or polynucleotide may be single-stranded), but may
contain both the sense and anti-sense strands (i.e., the
oligonucleotide or polynucleotide may be double-stranded).
[0124] Gene Expression
[0125] As used herein, the term "gene expression" refers to the
process of converting genetic information encoded in a gene into
RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of
the gene (i.e., via the enzymatic action of an RNA polymerase), and
for protein encoding genes, into protein through "translation" of
mRNA. Gene expression can be regulated at many stages in the
process. "Up-regulation" or "activation" refers to regulation that
increases the production of gene expression products (i.e., RNA or
protein), while "down-regulation" or "repression" refers to
regulation that decrease production. Molecules (e.g., transcription
factors) that are involved in up-regulation or down-regulation are
often called "activators" and "repressors," respectively.
[0126] Delivery of Nucleic Acids
[0127] The process of delivering a polynucleotide to a cell has
been commonly termed "transfection" or the process of
"transfecting" and also it has been termed "transformation". The
polynucleotide could be used to produce a change in a cell that can
be therapeutic. The delivery of polynucleotides or genetic material
for therapeutic and research purposes is commonly called "gene
therapy". The delivery of nucleic acid can lead to modification of
the DNA sequence of the target cell.
[0128] The polynucleotides or genetic material being delivered are
generally mixed with transfection reagents prior to delivery. The
term "transfection" as used herein refers to the introduction of
foreign DNA into eukaryotic cells. Transfection may be accomplished
by a variety of means known to the art including calcium
phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection,
polybrene-mediated transfection, electroporation, microinjection,
liposome fusion, lipofection, protoplast fusion, retroviral
infection, and biolistics.
[0129] The term "stable transfection" or "stably transfected"
refers to the introduction and integration of foreign DNA into the
genome of the transfected cell. The term "stable transfectant"
refers to a cell which has stably integrated foreign DNA into the
genomic DNA.
[0130] The term "transient transfection" or "transiently
transfected" refers to the introduction of foreign DNA into a cell
where the foreign DNA fails to integrate into the genome of the
transfected cell. The foreign DNA persists in the nucleus of the
transfected cell for several days. During this time the foreign DNA
is subject to the regulatory controls that govern the expression of
endogenous genes in the chromosomes. The term "transient
transfectant" refers to cells which have taken up foreign DNA but
have failed to integrate this DNA. The term "naked polynucleotides"
indicates that the polynucleotides are not associated with a
transfection reagent or other delivery vehicle that is required for
the polynucleotide to be delivered to a cell.
[0131] A "transfection reagent" or "delivery vehicle" is a compound
or compounds that bind(s) to or complex(es) with oligonucleotides,
polynucleotides, or other desired compounds and mediates their
entry into cells. Examples of transfection reagents include, but
are not limited to, cationic liposomes and lipids, polyamines,
calcium phosphate precipitates, histone proteins, polyethylenimine,
and polylysine complexes (polyethylenimine and polylysine are both
toxic). Typically, when used for the delivery of nucleic acids, the
transfection reagent has a net positive charge that binds to the
polynucleotide's negative charge. For example, cationic liposomes
or polylysine complexes have net positive charges that enable them
to bind to DNA or RNA.
[0132] Enzyme
[0133] Enzyme is a protein that acts as a catalyst. That is a
protein that increases the rate of a chemical reaction without
itself undergoing any permanent chemical change. The chemical
reactions that are catalyzed by an enzyme are termed enzymatic
reactions and chemical reactions that are not are termed
nonenzymatic reactions.
[0134] Complex
[0135] Two molecules are combined, to form a complex through a
process called complexation or complex formation, if the are in
contact with one another through noncovalent interactions such as
electrostatic interactions, hydrogen bonding interactions, and
hydrophobic interactions.
[0136] Modification
[0137] A molecule is modified, to form a modification through a
process called modification, by a second molecule if the two become
bonded through a covalent bond. That is, the two molecules form a
covalent bond between an atom form one molecule and an atom from
the second molecule resulting in the formation of a new single
molecule. A chemical covalent bond is an interaction, bond, between
two atoms in which there is a sharing of electron density.
[0138] Osmosis
[0139] Osmosis is the passage of a solvent through a semipermeable
membrane, a membrane through which solvent can pass but not all
solutes, separating two solutions of different concentrations.
There is a tendency for the separated solutions to become the same
concentration as the solvent passes from low concentration to high
concentration. Osmosis will stop when the two solutions become
equal in concentration or when pressure is applied to the solution
containing higher concentration. When the higher concentrated
solution is in a closed system, that is when system is of constant
volume, there is a build up of pressure as the solvent passes from
low to high concentration. This build up of pressure is called
osmotic pressure.
[0140] Salt
[0141] A salt is any compound containing ionic bonds, that is bonds
in which one or more electrons are transferred completely from one
atom to another.
[0142] Interpolyelectrolyte Complexes
[0143] An interpolyelectrolyte complexe is a noncovalent
interaction between polyelectrolytes of opposite charge.
[0144] Charge, Polarity, and Sign
[0145] The charge, polarity, or sign of a compound refers to
whether or not a compound has lost one or more electrons (positive
charge, polarity, or sign) or gained one or more electrons
(negative charge, polarity, or sign).
[0146] Cell Targeting Signals
[0147] Cell targeting signal (or abbreviated as the Signal) is
defined in this specification as a molecule that modifies a
biologically active compounds such as drug or nucleic acid and can
direct it to a cell location (such as tissue) 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
function of the biologically active compound can be enhanced.
[0148] The cell targeting signal can be a protein, peptide, lipid,
steroid, sugar, carbohydrate, (non-expresssing) polynucleic acid or
synthetic compound. The cell targeting signal enhances cellular
binding to receptors, cytoplasmic transport to the nucleus and
nuclear entry or release from endosomes or other intracellular
vesicles.
[0149] Nuclear localizing signals enhance the targeting of the
pharmaceutical 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. For example, karyopherin
beta itself could target the DNA to the nuclear pore complex.
Several peptides have been derived from the SV40 T antigen. These
include a short NLS (H-CGYGPKKKRKVGG-OH; SEQ ID 2) or long NLS's
(H-CKKKSSSDDEATADSQHSTPPKKKRKVEDPKDFPSELLS-OH; SEQ ID 3 and
H-CKKKWDDEATADSQHSTPPKKKRKVEDPKDFPSELLS-OH; SEQ ID 4). Other NLS
peptides have been derived from M9 protein
(CYNDFGNYNNQSSNFGPMKQGNF-GGRSSGPY; SEQ ID 5), E1A (H-CKRGPKRPRP-OH;
SEQ ID 6), nucleoplasmin (H-CKKAVKRPAATKKAGQAKKKKL-OH SEQ ID 7),and
c-myc (H-CKKKGPAAKRVKLD-OH; SEQ ID 8).
[0150] 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.
[0151] Cellular receptor signals are any signal that enhances the
association of the biologically active compound with a cell. This
can be accomplished by either increasing the binding of the
compound 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
asiologlycoproteins 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 thiol, 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.
[0152] Interaction Modifiers
[0153] An interaction modifier changes the way that a molecule
interacts with itself or other molecules, relative to molecule
containing no interaction modifier. The result of this modification
is that self-interactions or interactions with other molecules are
either increased or decreased. For example cell targeting signals
are interaction modifiers that change the interaction between a
molecule and a cell or cellular component. Polyethylene glycol is
an interaction modifier that decreases interactions between
molecules and themselves and with other molecules.
[0154] Reporter or Marker Molecules
[0155] Reporter or marker molecules are compounds that can be
easily detected. Typically they are fluorescent compounds such as
fluorescein, rhodamine, Texas red, cy 5, cy 3 or dansyl compounds.
They can be molecules that can be detected by infrared, ultraviolet
or visible spectroscopy or by antibody interactions or by electron
spin resonance. Biotin is another reporter molecule that can be
detected by labeled avidin. Biotin could also be used to attach
targeting groups.
[0156] Linkages
[0157] An attachment that provides a covalent bond or spacer
between two other groups (chemical moieties). The linkage may be
electronically neutral, or may bear a positive or negative charge.
The chemical moieties can be hydrophilic or hydrophobic. Preferred
spacer groups include, but are not limited to C1-C12 alkyl, C1-C12
alkenyl, C1-C12 alkynyl, C6-C18 aralkyl, C6-C 18 aralkenyl, C6-C 18
aralkynyl, ester, ether, ketone, alcohol, polyol, amide, amine,
polyglycol, polyether, polyamine, thiol, thio ether, thioester,
phosphorous containing, and heterocyclic.
[0158] Bifunctional
[0159] Bifunctional molecules, commonly referred to as
crosslinkers, are used to connect two molecules together, i.e. form
a linkage between two molecules. Bifunctional molecules can contain
homo or heterobifunctionality.
[0160] Crosslinking
[0161] Crosslinking refers to the chemical attachment of two or
more molecules with a bifunctional reagent. A bifunctional reagent
is a molecule with two reactive ends. The reactive ends can be
identical as in a homobifunctional molecule, or different as in a
heterobifucnctional molecule.
[0162] Amphiphilic and Amphipathic Compounds
[0163] Amphipathic, or amphiphilic, compounds have both hydrophilic
(water-soluble) and hydrophobic (water-insoluble) parts.
Hydrophilic groups indicate in qualitative terms that the chemical
moiety is water-preferring. Typically, such chemical groups are
water soluble, and are hydrogen bond donors or acceptors with
water. Examples of hydrophilic groups include compounds with the
following chemical moieties; carbohydrates, polyoxyethylene,
peptides, oligonucleotides and groups containing amines, amides,
alkoxy amides, carboxylic acids, sulfurs, or hydroxyls. Hydrophobic
groups indicate in qualitative terms that the chemical moiety is
water-avoiding. Typically, such chemical groups are not water
soluble, and tend not to hydrogen bonds. Hydrocarbons are
hydrophobic groups.
[0164] Detergent
[0165] Detergents or surfactants are water-soluble molecules
containing a hydrophobic portion (tail) and a hydrophilic portion
(head), which upon addition to water decrease water's surface
tension. The hydrophobic portion can be alkyl, alkenyl, alkynyl or
aromatic. The hydrophilic portion can be charged with either net
positive (cationic detergents), negative (anionic detergents),
uncharged (nonionic detergents), or charge neutral (zwitterionic
detergent). Examples of anionic detergents are sodium dodecyl
sulfate, glycolic acid ethoxylate(4 units) 4-tert-butylphenylether,
palmitic acid, and oleic acid. Examples of cationic detergents are
cetyltrimethylammonium bromide and oleylamine. Examples of nonionic
detergents include, laurylmaltoside, Triton X-100, and Tween.
Examples of zwitterionic detergents include
3-[(3-cholamidopropyl)dimthylammonio] 1-propane-sulfonate (CHAPS),
and N-tetradecyl-N,N-dimethyl-3-ammoniu-1-propanesulfonate.
[0166] Surface Tension
[0167] The surface tension of a liquid is the force acting over the
surface of the liquid per unit length of surface that is
perpendicular to the force that is acting of the surface. Surface
charge has the units force per length, e.g. Newtons/meter.
[0168] Membrane Active Compound
[0169] Membrane active agents or compounds are compounds (typically
a polymer, peptide or protein) that are able alter the membrane
structure. This change in structure can be shown by the compound
inducing one or more of the following effects upon a membrane: an
alteration that allows small molecule permeability, pore formation
in the membrane, a fusion and/or fission of membranes, an
alteration that allows large molecule permeability, or a dissolving
of the membrane. This alteration can be functionally defined by the
compound's activity in at least one the following assays: red blood
cell lysis (hemolysis), liposome leakage, liposome fusion, cell
fusion, cell lysis and endosomal release. An example of a membrane
active agent in our examples is the peptide melittin, whose
membrane activity is demonstrated by its ability to release heme
from red blood cells (hemolysis). In addition,
dimethylmaleamic-modified mellitin (DM-Mel) reverts to melittin in
the acidic environment of the endosome causes endosomal release as
seen by the diffuse staining of fluorescein-labled dextran in our
endosomal release assay.
[0170] More specifically membrane active compounds allow for the
transport of molecules with molecular weight greater than 50 atomic
mass units to cross a membrane. This transport may be accomplished
by either the total loss of membrane structure, the formation of
holes (or pores) in the membrane structure, or the assisted
transport of compound through the membrane. In addition, transport
between liposomes, or cell membranes, may be accomplished by the
fusion of the two membranes and thereby the mixing of the contents
of the two membranes.
[0171] Membrane Active Peptides
[0172] Membrane active peptides are peptides that have membrane
activity. There are many naturally occurring membrane active
peptides such as cecropin (insects), magainin, CPF 1, PGLa,
Bombinin BLP-1 (all three from amphibians), melittin (bees),
seminalplasmin (bovine), indolicidin, bactenecin (both from bovine
neutrophils), tachyplesin 1 (crabs), protegrin (porcine
leukocytes), and defensins (from human, rabbit, bovine, fungi, and
plants). Gramicidin A and gramicidin S (bacillus brevis), the
lantibiotics such as nisin (lactococcus lactis), androctonin
(scorpion), cardiotoxin I (cobra), caerin (frog litoria splendida),
dermaseptin (frog). Viral peptides have also been shown to have
membrane activity, examples include hemagglutinin subunit HA-2
(influenza virus), E1 (Semliki forest virus), F1 (Sendai and
measles viruses), gp41 (HIV), gp32 (SIV), and vp1 (Rhino, polio,
and coxsackie viruses). In addition synthetic peptides have also
been shown to have membrane activity. Synthetic peptides that are
rich in leucines and lysines (KL or KL.sub.n motif) have been shown
to have membrane activity. In particular, the peptide
H.sub.2N-KLLKLLLKLWLKLLKLLLKLL-CO.sub.2 (SEQ ID 9), termed
KL.sub.3, is membrane active.
[0173] Polymers
[0174] 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.
[0175] The main chain of a polymer is composed of the atoms whose
bonds are required for propagation of polymer length. For example
in poly-L-lysine, the carbonyl carbon, a-carbon, and a-amine groups
are required for the length of the polymer and are therefore main
chain atoms. The side chain of a polymer is composed of the atoms
whose bonds are not required for propagation of polymer length. For
example in poly-L-lysine, the .beta., .gamma., .delta., and
.epsilon.-carbons, and .epsilon.-nitrogen are not required for the
propagation of the polymer and are therefore side chain atoms.
[0176] To those skilled in the art of polymerization, there are
several categories of polymerization processes that can be utilized
in the described process. The polymerization can be chain or step.
This classification description is more often used that the
previous terminology of addition and condensation polymer. "Most
step-reaction polymerizations are condensation processes and most
chain-reaction polymerizations are addition processes" (M. P.
Stevens Polymer Chemistry: An Introduction New York Oxford
University Press 1990). Template polymerization can be used to form
polymers from daughter polymers.
[0177] Step Polymerization
[0178] In step polymerization, the polymerization occurs in a
stepwise fashion. Polymer growth occurs by reaction between
monomers, oligomers and polymers. No initiator is needed since
there is the same reaction throughout and there is no termination
step so that the end groups are still reactive. The polymerization
rate decreases as the functional groups are consumed.
[0179] Typically, step polymerization is done either of two
different ways. One way, the monomer has both reactive functional
groups (A and B) in the same molecule so that
A--B yields --[A--B]--
[0180] Or the other approach is to have two difunctional
monomers.
[0181] A--A+B--B yields --[A--A--B--B]--
[0182] Generally, these reactions can involve acylation or
alkylation. Acylation is defined as the introduction of an acyl
group (--COR) onto a molecule. Alkylation is defined as the
introduction of an alkyl group onto a molecule.
[0183] If functional group A is an amine then B can be (but not
restricted to) an isothiocyanate, isocyanate, acyl azide,
N-hydroxysuccinimide, sulfonyl chloride, aldehyde (including
formaldehyde and glutaraldehyde), ketone, epoxide, carbonate,
imidoester, carboxylate, or alkylphosphate, arylhalides
(difluoro-dinitrobenzene), anhyderides or acid halides,
p-nitrophenyl esters, o-nitrophenyl pentachlorophenyl esters, or
pentafluorophenyl esters. In other terms when function A is an
amine then function B can be acylating or alkylating agent or
amination.
[0184] If functional group A is a thiol, sulfflhydryl, then
function B can be (but not restricted to) an iodoacetyl derivative,
maleimide, aziridine derivative, acryloyl derivative, fluorobenzene
derivatives, or disulfide derivative (such as a pyridyl disulfide
or 5-thio-2-nitrobenzoic acid{TNB} derivatives).
[0185] If functional group A is carboxylate then function B can be
(but not restricted to) a diazoacetate or an amine in which a
carbodiimide is used. Other additives may be utilized such as
carbonyldiimidazole, dimethylaminopyridine, N-hydroxysuccinimide or
alcohol using carbodiimide and dimethylaminopyridine.
[0186] If functional group A is an hydroxyl then function B can be
(but not restricted to) an epoxide, oxirane, or an amine in which
carbonyldiimidazole or N, N'-disuccinimidyl carbonate, or
N-hydroxysuccinimidyl chloroformate or other chloroformates are
used.
[0187] 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 imine or iminium that may or may not
be reduced by reducing agents such as NaCNBH.sub.3) or hydroxyl
compound to form a ketal or acetal.
[0188] Yet another approach is to have one difunctional monomer so
that
A--A plus another agent yields --[A--A]--.
[0189] If function A is a thiol, sulthydryl, group then it can be
converted to disulfide bonds by oxidizing agents such as iodine
(I.sub.2) or NaIO.sub.4 (sodium periodate), or oxygen (O.sub.2).
Function A can also be an amine that is converted to a thiol,
sulfhydryl, group by reaction with 2-Iminothiolate (Traut's
reagent) which then undergoes oxidation and disulfide formation.
Disulfide derivatives (such as a pyridyl disulfide or
5-thio-2-nitrobenzoic acid {TNB} derivatives) can also be used to
catalyze disulfide bond formation.
[0190] Functional group A or B in any of the above examples could
also be a photoreactive group such as aryl azides, halogenated aryl
azides, diazo, benzophenones, alkynes or diazirine derivatives.
[0191] Reactions of the amine, hydroxyl, thiol, sulfhydryl,
carboxylate groups yield chemical bonds that are described as
amide, amidine, disulfide, ethers, esters, enamine, urea,
isothiourea, isourea, sulfonamide, carbamate, carbon-nitrogen
double bond (imine), alkylamine bond (secondary amine),
carbon-nitrogen single bonds in which the carbon contains a
hydroxyl group, thio-ether, diol, hydrazone, diazo, or sulfone.
[0192] Chain Polymerization: In chain-reaction polymerization
growth of the polymer occurs by successive addition of monomer
units to limited number of growing chains. The initiation and
propagation mechanisms are different and there is usually a
chain-terminating step. The polymerization rate remains constant
until the monomer is depleted.
[0193] Monomers containing vinyl, acrylate, methacrylate,
acrylamide, methaacrylamide groups can undergo chain reaction which
can be radical, anionic, or cationic. Chain polymerization can also
be accomplished by cycle or ring opening polymerization. Several
different types of free radical initiatiors could be used that
include peroxides, hydroxy peroxides, and azo compounds such as
2,2'-Azobis(-amidinopropane) dihydrochloride (AAP). A compound is a
material made up of two or more elements.
[0194] Types of Monomers: A wide variety of monomers can be used in
the polymerization processes. These include positive charged
organic monomers such as amines, imidine, guanidine, imine,
hydroxylamine, hydrozyine, heterocycles (like imidazole, pyridine,
morpholine, pyrimidine, or pyrene. The amines could be pH-sensitive
in that the pKa of the amine is within the physiologic range of 4
to 8. Specific amines include spermine, spermidine,
N,N'-bis(2-aminoethyl)-1,3-propanediamine (AEPD), and
3,3'-Diamino-N,N-dimethyldipropylammonium bromide.
[0195] Monomers can also be hydrophobic, hydrophilic or
amphipathic. Monomers can also be intercalating agents such as
acridine, thiazole organge, or ethidium bromide.
[0196] Other Components of the Monomers and Polymers: The polymers
have other groups that increase their utility. These groups can be
incorporated into monomers prior to polymer formation or attached
to the polymer after its formation. These groups include: Targeting
Groups--such groups are used for targeting the polymer-nucleic acid
complexes to specific cells or tissues. Examples of such targeting
agents include agents that target to the asialoglycoprotein
receptor by using asiologlycoproteins or galactose residues. Other
proteins such as insulin, EGF, or transferrin can be used for
targeting. Protein refers to a molecule made up of 2 or more amino
acid residues connected one to another as in a polypeptide. The
amino acids may be naturally occurring or synthetic. Peptides that
include the RGD sequence can be used to target many cells. Chemical
groups that react with thiol, 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 fatty
acids, cholesterol, dansyl compounds, and amphotericin
derivatives.
[0197] After interaction of the supramolecular complexes with the
cell, other targeting groups can be used to increase the delivery
of the drug or nucleic acid to certain parts of the cell. For
example, agents can be used to disrupt endosomes and a nuclear
localizing signal (NLS) can be used to target the nucleus.
[0198] A variety of ligands have been used to target drugs and
genes to cells and to specific cellular receptors. The ligand may
seek a target within the cell membrane, on the cell membrane or
near a cell. Binding of ligands to receptors typically initiates
endocytosis. Ligands could also be used for DNA delivery that bind
to receptors that are not endocytosed. For example peptides
containing RGD peptide sequence that bind integrin receptor could
be used. In addition viral proteins could be used to bind the
complex to cells. Lipids and steroids could be used to directly
insert a complex into cellular membranes.
[0199] The polymers can also contain cleavable groups within
themselves. When attached to the targeting group, cleavage leads to
reduce interaction between the complex and the receptor for the
targeting group. Cleavable groups include but are not restricted to
disulfide bonds, diols, diazo bonds, ester bonds, sulfone bonds,
acetals, ketals, enol ethers, enol esters, enamines and imines.
[0200] Polyelectrolyte
[0201] A polyelectrolyte, or polyion, is a polymer possessing
charge, that is the polymer contains a group (or groups) that has
either gained or lost one or more electrons. A polycation is a
polyelectrolyte possessing 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 polyelectrolyte
containing a net negative charge. 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 polyelectrolyte 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.
[0202] Chelator
[0203] A chelator is a polydentate ligand, a molecule that can
occupy more than one site in the coordination sphere of an ion,
particularly a metal ion, primary amine, or single proton. Examples
of chelators include crown ethers, cryptates, and non-cyclic
polydentate molecules. A crown ether is a cyclic polyether
containing (--X--(CR1-2)n)m units, where n=1-3 and m=3-8. The X and
CR1-2 moieties can be substituted, or at a different oxidation
states. X can be oxygen, nitrogen, or sulfur, carbon, phosphorous
or any combination thereof. R can be H, C, 0, S, N, P. A subset of
crown ethers described as a cryptate contain a second
(--X--(CR1-2)n)z strand where z=3-8. The beginning X atom of the
strand is an X atom in the (--X--(CR1-2)n)m unit, and the terminal
CH2 of the new strand is bonded to a second X atom in the
(--X--(CR1-2)n)m unit. Non-cyclic polydentate molecules containing
(--X--(CR1-2)n)m unit(s), where n=1-4 and m=1-8. The X and CR1-2
moieties can be substituted, or at a different oxidation states. X
can be oxygen, nitrogen, or sulfur, carbon, phosphorous or any
combination thereof.
[0204] Polychelator
[0205] A polychelator is a polymer associated with a plurality of
chelators by an ionic or covalent bond and can include a spacer.
The polymer can be cationic, anionic, zwitterionic, neutral, or
contain any combination of cationic, anionic, zwitterionic, or
neutral groups with a net charge being cationic, anionic or
neutral, and may contain steric stabilizers, peptides, proteins,
signals, or amphipathic compound for the formation of micellar,
reverse micellar, or unilamellar structures. Preferably the
amphipathic compound can have a hydrophilic segment that is
cationic, anionic, or zwitterionic, and can contain polymerizable
groups, and a hydrophobic segment that can contain a polymerizable
group.
[0206] Steric Stabilizer
[0207] A steric stabilizer is a hydrophilic group that prevents
aggregation of a polymer or particle by sterically hindering
particle to particle electrostatic interactions. Examples include:
alkyl groups, PEG chains, polysaccharides, hydrogen molecules,
alkyl amines. Electrostatic interactions are the non-covalent
association of two or more substances due to attractive forces
between positive and negative charges.
[0208] Buffers
[0209] Buffers are made from a weak acid or weak base and their
salts. Buffer solutions resist changes in pH when additional acid
or base is added to the solution.
[0210] Biological, Chemical, or Biochemical reactions
[0211] Biological, chemical, or biochemical reactions involve the
formation or cleavage of ionic and/or covalent bonds.
[0212] Reactive
[0213] A compound is reactive if it is capable of forming either an
ionic or a covalent bond with another compound. The portions of
reactive compounds that are capable of forming covalent bonds are
referred to as reactive functional groups.
[0214] Lipids
[0215] Lipids are compounds that are insoluble in water but soluble
in organic solvent which have the general structure composed of two
distinct hydrophobic sections, that is two separate sections of
uninterrupted carbon-carbon bonds. The two hydrophobic sections are
connected through a linkage that contains at least one heteroatom,
that is an atom that is not carbon (e.g. nitrogen, oxygen, silicon,
and sulfur). Examples include esters and amides of fatty acids and
include the glycerides (1,2-dioleoylglycerol (DOG)), glycolipids,
phospholipids (dioleoylphosphatidylethanolamine (DOPE)).
[0216] Hydrocarbon
[0217] Hydrocarbon means containing carbon and hydrogen atoms; and
halohydrocarbon means containing carbon, halogen (F, Cl, Br, I),
and hydrogen atoms.
[0218] Alkyl, Alkene, Alkyne, Aryl
[0219] Alkyl means any sp.sup.3 -hybridized carbon-containing
group; alkenyl means containing two or more sp.sup.2 hybridized
carbon atoms; aklkynyl means containing two or more sp hybridized
carbon atoms; aralkyl means containing one or more aromatic ring(s)
in addition containing sp.sup.3 hybridized carbon atoms; aralkenyl
means containing one or more aromatic ring(s) in addition to
containing two or more sp.sup.2 hybridized carbon atoms; aralkynyl
means containing one or more aromatic ring(s) in addition to
containing two or more sp hybridized carbon atoms; steroid includes
natural and unnatural steroids and steroid derivatives.
[0220] Steroid
[0221] A steroid derivative means a sterol, a sterol in which the
hydroxyl moiety has been modified (for example, acylated), or a
steroid hormone, or an analog thereof. The modification can include
spacer groups, linkers, or reactive groups.
[0222] Carbohydrate
[0223] Carbohydrates include natural and unnatural sugars (for
example glucose), and sugar derivatives (a sugar derivative means a
system in which one or more of the hydroxyl groups on the sugar
moiety has been modified (for example, but not limited to,
acylated), or a system in which one or more of the hydroxyl groups
is not present).
[0224] Polyoxyethylene
[0225] Polyoxyethylene means a polymer having ethylene oxide units
(--(CH.sub.2CH.sub.2O)n--, where n=2--3000).
[0226] Compound
[0227] A compound is a material made up of two or more
elements.
[0228] Electron Withdrawing and Donating Groups
[0229] Electron withdrawing group is any chemical group or atom
composed of electronegative atom(s), that is atoms that tend to
attract electrons. Electron donating group is any chemical group or
atom composed of electropositive atom(s), that is atoms that tend
to attract electrons.
[0230] Resonance Stabilization
[0231] Resonance stabilization is the ability to distribute charge
on multiple atoms through pi bonds. The inductive effective, in a
molecule, is a shift of electron density due to the polarization of
a bond by a nearby electronegative or electropositive atom.
[0232] Activated Carboxylate
[0233] An activated carboxylate is a carboxylic acid derivative
that reacts with nucleophiles to form a new covalent bond.
Nucleophiles include nitrogen, oxygen and sulfur-containing
compounds to produce ureas, amides, carbonates, carbamates, esters,
and thioesters. The carboxylic acid may be activated by various
agents including carbodiimides, carbonates, phosphoniums, uroniums
to produce activated carboxylates acyl ureas, acylphosphonates,
acid anhydrides, and carbonates. Activation of carboxylic acid may
be used in conjunction with hydroxy and amine-containing compounds
to produce activated carboxylates N-hydroxysuccinimide esters,
hydroxybenzotriazole esters,
N-hydroxy-5-norbomene-endo-2,3-dicarboximide esters, p-nitrophenyl
esters, pentafluorophenyl esters, 4-dimethylaminopyridinium amides,
and acyl imidazoles.
[0234] Nucleophile
[0235] A nucleophile is a species possessing one or more
electron-rich sites, such as an unshared pair of electrons, the
negative end of a polar bond, or pi electrons.
[0236] Cleavage and Bond Breakage
[0237] Cleavage, or bond breakage is the loss of a covalent bond
between two atoms. Cleavable means that a bond is capable of being
cleaved.
[0238] Substituted Group or Substitution
[0239] A substituted group or a substitution refers to chemical
group which is placed onto a parent system instead of a hydrogen
atom. For the compound methylbenzene (toluene), the methyl group is
a substituted group, substituent, or substitution on the parent
system benzene. The methyl groups on 2,3-dimethylmaleic anhydride
are substituted groups, or substitutions on the parent compound (or
system) maleic anhydride.
[0240] Primary and Secondary Amine
[0241] A primary amine is a nitrogen-containing compound which is
derived by monosubstitution of ammonia (NH.sub.3) by a
carbon-containing group. A primary amine is a nitrogen-containing
compound which is derived by disubstitution of ammonia (NH.sub.3)
by a carbon-containing group.
EXAMPLES
[0242] The following examples are provided in order to demonstrate
and further illustrate certain preferred embodiments and aspects of
the present invention and are not to be construed as limiting the
scope thereof.
Example 1
Synthesis of Succinyl-.beta.-cyclodextrin
[0243] .beta.-Cyclodextrin (0.5 gm, 0.38 mmol) and succinic
anhydride (0.5 gm, 5 mmol) were dissolved in anhydrous pyridine (10
mL) for 4 h. The succinyl-.beta.-cyclodextrin was then precipitated
by addition of 40 mL isopropyl alcohol. The precipitate was then
washed 3 times with 10 mL isopropyl alcohol.
Example 2
Synthesis of Poly-L-lysine-succinyl-.beta.-cyclodextrin
[0244] Succinyl-.beta.-cyclodextrin (75 mg, 0.05 mmol) and
poly-L-lysine (2 mg, MW 52,000, 0.01 mmol amines) were dissolved in
1 mL water. To this mixture was added
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (40
mg, 0.2 mmol) and the reaction was allowed to proceed overnight.
The reaction mixture was then placed into a dialysis bag (12,000
molecular weight cutoff) and dialyzed against 3.times.1 L water for
72 hr. Lyophilization resulted in 6.7 mg of
poly-L-lysine-succinyl-.b- eta.-cyclodextrin, which is 35%
modification of the amine residues. The polymer was then dissolved
in 0.2 mL of water.
Example 3
Synthesis of Oleoyl Poly-L-lysine
[0245] Poly-L-lysine (5 mg, 0.02 mmol amines) was dissolved in 0.5
mL water, to this solution was added oleoyl chloride (0.5 mg, 0.002
mmol) in 20 .mu.L of acetonitrile.
Example 3
Synthesis of Succinyloleoylpoly-L-lysine
[0246] To a solution of poly-L-lysine-oleoyl amide (2 mg) in 200 mL
water was added succinic anhydride (20 mg, 0.2 mmol) and potassium
carbonate (100 mg 0.7 mmol). After 5 minutes, the
succinylpoly-L-lysine -oleoyl amide was precipitated by addition of
1 mL isopropyl alcohol.
Example 3
Synthesis of Epichlorohydrin-.beta.-cyclodextrin Copolymer
[0247] .beta.-Cyclodextrin (0.5 gm, 0.38 mmol) and sodium hydroxide
(0.18 gm, 4.5 mmol) were dissolved in water (0.8 mL) and heated to
30.degree. C. To this solution was added epichlorohydrin (0.345 mL,
4.4 mmol) and the immiscible solutions were stirred at 30.degree.
C. for 3.5 h, during which time the epichlorohydrin dissolved in
the aqueous solution. The epichlorohydrin-.beta.-cyclodextrin
copolymer was then precipitated by the addition of acetone (10 mL).
The acetone was decanted and the precipitate was dissolved in water
(20 mL) and dialyzed in 14,000 molecular weight cutoff tubing
against 2.times.1 L water for 48 h. The polymer was then isolated
by lyophilization to yield 0.3 gm of polymer.
Example 4
Characterization of Particles Formed by Poly-L-lysine,
Epichlorohydrin-.beta.-cyclodextrin Copolymer, and 4-t-butylbenzoic
Acid
[0248] To a solution of epichlorohydrin-.beta.-cyclodextrin
copolymer (100 .mu.g/mL) and poly-L-lysine (100 .mu.g/mL) was added
4-t-butylbenzoic acid (3 mM). The size of the particle formed by
the three agents was 100 nm, measured by a Brookhaven ZetaPlus
Particle Sizer. Particle formation is observed only in the presence
of all three components and is independent of the order of addition
of each component.
Example 5
Characterization of Particles Formed by Plasmid DNA,
Epichlorohydrin-.beta.-cyclodextrin Copolymer, and Oleoylamine
[0249] To a solution of epichlorohydrin-.beta.-cyclodextrin
copolymer (50 .mu.g/mL) and plasmid DNA (10 .mu.g/mL) was added
oleoylamine (0.1 mM). The size of the particle formed by the three
agents was 78 nm, measured by a Brookhaven ZetaPlus Particle Sizer.
Particle formation is observed only in the presence of all three
components and is independent of the order of addition of each
component.
Example 6
Characterization of Particles Formed Between Plasmid DNA and
Poly-L-lysine-succinyl-.beta.-cyclodextrin.
[0250] To a solution of plasmid DNA (10 .beta.g/mL) was added
poly-L-lysine-succinyl-.beta.-cyclodextrin (30 .mu.g/mL). The size
of the particle formed was 88 nrm and its charge was 11.+-.7 mV,
measured by a Brookhaven ZetaPlus Particle Sizer. To these particle
was added Triton X-100 (0.2 mg/mL) resulting in a particle that was
140 nm in size with a charge of 22.+-.4 mV. Addition of sodium
chloride (100 mM) to these particles resulted in particles that
were 115 nm in size with a charge of 7.+-.2 mV. If Triton x-100 is
not added to the particles prior to the addition of sodium chloride
the particles become large, >200 nm.
Example 7
In vitro Transfection with
DNA-poly-L-lysine-succinylpoly-L-lysine-oleoyl Amide Particles in
the Presence of .beta.-cyclodextrin
[0251] To plasmid DNA pCILuc (10 .mu.g/mL, 2.6 .mu.g/.mu.L pCIluc;
prepared according to Danko I, Williams P, Herweijer H, Zhang G,
Latendresse J S, Bock I, Wolff J A Hum. Mol. Genet. 1997, 6, 1435)
in 0.5 mL of 0 or 3 mM aqueous .beta.-cyclodextrin was added
poly-L-lysine (30 .mu.g/mL). Subsequently, 0. 15 mg/mL of
succinyloleoylpoly-L-lysine was added. The DNA complexes were then
added (200 .mu.L) to a well containing 3T3 mouse embryonic
fibroblast cells in 290 mM glucose and 5 mM HEPES buffer pH 7.5.
After 1.5 h, the glucose solution was replaced with Dubelco's
modified Eagle Media and the cells were allowed to incubate for 48
h. The cells were then harvested and then assayed for luciferase
activity. Luciferase activity in the presence of
.beta.-cyclodextrin was 33-fold higher (324,305 relative light
units) than in the absence of .beta.-cyclodextrin (RLU=9,924).
Example 8
In vitro Transfection with
DNA-poly-L-lysine-epichlorohydrin-.beta.-cyclod- extrin Copolymer
in the Presence of P-t-butyl-benzoic Acid
[0252] To plasmid DNA pCIluc (10 .mu.g/mL, 2.6 .mu.g/.mu.L pCIluc)
in 0.5 mL of aqueous 0 or 3 mM 4-t-butylbenzoic acid was added
poly-L-lysine (30 .mu.g/mL). Subsequently, 0.15 mg/mL of
succinylated poly-L-lysine or epichlorohydrin-.mu.-cyclodextrin
copolymer was added. The DNA complexes were then added (200 .mu.L)
to a well containing 3T3 mouse embryonic fibroblast cells in
Dubelco's modified Eagle Media. After 1.5 h, the media was changed
and the cells were allowed to incubate for 48 h. The cells were
then harvested and then assay for luciferase activity. Luciferase
activity for the particles composed of epichlorohydrin-.mu.-cy-
clodextrin copolymer was 81 -fold higher (314166 relative light
units(RLU)) than those particles composed of succinylated
poly-L-lysine (3868 RLU).
Example 9
Characterization of Complexes of Plasmid DNA, Dodecylamine, and
.beta.-cyclodextrin-epichlorohydrin Copolymer
[0253] To a solution of plasmid DNA (10 .mu.g/mL) and
.beta.-cyclodextrin-epichlorohydrin copolymer (50 .mu.g/mL) was
added dodecylamine (100 .mu.M). The size of the particle formed was
181 nm as measured by a Brookhaven ZetaPlus Particle Sizer. Prior
to the addition of dodecylamine there were no particles formed and
solutions of .beta.-cyclodextrin epichlorohydrin copolymer and
dodecyl amine do no not form particles.
Example 10
Characterization of Complexes of Plasmid DNA, 1-adamantamine, and
.beta.-cyclodextrin-epichlorohydrin Copolymer
[0254] To a solution of plasmid DNA (10 .mu.g/mL) and
.beta.-cyclodextrin-epichlorohydrin copolymer (50 .mu.g/mL) was
added various amounts of 1-adamantanamine (100-600 .mu.M). The size
of the particle formed was 181 nm as measured by a Brookhaven
ZetaPlus Particle Sizer. Prior to the addition of dodecylamine
there were no particles formed and solutions of .beta.-cyclodextrin
epichlorohydrin copolymer and dodecyl amine do no not form
particles.
1 [1-adamantamine] (.mu.M) Size of particles (nm) 100 >30,000
200 125 300 85 400 78
Example 11
In vivo Expression of Complexes of Plasmid DNA, 1 -adamantamine,
and .beta.-cyclodextrin-epichlorohydrin Copolymer.
[0255] A complex of pCI Luc (50 .mu.g/mL), 250 .mu.g/mL
.beta.-cyclodextrin-epichlorohydrin copolymer, and 6 mM 1
-adamantamine in 0.2 mL were diluted to 2.5 mL in Ringers solution.
Tail vein injections of 2.5 mL of the complex were performed as
previously described (Zhang, G., Budker, V., Wolff, J. A. Hum. Gene
Ther. 1999, 10, 1735.) Luciferase expression was determined as
previously reported (Wolff, J. A., Malone, R. W., Williams, P.,
Chong, W., Acsadi, G., Jani, A. and Felgner, P. L. Direct gene
transfer into mouse muscle in vivo. Science, 1465-1468,1990.). A
Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany) luminometer
was used.
2 Organ Relative Light Units Liver 10,340,000 Spleen 103,631 Lung
102,851 Heart 50,350 Kidney 261,912
Example 12
In vivo Expression of Complexes of Digoxin-labeled Plasmid DNA and
.gamma.-cyclodextrin
[0256] Plasmid DNA was labeled with Mirus' LabelIt.RTM. digoxin
labeling kit according to protocol. A complex of digoxin-labeled
pCI Luc (2 .mu.g) and .gamma.-cyclodextrin (17 mg) were formulated
in 2.5 mL in Ringers solution. Tail vein injections of the complex
were performed as previously described (Zhang, G., Budker, V.,
Wolff, J. A. Hum. Gene Ther. 1999, 10, 1735.) Luciferase expression
was determined as previously reported (Wolff, J. A., Malone, R. W.,
Williams, P., Chong, W., Acsadi, G., Jani, A. and Felgner, P. L.
Direct gene transfer into mouse muscle in vivo. Science,
1465-1468,1990.). A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad,
Germany) luminometer was used.
3 Organ Relative Light Units Liver 9,450,000 Spleen 365,000 Lung
290,000 Heart 111,000 Kidney 166,000
[0257] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in cell biology,
chemistry, molecular biology, biochemistry or related fields are
intended to be within the scope of the following claims.
Sequence CWU 1
1
9 1 14 PRT Simian virus 40 1 Cys Gly Tyr Gly Pro Lys Lys Lys Arg
Lys Val Gly Gly Cys 1 5 10 2 13 PRT Simian virus 40 2 Cys Gly Tyr
Gly Pro Lys Lys Lys Arg Lys Val Gly Gly 1 5 10 3 37 PRT Simian
virus 40 3 Cys Lys Lys Lys Trp Asp Asp Glu Ala Thr Ala Asp Ser Gln
His Ser 1 5 10 15 Thr Pro Pro Lys Lys Lys Arg Lys Val Glu Asp Pro
Lys Asp Phe Pro 20 25 30 Ser Glu Leu Leu Ser 35 4 37 PRT Simian
virus 40 4 Cys Lys Lys Lys Trp Asp Asp Glu Ala Thr Ala Asp Ser Gln
His Ser 1 5 10 15 Thr Pro Pro Lys Lys Lys Arg Lys Val Glu Asp Pro
Lys Asp Phe Pro 20 25 30 Ser Glu Leu Leu Ser 35 5 31 PRT Homo
sapiens 5 Cys Tyr Asn Asp Phe Gly Asn Tyr Asn Asn Gln Ser Ser Asn
Phe Gly 1 5 10 15 Pro Met Lys Gln Gly Asn Phe Gly Gly Arg Ser Ser
Gly Pro Tyr 20 25 30 6 10 PRT Human adenovirus type 1 6 Cys Lys Arg
Gly Pro Lys Arg Pro Arg Pro 1 5 10 7 22 PRT Homo sapiens 7 Cys Lys
Lys Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln 1 5 10 15
Ala Lys Lys Lys Lys Leu 20 8 14 PRT Homo sapiens 8 Cys Lys Lys Lys
Gly Pro Ala Ala Lys Arg Val Lys Leu Asp 1 5 10 9 21 PRT Artificial
synthetic amphipathic peptide 9 Lys Leu Leu Lys Leu Leu Leu Lys Leu
Trp Leu Lys Leu Leu Lys Leu 1 5 10 15 Leu Leu Lys Leu Leu 20
* * * * *