U.S. patent application number 09/876904 was filed with the patent office on 2003-04-17 for encapsulation of plasmid dna (lipogenes.tm.) and therapeutic agents with nuclear localization signal/fusogenic peptide conjugates into targeted liposome complexes.
Invention is credited to Boulikas, Teni.
Application Number | 20030072794 09/876904 |
Document ID | / |
Family ID | 26905654 |
Filed Date | 2003-04-17 |
United States Patent
Application |
20030072794 |
Kind Code |
A1 |
Boulikas, Teni |
April 17, 2003 |
Encapsulation of plasmid DNA (lipogenes.TM.) and therapeutic agents
with nuclear localization signal/fusogenic peptide conjugates into
targeted liposome complexes
Abstract
A method is disclosed for encapsulating plasmids,
oligonucleotides or negatively-charged drugs into liposomes having
a different lipid composition between their inner and outer
membrane bilayers and able to reach primary tumors and their
metastases after intravenous injection to animals and humans. The
formulation method includes complex formation between DNA with
cationic lipid molecules and fusogenic/NLS peptide conjugates
composed of a hydrophobic chain of about 10-20 amino acids and also
containing four or more histidine residues or NLS at their one end.
The encapsulated molecules display therapeutic efficacy in
eradicating a variety of solid human tumors including but not
limited to breast carcinoma and prostate carcinoma. Combination of
the plasmids, oligonucleotides or negatively-charged drugs with
other anti-neoplastic drugs (the positively-charged cis-platin,
doxorubicin) encapsulated into liposomes are of therapeutic value.
Also of therapeutic value in cancer eradication are combinations of
encapsulated the plasmids, oligonucleotides or negatively-charged
drugs with HSV-tk plus encapsulated ganciclovir.
Inventors: |
Boulikas, Teni; (Mountain
View, CA) |
Correspondence
Address: |
Antoinette F. Konski
Baker & McKenzie
660 Hansen Way
Palo Alto
CA
94304
US
|
Family ID: |
26905654 |
Appl. No.: |
09/876904 |
Filed: |
June 8, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60210925 |
Jun 9, 2000 |
|
|
|
Current U.S.
Class: |
424/450 ; 264/4;
435/320.1; 435/458; 514/44A |
Current CPC
Class: |
A61K 9/1277 20130101;
A61K 9/1075 20130101; C12N 15/88 20130101; A61P 35/00 20180101;
A61K 9/1271 20130101; A61K 9/1278 20130101; A61K 9/127
20130101 |
Class at
Publication: |
424/450 ;
435/458; 435/320.1; 514/44; 264/4 |
International
Class: |
A61K 048/00; A61K
009/127; C12N 015/88 |
Claims
What is claimed is:
1. A method for producing micelles with entrapped therapeutic
agents, comprising: a) combining an effective amount of a
negatively charged therapeutic agent with an effective amount of a
cationic lipid in a ratio where about 30% to about 90% the
negatively charged atoms are neutralized by positive charges on
lipid molecules to form an electrostatic micelle complex in about
20% to about 80% ethanol; and b) combining the micelle complex of
step a) with an effective amount of a fusogenic-karyophilic peptide
conjugates in a ratio range of about 0.0 to about 0.3, thereby
producing micelles with entrapped therapeutic agents.
2. The method of claim 1, wherein the negatively charged
therapeutic agent is a therapeutic agent selected from the group
consisting of a polynucleotide and a negatively charged drug.
3. The method of claim 2, wherein the polynucleotide is a DNA
polynucleotide or an RNA polynucleotide.
4. The method of claim 2, wherein the polynucleotide is a DNA
polynucleotide.
5. The method of claim 4, wherein the DNA polynucleotide comprises
plasmid DNA.
6. The method of claim 1, further comprising combining an effective
amount of an anionic lipid in step a).
7. The method of claim 6, wherein the anionic lipid is dipalmitoyl
phosphatidyl glycerol (DDPG) or a derivative thereof.
8. The method of claim 4, further comprising combining an effective
amount of a DNA condensing agent selected from the group consisting
of spermine, spermidine, polylysine, polyarginine, polyhistidine,
polyornithine and magnesium or a divalent metal ion.
9. The method of claim 5, wherein the plasmid DNA comprises a
sequence encoding p53, HSV-tk, p21, Bax, Bad, IL-2, IL-12, GM-CSF,
angiostatin, endostatin and oncostatin.
10. The method of claim 1, wherein the cationic lipids are selected
from the group consisting of
3.beta.-(N--(N',N'-dimethylaminoethane)carbamoyl)- cholesterol,
dimethyldioctadecyl ammonium bromide (DDAB),
N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)
ammonium bromide (DMRIE), 1 ,2-dimyristoyl-3-trimethylammonium
propane (DMTAP), dioctadecylamidoglycylspermine (DOGS),
N-(1-(2,3-dioleoyloxy)propyl)-N,N,- N-trimethylammonium chloride
(DOTMA), 1,2- dipalmitoyl-3-trimethylammonium propane (DPTAP), 1
,2-disteroyl-3-trimethylammonium propane (DSTAP).
11. The method of claim 10, wherein the cationic lipids are
combined with the fusogenic lipid DOPE in a molar ratio from about
1:1 to about 2:1.
12. The method of claim 11, wherein the cationic lipids are
combined with the fusogenic lipid DOPE in a molar ratio of 1:1.
13. The method of claim 1, wherein the fusogenic-karyophilic
peptide is an NLS peptide.
14. The method of claim 13, wherein the NLS peptide is a peptide
selected from the group consisting of Seq. ID Nos. 20-622.
15. The method of claim 1, wherein the fusogenic-karyophilic
peptide conjugate is a sole fusogenic peptide.
16. The method of claim 1, wherein the NLS peptide component of the
fusogenic-karyophilic peptide conjugate is an NLS peptide selected
from the group consisting of Seq. ID Nos. 20-622.
17. The method of claim 1, wherein the fusogenic/NLS peptide
conjugates comprise amino acid sequences selected from the group
consisting of (KAWLKAF).sub.3 (SEQ ID NO:1), GLFKAAAKLLKSLWKLLLKA
(SEQ ID NO:2), LLLKAFAKLLKSLWKLLLKA (SEQ ID NO:3) as well as all
derivatives of the prototype
(Hydrophobic.sub.3Karyophilic.sub.1Hydrophobic.sub.2Karyophilic-
l).sub.2-3 where Hydrophobic is any of the A, I, L, V, P, G, W, F
and Karyophilic is any of the K, R, or H, containing a
positively-charged residue every 3rd or 4th amino acid, that form
alpha helices and direct a net positive charge to the same
direction of the helix.
18. The method of claim 1, wherein the fusogenic/NLS peptide
conjugate comprise an amino acid sequence selected from the group
consisting of GLFKAIAGFIKNGWKGMIDGGGYC (SEQ ID NO:4) from influenza
virus hemagglutinin HA-2 and YGRKKRRQRRR (SEQ ID NO:5) from TAT of
HIV.
19. The method of claim 1, wherein the fusogenic/NLS peptide
conjugate comprise an amino acid sequence selected from the group
consisting of MSGTFGGILAGLIGLL(K/R/H).sub.1-6 (SEQ ID NO:6),
derived from the N-terminal region of the S protein of duck
hepatitis B virus but with the addition of one to six
positively-charged lysine, arginine or histidine residues, and
combinations of these, GAAIGLAWIPYFGPAA (SEQ ID NO:7) derived from
the fusogenic peptide of the Ebola virus transmembrane protein;
residues 53-70 (C-terminal helix) of apolipoprotein (apo) All
peptide, the 23-residue fusogenic N-terminal peptide of HIV-1
transmembrane glycoprotein gp41, the 29-42-residue fragment from
Alzheimer's beta-amyloid peptide, the fusion peptide and N-terminal
heptad repeat of Sendai virus, the 56-68 helical segment of
lecithin cholesterol acyltransferase.
20. The method of any of claims 13 to 19, wherein the NLS peptide
component in fusogenic/NLS peptide conjugates are synthetic
peptides containing the above said NLS but further modified by
additional K, R, H residues at the central part of the peptide or
with P or G at the N- or C-terminus.
21. The method of claim 13, wherein the fusogenic peptide/NLS
peptide conjugates are linked to each other with a short amino acid
stretch representing an endogenous protease cleavage site.
22. The method of claim 1, wherein the structure of the preferred
prototype fusogenic/NLS peptide conjugate used in this invention
is: PKKRRGPSP(L/A/I).sub.12-20 (SEQ ID NO:8) where
(L/A/I).sub.12-20 is a stretch of 12-20 hydrophobic amino acids
containing A, L, I, Y, W, F and other hydrophobic amino acids.
23. The method of claim 1, wherein the fusogenic/NLS peptide
conjugates are added to the mixture of DNA/cationic lipid and are
incorporated into micelles.
24. The method of claim 1, further comprising combining an
effective amount of an encapsulating lipid solution to step b).
25. The method of claim 24, wherein the encapsulating lipid is a
lipid comprising cholesterol (40%),
dioleoylphosphatidylethanolamine (DOPE) (20%),
palmitoyloleoylphosphatidylcholine (POPC) (12%), hydrogenated soy
phosphatidylcholine (HSPC) (10%),
distearoylphosphatidylethanolamine (DSPE) (10%), sphingomyelin (SM)
(5%), and derivatized vesicle-forming lipid M-PEG-DSPE (3%).
26. The method of claim 24, wherein the encapsulating lipid is a
liposome.
27. The method of claim 26, wherein the liposomes comprises
vesicle-forming lipids and between about 1 to about 7 mole percent
of distearoylphosphatidyl ethanolamine (DSPE) derivatized with an
effective amount of polyethyleneglycol.
28. The method of claim 27, wherein the liposomes have a selected
average size of about 80 to about 160 nm.
29. The method of claim 27, wherein the polyethyleneglycol has a
molecular weight from about 1,000 to about 5,000 daltons.
30. A micelle with an entrapped therapeutic agent produced by the
method of claim 1.
31. A liposome encapsulated therapeutic agent produced by the
method of claim 24.
32. The method of claim 31, wherein the therapeutic agent further
comprises regulation by a liver, spleen or bone marrow regulatory
DNA sequence.
33. The method of claim 32, wherein the regulatory DNA sequence is
nuclear matrix DNA isolated from liver, spleen or bone marrow
cells.
34. A method for delivering a therapeutic agent in vivo, comprising
administration of an effective amount of the micelle of claim 30 to
a subject.
35. The method of claim 34, wherein the therapeutic agent further
comprises regulation by a tumor-specific regulatory DNA
sequence.
36. The method of claim 35, wherein the tumor-specific regulatory
sequence is nuclear matrix DNA isolated from specific tumor
cells.
37. A method for delivering a therapeutic agent in vivo, comprising
administration of an effective amount of the liposome encapsulated
agent of claim 31 to the subject.
38. The method of claim 34 or 37, wherein the administration is
intravenous administration or by injection.
39. A micelle with an entrapped DNA polynucleotide produced by the
method of claim 9.
40. A method for reducing tumor size in a subject comprising
administration of an effective amount of the micelle of claim 39 to
the subject.
41. The method of claim 40, further comprising administration of an
effective amount of a second therapeutic agent, wherein the agent
is selected from the group consisting of ganciclovir,
5-fluorocytosine, an antisense oligonucleotides a ribozyme, and a
triplex-forming oligonucleotide directed against genes that control
the cell cycle or signaling pathways.
42. The method of claim 41, further comprising administration of an
effective amount of a second therapeutic agent, wherein the second
therapeutic agent is selected from the group consisting of
adriamycin, angiostatin, azathioprine, bleomycin, busulfane,
camptothecin, carboplatin, carmustine, chlorambucile,
chlormethamine, chloroquinoxaline sulfonamide, cisplatin,
cyclophosphamide, cycloplatam, cytarabine, dacarbazine,
dactinomycin, daunorubicin, didox, doxorubicin, endostatin,
enloplatin, estramustine, etoposide, extramustinephosphat,
flucytosine, fluorodeoxyuridine, fluorouracil, gallium nitrate,
hydroxyurea, idoxuridine, interferons, interleukins, leuprolide,
lobaplatin, lomustine, mannomustine, mechlorethamine,
mechlorethaminoxide, melphalan, mercaptopurine, methotrexate,
mithramycin, mitobronitole, mitomycin, mycophenolic acid,
nocodazole, oncostatin, oxaliplatin, paclitaxel, pentamustine,
platinum-triamine complex, plicamycin, prednisolone, prednisone,
procarbazine, protein kinase C inhibitors, puromycine, semustine,
signal transduction inhibitors, spiroplatin, streptozotocine,
stromelysin inhibitors, taxol, tegafur, telomerase inhibitors,
teniposide, thalidomide, thiamiprine, thioguanine, thiotepa,
tiamiprine, tretamine, triaziquone, trifosfamide, tyrosine kinase
inhibitors, uramustine, vidarabine, vinblastine, vinca alcaloids,
vincristine, vindesine, vorozole, zeniplatin, zeniplatin, and
zinostatin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application Serial No. 60/210,925
filed Jun. 9, 2000. The contents of this application is hereby
incorporated by reference into the present disclosure.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of gene therapy
and is specifically directed toward methods for producing
peptide-lipid-polynucleotide complexes suitable for delivery of
polynucleotides to a subject. The peptide-lipid-polynucleotide
complexes so produced are useful in a subject for inhibiting the
progression of neoplastic disease.
BACKGROUND OF THE INVENTION
[0003] Throughout this application various publications, patents
and published patent specifications are referenced by author and
date or by an identifying patent number. Full bibliographical
citations for the publications are provided immediately preceding
the claims. The disclosures of these publications, patents and
published patent specifications are hereby incorporated by
reference into the present disclosure to more fully describe the
state of the art to which this invention pertains.
[0004] Gene therapy is a newly emerging field of biomedical
research that holds great promise for the treatment of both acute
and chronic diseases and has the potential to bring a revolutionary
era to molecular medicine. However, despite numerous preclinical
and clinical studies, routine use of gene therapy for the treatment
of human disease has not yet been perfected. It remains an
important unmet need of gene therapy to create gene delivery
systems that effectively target specific cells of interest in a
subject while controlling harmful side effects.
[0005] Gene therapy is aimed at introducing therapeutically
important genes into somatic cells of patients. Diseases already
shown to be amenable to therapy with gene transfer in clinical
trials include, cancer (melanoma, breast, lymphoma, head and neck,
ovarian, colon, prostate, brain, chronic myelogenous leukemia,
non-small cell lung, lung adenocarcinoma, colorectal,
neuroblastoma, glioma, glioblastoma, astrocytoma, and others),
AIDS, cystic fibrosis, adenosine deaminase deficiency,
cardiovascular diseases (restenosis, familial hypercholesterolemia,
peripheral artery disease), Gaucher disease, .alpha.1-antitrypsin
deficiency, rheumatoid arthritis and others. Human diseases
expected to be the object of clinical trials include hemophilia A
and B, Parkinson's disease, ocular diseases, xeroderma pigmentosum,
high blood pressure, obesity. ADA deficiency was the disease
successfully treated by the first human "gene transfer" experiment
conducted by Kenneth Culver in 1990. See, Culver, K. W. (1996) in:
Gene Therapy: A Primer for Physicians, Second Ed., Mary Ann
Liebert, Inc. Publ, New York, pp. 1-198.
[0006] The primary goals of gene therapy are to repair or replace
mutated genes, regulate gene expression and signal transduction,
manipulate the immune system, or target malignant and other cells
for destruction. See, Anderson, W. F. (1992) Science 256:808-813;
Lasic, D. (1997) in: Liposomes in Gene Delivery, CRC Press, pp.
1-295; Boulikas, T. (1998) Gene Ther. Mol. Biol. 1:1-172; Martin,
F. and Boulikas, T. (1998) Gene Ther. Mol. Biol. 1:173-214; Ross,
G. et al. (1996) Hum. Gene Ther. 7:1781-1790.
[0007] Human cancer presents a particular disease condition for
which effective gene therapy methods would provide a particularly
useful clinical benefit. Gene therapy concepts for treatment of
such diseases include stimulation of immune responses as well as
manipulation of a variety of alternative cellular functions that
affect the malignant phenotype. Although many human tumors are non
or weakly immunogenic, the immune system can be reinforced and
instructed to eliminate cancer cells after transduction of a
patient's cells ex vivo with the cytokine genes GM-CSF, IL-12,
IL-2, IL-4, IL-7, IFN-.gamma., and TNF-.alpha., followed by cell
vaccination of the patient (e.g. intradermally) to potentiate
T-lymphocyte-mediated antitumor effects (cancer immunotherapy). DNA
vaccination with genes encoding tumor antigens and immunotherapy
with synthetic tumor peptide vaccines are further developments that
are currently being tested. The genes used for cancer gene therapy
in human clinical trials include a number of tumor suppressor genes
(p53, RB, BRCA1, E1A), antisense oncogenes (antisense c-fos, c-myc,
K-ras), and suicide genes (HSV-tk, in combination with ganciclovir,
cytosine deaminase in combination with 5-fluorocytosine). Other
important genes that have been proposed for cancer gene therapy
include bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I, VEGF,
angiostatin, CFTR, LDL-R, TGF-.beta., and leptin. One major hurdle
preventing successful implementation of these gene therapies is the
difficulty of efficiently delivering an effective dose of
polynucleotides to the site of the tumor. Thus, gene delivery
systems with enhanced transfection capabilities would be highly
advantageous.
[0008] A number of different vector technologies and gene delivery
methods have been proposed and tested for delivering genes in vivo,
including viral vectors and various nucleic acid encapsulation
techniques. Alternative viral delivery vehicles for genes include
murine retroviruses, recombinant adenoviral vectors,
adeno-associated virus, HSV, EBV, HIV vectors, and baculovirus.
Nonviral gene delivery methods use cationic or neutral liposomes,
direct injection of plasmid DNA, and polymers. Various strategies
to enhance efficiency of gene transfer have been tested such as
fusogenic peptides in combination with liposomes or polymers to
enhance the release of plasmid DNA from endosomes.
[0009] Each of the various gene delivery techniques has been found
to possess different strengths and weaknesses. Recombinant
retroviruses stably integrate into the chromosome but require host
DNA synthesis to insert. Adenoviruses can infect non-dividing cells
but cause immune reactions leading to the elimination of
therapeutically transduced cells. Adeno-associated virus (AAV) is
not pathogenic and does not elicit immune responses but new
production strategies are required to obtain high AAV titers for
preclinical and clinical studies. Wild-type AAVs integrate into
chromosome 19, whereas recombinant AAVs are deprived of
site-specific integration and may also persist episomally.
[0010] Herpes Simplex Virus (HSV) vectors can infect
non-replicating cells, such as neuronal cells, and has a high
payload capacity for foreign DNA but inflict cytotoxic effects. It
seems that each delivery system will be developed independently of
the others and that each will demonstrate strengths and weaknesses
for certain applications. At present, retroviruses are most
commonly used in human clinical trials, followed by adenoviruses,
cationic liposomes and AAV.
[0011] As the challenges of perfecting gene therapy techniques have
become apparent, a variety of additional delivery systems have been
proposed to circumvent the difficulties observed with standard
technologies. For example, cell-based gene delivery using
polymer-encapsulated syngeneic or allogeneic cells implanted into a
tissue of a patient can be used to secrete therapeutic proteins.
This method is being tested in trials for amyotrophic lateral
sclerosis using the ciliary neurotrophic factor gene, and may be
extended to Factor VIII and IX for hemophilia, interleukin genes,
dopamine-secreting cells to treat Parkinson's disease, nerve growth
factor for Alzheimer's disease and other diseases. Other techniques
under development include, vectors with the Cre-LoxP recombinase
system to rid transfected cells of undesirable viral DNA sequences,
use of tissue-specific promoters to express a gene in a particular
cell type, or use of ligands recognizing cell surface molecules to
direct gene vehicles to a particular cell type.
[0012] Additional methods that have been proposed for improving the
efficacy of gene therapy technologies include designing p53 "gene
bombs" that explode into tumor cells, exploiting the HIV-1 virus to
engineer vectors for gene transfer, combining viruses with polymers
or cationic lipids to improve gene transfer, the attachment of
nuclear localization signal peptides to oligonucleotides to direct
genes to nuclei, and the development of molecular switch systems
allowing genes to be turned on or off at will. Nevertheless,
because of the wide range of disease conditions for which gene
therapies are required, and the complexities of developing
treatments for such diseases, there remains a need for improved
techniques for performing gene therapy. The present invention
provides methods and compositions for addressing these issues.
DISCLOSURE OF THE INVENTION
[0013] A method is disclosed for encapsulating DNA and negatively
charged drugs into liposomes having a different lipid composition
between their inner and outer membrane bilayers. The liposomes are
able to reach primary tumors and their metastases after intravenous
injection to animals and humans. The method includes micelle
formation between DNA with a mixture of cationic lipid and peptide
molecules at molar ratios to nearly neutralization ratios in 10-90%
ethanol; the cationic peptides specify nuclear localization and
have a hydrophobic moiety endowed with membrane fusion to improve
entrance across the cell membrane of the complex. These peptides
insert with their cationic portion directed toward condensed DNA
and their hydrophobic chain buried together with the hydrophobic
chains of the lipids in the micelle membrane monolayer. The
DNA/lipid/peptide micelles are converted into liposomes by mixing
with pre-made liposomes or lipids followed by dilution in aqueous
solutions and dialysis to remove the ethanol and allow liposome
formation and extrusion through membranes to a diameter below 160
nm entrapping and encapsulating DNA with a very high yield. The
encapsulated DNA has a high therapeutic efficacy in eradicating a
variety of solid human tumors including, but not limited to, breast
carcinoma and prostate carcinoma. A plasmid is constructed with DNA
carrying anticancer genes including, but not limited to p53, RB,
BRCA1, E1A, bcl-2, MDR-1, p21, pl6, bax, bcl-xs, E2F, IGF-I VEGF,
angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4,
IL-7, IFN-.gamma., TNF-.alpha., HSV-tk (in combination with
ganciclovir), E. coli cytosine deaminase (in combination with
5-fluorocytosine) and is combined with encapsulated cisplatin or
with other similarly systemically delivered antineoplastic drugs to
suppress cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 illustrates the structure of the cancer targeted
liposome complex.
[0015] FIG. 2 illustrates the results of plasmid DNA condensation
with various agents as well as various formulation of cationic
liposomes in affecting the level of expression of the reporter
beta-galactosidase gene after transfection of K562 human
erythroleukemia cell cultures.
[0016] FIG. 3 illustrates tumor targeting in SCID mice. FIG. 3A
shows a SCID mouse with a large and small human breast tumor before
and after staining with X-Gal to test the expression of the
transferred gene. Both tumors turn dark blue. The intensity of the
blue color is proportional to the expression of the
beta-galactosidase gene.
[0017] FIG. 3B shows that in the initial staining of the small
tumor, the skin and the intestines at the injection area are the
first organs to turn blue. FIG. 3C is a view of the back of the
animal. The two tumors are clearly visible after removal of the
skin (top). Dark staining of the small tumor and light blue
staining of the large tumor is evident at an initial stage of
staining (bottom). FIG. 3D is a view of the front side of the
animal. The two tumors are clearly visible after removal of the
skin. On the figure to the bottom the dark staining of both tumors
is evident at a later stage during staining.
[0018] FIG. 3E shows the front (top) and rear (bottom) higher
magnification view of the dark staining of both tumors at a later
stage during staining. Staining of the vascular system around the
small tumor can also be seen (bottom).
BRIEF DESCRIPTION OF THE TABLES
[0019] Table 1 is a list of molecules able to form micelles.
[0020] Table 2 lists several fusogenic peptides and describes their
properties, along with a reference.
[0021] Table 3 lists simple Nuclear Localization Signal (NLS)
peptides.
[0022] Table 4 shows a list of "bipartite" or "split" NLS
peptides.
[0023] Table 5 lists "nonpositive NLS" peptides lacking clusters of
arginines/lysines.
[0024] Table 6 lists peptides with nucleolar localization signals
(NoLS).
[0025] Table 7 lists peptides having karyophilic clusters on
non-membrane protein kinases.
[0026] Table 8 lists peptide nuclear localization signals on DNA
repair proteins.
[0027] Table 9 lists NLS peptides in transcription factors.
[0028] Table 10 lists NLS peptides in other nuclear proteins.
Modes for Carrying out the Invention
[0029] Definitions
[0030] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of immunology,
molecular biology, microbiology, cell biology and recombinant DNA.
These methods are described in the following publications. See,
e.g., Sambrook, et al. MOLECULAR CLONING: A LABORATORY MANUAL,
.sub.2 d Edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F.
M. Ausubel, et al. eds., (1987); the series METHODS IN ENZYMOLOGY
(Academic Press, Inc.); PCR: A PRACTICAL APPROACH, M. MacPherson,
et al., IRL Press at Oxford University Press (1991); PCR 2: A
PRACTICAL APPROACH, MacPherson et al., eds. (1995); ANTIBODIES, A
LABORATORY MANUAL, Harlow and Lane, eds. (1988); and ANIMAL CELL
CULTURE, R. I. Freshney, ed. (1987).
[0031] As used in the specification and claims, the singular form
"a," "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes
a plurality of cells, including mixtures thereof.
[0032] The term "comprising" is intended to mean that the
compositions and methods include the recited elements, but not
excluding others. "Consisting essentially of" when used to define
compositions and methods, shall mean excluding other elements of
any essential significance to the combination. Thus, a composition
consisting essentially of the elements as defined herein would not
exclude trace contaminants from the isolation and purification
method and pharmaceutically acceptable carriers, such as phosphate
buffered saline, preservatives, and the like. "Consisting of" shall
mean excluding more than trace elements of other ingredients and
substantial method steps for administering the compositions of this
invention. Embodiments defined by each of these transition terms
are within the scope of this invention.
[0033] The terms "polynucleotide" and "nucleic acid molecule" are
used interchangeably to refer to polymeric forms of nucleotides of
any length. The polynucleotides may contain deoxyribonucleotides,
ribonucleotides, and/or their analogs. Nucleotides may have any
three-dimensional structure, and may perform any function, known or
unknown. The term "polynucleotide" includes, for example, single-,
double-stranded and triple helical molecules, a gene or gene
fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,
recombinant polynucleotides, branched polynucleotides, plasmids,
vectors, isolated DNA of any sequence, isolated RNA of any
sequence, nucleic acid probes, and primers. A nucleic acid molecule
may also comprise modified nucleic acid molecules.
[0034] A "gene" refers to a polynucleotide containing at least one
open reading frame that is capable of encoding a particular
polypeptide or protein after being transcribed and translated.
[0035] A "gene product" refers to the amino acid (e.g., peptide or
polypeptide) generated when a gene is transcribed and
translated.
[0036] The following abbreviations are used herein: DDAB:
dimethyldioctadecyl ammonium bromide (same as
N,N-distearyl-N,N-dimethyla- mmonium bromide); DODAC:
N,N-dioleyl-N,N-dimethylammonium chloride; DODAP:
1,2-dioleoyl-3-dimethylammonium propane; DMRIE:
N-[1-(2,3-dimyristyloxy)p- ropyl]-N,N-dimethyl-N-(2-hydroxyethyl)
ammonium bromide; DMTAP: 1,2-dimyristoyl-3-trimethylammonium
propane; DOGS: Dioctadecylamidoglycylspermine; DOTAP (same as
DOTMA): N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium
chloride; DOSPA:
N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethy-
l ammonium trifluoroacetate; DPTAP: 1,2-
dipalmitoyl-3-trimethylammonium propane; DSTAP: 1
,2-disteroyl-3-trimethylammonium propane; DOPE,
1,2-sn-dioleoylphoshatidylethanolamine; DC-Chol,
3.beta.-(N--(N',N'-dimet- hylaminoethane)carbamoyl)cholesterol.
See, Gao et al., Biochem. Biophys. Res. Comm. 179:280-285
(1991).
[0037] As used herein, the term "pharmaceutically acceptable anion"
refers to anions of organic and inorganic acids that provide
non-toxic salts in pharmaceutical preparations. Examples of such
anions include the halides anions, chloride, bromide, and iodide,
inorganic anions such as sulfate, phosphate, and nitrate, and
organic anions. Organic anions may be derived from simple organic
acids, such as acetic acid, propionic acid, glycolic acid, pyruvic
acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic,
acid, fumaric acid, tartaric acid, citric acid, benzoic acid,
cinnamic acid, mandelic acid, methane sulfonic acid, ethane
sulfonic acid, p-toluenesulfonic acid, and the like. The
preparation of pharmaceutically acceptable salts is described in
Berge, et al., J. Pharm. Sci. 66:1-19 (1977), incorporated herein
by reference.
[0038] Physiologically acceptable carriers, excipients or
stabilizers are nontoxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, and other organic acids; antioxidants including ascorbic
acid; low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA: sugar alcohols such as mannitol or sorbitol; salt-forming
counter ions such as sodium; and/or nonionic surfactants such as
Tween, Pluronics or polyethylene glycol (PEG). PEG molecules also
contain a fusogenic peptide with an attached Nuclear Localization
Signal (NLS) covalently linked to the end of the PEG molecule.
[0039] The term "cationic lipid" refers to any of a number of lipid
species that carry a net positive charge at physiological pH. Such
lipids include, but are not limited to, DDAB, DMRIE, DODAC, DOGS,
DOTAP, DOSPA and DC-Chol. Additionally, a number of commercial
preparations of cationic lipids are available that can be used in
the present invention. These include, for example, LIPOFECTIN
(commercially available cationic liposomes comprising DOTMA and
DOPE, from GIBCO/BRL, Grand Island, N.Y., USA); LIPOFECTAMINE
(commercially available cationic liposomes comprising DOSPA and
DOPE, from GIBCO/BRL); and TRANSFECTAM (commercially available
cationic lipids comprising DOGS in ethanol from Promega Corp.,
Madison, Wis., USA).
[0040] This invention further provides a number of methods for
producing micelles with entrapped therapeutic drugs. The method is
particularly useful to produce micelles of drugs or compositions
having a net overall negative charge, e.g., DNA, RNA or negatively
charged small molecules. For example, the DNA can be comprised
within a plasmid vector and encode for a therapeutic protein, e.g.,
wild-type p53, HSV-tk, p21, Bax, Bad, IL-2, IL-12, GM-CSF,
angiostatin, endostatin and oncostatin. In one embodiment, the
method requires combining an effective amount of the therapeutic
agent with an effective amount of cationic lipids. Cationic lipids
useful in the methods of this invention include, but are not
limited to, DDAB, dimethyldioctadecyl ammonium bromide; DMRIE:
N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)
ammonium bromide; DMTAP: 1,2-dimyristoyl-3-trimethylammonium
propane; DOGS: Dioctadecylamidoglycylspermine; DOTAP (same as
DOTMA): N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium
chloride; DPTAP: 1,2-dipalmitoyl-3-trimethylammonium propane;
DSTAP: 1 ,2-disteroyl-3-trimethylammonium propane.
[0041] In one aspect, a ratio of from about 30 to about 90% of
phosphates contained within the negatively charged therapeutic
agent are neutralized by positive charges on lipid molecules
(negative charges are in excess) to form an electrostatic micelle
complex in an effective concentration of ethanol. In one aspect,
the ethanol solution is from about 20% to about 80% ethanol. In a
further aspect, the ethanol concentration is about 30%. The
ethanol/cationic lipid/therapeutic agent complex is then combined
with an effective amount of a fusogenic-karyophilic peptide
conjugate. In one aspect, an effective amount of the conjugate is a
ratio range from about 0.0 to about 0.3 (positive charges on
peptide to negative charges on phosphate groups) to neutralize the
majority of the remaining negative charges on the phosphate groups
of the therapeutic agents thereby leading to an almost complete
neutralization of the complex. The optimal conditions give to the
complex a slightly negative charge. However, when the positive
charges on cationic lipids exceed the negative charges on the DNA,
the excess of positive charges are neutralized by DPPG (dipalmitoyl
phosphatidyl glycerol) and its derivatives, or by other anionic
lipid molecules in the final micelle complex.
[0042] In an alternative embodiment, the above methods can be
modified by addition of DNA condensing agents selected from
spermine, spermidine, and magnesium or other divalent metal ions
neutralizing a certain percentage (1-20%) of phosphate groups.
[0043] In a further embodiment, the cationic lipids are combined
with an effective amount of fusogenic lipid DOPE at various molar
ratios for example, in a molar ratio of from about 1:1 cationic
lipid:DOPE. In an alternative embodiment, the cationic lipids are
combined with an effective amount of a fusogenic/NLS peptide
conjugate. Examples of fusogenic/NLS peptide conjugates include,
but are not limited to (KAWLKAF).sub.3 (SEQ ID NO:1),
GLFKAAAKLLKSLWKLLLKA (SEQ ID NO:2), LLLKAFAKLLKSLWKLLLKA (SEQ ID
NO:3), as well as all derivatives of the prototype
(Hydrophobic3-Karyophilic1-Hydrophobic2-Karyophilic1).sub.2-3 where
Hydrophobic is any of the A, I, L, V, P, G, W, F and Karyophilic is
any of the K, R, or H, containing a positively-charged residue
every 3rd or 4th amino acid, which form alpha helices and direct a
net positive charge to the same direction of the helix. Additional
examples include but are not limited to GLFKAIAGFIKNGWKGMIDGGGYC
(SEQ ID NO:4) from influenza virus hemagglutinin HA-2; YGRKKRRQRRR
(SEQ ID NO:5) from TAT of HIV; MSGTFGGILAGLIGLL(K/R/H).sub.1-6 (SEQ
ID NO:6), derived from the N-terminal region of the S protein of
duck hepatitis B virus, but with the addition of one to six
positively-charged lysine, arginine or histidine residues, and
combinations of these, able to interact directly with the phosphate
groups of plasmid or oligonucleotide DNA, compensating for part of
the positive charges provided by the cationic lipids.
GAAIGLAWIPYFGPAA (SEQ ID NO:7) is derived from the fusogenic
peptide of the Ebola virus transmembrane protein; residues 53-70
(C-terminal helix) of apolipoprotein (apo) AII peptide; the
23-residue fusogenic N-terminal peptide of HIV-1 transmembrane
glycoprotein gp41; the 29-42-residue fragment from Alzheimer's
.beta.-amyloid peptide; the fusion peptide and N-terminal heptad
repeat of Sendai virus; the 56-68 helical segment of lecithin
cholesterol acyltransferase. Included within these embodiments are
shorter versions of these peptides, that are known to induce fusion
of unilamellar lipid vesicles or all that are similarly derivatized
with the addition of one to six positively-charged lysine, arginine
or histidine residues (K/R/H).sub.1-6 able to interact directly
with the phosphate groups of plasmid or oligonucleotide DNA,
compensating for part of the positive charges provided by the
cationic lipids. The fusogenic peptides in the fusogenic/NLS
conjugates represent hydrophobic amino acid stretches, and smaller
fragments of these peptide sequences, that include all signal
peptide sequences used in membrane or secreted proteins that insert
into the endoplasmic reticulum. Alternatively, the conjugates
represent transmembrane domains and smaller fragments of these
peptide sequences.
[0044] In one aspect of the invention, the NLS peptide component in
fusogenic/NLS peptide conjugates is derived from the fusogenic
hydrophobic peptides. However, there is an addition of 5-6 amino
acid karyophilic Nuclear Localization Signals (NLS) derived from a
number of known NLS peptides, as well as from searches of the
nuclear protein databases, for stretches of five or more
karyophilic amino acid stretches in proteins containing at least
four positively-charged amino aids flanked by a proline (P) or
glycine (G). Examples of NLS peptides are shown in Tables 1-8. The
NLS peptide component in fusogenic/NLS peptide conjugates are
synthetic peptides containing the above said NLS, but further
modified by additional K, R, H residues at the central part of the
peptide or with P or G at the N- or C-terminus.
[0045] In a further aspect, the fusogenic/NLS peptide conjugates
are derived from the said fusogenic hydrophobic peptides but with
the addition of a stretch of H.sub.4-6 (four to six histidine
residues) in the place of NLS. Micelle formation takes place at pH
5-6 where histidyl residues are positively charged but lose their
charge at the nearly neutral pH of the biological fluids, thus
releasing the plasmid or oligonucleotide DNA from their
electrostatic interaction.
[0046] The fusogenic peptide/NLS peptide conjugates are linked to
each other with a short amino acid stretch representing an
endogenous protease cleavage site.
[0047] In a preferred aspect of the invention, the structure of the
preferred prototype fusogenic/NLS peptide conjugate used in this
invention is: PKKRRGPSP(L/A/I).sub.12-20 (SEQ ID NO:8), where
(L/A/I).sub.12-20 is a stretch of 12-20 hydrophobic amino acids
containing A, L, I, Y, W, F and other hydrophobic amino acids.
[0048] The micelles made by the above methods are further provided
by this invention by conversion into liposomes. An effective amount
of liposomes (diameter from about 80 to about 160 nm), or of a
lipid solution composed of cholesterol (from about 10% to about
50%), neutral phospholipid such as hydrogenated soy
phosphatidylcholine (HSPC) (from about 40% to about 90%), and the
derivatized vesicle-forming lipid PEG-DSPE (distearoylphosphatidyl
ethanolamine) from about 1-to about 7 mole percent, is added to the
micelle solution.
[0049] In a specific embodiment, the liposomes are composed of
vesicle-forming lipids and between from about 1 to about 7 mole
percent of distearoylphosphatidyl ethanolamine (DSPE) derivatized
with a polyethyleneglycol. The composition of claim 20, wherein the
polyethyleneglycol has a molecular weight is between about 1,000 to
5,000 daltons. Micelles are converted into liposomes with a
concomitant decrease of the ethanol concentration which can be
accomplished by removal of the ethanol by dialysis of the liposome
complexes through permeable membranes or reduced to a diameter of
80-160 nm by extrusion through membranes.
[0050] Liposome encapsulated therapeutic agents produced by the
above methods are further provided by this invention.
[0051] Also provided herein is a method for delivering a
therapeutic agent such as plasmid DNA or oligonucleotides to a
tissue cell in vivo by intravenous, or other type of injection of
the micelles or liposomes. This method specifically targets a
primary tumor and the metastases by the long circulating time of
the micelle or liposome complex because of the exposure of PEG
chains on its surface, its small size (80-160 nm) and the decrease
in hydrostatic pressure in the solid tumor from the center to its
periphery supporting a preferential extravasation through the tumor
vasculature to the extracellular space in tumors. A method for
delivering plasmid or oligonucleotide DNA across the cell membrane
barrier of the tumors using the micelle or liposome complexes
described herein is capable because of the presence of the
fusogenic peptides in the complex. In particular, a method for
delivering plasmid or oligonucleotide DNA to the liver, spleen and
bone marrow after intravenous injection of the complexes is
provided. Further provided is a method for delivering therapeutic
genes to the liver, spleen and bone marrow of cancer and noncancer
patients including but not limited to, factor VIII or IX for the
therapy of hemophilias, multidrug resistance, cytokine genes for
cancer immunotherapy, genes for the alleviation of pain, genes for
the alleviation of diabetes and genes that can be introduced to
liver, spleen and bone marrow tissue, to produce a secreted form of
a therapeutic protein.
[0052] The disclosed therapies also provide methods for reducing
tumor size by combining the encapsulated plasmid DNA carrying one
or more anticancer genes selected from the group consisting of p53,
RB, BRCA1, E1A, bc1-2, MDR-1, p21, p16, bax, bc1-xs, E2F, IGF-I
VEGF, angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2,
IL-4, IL-7, IFN-.gamma., TNF-.alpha., HSV-tk (in combination with
ganciclovir), E. coli cytosine deaminase (in combination with
5-fluorocytosine) with encapsulated antisense oligonucleotides
(antisense c-fos, c-myc, K-ras), ribozymes or triplex-forming
oligonucleotides directed against genes that control the cell cycle
or signaling pathways. These methods can be modified by combining
the encapsulated plasmid DNA carrying one or more anticancer genes
of with encapsulated or free antineoplastic drugs, consisting of
the group of adriamycin, angiostatin, azathioprine, bleomycin,
busulfane, camptothecin, carboplatin, carmustine, chlorambucile,
chlormethamine, chloroquinoxaline sulfonamide, cisplatin,
cyclophosphamide, cycloplatam, cytarabine, dacarbazine,
dactinomycin, daunorubicin, didox, doxorubicin, endostatin,
enloplatin, estramustine, etoposide, extramustinephosphat,
flucytosine, fluorodeoxyuridine, fluorouracil, gallium nitrate,
hydroxyurea, idoxuridine, interferons, interleukins, leuprolide,
lobaplatin, lomustine, mannomustine, mechlorethamine,
mechlorethaminoxide, melphalan, mercaptopurine, methotrexate,
mithramycin, mitobronitole, mitomycin, mycophenolic acid,
nocodazole, oncostatin, oxaliplatin, paclitaxel, pentamustine,
platinum-triamine complex, plicamycin, prednisolone, prednisone,
procarbazine, protein kinase C inhibitors, puromycine, semustine,
signal transduction inhibitors, spiroplatin, streptozotocine,
stromelysin inhibitors, taxol, tegafur, telomerase inhibitors,
teniposide, thalidomide, thiamiprine, thioguanine, thiotepa,
tiamiprine, tretamine, triaziquone, trifosfamide, tyrosine kinase
inhibitors, uramustine, vidarabine, vinblastine, vinca alcaloids,
vincristine, vindesine, vorozole, zeniplatin, zeniplatin, and
zinostatin.
[0053] The following examples are intended to illustrate, but not
limit the invention.
[0054] Liposome Composition
[0055] Liposomes are microscopic vesicles consisting of concentric
lipid bilayers. Structurally, liposomes range in size and shape
from long tubes to spheres, with dimensions from a few hundred
Angstroms to fractions of a millimeter. Vesicle-forming lipids are
selected to achieve a specified degree of fluidity or rigidity of
the final complex providing the lipid composition of the outer
layer. These are neutral (cholesterol) or bipolar and include
phospholipids, such as phosphatidylcholine (PC),
phosphatidylethanolamine (PE), phosphatidylinositol (PI), and
sphingomyelin (SM) and other type of bipolar lipids including but
not limited to dioleoylphosphatidylethanolamine (DOPE), with a
hydrocarbon chain length in the range of 14-22, and saturated or
with one or more double C.dbd.C bonds. Examples of lipids capable
of producing a stable liposome, alone, or in combination with other
lipid components are phospholipids, such as hydrogenated soy
phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine,
lysolecithin, lysophosphatidylethanol- amine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, cephalin, cardiolipin,
phosphatidic acid, cerebrosides, distearoylphosphatidylethan-
olamine (DSPE), dioleoylphosphatidylcholine (DOPC),
dipalmitoylphosphatidylcholine (DPPC),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoylphosphatidylethanolamine (POPE) and
dioleoylphosphatidylethanolamine
4-(N-maleimido-methyl)cyclohexane-1-carb- oxylate (DOPE-mal).
Additional non-phosphorous containing lipids that can become
incorporated into liposomes include stearylamine, dodecylamine,
hexadecylamine, isopropyl myristate, triethanolamine-lauryl
sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol
ricinoleate, hexadecyl stereate, amphoteric acrylic polymers,
polyethyloxylated fatty acid amides, and the cationic lipids
mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA),
DOSPA, DPTAP, DSTAP, DC-Chol). Negatively charged lipids include
phosphatidic acid (PA), dipalmitoylphosphatidylgly- cerol (DPPG),
dioleoylphosphatidylglycerol and (DOPG), dicetylphosphate that are
able to form vesicles. Preferred lipids for use in the present
invention are cholesterol, hydrogenated soy phosphatidylcholine
(HSPC) and, the derivatized vesicle-forming lipid PEG-DSPE.
[0056] Typically, liposomes can be divided into three categories
based on their overall size and the nature of the lamellar
structure. The three classifications, as developed by the New York
Academy Sciences Meeting, "Liposomes and Their Use in Biology and
Medicine," December 1977, are multi-lamellar vesicles (MLVs), small
uni-lamellar vesicles (SUVs) and large uni-lamellar vesicles
(LUVs).
[0057] SUVs range in diameter from approximately 20 to 50 nm and
consist of a single lipid bilayer surrounding an aqueous
compartment. Unilamellar vesicles can also be prepared in sizes
from about 50 nm to 600 nm in diameter. While unilamellar are
single compartmental vesicles of fairly uniform size, MLVs vary
greatly in size up to 10,000 nm, or thereabouts, are
multi-compartmental in their structure and contain more than one
bilayer. LUV liposomes are so named because of their large diameter
that ranges from about 600 nm to 30,000 nm; they can contain more
than one bilayer.
[0058] Liposomes may be prepared by a number of methods not all of
which produce the three different types of liposomes. For example,
ultrasonic dispersion by means of immersing a metal probe directly
into a suspension of MLVs is a common way for preparing SUVs.
[0059] Preparing liposomes of the MLV class usually involves
dissolving the lipids in an appropriate organic solvent and then
removing the solvent under a gas or air stream. This leaves behind
a thin film of dry lipid on the surface of the container. An
aqueous solution is then introduced into the container with
shaking, in order to free lipid material from the sides of the
container. This process disperses the lipid, causing it to form
into lipid aggregates or liposomes. Liposomes of the LUV variety
may be made by slow hydration of a thin layer of lipid with
distilled water or an aqueous solution of some sort. Alternatively,
liposomes may be prepared by lyophilization. This process comprises
drying a solution of lipids to a film under a stream of nitrogen.
This film is then dissolved in a volatile solvent, frozen, and
placed on a lyophilization apparatus to remove the solvent. To
prepare a pharmaceutical formulation containing a drug, a solution
of the drug is added to the lyophilized lipids, whereupon liposomes
are formed.
[0060] Preparing Cationic Liposome/Cationic Peptide/Nucleic Acid
Micelles
[0061] Cationic lipids, with the exception of sphingosine and some
lipids in primitive life forms, do not occur in nature. The present
invention uses single-chain amphiphiles which are chloride and
bromide salts of the alkyltrimethylammonium surfactants including
but not limited to C12 and C16 chains abbreviated DDAB (same as
DODAB) or CTAB. The molecular geometry of these molecules
determines the critical micelle concentration (ratio between free
monomers in solution and molecules in micelles). Lipid exchange
between the two states is a highly dynamic process; phospholipids
have critical micelle concentration values below 10.sup.-8 M and
are more stable in liposomes; however, single chain detergents,
such as stearylamine, may emerge from the liposome membrane upon
dilution or intravenous injection in milliseconds (Lasic,
1997).
[0062] Cationic lipids include, but are not limited to, DDAB:
dimethyldioctadecyl ammonium bromide (same as
N,N-distearyl-N,N-dimethyla- mmonium bromide); DMRIE:
N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2- -hydroxyethyl)
ammonium bromide; DODAC: N,N-dioleyl-N,N-dimethylammonium chloride;
DMTAP: 1,2-dimyristoyl-3-trimethylammonium propane; DODAP:
1,2-dioleoyl-3-dimethylammonium propane; DOGS:
Dioctadecylamidoglycylsper- mine; DOTAP (same as DOTMA):
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethyl- ammonium chloride;
DOSPA: N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarbo-
xamido)ethyl)-N,N-dimethyl ammonium trifluoroacetate; DPTAP:
1,2-dipalmitoyl-3-trimethylammonium propane; DSTAP:
1,2-disteroyl-3-trimethylammonium propane; DC-Chol,
3.beta.-(N--(N',N'-dimethylaminoethane)carbamoyl)cholesterol.
[0063] Lipid-based vectors used in gene transfer have been
formulated in one of two ways. In one method, the nucleic acid is
introduced into preformed liposomes made of mixtures of cationic
lipids and neutral lipids. The complexes thus formed have undefined
and complicated structures and the transfection efficiency is
severely reduced by the presence of serum. Preformed liposomes are
commercially available as LIPOFECTIN and LIPOFECTAMINE. The second
method involves the formation of DNA complexes with mono- or
poly-cationic lipids without the presence of a neutral lipid. These
complexes are prepared in the presence of ethanol and are not
stable in water. Additionally, these complexes are adversely
affected by serum (see, Behr, Acc. Chem. Res. 26:274-78 (1993)). An
example of a commercially available poly-cationic lipid is
TRANSFECTAM. Other efforts to encapsulate DNA in lipid-based
formulations have not overcome these problems (see, Szoka et al.,
Ann. Rev. Biophys. Bioeng. 9:467 (1980); and Deamer, U.S. Pat. No.
4,515,736).
[0064] The nucleotide polymers can be single-stranded DNA or RNA,
or double-stranded DNA or DNA--RNA hybrids. Examples of
double-stranded DNA include structural genes, genes including
control and termination regions, and self-replicating systems such
as plasmid DNA. Particularly preferred nucleic acids are plasmids.
Single-stranded nucleic acids include antisense oligonucleotides
(complementary to DNA and RNA), ribozymes and triplex-forming
oligonucleotides. In order to increase stability, some
single-stranded nucleic acids will preferably have some or all of
the nucleotide linkages substituted with stable, non-phosphodiester
linkages, including, for example, phosphorothioate,
phosphorodithioate, phosphoroselenate, methylphosphonate, or
O-alkyl phosphotriester linkages.
[0065] Encapsulating Cationic Liposome/Cationic Peptide/Nucleic
Acid Micelles into Neutral Liposomes
[0066] Cationic lipids used with fusogenic peptide/NLS conjugates
to provide the inner layer of the particle can be any of a number
of substances selected from the group of DDAB, DODAC, DMRIE, DMTAP,
DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol. The cationic
lipid is combined with DOPE. In one group of embodiments, the
preferred cationic lipid is DDAB:DOPE 1: 1.
[0067] Neutral lipids used herein to provide the outer layer of the
particles can be any of a number of lipid species that exist either
in an uncharged or neutral zwitterionic form at physiological pH.
Such lipids are selected from a group consisting of
diacylphosphatidylcholine, diacylphosphatidylethanolamine,
ceramide, sphingomyelin, cephalin, and cerebrosides. In one group
of embodiments, lipids containing saturated, mono-, or
di-unsaturated fatty acids with carbon chain lengths in the range
of C14 to C22 are preferred. In general, less saturated lipids are
more easily sized, particularly when the liposomes must be sized
below about 0.16 microns, for purposes of filter sterilization.
Consideration of liposome size, rigidity and stability of the
liposomes in the final preparation, its shelf life without leakage
of the encapsulated DNA, and stability in the bloodstream generally
guide the selection of neutral lipids for providing the outer
coating of our gene vehicles. Lipids having a variety of acyl chain
groups of varying chain length and degree of saturation are
available or may be isolated or synthesized by well-known
techniques. In another group of embodiments, lipids with carbon
chain lengths in the range of C14 to C22 are used. Preferably, the
neutral lipids used in the present invention are hydrogenated soy
phosphatidylcholine (HSPC), cholesterol, and
PEG-distearoylphosphatidyl ethanolamine (DSPE) or PEG-ceramide.
[0068] Methods for Preparing Liposomes
[0069] A variety of methods for preparing various liposome forms
have been described in several issued patents, for example, U.S.
Pat. Nos. 4,229,360; 4,224,179; 4,241,046; 4,737,323; 4,078,052;
4,235,871; 4,501,728; and 4,837,028, as well as in the articles
Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980) and Hope et
al., Chem. Phys. Lip. 40:89 (1986). These methods do not produce
all three different types of liposomes (MLVs, SUVs, LUVs). For
example, ultrasonic dispersion by means of immersing a metal probe
directly into a suspension of MLVs is a common way for preparing
SUVs.
[0070] Preparing liposomes of the MLV class usually involves
dissolving the lipids in an appropriate organic solvent and then
removing the solvent under a gas or air stream. This leaves behind
a thin film of dry lipid on the surface of the container. An
aqueous solution is then introduced into the container with
shaking, in order to free lipid material from the sides of the
container. This process disperses the lipid, causing it to form
into lipid aggregates or liposomes. Liposomes of the LUV variety
may be made by slow hydration of a thin layer of lipid with
distilled water or an aqueous solution of some sort. Alternatively,
liposomes may be prepared by lyophilization. This process comprises
drying a solution of lipids to a film under a stream of nitrogen.
The film is then dissolved in a volatile solvent, frozen, and
placed on a lyophilization apparatus to remove the solvent. To
prepare a pharmaceutical formulation containing a drug, a solution
of the drug is added to the lyophilized lipids, whereupon liposomes
are formed.
[0071] Following liposome preparation, the liposomes may be sized
to achieve a desired size range and relatively narrow distribution
of liposome sizes. Preferably, the preformed liposomes are sized to
a mean diameter of about 80 to 160 nm (the upper size limit for
filter sterilization before in vivo administration). Several
techniques are available for sizing liposomes to a desired size.
Sonicating a liposome suspension either by bath or probe sonication
produces a progressive size reduction down to small unilamellar
vesicles less than about 0.05 microns (50 nm) in size. Extrusion of
liposome through a small-pore polycarbonate is our preferred method
for reducing liposome sizes to a relatively well-defined size
distribution. The liposomes may be extruded through successively
smaller-pore membranes, to achieve a gradual reduction in liposome
size.
[0072] One way used to coat DNA with lipid is by controlled
detergent depletion from a cationic lipid/DNA/detergent complex.
This method can give complexes with stability in plasma. Hofland et
al. (1996), have prepared such complexes by dialysis of a mixture
of DOSPA/DOPE/DNA/octylglucoside.
[0073] Pharmaceutical compositions comprising the cationic
liposome/nucleic acid complexes of the invention are prepared
according to standard techniques and further comprise a
pharmaceutically acceptable carrier. Generally, normal saline will
be employed as the pharmaceutically acceptable carrier.
[0074] For in vivo administration, the pharmaceutical compositions
are preferably administered parenterally, i.e., intravenously,
intraperitoneally, subcutaneously, intrathecally, injection to the
spinal cord, intramuscularly, intraarticularly, portal vein
injection, or intratumorally. More preferably, the pharmaceutical
compositions are administered intravenously or intratumorally by a
bolus injection. In other methods, the pharmaceutical preparations
may be contacted with the target tissue by direct application of
the preparation to the tissue. The application may be made by
topical "open" or "closed" procedures. The term "topical" means the
direct application of the pharmaceutical preparation to a tissue
exposed to the environment, such as the skin, to any surface of the
body, nasopharynx, external auditory canal, ocular administration
and administration to the surface of any body cavities, inhalation
to the lung, genital mucosa and the like.
[0075] "Open" procedures are those procedures that include incising
the skin of a patient and directly visualizing the underlying
tissue to which the pharmaceutical preparations are applied. This
is generally accomplished by a surgical procedure, such as a
thoracotomy to access the lungs, abdominal laparotomy to access
abdominal viscera, or other direct surgical approach to the target
tissue.
[0076] "Closed" procedures are invasive procedures in which the
internal target tissues are not directly visualized, but accessed
via insertion of instruments through small wounds in the skin. For
example, the preparations may be administered to the peritoneum by
needle lavage. Likewise, the pharmaceutical preparations may be
administered to the meninges or spinal cord by infusion during a
lumbar puncture followed by appropriate positioning of the patient
as commonly practiced for spinal anesthesia or metrazamide imaging
of the spinal cord. Alternatively, the preparations may be
administered through endoscopic devices.
EXAMPLES
[0077] Materials and Methods
[0078] DDAB, DOPE (dioleoylphosphatidylethanolamine) and most other
lipids used here were purchased from Avanti Polar Lipids; PEG-DSPE
was from Syngena.
[0079] Engineering of Plasmid pLF
[0080] The pGL3-C (Promega) was cut with XbaI and blunt-end ligated
using the Klenow fragment of E. coli DNA polymerase. It was then
cut with HindIII and the 1689-bp fragment, carrying the luciferase
gene, was gel-purified. The pGFP-N1 plasmid (Clontech) was cut with
SmaI and HindIII and the 4.7 kb fragment, isolated from an agarose
gel, was ligated with the luciferase fragment. JM109 E. Coli cells
were transformed and 20 colonies were selected; about half of them
showed the presence of inserts; 8 clones with inserts were cut with
BamHI and XhoI to further confirm the presence of the luciferase
gene; seven of them were positive.
[0081] Radiolabeled plasmid pLF was generated by culturing
Escherichia coli in .sup.3H-thymidine-5'-triphosphate or .sup.32P
inorganic phosphate (5 mCi) (Dupont/NEN, Boston, Mass.) and
purified using standard techniques as described above.
[0082] DLS Measurements
[0083] A Coulter N4M light scattering instrument was used, at a
90.degree. angle, set at a run time of 200 sec, using 4 to 25
microsec sample time. The scan of the particle size distribution
was obtained in 1 ml sample volume using plastic cuvettes, at
20.degree. C. and at 0.01 poise viscosity.
[0084] In one aspect, this invention provides a method for
entrapping DNA into lipids that enhances the content of plasmid per
volume unit, and reduces the toxicity of the cationic lipids used
to trap plasmid or oligonucleotide DNA. The DNA becomes hidden in
the inner membrane bilayer of the final complex. Furthermore, the
gene transfer complex is endowed with long circulation time in body
fluids and extravasates preferentially into solid tumors and their
metastatic foci and nodules. The extravasation occurs through their
vasculature at most sites of the human or animal body after
intravenous injection of the gene-carrying vehicles. This occurs
because of their small size (100-160 nm), their content in neutral
to slightly negatively-charged lipids in their outer membrane
bilayers, and their coating with PEG. These gene delivery vehicles
are able to cross the cell membrane barrier after they reach the
extracellular tumor space because of the presence of fusogenic
peptides conjugated with karyophilic peptides. The vehicles assume
a certain predefined orientation in the lipid membrane with their
positive ends directed toward DNA and their hydrophobic tail buried
inside the hydrophobic lipid bilayer. The labile NLS-fusogenic
peptide linkage is cleaved after endocytosis and the remaining NLS
peptide bound to plasmid DNA aids its nuclear uptake. This occurs
especially when non-dividing cells are targeted, such as liver,
spleen or bone marrow cells that represent the major sites for
extravasation and concentration of these vehicles other than solid
tumors.
[0085] Organic Solvent
[0086] A suitable solvent for preparing a micelle from the desired
lipid components is ethanol, methanol, or other aliphatic alcohols
such as propanol, isopropanol, butanol, tert-butanol, iso-butanol,
pentanol and hexanol. Mixtures of two or more solvents may be used
in the practice of the invention. It is also to be understood that
any solvent that is miscible with an ethanol solution, even in
small amounts, can be used to improve micelle formation and its
subsequent conversion into liposomes, including chloroform,
dichloromethane, diethylether, cyclohexane, cyclopentane, benzene,
and toluene.
[0087] Cationic Lipids
[0088] In a further embodiment, the liposome encapsulated DNA
described herein further comprises an effective amount of cationic
lipids. Cationic lipids have been widely used for gene transfer; a
number of clinical trials (34 out of 220 total RAC-approved
protocols as of December, 1997) use cationic lipids. Although many
cell culture studies have been documented, systemic delivery of
genes with cationic lipids in vivo has been very limited. All
clinical protocols use subcutaneous, intradermal, intratumoral, and
intracranial injection as well as intranasal, intrapleural, or
aerosol administration but not I.V. delivery, because of the
toxicity of the cationic lipids and DOPE (see, Martin and Boulikas,
1998). Liposomes formulated from DOPE and cationic lipids based on
diacyltrimethylammonium propane (dioleoyl-, dimyristoyl-,
dipalmitoyl-, disteroyl-trimethylammonium propane or DOTAP, DMTAP,
DPTAP, DSTAP, respectively) or DDAB were highly toxic when
incubated in vitro with phagocytic cells (macrophages and U937
cells), but not towards non-phagocytic T lymphocytes. The rank
order of toxicity was DOPE/DDAB >DOPE/DOTAP >DOPE/DMTAP
>DOPE/DPTAP >DOPE/DSTAP; and the toxicity was determined from
the effect of the cationic liposomes on the synthesis of nitric
oxide (NO) and TNF-.alpha. produced by activated macrophages
(Filion and Phillips, 1997).
[0089] Another aspect to be considered before I.V. injection is
undertaken, is that negatively charged serum proteins can interact
and cause inactivation of cationic liposomes (Yang and Huang,
1997). Condensing agents used for plasmid delivery including
polylysine, transferrin-polylysine, a fifth-generation
poly(amidoamine) (PAMAM) dendrimer, poly(ethyleneimine), and
several cationic lipids (DOTAP, DC-Chol/DOPE, DOGS/DOPE, and
DOTMA/DOPE), were found to activate the complement system to
varying extents. Strong complement activation was seen with
long-chain polylysines, the dendrimer, poly(ethyleneimine), and
DOGS. Modifying the surface of preformed DNA complexes with
polyethyleneglycol (Plank et al., 1996) considerably reduced
complement activation.
[0090] Cationic lipids increase the transfection efficiency by
destabilizing the biological membranes, including plasma,
endosomal, and lysosomal membranes. Incubation of isolated
lysosomes with low concentrations of DOTAP caused a striking
increase in free activity of .beta.-galactosidase, and even a
release of the enzyme into the medium. This demonstrates that the
lysosomal membrane is deeply destabilized by the lipid. The
mechanism of destabilization was thought to involve an interaction
between cationic liposomes and anionic lipids of the lysosomal
membrane, thus allowing a fusion between the lipid bilayers. The
process was less pronounced at pH 5 than at pH 7.4, and anionic
amphipathic lipids were able to prevent partially this membrane
destabilization (Wattiaux et al., 1997).
[0091] In contrast to DOTAP and DMRIE that were 100% charged at pH
7.4, DC-CHOL was only about 50% charged as monitored by a
pH-sensitive fluorophore. This difference decreases the charge on
the external surfaces of the liposomes, and was proposed to promote
an easier dissociation of bilayers containing DC-CHOL from the
plasmid DNA, and an increase in release of the DNA-lipid complex
into the cytosol from the endosomes (Zuidam and Barenholz,
1997).
[0092] Although cationic lipids have been used widely for the
delivery of genes, very few studies have used systemic I.V.
injection of cationic liposome-plasmid complexes. This is because
of the toxicity of the lipid component in animal models, not
humans. Administration by I.V. injection of two types of cationic
lipids of similar structure, DOTMA and DOTAP, shows that the
transfection efficiency is determined mainly by the structure of
the cationic lipid and the ratio of cationic lipid to DNA; the
luciferase and GFP gene expression in different organs was
transient, with a peak level between 4 and 24 hr, dropping to less
than 1% of the peak level by day 4 (Song et al., 1997).
[0093] A number of different organs in vivo can be targeted after
liposomal delivery of genes or oligonucleotides. Intravenous
injection of cationic liposome-plasmid complexes by tail vein in
mice, targeted mainly the lung and to a smaller extent the liver,
spleen, heart, kidney and other organs (Zhu et al., 1993).
Intraperitoneal injection of a plasmid-liposome complex expressing
antisense K-ras RNA in nude mice inoculated i.p. with AsPC-1
pancreatic cancer cells harboring K-ras point mutations and PCR
analysis indicated that the injected DNA was delivered to various
organs except brain (Aoki et al., 1995).
[0094] A number of factors for DOTAP:cholesterol/DNA complex
preparation including the DNA:liposome ratio, mild sonication,
heating, and extrusion were found to be crucial for improved
systemic delivery; maximal gene expression was obtained when a
homogeneous population of DNA:liposome complexes between 200 to 450
nm in size were used. Cryo-electron microscopy showed that the DNA
was condensed on the interior of invaginated liposomes between two
lipid bilayers in these formulations, a factor that was thought to
be responsible for the high transfection efficiency in vivo and for
the broad tissue distribution (Templeton et al., 1997).
[0095] Steps to improve liposome-mediated gene delivery to somatic
cells include, persistence of the plasmid in blood circulation,
port of entry and transport across the cell membrane, release from
endosomal compartments into the cytoplasm, nuclear import by
docking through the pore complexes of the nuclear envelope,
expression driven by the appropriate promoter/enhancer control
elements, and persistence of the plasmid in the nucleus for long
periods (Boulikas, 1998a).
[0096] Plasmid Condensation With Spermine
[0097] In a further embodiment, the liposome encapsulated DNA
described herein is condensed with spermine and/or spermidine. DNA
can be presented to cells in culture as a complex with polycations
such as polylysine, or basic proteins such as protamine, total
histones or specific histone fractions, protamine (Boulikas and
Martin, 1997). The interaction of plasmid DNA with protamine
sulfate, followed by the addition of DOTAP cationic liposomes,
offered a better protection of plasmid DNA against enzymatic
digestion. The method gave consistently higher gene expression in
mice via tail vein injection as compared with DOTAP/DNA complexes.
50 .mu.g of luciferase-plasmid per mouse gave 20 ng luciferase
protein per mg extracted tissue protein in the lung, that was
detected as early as 1 h after injection, peaked at 6 h and
declined thereafter. Intraportal injection of protamine/DOTAP/DNA
led to about a 100-fold decrease in gene expression in the lung as
compared with I.V. injection. Endothelial cells were the primary
locus of lacZ transgene expression (Li and Huang, 1997). Protamine
sulfate enhanced plasmid delivery into several different types of
cells in vitro, using the monovalent cationic liposomal
formulations (DC-Chol and lipofectin). This effect was less
pronounced with the multivalent cationic liposome formulation,
lipofectamine (Sorgi et al., 1997).
[0098] Spermine is found to enhance the transfection efficiency of
DNA-cationic liposome complexes in cell culture and in animal
studies. This biogenic polyamine at high concentrations caused
liposome fusion most likely promoted by the simultaneous
interaction of one molecule of spermine (four positively charged
amino groups) with the polar head groups of two or more molecules
of lipids. At low concentrations (0.03-0.1 mM) it promoted
anchorage of the liposome-DNA complex to the surface of cells and
enhanced significantly transfection efficiency (Boulikas,
unpublished).
[0099] The polycations polybrene, protamine, DEAE-dextran, and
poly-L-lysine significantly increased the efficiency of
adenovirus-mediated gene transfer in cell culture. This was thought
to act by neutralizing the negative charges presented by membrane
glycoproteins that reduce the efficiency of adenovirus-mediated
gene transfer (Arcasoy et al., 1997).
[0100] Oligonucleotide Transfer
[0101] In a further embodiment, the liposome encapsulates
oligonucleotide DNA. Encapsulation of oligonucleotides into
liposomes increased their therapeutic index, prevented degradation
in cultured cells, and in human serum and reduced toxicity to cells
(Thierry and Dritschilo, 1992; Capaccioli et al., 1993; Lewis et
al., 1996). However, most studies have been performed in cell
culture, and very few in animals in vivo. There are still an
important number of improvements needed before these approaches can
move into clinical studies.
[0102] Zelphati and Szoka (1997), have found that complexes of
fluorescently labeled oligonucleotides with DOTAP liposomes,
entered the cell using an endocytic pathway mainly involving
uncoated vesicles. Oligonucleotides were redistributed from
punctate cytoplasmic regions into the nucleus. This process was
independent of acidification of the endosomal vesicles. The nuclear
uptake of oligonucleotides depended on several factors, such as
charge of the particle, where positively charged complexes were
required for enhanced nuclear uptake. DOTAP increased over 100 fold
the antisense activity of a specific anti-luciferase
oligonucleotide. Physicochemical studies of
oligonucleotide-liposome complexes of different cationic lipid
compositions indicated that either phosphatidylethanolamine or
negative charges on other lipids in the cell membrane are required
for efficient fusion with cationic liposome-oligonucleotide
complexes to promote entry to the cell (Jaaskelainen et al.,
1994).
[0103] Similar results were reported by Lappalainen et al. (1997).
Digoxigenin-labeled oligodeoxynucleotides (ODNs) complexed with the
polycationic DOSPA and the monocationic DDAB (with DOPE as a helper
lipid) were taken up by CaSki cells in culture by endocytosis. The
nuclear membrane was found to pose a barrier against nuclear import
of ODNs that accumulated in the perinuclear area. Although
DOSPA/DOPE liposomes could deliver ODNs into the cytosol, they were
unable to mediate nuclear import of ODNs. On the contrary,
oligonucleotide-DDAB/DOP- E complexes with a net positive charge
were released from vesicles into the cytoplasm. It was determined
that DDAB/DOPE mediated nuclear import of the oligonucleotides.
[0104] DOPE-heme (ferric protoporphyrin IX) conjugates, inserted in
cationic lipid particles with DOTAP, protected oligoribonucleotides
from degradation in human serum and increased oligoribonucleotide
uptake into 2.2.15 human hepatoma cells. The enhancing effect of
heme was evident only at a net negative charge in the particles
(Takle et al., 1997). Uptake of liposomes labeled with .sup.111In
and composed of DC-Chol and DOPE was primarily by liver, with some
accumulation in spleen and skin and very little in the lung after
I.V. tail injection. Preincubation of cationic liposomes with
phosphorothioate oligonucleotide induced a dramatic, yet transient,
accumulation of the lipid in lung that gradually redistributed to
liver. The mechanism of lung uptake involved entrapment of large
aggregates of oligonucleotides within pulmonary capillaries at 15
min post-injection via embolism. Labeled oligonucleotide was
localized primarily to phagocytic vacuoles of Kupffer cells at 24 h
post-injection. Nuclear uptake of oligonucleotides in vivo was not
observed (Litzinger et al., 1996).
[0105] Polyethylene Glycol (PEG)-Coated Liposomes
[0106] In a further embodiment, the liposome encapsulated DNA
described herein, further comprise coating of the final complex in
step 2 (FIG. 1) with PEG. It is often desirable to conjugate a
lipid to a polymer that confers extended half-life, such as
polyethylene glycol (PEG). Derivatized lipids that are employed,
include PEG-modified DSPE or PEG-ceramide. Addition of PEG
components prevents complex aggregation, increases circulation
lifetime of particles (liposomes, proteins, other complexes, drugs)
and increases the delivery of lipid-nucleic acid complexes to the
target tissues. See, Maxfield et al., Polymer 16:505-509 (1975);
Bailey, F. E. et al., in: Nonionic Surfactants, Schick, M. J., ed.,
pp. 794-821 (1967); Abuchowski, A. et al., J. Biol. Chem.
252:3582-3586 (1977); Abuchowski, A. et al., Cancer Biochem.
Biophys. 7:175-186 (1984); Katre, N. V. et al., Proc. Natl. Acad.
Sci. USA 84:1487-1491 (1987); Goodson, R. et al. Bio Technology
8:343-346 (1990).
[0107] Conjugation to PEG is reported to have reduced
immunogenicity and toxicity. See, Abuchowski et al., J. Biol. Chem.
252:3578-3581 (1977). The extent of enhancement of blood
circulation time of liposomes, by coating with PEG is described in
U.S. Pat. No. 5,013,556. Typically, the concentration of the
PEG-modified phospholipids, or PEG-ceramide in the complex will be
about 1-7%. In a particularly preferred embodiment, the
PEG-modified lipid is a PEG-DSPE.
[0108] Coating the surface of liposomes with inert materials
designed to camouflage the liposome from the body's host defense
systems was shown to increase remarkably the plasma longevity of
liposomes. The biological paradigm for this "surface modified"
sub-branch was the erythrocyte, a cell that is coated with a dense
layer of carbohydrate groups, and that manages to evade immune
system detection and to circulate for several months (before being
removed by the same type of cell responsible for removing
liposomes).
[0109] The first breakthrough came in 1987 when a glycolipid (the
brain tissue-derived ganglioside GM 1), was identified that, when
incorporated within the lipid matrix, allowed liposomes to
circulate for many hours in the blood stream (Allen and Chonn,
1987). A second glycolipid, phosphatidylinositol, was also found to
impart long plasma residence times to liposomes and, since it was
extracted from soybeans, not brain tissue, was believed to be a
more pharmaceutically acceptable excipient (Gabizon et al.,
1989).
[0110] A major advance in the surface-modified sub-branch was the
development of polymer-coated liposomes (Allen et al. 1991).
Polyethylene glycol (PEG) modification had been used for many years
to prolong the half-lives of biological proteins (such as enzymes
and growth factors) and to reduce their immunogenicity (e.g.
Beauchamp et al., 1983). It was reported in the early 1990s that
PEG-coated liposomes circulated for remarkably long times after
intravenous administration. Half-lives on the order of 24 h were
seen in mice and rats, and over 30 hours in dogs. The term
"stealth" was applied to these liposomes because of their ability
of evade interception by the immune system. The PEG hydrophilic
polymers form dense "conformational clouds" to prevent other
macromolecules from interaction with the surface, even at low
concentrations of the protecting polymer (Gabizon and
Papahadjopoulos, 1988; Papahadjopoulos et al., 1991; reviewed by
Torchilin, 1998). The increased hydrophilicity of the liposomes
after their coating with the amphipathic PEG5000 leads to a
reduction in nonspecific uptake by the reticuloendothelial
system.
[0111] Whereas the half-life of antimyosin immunoliposomes was 40
min, by coating with PEG, they increased their half-life to 1000
min after intravenous injection to rabbits (Torchilin et al.,
1992).
[0112] Micelles, Surfactants and Small Unilamellar Vesicles
[0113] In a further embodiment, the liposome encapsulated DNA
described herein, further comprise an initial step of micelle
formation between cationic lipids and condensed plasmid or
oligonucleotide DNA in ethanol solutions. Micelles are small
amphiphilic colloidal particles formed by certain kinds of lipid
molecules, detergents or surfactants under defined conditions of
concentration, solvent and temperature. They are composed of a
single lipid layer. Micelles can have their hydrophilic head groups
assembled exposing their hydrophobic tails to the solvent (for
example in 30-60% aqueous ethanol solution) or can reverse their
structures exposing their polar heads toward the solvent such as by
lowering the concentration of the ethanol to below 10% (reverse
micelles). Micelle systems are in thermodynamic equilibrium with
the solvent molecules and environment. This results in constant
phase changes, especially upon contact with biological materials,
such as upon introduction to cell culture, injection to animals,
dilution, contact with proteins or other macromolecules. These
changes result in rapid micelle disassembly or flocculation. This
is in contrast to the much higher stability of liposome
bilayers.
[0114] Single-chain surfactants are able to form micelles (see
Table 1, below). These include the anionic (sodium dodecyl sulfate,
cholate or oleate) or cationic (cetyl-trimethylammonium bromide,
CTAB) surfactants. CTAB, CTAC, and DOIC micelles yielded larger
solubility gaps (lower concentration of colloidally suspended DNA)
than corresponding SUV particles containing neutral lipid and CTAB
(1:1) (Lasic, 1997).
1TABLE 1 Molecules able to form micelles Molecule Reference CTAB,
CTAC, DOIC Lasic, 1997 Detergent/phospholipid micelles Lusa et al.,
```1998 Dodecyl betaine (amphoteric surfactant) de la Maza et al.,
1998 Dodecylphosphocholine cholate Lasic, 1997 Glycine-conjugated
bile salt (anionic Leonard and Cohen, 1998 steroid detergent-like
molecule) Lipid-dodecyl maltoside micelles Lambert et al., 1998
mixed micelles (Triton X-100 & Lopez et al., 1998
phosphatidylcholine) Octylglucoside (non-ionic straight Leonard and
Cohen, 1998 chain detergent) Oleate Lasic, 1997
PEG-dialkylphosphatidic Tirosh et al., 1998 acid
(dihexadecylphosphatidyl (DHP)-PEG2000) Phosphatidylcholine
(neutral zwitterionic) Schroeder et al., 1990 Polyethyleneglycol
(MW 5000)-distearoyl Weissig et al., 1998 phosphatidyl ethanolamine
(PEG-DSPE) sodium dodecyl sulfate (anionic straight Leonard and
Cohen, 1998 chain detergent) Sodium taurofusidate (conjugated
fungal Leonard and Cohen, 1998 bile salt analog) Taurine-conjugated
bile salts (anionic Leonard and Cohen, 1998 steroid detergent-like
molecule) Triton X-100 surfactant Lasic, 1997
[0115] There is a critical detergent/phospholipid ratio at which
lamellar-to-micellar transition occurs. For example, the
vesicle-micelle transition was observed for dodecyl maltoside with
large unilamellar liposomes. A striking feature of the
solubilization process by dodecyl maltoside was the discovery of a
new phase, consisting of a very viscous "gel-like" structure
composed of long filamentous thread-like micelles, over 1 to 2
microns in length.
[0116] A long circulating complex needs to be slightly anionic.
Therefore the liposomes used for the conversion of the micelles
into liposomes contain bipolar lipids (PC, PE) and 1-30% negatively
charged lipids (DPPG). The cationic lipids which are toxic, are
hidden in the inner liposome membrane bilayer. Those reaching the
solid tumor will exert their toxic effects causing apoptosis.
Apoptosis will be caused by the delivery of the toxic drug or
anti-neoplastic gene or oligonucleotide to the cancer cell but also
by the nuclear localization of the cationic lipids (along with
plasmid DNA) to the nucleus. Indeed, a number of studies suggest
that plasmid DNA is imported to nuclei; its translocation docks
cationic lipid molecules electrostatically attached to the DNA.
These cationic lipid molecules exert their toxicity by interfering
with the nucleosome and domain structure of the chromatin causing
local destabilization. This disturbance or aberrant chromatin
reorganization could be exerted at the level of the nuclear matrix
where plasmid DNA is attached for transcription, autonomous
replication, or integration via recombination.
[0117] Surfactants have found wide application in formulations such
as emulsions (including microemulsions) and liposomes. The most
common way of classifying and ranking the properties of the many
different types of surfactants, both natural and synthetic, is by
the use of the hydrophile/lipophile balance (HLB). The use of
surfactants in drug products, formulations and in emulsions has
been reviewed (Rieger, in: Pharmaceutical Dosage Forms, Marcel
Dekker, Inc., New York, 1988, p. 285).
[0118] Nonionic surfactants find wide application in pharmaceutical
and cosmetic products and are usable over a wide range of pH
values. In general, their HLB values range from 2 to about 18,
depending on their structure. Nonionic surfactants include,
nonionic esters such as ethylene glycol esters, propylene glycol
esters, glyceryl esters, polyglyceryl esters, sorbitan esters,
sucrose esters, and ethoxylated esters. Nonionic alkanolamides and
ethers, such as fatty alcohol ethoxylates, propoxylated alcohols,
and ethoxylated/propoxylated, block polymers are also included in
this class. The polyoxyethylene surfactants are the most popular
members of the nonionic surfactant class.
[0119] Anionic surfactants include carboxylates such as soaps, acyl
lactylates, acyl amides of amino acids, esters of sulfuric acid
such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates
such as alkyl benzene sulfonates, acyl isethionates, acyl taurates
and sulfosuccinates, and phosphates. The most important members of
the anionic surfactant class are the alkyl sulfates and the
soaps.
[0120] Cationic surfactants include quaternary ammonium salts and
ethoxylated amines. The quaternary ammonium salts are the most used
members of this class. If the surfactant molecule has the ability
to carry either a positive or negative charge, the surfactant is
classified as amphoteric. Amphoteric surfactants include acrylic
acid derivatives, substituted alkylamides, N-alkylbetaines and
phosphatides.
[0121] Classical micelles may not be effective as gene transfer
vehicles, but important intermediates in the formation of liposome
complexes encapsulating drugs or nucleic acids. The stability of
single chain surfactants-DNA-colloidal systems is lower than SUV
particles containing neutral lipid and CTAB (1:1). However, second
generation micelles are able to target tumors in vivo. Weissig and
co-workers (1998) used the soybean trypsin inhibitor (STI) as a
model protein to target tumors. STI was modified with a hydrophobic
residue of N-glutaryl-phosphatidyl-ethano- lamine (NGPE) and
incorporated into both polyethyleneglycol (MW 5000)-distearoyl
phosphatidyl ethanolamine (PEG-DSPE) micelles (<20 nm) and
PEG-DSPE-modified long-circulating liposomes (ca. 100 .mu.m). As
determined from the protein label by using ilIn attached to soybean
trypsin inhibitor via protein-attached diethylene triamine
pentaacetic acid, DTPA, PEG-lipid micelles accumulated better than
the same protein anchored in long-circulating PEG-liposomes in
subcutaneously established Lewis lung carcinoma in mice after tail
vein injection.
[0122] Loading a liposomal dispersion with an amphiphilic drug may
cause a phase transformation into a micellar solution. The
transition from high ratios of phospholipid to drug (from 2:1 to
1:1 downwards) were accompanied by the conversion of liposomal
dispersions of milky-white appearance (particle size 200 m) to
nearly transparent micelles (particle size below 25 nm). See,
Schutze and Muller-Goymann (1998).
[0123] Fusogenic Peptides
[0124] In a further embodiment, the liposome encapsulated DNA
described herein further comprises an effective amount of a
fusogenic peptide. Fusogenic peptides belong to a class of helical
amphipathic peptides characterized by a hydrophobicity gradient
along the long helical axis. This hydrophobicity gradient causes
the tilted insertion of the peptides in membranes, thus
destabilizing the lipid core and, thereby, enhancing membrane
fusion (Decout et al., 1999).
[0125] Hemagglutinin (HA) is a homotrimeric surface glycoprotein of
the influenza virus. In infection, it induces membrane fusion
between viral and endosomal membranes at low pH. Each monomer
consists of the receptor-binding HA1 domain and the
membrane-interacting HA2 domain. The NH2-terminal region of the HA2
domain (amino acids 1 to 127), the so-called "fusion peptide,"
inserts into the target membrane and plays a crucial role in
triggering fusion between the viral and endosomal membranes. Based
on the substitution of eight amino acids in region 5-14 with
cysteines and spin-labeling electron paramagnetic resonance, it was
concluded that the peptide forms an alpha-helix tilted
approximately 25 degrees from the horizontal plane of the membrane
with a maximum depth of 15 .ANG. from the phosphate group (Macosko
et al., 1997). Use of fusogenic peptides from influenza virus
hemagglutinin HA-2 enhanced greatly the efficiency of
transferrin-polylysine-DNA complex uptake by cells. The peptide was
linked to polylysine and the complex was delivered by the
transferrin receptor-mediated endocytosis (reviewed by Boulikas,
1998a). This peptide has the sequence: GLFEAIAGFI ENGWEGMIDG GGYC
(SEQ ID NO:9) and is able to induce the release of the fluorescent
dye calcein from liposomes prepared with egg yolk
phosphatidylcholine, which was higher at acidic pH. This peptide
was also able to increase up to 10-fold the anti-HIV potency of
antisense oligonucleotides, at a concentration of 0.1-1 mM, using
CEM-SS lymphocytes in culture. This peptide changes conformation at
the slightly more acidic environment of the endosome, destabilizing
and breaking the endosomal membrane (reviewed by Boulikas,
1998a).
[0126] The presence of negatively charged lipids in the membrane is
important for the manifestation of the fusogenic properties of some
peptides, but not of others. Whereas the fusogenic action of a
peptide, representing a putative fusion domain of fertilin, a sperm
surface protein involved in sperm-egg fusion, was dependent upon
the presence of negatively charged lipids, that of the HIV2 peptide
was not (Martin and Ruysschaert, 1997).
[0127] For example, to analyze the two domains on the fusogenic
peptides of influenza virus hemagglutinin HA, HA-chimeras were
designed in which the cytoplasmic tail and/or transmembrane domain
of HA was replaced with the corresponding domains of the fusogenic
glycoprotein F of Sendai virus. Constructs of HA were made in which
the cytoplasmic tail was replaced by peptides of human
neurofibromin type 1 (NF1) (residues 1441 to 1518) or c-Raf-1,
(residues 51 to 131) and were expressed in CV-1 cells by using the
vaccinia virus-T7 polymerase transient-expression system. Membrane
fusion between CV-1 cells and bound human erythrocytes (RBCS)
mediated by parental or chimeric HA proteins showed that, after the
pH was lowered, a flow of the aqueous fluorophore calcein from
preloaded RBCs into the cytoplasm of the protein-expressing CV-1
cells took place. This indicated that membrane fusion involves both
leaflets of the lipid bilayers and leads to formation of an aqueous
fusion pore (Schroth-Diaz et al., 1998).
[0128] A remarkable discovery was that the TAT protein of HIV is
able to cross cell membranes (Green and Loewenstein, 1998) and that
a 36-amino acid domain of TAT, when chemically cross-linked to
heterologous proteins, conferred the ability to transduce into
cells. The 11-amino acid fusogenic peptide of TAT (YGRKKRRQRRR (SEQ
ID NO:10)) is a nucleolar localization signal (see Boulikas,
1998b).
[0129] Another protein of HIV, the glycoprotein gp41, contains
fusogenic peptides. Linear peptides derived from the membrane
proximal region of the gp41 ectodomain have potential applications
as anti-HIV agents and inhibit infectivity by adopting a helical
conformation (Judice et al., 1997). The 23 amino acid residue,
N-terminal peptide of HIV-1 gp41 has the capacity to destabilize
negatively charged large unilamellar vesicles. In the absence of
cations, the main structure was a pore-forming alpha-helix, whereas
in the presence of Ca.sup.2+ the conformation switched to a
fusogenic, predominantly extended beta-type structure. The fusion
activity of HIV(ala) (bearing the R22.fwdarw.A substitution) was
reduced by 70%, whereas fusogenicity was completely abolished when
a second substitution (V2.fwdarw.E) was included, arguing that it
is not an alpha-helical but an extended structure adopted by the
HIV-1 fusion peptide that actively destabilizes
cholesterol-containing, electrically neutral membranes (Pereira et
al., 1997).
[0130] The prion protein (PrP) is a glycoprotein of unknown
function normally found at the surface of neurons and of glial
cells. It is involved in diseases such as bovine spongiform
encephalopathy, and Creutzfeldt-Jakob disease in humans, where PrP
is converted into an altered form (termed PrPSc). According to
computer modeling calculations, the 120 to 133 and 118 to 135
domains of PrP are tilted lipid-associating peptides inserting in a
oblique way into a lipid bilayer and able to interact with
liposomes to induce leakage of encapsulated calcein (Pillot et al.,
1997b).
[0131] The C-terminal fragments of the Alzheimer amyloid peptide
(amino acids 29-40 and 29-42) have properties related to those of
the fusion peptides of viral proteins inducing fusion of liposomes
in vitro. These properties could mediate a direct interaction of
the amyloid peptide with cell membranes and account for part of the
cytotoxicity of the amyloid peptide. In view of the epidemiologic
and biochemical linkages between the pathology of Alzheimer's
disease and apolipoprotein E (apoE) polymorphism, examination of
the potential interaction between the three common apoE isoforms
and the C-terminal fragments of the amyloid peptide showed that
only apoE2 and apoE3, not apoE4, are potent inhibitors of the
amyloid peptide fusogenic and aggregational properties. The
protective effect of apoE against the formation of amyloid
aggregates was thought to be mediated by the formation of stable
apoE/amyloid peptide complexes (Pillot et al., 1997a; Lins et al.,
1999).
[0132] The fusogenic properties of an amphipathic net-negative
peptide (WAE 11), consisting of 11 amino acid residues were
strongly promoted when the peptide was anchored to a liposomal
membrane. The fusion activity of the peptide appeared to be
independent of pH and membrane merging, and the target membranes
required a positive charge that was provided by incorporating
lysine-coupled phosphatidylethanolamine (PE-K). Whereas the coupled
peptide could cause vesicle aggregation via nonspecific
electrostatic interaction with PE-K, the free peptide failed to
induce aggregation of PE-K vesicles (Pecheur et al., 1997).
[0133] A number of studies suggest that stabilization of an
alpha-helical secondary structure of the peptide after insertion in
lipid bilayers in membranes of cells or liposomes is responsible
for the membrane fusion properties of peptides. Zn.sup.2+, enhances
the fusogenic activity of peptides because it stabilizes the
alpha-helical structure. For example, the HEXXH (SEQ ID NO:11)
domain of the salivary antimicrobial peptide, located in the
C-terminal functional domain of histatin-5, a recognized
zinc-binding motif is in a helicoidal conformation (Martin et al.,
1999; Melino et al., 1999; Curtain et al., 1999).
[0134] Fusion peptides have been formulated with DNA plasmids to
create peptide-based gene delivery systems. A combination of the
YKAKnWK (SEQ ID NO:12) peptide, used to condense plasmids into 40
to 200 nm nanoparticles, with the GLFEALLELLESLWELLLEA (SEQ ID NO:
13) amphipathic peptide, that is a pH-sensitive lytic agent
designed to facilitate release of the plasmid from endosomes
enhanced expression systems containing the beta-galactosidase
reporter gene (Duguid et al., 1998). See Table 2, below.
2TABLE 2 Fusogenic peptides Fusogenic peptide Source Protein
Properties Reference GLFEAIAGFIENGWEG Influenza virus Endowed with
membrane Bongartz et al., 1994 MIDGGGYC (SEQ ID hemagglutinin
fusion properties NO:9) HA-2 YGRKKRRQRRR (SEQ TAT of HIV Endowed
with membrane Green and ID NO:5) fusion properties Loewenstein,
1988 23-residue fusogenic N- HIV-1 trans- Was able to insert as an
Curtain et al., 1999 terminal peptide membrane alpha-helix into
neutral glycoprotein gp41 phospholipid bilayers 70 residue peptide
(SV- Fusion peptide Induced lipid mixing of egg Ghosh and Shai,
117) and N-terminal phosphatidylcholine- 1999 heptad repeat of
phosphatidyl`glycerol Sendai virus (PC/PG) large unilamellar
vesicles (LUVs) 23 hydrophobic amino S protein of A high degree of
similarity Rodriguez-Crespo et acids in the amino-terminal
hepatitis B virus with known fusogenic al., 1994 region (HBV)
peptides from other viruses. MSGTFGGILAGLIGLL N-terminal region Was
inserted into the Rodriguez-Crespo et (SEQ ID NO:6) of the S
protein of hydrophobic core of the al., 1999 duck hepatitis B lipid
bilayer and induced Virus (DHBV) leakage of internal aqueous
contents from both neutral and negatively charged liposomes
MSPSSLLGLLAGLQVV S protein of Was inserted into the
Rodriguez-Crespo et (SEQ ID NO:14) woodchuck hydrophobic core of
the al., 1999 hepatitis B virus lipid bilayer and induced (WHV)
leakage of internal aqueous contents from both neutral and
negatively charged liposomes N-terminus of Nef Nef protein of
Membrane-perturbing and Macreadie et al., human fusogenic
activities in 1997 inimuno- artificial membranes; causes deficiency
cell killing in E. coli and type 1 (HIV-1) yeast Amino-terminal
sequence F1 polypeptide of Can be used as a carrier Partidos et
al., 1996 F1 polypeptide measles virus system for CTL epitopes (MV)
19-27 amino acid segment Glycoprotein Adopts an amphiphilic Voneche
et al., 1992 gp51 of bovine structure and plays a key leukemia
virus role in the fusion events induced by bovine leukemia virus
120 to 133 and 118 to 135 Prion protein Tilted lipid-associating
Pillot et al., 1997b domains peptide; interact with liposomes to
induce leakage of encapsulated calcein 29-42-residue fragment
Alzheimer's beta- Endowed with capacities Lins et al., 1999 amyloid
peptide resembling those of the tilted fragment of viral fusion
proteins Non-aggregated amyloid Alzheimer's beta- Induces apoptotic
neuronal Pillot et al., 1999 beta-peptide (1-40) amyloid peptide
cell death LCAT 56-68 helical Lecithin Forms stable beta-sheets in
Peelman et al., 1999; segment cholesterol lipids Decout et al.,
1999 acyltransferase (LCAT) Peptide sequence B18 Membrane- Triggers
fusion between Ulrich et al., 1999 associated sea lipid vesicles; a
histidine- urchin sperm nch motif for binding zinc protein binding
is required for the fusogenic function 53-70 (C-terminal helix)
Apolipoprotein Induces fusion of Lambert et al., 1998 (apo) AII
unilamellar lipid vesicles and displaces apo AI from HDL and r-HDL
Residues 90-111 PH-30 alpha (a Membrane-fusogenic Nudome et al.,
1997 protein activity to acidic functioning in phospholipid
bilayers sperm-egg fusion) Casein signal peptides Alpha s2- and
Interact with Creuzenet et al., 1997 beta-casein
dimyristoylphosphatidyl- glycerol and -choline liposomes; show both
lytic and fusogenic activities Pardaxin Amphipathic Forms
voltage-gated, Lelkes and polypeptide, cation-selective pores;
Lazarovici, 1988 purified from the mediated the aggregation of
gland secretion of liposomes composed of the Red Sea
phosphatidylserine but not Moses sole of phosphatidylcholine
flatfish Pardachirus marmoratus Histatin-5 Salivary Aggregates and
fuses Melino et al., 1999 antimicrobial negatively charged small
peptide unilamellar vesicles in the presence of Zn2+ Gramicidin
(linear Antibiotic Induces aggregation and Massari and Colonna,
hydrophobic polypeptide) fusion of vesicles 1986; Toumois et al.,
1990 Amphipathic negatively Synthetic Forms an alpha-helix Martin
et al., 1999 charged peptide consisting inserted and anchored into
of 11 residues (WAE) the membrane (favored at 37.degree. C.)
oriented almost parallel to the lipid acyl chains; promotes fusion
of large unilamellar liposomes (LUV) A polymer of polylysine
Synthetic Histidyl residues become Midoux and (average 190)
partially cationic upon protonation of Monsigny, 1999 substituted
with histidyl the imidazole groups at pH residues below 6.0.;
disrupt endosomal membranes GLFEALLELLESLWELL Synthetic Amphipathic
peptide; a pH- Duguid et al., 1998 LEA (SEQ ID NO:4) sensitive
lytic agent to facilitate release of the plasmid from endosomes
(LKKL).sub.4 (SEQ ID NO: 15) Synthetic Amphiphilic fusogenic Gupta
and Kothekar, peptide, able to interact with 1997 four molecules of
DMPC Ac-(Leu-Ala-Arg-Leu).sub.3- Synthetic; basic Caused a leakage
of Suenaga et al., 1989; NHCH.sub.3 (SEQ ID NO: 16) amphipathic
contents from small Lee et al., 1992 peptides unilamellar vesicles
composed of egg yolk phosphatidylcholine and egg yolk phosphatidic
acid (3:1) Amphiphilic anionic Synthetic Can mimic the fusogenic
Murata et al., 1991 peptides E5 and E5L activity of influenza
hemagglutinin (HA) 30-amino acid peptide with Synthetic; Becomes an
amphipathic Parente et al., 1988 the major repeat unit Glu-
designed alpha-helix as the pH is Ala-Leu-Ala (GALA).sub.7 to mimic
lowered to 5.0; fusion of (SEQ ID NO:17) the behavior of
phosphatidylcholine small the fusogenic unilamellar vesicles
induced sequences of viral by GALA requires a peptide fusion
proteins length greater than 16 amino acids Poly Glu-Aib-Leu-Aib
Synthetic Amphiphilic structure upon Kono et al., 1993 (SEQ ID
NO:18) Aib the formation of alpha- represents 2- helix; caused
fusion of aminoisobutyric acid EYPC liposomes and
dipalmitoylphosphatidylcholine liposomes more strongly with
decreasing pH
[0135] Fusogenic Lipids
[0136] DOPE is a fusogenic lipid; elastase cleavage of
N-methoxy-succinyl-Ala-Ala-Pro-Val-DOPE (SEQ ID NO:19) converted
this derivative to DOPE (overall positive charge) to deliver an
encapsulated fluorescent probe, calcein, into the cell cytoplasm
(Pak et al., 1999). An oligodeoxynucleic sequence of 30 bases
complementary to a region of beta-endorphin mRNA elicited a
concentration-dependent inhibition of beta-endorphin production in
cell culture after it was encapsulated within small unilamellar
vesicles (50 nm) containing
dipalmitoyl-DL-alpha-phosphatidyl-L-serine endowed with fusogenic
properties (Fresta et al., 1998).
[0137] Nuclear Localization Signals (NLS)
[0138] In a further embodiment, the liposome encapsulated plasmid
or oligonucleotide DNA described herein further comprise an
effective amount of nuclear localization signal (NLS) peptides.
Trafficking of nuclear proteins from the site of their synthesis in
the cytoplasm to the sites of function in the nucleus through pore
complexes is mediated by NLSs on proteins to be imported into
nuclei (Tables 3-10, below). Protein translocation from the
cytoplasm to the nucleoplasm involves: (i) the formation of a
complex of karyopherin .alpha. with NLS-protein; (ii) subsequent
binding of karyopherin .beta.; (iii) binding of the complex to FXFG
peptide repeats on nucleoporins; (iv) docking of Ran-GDP to
nucleoporin and to karyopherin heterodimer by p10; (v) a number of
association-dissociation reactions on nucleoporins that dock the
import substrate toward the nucleoplasmic side with a concomitant
GDP-GTP exchange reaction transforming Ran-GDP into Ran-GTP and
catalyzed by karyopherin .alpha.; and (vi) dissociation from
karyopherin .beta. and release of the karyopherin
.alpha./NLS-protein by Ran-GTP to the nucleoplasm.
[0139] Karyophilic and acidic clusters were found in most
non-membrane serine/threonine protein kinases whose primary
structure has been examined (Table 6). These karyophilic clusters
might mediate the anchoring of the kinase molecules to transporter
proteins for their regulated nuclear import and might constitute
the nuclear localization signals. In contrast to protein
transcription factors that are exclusively nuclear possessing
strong karyophilic peptides composed of at least four arginines,
(R), and lysines, (K), within an hexapeptide flanked by proline and
glycine helix-breakers, protein kinases often contain one histidine
and three K+R residues (Boulikas, 1996). This was proposed to
specify a weak NLS structure resulting in the nuclear import of a
fraction of the total cytoplasmic kinase molecules, as well as in
their weak retention in the different ionic strength nuclear
environment. Putative NLS peptides in protein kinases may also
contain hydrophobic or bulky aromatic amino acids proposed to
further diminish their capacity to act as strong NLS.
[0140] Most mammalian proteins that participate in DNA repair
pathways seem to possess strong karyophilic clusters containing at
least four R+K over a stretch of six amino acids (Table 7).
[0141] Rules to predict nuclear localization of an unknown protein
Several simple rules have been proposed for the prediction of the
nuclear localization of a protein of an unknown function from its
amino acid sequence:
[0142] (i) An NLS is defined as four arginines (R) plus lysines (K)
within an hexapeptide; the presence of one or more histidines (H)
in the tetrad of the karyophilic hexapeptide, often found in
protein kinases that have a cytoplasmic and a nuclear function, may
specify a weak NLS whose function might be regulated by
phosphorylation or may specify proteins that function in both the
cytoplasm and the nucleus (Boulikas, 1996);
[0143] (ii) The K/R clusters are flanked by the a-helix breakers G
and P thus placing the NLS at a helix-turn-helix or end of a
a-helix. Negatively-charged amino acids (D, E) are often found at
the flank of the NLS and on some occasions may interrupt the
positively-charged NLS cluster;
[0144] (iii) Bulky amino acids (W, F, Y) are not present within the
NLS hexapeptide;
[0145] (iv) NLS signals may not be flanked by long stretches of
hydrophobic amino acids (e.g. five); a mixture of charged and
hydrophobic amino acids serves as a mitochondrial targeting
signal;
[0146] (v) The higher the number of NLSs, the more readily a
molecule is imported to the nucleus (Dworetzky et al., 1988). Even
small proteins, for example histones (10-22 kDa), need to be
actively imported to increase their import rates compared with the
slow rate of diffusion of small molecules through pores;
[0147] (vi) Signal peptides are stronger determinants than NLSs for
protein trafficking. Signal peptides direct proteins to the lumen
of the endoplasmic reticulum for their secretion or insertion into
cellular membranes (presence of transmembrane domains) (Boulikas,
1994);
[0148] (vii) Signals for the mitochondrial import of proteins (a
mixture of hydrophobic and karyophilic amino acids) may antagonize
nuclear import signals and proteins possessing both type of signals
may be translocated to both mitochondria and nuclei;
[0149] (viii) Strong association of a protein with large
cytoplasmic structures (membrane proteins, intermediate filaments)
make such proteins unavailable for import even though they posses
NLS-like peptides (Boulikas, 1994);
[0150] (ix). Transcription factors and other nuclear proteins
posses a great different number of putative NLS stretches. Of the
sixteen possible forms of putative NLS structures the most abundant
types are the 00.times.00, 000.times.0, 0000, and
00.times.0.times.0, where 0 is R or K, together accounting for
about 70% of all karyophilic clusters on transcription factors
(Boulikas, 1994);
[0151] (x) A small number of nuclear proteins seem to be void of a
typical karyophilic NLS. Either non karyophilic peptides function
for their nuclear import, as such molecules possess bipartite NLSs,
or these NLS-less proteins depend absolutely for import on their
strong complexation in the cytoplasm with a nuclear protein partner
able to be imported (Boulikas, 1994). This mechanism may ensure a
certain stoichiometric ratio of the two molecules in the nucleus,
and might be of physiological significance; and
[0152] (xi) A number of proteins may be imported via other
mechanisms not dependent on classical NLS.
[0153] A number of processes have been found to be regulated by
nuclear import including nuclear translocation of the transcription
factors NF-KB, rNFIL-6, ISGF3, SRF, c-Fos, GR as well as human
cyclins A and BI, casein kinase II, cAMP-dependent protein kinase
II, protein kinase C, ERKI and ERK2. Failure of cells to import
specific proteins into nuclei can lead to carcinogenesis. For
example, BRCA1 is mainly localized in the cytoplasm in breast and
ovarian cancer cells, whereas in normal cells the protein is
nuclear. mRNA is exported through the same route as a complex with
nuclear proteins possessing nuclear export signals (NES). The
majority of proteins with NES are RNA-binding proteins that bind to
and escort RNAs to the cytoplasm. However, other proteins with NES
function in the export of proteins; CRM1, that binds to the NES
sequence on other proteins and interacts with the nuclear pore
complex, is an essential mediator of the NES-dependent nuclear
export of proteins in eukaryotic cells. Nuclear localization and
export signals (NLS and NES) are found on a number of important
molecules, including p53, v-Rel, the transcription factor NF-ATc,
the c-Ab1 nonreceptor tyrosine kinase, and the fragile X syndrome
mental retardation gene product. The deregulation of their normal
import/export trafficking has important implications for human
disease. Both nuclear import and export processes can be
manipulated by conjugation of proteins with NLS or NES peptides.
During gene therapy, the foreign DNA needs to enter nuclei for its
transcription. A pathway is proposed involving the complexation of
plasmids and oligonucleotides with nascent nuclear proteins
possessing NLSs as a prerequisite for their nuclear import.
Covalent linkage of NLS peptides to oligonucleotides and plasmids
or formation of complexes of plasmids with proteins possessing
multiple NLS peptides was proposed (Boulikas, 1998b) to increase
their import rates and the efficiency of gene expression. Cancer
cells were predicted to import more efficiently foreign DNA into
nuclei, compared with terminally differentiated cells because of
their increased rates of proliferation and protein import.
[0154] Antineoplastic Drugs
[0155] In a further embodiment, the liposome encapsulated plasmid
or oligonucleotide DNA described herein, further comprises its use
for reducing tumor size or restricting its growth with combination
with encapsulated or free antineoplastic agents. Antineoplastic
agents preferably are: (i) alkylating agents having the
bis-(2-chloroethyl)-amin- e group such as chlormethine,
chlorambucile, melphalan, uramustine, mannomustine,
extramustinephosphat, mechlorethaminoxide, cyclophosphamide,
ifosfamide, or trifosfamide; (ii) alkylating agents having a
substituted aziridine group, for example tretamine, thiotepa,
triaziquone, or mitomycine; (iii) alkylating agents of the
methanesulfonic ester type such as busulfane; (iv) alkylating
N-alkyl-N-nitrosourea derivatives, for example carmustine,
lomustine, semustine, or streptozotocine; (v) alkylating agents of
the mitobronitole, dacarbazine, or procarbazine type; (vi)
complexing agents such as cis-platin; (vii) antimetabolites of the
folic acid type, for example methotrexate; (viii) purine
derivatives such as mercaptopurine, thioguanine, azathioprine,
tiamiprine, vidarabine, or puromycine and purine nucleoside
phosphorylase inhibitors; (ix) pyrimidine derivatives, for example
fluorouracil, floxuridine, tegafur, cytarabine, idoxuridine,
flucytosine; (x) antibiotics such as dactinomycin, daunorubicin,
doxorubicin, mithramycin, bleomycin or etoposide; (xi) vinca
alkaloids; (xii) inhibitors of proteins overexpressed in cancer
cells such as telomerase inhibitors, glutathione inhibitors,
proteasome inhibitors; (xiii) modulators or inhibitors of signal
transduction pathways such as phosphatase inhibitors, protein
kinase C inhibitors, casein kinase inhibitors, insulin-like growth
factor-1 receptor inhibitor, ras inhibitors, ras-GAP inhibitor,
protein tyrosine phosphatase inhibitors; (xiv) tumor angiogenesis
inhibitors such as angiostatin, oncostatin, endostatin,
thalidomide; (xv) modulators of the immune response and cytokines
such as interferons, interleukins, TNF-alpha; (xvi) modulators of
the extracellular matrix such as matrix metalloproteinase
inhibitors, stromelysin inhibitors, plasminogen activator
inhibitor; (xvii) hormone modulators for hormone-dependent cancers
(breast cancer, prostate cancer) such as antiandrogen, estrogens;
(xviii) apoptosis regulators; (xix) bFGF inhibitor; (xx) multiple
drug resistance gene inhibitor; (xxi) monoclonal antibodies or
antibody fragments against antigenes overexpressed in cancer cells
(anti-Her2/neu for breast cancer); (xxii) anticancer genes whose
expression will cause apoptosis, arrest the cell cycle, induce an
immune response against cancer cells, inhibit tumor angiogenesis
i.e. formation of blood vessels, tumor suppressor genes (p53, RB,
BRCA1, E1A, bc1-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I VEGF,
angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4,
IL-7, IFN-.gamma., and TNF-a); and (xxiii) antisense
oligonucleotides (antisense c-fos, c-myc, K-ras). Optionally these
drugs are administered in combination with chlormethamine,
prednisolone, prednisone, or procarbazine or combined with
radiation therapy. Future new anticancer drugs added to the arsenal
are expected to be ribozymes, triplex-forming oligonucleotides,
gene inactivating oligonucleotides, a number of new genes directed
against genes that control the cell proliferation or signaling
pathways, and compounds that block signal transduction.
[0156] Anti-cancer drugs include: acivicin, aclarubicin, acodazole
hydrochloride, acronine, adozelesin, adriamycin, aldesleukin,
altretamine, ambomycin, ametantrone acetate, aminoglutethimide,
amsacrine, anastrozole, anthramycin, asparaginase, asperlin,
azacitidine, azetepa, azotomycin, batimastat, benzodepa,
bicalutamide, bisantrene hydrochloride, bisnafide dimesylate,
bizelesin, bleomycin sulfate, brequinar sodium, bropirimine,
busulfan, cactinomycin, calusterone, caracemide, carbetimer,
carboplatin, carmustine, carubicin hydrochloride, carzelesin,
cedefingol, chlorambucil, cirolemycin, cisplatin, cladribine,
crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine,
dactinomycin, daunorubicin hydrochloride, decitabine,
dexormaplatin, dezaguanine, dezaguanine mesylate, diaziquone,
docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene,
droloxifene citrate, dromostanolone propionate, duazomycin,
edatrexate, eflomithine hydrochloride, elsamitrucin, enloplatin,
enpromate, epipropidine, epirubicin hydrochloride, erbulozole,
esorubicin hydrochloride, estramustine, estramustine phosphate
sodium, etanidazole, etoposide, etoposide phosphate, etoprine,
fadrozole hydrochloride, fazarabine, fenretinide, floxuridine,
fludarabine phosphate, fluorouracil, flurocitabine, fosquidone,
fostriecin sodium, gemcitabine, gemcitabine hydrochloride,
hydroxyurea, idarubicin hydrochloride, ifosfamide, ilmofosine,
interferon alfa-2a, interferon .alpha.-2b, interferon .alpha.-n1,
interferon .alpha.-n3, interferon .beta.-i a, interferon .gamma.-i
b, iproplatin, irinotecan hydrochloride, lanreotide acetate,
letrozole, leuprolide acetate, liarozole hydrochloride, lometrexol
sodium, lomustine, losoxantrone hydrochloride, masoprocol,
maytansine, mechlorethamine hydrochloride, megestrol acetate,
melengestrol acetate, melphalan, menogaril, mercaptopurine,
methotrexate, methotrexate sodium, metoprine, meturedepa,
mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin,
mitomycin, mitosper, mitotane, mitoxantrone hydrochloride,
mycophenolic acid, nocodazole, nogalamycin, ormaplatin, oxisuran,
paclitaxel, pegaspargase, peliomycin, pentamustine, peplomycin
sulfate, perfosfamide, pipobroman, piposulfan, piroxantrone
hydrochloride, plicamycin, plomestane, porfimer sodium,
porfiromycin, prednimustine, prednisone, procarbazine
hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin,
riboprine, rogletimide, safingol, safingol hydrochloride,
semustine, simtrazene, sparfosate sodium, sparsomycin,
spirogermanium hydrochloride, spiromustine, spiroplatin,
streptonigrin, streptozocin, sulofenur, talisomycin, taxol,
tecogalan sodium, tegaflir, teloxantrone hydrochloride, temoporfin,
teniposide, teroxirone, testolactone, thiamiprine, thioguanine,
thiotepa, tiazofurin, tirapazamine, topotecan hydrochloride,
toremifene citrate, trestolone acetate, triciribine phosphate,
trimetrexate, trimetrexate glucuronate, triptorelin, tubulozole
hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin,
vinblastine sulfate, vincristine sulfate, vindesine, vindesine
sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine
sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine
sulfate, vorozole, zeniplatin, zinostatin, zorubicin
hydrochloride.
[0157] Other anti-cancer drugs include: 20-epi-1,25
dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin,
acylfulvene, adecypenol, adozelesin, aldesleukin, ALL-TK
antagonists, altretamine, ambamustine, amidox, amifostine,
aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole,
andrographolide, angiogenesis inhibitors, antagonist D, antagonist
G, antarelix, anti-dorsalizing morphogenetic protein-1,
antiandrogen, antiestrogen, antineoplaston, antisense
oligonucleotides, aphidicolin glycinate, apoptosis gene modulators,
apoptosis regulators, apurinic acid, ara-CDP-DL-PTBA, arginine
deaminase, asulacrine, atamestane, atrimustine, axinastatin 1,
axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine,
baccatin III derivatives, balanol, batimastat, BCR/ABL antagonists,
benzochlorins, benzoylstaurosporine, beta lactam derivatives,
beta-alethine, betaclamycin B, betulinic acid, bFGF inhibitor,
bicalutamide, bisantrene, bisaziridinylspermine, bisnafide,
bistratene A, bizelesin, breflate, bropirimine, budotitane,
buthionine sulfoximine, calcipotriol, calphostin C, camptothecin
derivatives, canarypox IL-2, capecitabine,
carboxamide-amino-triazole, carboxyamidotriazole, CaRest M3, CARN
700, cartilage derived inhibitor, carzelesin, casein kinase
inhibitors (ICOS), castanospermine, cecropin B, cetrorelix,
chlorlns, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin,
cladribine, clomifene analogues, clotrimazole, collismycin A,
collismycin B, combretastatin A4, combretastatin analogue,
conagenin, crambescidin 816, crisnatol, cryptophycin 8,
cryptophycin A derivatives, curacin A, cyclopentanthraquinones,
cycloplatam, cypemycin, cytarabine ocfosfate, cytolytic factor,
cytostatin, dacliximab, decitabine, dehydrodidemnin B, deslorelin,
dexifosfamide, dexrazoxane, dexverapamil, diaziquone, didemnin B,
didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol,
9-dioxamycin, diphenyl spiromustine, docosanol, dolasetron,
doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen,
ecomustine, edelfosine, edrecolomab, eflornithine, elemene,
emitefur, epirubicin, epristeride, estramustine analogue, estrogen
agonists, estrogen antagonists, etanidazole, etoposide phosphate,
exemestane, fadrozole, fazarabine, fenretinide, filgrastim,
finasteride, flavopiridol, flezelastine, fluasterone, fludarabine,
fluorodaunorunicin hydrochloride, forfenimex, formestane,
fostriecin, fotemustine, gadolinium gallium nitrate texaphyrin,
galocitabine, ganirelix, gelatinase inhibitors, gemcitabine,
glutathione inhibitors, hepsulfam, heregulin, hexamethylene
bisacetamide, hypericin, ibandronic acid, idarubicin, idoxifene,
idramantone, ilmofosine, ilomastat, imidazoacridones, imiquimod,
immunostimulant peptides, insulin-like growth factor-1 receptor
inhibitor, interferon agonists, interferons, interleukins,
iobenguane, iododoxorubicin, ipomeanol, 4-, irinotecan, iroplact,
irsogladine, isobengazole, isohomohalicondrin B, itasetron,
jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide,
leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,
leukemia inhibiting factor, leukocyte alpha interferon,
leuprolide+estrogen+progesterone, leuprorelin, levamisole,
liarozole, linear polyamine analogue, lipophilic disaccharide
peptide, lipophilic platinum compounds, lissoclinamide 7,
lobaplatin, lombricine, lometrexol, lonidamine, losoxantrone,
lovastatin, loxoribine, lurtotecan, lutetium texaphyrin,
lysofylline, lytic peptides, maitansine, mannostatin A, marimastat,
masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase
inhibitors, menogaril, merbarone, meterelin, methioninase,
metoclopramide, MIF inhibitor, mifepristone, miltefosine,
mirimostim, mismatched double stranded RNA, mitoguazone,
mitolactol, mitomycin analogues, mitonafide, mitotoxin fibroblast
growth factor-saporin, mitoxantrone, mofarotene, molgramostim,
monoclonal antibody, human chorionic gonadotrophin, monophosphoryl
lipid A+myobacterium cell wall sk, mopidamol, multiple drug
resistance gene inhibitor, multiple tumor suppressor 1-based
therapy, mustard anticancer agent, mycaperoxide B, mycobacterial
cell wall extract, myriaporone, N-acetyldinaline, N-substituted
benzamides, nafarelin, nagrestip, naloxone +pentazocine, napavin,
naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid,
neutral endopeptidase, nilutamide, nisamycin, nitric oxide
modulators, nitroxide antioxidant, nitrullyn, 06-benzylguanine,
octreotide, okicenone, oligonucleotides, onapristone, ondansetron,
ondansetron, oracin, oral cytokine inducer, ormaplatin, osaterone,
oxaliplatin, oxaunomycin, paclitaxel analogues, paclitaxel
derivatives, palauamine, palmitoylrhizoxin, pamidronic acid,
panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase,
peldesine, pentosan polysulfate sodium, pentostatin, pentrozole,
perflubron, perfosfamide, perillyl alcohol, phenazinomycin,
phenylacetate, phosphatase inhibitors, picibanil, pilocarpine
hydrochloride, pirarubicin, piritrexim, placetin A, placetin B,
plasminogen activator inhibitor, platinum complex, platinum
compounds, platinum-triamine complex, porfimer sodium,
porfiromycin, propyl bis-acridone, prostaglandin J2, proteasome
inhibitors, protein A-based immune modulator, protein kinase C
inhibitor, protein kinase C inhibitors, microalgal., protein
tyrosine phosphatase inhibitors, purine nucleoside phosphorylase
inhibitors, purpurins, pyrazoloacridine, pyridoxylated hemoglobin
polyoxyethylene conjugate, raf antagonists, raltitrexed,
ramosetron, ras famesyl protein transferase inhibitors, ras
inhibitors, ras-GAP inhibitor, retelliptine demethylated, rhenium
Re 186 etidronate, rhizoxin, ribozymes, RII retinamide,
rogletimide, rohitukine, romurtide, roquinimex, rubiginone B 1,
ruboxyl, safingol, saintopin, SarCNU, sarcophytol A, sargramostim,
Sdi 1 mimetics, semustine, senescence derived inhibitor 1, sense
oligonucleotides, signal transduction inhibitors, signal
transduction modulators, single chain antigen binding protein,
sizofiran, sobuzoxane, sodium borocaptate, sodium phenylacetate,
solverol, somatomedin binding protein, sonermin, sparfosic acid,
spicamycin D, spiromustine, splenopentin, spongistatin 1,
squalamine, stem cell inhibitor, stem-cell division inhibitors,
stipiamide, stromelysin inhibitors, sulfinosine, superactive
vasoactive intestinal peptide antagonist, suradista, suramin,
swainsonine, synthetic glycosaminoglycans, tallimustine, tamoxifen
methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur,
tellurapyrylium, telomerase inhibitors, temoporfin, temozolomide,
teniposide, tetrachlorodecaoxide, tetrazomine, thaliblastine,
thalidomide, thiocoraline, thrombopoietin, thrombopoietin mimetic,
thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid
stimulating hormone, tin ethyl etiopurpurin, tirapazamine,
titanocene dichloride, topotecan, topsentin, toremifene, totipotent
stem cell factor, translation inhibitors, tretinoin,
triacetyluridine, triciribine, trimetrexate, triptorelin,
tropisetron, turosteride, tyrosine kinase inhibitors, tyrphostins,
UBC inhibitors, ubenimex, urogenital sinus-derived growth
inhibitory factor, urokinase receptor antagonists, vapreotide,
variolin B, velaresol, veramine, verdins, verteporfin, vinorelbine,
vinxaltine, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb,
zinostatin stimalamer.
[0158] pH-Sensitive Peptide-DNA Complexes
[0159] In a further embodiment of the invention, the genes in
plasmid DNA are brought in interaction with fusogenic peptide/NLS
conjugates. In a further embodiment the NLS moiety is a stretch of
histidyl residues able to assume a net positive charge at a pH of
about 5 to 6 and to show a reduction or loose completely this
charge at pH above 7. The electrostatic interaction of these
positively-charged peptides with the negatively-charged plasmid DNA
molecules, established at pH 5-6 is weakened at physiological pH
(pH-sensitive peptide-DNA complexes).
[0160] The first step of the present invention involves complex
formation between the plasmid or oligonucleotide DNA with the
histidyl/fusogenic peptide conjugate and lipid components in 10-90%
ethanol at pH 5.0 to 6.0. The conditions must be where the histidyl
residues have a net positive charge and can establish electrostatic
interactions with plasmids, oligonucleotides or negatively-charged
drugs. At the same time, the presence of the positively-charged
lipid molecules promotes formation of micelles. At the second step,
micelles are converted into liposomes by dilution with water and
mixing with pre-made liposomes or lipids at pH 5-6. This is
followed by dialysis against pH 7 and extrusion through membranes,
entrapping and encapsulating plasmids or oligonucleotides to with a
very high yield.
[0161] Whereas the composition of peptides and cationic lipids in
the first step provides the lipids of the internal bilayer, the
type of liposomes or lipids added at step 2 provide the external
coating of the final liposome formulation (FIG. 1). Examples for
the formulations of peptides include: HHHHHSPSL.sub.16 (SEQ ID
NO:623), and HHHHHSPS(LAI).sub.5 (SEQ ID NO:624).
[0162] These are added at a 1:0.5:0.5 molar ratio (negative charge
on DNA: cationic liposome: histidine peptide). The peptide inserts
in an alpha-helical conformation inside the lipid bilayer and not
only carries out DNA condensation but also endows membrane fusion
properties to the complex to improve entrance across the cell
membrane. The type of hydrophobic amino acids (for example, content
in aromatic amino acids), in the peptide chain is very important as
is the length of the peptide chain in ensuring integrity and
rigidity of the complexes. Coating the outer surface of the
complexes with polyethyleneglycol, hyaluronic acids and other
polymers conjugated to lipids gives the particles long circulation
properties in body fluids and the ability to target solid tumors
and their metastases after intravenous injection, and also the
ability to cross the tumor cell membrane.
[0163] Protease-Sensitive Linkages in Peptides Between the NLS and
Fusogenic Moieties
[0164] Conversion of Micelles Into Liposomes
[0165] An important issue of the present invention is the
conversion of micelles formed between the DNA and the cationic
lipids, in the presence of ethanol, into liposomes. This is done by
the direct addition of the micelle complex into an aqueous solution
of preformed liposomes. The liposomes have an average size of
80-160 nm or vice versa, leading to a solution of a final ethanol
concentration below 10%. A formulation suitable for pharmaceutical
use and for injection into humans and animals will require that the
liposomes are of neutral composition (such as cholesterol, PE, PC)
coated with PEG.
[0166] However, another important aspect is the research
application of the present invention, such as for transfection of
cells in culture. The composition of the aqueous solution of
liposomes is any type of liposomes containing cationic lipids and
suitable therefore for transfection of cells in culture such as
DDAB:DOPE 1: 1. These liposomes are preformed and downsized by
sonication or extrusion through membranes to a diameter of 80-160
nm. The ethanolic micelle preparations are then added to the
aqueous solution of liposomes with a concomitant dilution of the
ethanol solution to below 10%. This step will result in further
condensation of DNA or interaction of the negatively-charged
phosphate groups on DNA with positively charged groups on lipids.
Care must be taken so as only part of the negative charges on DNA
are neutralized by lipids in the micelle. The remaining charge
neutralization of the DNA is to be provided by the cationic
component of the preformed liposomes in the second step.
[0167] Regulatory DNA and Nuclear Matrix-Attached DNA
[0168] In a further embodiment of the present invention, the genes
in plasmid DNA are driven by regulatory DNA sequences isolated from
nuclear matrix-attached DNA using shotgun selection approaches.
[0169] The compact structural organization of chromatin and the
proper spatial orientation of individual chromosomes within a cell
are partially provided by the nuclear matrix. The nuclear matrix is
composed of DNA, RNA and proteins and serves as the site of DNA
replication, gene transcription, DNA repair, and chromosomal
attachment in the nucleus. Diverse sets of DNA sequences have been
found associated with nuclear matrices and is referred to as matrix
attachment regions or MARs. The MARs serve many functions, acting
as activators of gene transcription, silencers of gene expression,
insulators of transcriptional activity, nuclear retention signals
and origins of DNA replication. Current studies indicate that
different subsets of MARs are found in different tissue types and
may assist in regulating the specific functions of cells. The
presence of this complex assortment of structural and regulatory
molecules in the matrix, as well as the in situ localization of DNA
replication and transcription complexes to the matrix strongly
suggest that the nuclear matrix plays a fundamental, unique role in
nuclear processes. The structuring of genomes into domains has a
functional significance. The inclusion of specific MAR elements
within gene transfer vectors could have utility in many
experimental and gene therapy applications. Many gene therapy
applications require specific expression of one or more genes in
targeted cell types for prolonged time periods. MARs within vectors
could enhance transcription of the introduced transgene, prolong
the retention of that sequence within the nucleus or insulate
expression of that transgene from the expression of a cotransduced
gene (reviewed by Boulikas, 1995; Bode et al, 1996).
[0170] Various biochemical procedures have been used to identify
regulatory regions within genes. Traditionally, identification and
selection of regulatory DNA sequences depend on tedious procedures
such as transcription factor footprinting in vitro or in vivo, or
subcloning of smaller fragments from larger genomic DNA sequences
upstream of reporter genes. These methods have been used primarily
to identify regions proximal to the 5' end of genes. However, in
many instances, regulatory regions are found at considerable
distances from the proximal 5' end of the gene, and confer cell
type- or developmental stage- specificity. For example, studies
from the groups of Grosveld and Engel (Lakshmanan et al., 1999)
have shown that over 625 kb of genomic sequences surrounding the
GATA-3 locus are required for the correct developmental expression
of the gene in transgenic mice. Extensive DNA stretches at
distances 5-20 kb upstream of the gene were found to be responsible
for the central nervous system-specificity of expression. The
region between 20 to 130 kb upstream of the gene harbored
regulatory regions for urogenital-specific expression of GATA-3,
whereas sequences 90-180 kb downstream of the gene conferred
endocardial-specific expression.
[0171] The presently disclosed method has the potential of rapidly
identifying regulatory control regions. In cells, chromatin loops
are formed and different attachment regions are used in different
cell types or stages of development to modulate the expression of a
gene. The presently disclosed method for isolating regulatory
regions based on their attachment to the nuclear matrix can
identify regulatory regions irrespective of their distance from the
gene. Although the human genome project is expected to be almost
complete by the year 2000, information on the location and nature
of the vast majority of the estimated 500,000 regulatory regions
will not be available.
Example 1
[0172] Plasmid DNA condenses with various agents, as well as
various formulations of cationic liposomes. The condensation
affects the level of expression of the reporter beta-galactosidase
gene after transfection of K562 human erythroleukemia cell
cultures. Liposome compositions are shown in the Table below and in
FIG. 2. All lipids were from Avanti Polar Lipids (700 Industrial
Park Drive, Alabaster, Ala. 35007). The optimal ratio of lipid to
DNA was 7 mmoles total lipid/.mu.g DNA. The transfection reagent
(10 .mu.g DNA mixed with 70 mmoles total lipid) was transferred to
a small culture flask followed by the addition of 10 ml K562 cell
culture (about 2 million cells total); mixing of cells with the
transfection reagent was at 5-10 min after mixing DNA with
liposomes. Cells were assayed for beta-galactosidase activity
several times at 1-30 days post-transfection. The transfected cells
were maintained in cell culture as normal cell cultures.
[0173] Best results were obtained when the cells used for
transfection were at low number, not near confluence. In all
experiments the transfection material was added directly in the
presence of serum and antibiotics without removal of the
transfection reagent or washings of the cells. This simplifies the
transfection procedure and is suitable for lymphoid and other type
of cell cultures that do not attach to the dish, but grow in
suspension. All DNA condensing agents were purchased from Sigma.
They were suspended at 0.1 mg/ml in water. Plasmid pCMV.beta. was
purchased from Clontech and was purified using the Anaconda kit of
Althea Technologies (San Diego, Calif.). PolyK is polylysine, mw
9,400. PolyR is polyarginine. PolyH is polyhistidine.
[0174] To 100 .mu.l plasmid solution (10 .mu.g total plasmid DNA)
20 .mu.l or 50 .mu.l of polyK, polyR, polyH, were added; the volume
was adjusted to 250 .mu.l with water followed by addition of about
70 .mu.l liposomes (7 mnoles /.mu.g DNA). After incubation for 10
mm to 1 h at 20.degree. C. the transfection mixture was brought in
contact with the cell culture. The best DNA condensing reagent was
polyhistidine compared with the popular polylysine. The best
cationic lipid was DC-cholesterol (DC-CHOL:
[0175]
3.beta.[N--(N',N'-dimethylaminoethane)carbamoyl]cholesterol). SFV
is Semliki Forest virus expressing beta-galactosidase. The results
are shown in FIG. 2.
3 Liposome Molecular weight Composition Preparation L2 DDAB mw 631
DDAB 4.2 .mu.moles/ml 15 mg DDAB + DOPE mw 744 DOPE 4.2
.mu.moles/ml 0.88 ml 20 mg/ml DOPE L3 DOGS-NTA mw 1015.4 DOGS-NTA 1
.mu.mole/ml 5 mg DOGS DOPE 1 .mu.mole/ml 0.185 ml DOPE L4 DC-Chol
(mw 537) DC-Chol 1 .mu.mole/ml 0.106 ml DC-Chol (25 DOPE (mw 744)
DOPE 1 .mu.mole/ml mg/ml) + 0.185 ml DOPE (20 mg/ml) L5 DOTAP (mw
698) DOTAP 1.4 .mu.mole/ml 0.5 ml 10 mg/ ml DOTAP + DOPE (mw 744)
DOPE 1.3 .mu.mole/ml 0.25 ml DOPE (20 mg/ml) L6 DODAP (mw 648)
DODAP 1.54 .mu.moles/ml 0.5 ml 10 mg/ml DOPE 1.3 .mu.mole/ml DODAP
= 5 mg = 7.72 .mu.moles + 0.25 ml DOPE (20 mg/ml)
Example 2
[0176] Targeting Genes to Tumors Using Gene Vehicles
(Lipogenes).
[0177] As shown in FIG. 3, tumor targeting in SCID (severe combined
immunodeficient) mice were implanted subcutaneously, at two sites,
with human MCF-7 breast cancer cells. The cells were allowed to
develop into large, measurable solid tumors at about 30 days
post-inoculation. Mice were injected intraperitoneously with 0.2 mg
plasmid pCMV.beta. DNA (size of the plasmid is .about.4 kb) per
animal carrying the bacterial beta-galactosidase reporter gene.
Plasmid DNA (200 .mu.g, 2.0 mg/ml, 0.1 ml ) was incubated for 5 min
with 2001 neutral liposomes of the composition 40% cholesterol, 20%
dioleoylphosphatidylethanolamine(DOPE), 12%
palmitoyloleoylphosphatidylcholine (POPC), 10% hydrogenated soy
phosphatidylcholine (HSPC), 10% distearoylphosphatidylethanolamine
(DSPE), 5% sphingomyelin (SM), and 3% derivatized vesicle-forming
lipid M-PEG-DSPE.
[0178] At this stage, weak complexation of plasmid DNA with neutral
(zwitterionic) liposomes takes place. This ensures homogeneous
distribution of plasmid DNA to liposomes at the subsequent step of
addition of cationic liposomes. After complexation of plasmid DNA
with zwitterionic liposomes, 50 .mu.l of cationic liposomes
(DC-Chol 1 .mu.mole/ml:DOPE 1.4 .mu.mole/ml) were added and
incubated at room temperature for 10 min. At this stage, a mixed
liposome population is present and, most likely, formation of a
type of liposome-DNA complexes containing lipids from the
zwitterionic and cationic lipids takes place. The material was
injected (0.35 ml total volume) to the intraperitoneal cavity of
the animal. At 5 days post-injection the animal was sacrificed, the
skin was removed and the carcass was incubated into X-gal staining
solution for about 30 min at 37.degree. C. The animal was incubated
in fixative in X-gal staining for about 30 min (addition of 100
.mu.l concentrated glutaraldehyde to 30 ml X-gal staining solution)
and the incubation in staining solution continued. Photos were
taken in a time course during the incubation period revealing the
preferred organs where beta-galactosidase expression took
place.
[0179] Because of the tumor vasculature targeting shown in FIG. 3E,
the data imply that transfer of the genes of angiostatin,
endostatin, or oncostatin to the tumors (whose gene products
restrict vascular growth and inhibit blood supply to the tumor) is
expected to be a rational approach for cancer treatment. Also, a
combination therapy using anticancer lipogenes with encapsulated
drugs into tumor targeting liposomes appears as a rational cancer
therapy.
[0180] It is to be understood that while the invention has been
described in conjunction with the above embodiments, that the
foregoing description and the following examples are intended to
illustrate and not limit the scope of the invention. Other aspects,
advantages and modifications within the scope of the invention will
be apparent to those skilled in the art to which the invention
pertains.
4TABLE 3 Simple NLS Signal oligopeptide Protein and features
PKKKRKV (SEQ ID NO:20) Wild-type SV40 large T protein A point
mutation converting lysine- 128 (double underlined) to threonine
results in the retention of large T in the cytoplasm. Transfer of
this peptide to the N-terminus of .beta.- galactosidase or pyruvate
kinase at the gene level and microinjection of plasmids into Vero
cells showed nuclear location of chimeric proteins. PKKKRMV (SEQ ID
NO:21) SV40 large T with a K.fwdarw.M change. Site-directed
mutagenesis only slightly impaired nuclear import of large T.
PKKKRKVEDP (SEQ ID Synthetic NLS peptide from SV40 large T antigen
crosslinked to BSA NO:22) or IgG mediated their nuclear
localization after microinjec- tion in Xenopus oocytes. The
PKKGSKKA from Xenopus H2B was in- effective and PKTKRKV was less
effective. CGYGPKKKRKVGG (SEQ ID Synthetic peptide from SV40 large
T antigen conjugated to NO:23) various proteins and microinjected
into the cytoplasm of TC-7 cells. Specified nuclear localization up
to protein sizes of 465 kD (ferritin). IgM of 970 kD and with an
estimated radius of 25-40 nm was retained in the cytoplasm.
CYDDEATADSQHSTPPKKK SV40 large T protein long NLS. The long NLS but
not the short NLS, RKVEDPKDFESELLS was able to localize the bulky
IgM (970 kD) into the nucleus. (SEQ ID NO:24) Mutagenesis at the
four possible sites of phosphorylation (double underlined) impaired
nuclear import. CGGPKKKRKVG SV40 large T protein. This synthetic
peptide crosslinked to chicken (SEQ ID NO:25) serum albumin and
microinjected into HeLa cells caused nuclear localization. PKKKIKV
(SEQ ID NO:26) A mutated (R.fwdarw.I) version of SV40 large T NLS.
Effective NLS. MKx.sub.11CRLKKLKCSKEKPKC Yeast GAL4 (99 kD).
Fusions of the GAL4 AKCLKx.sub.5Rx.sub.3KTKR (SEQ ID gene portion
encoding the 74 N-terminal amino acid with E. coli NO:27)
.beta.-galactosidase introduced into yeast cells specify nuclear
localization. 74 N-terminal amino acid MKx.sub.11CRLKKLKCSKEKPKC
Yeast GAL4. Acted as an efficient nuclear localization sequence A
(SEQ ID NO:28) when fused to invertase but not to
.beta.-galactosidase introduced 29 N-terminal amino acid by
transformation into yeast cells. Polyoma large T protein.
Identified PKKARED (SEQ ID NO:29) by fusion with pyruvate kinase
cDNA and microinjection of Vero VSRKRPR (SEQ ID NO:30) African
green monkey cells. Mutually independent NLS. Can exert cooperative
effects. CGYGVSRKRPRPG Polyoma virus large T protein. This
synthetic peptide crosslinked (SEQ ID NO:31) to chicken serum
albumin and microinjected into HeLa cells caused nuclear
localization. APTKRKGS SV40 VP1 capsid polypeptide (46 kD). NLS (N
terminus) determined (SEQ ID NO:32) by infection of monkey kidney
cells with a fusion construct containing the 5' terminal portion of
SV40 VP1 gene and the complete cDNA sequence of poliovirus capsid
VP1 replacing the VP1 gene of SV40. APKRKSGVSKC (1-11) Polyoma
virus major capsid protein VP1 (11 N-terminal amino acid). (SEQ ID
NO:33) Yeast expression vectors coding for 17 N-terminal amino acid
of VP1 fused to .beta.-galactosidase gave a protein that was
transported to the nucleus in yeast cells. Subtractive constructs
of VP1 lacking A.sup.1 to C.sup.11 were cytoplasmic. This,
FITC-labeled, synthetic peptide crosslinked to BSA or IgG, caused
nuclear import after microinjection into 3T6 cells. Replacement of
K.sup.3 with T did not. PNKKKRK (SEQ ID NO:34) SV40 VP2 capsid
protein (39 kD). The 3' end of the SV40 VP2-VP3 (amino acid
position 317-323) genes containing this peptide when fused to
poliovirus VP1 capsid protein at the gene level resulted in nuclear
import of the hybrid VP1 in simian cells infected with the hybrid
SV40. EEDGPQKKKRRL (307-318) Polyoma virus capsid protein VP2. A
construct having truncated (SEQ ID NO:35) VP2 lacking the 307-318
peptide transfected into COS-7 cells showed cytoplasmic retention
of VP2. The 307-318 peptide crosslinked to BSA or IgG specified
nuclear import following their microinjection into NIH 3T6 cells.
GKKRSKA (SEQ ID NO:36) Yeast histone H2B. This peptide specified
nuclear import when fused to .beta.-galactosidase. KRPRP (SEQ ID
NO:37) Adenovirus E1a. This pentapeptide, when linked to the
C-terminus of E. coli galactokinase, was sufficient to direct its
nuclear accumulation after microinjection in Vero monkey cells.
CGGLSSKRPRP (SEQ ID Adenovirus type 2/5 E1a. This synthetic peptide
crosslinked NO:38) to chicken bovine albumin and microinjected into
HeLa cells caused nuclear localization. LVRKKRKTE.sub.3SP (NLS 1)
Xenopus N1 (590 amino acid). Abundant in X. laevis oocytes, (SEQ ID
NO:39) forming complexes with histones H3, H4 via two acidic
domains LKDKDAKKSKQE (NLS2) each containing 21 and 9 (D + E),
respectively. The NLS1 is (SEQ ID NO:40) required but not
sufficient for nuclear accumulation of protein N1. NLS 1 and 2 are
contiguous at the C-terminus. GNKAKRQRST v-Rel or p59.sup.v-rel the
transforming protein, product of the v-rel (SEQ ID NO:41) oncogene
of the avian reticuloendotheliosis retrovirus strain T (Rev-T).
v-Rel NLS added to the normally cytoplasmic .beta.-galactosidase
directed that protein to the nucleus. PFLDRLRRDQK NS1 protein of
influenza A virus, that accumulates in nuclei of virus- (SEQ ID
NO:42) infected cells. Determined to be an NLS by deletion
mutagenesis of PKQKRKMAR NS1 in recombinant SV40. The 1st NLS is
conserved among all NS1 (SEQ ID NO:43) proteins of influenza A
viruses. SVTKKRKLE (SEQ ID NO:44) Human lamin A. Dimerization of
lamin A was proposed to give a complex with two NLSs that was
transported more efficiently. SASKRRRLE Xenopus lamin A. NLS
inferred from its similarity to human lamin A (SEQ ID NO:45) NLS.
TKGKRKRID Xenopus lamin L.sub.I. NLS inferred from its sequence
similarity to (SEQ ID NO:46) human lamin A NLS. CVRTTKGKRKRIDV
Xenopus lamin L.sub.I. This synthetic peptide crosslinked to
chicken (SEQ ID NO:47) bovine albumin and microinjected into HeLa
cells caused nuclear localization. ACIDKRVKLD Human c-myc
oncoprotein. This synthetic peptide crosslinked to (SEQ ID NO:48)
chicken bovine albumin and microinjected into HeLa cells caused
nuclear localization. ACIDKRVKLD Human c-myc oncoprotein.
Conjugation of the M1 peptide to human (SEQ ID NO:49) serum albumin
and microinjection of Vero cells gives complete (M1, fully potent
NLS) nuclear accumulation. M2 gave slower and only partial nuclear
localization. RQRRNELKRSP (SEQ ID NO:50) (M2, medium potency NLS)
SALIKKKKKMAP Murine c-abl (IV) gene product. The p160.sup.gag/v-abl
has a cytoplasmic (SEQ ID NO:51) and plasma membrane localization,
whereas the mouse type IV c-abl protein is largely nuclear.
PPKKRMRRRIE Adenovirus 5 DBP (DNA-binding protein) found in nuclei
of infected (SEQ ID NO:52) cells and involved in virus replication
and early and late gene PKKKKKRP (SEQ ID NO:53) expression. Both
NLS are needed, and disruption of either site impaired nuclear
localization of the 529 amino acid protein. YRKCLQAGMNLEARKTKK Rat
GR, glucocorticoid receptor (795 amino acid) NLS1 determined by
KIKGIQQATA (497-524 amino fusion with .beta.-galactosidase (116
kD). NLS1 is 100% conserved acid) between human, mouse and rat GR.
Whereas the 407-615 amino acid (SEQ ID NO:54) fragment of GR
specifies nuclear location, the 407-740 amino acid fragment was
cytoplasmic in the absence of hormone, indicating that sequence
615-740 may inhibit the nuclear location activity. A second (NLS2)
is localized in an extensive 256 amino acid C-terminal domain. NLS
2 requires hormone binding for activity. RKDRRGGRMLKHKRQRDD Human
ER (estrogen receptor, 595 amino acid) NLS. NLS is between
GEGRGEVGSAGDMRAMINO the hormone-binding and DNA-binding regions;
ER, in contrast with ACIDNLWPSPLMIKRSKK GR, lacks a second NLS. Can
direct a fusion product with .beta.- (amino acid 256-303)
galactosidase to the nucleus. (SEQ ID NO:55) RKFKLKFNK Rabbit PG
(progesterone receptor). 100% homology in humans; F.fwdarw.L (SEQ
ID NO:56) change in chickens. When this sequence was deleted, the
receptor became cytoplasmic but could be shifted into the nucleus
by addition of hormone; in this case the hormone mediated the
dimerization of a mutant PG with a wild type PG molecule. GKRKNKPK
(SEQ ID NO:57) Chicken Ets1 core NLS. Within a 77 amino acid
C-terminal segment 90% homologous to Ets2. When deleted by deletion
mutagenesis at the gene level the mutant Ets1 became cytoplasmic.
PLLKKIKQ (SEQ ID NO;58) c-myb gene product; directs puruvate kinase
to the nucleus. PPQKKIKS (SEQ ID NO:59) N-myc gene product; directs
puruvate kinase to the nucleus. PQPKKKP (SEQ ID NO:60) p53; directs
puruvate kinase to the nucleus. SKRVAKRKL c-erb-A gene product;
directs puruvate kinase to the nucleus. (SEQ ID NO:61) CGGLSSKRPRP
Adenovirus type2/5 E1a. This synthetic peptide conjugated with a
(SEQ ID NO:62) bifunctional crosslinker to chicken serum albumin
(CSA) and microinjected into HeLa cells directed CSA to the
nucleus. MTGSKTRKHRGSGA Yeast ribosomal protein L29.
Double-stranded oligonucleotides (SEQ ID NO:63) encoding the 7
amino acid peptides (underlined) and inserted at the N-
MTGSKHRKHPGSGA terminus of the p-galactosidase gene resulted in
nuclear import. (SEQ ID NO:64) RHRKHP (SEQ ID NO:65) Mutated
peptides derived from yeast L29 ribosomal protein NLS, KRRKHP (SEQ
ID NO:66) found to be efficient NLS. The last two are less
effective NLS, KYRKHP (SEQ ID NO:67) resulting in both nuclear and
cytoplasmic location of .beta.-galactosidase KHRRHP (SEQ ID NO:68)
fusion protein. KHKKHP (SEQ ID NO:69) RHLKHP (SEQ ID NO:70) KHRKYP
(SEQ ID NO:71) KHRQHP (SEQ ID NO:72) PETTVVRRRGRSPRRRTPSP Double
NLS of hepatitis B virus core antigen. The two underlined
RRRRSPRRRRSQS (SEQ ID arginine clusters represent distinct and
independent NLS. Mutagenesis NO:73) showed that the antigen fails
to accumulate in the nucleus only when (One sequence, C-terminus)
both NLS are simultaneously deleted or mutated. ASKSRKRKL Viral
Jun, a transcription factor of the AP-1 complex. Accumulates in
(SEQ ID NO:74) nuclei most rapidly during G2 and slowly during G1
and S. The cell cycle dependence of viral but not of cellular Jun
is due to a C.fwdarw.S mutation in NLS of viral Jun. This NLS
conjugated to rabbit IgG can mediate cell cycle-dependent
translocation. GGLCSARLHRHALLAT Human T-cell leukemia virus Tax
trans-activator protein. The most (SEQ ID NO:75) basic region
within the 48 N-terminal segment. Missense mutations in this domain
result in its cytoplasmic retention. DTREKKKFLKRRLLRLDE Mouse
nuclear Mx1 protein (72 kD), Induced by interferons (among
(604-620) 20 other proteins) . Selectively inhibits influenza virus
mRNA (SEQ ID NO:76) synthesis in the nucleus and virus
multiplication. The cytoplasmic Mx2 has R.fwdarw.S and R.fwdarw.E
changes in this region. CGYGPKKKRKV (SV40 large Synthetic peptides
crosslinked to bovine serum albumin (BSA) and T) (SEQ ID NO:77)
introduced into MCF 7 or HeLa S3 cells with viral
co-internalization CGYGDRNKKKKE (human method using adenovirus
serotype 3B induced nuclear import of BSA. retinoic acid receptor)
(SEQ ID NO:78) CGYGARKTKKKIK (human glucocorticoid receptor) (SEQ
ID NO:79) CGYGIRKDRRGGR (human estrogen receptor) (SEQ ID NO:80)
CGYGARKLKKLGN (human androgen receptor) (SEQ ID NO:81)
RKRQRALMLRQAR Human XPAC (xeroderma pigmentosum group A
complementing 30-42 protein) involved in DNA excision repair. By
site-directed (SEQ ID NO:82) mutagenesis and immunofluorescence.
NLS is encoded by exon 1 which is not essential for DNA repair
function. EYLSRKGKLEL (SEQ ID T-DNA-linked VirD2 endonuclease of
the Agrobacterium NO:83) tumefaciens tumor-inducing (T.sub.i)
plasmid. A fusion protein with .beta.- (at the N-terminus)
galactosidase is targeted to the nucleus. The T-plasmid integrates
into plant nuclear DNA; VirD2 produces a site-specific nick for T
integration. VirD2 also contains a bipartite NLS at its C-terminus
(see Table 2). KKSKKKRC (SEQ ID NO:84) Putative core NLS of yeast
TRM1 (63 kD) that encodes the tRNA (95-102) modification enzyme
N.sup.2, N.sup.2-dimethylguanosine-specific tRNA methyltransferase.
Localizes at the nuclear periphery. The 70-213 amino acid segment
of TRM1 causes nuclear localization of .beta.- galactosidase fusion
protein in yeast cells. Site-directed mutagenesis of the 95-102
peptide resulted in its cytoplasmic retention. TRM1 is both nuclear
and mitochondrial. The 1-48 amino acid segment specifies
mitochondrial import. PQSRKKLR (SEQ ID NO:85) Max protein;
specifically interacts with c-Myc protein. Fusion of 126- 151
segment of Max to chicken pyruvate kinase (PK) gene, including this
putative NLS, followed by transfection of COS-1 cells and indirect
immunofluorescence with anti-PK showed nuclear targeting.
QPQRYGGGRGRRW (SEQ ID Gag protein of human foamy retrovirus; a
mutant that completely lacks NO:86) this box exhibits very little
nuclear localization; binds DNA and RNA in vitro.
[0181]
5TABLE 4 "Bipartite" or "split" NLS Signal Oligopeptide Protein and
features C-terminus Xenopus nucleoplasmin. Deletion analysis
demonstrated the presence of a signal responsible for nuclear
location. TKKAGQAKKK (SEQ ID NO:87) Xenopus nucleoplasmin
TKKAGQAKKKKLD Xenopus nucleoplasmin. Whereas these 17 amino acids
had NLS (SEQ ID NO:88) activity, shorter versions of the 17 amino
acid sequences were unable to locate pyruvate kinase to the
nucleus. TKKAGQAKKK(KLD) Xenopus nucleoplasmin. This 14 amino acid
segment was (SEQ ID NO:89) identified as a minimal nuclear location
sequence but was unable to locate puruvate kinase to the nucleus;
three more amino acids at either end (shown in parenthesis) were
needed. CGQAKKKKLD Xenopus nucleoplasmin-derived synthetic peptide;
crossliniked to (SEQ ID NO:90) chicken serum albumin and
microinjected to HeLa cells specified nuclear localization. This
suggests that nucleoplasmin may possess a simple NLS. KRPAMINO ACID
Xenopus nucleoplasmin bipartite NLS. Two clusters of basic
TKKAGQAKKKK (SEQ ID NO:91) amino acids (underlined) separated by 10
amino acid are half NLS components. HRKYEAPRHx.sub.6PRKR (SEQ ID
Yeast L3 ribosomal protein (387 amino acid) N-terminal 21 NO:92)
amino acid. Possible bipartite NLS. (Ribosomal proteins are
transported to the nucleus to assemble with nascent rRNA). Fusion
genes with .beta.-galactosidase were used to transform yeast cells
followed by fluorescence staining with b-gal antibody. The 373
amino acid of L3 fused to .beta.-gal failed to localize to the
nucleus, unless a 8 amino acid bridge containing a proline was
inserted between L3 and .beta.-gal. NKKKRKLSRGSSQKTKGTSASAK SV40
Vp3 structural protein. (35 amino acid C-terminus). By ARHKRRNRSSRS
(one sequence) DEAE-dextran-mediated transfection of TC7 cells with
mutated (SEQ ID NO:93) constructs. RVTIRTVRVRRPPKGKHRK Simian
sarcoma virus v-sis gene product (p28.sup.sis). The cellular (SEQ
ID NO:94) counterpart c-sis gene encodes a precursor of the PDGF
B-chain (platelet-derived growth factor). The NLS is 100% conserved
between v-sis gene product and PDGF. This protein is normally
transported across the ER; introduction of a charged ammo acid
within the hydrophobic signal peptide results in a mutant protein
that is translocated into the nucleus. Puruvate kinase-NLS fusion
product is transported less efficiently than cytoplasmic v-sis
mutant proteins to the nucleus. KRKIEEPEPEPKKAK Putative bipartite
NLS of Xenopus laevis protein factor xnf7. (SEQ ID NO:95) Inferred
by similarity to the bipartite NLS of nucleoplasmin. During oocyte
maturation xnf7 is cytoplasmic until mid-blastula- gastrula stage
due to high phosphorylation. Partial dephosphorylation results in
nuclear accumulation. KKYENVVIKRSPRKRGRPRKD Yeast SWI5 gene
product, a transcription factor. Underlined (SEQ ID NO:96) basic
amino acid show similarity to bipartite NLS of Xenopus
nucleoplasmin. The SWI5 gene is transcribed during S, G2 and M
phases, during which the SWI5 protein remains cytoplasmic due to
phosphorylation by CDC28-dependent histone H1 kinase at three
serine residues two near and one (double underlined) in the NLS.
Translocated at the end of anaphase/G1 due to dephosphorylation of
NLS. NLS confers cell cycle-regulated nuclear import of
SWI5-.beta.-galactosidase fusion protein. MKRKRNS 735-741 Bipartite
NLS of influenza virus polymerase basic protein 2 (SEQ ID NO:97)
(PB2). Mutational analysis of PB2 and transfection of BHK cells
GIESIDNVMGMIGILPDMTPSTEM showed that both regions are involved in
nuclear import. SMRGVRISKMGVDETSSAEKIV Deletion of 449-495 region
gives perinuclear localization to the 449-495 (SEQ ID NO:98)
cytoplasmic side. AHRARRLH (SEQ ID NO:99) "Tripartite" or "doubly
bipartite" NLS of adenovirus DNA 6-13 (BSI) polymerase (AdPol). BSI
and II functioned interdependently as PPRRRVRQQPP (SEQ ID NO:100)
an NLS for the nuclear targeting of AdPol, for which BSIII was
23-33 (BSII) dispensable. BSII-III was more efficient NLS than
BSI-II. PARARRRRAP (SEQ ID NO:101) 39-48 (BSIII) KRKx.sub.11KKKSKK
207-226 Human poly(ADP-ribose) polymerase (116 kD). The linear (SEQ
ID NO:102) distance between the two basic clusters is not crucial
for NLS activity in this bipartite NLS. Lysine 222 (double
underlined) is an essential NLS component. DNA binding and
poly(ADP- ribosyl)ating active site are independent of NLS.
GRKRAFHGDDPFGEGPPDKKGD Herpes simplex virus ICP8 protein
(infected-cell protein). This (SEQ ID NO:103) C-terminal portion of
ICP8 introduced into pyruvate kinase (PK) caused nuclear targeting
in transfected Vero cells. Inclusion of additional ICP8 regions to
PK led to inhibition of nuclear localization. KRPREDDDGEPSERKRARDDR
Bipartite NLS of VirD2 endonuclease of rhizogenes strains of (SEQ
ID NO:104) Agrobacterium tumefaciens. Within the C-terminal 34
amino acid. Each region (underlined) independently directs .beta.-
glucuronidase to the nucleus, but both motifs are necessary for
maximum efficiency. VirD2 is tightly bound to the 5' end of the
single stranded DNA transfer intermediate T-strand transferred from
Agrobacterium to the plant cell genome.
[0182]
6TABLE 5 "Nonpositive NLS" lacking clusters of arginines/lysines
Signal oligopeptide Protein and features QLVWMACNSAMINO Influenza
virus nucleoprotein (NP). The underlined region
ACIDFEDLRVLSFIRGTKVS (327-345) when fused to chimpanzee
a.sub.1-globin at the cDNA level and PRG 327-356 microinjected into
Xenopus oocytes specifies nuclear localization. (SEQ ID NO:105)
MNKIPIKDLLNPQ Yeast MAT a2 repressor protein, containing a
homeodomain. (NLS 1 at N-terminus) (SEQ ID The two NLS are
distinct, each capable of targeting .beta.-galactosidase to NO:106)
the nucleus. However, deletion of NLS2 results in a2 accumulation
at VRILESWFAKNIEN the pores. NLS1 and 2 may act at different steps
in a localization PYLDT (NLS2 at amino acid pathway. Part of the
homeodomain mediates nuclear localization in 141-159, part of the
addition to DNA binding. The core pentapeptide containing proline
and homeodomain) two other hydrophobic amino acids flanked by
lysines or arginines (SEQ ID NO:107) (underlined) was suggested as
one type of NLS core. Rx.sub.7Kx.sub.15KIPRx.sub.3HFY Drosophila
HP1 (206 amino acids) that binds to EERLSWYSDNED (SEQ ID
heterochromatin and is involved in gene silencing. NLS identified
by .beta.- NO:108) galactosidase/HP1 fusion proteins introduced by
P-element mediated 152-206 (C-terminal transformation into
Drosophila embryos. segment) FVx.sub.7- Adenovirus type 5 E1A
internal, developmentally-regulated .sub.20MxSLxYMx.sub.4MF NLS.
This NLS functions in Xenopus oocytes but not in somatic cells.
This NLS can be utilized up to the early neurula stage.
[0183]
7TABLE 6 Nucleolar localization signals (NoLS) Signal oligopeptide
Protein and features MPKTRRRPRRSQRKRPPTP Nucleolus localization
signal in amino terminus of human p27.sup.x- (SEQ ID NO:109)
.sup.III protein (also called Rex) of T cell leukemia virus type I
(HTLV-I). When this peptide is fused to N-terminus of .beta.-
galactosidase, directs it to the nucleolus. Deletion of residues 2-
8 (underlined), 12-18 (double-underline) or substitution of the
central RR (dotted-underlined) with TT abolish nucleolar
localization. Other amino acids between positions 20-80 increase
nucleolar localization efficiency. RLPVRRRRRRVP (SEQ ID NO:110)
Adenovirus pTP1 and pTP2 (preterminal proteins, 80 kD) between
amino acid residues 362-373. The 140 kD DNA polymerase of
adenovirus when it has lost its own NLS can enter the nucleus via
its interaction with pTP. The staining was nuclear and nucleolar
with some perinuclear staining as well. The NLS fused to the
N-terminus of E. coli .beta.-galactosidase was functional in
nuclear targeting. GRKKRRQRRRP HIV (human immunodeficiency virus)
Tat protein; localizes (SEQ ID NO:111) pyruvate kinase to the
nucleolus. Tat is constitutively nucleolar. RKKRRQRRR(AHQ) Tat
positive trans-activator protein of HIV-1 (human Nucleolar
localization signal immunodeficiency virus type 1). The 3 amino
acids shown in (SEQ ID NO:112) parenthesis are essential for the
localization of the .beta.- galactosidase to the nucleolus. The 9
amino acid basic region is able to localize .beta.-gal to the
nucleus but not to the nucleolus. KRVKLDQRRRP (SEQ ID NO:113)
Artificial sequence from c-Myc and HIV Tat NLSs that effectively
localizes pyruvate kinase to the nucleolus. FKRKHKKDISQNKRAVRR
Human HSP70 (heat shock protein of 70 kD); localizes pyruvate (SEQ
ID NO:114) kinase to the nucleus and nucleolus. HSP70 is
physiologically cytoplasmic but with heat-shock HSP70 redistributes
to the nucleoli, suggesting that the nucleolar targeting sequence
is cryptic at physiological temperature and is revealed under heat-
shock. RQARRNRRRRWRERQR (35-50) HIV-1 Rev protein (116 amino acid,
nucleolar). Mutations in (SEQ ID NO:115) either of the two regions
of arginine clusters severely impair nuclear localization.
.beta.-galactosidase fused to R.sub.4W was targeted to the nucleus,
and fused to the entire 35-50 region, was targeted to the
nucleolus. RQARRNRRRRWRERQRQ (35-51) HIV-1 Rev protein. A fusion of
this Rev peptide with .beta.- (SEQ ID NO:116) galactosidase became
nuclear but not nucleolar. The 1-59 amino acid segment of Rev fused
to .beta.-galactosidase localized entirely within the nucleolus.
Whereas the NRRRRW (bold) is responsible for nuclear targeting, the
RR and WRERQRQ (double underlined) specify nucleolar localization.
Rev may function to export HIV structural mRNAs from the nucleus to
the cytoplasm.
[0184]
8TABLE 7 Karyophilic clusters on non-membrane protein kinases
Non-membrane Karyophilic peptides protein kinase Species Features
73 FVVHKRCHE Protein kinase C (673 Bovine, human Known to
translocate to the (SEQ ID NO:117) aa) .beta. type nucleus
following treatment of 96 DDPRSKHKFKIH cells with mitogens. (SEQ ID
NO:118) 577 TKHPGKRLG (SEQ ID NO:119) 71 FVVHRRCHEF Protein kinase
C (697 bovine, human .gamma. (SEQ ID NO:120) aa) type 95
DDPRNKHKFRLH (SEQ ID NO:121) 591 TKHPAKRLG (SEQ ID NO:122) 72
FVVHKRCHE Protein kinase C (673 rabbit type .alpha. and (SEQ ID
NO:123) aa) .beta. 96 DDPRSKHKFKIH (SEQ ID NO:124) 577 TKHPGKRLG
(SEQ ID NO:125) 71 FVVHRRCHE PKC-I (701 aa) rat brain (SEQ ID
NO:126) 95 DDPRNKHKFRLH (SEQ ID NO:127) 594 TKHPGKRLG (SEQ ID
NO:128) 22 GENKMKSRLRKG Protein kinase C Drosophila 14 exons, 20
kb; 3 transcripts in (not conserved) (639 aa, 75 kDa) adult flies;
not expressed in 0-3 h (SEQ ID NO:129) Drosophila embryos; the
80SYVVHKRCHEYVT VVHKRCHE (SEQ ID (conserved) NO: 133)motif (or
VVHRRCHE (SEQ ID NO:130) (SEQ ID NO:134)) is conserved
211PDDKDQSKKKTR among all PKC known. TIK (not conserved) (SEQ ID
NO:131) 614PPFKPKIKHRKMC P (not conserved) (SEQ ID NO:132) 148
KKVLQDKRFK Glycogen synthase rat brain Phosphorylates glycogen
synthase, NRELQIMRKLD (SEQ kinase 3 c-Jun, c-Myb; two isoforms ID
NO:135) GSK-3.alpha. encoded by discrete genes; highly (483 aa)
expressed in brain; both .alpha. and .beta. forms are cytosolic but
also GSK-3.beta. associated with the plasma (420 aa) membrane
consistent with their role in signal transduction from the cell
surface. LQDRRFKNRELQ Zw3 Drosophila Product of the segment
polarity (SEQ ID NO:136) zeste-white 3 gene zw3; the protein
encoded has 34% homology to cdc2; mutations in zw3 give embryos
that lack most of the ventral denticles, differentiated structures
derived from the most anterior region of each segment.
289ECLKKFNARRKL Ca.sup.2+/calmodulin- rat brain Composed of nine 50
kDa .alpha.- KGAIL dependent protein subunits and three 60 kDa
.beta.- (SEQ ID NO:137) kinase II (CaM kinase subunits; both are
catalytic; II) .beta. subunit (542 aa, calmoduim- and ATP-binding
60.3 kDa) domains; highly expressed in forebrain neurons,
concentrated in postsynaptic densities; acts as a
Ca.sup.2+-triggered switch and could be involved in long-lasting
changes in synapses. 290LKKFNARRKL CaM kinase II (478 rat brain
This particular isoform is KGAILTTM (SEQ ID aa, 54 kDa) exclusively
expressed in the brain; NO:138) .alpha.-subunit high enzyme levels
in specific 450EETRVWHRRDGK brain areas; might be involved in (SEQ
ID NO:139) short- and long-term responses to transient stimuli. 185
GFAKRVKGRT CADPK catalytic bovine (cardiac By Edman degradation of
protein WTLCG subunit (349 aa, 40.6 muscle) fragments; mediates the
action of (SEQ ID NO:140) kDa) and is activated by cAMP; consists
of two regulatory (R) and two catalytic (C) subunits; cAMP releases
the C subunit from the inactive R.sub.2C.sub.2 cADPK; two cDNAs
were cloned encoding two isoforms of the catalytic subunit of cADPK
in mouse. 186 GFAKRVKGRTW CADPK bovine cDNA was isolated by
screening a TLCG (catalytic subunit) bovine pituitary cDNA library;
(SEQ ID NO:141) (350 aa) 93% sequence similarity to known bovine
cADPK; represents the second gene for the catalytic subunit of
cADPK. 29 EEEIQELKRKLH CGDPK (SEQ ID bovine lung By protein
sequencing; composed KCQSVLP (SEQ ID NO: 144) of two identical
subunits activated NO:142) (670 aa, 76.3 kDa) in an allosteric
manner by binding 389 KILKKRHIVDTR of cGMP and not by dissociation
(SEQ ID NO:143) of catalytic subunit as in cADPK; sequence similar
to cADPK 117 KTLKKHTIVK TPK3 S. cerevisiae cAMP-DPK is a tetrameric
protein (SEQ ID NO:145) (398 aa) with two catalytic and two cADPK
regulatory subunits; cAMP activates the kinase by dissociating the
catalytic subunits from the tetramer; all three TPK 1, 2, 3 are
catalytic subunits. 16S.sub.2H.sub.13GHG.sub.2 SNF1 (633 aa, 72
kDa) S. cerevisiae Ser/Thr kinase; 166 EYCHRHKIVHRD
autophosphorylated; plays a LKP (SEQ ID NO:146) central role is
carbon catabolite 495 PLVTKKSKTRWH repression in yeast required for
FG (SEQ ID NO:147) expression of glucose-repressible genes; region
60-250 shows high sequence similarity to cAMP- dependent protein
kinase (cADPK). 70 PVKKKKIKREIK Casein kinase II (.alpha.-
Drosophila CKIL is composed of a and 3 (SEQ ID NO:148) subunit,
catalytic) melanogaster subunits in a a2f32 130-150 kDa 269
DILQRHSRKRW (336 aa) protein; the a-subunit is the ERF (SEQ ID
NO:149) catalytic and the f3is 146 PKSSRHHHTDG CKII
(.beta.-subunit, Drosophila autophosphorylated. (SEQ ID NO:150)
regulatory) (215 aa) melanogaster 142 PKSSRHHHTDG CKII
(.beta.-subunit, bovine (lung) (SEQ ID NO:151) regulatory) (209 aa,
24.2 kDa) 108 PKQRHRKSLG KIN1 (1064 aa, 117 S. cereviszae 30% aa
similarity to bovine (SEQ ID NO:152) kDa) cADPK and 27% (KINi) or
25% 129 GSMCKVKLAK (KJN2) aa similarity to v-Src HRYTNE within the
kinase domain; the (SEQ ID NO:153) catalytic domains of KINi and
506 DRKHAKIRNQ KIN2 are near the N-terminus and (SEQ ID NO:154) are
structural mosaics with features 638 GNIFRKLSQRR characteristic of
both Tyr and KKTIEQ Ser/Thr kinases. (SEQ ID NO:155) 773
PPLNVAKGRKL HP (SEQ ID NO:156) 87 ELRQFHRRSLG KIN2 (1152 aa, 126 S.
cerevisiae (SEQ ID NO:157) kDa) 111 GKVKLVKHRQ TKE (SEQ ID NO:158)
217 GSLKEHHARKF ARG (SEQ ID NO:159) 807 LSVPKGRKLHP (SEQ ID NO:160)
60FLRRGIKKKLTLD STE7 (515 aa) S. cerevisiae Implicated in the
control of the (SEQ ID NO:161) three cell types in yeast: (a, a,and
472 PSKDDKFRHWC ala) of which a and a cells are RKIKSKIKEDKRIKRE
haploid and are specialized for (SEQ ID NO:162) mating whereas a/a
cells are diploid and are specialized for meiosis and sporulation;
with the exception of the mating type locus, MAT, all cells contain
the same DNA sequences. STE7 gene produces insensitivity to cell-
division arrest induced by the yeast mating hormone, a-factor. 722
QRRVKKLPSTTL S6KII.alpha. (733 aa) Xenopus (SEQ ID NO:163)
QRRVKKLPSITL S6KII .beta. Xenopus (SEQ ID NO:164) 742 QRRVKKLPSTTL
S6KII (752 aa) Chicken (SEQ ID NO:165) 713QRRVRKLPSTTL S6KII (724
aa) Mouse (SEQ ID NO:166) 16GVVYKGRHKTTG CDC2Hs Human Isolated by
expressing a human (SEQ ID NO:167) (297 aa) cDNA library in S.
pombe and 120 FCHSRRVLHRD p34.sup.cdc2 selecting for clones that
LKP (SEQ ID NO:168) complement a mutation in the cdc2 yeast gene;
the human CDC2 gene can complement both the inviability of a null
allele of S. cerevisiae CDC28 and cdc2 mutants of S. pombe; CDC2
mRNA appears after that of CDK2. GVVYKARHKLSGR cdc2 (297aa) S.
pombe High homology to S. cerevisiae (SEQ ID NO:169) CDC28.
119HSHRVLHRDLKP CDK2 (cell division Human The human CDK2 protein
has 65% (SEQ ID NO:170) kinase 2) (298 aa) sequence identity to
human p34.sup.cdc2 and 89% sequence identity to Xenopus Eg1 kinase;
human CDK2 was able to complement the inviability of a null allele
of S. cerevisiae CDC28 but not cdc2 mutants in S. pombe. CDK2 mRNA
appears in late G1/early S. 109 FCHSHRVLHRD Eg1 (297 aa) Xenopus
Cdk2-related LKP (SEQ ID NO:171) 125 GIAYCHSHRILH CDC28 (298 a) S.
cerevisiae The homolog of S. pombe Cdc2 RDLKP (SEQ ID NO:172) 119
HSHRVIHRDLKP cdk3 (305 aa) Human (SEQ ID NO:173) 56 KELKHKNIVR
PSSALRE (291 aa) Human cdc2-related kinase. (SEQ ID NO:174) (SEQ ID
NO:175) 1 MDRMKKIKRQ (N- PCTAIRE-1 (496 aa) Human cdc2-related
kinase. terminus) (SEQ ID NO:176) 141 DKPLSRRLRRV (SEQ ID NO:177) 1
MKKFKRR PCTAIRE-2 (523 aa) Human cdc2 related kinase. (SEQ ID
NO:178) 129 RNRIHRRIS (SEQ ID NO:179) 172 SRRSRRAS (SEQ ID NO:180)
304 HRRKVLHR (SEQ ID NO:181) 512 GHGKNRRQSM LF (SEQ ID NO:182) 163
HTRKILHR PCTAIRE-3 Human cdc2 related kinase. (SEQ ID NO:183) (380
aa) 369 PGRGKNRRQSIF (SEQ ID NO:184) 69 EVFRRKRRLH KKIALRE (358 aa)
Human cdc2-related kinase. (SEQ ID NO:185) (SEQ ID NO:187) 302
DKPTRKTLRKSR KHH (SEQ ID NO:186) 1 MVKRHKNT nim1.sup.+ gene product
S. pombe (SEQ ID NO:188) (new inducer of 87 DGELFHYIRKHGP mitosis);
protein (SEQ ID NO: 189) kinase (370 aa) 114 DAVAHCHRFRFR HRD (SEQ
ID NO:190) 295 KKSSSKKVVRRL QQRDD (SEQ ID NO:191) 194 PAQKLRKKNNFD
Wee1.sup.+ gene product S. pombe The Wee1.sup.+ gene functions as a
(SEQ ID NO:192) (877 aa) dose-dependent inhibitor that 388
KQHRPRKNTNFT delays the initiation of mitosis PLPP (SEQ ID NO:193)
until the yeast cell has attained a 592 KYAVKKLKVKF certain size;
Wee1 has a protein SGP (SEQ ID NO:194) kinase consensus probably
regulating cdc2 kinase. 266 PNETRRIKRAN CDC7 (497 aa) S. cerevisiae
Required for mitotic but not RAG (SEQ IDNO:195) meiotic DNA
replication presumably to phosphorylate specific replication
protein factors; implicated in DNA repair and meiotic
recombination; some homology with CDC28 and oncogene protein
kinases but differs in a large region within the phosphorylation
receptor domain. 48YDHVRKTRVAIKK ERK1 (MAP kinase) Rat Known to
translocate to the (SEQ ID NO:196) (367 aa; 42 kDa) nucleus
following their activation by phosphorylation at T-190, and Y-192
(T-183, Y-185 in ERK2). 59 ILKHFKHE FUS3 (353 aa) S. cerevisiae
MAP-(ERK1)-related. (SEQ ID NO:197) 252 QIKSKRAKEY KSS1 (368 aa) S.
cerevisiae MAP-(ERK1)-related. (SEQ ID NO:198) ELVKHLVKHGSN SW16 S.
cerevisiae Activator of CACGA-box with (SEQ ID NO:199) (803aa, 90
kDa) sequence similarity to cdc10; GKAKKIRSQLL required at START of
cell cycle. (SEQ ID NO:200) EQRIKRHRJDVSDED cdc10 S. pombe (SEQ ID
NO:201) SNIKSKCRRVV (SEQ ID NO:202) 37 PPKRIRTD CTD kinase (528 aa)
S. cerevisiae Consists of 3 subunits of 58, 38, (suggested by the
58 kDa subunit and 32 kDa; disruption of the 58 authors) (SEQ ID
(catalytic) kDa gene gives cells that lack CTD NO:203) kinase, grow
slowly, are cold 492 KLARKQKRP sensitive, but have different (SEQ
ID NO:204) phosphorylated forms of RNA pol II. 29 GVSSVVRRCIHKP
Phosphorylase kinase Rabbit (skeletal (SEQ ID NO:205) (catalytic
subunit) muscle) (386 aa) 489 KKYMARRKW Myosin light chain Chicken
gizzard Ca.sup.2+/calmodulin-activat- ed; QKTGHAV kinase (MLCK)
(669 phosphorylated by cADPK; first (SEQ ID NO:206) aa) described
as responsible for the phosphorylation of a specific class of
myosin light chains; required for initiation of contraction in
smooth muscle. 314 PWLNNLAEKAK Myosin light chain Rabbit (skeletal
By protein sequencing. RCNRRLKSQ kinase (partial 368 muscle) (SEQ
ID NO:207) carboxy-terminal aa 334 ILLKKYLMKJRR sequence) WKKNFIAVS
(SEQ ID NO:208) 28 GVSSVVRRCIHKP Phosphorylase kinase Mouse
(muscle) Glycogenolytic regulatory enzyme; (SEQ ID NO:209) (PhK)
(catalytic .gamma. undergoes complex regulation; subunit) (389 aa)
composed of 16 subunits containing equimolar ratios of .alpha.,
.beta., .gamma. and .delta. subunits; high levels in skeletal
muscle; isoforms in cardiac muscle and liver; cDNA probe does not
hybridize to X chromosome in mice and is thus distinct from the
mutant recessive PhK deficiency that results in glycogen storage
disease.
[0185]
9TABLE 8 Nuclear localization signals on DNA repair proteins Gene
Equivalent protein Putative NLS product in other species Features
HIGHER EUKARYOTES None ERCC1 RAD10 297 aa; DBD; interacts
(N-terminus) strongly with ERCC4 (XPF) MDPGKDKEGvpqpsgppaRKKF to
form an excision (bipartite NLS) endonuclease; unless the (SEQ ID
NO:210) KDKx.sub.11RKK is a bipartite NLS it may depend upon its
binding with ERCC4 for its nuclear import. None ERCC2 RAD3 (S. cer)
760 aa; DNA helicase 681DKRFARGDKRGKLPR (XPD) component of TFIIH,
(near the C-terminus) (four essential for cell viability; positive,
one negative over a contains one nucleotide- heptapeptide stretch)
binding, one DNA-binding, (SEQ ID NO:211) and seven domains
characteristic of helicases; 52% identity with S. cer RAD3 at the
amino acid level. 8 DRDKKKSRKRHYEDEE ERCC3 SSL2 (S. cer) 782 aa;
helicase, component (SEQ ID NO:212) (XPB) Haywire(Dros) of TFIIH
essential for cell 522 YVAIKTKKRILLYTM viability; helix-turn-helix,
(SEQ ID NO:213) DNA-BD, and helicase (weak NLS if at all,
hydrophobic domains environment) 769 PSKHVHPLFKRFRK (SEQ ID NO:214)
84 KKQTLVKRRQRKD ERCC5 RAD2; 1186 aa in human, 1196 in X. (SEQ ID
NO:215) (XPG) Rad13 laevis; 3' incision 210 EFTKRRRTL endonuclease;
involved in (SEQ ID NO:216) homologous recombination; 390
DESMIKDRKDRLP strongly nuclear (SEQ ID NO:217) 1170 GKKRRKLRRARGRK
RKT (SEQ ID NO:218) 253PQKQEKKPRKIMLNEASG ERCC6 RAD26 1493 aa;
involved in the (SEQ ID NO:219) CS-B preferential repair of active
314 PNKKARVLSKKEERLKK genes; nonessential for cell HIKKLQKR (SEQ ID
NO:220) viability 406 PLPKGGKRQKKVP (SEQ ID NO:221) 455
DGDEDYYKQRLRRWNK LRLQDKEKRLKLEDDSEESD (SEQ ID NO:222) 1028
DVQTPKCHLKRRIQP X.sub.8PKRKKFP (SEQ ID NO:223) 1180
KHKSKTKHHSVAEEETL EKHLRPKQKPKX.sub.15PHLVKK RRY (SEQ ID NO:224)
1324 PAGKKSRFGKKRN (SEQ ID NO:225) 21 PASVRASIERKRQRALM XPA RAD14
273 aa; zinc finger domain; LRGAR (SEQ ID NO:226) involved in
lesion 160 PPLKFIVKKNPHHSQW recognition GD (weak) (SEQ ID NO:227)
210 NREKMKQKKFDKKVKE (weak because of F) (SEQ ID NO:228) 72
YLRRAMKRFN (weak) XPC RAD4 (23% identity, 823 aas, 92.9 kDa; very
(SEQ ID NO:229) 44% similarity) hydrophilic protein; might be 262
PSAKGKRNKGGRKKRSK involved in lesion PSSSEEDEGPG (SEQ ID
recognition since XPC cells NO:230) (40% of all XP cases) can 297
QRRPHGRERR (weak) repair active parts of the (SEQ ID NO:231) genome
whereas inactive and 368 RTHRGSHRKDP (weak) the nontranscribed
strand of (SEQ ID NO:232) active genes are not repaired 384
SSSSSSSKRGKKMCSDG (SEQ ID NO:233) 531 ALKRHLLKYE (weak) (SEQ ID
NO:234) 594 SNRARKARLAEP (SEQ ID NO:235) 660 PNLHRVARKLD (weak)
(SEQ ID NO:236) 716 ERKEKEKKEKR (SEQ ID NO:237) 740 IRERLKRRYG (SEQ
ID NO:238) 801 GGPKKTKRERK (SEQ ID NO:239) 20 KSKAKSKARREEEEED XPC
940 aa; the first 117 aa are (SEQ ID NO:240) lacking in the
Legerski and 54 GKRKRG (SEQ ID NO:241) Peterson, (1992) XPC 69
GPAKKKVAKVTVK sequence (see above); the (SEQ ID NO:242) following
823 aa are 103 PSDLKKAHHLKRG identical. (SEQ ID NO:243) 82
EIDRRKKRPLENDGPVKK Rep-3 Swi4 (S. pom) 1137 aa; mismatch repair
KVKKVQQKE (SEQ ID (mouse) protein; Rep-3 is in the NO:244) Duc-1
immediate 5' flanking region 375 KENVRDKKKG (HeLa) of DHFR gene (89
bp) but (SEQ ID NO:245) transcribed from the opposite 571
FGRRKLKKWVT strand; a bidirectional (SEQ ID NO:246) promoter is
used for both 710 PLIKKRKDEIQG transcripts. (SEQ ID NO:247) 1091
KELEGLINTKRKRLKYF AKLW (SEQ ID NO:248) 422 EKHEGKHQKLL (weak) hMSH2
MSH2 (S. cer) human mismatch repair (SEQ ID NO:249) protein;
homologous to S. cerevisiae MSH2; associated with the hereditary
nonpolyposis colon cancer gene on chromosome 2p16. 397
PDIRRLTKKLNKRG MSH2 (SEQ ID NO:250) (S cer) 547 DAKELRKHKKYIE (SEQ
ID NO:251) 869 VKMAKRKANE (SEQ ID NO:252) 95 GELAKRSERRAEAE Human
Rad2 Rad2 (S. pom) 400 aa; required for fidelity (SEQ ID NO:253) of
chromosome separation at 354 KRKEPEPKGSTKKKAK mitosis; limited
similarity to TG (SEQ ID NO:254) RAD2 (ssDNA nuclease), 394 GKFKRGK
(SEQ ID rad 13, and XPG (ERCC5). NO:255) None mouse 339 aa;
recombination-repair RAD51 protein; 83% homology to S cerevisiae
RAD51 and 55% homology to E. coil RecA. None HHR23B/ RAD23 Subunit
of XPC (125 kDa) p58 None HHR23A RAD23 Subunit of XPC (125 kDa) 32
PSQAEKKSRARAQ RPA (34 kDa RPA (70, 34, and 14 kDa (SEQ ID NO:256)
subunit) subunits) might stabilize the helicase-melted DNA around
the lesion; antibodies against RPA 32 kDa subunit inhibit DNA
replication. GAKKRKIDDA ATPase Q1 RecQ (E. coli) 649 aa; altered in
XPC cells; (SEQ ID NO:257) undetermined role in repair PKKPRGKM
(SEQ ID NO:258) HMG-1 Calf thymus HMG 1 EHKKKHP (SEQ ID NO:259)
(259 aa); involved in the ETKKKFKDP (SEQ ID NO:260) recognition of
cisplatin EKSKKKK(E/D).sub.41 (SEQ ID lesions NO:261)
E.sub.3G.sub.2KKKKKFAK (SEQ ID NO:262) 512 RDEKKRKQLKKAKAK SSRP1
ABF (S. cer) 709 aa, 81 kDa, structure- MAKDRKSRKKP (SEQ ID
specific recognition protein NO:263) 1; involved in recognition of
619 GESSKRDKSKKKKKVKV cisplatin-induced lesions; KMEKK (SEQ ID
NO:264) also involved in Ig gene 674 GENKSKKKRRRSEDSEE
recombination; one HMG- EE (SEQ ID NO:265) box, similarity to SRY,
MTFII, LEF-1, TCF-1a, and ABF2. 1 MPKRGKKG (SEQ ID Ref-1 Redox
factor 1 from HeLa NO:266) (HAP1) cells; 37 kDa, 318 aa;
apurinic/apyrimidinic (AP) endonuclease for DNA repair but also of
redox activity stimulating Jun/Fos DNA binding. 1 MPKRGKKG HAP1
ExoIII 323 aa; apurinic/apyrimidinic (SEQ ID NO:267) (bovine) (E.
coli) (AP)-endonuclease ExoA (S. pneumoniae) DROSOPHILA 1
MGPPKKSRKDRSGGDKF Haywire ERCC3 (XPB) helicase with 66% identity to
GKKRRGQDE human ERCC3; flies (SEQ ID NO:268) SSL2 (S. cer)
expressing marginal levels of EMSYSRKRQRFLVNQG Haywire display
motor (weak) (SEQ ID NO:269) defects and reduced life span
YYEHRKKNIGSVHPLFK KFRG (bipartite) (SEQ ID NO:270) 77 ARGKKKQPK
(SEQ ID Rrp1 HAP1 Recombination repair protein NO:271) 1); 679 aa;
the 252 aa C- 98 KPKGRAKKA (SEQ ID terminal domain is NO:272)
homologous to AP- 157 QAKGRKKKELP (SEQ ID endonucleases, whereas
the NO:273) 1-426 aa domain is highly 179 EPPKQRARKE (SEQ ID
charged, carries all of the NO:274) putative NLSs. 241
PPKAASKRAKKGK (SEQ ID NO:275) 282 PKKRAKKTT (SEQ ID NO:276) 317
EPAPGKKQKKSAD (SEQ ID NO:277) 336 EEEAKPSTETKPAKGR KKAP (SEQ ID
NO:278) 372 KPARGRKKA (SEQ ID NO:279) 394 GSKTTKKAKKAE (SEQ ID
NO:280) S. CEREVISIAE 200 IEKLRRKLYISGG RAD1 ERCC4 1100 aa; 30%
sequence (SEQ ID NO:281) (XPF) identity to Rad16; RAD1 515
NKKRGVRQVLLN (SEQ Rad16 interacts strongly with ID NO:282) RAD10
565 KEQVTTKRRRTRG (conserved in Rad16) (SEQ ID NO:283) 1024
NLRKKIKSFNKLQ (SEQ ID NO:284) 89 RQRKERRQGKRE RAD2 XPGC 1031 aa,
117.8 kDa; ssDNA (SEQ ID NO:285) Rad13 endonuclease; rad mutants
907 ENKFEKDLRKKLVNNE are defective in incision (SEQ ID NO:286) 984
RDVNKRKKKGKQKRI (SEQ ID NO:287) 1017 KRISTATGKLKKRXM (SEQ ID
NO:288) 672 GKDDYGVMVLADRRF RAD3 ERCC2 or XPD; 778 aa, 89,779 Da;
30% SRKRSQLP (contains the bulky (S. cer) Rad15 or Rhp3 sequence
identity to rad16; F) (SEQ ID NO:289) ATP-dependent DNA helicase;
single-stranded DNA-dependent ATPase. 26 PLSRRRRVRRKNQPLPD RAD4 XPC
754 aa; mutations in RAD4 AKKKFKTG (SEQ ID NO:290) that that
inactivate the 134 NEERKRRKYFHMLYL excision repair function of (SEQ
ID NO:291) RAD4 result in truncated 160 EWINSKRLSRKLSNL proteins
missing the C- (weak) (SEQ ID NO:292) terminal one-third of RAD4.
254 EMSANNKRKFKTLKRSD weak (SEQ ID NO:293) 382 WMNSKVRKRRITKDDF GEK
(SEQ ID NO:294) 403 RKVITALHHRKRTKID DYED (SEQ ID NO:295) 504
KTGSRCKKVIKRTVGRP (SEQ ID NO:296) 150 FHPKRRRIYGFR (SEQ ID RAD5
1169 aa; helicase involved in NO:297) postreplication-repair (RAD6
215 DSRGRKKASM (SEQ ID epistasis group); binds DNA NO:298) with the
seven helicase 297 DGESLMKRRRTEGGNK motifs and with zinc fingers;
REK (SEQ ID NO:299) increases the instability of 1152 DEDERRKRRIEE
poly (GT) repeats in the yeast (SEQ ID NO:300) genome. 1
MSTPARRRLMRDFKRM RAD6 RAD6 mediates the KEDAPP (SEQ ID NO:301)
ubiquitination of H2A and H2B histones 15 GVAKLRKEKSGAD RAD10 ERCC1
210 aa; forms an (SEQ ID NO:302) endonuclease with RAD1; 76
DDYNRKRPFRSTRPGK the basic and tyrosine-rich (SEQ ID NO:303)
central domain was suggested to bind DNA by ionic interactions and
tyrosine intercalation. 172 EGKAHRREKKYE RAD14 XPAC 247 aa, 29.3
kDa; two zinc (SEQ ID NO:304) fingers; involved in lesion 200
NRLREKKHGKAHIHH recognition; 27% sequence (SEQ ID NO:305) identity
and 54% sequence similarity (if conserved residues are grouped
together) to human XPA; deletion of RAD14 gene generates high UV
sensitivity. 345 ERRKQLKKQGPKRP Ixr1 591 aa; two consecutive (SEQ
ID NO:306) (S. cer) HMG boxes; involved in 479 ETYKKRIKEWESCYPDE
recognition of 1,2-intrastrand (SEQ ID NO:307) d(GpG) and d(ApG)
cisplatin crosslinks. None RAD23 HHR23 483 LTCKKLKTHNRIILSG RAD26
ERCC6 1075 aa; disruption of the weak (SEQ ID NO:308) (yeast CS-B
(hum) RAD26 gene gives viable 934 NALRKSRKKITKQYEIGT ERCC6) yeast
cells unable to PX.sub.9GEIRKRDP preferentially repair the (SEQ ID
NO:309) actively transcribed strands; surprisingly, in contrast to
human CS-B cells, disruption of the RAD26 in yeast does not cause
sensitivity to UV, Cisplatin, or X-rays. 634 KPTSKPKRVRTATKKKIP
MRE11 Rad32 (S. pom) meiotic recombination (SEQ ID NO:310) protein;
functions in the 408 FYKKRSPVTRSKKSG same pathway with RAD51 (SEQ
ID NO:311) none; RAD51 RecA (E. coli) 402 aa; essential for repair
of 361 GFKKGKGCQR DSBs and recombination; (SEQ ID NO:312)
associates strongly with RAD52; self associates; neither RAD51 nor
RAD52 possess a typical simple NLS. none; RAD51 (K. 364 aa 328
GFKKGKGCQR lactis) (SEQ ID NO:313) none; RAD52 Rad22 504 aa; rad52
mutants are 155 ERAKKSAVTDALKRSLR defective in ionizing
GFGX.sub.gDKDFLAKIDKVKFD- P radiation, mitotic PD (tripartite)
recombination, mating-type (SEQ ID NO:314) switching, and repair of
DSDs. 1 MARRRLPDRPP RAD54 898 aa; recombination-repair (SEQ ID
NO:315) protein; ATP-binding motif; 65 GGRSLRKRSA helicase domains;
in the (SEQ ID NO:316) same subfamily of helicases 99 QLTKRRKD with
MOT1 and SNF2. (SEQ ID NO:317) 269 DETVFVKSKRVKASSS RAD55
Similarity to RecA, and (extremely weak if at all NLS) lower
similarity to RAD51, (SEQ ID NO:318) RAD57, and DMC1 317
GEDRKREGRNLKR (SEQ ID NO:319) 371 PISRQSKKRKFDYRVP RAD57 460 aa;
nucleotide-binding (SEQ ID NO:320) domain; limited similarity to
RAD51 62 GLKKPRKKTKSSRH SSL2 ERCC3 (XPB) 843 aa; putative helicase
that (SEQ ID NO:321) seems to function in repair 688 GRILRAKRRNDEG
but also in the removal of (SEQ ID NO:322) secondary structures in
the 5' 784 GRGSNGHKRFKS (weak) untranslated region of mRNA (SEQ ID
NO:323) to allow ribosome binding and scanning. 50 TRRHLCKIKGLSE
(weak) DMC1 RecA 334 aa; yeast homolog of (SEQ ID NO:324) RecA,
meiosis-specific; 277 DGRKPIGGHX.sub.12RKGRG dmc 1 mutants are
defective DER (bipartite) (SEQ ID in reciprocal recombmation
NO:325) and accumulate DSBs 11 ETEKRCKQKEQRY PMS 1 904 aa, 103 kDa;
mismatch- (SEQ ID NO:326) repair protein; MutL (Salmonella) and
HexB (Streptococcus) homolog None HRR25 Hhp1, Hhp1 (S. pom)
Mutations in HRR25 Ser/Thr 1 MDLRVGRKFRIGRKIG CR1 (mamm protein
kinase cause defects (SEQ ID NO:327) in DNA repair and 139
GRRGX.sub.8GLSKKYRDFNT retardation in cell cycling HRHIP (Bipartite
weak NLS) (SEQ ID NO:328) 96 HELTKRSSRRVETEK YKL510 383 aa;
structure-specific (SEQ ID NO:329) endonuclease; two domains of
about 100 aa with sequence similarity to N- and C-terminal regions
of RAD2. 200 MLAMARRKKKMSAK MOT1 Modifier of transcription 1; (SEQ
ID NO:330) 1867 aa; DNA helicase of S. 617 EHYKVKHTEK (weak
cerevisiae required for NLS) (SEQ ID NO:331) viability; increases
gene 670 LHPEKKRSISE (weak expression of several., but NLS) (SEQ ID
NO:332) not all, pheromone- responsive genes rn the absence of
STEl2; the 1257 to 1825 aa domain (568 aa residues) has homology to
SNF2 and RAD54 S. POMBE 60 SSIDEx.sub.5SIKRKRRI (SEQ ID Swi4 Duc-1
113 kDa; KCII sites are NO:333) Rep-3 upstream of NLS like in SV4O
large T; the homologous prokaryotic MutS and HexA lack NLS 96
GELAKRVARHQKARE Rad2 380 aa (weak NLS) (SEQ ID NO:334) 362
GSAKRKRDS (SEQ ID NO:335) 372 KGGESKKKR (SEQ ID NO:336) None Rad9
-- 427 aa; no homology to other DNA repair proteins; rad9 fission
yeast mutants are sensitive to both UV and ionizing radiation; may
be involved in recombination- repair. None Rhp3 or ERCC2 772 aa;
DNA helicase; 65% 681 DKRYGRSDKRTKLPK rad15 RAD3 identity to RAD3
and 55% (SEQ ID NO:337) identity to ERCC2; essential for viability
464 PPSKRRRVRGG Rad16 RAD 1 Function in repair of UV (SEQ ID
NO:338) damage for both cyclobutane dimer and (6-4) photoproduct
lesions; Rad 16 interacts with SwilO. 431 DFKQAILRKRKNESPE Rad21
628 aa, 67.8 kDa, acidic EVEP (SEQ ID NO:339) protein; a single
base substitution in mutant rad2 1- 45, changing an Ile into a Thr,
is responsible for the low efficiency in repair of DSBs after
g-radiation although capable of arresting atG2. 490 DKKAKKG (SEQ ID
Rad22 RAD52 496 aa; functions in NO:340) recombination-repair and
mating-type switching. 394 DVVQFYLKKKYTRSKRN Rad32 MRE11 (S. cer)
648 aa; meiotic DG (weak because of Y) (SEQ recombination protein;
rad32 ID NO:341) mutants are sensitive tog- 575 PSPALLKKTNKRRELP
and UV radiation; functions (SEQ ID NO:342) in the same pathway
with Rhp51 (RAD51). Rad51 recombination-repair GLAKKYRDHKTHLHIP
(weak Hhp1 CKI (mamm) Ser/Thr protein kinase; NLS because of Y and
H) (SEQ HRR25 (S. cer) mutation in this gene causes ID NO:343)
repair defects None Hhp2 CKI (mamm) Ser/Thr protein kinase;
GLAKKYRD KTHVHIP (H in HRR25 (S. cer) mutation in this gene causes
Hhp1 is replaced by F in Hhp2) repair defects (SEQ ID NO:344)
[0186]
10TABLE 9 NLS in Transcription factors NLS and Flanks Protein
factor and features highly basic HR.sub.4QRTRK.sub.7R Human GCF
(GC-factor) (SEQ ID NO:345) LRRKSRP (SEQ ID NO:346) SRRTKRRQ (SEQ
ID NO:347) GRKRKKRT Oct-6 protein transcription (SEQ ID NO:348)
factor from mouse cells GRRRKKRT Mouse Oct-2 protein transcription
(SEQ ID NO:349) factors (Oct-2.1 for Oct-2.6 isoforms) ARKRKRT
Oct-3 from mouse P19 (SEQ ID NO:350) embryonal carcinoma cells
NRRQKGKRS (SEQ ID NO:351) ECRRKKKE Human ATF-1. In basic (SEQ ID
NO:352) region/leucine zipper. ERKKRRRE Human ATF-3 (in basic
region (SEQ ID NO:353) that binds DNA) AKCRNKKKEKT (SEQ ID NO:354)
SKKKIRL Mouse Pu. 1 (Friend (SEQ ID NO:355) erythroleukemia cells).
Related QKGNRKKM to ets oncogene (SEQ ID NO:356) VKKVKKKL (SEQ ID
NO:357) VKRKKI Human PRDII-BF1 that binds (SEQ ID NO:358) to
IFN-.beta. gene promoter. CRNRYRKLE (The largest DNA-binding (SEQ
ID NO:359) protein known, of 298 kD). IRKRRKMK (SEQ ID NO:360)
PKKKRLRL (SEQ ID NO:361) GKKKKRKREKL Murine LEF-1 (397 aa). (within
the Lymphoid-specific with an HMG-box) HMG1-like box. NLS is
identical (SEQ ID NO:362) to that of human TCF-1.alpha..
GKKKKRKREKL Human TCF-1.alpha. (399 aa) (within the HMG-box) (T
cell-specific transcription factor that HMG-box) activates the T
cell receptor (SEQ ID NO:363) C.alpha.). Contains an HMG box. NLS
core is identical to that of murine LEF-1. GKKKRRSREKH Human TCF-1
(within the (uniquely T cell-specific). (SEQ ID NO:364) HMG box
containing. PKKCRARF (SEQ ID NO:365) FKQRRIKL Xenopus laevis
Oct-1(within (SEQ ID NO:366) POU-domain) NRRRKKRT (SEQ ID NO:367)
NRRQKEKRI (SEQ ID NO:368) DKRSRKRKRSK Drosophila Suvar (3) 7 gene
product (SEQ ID NO:369) involved in position-effect RLRIDRKRN
variegation (932 aas). Five widely (SEQ ID NO:370) spaced
zinc-fingers could help AKRSRRS condensation of the chromatin
fiber. (SEQ ID NO:371) IRKRRKMKSVGD.sub.2E.sub.2 Human MBP-1 (class
I MHC enhancer (SEQ ID NO:372) binding protein 1) mw 200 kD. (not
suggested Induced by phorbol esters and as NLS by the mitogens in
Jurkat T cells. authors; between the 1st and 2nd zinc finger)
PPKKKRLRLAE (suggested as NLS by the authors; just before 2nd zinc
finger) (SEQ ID NO:373) CRNRYRKLE (within 1st zinc finger) (SEQ ID
NO:374) PRRKRRV rat TTF-1 (thyroid nuclear factor (SEQ ID NO:375)
that binds to the promoter of HRYKMKRQ thyroid-specific genes). An
(SEQ ID NO:376) homeodomain protein. DGKRKRKN Human thyroid hormone
receptor .alpha. (SEQ ID NO:377) (c-erbA-1 gene). Belongs to the
DDSKRVAKRKL family of cytoplasmic proteins that (SEQ ID NO:378) are
receptors of hydrophobic NRERRRKEE ligands such as steroids, vitD,
(SEQ ID NO:379) retinoic acid, thyroid hormones. WKQRRKF The ligand
binding may expose the NLS for nuclear import of the (SEQ ID
NO:380) receptor-ligand complex. NRRKRKRS Drosophila gcl (germ
cell-less) gene (SEQ ID NO:381) product (569 aa, 65 kD), located
PKKKKL in nuclei, required for germ (SEQ ID NO:382) line formation.
ARRKRRRL C. elegans Sdc-3 protein (SEQ ID NO:383) (sex-determining
protein) LKFKKVRD (2,150 aas). A zinc finger protein. (SEQ ID
NO:384) FKKFRKF (SEQ ID NO:385) GKQKRRF (SEQ ID NO:386)
ERLKRDKEKREKE (SEQ ID NO:387) TRGRPKKVKE (SEQ ID NO:388)
SKKRGRRRKKT (SEQ ID NO:389) TRRQKRAKV (SEQ ID NO:390) SRKSKKRLRA
(SEQ ID NO:391) LKKIRRKIKNKI Drosophila BBF-2 (related to (SEQ ID
NO:392) CREB/ATF) ESRRKKKE (SEQ ID NO:393) Group 0000 DRNKKKKE
Xenopus RAR (retinoic acid receptor) (SEQ ID NO:394) ARRRRP (SEQ ID
NO:395) GRRRRA Human ATF-2 (the 2nd and (SEQ ID NO:396) 3rd NLS are
in basic region DEKRRKV that binds DNA) (SEQ ID NO:397) CRQKRKV
(SEQ ID NO:398) ERKRRD Myn (murine homolog of Max). Forms (SEQ ID
NO:399) a specific DNA-binding SRKKLRME complex with c-Myc
oncoprotein (SEQ ID NO:400) through a helix-loop-helix/ leucine
zipper. EEKRKRTYE human NF.kappa.B p65 (550 aa). (SEQ ID NO:401)
Not binding DNA; complexed with p50 that binds DNA. NF.kappa.B p50
also contains a NLS (Table 3b). GRRRRA Human HB16, a cAMP response
(SEQ ID NO:402) element-binding protein DEKRRKF (SEQ ID NO:403)
SRCRQKRKV (SEQ ID NO:404) SKKKKTKV Human TFIIE-.beta. (general (SEQ
ID NO:405) transcription initiation NRPDKKKI protein factor; forms
(SEQ ID NO:406) tetramer .alpha..sub.2.beta..sub.2 with
TFIIE-.alpha.) QRRKKP (SEQ ID NO:407) QKKRRFKT (SEQ ID NO:408)
SRKRKM Human kup transcriptional activator (SEQ ID NO:409) (433
aas). Two distantly spaced zinc fingers. Expressed in hematopoietic
cells and testis. ERKRLRNRLA Mouse Jun-B homologue to (SEQ ID
NO:410) avian sarcoma virus 17 oncogene v-jun product. One region
is similar ATKCRKRKL to yeast GCN4 and to Fos. (SEQ ID NO:411) (19
aa stretch) DKRx.sub.6ERKRRD (N-terminus) Max (specifically
associates with (SEQ ID NO:412) c-Myc, N-Myc, L-Myc). The Max-Myc
QSRKKLRME complex binds to DNA; neither (C-terminus) Max nor Myc
alone exhibit (SEQ ID NO:413) appreciable DNA binding. DKEKKIKLEEDE
Chicken VBP (vitellogenin gene- (within an binding protein).
Leucine zipper. acidic region) Related to rat DBP. (SEQ ID NO:414)
IKKAKKV (SEQ ID NO:415) TRRKKN (SEQ ID NO:416) TRDDKRRA Xenopus
borealis B1 factor. Closely (SEQ ID NO:417) related to the
mammalian USF. EVERRRRDK Binds to CACGTG in TFIIIA promoter (SEQ ID
NO:418) to developmentally regulate its expression. TRDEKRRA Human
USF (upstream stimulatory (SEQ ID NO:419) factor) activating the
major late EVERRRRDK adenovirus promoter (SEQ ID NO:420) YRRYPRRRG
YB-1, a protein that binds to the MHC (SEQ ID NO:421) class II Y
box. YB-1 is a negative QRRPYRRRRF regulator. (SEQ ID NO:422)
YRPRFRRG (SEQ ID NO:423) QRRYRRN (SEQ ID NO:424) YRRRRP (SEQ ID
NO:425) AKERQKKD Human TFEB Binds to IgH enhancer. (SEQ ID NO:426)
ERRRRF (SEQ ID NO:427) LKERQKKD Human TFE3 (536 aa). Binds to
.mu.E3 (SEQ ID NO:428) enhancer of IgH genes. IERRRRFN (SEQ ID
NO:429) YFRRRRLEKD (SEQ ID NO:430) KTVALKRRLKASSRL Human Dr1 (176
aa, 19 kD). (SEQ ID NO:431) Interacts with TBP (TATA-binding
protein) thus inhibiting association of TFIIA and/or TFIIB with
TBP. TBP-Dr1 association is affected by Dr1 phosphorylation to
repress activated and basal transcription. 1 LRRRGRQTY Drosophila
ultrabithorax (SEQ ID NO:432) protein (from the conserved 61 27
LTRRRRIEM amino acid homeodomain segment (SEQ ID NO:433) only).
Conserved in the antenappedia 51 QNRRMKLKKEI homeodomam protein.
(SEQ ID NO:434) SNRRRPDHR C. elegans sex-determining Tra-1 (SEQ ID
NO:435) protein. Zinc finger. Peaks in the VYRGRRRVRRE second
larval stage. (SEQ ID NO:436) P.sub.7AP.sub.2RRRRSADNKD.sub.2 (SEQ
ID NO:437) PKKPRHQF (SEQ ID NO:438) EKRKKERN Yeast NPS1
transcription protein (SEQ ID NO:439) factor (1359 aa) involved in
cell LLRRLKKEVE growth control at G2 phase. (SEQ ID NO:440) Has a
catalytic domain of protein kinases. EPLGRIRQKKRVY.sub.2D.sub.2
(SEQ ID NO:441) (EDAIKKRREARERRRLRQ) (SEQ ID NO:442)
DKETTASRSKRRSSRKKRT (SEQ ID NO:443) ESKKKKPKL (SEQ ID NO:444)
KKTAAKKTKTKS (SEQ ID NO:445) QRKRQKL Human 243 transcriptional (SEQ
ID NO:446) activator (968 aas), induced KAKKQK by mitogens (SEQ ID
NO:447) in T cells. N-terminal half is LRRKRQK homologous to
oncoprotein Rel and (SEQ ID NO:448) Drosophila Dorsal protein
involved in development. The C- terminal half contains repeats
found in proteins involved in cell-cycle control of yeast and
tissue differentiation in Drosophila. RDIRRRGKNKV Mouse NF-E2 (45
kD), an erythroid (SEQ ID NO:449) transcription factor from mouse
erythroleukemia (MEL) cells. Involved in globin gene regulation.
QNCRKRKLE Binds to AP-1-1ike sites. (SEQ ID NO:450) Homology to Jun
B, GCN4, Fos, ATF1 and CREB in basic region/leucine zipper (see
FIG. 2). Group 000x00 DKIRRKN Human glucocorticoid receptor (SEQ ID
NO:451) ARKTKKKI (SEQ ID NO:452) 473 DKIRRKNCP Mouse and human GR
(SEQ ID NO:453) (glucocorticoid recptor) EARKTKKKIKGIQ (SEQ ID
NO:454) Group 000x0 YRVRRERN C/EBP (CCAAT/enhancer (SEQ ID NO:455)
binding protein). VRKSRDKA Functions in liver-specific (SEQ ID
NO:456) gene expression. DRLRKRVE (SEQ ID NO:457) DKIRRKN Human
mineralocorticoid receptor (SEQ ID NO:458) ARKSKKL (SEQ ID NO:459)
DKIRRKN Human PR (progesterone receptor) (SEQ ID NO:460) GRKFKKF
(SEQ ID NO:461) EEVQRKRQKLMP Human and mouse NF.kappa.B 105 kD (SEQ
ID NO:462) precursor of p50 (968 aas) (first R is at 361 position).
EEVQRKRQKL Human NF-.kappa.B p50 (DNA-binding (SEQ ID NO:463)
subunit). Identical to protein KBF1, homologous to rel oncogene
product. NF-.kappa.B p65 also contains a NLS (Table 3a). GKTRTRKQ
Human TEF-1 (SV40 transcriptional (SEQ ID NO:464) enhancer factor
1). 426 aa. ARRKSRD (SEQ ID NO:465) QRKERKSKS Rat, mouse, human
IRF-1 (interferon (SEQ ID NO:466) regulatory factor-1). Induced in
TKSKTKRKL lymphoma T cells by the pituitary (SEQ ID NO:467) peptide
hormone prolactin. Regulates the growth-inhibitory interferon
genes. GKCRKKN Ehrlich ascites S-II transcription factor. (SEQ ID
NO:468) A general factor that acts at the elongation step.
ERSKKRSRE Tobacco TAF-1 transcriptional activator (SEQ ID NO:469)
ERELKREKRKQ (SEQ ID NO:470) ARRSRLRKQ (SEQ ID NO:471)
YKLDHMRRRIETDE Drosophila TFIIE.alpha. (433 aa), a general (SEQ ID
NO:472) transcription factor for RNA polymerase II. Composed of
subunits .alpha. and .beta.. DKNRRKS Human ER (estrogen receptor);
595 aa. (SEQ ID NO:473) IRKDRRG (SEQ ID NO:474) IKRSKKN (SEQ ID
NO:475) EQRRHRIE Yeast ADA2 (434 aa), a potential (SEQ ID NO:476)
transcriptional adaptor required TTRAEKKRLL for the function of
certain (SEQ ID NO:477) acidic activation domains. IDKKRSKEAKE (SEQ
ID NO:478) EAALRRKIRTISK Yeast GCN5 gene product (439 aa), (SEQ ID
NO:479) required for the function of GCN4 transcriptional activator
and for the activity of the HAP2-3-4 complex. Group 00x00 NKKMRRNRF
Mouse LFB3 (SEQ ID NO:480) NRRKx.sub.4RQK (SEQ ID NO:481) TKKGRRNRF
Mouse LFB1 (SEQ ID NO:482) NRRKx.sub.4RHK (SEQ ID NO:483)
NKKMRRNRFK rat vHNF1-A (SEQ ID NO:484) NKKMRRNR murine HNF-1.beta.
(SEQ ID NO:485) TKKGRRNRF mouse HNF-1 (SEQ ID NO:486) NKKMRRNRF
human vHNF1 (SEQ ID NO:487) TKKGRRNRF rat liver HNF1 (SEQ ID
NO:488) LRRQKRFK rat HNF-3.beta. (SEQ ID NO:489) QQH.sub.3SH.sub.4Q
(SEQ ID NO:490) LRRQKRFK rat HNF-3.gamma. (SEQ ID NO:491) LRRQKRFK
rat HNF-3.alpha. (SEQ ID NO:492) LKEKERKA rat DBP a protein factor
that (SEQ ID NO:493) binds to the D site of the albumin MKKARKV
gene promoter (SEQ ID NO:494) PRRERRY rat AT-BP1. Highly acidic
domain. Two (SEQ ID NO:495) zinc fingers. Binds to the B-domain of
.alpha..sub.1-antitrypsin gene promoter and to the NF-.kappa.B site
in the MHC gene enhancer. DRRVRKGKV A 19 kD Drosophila melanogaster
(SEQ ID NO:496) nonhistone associated with heterochromatin.
SKHGRRARRLDP murine EBF (early B-cell factor) (SEQ ID NO:497) of
591 aa. Regulates the pre-B and B lymphocyte-specific mb-1 gene.
Expressed in pre-B and B-cell lines but not in plasmocytomas,
T-cell and nonlymphoid cell lines. GRRTRRE human Sp1 (SEQ ID
NO:498) DEQKRAEKKAKE yeast SNF2, a transcriptional regulator (SEQ
ID NO:499) of many genes. IRRIHKVIRP (SEQ ID NO:500) LLRRLKKDVE
(SEQ ID NO:501) Group 0x00x0 AKAKAKKA mouse AGP/EBP (87% similarity
to (SEQ ID NO:502) C/EBP), ubiquitously expressed YKMRRERN (SEQ ID
NO:503) VRKSRDKA (SEQ ID NO:504) AKAKAKKA rat LAP, a 32-kD
liver-enriched (SEQ ID NO:505) transcriptional activator, also
present YKMRRERN in lung, with 71% sequence similarity (SEQ ID
NO:506) to C/EBP. Leucine zipper. VRKSRDKA Accumulates to maximal
levels (SEQ ID NO:507) around birth. YRQRRER Ig/EBP-1
(immunoglobulin gene (SEQ ID NO:508) enhancer-binding protein).
Forms VKKSRLKSKQK heterodimers with C/EBP. (SEQ ID NO:509)
EDPEKEKRIKELE mouse c-Myb (SEQ ID NO:510) MRRKV (SEQ ID NO:511)
DYYKVKRPKTD Drosophila eyes absent protein (760 aa), (SEQ ID
NO:512) a nuclear protein that functions in GRARGRRHQ early
development to prevent (SEQ ID NO:513) programmed cell death and to
allow the FRYRKIKDIY event that generate the eye to proceed. (SEQ
ID NO:514) Mutations cause programmed cell death of eye progenitor
cells. Group 0x0x00 AKAKAKKA rat IL-6DBP interacting with (SEQ ID
NO:515) interleukin-6 responsive elements. Has a leucine zipper
domain. DKRQRNRC mouse H-2RIIBP (MHC class I genes (SEQ ID NO:516)
H-2 region II binding protein). FkrtirkD Member of the nuclear
hormone receptor superfamily. FkrtirkD chicken RXR, related to RAR
(retinoic DKRQRNRC acid receptor), a nuclear (SEQ ID NO:517)
protein factor from the thyroid/steroid hormone receptor family
VKSKAKKT human NF-IL6 (345 aa). Specifically (SEQ ID NO:518) binds
to IL1-responsive YKIRRERN element in the IL-6 gene. (SEQ ID
NO:519) Leucine zipper. Homology to C/EBP. VRKSRDKA (SEQ ID NO:520)
QKKNRNKC mouse PPAR (peroxisome proliferator (SEQ ID NO:521)
activated receptor) Group 000xx00 EQIRKLVKKHG yeast RAP1 (SEQ ID
NO:522) It binds regulatory sites at FRRSMKRKA yeast mating type
silencers. (SEQ ID NO:523) human vitamin D receptor (427 aa) Group
00xx00 LKRHQRRH mouse WT1 (the murine homolog (SEQ ID NO:524) of
human Wilms' tumor predisposition gene WT1) LKRHQRRH human WT33
(Wilms' tumor (SEQ ID NO:525) predisposition) Group 000xx0
LKESKRKYDE yeast SWI3 99 kD, highly (SEQ ID NO:526) acidic protein.
Global transcription activator. EVLKVQKRRIYD human RBAP-1
(retinoblastoma- (SEQ ID NO:527) associated protein 1) factor (412
aa). A protein that binds to the pocket (functional domain) of the
retinoblastoma (RB) protein involved in suppression of cell growth
(tumor suppressor). The transcription factor E2F, implicated in
cell growth, binds to the same pocket of RB.
[0187]
11TABLE 10 NLS in other nuclear proteins Putative NLS Protein
YKSKKKA (SEQ ID NO:528) Yeast L3 TKKLPRKT (SEQ ID NO:529)
TRKKGGRRGRRL (SEQ ID NO:530) Yeast 59 ribosomal protein C-terminus
ARATRRKRCKG (SEQ ID NO:531) Yeast L16 ribosomal protein GKGKYRNRRW
(SEQ ID NO:532) yeast L2 ribosomal protein (homologous to Xenopus
L1). Encoded by intronless genes. GKGKMRNRRRIQRRG (SEQ ID NO:533)
Xenopus laevis L1 ribosomal protein (homologous NKKVKRRELKKN (SEQ
ID NO:534) to yeast L2) Encoded by intronless genes. AKTARRKA (SEQ
ID NO:535) IKAKEKKP (SEQ ID NO:536) GKPKAKKP (SEQ ID NO:537)
AKAKKRQ (SEQ ID NO:538) ERKRKS (SEQ ID NO:539) human S6 ribosomal
protein (homologous to yeast GKRPRTKA (SEQ ID NO:540) S10) HKRRRI
(SEQ ID NO:541) LKKQRTKKNKE (SEQ ID NO:542) PKMRRRTYR (SEQ ID
NO:543) Rat L17 ribosomal protein (184 aas) KKKISQKKLKK (SEQ ID
NO:544) YMRRRTYRA (SEQ ID NO:545) Podocoryne carnea (hydrozoan,
Coelenteratum) EVKKVSKKKL (SEQ ID NO:546) L17 ribosomal protein
(184 aas) highly homologous to rat L17. ERNRKDKDAKFR (SEQ ID
NO:547) human, rat ribosomal S13 protein ERKRKS (SEQ ID NO:548)
yeast S10 ribosomal protein (homologous to human QRLQRKRH (SEQ ID
NO:549) S6) IRKRRA (SEQ ID NO:550)
GRRRKKHRSRSRSRERRSRSRDRGRG.sub.12GRER 35 kD subunit of U2 small
nuclear DRRRSRDRER (SEQ ID NO:551) ribonucleoprotein auxiliary
factor (U2AF), an essential mammalian splicing factor. U2AF.sup.35
interacts with the 65 kD subunit (U2AF.sup.65). Both proteins are
concentrated in a small number of subnuclear organelles, the coiled
bodies. EFEDPRD (SEQ ID NO:552) human UsnRNP-associated 70 k
protein (437 aas) ETREERME (SEQ ID NO:553) that is phosphorylated
at Arg/Ser-rich domains; EAGDAPPDP (SEQ ID NO:554) involved in
splicing EERMERKRREK (SEQ ID NO:555)
HRDRDRDRERERRESRERDKERERRRSRSRD RRRRSRSRDKEERRRSRERSKDKDRDRKRRS
SRSRERARRERERKEE (SEQ ID NO:556) RDRDRERRRSHRSERERRRDRDRDRDRDR- EH
KRGER (SEQ ID NO:557) QKRNNKKSKKKRCAE (SEQ ID NO:558) yeast TRM1
enzyme for the N.sup.2, N.sup.2- EKLRKLKI (near C-terminus) (SEQ ID
NO:559) dimethylguanosine modification of both mitochondrial and
cytoplasmic tRNAs. TRM1 is both nuclear and mitochondrial. The
first motif is within a region (70-213 aa segment) known to cause
nuclear localization of .beta.-galactosidase. NKRKRV (SEQ ID
NO:560) Yeast nucleoporin NUP1 (1076 aa, 113 kD); an SLKINRSNRKRE
(SEQ ID NO:561) integral component of the pore complex. Involved
EPKRKRRLP (SEQ ID NO:562) in both binding and translocation steps
of nuclear ARMRHSKR (C-terminus) (SEQ ID NO:563) import.
KAEKEx.sub.3KVD.sub.2E.sub.2 (SEQ ID NO:564) Chicken, Xenopus No 38
nucleolar (38 kD); Kx.sub.3Kx.sub.5Kx.sub.3R (SEQ ID NO:565)
involved in intranuclear packaging of preribosomal particles.
Shuttles between nucleus and cytoplasm. KTEREAEKALEEKx.sub.7R (SEQ
ID NO:566) Chicken, hamster nucleolin (92 kD). Binds
Kx.sub.5Kx.sub.7Kx.sub.4RX3EDTTEETLR (SEQ ID NO:567) preribosomal
RNA. Shuttles between nucleus and
RG.sub.2RG.sub.2RG.sub.3RG.sub.2FG.sub.2RG.sub.3RGFG.sub.2RG.sub.3FRG.sub-
.2RG.sub.4 cytoplasm. DHKPQGKKIKFE (SEQ ID NO:568) (C-terminus)
WYKHFKKTKD (SEQ ID NO:569) human SATB1 (763 aa) which binds
selectively to AT-rich MARs with mixed A, T, C on one strand
excluding G. Binds to minor groove with little contact with bases.
QKKKQMKAD (SEQ ID NO:570) yeast CBF5p, a centromere-binding protein
(KKEKKE).sub.5 (SEQ ID NO:571) (55kDa, 483aa). The KKEKKRKSED (SEQ
ID NO:572) KKE repeat at its C-terminus EEKKSKKSKK (SEQ ID NO:573)
occurs in microtubule-binding domains; yeast cells containing only
three copies of the KKE repeat of CBF5p delay at G.sub.2/M;
depletion of CBE5p arrests cells at G.sub.1/S. TKKKSFKL (SEQ ID
NO:574) yeast CCE1, a cruciform cutting endonuclease
KSERERMLRESLKEERRRF (SEQ ID NO:575) rat nucleoporin 155 or Nup155
(1390 aas, 155 kDa), a protein of the nuclear pore complex;
contains 46 consensus sites for various kinases; associated with
both the nucleoplasmic and the cytoplasmic region of pores.
PKKGSKKA (SEQ ID NO:576) human H2B variant differentially expressed
during DGKKRKRSRKES (SEQ ID NO:577) the cell cycle GAKRHRKVLRD (SEQ
ID NO:578) Calf thymus histone H4 14-24 (102 aa) PAIRRLARRG (SEQ ID
NO:579) 32-41 EHARRKT (SEQ ID NO:580) 74-80 ARRIRGERA 127-135 (SEQ
ID NO:581) Calf thymus H3 (135 aa) GSHHKAKGK 121-129 (SEQ ID
NO:582) Calf thymus H2A (129 aa) RGKSGKARTKAKSRSSR 3-19 (SEQ ID Sea
urchin Psammechinus miliaris H2A (123 aa) NO:583) PKKGSKKA 10-17
(SEQ ID NO:584) Calf thymus H2B QKKDGKKRKRSRKES 22-36 (SEQ ID
NO:585) (125 aa) GGKKRHRKRKGSY (SEQ ID NO:586) Sea urchin
Psammechinus miliaris H2B (122 aa) 22-34 PRTDKKRRRKRKES 19-32 (SEQ
ID NO:587) Starfish H2B (121 aa) PAKAPKKKA 12-20 (SEQ ID NO:588)
Trout testis H1 EAKKPAKKA 104-112 (SEQ ID NO:589) (194 aa) AKKPKKV
128-134 (SEQ ID NO:590) AKKSPKKAKKP 142-152 (SEQ ID NO:591) PKKVKKP
183-189 (SEQ ID NO:592) PRRKAKRA 30-37 (SEQ ID NO:593) Sea urchin
Parechinus angulosus sperm H1 (248 PKKAKKT 119-125 (SEQ ID NO:594)
aa) AKAKKAKA 129-136 (SEQ ID NO:595) AKKARKAKA 139-147 (SEQ ID
NO:596) AKKAKKPKKKA 171-181 (SEQ ID NO:597) AKKAKKPAKK 182-191 (SEQ
ID NO:598) SPKKAKKP 192-199 (SEQ ID NO:599) AKKSPKKKKAKRS 200-212
(SEQ ID NO:600) PKKAKKA 213-219 (SEQ ID NO:601) AKKAKKS 227-233
(SEQ ID NO:602) PRKAGKRRSPKKARK 234-248 (SEQ ID NO:603) ARRRKTA 1-7
(SEQ ID NO:604) Annelid sperm H1a IRKFIRKA 55-61 (SEQ ID NO:605)
(119 aa) PKKKKA 83-88 (SEQ ID NO:606) AKKPKAKKVKKP 89-100 (SEQ ID
NO:607) AKKKTNRARKPKTKKNR 104-120 (SEQ ID NO:608) PKRKVSS 1-7 (SEQ
ID NO:609) Calf thymus HMG14 EEPKRRSARLS 14-24 (SEQ ID NO:610) (100
aa) PKRKAEGDAK 1-10 (SEQ ID NO:611) Calf thymus HMG17 PKGKKGKA
52-59 (SEQ ID NO:612) (89aa; 9,247 D) PKKPRGKM (SEQ ID NO:613) Calf
thymus HMG 1 EHKKKHP (SEQ ID NO:614) (259 aa) ETKKKFKDP (SEQ ID
NO:615) EKSKKKK(E/D).sub.41 (SEQ ID NO:616) E.sub.3G.sub.2KKKKKFAK
(SEQ ID NO:617) EHKKKHP (SEQ ID NO:618) Calf thymus HMG 2
PKGDKKGKKKDP (SEQ ID NO:619) (256 aa) E.sub.4G.sub.3KKKKKFAK (SEQ
ID NO:620) PKRKSATKGDEPARR 1-15 (SEQ ID NO:621) Trout testis H6 (60
aa) KPKKAAAPKKA 30-34 (SEQ ID NO:622)
REFERENCES
[0188] U.S. Patent Documents
[0189] U.S. Pat. No. 4,394,448 July, 1983 Szoka, Jr. et al.
[0190] U.S. Pat. No. 4,598,051 July, 1986 Papahadjopoulos et
al.
[0191] U.S. Pat. No. 5,013,556 May, 1991 Woodle et al.
[0192] Journal Articles
[0193] Allen, T. M. and Chonn, A. (1987) "Large unilamellar
liposomes with low uptake into the reticuloendothelial system" FEBS
Lett. 223:42-46.
[0194] Allen, T. M. et al. (1991) "Liposomes containing synthetic
lipid derivatives of polyethylene glycol show prolonged circulation
half-lives in vivo" Biochim. Biophys. Acta 1066:29-36.
[0195] Anderson, W. F. (1992) "Human gene therapy" Science
256:808-813.
[0196] Aoki, K. et al. (1995) "Liposome-mediated in vivo gene
transfer of antisense K-ras construct inhibits pancreatic tumor
dissemination in the murine peritoneal cavity" Cancer Res.
55:3810-3816.
[0197] Arcasoy, S. M. et al. (1997) "Polycations increase the
efficiency of adenovirus-mediated gene transfer to epithelial and
endothelial cells in vitro" Gene Ther. 4:32-38.
[0198] Beauchamp, C. O. et al. (1983) "A new procedure for the
synthesis of polyethylene glycol-protein adducts; effects on
function, receptor recognition, and clearance of superoxide
dismutase, lactoferrin, and alpha 2-macroglobulin" Anal. Biochem.
131:25-33.
[0199] Bongartz, J. -P. et al. (1994) "Improved biological activity
of antisense oligonucleotides conjugated to a fusogenic peptide"
Nucl. Acids Res. 22:4681-4688.
[0200] Boulikas, T. (1993) "Nuclear localization signals (NLS)"
Crit. Rev. Eukar. Gene Expression 3:193-227.
[0201] Boulikas, T. (1994) "Putative nuclear localization signals
(NLS) in protein transcription factors" J. Cell. Biochem.
55:32-58.
[0202] Boulikas, T. (1996a) "Cancer gene therapy and immunotherapy"
Intl. J. Oncol. 9:941-954.
[0203] Boulikas, T. (1996b) "Gene therapy to human diseases: ex
vivo and in vivo studies" Intl. J. Oncol. 9:1239-1251.
[0204] Boulikas, T. (1996c) "Liposome DNA delivery and uptake by
cells" Oncol. Rep. 3:989-995.
[0205] Boulikas, T. (1996d) "Nuclear import of protein kinases and
cyclins" J. Cell. Biochem. 60:61-82.
[0206] Boulikas, T. (1997a) "Gene therapy of prostate cancer: p53,
suicidal genes, and other targets" Anticancer Res.
17:1471-1506.
[0207] Boulikas, T. (1997b) "Nuclear import of DNA repair proteins"
Anticancer Res. 17:843-864.
[0208] Boulikas, T. (1997c) "Nuclear localization signal peptides
for the import of plasmid DNA in gene therapy" Int. J. Oncol.
10:301-309.
[0209] Boulikas, T. (1998a) "Status of gene therapy in 1997:
Molecular mechanisms, disease targets, and clinical applications"
Gene Ther. Mol. Biol. 1:1-172.
[0210] Boulikas, T. (1998b) "Nucleocytoplasmic trafficking:
implications for the nuclear import of plasmid DNA during gene
therapy" Gene Ther. Mol. Biol. 1:713-740.
[0211] Boulikas, T. and Martin, F. (1997) "Histones, protamine, and
polylysine but not poly(E:K) enhance transfection efficiency" Int.
J. Oncol. 10:317-322.
[0212] Capaccioli, S. et al. (1993) "Cationic lipids improve
antisense oligonucleotide uptake and prevent degradation in
cultured cells and in human serum" Biochem. Biophys. Res. Comm.
197:818-825.
[0213] Creuzenet, C. et al. (1997) "Interaction of alpha s2- and
beta-casein signal peptides with DMPC and DMPG liposomes" Peptides
18:463-472.
[0214] Culver, K. W. (1996) in: Gene Therapy: A primer for
physicians, Second Edition. Mary Ann Liebert, Inc. Publications,
NY, pp. 1-198.
[0215] Curtain, C. et al. (1999) "The interactions of the
N-terminal fusogenic peptide of HIV-1 gp41 with neutral
phospholipids" Eur. Biophys. J 28:427-436.
[0216] de la Maza, A. et al. (1998) "Solubilization of
phosphatidylcholine liposomes by the amphoteric surfactant dodecyl
betaine" Chem. Phys. Lipids 94:71-79.
[0217] Decout, A. et al. (1999) "Contribution of the hydrophobicity
gradient to the secondary structure and activity of fusogenic
peptides" Mol. Membr. Biol. 16:237-246.
[0218] Duguid, J. G. et al. (1998) "A physicochemical approach for
predicting the effectiveness of peptide-based gene delivery systems
for use in plasmid-based gene therapy" Biophys. J.
74:2802-2814.
[0219] Filion, M. C. and Phillips, N. C. (1997) "Toxicity and
immunomodulatory activity of liposomal vectors formulated with
cationic lipids toward immune effector cells" Biochim. Biophys.
Acta 1329:345-356.
[0220] Fresta, M. et al. (1998) "Liposomal delivery of a 30-mer
antisense oligodeoxynucleotide to inhibit proopiomelanocortin
expression" J. Pharm. Sci. 87:616-625.
[0221] Gabizon, A. and Papahadjopoulos, D. (1988) "Liposome
formulations with prolonged circulation time in blood and enhanced
uptake by tumors" Proc. Natl. Acad. Sci. USA 85:6949-6953.
[0222] Gabizon, A. et al. (1989) "Pharmacokinetics and tissue
localization of doxorubicin encapsulated in stable liposomes with
long circulation times" J. Natl. Cancer Inst. 81:1484-1488.
[0223] Ghosh, J. K. and Shai, Y. (1999) "Direct Evidence that the
N-Terminal Heptad Repeat of Sendai Virus Fusion Protein
Participates in Membrane Fusion" J. Mol. Biol. 292:531-546.
[0224] Green, M. and Loewenstein, P. M. (1988) "Autonomous
functional domains of chemically synthesized human immunodeficiency
virus tat transactivator protein" Cell 55:1179-1188.
[0225] Gupta, D. and Kothekar, V. (1997) "500 picosecond molecular
dynamics simulation of amphiphilic polypeptide Ac(LKKL).sub.4 NHEt
with 1,2 di-mysristoyl-sn-glycero-3-phosphorylcholine (DMPC)
molecules" Indian J. Biochem. Biophys. 34:501-511.
[0226] Hofland, H. E. J. et al. (1996) "Formation of stable
cationic lipid/DNA complexes for gene transfer" Proc. Natl. Acad.
Sci. USA 93:7305-7309.
[0227] Jaaskelainen, I. et al. (1994) "Oligonucleotide-cationic
liposome interactions. A physicochemical study" Biochim. Biophys.
Acta 1195:115-123.
[0228] Judice, J. K. et al. (1997) "Inhibition of HIV type 1
infectivity by constrained alpha-helical peptides: implications for
the viral fusion mechanism" Proc. Natl. Acad. Sci. USA
94:13426-13430.
[0229] Kono, K. et al. (1993) "Fusion activity of an amphiphilic
polypeptide having acidic amino acid residues: generation of fusion
activity by alpha-helix formation and charge neutralization"
Biochim. Biophys. Acta 1164:81-90.
[0230] Lambert, G. et al. (1998) "The C-terminal helix of human
apolipoprotein AII promotes the fusion of unilamellar liposomes and
displaces apolipoprotein AI from high-density lipoproteins" Eur. J.
Biochem. 253:328-338.
[0231] Lambert, O. et al. (1998) "A new "gel-like" phase in dodecyl
maltoside-lipid mixtures: implications in solubilization and
reconstitution studies" Biophys. J. 74:918-930.
[0232] Lappalainen, K. et al. (1997) "Intracellular distribution of
oligonucleotides delivered by cationic liposomes: light and
electron microscopic study" Histochem. Cytochem. 45:265-274.
[0233] Lasic, D. (1997) in: Liposomes in Gene Delivery, CRC Press,
pp. 1-295.
[0234] Lee, S. et al. (1992) "Effect of amphipathic peptides with
different alpha-helical contents on liposome-fusion" Biochim.
Biophys. Acta 1103:157-162.
[0235] Lelkes, P. I. and Lazarovici, P. (1988) "Pardaxin induces
aggregation but not fusion of phosphatidylserine vesicles" FEBS
Lett. 230:131-136.
[0236] Leonard, A. N. and Cohen, D. E. (1998) "Submicellar bile
salts stimulate phosphatidylcholine transfer activity of sterol
carrier protein 2" J. Lipid Res. 39:1981-1988.
[0237] Lewis, J. G. et al. (1996) "A serum-resistant cytofectin for
cellular delivery of antisense oligodeoxynucleotides and plasmid
DNA" Proc. Natl. Acad. Sci. USA 93:3176-3181.
[0238] Li, S. and Huang, L. (1997) "In vivo gene transfer via
intravenous administration of cationic lipid-protamine-DNA (LPD)
complexes" Gene Ther. 4:891-900.
[0239] Lins, L. et al. (1999) "Molecular determinants of the
interaction between the C-terminal domain of Alzheimer's
beta-amyloid peptide and apolipoprotein E alpha-helices" J.
Neurochem. 73:758-769.
[0240] Litzinger, D. C. et al. (1996) "Fate of cationic liposomes
and their complex with oligonucleotide in vivo" Biochim. Biophys.
Acta 1281:139-149.
[0241] Lopez, O. et al. (1998) "Direct formation of mixed micelles
in the solubilization of phospholipid liposomes by Triton X-100"
FEBS Lett. 426:314-318.
[0242] Lusa, S. et al. (1998) "Direct observation of lipoprotein
cholesterol ester degradation in lysosomes" Biochem. J
332:451-457.
[0243] Macosko, J. C. et al. (1997) "The membrane topology of the
fusion peptide region of influenza hemagglutinin determined by
spin-labeling EPR" J. Mol. Biol. 267:1139-1148.
[0244] Macreadie, I. G. et al. (1997) "Cytotoxicity resulting from
addition of HIV-1 Nef N-terminal peptides to yeast and bacterial
cells" Biochem. Biophys. Res. Commun. 232:707-711.
[0245] Martin, F. and Boulikas, T. (1998) "The challenge of
liposomes in gene therapy" Gene Ther. Mol. Biol 1: 173-214.
[0246] Martin, I. et al. (1999) "Membrane fusion induced by a short
fusogenic peptide is assessed by its insertion and orientation into
target bilayers" Biochemistry 38:9337-9347.
[0247] Martin, I. and Ruysschaert, J. M. (1997) "Comparison of
lipid vesicle fusion induced by the putative fusion peptide of
fertilin (a protein active in sperm-egg fusion) and the
NH2-terminal domain of the HIV2 gp41" FEBS Lett. 405:351-355.
[0248] Massari, S. and Colonna, R. (1986) "Gramicidin induced
aggregation and size increase of phosphatidylcholine vesicles"
Chem. Phys. Lipids 39:203-220.
[0249] Melino, S. et al. (1999) "Zn(2+) ions selectively induce
antimicrobial salivary peptide histatin-5 to fuse negatively
charged vesicles. Identification and characterization of a
zinc-binding motif present in the functional domain" Biochemistry
38:9626-9633.
[0250] Midoux, P. and Monsigny, M. (1999) "Efficient gene transfer
by histidylated polylysine/pDNA complexes" Bioconjug. Chem.
10:406-411.
[0251] Murata, M. et al. (1991) "Modification of the N-terminus of
membrane fusion-active peptides blocks the fusion activity"
Biochem. Biophys. Res. Commun. 179:1050-1055.
[0252] Niidome, T. et al. (1997) "Membrane interaction of synthetic
peptides related to the putative fusogenic region of PH-30 alpha, a
protein in sperm-egg fusion" J. Peptide Res. 49:563-569.
[0253] Pak, C. C. et al. (1999) "Elastase activated liposomal
delivery to nucleated cells" Biochim. Biophys. Acta
1419:111-126.
[0254] Papahadjopoulos, D. et al. (1991) "Sterically stabilized
liposomes: Improvements in pharmacokinetics and antitumor
therapeutic efficacy" Proc. Natl. Acad. Sci. USA
88:11460-11464.
[0255] Parente, R. A. et al. (1988) "pH-dependent fusion of
phosphatidylcholine small vesicles. Induction by a synthetic
amphipathic peptide" J. Biol. Chem. 263:4724-4730.
[0256] Partidos, C. D. et al. (1996) "Priming of measles
virus-specific CTL responses after immunization with a CTL epitope
linked to a fusogenic peptide" Virology 215:107-110.
[0257] Pecheur, E. I. et al. (1997) "Membrane anchorage brings
about fusogenic properties in a short synthetic peptide"
Biochemistry 36:3773-3781.
[0258] Peelman, F. et al. (1999) "Characterization of functional
residues in the interfacial recognition domain of lecithin
cholesterol acyltransferase (LCAT)" Protein Eng. 12:71-78.
[0259] Pereira, F. B. et al. (1997) "Permeabilization and fusion of
uncharged lipid vesicles induced by the HIV-1 fusion peptide
adopting an extended conformation: dose and sequence effects"
Biophys. J. 73:1977-1986.
[0260] Pillot, T. et al. (1999) "The nonfibrillar amyloid
beta-peptide induces apoptotic neuronal cell death: involvement of
its C-terminal fusogenic domain" J. Neurochem. 73:1626-1634.
[0261] Pillot, T. et al. (1997) "Specific modulation of the
fusogenic properties of the Alzheimer beta- amyloid peptide by
apolipoprotein E iso forms" Eur. J. Biochem. 243:650-659.
[0262] Pillot, T. et al. (1997) "The 118-135 peptide of the human
prion protein forms amyloid fibrils and induces liposome fusion" J.
Mol. Biol. 274:381-393.
[0263] Plank, C. et al. (1996) "Activation of the complement system
by synthetic DNA complexes: a potential barrier for intravenous
gene delivery" Hum. Gene Ther. 7:1437-1446.
[0264] Rodriguez-Crespo, I. et al. (1994) "Prediction of a putative
fusion peptide in the S protein of hepatitis B virus" J. Gen.
Virol. 75:637-639.
[0265] Rodriguez-Crespo, I. et al. (1999) "Fusogenic activity of
hepadenavirus peptides corresponding to sequences downstream of the
putative cleavage site" Virology 261:133-142.
[0266] Ross, G. et al. (1996) "Gene therapy in the United States: a
five-year status report" Hum. Gene Ther. 7:1781-1790.
[0267] Schroeder, F. et al. (1990) "Intermembrane cholesterol
transfer: role of sterol carrier proteins and phosphatidylserine"
Lipids 25:669-674.
[0268] Schroth-Diez, B. et al. (1998) "Fusion activity of
transmembrane and cytoplasmic domain chimeras of the influenza
virus glycoprotein hemagglutinin" J. Virol. 72:133-141.
[0269] Schutze, W. and Muller-Goymann, C. C. (1998) "Phase
transformation of a liposomal dispersion into a micellar solution
induced by drug-loading" Pharm. Res. 15:538-543.
[0270] Song, Y. K. et al. (1997) "Characterization of cationic
liposome-mediated gene transfer in vivo by intravenous
administration" Hum. Gene Ther. 8:1585-1594.
[0271] Sorgi, F. L. et al. (1997) "Protamine sulfate enhances
lipid-mediated gene transfer" Gene Ther. 4:961-968.
[0272] Suenaga, M. et al. (1989) "Basic amphipathic helical
peptides induce destabilization and fusion of acidic and neutral
liposomes" Biochim. Biophys. Acta 981:143-150.
[0273] Takle, G. B. et al. (1997) "Delivery of oligoribonucleotides
to human hepatoma cells using cationic lipid particles conjugated
to ferric protoporphyrin IX (heme)" Antisense Nucleic Acid Drug
Dev. 7:177-185.
[0274] Templeton, N. S. et al. (1997) "Improved DNA: liposome
complexes for increased systemic delivery and gene expression"
Nature Biotechnol. 15:647-652.
[0275] Thierry, A.R. and Dritschilo, A. (1992) "Intracellular
availability of unmodified, phosphorothioated and liposomally
encapsulated oligodeoxynucleotides for antisense activity" Nucl.
Acids Res. 20:5691-5698.
[0276] Tirosh, 0. et al. (1998) "Hydration of polyethylene
glycol-grafted liposomes" Biophys. J. 74:1371-1379.
[0277] Torchilin, V. P. (1998) "Polymer-coated long-circulating
microparticulate pharmaceuticals" J. Microencapsul. 15:1-19.
[0278] Torchilin, V. P. et al. (1992) "Targeted accumulation of
polyethylene glycol-coated immunoliposomes in infarcted rabbit
myocardium" FASEB J 6:2716-2719.
[0279] Toumois, H. et al. (1990) "Gramicidin A induced fusion of
large unilamellar dioleoylphosphatidylcholine vesicles and its
relation to the induction of type II nonbilayer structures"
Biochemistry 29:8297-8307.
[0280] Ulrich, A. S. et al. (1999) "Ultrastructural
characterization of peptide-induced membrane fusion and peptide
self-assembly in the lipid bilayer" Biophys. J. 77:829-841.
[0281] Voneche, V. et al. (1992) "The 19-27 amino acid segment of
gp51 adopts an amphiphilic structure and plays a key role in the
fusion events induced by bovine leukemia virus" J. Biol. Chem.
267:15193-15197.
[0282] Wattiaux, R. et al. (1997) "Cationic lipids destabilize
lysosomal membrane in vitro" FEBS Lett. 417:199-202.
[0283] Weissig, V. et al. (1998) "Accumulation of protein-loaded
long-circulating micelles and liposomes in subcutaneous Lewis lung
carcinoma in mice" Pharm. Res. 15:1552-1556.
[0284] Zelphati, O. and Szoka, Jr., F. C. (1997) "Intracellular
distribution and mechanism of delivery of oligonucleotides mediated
by cationic lipids" Pharm. Res. 13:1367-1372.
[0285] Zuidam, N. J. and Barenholz, Y. (1997) "Electrostatic
parameters of cationic liposomes commonly used for gene delivery as
determined by 4-heptadecyl-7-hydroxycoumarin" Biochim. Biophys.
Acta 1329:211-222.
* * * * *