U.S. patent application number 10/644080 was filed with the patent office on 2005-04-14 for methods for delivering compounds into a cell.
This patent application is currently assigned to ImaRx Pharmaceutical Corporation. Invention is credited to McCreery, Thomas, Unger, Evan C..
Application Number | 20050080029 10/644080 |
Document ID | / |
Family ID | 27417663 |
Filed Date | 2005-04-14 |
United States Patent
Application |
20050080029 |
Kind Code |
A1 |
Unger, Evan C. ; et
al. |
April 14, 2005 |
Methods for delivering compounds into a cell
Abstract
The present invention is directed, inter alia, to a method for
delivering a compound into a cell comprising administering to the
cell the compound to be delivered, an organic halide, and/or a
carrier. Ultrasound may also be applied, if desired.
Inventors: |
Unger, Evan C.; (Tucson,
AZ) ; McCreery, Thomas; (Alexandria, VA) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Assignee: |
ImaRx Pharmaceutical
Corporation
Tucson
AZ
85719
|
Family ID: |
27417663 |
Appl. No.: |
10/644080 |
Filed: |
August 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10644080 |
Aug 20, 2003 |
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09742938 |
Dec 21, 2000 |
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6638767 |
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09742938 |
Dec 21, 2000 |
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08841169 |
Apr 29, 1997 |
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6743779 |
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08841169 |
Apr 29, 1997 |
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08785661 |
Jan 17, 1997 |
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08785661 |
Jan 17, 1997 |
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08640554 |
May 1, 1996 |
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Current U.S.
Class: |
514/44R ;
424/45 |
Current CPC
Class: |
A61K 9/1272 20130101;
A61P 11/00 20180101; A61P 31/18 20180101; A61K 41/0028 20130101;
A61P 3/10 20180101; A61P 21/00 20180101; A61P 7/00 20180101; C12N
15/88 20130101; A61P 35/00 20180101; C12N 15/87 20130101; A61P 9/10
20180101; A61K 48/00 20130101 |
Class at
Publication: |
514/044 ;
424/045 |
International
Class: |
A61K 048/00; A61L
009/04 |
Claims
1. A method for delivering a compound into a cell comprising
administering to the cell a composition which comprises the
compound to be delivered and an organic halide.
2-104. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 08/785,661, filed Jan. 17, 1997, which in turn
is a continuation-in-part of U.S. application Ser. No. 08/640,554,
filed May 1, 1996, the disclosures of each of which are hereby
incorporated herein by reference in their entireties.
FIELD OF THE INVENTION
[0002] This invention relates to the field of intracellular
delivery, in particular, to the use of organic halides and/or
ultrasound to facilitate the delivery of a compound into a
cell.
BACKGROUND OF THE INVENTION
[0003] Cells are the basic structural and functional units of all
living organisms. All cells contain cytoplasm surrounded by a
plasma, or cell, membrane. Most bacterial and plant cells are
enclosed in an outer rigid or semi-rigid cell wall. The cells
contain DNA which may be arranged in 1) a nuclear membrane or 2)
free in cells lacking a nucleus. While the cell membrane is known
to contain naturally occurring ion channels, compounds that are
therapeutically advantageous to cells are usually too large to pass
through the naturally occurring ion channels. Conventional
interventional methods of delivery of compounds into cells have
proved difficult in view of the need for the compounds to pass
through the cell membrane, cell wall, and nuclear membrane.
[0004] Molecular biology has resulted in mapping the genomes of
many plants and animals including the mapping of much of the human
genome. The potential for advances in the understanding of the
genetic basis of diseases is great, as is the potential for the
development of therapies to treat such diseases. However, to fully
take advantage of these advancements and treatment therapies,
methods are needed which will allow for the delivery of desired
compounds into the target cells. Accordingly, researchers have
undertaken the development of a variety of intracellular delivery
methods for inserting genes and other compounds into both plant and
animal cells.
[0005] For example, calcium phosphate DNA precipitation has been
used to deliver genetic material into cells in cell culture.
However, one drawback of this method is that the resultant
efficiency of transfection (delivery of the genetic material into
the cells) and subsequent gene expression has been very low.
[0006] Improved transfection has been attained using viral vectors,
e.g., adenovirus and retrovirus, but again, difficulties with gene
expression have persisted. In addition, substantial concerns
regarding antigenicity and the potential of mutant viruses and
other possible deleterious effects exist.
[0007] Liposomes, manufactured more easily than viral vectors, have
shown promise as gene delivery agents. Liposomes have less
biological concerns (in that, for example, they are generally
non-antigenic) but the efficiency of transfection and gene
expression using liposomes has typically been lower than with
viruses.
[0008] Gene guns, wherein genes are attached to heavy metal
particles such as gold, have been used to fire the particles at
high speed into cells. However, while gene guns have resulted in
gene expression in culture systems, they have not worked well in
vivo. Furthermore, the blast of heavy metal particles may cause
damage to the cells and may result in the introduction of
undesirable foreign materials, e.g. gold particle fragments, into
the cells.
[0009] Electroporation is another method of delivering genes into
cells. In this technique, pulses of electrical energy are applied
to cells to create pores or openings to facilitate passage of DNA
into the cells. However, electroporation may damage cells, and
furthermore has not been shown to be highly effective in vivo.
[0010] Various publications disclose the use of lithotripsy shock
waves for effecting intracellular gene transfer, as well as the
delivery of other compounds, including, for example, Delius, M., et
al., "Extracorporeal Shock Waves for Gene Therapy," Lancet May 27,
1995, 345:1377; Lauer, U., et al., "Towards A New Gene Transfer
System: Shock Wave-Mediated DNA Transfer," J Cell Biochem 1994,
16A:226; Gambihier, S., et al., "Permeabilization of the Plasma
Membrane of L1210 Mouse Leukemia Cells Using Lithotripter Shock
Waves," J Membr Biol 1994, 141:267-75; and Mobley, T. B., et al.,
"Low Energy Lithotripsy with the Lithostar: Treatment Results with
19,962 Genal and Ureteral Calculi," J Urol 1993, 149:1419-24.
Lithotripsy delivers energy in the range of 200-380 bars, and a
frequency of 60-120 Hz, but may be as high as 1200 to 1300 bars.
The energy and frequency ranges are typically painful to a patient
and thus usually require patient sedation. Lithotripsy machines are
large and bulky and are typically cost prohibitive. Lauer et al.
disclose the delivery of 250 shock waves at 25 kV with a
lithotripter to deliver plasmid DNA which expressed hepatitis B
virus surface proteins in a HeLa cell suspension.
[0011] Gambihler et al. (cited above) teach the permeabilization of
mouse cells in vitro to deliver dextrans. The lithotripter shock
waves are delivered at 25 kV, at a discharge rate of 60/min. Mobley
et al. (cited above) disclose the use of lithotripsy to treat renal
and ureteral stones. The shock wave pressure was 200 to 380 bar and
a generator range of 10 to 29 kV.
[0012] Zhang, L., et al., "Ultrasonic direct gene transfer The
Establishment of High Efficiency Genetic Transformation System for
Tobacco," Sci Agric. Sin. 1991, 24:83-89 disclose increased gene
expression by tobacco using continuous wave ultrasound at 0.5
W/cm.sup.2 for 30 minutes. Zhang et al. do not disclose the
ultrasound frequency. The high energy level is in a range necessary
for poration to result in the cell wall of tobacco plants.
[0013] Rubin, et al., 31st Annual Meeting of the American Society
of Clinical Oncology, May 20-23, 1995, disclose the injection of
hepatic tumors with a plasmid/cationic lipid complex with
ultrasound guidance. Ultrasound is disclosed as a visual guide to
monitor the injection of the tumors, rather than as an aid to
deliver the complex to the liver tumors.
[0014] The present invention provides new and/or better methods for
delivering compounds, including genetic material, into a cell. The
methods of the present invention may provide a significant
advantage over prior art methodology, in that enhanced levels of
intracellular delivery, and in the case of nucleotides, gene
expression, may be achieved. In addition, the process of the
present invention may be performed in cell lines which may be
otherwise resistant to intracellular delivery and gene expression
using other conventional means. These and/or other aspects of the
present invention will become apparent from the further discussions
herein.
SUMMARY OF THE INVENTION
[0015] The present invention is directed, inter alia, to a method
for delivering a compound into a cell comprising administering to
the cell a composition which comprises the compound to be delivered
and an organic halide.
[0016] In addition, the invention provides a method of treating a
patient comprising administering to a patient a composition
comprising a therapeutically effective amount of a compound and an
organic halide.
[0017] The subject invention provides a method of effecting the
expression of a nucleotide sequence in a cell comprising
administering to said cell a composition which comprises a
nucleotide sequence and an organic halide.
[0018] If desired, the compositions may further comprise a carrier.
In addition, the method of the invention may further comprise the
application of ultrasound, as desired.
[0019] The present invention is also directed to a method for
delivering a compound into a cell comprising administering to the
cell the compound to be delivered, or a composition comprising the
compound to be delivered, and applying ultrasound.
[0020] Further, the invention pertains to a method of treating a
patient comprising administering to a patient a therapeutically
effective amount of a compound, or a composition comprising a
therapeutically effective amount of a compound, and applying
ultrasound.
[0021] Moreover, the subject invention provides a method of
effecting the expression of a nucleotide sequence in a cell
comprising administering to the cell a nucleotide sequence, or a
composition which comprises a nucleotide sequence, and applying
ultrasound.
[0022] If desired, the composition may further comprise
carrier.
[0023] Also included in the present invention are compositions and
kits comprising, for example, a therapeutically effective or
diagnostically effective amount of a compound to be delivered, an
organic halide, and/or a carrier, and, in the case of a kit,
optionally other conventional kit components.
[0024] These, as well as other, aspects of the invention are set
forth in greater detail below.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 depicts a test set-up for delivery of compounds to a
cell in vitro or ex vivo. In FIG. 1, A represents a standoff
platform. B is a six well plate, with individual wells B'. C
represents an ultrasound gel, while D represents a therapeutic
ultrasound transducer. In accordance with the invention, cells, the
compound to be delivered, an organic halide (if desired), and
optionally a carrier are placed in the wells. Using ultrasound
transducer D, ultrasound is then applied to cell culture plate B
such that the standoff platform (A) is cut (G) under each well (B')
for focusing ultrasound to the individual wells (B'). Ultrasound
transducer D may also be employed for the in vivo delivery of
compounds by applying transducer D, with ultrasound gel C, to a
patient instead of to a cell culture plate.
[0026] FIG. 2 displays the relationship between energy deposition,
ultrasound energy intensity, and ultrasound duty cycle (pulse
duration). The effect of attenuation as a function of tissue depth
is also portrayed as well as spatial peak temporal average
power.
[0027] FIG. 3 is a map of the pCAT.RTM. control vector (GenBank
accession number X65321) (Promega, Madison, Wis.) used in the
preparation of sequences introduced into cells in Examples 2 and
3.
[0028] FIG. 4 is a cutaway from FIG. 1, depicting one of the six
wells (B') of a six well plate B. In FIG. 4, A is a portion of the
standoff platform, B' is one well of six well plate B, C is the
ultrasound gel, D is the ultrasound transducer, E represents the
cells, and F represents microsphere carriers for the compound to be
delivered.
[0029] FIG. 5 is a prototype second harmonic transducer that emits
X and 2.times. frequencies and superimposes two beams at one focal
point for enhanced ultrasound effect. The transducer may be
employed in conjunction with in vitro, ex vivo and in vivo delivery
of compounds and compositions in accordance with the invention.
[0030] FIG. 6 is a map of the pCAT.RTM. basic vector (Promega,
Montgomeryville, Pa.) used in the preparation of sequences
introduced into cells in Example 10.
[0031] FIG. 7 depicts the results of the in vivo transfection
studies in mice set forth in Example 23.
DETAILED DESCRIPTION OF THE INVENTION
[0032] As employed above and throughout the disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings.
[0033] For purposes of the present invention, an "organic halide"
(also sometimes referred to as a halogenated organic compound)
denotes a compound which contains at least one carbon atom (or
optionally sulfur or selenium atom, such as in the case of SF.sub.6
and SeF6) and at least one halogen atom selected from the group
consisting of fluorine, chlorine, bromine, or iodine. Preferably
the halogen is fluorine (i.e., the compound is a fluorinated
compound).
[0034] Most preferably the organic halide is a fluorinated compound
which is perfluorinated (that is, fully fluorinated, e.g., a carbon
compound wherein all hydrogen atoms directly attached to the carbon
atoms have been replaced by fluorine atoms). The perfluorinated
organic halide (perfluorinated compound) is preferably a
perfluorocarbon or a perfluoroether. The organic halide may be in
the form of a gas, a liquid (including a gaseous precursor), or a
solid. Preferably the organic halide is a liquid, even more
preferably a liquid which is a gaseous precursor that converts to a
gas upon administration. Most preferably, the gaseous precursor
converts to a gas at the site of (in close or touching proximity
to) the cell.
[0035] "Gaseous precursor" refers to a liquid or solid which is
activated upon attaining a certain temperature or pressure to
convert to a gas. A gaseous precursor which is capable of
converting to a gas at the site of the cell may increase the
efficiency of cellular uptake of compounds, and is therefore
preferred.
[0036] Ideally, the gaseous precursors are liquid (or solid) at
ambient (room) temperature (e.g., 25.degree. C.), but will convert
to a gas either at physiological temperature (e.g., 37.degree. C.)
such as upon administration to a patient, or otherwise conveniently
at the site of the cell such as upon application of heat (such as,
for example, using ultrasound). If heat is applied, it should be
done so at a temperature sufficient to convert the gaseous
precursor to a gas, but insufficient to harm the cell (e.g.,
denature the proteins, etc.). Thus, ideally a gaseous precursor
becomes a gas at less than about 80.degree. C. Even more ideally,
the gaseous precursor becomes a gas at between about 30.degree. C.
and about 70.degree. C. Most ideally, the gaseous precursor becomes
a gas at between about 37.degree. C. and less than about 50.degree.
C.
[0037] A variety of different organic halides may be employed in
this invention. Where the organic halide is a carbon based halide
compound, the organic halide preferably contains from 1 to about 30
carbon atoms, more preferably 1 to about 24 carbon atoms, even more
preferably 1 to about 12 carbon atoms, still even more preferably
about 5 to about 12 carbon atoms, and most preferably about 6 to
about 10 carbon atoms. Thus, the number of carbon atoms in the
organic halide may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, carbon atoms, and
upwards. Sulfur or selenium based halide compounds, such as sulfur
hexafluoride and selenium hexafluoride, are also within the scope
of the invention and the phrase organic halide as used herein. The
organic halides contemplated herein may also, for example, have
carbon atoms interrupted by one or more heteroatoms, such as --O--
bonds (as in ether compounds) or have other substituents such as
amines, etc. Preferred organic halides of the present invention are
the perfluorinated organic halides such as perfluorocarbons and
perfluoroethers.
[0038] Table 1 lists representative organic halides useful in the
present invention. Other organic halides suitable for use in the
present invention will be readily apparent to one skilled in the
art, once armed with the present disclosure. All such organic
halides are intended to fall within the scope of the term organic
halide, as used herein.
1TABLE 1 Organic Halides Compound Boiling Point (.degree. C.) 1.
Mixed-halogenated Compounds 1-bromo-nonafluorobutane 43
perfluorooctyliodide 160-161 perfluoroocytlbromide 142
1-chloro-1-fluoro-1-bromomethane 38
1,1,1-trichloro-2,2,2-trifluoroethane 45.7
1,2-dichloro-2,2-difluoroethane 46 1,1-dichloro-1,2-difluoroethan-
e 45 1,2-dichloro-1,1,3-trifluoropropane 50.4
1-bromoperfluorobutane 43 1-bromo-2,4-difluorobenzene 44
2-iodo-1,1,1-trifluoroethane 53 5-bromovaleryl chloride 43
1,3-dichlorotetrafluoroacetone 43 bromine pentafluoride 40.3
1-bromo-1,1,2,3,3,3-hexafluoropropane 35.5 2-chloro
1,1,1,4,4,4-hexafluoro-2-butene 33 2-chloropentafluoro-1,3-butadi-
ene 37 iodotrifluoroethylene 30 1,1,2-trifluoro-2-chloroet- hane 30
1,2-difluorochloroethane 35.5 1,1-difluoro-2-chloroethane 35.1
1,1-dichlorofluoroethane 31.8 heptafluoro-2-iodopropane 39
bromotrifluoroethane -57.8 chlorotrifluoromethane -81.5
dichlorodifluoromethane -29.8 dibromofluoromethane 23
chloropentafluoroethane -38.7 bromochlorodifluoromethane -4
dichloro-1,1,2,2-tetrafluoroethane 3.1-3.6 2. Fluorinated Compounds
1,1,1,3,3-pentafluoropen- tane 40 perfluorotributylamine 178
perfluorotripropylamine 130 3-fluorobenzaldehyde 56
2-fluoro-5-nitrotoluene 53 3-fluorostyrene 40 3,5-difluoroaniline
40 2,2,2-trifluoroethylacrylate 45
3-(trifluoromethoxy)-acetophenone 49
1,1,2,2,3,3,4,4-octafluorobutane 44.8 1,1,1,3,3-pentafluorobutane
40 1-fluorobutane 32.5 1,1,2,2,3,3,4,4-octafluorobutane 44.8
1,1,1,3,3-pentafluorobutane 40 perfluoro-4 methylquinolizidine 149
perfluoro-N-methyl-decahydroquinone 150-155
perfluoro-N-methyl-decahydroisoquinone 150-155
perfluoro-N-cyclohexyl-pyrrolidine 145-152
tetradecaperfluoroheptane 76 dodecaperfluorocyclohexane 52 3.
Perfluorinated Compounds a. Perfluorocarbons perfluoromethane -129
perfluoroethane -78.3 perfluoropropane -36 perfluorobutane -2
perfluoropentane 29.5 perfluorohexane 59-60 perfluoroheptane 81
perfluorooctane 102 perfluorononane 125 perfluorodecane .about.143
perfluorododecane melting pt 75-77 perfluoro-2-methyl-2-pentene 51
perfluorocyclohexane 52 perfluorodecalin 142 perfluorododecalin --
perfluoropropylene -28 perfluorocyclobutane -6 perfluoro-2-butyne
-25 perfluoro-2-butene 1.2 perfluorobuta-1,3-diene 6 b.
Perfluoroether Compounds perfluorobutylethyl ether 60
bis(perfluoroisopropyl) ether 54 bis(perfluoropropyl) ether 59
perfluorotetrahydropyran 34 perfluoromethyl tetrahydrofuran 27
perfluoro t-butyl methyl ether 36 perfluoro isobutyl methyl ether
-- perfluoro n-butyl methyl ether 35.4 perfluoro isopropyl ethyl
ether -- perfluoro n-propyl ethyl ether 23.3 perfluoro cyclobutyl
methyl ether -- perfluoro cyclopropyl ethyl ether -- perfluoro
isopropyl methyl ether 36 perfluoro n-propyl methyl ether --
perflouro diethyl ether 3-4.5 perfluoro cyclopropyl methyl ether --
perfluoro methyl ethyl ether -23 perfluoro dimethyl ether -59 c.
Other sulfur hexafluoride m.p. -50.5, sublimes -63.8 selenium
hexafluoride m.p. -34.6 sublimes -46.6
[0039] Preferred organic halides include 1-bromo-nonafluorobutane,
1,1,1,3,3-pentafluoropentane, perfluorohexane,
perfluorocyclohexane, 1-bromo-1,1,2,3,3,3-hexafluoropropane,
heptafluoro-2-iodopropane, 1,1,2,2,3,3,4,4-octafluorobutane,
1-fluorobutane, tetradecaperfluorohepta- ne and
dodecaperfluorocylclohexane. Particularly preferred are
perfluorohexane (especially n-perfluorohexane) and
perfluorocyclohexane. A wide variety of other organic halides
useful in the present invention will be readily apparent to those
of skill in the art once armed with the present disclosure.
Suitable additional organic halides include those, for example,
disclosed in Long, Jr. in U.S. Pat. Nos. 4,987,154, 4,927,623, and
4,865,836, the disclosures of each of which are hereby incorporated
herein by reference in their entirety.
[0040] The amount of organic halide employed in the present
invention may vary, as one skilled in the art will recognize, once
armed with the present disclosure, and may be dependent on such
factors as the particular organic halide employed, type and nature
of the compound to be delivered, the age, weight, cells or patient
(animal) to be treated, the particular diagnostic, therapeutic or
other application intended (including the disease state, if any, to
be treated). Typically lower amounts are used and then increased
until the desired delivery effect is achieved. Representative
amounts are set forth in the examples herein. Of course, higher or
lower amounts may be employed, as will be recognized by the skilled
artisan.
[0041] Methods of introducing compounds into a cell (also referred
to variously herein as methods for delivering a compound into a
cell, methods of intracellular delivery, methods of promoting,
effecting, facilitating or enhancing the uptake of a compound into
a cell, and the like) include "transfection", which refers to the
introduction of genetic material, i.e., a nucleotide sequence
(e.g., DNA or RNA) into a host cell. Transfection is also sometimes
referred to as transformation. DNA (or RNA) which is new to the
cell into which it is incorporated is typically referred to as
heterologous DNA (or RNA) or exogenous DNA (or RNA). Some bacterial
species take up exogenous DNA and do not discriminate between
uptake of DNA from a similar or same species or from a completely
different species or organism. Exogenous DNA may also be taken up
by cells, but may or may not be incorporated into nuclear material
in a hereditable manner. The objective of transfection of a host
cell may be to effect expression of one or more carefully selected
sequences.
[0042] "Expression" and "gene expression" refer to the
transcription and/or translation of a nucleic acid sequence
resulting in the production of an amino acid, peptide and/or
protein. The nucleic acid sequence may or may not be incorporated
into the genetic material of the host cell. For example, the
nucleic acid sequence may be incorporated into the genome of a host
cell or may simply be introduced into the cell without
incorporation into the genome.
[0043] Gene expression, upon administration of the composition of
the present invention, may be effected (obtained., promoted,
facilitated or enhanced), and in fact may be enhanced in that the
expression of the nucleic acid sequences as compared to
conventional transfection techniques such as calcium phosphate
precipitation, viral vectors, microinjection, shock wave such as
for example lithotripsy, and electroporation, may be increased.
Methods of measuring enhanced gene expression will be known to
skilled artisans once armed with the present disclosure and include
enzyme-linked immunosorbent assay (ELISA) as well as methods
disclosed in Sambrook, et al., Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989), the disclosures of which are hereby
incorporated herein by reference in their entirety. Thus, as a
result of the methods of the present invention, a product (e.g., a
protein) may be produced. In addition, the prevention of the
production of a product (such as, as a result of an antisense
sequence delivered into the cell) by the host cell may also
result.
[0044] Without being bound by any theory of operation, it is
believed that delivery of nucleic acid sequences and other
compounds in accordance with the methods of the present invention
may induce a cell to take up the compound to be delivered thereto.
Included within the definition of delivery of a compound into a
cell in accordance with the methods of the present invention are
active and passive mechanisms of cellular uptake. Ion channels and
other means of transport utilized by cells to incorporate
extracellular materials, including compounds to be delivered
thereto, into the intracellular milieu are encompassed by the
present invention.
[0045] "Nucleotide sequence and nucleic acid sequence" refer to
single and double stranded DNA and RNA sequences, including and not
limited to oligonucleotide sequences of about 100 kb to about
1,000,000 kb (including whole chromosomes), preferably of about 4
kb to about 6 kb, more preferably about 1,000 nucleotides in
length, more preferably about 500 nucleotides in length, more
preferably about 250 nucleotides in length, more preferably about
100 nucleotides in length, more preferably about 50 nucleotides in
length, more preferably about 25 nucleotides in length, more
preferably about 10 nucleotides in length, even more preferably
about 3 to about 10 kbp in length. Embodied by the term "nucleotide
sequence" are all or part of a gene, at least a portion of a gene,
a gene fragment, a sense sequence, an antisense sequence, an
antigene nucleic acid, a phosphorothioate oligodeoxynucleotide, and
an alteration, deletion, mismatch, transition, transversion,
mutation, conservative substitution, and homolog of a sequence. The
phrase "at least a portion of," and "all or part of," as used
herein, means that the entire gene need not be represented by the
sequence so long as the portion of the gene represented is
effective to block or exhibit, depending on the type of sequence
used, gene expression. The sequences may be incorporated into an
expression vector such as, and not limited to, a plasmid, phagemid,
cosmid, yeast artificial chromosome (YAC), virus (e.g., adenovirus,
vaccinia virus, retrovirus), and defective virus (also known as a
"helper virus"). The nucleotide sequence may also be administered
naked, that is without an expression vector.
[0046] "Cell" and "host cell" refer to prokaryotic cells and
eukaryotic cells, including plant cells, animal cells, cells of
unicellular organisms, cells of multicellular organisms, etc.
Especially preferred are animal cells, more preferably mammalian
cells and most especially human cells, including but not limited to
living cells, tissues, and organs. Eukaryotic cells are cells of
higher organisms in which genetic material is enclosed by a nuclear
membrane. Prokaryotic cells are cells of lower organisms that lack
a well defined nucleus and contain genetic material that is not
enclosed within a membrane of its own. The cells may be present in
vivo or in vitro (e.g. in cell culture).
[0047] The invention has wide applications for effecting
(obtaining, promoting, facilitating or enhancing) and/or increasing
the efficiency of, intracellular delivery (e.g., transfection)
and/or, in the case of nucleotides, gene expression in both in
vitro and in vivo applications, and is particularly useful for
prokaryotic and eukaryotic animal cells, particularly mammalian
cells. Intracellular delivery includes delivery into the cells
through a cell membrane (plasma membrane), cell wall, and/or
nuclear membrane.
[0048] The phrase "cell membrane" (also termed "plasma membrane")
is used in its conventional sense as denoting the outer layer or
boundary of the cytoplasm of a living cell. Cell membranes are
typically comprised of protein and lipids, and are generally found
in animal cells.
[0049] The phrase "cell wall" is also used in its conventional
sense to denote a rigid or semi-rigid outer covering surrounding
the protoplasts of plant cells and most prokaryotes. Cell walls are
typically found, for example, in cells of bacteria, plants, algae,
and fungi. Cell walls are, on the other hand, generally not present
in animal cells. In plants, the wall typically comprises several
layers; a primary wall composed of cellulose microfibrils running
through a matrix of hemicelluloses and pectic substances surrounded
by a secondary wall composed of cellulose which is generally
lignified to a varying extent. Cell walls of fungi may contain
varying amounts of chitin. Cell walls of prokaryotes are typically
strengthened by mucopeptides and may be surrounded by a
mucilagenous capsule.
[0050] A wide variety of compounds can comprise the compounds to be
delivered to the cells in accordance with the invention, including
bioactive agents, diagnostic agents, pharmaceutical agents, and the
like, and include proteins, DNA and RNA (both single and
double-stranded), anti-sense and gene constructs, as well as other
organic or inorganic compounds. Whole genes, multiple gene
sequences, and gene fragments may be utilized as well as whole
chromosomes and chromosome fragments.
[0051] As noted above, the methods of the present invention may,
for example, be carried out in the presence of an organic halide,
with or without the application of ultrasound, or in the absence of
an organic halide but with the application of ultrasound. Where
bioactive agents other than nucleotides are employed as the
compound to be delivered, generally, for best results, an organic
halide is used, although use of an organic halide in such a
situation is not required.
[0052] If desired, the composition may further comprise a carrier.
The carrier employed may comprise a wide variety of materials.
Carriers may include, for example, lipids, polymers, proteins,
surfactants, inorganic compounds, metal ions, and the like, alone
or in combination with water and/or a solvent, or the carrier may
simply comprise water and/or a solvent. The lipids, proteins, and
polymers, for example, may be in liquid form or solid form (such
as, for example, the form of particles, fibers, sheets, layers,
etc.), or may take the form of a vesicle or other stable, organized
form, which may include but is not limited to, such forms commonly
referred to as, for example, liposomes, micelles, bubbles,
microbubbles, microspheres, lipid-, polymer-, and/or protein-coated
bubbles, microbubbles and/or microspheres, microballoons, aerogels,
hydrogels, clathrates, hexagonal HII phase structures, and the
like. The internal void of the vesicle or other stable form may,
for example, be filled with a liquid (including, for example, a
gaseous precursor), a gas, a solid, or solute material, or any
combination thereof, including, for example, the compound to be
delivered, the organic halide, and/or any targeting ligand, as
desired. Typically, the carrier is provided as an aqueous milieu,
such as water, saline (such as phosphate buffered saline), and the
like, with or without other carrier components, although other
non-aqueous solvents may also be employed, if desired. The carrier
may comprise a mixture in the form of an emulsion, suspension,
dispersion, solution, and the like. Lipid (including oil) in water
emulsions are especially preferred. As indicated above, the carrier
may also include buffers.
[0053] Thus, "vesicle", as used herein, refers to an entity which
is generally characterized by the presence of one or more walls or
membranes which form one or more internal voids. Vesicles may be
formulated, for example, from stabilizing compounds, such as a
lipid, including the various lipids described herein, a polymer,
including the various polymers described herein, or a protein,
including the various proteins described herein, as well as using
other materials that will be readily apparent to one skilled in the
art. Other suitable materials include, for example, any of a wide
variety of surfactants, inorganic compounds, and other compounds as
will be readily apparent to one skilled in the art. Also, as will
be apparent to one skilled in the art upon reading the present
disclosure, the organic halides may themselves act as suitable
carriers, and may in certain embodiments themselves form vesicles
and other organized structures. Thus the use of the organic halides
of the invention in combination with a compound to be delivered,
without an additional compound to serve as a carrier, is within the
scope of the invention. The lipids, polymers, proteins,
surfactants, inorganic compounds, and/or other compounds may be
natural, synthetic or semi-synthetic. Preferred vesicles are those
which comprise walls or membranes formulated from lipids. The walls
or membranes may be concentric or otherwise. In the preferred
vesicles, the stabilizing compounds may be in the form of a
monolayer or bilayer, and the mono- or bilayer stabilizing
compounds may be used to form one or more mono- or bilayers. In the
case of more than one mono- or bilayer, the mono- or bilayers may
be concentric, if desired. Stabilizing compounds may be used to
form unilamellar vesicles (comprised of one monolayer or bilayer),
oligolamellar vesicles (comprised of about two or about three
monolayers or bilayers) or multilamellar vesicles (comprised of
more than about three monolayers or bilayers). The walls or
membranes of vesicles prepared from lipids, polymers or proteins
may be substantially solid (uniform), or they may be porous or
semi-porous. The vesicles described herein include such entities
commonly referred to as, for example, liposomes, micelles, bubbles,
microbubbles, microspheres, lipid-, protein- and/or polymer-coated
bubbles, microbubbles and/or microspheres, microballoons,
microcapsules, aerogels, clathrate bound vesicles, hexagonal H II
phase structures, and the like. The vesicles may also comprise a
targeting ligand, if desired.
[0054] "Lipid vesicle", "polymer vesicle" and "protein vesicle"
refer respectively to vesicles formulated from one or more lipids,
polymers and proteins.
[0055] "Liposome" refers to a generally spherical or spheroidal
cluster or aggregate of amphipathic compounds, including lipid
compounds, typically in the form of one or more concentric layers,
for example, monolayers or bilayers. They may also be referred to
herein as lipid vesicles. The liposomes may be formulated, for
example, from ionic lipids and/or non-ionic lipids. Liposomes which
are formulated from non-ionic lipids may also be referred to as
"niosomes."
[0056] "Micelle" refers to colloidal entities formulated from
lipids. In certain preferred embodiments, the micelles comprise a
monolayer or hexagonal H2 phase configuration. In other preferred
embodiments, the micelles may comprise a bilayer configuration.
[0057] "Aerogel" refers to generally spherical or spheroidal
entities which are characterized by a plurality of small internal
voids. The aerogels may be formulated from synthetic or
semisynthetic materials (for example, a foam prepared from baking
resorcinol and formaldehyde), as well as natural materials, such as
polysaccharides or proteins.
[0058] "Clathrate" refers to a solid, semi-porous or porous
particle which may be associated with vesicles. In preferred form,
the clathrates may form a cage-like structure containing cavities
which comprise the vesicles. One or more vesicles may be bound to
the clathrate. A stabilizing material may, if desired, be
associated with the clathrate to promote the association of the
vesicle with the clathrate. Suitable materials from which
clathrates may be formulated include, for example, porous apatites,
such as calcium hydroxyapatite, and precipitates of polymers and
metal ions, such as alginic acid precipitated with calcium
salts.
[0059] "Emulsion" refers to a mixture of two or more generally
immiscible liquids and is generally in the form of a colloid. The
liquids may be homogeneously or heterogeneously dispersed
throughout the emulsion. Alternatively, the liquids may be
aggregated in the form of, for example, clusters or layers,
including mono- or bilayers.
[0060] "Suspension" or "dispersion" refers to a mixture, preferably
finely divided, of two or more phases (solid, liquid or gas), such
as, for example, liquid in liquid, solid in liquid, liquid in gas,
etc.) which can preferably remain stable for extended periods of
time.
[0061] "Hexagonal H II phase structure" refers to a generally
tubular aggregation of lipids, proteins, or polymers (especially
lipids) in liquid media, for example, aqueous media, in which any
hydrophilic portion(s) generally face inwardly in association with
an aqueous liquid environment inside the tube. The hydrophobic
portion(s) generally radiate outwardly and the complex assumes the
shape of a hexagonal tube. A plurality of tubes is generally packed
together in the hexagonal phase structure.
[0062] "Biocompatible" refers to materials which are generally not
injurious to biological functions and which will not result in any
degree of unacceptable toxicity, including allergenic responses and
disease states. The compositions of the present invention and/or
components thereof are typically biocompatible.
[0063] The nucleotide sequence or other compound to be delivered
may be administered, if desired, "in combination with" an organic
halide, and may further be administered, if desired, "in
combination with" a carrier, including a vesicle (or other stable
form). "In combination with" refers to the co-administration of the
compound to be delivered and the organic halide (and/or carrier, if
desired). The compound to be delivered and the organic halide
(and/or any carrier) may be combined in any of a variety of
different fashions, including simply being placed in admixture with
one another. In addition, for example, the nucleotide or other
compound to be delivered and/or the organic halide may be embedded,
encapsulated, or attached to, or with, one another, as desired
(including any and all combinations thereof). The phrase "in
admixture" includes solutions, suspensions, emulsions, dispersions,
mixtures, etc. The phrase "attached to" or variations thereof, as
used herein, denotes being linked in some manner, such as through a
covalent or ionic bond or other means of chemical or
electrochemical linkage or interaction. The phrase "encapsulated"
and variations thereof as used herein refers to a location inside
an internal void of a vesicle or other structure. The phrase
"embedded within" or variations thereof as used herein signifies a
positioning within the wall of a vesicle or other structure. Thus,
a nucleotide sequence, for example, can be positioned variably,
such as, for example, entrapped within the internal void of the
vesicle, situated on the internal wall of the vesicle, incorporated
onto the external surface of the vesicle, and/or enmeshed within
the vesicle structure itself. In addition, one or more vesicles may
be administered as a cavitator. In such case, the vesicles
accompany the administration of a compound and may serve to enhance
the efficiency of ultrasound.
[0064] Lipids may be used in the present invention as a carrier.
The lipids may be natural, synthetic or semisynthetic (i.e.,
modified natural). Lipids useful in the invention include, and are
not limited to, fatty acids, lysolipids, oils (including safflower,
soybean and peanut oil), phosphatidylcholine with both saturated
and unsaturated lipids including phosphatidylcholine;
dioleoylphosphatidylcholine; dimyristoylphosphatidylcholine;
dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine,
dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine;
distearoylphosphatidylcholine; phosphatidylethanolamines such as
dioleoylphosphatidylethanolamine; phosphatidylserine;
phosphatidylglycerol; phosphatidylinositol, sphingolipids such as
sphingomyelin; glycolipids such as ganglioside GM1 and GM2;
glucolipids; sulfatides; glycosphingolipids; phosphatidic acid;
palmitic acid; stearic acid; arachidonic acid; oleic acid; lipids
bearing polymers such as polyethyleneglycol, chitin, hyaluronic
acid or polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-,
oligo- or polysaccharides; cholesterol, cholesterol sulfate and
cholesterol hemisuccinate; tocopherol hemisuccinate, lipids with
ether and ester-linked fatty acids, polymerized lipids (a wide
variety of which are known in the art), diacetyl phosphate,
stearylamine, cardiolipin, phospholipids with short chain fatty
acids of about 6 to about 8 carbons in length, synthetic
phospholipids with asymmetric acyl chains (e.g., with one acyl
chain of about 6 carbons and another acyl chain of about 12
carbons),
6-(5-cholesten-3b-yloxy)-1-thio-.beta.-D-galactopyranoside,
digalactosyldiglyceride,
6-(5-cholesten-3.beta.-yloxy)hexyl-6-amino-6-deo-
xy-1-thio-.beta.-D-galactopyranoside,
6-(5-cholesten-3.beta.-yloxy)hexyl-6-
-amino-6-deoxyl-1-thio-.alpha.-D-mannopyranoside,
12-(((7'-diethylamino-co-
umarin-3-yl)carbonyl)methylamino)-octadecanoic acid;
N-[12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methyl-amino)
octadecanoyl]-2-aminopalmitic acid;
(cholesteryl).sub.4'-trimethyl-ammoni- o)butanoate;
N-succinyldioleoylphosphatidylethanolamine;
1,2-dioleoyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinyl-glycerol;
1,3-dipalmitoyl-2-succinylglycerol;
1-hexadecyl-2-palmitoyl-glycerophosph- oethanolamine and
palmitoylhomocysteine, and/or combinations thereof. Vesicles or
other structures may be formed of the lipids, either as monolayers,
bilayers, or multilayers and may or may not have a further
coating.
[0065] The preferred lipid carrier may be in the form of a
monolayer or bilayer, and the mono- or bilayer may be used to form
one or more mono- or bilayers. In the case of more than one mono-
or bilayer, the mono- or bilayers may be concentric. The carrier
may form a unilamellar vesicle (comprised of one monolayer or
bilayer), an oligolamellar vesicle (comprised of about two or about
three monolayers or bilayers) or a multilamellar vesicle (comprised
of more than about three monolayers or bilayers). The walls or
membranes of a vesicle may be substantially solid (uniform), or
they may be porous or semi-porous.
[0066] Lipids bearing hydrophilic polymers such as
polyethyleneglycol (PEG), including and not limited to PEG 2,000
MW, 5,000 MW, and PEG 8,000 MW, are particularly useful for
improving the stability and size distribution of organic
halide-containing composition. Dipalmitoylphosphatidylcholine
(DPPC) may be useful in the present invention at about 70% to about
90%, dipalmitoylphosphatidylethanolamine-- polyethylene glycol 5000
(DPPE-PEG 5000) may be useful at about 0% to about 20% and
dipalmitoylphosphatidic acid (DPPA) may be useful at about 0% to
about 20% (all percentages being in mole percent molecular weight).
A preferred product which is highly useful as a carrier in the
present invention contains about 82 mole percent DPPC, about 8 mole
percent DPPE-PEG 5,000 MW and about 10 mole percent DPPA. Various
different mole ratios of PEGylated lipid are also useful.
[0067] Additionally lipid moieties capable of polymerization are
embraced in the invention as coatings for the vesicles. Examples of
these include, but are not limited to, alkenyl and alkynyl
moieties, such as oleyl and linoleyl groups, diacetylene, acryloyl
and methacryloyl groups with or without polar groups to enhance
water solubility, cyanoacrylate esters optionally carrying
lipophilic esterifying groups or the compounds illustrated as A and
B, below. A number of such compounds are described, for example, in
Klaveness et al., U.S. Pat. No. 5,536,490. The disclosures of
Klaveness et al., U.S. Pat. No. 5,536,490, are hereby incorporated
herein by reference in their entirety.
[0068] Fluorinated or perfluorinated lipids may also be used in
this invention, either as the organic halide component or as an
additional carrier material. Examples of suitable fluorinated
lipids include but are not limited to compounds of the formula
C.sub.nF.sub.2n+1(CH.sub.2).sub.mC(O)OOP(OO.sup.-)O(CH.sub.2).sub.wN.sup.+-
(CH.sub.3).sub.3C.sub.nF.sub.2n+1(CH.sub.2).sub.mC(O)O
[0069] wherein: m is 0 to about 18, n is 1 to about 12; and w is 1
to about 8. Examples of and methods for the synthesis of these, as
well as other fluorinated lipids useful in the present invention,
are set forth in U.S. application Ser. No. 08/465,868, filed Jun.
6, 1995, Reiss et al. U.S. Pat. No. 5,344,930, Frezard, F., et al.,
Biochem Biophys Acta 1994, 1192:61-70, and Frezard, F., et al.,
Art. Cells Blood Subs and Immob Biotech. 1994, 22:1403-1408, the
disclosures of each of which are incorporated herein by reference
in their entirety. One specific example of a difluoroacyl
glycerylphosphatidylcholine, nonafluorinated diacyl
glycerylphosphatidylcholine, is represented by compound A, below.
Those skilled in the art will appreciate that analogous fluorinated
derivatives of other common phospholipids (diacylphosphatidyl
serine, diacylphosphatidyl ethanolamine, diacylphosphatidyl
glycerol, diacylphosphatidyl glycerol, etc.) as well as fluorinated
derivatives of fatty acyl esters and free fatty acids may also
function in accordance with the scope of the invention.
[0070] Additionally lipid based and fluorinated (including
perfluorinated) surfactants such as may be used as carriers in the
present invention.
[0071] A wide variety of such fluorinated compounds may be
employed, including, for example, the class of compounds which are
commercially available as ZONYL.RTM. fluorosurfactants (the DuPont
Company, Wilmington, Del.), including the ZONYL.RTM. phosphate
salts and ZONYL.RTM. sulfate salts, which are fluorosurfactants
having terminal phosphate or sulfate groups. Representative
compounds are disclosed, for example, in U.S. Pat. No. 5,276,145,
the disclosures of which are hereby incorporated herein by
reference in their entirety. Suitable ZONYL.RTM. surfactants also
include, for example, ZONYL.RTM. surfactants identified as Telomer
B, including Telomer B surfactants which are pegylated (i.e., have
at least one polyethylene glycol group attached thereto), also
known as PEG-Telomer B, available from the DuPont Company. Most
preferred are such pegylated fluorosurfactants.
[0072] Suitable polymerizable and/or fluorinated compounds include
123
[0073] In formula A, above, preferably x is an integer from about 8
to about 18, and n is 2.times.. Most preferably x is 12 and n is
24.
[0074] Cationic lipids and other derivatized lipids and lipid
mixtures also may be useful as carriers in the present invention.
Suitable cationic lipids include dimyristyl
oxypropyl-3-dimethylhydroxy ethylammonium bromide (DMRIE), dilauryl
oxypropyl-3-dimethylhydroxy ethylammonium bromide (DLRIE),
N-[1-(2,3-dioleoyloxyl)propal]-n,n,n-trime- thylammonium sulfate
(DOTAP), dioleoylphosphatidylethanolamine (DOPE),
dipalmitoylethylphosphatidylcholine (DPEPC),
dioleoylphosphatidylcholine (DOPC), polylysine, lipopolylysine,
didoceyl methylammonium bromide (DDAB),
2,3-dioleoyloxy-N-[2-(sperninecarboxamidoethyl]-N,N-di-methyl-1-p-
ropanaminium trifluoroacetate (DOSPA), cetyltrimethylammonium
bromide (CTAB), lysyl-PE,
3,.beta.-[N,(N',N'-dimethylaminoethane)-carbamoyl]chole- sterol
(DC-Cholesterol, also known as DC-Chol), (-alanyl cholesterol,
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA), dipalmitoylphosphatidylethanolamine-5-carboxyspermylamide
(DPPES), dicaproylphosphatidylethanolamine (DCPE),
4-dimethylaminopyridine (DMAP), dimyristoylphosphatidylethanolamine
(DMPE), dioleoylethylphosphocholine (DOEPC), dioctadecylamidoglycyl
spermidine (DOGS),
N-[1-(2,3-dioleoyloxy)propyl]-N-[1-(2-hydroxyethyl)]-N,N-dimethylammonium
iodide (DOHME), Lipofectin (DOTMA+DOPE, Life Technologies, Inc.,
Gaithersburg, Md.), Lipofectamine (DOSPA+DOPE, Life Technologies,
Inc., Gaithersburg, Md.), Transfectace (Life Technologies, Inc.,
Gaithersburg, Md.), Transfectam (Promega Ltd., Madison, Wis.),
Cytofectin (Life Technologies Inc., Gaithersburg, Md.). Other
representative cationic lipids include but are not limited to
phosphatidylethanolamine, phospatidylcholine,
glycero-3-ethylphosphatidylcholine and fatty acyl esters thereof,
di- and trimethyl ammonium propane, di- and tri-ethylammonium
propane and fatty acyl esters thereof. A preferred derivative from
this group is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimeth-
ylammonium chloride (DOTMA). Additionally, a wide array of
synthetic cationic lipids function as compounds useful in the
invention. These include common natural lipids derivatized to
contain one or more basic functional groups. Examples of lipids
which may be so modified include but are not limited to
dimethyldioctadecylammonium bromide, sphingolipids, sphingomyelin,
lysolipids, glycolipids such as ganglioside GM1, sulfatides,
glycosphingolipids, cholesterol and cholesterol esters and salts,
N-succinyldioleoylphosphatidylethanolamine,
1,2,-dioleoyl-sn-glycerol, 1,3-dipalmitoyl-2-succinylglycerol,
1,2-dipalmitoyl-sn-3-succinylglycerol,
1-hexadecyl-2-palmitoylglycerophos- phatidyl-ethanolamine and
palmitoylhomocystiene.
[0075] Other synthesized cationic lipids that are useful in the
present invention are those disclosed in pending U.S. patent
application Ser. No. 08/391,938, filed Feb. 2, 1995, and include,
for example, N,N'-Bis
(dodecyaminocarbonylmethylene)-N,N'bis((-N,N,N-trimethylammoniumethyl-ami-
nocarbonylmethylene)ethylenediamine tetraiodide; N,N"-Bis
(hexadecylaminocarbonylmethylene)-N,N',N"-tris((-N,N,N-trimethylammonium--
ethylaminocarbonylmethylenedi-ethylenetriamine hexaiodide; N,N'-Bis
(dodecylaminocarbonylmethylene)-N,N'-bis((-N,N,N-trimethylammoniumethylam-
ino-carbonylmethylene)cyclohexylene-1,4-diamine tetraiodide;
1,1,7,7-tetra-((-N,N,N,N-tetramethylammoniumethylaminocarbonylmethylene)--
3-hexadecylaminocarbonyl-methylene-1,3,7-triaazaheptane
heptaiodide; and
N,N,N'N'-tetra((-N,N,N-trimethylammoniumethylaminocarbonylmethylene)-N'-(-
1,2-di
oleoylglycero-3-phosphoethanolaminocarbonylmethylene)diethylenetria-
mine tetraiodide. Those of skill in the art will recognize that
countless other natural and synthetic variants carrying positive
charged moieties will also function in the invention.
[0076] Also useful as carriers in the present invention are a wide
variety of surfactants (i.e., surface-active agents), including
polyoxyalkylene fatty acid esters (such as polyoxyethylene fatty
acid esters), polyoxyalkylene fatty alcohols (such as
polyoxyethylene fatty alcohols), polyoxyalkylene fatty alcohol
ethers (such as polyoxyethylene fatty alcohol ethers),
polyoxyalkylene sorbitan fatty esters (such as, for example, the
class of compounds referred to as TWEEN.TM., commercially available
from ICI Americas, Inc., Wilmington, Del.), including
poly(oxyethylene)poly(oxypropylene) copolymers (such as Pluronics),
polysorbates (such as Tween20, Tween40, and Tween80),
polyoxyethylene alcohols (such as Brij), and plasmalogens, the term
applied to a number of a group of phospholipids present in
platelets that liberate higher fatty aldehydes, e.g. palmital, on
hydrolysis and may be related to the specialized function of
platelets in blood coagulation and plasmalogens are also present in
cell membranes of muscle and the myelin sheath of nerve fibers.
[0077] In the preferred embodiment of the invention the organic
halide is incorporated into the core of a vesicle which vesicle
carrier is also used to complex the compound to be delivered, such
as DNA.
[0078] A wide variety of oils may be preferably employed as
carriers in the present invention including, but not limited to,
safflower, soybean, and peanut oil. The composition may take the
form of an oil in water emulsion if desired.
[0079] The most preferred carrier is a cationic lipid (including a
cationic liposome), particularly as employed in an aqueous milieu.
A preferred cationic lipid is DPEPC in admixture with the neutral
fusogenic lipid dioleoylphosphatidylethanolamine (DOPE). A
preferred ratio of lipid to organic halide is 5:1 w/w. A preferred
embodiment is to formulate the lipid or polymer as an organic
halide-filled microsphere, such as a microsphere formed with the
lipids dipalmitoylphosphatidylcholine (DPPC),
dipalmitoylphosphatidylethanolamine coupled to polyethylene glycol
5000 (DPPE-PEG5000), and dipaInitoylphosphatidic acid (DPPA).
DPPC:DPPE-PEG5000:DPPA may be combined in a ratio of about
82%:8%:10% (mole %) or 83%:8%:5%. DPPE-PEG5000 is comprised of DPPE
and PEG5000 in a ratio of about 20%:80% (weight %). PEG5000 refers
to PEG having an average molecular weight of about 5000.
[0080] Proteins (including peptides) useful as carriers in
accordance with the present invention include molecules comprising,
and preferably consisting essentially of, .alpha.-amino acids in
peptide linkages. A wide variety of proteins may be employed as
carriers in the present invention, including natural, synthetic, or
semi-synthetic proteins. Included within the term "protein" are
globular proteins, such as albumins, globulins and histones, and
fibrous proteins such as collagens, elastins and keratins. Also
included are "compound proteins", wherein a protein molecule is
united with a nonprotein molecule, such as nucleproteins,
mucoproteins, lipoproteins, and metalloproteins. Preferable
proteinaceous macromolecules include for example, albumin,
collagen, polyarginine, polylysine, polyhistidine, .gamma.-globulin
and .beta.-globulin, with albumin, polyarginine, polylysine, and
polyhistidine being more preferred. Fluorinated peptides and
synthetic pseudopeptides are also useful as carriers. Fluorinated
peptides useful in the present invention include those described in
Lohrmann, U.S. Pat. No. 5,562,892, the disclosures of which are
hereby incorporated herein by reference in their entirety. Cationic
peptides may also be usefully employed as carriers in the present
invention. Various peptides suitable for use in the present
invention will be apparent to one skilled in the art based on the
present disclosure.
[0081] The methods of the present invention may also involve
vesicles or other organized stable form formulated from proteins,
peptides and/or derivatives thereof. Vesicles which are formulated
from proteins and which would be suitable for use in the methods of
the present invention are described, for example, in Feinstein,
U.S. Pat. Nos. 4,572,203, 4,718,433, and 4,774,958, and Cerny et
al., U.S. Pat. No. 4,957,656, all of the disclosures of each of
which are hereby incorporated by reference in their entirety. Other
protein-based vesicles, in addition to those described in the
aforementioned patents, would be apparent to one of ordinary skill
in the art, once armed with the present disclosure.
[0082] Included among the methods described in the aforementioned
patents for the preparation of protein-based vesicles are methods
which involve sonicating a solution of a protein. In preferred
form, the starting material may be an aqueous solution of a
heat-denaturable, water-soluble biocompatible protein. The
encapsulating protein is preferably heat-sensitive so that it can
be partially insolubilized by heating during sonication. Suitable
heat-sensitive proteins include, for example, albumin, hemoglobin,
collagen, and the like. Preferably, the protein is a human protein,
with human serum albumin (HSA) being more preferred. HSA is
available commercially as a sterile 5% aqueous solution, which is
suitable for use in the preparation of protein-based vesicles. Of
course, as would be apparent to one of ordinary skill in the art,
other concentrations of albumin, as well as other proteins which
are heat-denaturable, can be used to prepare the vesicles.
Generally speaking, the concentration of HSA can vary and may range
from about 0.1 to about 25% by weight, and all combinations and
subcombinations of ranges therein. It may be preferable, in
connection with certain methods for the preparation of
protein-based vesicles, to utilize the protein in the form of a
dilute aqueous solution. For albumin, it may be preferred to
utilize an aqueous solution containing from about 0.5 to about 7.5%
by weight albumin, with concentrations of less than about 5% by
weight being preferred, for example, from about 0.5 to about 3% by
weight.
[0083] The protein-based vesicles may be prepared using equipment
which is commercially available. For example, in connection with a
feed preparation operation as disclosed, for example, in Cerny, et
al., U.S. Pat. No. 4,957,656, stainless steel tanks which are
commercially available from Walker Stainless Equipment Co. (New
Lisbon, Wis.), and process filters which are commercially available
from Millipore (Bedford, Mass.), may be utilized.
[0084] The sonication operation may utilize both a heat exchanger
and a flow through sonicating vessel, in series. Heat exchanger
equipment of this type may be obtained from ITT Standard (Buffalo,
N.Y.). The heat exchanger maintains operating temperature for the
sonication process, with temperature controls ranging from about
65.degree. C. to about 80.degree. C., depending on the makeup of
the media. The vibration frequency of the sonication equipment may
vary over a wide range, for example, from about 5 to about 40
kilohertz (kHz), with a majority of the commercially available
sonicators operating at about 10 or 20 kHz. Suitable sonicating
equipment include, for example, a Sonics & Materials
Vibra-Cell, equipped with a flat-tipped sonicator horn,
commercially available from Sonics & Materials, Inc. (Danbury,
Conn.). The power applied to the sonicator horn can be varied over
power settings scaled from 1 to 10 by the manufacturer, as with
Sonics & Materials Vibra-Cell Model VL1500. An intermediate
power setting, for example, from 5 to 9, can be used. It is
preferred that the vibrational frequency and the power supplied be
sufficient to produce cavitation in the liquid being sonicated.
Feed flow rates may range from about 50 mL/min to about 1000
mL/min, and all combinations and subcombinations of ranges therein.
Residence times in the sonication vessel can range from about 1
second to about 4 minutes, and gaseous fluid addition rates may
range from about 10 cubic centimeters (cc) per minute to about 100
cc/min, or 5% to 25% of the feed flow rate, and all combinations
and subcombinations of ranges therein.
[0085] It may be preferable to carry out the sonication in such a
manner to produce foaming, and especially intense foaming, of the
solution. Generally speaking, intense foaming and aerosolating are
important for obtaining a contrast agent having enhanced
concentration and stability. To promote foaming, the power input to
the sonicator horn may be increased, and the process may be
operated under mild pressure, for example, about 1 to about 5 psi.
Foaming may be easily detected by the cloudy appearance of the
solution, and by the foam produced.
[0086] Such sonication methods may also be employed to prepare
lipid-based or other types of carriers as will be apparent to the
skilled artisan.
[0087] Suitable methods for the preparation of protein-based
vesicles may involve physically or chemically altering the protein
or protein derivative in aqueous solution to denature or fix the
material. For example, protein-based vesicles may be prepared from
a 5% aqueous solution of HSA by heating after formation or during
formation of the contrast agent via sonication. Chemical alteration
may involve chemically denaturing or fixing by binding the protein
with a difunctional aldehyde, such as glutaraldehyde. For example,
the vesicles may be reacted with 0.25 grams of 50% aqueous
glutaraldehyde per gram of protein at pH 4.5 for 6 hours. The
unreacted glutaraldehyde may then be washed away from the
protein.
[0088] The carriers may also be formulated with polymers, natural,
synthetic, or semisynthetic. A wide variety of polymers may be
utilized as carriers in the present invention, including synthetic
polymers including polyethylenes (such as, for example,
polyethylene glycol), polyoxyethylenes (such as, for example,
polyoxyethylene glycol), polypropylenes (such as, for example,
polypropylene glycol), pluronic acids and alcohols, polyvinyls
(such as, for example, polyvinyl alcohol), and
polyvinylpyrrolidone. Exemplary natural polymers suitable for use
in the present invention include polysaccharides. Polysaccharides
include, for example, arabinans, fructans, fucans, galactans,
galacturonans, glucans, mannans, xylans (such as, for example,
inulin), levan, fucoidan, carrageenan, galactocarolose, pectin
(including high methoxy pectin and low methoxy pectin; with low
methoxy pectin denoting pectin in which less than 40% of the
carboxylic acid groups are esterified and/or amidated, and high
methoxy pectin denoting pectin in which 40% or more of the
carboxylic acid groups are esterified and/or amidated), pectic
acid, amylose, pullulan, glycogen, amylopectin, cellulose,
carboxylmethylcellulose, hydroxypropyl methylcellulose, dextran,
pustulan, chitin, agarose, keratan, chondroitin, dermatan,
hyaluronic acid and alginic acid, and various other homopolymers or
heteropolymers such as those containing one or more of the
following aldoses, ketoses, acids or amines: erythrose, threose,
ribose, arabinose, xylose, lyxose, allose, altrose, glucose,
mannose, gulose, idose, galactose, talose, erythrulose, ribulose,
xylulose, psicose, fructose, sorbose, tagatose, glucuronic acid,
gluconic acid, glucaric acid, galacturonic acid, mannuronic acid,
guluronic acid, glucosamine, galactosamine and neuraminic acid. It
is recognized that some polymers may be prepared by chemically
modifying naturally occurring polymers. Such chemically modified
natural polymers also referred to as semisynthetic polymers. The
polymers employed may also comprise fluorinated polymers, including
those described in Lohrmann, U.S. Pat. No. 5,562,892, the
disclosures of which are hereby incorporated herein by reference in
their entirety. Furthermore, the polymers may be in the form of
vesicles, such as for example, those described in Unger, U.S. Pat.
No. 5,205,290, the disclosures of which are hereby incorporated
herein by reference in their entirety. As used herein, the term
"polymer" denotes molecules formed from the chemical union of two
or more repeating units, and include dimers, trimers, and
oligomers. In preferred form, the term "polymer" refers to
molecules which comprise 10 or more repeating units.
[0089] Metal ions may also be employed as carriers in the present
invention. Suitable metal ions include calcium ions, magnesium
ions, zinc ions, and the like, as well as a wide variety of
inorganic compounds. Other suitable metal ions as well as other
suitable inorganic compounds will be readily apparent to those
skilled in the art once armed with the present invention.
[0090] Other useful agents that may be employed in the carrier of
the present invention include osmotic agents, anti-microbials,
viscosity raising agents, suspending agents, humectants and
anti-humectants, depending upon the particular formulation
desired.
[0091] One or more emulsifying or stabilizing agents may also be
employed as or be included in the carrier. These agents help to
maintain the size of any discrete units (e.g., liquid droplets,
particles, gas bubbles, etc.) of the organic halide and/or
compounds to be delivered that may have formed the composition. The
size of these discrete units will generally affect the size of any
resultant gas bubbles that may form from any gaseous precursors.
The emulsifying and stabilizing agents also may be used to
generally coat or stabilize the organic halides, compounds to be
delivered, etc. Stabilization is desirable to maximize the
intracellular delivery effect. Although stabilization is preferred,
this is not an absolute requirement. Because any gas resulting from
organic halide gaseous precursors is more stable than air, they may
still be designed to provide useful delivery means; for example,
they pass through the pulmonary circulation following peripheral
venous injection, even when not specifically stabilized by one or
more coating or emulsifying agents. One or more coating or
stabilizing agents is preferred however, as are flexible
stabilizing materials. Also, it should be noted that compositions
stabilized by polysaccharides, gangliosides, and polymers are
generally more effective than those stabilized by albumin and other
proteins. Also, liposomes prepared using aliphatic compounds are
preferred, since microspheres stabilized with these compounds are
much more flexible and stable to pressure changes.
[0092] The carrier of the invention may also comprise a wide
variety of viscosity modifiers, including and not limited to
carbohydrates and their phosphorylated and sulfonated derivatives;
polyethers, preferably with molecular weight ranges between 400 and
8000; di- and trihydroxy alkanes and their polymers, preferably
with molecular weight ranges between 800 and 8000. Glycerol
propylene glycol, polyethylene glycol, polyvinyl pyrrolidone, and
polyvinyl alcohol may also be useful as carriers or stabilizers in
the present invention. Particles which are porous or semi-solid
such as hydroxyapatite, metal oxides and coprecipitates of gels,
e.g., hyaluronic acid with calcium may be used and may formulate a
center or nidus to stabilize compositions of the invention.
[0093] Emulsifying and/or solubilizing agents may also be used in a
carrier, particularly in conjunction with lipids or liposomes. Such
agents include and are not limited to, acacia, cholesterol,
diethanolamine, glyceryl monostearate, lanolin alcohols, lecithin,
mono- and di-glycerides, mono-ethanolamine, oleic acid, oleyl
alcohol, poloxamer, polyoxyethylene 50 stearate, polyoxyl 35 castor
oil, polyoxyl 10 oleyl ether, polyoxyl 20 cetostearyl ether,
polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate
60, polysorbate 80, propylene glycol diacetate, propylene glycol
monostearate, sodium lauryl sulfate, sodium stearate, sorbitan,
sorbitan mono-laurate, sorbitan mono-oleate, sorbitan
mono-palmitate, sorbitan monostearate, stearic acid, trolamine, and
emulsifying wax. All lipids with perfluoro fatty acids as a
component of the lipid in lieu of the saturated or unsaturated
hydrocarbon fatty acids found in lipids of plant or animal origin
may be used. Suspending and/or viscosity-increasing agents that may
be particularly useful with lipid or liposome solutions include but
are not limited to, acacia, agar, alginic acid, aluminum
mono-stearate, bentonite, magma, carbomer 934P,
carboxymethylcellulose, calcium and sodium and sodium 12, glycerol,
carrageenan, cellulose, dextrin, gelatin, guar gum, hydroxyethyl
cellulose, hydroxypropyl methylcellulose, magnesium aluminum
silicate, methylcellulose, pectin, polyethylene oxide, polyvinyl
alcohol, povidone, propylene glycol, alginate, silicon dioxide,
sodium alginate, tragacanth, and xanthum gum. A preferred product
of the present invention incorporates lipid as a mixed solvent
system in a ratio of 8:1:1 or 9:1:1 normal
saline:glycerol:propylene glycol.
[0094] The amount of carrier material employed in connection with
the subject invention may vary, as one skilled in the art will
recognize upon being placed in possession of the subject
disclosure, and may be dependent on such factors as the particular
carrier used, the type and nature of the compound to be delivered,
the age, weight, cells or patient (animal) to be treated, the
particular diagnostic, therapeutic or other application intended
(including the disease state, if any, to be treated), and the
organic halide (if any) used. Generally, smaller amounts of carrier
are employed, and increased until the desired delivery result is
obtained. Representative amounts are set forth in the examples
herein. Of course, higher or lower amounts may be employed, as will
be recognized by the skilled artisan.
[0095] A wide variety of different methods may be used to mix the
organic halide, compound to be delivered, and/or carrier, and
incorporate the compound to be delivered with or into any organic
halide and/or carrier. Methods include shaking by hand, vortexing,
mechanical shaking (e.g. with an Espe CapMix, Espe Medizin-Dental
GMBH, Seefeld, Germany), extruder (e.g. with a Lipex Biomembranes
Extruder Device, Vancouver, B.C., Canada), microemulsification
(e.g. with a Microfluidizer, Microfluidics Corp., Newton, Mass.),
mixing with static in line mixers (Cole-Parmer Instrument Co.,
Vernon Hills, Ill.), spray drying (e.g. with a Bucchi spray dryer,
Brinkmann Ind., Inc., Westbury, Mass.), mechanical stirring/mixing
(e.g. with a Silverson Mixer, Silverson Machines, Ltd., Waterside
Chesham Bucks, England) and sonication. In general it is desirable
to mix the carrier (e.g. lipids such as DPEPC and DOPE) together
with the organic halide prior to adding the compound to be
delivered (e.g., DNA). After adding the DNA, a carrier and organic
halide association will form with the DNA. If desired, additional
mixing may then be performed by one of the above techniques. In
some other situations, e.g. calcium precipitation, the DNA, organic
halide, and cations may be added together with one or more
stabilizing agents to form the precipitates of DNA/carrier/organic
halide in a single step process. Again, one of a variety of mixing
techniques as described above may be employed to decrease the size
of the resultant particles.
[0096] The carriers may be combined with the compound to be
delivered and the organic halide in varying amounts and
percentages, as will be understood by those skilled in the art once
armed with the present disclosure. Typically, smaller amounts of
all compositional components are employed, and increased
selectively in increments until the desired delivery effect is
achieved. Generally, when the compound to be delivered is employed
with a carrier, the ratio of organic halide and any carrier to the
compound to be delivered may be from about 6 to about 1, to about 1
to about 6, and variations therebetween. Preferably, the carrier to
compound to be delivered ratio is about 6 to about 1.
Representative ratios are provided by the examples herein. Of
course, other ratios can be suitably employed over a wide variety
of ranges as desired, as will be recognized by the skilled artisan,
and all such ratios are within the scope of the present
invention.
[0097] The resulting composition may be stored as a lyophilized, or
freeze dried, material for inhalation or hydration prior to use or
as a preformed suspension. Cryopreservatives known to skilled
artisans once armed with the present disclosure may be used in the
lyophilized form of the composition. To prevent agglutination or
fusion of vesicles as a result of lyophilization, it may be useful
to include additives which prevent such fusion or agglutination
from occurring. Additives which may be useful include sorbitol,
mannitol, sodium chloride, glucose, trehalose, polyvinylpyrrolidone
and poly(ethylene glycol) (PEG), for example, PEG polymers having a
molecular weight of from about 400 to about 10,000, with PEG
polymers having molecular weights of about 1000, 3000 (such as
PEG3350) and 5000 being preferred. These and other additives are
described in the literature, such as in the U.S. Pharmacopeia, USP
XXII, NF XVII, The United States Pharmacopeia, The National
Formulary, United States Pharmacopeial Convention Inc., 12601
Twinbrook Parkway, Rockville, Md. 20852, the disclosures of which
are hereby incorporated herein by reference in their entirety.
Lyophilized preparations generally have the advantage of greater
shelf life. As noted above, if desired, the lyophilized composition
may be (and preferably is) rehydrated prior to use.
[0098] The route of administration varies depending upon the
intended application. For cell culture applications, the
composition is typically contacted with the cells by, for example,
adding it to the cell culture media or applying it directly to the
cells. Advantages of this invention for transfection in cell
culture media include high activity in serum containing media and a
single step transfection process with higher efficiency
transfection than in other more complicated systems. Indeed, the
present invention makes it possible to obtain gene expression in
cells in which transfection was otherwise impossible or extremely
difficult. For in vivo administration the composition may simply be
injected, such as intravenously, intravascularly,
intralymphatically, parenterally, subcutaneously, intramuscularly,
intranasally, intrarectally, intraperitoneally, interstitially,
into the airways via nebulizer, hyperbarically, orally, topically,
or intratumorly, or otherwise administered.
[0099] One or more targeting ligands may be incorporated into the
carrier to facilitate uptake by selected cells. Targeting ligands
include, for example, peptides, antibodies, antibody fragments,
glycoproteins, carbohydrates, etc. Preferably, the targeting ligand
is covalently attached to the carrier, e.g., to a lipid. Preferably
the targeting ligand is attached to a linker which is attached to
the surface of the carrier. Preferred linkers are polymers, for
example, bifunctional PEG having a molecular weight of about 1,000
to about 10,000, most preferably 5,000. Generally, the targeting
ligand is incorporated into the carrier from about 0.1 mole % to
about 25 mole %, preferably about 1 mole % to about 10 mole %.
[0100] In this regard, the composition may be targeted to coated
pits of selected cells and taken up into endosomes via a process of
receptor mediated endocytosis. If desired ultrasound energy may be
applied to the target tissue to facilitate gene expression. For
inhalation the composition may be inhaled via a nebulizer or via an
inhaler. Also, oral or rectal routes may be utilized to administer
these composition. Transcutaneous application may be accomplished
by the use of penetration enhancing agents with or without the
application of sonophoresis (e.g. low frequency sound in range of
10 to 100 Khz) or iontophoresis. Also interstitial (e.g.
intratumoral) and subcutaneous injection may be performed to
administer the composition.
[0101] Also the invention may be practiced with gene gun techniques
or electroporation, or in combination with other transfection
techniques known in the art. In either case, ultrasound may be
applied to the cells before, after, and/or simultaneously with the
gene gun or electroporation procedure. The electric fields of
electroporation may also be pulsed in tandem with the ultrasound
energy to further increase the efficacy of transfection.
[0102] The compounds and compositions may, in accordance with the
present invention, be administered alone, or together with
ultrasound. If ultrasound is employed, it is administered at a
frequency and energy level sufficient to assist in inducing the
uptake of the compound to the cell. Where organic halide gaseous
precursors are employed, the ultrasound may be applied at a
frequency and energy level sufficient to convert the organic halide
gaseous precursor to a gas. For example, the present invention of
administering compounds to cells includes administering a
nucleotide sequence (or other compound of interest to be delivered)
to a cell and applying ultrasound to the cell for a time effective
to induce the uptake of the nucleotide sequence (or other
compound). Enhanced delivery of the compound (and expression of the
nucleotide sequence, in the case of nucleotide sequence being
administered) results. Ultrasound is carried out at a frequency,
energy level, and duty cycle for a therapeutically effective time
in which to induce delivery of the nucleotide sequence. Suitable
frequencies, energy levels and duty cycles are disclosed herein,
and other ranges will be readily apparent to one skilled in the art
once armed with the present disclosure.
[0103] The methods of the present invention permit the delivery of
sequences coding for the gene expression of a variety of proteins,
and antisense sequences which block gene expression of a variety of
proteins. As a result, a number of diseases may be treated with the
transfection methods of the present invention. In addition, the
methods of transfection of the present invention may be practiced
in vivo, ex vivo, and in vitro.
[0104] Administration of a nucleotide sequence by a microsphere
utilizes a nucleotide sequence attached to a microsphere in various
positions relative to the microsphere as set forth above. While not
intending to be bound by any particular theory or theories of
operation, the microsphere approach is believed to rely on the
fusion of the nucleotide sequence containing microsphere with the
plasma membrane of the host cell. The nucleotide sequence
subsequently traverses the cytoplasm and enters the nucleus. The
use of a microsphere results in little toxic effects to the host
cell, tissue, and the patient (in the case of in vivo use).
[0105] Intracellular delivery and transfection in accordance with
the methods of the present invention may be performed in vivo, ex
vivo, and in vitro. Included within the above three methods is
human gene therapy including wherein cells to be treated are
excised from a patient. The cells are treated with an appropriate
nucleotide sequence and transfection with ultrasound is carried out
in cell culture. The transfected cells are analyzed for gene
expression of the appropriate protein. The successfully transfected
cells, measured by gene expression, are then returned to the body
of the patient. Transfection with ultrasound thereby results in the
treatment of diseases by gene therapy. Diseases to be treated with
the methods of the present invention include and are not limited to
acquired immune deficiency syndrome, autoimmune diseases, chronic
viral infection, hemophilia, muscular dystrophy, cystic fibrosis,
diabetes, atherosclerosis, liver cancer, lung cancer, prostate
cancer, ovarian cancer, brain cancer, kidney cancer, melanoma,
neuroblastoma, and breast cancer. Many other diseases may, of
course, be treated with the methods of the present invention, as
will be apparent to the skilled artisan upon reading the present
disclosure, and the treatment of all such diseases are to be
considered within the scope of the present methods.
[0106] The use of heat, for example in the form of ultrasound,
lithotripsy shock waves, and increased body temperature, in the
present invention is useful in aiding the delivery of compounds,
such as, for example, nucleotide sequences, into cells for
therapeutic purposes. The introduction of a nucleotide sequence
into the cell is the first step in incorporating the sequence into
the genome. Such transfection techniques may be useful in
conjunction with testing the range of ultrasound frequency useful
in inducing the delivery of compounds, including nucleotide
sequences, to cells.
[0107] Each of the methods of the present invention include
administering all or part of a sense or an antisense sequence for
insulin (Giddings and Carnaghi, Mol. Endocrinol. 1990 4:1363-1369),
Bcl 2 (Tsujimoto, Y., et al., PNAS, USA 1986, 83:5214-5218), human
leukocyte antigen (Trucco, G., et al., Diabetes 1989, 38:1617-1622,
thymidine kinase (Axel, R., et al., J. Supramol. Struct. 1979, 8
(Suppl. 3):41), HLA-B7, Factor VIII (Higuchi, M., et al., Genomics
1990, 6:65-71, ras/p53 (Arai, N., et al., Mol Cell Biol 1986,
6:3232-3239, Mitsudomi, T., et al., Chest 1993, 104:362-365), high
density lipoprotein (hdl), leutinizing hormone releasing hormone
(Maier, C. C., et al., Cell Mol Neurobiol 1992, 12:447454) and
leutinizing hormone releasing hormone antagonist, antitumoral
agents such as and not limited to insulin-like growth factor-1
(IGF-1, Barnes, M., et al., Obstetrics and Gynecology 1997,
89:145-155), anti-IGF-1 (human IGF-1 gene fragment from published
patent application GB2241703 GenBank accession number A29119),
anti-k-ras (dog spleen mRNA 212 nucleotides GenBank accession
number S42999), anti-c-fos (Rattus norvegicus Sprague Dawley c-fos
gene, 5' flanking region GenBank accession number U02631), bcr-abl
(Barnes, M., et al., Obstetrics and Gynecology 1997, 89:145-155),
c-myc (mouse c-myc gene, exons 1 and 2 GenBank accession number
L00038, J00373, and J00374), c-myc promoter (Barnes, M., et al.,
Obstetrics and Gynecology 1997, 89:145-155), erbB-2 promoter
(Barnes, M., et al., Obstetrics and Gynecology 1997, 89:145-155),
erbB2 promoter-cytosine deaminase (human c-erb B2/neu protein gene,
partial cDNA (cds) GenBank accession number M95667), and antivirals
such as and not limited to anti-human papilloma virus (HPV),
anti-human immunodeficiency virus (HIV) such as HIVenv+rev (HIV
type 1, isolate BTSPR, env gene, C2V3 region, partial cds GenBank
accession number U53195), tar/Td-rev (HIV type 1 rev-1 gene, 5' end
GenBank accession number M38031, synthetic HIV1 TAR, 5' end GenBank
accession number M27943), ribozyme, zeta-chimpanzee receptor, and
the like, and all or part of a sequence encoding cytokines such as
and not limited to interleukin 2 (IL-2) (human brain MRNA 418
nucleotides GenBank accession number S77835), interleukin 4 (Arai,
N., et al., J Immunol 1989, 142:274-282), interleukin 7 (human
gene, exon 1 GenBank accession number M29048), interleukin 12
(mouse 5' flanking region of IL-12 p35 gene GenBank accession
number D63334), interleukin 4 (human IL-4 gene, complete cds
GenBank accession number M23442), interleukin 6 (human gene for
nuclear factor NF-IL-6 GenBank accession number X52560); gp130 (LIF
receptor/IL-6 receptor complex component MRNA 150 nucleotides
GenBank accession number S80479), interleukin 6 receptor,
granulocyte macrophage colony stimulating factor (GM-CSF) (human
GM-CSF gene, 5' flanking/promoter region GenBank accession number
U31279), interferon including interferon gamma (human immune
IFN-.gamma. gene, complete cds GenBank accession number J00219,
M37265, V00536), tumor necrosis factor beta, TNF-.beta., (human 5'
sequence of TNF-.alpha. gene GenBank accession number X59351)),
vascular endothelial growth factor (VEGF), human growth hormone
(hGH, Fidders, J. C., et al., Proc Natl Acad Sci (USA) 1979
76:4294-4298), colony stimulating factor, Factor VIII, Factor IX,
Factor X, and the like. Other sequences useful in the methods of
the present invention include ribozymes including catalytic RNA
which may have a hammerhead secondary structure (Bratty, et al.,
Biochim. Biophys. Acta 1993 1216:345-349 and McKay, D. B., RNA 1996
2:395-403), c-myc, c-myb, tumor suppressor genes such as and not
limited to human tumor antigen p53 (5' end GenBank accession number
M26864), genes offering chemoprotection such as and not limited to
those encoding multidrug resistance protein (MDR) (human mdr1 gene
GenBank accession number X78081), genes for antigen overexpression
such as and not limited to HLA-B7 (beta 2 microglobulin) (mouse MHC
class I HLA-B7 gene, 5' flanking region GenBank accession M35971),
carcinoembryonic antigen (CEA) (human 5' region GenBank accession
number U17131), suicide genes such as and not limited to thymidine
kinase (TK) (human TK gene encoding TK and promoter region GenBank
accession number M13643), Ras, gene complementation genes such as
and not limited to cystic fibrosis transmembrane conductance
regulator (CFTR) (human CFTR gene, exon 1 GenBank accession number
M55106 and M55499), adenosine deaminase (ADA) (human ADA gene,
complete cds GenBank accession number M13792), glucocerebrosidase,
IRAP/TK (human MRNA for IRAP GenBank accession number X53296),
vascular endothelial growth factor (VEGF) (mus musculus VEGF gene,
partial cds and promoter region GenBank accession number U41383),
LDLR (human LDL receptor gene fragment GenBank accession number
M60949), Fanconi Anemia Complementation Group C (FACC) (human FACC
gene, 5' region GenBank accession number X83116), p47-phox (human
P47 LBC oncogene MRNA, complete cds GenBank accession number
U03634), Factor IX (human Factor 1.times. gene, exon 1 GenBank
accession number K02048), .alpha.-1 antitrypsin (human .alpha.-1
antitrypsin gene S variant, complete cds GenBank accession number
K02212), .alpha.-1 iduronidase (human iduronidase gene sequence
GenBank accession number M88001), and iduronate sulfatase (Ids)
(Mus musculus Ids MRNA, complete cds GenBank accession number
L07921), and gene markers such as and not limited to NeoR and LacZ,
(bacteriophage T4 td gene, exon 2, 3' end; ORF2, complete cds and
ORF3, 5' end GenBank accession number M22627, and cloning vector
pZEO (isolate SVLacZ) .beta.-galactosidase (lacZ) gene,
phleomycin/zeocin-binding protein (ShBle) gene, (complete cds
GenBank accession number L36850).
[0108] DNA encoding certain proteins may be used in the treatment
of many different types of diseases. For example, adenosine
deaminase may be provided to treat ADA deficiency; tumor necrosis
factor and/or interleukin-2 may be provided to treat advanced
cancers; HDL receptor may be provided to treat liver disease;
thymidine kinase may be provided to treat ovarian cancer, brain
tumors, or HIV infection; HLA-B7 may be provided to treat malignant
melanoma; interleukin-2 may be provided to treat neuroblastoma,
malignant melanoma, or kidney cancer; interleukin-4 may be provided
to treat cancer; HIV env may be provided to treat HIV infection;
antisense ras/p53 may be provided to treat lung cancer; and Factor
VIII may be provided to treat Hemophilia B. See, for example,
Thompson, L., Science, 1992, 258, 744-746. Nucleotide sequences for
the above-identified proteins are available in the scientific
literature, including GENBANK, and are known to skilled
artisans.
[0109] In addition to a coding sequence or antisense sequence, the
nucleotide sequence administered to cells may have additional
sequences to assist in the expression of the sequence. Suitable
expression vectors, promoters, enhancers, and other expression
control elements are known in the art and may be found in Sambrook
et al., Molecular Cloning: A Laboratory Manual, second edition,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989). Promoters such as and not limited to SV40, RSV, CMV, cd5k,
IL5R.alpha. pgk-1, sr.alpha., TK, and the like are useful in the
present invention. Transcription and/or translation control
elements may be operatively linked to the sequence. For example, in
an upstream position, a promoter may be followed by a translation
initiation signal, comprising a ribosome binding site and an
initiation codon, and in a downstream position may be a
transcription termination signal. The transcription and translation
control elements may be ligated in any functional combination or
order. The transcription and translation control elements used in
any particular embodiment of the invention will be chosen with
reference to the type of cell into which the expression vector will
be introduced, so that an expression system is created. The
selection of promoters, enhancers, and other expression control
elements and the preparation of expression vectors suitable for use
in the present invention will be well within the ambit of one
skilled in the art once armed with the present disclosure. Also,
introduction of the expression vector incorporating a sequence into
a host cell can be performed in a variety of ways known in the
art.
[0110] Mammalian cells may be primed to be more susceptible to
uptake of DNA for gene therapy by the addition of various media,
buffers, and chemicals known to those of skill in the art and set
forth in Sambrook, supra. Administration of nucleotide sequences in
vivo may include, if desired, more than one sequence. For example,
a single carrier may contain more than one sequence or carriers
containing different sequences may be co-administered. In addition,
one sequence may be delivered in a carrier and another naked
sequence coadministered. Additional sequences, such as promoter
sequences, may be delivered together with a sequence for
therapeutic delivery, to increase expression thereof. For example,
a heat shock protein nucleic acid sequence is an example of an
upregulating gene sequence which may be used to increase expression
of a second gene sequence.
[0111] A wide variety of compounds (in addition to genetic
material) may also be delivered to cells in accordance with the
methods of the invention. Such other compounds include various
other bioactive agents. As used herein, "bioactive agent" refers to
any substance which may be used in connection with an application
that is therapeutic or diagnostic in nature, such as, for example,
in methods for diagnosing the presence or absence of a disease in a
patient or in methods for the treatment of disease in a patient. As
used herein, "bioactive agent" refers also to substances which are
capable of exerting a biological effect in vitro, in vivo, and/or
ex vivo. The bioactive agents may be neutral, or positively or
negatively charged, etc., as desired. Examples of suitable
bioactive agents include diagnostic and pharmaceutical agents,
including drugs, synthetic organic molecules, proteins, peptides,
vitamins, steroids, steroid analogs; and also include genetic
material, including nucleosides, nucleotides and
polynucleotides.
[0112] The phrase "diagnostic agent", as used herein, refers to any
agent which may be used in connection with methods for imaging an
internal region of a patient and/or diagnosing the presence or
absence of a disease in a patient. Exemplary diagnostic agents
include, for example, contrast agents for use in connection with
ultrasound imaging, magnetic resonance imaging or computed
tomography imaging of a patient. Diagnostic agents may also include
any other agents useful in facilitating diagnosis of a disease or
other condition in a patient, whether or not imaging methodology is
employed.
[0113] The terms "pharmaceutical agent" or "drug", as employed
herein, refer to any therapeutic or prophylactic agent which may be
used in the treatment (including the prevention, diagnosis,
alleviation, or cure) of a malady, affliction, disease or injury in
a patient. Therapeutically useful peptides, polypeptides and
polynucleotides may be included within the meaning of the term
pharmaceutical or drug, as are various other therapeutically useful
organic or inorganic compounds.
[0114] Particular examples of pharmaceutical agents which may be
delivered by the methods of the present invention include, but are
not limited to: mitotic inhibitors such as the vinca alkaloids,
radiopharmaceuticals such as radioactive iodine, phosphorus and
cobalt isotopes; hormones such as progestins, estrogens and
antiestrogens; anti-helminthics, antimalarials and antituberculosis
drugs; biologicals such as immune sera, antitoxins and antivenins;
rabies prophylaxis products; bacterial vaccines; viral vaccines;
aminoglycosides; respiratory products such as xanthine derivatives,
theophylline and aminophylline; thyroid therapeutics such as iodine
salts and anti-thyroid agents; cardiovascular products including
chelating agents and mercurial diuretics and cardiac glycosides;
glucagon; blood products such as parenteral iron, hemin,
hematoporphyrins and their derivatives; targeting ligands such as
peptides, antibodies, and antibody fragments; biological response
modifiers such as muramyl dipeptide, muramyl tripeptide, microbial
cell wall components, lymphokines (e.g. bacterial endotoxin such as
lipopolysaccharide and macrophage activation factor); subunits of
bacteria (such as Mycobacteria and Cornebacteria); the synthetic
dipeptide N-acetyl-muramyl-L-alanyl-D-i- soglutamine; antifingal
agents such as ketoconazole, nystatin, griseofulvin, flucytosine
(5-fc), miconazole, and amphotericin B; toxins such as ricin;
immunosuppressants such as cyclosporins; and antibiotics such as
.beta.-lactam and sulfazecin; hormones such as growth hormone,
melanocyte stimulating hormone, estradiol, beclomethasone
dipropionate, betamethasone, betamethasone acetate, betamethasone
sodium phosphate, betamethasone disodium phosphate, betamethasone
sodium phosphate, cortisone acetate, dexamethasone, dexarnethasone
acetate, dexamethasone sodium phosphate, flunisolide,
hydrocortisone, hydrocortisone acetate, hydrocortisone cypionate,
hydrocortisone sodium phosphate, hydrocortisone sodium succinate,
methylprednisolone, methylprednisolone acetate, methylprednisolone
sodium succinate, paramethasone acetate, prednisolone, prednisolone
acetate, prednisolone sodium phosphate, prednisolone tebutate,
prednisone, triamcinolone, triamcinolone acetonide, triamcinolone
diacetate, triamcinolone hexacetonide, fludrocortisone acetate,
oxytocin, and vasopressin, as well as their derivatives; vitamins
such as cyanocobalamin neionic acid; retinoids and derivatives such
as retinol palmitate and .alpha.-tocopherol; peptides and enzymes
such as manganese superoxide dismutase and alkaline phosphatase;
anti-allergens such as amelexanox; anti-coagulation agents such as
phenprocoumon and heparin; tissue plasminogen activators (TPA),
streptokinase, and urokinase; circulatory drugs such as
propranolol; metabolic potentiators such as glutathione;
antibiotics such as p-aminosalicyclic acid, isoniazid, capreomycin
sulfate cycloserine, ethambutol hydrochloride ethionamide,
pyrazinamide, rifampin, streptomycin sulfate dapsone,
chloramphenicol, neomycin, ceflacor, cefadroxil, cephalexin,
cephadrine erythromycin, clindamycin, lincomycin, amoxicillin,
ampicillin, bacampicillin, carbenicillin, dicloxicillin,
cyclacillin, picloxicillin, hetacillin, methicillin, nafcililn,
oxacillin, penicillin (G and V), ticarcillin rifampin and
tetracycline; antivirals such as acyclovir, DDI, Foscarnet,
zidovudine, ribavirin and vidarabine monohydrate; antianginals such
as diliazem, nifedipine, verapamil, erythritol tetranitrate,
isosorbide dinitrate, nitroglycerin (glyceryl trinitrate) and
pentaerythritol tetranitrate; antiinflammatories such as difluisal,
ibuprofin, indomethacin, meclofenamate, mefenamic acid, naproxen,
oxyphenbutazone, phenylbutazone, piroxicam, sulindac, tolmetin,
aspirin, and salicylates; antiprotozoans such as chloraquine,
hydroxychloraquine, metranidazole, quinine and meglumine
antimonate; antirheumatics such as penicillamine; narcotics such as
paregoric; opiates such as codeine, heroin, methadone, morphine,
and opium; cardiac glycosides such as deslanoside, digitoxin,
digoxin, digitalin, and digitalis; neuromuscular blockers such as
atracurium nesylate, gallamine triethiodide, hexaflorenium bromide,
metrocurine iodide, pancurium bromide, succinylcholine chloride
(suxamethonium chloride), tubocurarine chloride and vecuronium
bromide; sedatives such as amorbarital, amobarbital sodium,
aprobarbital, butabarbital sodium, chloral hydrate, ethchlorvynol,
ethinamate, flurazepam hydrochloride, glutethimide,
methotrimeprazine hydrochloride, methyprylon, midazolam
hydrochloride, paraldehyde, pentobarbital, pentobarbital sodium,
secobarbital sodium, tulbutal, temazepam and trizolam; local
anesthetics such as bupivacaine hydrochloride, chloroprocaine
hydrochloride, etidocaine hydrochloride, lidocaine hydrochloride,
mepivacaine hydrochloride, procaine hydrochloride, and tetracaine
hydrochloride; general anaesthetics such as droperidol, etamine
hydrochloride, methohexital sodium and thiopental sodium;
antineoplastic agents such as methotrexate, fluorouracil,
adriamycin, mitomycin, ansamitomycin, bleomycin, cystein
arabinoside, arabinosyl adenine, mercaptopolylysine, vincristine,
busulfan, chlorambucil, azidothymidine, melphalan (e.g. PAM, L-PAM
or phenylalanine mustard), mercaptopurine, mitotane, procarbazine
hydrochloride dactinomycin (actinomycin D), danorubicin
hydrochloride, dosorubicin hydrochloride, taxol, plicamycin
(mithramycin), aminoglutethimide, estramustine phosphate sodium,
flutamide, leuprolide acetate, leuprolide acetate, megestrol
acetate, tamoxifen citrate, testolactone, trilostane, amsacrine
(m-AMSA), asparaginase, etoposide (VP-16), interferon .alpha.-2a,
interferon .alpha.-2b, teniposide (VM-26), vinblastine sulfate
(VLB), vincristine sulfate, hydroxyurea, procarbaxine, and
dacarbazine.
[0115] Although a wide variety of compounds, including nucleotides,
may be delivered in accordance with the present invention,
preferably the nucleotides are less than about 10,000 bases (or
base pairs) in length, more preferably between about 20 to about
10,000 bases (or base pairs) in length, even more preferably
between about 2,000 and about 8,000 bases (or base pairs) in
length, and most preferably between about 4,000 and 6,000 bases (or
base pairs) in length. Other (non-nucleotide) compounds or
bioactive agents to be delivered are preferably less than about
5000 kilodaltons (5000 kD) in molecular weight, more preferably
between about 10 and about 1000 kD, even more preferably between
about 100 and about 500 kD. As one skilled in the art will
recognize, however, larger and smaller sized compounds may also be
delivered in accordance with the present invention.
[0116] The useful dosage of nucleotide sequences or other compounds
to be administered or delivered, as well as the mode of
administration, will vary depending upon type and nature of the
compound to be delivered, the age, weight, cells or patient
(animal) to be treated, the particular diagnostic, therapeutic, or
other application intended (including the disease state, if any, to
be treated), and the organic halide (if any) and carrier (if any)
employed. Typically, dosage is initiated at lower levels and may be
increased until the desired therapeutic effect is achieved. The
desired dosage, including any therapeutically or diagnostically
effective dosage amounts, will be well within the ambit of one
skilled in the art, armed with the prevailing medical literature
and with the present disclosure. Representative amounts are
provided in the examples herein. Of course, higher or lower amounts
may be employed, as will be recognized by the skilled artisan.
[0117] As one skilled in the art would recognize, administration of
compositions of the present invention may be carried out in various
fashions, such as intravascularly, intralymphatically,
parenterally, subcutaneously, intramuscularly, intranasally,
intrarectally, intraperitoneally, interstitially, into the airways
via nebulizer, hyperbarically, orally, topically, or intratumorly,
using a variety of dosage forms. One method of topical
administration is the addition of a nucleotide sequence (or other
compound to be delivered), preferably in a carrier such as and not
limited to a hydrogel, applied to the outside of a balloon
catheter. The catheter is inserted into the blood stream of a
patient. Once the balloon of the catheter reaches the location to
which the sequence is to be administered, the balloon is pumped up
and the sequence-containing hydrogel adheres to the blood vessel
surface thus delivering the sequence. In addition, ultrasound may
be applied to the cells endoscopically and intravascularly, for
example, as well as, of course, applied externally.
[0118] A number of transfection and other intracellular delivery
techniques are possible in accordance with the methods of the
present invention employing the subject methods and the organic
halides and/or carriers as disclosed herein. Two methods, using
calcium phosphate and viral vectors, are indirect methods of
introducing the nucleotide sequence into cells because they involve
the passive uptake of the nucleotide sequence by the cell which is
to be transfected.
[0119] Calcium phosphate coprecipitation is a chemical-mediated
indirect method of transfection. The nucleotide sequence (or other
compound to be administered) is introduced into mammalian cells,
for example, by coprecipitation of the sequence with calcium
phosphate, calcium chloride, calcium hydroxybutarate, and the like;
then the mixture is presented to the cells. The purified nucleotide
sequence is mixed with buffers containing phosphate and calcium
chloride which results in the formation of a very fine precipitate,
and the mixture is presented to the cells in culture. A protocol
for cells that grow attached to a substratum as set forth in Keown,
W. A., et al., "Methods for Introducing DNA into Mammalian Cells,"
in Methods in Enzymology, Vol. 185, Gene Expression Technology,
Ed., Goeddel, David V., pp. 527-537, Academic Press, Inc., New
York, N.Y., 1991 is incorporated herein by reference in its
entirety. Briefly, on day 1, cells are seeded at 2-3.times.10.sup.4
cells/cm.sup.2 in normal growth medium and allowed to attach. At
the time of transfecting, the cells should be 80-90% confluent. On
day 2, the nucleotide sequence-calcium phosphate copreciptate is
prepared, mixed and allowed to stand at room temperature for about
30 minutes. The nucleotide sequence is added to TE buffer (10 mM
tris, 1 mM EDTA Ph 8.0), 2.times.HBAS (Hanks' balanced slats, 1.4
mM Na.sub.2HPO.sub.4, 10 mM KCl, 12 mM glucose, 275 mM NaCl, and 40
mM HEPES, ph 6.95), and 2M CaCl.sub.2 (calcium chloride in 10 mM
HEPES, pH 5.8). The medium is removed from the cells and replaced
with fresh medium. The precipitate is mixed gently by shaking or
pipetting and added directly to the medium in dishes containing
cells. The cells are incubated at 37.degree. C. for 4 hours. The
medium containing the precipitate is removed and dimethyl sulfoxide
in 1.times.HBS. After 2 minutes, 4 ml of serum-free medium is added
to each dish. The mixture is aspirated, washed twice with
serum-free medium, and medium is added and incubated overnight at
37.degree. C. The cells are trypsinized and the contents of each
plate is split into 3-4 new plates. Selection may be applied for
stable transfectants, in which selective medium may be used at this
time or a day later.
[0120] The present invention employing the methods of the invention
and the organic halides and/or carriers may also be useful
concurrently with microinjection and electroporation.
Microinjection involves the direct microinjection of nucleotide
sequences into the nucleus of a host cell. Microinjection does not
expose the nucleotide sequence to the cytoplasm or organelles
within it. This is beneficial since considerable damage may result
to the DNA during transit from the cell exterior to the nucleus.
Electroporation involves electric field-mediated nucleotide
sequence transfection. When membranes are subjected to an electric
field of sufficiently high voltage, regions of the membrane undergo
a reversible breakdown, resulting in the formation of pores large
enough to permit the passage of nucleotide sequences.
Electroporated nucleotide sequences remain free in the cytosol and
nucleoplasm. Very few copies of transfected nucleotide sequences
may be introduced with electroporation. Cells susceptible to
electroporation include, for example, lymphocytes, hematopoietic
stem cells, and rat hepatoma cells.
[0121] "Ultrasound", "Sonoporation.TM.", and similar terms, refer
to pulses of sound energy, preferably repetitive pulses, sufficient
to assist in inducing the delivery of a compound into a cell, and,
if desired, the formation of a gas from a gaseous precursor.
Preferably, the ultrasound is in the frequency range of from about
10 kilohertz to less than about 50 megahertz and at an energy level
of from about 200 milliwatts/cm.sup.2 to about 10 watts/cm.sup.2.
While not intending to be bound to any particular theory of
operation, the ultrasound may assist in the delivery of the
compounds to the cells by inducing openings in the cell membrane,
or perhaps bursting endosomes inside a cell allowing compounds to
escape. Indeed, cells may be induced to take up (e.g., be
transfected with) compounds (e.g., nucleotide sequences) with ease
compared to conventional methods. Typically the ultrasound is
applied by external application, via a standard clinical ultrasound
device, but may also be applied in other fashions, such as
endoscopically and intravascularly, as described above. The use of
ultrasound in connection with the present invention may, in certain
embodiments, be preferred. However, as noted herein, the use of
ultrasound is not necessary or critical to the operation of the
methods of the invention. Thus, the subject methods may be carried
out with the application of ultrasound, or without the application
of ultrasound, as desired.
[0122] In accordance with the present invention, for in vivo
applications, a lower frequency of sound is usually selected for
cells of deep seated or thick tissues, e.g. transcutaneous
application of ultrasound to cells of the deep seated muscle or
organs in the abdomen or retroperitoneum. For cells of small
tissues a higher frequency of sound energy is applied, e.g. for the
eye. For intravascular applications, which may employ intravascular
catheters equipped with ultrasound transducers for endovascular
gene therapy, higher frequencies may be employed such as over about
20 megahertz. For most applications however the frequency of the
sound ranges from about 500 kilohertz to about 3 megahertz,
preferably from about 500 kilohertz to about 1 megahertz, more
preferably about 200 kilohertz, more preferably about 40 kilohertz
to about 25 megahertz, even more preferably about 10 megahertz.
Compared to lithotripsy, the frequency employed in the present
invention is more than about 2 or 3 orders of magnitude higher and
the energy levels of the present invention are lower.
[0123] The sound energy is applied in waves of sonic energy over a
given duty cycle (sometimes referred to as pulse duration) and
level of intensity. Generally continuous wave ultrasound which
applies a constant train of ultrasound pulses is employed. The duty
cycle is selected so that the level of energy output is in a
desired range. The duty cycle may be varied from between 1% and
100% meaning that the ultrasound energy will be pulsing from
between 1% and 100% of the time. For example, a period of
ultrasound treatment may take place over 25 minutes with three duty
cycles of ultrasound, each five minutes in duration, interrupted by
two periods of no ultrasound. Preferably the duty cycle is 100%,
more preferably about 75%, more preferably about 50%, even more
preferably about 20%, even more preferably about 15%, and even more
preferably about 10%.
[0124] Ultrasound for use in the present invention is typically
provided at a frequency lower than the frequency used for imaging
by ultrasound. The frequency of ultrasound which is selected will
vary depending upon the location of cells which are being
transfected, and or other factors that will be readily apparent to
one skilled in the art based upon the present disclosure.
[0125] In addition to frequency, the energy level (sometimes
referred to as power intensity or power level) also has a large
effect on total energy which is applied to the cells or tissue for
ultrasound enhanced transfection. Suitable energy levels will be
readily apparent to one skilled in the art based upon the present
disclosure. Typically, the energy level settings are somewhat
higher than employed in diagnostic ultrasound but may range from
about 500 milliwatts/cm.sup.2 to about 10 watts/cm.sup.2, more
preferably from about 200 milliwatts/cm.sup.2 to about 10
milliwatts/cm.sup.2, and more preferably of from about 50
milliwatts/cm.sup.2 to about 2 watts/cm.sup.2. The power level
which is applied is selected so that both peak spatial temporal
power and total energy deposition is generally below the cytotoxic
threshold for the cells or tissue. Generally, frequencies and
energy levels are applied at lower amounts, then increased until
the desired cellular uptake of the administered compound is
achieved.
[0126] As one skilled in the art would recognize, high energies of
ultrasound may be used for hyperthermia to heat the tissue and also
to directly ablate tissues with very high levels of energy. In the
ultrasound enhanced transfection and gene expression of the present
invention, energy levels are far below those which cause tissue
ablation and below those which cause a significant hyperthermic
effect. As one skilled in the art would recognize once armed with
the present disclosure, energy deposition is a function of both
power intensity and duty cycle. Higher spatial peak temporal
average power tends to shift the bioeffect curve such that lower
total energy may be applied to create a greater bioeffect. Higher
energy levels and lower ultrasonic frequencies are required for
penetration into deep seated tissues; conversely lower energy
levels and higher ultrasonic frequencies are needed for treatment
of superficial tissues or when the ultrasound transducer can be
applied directly to the tissue surface. Small volume cell culture
samples need less power for ultrasound enhanced transfection than
large volume bioreactor chambers which may be multiple liters in
size and therefore need higher energy levels to enhance gene
expression. The geometry of a cell culture container will also
affect the ultrasound energy requirements.
[0127] In accordance with the present invention, ultrasound energy
may be used to increase the efficiency of cellular uptake of a
compound (e.g. transfection) by inducing a cell to take up a
compound. In addition, gene expression of a nucleotide sequence is
enhanced by the application of ultrasound.
[0128] The ultrasound energy may be applied to the tissue or cells
either before, simultaneously with, or after administration of the
compound to the cell, preferably simultaneously with or after.
Typically the ultrasound energy is applied from no more than about
48 hours prior to administration of the compound or genetic
material to the cells and/or up to no more than about 48 hours
after the genetic material has been administered to the cell,
although longer or shorter times may be applied. More preferably,
the ultrasound energy is applied at some time or at various time
points from about 4 hours before administration of the compound or
genetic material up to about 24 hours after administration. Most
preferably the ultrasound energy is applied within about 1 hour
prior to transfection up to about 12 hours post transfection.
[0129] Either one or multiple applications of ultrasound energy may
be employed. The duration of ultrasound energy exposure (exposure
time) will vary depending upon the power level of the ultrasound
and the duty cycle. To determine the preferable duration,
ultrasound is typically applied at lower exposure times, and
increased until the desired cellular uptake of the compound
administered is achieved. A high intensity (high power level;
typically greater than about 2 watts/cm.sup.2, preferably over
about 5 watts/cm.sup.2, and also preferably over about 10
watts/cm.sup.2, depending on the pulse duration) ultrasound shock
wave may require only a few milliseconds of exposure. This may also
be the case when cavitation nuclei such as gas filled liposomes or
perfluorocarbon emulsions are present within the medium. A very
brief exposure to high energy ultrasound may be sufficient to
enhance transfection. The presence of cavitation nuclei in the
transfection medium will lower the cavitation threshold and
therefore potentially decrease energy requirements for ultrasound
enhanced transfection as well as to potentially decrease the
necessary exposure time. More typically the exposure time ranges
from about a few seconds to up to about an hour of ultrasound
energy application to the cell to achieve most effective ultrasound
enhanced gene tmnsfection. Even more preferably the duration of
ultrasound exposure ranges from about a few seconds to about a few
minutes and may be repeated at various intervals during
transfection. The duration of ultrasound energy exposure should be
sufficient to cause the desired effect but not so long that
significant cytotoxicity may result.
[0130] The effect of ultrasound enhanced transfection is
independent of hyperthermia. While the application of ultrasound
energy necessary to increase the efficiency of transfection may
result in a few degrees centigrade increase in temperature, any
increase in temperature is typically transient and the temperature
rapidly returns to baseline. More preferably the temperature does
not increase significantly during application of the ultrasound. An
increase in temperature is typically less than about 1.degree. C.
to about 2.degree. C. Progressively higher levels of ultrasound
energy will result in progressive rises in temperature but
temperature is preferably maintained below the level where
significant cytotoxicity will occur (e.g. 44.degree. C. or higher).
As one may note, the sample measures the temperature in a solution
of normal saline when exposed to ultrasound. The applied energy is
10 watts imparted through a 5.0 cm.sup.2 transducer, or 2 W
cm.sup.-2. Sound energy from the ultrasound transducer may be
simply converted to thermal energy in the aqueous milieu. The
amount of energy and/or the time of exposure may be modified so as
to prevent temperature-induced cell destruction.
[0131] The ultrasound energy may be applied with any of a variety
of commercially available ultrasound systems. For example a
Rich-Mar model 25 ultrasonic therapy apparatus (Rich-Mar
Corporation, Inola, Okla.) with the center frequency residing at
approximately 1.0 Mhz, in pulsed or continuous mode, may be used to
practice the invention. Conventionally available transducers, power
amplifiers and other component systems for practicing the invention
can also be readily assembled. Wave synthesizers and pulsers may
also be incorporated into the system to allow control over the
pulse repetition intervals, duty cycles, etc. Advantageously, these
components can also be used to modify the ultrasound pulses to
employ varying frequency and amplitude effects such as CHIRP
(increasing in frequency) and PRICH pulses (decreasing in
frequency) waveform patterns. Ultrasonic energies can also be
supplied from commercially available amplifiers, transducers and
frequency generators. By way of example, a power transducer with a
center frequency of 1.0 Mhz from Valpey-Fisher (Valpey-Fisher,
Hopkinton, Mass.), a power RF amplifier from ENI (ENI, Rochester,
N.Y.), and a function generator from Hewlett Packard (Hewlett
Packard, Sunnyvale, Calif.) may be a suitable setup to accomplish
the above goals. Alternatively, a pulse/function generator or an
arbitrary function generator may also be used to accomplish
variable pulse formats. In addition, methods that would allow for
gating the various signals together, could conceivably be
accomplished.
[0132] The high energy ultrasound system may also be incorporated
with ultrasound imaging such as described in U.S. patent
application U.S. Ser. No. 08/468,052, filed Jun. 6, 1995, and the
disclosures of which are hereby incorporated herein by reference in
their entirety. Also application of high energy ultrasound may be
performed under other forms of conventional imaging such as
endoscopy (e.g. fiberoptic), computed tomography, magnetic
resonance imaging, angiography, and nuclear medicine. Such imaging
may be employed, for example, to locate and identify in a patient
the cells to which the ultrasound induced (or other) heating should
be applied, or used to follow and/or locate the composition of the
invention after administration to a patient.
[0133] The ultrasound may be applied so as to effectively create
second harmonic superimposition on the target treatment zone of
tissue to increase the effectiveness of transfection. For example,
a prototype sector-vortex phased array transducer, depicted in FIG.
5, 120 mm in diameter, which generates 750 kHz and 1.5 MHz
ultrasound may be employed. As described in the reference by K.
Kawabata and S. Umemura, Ultrasonics Sonochemistry 1996, 3:1-5, a
transducer may be constructed with 32 piezoelectric (PZT)
transducer elements from lead zirconate PZT material. The
transducer may be constructed in two tracks such that there are 16
sectors in each track. The lower frequency ultrasound could be
applied from the outer track and the higher frequency, 1.5 MHz,
from the inner track. A shell may be constructed with a 120 mm
radius of curvature for geometric focusing. The beam profile
provided by the piezoelectric elements and spherical shape of the
transducer cell assembly can be designed so as to superimpose the
focal zones of the two different frequencies of ultrasound. This
may result in focal acoustic power with superimposition of the
lower frequency and higher frequency ultrasound sources. This
results effectively in second harmonic superimposition of the
ultrasound signal. While not intending to be bound by any
particular theory of operation, it is believed that this ultrasound
assembly will allow for improved transfection efficiency at lower
total amounts of energy and thus result in reducing damage to the
cell.
[0134] Skilled artisans would recognize, once armed with the
present disclosure, that the two ultrasound energy sources may be
at other frequencies such that the first source (low frequency) is
one half the frequency of the second source. For example, 500 kHz
in the outer assembly and 1 MHz in the inner assembly may be
employed. A range of different frequencies may be selected such
that the outer assembly is 1.times. and the inner assembly is
2.times.. Alternatively, the assemblies may be designed such that
the higher frequency is in the outer assembly and the lower
frequency is in the inner assembly. Alternatively still, odd
harmonics may be utilized such that the outer and inner tracks may
be represented by X and 3.times. frequencies or X and 5.times.
frequencies. The second harmonic, ultraharmonic, or subharmonic
frequencies are superimposed at the focal zone which is directed
towards the target tissues or cells to be transfected. Thus,
ultrasound may be administered simultaneously at two or more
frequencies to result in superimposition of ultrasonic frequencies,
including and not limited to second harmonic frequencies.
[0135] The present invention is also directed to a pharmaceutical
kit which comprises a compound to be delivered, an organic halide
and/or a carrier (including combinations thereof) for use to those
desirous of delivering to a cell a compound. The compound, organic
halide, and/or carrier may be mixed together or separately provided
(as in, for example, separate containers, such as separate vials or
packets). The pharmaceutical kit may further comprise conventional
kit components known to those skilled in the art once armed with
the present disclosure, such as, mixing vials, syringes, gauze,
etc.
[0136] The invention is further demonstrated in the following
actual Examples 1-3, 10-19, and 23, and prophetic Examples 4-9, and
20-22. The examples, however, are not intended to in any way limit
the scope of the present invention.
EXAMPLES
Example 1
Effect of Ultrasound on the Temperature of an Aqueous-Based Medium
in Culture Plate Well Phantoms and on Cell Viability
[0137] The first phase of evaluating the effect of ultrasound was
to measure the amount of heating caused by the ultrasound energy.
The experimental protocol was designed to evaluate the heating in
an individual well of a 6 well culture plate while exposed to
ultrasound. Ultrasound was applied for 30 seconds to each well and
the relationship between energy and heating is shown in Table 2
below.
2TABLE 2 Temperature increase Temperature increase Energy at 10%
Duty Cycle at 100% Duty Cycle 0.5 W/cm.sup.2 0.degree. C.
0.5.degree. C. 1 W/cm.sup.2 0.degree. C. 1.5.degree. C. 2
W/cm.sup.2 0.5.degree. C. 2.9.degree. C.
[0138] A follow-up experiment was carried out to assay the cell
viability after ultrasound exposure. A cell proliferation kit using
sodium
3'-[1-(phenylamino-carbonyl)3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzen-
e sulfonic acid hydrate (XTT, Boehringer-Mannheim, Indianapolis,
Ind.) as the cell viability indicator was carried out to evaluate
whether cell damage had occurred. In this experiment, higher
absorbance is due to viable cells causing uptake of XTT. Table 3
contains the results from this study.
3TABLE 3 Cell Viability as a Function of Ultrasound Power Input
Treatment Mean absorbance Standard Deviation No Ultrasound 7.83
0.41 0.5 W/cm.sup.2 10% Duty Cycle 7.83 0.41 0.5 W/cm.sup.2 100%
Duty Cycle 7.83 0.41 1 W/cm.sup.2 10% Duty Cycle 7.17 0.75 1
W/cm.sup.2 100% Duty Cycle 6.00 1.41 1.5 W/cm.sup.2 10% Duty Cycle
7.67 0.82 1.5 W/cm.sup.2 100% Duty Cycle 1.83 0.98 2 W/cm.sup.2 10%
Duty Cycle 6.83 0.75 2 W/cm.sup.2 100% Duty Cycle 1.60 0.55
[0139] As the data in Table 3 shows, the first noticeable change in
cell viability occurs with an energy of 1 W/cm.sup.2 and a 100%
Duty Cycle. At higher energies with a 100% duty cycle a
significantly larger number of cells are destroyed.
Example 2
Effect of Ultrasound on Gene Expression in Cell Culture
[0140] Materials and Methods for Transfection and Measurement of
Gene Expression
[0141] The DNA plasmid used was pCAT Control (GenBank accession
number X65321) (Promega, Madison, Wis.) (see FIG. 3).
[0142] The plasmid was transformed into DH5-.alpha. Escherichia
coli competent cells (Life Technologies, Gaithersburg, Md.). The
cells were plated on LB agar plates (Bio 101, Vista, Calif.) that
contained ampicillin (Boehringer Mannheim Biochemicals (BMB),
Indianapolis, Ind.). Resistant colonies were selected, grown up and
a Wizard mini-prep (Promega) plasmid DNA extraction was carried
out. Plasmid DNA was cut with restriction enzyme EcoRI (BMB) and
run on a 1% agarose gel (BMB). After fragments were evaluated for
size, the remaining culture was used to start a large culture and a
Wizard maxi-prep was carried out to produce DNA for transfection.
The DNA was quantified using a Hoefer TKO-100 mini-DNA fluorometer.
The DNA was then ready for use in transfections.
[0143] Cationic liposomes were made by mixing dipalmitoyl
ethylphosphocholine and dioleolyl phosphoethanolamine (Avanti Polar
Lipids Alabaster, Ala.). The lipid mixture was resuspended in water
and then sonicated to form small liposomes.
[0144] A human cervical cancer cell line (HeLa) was obtained from
the American Type Culture Collection (Rockville, Md.) and grown in
EMEM culture media (Mediatech, Washington, D.C.) supplemented with
calf serum (Life Technologies). These cells were used in the
transfections.
[0145] The DNA/lipid complex was formed in HEPES buffered saline
(HEPES 20 mmol/l, NaCl, 150 mmol/l, pH 7.4) (Sigma, St. Louis, Mo.)
by mixing the lipid and DNA at a ratio of 6 parts lipid to one part
DNA. This was incubated for 30 minutes at room temperature and then
used for transfection.
[0146] The pCAT control plasmid encodes for the enzyme
chloramphenicol acetyl transferase (CAT). This enzyme is not found
in mammalian systems. The CAT expression is assayed using a CAT
ELISA kit (BMB). This non-radioactive kit allows for sensitive
detection of CAT expression. The kit is based on a sandwich of
antibodies. A 96 well plate is coated with anti-CAT, this antibody
binds the CAT in the sample. The next antibody is the
anti-CAT-digoxigenin, digoxigenin is a hapten found only in the
digitalis plant. The rarity of this compound makes it ideal for
non-radioactive labelling. The next antibody added is an
anti-digoxigenin that has been labelled with horseradish
peroxidase. Horseradish peroxidase breaks down a substrate and
causes a color reaction which is then read with an SLT Spectra
Shell plate reader (SLT-Labinstnrunents Ges.m.b.h.,
Groedin/Salzburg, Austria). Using a standard curve, this plate
reader allows for measurement of protein concentration.
[0147] Protocol
[0148] The DNA complex was formed and added at a concentration of
30 .mu.g of lipid and 5 .mu.g of DNA per well in a 6 well plate
containing HeLa cells in 4 mls of EMEM. A Rich-Mar model 25
therapeutic ultrasound machine (Rich-Mar Corporation, Inola, Okla.)
was used to apply ultrasound to the wells of the 6 well plate. The
ultrasound was applied either 30 minutes before the DNA/lipid
complex was added, 1 hour after the complex was added or 4 hours
after the complex was added. In the first experiment, a standoff
pad was used that covered the entire base of the 6 well plate and
allowed sound to conduct from one well to another. The power
setting was 0.5 w/cm.sup.2 with a 10% duty cycle.
[0149] Three conditions were tested. No ultrasound, ultrasound
applied 30 minutes before the transfection and ultrasound applied 1
hour after the transfection.
[0150] The results set forth in Table 4 are from a transfection
with a 1:1 mix of dipalmitoylethyphosphocholine and dioleoyl
phosphoethanolamine (Avanti Polar Lipids, Alabaster, Ala.). The
transfection was carried out in the presence of serum. In addition,
a negative control was added which included cells grown up not
transfected with the 1:1 mix of dipalmitoylethyphosphocholine and
dioleoyl phosphoethanolamine and without ultrasound treatment.
4TABLE 4 Quantification of Gene Expression in Cells Exposed to
Ultrasound Treatment Cat expression (ng/ml) negative control 0 no
ultrasound 15.876 Ultrasound 20.529 30' pretreatment Ultrasound 1
hour 43.794 post treatment
[0151] This experiment was repeated with a standoff pad designed to
isolate the wells from each other, see FIG. 1 and FIG. 4. The base
of a 6 well plate was cut away to allow transducer access and a
second 6 well plate was inverted above it. 2% agarose was poured
onto this mold to form a standoff pad. The standoff pad was
constructed to allow for open (dead air) spaces between the
portions of the standoff pad that contacted the 6 well plate, such
that each well was raised above the standoff pad on a vertical
support with open spaces under each of the wells where the standoff
pad was cut away. The vertical supports were made of 2% agarose and
conducted the sound from the ultrasound transducer. The transducer
was placed below the standoff pad and sound was projected up
through the pad into the each of the wells of the 6 well plate. The
6 well plate was placed upright on the standoff pad such that the
cells on the bottom of the well were close to the transducer. In
both experiments, the expression of the CAT protein was measured by
CAT ELISA after 48 hours of incubation. Ultrasound was applied at 1
W/cm.sup.2 and 100% duty cycle. The ultrasound was applied for 30
seconds on each well.
[0152] The same transfection reagent was used as in the first
example. The test conditions were no ultrasound, 30 minutes before
transfection, 1 hour after transfection and 4 hours post
transfection. Again the transfection was carried out in the
presence of serum. In addition, a negative control was added which
included cells grown up not transfected with CAT/lipid complex and
without ultrasound treatment. The results are shown in Table 5.
5 TABLE 5 Treatment Cat expression (ng/ml) negative control 0 no
ultrasound 5.339 Ultrasound 5.339 30' pretreatment Ultrasound 1
hour post 10.078 treatment Ultrasound 4 hours post 4.988
treatment
Example 3
Application Using DNA with a Lipid Carrier and a Cavitator
[0153] The cells and the DNA/lipid complex were prepared as in
Example 2. Six well plates were seeded with HeLa cells and filled
with 16 ml of media as in Example 2. The lipid added was increased
to 135 .mu.g to allow for the increase in volume and the DNA was
also increased to 22.5 .mu.g per well. One hour after the complex
was added, 100 .mu.l of a liposome comprised of the lipids
dipalmitoylphosphatidylcholine (DPPC),
dipalmitoylphosphatidylethanolamine coupled to polyethylene glycol
5000 (DPPE-PEG5000), and dipalmitoylphosphatidic acid (DPPA), in a
ratio of about 82%:8%: 10% (mole %) was added to each well. The
DPPE-PEG5000 was comprised of DPPE and PEG5000 in a ratio of about
20%:80% (weight %). PEG5000 refers to PEG having an average
molecular weight of about 5000. In addition, a negative control was
added which included cells grown up not transfected with CAT/lipid
complex and without ultrasound treatment.
[0154] The six well plate was then covered with a sheet of parafilm
to prevent leakage, the lid was replaced and the plate was
inverted. By inverting the plate, the gas filled liposomes
(cavitator) were allowed to float up to the cells, now on top of
the standoff pad, plate construct. The ultrasound was applied from
the bottom, however, in this case, the sound was propagated through
the media to the cells. After the ultrasound exposure the plates
were returned to an upright position and the parafilm removed. The
plates were then incubated for 4 hours and the CAT ELISA performed
the results of which are set forth in Table 6.
6 TABLE 6 Treatment Cat expression in ng/ml Negative control 0 No
ultrasound 4.584 0.5 w/cm.sup.2/35% duty cycle 9.634 0.5
w/cm.sup.2/100% duty cycle 19.910 1 W/cm.sup.2/100% duty cycle
9.811
Example 4
Industrial Applications of Ultrasound Enhanced Transfection
[0155] A large scale bioreactor vessel containing a free suspension
of cells is equipped with a flow through chamber housing an
ultrasound transducer. Plasmid DNA containing the gene of interest
is added to the cell suspension with and without an organic halide.
As the cells circulate through the chamber 500 kilohertz ultrasound
is applied with a 10 percent duty cycle at an energy level of 200
milliwatts per cm.sup.2. Enhanced gene expression is attained, both
with and without the organic halide. By varying the rate of flow of
the cell suspension through the flow through chamber the proper
ultrasound exposure time is attained for optimal transfection
efficiency.
Example 5
Ex Vivo Enhancement of Gene Expression in Human Cells
[0156] Plasmaphoresis is used to harvest the T cells of a patient
with metastatic malignant melanoma. The T cells are placed in
tissue culture and incubated with granulocyte macrophage colony
stimulating factor (GMCSF) to increase multiplication of the
lymphocytes. After sufficient cell densities have been achieved the
gene for interleukin-2 (IL-2) is added to the cells with a cationic
liposomal vector, with and without an organic halide. One hour
later 1 megahertz ultrasound energy is applied at a power level of
0.5 watts with a 10% duty cycle for a duration of 5 minutes.
Twenty-four hours later the cells are infused back into the patient
in an effort to treat the metastatic tumors. Promising results in
the form of a perceptible decrease in tumor mass are observed, both
with and without the organic halide. Additional testing also
reveals enhanced IL-2 expression in the treated cells.
Example 6
Therapeutic Applications of Ultrasound Mediated Gene Delivery:
Duchenne's Muscular Dystrophy
[0157] In a patient with Duchenne's Muscular Dystrophy plasmid DNA
encoding the gene for dystrophin is injected at multiple sites into
the muscles of the thighs and legs, with and without an organic
halide. Ultrasound is then applied to the thighs and legs using
silicone gel as couplant between the transducer and the patient's
skin. The frequency is 200 kilohertz with a 10% duty cycle and a
power level of 1 watt. The transducer remains for about 2 to 3
minutes over any one location on the skin. Enhanced expression for
the gene for dystrophin is attained resulting in increased muscle
strength, both with and without the organic halide.
Example 7
Therapeutic Applications of Ultrasound Mediated Gene Delivery:
Atherosclerotic Heart Disease
[0158] A patient with atherosclerotic disease has marked narrowing
of the left anterior coronary artery. A balloon angioplasty
catheter coated with a hydrogel material binding the gene for
vascular endothelial growth factor (VEGF), both with and without an
organic halide, is placed at the site of arterial stenosis and the
balloon is inflated to a pressure of 9 atmospheres. An endovascular
ultrasound catheter is placed inside the vessel at the region where
the angioplasty was performed and ultrasound energy is applied. The
frequency is 20 megahertz at 1 watt per cm.sup.2 with 15% duty
cycle for 3 minutes. Enhanced gene expression of VEGF is observed
with diminished propensity to restenosis at the angioplasty site,
both with and without the organic halide.
Example 8
Therapeutic Applications of Ultrasound Mediated Gene Delivery
[0159] Similarly to Example 7 angioplasty is performed in a patient
using a balloon catheter coated with the gene for VEGF, and with
and without an organic halide, but in this case the ultrasound
energy is applied transcutaneously with a 1 megahertz focused
transducer equipped with both imaging and therapeutic elements. The
therapeutic 1 megahertz sound is applied at an energy level of 500
milliwatts/cm.sup.2 using a 20% duty cycle for a period of 5
minutes. The energy is focused upon the angioplasty site. Again
enhanced VEGF gene expression is observed and decreased propensity
to restenosis, both with and without the organic halide.
Example 9
Therapeutic Applications of Ultrasound Mediated Gene Delivery
[0160] Colonoscopy is performed in a patient with genetic
predisposition to colon cancer. A region of epithelial metaplasia
is identified in the descending colon. An ultrasound transducer
equipped with a semipermeable membrane and drug storage reservoir
containing the gene for Bcl 2 with a liposomal vector, both with
and without an organic halide, is positioned over the area of
epithelial metaplasia and ultrasound energy is applied at a
frequency of 500 kilohertz with an energy level of 500
milliwatts/cm.sup.2 and 10% duty cycle for a period of 3 to 5
minutes, to deliver the Bcl 2 gene to the cells in the region. On
follow-up colonoscopy 8 weeks later the epithelial metaplasia has
decreased significantly, particularly where an organic halide is
employed in the administration process. Further testing reveals
enhanced Bcl 2 expression in the region, both with and without the
organic halide employed in the delivery process.
Example 10
Transfection Efficiency of DPEPC/DOPE with and without Organic
Halides
[0161] Dipalmitoylethylphosphocholine (DPEPC) (Avanti Polar Lipids,
Alabaster, Ala.) was mixed with dioleoylphosphatidylethanolamine
(DOPE) (Avanti Polar Lipids, Alabaster, Ala.) at a 1:1 (w:w) ratio
in 10 ml of deionized water at a lipid concentration of 1 mg per
ml. 100 microliters of n-perfluorohexane (PCR, Inc., Gainesville,
Fla.) was added and mixed by shaking for 5 minutes on a Heavy Duty
#6 Wig-L-Bug (Crescent Dental, Lyons, Ill.). The mixture was then
extruded five times through two 0.8 .mu.m filters in a Lipex
Biomembranes Extruder Device (Vancouver, BC, Canada). Particles
without a fluorinated organic halide were sonicated for 10 minutes
at room temperature in a bath sonicator. The mixture was then
diluted in HEPES buffered saline at a ratio of 3011 of lipid mix to
701 of HBS per well. pCMVCAT (Life Technologies, Inc.,
Gaithersburg, Md.) containing the gene for chloramphenicol acetyl
transferase and the promoter from human cytomegalovirus (CMV) was
used to transfect HeLa cells. pCMVCAT may be produced in accordance
with the methods set forth in Foeeking and Hofstetter, Gene 1986
45:101-105, incorporated herein by reference in its entirety.
[0162] The pCAT.RTM. (Promega, Montgomeryville, Pa.) basic vector
lacks eukaryotic promoter and enhancer sequences. This allows
maximum flexibility in cloning any putative regulatory sequences
into the convenient multiple cloning sites.
[0163] Expression of CAT activity in cells transfected with this
vector is dependent on insertion of a functional promoter upstream
from the CAT gene. Enhancer elements can be inserted at the BamH I
site downstream from the CAT gene. The vector map sequence
reference points are multiple cloning sites (Hind III-Xba I)
2242-2271, SV40 small T antigen region 3064-3917, CAT gene start
site 2315, CAT gene stop site 2974, and .beta.-lactamase (Amp')
coding region 209-1069.
[0164] Plasmid DNA was diluted to 5 .mu.g per 100 .mu.l in HBS. The
DNA and lipid mixes were combined and incubated for 45 minutes at
room temperature. HeLa cells is (ATCC certified cell line 2 (CCL
2)) were plated at a concentration of 4.times.10.sup.5 per well in
Eagle's MEM with non-essential amino acids and Earle's BSS, 90%;
fetal bovine serum, 10%. 200 .mu.l of the lipid/DNA complex were
added to each well and incubated 48-72 hours in the presence of
serum. The process was repeated using n-perfluorohexane in volumes
of 50, 25, and 12.5 microliters. CAT expression was assayed using a
CAT ELISA from Boehringer Mannheim Biochemical (Indianapolis,
Ind.). The results are shown in Table 7.
7TABLE 7 Effect of Various Amount of Organic Halide on
Chloramphenical Transacetylase Expression in Various Cell Lines OH
Organic Halide Volume CAT Standard Lipids (OH) .mu.l Cell Line
Expression Deviation Lipofectin None HeLa 59.624 6.828
Lipofectamine None HeLa 27.917 5.112 Lipofectin None C127 0 1.582
Lipofectamine None C127 0 3.164 Lipofectin None COS1 104.424
117.883 Lipofectamine None COS1 305.392 23.254 Lipofectin None
NIH3t3 0 1.582 Lipofectamine None NIH3t3 5.766 8.809 DPEPC/DOPE
None HeLa 28.946 11.852 DPEPC/DOPE None C127 24.493 21.491
DPEPC/DOPE None COS1 4930.403 262.278 DPEPC/DOPE None NIH3t3 36.368
21.952 DPEPC/DOPE Perfluorohexane 12.5 HeLa 3642.958 229.17
DPEPC/DOPE Perfluorohexane 25 HeLa 126.976 19.126 DPEPC/DOPE
Perfluorohexane 50 HeLa 33.454 10.94 DPEPC/DOPE Perfluorohexane 100
HeLa 28.69 2.61 DPEPC/DOPE Bromononafluorobutane 12.5 HeLa 1795.29
1054.14 DPEPC/DOPE Bromononafluorobutane 25 HeLa 2725.611 1004.542
DPEPC/DOPE Bromononafluorobutane 50 HeLa 2634.161 456.709
DPEPC/DOPE Bromononafluorobutane 100 HeLa 2703.144 600.767
DPEPC/DOPE Perfluorohexane 12.5 C127 167.911 88.724 DPEPC/DOPE
Perfluorohexane 12.5 COS1 5348.326 93.809 DPEPC/DOPE
Perfluorohexane 12.5 NIH3t3 1968.405 316.894 * DPEPC/DOPE is
dipalmitoylethylphosphatidylcholine:dioleylphosphatidylethanolamine;
CAT expression units are ng/ml (protein).
Example 11
Transfection Efficiency of DPEPC with Organic Halides
[0165] An example of transfection of DPEPC with perfluorohexane and
1-bromononafluorobutane (BNFB, Fluoroseal, Houston, Tex.) was
carried out. Particles were prepared as set forth in Example 10
except that the BNFB sample was cooled before and after shaking on
the Wig-L-Bug to keep the BNFB in the liquid state. The results are
shown in Table 7.
Example 12
Improved Transfection in Cell Lines Normally Resistant to
Transfection with Perfluorohexane
[0166] COS-1 cells (ATCC cell repository line 1650 (CRL 1650)) were
propagated in Dulbecco's modified Eagle's medium, 90% and fetal
bovine serum, 10% and plated at a concentration of 1.times.10.sup.5
per well. NIH/3T3 cells (ATCC cell repository line 1658 (CRL 1658))
were propagated in Dulbecco's modified Eagle's medium with 4.5
g/liter glucose, 90% and calf serum, 10% and plated at a
concentration of 1.times.10.sup.5 per well. C127:LT cells (ATCC
cell repository line 1804 (CRL 1804)) were propagated in Dulbecco's
modified Eagle's medium with 4.5 g/liter glucose, 90% and fetal
bovine serum, 10% and plated at a concentration of 1.times.10.sup.5
per well. DPEPC:DOPE was prepared with perfluorohexane at a volume
of 12.5 microliters and without perfluorohexane as Example 10. The
lipid/DNA mixtures were incubated with the cells for 72 hours as
described in Example 10. Lipofectin and Lipofectamine (Life
Technologies, Gaithersburg, Md.), commercially available controls,
were used according to the manufacturer's instructions. The results
are shown in Table 7.
Example 13
Transfection Using Cationic Microspheres Filled with
Perfluorobutane Gas
[0167] A lipid solution with 1 mg per ml of lipid composed of 1:1
(w:w) DPEPC with dipalmitoylphosphatidylethanolamine (DPPE) was
prepared and placed in a 2 ml vial with a head space of
perfluorobutane gas. The samples were shaken for 1 minute of an
ESPE CapMix at 4500 RPM resulting in gas filled lipid coated
microspheres. To each sample pCMVCAT was added at a DNA
concentration of 1 .mu.g per 6 .mu.g lipid. The process was
repeated using 1:1 w:w DPEPC with DOPE. The gas filled microspheres
prepared from DPEPC/DPPE (gel state saturated lipids) formed stable
gas filled microspheres binding the DNA. When transfection
experiments were repeated substantially as outlined in Example 11,
however, there was no evidence of any appreciable gene expression.
When gas filled microspheres were prepared from the DPEPC/DOPE
lipids (DOPE is liquid crystalline state at physiologically
relevant temperature) the particle count was lower than for the
DPEPC/DPPE vesicles. When DNA was added to the lipids, the vesicles
fell apart, indicating how cationic lipid microspheres composed of
liquid crystalline state lipid are unstable to bind DNA when the
interior of the microsphere is filled with a gas PFC
(perfluorobutane) as opposed to a liquid PFC (see above).
Example 14
Transfection with Organic Halides
[0168] The experiment conducted in Example 12 was repeated except
that fluorinated organic halides were used in eight samples and
eight samples were subjected to ultrasound. Sixty minutes following
the incubation of the DNA/lipid complex with the cells, ultrasound
was applied by immersing the head of a lMz transducer directly to
the top of the cell culture well and ultrasound was applied for 5
to 30 seconds at a 10% duty cycle. Control groups were cells not
exposed to ultrasound with and without transfection materials.
Table 8 indicates that organic halides increase the efficiency of
ultrasound such that lower levels of energy are more effective.
1-bromononafluorobutane (BNFB) together with ultrasound results in
about a 50% enhancement of expression; whereas about a 30%
enhancement of expression is evident with perfluorohexane (PFC6)
and ultrasound.
8 TABLE 8 US Expression Lipids OH (sec.) Actual SD DPEPC/DOPE BNFB
0 7007 2084 DPEPC/DOPE BNFB 5 10442 610 DPEPC/DOPE BNFB 15 9910 656
DPEPC/DOPE BNFB 30 9381 140 none -- 0 -137 23 DPEPC/DOPE -- 0 1647
396 DPEPC/DOPE -- 5 6444 846 DPEPC/DOPE -- 30 8167 1206
Lipofectamine -- 0 389 125 Lipofectin -- 0 -118 14 DPEPC/DOPE PFC6
0 7290 2235 DPEPC/DOPE PFC6 5 9477 1017 DPEPC/DOPE PFC6 15 8545 401
DPEPC/DOPE PFC6 30 9630 827
[0169] In all cases above the cell line is NIH/3T3. Units of DNA
expression are ng/ml protein. Ultrasound was applied at 0.5
W/cm.sup.2, 10% duty cycle. OH-organic halide, US
(sec.)--ultrasound in seconds, BNFB--(1-bromonanofluorobutane),
PFC6--(perfluorohexane), SD--standard deviation.
[0170] As can be seen above the lipid mixture of DPEPC/DOPE
carrying the gene results in above background expression compared
to Lipofectin or Lipofectamine. This expression is enhanced with
the addition of either a fluorocarbon or by ultrasound. The best
results were obtained with fluorocarbons and ultrasound in
combination.
Example 15
Transfection with Perfluoroethers
[0171] The experiment described in Example 10 was repeated with the
following perfluoroethers: perfluoromethylbutyl ether (PFMBE),
perfluoro-4-methyl-tetrahydrofuran (PMTH) and
perfluorotetrahydropyran (PFTH). Transfection data is shown in
Table 9 below. Table 9 shows that perfluoroethers perform similar
to perfluorocarbons of the previous examples. Increased levels of
perfluoroethers do not appear to have a detrimental effect on
transfection and expression. Each of the perfluoroethers enhance
transfection greater than 100% over the control lipid (DODO).
9TABLE 9 Lipids Perfluoroether PFE (vol) CAT expression Std. Dev.
(none) none -- -54.773 4.957 DODO none -- 1490.801 183.278 DODO
PFMBE 0.125 mls 3578.363 55.702 DODO PFMBE 0.250 mls 3167.014
246.912 DODO PFMBE 0.500 mls 3509.693 109.497 DODO PFMTH 0.250 mls
3424.945 25.837 DODO PFMTH 0.500 mls 3373.024 36.736 DODO PFTH
0.250 mls 3306.029 67.332 DODO PFTH 0.500 mls 3344.886 235.041
[0172] In all cases above the cell line is HeLa, and the lipid
coating for the perfluoroether dioleyl-glycero-3 phosphocholine.
CAT expression units are ng/ml protein. Std. dev.--standard
deviation, PFE (vol)--perfluoroether in volume.
Example 16
Transfection Efficiency with Perfluorohexane
[0173] The experiment of Example 10 was repeated in HeLa cells
using DMRIE-C, DODO, DPDO, or other commercial cationic lipids with
and without perfluorohexane. The results are shown in Table 10
below. Table 10 shows that perfluorocarbons are effective with a
variety of lipids. Indeed, the enhancement of expression is
independent of the type of lipid used. DODO+PFC6 results in about 8
to about 10 fold increase in expression, DMRIE-C results in about a
40% enhancement of expression, and DPDO results in about a 4 fold
increase.
10TABLE 10 CAT Lipids PFC PFC (vol) expression Std. Dev. (none)
none -- -5.608 5.015 DMRIE-C none -- 1615.096 79.088 DMRIE-C PFC6
0.125 mls 2118.489 72.325 DODO none -- 611.594 228.548 DODO PFC6
0.250 mls 6331.664 443.727 DODO PFC6 0.500 mls 4829.148 379.244
DPDO none -- 426.652 238.019 DPDO PFC6 0.125 mls 1675.285 1146.279
Lipofectamine none -- -8.891 1.895 Lipofectin none -- 100.542
56.863
[0174] DODO--dioleyl-glycero-3 phosphoethylcholine. CAT expression
units are ng/ml protein. DPDO--dipalmitoyl-glycero-3
phosphocholine, Std. Dev.--standard deviation, PFC
(vol)--perfluorocarbon in volume.
Example 17
Effect of Ultrasound Alone on DMRIE and DODO Vesicle
Transfection
[0175] The experiments described above in the previous examples
were repeated using the DMRIE, DODO, and no lipids, in each case
without an organic halide, using the same procedures set forth
above in Example 16.
11 TABLE 11 US Expression Lipids OH (sec.) Actual SD none none 0 76
4 DMRIE none 0 426 49 DMRIE none 5 1450 317 DMRIE none 30 1204 120
DODO none 0 2719 102 DODO none 5 4073 61 DODO none 30 3914 53
[0176] In all cases above the cell line is HeLa. Units of DNA
expression are ng/ml protein. Ultrasound was applied at 0.5
W/cm.sup.2, 10% duty cycle. OH--organic halide,
PFC--perfluorocarbon, US (sec.)--ultrasound in seconds,
SD--standard deviation.
Example 18
Effect of Poly-L-Lysine (in Place of Lipids) on Transfection with
Perfluorohexane
[0177] The experiments described above in the previous examples
were repeated using the DODO with or without a fluorinated organic
halide, Poly-L-Lys with or without a fluorinated organic halide,
and no lipids and no organic halide, using the same procedures set
forth above in Example 16.
12 TABLE 12 CAT Carrier OH OH (vol) expression Std. Dev. (none)
none -- 1.378 1.012 DODO none -- 2190.158 684.125 DODO PFC6 0.125
3322.927 39.520 Poly-L-Lys none -- 37.901 2.072 Poly-L-Lys PFC6
0.125 50.909 5.128
[0178] DODO--dioleyl-glycero-3 phosphoethylcholine. CAT expression
units are ng/ml protein. OH--organic halide, OH (vol)--organic
halide in volume in mls, Std. Dev.--standard deviation.
Example 19
Comparison of Different Organic Halides as Transfection
Enhancers
[0179] The experiment was carried out as in Example 10 using HeLa
cells, dioleyl-glycero-3-phosphoethanolamine (DOPE) as the lipid
carrier and either perfluoropentane (PFC.sub.5),
1-bromoperfluorooctane (perfluoro-octylbromide, BrPFC.sub.8),
perfluorodecane (PFC.sub.10) or perfluorohexane (PFC.sub.6). The
Table 13 illustrates the transfection results.
13 TABLE 13 OH vol CAT Lipids OH (in mls) expression Std. Dev.
(none) none -- -11.434 1.478 DOPE none -- 295.447 18.828 DOPE PFC5
0.125 1204.762 198.020 DOPE PFC5 0.250 900.344 247.634 DOPE PFC5
0.500 1089.989 245.888 DOPE BrPFC8 0.125 276.236 57.468 DOPE BrPFC8
0.250 248.652 26.723 DOPE BrPFC8 0.500 114.175 65.473 DOPE PFC10
0.125 839.263 302.202 DOPE PFC10 0.250 811.678 171.344 DOPE PFC10
0.500 1186.044 322.248 DOPE PFC6 0.125 1108.215 164.989 DOPE PFC6
0.250 829.904 156.464 DOPE PFC6 0.500 441.745 33.219 OH--organic
halide, OH (vol)--organic halide in volume, Std. Dev.--standard
deviation.
[0180] The above results indicate that liquid (perfluorohexane or
perfluorodecane)perfluorocarbons work as well as perfluorocarbons
which would undergo at least a partial liquid to gas phase
transition at physiological temperatures (perfluoropentane). Only
the brominated compound, BrPFC.sub.8, was markedly less effective
in enhancing transduction.
[0181] The data also indicate that a preferred quantity of
perfluorocarbon for transfection generally is in the range of 0.125
mls to 0.250 mls or from about 1% to 3% v/v.
Example 20
Transfection in the Presence of Fluorinated Surfactants
[0182] The experiment described in Example 10 is repeated except
the lipids are suspended in varying amounts (1.25% to 5%) of
Zonyl.RTM. surfactant, (Du Pont Chemical Co., Wilmington, Del.). In
some samples perfluorohexane (0.125 mls, 0.250 mls or 0.500 mls) is
shaken with the surfactant. The sizes range from about 300 nm to
about 900 nm. Transfection efficiency where samples are prepared
from Zonyl.RTM. is about 10% to about 25% of the transfection where
samples are prepared without Zonyl.RTM..
Example 21
Transfection with Cationic Lipid Carrier and Lipid Suspension with
Organic Halides and with and without Ultrasound
[0183] The experiment described in Example 14 is modified such that
the control lipid mixture is DPPC/DPPA/PEG-5000+perfluoropropane
and results are compared to transfection using a suspension of the
cationic lipid DOTMA+perfluoropropane. After incubation ultrasound
is applied as in Example 14 to some of the samples. Results similar
to those obtained in Example 14 are observed.
Example 22
Transfection with Cationic Lipid Carrier and Lipid Suspension with
Ultrasound and 1-Bromononafluorobutane
[0184] Cationic liposomes are prepared from
dioleyolyethylphosphocholine and DOPE at a concentration of 1 mg/ml
lipid. To this is added 0.125 .mu.l 1-bromo-nonafluorobutane
(BNFB). The mixture is agitated with a Wig-L-Bug for 60 seconds and
the resulting vesicles are extruded with a 0.8 micron filter. The
resulting BFNB filled cationic liposomes are then complexed with
ribozymal RNA (hammerhead motif) encoding catalytic RNA specific
for vascular endothelial growth factor (VEGF) at a lipid to RNA
ratio of 1:6 using conventional methods. The liposomal RNA
preparation (1.0 ml) is injected IV into a patient with diabetic
retinopathy. A 5 megahertz ultrasound transducer is placed on the
patient's anesthetized cornea using a silicone acoustic coupling
gel. Ultrasound energy is focused onto the patient's retina using
2.0 Watts and a 10% duty cycle. As the liposomes enter the region
of ultrasound the gaseous precursor expands and pulsates. Local
shock waves are created. The ribozyme RNA is delivered to the
endothelial cells in the region of ultrasound application the
therapeutic RNA construct enters the target endothelial cells.
Catalytic RNA then causes reduction in VEGF production. The
patient's retinal deterioration is halted and blindness is
avoided.
Example 23
In Vivo Transfection Using Perfluorocarbons
[0185] Liposomes were prepared from dipalmitoylethylphosphocholine
(DPEPC) and dioleoylphosphatidylethanolamine (DOPE). Both lipids
were purchased from Avanti Polar Lipids (Alabaster, Ala.).
Lipofectin.RTM. and Lipofectamine.RTM. and DMRIE-C.RTM. were
obtained in a ready to use form from Life Technologies Inc.
(Gaithersburg, Md.). Plasmid pCMVCAT which contains the human
cytomegalovirus promoter and the chloramphenicol acetyl transferase
gene was provided by Life Technologies (Gaithersburg, Md.). Plasmid
DNA was prepared by large scale extraction from E. coli and
purified by CsCl banding by Lofstrand Labs Limited (Gaithersburg,
Md.). In a preliminary set of experiments the ratios of the two
lipids as well as the ratio of lipid to DNA was optimized by
measuring the levels of gene expression in HeLa cells following
transfection with the pCMVCAT plasmid using DPEPC/DOPE in a ratio
of 1:1, with a lipid to DNA ratio of 6:1 (the lipid and DNA
combination collectively being referred to as DPDO). The liposomes
were prepared by suspending the dried lipids in water and
lyophilizing the mixed lipids and resuspending the lipids in water.
The rehydrated lipids were agitated at about 2,000 r.p.m. on a
dental amalgamator (Heavy duty #6 Wig-L-Bug Crescent Dental, Lyons,
Ill.) for 5 minutes and then extruded through two 0.8 micron
polycarbonate filters (Nucleopore Costar, Cambridge, Mass.) using
an Extruder Device (Lipex Biomembranes, Vancouver, B.C., Canada) at
about 30 psi. For preparation of perfluorocarbon (PFC) filled
liposomes, 100 microliters, 50, 25 or 12.5 microliters of PFC was
added to the lipid solution prior to shaking on the dental
amalgamator. PFC's which were tested included perfluoropentane,
perfluorohexane, 1-bromononafluorobutane, perfluorooctylbromide and
perfluoromethylbutylether.
[0186] In vivo experiments were carried out in 50 male Balb/C mice.
The body weight of the mice was 15-20 g. The lipoplex was formed in
the same manner as for cell transfection using the DPEPC/DOPE lipid
mixture. The perfluorocarbon used in this experiment was
perfluorohexane. The mixture was injected intramuscularly at a does
of 200%1 per hind leg. The ultrasound (US) was applied at 1
W/cm.sup.2 for one minute to each leg in the animals that received
ultrasound. The animals were held for 2 days and then euthanized by
carbon dioxide asphyxiation. The hind legs were removed from the
animals and the muscle collected. The muscle was frozen in liquid
nitrogen and ground in a mortar and pestle. The tissue was
transferred to a pre-weighed 50 ml conical tube and the tissue
weight was recorded. The tissue was then placed into one ml of the
lysis buffer from the CAT ELISA kit. The CAT ELISA was then carried
out according to the manufacturer's protocol. The data was
transferred into Microsoft Excel and converted into ng CAT protein
per gram of tissue. Statistical analysis was carried out using the
JMP 3.1.5 statistical analysis package for the Macintosh.
[0187] The results are shown in FIG. 7. As the figure shows, the
use of a perfluorocarbon (with or without ultrasound) greatly
enhanced CAT expression in mice.
[0188] The disclosures of each patent, patent application and
publication cited or described in this document are hereby
incorporated herein by reference, in their entirety.
[0189] Various modifications of the invention, in addition to those
described herein, will be apparent to those skilled in the art from
the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims.
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