U.S. patent application number 10/864102 was filed with the patent office on 2005-03-17 for spatial and temporal control of gene expression using a heat shock protein promoter in combination with local heat.
This patent application is currently assigned to The GOV of the USA as represented by the Secretary of the Department of Health and Human Services, The GOV of the USA as represented by the Secretary of the Department of Health and Human Services. Invention is credited to Moonen, Chrit.
Application Number | 20050059623 10/864102 |
Document ID | / |
Family ID | 21819426 |
Filed Date | 2005-03-17 |
United States Patent
Application |
20050059623 |
Kind Code |
A1 |
Moonen, Chrit |
March 17, 2005 |
Spatial and temporal control of gene expression using a heat shock
protein promoter in combination with local heat
Abstract
The invention provides methods for using local heat to control
gene expression. The heat shock protein (hsp) gene promoter is
recombined with a selected therapeutic gene and expressed in
selected cells. Local controlled heating is used to activate the
hsp promoter, for example by using focused ultrasound controlled by
MRI.
Inventors: |
Moonen, Chrit; (Pessac,
FR) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
The GOV of the USA as represented
by the Secretary of the Department of Health and Human
Services
Rockville
MD
|
Family ID: |
21819426 |
Appl. No.: |
10/864102 |
Filed: |
June 9, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10864102 |
Jun 9, 2004 |
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10096549 |
Mar 11, 2002 |
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10096549 |
Mar 11, 2002 |
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09242244 |
Feb 29, 2000 |
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09242244 |
Feb 29, 2000 |
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PCT/US97/15270 |
Aug 14, 1997 |
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60024213 |
Aug 15, 1996 |
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Current U.S.
Class: |
514/44R ;
435/455; 604/20 |
Current CPC
Class: |
A61P 35/00 20180101;
C12N 2830/002 20130101; A61K 48/00 20130101; C12N 15/86 20130101;
C07K 14/54 20130101; C12N 15/85 20130101; C12N 2710/10343 20130101;
C07K 14/52 20130101; C12N 2830/85 20130101 |
Class at
Publication: |
514/044 ;
435/455; 604/020 |
International
Class: |
A61K 048/00; A61N
001/30; C12N 015/85 |
Claims
1-9. (canceled).
10. A method for the spatial and temporal control of the expression
of a genetically engineered gene of interest operably linked to a
heat shock promoter in host cells within a preselected region of a
mammal, comprising: selectively heating the region to non-lethal
supraphysiological temperatures for a period of time by applying
electromagnetic radiation to the region thereby spatially and
temporally controlling the expression of the gene of interest.
11. The method of claim 10, wherein the heat shock gene promoter is
the hsp70 promoter.
12. The method of claim 11, wherein the temperature distribution of
the region by is monitored by MRI temperature imaging; and the
heating is adjusted according to the monitoring so as to provide
from about a 3.degree. C. increase to about a 5.degree. C. increase
in the temperature of the region.
13. The method of claim 10, wherein the electromagnetic radiation
is microwave radiation.
14. The method of claim 10, wherein the electromagnetic radiation
is infrared radiation.
15. The method of claim 10, wherein the electromagnetic radiation
is radiofrequency radiation.
16. The method of claim 11, wherein the heat shock gene promoter is
the human hsp70B promoter and the mammal is a human.
17. The method of claim 10, wherein the temperature distribution of
the region is monitored by MRI temperature imaging; and the heating
is adjusted according to the monitoring so as to provide up to an
8.degree. C. temperature in the focal region.
18. A method of treating a mammal, the method comprising:
introducing a genetically engineered gene operably linked to a heat
shock promoter into host cells of a region of the mammal;
selectively heating the region to non-lethal supraphysiological
temperatures by applying electromagnetic radiation for a period of
time to the region; thereby spatially and temporally controlling
the expression of the gene and the amount of the protein produced
in the region.
19. The method of claim 18, wherein the heat shock gene promoter is
the hsp70 promoter.
20. The method of claim 19, wherein the heat shock gene promoter is
the human 70B promoter and the mammal is a human.
21. The method of claim 18, wherein the temperature distribution of
the region by is monitored by MRI temperature imaging; and the
heating is adjusted according to the monitoring so as to provide
from about a 3.degree. C. increase to about a 5.degree. C. increase
in the temperature of the region.
22. The method of claim 18, wherein the temperature distribution of
the region by is monitored by MRI temperature the heating is
adjusted according to the monitoring so as to provide up to an
8.degree. C. temperature in the focal region.
23. The method of claim 18, wherein the gene encodes cytosine
deaminase, interleukins 1-10, vascular endothelial growth factor
(VEGF), fibroblast growth factor (FGF), and platelet derived growth
factor (PDGF).
24. The method of claim 18, wherein the region comprises part of a
tumor.
25. The method of claim 23, wherein the tumor is a member of the
group consisting of prostate, glioma, ovarian, and mammary
tumors.
26. The method of claim 19, wherein the period of time is about 15
minutes.
27. The method of claim 18, wherein the electromagnetic radiation
is microwave radiation.
28. The method of claim 18, wherein the electromagnetic radiation
is infrared radiation.
29. The method of claim 18, wherein the electromagnetic radiation
is radiofrequency radiation.
30. The method of claim 18, wherein the gene encodes a wild-type
mammalian protein deficient in the mammal.
Description
BACKGROUND OF THE INVENTION
[0001] A. Field of the Invention
[0002] The present invention relates to the spatial and temporal
control of exogenous gene expression in genetically engineered
cells and organisms. In particular, the invention relates to the
use of heat-inducible promoters such as the promoter of heat shock
genes to control the expression of exogenous genes. More
particularly, the invention relates to the use of focused
ultrasound to heat cells that contain therapeutic genes under the
control of a heat shock promoter, thereby inducing the expression
of the therapeutic gene.
[0003] B. Description of Related Art
[0004] Disorders caused by a malfunctioning gene can-be treated by
stably transferring an exogenous functional gene into a host cell,
so that the gene product of that gene is produced in the host cell.
Gene transfer may also be used to express in a host cell exogenous
nucleic acids that kill the host cell, or that encode gene products
that alter the phenotype of the host cell and/or the metabolic
state of surrounding cells, or that suppress the expression of
selected genes in the host cell. Human diseases are amenable to
treatment by this approach, particularly those diseases where the
defect is with a single gene. For discussions on the application of
gene therapy towards the treatment of genetic as well as acquired
diseases See Miller, A. D. (1992) Nature 357:455-460, and Mulligan,
R. C. (1993) Science 260:926-932, both incorporated herein by
reference.
[0005] In many instances, it is desirable to express genetically
engineered genes only in certain tissues, and/or at will only at
certain times, and/or only to a certain degree. However, current
gene transfer and exogenous gene expression protocols do not
provide adequate means of simultaneously controlling which cells in
a heterogeneous population are transformed, and when, where and to
what degree the transferred genes are expressed.
[0006] One approach for the control of exogenous gene expression
that has received a great deal of attention is to transform host
cells with a gene that is under the control of an inducible
promoter, and then to switch the transferred gene on and off at
will by activating the inducible promoter. Inducible promoters
include: the metallothionine IIA promoter, the lacZ, tac, and trp
promoters, the phage T7 promoter/T7 RNA polymerase, the Candida
albicans MAL2 gene promoter. Some promoters are heat inducible:
e.g., the lambda PL promoter with a C1857 repressor, heat shock
protein promoters.
[0007] Heat shock proteins ("hsps") are a ubiquitous class of
proteins produced in response to stress, notably heat stress, as
well as a variety of other external agents. All cells that have so
far been tested contain hsps, and many different hsps have been
identified in a wide range of organisms. Many hsps are designated
according to their molecular mass, (e.g. hsp7O refers to a 7O
kDalton hsp; hsp56, hsp28). Additional examples of hsps include
ubiquitin, crystallin, rapamycin, P-glycoprotein, and others. The
following articles describe the properties of heat shock genes and
promoters: Yost et al., (1990) TIG, 6:222-226. RNA metabolism:
strategies for regulation in the heat shock response. Pennier,
(1994) Biochemie, 76:737-747. Translational control during heat
shock; Minowada and Welch, (1995) "The Clinical implications of the
stress response", J. Clin. Invest. 95:3-12; Lis and Wu, (1993) Cell
74:1-4. Protein traffic on the heat shock promoter: parking,
stalling, and trucking along; Holbrook and Udelsman, "Heat shock
protein gene expression in response to physiological stress and
aging, n in THE BIOLOGY OF HEAT SHOCK PROTEINS AND MOLECULAR
CHAPERONES" (Morimoto et al., (1994) Editors, Plainview, N.Y.: Cold
Spring Harbor Laboratory Press, pp. 577-593); Macario, (1995),
"Heat-shock proteins and molecular chaperones: implications for
pathogenesis, diagnostics, and therapeutics," Int. J. Chem. Lab
Res. 25:59-70.
[0008] Hsps participate in and influence a large variety of
cellular effects, including the assembly of newly formed
polypeptides (some HSPs function as chaperones), signalling
functions (e.g. response to steroid hormones), protein excretion,
DNA and RNA synthesis (see below). The synthesis of proteins during
heat shock is generally inhibited during stress, except for the
synthesis of the hsps.
[0009] Heat shock (and other forms of stress) result in the almost
immediate transcriptional activation of heat shock genes. The heat
shock response is quite dramatic. The heat shock messages appear in
the cytoplasm generally within minutes, and the translation of
message is carried out with a very high efficiency. For example, in
Drosophila cells, hsp genes are induced within just four minutes
after a temperature elevation of 4 to 9.degree. C. Within one hour,
there are several thousand transcripts per cell. These transcripts
are actively translated into hsp while at the same time, the
transcription of previously active genes is severely repressed.
Miller and Ziskin (1990) Ultrasound Med. Biol. 15:707-22 reported
that short exposures to sharply elevated temperature result in a,
protective effect against further thermal insult, and that the
generation of heat shock proteins by cells coincides with the onset
of such "thermal protection. "The level of synthesis of hsp70 in
cells during heat shock appears to be linearly related to their
thermotolerance. Li, G. C. (1985) Int. J. Radiat. Oncol. Biol.
Phys. 11:165-177. Two human hsp70 proteins have been
described-hsp70A (Wu, B., et al. (1985) Mol. Cell. Biol. 5:330-341;
Hunt, C., and Morimoto, R. I. (1985) Proc. Natl. Acad. Sci. USA
82:6455-6459) and hsp70B (Schiller, P., et al. (1988) J. Mol. Biol.
203:97-105). For a review of hsps, see, e.g., Morimoto et al.,
eds., Stress Proteins in Biology and Medicine (1990) Cold Spring
Harbor Press; Hightower, L. E. (1991) Cell 66:191-197; Craig, E.
A., and Gross, C. A. (1991) Trends Bioch. Sci. 16:135.
[0010] The heat shock genes of many organisms have been mapped and
sequenced. Heat shock genes are scattered at various chromosomal
locations. A remarkable feature of these genes is the general
absence of any intervening sequences.
[0011] Heat shock promoters from different sources have been have
been isolated, sequenced and used to express a variety of genes.
For example, Dreano, M., et al. (1986) Gene 49:1-8, describe the
use of the human hsp70B promoter, as well as a Drosophila hsp70
promoter, to direct the heat regulated synthesis of human growth
hormone, chicken lysozyme and a human influenza hemagglutinin. EPA
Publication No. 336,523 (Dreano et al., published 11 Oct. 1989)
describes the in vivo expression of human growth hormone using a
human hsp70 promoter. PCT Publication No. WO 87/00861 (Bromley et
al., published 12 Feb. 1987) describes the use of human and
Drosophila hsp promoters having 5'-untranslated region variants.
EPA Publication No. 118,393 (Bromley et al., published 12 Sep.
1984) and PCT Publication No. WO 87/05935 (Bromley et al.,
published 8 Oct. 1987) describe the expression of E. coli
beta-galactosidase and human influenza hemagglutinin, using a
Drosophila hsp70 promoter. See also U.S. Pat. Nos. 4,990,607,
4,797,359, 5,521,084, and 5,447,858. Overexpression of hsp-70 has
been accomplished in transgenic mice. Plumeer et al., (1995) J.
Clin. Inv. 95:1854-1860. See also Yost and Lindquist, (1986) Cell
45:185-193, Yost and Lindquist, (1988) Science 242:1544-1548, Garbe
et al. (1986) PNAS, 83:1812-1816, Blackman et al. (1986) J. Mol.
Biol. 188:499-515, Bond et al. (1986) Mol. Cell. Biol.
12:4602-4610, Kay et al. (1987) Nucl. Acids Res. 15:3723-3741,
Bond, (1988) EMBO J. 7:3509-3518. The published sequences of these
promoters are hereby incorporated by reference.
[0012] The inducible promoter systems which have been used to
control the expression of proteins typically have one or more of
the following limitations: they are restricted to a relatively
narrow range of host cells, or are only partially inducible, or are
derived from organisms, such as tumor viruses, which are inherently
dangerous.
[0013] More importantly, their use does not allow for finely tuned
localized expression of exogenous genes in transformed cells.
"Among the design hurdles for all vectors are . . . to enable the
transferred gene to be regulated". Crystal (1995), "Transfer of
genes to humans: Early lessons and obstacles to success."
SUMMARY OF THE INVENTION
[0014] The present invention provides a method for selective
temporal and spatial control of gene expression, comprising the
steps of:
[0015] a) linking a therapeutic gene with a heat shock protein
promoter to yield a genetically engineered construct in which the
selected therapeutic gene is under the control of the hsp
promoter;
[0016] b) inserting the hsp promoter-therapeutic gene construct
into a suitable vector and introducing the vector into a target
cell or organism,
[0017] c) selectively heating a predetermined discrete region of a
cell mass or organism that includes cells that contain the vector
construct,
[0018] d) repeating step c as many times as necessary.
[0019] In a preferred embodiment, the local heating is accomplished
using focused ultrasound. A magnetic resonance imaging instrument
is used to visualize the target tissue and to quantify and finely
control the level of heating (i.e., temperature).
[0020] The invention also supplies a method of providing a
therapeutic protein to selected cells in a multicellular organism,
comprising:
[0021] introducing into cells of a multicellular organism a DNA
molecule having a heat shock promoter sequence operably linked to
and exerting regulatory control over a sequence encoding a
therapeutic protein, and
[0022] activating said heat shock promoter sequence through the
application of a focused ultrasound so that said cells express a
therapeutically effective amount of said therapeutic protein.
[0023] In a number of embodiments, the invention comprises methods
of treating cancer, to induce local angiogenesis, and to treat
genetic diseases. In some embodiments, the gene of interest is
selected from the group of genes that encode toxic molecules or
pro-molecules.
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1 is a graph of attenuation of radiation by human
tissue.
[0025] FIG. 2 depicts a prototypic adenovirus vector.
[0026] FIG. 3 depicts an hsp promoter. FIG. 3A depicts a minimal
Drosophila hsp70 promoter containing HSEII, HSEI, GAGA and TATA
elements. +1 refers to the transcription start site. FIG. 3C
depicts the nucleotide sequence of the human hsp70B promoter.
[0027] FIG. 4 depicts a human hsp promoter obtained from the
Genbank database.
[0028] FIG. 5(a) is an intensity image of the rat leg in the focal
plane showing, from bottom to top, the transducer (in part), the
water bath with a high signal intensity (in the middle the inclined
table with the aperture for FUS can be seen), and finally the rat
leg. FIG. 5(b) is a temperature change map for the same slice after
one minute of heating. FIG. 5(c) depticts the temperature change
after three minutes of heating. Thermal diffusion is apparent. This
state was maintained throughout the 45 minute heating interval.
Temperature images were obtained during FUS application.
[0029] FIG. 6 shows the results of a Northern blot of locally
heated rat leg. Lanes 5, 6, and 7 are of samples taken from the
ultrasound focal region. Lane 5 shows markedly higher expression of
RNA at about 2.3 kb (arrow). Differential expression between this
sample and the surrounding samples ranges from a factor of 3 to
67.
DETAILED DESCRIPTION OF THE INVENTION
[0030] A. Definitions
[0031] The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. Unless specifically limited, the term
encompasses nucleic acids containing known analogues of natural
nucleotides which have similar binding properties as the reference
nucleic acid and are metabolized in a manner similar to naturally
occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence also implicitly encompasses conservatively
modified variants thereof (e.g. degenerate codon substitutions) and
complementary sequences and as well as the sequence explicitly
indicated. The term nucleic acid is used interchangeably with gene,
cDNA, and mRNA encoded by a gene.
[0032] The phrase "exogenous" or "heterologous nucleic acid"
generally denotes a nucleic acid that has been isolated, cloned and
ligated to a nucleic acid with which it is not combined in nature,
and/or introduced into and/or expressed in a cell or cellular
environment other than the cell or cellular environment in which
said nucleic acid or protein may typically be found in nature. The
term encompasses both nucleic acids originally obtained from a
different organism or cell type than the cell type in which it is
expressed, and also nucleic acids that are obtained from the same
cell line as the cell line in which it is expressed.
[0033] The phrase "a nucleic acid sequence encoding" refers to a
nucleic acid which contains sequence information for a structural
RNA such as rRNA, a tRNA, or the primary amino acid sequence of a
specific protein or peptide, or a binding site for a trans-acting
regulatory agent. This phrase specifically encompasses degenerate
codons (i.e., different codons which encode a single amino acid) of
the native sequence or sequences which may be introduced to conform
with codon preference in a specific host cell.
[0034] The term "recombinant" or "engineered" when used with
reference to a nucleic acid or a protein generally denotes that the
composition or primary sequence of said nucleic acid or protein has
been altered from the naturally occurring sequence using
experimental manipulations well known to those skilled in the art.
It may also denote that a nucleic acid or protein has been isolated
and cloned into a vector, or a nucleic acid that has been
introduced into or expressed in a cell or cellular environment
other than the cell or cellular environment in which said nucleic
acid or protein may be found in nature.
[0035] The term "recombinant" or "engineered" when used with
reference to a cell indicates that the cell replicates or expresses
a nucleic acid, or produces a peptide or protein encoded by a
nucleic acid, whose origin is exogenous to the cell. Recombinant
cells can express nucleic acids that are not found within the
native (nonrecombinant) form of the cell. Recombinant cells can
also express nucleic acids found in the native form of the cell
wherein the nucleic acids are reintroduced into the cell by
artificial means.
[0036] A cell has been "transformed" by an exogenous nucleic acid
when such exogenous nucleic acid has been introduced inside the
cell membrane. Exogenous DNA may or may not be integrated
(covalently linked) into chromosomal DNA making up the genome of
the cell. The exogenous DNA may be maintained on an episomal
element, such as a plasmid. In eucaryotic cells, a stably
transformed cell is generally one in which the exogenous DNA has
become integrated into the chromosome so that it is inherited by
daughter cells through chromosome replication, or one which
includes stably maintained extrachromosomal plasmids. This
stability is demonstrated by the ability of the eucaryotic cell to
establish cell lines or clones comprised of a population of
daughter cells containing the exogenous DNA.
[0037] "Heat-inducible promoter." A promoter is a nucleic acid
sequence associated with a gene that controls the transcription of
the gene by interacting with mainly transacting ligands such as
polymerases, transcription factors, transcription enhancers and
transcription suppressors. Promoters can be either constitutive or
inducible. A constitutive promoter promotes the constant
transcription of a gene, whereas an inducible promoter's activity
fluctuates as determined by the presence (or absence) of a specific
inducer. The regulatory elements of an inducible promoter are
usually located further upstream of the transcriptional start site
than the TATA box. Ideally, an inducible promoter should possess
the following properties: a low to nonexistent basal level of
expression in the absence of inductive stimulus, a high level of
expression in the presence of inductive stimulus, and an induction
scheme that does not otherwise alter the physiology of the cell. A
heat inducible promoter is one that is activated by exposing cells
that contain the promoter to a defined temperature increase.
[0038] A "host cell" is a cell which has been transformed by an
exogenous DNA sequence. Unless otherwise specified, the host cell
may be a plant or animal cell.
[0039] The term "tumor cell" or "cancer cell" or "neoplastic cell"
denotes a cell that demonstrates inappropriate, unregulated
proliferation. A "human" tumor is comprised of cells that have
human chromosomes. Such tumors include those in a human patient,
and tumors resulting from the introduction into a nonhuman host
animal of a malignant cell line having human chromosomes into a
nonhuman host animal.
[0040] "Selectively heating" means that only cells having
predetermined spatial coordinates in an organism, tissue or cell
mass are directly heated by the heat source, whereas cells that are
outside of these coordinates, even though adjacent, are not
directly heated. Heating of adjacent cells that may occur by normal
heat equilibration between the selectively heated cells and the
adjacent cells is a consequence of selective heating.
[0041] The term "pro-molecule" refers to a substance that is itself
not metabolically active as administered, but that is activated
either when chemically altered or metabolized by cells capable of
altering or metabolizing the pro-molecule, or when combined with
one or more other substances to form a complex that is
metabolically active. A pro-molecule may, upon chemical alteration
or combination with a second substance, become toxic to the host
cell. Examples include 5-fluorocytosine ("5FC"), which can be
converted to the lethal metabolite 5-fluorouracil ("5FU");
5-methoxypurine arabinoside; and gancyclovir. Pro-molecules
preferably cause no substantially no ill effects to an organism
except to cells that are capable of converting the pro-drug to a
toxic product (i.e., metabolite or complex). "Pro-drug activating
molecule" denotes a molecule such as an enzyme that is capable of
metabolizing the non-toxic pro-drug to its toxic metabolite, or a
molecule that combines (covalently or non-covalently) with a
pro-drug to yield a toxic product.
[0042] A "toxic molecule" is a molecule that inhibits cell growth,
or inhibits certain metabolic pathways, in some instances killing
the cell. See, e.g., WO 93/24136.
[0043] The phrase "therapeutic dose" or "therapeutic amount" or
"effective amount" means a dosage sufficient to produce a desired
result. The desired result can be subjective or objective
improvement in the recipient of the dosage, a decrease or an
increase in the number of a target population of cells, a decrease
in tumor size, a decrease in the rate of growth of cancer cells, a
decrease in metastasis, or any combination of the above.
[0044] B. The Invention
[0045] In accordance with the present invention, a nucleic acid
comprising a heat-inducible promoter, preferably an hsp promoter,
is obtained, coupled by known genetic engineering techniques to a
selected gene, the construct is introduced into host cells, and a
subset of transformed cells that occupy selected spatial
coordinates are heated to activate the promoter and express the
gene.
[0046] 1. Heat Shock Proteins and Heat Shock Protein Promoters
[0047] A number of heat inducible promoters are known (e.g., the
lambda PL promoter; and may be used herein. The preferred heat
inducible promoter for use in the present invention is a heat shock
protein promoter.
[0048] A region known as the heat shock element (HSE), is found
within the first 100 bp 5' of the RNA start site of eucaryotic heat
shock genes. Sorger, P. K. (1991) Cell 65:363. This region includes
the sequence nGAAn, repeated at least two times in head-to-head or
tail-to-tail orientation (nGAAnnTTCn or nTTCnnGAAn). Hsp70 genes
from different species differ in the number and orientation of HSEs
and in the types of other factor-binding sites found upstream. The
HSE functions in stress induced promoter activation by binding a
positive transactivating factor, the heat shock factor (HSF). The
binding constant of this factor to the heat shock element is about
a hundred fold higher than that of any other known mammalian
transcription factor to its respective binding site, rendering this
promoter one of the strongest.
[0049] The primary site for the regulation of protein synthesis is
the initiation of the polypeptide chain. In particular, the
activity of elF-2, and elF-4F, both initiation factors, is
modulated during heat shock. A hsp-related protein Idnase is
thought to be involved in the regulation of initiation factors.
Hsp-70 is thought to be a heat sensor by detecting the accumulation
of denatured proteins, and the production of eIF-2 is thought to
limit protein production. In turn, the factor eIF-4F could be
involved in the preferential synthesis of the hsps.
[0050] The specific transcription factor activated during heat
shock is often referred to as HSF-1. Recent reviews summarize its
action. HSP-1 trimerizes during stress (mediated by hsp-70) and
then binds to a consensus nucleotide sequence (the heat shock
element (HSE), located within the promoter element of the hsp
genes.
[0051] Heat shock promoters not specifically described herein are
nonetheless within the scope of the present invention if they meet
the following criteria:
[0052] when recombined with a reporter gene to form a construct
that is then introduced into a host cell that is capable of
expressing the reporter gene, the reporter gene is minimally or not
at all expressed at normal physiologic temperatures,
[0053] but when the transformed host cell is exposed to non-lethal
supraphysiological temperatures, the reporter gene is expressed at
a level which is at least five times (preferably 10 times, and most
preferably at least 100 times) the level of expression at
physiological temperatures.
[0054] 2. Genetic Engineering Methods for Obtaining a Heat Shock
Promoter, Operably Linking it to a Selected Gene, and Expressing it
in Cells.
[0055] In brief summary, the expression of natural or synthetic
nucleic acids is typically achieved by operably linking a nucleic
acid of interest to a promoter (which is either constitutive or
inducible), incorporating the construct into an expression vector,
and introducing the vector into a suitable host cell. Typical
vectors contain transcription and translation terminators,
transcription and translation initiation sequences, and promoters
useful for regulation of the expression of the particular nucleic
acid. The vectors optionally comprise generic expression cassettes
containing at least one independent terminator sequence, sequences
permitting replication of the cassette in eukaryotes, or
prokaryotes, or both, (e.g., shuttle vectors) and selection markers
for both prokaryotic and eukaryotic systems. Vectors are suitable
for replication and integration in prokaryotes, eukaryotes, or
preferably both. See, Giliman and Smith (1979), Gene, 8:81-97;
Roberts et al. (1987), Nature, 328:731-734; Berger and Kimmel,
Guide to Molecular Cloning Techniques, Methods in Enzymology,
volume 152, Academic Press, Inc., San Diego, Calif. (Berger);
Sambrook et al. (1989), MOLECULAR CLONING--A LABORATORY MANUAL (2nd
ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor
Press, N.Y., (Sambrook); and F. M. Ausubel et al., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, eds., Current Protocols, a joint
venture between Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc., (1994 Supplement) (Ausubel). Product information
from manufacturers of biological reagents and experimental
equipment also provide information useful in known biological
methods. Such manufacturers include the SIGMA chemical company
(Saint Louis, Mo.), R&D systems (Minneapolis, Minn.), Pharmacia
LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc.
(Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company
(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life
Technologies, Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika
Analytika (Fluka Chemie AG, Buchs, Switzerland), and Applied
Biosystems (Foster City, Calif.), as well as many other commercial
sources known to one of skill.
[0056] a. Nucleic Acids
[0057] The nucleic acids (including, promoters, genes and vectors)
used in the present method can be isolated from natural sources,
obtained from such sources as ATCC or GenBank libraries, or
prepared by synthetic methods. Synthetic nucleic acids can be
prepared by a variety of solution or solid phase methods. Detailed
descriptions of the procedures for solid phase synthesis of nucleic
acids by phosphitetriester, phosphotriester, and H-phosphonate
chemistries are widely available. See, for example, Itakura, U.S.
Pat. No. 4,401,796; Caruthers, et al., U.S. Pat. Nos. 4,458,066 and
4,500,707; Beaucage, et al., (1981) Tetrahedron Lett.,
22:1859-1862; Matteucci, (1981) et al., J. Am. Chem. Soc.,
103:3185-3191; Caruthers, et al., (1982) Genetic Engineering,
4:1-17; Jones, chapter 2, Atkinson, et al., chapter 3, and Sproat,
et al., chapter 4, in Oligonucleotide Synthesis: A Practical
Approach, Gait (ed.), IRL Press, Washington D.C. (1984); Froehler,
et al., (1986) Tetrahedron Lett., 27:469-472; Froehler, et al.,
(1986) Nucleic Acids Res., 14:5399-5407; Sinha, et al. (1983)
Tetrahedron Lett., 24:5843-5846; and Sinha, et al., (1984) Nucl.
Acids Res., 12:4539-4557, which are incorporated herein by
reference.
[0058] b. Vectors
[0059] A number of vectors may be used to operably linked selected
nucleic acids to heat shock promoters and mediate their
replication, cloning and/or expression. "Cloning vectors" are
useful for replicating and amplifying the foreign nucleic acids and
obtaining clones of specific foreign nucleic acid-containing
vectors. "Expression vectors" mediate the expression of the foreign
nucleic acid. Some vectors are both cloning and expression
vectors.
[0060] In general, the particular vector used to transport a
foreign gene into the cell is not particularly critical. Any of the
conventional vectors used for expression in the chosen host cell
may be used.
[0061] An expression vector typically comprises a eukaryotic
transcription unit or "expression cassette" that contains all the
elements required for the expression of exogenous genes in
eukaryotic cells. A typical expression cassette contains a promoter
operably linked to the DNA sequence encoding a desired protein and
signals required for efficient polyadenylation of the
transcript.
[0062] Eukaryotic promoters typically contain two types of
recognition sequences, the TATA box and upstream promoter elements.
The TATA box, located 25-30 base pairs upstream of the
transcription initiation site, is thought to be involved in
directing RNA polymerase to begin RNA synthesis. The other upstream
promoter elements determine the rate at which transcription is
initiated.
[0063] Enhancer elements can stimulate transcription up to 1,000
fold from linked homologous or heterologous promoters. Enhancers
are active when placed downstream or upstream from the
transcription initiation site. Many enhancer elements derived from
viruses have a broad host range and are active in a variety of
tissues. For example, the SV40 early gene enhancer is suitable for
many cell types. Other enhancer/promoter combinations that are
suitable for the present invention include those derived from
polyoma virus, human or murine cytomegalovirus, the long term
repeat from various retroviruses such as murine leukemia virus,
murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic
Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
1983, which is incorporated herein by reference.
[0064] In addition to a promoter sequence, the expression cassette
should also contain a transcription termination region downstream
of the structural gene to provide for efficient termination. The
termination region may be obtained from the same source as the
promoter sequence or may be obtained from a different source.
[0065] If the mRNA encoded by the selected structural gene is to be
efficiently translated, polyadenylation sequences are also commonly
added to the vector construct. Two distinct sequence elements are
required for accurate and efficient polyadenylation: GU or U rich
sequences located downstream from the polyadenylation site and a
highly conserved sequence of six nucleotides, AAUAAA, located 11-30
nucleotides upstream. Termination and polyadenylation signals that
are suitable for the present invention include those derived from
SV40, or a partial genomic copy of a gene already resident on the
expression-vector.
[0066] In addition to the elements already described, the
expression vector of the present invention may typically contain
other specialized elements intended to increase the level of
expression of cloned nucleic acids or to facilitate the
identification of cells that carry the transduced DNA. For
instance, a number of animal viruses contain DNA sequences that
promote the extra chromosomal replication of the viral genome in
permissive cell types. Plasmids bearing these viral replicons are
replicated episomally as long as the appropriate factors are
provided by genes either carried on the plasmid or with the genome
of the host cell.
[0067] The expression vectors of the present invention will
typically contain both prokaryotic sequences that facilitate the
cloning of the vector in bacteria as well as one or more eukaryotic
transcription units that are expressed only in eukaryotic cells,
such as mammalian cells. The prokaryotic sequences are preferably
chosen such that they do not interfere with the replication of the
DNA in eukaryotic cells.
[0068] Selected genes are normally be expressed when the DNA
sequence is functionally inserted into a vector. "Functionally
inserted" means that it is inserted in proper reading frame and
orientation and operably linked to proper regulatory elements.
Typically, a gene will be inserted downstream from a promoter and
will be followed by a stop codon, although production as a hybrid
protein followed by cleavage may be used, if desired.
[0069] The vector normally contains, in addition to the gene of
interest, one or more additional genes that encode selectable
markers. The selectable markers can be for positive selection
(e.g., the cells that express the marker gene survive whereas cells
that do not express the selected gene die; such genes can encode,
e.g., an antibiotic resistance) or for negative selection (e.g.,
the cells that express the marker gene die whereas cells that do
not express the selected gene survive; such genes can encode, e.g.,
cytosine deaminase).
[0070] While a variety of vectors may be used, it should be noted
that viral vectors such as retroviral vectors are useful for
modifying eukaryotic cells because of the high efficiency with
which the retroviral vectors transfect target cells and integrate
into the target cell genome. Additionally, the retroviruses
harboring the retroviral vector are capable of infecting cells from
a wide variety of tissues.
[0071] Retroviral vectors are produced by genetically manipulating
retroviruses. Retroviruses are called RNA viruses because the viral
genome is RNA. Upon infection, this genomic RNA is reverse
transcribed into a DNA copy which is integrated into the
chromosomal DNA of transduced cells with a high degree of stability
and efficiency. The integrated DNA copy is referred to as a
provirus and is inherited by daughter cells as is any other gene.
The wild type retroviral genome and the proviral DNA have three
genes: the gag, the pol and the env genes, which are flanked by two
long terminal repeat (LTR) sequences. The gag gene encodes the
internal structural (nucleocapsid) proteins; the pol gene encodes
the RNA directed DNA polymerase (reverse transcriptase); and the
env gene encodes viral envelope glycoproteins. The 5' and 3' LTRs
serve to promote transcription and polyadenylation of virion RNAs.
Adjacent to the 5' LTR are sequences necessary for reverse
transcription of the genome (the tRNA primer binding site) and for
efficient encapsulation of viral RNA into particles (the Psi site).
See Mulligan, R. C., (1983) In: Experimental Manipulation of Gene
Expression, M. Inouye (ed), 155-173; Mann, R., et al., (1983) Cell,
33:153-159; Cone, R. D. and R. C. Mulligan, (1984) Proceedings of
the National Academy of Sciences, U.S.A., 81:6349-6353.
[0072] Retroviral vectors are particularly useful for modifying
cells because of the high efficiency with which the retroviral
vectors transduce target cells and integrate into the target cell
genome. See Miller, A. D., (1992) supra. Retroviruses harboring the
retroviral vector are capable of infecting dividing cells from a
wide variety of tissues. These vectors have the ability to stably
integrate the transferred gene sequences into the chromosomal DNA
of target cells.
[0073] The design of retroviral vectors is well known to one of
skill in the art. See Singer, M. and Berg, P. supra. In brief, if
the sequences necessary for encapsidation (or packaging of
retroviral RNA into infectious virions) are missing from the viral
genome, the result is a cis acting defect which prevents
encapsidation of genomic RNA. However, the resulting mutant is
still capable of directing the synthesis of all virion proteins.
Retroviral genomes from which these sequences have been deleted, as
well as cell lines containing the mutant genome stably integrated
into the chromosome are well known in the art and are used to
construct retroviral vectors. Preparation of retroviral vectors and
their uses are described in many publications including European
Patent Application EPA 0 178 220, U.S. Pat. No. 4,405,712, Gilboa,
(1986) Biotechniques 4:504-512; Mann, et al., (1983) Cell
33:153-159; Cone and Mulligan, (1984) Proc. Natl. Acad. Sci. USA
81:6349-6353; Eglitis, M. A, et al. (1988) Biotechniques 6:608-614;
Miller, A. D. et al. (1989) Biotechniques 7:981-990, Miller, A. D.
(1992) Nature, supra, Mulligan, R. C. (1993), supra. and Gould, B.
et al., and International Patent Application No. WO 92/07943
entitled "Retroviral Vectors Useful in Gene Therapy". The teachings
of these patents and publications are incorporated herein by
reference.
[0074] In addition to the retroviral vectors mentioned above, cells
may be transformed with adenoviruses or adeno-associated viral
vectors. See, e.g., Methods in Enzymology, Vol. 185, Academic
Press, Inc., San Diego, Calif. (D. V. Goeddel, ed.) (1990) or M.
Krieger (1990), Gene Transfer and Expression--A Laboratory Manual,
Stockton Press, New York, N.Y., and the references cited therein.
Adenoviruses are double stranded, linear DNA viruses that cause the
"common cold", pneumonia, conjunctivitis and other illnesses. There
are 42 serotypes of adenovirus known to infect humans.
[0075] Adenoviruses typically enter cells by receptor-mediated
endocytosis. The specific receptor is unknown. Following
internalization, the genome of the vector likely does not integrate
into the host genome but instead functions episomally. This leads
to only transient gene expression and also avoids random genome
integration and its potential problems such as induced
tumorigenicity.
[0076] Adeno-associated viruses (AAVs) require helper viruses such
as adenovirus or herpes virus to achieve productive infection. In
the absence of helper virus functions, AAV integrates
(site-specifically) into a host cell's genome, but the integrated
AAV genome has no pathogenic effect. The integration step allows
the AAV genome to remain genetically intact until the host is
exposed to the appropriate environmental conditions (e.g., a lytic
helper virus), whereupon it re-enters the lytic life-cycle.
Samulski (1993), Current Opinion in Genetic and Development,
3:74-80, and the references cited therein provides an overview of
the AAV life cycle. See also West et al. (1987), Virology,
160:38-47; Carter et al. (1989), U.S. Pat. No. 4,797,368; Carter et
al. (1993), WO 93/24641; Kotin (1994), Human Gene Therapy,
5:793-801; Muzyczka (1994), J. Clin. Invest., 94:1351 and Samulski,
supra, for an overview of AAV vectors.
[0077] Recombinant AAV vectors (rAAV vectors) deliver foreign
nucleic acids to a wide range of mammalian cells (Hermonat &
Muzycka (1984) Proc Natl Acad Sci USA 81:6466-6470; Tratschin et
al. (1985) Mol Cell Biol 5:3251-3260), integrate into the host
chromosome (Mclaughlin et al. (1988) J Virol 62: 1963-1973), and
show stable expression of the transgene in cell and animal models
(Flotte et al. (1993) Proc Natl Acad Sci USA 90:10613-10617).
Moreover, unlike retroviral vectors, rAAV vectors are able to
infect non-dividing cells (Podsakoff et al. (1994) J Virol
68:5656-66; Flotte et al. (1994) Am. J. Respir. Cell Mol. Biol.
11:517-521). Further advantages of rAAV vectors include the lack of
an intrinsic strong promoter, thus avoiding possible activation of
downstream cellular sequences, and their naked icosahedral capsid
structure, which renders them stable and easy to concentrate by
common laboratory techniques.
[0078] rAAV vectors have several properties which make them
preferred gene delivery systems in clinical settings. They have no
known mode of pathogenesis and 80% of people in the United States
are currently seropositive for AAV (Blacklow et al. (1971) J Natl
Cancer Inst 40:319-327; Blacklow et al. (1971) Am J Epidemiol
94:359-366). Because rAAV vectors have little or no endogenous
promoter activity, specific promoters may be used, depending on
target cell type. rAAV vectors can be purified and concentrated so
that multiplicities of infection exceeding 1.0 can be used in
transduction experiments. This allows virtually 100% of the target
cells in a culture to be transduced, eliminating the need for
selection of transduced cells.
[0079] Plasmids designed for producing recombinant vaccinia, such
as pGS62, (Langford, C. L. et al. (1986), Mol. Cell. Biol.,
6:3191-3199) may also be used. Finally, nonpathogenic vectors
derived from HIV have been reported to transform non-dividing
cells, and may also be used.
[0080] Whatever the vector is used, generally the vector is
genetically engineered to contain, in expressible form, a gene of
interest. The particular gene selected will depend on the intended
treatment. Examples of such genes of interest are described below
at Section e below.
[0081] The vectors further usually comprise selectable markers
which result in nucleic acid amplification such as the sodium,
potassium ATPase, thymidine kinase, aminoglycoside
phosphotransferase, hygromycin B phosphotransferase,
xanthine-guanine phosphoribosyl transferase, CAD (carbamyl
phosphate synthetase, aspartate transcarbamylase, and
dihydroorotase), adenosine deaminase, dihydrofolate reductase, and
asparagine synthetase and ouabain selection. Alternatively, high
yield expression systems not involving nucleic acid amplification
are also suitable, such as using a baculovirus vector in insect
cells.
[0082] When nucleic acids other than plasmids are used the nucleic
acids can contain nucleic acid analogs, for example, the antisense
derivatives described in a review by Stein, et al., (1993) Science
261:1004-1011, and in U.S. Pat. Nos. 5,264,423 and 5,276,019, the
disclosures of which are incorporated herein by reference.
[0083] C. In Vitro Gene Transfer
[0084] There are several well-known methods of introducing nucleic
acids into animal cells, any of which may be used in the present
invention. These include: calcium phosphate precipitation, fusion
of the recipient cells with bacterial protoplasts containing the
DNA, treatment of the recipient cells with liposomes containing the
DNA, DEAE dextran, receptor-mediated endocytosis, electroporation,
micro-injection of the DNA directly into the cells, infection with
viral vectors, etc.
[0085] For in vitro applications, the delivery of nucleic acids can
be to any cell grown in culture, whether of plant or animal origin,
vertebrate or invertebrate, and of any tissue or type. In preferred
embodiments, the cells will be animal cells, more preferably
mammalian cells, and most preferably human cells.
[0086] Contact between the cells and the genetically engineered
nucleic acid constructs, when carried out in vitro, takes place in
a biologically compatible medium. The concentration of nucleic acid
varies widely depending on the particular application, but is
generally between about 1 .mu.mol and about 10 mmol. Treatment of
the cells with the nucleic acid is generally carried out at
physiological temperatures (about 37.degree. C.) for periods of
time of from about 1 to 48 hours, preferably of from about 2 to 4
hours.
[0087] In one group of preferred embodiments, a nucleic acid is
added to 60-80% confluent plated cells having a cell density of
from about 10.sup.3 to about 10.sup.5 cells/mL, more preferably
about 2.times.10.sup.4 cells/mL. The concentration of the
suspension added to the cells is preferably of from about 0.01 to
0.2 .mu.g/mL, more preferably about 0.1 .mu.g/mL.
[0088] d. In Vivo Gene Transfer
[0089] Alternatively, the compositions of the present invention can
also be used for the in vivo gene transfer, using methods which are
known to those of skill in the art. The insertion of genes into
cells for the purpose of medicinal therapy is a rapidly growing
field in medicine which has enormous clinical potential. Research
in gene therapy has been on-going for several years, and has
entered human clinical trials. Zhu, et al., (1993) Science
261:209-211, incorporated herein by reference, describes the
intravenous delivery of cytomegalovirus (CMV)-chloramphenicol
acetyltransferase (CAT) expression plasmid using DOTMA-DOPE
complexes. Hyde, et al., (1993) Nature 362:250-256, incorporated
herein by reference, describes the delivery of the cystic fibrosis
transmembrane conductance regulator (CFTR) gene to epithelia of the
airway and to alveoli in the lung of mice, using liposomes.
Brigham, et al., (1989) Am. J. Med. Sci. 298:278-281, incorporated
herein by reference, describes the in vivo transfection of lungs of
mice with a functioning prokaryotic gene encoding the intracellular
enzyme chloramphenicol acetyltransferase (CAT).
[0090] For in vivo administration, the pharmaceutical compositions
are preferably administered parenterally, i.e., intraarticularly,
intravenously, intraperitoneally, subcutaneously, or
intramuscularly. More preferably, the pharmaceutical compositions
are administered intravenously or intraperitoneally by a bolus
injection. For example, see Stadler, et al., U.S. Pat. No.
5,286,634, which is incorporated herein by reference. Intracellular
nucleic acid delivery has also been discussed in Straubringer, et
al., (1983) METHODS IN ENZYMOLOGY, Academic Press, New York.
101:512-527; Mannino, et al., (1988) Biotechniques 6:682-690;
Nicolau, et al., (1989) Crit. Rev. Ther. Drug Carrier Syst.
6:239-271, and Behr, (1993) Acc. Chem. Res. 26:274-278. Still other
methods of administering therapeutics are described in, for
example, Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat.
No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871;
Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat. No.
4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.
[0091] In certain embodiments, the pharmaceutical preparations may
be contacted with the target tissue by direct application of the
preparation to the tissue. The application may be made by topical,
"open" or "closed" procedures. By "topical", it is meant the direct
application of the pharmaceutical preparation to a tissue exposed
to the environment, such as the skin, oropharynx, external auditory
canal, and the like. "Open" procedures are those procedures which
include incising the skin of a patient and directly visualizing the
underlying tissue to which the pharmaceutical preparations are
applied. This is generally accomplished by a surgical procedure,
such as a thoracotomy to access the lungs, abdominal laparotomy to
access abdominal viscera, or other direct surgical approach to the
target tissue. "Closed" procedures are invasive procedures in which
the internal target tissues are not directly visualized, but
accessed via inserting instruments through small wounds in the
skin. For example, the preparations may be administered to the
peritoneum by needle lavage. Likewise, the pharmaceutical
preparations may be administered to the meninges or spinal cord by
infusion during a lumbar puncture followed by appropriate
positioning of the patient as commonly practiced for spinal
anesthesia or metrazamide imaging of the spinal cord.
Alternatively, the preparations may be administered through
endoscopic devices.
[0092] The nucleic acid can also be administered in an aerosol
inhaled into the lungs (see, Brigham, et al., (1989) Am. J. Sci.
298(4):278-281 or by direct injection at the site of disease
(Culver, (1994) HUMAN GENE THERAPY, MaryAnn Liebert, Inc.,
Publishers, New York. pp.70-71).
[0093] The methods of the present invention may be practiced in a
variety of hosts. Preferred hosts include mammalian species, such
as humans, non-human primates, dogs, cats, cattle, horses, sheep,
and the like.
[0094] The amount of nucleic acid administered will depend upon the
particular nucleic acid used, the disease state being diagnosed;
the age, weight, and condition of the patient and the judgement of
the clinician; but will generally be between about 0.01 and about
50 mg per kilogram of body weight; preferably between about 0.1 and
about 5 mg/kg of body weight or about 10.sup.8-10.sup.10 particles
per injection.
[0095] For in vivo gene transfer, pharmaceutical compositions
comprising selected vectors containing selected nucleic acids are
preferably administered parenterally, i.e., intraarticularly,
intravenously, intraperitoneally, subcutaneously, or
intramuscularly. More preferably, the pharmaceutical compositions
are administered intravenously or intraperitoneally by a bolus
injection. For example, see Stadler, et al., U.S. Pat. No.
5,286,634, which is incorporated herein by reference. Intracellular
nucleic acid delivery has also been discussed in Straubringer, et
al., (1983) METHODS IN ENZYMOLOGY, Academic Press, New York.
101:512-527; Mannino, et al., (1988) Biotechniques 6:682-690);
Nicolau, et al., (1989) Crit. Rev. Ther. Drug Carrier Syst.
6:239-271, and Behr, (1993) Acc. Chem. Res. 26:274-278. Still other
methods of administering therapeutics are described in, for
example, Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat.
No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871;
Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat. No.
4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.
[0096] Formulations suitable for administration include aqueous and
non-aqueous, isotonic sterile injection solutions, which
can-contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic with the blood of the intended
recipient, and aqueous and non-aqueous sterile suspensions that can
include suspending agents, solubilizers, thickening agents,
stabilizers, and preservatives. The formulations of packaged
nucleic acid can be presented in unit-dose or multi-dose sealed
containers, such as ampules and vials. Injection solutions and
suspensions can be prepared from sterile powders, granules, and
tablets of the kind previously described.
[0097] A vector dose which is sufficient to effect a beneficial
therapeutic response in the patient over time, or to inhibit
infection by a pathogen, is administered to a patient. A
therapeutically effective dose is an amount sufficient to cure, or
at least partially arrest, the symptoms of the disease and its
complications. Effective doses of the compositions of the present
invention, for the treatment of the above described conditions will
vary depending upon many different factors, including means of
administration, target site, physiological state of the patient,
and other medicants administered. Thus, treatment dosages will need
to be titrated to optimize safety and efficacy. In determining the
effective amount of the vector to be administered, the physician
evaluates the particular nucleic acid used, the disease state being
diagnosed; the age, weight, and condition of the patient,
circulating plasma levels, vector toxicities, progression of the
disease, and the production of anti-vector antibodies. The size of
the dose also will be determined by the existence, nature, and
extent of any adverse side-effects that accompany the
administration of a particular vector. Doses ranging from about 10
ng to 1 g, 100 ng to 100 mg, 1 .mu.g to 10 mg, or 30-300 .mu.g DNA
per patient are typical. Doses generally range between about 0.01
and about 50 mg per kilogram of body weight; preferably between
about 0.1 and about 5 mg/kg of body weight or about
10.sup.8-10.sup.10 particles per injection. In general, the dose
equivalent of a naked nucleic acid from a vector is from about 1
.mu.g to 100 .mu.g for a typical 70 kilogram patient, and doses of
vectors which include a retroviral particle are calculated to yield
an equivalent amount of inhibitor nucleic acid.
[0098] Prior to infusion, blood samples are obtained and saved for
analysis. Between 10.sup.8 and 1.times.10.sup.12 vectors are
infused intravenously over 60-200 minutes. Vital signs and oxygen
saturation by pulse oximetry are closely monitored. Blood samples
are obtained 5 minutes and 1 hour following infusion and saved for
subsequent analysis. At the physician's discretion, reinfusion is
repeated every 2 to 3 months for a total of 4 to 6 treatments in a
one year period. After the first treatment, infusions can be
performed on a outpatient basis at the discretion of the clinician.
If the reinfusion is given as an outpatient, the participant is
monitored for at least 4, and preferably 8 hours following the,
therapy.
[0099] If a patient undergoing infusion of a vector or transduced
cell develops fevers, chills, or muscle aches, he/she receives the
appropriate dose of aspirin, ibuprofen or acetaminophen. Patients
who experience reactions to the infusion such as fever, muscle
aches, and chills are premedicated 30 minutes prior to the future
infusions with either aspirin, acetaminophen, or diphenhydramine.
Meperidine is used for more severe chills and muscle aches that do
not quickly respond to antipyretics and antihistamines. Vector
infusion is slowed or discontinued depending upon the severity of
the reaction.
[0100] e. Expression of a Functional Copy of a Gene
[0101] Some methods of gene therapy serve to compensate for a
defect in an endogenous gene by integrating a functional copy of
the gene into the host chromosome. The inserted gene replicates
with the host DNA and is expressed at a level to compensate for the
defective gene. Diseases amenable to treatment by this approach are
often characterized by recessive mutations. That is, both copies of
an endogenous gene must be defective for symptoms to appear. Such
diseases include, for example, cystic fibrosis, sickle cell anemia,
.beta.-thalassemia, phenylketonuria, galactosemia, Wilson's
disease, hemochromatosis, severe combined immunodeficiency disease,
alpha-1-antitrypsin deficiency, albinism, alkaptonuria, lysosomal
storage diseases, Ehlers-Danlos syndrome, hemophilia,
glucose-6-phosphate dehydrogenase deficiency, agammaglobulimenia,
diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy,
Wiskott-Aldrich syndrome, Fabry's disease, fragile X-syndrome, and
the like. Other recessive mutations are known in the art, and the
use of the methods of the present invention to treat them is
contemplated herein.
[0102] There are several methods for introducing an exogenous
functional gene to compensate for the above genetic defects. In one
approach, cells are removed from a patient suffering from the
disease and contacted with a vector in vitro. Cells should be
removed from a tissue type in which disease symptoms are
manifested. If the cells are capable of replication, and the vector
used includes a selective marker, cells having internalized and
expressed the marker can be selected. Particularly if selection is
not performed, it is important that the frequency of gene transfer
into cells be high, for example, at least about 1, 5, 10, 25 or 50%
of cells.
[0103] After integration of the vector into the cellular genome,
and optionally, selection, cells are reintroduced into the patient.
In this application, and others discussed below (except
site-specific recombination to correct dominant mutations), it is
not necessary that the gene supplied be delivered to the same site
as is occupied by the defective gene for which it is
compensating.
[0104] Alternatively, the nucleic acid can be introduced directly
into a patient as a pharmaceutical composition. The complex is
delivered to the tissue(s) affected by the genetic disorder being
treated in a therapeutically effective dose. In this and other
methods, a therapeutically effective dose is an amount sufficient
to cure, or at least partially arrest, the symptoms of the disease
and its complications. Effective doses of the compositions of the
present invention, for the treatment of the above described
conditions will vary depending upon many different factors,
including means of administration, target site, physiological state
of the patient, and other medicants administered. Thus, treatment
dosages will need to be titrated to optimize safety and efficacy.
Doses ranging from about 10 ng to 1 g, 100 ng to 100 mg, 1 .mu.g to
10 mg, or 30-300 .mu.g DNA per patient are typical. Routes of
administration include oral, nasal, gastric, intravenous,
intradermal and intramuscular.
[0105] i. Stem Cell Therapy
[0106] The nucleic acids can also be used to transfect embryonic
stem cells or zygotes to achieve germline alterations. See
Jaenisch, (1988) Science, 240:468-1474; Gordon et al. (1984)
Methods Enzymol. 101:414; Hogan et al., (1986) Manipulation of the
Mouse Embryo: A Laboratory Manual, C.S.H.L. N.Y.; and Hammer et al.
(1985) Nature 315:680; Gandolfi et al. (1987) J. Reprod. Fert.
81:23-28; Rexroad et al. (1988) J. Anim. Sci. 66:947-953 and
Eyestone et al. (1989) J. Reprod. Fert. 85:715-720; Camous et al.
(1984) J. Reprod. Fert. 72:779-785; Heyman et al. (1987)
Theriogenology 27:5968. However, these methods are presently more
suitable for veterinary applications that human treatment due to
ethical and regulatory constraints in manipulating human
embryos.
[0107] As an example, cystic fibrosis (CF) is a usually fatal
recessive genetic disease, having a high incidence in Caucasian
populations. The gene responsible for this disease was isolated by
Riordan et al, (1989) Science 245:1059-1065. It encodes a protein
called the cystic fibrosis transmembrane conductance regulator
(CFTR) which is involved in the transfer of chloride ions
(Cl.sup.-) through epithelial cell membranes. Mutations in the gene
cause defects of Cl.sup.-- secretion in epithelial cells leading to
the various clinical manifestations. Although CF has a number of
symptoms including thickened exocrine gland secretions, pancreatic
deficiency, intestinal blockage and malabsorption of fat, the most
serious factor affecting mortality is chronic lung disease.
Accordingly, to treat a CF patient, a vector containing a coding
sequence for a functional CFTR gene product can be introduced into
the patient via nasal administration so that the nucleic acid
composition reaches the lungs. The dose of vector is preferably
about 10.sup.8-10.sup.10 particles.
[0108] As another example, defects in the .alpha. or .gamma. globin
genes (see McDonagh & Nienhuis in Hematology of Infancy and
Childhood (eds. Nathan & Oski, Saunders, Pa., 1992) at pp.
783-879) can be compensated for by ex vivo treatment of hemopoietic
stem cells with a nucleic acid containing a functional copy of the
gene. The gene integrates into the stem cells which are then
reintroduced into the patient. Defects in the gene responsible for
Fanconi Anemia Complement Group C can be treated by an analogous
strategy (see Walsh et al., (1994) J. Clin. Invest.
94:1440-1448).
[0109] ii. Cancer Therapy
[0110] Other applications include the introduction of a functional
copy of a tumor suppressor gene into cancerous cell or cells at
risk of becoming cancerous. D. Pardoll, (1992) "Immunotherapy with
cytokine gene-transduced tumor cells: the next wave in gene therapy
for cancer", Curr. Opin. Oncol. 4:1124-1129; Uckert and Walther,
(1994) "Retrovirus-mediated gene transfer in cancer therapy",
Pharmac. Ther. 63:323-347; Individuals having defects in one or
both copies of an endogenous tumor suppressor gene are particularly
at risk of developing cancers. For example, Li-Fraumeni syndrome is
a hereditary condition in which individuals receive mutant p53
alleles, resulting in the early onset of various cancers (Harris,
(1993) Science 262:1980-1981, Frebourg et al., (1992) PNAS
89:6413-6417; Malkin et al., (1990) Science 250:1233). Expression
of a tumor suppressor gene in a cancerous dell or a cell at risk of
becoming cancerous is effective to prevent, arrest and/or reverse
cellular proliferation and other manifestations of the cancerous
state. Suitable tumor suppressor genes for use in the invention
include p53 (Buchman et al., (1988) Gene 70:245-252), APC, DCC, Rb,
WT1, and NF1 (Marx, (1993) Science 260:751-752; Marshall, (1991)
Cell 64:313-326). Nucleic acid constructs bearing a functional copy
of a tumor suppressor gene are usually administered in vivo by the
route most proximal to the intended site of action. For example,
skin cancers can be treated by topical administration and leukemia
by intravenous administration. The methods of the invention are
useful for treating a wide variety of cancers, among them prostate,
glyoma, ovarian, and mammary tumors.
[0111] iii. Angiogenesis Therapy
[0112] Local disruptions in blood flow, e.g. coronary artery
disease, peripheral arterial occlusive disease, and cerebral
vascular disease (stroke) are among the most common causes of
morbidity and mortality. These disorders are all caused by
insufficient tissue perfusion.
[0113] The discovery of polypeptides capable of stimulating
angiogenesis has led to several investigations towards the ability
to improve local perfusion based on such angiogenic factors, e.g.
vascular endothelial growth factor (VEGF), fibroblast growth factor
(FGF), platelet derived growth factor (PDGF). L.-Q. Pu et al.
(1993) Circulation, 88:1-147; Symes and Sniderman, (1994) Curr.
Opin. Lipidol. 5:305-312; see also U.S. Pat. Nos. 5,219,759,
5,512,545, 5,491,220, 5,464,943, 5,464,774, 5,360,896, 5,175,383,
5,155,214. For example, it has been reported that revascularization
occurs after intravenous administration of ECGF for 10 days.
Injection in a remote site does not lead to increased
vascularization. A dose dependency was clearly established.
Angiogenic factors are toxic when used in high concentration, so
local application appears necessary. VEGF appears a better choice
since it can be secreted and it has mitotic activity.
[0114] M. Hoeckel et al. (1993) Arch. Surg. 128:423-429, has listed
the following criteria for therapeutic use of an angiogenic
factor:
[0115] 1) Able to stimulate neovascularization
[0116] 2) Negligible local and systemic side effects
[0117] 3) High efficiency in nanomolar and picomolar range
[0118] 4) Demonstrated dose-response relation
[0119] 5) Chemically defined and easy to handle
[0120] 6) Can be produced on a large scale
[0121] Localized heat induction of genetically engineered cells
provides the localized expression of angiogenic factors, and
minimizes systemic effects.
[0122] f. Suppression of Gene Expression
[0123] Methods of gene therapy using the nucleic acid constructs of
the invention can also be used for prophylactic or therapeutic
treatment of patients or cells, infected with or at risk of being
infected with, a pathogenic microorganism, such as HIV. The
effectiveness of antisense molecules in blocking target gene
functions has been demonstrated in a number of different systems
(Friedman et al. (1988), Nature 335:452-54, Malim et al., (1989)
Cell 58:205-14 and Trono et al., (1989) Cell 59:113-20). The vector
used includes a DNA segment encoding an antisense transcript, which
is complementary to a segment of the gene. Where the gene is from a
pathogenic microorganism, it should preferably play an essential
role in the life cycle of the and should also be unique to the
microorganism (or at least absent from the genome of the patient
undergoing therapy). For example, suitable sites for inhibition on
the HIV virus includes TAR, REV or nef (Chatterjee et al., (1992)
Science 258:1485-1488). Rev is a regulatory RNA binding protein
that facilitates the export of unspliced HIV pre mRNA from the
nucleus. Malim et al., (1989) Nature 338:254. Tat is thought to be
a transcriptional activator that functions by binding a recognition
sequence in 5' flanking mRNA. Karn & Graeble, (1992) Trends
Genet. 8:365. The nucleic acid is introduced into leukocytes or
hemopoietic stem cells, either ex vivo or by intravenous injection
in a therapeutically effective dose. The treatment can be
administered prophylactically to HIV.sup.- person, or to persons
already infected with HIV.
[0124] g. Cells to be Transformed
[0125] The compositions and methods of the present invention are
used to transfer genes into a wide variety of cell types, in vivo
and in vitro. Among those most often targeted for gene therapy are
precursor (stem) cells, especially hematopoietic stem cells. Other
cells include those of which a proportion of the targeted cells are
nondividing or slow dividing. These include, for example,
fibroblasts, keratinocytes, endothelial cells, skeletal and smooth
muscle cells, osteoblasts, neurons, quiescent lymphocytes,
terminally differentiated cells, slow or non-cycling primary cells,
parenchymal cells, lymphoid cells, epithelial cells, bone cells,
etc. The methods and compositions can be employed with cells of a
wide variety of vertebrates, including mammals, and especially
those of veterinary importance, e.g, canine, feline, equine,
bovine, ovine, caprine, rodent, lagomorph, swine, etc., in addition
to human cell populations.
[0126] To the extent that tissue culture of cells may be required,
it is well known in the art. Freshney (1994) (Culture of Animal
Cells, a Manual of Basic Technique, third edition Wiley-Liss, New
York); Kuchler et al. (1977) Biochemical Methods in Cell Culture
and Virology, Kuchler, R. J., Dowden, Hutchinson and Ross, Inc.,
and the references cited therein provides a general guide to the
culture of cells. Cultured cell systems often will be in the form
of monolayers of cells, although cell suspensions are also
used.
[0127] Gene therapy relies on the efficient delivery of therapeutic
genes to target cells. Most of the somatic cells that have been
targeted for gene therapy, e.g., hematopoietic cells, skin
fibroblasts and keratinocytes, hepatocytes, endothelial cells,
muscle cells and lymphocytes, are normally non-dividing. Retroviral
vectors, which are the most widely used vectors for gene therapy,
unfortunately require cell division for effective transduction
(Miller et al., (1990) Mol. Cell. Biol. 10:4239-4242). This is also
true with other gene therapy vectors such as the adeno-associated
vectors (Russell et al., (1991) Proc. Natl. Acad. Sci. USA
91:8915-8919; Alexander et al., (1994) J. Virol. 68:8282-8287;
Srivastrava, (1994) Blood Cells 20:531-538). Recently, HIV-based
vectors has been reported to transfect non-dividing cells.
Nonetheless, the majority of stem cells, a preferred target for
many gene therapy treatments, are normally not proliferating. Thus,
the efficiency of transduction is often relatively low, and the
gene product may not be expressed in therapeutically or
prophylactically effective amounts. This has led investigators to
develop techniques such as stimulating the stem cells to
proliferate prior to or during gene transfer (e.g., by treatment
with growth factors) Pretreatment with 5-fluorouracil, infection in
the presence of cytokines, and extending the vector infection
period to increase the likelihood that stem cells are dividing
during infection, but these have met with limited success.
[0128] h. Detection of Foreign Nucleic Acids
[0129] After a given cell is transduced with a nucleic acid
construct that encodes a gene of interest under the control of a
hsp promoter, it is important to detect which cells or cell lines
express the gene product and to assess the level of expression of
the gene product in engineered cells. This requires the detection
of nucleic acids that encode the gene products.
[0130] Nucleic acids and proteins are detected and quantified
herein by any of a number of means well known to those of skill in
the art. These include analytic biochemical methods such as
spectrophotometry, radiography, electrophoresis, capillary
electrophoresis, high performance liquid chromatography (HPLC),
thin layer chromatography (TLC), hyperdiffusion chromatography, and
the like, and various immunological methods such as fluid or gel
precipitin reactions, immunodiffusion (single or double),
immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked
immunosorbent assays (ELISAs), immunofluorescent assays, and the
like. The detection of nucleic acids proceeds by well known methods
such as Southern analysis, northern analysis, gel electrophoresis,
PCR, radiolabeling, scintillation counting, and affinity
chromatography.
[0131] The selection of a nucleic acid hybridization format is not
critical. A variety of nucleic acid hybridization formats are known
to those skilled in the art. For example, common formats include
sandwich assays and competition or displacement assays.
Hybridization techniques are generally described in NUCLEIC ACID
HYBRIDIZATION, A PRACTICAL APPROACH, Ed. Hames, B. D. and Higgins,
S. J., IRL Press, 1985.
[0132] The sensitivity of the hybridization assays may be enhanced
through use of a nucleic acid amplification system which multiplies
the target nucleic acid being detected. In vitro amplification
techniques suitable for amplifying sequences for use as molecular
probes or for generating nucleic acid fragments for subsequent
subcloning are known. Examples of techniques sufficient to direct
persons of skill through such in vitro amplification methods,
including the polymerase chain reaction (PCR) the ligase chain
reaction (LCR), Q.beta.-replicase amplification and other RNA
polymerase mediated techniques (e.g., NASBA) are found in Berger,
Sambrook, and Ausubel, as well as Mullis et al. (1987), U.S. Pat.
No. 4,683,202; PCR Protocols A Guide to Methods and Applications
(Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990)
(Innis); Arnheim & Levinson (Oct. 1, 1990), C&EN 36-47; The
Journal Of NIH Research (1991), 3: 81-94; (Kwoh et al. (1989),
Proc. Natl. Acad. Sci. USA, 86:1173; Guatelli et al. (1990), Proc.
Natl. Acad. Sci. USA, 87:1874; Lomell et al. (1989), J. Clin.
Chem., 35:1826; Landegren et al. (1988), Science, 241:1077-1080;
Van Brunt (1990), Biotechnology, 8:291-294; Wu and Wallace (1989),
Gene, 4:560; Barringer et al. (1990), Gene, 89:117, and Sooknanan
and Malek (1995), Biotechnology, 13:563-564. Improved methods of
cloning in vitro amplified nucleic acids are described in Wallace
et al., U.S. Pat. No. 5,426,039. Other methods recently described
in the art are the nucleic acid sequence based amplification
(NASBA.TM., Cangene, Mississauga, Ontario) and Q Beta Replicase
systems. These systems can be used to directly identify mutants
where the PCR or LCR primers are designed to be extended or ligated
only when a select sequence is present. Alternatively, the select
sequences can be generally amplified using, for example,
nonspecific PCR primers and the amplified target region later
probed for a specific sequence indicative of a mutation.
[0133] Oligonucleotides for use as probes, e.g., in in vitro
amplification methods, for use as gene probes, or as inhibitor
components are typically synthesized chemically according to the
solid phase phosphoramidite triester method described by Beaucage
and Caruthers (1981), Tetrahedron Letts., 22(20):1859-1862, e.g.,
using an automated synthesizer, as described in Needham-Van
Devanter et al. (1984), Nucleic Acids Res., 12:6159-6168.
Purification of oligonucleotides, where necessary, is typically
performed by either native acrylamide gel electrophoresis or by
anion-exchange HPLC as described in Pearson and Regnier (1983), J.
Chrom., 255:137-149. The sequence of the synthetic oligonucleotides
can be verified using the chemical degradation method of Maxam and
Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New
York, Methods in Enzymology, 65:499-560.
[0134] An alternative means for determining the level of expression
of the gene is in situ hybridization. In situ hybridization assays
are well known and are generally described in Angerer et al.
(1987), Methods Enzymol., 152:649-660. In an in situ hybridization
assay cells are fixed to a solid support, typically a glass slide.
If DNA is to be probed, the cells are denatured with heat or
alkali. The cells are then contacted with a hybridization solution
at a moderate temperature to permit annealing of specific probes
that are labelled. The probes are preferably labelled with
radioisotopes or fluorescent reporters.
[0135] i. Detection of Foreign Gene Products
[0136] The expression of the gene of interest under the control of
a n hsp promoter to produce a product may be detected or quantified
by a variety of methods. Preferred methods involve the use of
specific antibodies.
[0137] Methods of producing polyclonal and monoclonal antibodies
are known to those of skill in the art. See, e.g., Coligan (1991),
CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N.Y.; and Harlow and
Lane (1989), ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor
Press, NY; Stites et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th
ed.) Lange Medical Publications, Los Altos, Calif., and references
cited therein; Goding (1986), MONOCLONAL ANTIBODIES: PRINCIPLES AND
PRACTICE (2d ed.) Academic Press, New York, N.Y.; and Kohler and
Milstein (1975), Nature, 256:495-497. Such techniques include
antibody preparation by selection of antibodies from libraries of
recombinant antibodies in phage or similar vectors. See, Huse et
al. (1989), Science, 246:1275-1281; and Ward et al. (1989), Nature,
341:544-546. Specific monoclonal and polyclonal antibodies and
antisera will usually bind with a K.sub.D of at least about 0.1 mM,
more usually at least about 1 .mu.M, preferably at least about 0.1
.mu.M or better, and most preferably, 0.01 .mu.M or better.
[0138] The presence of a desired polypeptide (including peptide,
transcript, or enzymatic digestion product) in a sample may be
detected and quantified using Western blot analysis. The technique
generally comprises separating sample products by gel
electrophoresis on the basis of molecular weight, transferring the
separated proteins to a suitable solid support, (such as a
nitrocellulose filter, a nylon filter, or derivatized nylon
filter), and incubating the sample with labeling antibodies that
specifically bind to the analyte protein. The labeling antibodies
specifically bind to analyte on the solid support. These antibodies
are directly labeled, or alternatively are subsequently detected
using labeling agents such as antibodies (e.g., labeled sheep
anti-mouse antibodies where the antibody to an analyte is a murine
antibody) that specifically bind to the labeling antibody.
[0139] 3. Heat Induction of the hsp Promoter
[0140] Central to the present invention is the ability to
selectively activate heat-inducible promoters using localized heat.
In particular, it is important to controllably heat cells within a
target area located within deep tissue while minimizing heating of
surrounding cells.
[0141] Localized heating of deep lying tissues can be accomplished
by invasive or noninvasive methods (without opening the skin).
Among the invasive methods, the introduction of a catheter with a
heated tip can be used. Alternatively, a catheter with an optical
guide can be used. A laser beam can then be directed through the
catheter to the targeted tissue and heat can be deposited using
direct radiation (for example using infrared light). Although
irradiation by laser has been proposed for heating deep tissue, its
use in medicine has been limited by optical absorption and thermal
diffusion.
[0142] In the preferred embodiment, local heating is achieved by
noninvasive means. FIG. 1, taken from Ernst et al., PRINCIPLES OF
NUCLEAR RADIATION IN ONE AND Two DIMENSIONS, Oxford University
Press, 1987, illustrates the attenuation of electromagnetic and
ultrasound radiation. For heating of deep lying tissues, a
transition area in FIG. 1 is used (since both absorption and
penetration are needed). The X-ray region of the spectrum uses
ionizing radiation which is hazardous. The radiofrequency region
has a wavelength of more than 10 cm. Acoustical radiation is
strongly absorbed for wavelengths below 1 mm. Since the ability to
focus is limited to approximately half the wavelength, a focus
diameter of 5 cm or more can be attained by radiofrequency. This is
generally not localized enough to treat small lesions. Ultrasound
can be applied with a short enough wavelength to be localized and
can penetrate deeply and is to some extent absorbed by body
tissues. Therefore, the preferred method for noninvasive local
heating is focused ultrasound.
[0143] It is known that ultrasound can be aimed at a defined target
area, and that prolonged exposure of living tissues to ultrasound
can raise the temperature of the exposed tissue. In particular
focused ultrasound has been known to be very effective to locally
heat tissue so long as there is an acoustic path from the surface
to the lesion free of air and bone. Lele, L. L. (1962) "A simple
method for production of trackless focal lesions with focused
ultrasound: physical factors," J. Physiol 160:494-512; Fry et al.,
(1978) "Tumor irradiation with intense ultrasound", Ultrasound Med.
Biol. 4:337-341. Using an array of ultrasound transducers with high
precision of heat deposition, focused ultrasound can be delivered
at high intensity to a defined very small area of deep tissue.
Focus of the ultrasound is achieved by the shape of the transducer
(spherical, parabolical) and/or by combining several different
transducer elements and combining their ultrasound waves with
individually adjusted phases in order to provide a focal spot. The
principles of ultrasound can be found in, for example, Bushberg J.
T. et al., THE ESSENTIAL PHYSICS OF MEDICAL IMAGING, Williams and
Wilkins, Baltimore, 1994, pp. 367-526.
[0144] In general, published studies have either sought to use
ultrasound to deliberately burn tissue, or to image tissues without
significantly raising their temperature. See, e.g., McAllister et
al. (1994) Teratology 51:191; Cline et al. (1992) "MR-guided
focused ultrasound surgery" J. Comp. Asst. Tomog. 16:956-965.
Angles et al. (1991) Teratology 42:285 reported that ultrasound can
activate heat shock genes in the absence of any detectable rise in
temperature. In U.S. Pat. No. 5,447,858, a soybean hsp promoter was
recombined with a heterologous gene, introduced into plant cells,
and the hsp promoter was activated using the "heat of day" (column
11, lines 15-25) or incubation at, for example, 42.5.degree. C.
(column 11, line 37). High intensity focused ultrasound has been
used to ablate tumors in animal models (Lele (1962), J. Physiol.
160:494-512; Fry et al. (1978), Ultrasound Med. Biol. 4:337-341)
and is a proposed surgical technique for treating liver tumors (ter
Harr et al. (1991), Phys. Med. Biol. 36:1495-1501; ter Harr et al.
(1991), Min. Invas. Ther. 1:13-19).
[0145] In contrast, the present invention sets out to deliberately
heat tissue within a target volume, but in a finely controlled
fashion within a defined range of temperatures. In the past,
several factors have limited the use of ultrasound to locally heat
tissue: 1) the inability to precisely pinpoint the exact location
of heat deposition due to interference near air/water, water/bone,
and fat/water boundaries, 2) the inability to precisely quantify
temperature elevation, and 3) the inability to simultaneously
visualize the target tissue and surrounding tissues to monitor
extent and effects of ultrasound heating. One possibility is to use
a combination of focused ultrasound and magnetic resonance imaging
(MRI). Cline et al. (1994) Magn. Reson. Med. 31:628-636, Cline et
al. (1995) J. Comp. Asst. Tom. 16:956-965, De Poorter (1995) Magn.
Reson. Med. 33:74-81.
[0146] It should be noted that the heat shock promoter may be
activated by phenomena other than ultrasound that can raise body
temperature (e.g., fever, hot shower, stress). Thus, it is
appropriate to stringently control these variables (closely monitor
a patient's temperature, avoid hot showers, avoid stress-producing
environments) during the duration of treatment. Another approach is
to limit the duration of the gene therapy.
[0147] In addition, heat can activate endogenous heat shock genes
under the control of endogenous hsp promoters. The interaction
between the processes will be studied.
[0148] 4. Imaging of Temperature
[0149] An element of the noninvasive use of focused ultrasound is
that one needs to assure that 1) the heated area corresponds with
the target tissue, and 2) the temperature elevation corresponds
with the target temperature. The first problem requires anatomical
visualization, and the second one visualization of temperature
distribution.
[0150] Whereas ultrasound can in principle be used for both
purposes, its precision is considered inadequate for this
purpose.
[0151] Cline et al., (1992) J. Comp. Asst. Tomog. 16:956-963,
described the combination of Magnetic Resonance Imaging (MRI) and
focused ultrasound, in which MRI is used to visualize and map the
target area and to visualize and map the temperature distribution.
The principles of MRI can be found in for example Stark D. D. and
Bradley, W. G., MAGNETIC RESONANCE IMAGING, Mosby Year Book, 1992,
pp. 1-521.
[0152] Imaging of temperature can be accomplished by MRI in three
ways: 1) using the spin-lattice (Tl) relaxation dependence on
temperature; 2) using the diffusion constant dependence of water on
temperature; and 3) using the Larmor-precession frequency
dependence of water protons on temperature. It is increasingly
clear that the third-method is the preferred method since it is
rather independent of most intra- and extracellular processes, and
it can be measured very rapidly in an imaging method. Cline et al.
(1994), "MR temperature mapping of focused ultrasound surgery,"
Magn. Reson. Med. 31:628-636; De Poorter et al. (1995),
"Noninvasive MRI thermometry with the proton resonance frequency
(PRF) method: In vivo results in human muscle", Magn. Reson. Med.
33:74-819; Hall et al. (1985), "Mapping of pH and temperature
distribution using chemicalshift-resolved tomography," J. Maqn.
Reson. 65:501-505 (J. de Zwart et al. (1996), J. Magn. Reson.
Series B, 112:86-90 and references therein).
[0153] Proton resonance frequency (PRF) depends on temperature. PRF
information is obtained from the phase shift in gradient echo
images. MRI Thermometry based on PRF shows little if any dependence
on intra- and extracellular composition. Imaging speed is essential
for two reasons: avoiding motion artifacts, and limiting effects of
thermal conduction on quantitation of temperature increase. In
order to minimize total imaging time, the time between successive
excitations (repetition time, "TR") should be short. However, echo
time ("TE") should be long to allow phase accumulation. Using this
technology, it is feasible to acquire 3D images within 5 seconds
with a spatial resolution of about 3-4 mm, and a temperature
accuracy of about 2 degrees C.
[0154] In the preferred embodiment, a MRI guided focused ultrasound
is used as is described in Cline et al., (1995) Magn. Reson.
Imaging, 194:731-737. In future embodiments, the single ultrasound
transducer under mechanical control described by Cline et al. will
preferably be replaced with an array of transducers under
electronic control to steer the focus electronically as described
by Fan, X. and Hynynen, (1995) Med. Phys. 22(3):297-306.
EXAMPLES
[0155] The following examples are offered solely for the purposes
of illustration, and are intended neither to limit nor to define
the invention.
Example 1
[0156] Example 1 describes the use of focused ultrasound (FUS)
guided by MRI to heat a region of a transformed human tumor having
preselected three-dimensional coordinates, wherein the heating
activates a genetically engineered therapeutical gene which is
under the control of an hsp70 heat shock promoter.
[0157] A. Materials and Methods
[0158] 1. Preparation of a Vector Containing an hsp Promoter
Operably Linked to a Therapeutic Gene
[0159] Vectors derived from adenovirus serotype 5 are used in this
example. S. L. Brody and R. G- Crystal (1994), "Adenovirus-Mediated
In Vivo Gene Transfer," Ann. N.Y. Acad. Sci. 716:90-101. The ElA
and Elb region are optionally deleted to prevent replication (see
FIG. 2). The E3 region is also optionally be deleted to provide a
7.5 kb region for exogenous DNA.
[0160] The human hsp-70B promoter is used, as it is strictly heat
regulated and can promote a several thousand fold increase in
expression upon induction (M. Dreano et al. (1986), "High level
heat-regulated synthesis of proteins in eukaryotic cells," Gene
49:1). The sequence of the human hsp-70B promoter is given in FIG.
3C, together with its analogs from Drosophila and changes in
promoter activity using insertions. Voelmy (1994), Crit. Rev. Euk.
Gene Exp. 4: 1357. HSEII and HSEI refer to the common heat shock
elements II and I respectively. The insertions can alter promoter
efficiency (R. Voellmy et al. (1994), "Transduction of the stress
signal and mechanisms of transcriptional regulation of heat
shock/stress protein gene expression in higher eukaryotes," Crit.
Rev. in Eukar. Gene Expr. 4:357-401). It should be noted here that
hsp-70B is not activated by adenovirus gene products (M. C. Simon
et al. (1987), "Selective induction of human heat shock gene
transcription by the adenovirus ElA products, including the 13S ElA
product," Mol. Cell Biol. 7:2884.
[0161] The vector is constructed by inserting into a vector a
cassette containing the human interleukin-2, as described in
Addison et al. (1995) Proc. Nat. Acad. Sci. U.S.A. 92: 8522-6
(other lymphokines such as IL-1, IL-4, tumor necrosis factor, etc.
may be used; see, e.g., U.S. Pat. No. 4,992,367, and Furutani et
al. (1986), Nuc. Acids Res. 14:3167-79) except that the gene will
be under the control of the human hsp70 promoter in place of the E1
or E3 position of the adenovirus type 5 genome. The E. coli LacZ
gene is optionally included in the vector as a reporter gene. Its
product, .beta.-galactosidase, can be easily detected and
quantified by its specific substrate. An SV40 polyadenylation is
optionally used together with a inverse terminal repeat (ITR) as an
encapsidation signal and enhancer (see FIG. 1). Construction of the
vector is accomplished using a plasmid containing the cassette and
the adenovirus type 5 sequences used for homologous recombination
with the El- or E3-adenovirus genomic DNA.
[0162] The modified adenovirus is grown in 293 cells, a transformed
human embryonic kidney call line that expresses El proteins,
providing (in case of a replication-deficient adenovirus) El
functions to allow for the production of virus. Viral vectors are
produced in titers of up to 10.sup.12 plaque forming units per
mL.
[0163] 2. Administration of the Vector to a Patient
[0164] The vector is administered systemically to a patient that
has been diagnosed as having a mammary adenocarcinoma. Preferably,
1 .mu.g to 100 .mu.g vector DNA are injected in 0.1-2 mls of a
saline solution directly into the tumor. Alternatively,
approximately 10 .mu.g to 1 mg vector DNA are intravenously
injected in 1-5 mls of a saline solution.
[0165] The presence and/or of the vector is determined by obtaining
a biopsy of the cancerous tissue and demonstrating the presence of
the gene or gene product by well known Northern, Southern or
Western blotting techniques, or by detecting the activity of the
optional reporter LacZ gene.
[0166] 3. Focused Ultrasound Heating
[0167] A patient is placed on a special bed (e.g., General Electric
Co., Milwaukee, Wis., as described in Cline et al. 1994 and 1995,
supra) and moved into the magnet of a magnetic resonance imaging
(MRI) instrument (e.g., 1.5T MR Imaging system by Signa, GE Medical
Systems, Milwaukee, Wis.). The MRI instrument is equipped with a
focused ultrasound (FUS) device (Specialty Engineering Associates,
Milpitas, Calif.) under computer control. Specifically, the FUS
device can be incorporated in the bed of the MRI in such a way that
the transducer can be freely moved under the patient with motional
freedom in the three principal directions to allow the focus to be
placed anywhere in the human body. Alternatively, the focus can be
adjusted electronically by using a more complicated FUS transducer,
a so-called phased array FUS transducer, in fact a combination of
multiple transducers that can be controlled individually by
electronic means thus allowing to move the focus. Acoustic contact
between the focus and the FUS transducer is assured using
appropriate water, gel, or other means giving an uninterrupted
acoustic path from transducer to focus. A Sparc 10 (Sun
Microsystems, Mountain View, Calif.) workstation interfaced to the
motor controls, the FUS pulse generator and the MR imaging system
is used to program, plan, monitor and control therapy. Cline et
al., supra, and Zwart et al., supra.
[0168] The area of the target is immobilized by gentle straps to
the bed. (Note that the more accelerated the procedure, the less
the need for immobilization; with very accelerated procedures
immobilization is unnecessary.)
[0169] Highly detailed MRI images are obtained with a suitable
contrast to determine accurately the computer coordinates of the
target (e.g. tumor, or ischemic area) as per standard MRI
procedures. Based on i) coordinates of the target, ii) estimates of
ultrasound attenuation, iii) acoustic impedance transitions in the
ultrasound paths, the focus, power and exposure time of the FUS
device are targeted to give an increase in temperature of three
degrees Celsius in approximately 10 seconds at the target.
[0170] The FUS device is switched on for 10 seconds. Immediately
following the FUS exposure, a rapid MRI temperature image is taken
as per the procedure outlined in J. de Zwart et al. (1996), J.
Magn. Reson. Series B, 112:86-90 and references therein). An
evaluation is made as to the following criteria: i) Does the heated
spot correspond with the target (comparison of anatomical MRI and
temperature MRI), and ii) Is the temperature elevation indeed 3
degrees Celsius (quantification of temperature, see J. de Zwart et
al. under 5)? If not, the FUS target is moved in the first case,
and the power is adjusted in the second case. The trial heating is
repeated until location and power correspond with the target. Note
that, since hsp-70B promoter activity is linearly proportional with
the duration, gene expression in this adjustment procedure is
limited because of its short duration.
[0171] Once power and focus have been adjusted, the therapeutic
dose of the ultrasound is delivered. For the hsp-70B promoter, an
elevation by 3 degrees for 15 minutes gives rise to very large
expression of the gene under hsp-70B control. Therefore, the
initial exposure is 15 minutes. It can be increased or decreased at
the discretion of the attending physician, taking into
consideration the severity of the condition treated, the condition
(age, health) of the patient, and the size and location of the
target area.
[0172] The patient is then removed from the MRI. Evaluation of
therapy is performed by clinical examination and regular follow-up
of detailed anatomical MRI to evaluate tumor shrinkage.
Example 2
[0173] Example 2 is performed using the methods described in
Example 1, except that the gene for vascular endothelial growth
factor (VEGF) is transferred into cells of tissues suffering from
ischemic damage. The adenoviral vector that contains the VEGF gene
is injected directly into the vicinity of the affected tissue and
or into blood vessels that directly feed into the affected tissues.
Gene expression is induced and monitored as described above.
Evaluation of therapy is by clinical examination and regular
follow-up of detailed anatomical MRI to evaluate healing of
ischemic tissue.
Example 3
[0174] Example 3 describes the use of focused ultrasound (FUS)
guided by MRI to heat a predetermined area of the thigh muscle and
activate endogenous heat shock genes in a rat model.
[0175] Harlan Sprague-Dawley rats (n=6) with a mass of 428.+-.48 g
were studied under an approved National Institutes of Health animal
protocol. The relatively large size of the rat ensures that the
ultrasound will focus deep within the biceps femoris muscle in the
rat's right hind leg.
[0176] A polycarbonate rat holder was constructed that contained
both the FUS transducer and an MR surface coil. The holder was
placed in a fiberglass tube that was partially filled with water.
The ultrasound passes through a 38 mm aperture in the platform
supporting the animal.
[0177] The holder was inclined to insure that the rat hind leg was
underwater while maintaining its head at a safe level above the
water. The leg was supported by four 2-0 braided polyester fiber
sutures forming a grid across the aperture. The FUS transducer was
positioned so that the focus was 5 mm into the rat thigh.
[0178] Earlier tests with high intensity FUS performed on rats and
on fresh chicken legs had indicated that focusing close to the skin
may cause burns to the skin. The rat was anesthetized with an
intraperitoneal administration of ketamine and xylazine. Three
additional steps were taken to minimize the reflection and
disruption of the ultrasound caused by acoustic impedance
mismatches at interfaces. First, the right leg was shaved front and
back with electric shears. Shaving also helped to reduce potential
sources of RNA degradation in the harvesting of the tissue. The
skin was then cleaned with alcohol swabs and wet with a surfactant
to reduce the retention of air microbubbles along the skin.
Finally, after the rat had been immersed in water, large air
bubbles trapped under the leg were released. The rat was securely
positioned on its right side on the platform, and care was taken to
insure that the focal region of the ultrasound was not close to any
bones in the leg. Respiration was monitored to maintain a proper
level of sedation. Body temperature was measured rectally with a
pair of fiber optic temperature probes (Luxtron Fluoroptic Model
SMM, Santa Clara, Calif.). Another pair of probes monitored the
temperature of the water bath. The bath temperature was controlled
by heat exchange with a recirculating water heater. The target
rectal temperature was 37 to 38.degree. C. Deionized water was used
for the bath because it was free of microbubbles that could
interfere with the ultrasound.
[0179] Transmitting the ultrasound directly through water to the
leg was found to be more effective than other methods that used a
combination of ultrasound gel and water contained in a balloon or
condom. The FUS transducer (Specialty Engineering Associates,
Soquel, Calif.) was 38 mm in diameter with a radius of curvature
and nominal focal length of 25 mm. Its resonance frequency was
1.459 MHz. The predicted focal region, based on geometry and
defined as the full-width-half-maximum intensity area (Bamber J C,
Tristam M., "Diagnostic Ultrasound". In: Webb S, ed. THE PHYSICS OF
MEDICAL IMAGING. Bristol: Adam Hilger, 1988; 328-334), was an
ellipsoid with a major axis of 6 mm oriented along the transducer
axis and a minor axis of 1 mm. The rf surface coil was potted with
epoxy in a 58 mm diameter channel cut in the platform around the
FUS aperture. Tuning and matching capacitors were housed out of the
water above the rat.
[0180] Experiments were performed on a 4.7 T magnet controlled with
an Inova console (Varian NMR Instruments, Palo Alto, Calif.). MR
temperature mapping was performed using rf spoiled gradient echo
imaging (de Poorter et al. (1995), "Noninvasive MRI thermometry
with the proton resonance frequency (PRF) method: in vivo results
in human muscle", Magn. Reson. Med. 33:74-81 (1996); de Zwart et
al., "Fast magnetic-resonance temperature imaging." J. Magn. Reson.
B 112: 86-90). The gradient echo data allowed phase difference maps
to be reconstructed. The hydrogen nuclei in water demonstrate a
temperature dependent chemical shift that allows one to calculate
temperature changes from these phase differences. Echo and
repetition times were 12 and 75 ms, respectively. Five slices with
a thickness of 2 mm and a spacing of 3.5 mm were acquired
sequentially in an interleaved fashion. They were initially
centered around the nominal focal point. 128.times.128 maps were
calculated for the 10.times.10 cm.sup.2 field of view. Temperature
resolution was approximately 0.15.degree. C.
[0181] An initial "cold" reference image was acquired once the rat
was properly positioned in the magnet and its temperature
stabilized in the target range. This image was used to calculate
temperature changes in subsequent maps. Then, the ultrasound was
turned on at a low level (.sup..about.1 W electric) just long
enough to acquire a set of five slices. Data was exported to a Sun
workstation where user-written IDL code was used to construct
temperature maps (see also ref. 7). From the maps, the focal region
was identified. If necessary, adjustments to the slice locations
were made to center the focus in one slice. Heating of the leg
muscle by continuous FUS then occurred for 45 minutes. Real-time
temperature maps were used to keep the focal region elevated by
8.degree. C. at its center, giving about a 5.degree. C. rise at the
edge of the region. Thus, the focal region was heated to
42-45.degree. C. Expression of the hsp70 gene continued for an
additional 45 minutes after the heating period while MRI monitored
the leg temperature. Throughout the experiment the rat's core body
temperature as measured by the rectal probes was maintained within
one degree of the target range, and respiration was monitored to
insure proper anesthesia.
[0182] The rat was euthanized with pentobarbital and the right leg
muscle was frozen using a freeze clamp cooled in liquid nitrogen
(-196.degree. C.). The frozen sample was transported in a sterile
specimen jar immersed in liquid nitrogen to a lab where the tissue
samples were prepared for analysis. The samples were taken from a
three-by-three grid centered on the nominal axis of the FUS beam.
The center sample was subdivided by depth into three giving a total
of eleven samples. The size of each muscle sample was approximately
4 mm.times.4 mm.times.2 mm.
[0183] The samples were kept frozen until they were placed in
Eppendorf tubes containing 0.5 ml Trizol solution (Life
Technologies, Gaithersburg, Md.) and homogenized. The solution and
homogenized tissue were then stored at -.sub.80.degree. C. until
RNA was extracted. The RNA (30 .mu.g sample per lane) was separated
by gel electrophoresis, and transferred to a nylon membrane. The
integrity of the mRNA was assessed by visualization of the ethidium
bromide-stained RNA following transfer. The membranes were
hybridized with a .sup.32P-labeled cDNA that is complementary to
the inducible hsp70 mRNA. Autoradiographs were created and analyzed
for amount of inducible hsp70 in each sample.
[0184] An intensity image of the slice containing the focal region
is shown in FIG. 5(a). FIG. 5(b) shows the temperature change in
the same slice of the rat leg after one minute of heating. The
field of view is identical to FIG. 5(a), but temperatures were
calculated in a smaller region of interest only. In addition,
pixels falling below a threshold intensity were not calculated
because of poor signal-to-noise and appear black. Since there has
been little diffusion of heat into the surrounding tissue, FIG.
5(b) serves as a good indicator of the size of the focal region.
FIG. 5(c) is a temperature map acquired after about three minutes
of heating. Thermal diffusion is apparent, and subsequent data
showed that an approximately steady state had been reached.
[0185] FIG. 6 shows the Northern blot of total RNA prepared from
rat thigh muscle following exposure to MRI-guided FUS and reacted
with random primed labeled human hsp70 stress inducible gene probe.
The probe hybridizes strongly in lane 5 at a position of about 2.3
kb (arrow), as expected for a 70,000 dalton protein. RNA loaded
into lanes 8 and 11 was somewhat degraded. Measurements show that
the differential expression of heat-inducible hsp70 in the focal
region ranges from a factor of 3 to 67.
[0186] These results demonstrate that low level continuous FUS can
be used to elevate the expression of endogenous hsp70 mRNA in vivo,
and that the hsp70 promoter is a suitable target for use in conrol
of gene expression based on local heat. The results also show that
MRI can provide interactive temperature maps for monitoring local
heating of in vivo tissue by FUS. Incorporation of
three-dimensional fast imaging methods like PRESTO should allow for
faster temperature maps.
Example 4
[0187] Example 4 describes the use of focused ultrasound (FUS)
guided by MRI to heat a predetermined area of the thigh muscle and
activate endogenous heat shock genes in a mouse whose muscle tissue
has been transformed with an adenoviral vector.
[0188] A transgenic mouse which contains a genetically engineered
LacZ gene under control of the hsp7O promoter (For methods of
making transgenic mice, see, e.g., Charron et al. (1995) J. Biol.
Chem. 270: 30604-10; for adenoviral vectors useful in transforming
cells, see Addison et al. (1995) Proc. Nat. Acad. Sci. U.S.A. 92:
8522-6 and Wang et al. (1996) Proc. Nat. Acad. Sci. U.S.A. 93:
3932-6) is treated as described in Example 3 (i.e., regions of the
thigh muscle having preselected coordinates are heated using
FUS-MRI). A significant increase of the desired gene transcripts in
the FUS focal spot is observed.
[0189] All publications, patents and patent applications mentioned
in this specification are hereby incorporated by reference for all
purposes, to the same extent as if each individual publication,
patent or patent application had been specifically and individually
indicated to be incorporated by reference.
[0190] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
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