U.S. patent application number 10/027797 was filed with the patent office on 2002-10-03 for gene delivery and expression in areas inaccessible to direct protein delivery.
This patent application is currently assigned to Vanderbilt University. Invention is credited to Brigham, Kenneth, Canonico, Angelo, Meyrick, Barbara, Stecenko, Arlene.
Application Number | 20020142984 10/027797 |
Document ID | / |
Family ID | 21848061 |
Filed Date | 2002-10-03 |
United States Patent
Application |
20020142984 |
Kind Code |
A1 |
Brigham, Kenneth ; et
al. |
October 3, 2002 |
Gene delivery and expression in areas inaccessible to direct
protein delivery
Abstract
The present invention provides novel methods for gene delivery
and expression in areas that are currently inaccessible through the
use of conventional direct protein delivery techniques. In
particular, the methods and related products provided herein can be
used in the treatment of .alpha..sub.1 antitrypsin (AAT) related
disorders such as respiratory syncytial virus (RSV) infection.
Inventors: |
Brigham, Kenneth;
(Nashville, TN) ; Canonico, Angelo; (Nashville,
TN) ; Meyrick, Barbara; (Nashville, TN) ;
Stecenko, Arlene; (Nashville, TN) |
Correspondence
Address: |
Shari J. Corin, Ph. D.
NEEDLE & ROSENBERG, P.C.
Suite 1200, The Candler Building
127 Peachtree Street, N.E.
Atlanta
GA
30303-1811
US
|
Assignee: |
Vanderbilt University
|
Family ID: |
21848061 |
Appl. No.: |
10/027797 |
Filed: |
December 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10027797 |
Dec 20, 2001 |
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08951552 |
Oct 16, 1997 |
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6365575 |
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60029252 |
Oct 24, 1996 |
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Current U.S.
Class: |
514/44R ;
435/455 |
Current CPC
Class: |
A61P 31/12 20180101;
A61K 9/1272 20130101; A61P 11/00 20180101; A61K 48/00 20130101;
A61K 38/44 20130101; A61K 38/57 20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/44 ;
435/455 |
International
Class: |
A01N 043/04; C12N
015/63 |
Claims
What is claimed:
1. A method for delivering a nucleic acid molecule to a location in
an animal, wherein said location is inaccessible to direct protein
delivery, comprising the step of administering a positively charged
liposome to said animal, wherein said positively charged liposome
is associated with said nucleic acid molecule, wherein said nucleic
acid molecule is in operable association with a promoter.
2. The method of claim 1, wherein said nucleic acid molecule
encodes human .alpha..sub.1 antitrypsin.
3. The method of claim 1, wherein said nucleic acid molecule
encodes prostaglandin synthase.
4. The method of claim 1, wherein said location is selected from
the group consisting of an endothelial lung cell, a smooth muscle
cells adjacent to said endothelial lung cell, and lung
parenchyma.
5. The method of claim 1, wherein said location is selected from
the group consisting of a liver cell, a muscle cell, an osteogenic
cell, synoviocyte, and a lung cell.
6. The method of claim 1, wherein said animal is a mammal.
7. The method of claim 6, wherein said mammal is a human.
8. The method of claim 1, wherein said positively charged liposome
is Lipofectin.TM..
9. The method of claim 1, wherein said nucleic acid sequence
encodes a therapeutically effective protein and wherein said method
further comprises expressing said nucleic acid sequence to provide
said protein to said location.
10. The method of claims 9, wherein said protein is an
antiprotease.
11. The method of claim 1, wherein at least 10% of said nucleic
acid that is administered is delivered to said location.
12. The method of claim 11, wherein at least 50% of said nucleic
acid that is administered is delivered to said location.
13. The method of claim 12, wherein at least 90% of said nucleic
acid that is administered is delivered to said location.
14. A method for preventing or treating an animal having a
disorder, wherein at least one symptom associated with said
disorder is caused at least in part by an insufficient amount or
form of protein in a particular location of said animal, wherein
said method comprises the step of delivering a gene encoding said
protein to said location and expressing said gene.
15. The method of claim 14, wherein said gene encodes human
.alpha..sub.1 antitrypsin.
16. The method of claim 14, wherein said gene encodes prostaglandin
synthase.
17. The method of claim 14, wherein said location is selected from
the group consisting of an endothelial lung cell, a smooth muscle
cells adjacent to said endothelial lung cell, and lung
parenchyma.
18. The method of claim 14, wherein said location is selected from
the group consisting of a liver cell, a muscle cell, an osteogenic
cell, synoviocyte, and a lung cell.
19. The method of claim 14, wherein said animal is a mammal.
20. The method of claim 19, wherein said mammal is a human.
21. The method of claim 14, wherein said positively charged
liposome is Lipofectin.
22. The method of claim 14, wherein said gene encodes a
therapeutically effective protein.
23. The method of claims 22, wherein said protein is an
antiprotease.
24. The method of claim 14, wherein at least 10% of said nucleic
acid that is administered is delivered to said location.
25. The method of claim 24, wherein at least 50% of said nucleic
acid that is administered is delivered to said location.
26. The method of claim 25, wherein at least 90% of said nucleic
acid that is administered is delivered to said location.
27. A method for delivering a nucleic acid molecule to a location
in an animal, comprising the step of administering a positively
charged liposome to said animal, wherein said positively charged
liposome is associated with said nucleic acid molecule, wherein
said nucleic acid molecule is in operable association with a
promoter, wherein delivery of said gene is capable of producing a
therapeutic response but direct delivery of the protein encoded by
said gene does not produce a therapeutic response.
28. The method of claim 27 wherein delivery of said gene produces a
therapeutic response even when delivered at a 20-fold lower serum
concentration than said protein which does not produce a
therapeutic response when delivered directly as a protein.
29. The method of claim 27, wherein said therapeutic response is
elimination of an endotoxin induced increase in pulmonary vascular
resistance.
30. A method of producing an elevated therapeutic response relative
to the therapeutic response created by direct delivery of a protein
comprising the step of delivering a nucleic acid molecule encoding
said protein.
31. The method of claim 30, wherein the therapeutic response
created by direct delivery of said protein is non-existent or
immeasureable.
32. The method of claim 30, wherein said protein is an
antiprotease.
33. The method of claim 30, wherein said enhanced therapeutic
response is created in a patient suffering from a disorder selected
from the group consisting of adult respiratory distress syndrome,
cystic fibrosis, respiratory syncytial virus infection,
interstitial lung disease, and chronic obstructive pulmonary
disease.
34. The method of claim 1, wherein said method results in
generation of the protein encoded by the nucleic acid molecule, and
wherein said protein is in a location which is inaccessible to
direct protein deliver.
35. The method of claim 5 or 18 wherein said location is a lung
epithelial cell.
Description
RELATED APPLICATIONS
[0001] This application relates to U.S. patent application Ser. No.
60/029,252, filed Oct. 24, 1996, entitled "GENE DELIVERY AND
EXPRESSION IN AREAS INACCESSIBLE TO DIRECT PROTEIN DELIVERY" by
Brigham et al. (Lyon & Lyon Docket No. 222/246) which is
incorporated herein by reference in its entirety, including any
drawings.
INTRODUCTION
[0002] The present invention provides novel methods for gene
delivery and expression in areas that are currently inaccessible
through the use of conventional direct protein delivery techniques.
In particular, the methods and related products provided herein can
be used in the treatment of pulmonary disorders and delivery of
anti-viral proteins.
BACKGROUND OF THE INVENTION
[0003] The following review of the background of the invention is
merely provided to aid in the understanding of the present
invention and neither it nor any of the references cited within it
are admitted to be prior art to the present invention.
[0004] There are presently several approaches being studied for
delivering genes to humans. These approaches have either: (i)
removed somatic cells, permanently transformed them in vitro using
retrovirus vectors, and reinfused the transformed cells; or (ii)
used viruses to deliver the gene. Such therapy has been designed
for use with patients having inherited deficiency of gene products,
such as proteins, due to abnormalities of the gene during
development. Such therapies have also been used with disorders such
as emphysema, wherein it is thought that the disease process is a
result of a relative deficiency of an antiprotease over a long
period of time. Further, diseases such as acute lung injury
resulting in the adult respiratory distress syndrome (ARDS) are
thought to involve a relative deficiency of antiprotease activity.
In addition, cystic fibrosis (CF) is the most common lethal genetic
disease in Caucasians (Boat, T. F. et al., The Metabolic Basis of
Inherited Disease 2649-2680, 1989). Even though CF can affect
several organ systems, almost all patients develop chronic
obstructive pulmonary disease and chronic pulmonary infections with
resultant respiratory failure and early death.
[0005] Normally, the lung contains sufficient quantities of serine
antiproteases, principally .alpha..sub.1 antitrypsin
(.alpha..sub.1AT), to combat the effects of toxic substances
involved in such diseases, such as neutrophil elastase (NE).
However, in CF and other neutrophil-dominated inflammatory lung
diseases, the antiprotease defense system fails to prevent
proteolytic damage to lung tissue (Boat, T. F. et al., The
Metabolic Basis of Inherited Disease 2649-2680, 1989;
Richman-Eisenstat, J. B. Y. et al., Am. J. Phys. 264:L413-L418,
1993).
[0006] One attempt at solving some of the above problems is
described in International Patent Publication WO 92/19730 (hereby
incorporated in its entirety, including any drawings) which
describes means for the delivery of a gene encoding human
.alpha..sub.1 antitrypsin to the lungs for expression of the human
.alpha..sub.1 antitrypsin capable of alleviating the enzyme
deficiency. Further advances regarding cationic liposome mediated
antiprotease gene transfer to reduce the infectivity of RSV in
cultured cells is reported in M. Persmark et al., J. Investig. Med.
43 S:220, 1995 Other attempts to deliver particular genes to cells
of the lung or airway are described in International Patent
Applications with publication numbers WO 93/12756 and WO 93/12240,
both of which are incorporated herein by reference in their
entirety including any drawings.
[0007] Despite the progress and success that has been achieved by
such attempts, there still remains a need for a general method to
provide gene delivery and expression to areas currently
inaccessible to direct protein delivery. See R. C. Hubbard, et al.
P.N.A.S. 86:680-684, 1989 (describing attempt to deliver protein
via an aerosol and stating "In the present study,
.apprxeq.{fraction (1/500)}th of the administered dose was
recoverable in lung lymph, a value likely too low to provide
adequate protection for .alpha..sub.1-antitrypsin deficiency").
Such obstacles have previously prevented the successful in vivo
treatment of AAT related disorders such as congenital AAT
deficiency, as well as other disorders related to other proteins
(for example, PGH synthase see U.S. patent application Ser. No.
08/459,493, filed Jun. 2, 1995 incorporated herein by reference in
its entirety including any drawings) that could not previously be
treated by direct protein delivery to the desired target area using
conventional methods of protein delivery.
SUMMARY OF THE INVENTION
[0008] The present invention is based in part on the surprising
discovery that certain genes (preferred are genes encoding
antiproteases such as AAT) can be delivered and expressed in vivo
to certain target areas in animals (preferably mammals, more
preferably humans) which have previously been inaccessible (i.e.,
an insufficient amount or inappropriate form of the protein is able
to be provided to give a therapeutic response) to direct protein
delivery.
[0009] A significant and unexpected advantage of the present
invention is the ability of the delivered gene to provide a
therapeutic response in situations where direct delivery of the
protein (even when delivered at several fold higher serum
concentrations) does not produce a therapeutic effect. In
particular, delivery of the AAT gene has been found to essentially
eliminate endotoxin induced increase in pulmonary vascular
resistance, even when serum protein levels are approximately 20
fold lower than produced by protein delivery, which, (in contrast)
produces no discernable effect on endotoxin response. In addition,
delivery of the AAT gene is able to prevent respiratory syncytial
virus infection of lung epithelial cells, where direct delivery of
the encoded protein does not. Thus, such methods provide a method
for successfully treating disorders caused by a deficiency of the
product encoded by the gene of interest in the target area in the
particular organism suffering from such a disorder.
[0010] The .alpha..sub.1-antitrypsin protein is the major
antiprotease in the lungs of humans. It is believed that both acute
lung injury associated with inflammation and emphysema are a
consequence of protease/antiprotease imbalance and increasing the
antiprotease activity in the lungs is one possible approach to
prevention and therapy of these conditions. In addition, there is a
genetic form of .alpha..sub.1-antitrypsin deficiency where patients
develop emphysema at an early age.
[0011] Patients with .alpha..sub.1-antitrypsin deficiency are now
being treated with intravenous administration of the
.alpha.-antitrypsin protein. This intervention is expensive,
requires relatively frequent intravenous infusions, can cause
reactions in the patient and is of unproven efficacy. In addition,
since the protein is derived from human blood products, a risk of
infection by contaminating viruses, such as HIV is present. Gene
therapy by delivery of the DNA encoding .alpha..sub.1-antitrypsin
to the airway cells could provide a less invasive, safer, cheaper
and more effective therapy.
[0012] Thus, in one aspect the invention provides a method for
delivering a nucleic acid molecule to a location in an animal that
is inaccessible to direct protein delivery. Those skilled in the
art will understand that several conventional methods for directly
delivering proteins exist and are commonly used in the art. The
method involves the step of administering a positively charged
liposome to the animal. The positively charged liposome is
associated with the nucleic acid molecule, wherein said nucleic
acid molecule is in operable association with a promoter. Those
skilled in the art will recognize that a wide variety of promoters
may be used to assist in targeting the desired location.
[0013] In preferred embodiments, the nucleic acid molecule encodes
human .alpha..sub.1 antitrypsin, the location is selected from the
group consisting of an endothelial lung cell, a smooth muscle cells
adjacent to the endothelial lung cell, and lung parenchyma, or the
location is selected from the group consisting of a liver cell, a
muscle cell, an osteogenic cell, synoviocyte, and a lung cell.
Other preferred locations and genes are shown in Table I below.
1TABLE I Clinical Indication Gene Target Musculoskeletal Muscle
reconstruction and IGF-1 muscle rehabilitation Cachexia (muscle
wasting) IGF-1, GH, a HEH muscle, liver Osteoporosis calcitonin,
IGF-1 muscle Arthritis Osteoarthritis nexin joints Gouty arthritic
urate oxidase joints Wound healing (dermal, epidermal) FGF, EGF,
TGF.beta. skin Muscular Dystrophy Distrophin muscle Cardiovascular
Inotropic heart failure IGF-1 muscle Hypertension atrial naturetic
muscle factor Atherogenesis apo-A1, apo-B, Liver
Hypercholesterolemia apo-E, cholesterol 7-.alpha.hydroxylase Liver
LDL receptor Liver VLDL receptor Liver lipoprotein lipase Liver
Liver Restenosis injury IFN.alpha., k iNos, p53 Smooth muscle
Homocystinemia cystathione .beta.- Liver, synthase endo- thelium
Bleeding disorders FACTORS VII & IX MSV/ Liver Anemia
erythropoietin MSV/ Liver Inflammation Rheumatoid Arthritis
glucocortico- Joints mimetic receptor Inflammatory dermatitis TGFB
Skin (psoriasis Inflammatory disease (systemic) IL-1 (RA), IL-1
muscle (SR), TGFB, TNF (SR) Asthma glucocortico- Alveolar mimetic
receptor macro- phage Glomerulonephritis glucocortico- Kidney
mimetic receptor Myositis glucocortico- MSV mimetic receptor
Bronchopulmonary dysplasia superoxide Lung dismutase gluathione
Lung reductase/peroxidase Chronic active hepatitis glucocortico-
Liver mimetic receptor Cancer Various tumors IL-2, IL-12,
IFN.alpha., Tumor IFN.gamma., p53 cells Pulmonary ARDS AAT.sub.1,
PGH synthase, Lung (CO.alpha., CO.alpha.2) Cystic Fibrosis CFTR
Lung
[0014] In other preferred embodiments the animal is a mammal,
preferably a human, and the positively charged liposome is
Lipofectin.TM.. Other suitable liposomes are described in
International Patent Application with publication numbers WO
93/12756 and WO 93/12240, both of which are incorporated by
reference in their entirety, including any drawings. Preferably the
nucleic acid sequence encodes a therapeutically effective protein
(e.g., an antiprotease) and the method further involves expressing
the nucleic acid sequence to provide the protein to the
location.
[0015] In another aspect, the invention provides a method for
preventing or treating an animal having a disorder. At least one
symptom associated with the disorder is caused at least in part by
an insufficient amount or form of protein in a particular location
of the animal. The method involves the step of delivering a gene
encoding the protein to the location and expressing the gene.
Preferably at least 10%, more preferably at least 50%, most
preferably at least 90% of the nucleic acid that is administered is
delivered to said location.
[0016] In another aspect the invention provides a method for
delivering a nucleic acid molecule to a location in an animal. The
method involves the step of administering a positively charged
liposome to the animal. The positively charged liposome is
associated with the nucleic acid molecule, and the nucleic acid
molecule is in operable association with a promoter. Delivery of
the gene is capable of producing a therapeutic response, but direct
delivery of the protein encoded by the gene does not produce a
therapeutic response.
[0017] In preferred embodiments, delivery of the gene produces a
therapeutic response even when delivered at a 10-fold lower serum
concentration than the protein (which does not produce a
therapeutic response when delivered directly as a protein).
Preferably, the therapeutic response is elimination of an endotoxin
induced increase in pulmonary vascular resistance.
[0018] In another aspect, a method of producing an elevated
therapeutic response relative to the therapeutic response created
by direct delivery of a protein. The method involves the step of
delivering a nucleic acid molecule encoding the protein.
[0019] In preferred embodiments, the therapeutic response created
by direct delivery of the protein is non-existent or immeasurable,
and the protein is an antiprotease. Preferably, the enhanced
therapeutic response is created in a patient suffering from a
disorder selected from the group consisting of adult respiratory
distress syndrome, cystic fibrosis, respiratory syncytial virus
infection, and chronic obstructive pulmonary disease.
[0020] The composition is preferably capable of delivering the
nucleic acid or oligonucleotide into a cell. By "delivering the
nucleic acid or oligonucleotide into a cell" is meant transporting
a complexed and condensed nucleic acid or a complexed
oligonucleotide in a stable and condensed state through the
membrane of a cell (in vitro or in vivo), thereby transferring the
nucleic acid or oligonucleotide from the exterior side of the cell
membrane, passing through the lipid bilayer of the cell membrane
and subsequently into the interior of the cell on the inner side
(i.e., cytosol side) of the cell membrane and releasing the nucleic
acid or oligonucleotide once within the cellular interior.
[0021] In a preferred embodiment at least 1% of the nucleic acid or
oligonucleotide in the composition is delivered into the cell or
cells of the desired target location. In a more preferred
embodiment, at least 10% of the nucleic acid or oligonucleotide is
so delivered. In an even more preferred embodiment, at least 50% of
the nucleic acid or oligonucleotide is so delivered. In a most
preferred embodiment, at least 90% of the nucleic acid or
oligonucleotide is so delivered.
[0022] Furthermore, the composition may prevent lysosomal
degradation of the nucleic acid by endosomal lysis. In addition,
although not necessary, the composition may also efficiently
transport the nucleic acid through the nuclear membrane into the
nucleus of a cell.
[0023] By "nucleic acid" is meant both RNA and DNA including: cDNA,
genomic DNA, plasmid DNA, antisense molecule, polynucleotides or
olignucleotides, RNA or mRNA. In a preferred embodiment, the
nucleic acid administered is plasmid DNA which comprises a
"vector".
[0024] By "vector" is meant a nucleic acid molecule incorporating
sequences encoding polypeptide product(s) as well as, various
regulatory elements for transcription, translation, transcript
stability, replication, and other functions as are known in the art
and as described herein. Vector can include expression vector.
[0025] An "expression vector" is a vector which allows for
production or expressing a product encoded for by a nucleic acid
sequence contained in the vector. The product may be a protein or a
nucleic acid such as an mRNA which can function as an antisense
molecule.
[0026] A "transcript stabilizer" is a sequence within the vector
which contributes to prolonging the half life (slowing the
elimination) of a transcript.
[0027] A "DNA vector" is a vector whose native form is a DNA
molecule. By "non-viral" is meant any vector or composition which
does not contain genomic material of a viral particle.
[0028] An "antisense molecule" can be a mRNA or an oligonucleotide
which forms a duplex with a complementary nucleic acid strand and
can prevent the complementary strand from participating in its
normal function within a cell. For example, expression of a
particular growth factor protein encoded by a particular gene.
[0029] A "gene product" means products encoded by the vector.
Examples of gene products include mRNA templates for translation,
ribozymes, antisense RNA, proteins, glycoproteins, lipoproteins and
phosphoproteins.
[0030] "Post-translational processing" means modifications made to
the expressed gene product. These may include addition of side
chains such as carbohydrates, lipids, inorganic or organic
compounds, the cleavage of targeting signals or propeptide
elements, as well as the positioning of the gene product in a
particular compartment of the cell such as the mitochondria,
nucleus, or membranes. The vector may comprise one or more genes in
a linear or circularized configuration. The vector may also
comprise a plasmid backbone or other elements involved in the
production, manufacture, or analysis of a gene product. The nucleic
acid may be associated with a targeting ligand to effect targeted
delivery.
[0031] A "targeting ligand" is a component of the delivery system
or vehicle which binds to receptors, with an affinity for the
ligand, on the surface or within compartments of a cell for the
purpose of enhancing uptake or intracellular trafficking of the
vector. Glucans such as Tris-galactosyl residues, carnitine
derivatives, mannose-6-phosphate, monoclonal antibodies, peptide
ligands, and DNA-binding proteins represent examples of targeting
ligands which can be used to enhance uptake.
[0032] "Intracellular trafficking" is the translocation of the
vector within the cell from the point of uptake to the nucleus
where expression of a gene product takes place. Alternatively,
cytoplasmic expression of a nucleic acid construct utilizing, for
example, a T7 polymerase system may be accomplished. Various steps
in intracellular trafficking include endosomal release and
compartmentalization of the vector within various extranuclear
compartments, and nuclear entry.
[0033] "Endosomal release" is the egress of the vector from the
endosome after endocytosis. This is an essential and potentially
rate limiting step in the trafficking of vectors to the nucleus. A
lytic peptide may be used to assist in this process.
[0034] A "lytic peptide" is a peptide which functions alone or in
conjunction with another compound to penetrate the membrane of a
cellular compartment, particularly a lysosomal or endosomal
compartment, to allow the escape of the contents of that
compartment to another cellular compartment such as the cytosolic
and/or nuclear compartment.
[0035] "Compartmentalization" is the partitioning of vectors in
different compartments within a defined extracellular or
intracellular space. Significant extracellular compartments may
include, for example, the vascular space, hair follicles,
interstitial fluid, synovial fluid, cerebral spinal fluid, thyroid
follicular fluid. Significant intracellular compartments may
include endosome, potosome, lysosome, secondary lysosome,
cytoplasmic granule, mitochondria, and the nucleus.
[0036] "Nuclear entry" is the translocation of the vector across
the nuclear membrane into the nucleus where the gene may be
transcribed.
[0037] "Elimination" is the removal or clearance of materials
(vectors, transcripts, gene products) from a specific compartment
over time. This term may be used to reflect elimination from the
body, the vascular compartment, extracellular compartments, or
intracellular compartments. Elimination includes translocation
(excretion) from a particular compartment or biotransformation
(degradation).
[0038] The compounds which increase the efficacy of transfection of
a nucleic acid are suitable for internal administration. By
"suitable for internal administration" is meant that the compounds
are suitable to be administered within the tissue of an organism,
for example within a muscle or within a joint space, intradermally
or subcutaneously. Other forms of administration which may be
utilized are topical, oral, pulmonary, nasal and mucosal; for
example, buccal, vaginal or rectal. These substances may be
prepared as solutions, suspensions, gels, emulsions or
microemulsions. Oil suspensions of lyophilized nucleic acid, such
as plasmid DNA may be utilized. Delivery systems for these oil
suspensions include, but are not limited to, sesame oil, cottonseed
oil, soybean oil, lecithins, Tweens, Spans and Miglyols.
[0039] By "solutions" is meant water soluble substances and/or
surfactants in solution with nucleic acids. By "suspensions" is
meant water insoluble oils containing suspended nucleic acids. By
"gels" is meant high viscosity substances containing nucleic acids.
By "emulsion" is meant a dispersed system containing at least two
immiscible liquid phases. Emulsions usually have dispersed
particles in the 0.1 to 100 micron range. They are typically opaque
and thermodynamically unstable. Nucleic acids in the water phase
can be dispersed in oil to make a w/o emulsion. This w/o emulsion
can be dispersed in a separate aqueous phase to yield a w/o/w
emulsion. Alternatively, a suitable oil could be dispersed in an
aqueous phase to form an o/w emulsion.
[0040] A "microemulsion" has properties intermediate to micelles
and emulsions and is characterized in that they are homogenous,
transparent and thermodynamically stable. They form spontaneously
when oil, water, surfactant and co-surfactant are mixed together.
Typically, the diameter of the dispersed phase is 0.01 to 0.1
microns, usually of the w/o and o/w type. The sustained-release
compound containing a nucleic acid is administered to the tissue of
an organism, for example, by injection. In one embodiment the
tissue is preferably muscle tissue. In another embodiment the
tissue is preferably a joint space.
[0041] By "sustained-release compound" is meant a substance with a
viscosity above that of an isotonic saline solution (150 mM NaCl)
containing a nucleic acid; for example, DNA in saline at 1 mg/ml
has a viscosity of 3.01 mPa.multidot.sec, DNA in saline at 2 mg/ml
has a viscosity of 3.26 mPa.multidot.sec, DNA in saline at 3 mg/ml
has a viscosity of 5.85 mPa.multidot.sec (Viscosity measurements
were performed at 25.degree. C. in a Brookfield DV-III Rheometer
with a No. 40 Spindle at 75 rpm for 30 minutes). Preferably the
sustained-release compound has a viscosity in the range of about
0.1-20,000 mPa.multidot.sec above that of a complexation in which
isotonic saline is the delivery system for a nucleic acid. More
preferably the range is about 0.1-5000 mPa.multidot.sec above that
of a complexation in which isotonic saline is the carrier for a
nucleic acid. Even more preferably the range is about 0.1-1000
mPa.multidot.sec above that of a complexation in which isotonic
saline is the carrier for a nucleic acid.
[0042] "Targeted delivery" involves the use of targeting ligands
which specifically enhance translocation of a nucleic acid to
specific tissues or cells. A "target" is a specific organ, tissue,
or cell for which uptake of a vector and expression of a gene
product is intended. "Uptake" means the translocation of the vector
from the extracellular to intracellular compartments. This can
involve receptor mediated processes, fusion with cell membranes,
endocytosis, potocytosis, pinocytosis or other translocation
mechanisms. The vector may be taken up by itself or as part of a
complex. "Binding" is an intermediate step in uptake of some
compositions involving a high-affinity interaction between a
targeting ligand and a surface receptor on a target cell.
[0043] By "oligonucleotide" is meant a single-stranded
polynucleotide chain. In a preferred embodiment, the
oligonucleotide is less than 100 residues in length. In a more
preferred embodiment, the oligonucleotide is less than 50 residues
in length. In a most preferred embodiment, the oligonucleotide is
less than 30 residues in length.
[0044] In a preferred embodiment, the invention features a
composition capable of complexing and condensing the nucleic acid
or oligonucleotide. These compositions provide smaller, or
condensed, and more stable nucleic acid particles for delivery,
thereby enhancing the transfection rate of nucleic acid into the
cell and the subsequent expression therein.
[0045] By "complexing" is meant a high affinity interaction, based
upon non-covalent binding, between the chitosan-based substance and
the nucleic acid or oligonucleotide. By "affinity" is meant the
selective tendency of elements to combine with one, rather than
another element, when the physicochemical conditions are
appropriate. This interaction is most preferably an ionic
interaction but may be brought about wholly or in part by hydrogen
bonding, Van der Walls interactions or other chemical attractions
commonly recognized by those in the art. The compounds which
complex and condense a nucleic acid may also interact or associate
with the nucleic acid by intermolecular forces and/or valence bonds
such as: Van der Waals forces, ion-dipole interactions, ion-induced
dipole interactions, hydrogen bonds, or ionic bonds.
[0046] These interactions may serve the following functions: (1)
Stereo selectively protect nucleic acids from nucleases by
shielding; (2) facilitate the cellular uptake of nucleic acid by
"piggyback endocytosis". By "piggyback endocytosis" is meant the
cellular uptake of a drug or other molecule complexed to a delivery
system that may be taken up by endocytosis (C. V. Uglea and C.
Dumitriu-Medvichi, Medical Applications of Synthetic Oligomers. In:
"Polymeric Biomaterials." Edited by Severian Dumitriu. Marcel
Dekker, Inc. 1993) and incorporated herein by reference including
all drawings and figures. To achieve the desired effects set forth,
it is desirable, but not necessary, that the substances which
condense and complex nucleic acid have amphipathic properties; that
is, the substance has both hydrophilic and hydrophobic regions. The
hydrophilic region of the substance may associate with the largely
ionic and hydrophilic regions of the nucleic acid, while the
hydrophobic region of the substance may act to retard diffusion of
nucleic acid and to protect nucleic acid from nucleases.
Additionally, the hydrophobic region may specifically interact with
cell membranes, possibly facilitating endocytosis of the
composition and thereby nucleic acid associated with the compound.
This chitosan-based composition may increase the pericellular
concentration of nucleic acid.
[0047] By "condensing" is meant charge neutralization, exclusion of
water and compacting into colloidal particles. The composition
which condense and complex nucleic acid may also achieve one or
more of the following effects, due to their physical, chemical or
Theological properties: (1) Protect nucleic acid, for example
plasmid DNA, from nucleases; (2) increase the area of contact
between nucleic acid, such as plasmid DNA, through extracellular
matrices and over cellular membranes, into which the nucleic acid
is to be taken up; (3) concentrate nucleic acid, such as plasmid
DNA, at cell surfaces due to water exclusion; (4) indirectly
facilitate uptake of nucleic acid, such as plasmid DNA, either
increasing interaction with cellular membranes and/or by perturbing
cellular membranes due to osmotic, hydrophobic or lytic
effects.
[0048] By "increase the efficacy of transfection" is meant that a
nucleic acid or oligonucleotide when administered to an organism in
a composition comprising such a substance will be more readily
taken up into the interior of a cell by translocating across the
cellular membrane than if administered in a composition without
such a substance, for example when administered in a formulation
such as a saline solution. The increased efficiency of uptake of
nucleic acid, or oligonucleotide into cells could occur, for
example, due to a better steric fit between the composition
containing the nucleic acid and a pit on the surface of the
cellular membrane or due to protection of the nucleic acid from
attack by nucleases.
[0049] In another preferred embodiment, the composition has a net
positive charge ratio. By "net charge" is meant the resulting
positive, negative or neutral character of a compound which is
determined after balancing the total number of positive and
negative charges possessed by a molecule or compound. For example,
the DNA molecule, has a net negative charge due to the presence of
two anionic phosphate moieties on each base pair of the molecule.
The number of negatively charged phosphates exceed in number the
total number of positive charges on the DNA molecule. Thus the
surfeit of negative charges imparts a net negative character or
charge to DNA. The number of negative charges to positive charges
on compositions determines the net charge ratio. The net charge
ratio is symbolized by (-/+) where a dash, "-", stands for a
negative charge and a plus sign, "+", stands for a positive charge.
A net charge ratio of 1:1(-/+) is neutral; of 2:1(-/+) is negative
and of 1:2(-/+) is positive.
[0050] Another embodiment features the composition additionally
mixed with a cryoprotectant. By "cryoprotectant" is meant any
chemical or compound which will serve to protect nucleic acid and
oligonucleotides and the complexed particles during lyophilization,
storage, and subsequent rehydration. Examples of "cryoprotectants"
include, but are not limited to, such compounds as lactose,
sucrose, mannitol, and trehalose.
[0051] In another aspect, the nucleic acid or oligonucleotide is
delivered to a cell by the step of exposing the composition to the
cell. The method may be performed in vitro, in vivo, or on a cell
that has been removed from a living organism. If the method is
performed in vivo, then the exposing step may be performed by
administering the composition to an organism.
[0052] By "administering" is meant the route of introduction of the
composition into a body. Administration can be directly to a target
tissue or through systemic delivery. In particular, administration
may be by direct injection to the cells. Routes of administration
include, but are not limited to, intramuscular, aerosol, oral,
topical, systemic, nasal, ocular, intraperitoneal and/or
intratracheal, buccal, sublingual, oral, intradermal, subcutaneous,
pulmonary, intra-artricular, and intra-arterial. In a preferred
embodiment administration is by intravenous administration.
[0053] By "organism" is meant a living entity capable of
replication. In a preferred embodiment the organism is an animal,
in a more preferred embodiment a mammal, and in a most preferred
embodiment a human.
[0054] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] FIG. 1 shows the effects of AAT gene therapy vs. exogenous
protein administration on Respiratory Syncytial Virus (RSV)
replication. Transfection with the AAT gene 48 hours before RSV
infection significantly reduced RSV replication. AAT protein added
to the medium in amounts 50-100 times that achieved with AAT gene
therapy had no effect on RSV replication.
[0056] FIG. 2 shows pulmonary vascular resistance (PVR) normalized
to baseline following endotoxin exposure in a piglet in situ lung
preparation. Marked increase in PVR is seen in control piglets
(i.e., endotoxin alone) and in piglets that received exogenous AAT
protein before endotoxin. Intravenous transfection with the AAT
transgene blocks this effect.
[0057] FIG. 3 shows immunohistochemical localization of human AAT
in the pulmonary vasculature of the piglet. FIG. 3a shows that
following intravenous administration of the protein, AAT localizes
to the endothelium (arrow). FIG. 3b shows following gene transfer,
the AAT transgene product is seen in the endothelium, the adjacent
smooth muscle cells, and the lung parenchyma (arrows).
[0058] FIG. 4 shows expression of the alpha.sub.1 antitrypsin
transgene in the nasal mucosa of AAT deficient patents. Time course
and expression of the AAT transgene.
[0059] FIG. 5 shows expression of the alpha.sub.1 antitrypsin
transgene in the nasal mucosa of AAT deficient patents. Ratio of
AAT protein levels in lavages from transfected vs. control
nostril.
[0060] FIG. 6 shows the results of intravenous administration of
plasmid-liposome complexes: There was no effect of plasmid-liposome
compels infusion on lung mechanics (C.sub.dyn) or lung lymph flow
(Q.sub.L). A transient rise in pulmonary artery pressure (PA
Pressure) was seen which returned to baseline when the infusion was
stopped.
[0061] FIG. 7 shows the results of intravenous administration of
plasmid-liposome complexes: There was no effect of plasmid-liposome
complex infusion on white blood cell count (WBC) or oxygenation
(AaO.sub.2A-Baseline).
[0062] FIG. 8 shows the effects of endotoxin administration 72
hours after PGH synthase gene infusion: No effect on pulmonary
artery pressure was seen. A persistent improvement in lung
mechanics (C.sub.dyn) was noted.
[0063] FIG. 9 shows the effects of endotoxin administration 72
hours after PGH synthase gene infusion: A slight improvement in
oxygenation (AaO.sub.2A-Baseline) was seen. There was attenuation
in the late phase increase in lung microvascular permeability
(C.sub.LP).
[0064] FIG. 10 shows the effects of topical arachidonic acid on
PGE.sub.2 production by normal human nasal mucosa.
[0065] FIG. 11 shows the effects of topical IL-1 on arachidonate
simulated PGE.sub.2, production by normal human nasal mucosa.
[0066] FIG. 12 shows that preincubation of 25 nM NE with various
concentrations of .alpha..sub.1AT blocks the production of
chemotactic activity in the supernatant of 2CFSMEo- cells after 24
hour exposure to NE.
[0067] FIG. 13 shows a representative time course for the secretion
of the h.alpha.lAT gene product after transfer of the h.alpha.lAT
gene to 2CFSMEo- cells. Peak expression is seen at days 4-5.
DETAILED DESCRIPTION OF THE INVENTION
[0068] The present invention relates to a method for delivering a
nucleic acid molecule to a location in an animal that is
inaccessible to direct protein delivery. Such methods of delivery
allow for new methods of treating and preventing disorders based on
the deficiency of a certain protein in such a location. The
sections below provide examples of particular genes that can be
delivered to particular locations previously inaccessible via a
direct protein delivery. This detailed disclosure also presents new
diseased targets that are now treatable in view of the novel gene
delivery methods reported herein. Especially preferred embodiments
involving delivery of the AAT gene or prostaglandin synthase are
also described in detail and will be better understood in view of
the sections below.
[0069] I. AAT Gene Therapy Compared to Exogenous AAT Protein
(Prolastin)
[0070] The AAT protein, Prolastin, is available and in clinical use
and a number of other proteins have been suggested as therapeutics
for various disorders. The present invention, however, demonstrates
that certain locations (for example, the lung) might be better
protected by protein AAT (for example, produced in the cells for
which protection from proteolytic injury is needed, rather than by
the protein delivered intravenously (or via the airways). Provided
herein is experimental data substantiating that claim in cultured
lung epithelial cells and in vivo in a pig endotoxin model. In
particular, the data demonstrates that the location of AAT protein
delivered intravenously is different than that produced as a
consequence of in vivo gene delivery and that intravenously
delivered protein is not capable of achieving the same therapeutic
effect that is produced by the in vivo gene delivery.
[0071] II. AAT Gene Therapy Prevents Respiratory Syncytial Virus
(RSV) Infection of Lung Epithelial Cells, but Exogenous AAT
(Prolastin) Does Not.
[0072] As is true for many viruses, cell surface and intracellular
proteolytic events are involved in the ability of RSV to infect
respiratory epithelial cells. The effects of transfecting an RSV
susceptible lung epithelial cell line with the pCMV4AAT construct
was determined using cationic liposomes on RSV infectivity. Effects
of exogenous AAT added to the medium in a range of concentrations
was also determined. Transfection with the AAT plasmid resulted in
maximal levels of AAT in the medium of 300-800 ng/ml. FIG. 1 shows
viral replication in the cell line studied as plaque forming units
(PFU) in cells which received no treatment, cells exposed to RSV
after transfection with AAT and cells exposed to exogenous AAT at a
concentration of 30,000 ng/ml in the medium. The effects of the
exogenous protein and transfection with the AAT gene were
substantially different. Transfection with AAT markedly reduced RSV
infectivity, but exogenous protein had little effect.
[0073] III. AAT Gene Therapy Prevents Endotoxin Induced Lung
Toxicity in Piglets, but Exogenous AAT (Prolastin) Does Not.
[0074] Antiproteases, specifically AAT, have been suggested as
potential therapy for acute lung injury, but experimental efficacy
studies of with the exogenous protein have not been very
convincing. The present invention unexpectedly demonstrates that
AAT gene therapy is significantly more effective than the exogenous
protein as assessed in a piglet endotoxin model.
[0075] In young pigs (3-5 kg), the effects of in vivo transfection
with the pCMV4AAT construct using cationic liposomes delivered
intravenously was compared to the effects of AAT protein
(Prolastin) added to the circulation. 48 hours prior to endotoxin
administration, PCMV4AAT was delivered at a dose of 1 mg/kg body
weight complexed in a 1:3 (w:w) ratio with DOTMA/DOPE liposomes
(Lipofectin) by slow intravenous infusion. Results of the endotoxin
studies were compared with results from two other study groups. One
group received only endotoxin and the other group received
Prolastin one hour prior to endotoxin infusion. To study the
effects of endotoxin, an in situ perfused lung preparation (where
blood flow is kept constant and pulmonary vascular pressures are
measured was used.
[0076] Following transfection with the AAT plasmid, measurable
blood concentrations of human AAT ranging from 45.1-217.8 ng/ml
(average 105.5 ng/ml) were consistently observed. Blood
concentrations of AAT in the animals receiving Prolastin were in
excess of 2000 ng/ml. Five animals received the AAT gene, five
animals received the AAT protein, and six animals served as
controls. FIG. 2 shows pulmonary vascular resistance (PVR) over the
course of the endotoxin response in all three experimental groups.
Each line is the mean data for the animals in each group. Addition
of exogenous AAT had no discernible effect on the endotoxin
response. Surprisingly, and in contrast, transfection with AAT
essentially eliminated the endotoxin induced increase in PVR.
[0077] FIGS. 3A and 3B are photomicrographs showing
immunohistochemical staining of human .alpha..sub.1 antitrypsin in
the lungs of pigs 48 hours after transfection with the AAT gene
(FIG. 3A) and after intravenous infusion of Prolastin in the same
concentrations as given in the physiologic studies (FIG. 3B). There
are dramatic differences in the location of the protein. AAT
generated as a consequence of gene therapy is located throughout
the vascular wall and lung parenchyma, as well as on the surface of
the endothelium. In contrast, delivery of AAT protein localizes to
the vascular endothelium only; the exogenous protein does not
penetrate beyond the endothelium.
[0078] To our knowledge, this is the first demonstration that
protein such as AAT produced as a result of gene transfer is
present in a markedly different physiological space than is the
exogenous protein. It is also the first demonstration that the
therapeutic potential of proteins (such as AAT and other
antiproteases) produced as a consequence of gene transfer is
dramatically different than that of the exogenous protein. These
data indicate that gene therapy with AAT can achieve therapeutic
concentrations of the protein in the plasma, since plasma levels
are apparently irrelevant to some of the therapeutic effects
achieved with AAT gene therapy.
[0079] IV. Delivery of the AAT Gene to Humans with AAT
Deficiency
[0080] The AAT gene has now been delivered to the nasal epithelium
of two human patients with AAT deficiency. This protocol involves
removing the patients from Prolastin therapy for 1 month,
collecting baseline nasal lavage samples on several occasions and
then transfecting one nostril with the pCMV4AAT preparation using
DOTMA/DOPE cationic liposomes. The untransfected nostril serves as
the control. Following transfection, nasal lavage samples are
collected periodically for a month and nasal inflammation is
assessed by nasal scraping and staining of cells obtained in this
manner. Blood samples are taken for general screening to determine
if systemic distribution of the plasmid occurs.
[0081] The studies have presented no technical problems. No
abnormalities in blood tests have been identified and nasal
scrapings show no discernible effects of transfection as compared
to the untransfected nostril. FIG. 4 shows concentrations of AAT
measured by ELISA in the lavage from each nostril over time in the
second patient studied. There is a convincing increase in AAT
concentrations in the lavage fluid from the transfected nostril
with no consistent change in the untransfected nostril.
[0082] FIG. 5 summarizes the data from the two patients. It shows
expression of the AAT transgene as the ratio of AAT concentration
in the transfected nostril to that in the untransfected nostril in
each of the two patients studied. In each case there is a
convincing 3-6 fold increase in AAT in the transfected nostril
peaking at 3-5 days, and remaining about twice baseline at 7 days.
These data make a convincing case that the techniques of the
present invention effectively delivers the transgene to the nasal
mucosa. This clinical model thus appears ideal for testing
different preparations, delivery strategies, and the consequences
of in vivo gene delivery for the respiratory epithelium.
[0083] V. Disease Targets
[0084] There are several possible clinical targets for an AAT gene
medicine in addition to AAT deficiency. In view of the data in the
studies in piglets presented above, acute respiratory distress
syndrome (ARDS) is a realistic disease target. Further, a dose
ranging study in ARDS patients could be performed by those skilled
in the art may using techniques that are standard and conventional
in the art.
[0085] Patients with emphysema are another patient population that
would also be a target for AAT gene therapy. Although not a
consequence of a genetic AAT deficiency, the anatomy and physiology
of the disease in the patients who are candidates for lung
reduction are identical to patients with AAT deficiency. The most
popular hypothesis for development of emphysema is that it results
from a relative AAT deficiency (i.e. excessive protease burden). In
view of the new data presented herein showing that AAT gene therapy
is dramatically more effective than exogenous AAT protein an
evaluation of AAT gene therapy in this group is easily justified.
Extension to a broad group of patients with chronic obstructive
pulmonary disease (COPD) may also be justified.
[0086] Respiratory infections with respiratory syncytial virus
(RSV) are often lethal in immunocompromised patients, are a
precipitating cause of respiratory failure in patients with COPD,
and occur in annual epidemics in children. Thus, this a
particularly attractive clinical target, since the disease starts
in the nasal mucosa and AAT gene therapy could be targeted
there.
[0087] VI. Development of a Prostaglandin Synthase Gene Medicine
Studies in Chronically Instrumented Unanesthetized Sheep
[0088] The present invention also provides an animal model in
unanesthetized sheep for intravenous delivery of plasmid-liposome
complexes. This animal model can be used to define the effects of
the procedure on lung function, hemodynamics and general well-being
of the animals. In addition, the sheep lung lymph preparation
provides unique information relevant to the potential therapy for
acute lung injury (i.e. ARDS).
[0089] The current study protocol calls for two endotoxin studies
in each animal: One without PGH synthase gene transfection and the
other 72 hours following transfection with the gene. The two
studies must be done in random order in order to avoid historical
bias of the endotoxin responses.
[0090] Paired studies in 2 sheep (4 studies) have been completed.
In one of the animals, the PGH synthase gene was administered 72
hours prior to the initial endotoxin study. The second endotoxin
study was repeated one week later. The other animal had a baseline
endotoxin study performed before delivering the PGH synthase gene.
One week later, the PGH synthase gene was administered and the
endotoxin study repeated 72 hours after transfection.
[0091] No ill effects of infusing the plasmid-liposome complexes
were noted. FIG. 6 shows the effects of plasmid/liposome complex
infusion on lung mechanics, lung lymph flow, and pulmonary artery
pressure. FIG. 7 shows the effects of plasmid-liposome complex
infusion on the white blood count and oxygenation. Plasmid-liposome
complex infusion had no effect on blood cell counts, chemistries,
lung mechanics, arterial blood gases or lung lymph flow. The only
effect on physiology was a small rise in pulmonary artery pressure
during the infusion. This was a transient rise and returned
immediately to baseline when the infusion was stopped.
[0092] FIGS. 8 and 9 show the effects of PGH gene infusion on the
endotoxin response. There were no striking differences in pulmonary
vascular pressures between the endotoxin study and the endotoxin
study following transfection. A suggestion of improved oxygenation
and a persistent improvement in lung mechanics were noted.
Importantly, the late phase increase in lung microvascular
permeability, the basic injury in ARDS, was reduced in the
transfected animals. FIG. 9 shows lung lymph protein clearance (a
measure of permeability or leakiness of the lung microvasculature)
for the same studies. The persistent high permeability
characteristic of the endotoxin response in this preparation was
seen in the control studies, but, after transfection with PGH
synthase, this increase in permeability was markedly
attenuated.
[0093] These studies provide physiologic and biochemical data
critical to extension of these interventions to humans. In
addition, the data indicate that PGH synthase may be therapeutic in
acute lung injury.
[0094] VII. Eicosanoid Metabolism in the Human Nasal Mucosa
[0095] In view of the human AAT studies reported herein, the nasal
mucosa may be an ideal model for evaluating in vivo gene delivery.
This is especially true for studying effects of genes which may
alter arachidonic acid metabolism. Respiratory epithelium is
actively involved in eicosanoid metabolism and eicosanoid
metabolism in nasal epithelium is similar to respiratory epithelium
from the lower airway. In order to lay the groundwork for
evaluating PGH synthase gene therapy in initial human studies,
important data from human nasal lavage studies has been collected.
Those data demonstrate convincingly that eicosanoid metabolism is
easily studied in the nose and that effects on inflammation in this
relevant respiratory epithelium can be studied safely and with
minimal inconvenience to the patient.
[0096] FIG. 10 shows concentrations of prostaglandin E.sub.2
(PGE.sub.2), the principal prostanoid made by respiratory
epithelium, in nasal lavage fluid from normal subjects prior to and
after exposure to 50 .mu.M arachidonic acid. It is apparent that,
when normalized to protein content, there is a fairly constant
baseline concentration of PGE.sub.2 in nasal lavage fluid and that
after just 10 minutes exposure to arachidonic acid, PGE.sub.2
production increases. This protocol is virtually identical to that
used in cultured cells to test activity of PGH synthase. This
protocol in an appropriate target group of patients provides an
initial test of the function of the PGH synthase gene medicine in
human respiratory epithelium.
[0097] Effects of pro-inflammatory cytokines on eicosanoid
metabolism can also be studied in this model. FIG. 11 shows the
PGE.sub.2 production from the nasal mucosa following a brief
exposure to the cytokine interleukin-1 (IL-1). Five hours after
IL-1, arachidonic acid exposure resulted in a much larger increase
in PGE.sub.2 generation than occurred at baseline. Again, this is
very similar to studies which are routinely done in cultured cells
and implies induction of the cyclooxygenase enzyme by exposure to
this pro-inflammatory cytokine.
[0098] VIII. Disease Targets
[0099] In view of the above, it appears that ARDS is an appropriate
disease target for this gene medicine. The sheep studies show that
the complexes can be delivered intravenously without evident
toxicity and PGH synthase gene transfer appears to affect important
variables in the endotoxin response favorably. Those skilled in the
art can produce human quality reagents and perform initial studies
in nasal mucosa in patients with ARDS with detailed measurements of
eicosanoids, IL-1, IL-8, TNF, and assessment of inflammatory cells.
Whether or not nasal epithelium reflects the same dysfunctions as
epithelium lower in the respiratory tract, these studies would
provide a basis for proceeding to intravenous delivery of the
gene.
[0100] Interstitial pulmonary fibrosis (IPF) is an additional
target for PGH synthase gene therapy. IPF is a fatal disease for
which the only effective therapy right now is lung transplantation.
The perpetual inflammation and lung fibrosis in this disease may be
a consequence of a defect in PGH synthase so that the
anti-inflammatory eicosanoid, PGE.sub.2, is not produced in lung
cells in sufficient quantity to suppress this abnormal response to
injury.
[0101] Initial studies of both cultured lung fibroblasts and nasal
lavage eicosanoid metabolism have been completed in a few patients
with IPF. The nasal lavage fluid contains lower than normal
concentrations of PGE.sub.2. With the availability of human quality
reagents, initial studies with nasal instillation of the gene in
patients with IPF could be done and provide a basis for delivery of
the gene to the whole lung. Because this is a desperate disease,
proceeding rapidly to studies of efficacy is justified.
[0102] IX. AAT Blocks Chemotactic Activity
[0103] Human neutrophil elastase (NE) stimulates release of
neutrophil chemotactic activity by a bronchial epithelial cell line
and from nasal epithelial cells. NE stimulates the production of
neurophil chemotactic activity by 2CFSMEo- cells, a transformed
cystic fibrosis bronchial epithelial cell line. The production of
chemotactic activity is dose- and time-dependent and can be blocked
by preincubation of NE with .alpha..sub.1 antitrypsin
(.alpha..sub.1AT). Incubation of the NE-stimulated culture
supernatant with neutralizing concentrations of rabbit and human
interleukin 8 antibody completely neutralizes the chemotactic
activity.
[0104] Transfection of 2CFSMEo- cells with the eukaryotic
expression vector pCMV4.alpha..sub.1AT, complexed to cationic
liposomes in a 1:3 wt/wt/ratio, results in at least a 10-fold
increase in measured human .alpha..sub.1AT protein in culture
supernatant. Detection of human .alpha..sub.1AT mRNA by reverse
transcriptase polymerase chain reaction in total RNA from
transfected, but not untransfected cells, confirms successful gene
transfer. Compared with untransfected cells, transfer of the human
.alpha..sub.1AT gene decreases chemotactic activity in culture
supernatant and prevents cell detachment after NE exposure. This
data indicate that .alpha..sub.1AT gene transfer is capable of
blocking at least some of the biological effects of free elastase
on cultured epithelial cells.
[0105] The effect of a .alpha..sub.1AT gene transfer in cystic
fibrosis epithelial cells exposed to neutrophil elastase thus
demonstrates that plasmid-cationic liposome-mediated
h.alpha..sub.1AT gene transfer to a CF bronchial epithelial cell
line protects the cells from the toxic effects of elastase and
inhibits elastase-stimulated release of neutrophil chemotactic
activity from the epithelial cells.
[0106] Human .alpha..sub.1AT gene transfer to a CF epithelial cell
line, 2CFSMEo-, protects against NE-induced release of chemotactic
activity and cell detachment. Transfection of the 2CFSMEo- cells
with the h.alpha..sub.1AT gene results in production of
.alpha..sub.1AT protein for 1 wk after transfection with peak
expression seen at days 4 and 5. When transfected cells are exposed
to NE, transfer of the h.alpha..sub.1AT gene prevents NE-stimulated
production of a neutrophil chemotactic factor and cell
detachment.
[0107] This data (and the experimental examples and other
information in Canonico et al., Am. J. Respir. Cell Mol. Biol.
14:348-355, 1996, incorporated herein by reference in its entirety
including any drawings) indicate that Ne stimulates 2CFSMEo- calls
to release a neutrophilic chemotactic factor which could be
neutralized by treatment with anti-IL-8 antibody. Thus,
h.alpha..sub.1AT gene transfer using a plasmid-liposome delivery
system protects CF bronchial epithelial cells from NE-induced cell
detachment and prevents release of a neutrophil chemotactic factor,
or factors, which is either IL-8 or an immunogenically related
molecule. Our data do not exclude the possibility of other
chemoattractants, or complement fragments.
[0108] Neutrophil elastase is capable of upregulating production of
specific cytokines by bronchial epithelial cells and enzymatically
inactivating others (Ruef, C. et al., Eur. Respir. J. 6:1429-1436,
1993). Upregulation of IL-8 or other chemoattractants may play a
critical role in perpetuating the inflammatory cycle by recruiting
more neutrophils into the lung airspaces. In contrast to other
proinflammatory mediators, elastase-induced stimulation of cytokine
production is steroid resistant (Bedard, M. et al., Am. J. Respir.
Cell Mol. Biol. 9:455-462, 1993) but can be prevented by
.alpha..sub.1AT or secretory leukoprotease inhibitor (SLPI)
(Nakamura, H. et al., J. Clin. Invest. 89:1478-1484, 1992). Because
elastase's effect on epithelial cells is steroid resistant but can
be inactivated by the antiproteases .alpha..sub.1AT or SLPI, some
type of antielastase therapy may be beneficial in the treatment of
neutrophil-dominated lung diseases.
[0109] With the data accumulated implicating NE as a major agent of
lung destruction in various pulmonary diseases including CF,
antiprotease therapy seems rational. Although .alpha..sub.1AT, the
predominant antiprotease responsible for neutralizing NE, is
present in the lungs from CF patients, most of it is in an inactive
form (Meyer, K. C. et al. Science 245:1073-1080, 1989 and Suter, S.
et al., J. Infect. Dis. 153:902-909, 1986). Because of the chronic
inflammatory state in the lungs of CF patients, it is possible that
.alpha..sub.1AT could be oxidatively inactivated, but little data
exist to support this idea. Numerous studies have indicated that
.alpha..sub.1AT is inactivated by proteolysis rather than oxidation
and that this proteolysis is due to the overwhelming NE burden
found in the lungs of CF patients (Cantin, A. et al., Pediatr.
Pulmonol. 11:249-253, 1991 and Suter, S. and Chevalier, I., Eur.
Respir. J. 4:40-49, 1991). Available evidence suggests that
protease-antiprotease imbalance exists in the lungs of CF
patients.
[0110] In contrast to .alpha..sub.1AT deficiency, in which the lung
disease is the result of a systemic deficiency in antiprotease
protection 926), lung disease in CF is the result of a local
overwhelming protease burden. Consequently, it is rational to
direct antiprotease therapy locally rather than systemically. Even
though aerosol therapy with SLPI or .alpha..sub.1AT protein
corrects the protease-antiprotease imbalance in the epithelial
lining fluid (ELF) of CF patients (McElvaney, N. G. et al., J.
Clin. Invest. 90:1296-1301, 1992 and McElvaney, M. G. et al.,
Lancet 337:392-394 (1991), twice-daily therapy is necessary and
prohibitively expensive, h.alpha..sub.1AT gene therapy may permit
administration of the transgene at weekly or possibly longer
intervals with a more cost-effective profile. In addition, by
targeting the epithelial cells, h.alpha..sub.1AT aerosol gene
therapy may prevent elastolysis within the neutrophil-epithelial
cell interface, a microenvironment which excludes the exogenously
administered protein (Campbell, E. J. and Campbell, M. A., J. Cell
Biol. 106:667-676, 1988).
[0111] Plasmid-liposome mediated delivery of the .alpha..sub.1AT
gene to CF epithelial cells in vitro protects against
elastase-induced epithelial cell detachment and chemotactic release
from these cells. This approach to treating the inflammatory lung
injury in patients with CF or other inflammatory lung diseases
could complement current therapy directed at these diseases.
[0112] III. Administration
[0113] Administration as used herein refers to the route of
introduction of the composition into the body. Administration
includes but is not limited to intravenous, intramuscular,
systemic, subcutaneous, subdermal, topical, or oral methods of
delivery. Administration can be directly to a target tissue or
through systemic delivery.
[0114] In particular, the present invention can be used for
administering nucleic acid for expression of specific nucleic acid
sequence in cells. Routes of administration include intramuscular,
aerosol, olfactory, oral, topical, systemic, ocular,
intraperitoneal and/or intratracheal. A preferred method of
administering compositions is by oral delivery. Another preferred
method of administration is by direct injection into the cells or
by systemic intravenous injection.
[0115] Transfer of genes directly has been very effective.
Experiments show that administration by direct injection of DNA
into joints and thyroid tissue results in expression of the gene in
the area of injection. Injection of plasmids containing IL-1 into
the spaces of the joints results in expression of the gene for
prolonged periods of time. The injected DNA appears to persist in
an unintegrated extrachromosomal state. This means of transfer is
one of the preferred embodiments.
[0116] In addition, another means to administer the compositions of
the present invention is by using a dry powder form for inhalation.
Furthermore, administration may also be through an aerosol
composition or liquid form into a nebulizer mist and thereby
inhaled.
[0117] The special delivery route of any selected vector construct
will depend on the particular use for the nucleic acid associated
with the chitosan-based composition. In general, a specific
delivery program for each chitosan-based composition used will
focus on uptake with regard to the particular targeted tissue,
followed by demonstration of efficacy. Uptake studies will include
uptake assays to evaluate cellular uptake of the nucleic acid and
expression of the specific nucleic acid of choice. Such assays will
also determine the localization of the target nucleic acid after
uptake, and establishing the requirements for maintenance of
steady-state concentrations of expressed protein. Efficacy and
cytotoxicity is then tested. Toxicity will not only include cell
viability but also cell function.
[0118] The chosen method of delivery should result in cytoplasmic
accumulation and optimal dosing. The dosage will depend upon the
disease and the route of administration but should be between
0.1-1000 mg/kg of body weight/day. This level is readily
determinable by standard methods. It could be more or less
depending on the optimal dosing. The duration of treatment will
extend through the course of the disease symptoms, possibly
continuously. The number of doses will depend upon disease delivery
vehicle and efficacy data from clinical trials.
[0119] Establishment of therapeutic levels of nucleic acid or
oligonucleotide within the cell is dependent upon the rate of
uptake and degradation. Decreasing the degree of degradation will
prolong the intracellular half-life of the nucleic acid or
oligonucleotide.
[0120] V. Direct Delivery to the Liver
[0121] Compositions of the present invention can also be used in
reversing or arresting the progression of disease involving the
liver, such as liver cancer. One embodiment involves use of
intravenous methods of administration to delivery nucleic acid
encoding for a necessary molecule to treat disease in the liver.
Compositions which express a necessary protein or RNA can be
directly injected into the liver or blood supply so as to travel
directly to the liver.
[0122] VI. Direct DNA Delivery to Muscle
[0123] The muscular dystrophies are a group of diseases that result
in abnormal muscle development, due to many different reasons.
These diseases can be treated by using the direct delivery of genes
with the compositions of the present invention resulting in the
production of normal gene product. Delivery to the muscle using the
present invention is done to present genes that produce various
antigens for vaccines against a multitude of infections of both
viral, bacterial, and parasitic origin. The detrimental effects
caused by aging can also be treated using the compositions
described herein. Since the injection of the growth hormone protein
promotes growth and proliferation of muscle tissue, the growth
hormone gene can be delivered to muscle, resulting in both muscle
growth and development, which is decreased during the later
portions of the aging process. Genes expressing other growth
related factors can be delivered, such as Insulin Like Growth
Factor-1 (IGF-1). Furthermore, any number of different genes may be
delivered by this method to the muscle tissue.
[0124] IGF-1 can be used to deliver DNA to muscle, since it
undergoes uptake into cells by receptor-mediated endocytosis. This
polypeptide is 70 amino acids in length and is a member of the
growth promoting polypeptides structurally related to insulin. It
is involved in the regulation of tissue growth and cellular
differentiation affecting the proliferation and metabolic
activities of a wide variety of cell types, since the polypeptide
has receptors on many types of tissue. As a result, the
chitosan-based compositions of the present invention can utilize
IGF-1 as a ligand for tissue-specific nucleic acid delivery to
muscle. The advantage of a IGF-1/nucleic acid delivery system is
that the specificity and the efficiency of the delivery is greatly
increased due to a great number of cells coming into contact with
the ligand/composition with uptake through receptor-mediated
endocytosis. Using the nucleic acid described above in the
chitosan-based compositions of the present invention with the use
of specific ligands for the delivery of nucleic acid to muscle
cells provides treatment of diseases and abnormalities that affect
muscle tissues.
[0125] VII. Direct DNA Delivery to Osteogenic Cells
[0126] There are many other problems that occur during the aging
process, but one major problem is osteoporosis, which is the
decrease in overall bone mass and strength. The direct delivery of
compositions of the present invention can be used to deliver genes
to cells that promote bone growth. The osteoblasts are the main
bone forming cell in the body, but there are other cells that are
capable of aiding in bone formation. The stromal cells of the bone
marrow are the source of stem cells for osteoblasts. The stromal
cells differentiate into a population of cells known as Inducible
Osteoprogenitor Cells (IOPC), which then under induction of growth
factors, differentiate into Determined Osteoprogenitor Cells
(DOPC). It is this population of cells that mature directly into
bone producing cells. The IOPCs are also found in muscle and soft
connective tissues. Another cell involved in the bone formation
process is the cartilage-producing cell known as the
chondrocyte.
[0127] A factor identified to be involved in stimulating the IOPCs
to differentiate is known as Bone Morphogenetic Protein (BMP). This
19,000 MW protein was first identified from demineralized bone.
Another similar factor is Cartilage Induction Factor (CIF), which
also functions to stimulate IOPCs to differentiate thereby
initiating cartilage formation, cartilage calcification, vascular
invasion, resorption of calcified cartilage, and finally induction
of new bone formation. Cartilage Induction Factor has been
identified as being homologous to Transfecting Growth Factor
.beta..
[0128] Since osteoblasts are involved in bone production, genes
that enhance osteoblast activity can be delivered directly to these
cells. Genes can also be delivered to the IOPCs and the
chondrocytes, which can differentiate into osteoblasts, leading to
bone formation. BMP and CIF are the ligands that can be used to
deliver genes to these cells. Genes delivered to these cells
promote bone formation or the proliferation of osteoblasts. The
polypeptide, IGF-1 stimulates growth in hypophysectomized rats
which could be due to specific uptake of the polypeptide by
osteoblasts or by the interaction of the polypeptide with
chondrocytes, which result in the formation of osteoblasts. Other
specific bone cell and growth factors can be used through the
interaction with various cells involved in bone formation to
promote osteogenesis.
[0129] Nonlimiting examples of genes expressing the following
growth factors which can be delivered to these cell types are
Insulin, Insulin-Like Growth Factor-1, Insulin-Like Growth
Factor-2, Epidermal Growth Factor, Transfecting Growth
Factor-.alpha., Transfecting Growth Factor-.beta., Platelet Derived
Growth Factor, Acidic Fibroblast Growth Factor, Basic Fibroblast
Growth Factor, Bone Derived Growth Factors, Bone Morphogenetic
Protein, Cartilage Induction Factor, Estradiol, and Growth Hormone.
All of these factors have a positive effect on the proliferation of
osteoblasts, the related stem cells, and chondrocytes. As a result,
BMP or CIF can be used as conjugates to deliver genes that express
these growth factors to the target cells by the intravenous
injection of the nucleic acid/chitosan compositions of the present
invention. Using the nucleic acid described above in the
chitosan-based compositions of the present invention with the use
of specific ligands for the delivery of nucleic acid to bone cells
provides treatment of diseases and abnormalities that affect bone
tissues.
[0130] VIII. Direct DNA Delivery to the Synoviocytes
[0131] The inflammatory attack on joints in animal models and human
diseases may be mediated, in part, by secretion of cytokines such
as IL-1 and IL-6 which stimulate the local inflammatory response.
The inflammatory reaction may be modified by local secretion of
soluble fragments of the receptors for these ligands. The complex
between the ligand and the soluble receptor prevents the ligand
from binding to the receptor is normally present on the surface of
cells, thus preventing the stimulation of the inflammatory
effect.
[0132] Therapy consists of the construction of a vector containing
the soluble form of receptors for appropriate cytokines (for
example, IL-1), together with promoters capable of inducing high
level expression in structures of the joint and composition which
enables efficient uptake of this vector. This composition is then
used with the nucleic acid carried by the chitosan-based
compositions of the present invention. This DNA is injected into
affected joints where the secretion of an inhibitor for IL-1 such
as a soluble IL-1 receptor or natural IL-I inhibitor modifies the
local inflammatory response and resulting arthritis.
[0133] This method is useful in treating episodes of arthritis
which characterize many "autoimmune" or "collagen vascular"
diseases. This method can also prevent disabling injury of large
joints by inflammatory arthritis.
[0134] In addition to the above, the present invention can also be
used with the following method. Current therapy for severe
arthritis involves the administration of pharmacological agents
including steroids to depress the inflammatory response. Steroids
can be administered systemically or locally by direct injection
into the joint space.
[0135] Steroids normally function by binding to receptors within
the cytoplasm of cells. Formation of the steroid-receptor complex
changes the structure of the receptor so that it becomes capable of
translocating to the nucleus and binding to specific sequences
within the genome of the cell and altering the expression of
specific genes. Genetic modifications of the steroid receptor can
be made which enable this receptor to bind naturally occurring
steroids with higher affinity, or bind non-natural, synthetic
steroids, such as RU486. Other modifications can be made to create
steroid receptor which is "constitutively active" meaning that it
is capable of binding to DNA and regulating gene expression in the
absence of steroid in the same way that the natural steroid
receptor regulates gene expression after treatment with natural or
synthetic steroids.
[0136] Of particular importance is the effect of glucocorticoid
steroids such as cortisone, hydrocortisone, prednisone, or
dexamethasone which are the most important drugs available for the
treatment of arthritis. One approach to treating arthritis is to
introduce a vector in which the nucleic acid cassette expresses a
genetically modified steroid receptor into cells of the joint,
e.g., a genetically modified steroid receptor which mimics the
effect of glucocorticoids but does not require the presence of
glucocorticoids for effect. This is termed the glucocortico-mimetic
receptor. This is achieved by expression of a constitutively active
steroid receptor within cells of the joint which contains the DNA
binding domain of a glucocorticoid receptor. This induces the
therapeutic effects of steroids without the systemic toxicity of
these drugs.
[0137] Alternatively, steroid receptors which have a higher
affinity for natural or synthetic glucocorticoids, such as RU486,
can be introduced into the joint. These receptors exert an
increased anti-inflammatory effect when stimulated by non-toxic
concentrations of steroids or lower doses of pharmacologically
administered steroids. Alternatively, constitution of a steroid
receptor which is activated by a novel, normally-inert steroid
enables the use of drugs which would affect only cells taking up
this receptor. These strategies obtain a therapeutic effect from
steroids on arthritis without the profound systemic complications
associated with these drugs. Of particular importance is the
ability to target these genes differentially to specific cell types
(for example synovial cells versus lymphocytes) to affect the
activity of these cells.
[0138] As described in U.S. Pat. No. 5,364,791 to Vegeto, et al.,
entitled "Progesterone Receptor Having C Terminal Hormone Binding
Domain Truncations," and U.S. application Ser. No. 07/939,246,
entitled "Mutated Steroid Hormone Receptors, Methods for Their Use
and Molecular Switch for Gene Therapy," Vegeto, et al., filed Sep.
2, 1992, both hereby incorporated by reference (including
drawings), genetically modified receptors, such as the
glucocortico-mimetic receptor, can be used to create novel steroid
receptors including those with glucocortico-mimetic activity. The
steroid receptor family of gene regulatory proteins is an ideal set
of such molecules. These proteins are ligand activated
transcription factors whose ligands can range from steroids to
retinoids, fatty acids, vitamins, thyroid hormones and other
presently unidentified small molecules. These compounds bind to
receptors and either up-regulate or down-regulate
transcription.
[0139] The preferred receptor of the present invention is
modification of the glucocorticoid receptor, i.e., the
glucocorticoid-mimetic receptor. These receptors can be modified to
allow them to bind various ligands whose structure differs from
naturally occurring ligands, e.g., RU486. For example, small
C-terminal alterations in amino acid sequence, including
truncation, result in altered affinity and altered function of the
ligand. By screening receptor mutants, receptors can be customized
to respond to ligands which do not activate the host cells own
receptors.
[0140] A person having ordinary skill in the art will recognize,
however, that various mutations, for example, a shorter deletion of
carboxy terminal amino acids, will be necessary to create useful
mutants of certain steroid hormone receptor proteins. Steroid
hormone receptors which may be mutated are any of those receptors
which comprise the steroid hormone receptor super family, such as
receptors including the estrogen, progesterone,
glucocorticoid-.alpha., glucocorticoid-.beta., mineral corticoid,
androgen, thyroid hormone, retinoic acid, and Vitamin B3 receptors.
Furthermore, DNA encoding for other mutated steroids such as those
which are capable of only transrepression or of only
transactivation are also within the scope of the above embodiment.
Such steroids could be capable of responding to RU486 in order to
activate transrepression.
[0141] In addition to the above, the present invention can also be
used with the following method. Drugs which inhibit the enzyme
prostaglandin synthase are important agents in the treatment of
arthritis. This is due, in part, to the important role of certain
prostaglandin in stimulating the local immune response. Salicylates
are widely used drugs but can be administered in limited doses
which are often inadequate for severe forms of arthritis.
[0142] Gene transfer using the present invention is used to inhibit
the action of prostaglandin synthase specifically in affected
joints by the expression of an antisense RNA for prostaglandin
synthase. The complex formed between the antisense RNA and mRNA for
prostaglandin synthase interferes with the proper processing and
translation of this mRNA and lowers the levels of this enzyme in
treated cells. Alternatively RNA molecules are used for forming a
triple helix in regulatory regions of genes expressing enzymes
required for prostaglandin synthesis. Alternatively, RNA molecules
are identified which bind the active site of enzymes required for
prostaglandin synthesis and inhibit this activity.
[0143] Alternatively, genes encoding enzymes which alter
prostaglandin metabolism can be transferred into the joint. These
have an important anti-inflammatory effect by altering the chemical
composition or concentration of inflammatory prostaglandin.
[0144] Likewise, the present invention is useful for enhancing
repair and regeneration of the joints. The regenerative capacity of
the joint is limited by the fact that chondrocytes are not capable
of remodeling and repairing cartilaginous tissues such as tendons
and cartilage. Further, collagen which is produced in response to
injury is of a different type lacking the tensile strength of
normal collagen. Further, the injury collagen is not remodeled
effectively by available collagenase. In addition, inappropriate
expression of certain metalloproteinases is a component in the
destruction of the joint.
[0145] Gene transfer using promoters specific to chondrocytes
(i.e., collagen promoters) is used to express different collagens
or appropriate collagenase for the purpose of improving the
restoration of function in the joints and prevent scar
formation.
[0146] Gene transfer for these purposes is affected by direct
introduction of nucleic acid into the joint space where it comes
into contact with chondrocytes and synovial cells. Further, the
genes permeate into the environment of the joint where they are
taken up by fibroblasts, myoblasts, and other constituents of
periarticular tissue.
[0147] IX. Direct Delivery to the Lungs
[0148] Compositions of the present invention can also be used in
reversing or arresting the progression of disease involving the
lungs, such as lung cancer. One embodiment involves use of
intravenous methods of administration to delivery nucleic acid
encoding for a necessary molecule to treat disease in the lung.
Compositions which express a necessary protein or RNA can be
directly injected into the lungs or blood supply so as to travel
directly to the lungs. Furthermore, the use of an aerosol or a
liquid in a nebulizer mist can also be used to administer the
desired nucleic acid to the lungs. Finally, a dry powder form can
be used to treat disease in the lung. The dry powder form is
delivered by inhalation. These treatments can be used to control or
suppress lung cancer or other lung diseases by expression of a
particular protein encoded by the nucleic acid which is chosen to
be delivered.
[0149] Additional organs, tissues, cavities, cell or cells, spaces
for the administration of the molecules mentioned herein may be
found in "Nucleic Acid Transporters for Delivery of Nucleic Acids
into a Cell"; Smith et al., U.S. patent application Ser. No.
08/484,777, filed Dec. 18, 1995, incorporated herein by reference
in its entirety including any drawings.
EXAMPLES
[0150] The present invention will be more fully described in
conjunction with the following specific examples which are not to
be construed in any way as limiting the scope of the invention.
[0151] Materials and Methods
[0152] Preparation of pCMV4.alpha..sub.1AT Vector. Production and
design of the plasmid used for this study, pCMV4.alpha..sub.1AT,
has been previously described in International Patent Application,
Publication Number WO 92/19730, which is hereby incorporated herein
by reference in its entirety, including any drawings.
[0153] Isolation of Neutrophils for Chemotaxis Assay. A 30 ml
sample of heparinized whole blood was collected from a single
healthy adult donor into syringes. The collected blood was mixed
1:1 with normal saline, and each 30 ml of blood was layered over 15
ml Ficoll-Paquc (specific gravity 1.077) (Pharmacia, Piscataway,
N.J.) and centrifuged at 800.times.g for 30 min. After
centrifugation, the top layer and interface were removed and
discarded. The cell pellet was resuspended in phosphate-buffered
saline followed by centrifugation at 400.times.g. The washed cell
pellet was mixed 1:1 with 3% T-500 dextran (Pharmacia) prepared in
isotonic saline and allowed to sediment for 1 hour. The upper
fraction, containing predominantly neutrophils, was removed and
centrifuged at 400.times.g.
[0154] To lyse the remaining erythrocytes, the resulting pellet was
treated for 60 s with 0.2% saline and then made isotonic by adding
1.8% saline (1:1, vol/vol). The cells were washed, resuspended in
Dulbecco's modified Eagle's medium (DMEM)-f/12 (Gibco/BRL, Grand
Island, N.Y.) in polypropylene tubes to prevent cell adherence, and
placed on ice. The total cell yield was determined by counting a
portion of the final suspension in a hemocytometer. This method
yielded 5 to 8.times.10.sup.7 cells/30 ml whole blood. Visibility
was in excess of 95% as measured by trypan blue dye exclusion. The
differential cell count was determined by staining cytospin
preparations with Diff-Quik (Baxter Scientific Productions, McGaw
Park, Ill.) and counting across the entire slide. The differential
was consistently greater than 93% neutrophils, with <1%
monocytes, approximately 5% eosinophils, and 2% lymphocytes.
[0155] Measurement of Neutrophil Chemotactic Activity. Isolated
human neutrophils from a single donor were used as responding
cells. These cells were suspended in DMEM-F/12 culture medium at a
concentration of 1.times.10.sup.4 polymorphonuclear leukocytes/ml.
Chemotactic assays were performed utilizing the multiwell
microchamber as previously described (Falk, W. et al., J. Immunol.
Methods 33:239-247, 1980 and McCain, R. et al., Am. J. Respir. Cell
Mol. Biol. 8:28-34, 1993). Briefly, neutrophils were placed in the
upper chamber, and the potential chemoattractant was passed in the
lower chamber separated by polyvinylpyrrolidone (PVP)-free
polycarbonate filters with 3-.mu.m pore diameters. Cell migration
took place during a 30-min incubation period in a humidified
incubator at 37.degree. C. in a 5% CO.sub.2/air atmosphere.
[0156] After incubation, the filters were fixed and stained with
Diff-Quik, and the neutrophils that had migrated through the filter
were counted under oil immersion (.times. 1,000) light microscopy.
Chemotactic activity was expressed as the mean of triplicate
determinations of the number of neutrophils that migrated toward
the tested chemoattractant in 10 oil immersion fields. Fresh
DMEM-F/12 was used as a negative control; IL-8 was used as a
positive control. Cell culture supernatant was treated with
neutralizing concentration of rabbit anti-human IL-8 serum (Upstate
Biotechnology inc., Lake Placid, N.Y.) to assess the contribution
of IL-8 to the chemotactic bioactivity. A 1:5,000 dilution of
rabbit anti-human IL-8 serum was added to each sample and incubated
for 1 h at 37.degree. C., which is sufficient to neutralize
biologically relevant concentrations of natural IL-8 (McCain, R. et
al., Am. J. Respir. Cell Mol. Biol. 8:23-34, 1993). The nonimmune
rabbit serum used as a control had no effect on chemotaxis of
neutrophils, nor did it alter the chemotactic activity of
stimulated culture supernatant.
[0157] Cell Culture Techniques. The immortalized human CP bronchial
epithelial cell line 2CFSMEo- was used in these experiments. The
cell line was a gift from Dr. Ray Frizzell (University of Alabama,
Birmingham) (Cozens, A. L. et al., Proc. Natl. Acad. Sci USA
89:5171-5175, 1992). These cells were maintained on a plastic
surface in DMEM-F/12 with 10% feral bovine serum (Summit
Biotechnology, Ft. Collins, Colo.). Confluent cell cultures were
removed from passage with 1% trypsin-EDTA. For the NE dose-response
and the NE:.alpha..sub.1AT dose-response experiments,
4.times.10.sup.5 cells were placed in serum-conditioned medium onto
30-mm, 6-multiwell tissue culture wells (Becton Dickinson, Lincoln
Park, N.J.). In some experiments, in an effort to minimize
interwell variability during gene transfer, the cells were plated
onto 100-mm tissue culture dishes (Corning Glass Works, Corning,
N.Y.) and transfected with pCMV4.alpha..sub.1AT or pCMV4 complexed
to cationic liposomes. These cells were harvested 3 days after
transfection, and 4.times.10.sup.5 of the transfected cells were
subcultured onto the six multiwell tissue culture plate for further
experiments.
[0158] Experimental Protocols
[0159] Neurophil Elastase Exposure on Untransfected 2CFSMEo- Cells.
To determine neutrophil chemotactic activity and immunoreactive
levels of IL-8 produced by 2CFSMEo- cells following NE exposure, a
time- and dose-response experiment was performed prior to the gene
transfer experiments. 2CFSMEo- cells were plated onto six-multiwell
plates as described above. At 24 h after the wells were seeded, the
cells were washed 3 times with serum-free media, and serum-free
media was added to the cells (see below). Concentration of NE from
5 to 25 nM were added to the cells for 24 hours. After
determination of the optimum dose, a time course was performed to
determine the time of peak chemotactic activity production in
response to NE. After the time- and dose-response to Ne were
established, 25 nM NE was incubated with concentrations of
h.alpha..sub.1AT protein (Prolastin; Miles Inc., Elkhart, Ind.)
ranging from 0 to 50 nM to determine the effect of NE
neutralization on chemotaxis.
[0160] Protocol of h.alpha..sub.1AT Gene Transfer. Various amounts
of pCMV4.alpha..sub.1AT or pCMV4 were complexed to synthetic
cationic liposomes (Lipofectin, Bethesda Research Laboratories,
Gaithersburg, Md.) in a 1:3 wt/wt ratio. In preliminary studies, a
1:3 wt/wt ratio of plasmid to liposomes achieved maximal transgene
expression for this plasmid. The plasmid was brought up to equal
volume with the cationic liposome by the addition of sterile water.
The liposomes were added to the DNA, and complex formation occurred
at room temperature. The plasmid-liposome complexes were added to
2CFSMEo- cells in the presence of serum-containing medium (day 0).
Then, 24 h later (day 1), the media was removed and fresh media was
added. Subsequently, the media was changed at either 24- or 48-h
intervals. All transfection experiments were done in
triplicate.
[0161] Time Course of Gene Expression. To determine an optimal time
to expose the cells to NE with respect to transgene expression, a
time course of gene expression was performed. During the NE
exposure to transfected 2CFSMEo- cells, media was left on the cells
for 48 h (see below). Therefore, after the media was removed 24 h
following transfection, h.alpha..sub.1AT secretion was determined
in 48-h intervals.
[0162] h.alpha..sub.1AT RNA Analysis. To analyze for
h.alpha..sub.1AT with mRNA, 2CFSMEo- cells in 100-mm tissue culture
dishes were transfected with pCMV4.alpha..sub.1AT-liposome
complexes. At day 3 after transfection, RNA was isolated using RNA
STAT-60 (TEL-0TEST "H"; Friendswood, Tex.). RNA analysis was
performed directly from gene transfer experiments in the 100-mm
culture dishes to provide a large cell population for RNA
isolation. The quality of the RNA was determined by electrophoresis
in a 1% agarose denaturing gel; the quantity was determined by
optical density (Ultrospec Plus; Pharmacia, Piscataway, N.J.).
Reverse transcriptase polymerase chain reaction (RT-PCR) was
performed using 2 .mu.g of RNA from both control and transfected
cells. To ensure that the RT-PCR only amplified RNA, 2 .mu.g of
PCMV4.alpha..sub.1AT plasmid was used as a negative, internal
control. Prior to RT, the RNA and the PCMV4.alpha..sub.1AT plasmid
control were incubated with DNAse (Promega, Madison, Wis.) at
37.degree. C. for 15 min to digest any DNA that may have been
present. Immediately following the 37.degree. C. incubation, the
DNAse was heat-inactivated at 65.alpha..sub.1AT C for 15 min.
[0163] First-strand cDNA synthesis was performed using a
First-Strand cDNA Synthesis kit (Pharmacia). After first-strand
cDNA synthesis was complete, the samples were diluted 1:5; two
20-mer primers located 534 bases apart were added with 200
.mu.MdNTP and Taq polymerase (Boehringer Mannheim, Indianapolis,
Ind.). PCR was performed for 35 cycles (denatured at 95.degree. C.,
annealed at 45.degree. C., extended at 72.degree. C.). The
resultant 534-base pair PCR product was electrophorased through a
1% agarose gel and visualized by staining with ethidium
bromide.
[0164] As a positive control for PCR, 2 .degree.g of
PCMV4.alpha..sub.1AT plasmid was processed in an identical manner
to the RNA samples except without DNAse treatment.
[0165] NE Treatment of Transfected 2CFSMEo- Cells. Prior to the
addition of human NE (Elastin Products Co., Owensville, Mo.), the
cells were washed with serum-free media 3 times. After the washes,
serum-free media was placed on the cells and NE was added 24 h
later. This was done for two reasons: First, serum-free media was
used to minimize any potential neutralization of h.alpha..sub.1AT
or Ne with serum proteins. Second, a 24-h interval between the
addition of fresh media and the addition of NE provided an
opportunity for the transfected cells to replenish the supernatant
with h.alpha..sub.1AT before adding NE. Then, 24 h after the
addition of NE, the supernatant was collected and centrifuged at
13,000.times.g for 5 min to remove any cellular debris. The
supernatant was stored at -70.degree. C. for future measurement of
h.alpha..sub.1AT and IL-8 by ELISA and for chemotactic activity. In
addition, the effect of transfection and NE treatment on cell
morphology was evaluated utilizing an inverted-phase
microscope.
[0166] Statistical Analysis
[0167] Data were evaluated with analysis of variance followed by
contrast analysis of planned comparisons and, where appropriate,
post hoc comparisons by the Schefftest.
Example 1
Method of Plasmid Construction
[0168] The pCMV4-.alpha..sub.1-antitrypsin plasmid is constructed
by linkerprimer polymerase chain reaction (LP-PCR).
[0169] The coding sequence of .alpha..sub.1-antitrypsin is inserted
into the PCMV4 expression vector. This method consists of
synthesizing two oligonucleotide primers (20-30 nucleotides in
length). One oligonucleotide is homologous to the 5' untranslated
region immediately upstream (5') of the initiation codon and the
second oligonucleotide is complementary to the 3' untranslated
region immediately downstream (Y) of the stop transcription codon.
Both oligonucleotides have a one or two base substitution which
creates a unique restriction enzyme site in the untranslated
regions of the amplified gene. The 5' and 3' oligonucleotides were
designed such that the created restriction enzyme site is
approximately 8 nucleotides downstream from the 5' end of the
oligonucleotide. Both of these requirements are critical, the
former to insure a restriction enzyme site which is recognizable
and cleavable and the latter to insure that the reading frame of
the gene is not altered.
[0170] Except for the cDNA for .alpha..sub.1-antitrypsin and the
3'UTR of human growth hormone which were sequenced using the Sanger
dideoxy method, the sequence is compiled using the current version
of Genbank as source of the sequence information. The reading frame
of the .alpha..sub.1-antitrypsin gene is amplified using Vent DNA
polymerase, 100 ng of target DNA, a programmable temperature
cycler, and standard reaction conditions (denaturing at
93.5.degree., annealing at 56.degree. and extension at 75.degree.).
Vent DNA polymerase is used because it has a 3' to 5' proofreading
activity in addition to enhanced stability at high temperature and
a highly specific and processive 5' to 3' DNA polymerase activity.
After PCR amplification, the unique restriction sites were cleaved
with the appropriate restriction enzymes (ClaI; SmaI), the
amplified gene is separated from the small fragments released by
the action of the restriction enzymes and from unincorporated
primers and nucleotides by gel filtration through a S-400 spin
column. The amplified genes which now had cloning sites on each end
were ligated into PCMV4 which had been previously cleaved with the
same restriction enzymes which were utilized to prepare the cloning
sites on the amplified gene.
[0171] After ligation, the pCMV4-.alpha..sub.1-antitrypsin
construct is transfected into fresh competent bacteria (E. coli
NM522). The competent bacteria is prepared by standard methods. A
single bacterial colony is selected from a 14-day old refrigerated
storage plate. The colony is placed in 30 ml of LB broth and grown
to an O.D. of 0.600 at 600 nm. The bacterial suspension is cooled
on ice for 10 min and the bacteria were collected by centrifugation
for 10 min at 1,650.times.g. The bacterial pellet is resuspended in
10 ml transformation buffer, kept on ice for 10 min, and collected
again by centrifugation as above. The pellet is resuspended in 2 ml
transformation buffer; 70 .mu.l of dimethylsulfoxide (DMSO) is
added and the sample kept on ice for 10 min; 70 .mu.l of 1M
dithiothreitol (DDT) in KAc buffer is added and the sample
incubated for 5 min before a second 70.mu. aliquot of DMSO
added.
[0172] A 300 .mu.l aliquot of the bacteria is removed and an
aliquot of the ligated DNA added; this mixture is incubated on ice
for 45 min, heatshocked at 42.degree. C. for 3.5 min, diluted to 1
ml with 2XYT medium and incubated at 37.degree. C. for 1 hour.
Multiple aliquots of this mixture (routinely 25, 50, and 200 .mu.l)
were plated separately on LB agar plates containing 50 .mu.g/ml of
ampicillin which provides selection pressure for bacteria
containing the pCMV4 construct. The plasmid carries the gene for
ampicillin resistance. After the bacteria which harbor the plasmid
have grown into distinct colonies several of the colonies are grown
up as individual 5 ml liquid cultures in 2XYT medium containing
ampicillin. Aliquots, of the liquid cultures are stored frozen at
-7020 C.
Example 2
Small-Scale Preparation (2 L) of Plasmid
[0173] A pellet of frozen stock (E. coli strain NM522 containing
PCMV4-.alpha..sub.1-antitrypsin) is streaked onto an LB agar plate
containing 50 .mu.g/ml ampicillin with a sterile platinum loop. The
plate is incubated at 37.degree. C. overnight. Two liters of 2XYT
medium containing ampicillin are inoculated with a single colony
from this plate. Following overnight incubation at 37.degree. C.
the bacteria are collected by centrifugation for 20 min at
10,000.times.g. The bacterial pellet is processed and purified
using the Quiagen Mega-prep kit according to the manufacturer's
protocol. The isolated plasmid is precipitated with ethanol and
resuspended in sterile water. Demineralized water is treated with a
Millipore Milli-Q water purification system until conductivity
(built-in) is 18 mOhm or less, filtered through a 0.22.mu.
Millipore depth filter, and then steam autoclaved. Samples are
stored at -20.degree. C. in 1 ml aliquots.
[0174] Glassware, centrifuge bottles, microfuge tubes, magnetic
stir bars, water (see above), and bacterial growth media are steam
sterilized at 121.degree. C. for 45 minutes. Every three months,
the autoclave is validated using a Killit Kit (BBL) according to
the manufacturer's protocol.
Example 3
Scaled-Up Preparation (100 L) of Plasmid
[0175] A large batch of plasmid (1.3-1.4 kg cells) is prepared by
the University of Iowa Large Scale Fermentation Facility, 3-670
Bowen Science Building, Iowa City, Iowa 52242 (Lacy Daniels)
according to the following protocol.
[0176] I. Summary
[0177] One 100 liter fermentor run is performed with E. coli strain
NM522-PCMV4-.alpha.1-antitrypsin grown in medium composed of a
mixture of Luria Broth Base and Terrific Broth. An inoculum is
prepared from an isolated colony, scaled up by growth on plates and
grown finally in Ferribach flasks for the inoculum. All media
contain originally ca. 75-100 .mu.g ampicillin/ml. Cells are
harvested with a Sharples continuous centrifuge after absorbance
reached a near maximal point. Cells are frozen directly in liquid
nitrogen and shipped on dry ice. Cell yields are usually 1.3-14. kg
wet cells per fermentor run.
[0178] ii. Inoculum Preparation
[0179] The vials received from Dr. Canonico, Vanderbilt University,
are streaked onto Luria agar plates containing 100 .mu.g
ampicillin/ml. In a laminar flow cabinet, one isolated colony is
used to streak a lawn onto one whole plate of the same type; 8
plates are prepared. Plates are incubated overnight at ca.
35-37.degree. C., and then, in the laminar flow cabinet, removed
aseptically by cotton swab into a flask containing 50 ml of sterile
Luria broth containing 20% glycerol. This solution is transferred
to a series of sterile small plastic storage vials which are placed
in a -45.degree. C. freezer for future convenient use as
inoculurn.
[0180] For a 100 liter fermentor, six vials are used to inoculate
six 2.8 liter Fernbach flasks (500 ml Luria Broth base media in
each flask+100 .mu.g ampicillin/ml). The flasks are incubated
shaking at ca. 150 rpm at ca. 35-37.degree. C. for 9 hours. Flasks
are combined into two containers aseptically and inoculation is
done by peristaltic pump using a sterile stainless steal probe
connected with a sterile silicone rubber tubing leading into the
top of the fermentor vessel via a septum port.
2 iii. Fermentor Medium 20 g/L Luria Broth Base 15 g/L Terrific
Broth powder 75 .mu.g/ml. ampicillin added as liquid to sterile
medium 3 ml/L glycerol 10 ml Sigma Antifoam B
[0181] iv. Fermentor
[0182] The fermentor is a 100 liter working volume B. Braun 100D.
It is sterilized in place at 121.degree. C. using steam; the medium
is heated at 121.degree. C. for 45 min during which all critical
connections and components are also sterilized. External items are
sterilized in an autoclave. Incoming air and exiting gas are
sterilized by filtration. Fermentation conditions are:
[0183] 37.degree. C.
[0184] 300 rpm (16 cm Rushton-type impeller)
[0185] air at 30 liters/min (0.3 vvm); internal pressure 0.3
bar
[0186] pH monitored with internal pH probe; confirmed with a lab pH
meter; pH is initially 6.8 and varies between 6.7 and 7.0 (no pH
control is used since there is predictably little change)
[0187] dissolved oxygen is monitored with an oxygen probe, and
varied between 100% of saturation at the start to a minimum of ca.
67% during the run an automatic foam control system and silicone
antifoam is provided to control foaming problems, but generally
very little foam is produced during the run; a total of ca. 20-40
ml of Sigma Antifoam B is used during the run.
[0188] Culture samples are taken each 1-2 hours to follow growth by
monitoring the absorbance at 600 nm. Samples are removed via a
sample port that is sanitized with steam following each sampling. A
plot of a typical growth curve is appended. The initial medium has
75 .mu.g/ml ampicillin; additional ampicillin is added after
approximately 3 and 6 hours to make an additional 25 or 50
.mu.g/ml, respectively.
[0189] v. Harvesting
[0190] When cells reach near maximum absorbance (after ca. 5-7.5
hours of fermentor growth), vessel cool-down begins and harvesting
is initiated when the medium reaches 25.degree. C.; cooling
continues during harvesting and the contents reach 17.degree. C. by
the end of harvesting. Harvesting is done with a Sharples tubular
bowl continuous centrifuge capable of harvesting a maximum of ca.
1.6 kg wet cells. The cells are fed via a Tygon hose from the
sample port into the bottom of the rotating stainless steel bowl.
When all of the vessel contents are centrifuged, the centrifuge
bowl is removed and the contents removed by spatula and placed as
chunks of a paste into liquid nitrogen. The frozen chunks are then
transferred to clean plastic bottles and kept in a -45.degree. C.
freezer until they are shipped on dry ice. Cell yields per
fermentor are 1.3-1.4 kg.
[0191] Although certain embodiments and examples have been used to
describe the present invention, it will be apparent to those
skilled in the art that changes to the embodiments and examples
shown may be made without departing from the scope or spirit of the
invention.
[0192] Those references not previously incorporated herein by
reference, including both patent and non-patent references, are
expressly incorporated herein by reference for all purposes.
[0193] Other embodiments are within the following claims.
* * * * *