U.S. patent application number 10/172388 was filed with the patent office on 2003-04-24 for delivery of polypeptides by secretory gland expression.
Invention is credited to German, Michael S., Goldfine, Ira D., Rothman, Stephen S..
Application Number | 20030078226 10/172388 |
Document ID | / |
Family ID | 27384068 |
Filed Date | 2003-04-24 |
United States Patent
Application |
20030078226 |
Kind Code |
A1 |
German, Michael S. ; et
al. |
April 24, 2003 |
Delivery of polypeptides by secretory gland expression
Abstract
The invention features methods for delivering a polypeptide to
the bloodstream of a subject by introduction of a nucleic acid
construct into secretory gland cells(e.g., cells of salivary gland,
pancreas, or liver). In general, the method involves introduction
of a nucleic acid construct into a secretory gland duct, which
introduction results in expression of a gene product encoded by the
introduced construct and delivery of the gene product into the
bloodstream of the subject.
Inventors: |
German, Michael S.; (Daly
City, CA) ; Goldfine, Ira D.; (Kentfield, CA)
; Rothman, Stephen S.; (Berkeley, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD RD
SUITE 200
MENLO PARK
CA
94025
US
|
Family ID: |
27384068 |
Appl. No.: |
10/172388 |
Filed: |
June 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10172388 |
Jun 14, 2002 |
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09550302 |
Apr 14, 2000 |
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09550302 |
Apr 14, 2000 |
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09130886 |
Aug 7, 1998 |
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6255289 |
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09130886 |
Aug 7, 1998 |
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08591197 |
Jan 16, 1996 |
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5885971 |
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08591197 |
Jan 16, 1996 |
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08410660 |
Mar 24, 1995 |
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5837693 |
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Current U.S.
Class: |
514/44R ;
435/455 |
Current CPC
Class: |
A61K 38/28 20130101;
A61K 38/1709 20130101; A61K 38/37 20130101; A61K 38/1816 20130101;
A61K 38/27 20130101; A61K 48/00 20130101 |
Class at
Publication: |
514/44 ;
435/455 |
International
Class: |
A61K 048/00; C12N
015/85 |
Claims
What is claimed is:
1. A method of delivering a protein to the bloodstream of a mammal,
the method comprising the step of: introducing a DNA construct into
a pancreas cell in vivo, wherein the DNA construct comprises a DNA
sequence of interest which encodes a protein and a eukaryotic
promoting sequence operably linked to the DNA sequence of interest,
wherein the introduced DNA construct is expressed and the protein
encoded by the introduced DNA construct is delivered into the blood
stream of the mammal.
2. The method of claim 1, wherein said introducing is by delivery
of the DNA construct into a lumen of a pancreatic duct.
3. The method of claim 1, wherein the DNA construct is not
contained within a viral particle.
4. The method of claim 1, wherein said introducing is by delivery
of the DNA construct into a lumen of a pancreatic duct, and the DNA
construct is not contained within a viral particle.
5. The method of claim 1, wherein the protein is insulin.
6. The method of claim 1, wherein the protein is growth
hormone.
7. The method of claim 1, wherein the protein is clotting factor
VIII.
8. The method of claim 1, wherein the protein is
erythropoietin.
9. The method of claim 1, wherein the mammal is a human and the
protein is a human protein.
10. The method of claim 5, wherein the protein is selected from the
group consisting of human growth hormone and human insulin.
11. A method of delivering a protein to the bloodstream of a
mammal, the method comprising the step of: introducing a DNA
construct into a liver cell in vivo, wherein the DNA construct
comprises a DNA sequence of interest which encodes a protein and a
eukaryotic promoting sequence operably linked to the DNA sequence
of interest, wherein the introduced DNA construct is expressed and
the protein encoded by the introduced DNA construct is delivered
into the blood stream of the mammal.
12. The method of claim 11, wherein said introducing is by delivery
of the DNA construct into a lumen of a hepatic duct.
13. The method of claim 11, wherein the DNA construct is not
contained within a viral particle.
14. The method of claim 11, wherein said introducing is by delivery
of the DNA construct into a lumen of a hepatic duct, and the DNA
construct is not contained within a viral particle.
15. The method of claim 11, wherein the protein is insulin.
16. The method of claim 11, wherein the protein is growth
hormone.
17. The method of claim 11, wherein the protein is clotting factor
VIII.
18. The method of claim 11, wherein the protein is
erythropoietin.
19. The method of claim 11, wherein the mammal is a human and the
protein is a human protein.
20. The method of claim 15, wherein the human protein is selected
from the group consisting of human growth hormone and human
insulin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 09/130,888, filed Aug. 7, 1998, which is a
continuation of U.S. application Ser. No. 08/591,197, filed Jan.
16, 1996, which is a continuation-in-part of U.S. application Ser.
No. 08/410,660, filed Mar. 24, 1995, which applications are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates generally to delivery of a substance
to the bloodstream of a subject, in particular to
bloodstream-directed delivery of a polypeptide.
BACKGROUND OF THE INVENTION
[0003] The ability to replace defective or absent genes has
attracted wide attention as a method to treat a variety of human
diseases (Crystal 1995 Science 270:404), Lever et al. 1995 Gene
Therapy. Pearson Professional, New York p. 1-91; Friedmann 1996
Nature Med. 2:144). Although originally intended as a means of
correcting inherited disorders in certain populations of somatic
cells, gene-based therapy can be a useful means to supply exogenous
gene products to the circulatory system for the treatment of a wide
range of systemic disorders that involve deficiencies in
circulating proteins, such as hormones, growth factors, and
clotting proteins (Lever et al. 1995 supra; Buckel 1996 TiPS
17:450), as well as a means of administering other polypeptide
drugs. The success of this application depends upon developing
effective methods to both manufacture the desired protein in vivo
and then secrete it into blood (Crystal 1995 supra; Lever et al.
1995 supra).
[0004] Currently, DNA-based therapy (i.e., gene therapy) is carried
out in a variety of ways but involves two general protocols. In the
first method, referred to as ex vivo gene therapy, cells are
extracted from an individual and subjected to genetic manipulation.
After genetic material has been properly inserted into the cells,
the cells are implanted back into the individual from which they
were removed. Persistent, in vivo expression of the newly implanted
genetic material after transplantation of the transformed cells has
been successful (see Morgan et al., Science 237:1476 (1987); and
Gerrard et al., Nat. Genet. 3:180 (1993)). In the second approach
to DNA-based therapy, referred to as in vivo gene therapy, cells
within a living organism are transformed in situ with exogenous
genetic material.
[0005] Several different methods for transforming cells can be used
in accordance with either the ex vivo or in vivo transfection
procedures. For example, various mechanical methods can be used to
deliver the genetic material, including the use of fusogenic lipid
vesicles (liposomes incorporating cationic lipids such as
lipofection; see Felgner et al., Proc. Natl. Acad. Sci. U.S.A.
84:7413-7417 (1987)); direct injection of DNA (Wolff, et al.,
Science (1990) 247:1465-1468); and pneumatic delivery of DNA-coated
gold particles with a device referred to as the gene gun (Yang et
al., Proc. Natl. Acad. Sci. U.S.A. 1990; 87:1568-9572). Morsy et
al. reviews several of the different techniques useful in
transformation of cells ex vivo or in vivo and provides citations
of numerous publications in each area (Morsy et al., JAMA
270:2338-2345 (1993)).
[0006] One method of particular interest for delivery of genetic
material involves use of recombinant viruses to infect cells in
vivo or ex vivo. In these methods, a virus containing the desired
genetic material is allowed to infect target cells within the
subject. Upon infection, the virus injects its genetic material
into the target cells. The genetic material is then expressed
within the target cell, providing for expression of the desired
genetic material. However, it would be preferable to avoid
introduction of the desired genetic material by viral infection for
a number of reasons. For example, viral infection results in
delivery of viral DNA in addition to the desired genetic material,
which may in turn result in undesirable cellular effects such as,
adverse immune reactions, productive viral replication, and adverse
integration events.
[0007] There is a need in the field for a method for delivery of
genetic material into a cell in vivo to provide for expression of
the introduced polynucleotide and secretion of the gene product it
encodes into the bloodstream. The present invention addresses this
problem.
SUMMARY OF THE INVENTION
[0008] The invention features methods for delivering a polypeptide
to the bloodstream of a subject by introduction of a nucleic acid
construct into secretory gland cells(e.g., cells of salivary gland,
pancreas, or liver). In general, the method involves introduction
of a nucleic acid construct into a secretory gland duct, which
introduction results in expression of a gene product encoded by the
introduced construct and delivery of the gene product into the
bloodstream of the subject.
[0009] A primary object is to provide a method of delivering a
polypeptide to the bloodstream of a subject by introducing a
nucleic acid construct into cells of a secretory gland, e.g.,
liver, pancreatic or salivary gland (e.g., parotid gland) cells,
preferably by introduction of the construct into a duct of a
secretory gland. The secretory gland cells subsequently express a
biologically active protein, which protein is secreted into the
circulatory system.
[0010] Another object is to provide a non-invasive method of
protein delivery (i.e., the method involves introduction of the
nucleic acid of interest from outside the body (i.e., from the duct
system of particular glands) wherein cells of a secretory gland,
preferably the pancreas, salivary gland, or liver of a mammal are
genetically modified to express a biologically active and
therapeutically useful polypeptide, which polypeptide is secreted
into the circulatory system of the individual.
[0011] Another object is to produce genetically transformed
secretory gland cells which cells have incorporated into their
genome genetic material which, when expressed, produces a
biologically active and therapeutically useful protein which is
secreted into the circulatory system.
[0012] An advantage of the present invention is that polypeptides
can be delivered into the bloodstream on either a long term basis
(e.g., by repeated administration of the construct) or on a short
term basis. Thus, the invention is useful for treatment of diseases
or conditions wherein individuals are suffering from a deficiency
in a particular protein and/or can benefit from administration of
an exogenous protein having a desired activity (e.g, antimicrobial
activity).
[0013] Another advantage of the invention is that, in one
embodiment, the nucleic acid constructs can be introduced for
expression in a secretory gland cell without the need to contain
the construct within a virion (e.g., the method does not require
the use of a viral vector containing the construct to introduce the
nucleic acid of interest into the secretory gland cell).
[0014] These and other objects, advantages and features of the
present invention will become apparent to those persons skilled in
the art upon reading the details of the vectors, cell lines and
methodology as more fully set forth below.
BRIEF DESCRIPTION OF THE DRAWING
[0015] FIG. 1 is a schematic view of pBAT14hins construct useful in
producing recombinant insulin in secretory gland cells according to
the invention;
[0016] FIG. 2 is a map of the pFGH construct, which contains the
human growth hormone genomic sequence;
[0017] FIG. 3 is a map of the pFGH.CMV construct, which contains
the human growth hormone genomic sequence operably linked to the
CMV promoter;
[0018] FIG. 4 is a map of the pFGH.chymo construct, which contains
the human growth hormone genomic sequence operably linked to the
chymotrypsin B promoter;
[0019] FIG. 5 is a graph showing tissue expression of hGH following
transformation of salivary gland cells by intraductal
introduction.
[0020] FIG. 6 is a graph showing regulation of plasma levels of
recombinant hGH expressed from salivary gland cells.
[0021] FIG. 7 is graph showing the relative levels of hGH
expression in salivary gland tissue in rats that received either no
DNA (control rats), pFGH.CMV, pFGH.CMV premixed with lipofectin, or
pFGH.CMV premixed with adenovirus.
[0022] FIG. 8 is a graph showing the levels of tissue expression of
human growth hormone expression in the pancreas of rats after
intraductal retrograde introduction with either a control
containing no DNA or a test sample containing a human growth
hormone construct;
[0023] FIG. 9 is a graph showing the serum levels of human growth
hormone in rats after intraductal retrograde introduction into the
pancreas with either a control containing no DNA or a test sample
containing a human growth hormone construct; and
[0024] FIG. 10 is a graph showing the correlation between
pancreatic tissue expression and serum levels of human growth
hormone.
[0025] FIG. 11 is a graph showing regulation of plasma levels of
recombinant hGH expressed from pancreatic cells.
[0026] FIG. 12 is a map of the pBAT16.hInsG1.M2 construct, which
contains DNA encoding an altered form of human insulin.
[0027] FIG. 13 is a graph showing the glucose response in
streptozotocin-treated rats having pancreatic cells transformed
with either human insulin (open bars) or green fluorescent protein
(GFP; striped bars).
[0028] FIG. 14 is a graph showing the blood glucose levels in
control rats (mock-treated; closed squares), streptozotocin-treated
rats (open squares), and streptozotocin rats treated by
transformation of pancreatic cells with DNA encoding human insulin
(closed circles).
[0029] FIG. 15 is a graph showing the relative amounts of hGH in
the pancreatic tissue of rats that received either pFGH (control),
pFGH.chymo, pFGH.RSV, pFGH.RSV, or pFGH.CMV by intraductal
administration to the pancreas.
[0030] FIG. 16 is a graph showing the relative amounts of hGH in
the pancreatic tissue of rats that received either no DNA
(mock-transformed), pFGH.CMV, pFGH.CMV premixed with lipofectin, or
pFGH.CMV premixed with adenovirus.
[0031] FIG. 17 is a graph showing the relative levels of plasma hGH
in rats that received either pFGH (control), pFGH.chymo, pFGH.RSV,
pFGH.RSV, or pFGH.CMV by intraductal administration to the
pancreas.
[0032] FIG. 18 is a graph showing the relative amounts of plasma
hGH in rats that received either no DNA (mock-transformed),
pFGH.CMV, pFGH.CMV premixed with lipofectin, or pFGH.CMV premixed
with adenovirus by intraductal administration to the pancreas.
[0033] FIG. 19 is a graph showing the blood glucose levels of
streptozotocin-treated rats (diabetic) that received either no DNA
(open squares) or received human insulin-encoding DNA by
intraductal administration into the pancreas (closed squares) over
a three day period.
[0034] FIG. 20 is a graph showing the plasma insulin levels of
streptozotocin-treated rats (diabetic) that received either no DNA
(open squares) or received human insulin-encoding DNA by
intraductal administration into the pancreas (closed squares) over
a three day period.
[0035] FIG. 21 is a graph showing the blood glucose levels (over a
six day period) of streptozotocin-treated rats (diabetic) that
received either no DNA (open squares) or received human
insulin-encoding DNA by intraductal administration into the
pancreas (closed squares).
[0036] FIG. 22 is a graph showing expression of hGH in the plasma
of control rats (no DNA) and of rats in which hGH-encoding DNA was
introduced into the liver by intraductal administration.
[0037] FIG. 23 is a graph showing stimulation of hGH secretion into
the plasma of rats that received hGH-encoding DNA by intraductal
injection into the pancreas.
[0038] FIG. 24 is a graph showing the relative levels of plasma hGH
in rats that received no DNA (control), received hGH-encoding DNA
via intraductal delivery to the liver, received hGH-encoding DNA
via intraductal delivery to the pancreas, or received hGH-encoding
DNA via intraductal delivery to both the liver and pancreas.
[0039] FIG. 25 is a graph showing the relative levels of hGH
expression in pancreas tissue following administration of DNA to
both pancreas and liver or to pancreas alone. The graph shows
tissue levels of hGH after administration of a control (no DNA) to
both pancreas and liver (left-most bar); administration of pFGH.CMV
to both pancreas and liver (center bar); and pFGH.CMV to pancreas
alone (right-most bar). Adenovirus was admixed with the construct
as an adjuvant.
DETAILED DESCRIPTION OF THE INVENTION
[0040] Before the present method of genetically transforming
secretory gland cells and methods for delivering a polypeptide to
the bloodstream of a subject are described, it is to be understood
that this invention is not limited to the particular methodology,
protocols, cell lines, secretory glands, vectors and reagents
described as such may, of course, vary. It is also to be understood
that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to limit the scope
of the present invention which will be limited only by the appended
claims.
[0041] It must be noted that as used herein and in the appended
claims, the singular forms "a", "and", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a secretory gland cell" includes a plurality
of such cells and reference to "the transformation vector" includes
reference to one or more transformation vectors and equivalents
thereof known to those skilled in the art, and so forth.
[0042] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. Although
any methods, devices and materials similar or equivalent to those
described herein can be used in the practice or testing of the
invention, the preferred methods, devices and materials are now
described.
[0043] All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing the cell
lines, vectors, and methodologies which are described in the
publications which might be used in connection with the presently
described invention.
[0044] Definitions
[0045] By "secretory gland" is meant an aggregation of cells
specialized to secrete or excrete materials not related to their
ordinary metabolic needs. Secretory glands include salivary glands,
pancreas, mammary glands, thyroid gland, thymus gland, pituitary
gland, liver, and other glands well known in the art.
[0046] By "exocrine gland" is meant a ducted gland or portion of a
ducted gland that releases its products externally relative to the
body, e.g., either into the internal cavities such as the ocular
and nasal cavities, the lumen of the gastrointestinal tract, or
onto the surface of the body.
[0047] By "salivary gland" is meant a gland of the oral cavity
which secretes saliva, including the glandulae salivariae majores
of the oral cavity (the parotid, sublingual, and submandibular
glands) and the glandulae salivariae minores of the tongue, lips,
cheeks, and palate (labial, buccal, molar, palatine, lingual, and
anterior lingual glands).
[0048] By "pancreas" is meant a large, elongated, racemose gland
situated transversely behind the stomach, between the spleen and
the duodenum. The pancreas is composed of an endocrine portion (the
pars endocrina) and an exocrine portion (the pars exocrina). The
pars endocrina, which contains the islets of Langerhans, produces
and secretes proteins, including insulin, directly into the
bloodstream. The pars exocrina contains secretory units and
produces and secretes a pancreatic juice, which contains enzymes
essential to digestion, into the duodenum.
[0049] By "transformation" is meant a permanent or transient
genetic change induced in a cell following incorporation of new DNA
(i.e., DNA exogenous to the cell).
[0050] By "transfection" is meant the transformation of a cell with
DNA from a virus.
[0051] By "transformed cell" is meant a cell into which (or into an
ancestor of which) has been introduced, by means of recombinant DNA
techniques, a DNA molecule encoding a protein of interest.
[0052] By "nucleic acid of interest" is meant any DNA or RNA
molecule which encodes a polypeptide or other molecule which is
desirable for administration to a mammalian subject for expression
of the product encoded by the nucleic acid of interest and delivery
of the encoded product into the bloodstream of the mammalian
subject. The nucleic acid is generally operatively linked to other
sequences which are needed for its expression such as a promoter.
The term "DNA of interest" is used to refer to the nucleic acid of
interest.
[0053] By "vector" is meant any compound, biological or chemical,
which facilitates transformation of a target cell (e.g., a
secretory gland cell) with a DNA of interest. Exemplary biological
vectors include viruses, particularly attenuated and/or
replication-deficient viruses. Exemplary chemical vectors include
lipid complexes and naked DNA constructs.
[0054] By "naked DNA" or "naked nucleic acid" or DNA sequence and
the like is meant a nucleic acid molecule that is not contained
within a viral particle, bacterial cell or other encapsulating
means that facilitates delivery of nucleic acid into the cytoplasm
of the target cell. Naked nucleic acid can be associated with means
for facilitating delivery of the nucleic acid to the site of the
target cell (e.g., means that facilitate travel into the target
cell of the nucleic acid through the alimentary canal, protect the
nucleic acid from stomach acid, and/or serve to penetrate
intestinal mucus) and/or to the surface of the target epithelial
cell.
[0055] By "promoter" is meant a minimal DNA sequence sufficient to
direct transcription.
[0056] "Promoter" is also meant to encompass those nucleic acid
elements sufficient for promoter-dependent gene expression
controllable for cell-type specific, tissue-specific or inducible
by external signals or agents; such elements may be located in the
5' or 3' regions of the native gene. By "eukaryotic promoter" is
meant a promoter that is functional in eukaryotic cells, which
promoters include, but are not limited to, promoters obtained from
a eukaryotic gene.
[0057] By "secretory gland specific promoter" is meant a promoter
which directs expression of an operably linked DNA sequence when
bound by transcriptional activator proteins, or other regulators of
transcription, which are unique to a specific type of secretory
gland cell. For example, by "salivary gland specific promoter" is
meant a secretory gland specific promoter which directs expression
in a salivary gland cell. A salivary amylase promoter is an example
of a salivary gland specific promoter. By "pancreas specific
promoter" is meant a secretory gland specific promoter which
directs expression in a pancreatic cell. Examples of pancreas
specific promoters include a pancreatic amylase promoter and an
insulin promoter.
[0058] By "operably linked" is meant that a DNA sequence and a
regulatory sequence(s) are connected in such a way as to permit
gene expression when the appropriate molecules (e.g.,
transcriptional activator proteins) are bound to the regulatory
sequence(s).
[0059] By "operatively inserted" is meant that the DNA of interest
introduced into the cell is positioned adjacent a DNA sequence
which directs transcription and translation of the introduced DNA
(i.e., facilitates the production of, e.g., a polypeptide encoded
by a DNA of interest).
[0060] By "retrograde ductal introduction" is meant the
administration of a liquid or other material into the fluid
contents of the duct system of an exocrine gland in a direction
opposite to the normal flow of that fluid, either at the external
orifice of the duct system or through its wall. "Retrograde ductal
introduction" can be a single, discontinuous administration or
continuous administration (i.e., perfusion), and can be
accomplished by introduction of the material by convection (e.g.,
infusion), diffusion, or both .
[0061] By "mammalian subject" or "mammalian patient" is meant any
mammal for which delivery of a polypeptide or other gene product to
the bloodstream is desired, including human, bovine, equine,
canine, and feline subjects.
[0062] As used herein, the terms "treatment", "treating", and the
like, refer to obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a disease or symptom thereof and/or may be
therapeutic in terms of a partial or complete cure for a disease
and/or adverse affect attributable to the disease. "Treatment", as
used herein, covers any treatment of a disease in a mammal,
particularly in a human, and includes: (a) preventing the disease
from occurring in a subject which may be predisposed to the disease
but has not yet been diagnosed as having it; (b) inhibiting the
disease, i.e., arresting its development; and (c) relieving the
disease, i.e., causing regression of the disease. "Treatment" is
also mean to encompass delivery of an agent in order to provide for
a pharmacologic effect, even in the absence of a disease or
condition. For example, "treatment" encompasses delivery of growth
factors that can provide for enhanced or desirable effects in the
subject (e.g., enhanced milk production in cattle, enhanced weight
gain in livestock, etc.).
[0063] By "euglycemia" or "euglycemic state" is meant a state
associated with a level of blood glucose that is normal or nearly
normal, particularly relative to the levels of blood glucose in a
subject having a disease or condition associated with
hyperglycemia. In humans, euglycemia correlates with blood glucose
levels in the range of 70 mg/dl to 130 mg/dl.
[0064] The terms "synergistic," "synergistic effect," and the like
are used herein to describe improved effects (e.g., an increase in
tissue expression levels in one or more secretory glands, an
increased responsiveness to hormonal stimulation to elicit
secretion of a polypeptide of interest, or a decrease in an
undesirable phenotype) by combining one or more aspects of the
invention (e.g., by transformation of more than one secretory gland
in a single subject, or by transformation of a secretory gland(s)
with multiple constructs encoding the same or different
polypeptides).
[0065] Overview of the Invention
[0066] The present invention features methods for delivering a
protein to the bloodstream of a subject, and use of such methods to
produce genetically altered secretory gland cells. More
specifically the invention features methods for delivery of a
protein or other product encoded by a nucleic acid sequence of
interest to a mammalian subject by expression of a DNA of interest
in cells within a secretory gland of a mammalian patient.
Preferably, the transformed secretory gland cells expressing the
protein encoded by the DNA of interest secrete a therapeutically
effective amount of the protein into the bloodstream of the
mammalian patient. Preferably, the secretory gland into which the
DNA of interest is introduced and expressed is the pancreas, a
salivary gland, or the liver. In general, the methods of the
invention result in expression of the gene product (e.g.,
polypeptide) encoded by the construct in secretory gland cells
(e.g., acinar cells of the salivary gland, acinar cells of the
pancreas, or hepatocytes or parenchymal cells of the liver) to
effect delivery of the gene product to the bloodstream. In short,
the invention features a delivery system that involves introduction
of a nucleic acid sequence encoding a product of interest (e.g., a
protein) into a secretory gland cell (e.g., a salivary gland cell,
hepatocyte, or pancreatic cell, particularly exocrine cells of
salivary gland, liver, or pancreas), expression of the encoded
protein, and delivery of the protein into the bloodstream by
secretion of the protein by the transformed secretory gland
cell.
[0067] In one embodiment of particular interest, the present
invention preferably uses either naked DNA or DNA premixed with
adjuvants (e.g., lipofectin or viral particles). It is not
necessary to incorporate the DNA into viral particles in order to
achieve transformation of secretory gland cells and provide
expression of the polypeptide of interest at
physiologic/therapeutic levels in the bloodstream.
[0068] An important feature of the invention is the use of exocrine
cells of glands of the gastrointestinal tract (i.e., pancreas,
liver, salivary gland) to produce and secrete therapeutic proteins
into blood. While it is well understood that exocrine cells secrete
into the lumen of the glands' ducts (i.e. in an exocrine
direction), with the exception of the liver (i.e., the hepatocytes
secrete cellular products in both directions, e.g. blood proteins
into blood and bile salts into the intestinal lumen), it is not
widely appreciated that exocrine cells can also secrete significant
amounts of protein into the systemic circulation. For example,
exocrine proteins such as .alpha.-amylase (salivary glands),
pepsinogen (gastric glands), various digestive enzymes from the
exocrine pancreas, salivary gland kallikreins and nerve growth
factor (Liebow, 1988 Pancreas 3:343-351) are normal constituents of
blood. In the pancreas, substantial quantities of digestive enzymes
are released into the circulation (Saito et al., 1973 Jpn. J.
Physiol. 23:477-95; Isenman et al. 1977 Proc. Natl. Acad. Sci (USA)
74:4068-4072; Papp et al. 1980 Acta Physiol. Acad. Sci. Hung.
56:401-410; Geokas et al., 1980 Am. J. Physiol. 238:238-246;
Miyasaka et al. 1981 Am. J. Physiol. 241:170-175; Grendell et al.
1982 Am. J. Physiol. 243:54-59). Endocrine secretion can be greatly
enhanced by common secretory stimulants (Saito et al., supra;
Isenman et al. supra; Miyasaka et al. supra; Grendell et al.
supra). As much as 20-25% of the total secreted product can be
released into blood as a consequence of stimulation (Grendell et
al. supra). The present invention takes advantage of the discovery
that exocrine gland cells can be transformed with a desired DNA
sequence and secrete the encoded polypeptide into the bloodstream
rather than only or primarily into the gastrointestinal tract.
[0069] In addition to the advantages described above, the invention
also permits access to the cells of secretory glands without
invasive procedures. For example, it is possible to cannulate
either the collecting duct of a major salivary gland through its
orifice in the mouth, or the common bile or pancreatic duct by
means of endoscopic retrograde cholangiopancreatography (ERCP).
These are common diagnostic procedures performed on awake patients.
The non-invasive methods of the invention allow delivery of the DNA
of interest in a safe manner that substantially avoids the
inflammatory and immunological responses associated with other
means of DNA delivery.
[0070] The invention also takes advantage of the protein-producing
capacity of secretory gland cells. This advantage is particularly
useful for the production of hormones such as hGH and insulin,
which have short half-lives in blood and are cleared quickly. The
cells of the exocrine glands are the body's major protein
synthesizing and secreting systems. For example, the human exocrine
pancreas manufactures and secretes approximately 20 g of protein
daily. According to the present invention, even a small proportion
of protein synthesized by secretory glands provides enough secreted
product to provide therapeutic protein levels for the treatment of
most diseases of circulating proteins.
[0071] The invention will now be described in further detail.
[0072] Constructs
[0073] Any nucleic acid construct having a eukaryotic promoter
operably linked to a DNA of interest can be used in the invention.
The constructs containing the DNA sequence (or the corresponding
RNA sequence) which may be used in accordance with the invention
may be any eukaryotic expression construct containing the DNA or
the RNA sequence of interest. For example, a plasmid or viral
construct (e.g. adenovirus) can be cleaved to provide linear DNA
having ligatable termini. These termini are bound to exogenous DNA
having complementary, like ligatable termini to provide a
biologically functional recombinant DNA molecule having an intact
replicon and a desired phenotypic property. Preferably the
construct is capable of replication in both eukaryotic and
prokaryotic hosts, which constructs are known in the art and are
commercially available.
[0074] The exogenous (i.e., donor) DNA used in the invention is
obtained from suitable cells, and the constructs prepared using
techniques well known in the art. Likewise, techniques for
obtaining expression of exogenous DNA or RNA sequences in a
genetically altered host cell are known in the art (see, for
example, Kormal et al., Proc. Natl. Acad. Sci. USA, 84:2150-2154,
1987; Sambrook et al. Molecular Cloning: a Laboratory Manual, 2nd
Ed., 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.; each of which are hereby incorporated by reference with
respect to methods and compositions for eukaryotic expression of a
DNA of interest).
[0075] Preferably, the DNA construct contains a promoter to
facilitate expression of the DNA of interest within a secretory
gland cell. Preferably the promoter is a strong, eukaryotic
promoter such as a promoter from cytomegalovirus (CMV), mouse
mammary tumor virus (MMTV), Rous sarcoma virus (RSV), or
adenovirus. More specifically, exemplary promoters include the
promoter from the immediate early gene of human CMV (Boshart et
al., Cell 41:521-530, 1985) and the promoter from the long terminal
repeat (LTR) of RSV (Gorman et al., Proc. Natl. Acad. Sci. USA
79:6777-6781, 1982). Of these two promoters, the CMV promoter is
preferred as it provides for higher levels of expression than the
RSV promoter.
[0076] Alternatively, the promoter used may be a tissue-specific
promoter. For example, where the secretory gland is the pancreas,
the promoter used in the vector may be a pancreas specific
promoter, e.g., an insulin promoter or a pancreas .alpha.-amylase
promoter; where the secretory gland is a salivary gland, the
tissue-specific promoter may be a salivary .alpha.-amylase promoter
or mumps viral gene promoter. Both pancreatic and salivary
.alpha.-amylase genes have been identified and characterized in
both mice and humans (see, for example, Jones et al., Nucleic Acids
Res., 17:6613-6623; Pittet et al., J. Mol. Biol., 182:359-365,
1985; Hagenbuchle et al., J. Mol. Biol., 185:285-293, 1985;
Schibler et al., Oxf. Surv. Eukaryot. Genes, 3:210-234, 1986; and
Sierra et al., Mol. Cell. Biol., 6:4067-4076, 1986 for murine
pancreatic and salivary .alpha.-amylase genes and promoters;
Samuelson et al., Nucleic Acids Res., 16:8261-8276, 1988; Groot et
al., Genomics, 5:29-42, 1989; and Tomita et al., Gene, 76:11-18,
1989 for human pancreatic and salivary .alpha.-amylase genes and
their promoters; Ting et al., Genes Dev. 6:1457-65, 1992 for human
salivary .alpha.-amylase AMY1C promoter sequences).
[0077] The-constructs of the invention may also include sequences
in addition to promoters which enhance secretory gland specific
expression. For example, where pancreas specific expression of the
DNA of interest is desired, the construct may include a PTF-1
recognition sequence (Cockell et al., Mol. Cell. Biol.,
9:2464-2476, 1989). Sequences which enhance salivary gland specific
expression are also well known in the art (see, for example, Robins
et al., Genetica 86:191-201, 1992).
[0078] Other components such as a marker (e.g., an antibiotic
resistance gene (such as an ampicillin resistance gene) or
.beta.-galactosidase) to aid in selection of cells containing
and/or expressing the construct, an origin of replication for
stable replication of the construct in a bacterial cell
(preferably, a high copy number origin of replication), a nuclear
localization signal, or other elements which facilitate production
of the DNA construct, the protein encoded thereby, or both.
[0079] For eukaryotic expression, the construct should contain at a
minimum a eukaryotic promoter operably linked to a DNA of interest,
which is in turn operably linked to a polyadenylation sequence. The
polyadenylation signal sequence may be selected from any of a
variety of polyadenylation signal sequences known in the art.
Preferably, the polyadenylation signal sequence is the SV40 early
polyadenylation signal sequence. The construct may also include one
or more introns, which can increase levels of expression of the DNA
of interest, particularly where the DNA of interest is a cDNA
(e.g., contains no introns of the naturally-occurring sequence).
Any of a variety of introns known in the art may be used (e.g, the
human .beta.-globin intron, which is inserted in the construct at a
position 5' to the DNA of interest).
[0080] The DNA of interest may be inserted into a construct so that
the therapeutic protein is expressed as a fusion protein (e.g., a
fusion protein having .beta.-galactosidase or a portion thereof at
the N-terminus and the therapeutic protein at the C-terminal
portion). Production of a fusion protein can facilitate
identification of transformed cells expressing the protein (e.g.,
by enzyme-linked immunosorbent assay (ELISA) using an antibody
which binds to the fusion protein).
[0081] The Nucleic Acid (DNA) of Interest
[0082] The DNA of interest can be any DNA encoding any protein for
which intravenous delivery is desirable. For example, intravenous
protein delivery is appropriate in, for example, treating a
mammalian subject having an inherited or acquired disease
associated with a specific protein deficiency (e.g., diabetes,
hemophilia, anemia, severe combined immunodeficiency). Such protein
deficient states are amenable to treatment by replacement therapy,
i.e., expression of a protein to help restore the bloodstream
levels of the protein to substantially normal levels.
[0083] Alternatively, the DNA of interest may encode a polypeptide
that is either normally present in a healthy mammalian subject or
which is foreign to the mammalian subject, and which polypeptide is
effective in providing a desired effect (e.g., enhanced weight
gain, enhanced growth), in the production of a polypeptide for
subsequent isolation (e.g., to produce human proteins in a mammal),
and/or treatment of a condition by expression or over-expression of
the polypeptide. For example, the DNA of interest can encode
antimicrobial, antiparasitic, antifungal, or antiviral polypeptides
for treatment of a mammalian subject having a viral (e.g., human
immunodeficiency virus (HIV), Epstein-Barr virus (EBV), herpes
simplex virus (HSV), bacterial, fungal, and/or parasitic infection,
particularly where the infection is chronic, i.e., persisting over
a relatively long period of time. The methods of the invention may
also be used to enhance expression of a protein present in a normal
mammal, or to express a protein not normally present in a normal
mammal, in order to achieve a desired effect (e.g., to enhance a
normal metabolic process, to provide for a desired immunologic
effect (e.g., enhanced immunity or immune responsivity)). For
example, a secretory gland of a dairy cow may be transformed with
DNA encoding bovine growth hormone (BGH) in order to enhance levels
of BGH in the bloodstream and enhance milk production.
[0084] The DNA of interest is preferably obtained from a source of
the same species as the mammalian subject to be treated (e.g. human
to human), but this is not an absolute requirement. DNA obtained
from a species different from the mammalian subject can also be
used, particularly where the amino acid sequences of the proteins
are highly conserved and the xenogeneic protein is not highly
immunogenic so as to elicit a significant, undesirable antibody
response against the protein in the mammalian host.
[0085] Exemplary, preferred DNAs of interest include DNA encoding
insulin, growth factors (e.g., growth hormone, insulin-like growth
factor-1 (IGF-I), platelet-derived growth factor (PDGF), epidermal
growth factor (EGF), acidic fibroblast growth factor, basic
fibroblast growth factor, or transforming growth factor .beta.),
cytokines (e.g., interferon (INF) (e.g., INF-.alpha.2b,
INF-.alpha.2a, INF-.alpha.N1, INF-.beta.1b, INF-.gamma.),
interleukin (e.g, IL-2, IL-8), or tumor necrosis factor (TNF) (e.g,
TNF-.alpha., TNF-.beta.)), clotting factors (e.g., clotting factor
VIII), hormones (e.g, GP-1), antimicrobial polypeptides (e.g.,
antibacterial, antifungal, antiviral, and/or antiparasitic
polypeptides), enzymes (e.g., adenosine deaminase), filgastim
(Neupogen), hemoglobin, erythropoietin, insulinotropin,
imiglucerase, sarbramostim, antigens, tissue plasminogen activator
(tPA), urokinase, streptokinase, endothelian, soluble CD4, and
antibodies and/or antigen-binding fragments (e.g, FAbs) thereof
(e.g., orthoclone OKT-e (anti-CD3), GPIIb/IIa monoclonal antibody).
Preferably, the mammalian subject is a human subject and the DNA
expressed encodes a human protein.
[0086] Table 1 provides a list of exemplary proteins and protein
classes which can be delivered to the bloodstream of a mammalian
subject via the method of secretory gland cell transformation of
the invention.
1TABLE 1 Exemplary Proteins and Protein Classes for Expression in
and Secretion by Secretory Gland Cells SPECIFIC EXEMPLARY PROTEINS
insulin interferon-.alpha.2B human growth hormone (hGH)
transforming growth factor (TGF) erythropoietin (EPO) ciliary
neurite transforming factor (CNTF) clotting factor VIII
insulin-like growth factor-1 (IGF-1) bovine growth hormone (BGH)
granulocyte macrophage colony stimulating factor (GM-CSF) platelet
derived growth factor interferon-.alpha.2A (PDGF) clotting factor
VIII brain-derived neurite factor (BDNF) thrombopoietin (TPO)
insulintropin IL-1 tissue plasminogen activator (tPA) IL-2
urokinase IL-1 RA streptokinase superoxide dismutase (SOD)
adenosine deamidase catalase calcitonin fibroblast growth factor
(FGF) arginase (acidic or basic) neurite growth factor (NGF)
phenylalanine ammonia lyase granulocyte colony stimulating
.gamma.-interferon factor (G-CSF) L-asparaginase pepsin uricase
trypsin chymotrypsin elastase carboxypeptidase lactase sucrase
intrinsic factor calcitonin parathyroid hormone(PTH)- like hormone
Ob gene product cholecystokinin (CCK) glucagon insulinotrophic
hormone EXEMPLARY CLASSES OF PROTEINS enzymes (e.g., proteases,
pituitary hormones phospholipases, etc.) protease inhibitors growth
factors cytokines somatomedians chemokines immunoglobulins
gonadotrophins interleukins chemotactins interferons lipid-binding
proteins
[0087] Numerous proteins that are desirable for intravenous protein
therapy are well known in the art and the DNA encoding these
proteins has been isolated. For example, the sequence of the DNAs
encoding insulin, human growth hormone, intrinsic factor, clotting
factor VIII, and erythropoietin are available from Genbank and/or
have been described in the scientific literature (e.g., human
clotting factor VIII gene: Gitschier et al., Nature 312:326-330,
1984; Wood et al., Nature 312:330-337, 1984; human intrinsic
factor: Hewitt et al., Genomics 10:432-440, 1991). Moreover,
proteins commonly used in treatments can be used in the procedures
of the present invention. Such proteins are disclosed in, for
example, the Physicians' Desk Reference (1994 Physicians' Desk
Reference, 48th Ed., Medical Economics Data Production Co.,
Montvale, N.J.; incorporated by reference) and can be dosed using
methods described in Harrison's Principles of Internal Medicine
and/or the AMA "Drug Evaluations Annual" 1993, all incorporated by
reference.
[0088] Where the DNA encoding a protein of interest has not been
isolated, this can be accomplished by various, standard protocols
well known to those of skill in the art (see, for example, Sambrook
et al., ibid; Suggs et al., Proc. Natl. Acad. Sci. USA
78:6613-6617, 1981; U.S. Pat. No. 4,394,443; each of which are
incorporated herein by reference with respect to identification and
isolation of DNA encoding a protein of interest). For example,
genomic or cDNA clones encoding a specific protein can be isolated
from genomic or cDNA libraries using hybridization probes designed
on the basis of the nucleotide or amino acid sequences for the
desired gene. The probes can be constructed by chemical synthesis
or by polymerase chain reaction (PCR) using primers based upon
sequence data to amplify DNA fragments from pools or libraries
(U.S. Pat. Nos. 4,683,195 and 4,683,202). Nucleotide substitutions,
deletions, additions, and the like can also be incorporated into
the polynucleotides, so long as the ability of the polynucleotide
to hybridize is not substantially disrupted. (Sambrook et al.
ibid). The clones may be expressed or the DNA of interest can be
excised or synthesized for use in other constructs. If desired, the
DNA of interest can be sequenced using methods well known in the
art.
[0089] It may also be desirable to produce altered forms of the
therapeutic proteins that are, for example, protease resistant or
have enhanced activity relative to the wild-type protein. For
example, where the therapeutic protein is a hormone, it may be
desirable to alter the protein's ability to form dimers or
multimeric complexes. For example, insulin may be modified so as to
prevent its dimerization has a more rapid onset of action relative
to wild-type, dimerized insulin.
[0090] Vectors for Delivery of the DNA of Interest to the Secretory
Gland Cell
[0091] The vectors for delivery of the DNA of interest can be
either viral or non-viral, or may be composed of naked DNA admixed
with an adjuvant such as viral particles (e.g, adenovirus) or
cationic lipids or liposomes. An "adjuvant" is a substance that
does not by itself produce the desired effect, but acts to enhance
or otherwise improve the action of the active compound. The precise
vector and vector formulation used will depend upon several factors
such as the secretory gland targeted for gene transfer.
[0092] Non-Viral Vectors
[0093] The DNA of interest may be administered using a non-viral
vector. "Non-viral vector" as used herein is meant to include naked
DNA, chemical formulations containing naked DNA (e.g, a formulation
of DNA and cationic compounds (e.g., dextran sulfate)), and naked
DNA mixed with an adjuvant such as a viral particle (i.e., the DNA
of interest is not contained within the viral particle, but the
transforming formulation is composed of both naked DNA and viral
particles (e.g., adenovirus particles) (see, e.g., Curiel et al.
1992 Am. J. Respir. Cell Mol. Biol. 6:247-52). Thus "non-viral
vector" can include vectors composed of DNA plus viral particles
where the viral particles do not contain the DNA of interest within
the viral genome.
[0094] In one preferred embodiment, the formulation comprises viral
particles which are mixed with the naked DNA construct prior to
administration. Preferably, about 10.sup.8 to 10.sup.10 viral
particles (preferably about 1.times.10.sup.8 to 5.times.10.sup.10,
more preferably about 3.times.10.sup.10 particles) are mixed with
the naked DNA construct (about 5 .mu.g to 50 .mu.g DNA, more
preferably about 8 .mu.g to 25 .mu.g DNA) in a total volume of
about 100 .mu.l. Preferably the viral particles are adenovirus
particles (Curiel et al., 1992 supra).
[0095] Alternatively or in addition, the DNA of interest can be
complexed with polycationic substances such as poly-L-lysine or
DEAC-dextran, targeting ligands, and/or DNA binding proteins (e.g,
histones). DNA- or RNA-liposome complex formulations comprise a
mixture of lipids which bind to genetic material (DNA or RNA) and
facilitate delivery of the nucleic acid into the cell. Liposomes
which can be used in accordance with the invention include DOPE
(dioleyl phosphatidyl ethanol amine), CUDMEDA
(N-(5-cholestrum-3-.beta.-ol 3-urethanyl)-N',N'-dimethylethylene
diamine).
[0096] Lipids which can be used in accordance with the invention
include, but are not limited to, DOPE (Dioleoyl
phosphatidylethanolamine), cholesterol, and CUDMEDA
(N-(5-cholestrum-3-ol 3 urethanyl)-N',N'-dimethy-
lethylenediamine). As an example, DNA can be administered in a
solution containing one of the following cationic liposome
formulations: Lipofectin.TM. (LTI/BRL), Transfast.TM. (Promega
Corp), Tfx50.TM. (Promega Corp), Tfx10.TM. (Promega Corp), or
Tfx20.TM. (Promega Corp). The concentration of the liposome
solutions range from about 2.5% to 15% volume:volume, preferably
about 6% to 12% volume:volume. Further exemplary methods and
compositions for formulation of nucleic acid (e.g., DNA, including
DNA or RNA not contained within a viral particle) for delivery
according to the method of the invention are described in U.S. Pat.
Nos. 5,892,071; 5,744,625; 5,925,623; 5,527,928; 5,824,812;
5,869,715.
[0097] The DNA of interest can also be administered as a chemical
formulation of DNA or RNA coupled to a carrier molecule (e.g., an
antibody or a receptor ligand) which facilitates delivery to host
cells for the purpose of altering the biological properties of the
host cells. By the term "chemical formulations" is meant
modifications of nucleic acids to allow coupling of the nucleic
acid compounds to a carrier molecule such as a protein or lipid, or
derivative thereof. Exemplary protein carrier molecules include
antibodies specific to the cells of a targeted secretory gland or
receptor ligands, i.e., molecules capable of interacting with
receptors associated with a cell of a targeted secretory gland.
[0098] Viral Vectors
[0099] In general, viral vectors used in accordance with the
invention are composed of a viral particle derived from a
naturally-occurring virus which has been genetically altered to
render the virus replication-defective and to express a recombinant
gene of interest in accordance with the invention. Once the virus
delivers its genetic material to a cell, it does not generate
additional infectious virus but does introduce exogenous
recombinant genes into the cell, preferably into the genome of the
cell.
[0100] Numerous viral vectors are well known in the art, including,
for example, retrovirus, adenovirus, adeno-associated virus, herpes
simplex virus (HSV), cytomegalovirus (CMV), vaccinia and poliovirus
vectors. Retroviral vectors are less preferred since retroviruses
require replicating cells and secretory glands are composed of
mostly slowly replicating and/or terminally differentiated cells.
Adenovirus and AAV are preferred viral vector since this virus
efficiently infects slowly replicating and/or terminally
differentiated cells. The viral vector may be selected according to
its preferential infection of the targeted secretory gland (e.g.,
where the secretory gland is a salivary gland, the viral vector may
be derived from an attenuated (i.e., does not cause significant
pathology or morbidity in the infected host, e.g, the virus is
nonpathogenic or causes only minor disease symptoms) and/or
replication-deficient mumps virus or other attenuated and/or
replication-deficient virus which is substantially specific for
salivary gland cell).
[0101] Where a replication-deficient virus is used as the viral
vector, the production of infective virus particles containing
either DNA or RNA corresponding to the DNA of interest can be
produced by introducing the viral construct into a recombinant cell
line which provides the missing components essential for viral
replication. Preferably, transformation of the recombinant cell
line with the recombinant viral vector will not result in
production of replication-competent viruses, e.g., by homologous
recombination of the viral sequences of the recombinant cell line
into the introduced viral vector. Methods for production of
replication-deficient viral particles containing a nucleic acid of
interest are well known in the art and are described in, for
example, Rosenfeld et al., Science 252:431-434, 1991 and Rosenfeld
et al., Cell 68:143-155, 1992 (adenovirus); U.S. Pat. No. 5,139,941
(adeno-associated virus); U.S. Pat. No. 4,861,719 (retrovirus); and
U.S. Pat. No. 5,356,806 (vaccinia virus). Methods and materials for
manipulation of the mumps virus genome, characterization of mumps
virus genes responsible for viral fusion and viral replication, and
the structure and sequence of the mumps viral genome are described
in Tanabayashi et al., J. Virol. 67:2928-2931, 1993; Takeuchi et
al., Archiv. Virol., 128:177-183, 1993; Tanabayashi et al., Virol.
187:801-804, 1992; Kawano et al., Virol., 179:857-861, 1990; Elango
et al., J. Gen. Virol. 69:2893-28900, 1988.
[0102] Conditions or Diseases Amenable to Treatment Using the
Method of the Invention
[0103] Various disease conditions are amenable to treatment using
the methods of the invention. One skilled in the art can recognize
the appropriate protein which should be produced by the invention
for treating specific disease conditions. Exemplary diseases which
are amenable to treatment using the subject invention, and
exemplary, appropriate proteins which can be used in treating these
diseases, are shown in Table 2.
2TABLE 2 Exemplary Disease Conditions Amenable to Treatment Using
the Invention Enzyme Deficiency Endotoxic Shock/Sepsis Adenosine
deaminase.sup.1 Lipid-binding protein (LBP) Purine nucleotide
phosphorylase Galactosidase .beta.-glucuronidase Antioxidants for
Cancer Therapy Anemia Superoxide dismutase Erythropoietin Catalase
Cancer Growth Factors (for use in wound .alpha.-Interferon healing,
induction of red blood cell formation, etc.) .gamma.-Interferon
Epidermal growth factor .alpha.-IL1 G-CSF Phenylalanine ammonia
lyase .gamma.-Interferon Arginase Transforming growth factor
L-asparaginase Erythropoietin Uricase Thrombopoietin Granulocyte
colony stimulating Insulin-like growth factor-1 factor (G-CSF)
Monoclonal antibodies Insulin Tissue necrosis factor Human growth
hormone Cardiovascular Disease Diabetes Tissue plasminogen
activator Insulin Urokinase (native or chimeric) Glucagon
.alpha..sub.1-antitrypsin Insulinotrophic hormone Antithrombin-III
Other proteases or protease inhibitors Clotting disorders
Apolipoproteins (particularly B-48) Clotting factor VIII
Circulating Scavenger Receptor APO A1.sup.2 Obesity and Feeding
Gastrointestinal and Pancreatic Ob gene product Deficiencies
Cholecystokinin (CCK) Pepsin (for esophageal reflux) Trypsin
Chymotrypsin Bone diseases Elastase Calcitonin Carboxypeptidase
PTH-like hormone Lactase (for lactose deficiency) Sucrase;
Intrinsic Factor (pernicious anemia) Organ-Specific Autoimmune
diseases (target of antibody in parentheses) Myasthenia gravis
(acetylcholine receptors) Graves' disease (thyroid-stimulating
hormone receptor) Thyroiditis (thyroid, peroxidase)
Insulin-resistant diabetes with acanthosis nigricans or with ataxia
telangiectasia (Insulin receptor) Allergic rhinitis, asthma
(Beta.sub.2-adrenergic receptors) Juvenile insulin-dependent
diabetes (insulin, GAD65) Pernicious anemia (gastric parietal
cells, vitamin B.sub.12 binding site of intrinsic factor) Addison's
disease (adrenal cells) Idiopathic hypoparathyroidism (parathyroid
cells) Spontaneous infertility (sperm) Premature ovarian failure
(interstitial cells, corpus luteum cells) Pemphigus (intercellular
substance of skin and mucosa) Bullous pemphigoid (basement membrane
zone of skin and mucosa) Primary biliary cirrhosis (mitochondria)
Autoimmune hemolytic anemia (erythrocytes) Idiopathic
thrombocytopenic purpura (platelet) Idiopathic neutropenia
(neutrophils) Vitiligo (melanocytes) Osteosclerosis and Meniere's
disease (type II collagen) Chronic active hepatitis (nuclei of
hepatocytes) Systemic Autoimmune Diseases (defect/organ affected in
parentheses) Goodpasture's syndrome (basement membranes)
Rheumatoid-arthritis (.gamma.-globulin, EBV-related antigens,
collagen types II and III) Sjogren's syndrome (.gamma.-globulin,
SS-A (Ro), SS-B (La)) Systemic lupus erythematosus (nuclei,
double-stranded DNA, single- stranded DNA, Sm ribonucleoprotein,
lymphocytes, erythrocytes, neurons, .gamma.-globulin) Scleroderm
(nuclei, Scl-70, SS-A (Ro), SS-B (La), centromere) Polymyositis
(nuclei, Jo-1, PL-7, histadyl-tRNA or threonyl-tRNA synthetases,
PM-1, Mi-2) Rheumatic fever (myocardium heart valves, choroid
plexus) .sup.1For treatment of severe combined immunodeficiency
.sup.2Converts low-density lipoproteins to high-density
lipoproteins
[0104] Transformation of Secretory Gland Cells
[0105] The DNA of interest-containing vector (i.e., either a viral
or non-viral vector (including naked DNA)) is introduced into the
secretory gland in vivo via the duct system (i.e., by retrograde
intraductal administration, which may be accomplished by perfusion
(i.e., continuous injection), or by a single, discontinuous
injection). Intraductal administration can also be accomplished by
cannulation, which can be accomplished for the pancreas and the
liver by, for example, insertion of the cannula through a lumen of
the gastrointestinal tract, by insertion of the cannula through an
external orifice, insertion of the cannula through the common bile
duct. Retrograde ductal administration may be accomplished in the
pancreas and liver by endoscopic retrograde
chalangio-pancreatography (ECRP). The methods of the invention can
involve delivery to both the pancreas, the liver, the salivary
gland, or to any combination thereof. Ductal administration
provides several advantages. Because the vector is presented to the
cells from "outside" the body (from the lumen), the immunological
and inflammatory reactions that are commonly observed as a result
of the administration of transforming formulations and their
adjuvants into blood and interstitial fluid may be avoided.
[0106] Moreover, the cells of secretory glands from a monolayer
that encloses the duct system. As a consequence, virtually all of
the cells of the glands can be accessed by a single administration
into the duct. In this way it is possible to transfect large masses
of cells in a relatively simple manner with a single procedure. The
DNA of interest can thus also be administered without substantial
dilution (it is only diluted by the fluid in the duct system) and
without the-need to develop organ specific targeting signals. In
contrast, intravenous administration necessarily greatly dilutes
the material and requires that it be targeted to the organ of
interest in some fashion.
[0107] The amount of DNA to transform a sufficient number of
secretory gland cells and provide for expression of therapeutic
levels of the protein can be readily determined using an animal
model (e.g., a rodent (mouse or rat) or other mammalian animal
model) to assess factors such as the efficiency of transformation,
the levels of protein expression achieved, the susceptibility of
the targeted secretory gland cells to transformation, and the
amounts of DNA required to transform secretory gland cells.
[0108] The precise amount of DNA administered will vary greatly
according to a number of factors including the susceptibility of
the target cells to transformation, the size and weight of the
subject, the levels of protein expression desired, and the
condition to be treated. For example, the amount of DNA introduced
into a secretory gland of a human is generally from about 1 .mu.g
to 200 mg, preferably from about 100 .mu.g to 100 mg, more
preferably from about 500 .mu.g to 50 mg, most preferably about 10
mg. Specifically, the amount of DNA introduced into the pancreas of
a human is, for example, generally from about 1 .mu.g to 100 mg,
preferably about 100 .mu.g to 10 mg, more preferably from about 250
.mu.g to 5 mg, still more preferably from about 500 .mu.g to 1.5
mg, most preferably about 1 mg. The amount of DNA introduced into
the salivary gland of a human is, for example, generally from about
2.5 .mu.g to 30 mg, more preferably from about 25 .mu.g to 3 mg,
still more preferably from about 100 .mu.g to 1 mg, most preferably
about 250 .mu.g. The amount of DNA introduced into the liver of a
human is, for examples, generally from about 10 .mu.g to 500 mg,
more preferably from about 100 .mu.g to 300 mg, still more
preferably from about 150 .mu.g to 100 mg, most preferably about 1
mg Generally, the amounts of DNA for human therapy according to the
invention can be extrapolated from the amounts of DNA effective for
therapy in an animal model. For example, the amount of DNA for
therapy in a human is roughly 100 times the amount of DNA effective
in therapy in a rat. The amount of DNA necessary to accomplish
secretory gland cell transformation will decrease with an increase
in the efficiency of the transformation method used.
[0109] The methods of the invention can be used to accomplish
delivery of a polypeptide to the bloodstream on either a long term
basis (e.g., by repeated administration of the construct) or on a
short term basis (e.g., for several hours or a few days). In this
regard, the invention takes advantage of the normal turnover of the
cells that are transformed by the introduced construct in order to
provide a means for controlling dosage of the polypeptide to the
bloodstream. In another aspect, where substantially constitutive
delivery is desired, then the construct can be introduced into a
duct for expression by and delivery from the liver. Furthermore,
and without being held to theory, it may be more desirable to
introduce the construct for expression by and delivery from the
liver where longer term delivery (e.g., weeks to months (e.g.,
about 3 weeks to about 3 months or more)), while shorter term
delivery can be accomplished using the salivary glands or pancreas
versus (e.g., hours to days (e.g., about 24, 36, or 48 hours to
about 3, 6, or 10 days).
[0110] Intravenous Protein Therapy by Transformation of Salivary
Gland, Pancreatic, and Liver Cells
[0111] Secretory glands transformed according to the invention
facilitate high levels expression of a DNA of interest,
particularly where the DNA of interest is operably linked to a
strong eukaryotic promoter (e.g., CMV, MMTV). The expressed protein
is then secreted at high levels into the bloodstream. The protein
so expressed and secreted is thus useful in treating a mammalian
subject having a variety of conditions.
[0112] In a preferred embodiment, the proteins are secreted into
the bloodstream at levels sufficient for intravenous protein
therapy. For example, the amount of a specific protein normally
released into the blood from the pancreas can be substantial, e.g.,
a specific endogenous protein released into the bloodstream can be
as much as 25% of the amount of duct-directed secretion of that
protein. This amounts to as much as 1-2 mg of protein/gram of
tissue being directed into the blood per hour.
[0113] Bloodstream levels of the therapeutic protein may be
enhanced by several different methods. For example, bloodstream
levels can be enhanced by increasing the overall level of
expression of the desired protein, e.g., by integration of multiple
copies of the DNA of interest into the genome of the target cells,
by operably linking a strong promoter (e.g., a promoter from CMV)
and/or enhancer elements to the DNA of interest in the construct,
or by transformation of a greater number of target cells in the
subject (e.g., by administration of multiple doses of the
transforming material).
[0114] Secretion of the therapeutic protein into the bloodstream
can also be enhanced by incorporating leader sequences, amino acid
sequence motifs, or other elements that mediate
intravenous-directed secretion into the sequence of the therapeutic
protein. For example, the DNA of interest can be engineered to
contain a secretion signal that directs secretion of the protein
primarily into the bloodstream, thereby increasing the amount of
the protein produced in the secretory gland that reaches in the
bloodstream. Intravenous-directed secretion signals can be
identified by, for example, site-directed mutagenesis of DNA
encoding a bloodstream-targeted protein (e.g., insulin). The
mutants can be screened by expression of the mutated DNA in
secretory gland cells and subsequently determining the ratio of,
for example, salivary to intravenous expression.
[0115] Alternatively, intravenous-directed secretion signals can be
identified by constructing recombinant, chimeric proteins composed
of, for example, a putative intravenous secretion signal inserted
into a saliva-directed protein. Intravenous secretion signals would
then be identified by their ability to re-direct expression of the
saliva-directed protein into the bloodstream. Putative intravenous
secretion signals and duct system secretion signals can also be
identified by comparison of DNA and amino acid sequences of
proteins which are preferentially secreted into the bloodstream.
Areas of homology or common motifs among the proteins could then be
tested as described above.
[0116] Overall secretion from salivary gland and the pancreas can
be augmented by hormonal stimulation. For example, where the
protein is primarily secreted into the duct system and is secreted
at lower levels into the bloodstream, hormonal stimulation enhances
intravenous secretion as well as secretion into the duct. Thus,
therapeutically effective levels of the protein the bloodstream may
be achieved or enhanced by administration of an appropriate,
secretory gland specific hormone. For example, secretory gland
secretion can be enhanced by administration of a cholinergic
agonist such as acetyl-.beta.-methyl choline, or can be augmented
or further augmented by control of diet (i.e., eating stimulates
pancreatic and salivary gland secretion). Thus, because eating a
meal can elicit a secretory response, adjustment of meals (e.g.,
frequency of meals and/or amounts eaten) can be used as a dosing
mechanism for delivery of the desired protein, and can be
accomplished without administration of additional protein-encoding
DNA.
[0117] Bloodstream-directed secretion can also be regulated at
either the level of transcription, translation, or secretion.
Transcriptional regulation involves the timing and level of
transcription directed from the DNA of interest, while
translational regulation involves the production of polypeptides
from transcribed RNA. Secretory regulation involves the release of
polypeptides from the cell (e.g., from secretory cells in which the
polypeptides to be secreted are stored within intracellular
vacuoles). Methods for providing transcriptional and/or
translational regulation of a DNA of interest are well known in the
art (e.g, transcriptional regulation through the use of inducible
promoters).
[0118] Secretory regulation can be achieved by, for example,
administration of a hormone that elicits a secretory response in
the desired secretory gland, or by activity that stimulates
production of such hormone(s) (e.g., eating to stimulate pancreatic
secretion). Unlike regulation at the level of transcription or
translation, which can take many hours to become effective,
regulation of secretion occurs within minutes after stimulation.
Moreover, endocrine secretion from the pancreas and salivary glands
is stimulated by hormones and neurotransmitters that are natural
components of the feeding response; thus feeding itself can act as
a dosing mechanism.
[0119] The actual number of transformed secretory gland cells
required to achieve therapeutic levels of the protein of interest
will vary according to several factors including the protein to be
expressed, the level of expression of the protein by the
transformed cells, the rate of protein secretion, the partitioning
of the therapeutic protein between the gastrointestinal tract and
the bloodstream, and the condition to be treated. For example, the
desired intravenous level of therapeutic protein can be readily
calculated by determining the level of the protein present in a
normal subject (for treatment of a protein deficiency), or by
determining the level of protein required to effect the desired
therapeutic result.
[0120] Application of the Method of the Invention to Achieve
Euglycemia in a Diabetic Syndrome
[0121] In another preferred embodiment of the invention, pancreatic
cells are transformed using insulin-encoding DNA to provide for
expression and secretion of insulin into the bloodstream of a
mammalian subject. Transformation of pancreatic cells with insulin
encoding DNA not only provides for regulated expression of insulin
in a mammalian subject, but also provides for maintenance of a
euglycemic state (i.e., normal blood glucose levels) in diabetic
subjects for extended periods of time (e.g., up to 6 to 7 days post
transformation). Thus, not only is the exocrine pancreas secreting
insulin to reduce blood sugar, but regulating its secretion so that
blood levels are maintained at normal levels, e.g., are regulated.
Thus, pancreatic transformation with insulin-encoding DNA can be
used in the therapy of individuals having a disease or condition
associated with elevated blood glucose levels (e.g., diabetes
(e.g., type I or type II diabetes), and hyperglycemia). This aspect
of the invention may be applied to regulate levels of other
proteins in the bloodstream.
[0122] Assessment of Protein Therapy
[0123] The effects of expression of the protein encoded by the DNA
of interest following in vivo transfer of the DNA of interest can
be monitored in a variety of ways. Generally, a sample of blood
from the subject can be assayed for the presence of the therapeutic
protein. Appropriate assays for detecting a protein of interest in
blood samples are well known in the art. For example, a sample of
blood can be tested for the presence of the polypeptide using an
antibody which specifically binds the polypeptide in an ELISA
assay. This assay can be performed either qualitatively or
quantitatively. The ELISA assay, as well as other immunological
assays for detecting a polypeptide in a sample, are described in
Antibodies: A Laboratory Manual (1988, Harlow and Lane, eds. Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
[0124] Alternatively, or in addition, the efficacy of the
polypeptide therapy can be assessed by testing a sample of blood
for an activity associated with the polypeptide (e.g., an enzymatic
activity). Furthermore, the efficacy of the therapy using the
methods of the invention can be assessed by monitoring the
condition of the mammalian subject for improvement. For example,
where the polypeptide is erythropoietin, the subject's blood is
examined for hematocrit, iron content or other parameters
associated with anemia.
EXAMPLES
[0125] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to carry out the invention and is not intended
to limit the scope of what the inventors regard as their invention.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g., amounts, temperatures, etc.), but some experimental
error and deviation should be accounted for. Unless indicated
otherwise, parts are parts by weight, molecular weight is weight
average molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Example 1
In vivo Gene Transfer to Salivary Glands by Administration of Naked
DNA Encoding Insulin
[0126] Four adult rats weighing approximately 300 g each were
anesthetized with an intraperitoneal injection of sodium
pentobarbital. An incision was made parallel to the line of the
mandible and both parotid glands exposed by dissection. Two rats
received a total of 100 .mu.l of 0.5 .mu.g/.mu.l pBAT14.hIns
plasmid which contains cDNA encoding human insulin (FIG. 1), while
the remaining two rats received 100 .mu.l 0.9% saline (sham
animals). The test and control samples were administered by
multi-site subcapsular injection to each parotid gland of each
animal. No significant leakage of material or bleeding occurred.
The wound was closed after administration. After 3 hours, the
animals were awake, drinking water, and appearing normal.
[0127] Approximately 24 hours after cDNA injection, the animals
were again anesthetized and a tracheostomy performed. A control
blood sample was drawn from the femoral vein of each animal. The
cholinergic agonist acetyl-p-methyl choline (McH) was injected into
each subcutaneously at 0.8 mg/kg body weight into each animal. The
salivary glands appeared normal and showed no signs of
inflammation. Twenty minutes after McH injection, saliva and blood
samples were collected from each animal. The blood samples were
collected from the inferior vena cava and by heart puncture. Serum
was separated from the blood of all samples after clotting, and
kept at -20.degree. C. prior to assay. In addition, blood was
collected from 10 normal rats, and serum prepared to determine the
blood level of insulin in untreated rats.
[0128] The results of this experiment are shown in Table 3. The
level of insulin in the blood of transfected animals and in blood
of untransfected animals was essentially the same. Administration
of McH induced an increase in serum insulin levels in both the
control and transfected animals. The concentration of insulin was
higher after McH stimulation in the two transfected animals than in
the McH-stimulated control animals.
3 TABLE 3 Treatment Insulin (.mu.U/ml) Normal (untreated) 2.6 cDNA
1 -McH 1.4 +McH 10.0 cDNA 2 -McH 2.5 +McH 11.6 Control 1 -McH 2.0
+McH 5.6 Control 2 -McH 2.0 +McH 9.2
Example 2
In vivo Gene Transfer to Salivary Glands by Administration of an
Increased Dosage Naked DNA Encoding Insulin
[0129] In a second experiment, four adult rats weighing
approximately 300 g each were anesthetized with an intraperitoneal
injection of sodium pentobarbital. Two rats received a total of 100
.mu.l of 1-1.2 .mu.g/.mu.l pBAT14.hIns plasmid containing cDNA
encoding human insulin, while the remaining two rats received 100
.mu.l 0.9% saline (control animals). The test and control samples
were administered by multi-site subcapsular injection to each
parotid gland of each animal as described above in Example 1, and
the wound closed after administration.
[0130] Approximately 24 hours after injection, the animals were
again anesthetized, blood samples drawn from each animal, and the
cholinergic agonist McH administered at 0.8 mg/kg body weight by
subcutaneous injection. Twenty minutes after McH injection, blood
samples were drawn from the inferior vena cava and by heart
puncture of each animal. Serum was separated from the blood of all
samples after clotting, and kept at -20.degree. C. prior to assay
for insulin. The parotid glands of all animals looked normal and
showed no signs of inflammation.
[0131] As shown in Table 4, the levels of insulin in the blood of
the transfected animals was substantially higher than in the
previous experiment, suggesting that the increased dosage of cDNA
resulted in increased insulin production. Insulin levels in the
transfected animals were elevated by McH stimulation. Moreover, the
animals transfected with 100 .mu.g-120 .mu.g cDNA had about 50%
greater insulin levels after McH stimulation than the animals
transfected with 50 .mu.g cDNA described above.
4 TABLE 4 Treatment Insulin (.mu.U/ml) cDNA 3 -McH 6.4 +McH 15.2
cDNA 4 -McH 7.2 +McH 15.2
Example 3
Effects of Isoprenaline Stimulation on Expression of Insulin
Following in vivo Transformation of Salivary Glands
[0132] Two transfected rats and two control rats were treated using
the same protocol, vector, and dose as in Example 2. Approximately
24 hours after injection, the animals were again anesthetized,
blood samples drawn from each animal, and the adrenergic agonist
isoprenaline (IsO) was administered at 0.1 .mu.g/kg body weight by
subcutaneous injection. Twenty minutes after Iso injection, blood
samples were drawn from the inferior vena cava and by heart
puncture of each animal. Serum was separated from the blood of all
samples after clotting, and kept at -20.degree. C. prior to assay
for insulin.
[0133] As shown in Table 5, the blood insulin levels in the
transfected animals was substantially elevated relative to control
values. Injection of Iso had no effect upon blood insulin
levels.
5 TABLE 5 Treatment Insulin (.mu.U/ml) cDNA 5 -IsO 8.5 +IsO 8.0
cDNA 6 -IsO 6.4 +IsO 8.0
Example 4
Effects of Streptozotocin on Insulin Levels in Rats Following in
vivo Transfer of cDNA Encoding Insulin to Salivary Glands
[0134] Streptozotocin, which induces diabetes mellitus in rats, was
administered to three adults rats weighing approximately 220-230 g
at 70 mg/kg body weight by intraperitoneal injection. The animals
were anesthetized by intraperitoneal injection of sodium
pentobarbital. Two of the animals received a 50 .mu.l volume of 2
.mu.g/.mu.l pBAT14.hIns plasmid which contains cDNA encoding human
insulin. The remaining rat received 100 .mu.l 0.9% saline (control
animal). The test and control samples were administered by
multi-site subcapsular injection to each parotid gland of each
animal as described in Example 1.
[0135] Approximately 48 hours after cDNA or saline injection, the
animals were again anesthetized and a tracheostomy performed. A
control blood sample was drawn from the femoral vein of each
animal. McH was administered at 0.8 mg/kg body weight by
subcutaneous injection. Twenty minutes after McH injection, saliva
and blood samples were collected from each animal. The blood
samples were collected from the inferior ven cava and by heart
puncture. Serum was separated from the blood of all samples after
clotting, and kept at -20.degree. C. prior to assay. In addition,
the salivary glands and a portion of the pancreas were removed and
homogenized in 50 mM phosphate buffer (pH 8.0) 1:10 w/v. The
homogenates were spun at 50,000.times.g for 1 h and the supernatant
stored at -20.degree. C. A small portion of parotid salivary glands
were fixed in 10% buffered formalin and saved for histologic
examination. The parotid glands showed no observable signs of
inflammation as a result of cDNA injection.
[0136] As shown in Table 6, streptozotocin administration decreased
the blood levels of insulin in the transfected animals. Stimulation
with McH was effective in increasing serum insulin levels in one of
the two transfected animals, but not in the control animal.
6 TABLE 6 Treatment Insulin (.mu.U/ml) Glucose (mg/dl) cDNA -McH
0.5 268 +McH 8.8 385 cDNA -McH 1.6 321 +McH 1.0 413 Control -McH
5.2 230 +McH 4.8 335
Example 5
Summary of Results of in vivo Gene Transfer to Salivary Glands by
Percutaneous Administration of Naked DNA Encoding Insulin
[0137] Nine adult rats were anesthetized with an intraperitoneal
injection of sodium pentobarbital. Six rats were injected
percutaneously with DNA encoding insulin. Two rats received a 100
.mu.l volume of 0.5 .mu.g/.mu.l pBAT14.hIns plasmid which contains
cDNA encoding human insulin (low dose animals), while the four
other transfected rats received a 100 .mu.l volume of 1.0
.mu.g/.mu.l pBAT14.hIns (high dose animals). The remaining three
rats received 100 .mu.l 0.9% saline (sham animal).
[0138] Approximately 24 hours after injection, the animals were
again anesthetized. Control blood samples were drawn from the
femoral vein of each animal. Two of the control animals, two of the
low dose animals, and two of the high dose animals received a
subcutaneous injection of 0.8 mg/kg body weight McH. Two of the
high dose transfected animals received a subcutaneous injection of
the adrenergic agonist IsO at 0.1 .mu.g/kg body weight. Twenty
minutes after McH or Iso injection, saliva and blood samples were
collected from each animal. The blood samples were collected from
the inferior ven cava and by heart puncture. Serum was separated
from the blood of all samples after clotting, and kept at
-20.degree. C. prior to assay.
[0139] The results of this experiment are shown in Table 7. The
serum insulin levels were highest in the high dose transfected
animal group. The serum insulin levels of the low dose transfected
group and the control group were similar. After stimulation with
McH, serum insulin levels were again markedly higher in the high
dose transfected group than in either the low dose transfected or
the control groups. Serum insulin levels after McH stimulation were
higher in the low dose transfected group than in the control group.
Iso injection of high dose transfected rats had no significant
effect upon serum insulin levels as compared to serum insulin
levels in the absence of agonist. These data show that high dose
cDNA increase both unstimulated (-McH) and McH-stimulated insulin
responses.
7TABLE 7 Average values for the effect percutaneous administration
of human insulin cDNA to parotid gland on serum insulin Treatment
Insulin (.mu.U/ml) Control 2.5 (12) Low dose cDNA (0.5 .mu.g/.mu.l)
2.0 (2) High dose cDNA (1.0 .mu.g/.mu.l) 7.4 (6) With cholinergic
stimulation (McH) Control 7.6 (2) Low dose cDNA (0.5 .mu.g/.mu.l)
13.0 (2) High dose cDNA (1.0 .mu.g/.mu.l) 15.2 (2) () = # of
animals
Example 6
In vivo Gene Transfer of DNA Encoding Human Growth Hormone by
Retrograde Intraductal Administration of DNA
[0140] A DNA fragment encoding human growth hormone (hGH) is
operably linked to the LTR of Rous sarcoma virus, which serves as a
promoter, and the SV40 type T antigen, which serves as a nuclear
localization signal. This promoter-localization signal-hGH DNA
cassette is then inserted into the bacterial plasmid pBR322.
Escherichia coli is then transformed with the plasmid using
conventional transformation procedures. E. coli containing the
plasmid are selected by virtue of the tetracycline or ampicillin
resistance encoded by pBR322, and the transformed bacterial cells
propagated in culture. Plasmid DNA is then isolated from the
transformed bacterial cell culture and the DNA purified by cesium
gradient.
[0141] Approximately 10 mg to 20 mg of the purified plasmid DNA
containing hGH DNA is administered into the salivary gland of a
human patient by retrograde administration via a salivary gland
duct. Expression and intravenous secretion of the protein is
assessed using the method described above.
Example 7
In Vivo Transformation of Salivary Glands by Retrograde Intraductal
Administration of DNA Encoding Human Growth Hormone
[0142] Four constructs for expression of human growth hormone (hGH)
were prepared using techniques well known in the art (see, for
example, Sambrook et al. ibid). The first construct, pFGH, contains
the genomic hGH DNA sequence inserted in the commercially available
vector pBLUESCRIPT SK+.TM. (Stratagene, LaJolla Calif.) (FIG. 2).
Because the hGH coding sequence is not linked to a promoter, this
vector provides for no or only low-level hGH expression. Thus, the
pFGH construct serves as a negative control for hGH expression in
the pancreas. The second construct, pFGH.CMV, was constructed by
operably inserting the promoter from the immediate early gene of
human CMV upstream of the genomic hGH sequence of the pFGH vector
(FIG. 3). The third construct, pFGH.chymo, was constructed by
operably inserting the rat chymotrypsin B gene promoter upstream of
the genomic hGH sequence of the pFGH vector (FIG. 4). The fourth
construct, pFGH.RSV, was constructed by operably inserting the
promoter from the long terminal repeat (LTR) of RSV upstream of the
genomic hGH sequence of the pFGH vector.
[0143] Twelve adult rats weighing approximately 300 g each were
anesthetized with an intraperitoneal of sodium pentobarbital. A
total volume of 50 .mu.l containing 4 .mu.g of the pFGH.CMV
plasmid, which contains cDNA encoding human growth hormone (hGH),
was introduced into each submandibular gland of 8 rats by
retrograde ductal administration via the ducts leading from the
oral mucosa to the salivary gland. Briefly, both the left and right
Wharton's duct were cannulated intraorally with polyethylene (PE)
10 tubing, and the DNA introduced into the duct system of each
gland in a retrograde fashion (4 .mu.g/50 .mu.l of PBS). The
material was kept in place for two minutes before normal flow was
reestablished.
[0144] For three of these animals the DNA was mixed in a 6%
solution of the cationic lipid Lipofectin (labeled "liposomes")
from Life Technologies (Gaithersburg, Md.). For four of these
animals, the DNA was mixed with a 1:50 dilution of
replication-defective human adenovirus (Ad5-di 342) supernatant.
Control rats (4 rats) received 50 .mu.l 0.9% saline (control)
without plasmid. No significant leakage of material or bleeding
occurred. After 3 hours, the animals were awake, drinking water,
and appearing normal.
[0145] Approximately 48 hours after cDNA administration, the
animals were sacrificed. The right and left submandibular glands
were removed and were homogenized in cold 0.2 M (pH 8.0) sodium
phosphate buffer (1:10 w/v) containing the protease inhibitors
aprotinin, leupeptin, pepstatin, and PEFABLOC SC.TM..
Homogenization was completed by shearing after 10 passes with a
motorized pestle at approximately 4000 rpm in a glass homogenizer.
The homogenates were centrifuged at 1000 g for 15 min, and the
supernatant collected and stored at -80.degree. C. until analysis.
The levels of hGH in the protein samples were measured using the
hGH radioimmune assay (Nichols Institute). Each assay was performed
in duplicate and compared to a set of control samples.
[0146] Each of the submandibular glands into which the pFGH.CMV
vector was introduced expressed hGH in the salivary gland tissue;
hGH expression was undetectable in the control rats' salivary
glands (FIG. 5).
Example 8
In Vivo Transformation of Salivary Glands by Retrograde Intraductal
Administration of hGH-Encoding DNA and Regulation of hGH
Secretion
[0147] Three adult rats weighing approximately 300 g each were
anesthetized with an intraperitoneal injection of sodium
pentobarbital. A control blood sample (prior to DNA) was drawn from
the femoral vein of each animal. A total of 4 .mu.g of the
hGH-encoding plasmid pFGH.CMV in 50 .mu.l, was introduced into each
submandibular gland of each rat by retrograde ductal administration
via the ducts leading from the oral mucosa to the salivary gland as
described above. No significant leakage of material or bleeding
occurred. After 3 hours, the animals were awake, drinking water,
and appearing normal.
[0148] Forty-eight hours after cDNA administration, the animals
were again anesthetized and a control blood sample was drawn from
the femoral vein of each animal (unstimulated serum level). The
cholinergic agonist acetyl-.beta.-methyl choline (McH) was injected
subcutaneously at 0.8 mg/kg body weight into each animal. Blood
samples were collected from the femoral vein of each animal at 10
min, 20 min, 40 min, and 50 min after McH injection. Serum was
separated from the blood of all samples after clotting, and kept at
-20.degree. C. prior to assay.
[0149] As shown in FIG. 6 (one representative animal), secretion of
hGH into the bloodstream was dramatically increased in response to
administration of McH, peaking at 40 min. Thus, these data
demonstrate that introduction of hGH-encoding DNA into the salivary
gland results in bloodstream-directed secretion of hGH and
regulation by cholinergic stimulation. Moreover, regulation is at
the level of secretion, not transcription, since transcriptional
regulation would not result in increased hGH bloodstream levels in
such a short period.
Example 9
Human Growth Hormone (hGH) Expression in Rat Salivary Gland
[0150] Four micrograms of the pFGH.CMV construct, premixed with
either Lipofectin or adenovirus, was introduced into each
submandibular gland via retrograde ductal administration (via
Wharton's duct) as described above. Two days later, each gland was
harvested and hGH content was measured
[0151] As shown in FIG. 7, tissue levels of hGH averaged about 50
ng/g tissue wet weight. Plasma hGH levels were in the 20-40 pg/ml
range. As in the pancreas, the addition of adenovirus increased
tissue hGH levels, in this case to 100 ng/g (FIG. 21).
Example 10
In vivo Gene Transfer of DNA Encoding Human Growth Hormone by
Retrograde Intraductal Administration of DNA into the Pancreas
[0152] Each of the vectors of FIGS. 2-4 were used to transfect the
pancreas of approximately 300 g adult rats (pFGH+lipofectin, 4
rats; pFGH.chymo+lipofectin, 4 rats; pFGH.RSV+lipofectin, 4 rats;
pFGH.CMV+lipofectin, 10 rats; pFGH.CMV without lipofectin, 7 rats;
negative control (no DNA, no lipofectin), 3 rats). Pancreatic
transfection was accomplished by first anesthetizing the rats and
performing a laparotomy to expose the duodenum. The pancreas and
the associated common bile duct were identified, and the common
bile duct was cannulated either extraduodenally or through the
papilla of Vater. The hepatic duct was occluded, and 100 .mu.l of
phosphate-buffered saline (PBS) containing one of the four vectors,
or 100 .mu.l of PBS alone as a negative control, were slowly
introduced into the pancreatic duct in a retrograde direction. The
vector-containing solutions were composed of 8 .mu.g DNA per 100
.mu.l in PBS, either with or without 6% lipofectin, a cationic
lipid used to increase transfection efficiency. The solution was
left in place for 5 min before secretory flow was allowed to resume
and hepatic duct blockage removed. The catheter was left in place
and inserted into the duodenum through a small hole to ensure
adequate biliary and pancreatic flow post-operatively. The abdomen
was then closed with sutures. The animals recovered fully and
rapidly from the surgery without obvious side effects. This
transfection method provides direct access of the vector to over
90% of the pancreatic gland cells.
[0153] At 48 hr after surgery, a blood sample was obtained to
measure serum hGH levels, and the rats were sacrificed. At autopsy,
the pancreas of both control and test rats appeared normal, and
exhibited no gross or microscopic pathology.
[0154] The pancreas was dissected free from the mesenteric surface
and was homogenized in cold 0.2 M (pH 8.0) sodium phosphate buffer
(1:10 w/v) containing protease inhibitors aprotinin, leupeptin,
pepstatin, and PEFABLOC SC.TM.. Homogenization was completed by
shearing after 10 passes with a motorized pestle at approximately
4000 rpm in a glass homogenizer. The homogenate was then
centrifuged at 1000 g for 15 min. The supernatant was collected and
stored at -80.degree. C. until analysis. The levels of hGH in the
serum and pancreatic protein samples were measured using the hGH
radioimmune assay (Nichols Institute). Each assay was performed in
duplicate and compared to a set of control samples.
[0155] Rats that received the pFGH.CMV vector expressed higher
levels of hGH in the pancreatic tissue (FIG. 8), compared to
background levels of pancreatic hGH expression in rats that
received either no DNA (PBS alone) or the pFGH vector (hGH DNA with
no promoter). The addition of lipofectin modestly increased hGH
expression in rats that received the pFGH.CMV construct. In
addition, rats that received the pFGH.CMV vector secreted hGH in
the serum at levels increased relative to hGH secretion levels in
rats treated with either control samples (no DNA or pFGH, or with
samples containing hGH DNA linked to either the chymotrypsin B or
RSV promoters (FIG. 9). In FIG. 10, all data from the above
experiments (including all controls and vectors) are analyzed by
plotting the hGH serum levels against the hGH tissue levels. This
graph shows that higher tissue levels result in higher levels of
secretion into the blood. Thus, retrograde intraductal pancreatic
administration of the pFGH.CMV vector successfully transfected
pancreatic cells to provide both hGH pancreatic tissue expression
and hGH secretion into the bloodstream.
Example 11
In Vivo Transformation of Pancreatic Cells by Retrograde
Intraductal Administration of hGH-Encoding DNA and Regulation of
hGH Secretion
[0156] Eight rats were anesthetized and control blood samples (no
DNA) were collected from the femoral vein of each animal.
Pancreatic transfection was accomplished by exposing the duodenum
by laparotomy and identifying the pancreas and the associated
common bile duct. The common bile duct was cannulated either
extraduodenally or through the papilla of Vater, and the hepatic
duct was occluded. A 1:50 dilution of replication-defective human
adenovirus (Ad5-di 342) supernatant in 100 .mu.l of
phosphate-buffered saline (PBS) containing 8 .mu.g of the
hGH-encoding plasmid pFGH.CMV (FIG. 3) was slowly infused into the
pancreatic duct in a retrograde direction. The solution was left in
place for approximately 5 min before secretory flow was allowed to
resume and the hepatic duct blockage removed. The catheter was left
in place and inserted into the duodenum through a small hole to
ensure adequate biliary and pancreatic flow post-operatively. The
abdomen was then closed with sutures. The animals recovered fully
and rapidly from the surgery without obvious side effects.
[0157] At 48 hr after surgery, a blood sample was obtained to
measure serum hGH levels (unstimulated serum levels). The
cholinergic agonist McH was injected subcutaneously into each rat
at 0.8 mg/kg body weight. Blood samples were collected from the
inferior vena cava of each animal at 15 min intervals following McH
injection. Serum was separated from the blood of all samples after
clotting, and kept at -20.degree. C. prior to assay.
[0158] As shown in FIG. 11 (one representative animal) plasma
levels of hGH increased markedly following McH injection,
demonstrating that secretion of hGH expressed by transformed
pancreatic cells is regulated by agonist stimulation. Moreover,
bloodstream-directed secretion of hGH from the transformed
pancreatic cells occurred at relevant, physiological levels useful
in therapeutic administration (i.e., at the ng/ml level).
Example 12
Treatment of Diabetes Mellitus Over a Three Day Period by In Vivo
Transformation of Pancreatic Cells by Retrograde Intraductal
administration with Insulin-Encoding DNA
[0159] Streptozotocin, which induces diabetes mellitus in rats, was
administered to 8 male Sprague-Dawley rats (260-280 g) after
overnight fasting by intraperitoneal injection in 1 mM citrate
buffer (pH 4.5) (Sigma) at 65 mg/kg of body weight. One hour later,
animals were anesthetized with Nembutal and the body cavity opened
to expose the gastrointestinal tract. Each animal was given the
appropriate DNA construct directly by retrograde administration in
the pancreatic duct in a 100 .mu.l volume containing 8 .mu.g DNA
plus adenovirus (Ad5-di 342)(3.times.10.sup.10 viral particles) as
described above. Test animals (4 rats) received the human
insulin-encoding construct pBat16.hInsG1.M2. The pBAT16.hInsG1.M2
construct (FIG. 12) encodes an insulin gene containing a
site-directed mutation of the second protease site to create a
furin recognition site; this construct provides for enhanced
expression of processed insulin in non-neuroendocrine cells. In
addition, the human .beta.-globin first intron replaces the first
insulin gene intron which is inefficiently spliced. Control animals
(4 rats) received the control construct CMV-GFP, which contains a
green fluorescent protein (GFP)-encoding sequence operably linked
to a CMV promoter. The animals recovered fully and rapidly from the
surgery without obvious side effects. Body weight and blood glucose
were monitored daily for three days post-administration. Blood
glucose was measured by the glucose oxidase method (Lifescan,
Milpitas, Calif.).
[0160] As shown in FIG. 13, treatment of the streptozotocin-induced
diabetic rats with the insulin-encoding construct resulted in
maintenance of almost complete euglycemia for 3 days. In contrast,
control animals that received the GFP-encoding construct remained
hyperglycemic throughout the test period. The data show that
introduction of insulin-encoding DNA into the pancreas results in
pancreatic cell transformation, as well as secretion of insulin by
the transformed pancreatic cells at levels sufficient to overcome
diabetes in an animal model. Moreover, these results show that the
method of the invention provides regulated and relatively normal
blood glucose levels. Surprising, the exocrine pancreas regulates
the release of insulin such that blood sugar levels are maintained
at regulated levels (normally the endocrine pancreas is responsible
for regulation of bloodstream-directed secretion).
Example 13
Treatment of Diabetes Mellitus Over a Six Day Period by In Vivo
Transformation of Pancreatic Cells by Retrograde Intraductal
Administration with Insulin-Encoding DNA
[0161] Streptozotocin was administered to 14 rats at 70 mg/kg body
weight by intraperitoneal injection to induce diabetes mellitus.
The animals were then anesthetized by intraperitoneal injection of
sodium pentobarbital. Two rats did not receive streptozotocin and
served as one negative control. Insulin-encoding DNA in the
pBAT16.hInsG1.M2 construct (FIG. 12) was administered to 8 of the
streptozotocin-injected rats by retrograde ductal administration as
described above. Six streptozotocin-treated rats received either
100 .mu.l of saline without DNA (2 animals) or a control DNA
without the human insulin gene (4 animals) by pancreatic retrograde
ductal administration as additional negative controls. The animals
recovered fully and rapidly from the surgery without obvious side
effects. Blood samples were collected from the femoral vein of each
animal at 24 hr intervals for 6 days. Human insulin was measured
using a double antibody radioimmunoassay (Linco Laboratories, Saint
Louis, Mo.).
[0162] As shown in (FIG. 14), blood glucose levels were
significantly decreased in the diabetic rats that received the
insulin-encoding DNA (+Strep, +DNA) relative to diabetic the rats
that received no DNA (+Strep, No DNA). Furthermore, these decreased
blood glucose levels were observed throughout the entire 6 day
course of the experiment. Thus, these data show that introduction
of insulin-encoding DNA into the pancreas results in persistent
expression of insulin, and that the insulin expressed by the
transformed pancreatic cells is secreted into the bloodstream and
can function in regulation of blood glucose at levels sufficient to
overcome diabetes in an animal model. As shown in FIG. 14, elevated
insulin levels for such an extended period additionally demonstrate
prolonged expression from the DNA introduced into the pancreatic
cells.
Example 14
In Vivo Transformation of Pancreatic Cells by Retrograde
Intraductal Administration of Green Fluorescent Protein-Encoding
DNA and Expression in Pancreatic Cells
[0163] To identify the pancreatic cells that expressed the
recombinant protein, DNA encoding green fluorescent protein (GFP)
was used to transform pancreatic cells according to the methods of
the invention. EGFP cDNA from plasmid pEGFP.C2 (Clontech) was
inserted into pFOX. The EGFP sequence was modified to contain an
SV40 nuclear localization signal, in-frame at the 3' end. This
addition allowed for partial nuclear localization and facilitated
immunohistochemical detection. The CMV immediate early promoter was
positioned upstream of the first intron of human .beta.-globin to
create the expression vector pFOX.EGFP.N2.CMV.
[0164] After fasting overnight, Male Sprague-Dawley rats (260-280
g) were anesthetized and the body cavity opened to expose the
gastrointestinal tract. The green fluorescent protein
(GFP)-encoding construct pFOX.EGFP.N2.CMV was administered to each
animal by retrograde administration in the pancreatic duct in a 100
.mu.L volume containing 8 .mu.g DNA premixed with adenovirus
(3.times.10.sup.10 viral particles) as described above. The animals
recovered fully and rapidly from the surgery without obvious side
effects.
[0165] Seventy-two hours post-treatment, the animals were
sacrificed, and pancreases were removed and weighed (wet weight).
Samples of each pancreas were fixed in 5% buffered formalin for
24-48 hours at room temperature. Fixed tissues were dehydrated and
imbedded in paraffin, and 5 .mu.m sections were processed for
immunohistochemistry using standard techniques. Endogenous
peroxidase was quenched in 0.7% H.sub.2O.sub.2/MeOH, and antigen
retrieval was performed using Citra solution (Biogenex, San Ramon,
Calif.) according to the manufacturers' instructions. Sections were
preincubated for 30 minutes in 5% goat serum/phosphate-buffered
saline (PBS), and then incubated overnight at 4.degree. C. with
primary antisera diluted in 5% goat serum/PBS.
[0166] The primary antisera were selected from either anti-GFP
antisera (1:1500; Clontech, Palo Alto, Calif.), anti-insulin
antisera (1:500; Dako, Carpenteria, Calif.), or non-specific rabbit
sera (1:1500). The following day all sections were incubated with
biotinylated goat anti-rabbit antiserum (5 .mu.g/ml; Vector,
Burlingame, Calif.) for 30 minutes at room temperature, and then
incubated with streptavidin-aminohexanol-biotin horseradish
peroxidase (HRP) complex (Vectastain-Elite, Vector). Protein was
visualized by reaction with the peroxidase substrate
3,3-diamino-benzidine tetrahydrochloride (DAB; Sigma). The color
reaction was followed by a brief counter stain in 1% methyl green
(Sigma) prior to mounting. Negative controls included staining of
sections from pancreas with no CMV-GFP administration, and omission
of primary antiserum.
[0167] Staining for GFP was observed in the pancreas of animals
treated with GFP DNA, but not in control animals. GFP expression
was restricted to exocrine cells; there was no staining in either
ductal or islet cells. Moreover, expression was observed in
0.1-1.0% of exocrine cells. Endogenous insulin was detected in
adjacent sections; but GFP expression did not co-localize with
insulin expression, suggesting that the pancreatic cells primarily
transformed are exocrine, not endocrine cells. Under the conditions
studied there was no histological indication of inflammatory
infiltration as a consequence of ductal administration of the
vector.
[0168] These data show that introduction of the DNA construct
results in successful transformation of pancreatic cells, despite
the introduction of the construct against the flow of pancreatic
juice and the high concentrations of DNase in the pancreatic juice.
Moreover, these data, combined with the data above showing that
transformation of the pancreas results in bloodstream-directed
secretion of the encoded protein, and suggest that transformation
of exocrine pancreatic cells results in bloodstream-directed
secretion of the protein encoded by the introduced construct.
Furthermore, because insulin staining and GFP staining did not
co-localize, introduction of the GFP-encoding construct resulted in
transformation of exocrine tissue, which is normally associated
with protein secretion into the gastrointestinal tract, rather than
endocrine tissue, which is normally associated with
bloodstream-directed secretion. Despite this, bloodstream-directed
secretion was still obtained at physiologically relevant levels
sufficient to treat diabetes mellitus in an animal model as
evidenced in the examples above.
Example 15
In Vivo Transformation of Pancreatic Cells with hGH-Encoding DNA
and Expression in Rat Exocrine Pancreas and Plasma
[0169] Following overnight fasting and anesthesia with
pentobarbital, the abdominal cavity of the rats was opened and the
pancreatic duct cannulated external to the duodenum with PE 10
tubing as described above. Eight to twenty-five micrograms of each
of pFGH (promoter less construct), pFGH.chymo (construct with the
chymotrypsin promoter), pFGH.RSV (construct with the RSV promoter),
and pFGH.CMV (construct with the CMV promoter) was administered in
a total volume of 100 .mu.l of PBS into the pancreas via the
pancreatic duct as described above. Immediately prior to
administration construct samples were optionally premixed with
either Lipofectin (6-12% vol:vol) or adenovirus (3.times.10.sup.10
viral particles). The material was kept in the duct for 5 min prior
to establishing normal flow. The abdomen was the closed and the
animals allowed to recover.
[0170] Forty-eight hours later the pancreas was harvested, plasma
obtained, and human growth hormone measured. The animals were
anesthetized, blood samples taken (either from the femoral vein or
inferior vena cava), and the transfected tissue removed. The tissue
was homogenized in PBS containing 5 mM Na2HPO.sub.4 (pH 7.8) at a
tissue to fluid ratio of 1:10 using a motorized mortar and pestle.
Large particulate material in the homogenate was removed by
sedimentation at 10,000.times.g for 30 minutes, and the supernatant
assayed for the protein of interest. The results are shown in FIGS.
15-18. All data shown are the mean .+-. the SEM.
[0171] The effects of the various promoters upon tissue expression
of hGH are shown in FIGS. 15 and 16, respectively. In these
experiments, the constructs were mixed with lipofection prior to
administration. Of the promoters tested, the CMV promoter was by
far the most effective, and produced high levels of hGH in tissue
(in the range of 150 ng/g tissue wet weight) when compared to
either promoter less controls, or plasmids containing RSV and
chymotrypsin promoters (FIG. 15). The cationic lipid adjuvant
Lipofectin increased expression by about 50%, and pre-mixing the
plasmid with adenovirus enhanced tissue expression five fold (FIG.
16). Expression of hGH at 24, 48 or 72 hours after administration
was similar under all conditions studied.
[0172] As shown in FIGS. 17 and 18, hGH was secreted into plasma.
Plasmids containing the CMV promoter increased circulating levels
of hGH five times above background (FIG. 17). With plasmid alone,
plasma hGH concentrations in the range of 60 to 80 pg/ml were
routinely observed. Premixing the plasmids with adjuvants also
increased circulating hGH levels (FIG. 18). Lipofectin increased
plasma levels by an additional 50%, and adenovirus by 75%, when
compared to plasmid alone.
Example 16
Human Insulin Expression and Secretion in Diabetic Rat Pancreas
[0173] In an attempt to treat a disease state, diabetes mellitus,
we expressed human insulin in the exocrine pancreas. Fasted
experimental and control animals received intra-peritoneal
streptozotocin (Sigma; 65 mg/kg body weight, in 1 mM citrate
buffer, pH 4.5) on day zero one hour prior to administration of the
insulin-encoding construct. The experimental animals subsequently
received 8 .mu.g of the insulin plasmid (pBAT16.hInsG1.M2) premixed
with adenovirus and introduced into the pancreatic duct, also on
day zero. The pBAT16.hInsG1.M2 construct contains the human insulin
cDNA linked to a CMV immediate early promoter, which is positioned
upstream of the first intron of human P-globin. The human insulin
cDNA was mutated to convert the second protease site, between
peptides C and A, to a furin recognition site. This allows for
correct proteolytic processing of mature insulin in non-endocrine
cells.
[0174] Plasma insulin and glucose levels were determined for up to
six days. Plasma glucose levels in diabetic rats (n=3), and
diabetic rats treated with the pBAT16.hInsG1.M2 plasmid (n=3),
measured over a three day period, are shown in FIG. 19. Plasma
insulin levels in diabetic rats (n=3), and diabetic rats treated
with the pBAT16.hInsG1.M2 plasmid (n=3), measured over a three day
period, are shown in FIG. 20. Plasma glucose levels in individual
diabetic (n=3) and pBAT16.hInsG1.M2 plasmid-treated diabetic rats
(n=3), measured over a six day period, are shown in FIG. 21.
[0175] As a consequence of streptozotocin administration, blood
glucose levels rose from the normal level of 100 mg/dl to 300-400
mg/dl within 24 hours and remained elevated for the duration of the
study (FIG. 19). Treatment with the human insulin plasmid reduced
blood glucose levels in diabetic rats to the normal range (FIGS. 19
and 21), and concentrations of insulin remained near pre-treatment
values (FIG. 20). Blood glucose levels were euglycemic for the
duration of the study (6 days; FIG. 21). Animals transfected with a
control plasmid remained diabetic (data not shown). These data show
that regulation of insulin secretion in response to feeding was
effective.
Example 17
In Vivo Transformation of Liver Cells by Retrograde Intraductal
Administration of hGH-Encoding DNA and Bloodstream-Directed hGH
Secretion
[0176] Four rats were anesthetized and control blood samples (no
DNA) were collected from the femoral vein of each animal.
Expression of the construct in liver cells was accomplished by
exposing the duodenum by laparotomy and identifying the liver and
the associated common bile duct. The common bile duct was
cannulated either extraduodenally or through the papilla of Vater.
The tubing was advanced to the bifurcation of the hepatic duct in
order to prevent introduced material from entering the distally
located pancreatic drainage. A 1:50 dilution of
replication-defective human adenovirus supernatant in 100 .mu.l of
phosphate-buffered saline (PBS) containing 8 .mu.g of the
hGH-encoding plasmid pFGH.CMV or 100 .mu.l of PBS alone (no DNA)
were slowly infused into the hepatic duct in a retrograde
direction. The solution was left in place for approximately 2 min
to 5 min before secretory flow was allowed to resume and the
pancreatic duct blockage removed. The catheter was left in place
and inserted into the duodenum through a small hole to ensure
adequate biliary and pancreatic flow post-operatively. The abdomen
was then closed with sutures. The animals recovered fully and
rapidly from the surgery without obvious side effects.
[0177] Plasma hGH levels were measured 2 days after treatment; the
results are shown in FIG. 22. Each data point in FIG. 22 represents
the mean.+-.standard error of the mean (SEM) for three animals.
These data demonstrate that liver cells were transformed with the
hGH-encoding DNA. Furthermore, hGH was secreted by the transformed
liver cells into the bloodstream at physiologically relevant
levels.
Example 18
Stimulation of Human Growth Hormone (hGH) Secretion
[0178] Even when exocrine secretory cells store large amounts of
protein, such as after a period of fasting, they secrete these
proteins at a low rate under unstimulated conditions (i.e. basal or
constitutive secretion). Greater rates are achieved when exogenous
stimulants (e.g., hormonal stimulants and/or stimulation associated
with eating) are applied. To determine whether secretion of the
engineered protein would be enhanced during feeding, pancreatic
secretion was stimulated with a secretory stimulant. For these
experiments we used animals in which both pancreas and liver were
transfected. Eight micrograms of the pFGH.CMV construct were
introduced into ducts of both the pancreas and liver of four rats
as described above. A blood sample was taken prior to
administration as a control. Two days after transfection, a second
control blood sample was taken and the rats were treated with the
cholinergic agonist, acetyl-.beta.-methylcholine (McH) (0.8 mg/kg
body weight).
[0179] As shown in FIG. 23, hGH secretion was increased three fold
within 30 minutes of stimulation, with plasma levels approaching
1.0 ng/ml. Similar enhancement of hGH secretion was observed when
either the pancreas was studied alone, or when the salivary glands
were studied alone. These data show that hGH secretion is enhanced
by stimulation with a cholinergic agonist. Thus secretion of hGH is
regulated in a manner similar to secretion of endogenous
proteins.
[0180] Although the concentration of hGH in plasma was correlated
to the level of hormone in the pancreas (r=0.55, p<0.01, n=41),
at high tissue levels, plasma concentration was not linearly
proportional to tissue content. For example, addition of adenovirus
to the hGH vector produced a five fold increase in tissue levels
relative to the plasmid alone (FIG. 7), but only about a two fold
increase in plasma concentration (see, e.g., FIGS. 17 and 18
described above). This lack of proportionality indicates that it is
not the concentration of product in the cells alone that determines
the rate of secretion into blood, but that at high tissue levels,
secretion is limited by other factors. This result is similar to
what is observed for endogenous protein secretion and suggests that
secretion of the engineered protein is regulated in much the same
manner.
Example 19
Comparison of hGH Secretion by Rat Liver, Pancreas, and Combined
Liver and Pancreas Transformed with hGH-Encoding DNA
[0181] Eight micrograms of the pFGH.CMV construct premixed with
adenovirus as described above, was introduced into the ducts of
either the liver, the pancreas, or both organs of the same animal.
Where only the liver or the pancreas was transformed (liver alone
or pancreas alone), the DNA was introduced according to the methods
described above. Where both the liver and pancreas received the
formulation, the DNA-containing formulation was introduced into the
hepatic duct first, and then the tubing partially withdrawn to
provide access to the pancreatic duct system. A temporary ligature
was then placed around the hepatic duct to prevent the second
infusion from entering the parenchyma of the liver. Thus, animals
in which both the pancreas and liver were transformed received two
doses of the DNA-containing formulation. Plasma hGH levels were
measured two days later.
[0182] In animals having transformed liver (liver alone) or
pancreas (pancreas alone), hGH was expressed in liver or pancreatic
tissue, respectively, and hGH detected in plasma under both
circumstances. Tissue levels in liver when transformed alone were
far lower than in the pancreas when transformed alone (less than 1
ng/g, as compared to about 500 ng/g), but hGH concentration in
plasma of animals in which only the liver was transformed was
nonetheless comparable to hGH plasma levels in animals having only
the pancreas transformed (in the range of 0.15 ng/ml; FIG. 24).
These results are consistent with the observation that, in contrast
to the exocrine cells of the pancreas and salivary glands,
hepatocytes secrete most of what they produce soon after synthesis.
Thus the liver can provide for substantially constitutive delivery
of a polypeptide into the bloodstream.
[0183] When pancreas and liver were both transfected, plasma levels
were higher than seen when the glands were treated individually
(nearly 0.3 ng/ml)--a value approximately equal to the sum of that
observed for the two organs separately. Surprisingly,
transformation of both liver and pancreas resulted in tissue levels
in the pancreas being significantly increased relative to tissue
levels in the pancreas when the pancreas was transformed alone
(FIG. 25)
Example 20
In vivo Gene Transfer of DNA Encoding Human Growth Hormone by
Retrograde Administration of DNA into the Salivary Gland
[0184] A DNA expression construct encoding human growth hormone
(hGH) is prepared by operably linking a CMV promoter to
hGH-encoding DNA. The expression cassette is then inserted into a
construct such as the bacterial plasmid pBR322. Escherichia coli is
then transformed with the plasmid using conventional transformation
procedures. E. coli containing the plasmid are selected by virtue
of the tetracycline or ampicillin resistance encoded by pBR322, and
the transformed bacterial cells propagated in culture. Plasmid DNA
is then isolated from the transformed bacterial cell culture and
the DNA purified by cesium gradient.
[0185] Approximately 250 .mu.g of the purified plasmid DNA
containing hGH DNA is introduced into the salivary gland of a human
patient by retrograde ductal administration via a salivary gland
duct. Expression and intravenous secretion of the protein is
assessed using the method described above.
Example 21
In vivo Gene Transfer of DNA Encoding Human Growth Hormone by
Retrograde Ductal Administration of Naked DNA into the Pancreas
[0186] A construct containing hGH-encoding DNA (Marshall et al.,
Biotechnology 24:293-298, 1992) operably linked to the CMV promoter
is resuspended in 0.9% saline and a volume of the DNA solution is
administered to a human patient. Approximately 1 mg of DNA is
delivered to the pancreas of the patient by cannulation of the
pancreatic duct by duodenal intubation using endoscopic retrograde
cholangio-pancreatography- . Expression and secretion of human
growth hormone into the bloodstream is assessed by detection of the
protein in the patient's blood.
Example 22
In vivo Gene Transfer of DNA Encoding Human Insulin by Cannulation
of Naked DNA into the Liver
[0187] A construct containing human insulin-encoding DNA operably
linked to the CMV promoter is resuspended in 0.9% saline and a
volume of the DNA solution is administered to a human patient.
Approximately 1 mg of DNA is delivered to the patient's liver by
cannulation of the hepatic duct. Expression and secretion of human
growth hormone into the bloodstream is assessed by detection of the
protein in the patient's blood.
[0188] Following procedures similar to those described above, other
proteins and gene products can be expressed from DNA inserted in a
secretory gland cell according to the invention.
[0189] The invention now being fully described, it will be apparent
to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the appended claims.
* * * * *