CA2304803A1

CA2304803A1 - Protein delivery by secretory gland expression

Info

Publication number: CA2304803A1
Application number: CA002304803A
Authority: CA
Inventors: Stephen S. Rothman; Michael S. German; Ira D. Goldfine
Original assignee: Individual
Current assignee: University of California San Diego UCSD
Priority date: 1997-10-02
Filing date: 1998-09-24
Publication date: 1999-04-15
Also published as: AU744279B2; EP1030557A4; JP2001518316A; EP1030557A1; AU9668998A; WO1999017610A1

Abstract

Secretory gland cells, particularly pancreatic, hepatic, and salivary gland cells, are genetically altered to operatively incorporate a gene which expresses a protein which has a desired therapeutic effect on a mammalian subject. The expressed protein is secreted directly into the bloodstream to obtain therapeutic levels of the protein thereby treating the patient in need of the protein. The transformed secretory gland cells provide long term or short term therapies for diseases associated with a deficiency in a particular protein or which are amenable to treatment by overexpression of a protein.

Description

This invention relates generally to the field of genetic transformation of cells in vivo, more particularly to in vivn transformation of secretory gland cells by introduction of the transforming nucleic acid into a secreton~ gland via a secretorc~ gland duct.
The ability to replace defective or absent genes has attracted wide attention as a method to treat a variey of human diseases {Cwstal 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 circulaton~ 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; Bucket 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).
Currently. DNA-based therapy (i.e., gene therapy) is carried out in a variety of ways but involves hvo general protocols. In the first method, referred to as ex vivo gene therapy, cells are eWracted 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 viva 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 sit:~ with erogenous genetic material.
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 fitsogenic 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 deliven~ 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 ofthe different techniques useful in transformation of cells ex viva or in viva and provides citations of numerous publications in each area (horsy et al., JAMA 270:2338-2345 (1993)).
One method of particular interest for deiiven~ of genetic material involves use of recombinant viruses to infect cells in viva or ex viva. 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 delivew 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.
There is a need in the field for a method for deliyey of genetic material into a cell in viva to provide for expression of the introduced polynucieotide and secretion of the gene product it encodes into the bloodstream. The present invention addresses this problem.
l5 ~~umman~ of the Invention Secreton~ gland cells are genetically altered to operatively incorporate a gene which is expressed by the genetically altered secretary gland cell to produce a polypeptide which is subsequently secreted into the bloodstream. Specifically. the invention involves introduction of a nucleotide of interest into a secretow gland via the duct system (e.g., by retrograde ductal administration) to transform a secreton~ gland cell.
In one embodiment the invention features genetic alteration of cells of nvo secretoy glands (e.~.. the liver and the pancreas).
In another embodiment. the invention features transformation of pancreatic cells with insulin-encoding nucleic acid to provide for expression and secretion of insulin at levels sufficient to maintain a substantially euglycemic state in a subject having a diabetic syndrome.
A primaw object is to provide a non-invasive method of protein delivew (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 secretary gland. preferably the pancreas. salivary gland. or liver of a mammal are genetically modified to e~cpress a biologically active and therapeutically useful polypeptide, which polypeptide is secreted into the circulatory system of the individual.
Another object is to produce genetically transformed secreton~ 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 circulatow system.

An advantage of the present invention is that both long and short term therapy can be provided for diseases wherein individuals are suffering from the disease due to a deficiency in a particular protein. or by supplying an exogenous protein having a desired activit<~ (e.g, antimicrobial activity).
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 methodology and compositions as more fully set forth below.
Fig. 1 is a map of the pFGH construct, which contains the human growrth hormone (hGH) genomic sequence.
Fig. 2 is a map of the pFGH.CMV construct, which contains the hGH genomic sequence operably linked to the CMV promoter.
Fig. 3 is a map of the pFGH.chymo construct. which contains the hGH genomic sequence operably linked to the chymotrypsin B promoter.
Fig. ~l is a graph showing the levels of tissue expression of hGH expression in the pancreas of rats after retrograde injection with either a control containing no DNA or a test sample containing a hGH construct.
Fig. ~ is a graph showing the serum levels of hGH in rats after retrograde pancreatic injection with either a control containing no DNA or a test sample containing a human growth hormone construct.
Fig. 6 is a graph showing the correlation bet<veen pancreatic tissue expression and serum levels of hGH.
Fig. 7 is a graph showing tissue expression of hGH following transformation of salivary gland cells by intraductal injection.
Fig. 8 is a graph showing regulation of plasma levels of hGH expressed from transformed salivary gland cells.
Fig. 9 is a graph showing regulation of plasma levels of hGH expressed from transformed pancreatic cells.
Fig. 10 is a map of the pBATI6.hInsG1.M2 construct, which contains DNA
encoding an altered form of human insulin.
Fig. 11 is a graph showing the glucose response in streptomtocin-treated rats having pancreatic cells transformed with either human insulin (open bars) or green fluorescent protein (GFP:
striped bars).

Fig. 12. is a graph showing the blood glucose levels in control rats (mock-treated; closed squares), streptowtocin-treated rats (open squares), and streptozotocin rats treated by transformation of pancreatic cells with DNA encoding human insulin {closed circles).
Fig. 13 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 injection.
Fig. 14 is a graph showing the relative amounts of hGH in the pancreatic tissue of rats that received either pFGH (control), pFGH.ch~~mo, pFGH.RSV, pFGH.RSV, or pFGH.CMV
by intraductal administration to the pancreas.
Fig. 15 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.
Fig. 16 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.
Fig. 17 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..
Fig. 18 is a graph showing the relative levels of plasma fiGH 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 deliven~ to both the liver and pancreas.
Fig. 19 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.
Fig. 20 is graph showing the relative levels of hGH expression in salivay gland tissue in rats that received either no DNA (control rats), pFGH.CMV, pFGH.CMV premixed with lipofectin, or pFGH.CMV premixed with adenovirus.
Fig. 21 is a graph showing stimulation of hGH secretion into the plasma of rats that received hGH-encoding DNA by intraductal injection into both the pancreas and liver.
Fig. 22 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 intraducta!
injection into the pancreas (closed squares) over a three day period.

Fig. 23 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 injection into the pancreas (closed squares)over a three day period.
Fig. 24 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 intraductai injection into the pancreas (closed squares).
j~escription of the Preferred Embodiments Before the present method of genetically transforming secretory gland cells and methods for protein delivery 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 mat'. of course, vary. It is also to be understood that the terminology used herein is for the propose of describing particular embodiments only. and is not intended to limit the scope of the present invention which will be limited onlv by the appended claims.
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.
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.
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. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such a disclosure by virtue of prior invention.
Definitions 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.
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.
By "salivary gland" is meant a gland of the oral cavity which secretes saliva, including the glandulae sellyarixe majores of the oral cavity (the parotid, sublingual. and submandibular glands) and the glandulae sellyarixe minores of the tongue, lips, cheeks. and palate (labial. buccal, molar, palatine. lingual, and anterior lingual glands).
!0 By "pancreas" is meant a large, elongated, racemose gland situated transversely behind the stomach. bet<veen the spleen and the duodenum. The pancreas is composed of an endocrine portion (the pars endocrine) and an exocrine portion (the pars exocrine). The pans endocrine. which contains the islets of Langerhans, produces and secretes proteins. including insulin.
directly into the bloodstream. 'The pars exocrine contains sceretory units and produces and secretes a pancreatic IS juice. which contains enzymes essential to protein digestion. into the duodenum.
Bv "retrograde ductal injection" 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 injection" can be a single. discontinuous administration or continuous administration (i.e., 20 perfusion).
By "transformation" is meant a genetic change induced in a cell following incorporation of new DNA (i.e.. DNA exogenous to the cell). Where the cell is a mammalian cell.
the genetic change may be achieved by introduction of the DNA into the genome of the cell.
Bv "transfection" is meant the transformation of a cell with DNA from a virus.
25 By "transformed cell" is meant a cell into which (or. where the introduced DNA is incorporated into the genome. into an ancestor of which) has been introduced.
b~~ means of recombinant DNA techniques, a DNA molecule encoding a protein of interest.
Bv "nucleic acid of interest'' is meant any DNA or RNA molecule which encodes a polvpeptide or other molecule which is desirable for administration to a mammalian subject for 30 expression of the product encoded by the nucleic acid of interest and delivery of the encoded product into the blood stream 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 as shorthand herein to refer to the nucleic acid of interest.
By "construct" is meant a nucleic acid molecule which contains the nucleic acid of interest 35 (e.g., the DNA of interest), generally operably linked to a promoter for expression of the polypeptide WO 99/17610 PCT/US98l20199 encoded by the nucleic acid of interest. "Constructs" as used herein is generally meant to refer a nucleic acid molecule that facilitates expression of a polypeptide encoded by the nucleic acid to be introduced into a secretory gland cell.
By ''vector is meant any compound. biological or chemical, that facilitates transformation of a secretory gland cell with a DNA of interest. Exemplary biological vectors include vinrses, particularly attenuated and/or replication-deficient viruses. Exemplary chemical vectors include lipid complexes and naked DNA constructs.
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 cyoplasm 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.
IS By "promoter" is meant a minimal DNA sequence sufficient to direct transcription.
"Promoter" is also meant to encompass those promoter 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 ~' or 3' regions of the native gene.
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 h~pe of secretory gland cell.
For example, by "salivary gland specific promoter" is meant a secretow 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. E.vamples of pancreas specific promoters include a pancreatic amylase promoter and an insulin promoter.
By "operably linked" is meant that a DNA sequence and a regulatory sequences) 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).
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 poiypeptide encoded by a DNA
of interest).
By "mammalian subject" or "mammalian patient" is meant any mammal to which intravenous protein delivery is desired, including human, bovine, equine, canine, and feline subjects.
_7_ By ''euglycemia" or ''euglycemic state" is meant a state associated with a level of blood glucose that is normal or nearly normal, particuiarly 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.
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 glands) with multiple constructs encoding the same or different polypeptides).
Overview of the Invention The present invention features methods for genetically altering a secretow gland cell (i.e., secreton~ gland cell transformation) and methods of delivering a protein using the methods of genetically altering secretors 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 secretorv gland of a mammalian patient (i.e., by in vivo gene therapy). 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 secretow gland into which the DNA of interest is introduced and expressed is the pancreas. a salivaw gland. or the liver. 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 blood stream by secretion of the protein by the transformed secretory gland cell.
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/ther;peutic levels in the bloodstream.
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 satls into the intestinal lumen), it _g_ is not widely appreciated that exocrine cells can also secrete significant amounts of protein into the systemic circulation. For example, exocrine proteins such as a-amylase (salivan~ glands), pepsinogen (gastric glands). various digestive enzymes firm the exocrine pancreas. salivary gland kallikreins and nerve growth factor (Liebow, 1988 Pancreas 3:343-3~ 1 ) 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. 1997 Proc.
Natl. Acad. Sci (USA) 74:4068-4072; Papp et x1.1980 Acta Physiol. Acad. Sci. Hung. X6:401-X10:
Geokas et al.. 1980 Am. J. PhysioI. 238:238-246: Miyasaka et al. 1981 Am. J. Physiol. 241:170-17~:
Grendell et al.
1982 Am. J. Physiol. 243:~~1-~9). Endocrine secretion can be greatly enhanced by common secretory stimulants (Saito et al., supra; Isenman et al. s:rpra; 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. s:rpra). The present invention takes advantage of the discovety.~ that exocrine gland cells can be transformed with a desired DNA
sequence and secrete the encoded polvpeptide into the bloodstream rather than only or primarily into the gastrointestinal tract.
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 salivaw 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 deliven~ of the DNA of interest in a safe manner that substantially avoids the inflammatow and immunoiogical responses associated with other means of DNA
deliver.
The invention also takes advantage of the protein-producing capacity of secreton~ 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 secreton~ glands provides enough secreted product to provide therapeutic protein levels for the treatment of most diseases of circulating proteins.
The invention will now be described in further detail.
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 DIv'A 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. .
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. ~ol~cular Cloning: a Laboratory Manual, 2nd Ed., 1989. Cold Spring Harbor Laboratory Press, Cold Spring Harbor; NY; each of which are hereb~~ incorporated by reference with respect to methods and compositions for eukan~otic expression of a DNA of interest).
Preferably, the DNA construct contains a promoter to facilitate expression of the DNA of interest within a secreton- gland cell. Preferable the promoter is a strong.
eukaryotic promoter such I5 as a promoter from cy~tomegalovirus (CMV), mouse mamman~ tumor virus (MhITV), 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.
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 is preferably a pancreas specific promoter. e.g., an insulin promoter or a pancreas a-amylase promoter: where the secretory gland is a saiivan- gland, the tissue-specific promoter may be a salivary a-amylase promoter or mumps viral gene promoter. Both pancreatic and salivary a-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. Biul., 182:359-365, 1985: Hagenbuchle et al., J. Mol.
Biol., 185:285-293, 1985;
Schibler et al., Oxf. Si~rv. Eukaryor. Genes, 3:210-234, 1986; and Sierra et al., Mol. Cell. Biol. , 6:4067-4076. 1986 for marine pancreatic and salivan~ a-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 a-am~~lase genes and their promoters: Ting et al., Genes Deu 6:1457-65; 1992 for human salivary a-amylase AMY 1 C
promoter sequences).
The constructs of the invention may also include sequences in addition to promoters which enhance secretory gland specific expression. For etample, where pancreas specific expression of the DNA of interest is desired, the construct may include a PT1~-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:
x91-201, 1992).
Other components such as a marker (e.g., an antibiotic resistance gene (such as an ampicillin resistance gene) or ~3-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.
For eukatyotic expression, the construct should contain at a minimum a eukanotic promoter operably linked to a DNA of interest, which is in turn operably linked to a potadenylation sequence. The polyadenylation signal sequence may be selected from any of a variety of polvadenylation signal sequences known in the art. Preferably. the poh~adenylation 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 p-globin intron. which is inserted in the construct at a position ~' to the DNA of interest).
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 p-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 oftransformed cells expressing the protein (e.g.;
b~~ enzyme-linked immunosorbent assay (ELISA) using an antibody which binds to the fusion protein).
The DNA of interest can be any DNA encoding any protein for which intravenous therapy is desirable. For example, intravenous protein therapy is appropriate in 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 restore the normal bloodstream levels of the protein to at least normal levels.
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 treatment of a condition by expression or over-expression of the polypeptide. For example, the DNA of interest can encode antimicrobial, antiparasitic, antifimgal, or antiviral polypeptides for treatment of a mammalian subject having a viral (e.g., human WO 99/17610 PCT/US98l10199 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). 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.
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.
Exemplary, preferred DNAs of interest include DNA encoding insulin. growth factors (e.g., growth hormone. insulin-like growth factor-I (IGF-I), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), acidic fibroblast growth factor, basic fibroblast growth factor, or transforming growth factor (i), cyokines (e.g., interferon {INF) (e.g., INF-a2b, INF-a2a, INF-aNl, INF-~i Ib. INF-y). interleukin (e.g, IL-2, IL-8), or tumor necrosis factor (TNF) (e.g, TNF-a. TNF-~3)), clotting factors (e.g., clotting factor VIII), hormones (e.g, GP-1), antimicrobial poh~peptides (e.g., antibacterial. antifungah antiviral, and/or antiparasitic polypeptides), enzymes (e.g.. adenosine deaminase), filgastim (Neupogen). hemoglobin. ervthropoietin, 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.
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.

WO 99/17610 PC'T/US98/20199 SPECIFIC E7~EMPLARY PROTEINS

insulin interferon-a2B

human growth hormone (hGH) transforming growth factor (TGF) erythropoietin (EPO) ciliarv neurite transforming factor (CN'TF) clotting factor VIII insulin-like growth factor-1 (IGF-1) bovine growth hormone (BGH) granulocyte macrophage colony stimulating factor (GM-CSF) platelet derived growth factorinterferon-a2A
(PDGF) clotting factor VIII brain-derived neurite factor (BDNF) thrombopoietin fIPO) insulinwpin 1L-1 tissue plasminogen activator (tPA) IL-2 urokinase _ IL-1 RA streptokinase superoxide dismutase (SOD) adenosine deamidase catalase calcitonin fibmblast growth factor (FGF)arginase (acidic or basic) neurite growth factor (NGF) phenvlalanine ammonia 1~-ase granulocvte colony stimulatingy-interferon factor (G-CSF) L-asparaginase pepsin 2U uricase trvpsin chvmo sin elastase cafioxvpeptidase lactase sucrase intrinsic factor calcitonin parathyroid hormone(PTH)-like hormone Ob gene product cholecvstokinin (CCK) luca on insulinotro hic honmone E.\EV1PLARY CLASSES OF PROTEINS

enzymes (e.g., proteases. pituitary hormones phospholipases. etc.) .

protease inhibitors growth factors cvtokines somatomedians chemokines immunoglobulins gonadotrophins interleukins chemotactins interferons lipid-binding proteins 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 etytttropoietin are available from Genbank and/or have been described in the scientific literature (e.g., human clotting factor VIII gene: Gitschier et al., Nature 311:326-330, 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, NJ: 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.
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:
USPN 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 (USPNs 4.683.19 and 4,683.202). Nucleotide substitutions. deletions. additions. and the like can also be incorporated into the polynuclcotides. 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.
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-h~pe protein. For example, ~yhere the therapeutic protein is a hormone. it may be desirable to alter the protein's abiliy 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-tc~pe, dimerized insulin.
Y~ctors for Deliven~ of the DNA of Interest to the Secreton~ Gla_r di Cell The vectors for deliven~ 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 othervise improve the action of the active compound. The precise vector and vector formulation used will depend upon several factors such as the secreton~ gland targeted for gene transfer.
Non-viral vectors 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.
In one preferred embodiment, the formulation comprises viral particles which are mixed with the naked DNA construct prior to administration. Preferably, about 108 to 10'° viral particles (preferably about 1 x 10'° to ~ x 10'°, more preferably about 3 x 10'° particles) are mixed with the naked DNA construct (about ~ pg to ~0 pg DNA. more preferably about 8 pg to 25 ug DNA) in a total volume of about 100 Nl. Preferably the viral particles are adenovirus particles (Curiel et al., 1992 supra).
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 delivew 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-~3-0l 3-urethany!)-N'.N'-dimethylethylene diamine).
For example. the naked DNA can be administered in a solution containing LipofectinT"
(LTI/BRL) at a concentrations ranging from about 2.~ % to 1~% volume:volume, preferably about 6% to 12% volume:volume. Preferred methods and compositions for formulation of DNA for delivery according to the method of the invention are described in USPN

5.27.928. incorporated herein by reference.
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 cannier 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.
Vii< vectors 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.
'Numerous viral vectors are well known in the art, including, for example, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus (HSV), cvtomegalovirus (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 is a 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 cells).
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 252:431-431. 1991 and Rosenfeld et al., Cell 68:143-155. 1992 (adenovirus):
USPN 5,139.941 (adeno-associated virus); USPN 4.861,719 (retrovirus); and USPN 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., Yirol. 187:801-804.
1992; Kawano et al., Yirol., 179:857-861, 1990; Elango et al., J. Gen. Yirol. 69:2893-28900, 1988.
Con iti~ns or Disease_c Amenable to Trea_trraent Using the Method of the Invention 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.

TABLE 2: Frem~l~a~,~~'Zsease Conyi~S Amen_a_ble to Treatment Usine Lhe Ln_vention Enzvme Deficiency Endotoxic ShocWSeOsis Adenosine deaminase~ Lipid-binding protein (LBP) Purine nucleotide phosphoryiase Galactosidase (i-glucuronidase ~3gQxidants for Cancer Theranv0pgmj~

Superoxide dismutase Erythropoietin Catalase Growth Factors (for use in wound healieg.

a-Interferon induction of red blood cell formation. etc.) y-Interferon Epidermal growth factor a-IL I G-CSF

Phemlalanine ammonia lyase y-Interferon Arginase Transforming growth factor L-asparaginase Ervthropoietin Uricase Thrombopoietin Granulocye colony stimulatingInsulin-like grownh factor-1 factor (G-CSF) Monoclonal antibodies Insulin Tissue necrosis factor Human growth hormone Cardiovascular Disecase Diabetes Tissue plasminogen activator Insulin Urokinase (native or chimeric)Glucagon a~-antitnpsin Insulinotrophic hormone 2~ Antithmmbin-III

Other proteases or protease Clotting, disorders inhibitors Clotting factor VIII
Apolipoproteins (particularly B-~8) Circulating Scavenger Receptor APO Al=

QJ esitv and Feeding Gastrointestinal and P~,ncreatic Ob gene product Deficiencies Cholecystokinin (CCK) Pepsin (for esophageal reflux) Tnpsin Chymotwpsin Bone diseases Elastase Calcitonin Carbowpeptidase PTH-like hormone Lactase (for lactose deficiency) Sucrase: Intrinsic Factor (pernicious anemia) -1 ~-TABLE 2 (cont.) Organ- ~ecific 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 teiangiectasia (Insulin receptor) Allergic rhinitis, asthma (Beta=-adrenergic receptors) Juvenile insulin-dependent diabetes (insulin. GAD65) Pernicious anemia (gastric parietal cells, vitamin B,= binding site of intrinsic factor) Addison's disease (adrenal cells) Idiopathic hyoparathyroidism (parathyroid cells) Spontaneous infertility (sperm) Premature ovarian failure (interstitial cells. corpus luteum cells) I Pemphigus (intercellular substance of skin and mucosa) ~

Bullous pemphigoid (basement membrane zone of skin and mucosa) Primary biliary cirrhosis (mitochondria) Autoimmune hemolytic anemia (enthrocyes) Idiopathic thrombocytopenic pmpura (platelet) Idiopathic neutropenia (neutrophils) Vitiligo (melanocytes) Osteosclerosis and Meniere's disease (type II collagen) Chronic active hepatitis (nuclei of hepatocytes) Systemls ~,g,~pmimune Diseases (defecUorgan affected in parentheses) Goodpasture's syndrome (basement membranes) Rheumatoid arthritis (y-globulin. EBV-related antigens. collagen types II and III) SjBgren's syndrome (y-globulin. SS-A (Ro), SS-B (La)) Systemic lupus enthematosus (nuclei. double-stranded DNA, single-stranded DNA. Sm ribonucleoprotein, lymphocytes. ewthrocyes, neurons. Y-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) For treatment of severe combined immunodeficience = Concerts love-density lipoproteins to high-density lipoproteins Tran~~f~r~atiQn of Secretor~~ GI~~~Cells 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 ductal injection. which may be accomplished by perfusion (i.e., continuous injection), or by a single, discontinuous injection). Retrograde ductal injection may be accomplished in the pancreas and liver by endoscopic retrograde chalangio-pancreatography (ECRP). 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 obser~~ed as a result of the administration of transforming formulations and their adjuvants into blood and interstitial fluid may be avoided.
Moreover, the cells of secreton~ glands form 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.
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.
The precise amount of DNA administered will van' 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 pg to 200 mg, preferably from about 100 pg to 100 mg. more preferably from about 500 lrg 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 ~g to 100 mg, preferably about 100 pg to 10 mg, more preferably from about 2~0 pg to 5 mg, still more preferably from about 500 lrg to 1.5 mg, most preferably about 1 mg. The amount of DNA introduced into the salivan~ gland of a human is, for example, generally from about 2.5 pg to 30 mg, more preferably from about 25 pg to 3 mg, still more preferably from about 100 pg to 1 mg, most preferably about 2~0 pg. The amount of DNA
introduced into the liver of a human is, for examples. generally from about 10 pg to X00 mg, more preferably from about 100 ug to 300 mg, still more preferably from about 1~0 pg 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.
In a preferred embodiment of the invention, at least t<vo secretory glands are transformed according to the methods of the invention. Any two secretory glands can be transformed concurrently. For example, the DNA of interest can be administered to both the pancreas and the WO 99/17610 PCT/US98l20199 liver, or to both the salivary gland and the pancreas, or to both the salivary gland and the liver, or to all three. Preferably, cells of the pancreas and the liver are concurrently transformed.
Concurrent transformation of the secretory glands can be carried out several hours to several days apart or, preferably, simultaneously (i.e., DNA is introduced into the two secretory glands during the same procedure. For example, where the liver and pancreas are to be concurrently transformed, the DNA formulation can be introduced simultaneously via a common duct, or separately (e.g., first via the pancreatic duct with occlusion of the hepatic duct, then vice versa).
Concurrent transformation of at least two or more secretory glands can advantageously provide higher levels of expression of the polypeptide of interest in a secretory gland tissue and/or in the bloodstream and can, unexpectedly. provide for synergy between the organs (e.g., to provide for higher levels of tissue expression in a secretory gland than when the secretory gland is transformed alone). For example, concurrent transformation of the pancreas and the liver results in increased levels of tissue expression in the pancreas relative to tissue levels in pancreas when it is transformed alone.
Moreover. the liver releases the polypeptide of interest in a continuous fashion that is not regulated by hormonal stimulation. The pancreas provides a relatively lower level of constitutive secretion and stores most of the polypeptide of interest and only releases large amounts after stimulation (e.g., after the individual eats). Therefore. transformation of both liver and pancreas has the advantage of providing both constitutive secretion primarily form the liver, and hormonally-regulated secretion from the pancreas.
Intravenoes protein theraw by tr3nsforrnatjyn of sa )yes gland. pancreatic, and liver cells Secreton~ 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.
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 protein that is released into the bloodstream can be as much as 25% of the amount of duct-directed secretion of that specifrc protein.
This amounts to as much as 1-2 mg of protein/gram of tissue being directed into the blood per hour.
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 WO 99/17b10 PCT/US98/20199 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).
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 o~ for example. salivaw to intravenous expression.
Alternatively, intravenous-directed secretion signals can be identified b~-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 s~-stem 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.
Overall secretion from secretory glands 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. secretor~~ gland specific hormone. For example, secretory gland secretion can be enhanced by administration of a cholinergic agonist such as acetyl-p-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.
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 finm the DNA of interest. while translational regulation involves the production of 3~ 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).
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 honmone(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 salivan~ glands is stimulated by hormones and neurotransmitters that are natural components of the feeding response: thus feeding itself can act as a dosing mechanism.
The actual number of transformed secretow 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 l5 partitioning of the therapeutic protein behveen 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.
A~pdication of the Method of the Invention to Achieve Euglycemia in a Diabetic Symdrome 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). 'Ibis aspect of the invention may be applied to regulate levels of other proteins in the bloodstream.

A~,,g~,~,~ent of Protein Thera~nv 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 poIypeptide in a sample. are described in A~libodies: A Laboratory Manual (1988, Harlow and Lane. eds. Cold Spring Harbor Laboraton~. Cold Spring Harbor. NY).
Alternatively. or in addition. the et~cacy 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 1 ~ polypeptide is en~thropoietin, the subject's blood is examined for iron content or other parameters associated with anemia.
The follon-ing examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of ho«~ to cam' 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 othern~ise. parts are pans b~~ weight. molecular weight is weight average molecular weight. temperature is in degrees Centigrade. and pressure is at or near atmospheric.
)~~ l~n vivo gene transfer of DNA encoding human growth hormone by retrograde infection Four constructs for expression of human growth hormone (hGH) were prepared using techniques well known in the art (see, for example, Sambrook et al. ibic~. The first construct, pFGH, contains the genomic hGH DNA sequence inserted in the commercially available vector pBLUESCRIPT SK+T'~' (Stratagene, LaJolla California) (Fig. 1). 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 constnrcted by operably inserting the promoter from the immediate early gene of human CMV upstream of the genomic hGH sequence of the pFGH
vector (Fig. 2).
The third construct, pFGH.chymo, was constructed by operably inserting the rat chymotrvpsin B
gene promoter upstream of the genomic hGH sequence of the pFGH vector (Fig.
3). 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.
Each of the four vectors was used to transfect the pancreas of appm~cimately 300 g adult male, Sprague-Dawley 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 pl of phosphate-buffered saline (PBS) containing one of the four vectors. or 100 pl of PBS alone as a negative control. were slowly injected or infused into the pancreatic duct in a retrograde direction.
IS The vector-containing solutions were composed of 8 pg DNA per 100 pl in PBS. either with or without 6% lipofectin. a cationic lipid used to increase transfection efficiency. The solution was left in place for ~ min before secretors 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 surgen~ without obvious side effects. This transfection method provides direct access of the vector to over 90% of the pancreatic gland cells.
At 48 hr after surgen~, a blood sample was obtained to measure senrm 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.
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 SCT'~. Homogenization was completed b~~
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 ° 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.
Rats injected with the pFGH.CMV vector e~cpressed higher levels of hGH in the pancreatic tissue (Fig. d), compared to background levels of pancreatic hGH e~cpression in rats injected with either no DNA (PBS alone) or the pFGH vector (hGH DNA with no promoter). The addition of lipofectin modestly increased hGH expression in rats injected with the pFGH.CMV construct. In addition, rats transfected with the pFGH.CMV vector secreted hGH in the senior at levels increased relative to background levels and to hGH secretion levels in rats injected with either control samples (no -DNA or pFGH) or vm~ith samples containing hGH DNA linked to either the chymotrvpsin B or RSV promoters (Fig. 5). In Fig. 6, all data from the above experiments (including all controls and vectors) are analyzed by plotting the hGH serum levels against the hGH tissue levels. 'Ibis graph shows that higher tissue levels result in higher levels of secretion into the blood. Thus, retrograde pancreatic injection of the pFGH.CMV vector successfully transfected pancreatic cells to provide both hGH pancreatic tissue expression and hGH secretion into the bloodstream.
F~pJe 2~ In Vivo Transformation of Pancreatic Cells by Retrograde Ductal Infection ofhGH-Fncoding DNA a_~d Regulation of hGH Secretion 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 laparotomv and identift~ing 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 (Ads-di 342) supernatant in 100 wl of phosphate-buffered saline (PBS) containing 8 pg ofthe hGH-encoding plasmid pFGH.CMV (Fig. 2) was slowly infused into the pancreatic duct in a retrograde direction. The solution was left in place for approximatey ~ 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-operativel. The abdomen was then closed with sutures. The animals recovered fully and rapidly from the surgey without ob~~ious side effects.
At 48 hr after surgen~. a blood sample was obtained to measure scrum 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 l~ min intervals following MeH injection. Serum was separated from the blood of all samples after clotting, and kept at -20°C prior to assay.
As show in Fig. 9 (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).

Examnle 3: In Yivo Transforma~xion of Salivary Glands by Re a Du ~ j~jection of DNA
Encoding Human Growth Hormone Twelve adult rats weighing approximately 300 g each were anesthetized with an intraperitoneal injection of sodium pentobarbital. A total volume of ~0 pl containing 4 pg of the .
pFGH.CMV plasmid, which contains cDNA encoding human growth hormone (hGH) (Fig. 2). was introduced into each submandibular gland of 8 rats by retrograde ductal injection via the ducts leading from the oral mucosa to the salivar~~ gland. Briefly, both the left and right Wharton's duct were cannulated intraorally with polyethylene (PE) 10 tubing, and the DNA
injected into the duct system of each gland in a retrograde fashion (4 .ug/~0 pl of PBS). The material was kept in place for t<vo minutes before normal flow was reestablished.
For three of these animals the DNA was mixed in a 6% solution of the cationic lipid Lipofectin (labeled "liposomcs") from Life Technologies (Gaithersburg. MD).
For four of these animals. the DNA was mixed with a I :~0 dilution of replication-defective human adenovirus (Ad5-l~ di 342) supernatant. Control rats (4 rats) received ~0 pl 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.
Approximately 48 hours after cDNA injection; 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 SCT''i. 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 l~ min. and the supernatant collected and stored at -80°C
until analysis. The levels of hGH in the protein samples were measured using the hGH radioimmune assay (Nichols Institute).
Each assaw was performed in duplicate and compared to a set of control samples.
Each of the submandibular glands of the rats injected with the pFGH.CMV vector expressed hGH in the salivarv_ gland tissue: hGH expression was undetectable in the control rats' salivaw glands (Fig. 7).
Examvle 4: In Yivo Transformation of Salivan~ Glands by Retrograde Ductal Infection of hGH-Encoding; DNA and Regdlation of hGH Secretion 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 draw from the femoral vein of each animal. A total of 4 pg of the hGH-encoding plasmid pFGH.CMV
(Fig. 2) in ~0 pl, was introduced into each submandibular gland of each rat by retrograde ductal WO 99/17610 PGT/US98/201.99 a injection via the ducts leading from the oral mucosa to the salivary gland as described above in Example 2. No significant leakage of material or bleeding occurred. After 3 hours. the animals were awake, drinking water. and appearing normal.
Forty-eight hours after cDNA injection. 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-p-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 ~0 min after McH injection. Serum was separated from the blood of all samples after clotting. and kept at -20°C prior to assay.
As show in Fig. 8 (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 salivan~ 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 l5 hGH bloodstream levels in such a short period.
Example ~: Treatment of Diabetes Mellitus Over a Three Day Period b~~n Lwo Transformation of Pancreatic Cells b~~ Retrograde Duct at I IO,jection with Insulin-Encoding DNA
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.~) (Sigma) at 6~ 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 injection in the pancreatic duct in a 100 pl injection volume containing 8 pg DNA plus adenovirus (Ads-c!i 342)(3 ~
10'° viral particles) as described above. Test animals (4 rats) received the human insulin-encoding construct pBatl6.hlnsGl.M2. The pBATI6.hInsGl.M2 construct (Fig. 10) 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 (3-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-injection. Blood glucose was measured by the glucose oxidase method (Lifescan, Milpitas, CA).

As show in Fig. 11, 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).
ple 6' Treatment of Diabetes Mel]jtus Over a Siv D~,v Period by In Yivo Transformation of Pancreatic Cells by R rograde Ductal Injection wit_h_ Inylin-Encoding DNA
Streptozotocin vcas 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 negati~~e control. Insulin-encoding DNA in the pBATI6.hInsGI.M2 construct (Fig. 10) tvas administered to 8 of the streptozotocin-injected rats by retrograde ductal injection as described above. Sit streptozotocin-treated rats received either 100 lrl of saline without DNA (2 animals) or a control DNA without the human insulin gene (4 animals) by pancreatic retrograde ductal injection 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 inten~als for 6 days. Human insulin was measured using a double antibody radioimmunoassay (Linco Laboratories. Saint Louis. MO).
As shown in (Fig. 12), 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. 12. elevated insulin levels for such an extended period additionally demonstrate prolonged expression from the DNA introduced into the pancreatic cells.

Example 7: In Vivo Trangformation of Pancreatic Cells by Retroerade Ductal Infection of Green Fluorescent Protein-Enyodi~ DNA and Expression in Pancreatic Cells To identiy 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 p-globin to create. the expression vector pFOX.EGFP.N2.CMV.
After fasting overnight. Male Sprague-Dawiev rats (260-280 g) were anesthetized and the body caviy opened to expose the gasuointestinal tract. The green fluorescent protein (GFP)-encoding construct pFOX.EGFP.N2.CMV was administered to each animal by retrograde injection in the pancreatic duct in a I00 pl injection volume containing 8 pg DNA
premixed with adenovirus (3 x 10"'viral particles) as described above. The animals recovered full and rapidly from the surgen~ without obvious side effects.
Seventy-t<vo hours post-treatment. the animals were sacrificed. and pancreases were removed and weighed (wet weight). Samples of each pancreas were fixed in ~%
buffered fonmalin for 24-48 hours at room temperature. Fined tissues were dehydrated and imbedded in paraffin, and 5 pm sections were processed for immunohistochemistry using standard techniques. Endogenous peroxidase was quenched in 0.7% H,O,/MeOH, and antigen retrieval was performed using Citra solution (Biogenex, San Ramon, CA) according to the manufacturers' instructions. Sections were preincubated for 30 minutes in ~% goat serum/phosphate-buffered saline (PBS).
and then incubated overnight at d°C with primaw antisera diluted in ~% goat senum/PBS.
The primary antiscra were selected from either anti-GFP antisera ( 1:1500:
Clontech. Palo Alto. CA), anti-insulin antisera ( 1:500: Dako, Carpenteria, CA), or non-specific rabbit sera ( (:1500).
The following day all sections were incubated with biotinylated goat anti-rabbit antiserum (5 pg/ml;
Vector. Burlingame, CA) 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 not injected with CMV-GFP. and omission of priman~ antiserum.
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 obsen~ed in 0.1-! .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 inflammator<~ infiltration as a consequence of ductal injection of the vector.
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 juices and the high concentrations of DNase in the pancreatic juice. Moreover, these data. combined with the data above showing that transfom:ration 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 normalh~
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 I~ at physiologically relevant levels sufficient to treat diabetes mellitus in an animal model as evidenced in the examples above.
Example 8: In Yivn Transformation of Liver Cells by Retrograde Ductal Infection of hGH-Encoding DNA and !~3loorj~r~a-Directed hGH Secretion Four rats were anesthetized and control blood samples (no DNA) were collected from the femoral vein of each animal. Transfection of liver cells was accomplished by exposing the duodenum by laparotomy and identift~ing the liver and the associated common bile duct. The common bile duct was cannulated either estraduodenall or through the papilla of Vatcr. The tubing was advanced to the bifurcation of the hepatic duct in order to prevent injected material from entering the distally located pancreatic drainage. A 1:~0 dilution of replication-defective human adenovirus supernatant in 100 pl of phosphate-buffered saline (PBS) containing 8 pg of the hGH-encoding plasmid pFGH.CMV (Fig. 2) or 100 pl 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 ~ 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 surgen~ without obvious side effects.
Plasma hGH levels were measured 2 days after treatment; the results are shown in Fig. 13.
Each data point in Fig. 13 represents the mean t 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 9: In Vivo Transformation of Pancreatic Cells with hGH-Encoding DNA
and Exioression in Rat Exocrine Pancreas and Plasma Following overnight fasting and anesthesia with pentobarbital. the abdominal cavit<~ of the rats was opened and the pancreatic duct cannulated external to the duodenum with PE 10 tubing as described above. Eight to t<venty-five micrograms of each of pFGH (promoter less construct), pFGH.chymo (construct with the chymotn~psin promoter), pFGH.RSV (construct with the RSV
promoter), and pFGH.CMV (construct with the CMV promoter) was injected in a total volume of 100 pl of PBS into the pancreas via the pancreatic duct as described above.
Immediately prior to injection construct samples were optionally premixed with either Lipofectin (6-12% vol:vol) or adenovirus (3 X 10'° viral particles). The material was kept in the duct for ~ min prior to establishing normal flow. The abdomen was the closed and the animals allowed to recover.
1 ~ 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 ~ mM Na2HP0, (pH 7.8) at a tissue to fluid ratio of I :10 using a motorized mortar and pestle. Large particulate material in the homogenate was removed by sedimentation at 10.000 x g for 30 minutes, and the supernatant assayed for the protein of interest. The results are shown in Figs. 14-17. All data shown are the mean t the SEM.
The effects of the various promoters upon tissue expression and secretion of hGH into the bloodstream are shown in Figs. l~4 and 1~. 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 1 SO ng/g tissue wet weight) when compared to either promoter less controls. or plasmids containing RSV and chymotrypsin promoters (Fig. 14). The cationic Lipid adjuvant Lipofectin increased expression by about 50%, and pre-mixing the plasmid with adenovirus enhanced tissue expression five fold (Fig.
I S). Expression of hGH at 24, 48 or 72 hours after injection was similar under all conditions studied.
As shown in Figs. 16 and 17, hGH was secreted into plasma. Plasmids containing the CMV
promoter increased circulating levels of hGH five times above background (Fig.
16). 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. 17). Lipofectin increased plasma levels by an additional 50%. and adenovirus by 75%, when compared to plasmid alone.
Pancreas Transformed with hGH-Enco~jing DNA
Eight micrograms of the pFGH.CMV construct premixed with adenovirus as described above in Example 9. was injected 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 were transformed, 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.
In animals having transformed liver (liver alone) or pancreas (pancreas alone), hGH was expressed in Iiver 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 X00 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 0.1 ~ ng/ml: Fig. 18).
These results are consistent with the observation that. in contrast to the exocrine cells of the pancreas and salivary glands. hepatocvtes secrete most of what they produce soon after synthesis.
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 t<vo 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. 19) Example 11: Human ~rowrt hormone (hGjj,) exyression in rat salivarv$land.
Four micrograms of the pFGH.CMV construct, premixed with either Lipofectin or adenovirus, was injected into each submandibular gland via retrograde ductal injection (via Wharton's duct) as described above. Two days later. each gland was harvested and hGH content was measured As shown in Fig. 20. 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. 20).
~ Examyle 12: Stimulat~n of Human growth hormone (hGH) secretion.
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 l0 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 injected into ducts of both the pancreas and liver of four rats as described above. A blood sample was taken prior to injection as a control. Two days after transfection. a second control blood sample was taken and the rats were 1~ treated with the cholinergic agonist. aceyl-(i-methvlcholine (McH) (0.8 mg/kg body weight).
As shown in Fig. 21. hGH secretion was increased three fold within 30 minutes of stimulation. with plasma levels approaching I .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.
20 Thus secretion of hGH is regulated in a manner similar to secretion of endogenous proteins.
Although the concentration of hGH in plasma was correlated to the level of hormone in the pancreas (r-0.», p<0.01. n=~l l). 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. 20).
but only about a t<vo fold 25 increase in plasma concentration (see. e.g.. Figs. 16 and 17, Example 9 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.
FY~,mple 13: Human Insulin Expression and Secretion in Diabetic Rat Pancrr;as 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; 6~ mg/kg body weight. in 1mM citrate buffer, pH4.5) on day zero one hour prior to administration of the insulin-encoding construct. The e~.perimental animals subsequently received 8 pg of the insulin plasmid (pBATI6.hInsGl.M2) premixed with adenovirus and injected into the pancreatic duct. also on day zero. The pBATI6.hInsGl.M2 construct contains the human insulin cDNA linked to a CMV immediate early promoter. which is positioned upstream of the first intron of human ~i-globin. The human insulin cDNA was mutated to convert the second protease site, bet<veen peptides C and A. to a furin recognition site. This allows for correct proteolWic processing of mature insulin in non-endocrine cells.
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 pBATI6.hInsGl.M2 plasmid (n=3), measured over a three day period, are shown in Fig. 22. Plasma insulin levels in diabetic rats (n=3).
and diabetic rats treated with the pBATl6.hInsGl.M2 plasmid (n = 3). measured over a three day period, are shown in Fig. 23. Plasma glucose levels in individual diabetic (n=3) and pBAT16.h1nsG 1.M2 plasmid- treated diabetic rats (n=3). measured over a six day period. are shown in Fig. 24.
As a consequence of streptozotocin administration. blood glucose levels rose from the normal level of 100 mg/dl to 300-:.100 mg/dl within 2=1 hours and remained elevated for the duration of the study (Fig. 22). Treatment with the human insulin plasmid reduced blood glucose levels in diabetic rats to the normal range (Figs. 22 and 24). and concentrations of insulin remained near pre-treatment values (Fig. 23). Blood glucose levels were euglycemic for the duration of the study (6 days: Fig. 24). 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 14: In vivo Pene transfer of DNA encoding human growth hormone ~v r r grade injection of DNA into the salivaw eland A DNA expression construct encoding human growth hormone (hGH) is prepared b~~
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. cull 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.
Approximately 250 ug of the purified plasmid DNA containing hGH DNA is injected into the saiivay gland of a human patient by retrograde ductal injection via a salivan~ gland duct.
Expression and intravenous secretion of the protein is assessed using the method described above.

~ple I~: In viva gene transfer of DNA encoding human ero«th hormone by retrograde ductal 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 I mg of DNA is delivered to the pancreas of the patient by cannulation of the pancreatic duct by duodenal intubation using endoscopic retrograde choiangio-pancreatography. Expression and secretion of human growth hormone into the bloodstream is assessed by detection of the protein in the patient's blood.
~nnle 16' In vivyg~ne transfer of DNA encodine human insuljn ~v cannuiatinn of naked DNA
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.
1~ Expression and secretion of human groWh hormone into the bloodstream is assessed by detection of the protein in the patient's blood.
7' In viv r a liver of a atn ient A construct containing human insulin-encoding DNA operable linked to the CMV
promoter is resuspended in 0.9% saline. A volume of the DNA solution is administered to a human patient so as to transform both pancreatic and hepatic cells (e.g., by introducing the DNA solution into the common bile duct before it splits into the hepatic and pancreatic ducts).
Approximately 2 mg of DNA is delivered to the patient's liver by cannulation of the hepatic duct: in addition. approximately 2~ 1 mg of DNA is delivered to the pancreas via retrograde injection via the pancreatic duct.
Expression and secretion of human growh hormone into the bloodstream is assessed by detection of the protein in the patient's blood.
Following procedures similar to those described above, other therapeutic proteins can be expressed from DNA inserted in the genome of a secretor<~ gland cell by gene transfer according to the invention.
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.

Claims

What is claimed is:

1. A method of delivering a polypeptide to a mammalian subject, the method comprising:
introducing in viva into at least two secretory glands of a mammal a construct comprising a DNA of interest that encodes a desired polypeptide and a eukaryotic promoting sequence operably linked to the DNA of interest, said introducing being by retrograde ductal injection:
wherein the introduced construct genetically transforms cells of each secretory gland and wherein expression of the DNA by the genetically transformed cells of the secretory glands results in bloodstream-directed secretion of the protein encoded by the DNA of interest.

2. The method of claim 1, wherein the secretory glands are selected from the group consisting of pancreas, liver, and salivary gland.

3. The method of claim 1, wherein the secretory glands are liver and pancreas.

4. The method of claim 1, wherein the DNA of interest encodes a polypeptide selected from the group consisting of insulin, growth hormone, interferon-alpha 2b, Interferon-alpha 2a, interferon-alpha N1, filgastim, insulinotropin, imiglucerase, clotting factor VIII, interferon-beta b, erythropoietin, sargramostim, interleukin-2, interferon-gamma, anti-CD3 antibody, GPIIb/IIIa monoclonal antibody, adenosine deaminase, interleukin-8, insulin-like growth factor-1, platelet-derived growth factor, epidermal growth factor, hemoglobin.

5. The method of claim 1, wherein the DNA of interest encodes human insulin.

6. A method of controlling blood glucose levels in a mammalian subject having a disease or condition associated with hyperglycemia, the method comprising:
introducing into a mammalian patient's pancreas a construct comprising insulin-encoding DNA and a eukaryotic promoting sequence operably linked to the insulin-encoding, said introducing being by intraductal injection. thereby accomplishing genetic transformation of a pancreatic cell: and allowing the genetically transformed cell to express the protein and secrete the protein into the bloodstream of the patient in a therapeutically effective amount thereby treating the patient;
wherein blood glucose levels in the mammalian subject are decreased to achieve a euglycemic state.

7. The method of claim 6, wherein the euglycemic state is maintained for at least three days.

8. The method of claim 6, wherein the euglycemic state is maintained for at least 6 days.

9. The method of claim 6, the method further comprising enhancing insulin expression by administration of a cholinergic agonist to the mammalian subject.

10. The method of claim 6, the method further comprising enhancing insulin expression by ingestion of a meal.