Co-precipitates of adenovirus with metal salts

The present invention provides co-precipitates of metal salts and adenoviral vectors containing a transgene. The co-precipitates of the invention exhibit increased efficiency of gene transfer to a target cell relative to adenoviral vectors alone. Methods of making and using the co-precipitates are also provided. A method of delivering cystic fibrosis transmembrane conductance regulator to an individual with CF utilizing a co-precipitate of cationic molecules and adenoviral vectors containing a transgene encoding a CFTR protein is provided.

FIELD OF THE INVENTION 
This invention was made with government support under HL5 1670 awarded by 
the National Heart, Lung and Blood Institute of the National Institutes of 
Health. The government has certain rights in the invention. 
The present invention is directed to co-precipitates of metal salts and 
adenoviral vectors containing transgenes. The co-precipitates of the 
invention exhibit increased efficiency of gene transfer relative to 
adenovirus alone, particularly in cells that are normally poorly infected 
by adenovirus. 
BACKGROUND OF THE INVENTION 
Effective use of transgenes for the treatment of inherited and acquired 
disorders requires efficient delivery of transgenes. Various vector 
systems have been developed that are capable of delivering a transgene to 
a target cell. However, there remains a need to improve efficiency of 
available gene transfer methods. Improved efficiency is desirable both to 
increase the ability of the vector to correct the cellular defect, and to 
decrease the required amount of the vector and thereby reduce toxicity. 
Adenoviral vectors, have been designed to take advantage of the desirable 
features of adenovirus which render it a suitable vehicle for nucleic acid 
transfer. Adenovirus is a non-enveloped, nuclear DNA virus with a genome 
of about 36 kb, which has been well-characterized through studies in 
classical genetics and molecular biology (Horwitz, M. S., "Adenoviridae 
and Their Replication," in Virology, 2nd edition, Fields et al., eds., 
Raven Press, New York, 1990). The viral genes are classified into early 
(known as E1-E4) and late (known as L1-L5) transcriptional units, 
referring to the generation of two temporal classes of viral proteins. The 
demarcation between these events is viral DNA replication. The human 
adenoviruses are divided into numerous serotypes (approximately 47, 
numbered accordingly and classified into 6 subgroups: A, B, C, D, E and 
F), based upon properties including hemaglutination of red blood cells, 
oncogenicity, DNA base and protein amino acid compositions and homologies, 
and antigenic relationships. 
Recombinant adenoviral vectors have several advantages for use as gene 
transfer vectors, including tropism for both dividing and non-dividing 
cells, minimal pathogenic potential, ability to replicate to high titer 
for preparation of vector stocks, and the potential to carry large inserts 
(Berkner, K. L., Curr. Top. Micro. Immunol. 158:39-66, 1992; Jolly, D., 
Cancer Gene Therapv 1:51-64, 1994). 
The cloning capacity of an adenovirus vector is proportional to the size of 
the adenovirus genome present in the vector. For example, a cloning 
capacity of about 8 kb can be created from the deletion of certain regions 
of the virus genome dispensable for virus growth, e.g., E3, and the 
deletion of a genomic region such as E1 whose function may be restored in 
trans from 293 cells (Graham, F. L., J. Gen. Virol. 36:59-72, 1977) or 
A549 cells (Imler et al., Gene Therapy 3:75-84, 1996). Such E1 deleted 
vectors are rendered replication-defective. The upper limit of vector DNA 
capacity for optimal carrying capacity is about 105%-108% of the length of 
the wild-type genome. Further adenovirus genomic modifications are 
possible in vector design using cell lines which supply other viral gene 
products in trans, e.g., complementation of E2a (Zhou et al., J. Virol. 
70:7030-7038, 1996), complementation of E4 (Krougliak et al., Hum. Gene 
Ther. 6:1575-1586, 1995; Wang et al., Gene Ther. 2:775-783, 1995), or 
complementation of protein IX (Caravokyri et al., J. Virol. 69:6627-6633, 
1995; Krougliak et al., Hum. Gene Ther. 6:1575-1586, 1995). 
Adenoviral vectors for use in gene transfer to cells and in gene therapy 
applications commonly are derived from adenoviruses by deletion of the 
early region 1 (E1) genes (Berkner, K. L., Curr. Top. Micro. Immunol. 
158:39-66, 1992). Deletion of E1 genes renders the vector replication 
defective and significantly reduces expression of the remaining viral 
genes present within the vector. However, it is believed that the presence 
of the remaining viral genes in adenovirus vectors can be deleterious to 
the transfected cell for one or more of the following reasons: (1) 
stimulation of a cellular immune response directed against expressed viral 
proteins, (2) cytotoxicity of expressed viral proteins, and (3) 
replication of the vector genome leading to cell death. 
Transgenes that have been expressed to date by adenoviral vectors include 
p53 (Wills et al., Human Gene Therapy 5:1079-188, 1994); dystrophin 
(Vincent et al., Nature Genetics 5:130-134, 1993; erythropoietin (Descamps 
et al., Human Gene Therapy 5:979-985, 1994; omithine transcarbamylase 
(Stratford-Perricaudet et al., Human Gene Therapy 1:241-256, 1990; We et 
al., J. Biol. Chem. 271:3639-3646, 1996;); adenosine deaminase (Mitani et 
al., Human Gene Therapy 5:941-948, 1994); interleukin-2 (Haddada et al., 
Human Gene Therapv 4:703-711, 1993); and 60 1-antitrypsin (Jaffe et al., 
Nature Genetics 1:372-378, 1992); thrombopoietin (Ohwada et al., Blood 
88:778-784, 1996); and cytosine deaminase (Ohwada et al., Hum. Gene Ther. 
7:1567-1576, 1996). 
The particular tropism of adenoviruses for cells of the respiratory tract 
has relevance to the use of adenovirus in gene transfer for cystic 
fibrosis (CF), which is the most common autosomal recessive disease in 
Caucasians. Mutations in the cystic fibrosis transmembrane conductance 
regulator (CFTR) gene that disturb the cAMP-regulated Cl.sup.- channel in 
airway epithelia result in pulmonary dysfumction (Zabner et al., Nature 
Genetics 6:75-83, 1994). Adenovirus vectors engineered to carry the CFTR 
gene have been developed (Rich et al., Human Gene Therapy 4:461-476, 1993) 
and studies have shown the ability of these vectors to deliver CFTR to 
nasal epithelia of CF patients (Zabner et al., Cell 75:207-216, 1993), the 
airway epithelia of cotton rats and primates (Zabner et al., Nature 
Genetics 6:75-83, 1994), and the respiratory epithelium of CF patients 
(Crystal et al., Nature Genetics 8:42-51, 1994). Recent studies have shown 
that administering an adenoviral vector containing a DNA sequence encoding 
CFTR to airway epithelial cells of CF patients can restore a functioning 
chloride ion channel in the treated epithelial cells (Zabner et al., J. 
Clin. Invest. 97:1504-1511, 1996; U.S. Pat. No. 5,670,488, issued Sep. 23, 
1997). 
Transfer of the cystic fibrosis transmembrane conductance regulator (CFTR) 
cDNA to airway epithelia of patients with cystic fibrosis (CF) thus 
provides an example of successful use of gene transfer to correct a 
cellular defect, i.e., the CF defect in electrolyte transport. Vector 
systems including adenoviral vectors (Zabner et al. (1993) Cell 75: 207; 
Knowles et al. (1995) New Engl. J. Med. 333: 823; Hay et al. (1995) Hum. 
Gene. Ther. 6: 1487; Zabner et al. (1996) J. Clin. Invest. 97: 1504 and 
U.S. Pat. No. 5,670,488) and cationic lipids (Caplen et al. (1995) Nat. 
Med. 1: 39 and U.S. Pat. No. 5,650,096) have been shown to be capable of 
transferring the CFTR cDNA and expressing CFTR in mature ciliated human 
airway epithelia. The successful delivery of CFTR in such cells is 
manifest in the appearance of a functional chloride ion channel in the 
treated cells. 
While CFTR cDNA can be delivered to target cells for expression, current 
adenoviral vectors are less than optimal in delivering the CFTR cDNA to 
airway epithelia because the binding of the virus to the apical (exposed) 
surface of the epithelium is limited. Grubb et al. (1994) Nature 371: 802. 
The limited infection can be partially overcome by increasing the contact 
time between the virus and the apical surface. Zabner et al. (1996) J. 
Virol. 70: 6994. 
Cationic lipid vector-mediated gene transfer to mature human airway 
epithelia is also suboptimal. Caplen et al. (1995) Nat. Med. 1: 39. While 
it appears that cationic molecules bind to the cell surface and in some 
cases are taken up by the cell, important barriers to transgene expression 
may be release of DNA from the endosome, entry into the nucleus, release 
of DNA from the cationic molecule, and transcription of the DNA. Zabner et 
al. (1995) J. Biol. Chem. 270: 18997. 
Gene transfer systems that combine viral and nonviral components have been 
reported. Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90: 11548; Wu 
et al. (1994) J. Biol. Chem. 269: 11542; Wagner et al. (1992) Proc. Natl. 
Acad. Sci. USA 89: 6099; Yoshimura et al. (1993) J. Biol. Chem. 268: 2300; 
Curiel et al. (1991) Proc. Natl. Acad. Sci USA 88: 8850; Kupfer et al. 
(1994) Hum. Gene Ther. 5: 1437; and Gottschalk et al. (1994) Gene Ther. 1: 
185. In most cases, adenovirus has been incorporated into the gene 
delivery systems to take advantage of its endosomolytic properties. The 
reported combinations of viral and nonviral components generally involve 
either covalent attachment of the adenovirus to a gene delivery complex or 
co-internalization of unbound adenovirus with cationic lipid: DNA 
complexes. Further, the transferred gene is contained in plasmid DNA that 
is exogenous to the adenovirus. In these formulations, large amounts of 
adenovirus are required, and the increases in gene transfer are often 
modest. 
Calcium phosphate is often used to facilitate entry of DNA per se into 
cells in transformation or transfection procedures. Transfection with 
calcium phosphate co-precipitates containing plasmid DNA or naked 
adenoviral DNA is well known for cultured cell lines (Chen et al. (1987) 
Mol. Cell. Biol. 7:2745-2752; Graham et al. (1973) Virol. 52: 465-477). 
However, such DNA calcium phosphate co-precipitates are generally 
ineffective in primary cell cultures and in vivo. To date, there have been 
no reports of calcium phosphate being used to enhance transfection with 
recombinant viral vectors, including adenovirus. 
Accordingly, there is a need in the art for improved vector systems for the 
efficient delivery of transgenes to target cells. The present invention 
overcomes certain limitations associated with adenoviral vectors and while 
retaining the desirable features of the vector system. 
SUMMARY OF THE INVENTION 
The present invention provides co-precipitates of metal salts and 
adenoviral vectors in which the adenoviral vectors comprises a transgene 
to be delivered to a target cell or tissue. In a preferred embodiment, the 
cation of the metal salt is a divalent cation such as alkaline earth 
metals or transition-state metals, with alkaline metals being more 
preferred. Preferred metals include calcium, magnesium, manganese, cobalt, 
selenium, and zinc, with calcium being more preferred. Preferred anions of 
the metal salt include phosphates, carbonates and sulfides, with 
phosphates being more preferred. One particularly preferred metal salt 
precipitate to be utilized in accordance with the present invention is 
calcium phosphate (CaPi). Compositions comprising the co-precipitates of 
the invention dispersed in a carrier are also provided. 
In another embodiment, the present invention is directed to a method of 
making a co-precipitate of the metal salt and adenoviral vectors 
containing a transgene by precipitating the metal salt in the presence of 
the adenoviral vectors to form a co-precipitate having the adenoviral 
vectors dispersed therein. In a preferred embodiment, the metal salt and 
adenoviral vectors are co-precipitated at a ratio at which gene transfer 
by the co-precipitate to a host cell is optimal. 
In another embodiment, the present invention provides a method of 
delivering a transgene to a cell. The method of delivering the transgene 
to a cell is accomplished by precipitating metal salt in the presence of 
adenoviral vectors containing the transgene, and administering the 
co-precipitate to a cell whereby the co-precipitate facilitates cell 
infection and transgene expression. 
The present invention also provides a method of introducing a transgene 
encoding a cystic fibrosis transmembrane conductance regulator (CFTR) 
protein into the cells of an individual with cystic fibrosis (CF). The 
method includes preparing a co-precipitate of metal salt and adenoviral 
vectors containing a transgene encoding a CFTR protein, and administering 
said co-precipitate to the cells of a CF patient. In a preferred 
embodiment the cells are airway epithelial cells. 
In another embodiment, a method of providing CFTR to the airway epithelial 
cells of an individual with CF is provided. The method includes 
administering a therapeutically effective amount of a co-precipitate of 
the metal salt and adenoviral vectors containing a transgene encoding a 
CFTR protein to the airway epithelial cells of a CF patient. 
BRIEF DESCRIPTION OF THE DRAWINGS 
FIG. 1, Panels A-E, are bar graphs showing .beta.-galactosidase activity of 
NIH 3T3 cells 24 hours after addition of adenoviral vector:calcium 
phosphate (Ad:CaPi) co-precipitates with one of the following variables 
tested over a range of values: Panel A) Ca.sup.2+ concentration; Panel B) 
Pi concentration; Panel C) pH during precipitate formation; Panel D) 
duration of precipitate formation before addition to cells; and Panel E) 
MOI of adenovirus. 
FIG. 2, Panels A-C, are bar graphs showing the effects of serum and Ad:CaPi 
co-precipitation on transgene expression in NIH 3T3 cells treated with 40 
MOI adenovirus for 20 min. 
FIG. 3, Panels A and B, are bar graphs showing the effect of 
Cy3-labeled-Ad:CaPi co-precipitates on adenovirus association with NIH 3T3 
cells: Panel A shows expression (top, n=3) and binding (bottom, n=5); and 
Panel B shows binding at 4.degree. C. and 37.degree. C. (n=5). 
FIG. 4, Panels A and B, are bar graphs showing the following: Panel A the 
effect of fiber knob protein on transgene expression; Panel B the effects 
of heat inactivation, antibody inactivation, and substitution of plasmid 
DNA for adenovirus on transgene expression. 
FIG. 5, Panels A and B, are bar graphs showing expression of 
.beta.-galactosidase in normal airway epithelia in vitro and CFTR Cl.sup.- 
current in CF airway epithelia in vitro: Panel A shows expression versus 
total Ca.sup.2+ concentration; and Panel B shows CFTR Cl.sup.- current 
of cells transfected with either Ad2/CFTR-16:CaPi at a 5.8mM Ca.sup.2+ 
concentration or Ad2/CFTR-16 alone. 
FIG. 6, Panels A-C, are photomicrographs of whole mouse lung stained with 
X-gal reagent: Panel A shows mouse lung treated with vehicle control; 
Panel B shows mouse lung treated Ad2/.beta.pGal-2 (2.times.10.sup.8 IU) 
alone; and Panel C shows mouse lung treated with Ad2/.beta.Gal-2 
(2.times.10.sup.8 IU) as a Ad: CaPi co-precipitate. 
FIG. 7, Panels A-C, are photomicrographs of sections of the blue stained 
mouse lungs shown in FIG. 6: Panel A shows mouse lung treated with the 
vehicle control; Panel B shows mouse lung treated Ad2/.beta.Gal-2 
(2.times.10.sup.8 IU) alone; and Panel C shows mouse lung treated with 
Ad2/.beta.Gal-2 (2.times.10.sup.8 IU) as a Ad:CaPi co-precipitate. 
FIG. 8 is a bar graph showing the percentage of mouse airway epithelium 
cells in vivo stained with X-gal reagent according to airway diameter and 
treatment with Ad2/.beta.Gal-2 alone or Ad2/.beta.Gal-2:CaPi 
co-precipitate.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides co-precipitates of metal salts and 
adenoviral vectors that are useful for the delivery of a transgene to a 
cell. These co-precipitates increase the binding and subsequent uptake of 
the adenoviral vector by a target cell or tissue. The co-precipitates are 
particularly useful for adenovirus-mediated gene transfer to cells that 
are not easily infected by adenovirus alone (i.e., adenoviral resistant 
cells), for example cells that do not express a cell surface receptor that 
binds adenovirus fiber. The adenovirus fiber molecules are found on the 
surface of adenovirus and are believed, inter alia, to mediate viral 
infectivity. 
In addition, the present co-precipitates are useful for delivering an 
adenovirus vector to a specific cell type. In the co-precipitates of the 
present invention in which the metal salt masks part of the adenovirus, in 
particular the adenovirus fiber, the specificity of the adenovirus for its 
widely distributed natural targets may be reduced or eliminated. Since the 
adenoviral vectors within the co-precipitates retain infectivity despite 
masking of the fiber, the co-precipitates of the invention can be 
delivered to a desired target for effective transgene delivery. 
In the co-precipitates of the present invention, the adenoviral vectors 
maintain their ability to facilitate delivery of a transgene. Transfer of 
a transgene to cells that are not easily infected by adenovirus alone 
(i.e., adenoviral resistant cells) is enhanced by the present 
co-precipitates. 
The metal salt component of the co-precipitate of the present invention is 
a metal salt that forms a precipitate, in which the precipitate enhances 
gene transfer by an adenoviral vector to a cell type in which infection by 
adenovirus alone is limited. Adenoviral resistant cells include, but are 
not limited to, NIH-3T3 and 9L gliosarcoma cells, which are useful as in 
vitro model systems for assays of the co-precipitates of the invention. 
Other cells that are poorly infected by adenovirus, such as human 
endothelial cells, readily express a transgene product when treated with 
the co-precipitates of the present invention. A simple and convenient 
assay to determine the ability of a metal salt to enhance gene transfer is 
provided hereinbelow. 
Examples of metal salt precipitates are known to those of ordinary skill in 
the art. In accordance with the present invention, any metal salt that 
forms a precipitate can be utilized as long as the precipitate is 
non-toxic or exhibits minimal toxicity to mammals. In a preferred 
embodiment, the metal is a divalent cation capable of forming a 
precipitate with the salt anion. Preferred divalent cations are alkaline 
earth metals such as calcium and magnesium, with calcium being more 
preferred. Other preferred divalent cations are transition-state metals 
such as manganese, cobalt, selenium and zinc. The anion of the metal salt 
is preferably phosphate, carbonate or a sulfide, with phosphate being 
particularly preferred. One particularly preferred metal salt precipitate 
that has been found to be very effective in enhancing viral infectivity 
and transgene expression is calcium phosphate (CaPi). 
As will be apparent to one skilled in the art, the ratio of metal cations 
to salt anions for precipitate formation is variable and dependent on 
parameters such as pH of the solution, temperature and on the cations and 
anions selected. These parameters can easily be established from a 
precipitate's known solubility product (K.sub.sp) or if unavailable by 
solubility curves ascertained through routine titration studies. 
The co-precipitate is formed by precipitating the metal salt out of 
solution in the presence of the adenoviral vectors thereby forming a 
co-precipitate of the metal salt having the adenoviral vectors dispersed 
therein. For example, the adenoviral vectors can be suspended in phosphate 
buffered saline, at room temperature, to which a sufficient amount of a 
metal halide (e.g., calcium chloride) is added to precipitate the metal 
salt out of solution. During precipitation, the adenoviral vectors in the 
solution are entrapped by the forming precipitate thereby producing the 
co-precipitate of the invention. 
Preferably, the precipitation of the metal salt is conducted using 
serumfree media, such as phosphate or carbonate buffered saline. The 
presence of serum during precipitation is disadvantageous in that it 
interferes with precipitate formation which significantly reduces enhanced 
transgene expression. While not wishing to be bound by theory, it is 
believed that the presence of non-adenovirus proteins and peptides in the 
serum interferes with precipitate formation thereby reducing the 
effectiveness of the co-precipitate. Thus, it is preferable to precipitate 
the metal salt in serum-free media to obtain a metal salt/adenoviral 
vector co-precipitate free of non-adenoviral proteins and peptides. 
In the calcium phosphate embodiment of the present invention, precipitation 
is preferably conducted at a solution pH greater than 6.5, since transgene 
expression is generally minimalized at lower pH values as evidenced by the 
CaPi co-precipitates illustrated in the examples below. More preferable, 
the solution should be at a pH of at least 7 or greater (e.g., 8). Values 
of hydrogen ion concentration greater than 11, and more preferably 10, 
should be avoided to minimize irritation of an individual's mucosal 
membranes. 
However, metal salt precipitates, other than calcium phosphate, that 
provide enhanced viral infectivity and transgene expression can be 
achieved at pH values of less than 6.5. Following the teachings of the 
present invention, metal precipitates formed at a pH less than 6.5 that 
provide enhanced viral infectivity and transgene expression can be easily 
ascertained through routine experimentation. 
As will be apparent to the skilled artisan, the size of the co-precipitate 
is inversely proportional with reaction time. Preferably, the reaction is 
stopped when the precipitate has a relatively small particle size. It is 
believed that small partifacilitatefacilitates enhanced viral infection 
and transgene expression due the increased surface area provided by the 
particulate in comparison to the larger particulate formed if the reaction 
is allowed to continue where the metal salt precipitate falls to the 
bottom of the flask. The reaction is stopped when the solution has 
developed a slight haze, which indicates the formation of small 
particulate, by adding serum (e.g., Fetal Calf Serum) to the solution. 
The adenoviral vector component of the co-precipitate of the present 
invention contains adenoviral DNA and a transgene of interest. The 
transgene is operably linked to a promoter and regulatory sequences to 
effect expression of the transgene in the target cell. 
Adenovirus-based vectors for the delivery of transgenes are well known in 
the art and may be obtained commercially or constructed by standard 
molecular biological methods. Recombinant adenoviral vectors containing 
exogenous genes (transgenes) for transfer are generally derived from 
adenovirus type 2 (Ad2) and adenovirus type 5 (Ad5). They may also be 
derived from other non-oncogenic serotypes. See, e., Horowitz, 
Adenoviridae and Their Replication, in Virology, Second Edit., Fields, et 
al, Eds., Raven Press Ltd., New York, 1990, incorporated herein by 
reference. 
The adenoviral vectors in the present invention are incapable of 
replicating, have minimal viral gene expression and are capable of 
expressing the transgene in target cells. Adenoviral vectors are generally 
made replication-defective by deletion of the E1 region genes. The 
replication-defective vectors may be produced in the 293 cell (ATCC CRL 
1573), a human embryonic kidney cell line expressing E1 functions. The 
deleted El region may be replaced by the transgene of interest under the 
control of an adenoviral or non-adenoviral promoter. The transgene may 
also be placed in other regions of the adenovirus genome. Graham et al. 
"Adenovirus-based expression vectors and recombinant vaccines" in 
Vaccines: New Approaches to Immunological Problems, 363-390, Ellis, ed., 
Butteworth-Heinemann, Boston, 1992 provide a review of the production of 
replication-defective adenoviral vectors, and is incorporated herein by 
reference. 
The skilled artisan is also aware that other non-essential regions of the 
adenovirus can be deleted or repositioned within the viral genome to 
provide an adenoviral vector suitable for delivery of a transgene in 
accordance with the present invention. For example, U.S. Pat. No. 
5,670,488, the disclosure of which is incorporated herein by reference, 
discloses that some or all of the E1 and E3 regions may be deleted, and 
non-essential open reading frames of E4 can be deleted. Other 
representative adenoviral vectors are disclosed, for example by Rich et 
al. (1993) Human Gene Therapy 4: 461, Brody et al (1994), Ann NY Acad Sci. 
716: 90, Wilson (1996) New Engl. J. Med. 334: 1185, Crystal (1995) Science 
270: 404; O'Neal et al. (1994) Hum. Mol. Genet. 3: 1497; and Graham et al. 
(1992) in "Vaccines: New Approaches to Immunologic Problems", 
Butterworth-Heinemann, Boston, 363-390, the disclosures of which are 
incorporated herein by reference. In a preferred embodiment of the present 
invention, the adenoviral vector is an E1 deleted Ad2- or Ad5-based 
vector. 
A transgene is defined herein as any nucleic acid or gene that is not 
native to adenovirus. Any nucleic acid that can be transcribed in the 
adenoviral vector is contemplated. In a preferred embodiment, the 
transgene encodes a biologically functional protein or peptide. A 
biologically functional protein or peptide is a protein or peptide that 
affects the cellular mechanism of a cell in which it is expressed, or the 
function of a tissue or an organism. For example, the biologically 
functional protein or peptide may be essential for normal growth or repair 
of a cell, for maintaining the health of an organism, or for producing a 
secreted protein that acts at a site distant from the cell or tissue in 
which it was produced. The protein or peptide may maintain or improve the 
health of a mammal by supplying a missing protein, by providing increased 
quantities of a protein that is under-produced, or by providing a protein 
or peptide that inhibits or counteracts an undesired molecule. Transgenes 
that express a biologically functional protein or peptide useful in the 
prevention or treatment of an inherited or acquired disorder in a mammal 
are particularly preferred. Examples of such biologically functional 
proteins include cytokines, growth factors, tumor suppressors, and 
clotting factors. 
In one embodiment of the present invention, the transgene is DNA encoding 
functional cystic fibrosis transmembrane conductance regulator (CFTR) 
protein. CFTR is a phosphorylation and nucleoside triphosphate-regulated 
Cl.sup.- channel located in the apical membrane of epithelial cells in 
the lung, intestine, pancreas and sweat glands. For a review, see Welsh et 
al., (1992) Neuron 8: 821, incorporated herein by reference. Cystic 
fibrosis (CF) results from a non-functional Cl.sup.- channel in an 
individual's epithelial cells caused by mutations in the gene encoding 
CFTR. Such mutations result in loss of function of the chloride channel 
and thus defective electrolyte transport in affected epithelial cells. DNA 
encoding wild-type CFTR is known in the art; the sequence is disclosed, 
for example, in U.S. Pat. No. 5,670,488, incorporated herein by reference. 
A deletion mutant of CFTR that encodes a regulated Cl.sup.- channel is 
disclosed by Sheppard et al. (1994) Cell 76: 1091, and in, U.S. Pat. No. 
5,639,661, the disclosures of which are incorporated herein by reference. 
In accordance with the present invention, DNA encoding a CFTR protein 
includes the foregoing published sequences as well as other DNA encoding 
CFTR known to those of skill in the art. Further included are 
modifications of the known DNA molecules, for example mutations, 
substitutions, deletions, insertions and homologs, that encode a 
functional CFTR protein, i.e., a chloride channel. 
DNA encoding a CFTR protein can be identified by those of ordinary skill in 
the art by its ability, upon expression in host cells, to correct the 
Cl.sup.- channel defect in cultured CF airway epithelia, for example by 
the methods described by Rich et al. (1990) Nature 347: 358, incorporated 
herein by reference. Briefly, cultured CF airway epithelial cells are 
infected with adenoviral vectors containing DNA encoding a CFTR protein. 
Virus-mediated expression of functional CFTR protein is assessed using an 
SPQ [6-methoxy-N-(3-sulfopropyl)-quinolinium, Molecular Probes, Eugene, 
OR] halide efflux assay. SPQ is a halide-sensitive fluorophore, the 
fluorescence of which is quenched by halides. In this assay, cells are 
loaded with SPQ, CFTR is activated by cAMP agonists, the CFTR Cl.sup.- 
channel opens, halides exit the cell, and SPQ fluorescence in the cell 
increases rapidly. Thus increases in intracellular fluorescence in 
response to cAMP provide a measure of a functional Cl.sup.- channel. 
In another assay suitable to identify functional CFTR proteins, CF 
epithelial cells are infected with adenoviral vectors containing DNA 
encoding a CFTR protein, and secretion of Cl.sup.- from infected cells is 
measured in response to cAMP stimulation. The secretion of Cl.sup.- can 
be measured as an increase in transepithelial short-circuit current with 
addition of cAMP agonists, as described for example by Rich et al. (1993) 
Human Gene Therapy 4: 461, the disclosure of which is incorporated herein 
by reference. Expression of a functional CFTR protein can also be assessed 
by patch clamp techniques that detect reversibly activated whole-cell 
currents in response to addition of cAMP agonists, or single-channel 
currents in excised, cell-free patches of membrane in response to 
cAMP-dependent protein kinase and ATP. Patch clamp techniques are 
described for example by Sheppard et al. (1994) Cell 76: 1091, and U.S. 
Pat. No. 5,639,661, the disclosures of which are incorporated herein by 
reference. 
In another embodiment, the transgene is a nucleic acid that is capable of 
being transcribed into an RNA molecule that is sufficiently complementary 
to hybridize to an mRNA or DNA of interest. Such an RNA molecule is 
referred to herein as an antisense molecule. Antisense molecules are 
useful in preventing or limiting the expression of overproduced, 
defective, or otherwise undesirable nucleic acid molecules. 
In the adenoviral vectors of the present invention, the transgene is 
operably linked to expression control sequences, e.g., a promoter that 
directs expression of the transgene. The promoter may be an endogenous 
adenovirus promoter, for example the E1 a promoter or the Ad2 major late 
promoter (MLP) or a heterologous eucaryotic promoter, for example a 
phosphoglycerate kinase (PGK) promoter or a cytomegalovirus (CMV) 
promoter. Similarly, those of ordinary skill in the art can construct 
adenoviral vectors utilizing endogenous or heterologous poly A addition 
signals. 
In accordance with the present invention, the co-precipitate is prepared by 
mixing the necessary components to initiate precipitation of the metal 
salt in the presence of the adenoviral vectors containing a transgene. The 
co-precipitate once formed can be dispersed with a carrier or diluent, for 
example, a physiological buffer, such as saline, phosphate buffered saline 
(PBS), water, dextrose, serum and solutions with excipients such as 
polyethylene glycol (PEG), propylene glycol and glycerol. Those of 
ordinary skill in the art appreciate that the choice of a diluent is 
dictated by the intended use and the route of administration for the 
co-precipitate. Thus, one skilled in the art can choose an appropriate 
diluent in accordance with known formulation principles. 
The adenoviral vectors containing a transgene and the metal salt are 
preferably co-precipitated at a ratio that achieves optimal transfer of 
the transgene to a target cell or tissue. Generally, greater loading of 
the co-precipitate with adenoviral vectors results in greater transgene 
expression. The effectiveness of a specific ratio can be conveniently 
determined by utilizing a reporter gene (e.g., lacZ) as the transgene, 
preparing co-precipitates of varying ratios of adenoviral vector to metal 
salt, and infecting target cells with the co-precipitates. Transfer of the 
transgene to the target cell is evaluated by measuring the level of the 
transgene product in the target cell. The level of transgene product in 
the host cell directly correlates with the efficiency of transfer of the 
transgene by the co-precipitate. Expression of the transgene can be 
monitored by a variety of methods including, inter alia, immunological, 
histochemical and activity assays, depending upon the selected transgene. 
For example, if the transgene encodes .beta.-galactosidase (lacZ), 
activity can be measured by methods well known in the art, for example by 
using a commercially available method such as a Galacto-Lite kit (Tropix, 
Inc., Bedford, Mass.) as disclosed by Zabner et al. (1996) Gene Therapy 3: 
458. When the transgene is DNA encoding a CFTR protein, the presence of a 
functional regulated chloride channel in host cells can be determined by 
the methods disclosed by Sheppard et al. (1994) Cell 1: 1091 and U.S. Pat. 
No. 5,639,661, incorporated herein by reference. The foregoing assay for 
optimizing ratios is also useful for identifying cationic molecules that 
maximize transgene delivery. 
Thus, by measuring the expression of the transgene transferred by 
co-precipitates of varying ratios of metal salt to adenoviral vector, a 
suitable ratio of a specific metal salt (e.g., CaPi) to adenoviral vectors 
can be determined. The ratios herein are described in terms of the total 
concentration of metal and salt ions per a specified number adenoviral 
particles. The useful ratios of metal salts per adenoviral particle vary 
depending upon the choice of metal salt used (e.g., Ca.sub.3 
(PO.sub.4).sub.2 versus Mg.sub.3 (PO.sub.4).sub.2). Preferably, the metal 
salt precursors and adenoviral vectors are diluted and then mixed in the 
absence of serum at the selected ratio to form the co-precipitates of the 
invention. The co-precipitates are then used to transfer a transgene to a 
cultured cell or to a cell or tissue or organ in vivo. In a preferred 
embodiment, the co-precipitates are used within about five hours from the 
time of preparation. In a more preferred embodiment, the co-precipitates 
are used within from about fifteen minutes to about one hour from the time 
of preparation. Those skilled in the art can readily determine suitable 
methods for stabilizing the co-precipitates, for example using excipients 
such as polyethyleneglycol (PEG). 
The co-precipitates of the present invention are useful for transferring a 
transgene to a target cell. The co-precipitates are particularly useful 
for the transfer of a transgene to a cell that is not easily infected by 
adenovirus alone (i.e., an adenoviral resistant cell). The target cell may 
be in vitro or in vivo. Use of the co-precipitates in vitro allows the 
transfer of a transgene to a cultured cell and is useful for the 
recombinant production of the transgene product. The co-precipitates may 
provide delivery of a transgene to a cell in vivo, and expression therein, 
for example where the transgene product is absent, insufficient, or 
nonfunctional. Alternately, the expression of the transgene may serve to 
block the expression or function of an undesired gene or gene product in 
the target cell. 
Accordingly, the present invention provides a method of delivering a 
transgene to a cell. The method includes co-precipitating a metal salt 
with adenoviral vectors containing the transgene to form the 
co-precipitate, and administering the co-precipitate to a cell. In a 
preferred embodiment, the cell is one that is not easily infected by 
adenovirus alone. The co-precipitate may be administered to the cell by 
methods known in the art that facilitates infection of the cell. Infection 
of a target cell in culture is accomplished by incubating the target cell 
with the co-precipitate. Conditions of time, temperature, environment and 
culture media are standard conditions for infection of cultured cells and 
are within the skill of those in the art. For example, representative 
conditions for the infection of cultured airway epithelial cells are 
infection with 5,000 to 10,000 viral particles/cell, for fifteen minutes 
to six hours in a 5% CO.sub.2 humidified environment at 37.degree. C. 
Effective delivery of the transgene to the target cell can be confirmed by 
detecting the transgene product as described above. 
Infection of a target cell in vivo is accomplished by contacting the target 
cell with the co-precipitate. The co-precipitate is delivered as a 
composition in combination with a carrier, which includes any and all 
solvents, diluents, isotonic agents, and the like. The use of such media 
and agents for preparing compositions, as provided for herein, is well 
known in the art. The co-precipitates of the invention may be delivered to 
the target cell by various delivery routes appropriate for the target 
cell, including for example by ingestion, injection, aerosol, inhalation, 
and the like. The co-precipitates may be delivered intravenously, by 
injection into tissue, such as brain or tumor, or by injection into a body 
cavity such as pleura or peritoneum. In a preferred embodiment, the 
transgene is a DNA molecule encoding CFTR or an analog or variant thereof 
(see, e.g., U.S. Pat. No. 5,639,661) which provides functional regulated 
chloride channel activity in target cells, and the co-precipitate is 
delivered to the airway epithelia by inhalation. 
The present invention further provides a method of delivering a transgene 
encoding CFTR or variant thereof capable of forming a functional chloride 
channel to the cells of an individual with CF. The method includes 
co-precipitating the metal salt with adenoviral vectors containing the 
transgene encoding CFTR to form a co-precipitate, and thereafter 
administering said co-precipitate to the cells of an individual with CF. 
In a preferred embodiment the cells are airway epithelial cells. The 
co-precipitate may be delivered to the target cells as a composition 
including the co-precipitate and an acceptable carrier. The co-precipitate 
may be delivered to airway epithelial cells by methods known in the art, 
for example by inhalation or intubation and lavage. In a preferred 
embodiment, delivery of the co-precipitate is by inhalation. 
The present invention further provides a method of providing CFTR to airway 
epithelial cells of individuals with CF. The method comprises combining 
the selected anions and cations with an adenoviral vector containing a 
transgene encoding CFTR to form a co-precipitate and administering the 
co-precipitate to epithelial cells of an individual with CF in a fashion 
and under conditions whereby functional Cl.sup.- channel activity is 
produced in the treated cells. Production of functional Cl.sup.- channels 
in CF patients can be evaluated by the alleviation of the symptoms 
associated with CF such as abnormal mucous secretion, bacterial infection, 
inflammation, tissue damage and fibrosis. The term "transgene encoding 
CFTR" includes DNA molecules that encode a Cl.sup.- channel that, when 
expressed in an airway epithelial cells of a CF patient, alleviate the 
chloride channel defect in the airway epithelial cells. In a preferred 
embodiment, the transgene has the sequence disclosed in U.S. Pat. No. 
5,670,448 or U.S. Pat. No. 5,639,661, the disclosures of which are 
incorporated herein by reference. 
In accordance with the present invention, co-precipitates can be formed 
using the adenoviral vector Ad2/CFTR-2 and administered to patients (i.e., 
humans) as set forth in Zabner et al. (1996) J. Clin. Invest. 
97(6):1504-151 1, which is herein incorporated by reference. As 
demonstrated by Zabner et al. (1996), repeated intranasal administration 
of Ad2/CFTR-2 alone corrected the defect in airway epithelial Cl.sup.- 
transports in individuals. Ad2/CFTR-2 is an adenovirus 2-based E1 
replacement vector containing the CFTR cDNA, the PGK promoter, and a 
synthetic bovine growth hormone poly A addition site. The vector contains 
the E3 region but lacks all of E4 with the exception of Open Reading Frame 
6. 
A metal salt co-precipitate having Ad2/CFTR-2 dispersed therein can be 
prepared in the following manner. Approximately 1+10.sup.10 infectious 
units (I.U.) is dispersed in a serum-free media such as phosphate buffered 
saline (PBS). While gently stirring the solution a pre-determined amount 
of calcium chloride (CaCl.sub.2) is added to the solution. Approximately 
15-30 minutes after the addition of CaCl.sub.2, the solution develops a 
slight haze indicating that small particles of Ad2/CFTR-2:CaPi 
co-precipitate have formed. Serum (e.g., Fetal Calf Serum) is added to the 
solution to stopped further precipitation. Samples of the co-precipitate 
containing media can be removed and applied to the nasal epithelia of 
individuals suffering from CF. 
The co-precipitate may be administered to the epithelial cells as a 
composition comprising the co-precipitate and an carrier. The 
co-precipitate may be administered by acceptable routes for delivery to 
epithelial cells. In a preferred embodiment the cells are airway 
epithelial cells and the co-precipitate is delivered by inhalation or 
intubation and lavage. For example, the composition may be administered to 
nasal epithelium using a modified Foley catheter, which is introduced 
under endoscopic guidance to the area beneath the inferior turbinate as 
described by Zabner et al. (1996) J. Clin. Invest. 97(6) 1504. Intubation 
and lavage is described by Welsh et al. (1995) Human Gene Therapy 6: 205, 
incorporated herein by reference. In a preferred method, the composition 
comprises the co-precipitate and phosphate buffered saline or other 
carrier and is administered by inhalation of aerosol, dry powder, or by 
instillation, for example by bronchoscopy. 
The present invention further provides compositions comprising 
co-precipitates of metal salts and adenoviral vectors containing a 
transgene and further comprising a carrier. In a preferred embodiment the 
metal salt is calcium phosphate with the transgene encoding CFTR. 
Where the compositions are pharmaceutical compositions, they may be 
prepared by techniques known in the art. The formulation of pharmaceutical 
compositions is generally known in the art and reference can conveniently 
be made to Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing 
Co., Easton, Pa. The pharmaceutical forms of the present co-precipitates 
suitable for administration include sterile aqueous solutions and 
dispersions. The subject co-precipitates are compounded for convenient and 
effective administration in pharmaceutically effective amounts with a 
suitable pharmaceutically acceptable carrier and/or diluent in a 
therapeutically effective dose. 
The precise effective amount of co-precipitate to be used in the methods of 
this invention applied to humans can be determined by the ordinary skilled 
artisan with consideration of individual differences in age, weight, 
extent of disease and condition of the individual. It can generally be 
stated that the preparation of the present invention should be preferably 
administered in an amount of at least about 1 plaque forming unit (PFU) 
per desired target cell. 
It is especially advantageous to formulate parenteral compositions in 
dosage unit form for ease of administration and uniformity of dosage. 
Dosage unit form as used herein refers to physically discrete units suited 
as unitary dosages for the mammalian subjects to be treated, each unit 
containing a predetermined quantity of active material calculated to 
produce the desired phenotypic effect in association with the required 
carrier. The specification for the novel dosage unit forms of the 
invention are dictated by and directly depend on the unique 
characteristics of the co-precipitate and the limitations inherent in the 
art of compounding. 
The co-precipitate is compounded for convenient and effective 
administration in effective amounts with a suitable carrier in dosage unit 
form as described above. In the case of compositions containing 
supplementary active ingredients, the dosages are determined by reference 
to the usual dose and manner of administration of the ingredients. 
In the method of treatment according to the present invention, the 
co-precipitates may be administered in a manner compatible with the dosage 
formulation, in such amount as will be therapeutically effective, and in 
any way which is medically acceptable for the treatment of CF. 
The invention is further illustrated by the following specific examples 
which are not intended in any way to limit the scope of the invention. 
EXAMPLE 1 
Materials and Methods 
A. Cell Cultures 
NIH-3T3 and COS-1 cells were cultured on 24-well plates (Corning, 25820) in 
Dulbecco's minimal essential media (DMEM, high glucose) supplemented with 
10% fetal calf serum (FCS) (Sigma Chemical Co., St. Louis), 100 U/ml 
penicillin and 100 .mu.g/ml streptomycin (P/S). HeLa cells were cultured 
in Eagle's minimal essential media (EMEM, Life Technologies, Inc.) 
supplemented with 10% FCS, 10 mM nonessential amino acids (Sigma) and P/S. 
Cultured cell lines were infected 18-24 hours after seeding when the cells 
were approximately 70% confluent. Primary cultures of normal and CF human 
airway epithelia were isolated and grown as described previously (Zabner 
et al. (1996) J. Virol. 70:6994-7003; Smith et al. (1996) Cell 
85:229-236). The culture media consisted of a 1:1 mix of DMEM/Ham's F 12, 
5% Ultraser G (Biosepra SA, Cedex, France), 100 U/ml penicillin and 100 
.mu.g/ml streptomycin, 1% nonessential amino acids, and 0.12 units/ml 
insulin. Cells were seeded on Millicell polycarbonate filters (Millipore). 
Twenty four hours after seeding, the cells were switched to the air-liquid 
interface and then grown with a dry apical surface. Epithelia were 
infected at least 2 weeks after seeding; transgene expression was measured 
2-4 days later. 
B. Vectors and Vector-Related Reagents 
Recombinant adenovirus vectors (Ads) expressing .mu.-galactosidase, 
Ad2/.mu.Gal-2, and CFTR, Ad2/CFTR-16, were prepared as previously 
described (Zabner et al. (1996) J. Virol. 70:6994-7003) by the University 
of Iowa Gene Transfer vector Core at titers of approximately 
1.times.10.sup.10 I.U./ml. Ad2/CFTR-16 which is disclosed in U.S. Pat. 
application Ser. No. 08/839,552 filed Apr. 14, 1997, incorporated herein 
by reference is a recombinant Ad2 vector having an E1 and E3B deletion 
(but is E4+), in which the CFTR gene is under control of a CMV promoter. 
Other Ad vectors, e.g. Ad2/CTFR-8 (disclosed in U.S. Pat. No. 5,707,618, 
incorporated herein by referenced may also be used. Fiber knob protein was 
a gift of Dr. Paul Freimuth (Brookhaven National Laboratories, Upton, 
N.Y.). In some studies, adenovirus was complexed with poly-L-lysine (55 
kDa, Sigma) or the cationic lipid GL-67 (a gift of Drs. Seng Cheng and 
David Harris, Genzyme), as described in Fasbender et al. (1997) J. Biol. 
Chem. 272:6479-6489, and U.S. patent application Ser. No. 08/755,035, 
filed Nov. 22, 1996. Neutralizing antibody (Virostat, 1401, Portland, Me.) 
or non-neutralizing antibody (MAB8052, Chemicon International Inc., 
Temecula, Calif.) was applied to virus in some studies. A 1:100 dilution 
of antibody was incubated with 2.times.10.sup.9 particles for 30 minutes 
at room temperature before formation of co-precipitates. 
C. Ad:CaPi Co-Precipitate Formation 
For the majority of the examples, CaPi co-precipitates containing 
adenovirus were formed by placing 8.times.10.sup.9 particles of 
recombinant adenovirus in 1 ml of EMEM (M-0268, Sigma), which contains 1.8 
mM Ca.sup.2+ and 0.86 mM Pi. An aliquot of a 2 M CaCl.sub.2 (CaCl.sub.2 
.times.2H.sub.2 O, E1200, Promega, Madison, Wis.) solution was added to 
achieve the desired Ca.sup.2+ concentration. However, a wide range of 
adenovirus particles and concentrations of Ca.sup.2+ and Pi were used as 
described in the subsequent examples and illustrated in the Figures. The 
solution was mixed by vortex or gentle pipette tip aspiration. Reference 
to Ca.sup.2+ and Pi concentrations refers to total ion concentration and 
not free ion concentration. Unless otherwise noted, the mixture was 
allowed to incubate for 20-30 minutes at room temperature. Then 250 .mu.l 
was applied to cells for 20 minutes followed by washing the surface to 
remove the co-precipitate. 
D. Adenovirus Tracking with Carbocvanine Dye 
To evaluate virus association with cells, adenovirus was labeled with the 
carbocyanine dye, Cy3 (Amersham Inc., Arlington Heights, Ill.) using 
methods described by Drs. P. L. Leopold and R. G. Crystal (Leopold et al. 
(1998) Hum. Gene Ther. 9:367-378). Cy3 was covalently conjugated to capsid 
proteins of Ad2/.beta.Gal-2 by mixing 5 nmoles of Cy3 with 10.sup.12 
particles of virus in 1.5 ml of Na.sub.2 CO.sub.3 at pH 9.0 for 2 hours at 
4.degree. C. The solution was subsequently transferred to a dialysis 
chamber (Slide-A-Lyzer, 10,000 MW cutoff, Pierce Co., Rockford, Ill.) and 
dialyzed against two changes of phosphate-buffered saline (PBS), 3% 
sucrose, pH 7.4 at 4.degree. C. for 24 hours. Expression and infection 
studies were performed immediately after dialysis of the conjugate. This 
labeling procedure decreased the I.U./particle ratio by 5-35%. 
E. Evaluation of Transgene Expression and Toxicity 
.beta.-galactosidase activity was measured 24 hours after application of 
vector as previously described (Fasbender et al. (1997) J. Biol. Chem. 
272:6479-6489, U.S. application Ser. No. 08/755,035) with X-gal staining 
of epithelia as previously described (Zabner et al. (1996) J. Virol. 
70:6994-7003). To evaluate transepithelial electrolyte transport, 
epithelia were mounted in modified Ussing chambers (Zabner et al. (1996) 
J. Virol. 70:6994-7003). Short-circuit current (Isc) was measured under 
baseline conditions, and after addition of amiloride (10 .mu.M), cAMP 
agonists (10 .mu.M forskolin and 100 .mu.M IBMX), and bumetanide 100 
.mu.M. Individual experiments were performed using 3 sets of cells and all 
experiments were repeated at least 3 times. Statistical significance was 
evaluated using a paired or unpaired t-test. 
F. Transmission Electron Microscopy 
Ad:CaPi coprecipitates were processed for transmission electron microscopy 
(TEM) using a negative stain technique. Fifteen-.mu.l drops of freshly 
prepared samples were placed on glow-discharged collodion/carbon-coated 
400-mesh copper grids for 3) minutes. The solution was wicked off with 
filter paper and replaced with 1% aqueous uranyl acetate for 30 s. After 
removal of this solution, grids were allowed to dry and imaged in a 
Hitachi H-7000 transmission electron microscope. 
G. Murine Studies 
For in vivo analysis, 6-8 week old C57BL/6 mice (Jackson Laboratories, Bar 
Harbor, Me.) were used. Mice were lightly anesthetized using a 
methoxyflurane chamber. Ad2/.beta.Gal-2 (2.times.10.sup.8 I.U., 
5.times.10.sup.9 particles) was administered alone or as Ad:CaPi 
coprecipitates intranasally in two 62.5 .mu.l instillations delivered 5 
minutes apart. The experiment was performed twice with 6 animals in each 
group. Three days after vector administration, animals were sacrificed. 
Phosphate buffered saline (PBS, 10 ml) was instilled into the right 
ventricle and then the lungs and heart were removed intact. The trachea 
was intubated and instilled at 10 cm of pressure with the following 
solutions in order: PBS, 4% paraformaldehyde and 0.2% gluteraldehyde, PBS, 
and finally X-Gal reagent for an overnight incubation at room temperature. 
After photography, lungs were embedded in paraffin and serially sectioned. 
Lung sections for each condition (n=3) were analyzed by counting all 
airways that were cut at a perpendicular angle. For each such airway, the 
airway diameter and number of blue cells (positive for B-galactoside) was 
determined. The percentage of blue cells was calculated as: number of blue 
cells.div.(diameter.times..pi..div.4.9 .mu.m). The average width of an 
airway cell (4.9 .mu.m) was determined in separate experiments using 
hematoxylin and eosin-stained sections and is in excellent agreement with 
earlier studies (Mariassy, A. T. (1992) Epithelial cells of trachea and 
bronchi. In Comparative Biology of the Normal Lung. R. A. Parent, editor. 
CRC Press, Boca Raton. 63-76). 
EXAMPLE 2 
To ascertain whether incorporation of Ad2/.mu.Gal-2 in a precipitate would 
enhance adenovirus-mediated gene transfer, NIH 3T3 cells were transfected 
because they show little fiber receptor activity and are resistant to 
adenovirus infection (Seth, et al., (1994) J. Virol. 68:933-940). 
Accordingly, NIH 3T3 cells resemble the apical surface of differentiated 
airway epithelia and provide an in vitro model. 
Standard conditions for ascertaining transgene expression of 
.beta.-galactosidase were a Ca.sup.2+ concentration of 5.8 mM, Pi 
concentration of 0.86 mM, pH 7.4, duration of complex formation before 
addition to cells of 15-30 minutes, and 40 MOI Ad2/.beta.Gal-2. The 
co-precipitate was applied to cells for approximately 20 minutes in each 
case. One of the following conditions was varied to ascertain its effect 
on transgene expression: A) Ca.sup.2+ concentration; B) Pi concentration; 
C) pH during complex formation; D) duration of complex formation before 
addition to cells; and E) MOI of adenovirus. .beta.-galactosidase activity 
(i.e., blue staining) was measured 24 hours after vector addition. The 
results of these studies are shown in FIG. 1, Panels A-E, in which each 
panel represents results from an experiment with n=3 and each experiment 
was repeated at least twice. 
FIGS. 1, Panel A and Panel B show that when Ca.sup.2+ and Pi 
concentrations were increased during complex formation, 
.beta.-galactosidase expression increased, reached a maximum, and then 
decreased. These data illustrate that formation of an effective 
co-precipitate requires optimal stoichiometry of Ca.sup.2+ to Pi, which 
can easily be ascertained by one skilled in the art. FIG. 1, Panel C shows 
that Ad:CaPi coprecipitates formed at acidic pH (below pH 7) provided 
little enhancement of gene transfer As a control for the effect of pH on 
the virus, HeLa cells were infected with adenovirus alone that had been 
exposed to the same pH as the Ad:CaPi coprecipitates; there was no effect 
of pH on gene transfer (not shown). 
FIG. 1, Panel D shows that formation time during which Ca.sup.2+, Pi, and 
adenoviral vectors were incubated before they were applied to cells 
influences gene transfer and transgene expression. As shown in Panel D, 
with increasing durations of the pre-incubation, transfection efficiency 
increased reaching a maximum level of efficiency at about 60 minutes. 
After 60 minutes, expression decreased with significantly reduced levels 
after 120 minutes. A time-dependent effect on precipitate formation was 
observed with light microscopy and visual inspection. The formation of 
fine precipitates was observed when infection was maximal (i.e., between 
30 to 60 minutes). The development of fine precipitates provided the 
solution with a slight haze due to the suspended fine particles. However, 
with longer formation periods, macro-precipitates formed as evidenced by 
relatively larger particles that fell to the bottom of the vessel and gene 
expression decreased. 
FIG. 1, Panel E shows that when the amount of virus added during 
precipitate formation increased (without a change in volume, Ca.sup.2+, or 
Pi), gene expression increased. Thus, greater loading of the 
co-precipitates with adenoviral vectors resulted in greater transgene 
expression. 
In summary, the increase in total transgene expression shown in FIG. 1, 
Panels A-E, was dramatically greater than the increase in the percent of 
3T3 cells expressing .beta.-galactosidase transgene, after applying 
adenovirus alone for 20 minutes. With the Ad2/.beta.Gal-2 alone only 3.5% 
of the cells stained blue (n=607 cells) after staining for 
.beta.-galactosidase, whereas with Ad2/.beta.Gal-2:CaPi coprecipitates 99% 
of cells stained blue (n=800 cells). Ad:CaPi coprecipitates also enhanced 
expression in other cells that are relatively resistant to adenovirus 
infection (9 L gliosarcoma cells, a 130-fold increase compared to 
adenovirus alone, and primary cultures of human umbilical vein endothelial 
cells, a 150-fold increase). Likewise in cells that are easily infected 
Ad:CaPi coprecipitates provided a significant advantage over adenovirus 
alone (HeLa cells, a 9-fold increase, and COS cells, a 12-fold increase). 
EXAMPLE 3 
The effects of serum on the co-precipitates of the present invention were 
ascertained. In all experiments NIH 3T3 cells were treated with 40 MOI 
adenovirus for 20 minutes with the following variations: A) Ad:CaPi 
co-precipitates were formed in the absence of serum and then added to 
cells in the presence (+) or absence (-) of 10% FCS; B) Ad:CaPi 
co-precipitates were formed in the presence (+) of 10% FCS, or 10% FCS was 
added after formation of the precipitate (-); and C) Ad:CaPi 
co-precipitates or adenovirus and CaPi precipitates were added separately 
to cells in presence of 10% FCS. For A, B, and C, the Ca.sup.2+ 
concentration was 5.8 mM, Pi concentration was 0.86 mM, and the 
precipitates were formed for 15-30 minutes. 
Data for these experiments are shown in FIG. 2, Panels A-C (asterisk 
indicates p&lt;0.05; data are from one experiment (n=3) and each experiment 
was repeated at least 3 times). Panel A shows that co-precipitates can be 
added to serum without loss of efficacy. This illustrates that once the 
precipitate is generated, it is relatively stable. Panel B shows that when 
serum is added to the precipitating solution prior to precipitate 
formation the enhancement in gene transfer was abolished. This result 
suggests that serum interferes with formation of the Ad:CaPi 
co-precipitate. Panel C shows that CaPi precipitate when separately 
applied to cells in combination with adenovirus provided little 
enhancement of expression. Thus, the above experiments indicate that 
enhanced infection efficiency requires that adenovirus be included in the 
metal salt precipitate. 
EXAMPLE 4 
To assess binding of the co-precipitated adenoviral vectors, NIH 3T3 cells 
were treated with 2.times.10.sup.9 particles of Cy3-labeled 
Ad2/.beta.Gal-2 for 60 minutes, rinsed with 3T3 media to remove unbound 
virus, and fixed with 4% paraformaldehyde. Cy3-labeled 
Ad2/.beta.Gal-2:CaPi coprecipitates were formed with 5.8 mM Ca.sup.2+ and 
0.86 mM Pi and applied to cells at 37.degree. C. To ascertain the effects 
of temperature, binding studies of the adenovirus alone and as an Ad:CaPi 
co-precipitate were conducted at 4.degree. C. and 37.degree. C. Incubation 
at 4.degree. C. was chosen since endocytosis is blocked at this 
temperature. In both experiments virus was placed on cells for 60 minutes 
and then removed by washing. 
The results are shown in FIG. 3, Panels A and B. Panel A shows that an 
increase in transgene expression (top)(n=3) was parallel to an increase in 
viral binding (bottom)(n=5) for the Ad:CaPi co-precipitate, which were 
both dramatically improved over adenovirus alone. Panel B shows that 
incubation temperature has little effect on adenovirus binding, 
illustrating that adenoviral association is dependent on cellular 
mechanisms other than conventional endocytosis as with DNA:CaPi 
co-precipitates. 
EXAMPLE 5 
The ability of Ad:CaPi co-precipitates to enhance gene transfer to NIH 3T3 
cells, which express little fiber receptor, suggested that binding of 
adenovirus fiber to its cell surface receptor was not required. To test 
this hypothesis directly, COS cells which are readily infected by 
adenovirus were studied. Fiber knob (0.7 .mu.pg/ml) was added to cells for 
10 minutes and then Ad:CaPi co-precipitates were applied in the continued 
presence of fiber knob. Cells treated with adenovirus received 40 MOI 
(2.times.10.sup.9 particles) and Ad-CaPi co-precipitates were prepared 
with 5.8 mM Ca.sup.2+ and 0.86 mM Pi incubated for 15-30 minutes. The 
results are shown in FIG. 4, Panel A, with cells having fiber knob present 
depicted by shaded bars and cells absent fiber knob depicted by black bars 
(asterisk indicates p&lt;0.05). Panel A shows that fiber knob protein 
inhibited transgene expression by adenovirus alone by 94%, as expected by 
conventional wisdom. However, no appreciable effect on transgene 
expression by Ad:CaPi co-precipitates was detected. These results show 
that adenovirus fiber is not required for the enhanced efficiency of 
infection utilizing the metal salt precipitates of the invention. 
NIH 3T3 cells were treated with the following for 20 minutes to ascertain 
the effects: (1) adenovirus to be used alone or as a co-precipitate was 
heat inactivated at 60.degree. C. for 30 minutes where indicated; (2) 
antibodies (neutralizing and non-neutralizing) were incubated with 
adenovirus for 30 minutes before co-precipitation; (3) Plasmid DNA 
(pCMV-.beta.Gal, 16.6 ng) was utilized alone or co-precipitated with CaPi 
under the same conditions used for adenovirus. The amount of plasmid DNA 
equaled approximately 2.times.10.sup.9 plasmids. Cells treated with 
adenovirus received 40 MOI (2.times.10.sup.9 particles) and Ad-CaPi 
coprecipitates were prepared with 5.8 mM Ca.sup.2+ and 0.86 mM Pi 
incubated for 15-30 minutes. Following intervention by one of the above 
described treatments, the vectors were removed and .beta.-galactosidase 
activity was measured 24 hours later. Data are from one experiment (n=3) 
and each experiment was repeated 3 times. 
The results are shown in Panel B. As is readily apparent from Panel B, heat 
inactivation, which disrupts viral proteins, inhibited transgene 
expression for adenovirus alone or as a CaPi co-precipitate. Similar 
results were obtained when adenovirus, before co-precipitate formation, 
was treated with a neutralizing anti-hexon antibody that presumably 
inhibits infection by interfering with steps subsequent to binding such as 
endosomal escape and traffic of viral DNA to the nucleus. Moreover, cells 
transfected with CaPi coprecipitates containing 2.times.10.sup.9 plasmids 
encoding .beta.-galactosidase exhibited little expression in comparison to 
precipitates formed with 2.times.10.sup.9 particles of Ad2/.beta.Gal-2. 
These data demonstrate that although fiber is not necessary for infection 
with Ad:CaPi co-precipitates, other adenoviral proteins are required to 
facilitate gene transfer and expression. These results also explain why 
CaPi precipitates that contain adenovirus produce much more transgene 
expression than those containing DNA alone. 
EXAMPLE 6 
To evaluate gene transfer to airway epithelia, we studied primary cultures 
of human airway epithelia grown at the air-liquid interface were studied. 
Under these conditions, the epithelia differentiate and form a ciliated 
epithelium that is resistant to gene transfer by adenovirus and cationic 
lipid vectors (Zabner et al.(1996) J. Virol. 70:6994-7003; Fasbender, et 
al. (1997) Gene Ther. 4:1173-1180). Primary cultures of normal airway 
epithelia grown at the air-liquid interface were studied 14-20 days after 
seeding. Ad2/.beta.Gal-2 (at an MOI of 50) was applied to the apical 
surface of the epithelia for 20 minutes and then removed by washing. Three 
or 4 days later, .beta.-galactosidase activity was measured as described 
in Example 1. The Ca.sup.2+ concentration was varied between 1.8 to 17.7 
mM with Pi concentration was 0.86 mM (n=9). The results of transgenic 
expression versus Ca.sup.2+ concentration are shown FIG. 5, Panel A. FIG. 
5, Panel A shows that when vector was applied to the apical surface for a 
short exposure time (20 minutes), Ad:CaPi co-precipitates enhanced 
transgene expression. 
The ability of Ad:CaPi co-precipitates to transfer CFTR cDNA to CF airway 
epithelia grown at the air-liquid interface was tested by applying 
Ad2/CFTR-16 (at an MOI of 50) for 20 minutes. The Ca.sup.2+ concentration 
was 5.8 mM with a Pi concentration of 0.86 mM. CFTR Cl.sup.- current was 
measured as described in Example 1. Briefly, the current was measured by 
inhibiting Na+ current with amiloride (10.sup.-5 M), applying cAMP 
agonists, and then measuring the current inhibited by bumetanide (100 
.mu.M) applied to the basolateral surface. The results are shown in FIG. 
1, Panel B (n=6, asterisk indicates p&lt;0.05 compared to adenovirus alone 
for 20 minutes). Untreated CF epithelia which was used as a negative 
exhibited no Cl.sup.- current. As a positive control, Ad2/CFTR-16 was 
allowed to remain on the mucosal surface for 24 hours; this long 
incubation period allowed significant transgene expression and 
transepithelial Cl.sup.- transport increases into the normal range 
(Zabner et al.(1996) J. Virol. 70:6994-7003). As a comparative standard, 
adenovirus alone was applied to the mucosal surface for only 20 minutes, 
in which little Cl.sup.- current was exhibited, as previously reported 
(Zabner, et al. (1997) J. Clin. Invest. 100:1144-1149, Zabner et al.(1996) 
J. Virol. 70:6994-7003). However, the application of Ad:CaPi 
co-precipitates for 20 minutes resulted in Cl.sup.- current that was at 
least as large as that obtained following a 24 hours incubation with 
adenovirus alone. These data demonstrate that Ad:CaPi co-precipitates are 
much more efficient than adenovirus alone for the transfer of CFTR cDNA to 
differentiated airway epithelia and the generation of CFTR Cl.sup.- 
current indicating that a functioning chloride ion channel was present. 
EXAMPLE 7 
To investigate Ad:CaPi co-precipitates in vivo, Ad2/.beta.Gal-2 
(2.times.10.sup.8 IU) was administered as virus alone or as Ad:CaPi 
co-precipitates to mouse lungs. The co-precipitates had Ca.sup.2+ 
concentrations that ranged from 5.8, 12, 18, and 36 mM with a Pi 
concentration of 0.86 mM. In both experiments Ad2/.beta.Gal-2 
(2.times.10.sup.8 I.U.) delivered by intranasal administration. This dose 
is lower than usually applied to obtain significant pulmonary gene 
transfer in order to ascertain whether delivery as a co-precipitate would 
enhance gene transfer. Three days following intranasal administration 
whole lungs were stained with X-gal reagent following the procedure set 
forth in Example 1. The results of X-gal staining is shown in FIG. 6, 
Panels A-C. Panel A shows that upon gross examination there was little 
evidence of staining. Panel B shows lung that received Ad2/.beta.Gal-2 
alone appeared similar to lungs not treated with virus. In contrast, Panel 
C shows lungs treated with the same dose of virus delivered as Ad:CaPi 
coprecipitates (12 mM Ca.sup.2+ and 0.86 mM Pi concentrations) exhibited 
significant X-Gal staining as readily apparent from the photomicrograph. 
Particularly striking was the pattern of X-Gal staining which traced the 
airways, rather than the parenchyma. Out of the co-precipitates with 
varying Ca.sup.2+ concentrations (5.8, 12, 18, and 36 mM) tested, the 
co-precipitate that contained 12 mM Ca.sup.2+ and 0.86 mM Pi was the most 
effective. Similar results were obtained with 6 animals in each group. 
Sections of the X-gal stained lungs described above were taken. 
Representative photomicrographs from lungs treated with vehicle, 
adenovirus alone or Ad:CaPi coprecipitates are shown in FIG. 7, Panels 
A-C. Panel B shows that in sections from lungs treated with adenovirus 
alone, there were a few blue-stained cells in airways and in the 
parenchyma. In contrast, Panel C shows a field from a lung treated with 
Ad:CaPi co-precipitate of the present invention in which most small and 
medium-sized airways showed positively-stained cells. Staining was 
predominantly in the airways with only rare positive cells in the 
parenchyma. 
Blue-stained cells were counted in sections of lungs following the 
procedure set forth in Example 1. Percentage blue cells was determined for 
airways of airway diameters of 0-80 .mu.m, 80-200 .mu.m and over 200 
.mu.m. Data was compiled from 50-200 airways. The quantitative assessment 
of expression is shown in FIG. 8 (asterisk indicates p&lt;0.05). Readily 
apparent from FIG. 8 is that in each airway region more cells expressed 
the transgene after administration of Ad:CaPi coprecipitates than 
adenovirus alone. Thus, FIG. 8 conclusively shows that the adenoviral 
vector/metal salt co-precipitates of the present invention enhance 
transgene expression in vivo.