in livers of several animals, and maximal levels of expression of the fully processed human factor IX were detected 30 days after introduction. The concentra-.
METHODOLOGY
Regilation of th. phosphoenolpyruvate carboxykinase/humai factor IX gen introduced into the livers of adult rats by receptor-mediited THOMAS
gene transfer
FERKOL,
MRY L. :LINDERRGh1 JIAN CHEN,t.* JOSE C. PERALZS,t L CRAW] ORD,t ()5(R D RATNOFV,1 AND RICHARD W HANSONtS of Medicinet at University Hospitals Department of Pediatri , R.ainbow Babies and Childrena Hospital; tDepartnint and the w Center for Molecular Nutrition, Case Western Reserve of Cleveland; TDepartim at of Btochemsstry - _____ University School of M dicine, Cleveland, Ohio 44106.4935, USA
DEBORAH
ABSTRACT
glycoprotein functional animals.
structed
cDNA
Gene receptor
systems targeting the asialo#{149} been developed to introduce genes into cells in culture and livers of intact A synthetic neoglycoprotein carrier was conand complexed to a chimeric gene containing the
for human
transfer have
factor
IX ligated
to the
promoter-
regulatory region of the gene for phosphoenolpyruvate carboxykinase from the rat. The complex was used to transfect human hepatoma cells that express the asialoglycoprotein receptor. Human factor IX DNA sequences were found in cells 10 days after treatment. A 1.4 IcR mRNA transcript was detected by Northern blot
hybridization,
which
dexamethasone hybridization
or cAMP with theophylline. of proteins secreted into
was inducible
by treatment
with
Western blot the culture
medium detected human factor IX. The chimeric gene was also transferred into livers of rats using the neoglycoprotein carrier system after partial hepatectomy. Although the was transcribed
results
were
variable,
the
exogenous
gene
in livers of several animals, and maximal levels of expression of the fully processed human factor IX were detected 30 days after introduction. The concentration of factor IX in the blood returned to control levels 60 days after transfection. Factor IX production was induced as late as 96 days after treatment by feeding transfected animals a diet high in protein but devoid of carbohydrates.
This
DNA
carrier
system
can
be
used
to
introduce functional genes into the livers of rats, and may be a useful technique for gene therapy targeting the liver.Ferkol, T., Lindberg, G. L., Chen,J., Perales,J. C.,. Crawford, D. R., Ratnoff, 0. D., Hanson, R. W. Regulation of the phosphoenolpyruvate carboxykinase human factor IX gene introduced into the livers of adult rats by receptor-mediated gene transfer. FASEB J. 7: 1081-1091; 1993. Key Words: asialoglycoprotein receptor . human coagulation factor IX phosphoenolpynwate carboxykinasepivmoter gene thriupy’ liver
DISEASE, OR HEMOPHILIA B, is a sex-linked recessive bleeding disorder caused by a deficiency of functional coagulation factor IX in the circulation. Human factor IX is a plasma glycoprotein, normally synthesized in the liver, that plays an integral role in the intrinsic coagulation pathway (1). Once converted to its serine protease form (IXa) by activated plasma thromboplastin antecedent (factor XIa), the activated protein interacts with coagulation factor Villa,
calcium
ions,
and
phospholipids
to produce
a complex
that
X to Xa. Factor IX undergoes several posttranslational modifications in the liver that are essential for its function before secretion into the blood. These include vitamin K-dependent ‘y-carboxylation of amino-terminal glutamic acid residues and -hydroxylation of aspartic acid (1). The gene that encodes human factor IX is 1,248 base pairs in length; after posttranslational modifications, the mature protein has a molecular weight of approximately 54,000 Da (2). Christmas disease accounts for approximately 10 to 20% of all inherited clotting disorders (3). Affected individuals exhibit a wide range of clinical severity that correlates generally with the concentration of circulating factor IX. Patients with severe deficiencies of functional factor IX may bleed spontaneously into soft tissues and joints or after minor trauma. Transfusions of plasma or plasma concentrates rich in factor IX are used to abort bleeding episodes by temporarily correcting the deficiency (3). Unfortunately, clinical management of this disease has been confounded by viral contamination of pooled plasma. Blood-borne infections with hepatitis and human immunodeficiency viruses have become significant problems in the treatment of the hereditary clotting disorders (4-6). These complications underscore the importance of developing alternative therapeutic approaches. In considering genetic correction of Christmas disease, several issues must be addressed. First, the method of gene transfer to be used and the optimal cells for transfection must be determined. The gene must be delivered efficiently to the target cells regardless of the method of gene transfer. Transfected cells must be characterized in culture to determine if the exogenous DNA introduced into the target cell is expressed appropriately and regulated in vitro. The level, regulation, and duration of factor IX expression in traxisfected cells in vivo are important practical considerations, as any system that is used must have clear therapeutic benefits. Finally, the gene transfer system must not place either the patient or the population at risk (7). converts
factor
CHRISTMAS
0892-6638/93/0007-1081/$01.50.
© FASEB
‘Current
Animal 2Current
address: Nutritional Physiology Group, Department of Science, Iowa State University, Ames, Iowa 50011, USA. address:
Department
of Exercise
Science,
University, Sudbury, Ontario P3E 2C6. 3To whom correspondence should be addressed, of Biochemistry, Case Western Reserve University cine, Cleveland, OH 44106-4935, USA
Laurentian
at: Department School of Medi-
1081
METHODOLOGY A plasmid
vector
IX promoter-regulatory tion
that
expresses
(pPFIXp)4 region
factor
was of the
normal
human
constructed, gene
for the
2.6kB
coagula-
using
the
cytosolic
report,
the
carboxykinase
delivery,
expression,
and
regulation
of human
factor IX produced in transfected human hepatoma cell lines (HepG2) are described. In addition, the functional chimeric PEPCK/human factor IX gene was transferred successfully into the livers of intact rats using a neoglycoprotein DNA carrier, levels
resulting
in the
of human
MATERIALS
I
form
(GTP) (EC 4.1.1.32) (PEPCK) from the rat to drive expression of the full-length cDNA encoding human factor IX. Because of its high level of expression in the liver, the PEPCK promoter is particularly suited for hepatic gene transfer (8). In addition, transcription from the promoter can be induced in animals by glucagon and glucocorticoids or by feeding the animals a diet high in protein but deficient in carbohydrate (8). In this of phosphoenolpyruvate
of significant
circulating
IX.
factor
AND
production
METHODS
Bgl II v
EcoRI..-j,
1.4kB
PstI
,-EcoRI
,
H
PEPCK
Ava I
PstI
Human Factor IX
PoIyA
FIX probe Figure 1. pPFIXp. ligated to solic form
Schematic
diagram
of the structure
of the chimeric
gene
The complementary DNA for human factor IX was the promoter-regulatory region of the gene for the cytoof PEPCK from the rat (-4491+ 73). The bovine growth polyadenylation signal was inserted into the Hind!!! site
hormone in the same transcriptional orientation as the human factor IX cDNA. Digestion with EcoRI releases the entire chimeric gene (2.6 kB), whereas digestion with BglII linearizes the pPFIXp plasmid (4.5 kB). The human factor IX cDNA probe (FIX) used in these studies is indicated at the bottom of the figure.
Materials All DNA-modifying enzymes and nucleotides were chased from Boehringer Mannheim (Indianapolis, [a32P]dCTP (3000 Ci/mmol), [‘4C]chloramphenicol mCi/mmol),
and
GeneScreen
Plus
were
obtained
pur-
md.); Du
Conn.).
The
enhanced
chemiluminescence
detec-
from Amersham Corporation (Arlington Heights, Ill.). All media, sera, and antibiotics were purchased from Gibco Laboratories (Grand Island, N.Y.). Bovine serum albumin, poly (L-lysine), D-galactose, insolubilized neuraminidase (type X-A), orosomucoid, and acetyl coA were obtained from Sigma Chemical Company (St. Louis, Mo.); l-ethyl-3-(3-dimethylaminopropyl) carbodiimide was purchased from Pierce Chemical Company (Rockford, Ill.). tion
system
Construction
was
purchased
of plasmids
An Escherichia coli cloning vector derived from the pBR322 plasmid was digested by EcoR I and Bgl II, thus removing a segment of the multiple cloning site, and the promoterregulatory region of the PEPCK (EcoRI-Bgl II restriction fragment, positions -449 to + 73) was inserted. The Psi I-Psi I fragment from the cDNA for the human factor IX gene was inserted into the Psi I site of the multiple cloning site in the same transcriptional orientation as the PEPCK promoter regulatory region. The human factor IX cDNA was generously provided by Dr. Earl Davie (University of Washington, Seattle). The pPFIX chimeric gene was digested with Hind III, and the ends were made blunt with the Klenow fragment of DNA polymerase I. The Pvu II-Pvu II fragment of the structural gene of bovine growth hormone, which contained the polyadenylation signal, was ligated into the Hind III site (Fig. 1). The size of the plasmid was 4.5 kB. No episomal origin of replication was present. The plasmid pRSVCAT contained the structural gene for E. coli chloramphenicol acetyltransferase (CAT) ligated to the 5 long terminal repeat of Rous sarcoma virus (9).
1082
Vol. 7
August
1993
of the neoglycoprotein
and asialoorosomucoid
(57.9
from
Pont-New England Nuclear (Boston, Mass,). Restriction enzymes were used according to the specifications of the manufacturer. Reverse transcriptase and polymerase chain reaction reagents were obtained from Perkins Elmer Cetus (Norwalk,
Production carriers A synthetic vine serum
glycoprotein carrier was developed in which boalbumin (BSA) was covalently bound to poly (L-
lysine) using 1-ethyl-3-(3-dimethylaminopropyl) mide (EDC). The entire structure glycosylated using D-galactose. Briefly,
carbodiiwas subsequently the reaction mixture
used to conjugate DNA contained 167 mM galactose, 4 mM poly (L-lysine) (Mr 3800 Da), 160 mM BSA, and 10 mM EDC in water (pH 7.5); this mixture was incubated for 48 h at 22#{176}C. The components were mixed from stock solutions, except for EDC, which was prepared immediately before use. The neoglycoprotein conjugate was complexed to plasmid DNA using a molar ratio of carrier (BSA) to DNA of approximately 360:1, based on the results of gel retardation
assays.
The
carrier-DNA
complexes
were
dialyzed
against 150 mM sodium chloride at 4#{176}C for 16 h before transfection. A more detailed description of the development of this conjugate will be presented elsewhere (J. Chen, D. R. Crawford, G. L. Lindberg, J. C. Perales, T. Ferkol, and R. W. Hanson, unpublished observations). The asialoorosomucoid carrier raminidase,
was prepared and linking
by treating
orosomucoid
with
neu-
the resultant asialoglycoprotein with poly (L-lysine) essentially as described by Wu and colleagues (10). The conjugate was purified, and stored at - 20#{176}C before
use.
Cell culture HepG2 human gle’s medium,
tal calf
serum.
and hormonal hepatoma supplemented Transfection
induction cells were grown in modified Eawith 5% calf serum and 5% fewas
performed
when
the
cells
4Abbreviations: BSA, bovine serum albumin; Bt2cAMP, N6, 20-dibutyryladenosine 3-5-cyclic monophosphate; CAT, chloramphenicol acetyltransferase; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; kB, kiobases; kDa, kiodaltons; PBS, phosphate buffered saline; PEPCK, phosphoenolpyruvate carboxykinase; PCR, polymerase chain reaction; RT, reverse transcriptase; pPFIXp, normal human coagulation factor IX.
The FASEB Journal
FERKOL ET AL.
METHODOLOGY by Southern
reached approximately 50% confluence. Each transfection experiment used up to 20 jig of plasmid in a total volume of 400 ji1. Fifty microliters of 1.0 M CaCl2 was added to the
analyzed
culture
Detection
medium
at
the
time
of
transfection
with
the
ne-
oglycoprotein of transfected
carrier-DNA complexes. Hormonal treatment and control cells was performed in serum-free media and in the presence of 0.5 mM Bt2cAMP and 1.0 mM theophylline or 1 jiM dexamethasone. Menadione (2-methyl-1,4-naphthoquinone), 500 ng/ml, was added to the media before induction because vitamin K is required for posttranslational modifications of factor IX. The cells were incubated for 3 to 24 h in the presence of the various hormones.
Animals Adult, male Sprague-Dawley rats, approximately 250 g in weight, were anesthetized with either. Using aseptic technique, 2.0 to 3.0 ml of a solution containing 400 to 500 jig of either plasmid pRSVCAT or pPFIXp complexed to the neoglycoprotein and asialoorosomucoid-based carrier was injected
into
pRSVCAT various
the portal vein. plasmid, rats were tissues were removed
ramphenicol
In the case of transfer of the killed 1 day after infusion and for the determination of chlo-
acetyltransferase
was performed immediately animals by removal of the
activity.
Partial
before injection medial lobe, which
protocol
Western mittee.
Reserve
was
reviewed
University
and
approved
Institutional
Animal
of human
polymerase The
chain
factor
using
a radiolabeled
presence
IX mRNA
the
reaction
of mRNA
transcripts
for
human
factor
IX in
the livers of rats transfected with pPFIXp Was determined after treatment of total cellular hepatic RNA with Moloney murine leukemia virus reverse transcriptase and amplifica-
tion of the resultant cDNA by the polymerase chain reaction. Briefly, 1 jig of total rat liver RNA was treated with 10 U DNase I (RNAse-free) and added to a solution containing 500 nM of(dT)16 oligonucleotide primer and 500 nM of each dNTP, and heated to 42#{176}C for 2 mm. Reverse transcriptase was added, the mixture was incubated for 30 mm at 42#{176}C, and I jil of the cDNA pool was amplified by the polymerase chain reaction, using primers within the hFIX cDNA (primers hFIX I and hFIX II). As a control, rat PEPCK mRNA transcripts were also amplified under similar conditions using primer TTGGCCAACAGGGGAAAG (PEPCK A), which corresponds to positions 32-50 in exon 1 of the PEPCK gene, and primer CCTGGCGTTGAATGCTTT (PEPCK C), which corresponds to positions 1057-1075 in exon 3. The expected size of the amplified cDNA generated
of selected accounts for
by
D
the
Care
.(J)
G)_ .2’
Case
Corn-
D
D
D
4-
9-
l-
ci)
(I)
and animal
ci)
(I)
cp_ .2C
Dr
DO
C
C()
DUJ DNA
with
probe.
hepatectomy
approximately 40% of the hepatic mass. Nontransfected mock transfected animals were used as controls. The research
cDNA
blot hybridization
DW
U)
cl)_ D0 CQ
W
Cl) (I)
cD_
.g’cc D C
LLi
probes
The following radiolabeled cDNA probes were used for DNA and RNA hybridization experiments: PEPCK, a 620 base pair Barn HI/Bgl II fragment of the 5’ region of the PEPCK gene; actin, a 1150 base pair Psi I digestion fragment of mouse /3-actin; and FIX, a 700 base pair Ava I/Hind III fragment from the 5’ region of the human factor IX cDNA. All DNA probes were labeled with [&2PJdCTP using the random oligo priming method as described by the manufacturer. The specific activity of probes labeled by this method was approximately 108_109 cpm/jig DNA.
Isolation
and analysis
of cellular
DNA
plasmid
3.5
and RNA
Genomic DNA and cellular RNA were isolated using standard techniques (11). Southern and Northern blot analysis and agarose gel electrophoresis were performed by established methods (12, 13).
Detection of the exogenous polymerase chain reaction
4.2
DNA using
4-2.6
kB.
2.0
the
One microgram
of genomic DNA was amplified in 100 il of buffer solution as recommended by the manufacturer. After incubating the solution at 94#{176}C for 7 mm to denature the genomic DNA, the DNA was amplified through 30 cycles using the following primers for human factor IX: GGTAAAT’IUGAAGAGTTTGTT (hFIXI), a primer to detect the 5’ end of the human factor IX cDNA corresponding to nucleotide positions 159-180, and A1DTTCTGTCTCCTCAATATT (hFIX Ii), a human factor IX antisense primer that corresponds to positions 857-878. Twenty microliters of amplified DNA was separated by electrophoresis using a 1.6% agarose gel, transferred to a nitrocellulose filter, and HUMAN
FACTOR IXGENE TRANSFER INTO HEPATOCYES
N
5d
lOd
15d
2. Analysis of transfected HepG2 human hepatoma cells for recombinant gene transfer. Total cellular DNA was extracted from HepG2 cells at 5, 10, and 15 days after transfection. Genomic DNA samples (20 g) from nontransfected and transfected cells were digested with Eco RI, and analyzed for the presence of foreign sequences by Southern blot hybridization using a radiolabeled FIX cDNA probe. Undigested genomic DNA samples were also examined. The autoradiograph was exposed to radiographic fIlm for 2 h. Figure
1083
METHODOLOGY from the rat PEPCK mRNA was 426 base pairs. The RNA isolated from livers of transfected, mock transfected, and nontransfected rats were evaluated. In addition, the same RNA samples not converted to cDNA by reverse transcriptase were also used as polynierase chain reaction templates as controls to ensure that contaminating plasmid DNA had not been amplified. The products were separated by agarose gel electrophoresis and Southern blot hybridization using a radiolabeled human factor IX or PEPCK cDNA probes. Measurement
of human
factor
IX
The relative concentration of human factor IX in the blood of animals treated with the neoglycoprotein complex was evaluated by Western blot analysis. Plasma samples (1.0 jil) were subjected to electrophoresis in SDS/I0% polyacrylamide gels and transferred onto nitrocellulose membrane filters using standard techniques. The blots were blocked with lx PBS, pH 7.4, 0.03% polyoxyethylenesorbitan monolaurate (Tween 20), and 10% (w/v) dry skim milk for 2 h at room temperature, followed by incubation with a 1/1000 dilution of a monoclonal murine anti-human factor
IX antibody
(3 jig/ml)
for 2 h at room temperature.
The
monoclonal antibody was kindly provided by Dr. Kenneth Smith (United Blood Services, Albuquerque, N. Mex.). The membrane was washed three times in lx PBS, pH 7.4 and 0.03% Tween 20, then incubated with a 1/500 dilution of goat anti-murine IgG (H + L)-horseradish peroxidase conjugate. The membrane was then washed vigorously four times with lx PBS, pH 7.4 and 0.03% Tween 20, and 10 ml of Western blot enhanced chemiluminescence detection solution was applied for 1 mm. The luminescence emitted from the filter was detected by a 10 s exposure to photographic film. The procoagulant activity of human factor IX was determined using a modification of the one stage, kaolin-
A
activated, partial thromboplastin time with factor IXdeficient human plasma (14). Blood samples were obtained from the experimental animals by venipuncture. One-fiftieth volume of 500 mM sodium citrate, pH 5.0, was added to prevent coagulation, and the plasma was stored at - 20#{176}C. The samples were assayed in duplicate, and their activity was compared to the functional activity of pooled plasma from 24 normal adult human males. In all calculations, one unit of factor IX activity in 1 ml of normal human plasma is equivalent to 100% functional activity or approximately 3 jig of factor IX per milliliter. Assay
for chloramphenicol
Chloramphenicol acetyltransferase activity was determined as described by Gorman et al. (15). After thin-layer chromatography, the radioactivity in the acetylated and nonacetylated chloramphenicol species was measured, and chloramphenicol acetyltransferase activity was expressed as the ratio of acetylated to nonacetylated radioactivity. Statistical
evaluation
Differences in human factor IX production between animals were compared using a paired Student’s I test. Data are expressed as the means ± standard deviations for the number of animals indicated in the figure legends.
RESULTS Transfer
of the PEPCK/human
hepatoma
factor
IX gene to HEPG2
cells
Initial experiments involved the in vitro transfer of plasmids expressing the human factor IX gene into human hepatoma
B
1234
28S
acetyltransferase
1
2
3
4
____ 69
.
FIX
60 kDa. -4
18S
F ‘4-
46
1.4kB.
30
Actin
Figure
‘4-
3. A) Analysis from
1084
Vol. 7
HepG2
cells 2 days
August
1993
HepG2
kB.
factor IX gene. Total cellular RNA was IX mRNA by Northern blot hybridization using radiolabeled FIX or actin cDNA probes. The following RNA samples (20 g) were analyzed: nontransfected HepG2 cells (lane 1), transfected cells (lane 2), and transfected cells 4 h after treatment with either dexamethasone (lane 3) or Bt2cAMP with theophylline (lane 4). Sizes based on the migration distances of the ribosomal RNA bands are noted in the left margin. The autoradiograph was exposed to radiographic film for 24 h. Densitometric scanning was used to quantitate the relative level of expression of human factor IX mRNA. B) Western blot analysis of human factor IX secreted into the culture medium of transfected HepG2 cells. After the addition of vitamin K3, media from three plates of identically treated cells was collected during a 24 h period and combined. Proteins in the pooled media were separated in a 10% SDS-polyacrylamide gel. The following media samples (50 jil) were analyzed: nontransfected HepG2 cells (lane 1), transfected cells (lane 2), and transfected cells after treatment with either dexamethasone (lane 3) or Bt2cAMP with theophylline (lane 4). Protein molecular weight standards are indicated in the right margin. isolated
of transfected
2.8
after
cells for expression
transfection
and examined
of the chimeric
for recombinant
The FASEB Journal
PEPCKJhuman human
factor
FERKOL E AL.
METHODOLOGY V
A
I-
.-
CM
.-
CM
B
NT
T 2h
.C V 0 CM CM .-
C
720
z-i-i--i.-
V
0 C’)
b.-*
T1d
T 2d C
4.2 13d
3.5
_j . I--J-JCI) D
T 1Od
z-i--
0.
947
831 720 b.-’ 564
T 30d
2.0
4-2.6kB.
r,
Undag
10 copies
EcoRl
Figure 4. A) Analysis of livers for the presence of the PEPCK/human factor IX gene. Total cellular DNA was extracted from the livers of rats at several times after infusion of the neoglycoprotein complex. Genomic DNA (20 g) from two transfected animals (Ti and T2), isolated one day after transfection, was digested with Eco RI and examined for the presence of the human factor IX gene by Southern blot hybridization using the radiolabeled FIX cDNA probe. Undigested DNA samples were also examined. Genomic DNA from a nontransfected rat (NT) was analyzed as a control. DNA size markers are reported in base pairs along the left margin. The autoradiograph was exposed to radiographic film for 48 h. B) Analysis of the PEPCKJhuman factor IX gene in livers of transfected rats by slot blot hybridization. Total cellular DNA (10 tg) from livers of rats 2 h (T 2h), 1 day (T id), 2 days (T 2d), 3 days (T 3d), 10 days (T lOd), and 30 days (T 30d) after injection of the neoglycoprotein complex were analyzed for the presence of the exogenous gene by slot blot hybridization. The latter two rats were also subjected to partial hepatectomy at the time of transfection. Genomic DNA from the liver of a nontransfected animal (NT) was also examined. Plasmid standard corresponding to approximately 10 copies per cell is shown. The autoradiograph was exposed to radiographic film for 24 h. C) Polymerase chain reaction amplification of hepatic DNA. One microgram of genomic DNA from livers of the rats 2 h (T 2h), 2 days (T 2d), 10 days (T lOd), and 30 days (T SOd) was amplified by PCR using specific human factor IX primers transferred to a nitrocellulose filter, and hybridized to a radiolabeled cDNA probe. In addition, genomic DNA from the liver of a nontransfected animal (NT) was examined. Molecular weights are reported in base pairs in the right margin. The autoradiograph was exposed to radiographic film for 1 h. D) Polymerase chain reaction amplification of genomic DNA from several tissues. One microgram of genomic DNA from liver (T Liver), lung (T Lung) and spleen (T Spleen) of a rat 2 days after transfection was amplified by PCR using human factor IX primers, transferred to a nitrocellulose filter, and hybridized to a radiolabeled cDNA probe. Genomic DNA from the liver of a nontransfected animal (NT Liver) was evaluated. Molecular weights are reported in base pairs in the right margin.
The
autoradiograph
was exposed
to radiographic
film
for 2 h.
cells using the neoglycoprotein carrier. HepG2 cells used in these experiments express the asialoglycoprotein receptor (J. C. Perales, T Ferkol, and R. W. Hanson, unpublished observations), permitting introduction of the neoglycoprotein carrier-DNA complex. Twenty micrograms of plasmid DNA was conjugated to the carrier and applied to cells in culture for 48 h, at which time genomic DNA was isolated from the transfected cells and examined by Southern blot analysis. Undigested genomic DNA produced distinct bands that corresponded in size to supercoiled, linear, and nicked forms of the plasmid (Fig. 2). Digestion with the Eco RI released the expected 2.6 kB fragment. The endogenous human factor IX gene was detected after longer exposure to photographic film. Otherwise, nontransfected and mock transfected controls did not have bands that were detectable by hybridization. The plasmid persisted in HepG2 cells for 10 days after transfection (Fig. 2).
HUMAN
FACTOR IX GENE TRANSFER INTO HEPATOCYTES
The expression and hormonal regulation of the PEPCK/human factor IX chimeric gene in transfected HepG2 cells were analyzed by Northern blot techniques. A 1.4 kB transcript, the expected size of human factor IX mRNA if transcription was initiated at the appropriate start site by the PEPCK promoter, was detected in total cellular RNA (Fig. 3a). Neither the transcript from the chimeric gene nor the endogenous PEPCK mRNA hybridized to the PEPCK cDNA probe. The FIX cDNA probe did not hybridize to RNA isolated from nontransfected and mock transfected control cells. Treatment with dexamethasone had a negligible effect on the expression of the chimeric PEPCKJhuman factor IX gene, whereas the addition of Bt2cAMP and theophylline induced expression 16-fold during the same time period. Western blot analysis of the culture media of transfected cells demonstrated the presence of the mature, fully processed human factor IX (Fig. 3b). The
1085
METHODOLOGY
A
Huma Fa
-__PEPCK
B
J-
IX
PolyA mRNAtransoipt
I
Non +
Moc2 -
+
-
cONA PCR
pdmers
I
-
Amptifled fragment
2
-
720 b.-*
720 b -1
Oro2 +
‘.
Neo2 +
-
-
947 831
#{149} 0;
Internal probe i-
Figure sion
5. The specificity
in the livers
of rats
564
376b-J
and duration of human factor IX expresafter transfection with the neoglycoprotein
C
complex. Hepatic mRNA from transfected rats was incubated in the presence (+) or absence (-) of reverse transcriptase. Using primers specific for either human factor IX or PEPCK, the resultant cDNA was amplified by polymerase chain reaction (PCR). The reaction products were separated by electrophoresis in a 1.6% agarose gel, and analyzed by Southern blot hybridization using a human factor IX or PEPCK radiolabeled cDNA probe. The sizes
of the amplification
products
are indicated
in the left margin.
Non
NeolO
Neo3O
831 720 b.-*
,
A)
Schematic diagram of the PEPCKJhuman factor IX gene, polymerase chain reaction amplification primers, and human factor IX probe. B) Evaluation of human factor IX mRNA. Non: hepatic
564
RNA from nontransfected rat; Moc2: hepatic RNA from mock transfected rat; Oro2: hepatic RNA from rat 2 days after transfection with asialoglycoprotein complex; Neo2: hepatic RNA from rat 2 days after transfection with neoglycoprotein complex. C) The duration of expression of human factor IX. Non: hepatic RNA from nontransfected rat after partial hepatectomy; NeolO: hepatic RNA from rat 10 days after partial hepatectomy and transfection with neoglycoprotein complex; Neo3O: hepatic RNA from rat 30 days after transfection with neoglycoprotein complex and partial hepatectomy. D) Evaluation of PEPCK mRNA. Non: hepatic RNA from nontransfected rat after partial hepatectomy; NeolO: hepatic RNA from rat 10 days after partial hepatectomy and transfection with neoglycoprotein complex; NeoSO: hepatic RNA from rat 30 days after transfection with neoglycoprotein complex and partial hepatectomy.
amount of human factor IX in the media detected by Western blot hybridization served changes in the factor IX message.
Introduction
of the PEPCK/human
of transfected cells paralleled the ob-
factor
IX gene into
rats The neoglycoprotein DNA packaging system was used to transfer the chimeric PEPCKJhuman factor IX gene into the livers of intact animals. Rats were anesthetized, and 400-500 jig of plasmid was complexed to the neoglycoprotein carrier and infused slowly into the hepatic portal vein over several minutes. For comparison the plasmid was also complexed to an asialoglycoprotein carrier, as described by Wu and his colleagues (10, 16-19), and injected into the portal vein. Mock transfections of animals (carrier alone, DNA alone, polylysine complexed to the plasmid, and the carrier complexed to an irrelevant plasmid) were also performed. We established the presence of human factor IX DNA in the liver 1 day after injection of the neoglycoprotein-DNA complex by Southern blot analysis of total cellular DNA digested with Eco RI, which yields the intact PEPCKIhuman factor IX gene (Fig. 4a). Undigested DNA showed three prominent bands corresponding to the nicked, linear, and supercoiled forms of the vector, indicating that much of the retained DNA that was transferred into the liver existed as
1086
Vol. 7
August
1993
D
Non +
NeolO +
-
-
Neo3O +
517 453 426b.-* 394
an episome. A portion of the transferred DNA also seemed to be degraded. The digested DNA produced the expected 2.6 kB band that hybridized with the FIX cDNA probe. The difference in the intensity of the signal shown in Fig. 4a is most likely due to the variability in the efficiency of gene transfer between the two animals. Large amounts of hybridizable DNA were present in the livers of rats 2 h after transfection, whereas tained markedly less
analysis plasmid,
portion
DNA
of transferred
at subsequent time points conimplying that a considerable was
destroyed
in the
hepatocytes
(Fig. 4b). Human factor IX DNA sequences were detected as long as 30 days after injection in the livers of transfected animals by polymerase chain reaction amplification, using primers specific for the human factor IX cDNA (Fig. 4c). Amplification of DNA extracted from the lungs of rats 2 days after transfection using polymerase chain reaction did not demonstrate the transferred gene. However, human factor IX sequences were found in the spleen, which suggests that a portion of the DNA-carrier complex may be extracted from the circulation by the reticuloendithelial system. No human factor IX DNA was found in the livers of nontransfected controls. The mRNA transcripts for human factor IX were detected in the livers of transfected rats by treating total cellular RNA with reverse transcriptase and amplifying the cDNA generated by the polymerase chain reaction (Fig. 5a),
The FASEB Journal
FERKOL Er AL.
METHODOLOGY sient
of transgene expression is consistent with the g’ observations by Wu and colleagues (17). Furthermore, deliv.#{231} ery of the exogenous gene to hepatocytes appears to be a .2 specific process, because mock transfections using polylysine-DNA complexes injected into the hepatic portal
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vein of rats failed to result in the uptake and expression of the transgene. In a single experiment, exp,ression of the chimeric
PEPCK/human
factor
IX gene was blocked
by infus-
ing a fourfold molar excess of galactosylated albumin, which competes with the carrier for the asialoglycoprotein receptors on the surface of hepatocytes, before injection of the complex. A factor IX activity level of 13.8% was measured in the plasma of this animal 2 days after treatment. The pPFIXp plasmid was complexed to the neoglycoprotem conjugate and infused into the hepatic portal vein of rats after partial hepatectomy. Functional human fac-
.
tor IX was measured
.
at significant
levels in several
days after injection. The levels returned to baseline day 60 (Fig. 8a and Fig. 8b). No significant increase
IX activity
rats 30 values by in factor
was found in the blood of nontransfected,
control
animals results
Figure
6. Tissue specificity and expression of pRSVCAT ininto rats using the neoglycoprotein carrier. Five hundred micrograms of the plasmid pRSVCAT complexed to the neoglycoprotein carrier was infused into the portal vein of a rat that was not subjected to partial hepatectomy. The animal was killed I day after transfection, and the tissues indicated in the figure were assayed for CAT activity as described in the Methods. No CAT activity was detected in the tissues of nontransfected animals. troduced
using oligonucleotide or for PEPCK. All presence and absence
IX mRNA days after transfected
primers specific for human factor IX RNA samples were evaluated in the of reverse transcriptase. Human factor was detected in the livers of rats 2, 10, and 30
transfection, animals
IX mRNA
transcripts
but
(Fig.
not
in
5b and
were noted
NT
nontransfected or mock 5c). No human factor
Fig.
in the absence
of reverse
transcriptase. The mRNA from the endogenous PEPCK gene was identified in the corresponding samples (Fig. 5d). In our initial in vivo experiments, the neoglycoprotein carrier was complexed to pRSVCAT and injected into the portal vein of rats, and various tissues were analyzed for CAT activity 1 day after transfection (Fig. 6). Chloramphenicol acetyltransferase activity was found exclusively in the liver of transfected animals; no activity was found in the other tissues examined. The plasmid pPFIXp was also complexed to the neoglycoprotein carrier and injected into the hepatic portal vein of rats. Baseline factor IX levels of 13.2% and 10.7% activity was detected in the blood of nontransfected and mock transfected control animals, respectively, which probably reflects the endogenous factor IX in the rat. The degree of sequence identity of factor IX among mammalian species is relatively high (20). Nevertheless, the factor IX levels measured in the blood of rats transfected with either the neoglycoprotein (P = 0.01) or asialoorosomucoid carrier (P = 0.02) were significantly greater than that found in nontransfected or mock transfected animals as measured by the functional activity assay (Fig. 7) and Western blot hybridization. No statistically significant difference in the production of factor IX was detected using the two methods of receptormediated gene transfer (Fig. 7). The duration of expression of the transferred gene in rats was relatively short-lived in animals with intact livers, lasting less than 5 days. The tranHUMAN
after partial hepatectomy. Figure 8b shows the of transfection experiments in which three different preparations of the neoglycoprotein carrier were used and demonstrates the variability of this method in vivo. Only 50% of the transfected rats had factor IX levels greater than the 95% confidence interval of the control animals. However, this variability in transgene expression using the neoglycoprotein conjugate was similar to what we found using the asialoorosomucoid-based carrier. The onset of expression was also delayed in these animals, the cause of which is unclear. This lag in expression has been observed with other chimeric genes driven by the PEPCK
FACTOR IX GENE TRANSFER INTO HEPATOCYTES
MT
Neo
Oro 0
5
10
15
20
Factor IX Percent Functional Activity Figure
7. Expression of the gene for PEPCK/human factor IX in rats treated with the neoglycoprotein carrier. The biological activity
of human factor IX was measured in plasma from animals two days after injection with the either the neoglycoprotein (neo; is - 4) or asialoorosomucoid-based (oro; is 3) carrier complexed to 400 ig of the plasmid pPFIXp. Plasma from animals that were not transfected (NT; is 8) or mock transfected (MT; is - 4) with an irrelevant plasmid (pRSVCAT) were also analyzed. Levels of circulating human factor IX were determined by a one-stage clotting assay as described in the Methods. The values are expressed as the mean ± standard deviation for the number of animals indicated. 1087
METHODOLOGY
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