(Received for publication, October 12, 1994)
From the
In this report we describe a novel gene delivery system using
malaria circumsporozoite (CS) protein as a specific ligand. The CS
protein covers the entire surface of sporozoites of malaria parasites.
Previous studies have demonstrated that intravenously injected CS
protein binds specifically to the basolateral surface of hepatocytes
within minutes, indicating the high hepatocyte specificity of CS
protein. This characteristic of CS protein prompted us to explore the
possibility of using this protein as a liver-specific ligand for
hepatic gene delivery vehicle. As an initial step, we investigated the
efficacy of CS protein-mediated gene transfer into primary hepatocytes
as well as established cell lines. Recombinant CS proteins were
chemically conjugated to poly(L-lysine). The CS conjugates
were complexed with recombinant plasmid DNA carrying a reporter gene.
When the DNA complex was used to transfect primary hepatocytes, a very
low level of expression of the reporter gene was observed. The level of
expression was greatly enhanced when the cells were cotransfected with
adenovirus, which presumably releases the internalized DNA from
endosomal entrapment. The CS-mediated gene transfer into the cells
required region II+, an evolutionarily conserved amino acid
sequence conferring the binding of CS protein to its receptor. CS
protein also efficiently mediated gene transfer into a number of cell
lines, i.e. HepG, HeLa, NIH3T3, and K562, but not
HL-60, which contains low levels of receptor. Thus, the CS conjugate
can be used to deliver DNA into many different cultured cells. Most
importantly, the CS conjugate has a potential to be further developed
into a liver-specific gene delivery vehicle in vivo.
There has been an increasing interest in the development of gene therapy using receptor-mediated gene delivery systems in recent years (1) . Receptor-mediated gene targeting takes advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific(2) . Furthermore, in comparison with viral delivery systems(1) , receptor-mediated gene delivery allows a greater flexibility of DNA in terms of size and sequence selections because the DNA to be delivered does not need to be packaged into viral capsids, avoiding tedious clonal selection and virus production processes. These characteristics make the system attractive to human gene therapy.
Receptor-mediated gene targeting vehicles consist of two components:
a cell receptor-specific ligand and a polycationic moiety, e.g. polylysine. The polycationic moiety serves as an intermediate for
electrostatic interaction with DNA, resulting in the formation of
toroid complex when the negative charge of the DNA molecule is
completely neutralized. The toroid complex can be internalized via
normal receptor-mediated endocytosis. Several ligands have been used
for receptor-mediated gene transfer. The most extensively characterized
ligands are asialoorosomucoid (ASOR) ()(3) and
transferrin(4) . Recently, a synthetic neoglycoprotein that
recognizes the same receptor as ASOR has been used as a gene delivery
vehicle(5, 6) . In vivo, the distributions of
ASOR and transferrin receptors are very different. Transferrin
receptors are found in many different cell types, while ASOR receptors
are almost exclusively distributed on the sinusoidal domain of the
hepatocytes(3) . Because of its unique distribution, much
attention has been focused on the development of ASOR as a hepatic gene
delivery vehicle; however, the efficiency of ASOR-mediated hepatic gene
delivery system has not been perfected.
To fully develop the capacity of receptor-mediated gene targeting, it is necessary to explore as many cell-specific ligands as possible. The malaria circumsporozoite (CS) protein is a potential candidate for hepatic gene delivery. Malaria is transmitted by the bite of infected female anopheles. Minutes after infection, the malaria sporozoites invade hepatocytes. This liver-specific invasion is mainly attributed to the circumsporozoite protein (CS), which densely coats the outer surface of sporozoite(7, 8) . CS protein recognizes its receptor that is predominantly distributed at the basolateral domain of hepatocytes (9) . The specificity of cell recognition and rapid invasion prompted us to explore the possibility of using this protein as a liver-specific ligand for hepatic gene delivery vehicle.
The initial work has to establish the formulations of CS-polylysine carrier for the efficient delivery of DNA into primary hepatocytes. We report here that CS conjugates can efficiently deliver recombinant plasmid DNA into primary hepatocyte cultures as well as into a number of established cell lines, demonstrating the potential application of this molecular conjugate for in vitro and in vivo gene delivery.
CHO, HeLa, NIH3T3, and HepG2
cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% FCS. HL-60 and K562 cells were maintained in RPMI
1640 medium containing 10% FCS. Cells were maintained in a 37 °C
incubator containing 5% CO in air.
Prior to protein
uptake assay, the culture medium was replaced with 0.5 ml of serum-free
medium. 2.5 µg of [H]CS27IVC-His
were added to the culture. The cells were incubated at 37 °C
in 5% C0
incubator. At indicated time intervals, the medium
was removed. The cells were washed three times with phosphate-buffered
saline and lysed with 0.5 ml of 0.5 N NaOH. The radioactivity
of cell lysate was counted in Scintiverse (Fisher) by a liquid
scintillation counter (Beckman LS3801).
To determine uptake of CS
conjugate-DNA complex, pCMV--gal plasmid DNA was nick-translated
with [
P]dCTP using nick translation kit (Life
Technologies, Inc.) according to the protocols provided by the vender.
The labeled DNA was purified by Sephadex G-25 chromatography. Ten ng of
P-labeled DNA was mixed with 2.99 µg of unlabeled
pCMV-
-gal DNA. The DNA was then mixed with 3.7 µg of CS
conjugate in a total volume of 500 µl of HBS. The formation of DNA
complex was checked by gel retardation assay on a 0.8% agarose gel. The
DNA complex was then diluted to 1 ml with serum-free medium and added
to 5
10
primary hepatocytes or HL-60 cells. The
cells were incubated at 37 °C in 5% CO
incubator. At
different time intervals, the medium was removed and the
radioactivities in the cultured cells were determined.
Figure 1:
Schematic representation of the
recombinant CS proteins. The major functional regions of CS protein,
region I (RI), region (II+), and repeat region (REPEATS), which contains 21 (NANP) amino acid residues are
indicated. The numbers shown above the constructs represent
amino acid sequences from P. falciparum CS protein.
DHFR-CSF1-His is the fusion protein of mouse DHFR and CS
peptides. The 18 amino acid residues shown below RII+ are the
epitope recognized by receptor.
Figure 2:
Purification of CS/polylysine and
transferrin/polylysine conjugates. CS27IVC-His/polylysine
and transferrin/polylysine were conjugated by EDC. The reaction
mixtures were then passed through a Superose 6 column. Details are
described under ``Materials and Methods.'' A,
chromatographic eluting profiles of
[
H]CS27IVC-His
and
CS27IVC-His
/polylysine conjugate. B, eluting
profiles of FITC-polylysine and FITC-polylysine conjugated by EDC. The
fluorescence intensity was determined by a fluorometer at excitation
490 nm and emission 530 nm. C, the elution profiles of
transferrin and transferrin/polylysine conjugate. D and F, acid-urea gel electrophoretic analyses of proteins eluted
from the CS/polylysine and transferrin/polylysine, respectively.
Unconjugated CS protein transferrin (Tf), polylysine (PLL), and a mixture of CS and PLL (CS/PLL)
were run as references. E and G, transfection
efficiencies of protein fractions corresponding to the eluting profiles
shown in D and F. Six µg of pCMV-
-gal
plasmid DNA were complexed with 5.8 µg of CS-PLL conjugate from
each fraction in D and 4.2 µg of transferrin-PLL from each
fraction in E. DNA complexes were used to transfected HepG2
cells in the presence of adenovirus (5
10
particles/cell). The results were calculated from duplicated
samples, and 800-1,000 cells in three random fields were
counted.
Aliquots from each fractions were determined by acid-urea polyacrylamide gel electrophoresis. Under these electrophoretic conditions, free polylysine and polylysine-polylysine conjugates (not shown) migrated more rapidly than did unconjugated CS protein and CS-polylysine conjugate (Fig. 2D), because of their high contents of positive charges. As shown in Fig. 2D, fractions eluted in the front peak (fractions 7-10) of the CS conjugate sample contained no detectable free polylysine, whereas, as expected, polylysine and/or polylysine-polylysine conjugates were only seen in the second peak (fractions 11-20). Proteins eluted in the front peak had a very slow mobility in this gel system, i.e. barely entering the stacking gel. These results suggest that the molecular size of these protein conjugates are rather large. The second peak also contained CS-polylysine conjugates, as suggested from their mobilities in the acid-urea gel electrophoresis (Fig. 2D, arrows). However, under the optimized transfection conditions (see below), only the conjugates from the front peak showed transfection activities (Fig. 2E).
The exact
molecular mass of the conjugates in these two peaks have not been
vigorously determined. The molar ratios of CS:polylysine in these types
of conjugate preparations were not known. However, the results shown
here were highly reproducible (five independent experiments).
Furthermore, similar results were obtained in the preparations of
functional transferrin- (Fig. 2C, F, and G) and ASOR-poly-L-lysine ()conjugates.
Thus, we conclude that this simple gel filtration chromatographic
technique can be used to prepare functional molecular carriers for gene
delivery.
To demonstrate that the CS-mediated DNA transfection seen in the cultured primary hepatocytes was ligand-dependent and to optimize conditions for the transfection efficiency. Two parameters were investigated: CS conjugate to DNA ratio and the concentration of adenovirus. Six µg of reporter DNA were complexed with various amounts of CS conjugate. Hepatocyte cultures were transfected with these complexes. Fig. 3shows that a maximal transfection efficiency of about 80% was achieved when 0.14 nM of CS conjugate was used, corresponding to a CS conjugate:DNA ratio of 116:1. This optimal ratio of CS conjugate to DNA was comparable with those published using transferrin (4) and ASOR (15) conjugates. In contrast, within the same ranges of carrier:DNA ratios, BSA conjugates prepared under the same conditions showed no better than 10% of transfection efficiency. Likewise, less than 10% of transfection efficiency was seen with polylysine as a carrier. These results suggested that the transfection efficiency seen in the CS carrier is ligand-dependent.
Figure 3:
Effects of CS-polylysine conjugate
concentrations on the expression of -galactosidase in primary
hepatocytes. Different amounts of CS conjugate, BSA conjugate, and
polylysine were complexed with 6 µg of pCMV-b-gal DNA as described
under ``Materials and Methods.'' Primary hepatocytes (3
10
) were transfected with the complexes in the
presence of adenovirus (4
10
particles/cell). The
cells were stained with X-gal, and percentage of blue cells were
determined by visually counting 800-1000 cells from three random fields
for each duplicated samples.
It is of importance to investigate whether conjugates prepared by various ligand:polylysine:EDC ratios would affect the biological activities. To this end, we carried out conjugation reactions with fixed concentration of CS protein with increased concentrations of polylysine. To derive the reactions to completion because of increased polylysine, EDC concentrations were increased proportionally. Table 1showed that conjugates prepared at the 1:1 to 1:2.5 molar ratios of CS protein:polylysine, greater than 80% of transfection efficiencies were achieved. However, the transfection efficiency was drastically reduced at molar ratio 1:5.0. The reason of this reduced transfection efficiency could be due to overmodification of the CS protein with polylysine or EDC, thereby reducing the affinity of CS protein to its receptors.
To determine the optimal viral concentration for
transfection, conjugate:DNA complex at a ratio of 116 to 1 was added to
primary hepatocyte cultures followed by addition of different amounts
of virus particles. As shown in Fig. 4, increasing adenovirus
particles resulted in increased transfection efficiency with optimal
amount of viral concentration of 4 10
particles/cell under the transfection conditions. Additional
viral particles resulted in decreased transfection efficiency, probably
due to excess cytotoxicity of viral infection. The viabilities of
transfected cells decreased as the viral concentrations increased.
Under the optimal transfection conditions, about 58% of cells were
viable (or 66% after correcting against the plating efficiency, which
was about 90%).
Figure 4:
Effects of adenovirus concentration on the
expression of -galactosidase in primary hepatocytes.
CS27IVC-His
conjugate (7.4 µg) was complexed with 6
µg of pCMV-
-gal DNA. Indicated amount of adenovirus was added
to the cells after the addition of the complex. Eighteen hours after
transfection, cell viability, and
-galactosidase activity were
determined by trypan blue exclusion and X-gal stains, respectively.
Data were collected by counting 800-1000 cells from three random fields
for each triplicated sample.
Since substantial cell death was found at the
optimal adenovirus concentration of CS-mediated gene transfer, it was
of importance to investigate whether there is a causal relationship
between adenovirus-induced cytotoxicity and the reporter gene
expression. To this end, we carried out the following kinetics study.
Primary hepatocytes in cultures were transfected with CS conjugate-DNA
complex in the presence of adenovirus. At different time intervals, the
cultures were terminated and expression of reporter gene and cell
viability were determined. As shown in Fig. 5, expression of
-galactosidase appeared 6 h after transfection, reached its
maximum around 15 h, and decreased thereafter. On the other hand, cell
viability decreased as the transfection time increased in a linear
pattern. At the time when the reporter gene started to express (6 h
after transfection), 80% of cells were still viable, while at the
maximal levels of expression, 60%. Virtually no viable cells were
detected after 48 h of transfection. These results suggest that the use
of adenovirus as an enhancer for reporter gene expression in
receptor-mediated gene delivery caused important toxicity.
Figure 5:
Kinetics of -galactosidase expression
and cell viability after transfection with DNA complex.
CS27IVC-His
conjugate (7.4 µg) was complexed with 6
µg of pCMV-
-gal DNA. The transfection of primary hepatocytes
was initiated (time 0) by the addition of the DNA complexes and
adenovirus (4
10
particles/cell). At different time
intervals, percentage of blue cells (
-galactosidase expression)
and cell viability were determined as described in the legend to Fig. 4. The plating efficiencies of hepatocytes were also
included. The data were not normalized against the plating
efficiencies.
Figure 6:
Expression of -galactosidase in
primary hepatocytes after transfected with DNA complexes containing
different recombinant CS proteins. CS conjugates containing
CS27IVC-His
(b), CS27IC-His
(c), and DHFR-CSF1-His
(d) were
complexed with 6 µg of pCMV-
-gal DNA. The amount of CS
conjugate used were experimentally determined in pre-experiments.
Transfection was carried out in the presence of adenovirus (4
10
particles/cell).
-Galactosidase was determined by
X-gal staining. Six µg of pCMV-
-gal DNA was used as control (a).
To confirm the importance of region
II+ in CS-mediated gene delivery, a peptide containing region
II+ sequence of CS protein (E35) was used as a competitor for the
CS-mediated gene transfer. Different amounts of E35 peptide, ranging
from 0.031 to 0.248 µM, were added to the transfection
mixture. The efficiencies of transfection were analyzed in comparison
with those using an unrelated peptide (A128) with different sequence
but the same length as competitor. In the presence of 0.125 µM E35 peptide, greater than 50% of -galactosidase activity was
inhibited and the activity was completely ablated when 0.248 µM of E35 was added. However, at the comparative concentrations,
peptide A128 showed no competitive effect (Fig. 7). These
results further supported the notion that region II+ of CS protein
contains functional residues for gene transfer.
Figure 7:
Influence of region II CS peptide on the
expression of -galactosidase in primary hepatocytes.
CS27IVC-His
conjugate (7.4 µg) was conjugated to 6
µg of pCMV-
-gal DNA. Different amounts of E35 and A128
peptides were added to cells after the addition of DNA complexes and
adenovirus (4
10
particles/cell).
-Galactosidase activity was quantitated using ONPG as a substrate.
Results were from triplicate experiments.
Figure 8:
Expression of -galactosidase in
different cell lines transfected with CS carrier. CS27IVC-His
conjugate (7.4 µg) was complexed with 6 µg of
pCMV-
-gal DNA. The DNA complex was used to transfect HeLa, NIH3T3,
CHO, Hep G2, and HL-60. The viral concentration for each cell line was
optimized.
-Galactosidase activity was quantified as described
under ``Materials and Methods'' using ONPG as a substrate.
Data were average of triplicated
experiments.
The inability of CS
protein-mediated gene transfer into HL-60 cells could be due to the
absence of either adenovirus receptor or CS receptor, or both. To
address the adenovirus receptor issue, we transfected HL-60 cells with
recombinant -galactosidase adenovirus (ranging from 5
10
to 1
10
particles/5
10
cells). We found that this recombinant adenovirus has very poor
transfection efficiency (<1%) in HL-60 cells, in comparison with
greater than 95% transfection efficiency in HepG2 cells (data not
shown). Although there may be other reasons for the inability of the
expression of reporter gene in the adenovirus-infected HL-60 cells, the
possibility of lacking adenovirus receptors in these cells could not be
formally ruled out.
To address the issue of CS receptors in HL-60
cells, we determined the rates of
[H]CS27IVC-His
uptake in primary
hepatocytes and in HL-60 cells. Since it has been demonstrated that
multimeric CS proteins were more efficiently uptaken than monomeric
proteins by the liver parenchyma(29) ,
H-labeled
multimeric CS fractions (the first peak, Fig. 2A, dashed line) were used for the uptake study. As shown in Fig. 9, the levels of labeled CS protein uptake by HL-60 cells
were significantly lower than those in the primary hepatocytes,
suggesting that HL-60 cells containing reduced levels of CS receptors.
To substantiate these observations, we also measured the uptakes of DNA
mediated by CS protein conjugates in these two different cell types,
using transferrin-conjugates and BSA conjugates as positive and
negative controls, respectively. As shown in Fig. 10, the
transferrin-DNA complexes were efficiently uptaken by both primary
hepatocytes and HL-60 cells. However, the levels of CS-DNA uptake were
significantly lower in HL-60 cells than those in primary hepatocytes.
These results are consistent with the findings that the cell-type
specificities of CS-mediated gene transfer are correlated with the
presence of functional CS receptors.
Figure 9:
Uptake of CS27IVC-His by
primary hepatocytes and HL-60 cells.
[
H]CS27IV-His
(2.5 µg) was mixed
with 0.5 ml of serum-free medium and added to 5
10
cells. At different time intervals, the medium was removed. The
cells were washed three times with phosphate-buffered saline and lysed
with 0.5 ml of 0.5 N NaOH. The radioactivity was measured by a
liquid scintillation counter.
Figure 10:
Uptake of DNA complex by primary
hepatocytes (A) and HL-60 cells (B).
CS27IVC-His (3.7 µg), transferrin (2.9 µg), and BSA
(2.75 µg) conjugates were complexed with 3 µg of
P-labeled DNA prepared by mixing 10 ng of nick-translated
DNA with 2.99 µg of cold DNA. The complexes were added to 5
10
cells. Data were from triplicate
samples.
Previous study has demonstrated that CS protein binds to heparan sulfate proteoglycans associated with the surface membrane of hepatocytes, and the binding can be abolished by heparinase treatment (26, 27) . These observations suggested that the CS receptor(s) may be heparan sulfate-related proteoglycans. In an attempt to determine whether the CS-mediated gene transfer into primary hepatocytes could be blocked by heparin and dextran sulfate (by competing CS-conjugate binding to receptors), however, we found that these chemicals cause dissociation of DNA from the CS conjugate, as judged by gel-retardation assay (not shown).
CS protein has been known to play an important role in the invasion of liver by malaria parasite(7, 8) . It is known that this hepatic invasion is nonpathogenic and malaria symptom manifests during erythrocyte cycle(8) . Much attention to this protein in the malaria field is in its potential use as a target for intervention of malaria infection, particularly in the vaccine development(28) . The specificity of CS protein for hepatocytes described in those studies (7, 29) raised a distinct possibility that CS protein may be used as an effective vehicle for hepatic gene targeting. For this reason, we initiated the present study using primary hepatocyte cultures. We believe that this in vitro study is a prerequisite for future in vivo experimentation. Using this in vitro system, we have made several important observations.
First, we demonstrated that CS
protein can be utilized as a carrier to deliver recombinant DNA into
primary hepatocytes. The expression of -galactosidase reporter
gene required the presence of adenovirus. Adenovirus is believed to
function as an endosomal disrupting tool, facilitating the release of
internalized DNA after endocytosis. These results strongly suggest that
CS protein, like the other molecular carriers such as ASOR and
transferrin, undergoes a similar endosome-lysosome pathway after
internalization by endocytosis. Although it has been generally believed
that malaria infection is receptor-mediated, there is no direct
evidence regarding how the sporozoites invade hepatocytes.
Understanding this endocytic pathway of invasion may facilitate
research aiming at control of malaria infection.
Second, we demonstrated that CS-mediated gene transfer into primary hepatocytes require the presence of region II+ but not the NANP repeats. These observations have important implications in the use of CS protein as a liver-specific gene targeting carrier. These repeats are immunodominant and confer the major antigenicity of CS protein. Human sera from P. falciparum malaria parasite-infected individuals in endemic areas contained mainly antibodies against the CS repeats(7, 8) . On the other hand, antibody against region II+ has not been detected in the human sera of malaria-infected individuals. Moreover, antibodies to region II have been difficult to elicit by immunization of mice and rabbits(31) . The fact that deleting the immunodominant domain did not compromise the ability of gene transfer (Table 2) suggests that repeat-free CS protein can be used as a ligand for gene delivery with reduced immunogenic complications to the hosts.
Region II+ is the epitope for receptor binding(7) . It contains evolutionarily conserved amino acid residues with homology to cell adhesive motif found in several other proteins, including thrombospondin(32) , properdin(33) , and a blood-stage antigen of P. falciparum(33) . The observation that region II+ peptide can block the CS-mediated gene transfer (Fig. 7) raises the possibility that the synthetic peptide containing region II+ residues may be used as a ligand for gene targeting via CS receptor. Experiments testing this possibility are under way.
Third, we showed that in addition to primary hepatocytes, CS-protein could elicit gene transfer into many established cell lines, including HeLa, NIH3T3, CHO, HepG2, and K562 cells (Fig. 8). We believe that this is not due to alteration of CS protein configuration by polylysine conjugation that resulted in changes in ligand specificity for the following reasons. (i) The transfection is ligand-dependent; very low transfection efficiency was seen using BSA conjugates or polylysine as carriers (Fig. 5). (ii) The transfection efficiencies in different cell types were correlated with the levels of cellular receptor(s); very low transfection efficiency was found in HL-60 cells in which receptor levels were low ((25) , and Fig. 9and Fig. 10). (iii) The transfection efficiencies were region II+-dependent, consistent with the binding specificity of CS protein to its receptor(7) . These observations suggest a broad applicability of CS protein as gene delivery vehicle for cultured cells. In this regard, CS protein may be utilized for gene transfer into patient-derived cell cultures in the ex vivo gene therapy.
The observation that CS protein could
effectively deliver recombinant DNA into primary hepatocytes described
in this report suggests that it can be a potential carrier for hepatic
gene targeting. Although the present study showed that the efficient
delivery requires adenovirus with the titer as high as 10 cytotoxic particles/cell, the same efficacy of gene delivery may
be achieved by coupling of adenovirus to DNA
complex(22, 24, 34, 35) , or using
cripple adenovirus particles (36) or less toxic adenovirus (37) thereby reducing the requirement of viral particles for
endosomal lysis. Another concern of using adenovirus is the safety
issue and should be critically evaluated, although the recent study
showed that no major cytotoxic effects were found in the rat liver
after adenovirus-mediated in vivo gene transfer(38) .
However, a recent human trial of recombinant adenovirus containing the
human cystic fibrosis transmembrane conductance regulator gene into the
CF respiratory epithelium with dose up to 2
10
plaque-forming units showed toxic effects to one of the four
patients, including local and systemic inflammatory
syndrome(39) . Alternatively, viral fusogenic peptides can be
used for substitution of adenovirus, since influenza hemagglutinin HA-2
terminal peptides have been demonstrated to augment gene transfer
efficiency greater than 100-fold by the receptor-mediated gene transfer
into HeLa cells(40, 41) . In addition, recent studies
in protein trafficking and endosomal physiology have identified several
endogenous proteins associated with vesicle budding, membrane fusion (42) , and protein translocation(43) . Upon further
development, these proteins could be potential tools for enhancing the
expression of transgenes in the receptor-mediated gene delivery system.
Additional concern regarding hepatic DNA delivery systems is the structural requirements of the delivery vehicles. In the in vivo setting, the size of the DNA complex must be <200 nm to pass the fenestrations in the liver parenchyma. Although the exact size of CS carrier-DNA complexes used in the present study was not determined, previous study using ASOR-polylysine-DNA complex prepared under the similar procedure gave molecular sizes of 80-100 nm(15) . This size range may be acceptable for in vivo trails. Perales et al.(6) have recently reported that, by changing salt conditions, it is possible to modulate the sizes of galactosylated poly(L-lysine)-DNA complex (to about 10 nm). The formulation of such small DNA delivery system was found to correlate with the prolonged expression of transgene in the livers of intact animals.
Most of our current knowledge about receptor-mediated gene targeting to livers has been derived from the studies of ASOR receptors. Using this delivery system, Wu and associates (44) have demonstrated a partial correction of genetic analbuminemia in Nagase rats by intravenous injection of complexed DNA containing the human serum albumin gene under the control of the mouse albumin enhancer/promoter. In another study, target expression of low density lipoprotein receptor gene into the receptor-deficient rabbits was demonstrated(45) . The levels of expression of the transgenes in these animals remained relatively low. It is conceivable that the efficacy of ASOR-mediated hepatic gene transfer is influenced by the physiology of ASOR and its receptors(46, 47) . The synthesis and bioavailability of ASOR receptors and their circulating ligands can drastically affect the efficacy of gene delivery. The use of CS protein, which is not normally present in the host, may minimize possible interference by the endogenous ligand (if there is any), thereby enhancing the targeting efficacy. The CS protein carrier may be particularly useful in individuals with hepatitis(48) , liver cirrhosis(49) , hepatocellular carcinomas(49, 50) , and diabetes mellitus(51) . These individuals suffer from accumulation of ASOR due to down-regulation of ASOR receptor synthesis.
The development of CS conjugate as DNA carrier, in complement to the existing ASOR carrier system, should allow one to test the synergism in the targeting specificity and enhancement of expression of the dual delivery systems. The availability of additional hepatic DNA targeting system would allow one to distribute the ``payloads'' into different vehicles and thus facilitate the efficacy of targeting. From the economic point of view, the production of CS protein carriers should be considerably less expensive than the ASOR carrier. The gene encoding the CS protein has been cloned and bacterially expressed recombinant proteins are available. In contrast, because of the low abundance of ASOR in blood, a large volume of blood is required to extract sufficient quantities of the ligand. This not only increases the cost of production, it also creates the potential risk of viral contamination in the ligand preparations, as in the case of many other blood-derived products. The use of CS conjugates should impose minimal risk to patients and to the general population.
In conclusion, the present study established that the CS protein can be an effective system for gene transfer into primary hepatocyte cultures as well as into many different cultured cell lines. These findings provide a basis for further in vivo studies of hepatic gene delivery. These experiments are currently under way.