©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Malarial Circumsporozoite Protein Is a Novel Gene Delivery Vehicle to Primary Hepatocyte Cultures and Cultured Cells (*)

(Received for publication, October 12, 1994)

Zhi-Ming Ding (1) Richard J. Cristiano (2) Jack A. Roth (2) Bela Takacs (3) M. Tien Kuo (1)(§)

From the  (1)Department of Molecular Pathology and (2)Section of Thoracic Molecular Oncology, Thoracic and Cardiovascular Surgery, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 and (3)Pharmaceutical Research, Preclinical Dermatology, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(2), 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.


INTRODUCTION

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) (^1)(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.


MATERIALS AND METHODS

Recombinant CS Proteins and Peptides

Bacterially derived CS recombinant proteins CS27IVC-His(6) (27-123(NANPNVDP)(3)(NANP) 300-411), CS27IC-His(6)(27-123(NANPNVDP)(3)(NANP)(1)300-411) containing P. falciparum CS sequences from the T4 isolate were prepared according to the procedure previously described(10, 11) . The recombinant plasmids were constructed by inserting the corresponding encoded DNA sequences into PDS56/RBSII vector. A synthetic oligonucleotide encoding hexahistidine was ligated to the 3` end of the CS genes to facilitate the recombinant protein purification by metal chelate affinity chromatography. The constructs were transformed into Escherichia coli strain M15. Recombinant protein DHFR-CSF1-His(6) ((DHFR)6-146), which contains a mouse dihydrofolate reductase (DHFR) cDNA linked to amino acid residues 6-146 of the CS sequence and the engineered hexahistidine, has been described previously(11) . Peptide E35 (EWSPCSVTCGNGIZVRIKPGSAN) and A128 (GNEIEPGNNAYGSQSDTDASELT), both having 23 amino acid residues, were synthesized on a Vega Coupler 250C synthesizer using tert-butocycarbonyl chemistry and deprotected as described previously(12) . The peptides were purified by high performance liquid chromatography. The amino acid composition of the synthesized peptides were confirmed by amino acid analysis.

Cells and Cell Culturing

Hepatocytes were isolated from adult male C57BL/6 mice by the procedure previously described (13) with slight modification. Briefly, the livers were perfused with a solution containing collagenase (100 units/ml), trypsin inhibitor, 120 µg/ml (both from Worthington) and Hank's A balanced salts by cannulating the vena cava and releasing the perfusate via the portal vein. The resulting crude hepatocytes were suspended in 36% Percoll and further purified by centrifugation at 20,000 times g for 20 min(14) . The purified hepatocytes usually had >95% viability at the time of plating as determined by trypan blue exclusion. The cells were suspended in Waymouth's MD 705/1 medium (Life Technologies, Inc.) containing 10% fetal calf serum (FCS) (Life Technologies, Inc.), penicillin (100 units/ml) and streptomycin (100 mg/ml) and plated onto Costar six-well tissue culture plate 3506 at 3 times 10^5 cells/well.

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(2) in air.

Preparation of CS Conjugate

CS protein was conjugated to poly(L-lysine) (molecular mass = 26.3 kDa, Sigma) by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, Sigma) according to the protocol described by Cristiano et al.(15) . Standard reaction mixture (1.5 ml) contained 1 ml of CS protein (in 100 mM MOPS buffer, pH 7.3) and 0.5 ml of poly(L-lysine) also in 100 mM MOPS, pH 7.3. EDC powder was then directly added to the mixture so that the final concentrations of CS protein, poly(L-lysine), and EDC were 94.3 µM, 141.05 µM, and 90.90 mM, respectively. This molar ratio is comparable to those used by other investigators in the preparation of ASOR conjugates(3, 15) . FITC-polylysine (3.72 mg) was dissolved in 100 mM MOPS, pH 7.3, and EDC was added to the sample to a final concentration of 90.90 mM to mimic the conditions for the preparation of the CS conjugate. The reaction mixture was incubated at 25 °C for 18 h. The mixture was applied to a Superose 6 column (16 times 30 cm) pre-equilibrated in HBS (150 mM NaCl, 20 mM Hepes, pH 7.3). The samples were eluted with the same buffer at the velocity of 0.17 ml/min and collected in 1-ml fractions. The chromatography was monitored by UV absorption at 280 nm. Protein concentrations in each fraction were determined by Bio-Rad protein assay system according to vendor's specifications, using immunoglobulin G (IgG) as reference. The amounts of conjugates used throughout this study were based on this protein determination unless otherwise indicated. Conjugates were analyzed by acid-urea gel electrophoresis according to the procedure described by Meckee et al.(16) . Transferrin, ASOR, and BSA (Sigma) conjugates were prepared by the same procedure.

Preparation of Adenovirus

Adenovirus dl312 was propagated in 293 cell and purified twice on CsCl density gradient according to the procedure described(17) . The purified virus was dialyzed against HBS buffer (three times, 1 liter each). Viral DNA was extracted from aliquots of viral preparation by 1 h of SDS (0.1%) and proteinase K (100 µg/ml) digestion, followed by phenol/chloroform extraction and ethanol precipitation. The yield of DNA was spectrophotometrically determined at 260 nm absorbance, and used for calculation of the number of viral particles(15) . The viruses were then aliquoted, and stored in HBS containing 10% glycerol at -80 °C until use. Recombinant adenoviruses harboring beta-galactosidase linked to the cytomegalovirus promoter (18) were prepared by the same procedure.

Formation of DNA Complex and Transfection

The reporter construct, pCMV-beta-gal(19) , which contains a bacterial beta-galactosidase gene under the transcriptional control of cytomegalovirus enhancer/promoter was used for transfection assay. Plasmid DNA was prepared by 2 times CsCl gradient ultracentrifugation. Six µg of pCMV-beta-gal DNA in 350 µl of HBS was mixed with various amounts of CS protein conjugate in 150 µl of HBS. The mixture was incubated at 25 °C for 30 min. Before transfection, culture medium was removed and replaced by 0.5 ml of the medium containing 2% FCS. CS protein conjugate-DNA complex was added to the cells, immediately followed by addition of an appropriate amount of adenovirus. The cells were incubated at 37 °C for 2 h. After addition of 2 ml of complete medium supplemented with 10% FCS, cells were incubated for an additional 16 h.

beta-Galactosidase Activity Assay

beta-Galactosidase activity was analyzed by the methods described by McGregor et al.(20) . Briefly, The medium was removed from transfected cells. The cells were washed three times with phosphate-buffered saline. The expression of beta-galactosidase were determined either by histochemical staining (X-gal stains) or by measuring the enzymatic activity of cell extracts using ONPG as substrate. The unit of ONPG assay was defined as described by McGregor et al.(20) .

Uptakes of CS27IVC-His(6) and Conjugate-DNA Complex

CS27IVC-His(6) was tritium-labeled by sodium cyanoboro[^3H]hydride according to the procedure previously described(21) . Briefly, 1 mg of CS protein in 1 ml of HBS was added to 10 mCi of sodium cyanoboro[^3H]hydride in the presence of 12 mM formaldehyde. The reaction proceeded at 4 °C for 30 min. Labeled CS protein was purified by Sephadex G-25 chromatography. The specific activity of labeled protein was 68,000 cpm/µg counted in a Beckman LS 3801 liquid scintillation counter.

Prior to protein uptake assay, the culture medium was replaced with 0.5 ml of serum-free medium. 2.5 µg of [^3H]CS27IVC-His(6) were added to the culture. The cells were incubated at 37 °C in 5% C0(2) 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-beta-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-beta-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 times 10^5 primary hepatocytes or HL-60 cells. The cells were incubated at 37 °C in 5% CO(2) incubator. At different time intervals, the medium was removed and the radioactivities in the cultured cells were determined.


RESULTS

Preparation of CS-Poly(L-lysine) Conjugate

When ^3H-recombinant CS27IVC-His(6) protein (molecular mass = 53 kDa, Fig. 1) was passed through a Superose 6 column and the eluents were monitored by radioactivity, two distinct peaks were resolved (Fig. 2A, dashed line). These two peaks may correspond to oligomeric (but predominantly dimeric) and monomic CS proteins, respectively, since it has been reported that CS protein can oligomerized by cysteine formation(9) . Recombinant CS27IVC-His(6) protein was conjugated to poly(L-lysine) (26.3 kDa) by EDC, and fractionated by the same column. Two distinct peaks were also eluted (Fig. 2A, solid line). The front peak had an apparent molecular mass of greater than 100 kDa, whereas the second peak, approximately 80 kDa, using the eluting profiles of presumptive multimeric CS protein (Fig. 2A) and unconjugated transferrin (80 kDa) (Fig. 2C, broken line) as references. These results suggested that the front peak contained CS conjugate with very little contamination of free CS protein. When poly(L-lysine) alone was treated with EDC and fractionated through the same column, there was a shift in the eluting profile as compared with that of the untreated sample (Fig. 2B), indicating the formation of polylysine-polylysine conjugates. However, both the conjugated polylysine and free polylysine were eluted behind the front peak of the CS conjugate (compare panels A and B). These results suggest that the front peak in the CS conjugate preparation should contain only negligible amount of polylysine-polylysine conjugates. Thus, these chromatographic conditions apparently are effective in the preparation of CS conjugates without significant contamination of free CS protein and self-conjugated polylysine. We consider this very important, because these contamination would have an adverse effect on the efficacy of receptor-mediated gene delivery.


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(6) 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(6)/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 [^3H]CS27IVC-His(6) and CS27IVC-His(6)/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-beta-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 times 10^3 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 (^2)conjugates. Thus, we conclude that this simple gel filtration chromatographic technique can be used to prepare functional molecular carriers for gene delivery.

Optimization of Transfection Conditions for Primary Hepatocytes

To test whether the CS-polylysine conjugates could deliver recombinant DNA into primary hepatocytes, we carried out a set of pilot experiments. CS27IVC-His(6) protein conjugates from the front peak were complexed with recombinant pCMV-beta-gal DNA. The DNA complexes were used to transfect primary hepatocyte cultures. Eighteen hours after transfection, cells were stained with X-gal. Less than 0.1% of positive cells were found (data not shown). It is possible that the DNA complexes were internalized but not able to escape from endosomal entrapment. Previous studies of ASOR and transferrin carriers demonstrated that adenovirus infection could destabilize endosomes, presumably allowing endosomally contained plasmid DNA to escape(15, 22, 23, 24) . Therefore, we cotransfected hepatocytes with a replication defective adenovirus (dl312). The application of adenovirus greatly enhanced the frequency of X-gal-positive cells, indicating that the efficiency of CS-mediated gene transfer also required endosomal destabilizing agents. Protein conjugates from the fractions of second peak were similarly analyzed, however, no more than 20% of blue cells were seen under various ``optimization'' conditions (see below). Thus, all the data presented here were using CS27IVC-His(6) conjugates collected from the front peak of the Sepharose 6 column (for simplicity, hereafter referred to as CS conjugate unless other specifications).

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 beta-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 times 10^5) were transfected with the complexes in the presence of adenovirus (4 times 10^4 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 times 10^4 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 beta-galactosidase in primary hepatocytes. CS27IVC-His(6) conjugate (7.4 µg) was complexed with 6 µg of pCMV-beta-gal DNA. Indicated amount of adenovirus was added to the cells after the addition of the complex. Eighteen hours after transfection, cell viability, and beta-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 beta-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 beta-galactosidase expression and cell viability after transfection with DNA complex. CS27IVC-His(6) conjugate (7.4 µg) was complexed with 6 µg of pCMV-beta-gal DNA. The transfection of primary hepatocytes was initiated (time 0) by the addition of the DNA complexes and adenovirus (4 times 10^4 particles/cell). At different time intervals, percentage of blue cells (beta-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.



Dependence of CS Protein-mediated Gene Delivery upon the Evolutionarily Conserved Region II+ Epitope

To investigate the functional domain in CS protein for mediating the DNA transfer, we tested recombinant constructs containing deletions in various regions of the CS protein (Fig. 1). Recombinant CS27IC-His(6), which contains regions I and II+ but only one copy of the NANP repeat (instead of the 21 copies as in CS27IVC-His(6)), had 84% transfection efficiency in the primary hepatocyte cultures (Table 1). This transfection efficiency was consistent with that using the CS27IVC-His(6) conjugates (Table 1). However, recombinant DHFR-CSF1 (Fig. 1), which lacks region II+ and the entire NANP repeats, but retains only region I, showed poor gene transfer ability (Table 1). Histochemical staining of the transfected cells using these various conjugates is shown in Fig. 6. These results suggest that region II+ is important for CS-mediated gene delivery.


Figure 6: Expression of beta-galactosidase in primary hepatocytes after transfected with DNA complexes containing different recombinant CS proteins. CS conjugates containing CS27IVC-His(6) (b), CS27IC-His(6) (c), and DHFR-CSF1-His(6) (d) were complexed with 6 µg of pCMV-beta-gal DNA. The amount of CS conjugate used were experimentally determined in pre-experiments. Transfection was carried out in the presence of adenovirus (4 times 10^4 particles/cell). beta-Galactosidase was determined by X-gal staining. Six µg of pCMV-beta-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 beta-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 beta-galactosidase in primary hepatocytes. CS27IVC-His(6) conjugate (7.4 µg) was conjugated to 6 µg of pCMV-beta-gal DNA. Different amounts of E35 and A128 peptides were added to cells after the addition of DNA complexes and adenovirus (4 times 10^4 particles/cell). beta-Galactosidase activity was quantitated using ONPG as a substrate. Results were from triplicate experiments.



Dependence of CS Protein-mediated Gene Delivery upon Its Receptors

Although in vivo study has demonstrated that CS protein almost exclusively binds to hepatocytes, using an cell adhesion assay, Rich et al.(25) implicated that CS receptors are present in a broad range of established cell lines, particularly CEM, HSB-2, K562, and KG-1 lymphocytic cell lines. Therefore, we investigated whether the efficiency of CS-mediated gene transfer could be correlated with the presence of receptors on these various cell lines. We chose HepG2 (7) and CHO (26, 27) cells, which have been reported to contain high levels of CS receptor, and HL-60 cells, which contain low levels of receptors(25) . pCMV-beta-gal reporter gene was transfected into these cells using conjugates prepared from CS27IVC-His(6) protein. Under the optimal conditions, CHO and HepG2 cells exhibited beta-galactosidase activities 130 times higher than that of the HL-60 cells (Fig. 8).


Figure 8: Expression of beta-galactosidase in different cell lines transfected with CS carrier. CS27IVC-His(6) conjugate (7.4 µg) was complexed with 6 µg of pCMV-beta-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. beta-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 beta-galactosidase adenovirus (ranging from 5 times 10^6 to 1 times 10^9 particles/5 times 10^5 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 [^3H]CS27IVC-His(6) 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) , ^3H-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(6) by primary hepatocytes and HL-60 cells. [^3H]CS27IV-His(6) (2.5 µg) was mixed with 0.5 ml of serum-free medium and added to 5 times 10^5 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(6) (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 times 10^5 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).

Gene Delivery by CS Conjugates in Various Cultured Cells

Since CS receptor has been found broadly distributed in established cell lines(25) , CS conjugate may be used as a conventional tool for DNA transfer in different cell lines. To explore this possibility, we chose NIH3T3 (normal fibroblast cell line), HeLa (tumor cell line), and K562 (suspension culture) for transfection using CS conjugates. HeLa, NIH3T3, and K562 all showed high transfection efficacies (Fig. 8). However, a number of human melanoma cell lines (A-375, HT-144, and WM115), and freshly prepared human bone marrow cells (gifts of Dr. Albert Deiseroth, M. D. Anderson Cancer Center) showed very low levels of transfection efficiencies (data not shown). Whether the poor transfection efficiencies in these cells were due to the lack of adenovirus and/or CS receptors have not been determined. Nonetheless, these results demonstrated the CS conjugate can be used as a gene delivery carrier in some cultured cells.


DISCUSSION

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 beta-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 10^4 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 times 10^9 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.


FOOTNOTES

*
This research was supported in part by National Institutes of Health Grants CA 55813 and DK49091 (to M. T. K.) and CA11672 (to the core facility of M. D. Anderson Cancer Center). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Pathology (Box 89), University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-3214; Fax: 713-792-4672.

(^1)
The abbreviations used are: ASOR, asialoorosomucoid; CS, circumsporozoite; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; MOPS, 3-(N-morpholino)propanesulfonic acid; DHFR, dihydrofolate reductase; ONPG, O-nitrophenyl beta-D-galactoside; X-gal, 5-bromo-4-chloro-3-indolyl beta-D-galactoside; FCS, fetal calf serum; BSA, bovine serum albumin; CHO, Chinese hamster ovary; FITC, fluorescein isothiocyanate; HBS, Hepes-buffered saline.

(^2)
Z.-M. Ding, R. J. Cristiano, J. A. Roth, B. Takacs, and M. T. Kuo, unpublished results.


ACKNOWLEDGEMENTS

We are grateful to Dr. T. C. Liang for valuable comments on the manuscript.


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