Multicomponent DNA Carrier with a Vesicular Stomatitis Virus G

Farmington, Connecticut 06030. Received June 9, 1999; Revised Manuscript Received September 10, 1999. Genes can be targeted to hepatocytes in vitro an...
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Bioconjugate Chem. 1999, 10, 1075−1083

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Multicomponent DNA Carrier with a Vesicular Stomatitis Virus G-Peptide Greatly Enhances Liver-Targeted Gene Expression in Mice† Martin J. Schuster, George Y. Wu, Cherie M. Walton, and Catherine H. Wu* Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut School of Medicine, Farmington, Connecticut 06030. Received June 9, 1999; Revised Manuscript Received September 10, 1999

Genes can be targeted to hepatocytes in vitro and in vivo by the use of asialoorosomucoid-polylysine conjugates. After systemic application, this nonviral vector is recognized by highly selective asialoglycoprotein (AsGP) receptors on the sinusoidal liver cell membrane and is taken up via receptormediated endocytosis. As most of the DNA is rapidly transferred to lysosomes where it is degraded, transfection efficiency is low and gene expression transient. To address this problem, we incorporated a pH-dependent synthetic hemolytic peptide derived of the G-protein of Vesicular Stomatitis Virus (VSV) into the gene transfer system, to increase endosomal escape of internalized DNA. The multicomponent carrier binds DNA in a nondamaging way, is still recognized by the AsGP receptor, and is targeted to the liver in vivo. Injection of DNA complexes containing a luciferase marker gene resulted in luciferase expression of 29 000 pg/g liver which corresponded to an increase of a factor of 103 overexpression after injection of DNA complexes without endosomolytic peptide. Furthermore, the amount of intact transgene within isolated liver cell nuclei was increased by a factor of 101-102 by the use of the multicomponent carriers. These results demonstrate that incorporation of a hemolytic peptide into a nonviral vector can greatly increase gene expression while retaining cell type targetability in vivo.

We have shown previously that DNA can be delivered specifically to the liver based on the presence of receptors on hepatocytes that recognize galactose-terminal (asialo)glycoproteins (AsGP).1 Binding of the soluble DNAprotein complex by the AsGP receptor results in internalization of the ligand into the endosomal compartment and ultimately degradation in lysosomes. In early studies, this strategy was used to deliver foreign genes to hepatocytes in vitro (1) as well as in vivo (2), resulting in short term, low level gene expression. Targeted transfer to hepatocytes by this technology was successfully applied to temporarily correct metabolic defects in animal models of human disease (3, 4). Subsequent experiments were performed to prolong the duration as well as to increase the efficiency of targeted gene expression. Partial hepatectomy immediately after intravenous injection of the complex resulted in extended persistence and expression of the transgene (5, 6). The majority of the persisting DNA was shown to exist in cytoplasmic vesicles for many weeks (7, 8). Disruption of the microtubule network, which is required for the translocation of endosomes to lysosomes (9) was shown to be responsible for this effect (4). To circumvent the natural degradative process, in a noninvasive manner, we sought to imitate survival † This work was presented, in part, at the annual American Association for the Study of Liver Diseases meeting in Chicago, IL, 7-11 November, 1997, and has been published in abstract form [Hepatology (1997) 26, 129A]. * To whom correspondence should be addressed. Phone: (860) 679-3158. Fax: (860) 679-3159. E-mail: [email protected]. 1 Abbreviations: AsGP, asialoglycoprotein; AsOR, asialoorosomucoid; OR, orosomucoid; VSV, vesicular stomatitis virus; VSVG, peptide derived from vesicular stomatitis virus Gprotein; lysine-OMe, L-lysine-methyl ester; H and E, hematoxylin and eosin.

techniques of natural pathogens. We noted that vesicular stomatitis virus (VSV) has evolved a mechanism for survival that depends on release from endocytotic vesicles after cell entry. Membrane fusion is mediated by the VSV glycoprotein (G-protein) in a pH-dependent manner. After internalization within endosomes, the decrease in pH triggers a conformational change in the G-protein which results in insertion into the endosomal membrane and release of the virus from the endosome. It was demonstrated that G-protein-mediated fusion depends on an internal domain (10, 11) as well as membrane anchoring (12) of the protein. A synthetic peptide corresponding to the amino-terminal 25 amino acids of VSV G-protein has been shown to be hemolytic at pH 5, but inactive at physiological pH, and thought to be the fusogenic domain (13, 14). Although this peptide was later proven to be dispensable for G-protein membrane fusion (15), its properties appeared favorable for achieving release of endosomal contents following receptor mediated endocytosis. In the following report, we demonstrate for the first time, that a hemolytic peptide corresponding to the amino-terminal 25 amino acids of the VSV G-protein (VSVG) increases the efficiency of receptor-mediated gene transfer in vivo. We found that this carrier was still highly liver selective and greatly enhanced receptormediated gene expression in vivo. MATERIALS AND METHODS

Animals. Balb C female mice (∼20 g body weight) were obtained from Charles River Laboratory, Wilmington, MA, housed under controlled conditions of temperature and humidity, and fed normal chow ad libitum. All experimental procedures were performed in accordance with protocols approved by our Institutional Animal Care Committee.

10.1021/bc990071r CCC: $18.00 © 1999 American Chemical Society Published on Web 10/21/1999

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Materials. A 878 bp BglII-HindIII fragment of pRcCMV (Invitrogen, Carlsbad, CA) containing CMV immediate-early gene enhancer-promoter sequences was cloned into the BglII-HindIII sites of pGL3-Basic (Promega, Madison, WI) to create pGL3CMVluc. pEGFP-C1 was obtained from Clontech (Palo Alto, CA). Plasmid DNA was prepared as described previously (16). All chemicals used were of analytical grade (Sigma, St. Louis, MO), if not mentioned otherwise. DNA-modifying enzymes were obtained from Life Technologies (Gaithersburg, MD). Preparation of DNA Carriers. Asialoorosomucoid (AsOR) was produced by acid hydrolysis (17) of orosomucoid isolated from pooled human serum (American Red Cross) (18) and covalently linked to L-lysine methyl ester (lysine-OMe) using 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC, Pierce, Rockford, IL) to incorporate positive charges for DNA binding. Ten milligrams of AsOR was dissolved in 1 mL of water and filtered through a 0.45 µm syringe-tip filter (Acrodisc, Gelman Sciences, Ann Arbor, MI). Fifty milligrams of lysine-OMe was dissolved in 1 mL of water and added to the AsOR, and the pH adjusted to 6.5 using NaOH at 0.1 N. To this mixture was added 4 mg of EDC, dissolved in 1 mL of water and incubated with stirring at 37 °C for 5 h, followed by dialysis of the reaction mixture through membranes with 12-14 kDa exclusion limits (Spectra/ Por, Spectrum Medical Industries, Houston, TX) against 20 L of water at 4 °C for 24 h. The dialyzate was lyophilized and then redissolved in 0.15 M NaCl (10 mg/ mL), filtered through a 0.45 µm syringe-tip filter, and applied on a Waters HPLC system using a Shodex KW804 column (300 × 8 mm, Waters Corporation, Milford, MA). Samples of 250 µL were injected and eluted with 0.15 M NaCl at a flow rate of 0.12 mL/min. Samples 0.6 mL each were collected, and absorption was monitored at 280 nm. Samples of the two peaks in the effluent were analyzed by 12% SDS-PAGE. The second peak was used for further conjugation and corresponding samples were lyophilized. Purified conjugates were hydrolyzed in constant boiling HCl and submitted for amino acid analysis to determine the ratios of components present. The total number of lysine residues minus the lysine residues expected from AsOR alone provided quantitation of the amount of lysine-OMe present. The number of aspartic acid residues was used to determine the amount of AsOR in each conjugate, and a lysine-OMe to AsOR molar ratio was calculated. VSVG peptide (KFTIVFPHNQKGNWKNVPSNYHYCP) was obtained from Immune Response Corporation (Carlsbad, CA) and shown to be hemolytic in a pHdependent manner as described previously (13, 14). The cysteine residue was used to couple peptides to AsORlysine-OMe with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP, Pierce). Five milligrams of purified AsORlysine-OMe was dissolved in 1 mL of PBS (pH 7.4), and 0.18 mL of freshly prepared SPDP (3.1 mg/mL in DMSO) was added to give a 30-fold molar excess of SPDP over AsOR-lysine-OMe. The mixture was incubated with stirring for 1 h at 25 °C. The reaction mixture was then dialyzed in membranes with 12-14 kDa exclusion limits against 20 L of water for 24 h at 4 °C and lyophilized. To remove any free SPDP, the conjugate was dissolved in water and applied to a Sephadex G25 column (Amersham Pharmacia Biotech, Piscataway, NJ), followed by elution with water. The eluate was checked for absorption at 280 nm and lyophilized. To determine the molar ratio of AsOR-lysine-OMe to SPDP, the absorption at 343 nm was measured with and without reduction using dithiothreitol

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(DTT) as described previously (19). To activate the sulfhydryl group, VSVG peptide was dissolved in PBS and incubated with a 10-fold molar excess of DTT for 60 min at 25 °C. Excess DTT was separated by HPLC using an Aquapore Cation Exchange CX-300 column (250 × 10 mm, Rainin Instrument) and elution with 0.3 M NaCl at a flow rate of 1 mL/min. VSVG was then conjugated to AsOR-lysine-OMe-SPDP. Two milligrams of AsORlysine-OMe-SPDP and 10 mg of activated VSVG peptide were each dissolved in 1 mL of water. VSVG was added in a 20-fold molar excess over AsOR to the conjugate. NaCl at 1.5 M was added to make a final concentration of 0.15 M, and the reaction mixture was passed through a 0.45 µm syringe-tip filter and incubated on a nutator at 25 °C for 18 h, followed by lyophilization. Five milligram samples were then dissolved in 0.5 mL of water, applied to a Sephadex G25 column, and eluted with water. The eluate was monitored for its absorbance at 280 nm, and the first peak was collected. The conjugates were analyzed on a 12% SDS-polyacrylamide gel with and without the addition of DTT as described previously (19). Amino acid analysis was performed to permit calculation of the molar ratio of VSVG to AsOR from tyrosine (present in AsOR and VSVG) and glutamic acid (present only in AsOR) content. AsOR-polylysine (26 kDa) was prepared as described (2), using poly-L-lysine, 26 kDa. Formation of Protein Conjugate-DNA Complexes. Ethidium bromide exclusion was used to determine the DNA-binding capacity of protein conjugates. Sixty micrograms of pGL3CMVluc was dissolved in 2.5 mL of 0.15 M NaCl containing 2.5 µM ethidium bromide. Conjugates (0.6 mg/mL) were dissolved in 0.15 M NaCl and added in 50 µL increments to the DNA solution, while fluorescence was measured with excitation at 260 nm and emission at 591 nm on a fluorimeter. The minimum amount of protein conjugate resulting in maximum ethidium bromide exclusion was selected, and DNA complexation was confirmed by agarose gel retardation as described previously (1). DNA-protein conjugate complexes for injection were made starting with 40 µg/mL of DNA in 0.15 M NaCl. While continuously vortexing, 25 µL aliquots of protein conjugate were added over 30 min at 25 °C, followed by incubation for 30 min at 25 °C. Absorption was measured at 260, 340, and 400 nm to detect complexes. The purified products were filtered through a 0.22 µm syringe top filter (Gelman Sciences). Recovery of filtered complexes was measured by absorption at 260 nm and brought to a concentration of 10 µg with respect to DNA in 0.5 mL by adding sterile 0.15 M NaCl. Dissociability and Stability of DNA-Protein Conjugate Complexes. To investigate dissociability, DNA was complexed with either AsOR-lysine-OMe, AsORlysine-OMe-VSVG, or AsOR-polylysine as described above and mixed with equal amounts of either 0.15 M NaCl, 8 M urea, or 0.15 M NaCl containing 200 units of heparin and incubated at 37 °C. Aliquots were withdrawn at 0, 10, and 30 min, respectively, transferred on ice, and analyzed on a 0.8% agarose gel containing 0.5 µg/mL EtBr. To determine if DNA complexed by protein conjugates were protected from degradation by serum nucleases, DNA complexes were incubated at 37 °C in the presence of 0.33 milliunits of DNase I (Boehringer Mannheim). Aliquots were withdrawn after 0, 2, 5, 10, and 30 min, respectively, and reactions stopped by adding Na2EDTA up to 12.5 mM, followed by heat inactivation for 20 min at 75 °C. To release DNA from protein conjugates, 200 units of heparin (for AsOR-polylysine, AsOR-lysine-OMe) or an equal volume of 8 M urea (for

Liver-Targeted Gene Expression in Mice

AsOR-lysine-OMe-VSVG) were added. Finally, all samples were analyzed on a 0.8% agarose gel containing 0.5 µg/ mL EtBr. Following electrophoresis, DNA was visualized by UV transillumination (302 nm). Organ Distribution of AsOR-Lysine-OMe-VSVG Conjugates. To determine whether the multicomponent carrier retained its ability to be recognized by hepatocyte AsGP receptors in vivo, conjugates were iodinated as described (20). Two micrograms of [125I]AsOR-lysineOMe-VSVG or AsOR-lysine-OMe-[125I]VSVG with a specific activity of 106 cpm/µg, respectively, was injected intravenously into tail veins of mice. Animals were sacrificed after 10 min, and 125I content of various organs was determined using a γ counter. Experiments were performed in triplicate, and results were expressed as means ( SEM. To determine whether the organ distribution of the radiolabeled carrier corresponded to luciferase expression found in the solid organs, liver, spleen, kidneys, and lungs were removed 24 h after injection, homogenized, and assayed for luciferase expression. Assay for Targeted Gene Expression. Complexes containing 10 µg of pGL3CMVluc in 0.5 mL of 0.15 M NaCl were passed through 0.2 µm syringe-tip filters and immediately injected into the tail veins of mice over the course of 10-30 s. After 24 h and after 7 days, animals were sacrificed, and livers removed, washed with ice-cold PBS, and weighed. A liver section (∼100 mg) was removed, weighed, and homogenized in lysis buffer (100 mg/mL, Luciferase Assay System, Promega), and liver luciferase activity determined by luminometry. A standard curve for luciferase using firefly luciferase (Analytical Luminescence Laboratory, Ann Arbor, MI) was performed along with the test samples. For each experiment, injections were performed under identical conditions in three animals in parallel, using free DNA, or DNA complexed by AsOR-lysine-OMe, AsOR-polylysine, or AsOR-lysine-OMe-VSVG, respectively. All experiments were performed at least in triplicate, and results were expressed as means ( SEM in units of picograms of luciferase per gram of liver tissue (1 pg corresponds to 200 000 LU/30 s). The remaining liver tissue was used for isolation of liver cell nuclei. Competition experiments were performed using AsOR or orosomucoid (OR) and mixed with the DNA-protein conjugates complexes in 100-fold molar excess immediately before injection. To determine whether an adverse immune response could occur due to antigenicity of complexes, second injections of the identical complexes were performed 1 week after the first. Targeted gene expression was evaluated as described above for animals that received only single injections. To determine the efficiency of liver cell transduction, immunohistochemical analysis for luciferase protein was performed 24 h after injection of DNA complexed with AsOR-lysine-OMe-VSVG. Animals were sacrificed, and livers were removed and frozen in Tissue Freezing Medium (Triangle Biomedical Medium, Durham, NC). Cryosections were fixed with 4% p-formaldehyde, incubated for 30 min with blocking solution (5% dry milk, 150 mM NaCl, 10 mM Tris Cl, pH 7.5, and 0.5% Tween 20), followed by incubation with anti-luciferase antibody (1:500, polyclonal, Cortex Biochemicals, San Leandro, CA) for 2 h, followed by washing and incubation with anti-rabbit-IgG Texas Red (1:5000, Amersham) for 30 min. Following treatment with antifade (Dabco, Sigma), samples were subjected to fluorescence laser scanning microscopy (Zeiss CLSM, Germany). For direct detection of green fluorescent protein (GFP) in liver sections from mice injected with pEGFP-C1 complexed with multicom-

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ponent carrier, cryosections were fixed with 4% pformaldehyde, treated with antifade, and subjected to fluorescence laser scanning microscopy. Assay for Acute Liver Toxicity. To investigate whether injection of DNA complexed by AsOR lysineOMe-VSVG conjugates led to liver cell damage, blood samples were collected from the retroorbital plexus 24 h after injection, and serum was subjected to ALT analysis (kinetic ALT kit, Sigma). Liver sections were stained with H and E and examined by light microscopy to determine signs of liver cell necrosis or cellular infiltration. Isolation of Nuclei and Extraction of Episomal DNA. Twenty four hours after injection of complexed DNA in the mice, livers were removed and homogenized in 4 mL of buffer (sucrose at 0.32 M, CaCl2 at 3 mM, magnesium acetate at 2 mM, Na2EDTA at 0.1 mM, Tris HCl, pH 8.0, at 10 mM, DTT at 1 mM, and NP40 at 0.5%) using a Dounce Tissue Grinder (Kontes) and applying five strokes with a loose, and then a tight-fitting pestle. The homogenate was loaded on a gradient consisting of four layers with 1.0, 1.4, 1.6, and 1.8 M sucrose, respectively, in buffer 2 (magnesium acetate at 5 mM, Na2EDTA at 0.1 mM, Tris HCl, pH 8.0, at 10 mM, and DTT at 1 mM) and centrifuged for 40 min, 34000g at 4 °C (Beckmann SW28 rotor). The nuclei containing pellet was resuspended in 1 mL of buffer 1 (without NP40) and pelleted again by low-speed centrifugation (750g, 2 min). Finally, nuclei were resuspended in 1 mL of TE50/10 (Tris HCl, pH 7.5, at 50 mM and Na2EDTA at 10 mM) and lysed with 1.5 mL of lysis buffer (SDS at 1% and NaOH at 0.2 N). To obtain episomal DNA, a modified Hirt extraction was performed by adding 1.25 mL of 3-5 M potassium acetate. After incubation for 15 min on ice, the precipitate was pelleted and the supernatant was extracted with phenol/chloroform. Following precipitation with ethanol, samples were resuspended in 100 µL of TE (Tris HCl, pH 7.5, at 10 mM and Na2EDTA at 1 mM) and subjected to PCR analysis. Semiquantitative PCR of Full-Length Luciferase Gene. A semiquantitative PCR assay was performed as described (21) to determine the amount of intact luciferase cDNA in isolated nuclei. Primers used were S1 (5′-3′) cat aaa gaa agg ccc ggc gcc att and AS1 caa aca caa ctc ctc cgc gca act, resulting in the amplification of a 1538 bp fragment, corresponding to nt 105-1642 of pGL3-Basic and comprising 93% of the luciferase coding sequence. As a competitive internal standard, a plasmid pLUC-KpnI was constructed containing the 1538 bp luciferase PCR fragment cloned into pGEM-T vector (Promega) with an additional KpnI restriction site at position 595 (with respect to the PCR product). The new KpnI site was created by PCR site-directed mutagenesis, using S2 (5′-3′) ctg gat cta ctg ggg tac cta agg gtg tg and AS2 cac acc ctt agg tac ccc agt aga tcc ag as primers. After KpnI digestion of PCR samples, PCR products generated from target sequences (1538 bp) or internal standard (596/942 bp), respectively, could be discriminated. DNA amplification experiments were carried out with 2.5 units of Taq polymerase/reaction and 1.5 mM MgCl2. A hot start protocol was used with annealing at 60 °C (45 s), elongation at 72 °C (3 min), and denaturation at 94 °C (45 s) for 35 cycles, followed by a final elongation at 72 °C for 10 min. For each sample of episomal DNA extracted from one animal, eight PCR reactions were performed in parallel, containing 5 µL of sample as template and an increasing amount (0-250 fg) of internal standard pLuc-KpnI as competitive template. Fifteen microliters of each reaction was digested with KpnI and loaded onto a 0.8% agarose gel containing 0.5 µg/mL EtBr

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internal standard and target sequences was used to calculate the relative increase of the amount of transgene in the isolated nuclei. Assays were performed in parallel for mice injected with either DNA alone or DNA complexed with AsOR-polylysine or AsOR-lysine-OMe-VSVG. RESULTS

Figure 1. SDS-PAGE of AsOR-lysine-OMe-VSVG. Conjugates were analyzed with or without the addition of DTT to reduce disulfide bonds between peptides and AsOR-lysine-OMe. Lane 1, AsOR; lane 2, AsOR-lysine-OMe; lane 3, VSVG peptide (25 aa); lane 4, AsOR-lysine-OMe-VSVG; lane 5, AsOR-lysine-OMeVSVG plus DTT.

and subjected to electrophoresis. DNA was visualized by UV transillumination (302 nm), and the intensity of bands was measured using IPLab Gel software. The PCR reaction with equal density of bands generated from

After covalently linking lysine-OMe to AsOR and purification by HPLC, the effluent fractions showed two peaks regarding absorbance at 280 nm. Analysis by SDS-PAGE demonstrated that the first peak contained aggregates and the second peak, which was used for further conjugation, was mostly monomeric conjugate. The molar ratio between linked lysine-OMe residues and AsOR was calculated to be 8:1 based on amino acid analysis of the purified material. AsOR-lysine-OMe migrated slightly slower on a 12% SDS-polyacrylamide gel compared to AsOR (Figure 1, lane 1 and 2). After conjugation of AsOR-lysine-OMe with SPDP and analysis of the product after reduction with DTT, the molar ratio of SPDP to AsOR-lysine-OMe was calculated to be 2224:1. Conjugates of AsOR-lysine-OMe with VSVG were analyzed on a 12% SDS polyacrylamide gel and showed a single band that had no detectable free VSVG (Figure 1, lane 4). That the VSVG peptide was indeed linked by disulfide bonds was shown by reduction of the conjugates with DTT which resulted in the appearance of free peptide at the appropriate location (Figure 1, lane 5). On the basis of amino acid analysis, the ratio of AsOR:VSVG was calculated to be 1:8. The average minimum amount of conjugate resulting in maximum EtBr exclusion was approximately 1.6 µg/µg of DNA, with some variation between different batches. Binding of the DNA was confirmed by complete retardation on an agarose gel (Figure 2A), and complexes composed of this ratio were used in all subsequent experiments.

Figure 2. Agarose gel electrophoresis of DNA-protein conjugate complexes. Complexes were formed as described in the text containing 1 µg of DNA and incubated at 37 °C for the indicated time. (A) Incubation in saline; (B) Incubation with heparin; (C) Incubation with urea. Samples were analyzed on a 0.8% agarose gel containing 0.5 µg/mL EtBr and DNA was visualized by UV transillumination.

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Table 1. Distribution of Radioactivity after Injection of Different Organsa organs

[125I]AsOR (ref 2)

blood liver heart spleen lung kidney

8.5 ( 1.0 89 ( 3.5 0.1 ( 0.1 0.5 ( 0.1 1.6 ( 0.1 0.2 ( 0.1

125I-Labeled

Protein Conjugates and Luciferase Expression in

fraction of injected counts (%) [125I]AsOR-lysine-OMe-VSVG AsOR-lysine-OMe-[125I]VSVG 13.3 ( 4.1 78.6 ( 10.1 0.6 ( 0.4 0.6 ( 0.3 5.1 ( 5.4 1.9 ( 0.6

12.3 ( 3.5 79 ( 8.0 0.7 ( 0.4 2.7 ( 1.2 6.1 ( 4.4 1.4 ( 0.9

luciferase (pg/g tissue); pCMVluc AsOR-lysine-OMe-VSVG ND 15 351 ( 484 ND 11.6 ( 6.2 9.7 ( 2.5 11.3 ( 4.3

a Two micrograms of [125I]AsOR, [125I]AsOR-lysine-OMe-VSVG, or AsOR-lysine-OMe-[125I]VSVG with a specific activity of 106 cpm/µg, respectively, was injected intravenously into mice. Animals were sacrificed after 10 min, and 125I content of the organs was determined using a γ counter. Luciferase activity is shown in units of picograms per gram tissue 24 h after mice were injected with 10 µg of DNA complexed by AsOR-lysine-OMe-VSVG. Experiments were performed in triplicate, and results were expressed as means ( SEM (1 pg corresponds to 200 000 LU/30 s). ND, not done.

Figure 3. Protection of DNA complexed by protein conjugates against degradation. Complexes were formed as described in the text containing 1 µg of DNA and incubated at 37 °C in the presence of DNase I for the indicated time. After aliquots were withdrawn, reactions were stopped by adding Na2EDTA up to 12.5 mM, followed by heat inactivation for 20 min at 75 °C. To release DNA from protein conjugates, 200 units of heparin (AsOR-polylysine, AsOR-lysine-OMe) or an equal volume of 8 M urea (AsOR-lysine-OMeVSVG) was added. Samples were analyzed on a 0.8% agarose gel containing 0.5 µg/mL EtBr and DNA was visualized by UV transillumination.

The organ distribution of radioactivity after intravenous injection of iodinated AsOR-lysine-OMe-VSVG into mice showed a slight difference compared to the organ distribution of iodinated AsOR alone (2). When either AsOR or VSVG peptide was labeled with 125I, about 80% of recovered radioactivity was found in the liver, while about 12-13% was still in the circulation (Table 1). An average of 96 ( 2% of total radioactivity injected was recovered. In accordance with the distribution of radioactivity, no substantial luciferase activity was detected in solid organs other than the liver (Table 1). Dissociability of DNA from protein conjugates showed a notable difference between AsOR-polylysine, AsORlysine-OMe, and AsOR-lysine-OMe-VSVG conjugates. When incubated in the presence of heparin, DNA was dissociated from AsOR-polylysine conjugates completely, while DNA was only slightly released from AsOR-lysineOMe-VSVG conjugates (Figure 2B). When incubated in the presence of 4 M urea, DNA was dissociated completely from the latter conjugate, but not from AsORlysine-OMe or AsOR-polylysine (Figure 2C). These differences suggest that interactions in addition to electrostatic ones between DNA and protein conjugates are likely involved with AsOR-lysine-OMe-VSVG conjugates. Figure 2 shows that DNA-protein conjugate complexes were stable when incubated at 37 °C in normal saline over 30 min, and DNA was completely retarded after agarose gel electrophoresis (Figure 2A). Incubation of

AsOR-lysine-OMe-VSVG complexes in the presence of DNase I showed greater protection of DNA against degradation over 5 min, compared to DNA alone which was already completely degraded in the same incubation period (Figure 3). AsOR-polylysine provided greater protection to complexed DNA over 30 min, compared to AsOR-lysine-OMe (lacking VSVG peptide), which had no obvious protective effect (Figure 3). Liver luciferase activity determined by luminometry performed 24 h after injection of pGL3CMVluc in the form of complexes is shown in Table 2. Luciferase activity after injection of DNA complexed with multicomponent protein conjugates was, on average, 3 orders of magnitude higher than luciferase activity after injection of AsOR-polylysine-DNA complexes containing the same amount of DNA, and more than 4 orders of magnitude increased over gene expression after injection of free DNA. Expression from multicomponent complexes was 99% blocked by competition with a 100-fold molar excess of AsOR, but essentially unaffected by coadministration of a 100-fold molar excess of OR, which has an identical amino acid sequence, but lacks exposed terminal galactose residues and, therefore, is not a ligand for the AsGP receptor (Table 2). The duration of gene expression was short, with only 1.0 ( 0.9% (n ) 3) of luciferase activity remaining after 1 week. A second injection of DNA complexed by AsOR lysine-OMe-VSVG in the same animal, 1 week after the first injection, resulted in

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Table 2. Luciferase Activity in Liver Tissue 24 h after Mice Were Injected with 10 µg of DNA Either Alone or Complexed by Protein Conjugatesa competitionc (% inhibition)

luciferase (pg/g liver) injection

mean

minimum

maximum

DNA alone AsOR-lysine-OMe AsOR-polylysine AsOR-lysine-OMe-VSVG

2.2 ( 1.9 2.2 ( 2.0 12.2 ( 11.4 29 000 ( 28 000

0.1 0.2 0.7 522

5.5 8.0 29.7 79 600

fold

increaseb

NA 1.0 11.1 18 000

AsOR ND ND ND 99.2 ( 0.9

OR ND ND ND 3.0 ( 3.1

a For each experiment, injections were performed under identical conditions in three animals in parallel, using free DNA or DNA complexed by AsOR-lysine-OMe, AsOR-polylysine, or AsOR-lysine-OMe-VSVG, respectively. A standard curve for luciferase using firefly luciferase was performed along with the test samples. All experiments were performed at least in triplicate, and results were expressed as means ( SEM in units of picograms of luciferase per gram of liver tissue (1 pg corresponds to 200 000 LU/30 s). NA, not applicable. ND, not done. bFold increase gives the average increase compared to the expression of luciferase after injection of free DNA. cCompetition experiments were performed using AsOR or OR, mixed with the DNA-protein complexes in 100-fold molar excess immediately before injection. Results are shown as percent inhibition.

Figure 4. Immunohistochemical analyses of liver sections for luciferase gene expression (panels A and B) and green fluorescent protein gene expression (GFP, panels C and D). Twenty-four hours after injection of DNA (pCMVluc, panels A and B or pEGFP-C1, panels C and D) either alone (panels A and C), or complexed with AsOR-lysine-OMe-VSVG (panels B and D), animals were sacrificed and cryosections of the liver fixed, and treated as described in the the Materials and Methods. Representative sections of the liver are shown. Panels A and B show analyses of Texas Red signal for luciferase, and panels C and D show analyses for GFP expression.

luciferase expression of the same magnitude as the first injection (7695 ( 1777 pg of luciferase/g of liver, n ) 3). Immunohistochemical analysis of liver sections using anti-luciferase antibodies and laser scanning fluorescence microscopy showed a transfection efficiency of about 1.0% of cells. A representative section is shown in Figure 4B. Direct fluorescence analysis after delivery of a green fluorescent protein expression vector confirmed these results (Figure 4D). Animals injected with DNA alone, showed no expression of luciferase or GFP (Figure 4, panels A and C). Injected animals showed no signs of acute toxicity, as shown by normal serum ALT values, and normal liver

histology in H and E stained liver sections (data not shown). The transgene could not be demonstrated in the episomal fraction of nuclear DNA 24 h after injection by Southern Blot analysis. However, semiquantitative competitive PCR assay, of which Figure 5 is a representative of six separate experiments, clearly demonstrated the presence of intact luciferase gene in liver cell nuclei after delivery by the various protein conjugates. The amount of DNA was approximately 1-2 orders of magnitude larger after injection of the multicomponent conjugate (panel D), compared to injection of the same amount of DNA in the form of AsOR-polylysine complexes (panel

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Figure 5. Semiquantitative competitive PCR analysis of episomal DNA extracted from isolated liver cell nuclei 24 h after injection of free DNA or DNA complexed with the various protein conjugates. (A) Negative control; (B) free DNA; (C) AsOR-polylysine; (D) AsOR-lysine-OMe-VSVG. Each PCR reaction contained the same amount of extracted episomal DNA (5 µL of a total of 100 µL) and an increasing amount of competitive template from lane 1 to lane 8 of 0, 0.25, 1.25, 2.5, 12.5, 25, 125, and 250 fg, respectively. After digestion with KpnI, the PCR product amplified from wild-type luciferase DNA appeared as a band at 1538 bp. The PCR product amplified from competitive template sequences appeared as bands at 596 and 942 bp, respectively.

C). There was no intact luciferase gene present after injection of DNA alone (panel B). DISCUSSION

In contrast to viral vectors, synthetic vectors resemble classical pharmaceutical agents in which defined compounds are produced by chemical means. A broad spectrum of genes in the form of plasmid DNA has been delivered to a large variety of cells by receptor-mediated endocytosis using nonviral vectors (22). So far, only a few studies have used this technique to successfully deliver polynucleic acids in vivo to the liver (2-8, 23-25) and other organs (26-29). When linked to a target cellspecific ligand, the DNA is cointernalized by the target cell into the endosomal compartment, from which release is required into the cytoplasm in order to escape degradation in the lysosome. While disruption of the microtubular network, either through partial hepatectomy (6) or by administration of colchicine (4), has been used to increase and prolong gene expression, these methods are not likely to be practical clinically. In the current study, a DNA-binding component composed of lysine-OMe, and AsOR was used instead of polylysine for several reasons. First, it combines the cell-targeting component and DNAbinding component in a single molecule. Negatively charged amino acids on AsOR that react with lysine-OMe are converted into positively charged residues. For each lysine-OMe linked, there is a net increase of two positive charges per residue. Second, the dissociability of conju-

gate from DNA once inside the cells was hypothesized to be likely improved with a DNA carrier having dispersed positive charges rather than the concentrated multiple positive charges of polylysine-based carriers. Finally, it was possible that reproducibility of synthesis of conjugates might be improved by avoiding commercial polylysines that have substantial heterogeneity of chain length within the same batch, and especially from batch to batch (30). Many viruses have evolved mechanisms to escape the degradative endosomal-lysosomal pathway. We and others have taken advantage of the endosomolytic property of whole viruses such as adenoviruses (31-37), rhinoviruses (38), or short synthetic fusogenic peptides derived from Influenza Virus hemagglutinin (39-42) to enhance DNA escape from endocytotic vesicles and augment gene expression after receptor-mediated gene transfer in vitro. The coadministration of endosomolytic viral particles, like adenoviruses, showed impressive enhancement of gene expression in vitro (35). Direct linkage between viral particle and the receptor ligand, substantially increased gene expression (33, 36). The addition of VSVG protein partially purified from VSV-infected cells to cationic lipids enhanced gene transfer in vitro by a factor of 10 (43). The attachment of virus-derived or synthetic endosomolytic peptides to polylysine-based nonviral vectors has also been shown to be feasible in increasing gene expression in different cell types in vitro (39-42). However, until now, it has not been shown that viral peptides

1082 Bioconjugate Chem., Vol. 10, No. 6, 1999

can increase receptor-mediated gene delivery in vivo. While the incorporation of VSVG peptides into the DNA carrier was designed to enhance endosomal escape, the data on DNA stability of the complex suggest that the addition of the peptide also had the unexpected effect of increasing resistance to nuclease degradation compared to the AsOR-lysine-OMe parent conjugate, Figure 2. As there has not been described any inherent nuclease inhibitory activity for VSVG peptide, it is likely that the protective effect is related to the relative inaccessibility of the DNA when in the form of AsOR-lysine-OMe-VSVG complexes. There was considerable variability in the liver assays particularly in Table 2. Different batches of conjugate were used for Table 1 and Table 2 experiments, which could account for the differences in the results between the values in the tables and the variability. As a result, there was no significant difference between the liver value in Table 1 (15 351 ( 484) and Table 2 (29 000 ( 28 000). Similarly, while the mean level of expression on reinjection was only approximately 25% (7695 ( 1777) of the initial value, because of the variability, this difference also did not reach statistical significance at the 0.05 level. Our results demonstrate that it is possible to link a hemolytic peptide to nonviral vectors while retaining receptor recognition and internalization of the ligand. The present study demonstrates that this results in a large increase of gene expression and a higher amount of the intact transgene inside the liver cell nuclei. While increasing the level, the duration of gene expression was not extended and lasted not longer than 7 days. DNA delivered by asialoglycoprotein-targeting to the liver has been shown previously to remain episomal (44). The incorporation of the VSV peptide would not be expected to change the episomal status of the targeted DNA, and so an increase the duration of gene expression was not expected. Although the data do not directly prove the mechanism of increased gene expression, the results are consistent with an improved endosomal release of delivered DNA by documented pH-dependent hemolytic properties of the VSVG peptide. Regarding the magnitude of increase of reporter gene expression, our results are in accordance with previously reported in vitro studies (31, 35, 39). The difference between the increase of nuclear transgene by a factor of 101-102 and the increase of gene expression can be explained by the fact that a single mRNA is translated into multiple copies of corresponding protein. In agreement with the pH-dependent hemolytic activity of the VSVG peptide (13, 14), we neither observed physical distress in injected animals nor noticed any signs of hemolysis. Accordingly, neither elevations of serum ALT levels nor signs of liver cell necrosis or cellular infiltrates at the light microscopic level were found. In conclusion, our results demonstrate, that DNA can be targeted to the liver in vivo using a multicomponent carrier. The protein conjugate-DNA complexes are taken up by receptor-mediated endocytosis via the AsGP receptor. The incorporation of a hemolytic peptide derived from VSV G-protein into an AsOR-based nonviral vector greatly increases gene expression in vivo. This concept could be extended to other DNA carriers based on other targeting ligands, which may permit enhanced DNA delivery and gene expression to a variety of nonhepatocytic cells.

Schuster et al. ACKNOWLEDGMENT

We thank Dr. Ellen Carmichael for helpful advice. The technical assistance of Peili Zhan and the secretarial assistance of Mrs. Martha Schwartz is gratefully acknowledged. This work was supported in part by grants from the NIDDK: DK-42182 (G.Y.W.), the Immune Response Corporation (C.H.W.), and the Herman Lopata Chair in Hepatitis Research (G.Y.W.), and the Deutsche Forschungsgemeinschaft (Schu 1112/1-2 to M.J.S). LITERATURE CITED (1) Wu, G. Y., and Wu, C. H. (1987) Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J. Biol. Chem. 262, 4429-4432. (2) Wu, G. Y., and Wu, C. H. (1988) Receptor-mediated gene delivery and expression in vivo. J. Biol. Chem. 263, 1462114624. (3) Wilson, J. M., Grossman, M., Wu, C. H., Roy Chowdhury N., Wu, G. Y., and Roy Chowdhury J. (1992) Hepatocytedirected gene transfer in vivo leads to transient improvement of hypercholesterolemia in low-density lipoprotein receptordeficient rabbits. J. Biol. Chem. 26, 963-967. (4) Roy Chowdhury, N., Hays, R. M., Bommineni, V. R., Franki, N., Roy Chowdhury, J., Wu, C. H., and Wu, G. Y. (1996) Microtubular disruption prolongs the expression of human bilirubin-uridinediphosphoglucuronate-glucuronosyltransferase-1 gene transferred into Gunn rat livers. J. Biol. Chem. 271, 2341-2346. (5) Wu, C. H., Wilson, J. M., and Wu, G. Y. (1989) Targeting genes: Delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo. J. Biol. Chem. 264, 16985-16987. (6) Wu, G. Y., Wilson, J. M., Shalaby, F., Grossman, M., Shafritz, D. A., and Wu, C. H. (1991) Receptor-mediated gene delivery in vivo. Partial correction of genetic analbuminemia in Nagase rats. J. Biol. Chem. 266, 14338-14342. (7) Roy Chowdhury N., Wu, C. H., Wu, G. Y., Yerneni, P. C., Bommineni, V. R., and Roy Chowdhury, J. (1993) Fate of DNA targeted to the liver by asialoglycoprotein receptor-mediated endocytosis in vivo. J. Biol. Chem. 268, 11265-11271. (8) Bommineni, V. R., Roy Chowdhury, N., Wu, G. Y., Wu, C. H., Franki, N., Hays, R. M., and Roy Chowdhury, J. (1994) Depolymerization of hepatocellular microtubules after partial hepatectomy. J. Biol. Chem. 269, 25200-25205. (9) Graham, W. (1990) Trawling for receptors. Nature 346, 318319. (10) Zhang, L., and Ghosh, H. P. (1994) Characterization of the putative fusogenic domain in vesicular stomatitis virus glycoprotein G. J. Virol. 68, 2186-2193. (11) Fredericksen, B. L., and Whitt, M. A. (1995) Vesicular stomatitis virus glycoprotein mutations that affect membrane fusion activity and abolish virus infectivity. J. Virol. 69, 1435-1443. (12) Odell, D., Wanas, E., Yan, J., and Ghosh, H. P. (1997) Influence of membrane anchoring and cytoplamic domains on the fusogenic activity of vesicular stomatitis virus glycoprotein G. J. Virol. 71, 7996-8000. (13) Schlegel, R., and Wade, M. (1984) A synthetic peptide corresponding to the NH2 terminus of vesiculo stomatitis virus glycoprotein is a pH-dependent hemolysin. J. Biol. Chem. 259, 4691-4694. (14) Schlegel, R., and Wade, M. (1985) Biologically active peptides of the vesiculo stomatitis virus glycoprotein. J. Virol. 53, 319-323. (15) Woodgett, C., and Rose, J. K. (1986) Amino-terminal mutation of the vesicular stomatitis virus glycoprotein does not affect its fusion activity. J. Virol. 59, 486-489. (16) Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning. A laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Plainview, NY. (17) Schmidt, K., Polis, A., Hunziker, K., Fricke, R., and Yayoshi, M. (1967) Partial characterization of the sialic acidfree forms of R1-acid glycoprotein from human plasma. Biochem. J. 104, 361-368.

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