Intracellular Signal-Responsive Artificial Gene Regulation for Novel

Molecular Mechanism of Caspase-3-Induced Gene Expression of Polyplexes Formed from Polycations Grafted with Cationic Substrate Peptides. Kenji Kawamur...
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Biomacromolecules 2002, 3, 905-909

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Intracellular Signal-Responsive Artificial Gene Regulation for Novel Gene Delivery Yoshiki Katayama,*,†,‡ Kenji Fujii,† Etsuko Ito,† Shigeki Sakakihara,† Tatsuhiko Sonoda,† Masaharu Murata,† and Mizuo Maeda† Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, and PRESTO, Japan Science and Technology Corporation, Japan Received March 6, 2002; Revised Manuscript Received May 28, 2002

We describe two types of artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) or caspase-3. These molecular systems use newly synthesized cationic polymers, PAK and PAC. The PAK polymer includes substrate oligopeptide for PKA, ARRASLG, as receptor of PKA signal, while the PAC polymer possesses oligopeptide that is comprised of a substrate sequence of caspase-3, DEVD, and a cationic oligolysine, KKKKKK. These polymers formed stable complexes with DNA to totally suppress the gene expression. However, PKA or caspase-3 signal disintegrates the PAK-DNA or the PAC-DNA complex, respectively. This liberates the DNA and activated the gene expression. These systems are the first concept of an intracellular signal-responsive gene-regulation system using artificial polymer. We expect that these systems can be applied to the novel highly cell specific gene delivery strategy that is involved in our previously proposed new drug delivery concept, the drug delivery system based on responses to cellular signals. Introduction Developments in molecular biology have provided many novel drug targets relating to various diseases and made it possible to design a number of new drugs. However, those drugs have to be delivered only to target cells, which fall into a disordered state. Otherwise, an undesired side effect may occur. Such side effects are sometimes fatal. To avoid this outcome, many drug-targeting strategies have been developed.1-5 Those systems often use a molecular marker that is specific for disordered cells on the cellular surface.6-9 This strategy effectively mimics the intrinsic recognition mechanism of immune cells to the abnormal cells. However, such a so-called active targeting strategy is sometimes not entirely successful, because effective molecular markers on the cellular surface are not always available. Living cells, on the other hand, possess elaborate molecular reaction cascades, an intracellular signal transduction system in the intracellular space, to regulate the cellular functions and responses. When living cells fall into the disordered state, corresponding intracellular signals are certainly hyper- or hypoactivated.10-18 If such unusual intracellular signals can be used for purposes of switching the pharmacological activity of the drug, a novel strategy for the dosage of drugs, which is completely cell selective, can be designed. We recently reported on a drug capsule that disintegrated when exposed to the protein kinase A signal as an example of such a new strategy of the drug delivery.19 Here we attempted to apply this concept to gene delivery. For this purpose, we * To whom correspondence may be addressed. [email protected]. † Kyushu University. ‡ PRESTO, Japan Science and Technology Corporation.

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designed two artificial gene regulation systems using cationic polymers. Each polymer formed a complex with DNA to suppress the gene expression. However, those complexes were disintegrated by protein kinase A or caspase-3, which are important for live20,21 and dead22-25 intracellular signals, respectively, and activated the gene expression. Extraordinary activation of these enzymes is known to cause many diseases such as melanoma,5 prostate tumor,13 or colon cancer14 for PKA and hepatitis, Alzheimer’s disease, Perkinson’s disease, or other various nerve-denaturing diseases for caspase-3. Thus, these enzymatic activities are important criterions for the cellular condition. These systems we reported here are the first examples of an artificial gene regulation system via a cationic polymer. Results encouraged us to develop a novel basic concept of gene delivery, and we propose such a new concept of drug delivery as a new drug delivery system based on responses to cellular signals (D-RECS). Materials and Methods Preparation of PAK and PAC. The N-methacryloylpeptide monomer (N-methacryloyl-ALRRASLG and N-methacryloyl-AGDEVDGKKKKKK) was synthesized with automatic peptide synthesizer by Fmoc chemistry using corresponding Fmoc-amino acids and N-methacryloyl-alanine as the N-terminus amino acid. The N-methacryloyl-alanine was easily obtained from alanine (2.7 g, 30 mmol) and methacryloyl chloride (2.9 mL, 30 mmol) in THF/water (1/1 v/v) in the presence of an equimolar amount of NaOH at 0 °C followed by recrystallization from chloroform (white powder, yield 1.17 g, 25%). The obtained peptide was purified with reverse-phase high-performance liquid chromatography

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(HPLC) using acetonitrile/0.1% TFA (20% v/v to 80% v/v) and then lyophilized. The PAK polymer was synthesized by ordinary radical copolymerization of acrylamide (16 mg, 0.23 mmol) and N-methacryloylpeptide (6 mg, 6.7 µmol) in water (2.0 mL) at room temperature for 1 h under nitrogen atmosphere using ammonium persulfate (1.5 mg, 1.4 mmol) and N,N,N′,N′-tetramethylethylenediamine (2 µL, 2.9 mmol) as a redox initiator couple. Then it was purified with dialysis using a semipermeable membrane bag (with a molecular weight cutoff of 50 000) against water overnight followed by lyophilizing. We obtained 16 mg of the polymer, and the peptide content in the polymer chain was 1.95 mol % calculated from the value of the elemental analysis. The PAC polymer was synthesized in a manner similar to that of PAK using N-methacryloyl-peptide (25 mg, 1.7 µmol) and Nacrylamide (14 mg, 0.2 mmol) to obtain 14 mg of a white powder. The purity of the polymers was checked with reverse-phase HPLC after the dialysis using water-acetonitrile (95/5 to 70/30 for 30 min). The mean molecular weight of PAK or PAC was estimated by gel-permeation chromatography using OHPak SB-804 HQ and OHPak SB-806M HQ (Pharmacia Biothech). The calibration line was made using the results obtained from some poly(ethylene glycol)s that had known molecular weight. The aqueous sample polymer solution (3 mg/mL, 25 µL) was injected and analyzed with reflective indicator. Agarose Gel Electrophoresis of the Polymer-DNA Complex in the Absence or Presence of Activated PKA and caspase-3. pQBI63 (Promega, 0.1 µg), which is a T7 plasmid coding enhanced green fluorescent protein, was dissolved in Transcription 5X buffer (400 mM HEPES (pH 7.5), 160 mM MgCl2 and 200 mM DTT). Then various concentrations of PAK polymer were added to the solution. All the solutions were diluted to 10 µL with the same buffer and allowed to stand at room temperature for 15 min. For the disintegration of the complex, PKA C-subunit (10 U, Daiichi Pure Chemicals) was added to each solution and incubated at 37 °C for 30 min. The formation and the disintegration of the complex were assayed with 1% agarose gel electrophoresis using Tris-borate buffer (pH 8.0). In the case of the PAC polymer, experimental protocols were similar to that in the case of the PAK polymer but used 1234 bp linear DNA (0.1 µg), which was a restriction fragment from pRLnull with BamH-I and Afl-II and active caspase-3 (2U, CHEMICON) as a DNA and a cellular signal, respectively. Imaging by Atomic Force Microscopy. An atomic force microscope JSPM4210 (JEOL) was used to capture topographical images of plasmid DNA, the DNA-PAC complex before and after the treatment of caspase-3 immobilized on the mica surface. The tapping mode was used to reduce any damage caused by physical contact with the tip, and tapping frequency was set to 150 kHz. Atomic force microscopy (AFM) was operated in the ambient air at 15-25% humidity. A scanning field of view was 1 µm × 1 µm (coarse scanning) or 500 nm × 500 nm (fine scanning) with the scanning rate of 0.66 Hz and 512 scanning line. The silicon tips we used had an estimated curvature of 10 nm. Height images in the range of 0-5 nm were flattened to remove the background

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Figure 1. Synthetic scheme and chemical structures of the PAK and PAC polymers.

curvature of the mica surface, and the images were analyzed using JEOL WinSPM image analysis software. In Vitro Expression of the DNA. All experiments were performed using an in vitro expression system (T7 S30 Extract System for Circular DNA; Promega) containing T7 S30 extract and an amino acids mixture. Thus “T7 Quick Master Mix” (16 µL) and 1 mM methionine (0.5 µL) were added to a solution (10 µL as a final volume) containing the DNA (pRL-CMV, 1 µg) and was incubated at 30 °C for 30 min. Then luciferine solution was added to the 1 µL of the solution and the chemiluminescence was measured using multilabel counter ARVO SX (Wallac Inc.). When PKA C-subunit (10 U) or caspase-3 (2 U) was added to the polymer-DNA solution as the PBS solution, the reaction mixture was incubated at 30 °C for 30 min. If caspase-3 was used, its inhibitor, Ac-DEVD-CHO (5 µg, 10 nmol), was added to the reaction mixture before the expression was initiated. Results and Discussion Our strategy for designing intracellular signal-responsive polymers was to use the substrate peptide of cyclic AMPdependent protein kinase (protein kinase A, PKA) or caspase-3 (Figure 1). Both polymers, PAK and PAC,26 are graft-type copolymers that were synthesized using each methacryloyl-peptide monomer and acrylamide with radical copolymerization.19 The resulting PAK and PAC polymers contained the peptide side chain at concentrations of 1.95 and 6.18 mol %, respectively. Both polymers did not contain any unpolymerized peptide or acrylamide after the dialysis. This is important because a contamination of acrylamide monomer tends to inhibit the gene expression system. The mean moleculr weights of PAK and PAC were estimated to ca. 150 000 by gel-permeation chromatography. The peptide in the PAK polymer contained a consensus amino acid sequence, RRXSL, for the selective phosphorylation site of

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PKA.27,28 On the other hand, the peptide on the PAC polymer included the DEVD sequence for a specific substrate for caspase-329,30 and the cationic portion KKKKKK. These peptides introduced into the polymers possess cationic net charge at physiological pH. First, we investigated whether the polymers actually worked as substrates for each enzyme. It was found that PAK acted as a good substrate for PKA in the coupled enzyme assay using pyruvate kinase and lactate dehydrogenase.31 The time course of the phosphorylation in PAK with PKA (10 U) was nearly the same as that in the free substrate peptide (ALRRASLG) at 37 °C and pH 7.4 (data not shown). On the other hand, both PAC and the free peptide (AGDEVDGKKKKKK) were not cleaved with activated caspase-3 (2 U) in a competition assay performed with a colorimetric substrate, Ac-DEVD-pNA (pNA ) p-nitroanilino-). Interestingly, the polymer acted as a substrate of caspase-3 in the presence of DNA (data not shown). Presumably, the electrostatic interaction of the cationic portion of the pendant peptides with anionic charges in DNA suppressed the inhibitory effect of the cationic charge in the peptide to be cleaved with caspase-3. Actually, the cleaved fragment of the peptide whose mass number was 844.91 was seen after the treatment of the PAC-DNA complex with caspase-3. On the other hand, any fragment was not seen if DNA was not used (data not shown). Second, we examined the interaction of the polymers with DNA to form an electrostatic complex. When the PAK polymer was added to the DNA solution, the migration of the DNA was gradually suppressed in a gel-electrophoresis experiment, depending on the concentration of the polymer (the left panel in Figure 2a). An addition of the PAC polymer also suppressed the migration of the DNA in a concentrationdependent manner (lanes 1-3 in Figure 2b). These results mean that the PAK or PAC polymer actually formed a polymer complex with DNA through an electrostatic interaction. On the other hand, when activated PKA (10 U) was added to each solution of the PAK-DNA complex, the band of the original DNA was completely recovered (the right panel in Figure 2a). In the PAC-DNA complex, addition of activated caspase-3 (2 U) also tended to recover the band of original DNA (lanes 5 and 6 in Figure 2b). These results clearly indicate that the PKA signal and caspase-3 signal can each disintegrate the DNA-polymer complex to release the DNA. Figure 3 shows atomic force microscopy (AFM) images of the DNA-PAC complex in the absence or presence of caspase-3 (2 U). These images also supported the formation of a loosely condensed complex (mean diameter of 200-300 nm) of PAC-DNA and the disintegration of the complex with caspase-3 signal. Since the PKA or caspase-3 signal actually decreased the stability of the polymer-DNA complex, we then investigated whether this stability change in the complex is directly related to the regulation of the gene expression by using an in vitro expression system.32,33 Figure 4 shows the regulation of the luciferase expression with the PKA or caspase-3 signal. The luciferase expression was significantly and totally suppressed in PAK-DNA and PAC-DNA complexes, respectively.

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Figure 2. Formation of the polymer-DNA complex and its disintegration in response to the PKA (a) or caspase-3 (b) signal: (a) Left panel shows the result of 1% agarose gel electrophoresis of PAKDNA (pQBI63) complex. The ratios of the concentration of arginine residue in PAK versus that of phosphate residue in DNA in the respective lanes (1-7) were 0, 0.5, 1.0, 2.0, 3.0, 5.0, and 10.0. After the addition of PKA C-subunit (10 U), the pattern of the gel electrophoresis changed to that shown in the right panel. (b) 1234 bp linear DNA (0.1 µg), which was a restriction fragment from pRLnull with BamH-I and Afl-II, was dissolved in Transcription 5X buffer. Then various concentrations of PAC polymer were added and diluted to 10 µL. Active caspase-3 (2 U) was added in some of the solutions. Lanes 2-4 and 5-7 show the electropherograms of the DNApolymer mixture in the absence and presence of caspase-3, respectively. The ratio of cationic charge of the PAC polymer (the net charge of each peptide side-chain was assumed to be +2) to the phosphate residue in the DNA was 1.0 in lanes 3 and 6 and 5.0 in lanes 4 and 7. Lanes 2 and 5 did not contain PAC polymer. Lane 1 shows the 1 kb DNA ladder.

However, the complex formed by DNA-polymer mixture at 1:1 charge ratio also suppressed the gene expression quite effectively despite the result of the Figure 2 in which most of DNA did not form the complex with the polymer at 1:1 charge ratio. This reason is unclear. However, the DNApolymer complex should weaken in the gel electrophoresis, because DNA and the polymer tend to move to the opposite direction. This factor may cause the difference between the results in the electrophoresis and in vitro experiment experiments. Migration of DNA would not be retarded if the interaction were quite weak. On the other hand, RNApolymerase has to move along the entire region of DNA strand coding the protein. Thus, the suppression of the gene expression by the polymer is easier than the retardation of the DNA migration in the gel electrophoresis. On the other hand, polyacrylamide or poly-L-lysine did not suppress the expression of luciferase at all (data not shown). Thus, the suppression of the gene was not caused by an ambiguous inhibitory effect of acrylamide on the expression system or by simple electrostatic interaction. Probably, an electrostatic mooring of the neutral polyacrylamide chain refused the access of the transcriptional factors, in conjunction with steric hindrance. However, an addition of PKA(10 U) or activated caspase-3 (2 U) recovered the gene expression up to 80% in the PAK-DNA system and 100% in the PAC-DNA system

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Figure 3. AFM height image of DNA and the DNA-PAC complex before and after the treatment of activated caspase-3. Height is indicated by a color code with dark (0 nm) and light (3 nm). (a) pQBI63 plasmid DNA, (b) the pQBI63-PAC complex. The ratio of cationic charge of the PAC polymer (the net charge of each peptide side chain was assumed to be +2) to the phosphate residue in the DNA was 3.0. (c) The pQBI63-PAC complex after the treatment of caspase-3 (2 U) for 30 min at room temperature.

compared with that of free DNA. On the other hand, if other kinases (MAPK or Akt) or protease (factor Xa) are used instead of PKA or caspase-3 in the system using PAK or PAC, respectively, the recovery of the luciferase expression was not seen in the same conditions (data not shown). Thus the response of the system is specific to the activity of the target enzyme. Obtained results all supported the diagram of the gene-switching mechanisms shown in Figure 5. Both cationic polymers, PAK and PAC, form stable complexes with DNA through electrostatic interaction to suppress the accessibility of RNA polymerase. If PKA is continuously activated, the serine residues in the peptide included in PAK are phosphorylated. This causes the cancellation of the net cationic charge of the entire polymer and renders the complex unstable, whereas the cationic portion of the peptide is cleaved with activated caspase-3 in the DNA-PAC complex. In this case, the net charge of the polymer changes from cationic to anionic. This also labilizes the polymer-DNA complex and renders the DNA free. As a result, gene expression should be accelerated by the PKA or caspase-3 signal. The question, why PKA or caspase-3 was not refused by the DNA-polymer complex despite the effective refusal to RNA-polymerase, may be able to be explained as follows. The interaction manner between the polymer-DNA complex and RNA-polymerase should be different from that between the complex and other enzymes such as PKA or caspase-3. RNA-polymerase has to move along the entire region of the DNA strand for the transcription. On the other hand, PKA or caspase-3 can act if it can interact just with the peptide on the polymer. Thus, PKA or caspase-3 should be able to act from the outside part of the complex. In fact, a similar phenomenon can be seen in the transcriptional regulation in living cell. Recently the mechanism of the transcriptional

Figure 4. Suppression of luciferase expression with the polymers and its cancellation in response to the PKA (a) or caspase-3 (b) signal. (a) The left column (DNA) shows the relative luminescence intensity of the expressed luciferase in the solution containing 0.1 µg of the DNA (0.48 nmol/µL as phosphate residue) only. In the DNA-PAK, PAK polymer (0.48 nmol/µL as arginine residue) and the DNA (0.48 nmol/µL as phosphate residue) were mixed and allowed to stand at room temperature for 15 min before in vitro expression. Then, in the DNA-PAK + PKA C-subunit, PKA C-subunit (10 U) was added to the DNA-PAK solution, which was then allowed to stand at 37 °C for 2 h before the expression experiment. (b) The left column (DNA) shows the relative luminescence intensity of the expressed luciferase in the solution containing 1 µg of the DNA (4.8 nmol/µL as phosphate residue) only. In the DNA-PAC (+/- ) 1.0) and (+/- ) 3.0), PAC polymer was mixed with the DNA (1 µg) at a concentration in which the ratios of cationic charge of the PAC polymer (the net charge of each peptide side chain was assumed to be +2) to the phosphate residue in the DNA were 1.0 and 3.0, respectively, before the expression experiment. In DNA-PAC (+/- ) 1.0) + caspase-3 and (+/- ) 3.0) + caspase-3, each DNA-PAC solution was incubated with activated caspase-3 (2 U) at 37 °C for 2 h before the expression experiment.

regulation of gene has received much attention. The gene is generally suppressed its transcription through the electrostatic complexation with cationic histone complex. The transcription is regulated by the change of the content of acetylation on the lysine residue, because acetylation tends to cancel the cationic charges of histone.34,35 In this case, RNApolymerase cannot access to the DNA. On the other hand, histone-acetyltransferase can act for the DNA-histone complex. Thus, our system is also interesting as a model of gene suppression and gene regulation with the histone complex.

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Figure 5. Diagram of the artificial gene regulation in response to protein kinase (a) and caspase (b) signals. The PAK and PAC polymers, which include cationic peptide side chains, form stable complexes with DNA through an electrostatic interaction and suppress the gene expression. Protein kinase A (PKA) phosphorylates the peptide side chain of PAK to introduce anionic charges into the polymer. Caspase-3 cleaved the cationic portions of the peptide in PAC, which changed the charge of the polymer from cationic to anionic. These events cause the disintegration of the complexes, releasing the DNA to activate transcription of the gene.

We have established here the first example of artificial gene regulation systems that potentially satisfy the condition of the “D-RECS” concept in gene delivery by using cationic peptide-polymer conjugates. The use of an enhancer sequence in the delivered DNA is able to selectively induce gene expression in the target cell.36,37 However, this strategy can only use nuclear signals. In comparison, our strategy can be applied to a wider range of intracellular signals including signals in the cytosol. This characteristic offers another important advantage for a gene delivery system. Generally, the gene carrier has to form tight complexes with DNA, but the complex has to be disintegrated easily once the complex is delivered into a cell.38,39 These somewhat conflicting requirements have been an impediment in the development of a gene carrier that has high efficiency of gene expression. Our system can potentially resolve this issue, because the complex should be disintegrated actively in response to a target cellular signal. We have also succeeded in delivering the PAK-DNA complex into NIH 3T3 cells, and this application of the regulation system to living cells will be reported in due course. Acknowledgment. This work was financially supported by PRESTO, Japan Science Corporation. References and Notes (1) Meers, P. AdV. Drug DeliVery ReV. 2001, 53, 265-272. (2) Torchilin, V. P. Eur. J. Pharm. Sci. 2000, 11, S81-S91. (3) Yasugi, K.; Nagasaki, Y.; Kato, M.; Kataoka, K. J. Controlled Release 1999, 62, 89-100. (4) Kohori, F.; Sakai, K.; Aoyagi, T.; Yokoyama, M.; Sakurai, Y.; Okano, T. J. Controlled Release 1998, 55, 87-98. (5) Nabel, G. J.; Nabel, E. G.; Yang, Z. Y.; Fox, B. A.; Plautz, G. E.; Gao, X.; Huang, L.; Shu, S.; Gordon, D.; Chang, A. E. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11307-11311.

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