Gene Therapy (2005) 12, 1581–1590 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt RESEARCH ARTICLE The metabolizes peptide-mediated nonviral gene delivery systems

J Kim, C-P Chen and KG Rice Division of Medicinal and Natural Product Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA, USA

The proteasome is a multisubunit cytosolic protein complex directly with proteasome inhibition, and was not the result of responsible for degrading cytosolic proteins. Several studies lysosomal enzyme inhibition. The enhanced transfection was have implicated its involvement in the processing of viral specific for peptide DNA condensates, whereas Lipofecta- particles used for gene delivery, thereby limiting the mine- and polyethylenimine-mediated gene transfer were efficiency of gene transfer. Peptide-based nonviral gene significantly blocked. A series of novel gene transfer peptides delivery systems are sufficiently similar to viral particles in containing intrinsic GA proteasome inhibitors (CWK18(GA)n, their size and surface properties and thereby could also be where n ¼ 4, 6, 8 and 10) were synthesized and found to recognized and metabolized by the proteasome. The present inhibit the proteasome. The gene transfer efficiency mediated study utilized proteasome inhibitors (MG 115 and MG 132) by these peptides in four different cell lines established that a to establish that peptide DNA condensates are metabolized GA repeat of four is sufficient to block the proteasome and by the proteasome, thereby limiting their gene transfer significantly enhance the gene transfer. Together, these efficiency. Transfection of HepG2 or cystic fibrosis/T1 (CF/T1) results implicate the proteasome as a previously undiscov- cells with CWK18 DNA condensates in the presence of ered route of metabolism of peptide-based nonviral gene MG 115 or MG 132 resulted in significantly enhanced gene delivery systems and provide a rationale for the use of expression. MG 115 and MG 132 increased luciferase proteasome inhibition to increase gene transfer efficiency. expression 30-fold in a dose-dependent manner in HepG2 Gene Therapy (2005) 12, 1581–1590. doi:10.1038/ and CF/T1. The enhanced gene expression correlated sj.gt.3302575; published online 30 June 2005

Keywords: peptide-mediated gene delivery; metabolism; proteasome; gene transfer

Introduction subunits having multiple proteolytic activities.11,12 The chymotrypsin-like activity, trypsin-like activity and The premature metabolism of plasmid DNA limits both peptidyl-glutamyl peptide hydrolyzing activity residing the level and duration of transient gene expression by within the 20S proteasome are responsible for degrading nonviral gene delivery systems.1 However, no prior studies polypeptides.13–15 have determined which enzymes are involved in dissociat- Recently, proteasomal processing has been implicated ing and degrading nonviral gene delivery polymers prior in the intracellular trafficking and metabolism of to the metabolism of plasmid DNA. The accepted dogma is viral vectors used for gene delivery. In the presence that peptide-based nonviral gene delivery systems are of proteasome inhibitors, adeno-associated virus (AAV) degraded by lysosomal proteases.2 Since nonviral gene mediated 50-fold higher gene expression in cell culture delivery systems represent unique combinations of poly- and enhanced the level of AAV-mediated transduction mer, lipid, peptide and DNA, there are likely multiple in the liver and lung.16 pathways that account for the intracellular metabolism of Based on the evidence that viruses are metabolized individual nonviral gene delivery systems. by the proteasome, we speculated that peptide DNA are multisubunit protease complexes condensates could also undergo a similar route of that play a major role in the selective degradation metabolism due to their comparable size and suscept- of intracellular proteins3–5 such as proteins that are miss ibility to serine proteases. In support of this hypothesis, folded or oxidatively damaged, proteins involved in the following investigation demonstrates that low mole- signal transduction, transcription factors and the proces- cular weight proteasome inhibitors (MG 115 and MG sing of foreign proteins for antigen presentation.6–10 The 132) block peptide DNA condensate metabolism and proteolytic core, 20S proteasome, has a cylindrical simultaneously enhance gene transfer efficiency. Further structure composed of a stack of four heptameric support for this hypothesis comes from the finding that proteasome inhibition enhances peptide-mediated gene transfer but not gene transfer mediated by other carriers Correspondence: Dr KG Rice, Division of Medicinal and Natural Product such as polyethylenimine (PEI) and Lipofectamine. This Chemistry, College of Pharmacy, 115 S. Grand Ave., University of Iowa, Iowa City, IA 52242, USA could explain differences in gene transfer efficiency Received 22 December 2004; accepted 16 May 2005; published observed between nonviral delivery systems and also online 30 June 2005 provide a working hypothesis on how to improve the Proteasome metabolism of nonviral gene delivery systems J Kim et al 1582 gene transfer efficiency of peptide-mediated gene deliv- There are also mechanisms by which nonubiquinated ery by blocking metabolism. peptides are degraded by the proteasome. Peptide DNA condensates composed primarily of polylysine se- Results quences may also be recognized as foreign and processed by this pathway resulting in their metabolism. To Proteasomes degrade a variety of proteins by recogniz- investigate this possibility, proteasome inhibitors were ing polyubiquitin attached to their lysine side chains.17 applied to mammalian cells during in vitro gene transfer 18 with CWK18 DNA condensates. Proteasome inhibition was achieved using two tripeptidyl aldehyde analogues, MG 115 and MG 132, that block both the chymotryptic and tryptic-like activity19 and by epoxomicin, which selectively blocks the chymotrypsin-like activity of 20S proteasomes (Figure 1).20 Since proteasome inhibitors are known to induce ,21–23 the relationship between proteasome inhibitor concentration and cell viability was deter- mined. Treatment of HepG2 cells for 24 h with MG 115, MG 132 or epoxomicin resulted in a dose-dependent decrease in cell viability measured by the MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

assay (Figure 2a–c). The LD50 was approximately 2 mM for MG 115 and MG 132 and was 0.5 mM for epoxomicin. Figure 1 Schematic of the proteasome and the site of inhibition by MG The percent inhibition of the proteasomal chymo- 115, MG 132 and epoxomicin. tryptic-like activity measured in HepG2 cell lysate was

Figure 2 Influence of proteasome inhibitors on cell viability. HepG2 cells were treated in triplicate with varying concentrations of MG 115 (a), MG 132 (b) or epoxomicin (c) for 24 h and assessed for cytotoxicity by the MTT assay. The cytotoxicity of a cathepsin B inhibitor, CA-074 Me, on HepG2 cells was evaluated (d). Likewise, CF/T1 cells were treated with varying concentrations of MG 115 (e), MG 132 (f) for 24 h and assessed for cytotoxicity by the MTT 7 assay. The LD50 was estimated from the concentration of proteasome inhibitor that caused 50% apoptosis. Values are the mean s.d. (n ¼ 3).

Gene Therapy Proteasome metabolism of nonviral gene delivery systems J Kim et al 1583

Figure 3 Inhibition of proteasomal activity. HepG2 cells were treated in triplicate with varying concentrations of MG 115 (a), MG 132 (b) or epoxomicin (c) for 24 h and assessed for proteasome activity. The cathepsin B activity and proteasome activity were determined following treatment of HepG2 cells with CA-074 Me (d). Likewise, CF/T1 cells were treated with varying concentrations of MG 115 (e) and MG 132 (f) for 24 h and assessed for proteasome activity. The proteasomal activity is expressed as the released AMC per milligram of total cell protein. Values are the mean7s.d. (n ¼ 3).

directly related to the inhibitor dose applied to cells peptide specific, CWK18 was substituted with CWK17C, a (Figure 3a–c). Normalization of the proteasome activity more potent gene transfer peptide that undergoes DNA on a per mg of protein basis accounted for losses due template sulfhydryl crosslinking to form DNA conden- 18 to cell apoptosis. MG 115 and MG 132 produced an IC50 sates. Transfection of HepG2 cells with CWK17C DNA of 2 and 5 mM, respectively, whereas epoxomicin was in the presence of 10 mM MG 132 resulted in a 50-fold significantly more potent with an IC50 of 0.5 mM. enhancement in gene transfer efficiency (not shown). The

The IC50 of each proteasome inhibitor was further relative magnitude change in gene expression was analyzed by adding inhibitors directly to HepG2 cell independently reproduced twice with triplicate analysis lysates. MG 115, MG 132 and epoxomicin each possessed for each experiment; however, the absolute magnitude in an IC50 of approximately 0.1 mM, indicating the cell gene expression was somewhat variable between experi- membrane was an uptake barrier to proteasome inhibi- ments which is a function of cell seeding number and cell tors. confluency at the time of transfection.

The reporter gene expression mediated by CWK18 The influence of proteasome inhibitors on lysosomal DNA condensates was amplified by treating cells with enzyme activity was analyzed to determine if inadver- MG 115, 132 and epoxomicin as a function of increasing tent cathepsin B inhibition could be responsible for the proteasome inhibitor dose (Figure 4a–c). The luciferase increased luciferase expression. Treatment of HepG2 expression increased 30-fold when treating HepG2 cells cells with up to 10 mM of a cathepsin B inhibitor (CA-074 with 10 mM of MG 115 or MG 132 (Figure 4a and b). The Me) did not alter cell viability as measured by the MTT increase in gene expression was less significant when assay (Figure 2d). CA-074 Me concentrations of 0.05 mM treating cells with epoxomicin, which only amplified and higher inhibited cathepsin B to less than 50% gene expression by 10-fold at 1 mM (Figure 4c). In order to maximal activity without significantly altering protea- determine if the observed increase in gene expression is some activity (Figure 3d). In contrast to the amplified

Gene Therapy Proteasome metabolism of nonviral gene delivery systems J Kim et al 1584

Figure 4 Influence of proteasome inhibitors on peptide mediated gene delivery. HepG2 cells were treated with varying concentrations of MG 115 (a), MG

132 (b), epoxomicin (c) or CA 074 Me (d) for 24 h during transfection with CWK18 DNA condensates encoding luciferase. Likewise, CF/T1 cells were

treated with varying concentrations of MG 115 (e) and MG 132 (f) during CWK18 DNA transfection. Luciferase intensities were converted to fmol using a standard curve and normalized for total cell protein. Values are the mean7s.d.viations of three determinations.

gene expression observed by proteasome inhibition, To establish if proteasome inhibition also amplify gene

the gene expression mediated by CWK18 DNA was not expression in cystic fibrosis/T1 (CF/T1) cells, CWK18 influenced by cathepsin B inhibition, suggesting that DNA condensates were transfected in the presence of lysosomal degradation was not responsible (Figure 4d). increasing concentrations of MG 115 and MG 132. Protea- Likewise, the effect of MG 132 on the cathepsin B activity some inhibitors caused apoptosis of CF/T1 cells with an

was tested at 0–20 mM. Approximately 80–120% of LD50 of approximately 50 mM (Figure 2e and f). Treatment cathepsin B activity was recovered in the presence of of CF/T1 cells with MG 115 or MG 132 inhibited the 2–20 mM MG 132, respectively, indicating that this proteasome activity in a dose-dependent manner with a proteasome inhibitor does not block cathepsin B activity. maximal inhibition of 40% at 10 mM (Figure 3e and f). The To determine if proteasome inhibition influences decrease in proteasome activity correlated with a 10-fold the gene expression mediated by nonpeptide gene increase in gene expression when treating cells with MG transfer agents, HepG2 cells were transfected with 115 and MG 132 (Figure 4e and f). Lipofectamine and PEI in the presence of MG 115, MG The amplified luciferase expression could either be the 132 and epoxomicin (Figure 5a and b). Inhibitor concen- result of an increased number of transfectants or due trations of 5 mM of MG 132 and 115, and 1 mM epoxomicin to an increase in the transduction level of transfectants.

were chosen to achieve 50% proteasomal inhibition CWK18 DNA condensates encoding nuclear-targeted in HepG2 cells. The luciferase expression mediated b-galactosidase (NTbGal) were used to distinguish bet- by Lipofectamine gene transfer was slightly decreased ween these possibilities. Analysis of NTbGal-expressing by MG 115 and epoxomicin and significantly decreased cells transformed in the presence and absence of by MG 132 (Figure 5a). Similarly, the gene transfer proteasome inhibitors established that the number of efficiency mediated by PEI was reduced by nearly transformed cells was the same (number of expressing 10-fold when treating with each proteasome inhibitor cells per field), but that the transduction level (intensity (Figure 5b). of blue in cells) was significantly increased by the

Gene Therapy Proteasome metabolism of nonviral gene delivery systems J Kim et al 1585

Figure 7 Influence of proteasome inhibition on plasmid DNA metabolism. 125 CWK18 I-DNA condensates were used to transfect HepG2 cells in the presence of 0, 0.1, 2, 5, 10 or 20 mM of MG 132. Cells were harvested 2 h after the transfection and DNA was extracted from the cell lysate, resolved by agarose gel electrophoresis and detected by autoradiography.

influence the ability of proteasome inhibition to amplify gene expression. To directly determine if plasmid DNA metabolism was influenced by incubation with proteasome inhibi- 125 tors, HepG2 cells were transfected with CWK18 I-DNA condensates. During a 2 h incubation, approximately 12% of the DNA dose remained cell associated following extensive washing with phosphate-buffered saline (PBS). Analysis of the cell-associated DNA recovered by agarose gel electrophoresis and autoradiography estab- Figure 5 Influence of proteasome inhibition on polyethyleneimine and lished that the amount of supercoiled and open circular Lipofectamine-mediated transfection. HepG2 cells were treated with 5 mM DNA increased five-fold as a function of protea- MG 115 or MG 132, or 1 mM epoxomicin for 24 h during transfection with 5 mg of plasmid DNA complexed with Lipofectamine (a) or PEI (b). some inhibitor concentration (Figure 7). A similar result Luciferase intensities were normalized for total cell protein. Values are occurred when harvesting cells after a 4 h transfection. mean7s.d. of three determinations. To establish if an intrinsic peptide proteasome inhibitor derived from the Epstein–-Barr virus nuclear antigen-1 would enhance gene expression, a panel of

four peptides was synthesized by extending CWK18

on the C-terminal side with (GA)n repeats of 4, 6, 8 or 10.24 A fluorescent displacement assay confirmed that

CWK18(GA)n peptides bound to DNA with comparable

affinity as CWK18. Likewise, QELS (quasielastic light

scattering) analysis of CWK18(GA)n DNA condensates established the formation of particles of approximately 80 nm in diameter that were indistinguishable from

those formed using CWK18.

CWK18(GA)n peptides were each studied for their ability to directly inhibit the proteasome derived from

HepG2 cells. CWK18(GA)4,CWK18(GA)6 and CWK18(GA)8 each produced a comparable dose-dependent inhibition of proteasomal lysis of Suc-LLVY-AMC (7-amino-4- methylcoumarin), with a maximal inhibition of 30%

observed at 10 mM.CWK18(GA)10 was a more potent inhi- bitor, resulting in 65% proteasome inhibition at 10 mM (Figure 8). Figure 6 Influence of proteasome inhibition on transduction. HepG2 cells In vitro transfection of CWK18(GA)n DNA condensates were transfected with CWK18 DNA condensates encoding NTbGal in the in HepG2, COS-7, 3T3 and CHO cells established that presence of 10 mM MG 115 or MG 132, or 1 mM epoxomicin for 24 h, and CWK18(GA)4 and CWK18(GA)6 were significantly more then grown for an additional 24 h. Cells and then treated with X-gal potent gene transfer peptides relative to a control trans- and photographed. fection using CWK18 DNA (Figure 9) COS-7 and 3T3 cells

were transformed by CWK18(GA)4 and CWK18(GA)6 with approximately eight-fold greater efficiency over control, proteasome inhibition by MG 115 or MG 132, but less so whereas CWK18(GA)8 and CWK18(GA)10 were equal or by epoxomicin (Figure 6). Quantitative analysis of less efficient than control. Transfection of HepG2 cells

NTbGal expression revealed a six-fold enhancement mediated by CWK18(GA)4,CWK18(GA)6 and CWK18(GA)8 when treating with 10 mM MG 115 or 132, establishing was nearly 20-fold greater than control. The transfection that the nature of the reporter gene did not greatly of CHO cells was maximal using CWK18(GA)8.

Gene Therapy Proteasome metabolism of nonviral gene delivery systems J Kim et al 1586 The finding that CWK18(GA)10 was the most potent ing gene transfer could be due to less efficient cell uptake

proteasome inhibitor (Figure 8) but less efficient than of DNA CWK18(GA)10 as the result of steric repulsion of

CWK18(GA)4, CWK18(GA)6 and CWK18(GA)8 at mediat- the GA repeat blocking electrostatic binding of the DNA condensates to the cell surface. To examine this possibi- lity, the percent of cell-associated DNA was measured following treatment of HepG2 cells with 125I-DNA

condensates. Condensates prepared with CWK18(GA)4,

CWK18(GA)6 and CWK18(GA)8 resulted in approximately 17% of the DNA remaining cell associated in 2 h,

whereas condensates prepared with CWK18(GA)10 only afforded 11%. These results are consistent with increased

shielding of the DNA condensates with CWK18(GA)10, which limits its ability to mediate gene transfer electro- statically.

Discussion The proteasome is a major intracellular proteolytic regulatory element involved in many cellular pro- cesses.25 The majority of substrates for the proteasome are denatured ubiquitinated proteins; however, large Figure 8 Proteasome inhibition using CWK18(GA)n. Incubation of either macromolecular particles such as viruses are also 16,26 CWK18 or CWK18(GA)n (0, 5 or 10 mM) and Suc-Leu-Leu-Val-Tyr-AMC in recognized and degraded. It therefore stands to 250 ml of the proteolysis buffer containing 5 mM ATP and 50 mloftheHepG2 reason that peptide DNA particles of similar size to lysate (0.4 mg/ml protein) at 371C for 3 h resulted in partial inhibition of the proteasome. The proteolysis product, AMC, was quantified by RP-HPLC and viral particles may also be recognized by the proteasome, used to calculate the percent proteasome inhibition. The results establish that especially since lysine residues predominate on the

CWK18(GA)4,CWK18(GA)6 and CWK18(GA)8 possess indistinguishable surface of these particles and are the major recognition inhibitory potency, whereas CWK18(GA)10 is significantly more potent. determinant for the ubiquination pathway.

Figure 9 In vitro gene transfer efficiency using CWK18(GA)n. HepG2, COS-7, 3T3 and CHO cells were transfected for 24 h with CWK18(GA)n DNA and

compared to CWK18 DNA condensates as a control. The gene transfer efficiency increased eight- to 30-fold over control, depending on cell type, when using

CWK18(GA)4,CWK18(GA)6 and CWK18(GA)8 as the gene transfer peptide, whereas the gene transfer of CWK18(GA)10 was not significantly increased.

Gene Therapy Proteasome metabolism of nonviral gene delivery systems J Kim et al 1587 Proteasomal inhibitors were first reported to enhance We incorporated GA repeats into DNA-condensing the gene expression mediated by a viral vector in CF/T1, peptides and tested these as gene delivery agents a human cell line that display the phenotype of CF containing intrinsic proteasome inhibitors. The results 16 endothelial cells. In the present study, we demonstrate demonstrate that CWK18(GA)n peptides can directly that proteasome inhibition also enhances peptide- inhibit the proteasome prepared from HepG2 cells mediated nonviral gene delivery in CF/T1 cells and (Figure 8). Although the potency of this inhibition is have extended these results to include other cell lines. As weaker than MG 115, MG 132 and epoxomicin, the effect illustrated in both HepG2 and CF/T1 cells, proteasome of a GA peptide may be amplified by its coincident inhibition by MG 115 or MG 132 resulted in a significant intracellular targeting with DNA, achieving significant enhancement in gene expression (Figure 4). The magni- concentrations in transformed cells. Likewise, GA pep- tude of enhanced gene expression was highly inhibitor tides may be capable of more selective inhibition of one dose dependent and correlated with the percent protea- of the three proteolytic activities of the proteasome. somal inhibition (Figure 3). Conversely, lysosomal In agreement with this hypothesis, the gene transfer enzyme inhibition failed to enhance CWK18 DNA gene efficiency mediated by CWK18(GA)4 or CWK18(GA)6 transfer. Nonpeptide gene delivery systems such as PEI DNA condensates was significantly greater in four and Lipofectamine were negatively influenced by pro- different cell lines relative to a control of CWK18 DNA teasomal inhibition (Figure 5). Likewise, the enhanced (Figure 9). This was not the result of increased cell gene expression was independent of reporter gene, since uptake, since longer GA sequences of 10 repeats partially luciferase and NTbGal reporter genes both demonstrated blocked cell entry of 125I-DNA. The enhanced gene amplified gene expression as a result of proteasome transfer efficiency is very likely the result of proteasomal inhibition. Collectively, these data support a working inhibition causing an increased stability of peptide DNA hypothesis that proteasomal inhibition blocks the meta- condensates. Inhibition of peptide DNA condensate bolism of CWK18 DNA condensates leading to retention metabolism using this novel strategy may also increase of plasmid DNA and higher levels of gene expression. the level and duration of gene expression in vivo. In support of this hypothesis, proteasome inhibition A detailed understanding of how nonviral gene caused an increase in cell transduction, but not the delivery systems gain entry into the cell, stabilize DNA number of transfectants, as observed by the intensity of from metabolism and mediate delivery to the nucleus is blue-stained cells following transformation with NTbGal essential toward finding ways to optimize expression. (Figure 6). This observation was likely the result of The proteasome is another important barrier that limits an increase in the quantity of intracellular plasmid DNA the effectiveness of peptide DNA gene delivery systems. in transformed cells. A direct measurement of the intracellular plasmid DNA recovered 2 h after transfec- tion demonstrated a proteasome inhibitor dose-depen- Materials and methods dent five-fold increase in plasmid DNA (Figure 7). Thus, the increase in intracellular plasmid DNA correlates HepG2 cells were provided by the Center for Gene directly with the increase in gene expression, which Therapy of CF in the University of Iowa. CF/T1 cells relates to the inhibition of proteasomal activity. These were kindly provided by Dr Englehardt at The Uni- data strongly suggest a mechanism in which internalized versity of Iowa. DMEM, MEM, Hams-F12, L-glutamine,

CWK18 DNA condensates are metabolized by the protea- sodium pyruvate, penicillin/streptomycin, inactivated some to release naked DNA, which is degraded by fetal bovine serum (FBS) and Lipofectamine 2000 were endonucleases (Figure 1). In the absence of proteasome from Invitrogen Life Technologies Inc. (Carlsbad, CA, inhibition, very little intact plasmid DNA can be detected USA). A 5.6 kbp plasmid (pCMVL) encoding the reporter in association with cells within 2 h following transfec- gene luciferase was a gift from Dr Hickman at the tion (Figure 7). Proteasome inhibition apparently blocks University of California (Davis, CA, USA). The 7.5 kbp the digestion of CWK18, which leads to a stabilization of plasmid expressing NTbGal under the control of the

DNA condensates, since intact CWK18 DNA condensates CMV promoter was obtained from the University of are resistant to digestion by DNAse.27,28 The stabilization Michigan core vector laboratory (Ann Arbor, MI, USA). of CWK18 DNA apparently improves the efficiency Endotoxin-free plasmids were purified from Escherichia of gene transfer, presumably by slower controlled coli on a Qiagen ultrapure column according to the release of DNA into the cytosol. Lipofectamine and manufacturer’s instructions. Luciferase from Photinus PEI are not peptide substrates that are susceptible to pyralis, D-luciferin, ATP, b-galactosidase, CPRG (chloram- the proteasome. Their ability to avoid metabolism by the phenicol-b-D-galactopyranoside) were purchased from proteasome in the cytosol may be related to their greater Roche Diagnostics Corporation (Indianapolis, IN, USA). inherent gene transfer efficiency relative to peptide gene BCA assay kit was from Pierce Biotechnology Inc. delivery. (Rockford, IL, USA). Bovine insulin, hydrocortisone, Organisms such as the Epstein–Barr virus have also endothelial cell growth supplement, epidermal growth evolved peptide sequences that serve to block the action factor, 3,30-5-triiodo-L-thyronine, apo-transferrin, cholera of the proteasome.24,29,30 Earlier investigations have toxin and polyethyleneimine (25K, branched) were demonstrated that a GA dipeptide with as few as four purchased from Sigma-Aldrich Chemical Co. (St Louis, repeats serves as a transferable element to protect MO, USA). MG 115 (N-Cbz-Leu-Leu-Nva-CHO), MG 132 proteins from proteasome degradation.31 Extrapolation (N-Cbz-Leu-Leu-Nle-CHO), epoxomicin, proteasome of these findings would suggest that peptide DNA substrate III (Suc-Leu-Leu-Val-Tyr-AMC), CA-074 Me, condensates containing GA repeats of various lengths a cathepsin B inhibitor, and cathepsin B substrate III may also protect DNA from metabolism and increase (N-Cbz-Arg-Arg-AMC) were purchased from Calbio- their gene transfer efficiency. chem (San Diego, CA, USA). X-gal (5-bromo-4-chloro-3-

Gene Therapy Proteasome metabolism of nonviral gene delivery systems J Kim et al 1588 indolyl-b-galactopyranoside) was purchased from Boeh- media were replaced with 2 ml of fresh culture media ringer Mannheim Corporation (Indianapolis, IN, USA). and 500 ml of 0.5% (w/v) MTT in PBS solution, and then All the other agents were reagent grade and used incubated for 2 h to let formazan crystals form. The without further purification. crystals were dissolved by adding 2 ml of dimethylsulf- oxide (DMSO) then measured spectrophotometrically at Preparation of DNA-condensing peptides 595 nm on a microplate reader (Bio-Rad, Bethesda, MD,

The alkylated-Cys-Trp-Lys18 (CWK18) DNA-condensing USA). The percent viability was determined relative to peptide was synthesized as reported previously.32 untreated cells.

CWK18(GA)n peptides (n ¼ 4, 6, 8 or 10) were synthesized CF/T1 cells were grown in Hams-F12 supplemented using standard Fmoc procedures with 9-hydroxybenzo- with 10 mg/ml of insulin, 0.3 mM hydrocortisone, 4 mg/ml triazole and diisopropylcarbodiimide double couplings of endothelial cell growth supplement, 25 ng/ml of at 30 mmol scale on an Apex 396 Advanced ChemTech epidermal growth factor, 20 pg/ml triiodothyronine, solid-phase peptide synthesizer. Peptides were cleaved 5 mg/ml of apotransferrin and 10 ng/ml of cholera toxin

from the resin and side chain protecting groups were at 371C in a humidified 5% CO2 incubator. Cells were removed by reaction with TFA/ethane dithiol/water plated on 6 Â 35-mm wells at 1.5 Â 105 cells/well (95:2.5:2.5 (v/v/v)) for 3 h at room temperature. Cleaved and grown for a week to reach 60% confluency. MG peptides were precipitated with cold ether. The pre- 115 or MG 132 were added in the culture media at the cipitates were washed twice with cold ether and concentration of 10, 25 or 50 mM and incubated with the dissolved in 0.1% TFA for purification. Peptides were cells for 6 h, and then incubated further with 5 mM MG purified to homogeneity on RP-HPLC by injecting 115 or MG 132 for another 18 h at 371C. After the 24 h

2 mmol onto a Vydac C18 semipreparative column treatment, cells were analyzed by the MTT assay as (Vydac218TP1022, 2 Â 25 cm) eluted at 10 ml/min with described above. a gradient of acetonitrile of 10–25% in 0.1% TFA over 30 min while monitoring tryptophan absorbance at Proteasomal activity 280 nm. The major peak was collected and pooled from HepG2 or CF/T1 cells were treated with proteasome multiple runs, concentrated by rotary evaporation and inhibitors as described above, and then lysed by three lyophilized. Purified peptides were reconstituted in 0.1% freezing/thawing cycles in 300 ml of distilled water. The TFA (degassed with argon) and quantified by tryptophan lysates were centrifuged at 13 000 g at 41C for 30 min. À1 À1 absorbance (e280 ¼ 5600 M cm ) to determine the iso- The supernatant (50 ml) was diluted with 245 mlof lated yield, which was typically 10–15%. The purity was proteolysis buffer (50 mM Hepes, 20 mM KCl, 5 mM 34 495%. The peptides were characterized on Agilent 1100 MgCl2 and 1 mM DTT, pH 7.8) containing 5 mM ATP. LC-ESI-ion trap to provide m/z (calculated/found): The reaction was initiated by adding 5 mlof2mM Suc- CWK18(GA)4 (3126.0/3127.6), CWK18(GA)6 (3383.3/ LLVY-AMC in DMSO (o2% DMSO final concentration) 3384.4), CWK18(GA)8 (3639.5/3640.8) and CWK18(GA)10 to the diluted cell lysates. After a 24 h incubation at 371C, (3895.8/3897.6). the reaction was stopped by adding 200 ml of ice-cold The Cys residue on the purified peptide was alkylated ethanol and 2 ml of 0.125 M borate buffer, pH 9. The by reaction with 100 mol excess of sodium iodoacetate in fluorescent proteolysis product, AMC, was quantified on

50 mM Tris (pH 7.5) at room temperature for 4 h. The RP-HPLC by injecting 30 ml onto a Vydac C18 analytical alkylated peptides were purified and characterized as column (4.6 Â 250 mm, pore size 300 A˚ , bead diameter described above. The concentration of stock solutions 10 mm) eluted at 1 ml/min with 0.1% TFA and a gradient À1 À1 were determined by Abs280 nm e ¼ 5600 M cm . of acetonitrile (5–55% over 35 min) while monitoring

fluorescence at lex of 380 nm and lem of 460 nm. The Preparation of peptide DNA condensates peak area was converted to nanograms of AMC using

CWK18 DNA and CWK18(GA)n DNA condensates were a standard curve prepared by injecting AMC standards. prepared by combining 50 mg of pCMVL in 500 mlof The percent proteasome activity was calculated by Hepes-buffered mannitol (HBM, 5 mM Hepes, 0.27 M comparing to the AMC generated by control cells. mannitol, pH 7.5) with 20 nmol of peptide in 500 mlof The influence of proteasome inhibitors on lysosomal HBM while vortexing to create DNA condensates enzymatic activity was also evaluated. Cell lysates (50 ml) + À possessing a charge ratio (NH4:PO4 ) of 2:1. The particle were diluted with 245 ml of proteolysis buffer and 5 mlof size and zeta potential of DNA condensates were 2mM cathepsin B substrate III (N-Cbz-Arg-Arg-AMC), measured by quasielastic light scattering on a Brookha- and then incubated at 371C for 24 h. The AMC peak area ven ZetaPluss particle sizer (Brookhaven Instruments was measured by RP-HPLC as described above. Corporation, NY, USA). Gene transfer and proteasomal inhibition Cell culture and cytotoxicity assay HepG2 cells (3 Â 105) were plated on 6 Â 35-mm wells HepG2 cells were grown in DMEM supplemented with and grown to 40–70% confluency. Transfections were 50% Hams-F12, 10% FBS, 250 U/ml penicillin and performed in MEM (2 ml/35-mm well) supplemented 250 mg/ml streptomycin at 371C in a humidified 5% with 2% FBS, sodium pyruvate (1 mM), penicillin and

CO2 incubator. The in vitro toxicity of proteasome streptomycin (100 U and 100 mg/ml) and 0–10 mM MG 33 inhibitors was evaluated by MTT reduction assay. 115, MG 132 or epoxomicin. CWK18 DNA condensates Briefly, HepG2 cells were plated on 6 Â 35-mm wells at (10 mg of DNA in 0.2 ml of HBM) were added dropwise 3 Â 105 cells/well and grown to 40–70% confluency. The to wells in triplicate. After 24 h at 371C, cells were culture media were replaced with 2 ml of fresh MEM washed twice with 2 ml of ice-cold PBS (Ca2+ and Mg2+ supplemented with 2% FBS and different concentrations free) and then treated with 0.5 ml of lysis buffer (25 mM of proteasome inhibitors. After 24 h of incubation, the Tris hydrochloride, pH 7.8, 1 mM EDTA, 8 mM magne-

Gene Therapy Proteasome metabolism of nonviral gene delivery systems J Kim et al 1589 sium chloride, 1% Triton X-100) for 10 min at 41C. The ated using a plasmid encoding NTbGal. HepG2 cells cell lysates were scraped, transferred to 1.5-ml micro- were transfected for 24 h with 10 mg CWK18 DNA in the centrifuge tubes and centrifuged for 10 min at 13 000 g presence of 10 mM MG 115, MG 132 or 1 mM epoxomicin. at 41C to pellet cell debris. Luciferase relative light units Cells were washed twice with 1 ml of PBS and then fixed were measured by a Lumat LB 9501 (Berthold Systems, with 2 ml of PBS containing 2 v/v% formaldehyde Germany) with 10 s integration after automatic injection and 0.2 v/v% glutaraldehyde at room temperature for of 100 ml of 0.5 mMD-luciferin. The relative light units 10 min. Fixed cells were then stained by 0.1 w/v% X-gal, were converted to fmol using a standard curve generated 2mM magnesium chloride, 4 mM potassium ferricyanide by adding a known amount of luciferase to 35-mm wells and 4 mM potassium ferrocyanide in PBS at 371C for containing 40–70% confluent HepG2 cells. The resulting 4 h. Cells containing blue nuclei were counted using an standard curve had an average slope of 1 Â104 relative Olympus BX-51 Light Microscope under  10 magnifi- light units/fmol of enzyme. Protein concentrations were cation. measured by BCA assay using bovine serum albumin as a standard.35 The amount of luciferase recovered in each DNA metabolism sample was normalized to milligrams of protein and The intracellular metabolism of plasmid DNA was reported as the mean and s.d. obtained from triplicate analyzed in the presence and absence of proteasome transfections. inhibitors. Iodinated pCMVL was prepared with a Gene transfer efficiency in CF/T1 cells was deter- specific activity of 200 nCi/mg of DNA.36 HepG2 cells 5 mined by plating 1.5  10 cells on 6  35-mm wells 1 were grown in 35 mm wells and incubated with CWK18 week prior to transfection. Cells that were 60% confluent 125I-DNA condensates in MEM supplemented with 2% were treated with Hams-F12 supplemented with insulin, FBS in the presence of 0, 0.1, 2, 5, 10 and 20 mM MG 132. hydrocortisone, endothelial cell growth supplement, Cells were harvested with 0.5 ml of lysis buffer at epidermal growth factor, triiodothyronine, transferrin, predetermined time points. The amount of radioactivity cholera toxin and 10, 25 or 50 mM MG 115 or MG 132. in each cell lysate was g-counted followed by extraction

CWK18 DNA condensates (10 mg of DNA in 0.2 ml of of the DNA, as described below, and then analyzed HBM) were added to triplicate sets of cells for 6 h. Cells by agarose gel electrophoresis and autoradiography. were further incubated for 18 h after replacing the Plasmid DNA was extracted from cell lysates (250 ml) transfection media with fresh culture media containing following incubation with proteinase K (250 mlof 5 mM MG 115 or MG 132. The cells were harvested and 1.0 mg/h proteinase K in 100 mM sodium chloride, 1% the luciferase assays were performed as described above. SDS and 50 mM Tris-HCl, pH 8.0) for 18 h at 371Cin PEI–DNA complexes were prepared by mixing 50 mg order to digest endonucleases. DNA was extracted with of DNA in 500 ml of HBM (5 mM Hepes, 0.27 M mannitol, 500 ml of phenol/chloroform/isoamyl alcohol (24:25:1 pH 7.5) with 50 mg PEI in 500 ml of HBM while vortexing v/v/v%) and then precipitated with 1 ml of ethanol to create DNA complexes possessing a charge ratio and centrifuged at 13 000 g for 15 min. The DNA pellet + À (NH4:PO4 ) of 8:1. HepG2 cells were transfected in the was air-dried, dissolved in 10 ml of Tris-EDTA buffer, presence of proteasome inhibitors with 5 mg of PEI–DNA g-counted, and then the entire sample was loaded on for 24 h as described above, and then processed to 1% agarose gel and electrophoresed for 1 h at 80 V. The determine luciferase expression. gel was dried on a zeta probe membrane and auto- Lipofectamine–DNA complexes were prepared by radiographed on a PhosphorImager (Molecular Devices, mixing 25 mg of DNA in 500 ml of distilled water with Sunnyvale, CA, USA) following a 36 h exposure. The 25 mg of Lipofectamine in 500 ml of distilled water while density of DNA bands was analyzed with ImageQuaNT vortexing to create DNA lipoplexes. HepG2 cells were 4.1 software, and normalized by the protein concentra- transfected in the presence of proteasome inhibitors by tion of each sample. 5 mg of Lipofectamine–DNA complexes in serum-free MEM supplemented with sodium pyruvate (1 mM)and Proteasomal inhibition using CWK18(GA)n penicillin and streptomycin (100 U and 100 mg/ml) for 24 h The inhibitory effect of CWK18(GA)n on the proteasomal and then processed to determine luciferase expression. hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC was studied. HepG2 cell lysate (10 cm diameter culture plate of Lysosomal enzyme inhibition 40–70% confluent cells) was prepared in distilled water HepG2 cells were transfected in the presence of increas- by three freezing/thawing cycles and diluted to 0.4 mg ing concentration of a cathepsin B inhibitor (CA-074 Me). total protein/ml concentration. Suc-LLVY-AMC (10 nmol) m Cells (3 Â 105) were plated on 6 Â 35-mm wells and was combined with 0–3 nmol of CWK18(GA)n in 250 lof grown to 40–70% confluency. CWK DNA condensates the proteolysis buffer containing 5 mM ATP and then 18 m (10 mg of DNA in 0.2 ml of HBM) were added to HepG2 mixed with 50 l of proteasomal prep and incubated at 1 m cells in MEM (2 ml/35-mm well) supplemented with 2% 37 C for 3 h. The reaction was quenched by adding 200 l of ice-cold ethanol and 2 ml of 0.125 M borate buffer, FBS, sodium pyruvate (1 mM), penicillin and strepto- pH 9. The proteolysis product, AMC, was quantified by mycin (100 U and 100 mg/ml) and 0–10 mM CA-074 Me. Luciferase and BCA assays were performed as described HPLC as described above. above. Transfection efficiency of CWK18(GA)n DNA HepG2 cells were grown as described above. 3T3, CHO Influence of proteasome inhibition on gene and COS-7 cells were grown in DMEM supplemented transduction with 10% FBS, 100 U/ml penicillin and 100 mg/ml

The influence of proteasome inhibition on the transduc- streptomycin at 371C in a humidified 5% CO2 incubator. 5 5 5 tion efficiency of CWK18 DNA condensates was evalu- 3T3 (1.5 Â 10 ), CHO (1.0 Â 10 ), COS-7 (1.0 Â 10 ) and

Gene Therapy Proteasome metabolism of nonviral gene delivery systems J Kim et al 1590 HepG2 cells (5 Â 105) were plated onto 6 Â 35-mm wells 17 Goettsch S, Bayer P. Structural attributes in the conjugation of and grown to 50% confluency. Transfections were perfor- ubiquitin, SUMO and RUB to protein substrates. Front Biosci med by adding 10 mg of pCMVL condensed with 4 nmol 2002; 7: a148–162.

of CWK18(GA)n as described above. CWK18 was used 18 McKenzie DL, Kwok KY, Rice KG. A potent new class of as a control gene delivery peptide to compare with reductively activated peptide gene delivery agents. J Biol Chem 2000; 275: 9970–9977. CWK18(GA)4,CWK18(GA)6,CWK18(GA)8 and CWK18(GA)10. 19 Bogyo M, Wang EW. Proteasome inhibitors: complex tools for a complex enzyme. Curr Top Microbiol Immunol 2002; 268: Acknowledgements 185–208. 20 Meng L et al. Epoxomicin, a potent and selective proteasome We gratefully acknowledge support for this work from a inhibitor, exhibits in vivo antiinflammatory activity. Proc Natl seed grant from the Cystic Fibrosis Foundation through Acad Sci USA 1999; 96: 10403–10408. The University of Iowa Center for Gene Therapy and 21 Almond JB, Cohen GM. The proteasome: a novel target for from NIH DK066212. . Leukemia 2002; 16: 433–443. 22 Adams J et al. Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res 1999; 59: 2615–2622. 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