ISSN 1990519X, Cell and Tissue Biology, 2010, Vol. 4, No. 4, pp. 362–371. © Pleiades Publishing, Ltd., 2010. Original Russian Text © A.M. Efremov, I.V. Dukhovlinov, E.B. Dizhe, S. V. Burov, M.V. Leko, B.N. Akifiev, D.A. Mogilenko, I.A. Ivanov, A.P. Perevozchikov, S.V. Orlov, 2010, published in Tsitologiya, Vol. 52, No. 5, 2010, pp. 371–379.
Nonviral Regenerative Gene Therapy of Mammalian Cutaneous Damages A. M. Efremova, b, c, I. V. Dukhovlinova, E. B. Dizhea, c, S. V. Burovd, e, M. V. Lekod, e, B. N. Akifieva, c, D. A. Mogilenkoa, b, c, I. A. Ivanova, A. P. Perevozchikova, b, c, and S. V. Orlova, b, c* a
Pharma Gen Ltd., St. Petersburg, Russia St. Petersburg State University, St. Petersburg, Russia c Institute of Experimental Medicine, Northwestern Branch of Russian Academy of Medical Sciences, St. Petersburg, Russia d Institute of Macromolecular Compounds, Russian Academy of Sciences, St. Petersburg, Russia e Diapharm Ltd., St. Petersburg, Russia *email:
[email protected] b
Received August 03, 2009
Abstract—The rate and character of superficial tissue regeneration after wounds, burns, and other traumas depend on cell proliferation within the damaged area. The acceleration of wound healing via the stimulation of cell proliferation and extracellular matrix synthesis is one of the most important tasks of modern medicine. There are gene therapy approaches to wound treatment, such as the transfer of genes that encode mitogenic growth factors to the wound area. The most important step in the development of the gene therapy approaches is the design of gene delivery tools. Despite the high efficiency of viral vectors, the nonviral approaches have some advantages (low toxicity, low immunogenity, safety, and the absence of side effects). Among the nonviral gene delivery tools molecular conjugates are the most popular due to their efficiency, simplicity, and the capacity for targeted gene transfer. In the present work, we have developed two molecular conjugates, NLSTSF7 and NLSTSF12, which consist of the modified signal of the nuclear localization of the Tantigen of the SV40 virus (cationic part) and the peptide ligands of the mammalian transferrin receptor (ligand part). Those conjugates bind to plasmid DNA via the formation of polyelectrolytic complexes and are able to deliver plasmid DNA into cells that express transferrin receptors through receptormediated endocy tosis. The transfer of the expression vector of the luciferase gene in the complex with the molecular conjugate NLSTSF7 to murine surface tissues led to about the 100fold increase of luciferase activity in comparison with the transfer of the free expression vector. The treatment of mice with incised wounds with complexes of the expression vector of the synthetic human gene that encodes insulinlike growth factor 1 with molecular conjugate NLSTSF7 led to the acceleration of wound healing in comparison with mice treated with the free expression vector. The obtained results confirm the high efficiency of the developed approach to regenerative gene therapy for treating the superficial tissue damage of mammals. Key words: molecular conjugates, gene transfer, regenerative gene therapy, transferrin receptor, insulinlike growth factor 1. DOI: 10.1134/S1990519X10040097
INTRODUCTION Damages of superficial tissues (burns and wounds) are dangerous traumas and are often associated with longterm regeneration process, chronic wounds, numerous scars, and severe infections. The regenera tion of damaged tissue depends mainly on the prolifer ation of various types of cells present in the damage zone and on their participation in the synthesis of intercellular matrix components (Bowler, 2002). The acceleration of wound healing and the treatment of other damages by the stimulation of regeneration is one of the most important tasks in practical medicine. The majority of the current methods of stimulating regeneration are based on either improving the
trophics of tissues surrounding the damaged area or an increase in the proliferation rate of their constituent cells (this concerns mainly connective tissue cells) (Bowler, 2002). The most efficient agents turned out to be those based on mitogenic growth factors able to specifically trigger the division of target cells (Macri and Clark, 2009; Ferguson et al., 2009). It is worth noting the brief lifespan in the organism of signal mol ecules, which also includes mitogenic growth factors, which necessitates frequent repeated administrations of therapeutic agents. Therefore, it seems important to develop gene therapy methods of stimulating mam malian wound healing. In this case, the expression vector that contains the gene encoding some particular
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healingstimulating factor is administered into the tis sues that surround the damages. The expression of this gene allows one to maintain a constant level of the growth factor in the damage site, thereby stimulating the process of regeneration (Makinen et al., 2002). The key moment that determines the efficiency of gene therapy is the choice of the way of the DNA transfer into target cells. The use of viral vectors for this purpose has significant limitations (Molas et al., 2003). Therefore, the development of nonviral meth ods of DNA transfer is important. Despite the lower efficiency of the single use, the nonviral agents do not have several principal drawbacks of viral vectors (high immunogeneity and toxicity) and can be used many times. The most prospective approach in this field is the use of molecular conjugates, i.e., cation carriers, for DNA condensation due to electrostatic interac tions with the subsequent delivery of DNA–carrier polyelectrolyte complexes into mammalian cells. The specificity of the transfer of DNA–polycationic com plexes into target cells is achieved by the administra tion of ligands to cell receptors into their composition, which leads to the receptormediated endocytosis of the complexes (Molas et al., 2003). Thus, the effi ciency of therapy is due, to a significant degree, to the selectivity of ligands used in the development of cation carriers with respect to the receptors of target cells. In the process of regenerating mammalian skin integu ment, the proliferation of skin fibroblast in the areas directly adjacent to the wound plays a particular role. The development of highly efficient and specific methods of DNA transfer to fibroblasts of mammalian superficial tissues are a significant contribution to the development of methods of wound therapy. When developing molecular conjugates which are able to transfer efficiently and specifically the genes of “interest” into fibroblasts of mammalian superficial tissues, two factors seem extremely important— choice of the ligand with significant amounts of recep tors on the surface of target cells and the cation carrier providing the efficient DNA compaction with forma tion of polyelectrolyte complexes unable to penetrate into cells by absorptional endocytosis. The latter cir cumstance is importance for the specificity of gene transfer. Transferrin receptors have long attracted the attention of researchers as targets for delivering phar macologically active substances to cells (Thorstensen and Romslo, 1993; Jones and Shusta, 2007). One of the important advantages of transferrin receptors in the development of systems of regenerative and antitu mor gene therapy is the correlation of the number of receptors on the surface cells with proliferation rate. The more active the cell proliferation, the higher the number of transferrin receptors present on their sur face (Ponka and Lok, 1999; Inoue et al., 1993). Dur ing the transfer of genes of growth factors that stimu late proliferation, the appearance of positive feedback can be expected, i.e., the acceleration of cell division will lead to an increased efficiency of gene delivery. CELL AND TISSUE BIOLOGY
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Significant drawbacks of transferrin receptors as tar gets for gene transfer are the relatively high molecular mass of their ligand transferrin and the dependence of ligandreceptor interactions on the saturation of transferrin with iron ions (Ponka and Lok, 1999). An increase in the sizes of DNA–conjugate complexes up to 100 nm is known to lead to a marked deterioration of DNA penetration into cell nuclei and, as a conse quence, to a drop in the level of the transferred gene expression (Erbacher et al., 1995). Dependence of efficiency of endocytosis on saturation of transferrin with iron produces additional difficulties, particularly at transfer of genes in vivo. These limitations of molec ular conjugates based on transferrin can be avoided by replacing transferrin with low molecular peptide ligands that have a high affinity to transferrin recep tors. Based on the literature (Lee et al., 2001), in the present work, we report on the synthesis of molecular conjugates that achieve efficient delivery to proliferat ing cells of genes of interest that express significant amounts of transferrin receptors and the creation of a gene therapy system for mammalian skin integuments based on these conjugates. MATERIAL AND METHODS Reagents. Reagents and amino acid derivatives from the following companies have been used in this work: Sigma Chemical Co., Fisher Scientific, Bachem (United States); Reanal (Hungary); Saxon Biochemi cals GmbH (Germany); and Amersham Bioscience, Roche Applied Science, Invitrogen, Promega, Gibco (United States), as well as of the production of quali fication ch.p. and p.f.a.;4(2',4'dimethoxyphenyl Fmocaminomethyl)phenoxyacetoamidonorleucyl aminomethyl polymer (Gl. Biochem., China) by Rus sian companies and enzymes for geneengineering works (Fermentas, Lithuania and NPO SibEnzim, Novosibirsk). Dimethylformamide was distillated before use in a vacuum and stored above molecular sieves 4 Å. Individualities of derivative amino acids was controlled by the TLC methods on Merck F 254 plates (Germany) in the following solvent systems: 1% ammonium–fluorobutyl alcohol, 1 : 3 (A); chloro form–methanol, 9 : 2 (B); chloroform–methanol– acetic acid, 90 : 7 : 3 (C). Chromatograms were visual ized by UV irradiation at 254 nm and by the carbon ization in sulfuric acid. Animals. Male DBA2 mice aged 6 weeks were obtained from the Rappolovo animal nursery of the Russian Academy of Medical Sciences. Cell cultures. The cell strain of human hepatocellu lar carcinoma (hepatoma), HepG2, was obtained from the Department of Cell Cultures of Institute of Cytology of the Russian Academy of Sciences (St. Petersburg). The cells were grown in DMEM medium containing 10% fetal bovine serum (Biolot, Russia) at 37°C in a CO2 incubator with the content of CO2 of 5%.
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Plasmids. The plasmid pCMVluc is an expression vector for the cDNA gene of firefly luciferase Photi nus piralis (luc) driven by the promoter of early genes of human cytomegalovirus (CMV) and was described previously (Alkifiev et al., 2004). Plasmid pCMVIGF1s contains a synthetic gene that encodes a human insulinlike growth factor 1 controlled by the CMV promoter and was described previously (Ivanov, 2007). Plasmid pΔIGF1s differs from pCMVIGF1s by the absence of the CMV promoter sequence. All works on obtaining genetic construc tions were performed in compliance with the described methods (Maniatis et al., 1984). Synthesis of peptides. Cationic peptides K8, Tat, NLS, NLSArg were synthesized by the solid phase method using the BOC/Bzl strategy on an NPS4000 semiautomatic synthesizer (Neosystem Laboratoires, France) on 4methylbenzhydroaminopolymer (0.55 mM/g, 0.25 mM). Upon the completion of syn thesis, the final product was detached from the poly mer carrier with simultaneous deblocking by 1 M trif luoromathansulfoacid in trifluoroacetic acid in com pliance with the standard manufacturer’s protocol (Applied Biosystems Inc., United States). Peptides were purified using gel filtration on Sephadex G15 in 50% acetic acid and subsequent highefficiency reversephase chromatography (HELC) on a Delta Pak C18 column (Waters Chromatography, United States) in the system H2O/acetonitryl/0.01% trifluo roaceic acid. Molecular conjugates NLSTSF7 and NLSTSF12 were synthesized on 4(2',4'dimethox yphenylFmocaminomethyl)phenoxyacetoamido norleucylaminomethylpolymer with a content of amino groups 0.5 mM/g. To protect the side function of Ser, Thr, and Tyr, we used the But group; for Arg, we used Pbf; for Lys and Trp, we used BOC; and for His, we used Trt. The Fmoc group served as a temporary Nαprotection. The detachment of synthesized peptides from polymer with simultaneous deblocking was performed by the action of a TIS : H2O : TFA (2.5 : 2.5 : 95; 5 ml/0.5 g resin) mixture for 2 h. Subsequent purifica tion was performed using reversephase HELC. The obtained peptides were analyzed by HELC using a System Gold chromatograph (Beckman, United States) on Zorbax 300SBC18 columns (4.6 × 150 mm, 5 μm) for analytical chromatography and on a Discov ery C18 (10 × 250 mm, 5 μm) for preparative chroma tography. The structure of the obtained peptides was confirmed by the data of amino acid analysis and ESI MS massspectrometry. The amino acid analysis was performed on a Microtechna T339M analyzer (Czech Republic) after the hydrolysis of peptides with 6 N HCl at 100°C for 24 h. Mass spectra were recorded on a MX5303 timepassing massreflectron with the source of ions of the Electrospray type (Institute of Problems of Chemical Physics, Russian Academy of Sciences). The amino acid compositions and molecu lar masses calculated were from the data of massspec
trometry that corresponded to theoretical values for all synthesized peptides. Preparation of complexes of plasmid DNA with cat ionic peptides and molecular conjugates. The forma tion of DNA complexes with molecular conjugates was performed in phosphatesalt buffer (PBS) with different ratio of DNA and peptide charges in the reaction medium according (Ignatovich et al., 2002). The dependence of the stability of the DNA–conju gate complexes on ion strength of reaction mixture was revealed by adding the necessary amounts of NaCl to the reaction. The formation of the DNA–conjugate complexes was controlled by the method of retarda tion in agarose gel. Transfection of cells and analysis of expression of the genereporter encoding luciferase. The HepG2 cells were transfected by preparations of complexes K8/pCMVluc, Tat/pCMVluc, NLS/pCMVluc, NLS Arg/pCMVluc, NLSTSF7–pCMVluc, and NLS TSF12–pCMVluc as described earlier (Dizhe et al., 2006). The cells were seeded with density of 104 cells/cm2 onto Petri dishes (30 mm in diameter) and grew until the state of 60–70% monolayer in the DMEM medium with addition of 10% embryonic calf serum (Biolot, Russia) at 37°C in atmosphere of 5% CO2. The DNA–peptide complexes with different charge ratios were prepared as 12 μg DNA per Petri dish in 500 μl PBS. Upon their addition to cells, CaCl2 was added to the medium until a concentration of 4 mM. After incubation with complexes for 2 h the cells were cleaned of noninternalized complexes with heparin solution (58 μg/ml in PBS) and incubated in the DMEM medium with 10% serum for 24 h. The activity of luciferase was measured using the Luciferase Assay System, a commercial system of esti mating the luciferase activity (Promega, United States), in keeping with the manufacturer’s instruc tions. Luminescence was measured on a Turner Bio system 20/20 luminometer. The concentration of pro tein in cell lysates was determined by Bradford’s method. The luciferase activity was expressed in rela tive light units (RLUs) that represent the number of flashes per minute per 1 mg of total protein of cell extracts. The background values of luminescence did not exceed 120 RLU. Transfer of complexes of plasmid DNA with molec ular conjugates into superficial mouse tissues. Incised wounds were cut with a surgical scalpel to a depth of 2 mm with the involvement of underlying muscle tis sue in the lower back area. Puncture wounds were cre ated by ten punctures with an insulin syringe into the lower back. Experimental preparations were injected with an insulin syringe as ten punctures around each wound. The plasmid DNA and complexes of plasmid DNA with molecular conjugate were introduced in the amount of 50 μg DNA per mouse in 100 μl PBS con taining 4 mM CaCl2. To measure luciferase activity on the third day after transferring mice of plasmid pCM Vluc, the animals were sacrificed and the tissue area, CELL AND TISSUE BIOLOGY
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including the wound (about 100 mg), was dissected out. Samples were homogenized in Reporter Lysis Buffer (Promega, United States), frozen, and centri fuged for 3 min at 10000 g. Supernatant was used to measure the luciferase activity and the protein con centration as described above. The expression of the synthetic gene of insulinlike growth factor 1 (IGF1) was estimated on the 1st, 3rd, and 7th days after the mice were transferred to pCM VIGF1s by RealTime RTPCR. Tissue samples were obtained as described above and were used for isola tion of RNA. As a negative control, the mice not treated with pCMVIGF1s were used. RNA was iso lated from tissues with use of the TRI Reagent kit (Sigma, United States) according to the manufac turer’s instructions. Two milliliters of TRI Reagent were used per 100 mg of cells. The concentration of RNA was determined spectrophotometrically. The isolated RNA was treated with DNAse I. The absence of contamination of the isolated RNA with plasmid was checked by the PCR method using primers for the IGF1s gene. Synthesis of cDNA was performed with use of a Revert Aid® First Strand cDNA Synthesis Kit (Fermentas, Lithuania). The relative mRNA content was measured by RTPCR with use of a SYBR GREEN Supermix (BioRad, United States). All samples were normalized by the level of expression of the housekeeping βactin gene. The reaction was performed in IQ5 thermocycler (BioRad, United States); the data were preliminarily analyzed using the supplied software. The efficiency of amplification was determined from the external stan dard curve, which was plotted by an analysis of a series of dilutions (5, 10, 30, and 50 ng) of the control plas mid. The calculated efficiency of amplification for the used primers was about 0.98 rel. units. The reaction without the addition of DNA was used as a negative control. The data were normalized by the amount of tissue and concentration of RNA and DNA. An increase in the amount of the PCR product after n cycles was calculated by the ΔCt method using the for mula R = (1 + E)ΔCt, where Ct is the threshold cycle, E is the efficiency of amplification, and ΔCt = Ct(Mod)norm is Ct(Nat)norm. The area of the wound on the bodies of experimen tal animals was measured using photography of the damaged region. The photographed material was ana lyzed using the ImageTool system of computer image analysis for Windows 2.0 (United States), according to the main principles of stereology in morphometry. Histological study of bioptates. Tissue samples obtained as described above were fixed for 4 days in 4% formalin in PBS and dehydrated in alcohols of ascend ing concentration and xylene. Then, the samples were embedded in Histomix commercial paraffin and 6μm thick sections were cut. For subsequent work, Super CELL AND TISSUE BIOLOGY
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frost object glasses were used. Paraffin sections were dehydrated in xylene and a series of alcohols of descending concentration, then stained with hema toxylineosin by the standard procedure. The obtained preparations were examined and photographed using a Leica DFC500 microscope. Mitotic index for each animal was determined in the wound preparations stained with hematoxylineosin (see above) in ten fields of vision in three wound sections with a total content of at least 1000 nuclei. The statistical treatment of results was performed using the Statistica Release 5.0 software package (StatSoft Inc., United States). Diagrams present the mean values from three to five repeats. The error rods correspond to the standard error of the mean. Differ ences were considered to be statistically significant at p < 0.05 in Student’s paired test with unequal devia tions. RESULTS AND DISCUSSION Molecular conjugates based on peptide ligands to transferrin receptor. Two peptides were used as a ligand component for synthesis of molecular conjugates, i.e., TSF7 (HisAlaIleTyrProArgHis) and TSF12 (ThrHisArgProProMetTrpSerProValTrp Pro), which were chosen earlier by the method of phage display from the capability of binding to trans ferrin receptors (Lee et al., 2001). Previously, we showed that the arginine and lysinerich cationic peptides form polyelectrolyte complexes with plasmid DNA and enable its delivery to cells by adsorptional endocytosis (Ignatovich et al., 2003; Dizhe et al., 2006). For the development of receptormediated gene delivery system, these paths of internalization should be avoided, since they lead to a drop in the specificity of the transfer (see above). Therefore, par ticular attention was paid to chosing the cationic com ponent. As candidates, we considered the modified signal of the nuclear localization of Tantigen of SV40 (NLS), ProLysLysLysArgLysVal, and the signal of nuclear localization of protein Rev of HIV1 (NLS Arg), ArgArgAsnArgArgArgArg. Both cationic peptides are efficiently bound to plasmid DNA via the formation of polyelectrolyte complexes (Fig. 1a). Fig ure 1b presents the results of the transfection of culti vated human hepatoma cells HepG2 by the pCMVluc expression vector (contains the reporter gene encod ing luciferase under the control of promoter of human cytomegalovirus early genes) in complexes with vari ous cationic peptides. In keeping with the previously published data (Ignatovich et al., 2002, 2003; Dizhe et al., 2006), the lysinerich peptide K8 and arginine rich fragment of protein Tat of HIV1, as well as the argininerich peptide NLSArg, enables the efficient transfection of HepG2 cells by adsorptional endocy tosis. Under the same experimental conditions, the NLS–pCMVluc complexes turned out to be transfec tionally inactive. The ability to bind plasmid DNA and
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K 7 8 (b) Relative luciferase activity, RLU 14000000 12000000 10000000 8000000 6000000 4000000 2000000 0 K8 Tat NLSarg 1
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Fig. 1. Complexes of plasmid DNA with cationic peptides NLS and NLSArg. (a) NLS/pCMVluc and NLSarg/pCMVluc complex for mation at various DNA : peptide charge ratios: 1. pCMV luc : NLS charge ratio of 1 : 2; 2. pCMVluc : NLS charge ratio of 1 : 1.5; 3. pCMVluc : NLS charge ratio of 1 : 1; 4. pCMVluc : NLS charge ratio 1 : 0.75; 5. pCMVluc : NLS charge ratio of 1 : 0.5; 6. pCMVluc : NLS charge ratio of 1 : 0.25; K, free pCMVluc; 7. pCMVluc : NLSArg charge ratio of 1 : 2; 8. pCMVluc : NLSArg charge ratio of 1 : 1.5; 9. pCMVluc : NLSArg charge ratio of 1 : 1; 10. pCMVluc : NLSArg charge ratio of 1 : 0.75; 11. pCMVluc : NLSArg charge ratio of 1 : 0.5; 12. pCMVluc : NLSArg charge ratio of 1 : 0.25; (b) the transfection efficiency of complexes of pCMVluc with cationic peptides (luciferase assay): HepG2 cells were transfected by complexes of pCMVluc with cationic peptides K8, Tat, NLSArg and NLS. Values are mean ± standard error of mean of three independent experiments.
the low level of adsorptional endocytosis of the NLS– DNA complexes enabled us to choose NLS as the cat ionic component to prepare molecular conjugates based on peptide ligands of the transferrin receptor. Two conjugates were synthesized, i.e., NLSTSF7 (ProLysLysLysArgLysValbAlaHisAlaIle TyrProArgHisNH2) and NLSTSF12 (ProLys LysLysArgLysValbAlaThrHisArgProPro MetTrpSerProValTrpProNH2). In both cases, binding the ligand and cationic parts of conjugates was achieved via the βalanine residue. Figure 2 presents the results of study of binding of molecular conjugates to the plasmid DNA pCMVluc. The binding of pep tide ligands did not affect the NLS capability of bind ing plasmid DNA under isotonic conditions (Fig. 2a). At the same time, a decrease in the stability of the complexes at high values of the solution ion strength was observed as compared with the free cationic pep tide NLS (Fig. 2b). Neither of the NLSTSF7–pCM Vluc and NLSTSF12–pCMVluc complexes are sta ble at a solution ion strength exceeding 300 mM. On
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(b) Multiple excesses of conjugate “+” charges versus the “–” charges of DNA 3.5 3.0 1 2.5 2 2.0 3 1.5 1.0 0.5 150 300 500 mM 0 Ionic strenght of reaction medium Fig. 2. Formation of complexes NLSTSF7–pCMVluc and NLSTSF12–pCMVluc. (a) optimization of the DNA : peptide charge ratio using the gel retardation method: 1. pCMVluc : NLSTSF7 charge ratiov 1 : 0.25; 2. pCMVluc : NLSTSF7 charge ratio of 1 : 0.38; 3. pCMVluc : NLSTSF7 charge ratio of 1 : 0.5; 4. pCMVluc : NLSTSF7 charge ratio of 1 : 0.63; 5. pCMVluc : NLSTSF7 charge ratio of 1 : 0.75; 6. pCM Vluc : NLSTSF7 charge ratio of 1 : 1; 7. free pCMVluc; 8. pCMVluc : NLSTSF12 charge ratio of 1 : 0.25; 9. pCMVluc : NLSTSF12 charge ratio v1 : 0.38; 10. pCMVluc : NLSTSF12 charge ratio of 1 : 0.5; 11. pCMVluc : NLSTSF12 charge ratio of 1 : 0.63; 12. pCMVluc : NLSTSF12 charge ratio of 1 : 0.75; 13. pCMVluc : NLSTSF12 charge ratio of 1 : 1; (b) depen dence of electrostatic interactions between molecular con jugates and plasmid DNA on the ionic strength of reaction medium. Ordinate shows multiple excesses of conjugate positive charges versus negative charges of DNA. 1— NLSTSF12pCMVluc complexes; 2—NLSTSF7 pCMVluc complexes; 3—NLSpCMVluc complexes.
the whole, the NLSTSF7–pCMVluc complex turned out to be more stable than NLSTSF12–pCM Vluc, which seems to be due to the higher density of the positive charge in the case of NLSTSF7 (the ratio of mass to charge is 304.71) as compared with NLS TSF12 (masstocharge ratio is 404.3). Transfectional activity of pCMVluc complexes with molecular conjugates NLSTSF7 and NLSTSF12. The transfectional activity of NLSTSF7–pCMVluc and NLSTSF12–pCMVluc complexes was esti mated on HepG2 cells that express significant amounts of transferrin receptors. Both complexes turned out to be able to provide the expression of the transferred gene (Fig. 3a). Taking into account the CELL AND TISSUE BIOLOGY
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Fig. 3. Transfection activity of NLSTSF7–pCMVluc and NLSTSF12–pCMVluc complexes (luciferase assay). (a) Dependence of luciferase activity on presence of free molecular conjugates in transfection medium; (b) effect of large excesses of molecular conjugates on the luciferase gene expression level. HepG2 cells were transfected by the NLSTSF7–pCMVluc and NLSTSF12–pCMVluc complexes prepared at the DNA : peptide charge ratio of 1 :1 or 1 : 5. In competition experiments, a 50fold excess of free molecular conjugate was added to cells during transfection. Values are mean ± standard error of the mean of three independent experiments.
almost complete absence of adsorption endocytosis (see Fig. 1b), it can be concluded that transfection was achieved through receptormediated endocytosis via transferrin receptors. One more indication of the role of receptormediated endocytosis in the internaliza tion of the complexes of molecular conjugates with DNA is the absence of a stimulating effect of free con jugates not connected with DNA on the level of cell transfection (Fig. 3a). Previously, we showed that, for complexes of plasmid DNA with cationic peptides K8 and Tat (which penetrate cells through adsorption endocytosis), the addition of excess of free peptides to cells during transfection leads to a significant increase in the efficiency of transfection, which is apparently due to the protection of complexes from destruction by negatively charged proteoglycans of the cell surface (Ignatovich et al., 2003). This dependence is absent in the case of molecular conjugates based on luliberine analogs that penetrate cells with the participation of the corresponding receptors (Burov et al., 2007; Efre mov et al., 2010). Direct evidence of the role of recep CELL AND TISSUE BIOLOGY
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Fig. 4. Factors that affect NLSTSF7–pCMVluc and NLSTSF12–pCMVluc transfection efficiency in HepG2 cells (luciferase assay). K, transfection by NLSTSF7–pCMVluc and NLS TSF12–pCMVluc complexes prepared at the DNA : pep tide charge ratio 1 : 1; –CaCl2, transfection without 4 mM CaCl2; +10% FCS, transfection in the presence of 10% FCS. Values are mean ± standard error of the mean of three independent experiments.
tormediated endocytosis were obtained in experi ments on the transfection of the HepG2 cells in the presence of significant excesses of free molecular con jugates. The addition to cells of a 50fold excess of free conjugate led to a fall of the cell transfection level both by the NLSTSF7–pCMVluc and by the NLS TSF12–pCMVluc complexes (Fig. 3b). When developing systems of gene transfer, it is important to take into account the effects of several factors on the efficiency of delivery. Figure 4 presents the results of experiments on studying the action of Ca2+ and fetal calf serum (FCS) on the efficiency of HepG2 cell transfection by pCMVluc complexes with molecular conjugates NLSTSF7 and NLSTSF12. At low concentrations or in the absence of Ca2+ ions (lower than 2 mM), the level of expression of the trans ferred gene decreased to almost zero. The obtained results agree well with the notion of the receptor mediated character of penetration of complexes into cells because Ca2+ ions play a significant role in this process. Another important factor that considerably affects the efficiency of cell transfection with polyelec trolyte complexes is the presence of blood serum pro teins in cultural medium. Previously, we showed that cell transfection with complexes of DNA with the cat ionic peptide Tat and with molecular conjugates based on luliberin analogs in the presence of blood serum proteins leads to a significant decrease in the expres sion of the transferred gene (Ignatovich et al., 2003; Burov et al., 2007). This can be of decisive significance in the development of systems of gene transfer in vivo. Similar experiments were also carried out with the
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molecular conjugates. To solve the tasks of the present work, i.e., to develop tools for the regenerative gene therapy of superficial damage in mammalian tissues, the molecular conjugate NLSTSF7 seems to be more optimal; however, if the gene delivery system requires a systematic administration, NLSTSF12 might become preferable. Transfer of genereporter encoding luciferase in complex with molecular conjugate NLSTSF7 into mouse cutaneous integuments. The ability of com plexes of DNA and molecular conjugate NLSTSF7 to penetrate into cells of superficial tissues was checked on DBA2 mice (males, 2 weeks). NLS TSF7–pCMVluc complexes (1 : 1 charge ratio) were either introduced into the skin of healthy animals (ten punctures around the area ~60 mm2) or punctured arround a wound of similar area. The amount of com plexes corresponded to 50 μg DNA per mouse. The control mouse group was submitted to an injection of the same amount of pCMVluc free plasmid. Luciferase activity was measured in lysates obtained from bioptates of mouse superficial tissues in the area of injections on the 3rd day after the beginning of experiment and equated by the concentration of total protein in lysates; results are presented in Fig. 5. The use of NLSTSF7–pCMVluc complexes led to an increase in the luciferase activity by more than 100 times compared to free DNA injections. No signifi cant difference was noted in the level of the transferred gene expression at injections to healthy animals and animals with punctured wounds (Fig. 5). Thus, the synthesized molecular conjugate NLSTSF7 is able to transfer genes efficiently into superficial mammalian tissues. The obtained results have allowed us to begin the development of a system of regenerative gene ther apy of superficial mammalian damages. Model of regenerative gene therapy of damages of mouse cutaneous integuments. During the develop ment of the regenerative gene therapy model, we used our previously obtained synthetic gene that encodes human insulinlike growth factor 1 (IGF1). The syn thetic gene differs from the natural one by multiple nucleotide replacements that do not change the amino acid sequence of the encoded protein, but eliminate
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NLSTSF7/pCMVIuc
Fig. 5. Delivery of pCMVluc into murine surface tissues within complexes NLSTSF7–pCMVluc (luciferase assay): healthy mouse, ten injections of free pCMVluc (50 μg per mouse) or NLSTSF7–pCMVluc complex pre pared at DNA : peptide charge ratio of 1 : 1 (50 μg of DNA per mouse) around the area (S ~ 60 mm2) of lower part of the back; cut wound, 10 injections of free pCMVluc (50 μg per mouse) or NLSTSF7–pCMVluc complex prepared at DNA : peptide charge ratio of 1 : 1 (50 μg of DNA per mouse) around cut wound (S ~ 60 mm2) on lower part of the back. Luciferase activities were measured 3 days after injections and adjusted according to the concentration of total protein in lysates. Values are mean ± standard error of mean of six independent experiments.
NLSTSF7 and NLSTSF12 molecular conjugates. It has been established that, during cell transfection, the introduction of 10% FCS into cultural medium leads to a decrease in the level of transferred gene expression (Fig. 4). It is interesting to note that, in the presence of serum, the level of cell transfection with complexes of DNA with NLSTSF12 was higher than in the case of complexes of DNA with NLSTSF7, despite that, in the absence of serum, the reverse situation was observed. It is necessary to take this circumstance into account during the subsequent modernization of non viral systems of gene transfer based on synthesized
Dynamics of wound regeneration of mouse cutaneous integuments Area of wounds (mm2) at terms
Treatment of wounds with genetic constructions
1 days
7 days
10 days
14 days
21 days
pCMVIGF1s NLSTSF7/pCMVIGF1s pΔIGF1s NLSTSF7
60 ± 13.5 57 ± 11.2 66 ± 20 62 ± 13.5
28.5 ± 6 24 ± 7.5 42 ± 9 46.5 ± 8
12 ± 3 6±2 24 ± 5.5 28.5 ± 6
0 0 10.5 ± 6 10.5 ± 4.5
0 0 0 0
Note: pCMVIGF1s refers to mice injected with free vector of expression of the human insulinlike growth factor; NLSTSF7/pCMVIGF1s refers to mice injected with complexes NLSTSF7/pCMVIGF1s; pΔIGF1s refers to mice injected with a free vector of expression of human insulinlike growth factor with deleted promoter; NLSTSF7 refers to mice injected with free molecular conjugate NLSTSF7. CELL AND TISSUE BIOLOGY
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NLSTSF7/pCMVIIGF1s pCMVIGF1s
20 (а)
(b)
15 Fig. 7. Visual estimate of wound of mice on 14th day after beginning of experiment: (a) mouse was injected with NLSTSF7/pCMVIGF1s complexes; (b) mouse was injected with free NLSTSF7 molecular conjugate. Com plete healing of the wound occurs in case of mouse injected with NLSTSF7/pCMVIGF1s complexes.
10 5 0
3
5 7 Days after the gene transfection
Fig. 6. Expression of human synthetic IGF1 gene in mice after delivery of expression vector pCMVIGF1s within complex NLSTSF7/pCMVIGF1s (RealTime RTPCR assay). White columns correspond to transfer of free pCMVIGF 1s, black columns correspond to the transfer of the NLS TSF7/pCMVIGF1s complexes. Ordinate shows the number of cycles required to reach the threshold level of fluorescence (approximately ten standard deviations of fluctuations in the background fluorescence). Difference in one cycle corresponds to twofold difference in IGF1 mRNA level. Values are mean ± standard error of average of three independent experiments.
the AUrich sites responsible for destabilization of the corresponding mRNA. These replacements have led to a rise in the level of gene expression by more than 100 times (Ivanov, 2007). Mice with incised wounds were submitted to the transfer of the expression vector pCMVIGF1s, which contains the synthetic IGF1 gene under the control of a promoter of human cytomegalovirus early genes. Sixweekold DBA2 mice (males) were used in these experiments. In the experiment, four groups of animals were formed, ten
(а)
mice in each. The experimental groups were injected with either free pCMVIGF1s or NLSTSF7/pCM VIGF1s complexes (1 : 1 charge ratio). The injections were performed ten times in the area surrounding the wound. 50 μg DNA per mouse in 100 μl PBS contain ing 4 mM CaCl2 were used. Control groups were injected with plasmid DNA with deleted promoter (pΔIGF1s) or with free conjugate NLSTSF7. On the 3rd, 5th, and 7th days, two mice from each group were sacrificed and RNA was isolated from the areas adjacent to the wound, which was used for the quanti tative estimation of IGF1 expression by RTPCR. The results are presented in Fig. 6. The expression level of the IGF1 gene on the 3rd and 5th days after the beginning of the experiment was 64–128 times higher following injections of complexes NLS TSF7/pCMVIGF1s as compared with injections of free pCMVIGF1s. In the control groups injected with plasmid with deleted promoter or free molecular conjugate NLSTSF7, no expression of human IGF 1 was recorded. The obtained data confirm the results presented in Fig. 5 and indicate the high efficiency of the molecular conjugate NLSTSF7 for the delivery of genes into the superficial mammalian tissues.
(b)
Fig. 8. Paraffin slides (6 μm, hematoxylin and eosin staining, 40fold magnification) through the wound of mice injected with free NLSTSF7 (negative control) (a) or NLSTSF7/pCMVIGF1s complexes (b). Biopsies were performed on 10th day after begin ning of experiment. Black arrows show vessels; red arrow indicates part of undamaged epidermis; blue arrow shows wound. CELL AND TISSUE BIOLOGY
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EFREMOV et al.
The rate of regeneration was estimated by measur ing the area of wounds on the 7th, 10th, 14th, and 21st days after the beginning of the experiment (see Mate rials and Methods). The results are summarized in the table. Photographs of mice injected with free NLS TSF7 and complexes NLSTSF7/pCMVIGF1s on the 14th day after the beginning of the experiment are presented in Fig. 7. Injections of both free pCM VIGF1s and NLSTSF7/pCMVIGF1s complexes led to the significant acceleration of the wound healing process compared to wound healing in control groups. Fourteen days after the beginning of the experiment, in mice injected with NLSTSF7/pCMVIGF1s complexes, complete wound healing occurred, whereas, in the control group, the wound area amounted to about 10 mm2. In mice injected with NLSTSF7/pCMVIGF1s complexes, on the 10th day after the beginning of the experiment, the size of the wound was two times lower than in mice injected with free plasmid and four times lower than that in control groups. On the 10th day after the beginning of experiment, a histological study of wound bioptates was performed (Fig. 8). It has been established that, in mice injected with NLSTSF7/pCMVIGF1s com plexes, granulation tissue is more pronounced (the thicker the layer, the higher the number of vessels) compared to the control group of animals injected with the NLSTSF7free conjugate. Besides, in mice injected with the NLSTSF7/pCMVIGF1s complex, the epidermis layer on the border of the wound is better developed. In parallel, the mitotic index of fibroblasts in the wound area was estimated. The transfer to mice of pCMVIGF1s in the complex with the NLSTSF7 molecular conjugate leads to a rise in the number of proliferating fibroblasts by 2.6 times (mitotic index 1.6) compared to the control group of animals injected with the free conjugate NLSTSF7 (mitotic index 0.6). The obtained results indicate a high efficiency of the developed system of the nonviral regenerative gene therapy of mammalian cutaneous integuments based on the use of molecular conjugates with a high affinity to transferrin receptors. ACKNOWLEDGMENTS The work is supported by the Federal Agency of Science and Innovations (State Contract 02.512.11.2274). REFERENCES Akifiev, B.N., Dizhe, E.B., Efremov, A.M., Mogi lenko, D.A., Oleinikova, G.N., Lapikov, I.A., Zhdano va, O.A., Kidgotko, O.V., Orlov, S.V., and Perevozchi kov, A.P., HydrodynamicsBased Transfer of Human Apoli poprotein AI Gene into Mice: Study of Factors Involving an Efficacy and Duration of the Transferred Gene Expres sion in Animals’ Liver, Mol. Biol., 2004, vol. 38, no. 6, pp. 1076–1084.
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