enhancement of tumor-specific immune response with plasmid dna replicon vectors

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Enhancement of Tumor-specific Immune Response with Plasmid DNA Replicon Vectors Wolfgang W. Leitner 1,2 , Han Ying 1 , David A. Driver, Thomas W. Dubensky, and Nicholas P. Restifo National Cancer Institute, NIH, Bethesda, Maryland 20892-1502 [W. W. L., H. Y., N. P. R.], and Center for Gene Therapy, Chiron Corporation, Emeryville, California 94608 [D. A. D., T. W. D.] Abstract To enhance the immunogenicity of nucleic acid vaccines, we used plasmid DNA vectors that contained replicons derived from the prototype alphavirus, Sindbis, and another alphavirus, Semliki Forest virus. When transfected into cells or injected directly into animal muscle, these plasmids launch a self-replicating RNA vector (replicon) which in turn directs the expression of a model tumor antigen. Immunization with plasmid DNA replicons elicited immune responses at doses 100 to 1000- fold lower than conventional DNA plasmids and effectively treated mice bearing an experimental tumor expressing the model antigen. Significantly, replicon-based DNA plasmids did not produce a greater quantity of antigen; instead, antigen production differed qualitatively. Plasmid DNA replicons mediated antigen production that was homogeneous in all transfected cells and associated with the apoptotic death of the host cells. Because of their safety and efficacy, plasmid DNA replicons may be useful in the development of recombinant vaccines for infectious diseases and cancer. Introduction Naked” nucleic acids are attractive candidate vectors for the development of cancer vaccines encoding tumor-associated antigens. They are relatively simple to generate and safe to administer. Because they are not associated with a viral coat, “naked” nucleic acids are not generally subject to neutralizing antibody reactions that can hamper the clinical efficacy of vaccines based on recombinant viruses such as adeno- and vaccinia viruses (reviewed in Refs. 1 and 2). In preclinical tumor models, nucleic acid vaccines encoding model antigens can elicit antigen- specific antibody and CD8 + T-cell responses and can be therapeutically effective when combined with immunomodulatory molecules such as CD40 ligand or interleukin 2 or interleukin 12 (3-6). DNA vectors have also been enhanced by optimizing promoters, introns, and polyadenylation signals. Immunostimulatory sequences have also been shown to enhance the function of some plasmid DNA immunization (7). One improvement upon plasmid DNA vectors was the incorporation of alphavirus replicons (8,9). In animal models of infectious disease, these plasmid DNA replicons are substantially more efficient at stimulating antigen- specific immune responses, particularly cellular responses, as compared with conventional plasmid DNA expression vectors (10,11). Alphavirus replicons, in the form of RNA, DNA, or infectious particles, are generally potent inducers of broad immune responses in both rodents and primates (reviewed in Refs. 12). 1 W. W. L. and H. Y. contributed equally to this work. 2 To whom requests for reprints should be addressed, at the National Cancer Institute, Building 10, Room 2B46, Bethesda, MD 20892-1502. Phone: (301) 496-6357; Fax: (301) 496-0011; E-mail: [email protected]. NIH Public Access Author Manuscript Cancer Res. Author manuscript; available in PMC 2008 February 22. Published in final edited form as: Cancer Res. 2000 January 1; 60(1): 51–55. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

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Enhancement of Tumor-specific Immune Response with PlasmidDNA Replicon Vectors

Wolfgang W. Leitner1,2, Han Ying1, David A. Driver, Thomas W. Dubensky, and Nicholas P.RestifoNational Cancer Institute, NIH, Bethesda, Maryland 20892-1502 [W. W. L., H. Y., N. P. R.], and Center forGene Therapy, Chiron Corporation, Emeryville, California 94608 [D. A. D., T. W. D.]

AbstractTo enhance the immunogenicity of nucleic acid vaccines, we used plasmid DNA vectors thatcontained replicons derived from the prototype alphavirus, Sindbis, and another alphavirus, SemlikiForest virus. When transfected into cells or injected directly into animal muscle, these plasmidslaunch a self-replicating RNA vector (replicon) which in turn directs the expression of a model tumorantigen. Immunization with plasmid DNA replicons elicited immune responses at doses 100 to 1000-fold lower than conventional DNA plasmids and effectively treated mice bearing an experimentaltumor expressing the model antigen. Significantly, replicon-based DNA plasmids did not produce agreater quantity of antigen; instead, antigen production differed qualitatively. Plasmid DNA repliconsmediated antigen production that was homogeneous in all transfected cells and associated with theapoptotic death of the host cells. Because of their safety and efficacy, plasmid DNA replicons maybe useful in the development of recombinant vaccines for infectious diseases and cancer.

Introduction“Naked” nucleic acids are attractive candidate vectors for the development of cancer vaccinesencoding tumor-associated antigens. They are relatively simple to generate and safe toadminister. Because they are not associated with a viral coat, “naked” nucleic acids are notgenerally subject to neutralizing antibody reactions that can hamper the clinical efficacy ofvaccines based on recombinant viruses such as adeno- and vaccinia viruses (reviewed in Refs.1 and 2).

In preclinical tumor models, nucleic acid vaccines encoding model antigens can elicit antigen-specific antibody and CD8+ T-cell responses and can be therapeutically effective whencombined with immunomodulatory molecules such as CD40 ligand or interleukin 2 orinterleukin 12 (3-6). DNA vectors have also been enhanced by optimizing promoters, introns,and polyadenylation signals. Immunostimulatory sequences have also been shown to enhancethe function of some plasmid DNA immunization (7). One improvement upon plasmid DNAvectors was the incorporation of alphavirus replicons (8,9). In animal models of infectiousdisease, these plasmid DNA replicons are substantially more efficient at stimulating antigen-specific immune responses, particularly cellular responses, as compared with conventionalplasmid DNA expression vectors (10,11). Alphavirus replicons, in the form of RNA, DNA, orinfectious particles, are generally potent inducers of broad immune responses in both rodentsand primates (reviewed in Refs. 12).

1W. W. L. and H. Y. contributed equally to this work.2To whom requests for reprints should be addressed, at the National Cancer Institute, Building 10, Room 2B46, Bethesda, MD20892-1502. Phone: (301) 496-6357; Fax: (301) 496-0011; E-mail: [email protected].

NIH Public AccessAuthor ManuscriptCancer Res. Author manuscript; available in PMC 2008 February 22.

Published in final edited form as:Cancer Res. 2000 January 1; 60(1): 51–55.

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We have demonstrated recently that an RNA vaccine encoding a model tumor-associatedantigen together with the gene for RNA replicase from the Semliki Forest virus was effectivein the treatment of an experimental tumor (13). In the present study, we extend this work toDNA plasmids, using a CMV3 promoter to “prime the pump” and generate a long positivestrand of RNA (replicon) which, like the alphaviral genome itself, is then capable of self-replication. We then compared these vectors, quantitatively and qualitatively, to conventionalplasmid-based vaccines.

Materials and MethodsVaccines

Plasmid DNA replicons encoding β-gal and conventional CMV promoter-based β-gal plasmidsare shown in Fig. 1A. pSIN1.5-β-gal (Chiron Technologies, San Diego, CA) uses the Sindbisvirus replicon, whereas a Semliki Forest virus replicase drives mRNA replication in the pRep-LacZ plasmid. pSPORT-β-gal was obtained from Life Technologies, Inc. (Bethesda, MD), andpCMV-β-gal was constructed at Powderject (Middleton, WI) by cloning a CMV-β-gal-cassetteinto the PGEM plasmid backbone (Promega Corp., Madison, WI; Ref. 4). A Semliki Forestvirus replicase-based self-replicating RNA construct encoding β-gal (Rep-RNA) was used insome in vitro studies as a positive control (13). To monitor antigen expression on a cellularlevel, two plasmids were used that express EGPF either under the control of a CMV promoter(pEGFP-C1; Clontech Laboratories, Palo Alto, CA) or encode a Sindbis replicon (pSIN1.5-EGFP, provided by Dr. Rong-Fu Wang, National Cancer Institute, Bethesda, MD).

Peptides and Cell LinesThe synthetic peptide TPHPARIGL, representing the naturally processed H-2 Ld-restrictedpeptide 876–884 of β-gal, and the peptide LPYLGWLVF, representing residues 35–43 of theP815A protein (P-1A), were synthesized and purified by Peptide Technologies (Washington,DC) and assayed by high-performance liquid chromatography and amino acid analysis. A β-gal-expressing clone (CT26.CL25) was generated from CT26.WT transduced with a LacZretrovirus and maintained in complete medium based on RPMI 1640 with 10% fetal bovineserum, as described (14). BHK-21 (American Type Culture Collection, Manassas, VA) cellswere used for in vitro transfection experiments.

ImmunizationsFemale BALB/c mice (6–10 weeks of age; Jackson Laboratories, Bar Harbor, ME) wereimmunized with 50 μl of plasmid DNA in PBS into each quadricep, and immune responseswere evaluated 3 weeks later. Sera antibody titers were determined individually in four mice/group (mean titers are shown) in a β-gal-ELISA assay as described previously (13). CD81 T-cell function was assessed in pooled spleen cells (four mice/group) cultured in the presence of1 μg/ml β-gal-peptide for 6 days. Subsequently, 105 effector cells/well were incubated with105 target cells (CT26.WT alone or pulsed with 1 μg/ml peptide, or CT26.CL25) for 24 h.Supernatants were diluted 1:10 and tested for IFN-γ release using a mIFN-γ ELISA kit(Endogen, Cambridge, MA). For tumor prevention experiments, at least five mice/group wereinjected i.v. with 5 × 105 CT26.CL25 cells 3 weeks after a single immunization with DNA.For treatment experiments, 10 mice/group were injected with 1 × 105 CT26.CL25 cells 2 daysprior to treatment with DNA. The number of pulmonary metastases was determined in a blindedfashion 12 days after challenge, and data were analyzed using the Kruskal-Wallis test.

3The abbreviations used are: CMV, cytomegalovirus; βgal, β-galactosidase; EGFP, enhanced green fluorescent protein.

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In Vitro Transfection and Analysis of Antigen ExpressionBHK cells seeded at 1 × 105 cells/well in a six-well plate were cultured for 24 h and transfectedwith 1 μg of DNA/well using LipofectAMINE PLUS (Life Technologies, Inc.), according tothe manufacturer's instructions. Twenty-four h after transfection, cells were washed, fixed in0.5% glutaraldehyde for 10 min, and incubated in 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Life Technologies, Inc.; 1:50) at 37°C for 1–2 h to determine transfectionefficiency. Total β-gal production in lysed cells was determined using a β-gal ELISA(Boehringer Mannheim, Mannheim, Germany) as described (15). Expression of β-gal isreported as percentage of β-gal protein corrected for the transfection efficiency for theparticular plasmid based on the total amount of cellular protein, determined with a BCA ProteinAssay (Pierce, Rockford, IL). Cells transfected with EGFP plasmids were analyzed for antigenexpression on a FACScan (Becton Dickinson, San Jose, CA).

Apoptosis in Transfected Cells in VitroBHK cells were transfected with SIN-EGFP as described above. After 24 h in the presence orabsence of 20 μM/ml caspase inhibitors (peptides z-VAD.fmk and z-FA.fmk; Enzyme SystemsProducts, Livermore, CA), cells were harvested and seeded in quadruplicates at 50–100 cells/well in 96-well plates. Fresh caspase inhibitor was added every 24 h, and living cells werecounted daily using fluorescence microscopy.

Results and DiscussionPlasmid DNA Replicons Induce Stronger Humoral and Cellular Immune Responses ThanConventional DNA Vaccines

To evaluate the effects of the alpha-viral replicon on immune responses elicited by DNAvaccines encoding LacZ, we compared two plasmid DNA replicons encoding LacZ and twoconventional CMV promoter-based LacZ plasmids. pSIN uses the Sindbis nonstructuralproteins, whereas pRep uses nonstructural proteins derived from Semliki Forest virus. Theconventional plasmids had been optimized for antigen expression (pSPORT-β-gal) or havesuccessfully been used as a vaccine before in preclinical tumor vaccine trials (pCMV-β-gal;Ref. 4). After a single immunization, pSIN-β-gal produced strong antibody responses at alldoses, whereas detectable humoral responses with all other plasmids were lost with between10 and 1 μg (Fig. 1B). Note that the plasmid DNA replicons, pSIN and pRep, are about twotimes larger than the conventional CMV-based plasmids, pSPORT and pCMV, and thus onlyhalf the number of moles of plasmid DNA replicon were delivered per μg of DNA.

Cellular responses were superior in mice immunized with the plasmid DNA replicon, asmeasured by antigen-specific IFN-γ release (Fig. 1C). At the 1-μg dose, only mice immunizedwith the plasmid DNA replicons, pSIN and pRep, produced an IFN-γ response. Conventionalplasmids were not immunogenic at doses <10 μg in repeated experiments. Interestingly, thestrongest response of all groups was obtained with the lowest dose of pSIN, and similar to theantibody response, no direct correlation between the injected dose and the strength of theimmune response was observed.

Plasmid DNA Replicon Vaccines Effectively Immunize against Tumor ChallengeProtection from tumor challenge was evaluated in experiments in which mice were immunizedwith a single dose of vector injected i.m. Immunization with 50 μg resulted in completeprevention with all plasmids used (not shown) and protection that was nearly complete at the10-μg dose (Fig. 2A). However, at 1 and 0.1 μg/mouse, only immunization with pSINcompletely protected from tumor challenge. Although pRep showed some protection, the twonon-replicon-coding plasmid vaccines completely lost their protective effect.

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A Plasmid DNA Replicon Treats Established TumorA more challenging evaluation of the in vivo efficacy than tumor prevention is the treatmentof established tumor. On the basis of the immunogenicity of the plasmids in the above studies,pSIN was selected for treatment studies. At all doses, the plasmid DNA replicon pSIN yieldedsignificant treatment of established CT26.CL25 tumors. Of the conventional DNA plasmids,only pSPORT treated tumors and only at doses <10 μg (Fig. 2B; only 10-μg dose shown),whereas pCMV-β-gal had no therapeutic effect at any dose used (0.1 to 50 μg, not shown).pSIN was most efficacious at the lowest dose (median number of metastases at 0.1-μg dose,43), whereas its efficacy slightly decreased with increased dose (median at 1 μg, 76; medianat 10 μg, 124). To study the increase in survival after tumor challenge, mice with establishedlung metastasis were treated with a single dose of pSIN or pSPORT (Fig. 2C). Although bothplasmids prolonged survival at very high doses (50 or 100 μg), there was no significantdifference between the plasmids (data not shown). However, survival was significantlyprolonged by treatment with 10 or 1 μg pSIN (compared with control P < 0.00001 and P =0.0001, respectively), whereas the same doses of pSPORT were ineffective compared withcontrol (P = 0.12 and P = 0.09, respectively).

In Vitro Antigen Expression Does Not Correlate with in Vivo Immunogenicity of the IndividualPlasmids

One possible explanation for the enhanced immunogenicity of replicon-based systems was thatthey mediated increased expression of the encoded antigen. However, we found no correlationbetween antigen expression and immunogenicity (Fig. 3). Despite the presence of the alphaviralreplicon in pSIN and pRep, a conventional CMV-based plasmid yielded the highest β-galproduction. To test antigen expression on a single-cell basis, cells were transfected with aconventional, CMV (pEGFP-C1)- or replicon (pSIN1.5-EGFP)-based plasmid encoding EGFPfor analysis of the transfected cells by flow cytometry. pEGFP-C1-transfected cells variedwidely in their expression of the transgene, whereas pSIN-transfected cells displayed ahomogenous EGFP expression profile (Fig. 4A). This phenomenon is a direct result of theplasmid-encoded alphaviral replicon, because the same staining pattern was obtained with aself-replicating RNA construct (Rep-RNA-EGFP, data not shown). To test whether thisstriking effect was merely attributable to a choice of a particular concentration of DNA, weassessed antigen expression in cells transfected with limiting amounts of plasmid. For theconventional CMV-based plasmid, pEGFP-C1, the overall expression level directly correlatedwith the amount of DNA used, leading to the disappearance of highly transfected cells at lowDNA concentrations. In contrast, pSIN-EGFP-transfected cells express the same amount ofantigen independent of the amount of plasmid used, establishing a quantitative as well as aqualitative difference between the two systems (Fig. 4B). This phenomenon could explain whyimmune responses induced by CMV-based plasmids correlate with the dose of injected DNA,whereas plasmid DNA replicons do not show a dose titration.

Plasmid Transfection of Cells in Vitro Induces ApoptosisAlphaviral infection of cells is known to induce their apoptotic death. Likewise, transfectionwith “self-replicating” RNA constructs cause the quantitative death of cells by apoptosis(16-18). This death is likely caused by the requisite double-stranded RNA intermediates thatare generated during the replication of the alphavirus. To test whether the DNA constructs alsomediated apoptotic death, BHK cells were transfected with the plasmid DNA replicon, pSIN-EGFP (Fig. 4C). As a positive control, cells were transfected with a self-replicating RNAconstruct, Rep-RNA-EGFP (13). Cells transfected with pSIN-EGFP start to disappear after abrief proliferation phase with a peak at 48 h after transfection, following the same kinetics ascells transfected with a self-replicating RNA construct. Survival of transfected BHK cells canbe significantly prolonged, but not completely prevented, by the addition of the caspase

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inhibitor z-VAD.fmk but not the control fluro-methyl-ketone peptide inhibitor, z-FA.fmk (Fig.4D).

Implications for Vaccine DevelopmentOn the basis of the notion that “more antigen is better,” vaccinologists have spent a great dealof effort optimizing antigen production by recombinant vectors. However, we have shownpreviously that the optimization of antigen expression is best conducted in dendritic cells andthat late promoters, which mediate the highest expression of antigens by vaccinia viralconstructs, were not the most immunogenic (15). The findings presented here suggest that thequalitative aspects of antigen expression may be more important than high-level antigenproduction. Thus, although we found that the replicon-based vectors did not necessarilyproduce more antigen (Fig. 3), the immunogen was produced at consistent levels in alltransfected cells and was associated with the apoptotic death of host cells (Fig. 4). This caspase-dependent death is likely mediated by the double-stranded RNA-dependent kinase PKR, aswell as RNaseL, both enzymes involved in cellular defense against viral infection (19,20).

Apoptotic death associated with the production of double-stranded RNA intermediates is likelyto have immunological consequences that are of benefit to the vaccinologist: (a) death byapoptosis may increase the uptake of antigen by dendritic cells for subsequent processing andclass I-restricted presentation to CD8+ T cells (21). This uptake is mediated primarily byαVβ5 receptors on the surfaces of dendritic cells and is restricted to dendritic cells (22). Wehave demonstrated previously an increased uptake of cells undergoing apoptosis as a result oftransfection with “self-replicating” RNA (13), confirming these observations; and (b) double-stranded RNA itself is a “danger signal,” functioning as an adjuvant to the T-cell-specificstimulus of the encoded antigen (23). Plasmid DNA replicons thus appear to mimic a host cellinfection by an alphavirus because the replicons mediate the production of double-strandedRNA, which in turn activates dendritic cells (24).

The present work reveals that factors in addition to the amount of expressed antigen canprofoundly affect the immunogenicity of the recombinant vaccine. Indeed, more antigen is notnecessarily better. Instead, “danger signals” provided by the “naked” plasmid DNA repliconsmight be the key to their enhanced immunogenicity.

Acknowledgments

We thank M. Blalock for graphics, P. Spiess for help with animal experiments, Dr. Rong-Fu Wang for the constructionof pSIN1.5-EGFP, and Dr. S. A. Rosenberg for helpful discussion.

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Fig. 1.Plasmid DNA replicons have superior immunogenicity compared with two conventionalplasmids. A, structure of plasmids. The two plasmid DNA replicons, pSIN and pRep, encodealphaviral replicons from Sindbis virus and Semliki Forest virus, respectively. The LacZ geneis inserted between the subgenomic start sequence and a 3′ replicase-recognition sequence plusintron. Because the plasmid DNA replicons are twice the size of the conventional plasmidsused, only half the number of plasmid copies of plasmid DNA replicon were injected comparedwith conventional plasmids. B, β-gal-specific IgG responses elicited by a single i.m.immunization with plasmid DNA replicons versus conventional plasmid DNA. Sera werecollected from four mice/group 21 days after immunization and diluted 1:20, 1:100, 1:250,1:2500. A490 nm shown are averages of individual determinations. Similar results were obtainedin two additional experiments. C, plasmid DNA replicons elicit superior cellular immuneresponses. Pooled splenocytes (four mice/group) from mice immunized 21 days earlier werecultured for 6 days in the presence of β-gal876–884 peptide and then tested for antigen-specificrecognition as measured by IFN-γ released/105 cells. Shown are values obtained in response

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to CT26 pulsed with β-gal peptide, but similar results were obtained using the LacZ-transfectedtarget CT26.CL25 (not shown). The response to control targets (CT26.WT alone or pulsedwith the P-1A35–43 peptide) was < 100 pg for all groups. This experiment was repeated withsimilar results.

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Fig. 2.Therapeutic efficacy of “naked” DNA immunization. A, prevention of tumor challenge withplasmid DNA replicons is superior to conventional plasmids at limiting amounts of DNA. Micewere immunized once and challenged 21 days later with 5 × 105 CT26.CL25 tumor cells.Pulmonary metastasis were enumerated 12 days after challenge. B, only p-SIN-β-gal treatsestablished pulmonary metastasis after challenge with 1 × 105 CT26.CL25. The mean numberof pulmonary metastasis is indicated. C, a single treatment with 1 μg of p-SIN-β-galsignificantly prolongs survival of CT26.CL25 tumor-bearing mice. *, P compares pSIN-β-galwith PBS control. There was no significant difference between PBS and pSPORT-β-gal.Treatment with 10 μg of either plasmid yielded almost identical results as with 1 μg (not shown).

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Fig. 3.Plasmid DNA replicons do not necessarily induce more antigen expression than conventionalplasmids. The amount of β-gal produced by transfected BHK-21 cells within 24 h is expressedas percentage of β-gal based on the total cellular protein of the transfected cells, after correctingfor the different transfection efficiencies of the various plasmids. SDs (bars) were calculatedfrom replicate transfections. The highest expression was obtained with the least immunogenicplasmid, pCMV-β-gal, whereas the most immunogenic plasmid, pSIN-β-gal, yielded thelowest antigen production.

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Fig. 4.Plasmid DNA replicons are qualitatively different than conventional plasmids. A, BHK-21cells transfected with pEGFP-C1 or pSIN-EGFP produce a distinct and significantly differentstaining pattern, as analyzed by flow cytometry. B, altering the amount of pSIN-EGFP usedfor transfection does not affect the EGFP expression profile. However, transfection withvarious amounts of pEGFP-C1 plasmid causes a shift in the subpopulations of EGFP-expressing cells, resulting in a decrease in the high-expressor cell population when reducingthe amount of plasmid. Transfected cells are grouped into low, medium, and high expressors.Categories are based on expression level of EGFP by pSIN-EGFP-transfected cells, whichequals medium expressors. C, alphaviral replicase causes cell death in transfected cells in

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vitro, whereas cells transfected with a conventional DNA construct continue to proliferate.BHK-21 cells were transfected with pSIN-EGFP, pEGFP-C1, or a self-replicating RNAconstruct expressing EGFP (Rep-RNA-EGFP). D, the decrease in the number of pSIN-transfected cells is attributable to apoptosis, which can be delayed but not completely preventedby using the caspase inhibitor z-VAD.fmk. The control-peptide z-FA.fmk does not affect cellsurvival. The number of cells is reported as %EGFP+ cells based on the number of cells plated24 h after transfection (= 100%).

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