Rakku sisenevad peptiidid: tsütotoksilisus, immunogeensus ning rakendamine tuumor-spetsiifilise transportvektorina

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Viimastel aastatel, suur tähelepanu kasvajate ravis on pööratud mitmesugustele terapeutilise toimega ravimainete sisestamise strateegiatele, mis ühendavad endas nii ravimite spetsiifilist edastamist kui ka vähivastase kemoteraapiat. Rakku sisenevad peptiidid (RSPd või CPP-s, cell-penetrating peptides) osutunud väga tõhusateks vektoriteks selliste terapeutiliste ravimite sisseviimisel nagu proteiine ning nukleiinhappeid (plasmiidne DNA (pDNA) ja splaissingut korrigeerivaid oligonukleotiide (SKO)). RSPde kasutamine võib olla piiratud nende poolt põhjustatud kõrvaltoimete tõttu: tsütotoksilised efektid ning immunvastuse esilekutsumine. Käesoleva töö peamiseks eesmärgiks oli analüüsida uute RSPde, PepFect-ide immunogeenset ja tsütotoskilist aktiivsust in vitro ja in vivo; uurida p53 analoogsete peptiidide tsütotoksilisust ning apoptootilist toimet rinnavähi rakumudelil ning välja töötada uus RSP, mis on võimeline sisenema ja spetsiifiliselt ära tundma peaajuvähi rakke gliobalstoomi (U87 MG) rakumudelil nii in vitro kui ka in vivo. Esimeses töös näidati, et uued RSPd PepFect-id, mis põhinevad peptiidi transportaan järjestusel, ei avaldanud tsütotoksilist ega immunogeenset toimet immuunsüsteemi rakkudele peptiidi kontsentratsioonidel 5 µM ja 10 µM in vitro tingimustes. Sarnane tulemus saadi ka immunokompetentsete hiirte süstimisel, kui kasutatud annused olid suuremad kui 5 mg/kg kohta. Teises töös disainiti ning sünteesiti lühikesi p53-analoogseid peptiide, mis lisaks efektiivsele rakku sisenemisele avaldasid ka apoptootilist toimet rinnavähi rakkudele in vitro (seerumivabas keskkonnas). Lühikesed p53-analoogsed peptiidid omavad head perspektiivi vähivastase strateegia arendamiseks. Kolmandas töös kirjeldati uue transportvektori, gHoPe2, väljatöötamist, mis on võimeline spetsiifiliselt sihtmärkrakkudesse sisenema. Demonstreeriti ka selle potentsiaalseid kasutusvõimalusi ning toimet ainete transpordil vähirakkudesse. Neljandas töös näidati, et mitteamfipaatse ja sünteetilise (RxR)4 peptiidi stearüülhappega N-terminaalse modifitseerimine tagab efektiivse ning mitte toksilise nukleiinhappete transporti rakkudesse. Käesolev töö annab täiendava ülevaate vähispetsiifilistel RSPdel põhinevatest transportsüsteemidest, hinnates nende toksilisust, immunogeenset aktiivsust ning spetsiifilist transporti kasvajarakkudesse.The delivery of therapeutic agents for the hard-to-treat tumors which comprise both targeting and anticancer chemotherapy strategies, have received much attention. Application of cell-penetrating peptides is one of very promising strategies for targeted drug delivery. However, similar issues as for any other drug delivery system: cytotoxicity and the tendency to induce innate immune response may limit their use. The current thesis is focused on the characterization of immunogenic and cytotoxic activities of cell-penetrating peptides PepFects, on the cytotoxic and apoptotic activities of newly designed cell-penetrating p53 analogues and on the development of a peptide-based glioma-targeted drug delivery vector. In Paper I, the cytotoxic and immunogenic activities of PepFect peptides and PepFect/nucleic acid complexes were analyzed in vitro and in vivo. The current study confirmed that PepFect peptides possess promising potential in nucleic acid delivery without evidence of undesired cytotoxicity and inflammation at the concentrations of 10 μM and 5 μM, respectively in vitro and at a dose of 5 mg/kg in vivo. In Paper II, we generated short p53-derived cell-penetrating protein analogues and analyzed their internalization efficacy, cytotoxic and apoptotic activities in the neuroblastoma and breast cancer cell lines. Even though the effect was observed under serum-free conditions, the novel protein analogues demonstrated selective apoptotic activity in the p53-non-active breast cancer cell line suggesting promising perspectives for a future anticancer strategy. In Paper III, a novel tumor-selective peptide-based drug delivery platform gHoPe2 has been developed demonstrating potential application of CPP-s for tumor-targeted delivery. In Paper IV, we showed that N-terminal modification of non-amphipathic (RxR)4 with stearic acid moiety resulted in efficient peptide vector for nucleic acids delivery in non-toxic manner. In conclusion, the current work provides evidence for the development of apoptotic protein-derived cell-penetrating peptides and tumor-targeted cell-penetrating peptide-based drug delivery vector with reduced toxicity and immunogenicity

Fibrinogen beta variants confer protection against coronary artery disease in a Greek case-control study

<p>Abstract</p> <p>Background</p> <p>Although plasma fibrinogen levels are related to cardiovascular risk, data regarding the role of fibrinogen genetic variation in myocardial infarction (MI) or coronary artery disease (CAD) etiology remain inconsistent. The purpose of the present study was to investigate the effect of <it>fibrinogen A (FGA)</it>, <it>fibrinogen B (FGB) </it>and <it>fibrinogen G (FGG) </it>gene SNPs and haplotypes on susceptibility to CAD in a homogeneous Greek population.</p> <p>Methods</p> <p>We genotyped for rs2070022, rs2070016, rs2070006 in <it>FGA </it>gene, the rs7673587, rs1800789, rs1800790, rs1800788, rs1800787, rs4681 and rs4220 in <it>FGB </it>gene and for the rs1118823, rs1800792 and rs2066865 SNPs in <it>FGG </it>gene applying an arrayed primer extension-based genotyping method (APEX-2) in a sample of CAD patients (n = 305) and controls (n = 305). Logistic regression analysis was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs), before and after adjustment for potential confounders.</p> <p>Results</p> <p>None of the <it>FGA </it>and <it>FGG </it>SNPs and <it>FGA, FGB, FGG </it>and <it>FGA-FGG </it>haplotypes was associated with disease occurrence after adjustment. Nevertheless, rs1800787 and rs1800789 SNPs in <it>FGB </it>gene seem to decrease the risk of CAD, even after adjustment for potential confounders (OR = 0.42, 95%CI: 0.19-0.90, p = 0.026 and OR = 0.44, 95%CI:0.21-0.94, p = 0.039, respectively).</p> <p>Conclusions</p> <p><it>FGA </it>and <it>FGG </it>SNPs as well as <it>FGA, FGB, FGG </it>and <it>FGA-FGG </it>haplotypes do not seem to be important contributors to CAD occurrence in our sample. On the contrary, <it>FGB </it>rs1800787 and rs1800789 SNPs seem to confer protection to disease onset lowering the risk by about 50% in homozygotes for the minor alleles.</p

Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential

ROS1 Asp2213Asn polymorphism is not associated with coronary artery disease in a Greek case-control study

Background: Rs619203 (Cys2229Ser) and rs529038 (Asp2213Asn) polymorphisms in the ROS1 gene have been studied in relation to myocardial infarction (MI) yielding inconsistent results. We investigated the role of ROS1 rs529038 polymorphism in coronary artery disease (CAD) in Greeks using a case-control study. Methods: Genotyping for rs529038 polymorphism was performed using a multiplex PCR technique in patients with CAD (n=294) and controls (n=311). Logistic regression analysis was used to calculate crude and adjusted odds ratios (ORs). Results: Logistic regression analysis did not show any statistically significant effect of ROS1 polymorphism in the occurrence of CAD (AG vs. AA, OR: 1.08, p=0.635; GG vs. AA, OR: 0.62, p=0.220). Adjustment for confounding factors gave similar results, irrespective of the type of disease (i.e., stable coronary artery disease vs. acute coronary syndrome). Conclusions: Our findings do not support the hypothesis that ROS1 rs529038 polymorphism is an important contributing factor in the etiology of CAD in the Greek population. Clin Chem Lab Med 2009;47:1471–3.Peer Reviewe

A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo

Finding suitable nonviral delivery vehicles for nucleic acid–based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice