Studies on inducible alphavirus-based antitumour strategy mediated by site-specific delivery with activatable cell-penetrating peptides
Date
2015-08-04
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Abstract
Kasvajate ravi on tänaseni osutunud suureks väljakutseks nii arstidele, kes tegelevad otsese raviga, kui ka teadlastele, kes erinevaid terapeutilisi lähenemisi välja töötavad. Kirurgiline sekkumine on enimrakendatud lähenemine vähiravis, mida tihtipeale kombineeritakse nii kiirituse kui ka kemoteraapiaga, kuid kahjuks näeme siiani suurt hulka patsiente, kellel tekib kasvaja mõne aja pärast uuesti. Siinkohal tuleb hakata mõtlema uudsetele lahendustele – äkki ei piisa ainult sellest, et toimetada sobiv ravimolekul kasvajasse ja lasta seal mõjuda, kas on olemas ka mõni teine lähenemine?
Uurimustöös pakuti välja seni proovimata uudne lahendus kemoteraapiasüsteemi loomiseks, mis koosneb kahest erinevast osast. Palju on räägitud erinevate geenijärjestuste transpordist haigetesse kudedesse, eesmärgiga neid ravida, kuid palju vähem kõlapinda on saanud geneetiliselt modifitseeritud viiruste kasutamine sarnaseks eesmärgiks. Kuna tegemist on ikkagi kasvajarakkudega, ei ole neid praeguste tehnoloogiate juures enam võimalik tagasi normaalseteks rakkudeks muuta, seega ei ole ka nende ellu jäämine väga oluline. Siinkohal tulevadki mängu viirused, mille heaks näiteks on alfaviiruste hulka kuuluv Semliki Forest viirus. Sellise viiruse kasvajarakku sisestamine võimaldaks saavutada kaks eesmärki korraga: esiteks oleks viiruse genoomi sisestatud järjestuselt võimalik toota terapeutilise efektiga valke, teiseks võimaldab viiruse omadus oma peremeesrakk hävitada, efektiivselt võidelda kasvajarakkudega. Samas ei ole kõik nii lihtne, nagu esmapilgul tundub, kuna viirustel on tihtipeale omadus hakata oma peremeesorganismis paljunema, mille tagajärjeks on nakatumine. Selle piiramiseks on välja mõeldud kontrollsüsteem, mis moodustab ka ravimehhanismi esimese poole, mille puhul sisestatakse viirustesse defektsed intronid, mis võimaldavad neil paljuneda ainult sellistes rakkudes, mis sisaldavad aktiveerivat molekuli, mis neid siis läbi defekti blokeerimise sisse lülitab.
Antud süsteemi täielikult toimimiseks on vaja ka teist poolt, ehk siis efektiivset meetodit nii eelpool mainitud viiruse kui ka selle aktivaatori transpordiks soovitud rakkudesse, mis antud kontekstis on kasvajarakud. Selle saavutamiseks modifitseeriti ühte rakku sisenevat peptiidi (klass efektiivseid ja võrdlemisi ohutuid transpordimolekule), milleks oli PepFect14. Kuna antud molekul oli eelnevalt demonstreerinud suutlikkust transportida erinevaid lastmolekule erinevatesse kudedesse, eelistatuna kopsu, siis tuli esmalt leida moodus aktiivsuse maha surumiseks. Selleks kasutati molekuli, PEG, mis käitub nagu kaitsekilp, takistades peptiidil erinevate rakkudega kontakti astumast. Siinkohal võib tekkida küsimus, et kuidas molekuli varjestamine aitab kaasa kasvajarakkudesse jõudmisele, kas nimetatud molekul ei blokeeri kõiki funktsioone ära? Lahenduseks on „kilbi“ kinnitamine läbi sellise vahelüli, mis lagundatakse spetsiifiliste kasvajarakkude keskkonnas avalduvate ensüümide poolt, muutes eelnevalt uinunud olekus süsteemi aktiivseks ainult kasvajakoes, võimaldades kompleksidel (transportiv peptiid ning selle lastiks olev viiruse järjestus või siis aktiveeriv molekul) seeläbi valikuliselt kasvajarakkudesse siseneda.
Kokkuvõttes võimaldab antud strateegia tappa kasvajakudesid kontrollitava viirusega, mis samas toodab ravimolekule, mis, sõltuvalt täpsest funktsioonist, kas stimuleerivad immuunsüsteemi või toimivad mõnda muud rada pidi. Kindlustamaks, et viirus ja selle aktiveerimiseks vaja minev molekul jõuaksid just kasvajatesse ja mitte kuhugile mujale, on nende transpordiks rakendatud kasvajate poolt aktiveeritav peptiidil põhinev geenitranspordisüsteem.
The treatment of tumours is still a great challenge both for the doctors who work with the direct treatment and for the scientist who develop various therapeutic approaches. The most common strategy for cancer treatment is the surgical intervention which is often combined with either radio- or chemotherapy. Unfortunately we still see a significant amount of patients who have a recurring tumour after a period of time. This gives a reason to start thinking about novel approaches–perhaps it not enough just to deliver the suitable therapeutic molecule into the tumour and let it have its effect, perhaps there is a different approach available? In the research, a novel concept for the development of a chemotherapy system was suggested, comprised of two different parts. There has been a lot of discussion about the delivery of various genomic sequences into the diseased tissues with the broader goal of curing them but there has been significantly less talk about the use of genetically modified viruses to achieve a similar effect. As the focus is on tumour cells, it is not possible to turn them back into normal cells using the technology currently available, therefore it is not important to ensure their survival. At this point the viruses come into play, a good example of which is the Semliki Forest virus, belonging to the alphavirus genus. The introduction of this virus to the tumour cell would allow to accomplish two goals at once: first, it would be possible to produce proteins with a therapeutic effect from the transgene sequence introduced into the genome of the virus, secondly, the tendency of the virus to destroy its host cell allows to efficiently combat the malignant cells. It should be noted that everything is not as simple as it might seem as the viruses tend to replicate in their host, resulting in an infection. To control this, a system has been devised, which also forms the first half of the therapeutic system, upon which defective introns will be introduced to the genome of the virus, allowing them to replicate only in the cells containing the activating molecule which turns them on through the blockage of the defective site. For this system to work, there needs to be a second half, which is an efficient method for the delivery of virus and its activator into the desired cells, which, in the current context, are the tumour cells. To achieve this, a cell-penetrating peptide (a class of efficient and relatively harmless transport molecules), PepFect14, was modified. As the molecule had previously demonstrated the capability to transport its cargo molecules into various tissues, especially to lungs, a way to inhibit its activity first had to be found. For this, a molecule, PEG, was applied, which acts as a shield, preventing the peptide to come into contact with various cells. At this point a question might arise–how does the shielding of the molecule help to reach the tumour cells, won’t the described molecule block all of the functions? The solution is the attachment of the “shield” via a linker molecule, which will be dissolved by the specific enzymes expressed in the tumour environment, turning the previously dormant system into an active one specifically in the tumour tissue, thereby allowing the complexes (the transporting peptide and its cargo, which can be either the virus or its activator molecule) to enter specifically into the tumour cells. As a result, the presented strategy allows to kill tumour tissues with a controllable virus, which also produces therapeutic molecules which, depending on the specific function, either stimulate the immune system or act through some other pathway. To ensure that the virus and its activator would reach the tumour and not anywhere else, a peptide-based delivery system, activated in tumours, has been applied
The treatment of tumours is still a great challenge both for the doctors who work with the direct treatment and for the scientist who develop various therapeutic approaches. The most common strategy for cancer treatment is the surgical intervention which is often combined with either radio- or chemotherapy. Unfortunately we still see a significant amount of patients who have a recurring tumour after a period of time. This gives a reason to start thinking about novel approaches–perhaps it not enough just to deliver the suitable therapeutic molecule into the tumour and let it have its effect, perhaps there is a different approach available? In the research, a novel concept for the development of a chemotherapy system was suggested, comprised of two different parts. There has been a lot of discussion about the delivery of various genomic sequences into the diseased tissues with the broader goal of curing them but there has been significantly less talk about the use of genetically modified viruses to achieve a similar effect. As the focus is on tumour cells, it is not possible to turn them back into normal cells using the technology currently available, therefore it is not important to ensure their survival. At this point the viruses come into play, a good example of which is the Semliki Forest virus, belonging to the alphavirus genus. The introduction of this virus to the tumour cell would allow to accomplish two goals at once: first, it would be possible to produce proteins with a therapeutic effect from the transgene sequence introduced into the genome of the virus, secondly, the tendency of the virus to destroy its host cell allows to efficiently combat the malignant cells. It should be noted that everything is not as simple as it might seem as the viruses tend to replicate in their host, resulting in an infection. To control this, a system has been devised, which also forms the first half of the therapeutic system, upon which defective introns will be introduced to the genome of the virus, allowing them to replicate only in the cells containing the activating molecule which turns them on through the blockage of the defective site. For this system to work, there needs to be a second half, which is an efficient method for the delivery of virus and its activator into the desired cells, which, in the current context, are the tumour cells. To achieve this, a cell-penetrating peptide (a class of efficient and relatively harmless transport molecules), PepFect14, was modified. As the molecule had previously demonstrated the capability to transport its cargo molecules into various tissues, especially to lungs, a way to inhibit its activity first had to be found. For this, a molecule, PEG, was applied, which acts as a shield, preventing the peptide to come into contact with various cells. At this point a question might arise–how does the shielding of the molecule help to reach the tumour cells, won’t the described molecule block all of the functions? The solution is the attachment of the “shield” via a linker molecule, which will be dissolved by the specific enzymes expressed in the tumour environment, turning the previously dormant system into an active one specifically in the tumour tissue, thereby allowing the complexes (the transporting peptide and its cargo, which can be either the virus or its activator molecule) to enter specifically into the tumour cells. As a result, the presented strategy allows to kill tumour tissues with a controllable virus, which also produces therapeutic molecules which, depending on the specific function, either stimulate the immune system or act through some other pathway. To ensure that the virus and its activator would reach the tumour and not anywhere else, a peptide-based delivery system, activated in tumours, has been applied
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Keywords
alfaviirus, peptiidid, kasvajarakud, alphavirus, peptides, tumor cells