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Elektrogenska terapija z interlevkinom 12 za zdravljenje kožnih in oralnih tumorjev pri psih
Lampreht Tratar, Urša (Avtor), Čemažar, Maja (Mentor) Več o mentorju... Povezava se odpre v novem oknu

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Izvleček
Znanstveno izhodišče: Spontani tumorji pri psih predstavljajo odličen model za preizkušanje novih terapij in za njihov prenos v humano onkologijo. Ena izmed novih terapij, ki se je izkazala za izredno učinkovito za zdravljenje tumorjev pri psih in ljudeh je elektrokemoterapija. Elektrokemoterapija je lokalno zdravljenje, katera ima velik uspeh na lokalnem nivoju, vendar ne na sistemskem. Kot dodatno terapijo s sistemskim učinkom, bi lahko uporabili elektrogensko terapijo z interlevkinom-12 (IL-12), ki je v nekaterih predkliničnih in kliničnih študijah izboljšala odgovor na zdravljenje. IL-12 je provnetni citokin, ki preko interferona gama (IFNɣ) poveča delovanje protitumorskega imunskega odziva. V kliničnih študijah, kjer so uporabili kombinacijo elektrokemoterapije in elektrogenske terapije z IL-12 za zdravljenje spontanih tumorjev pri psih, so dokazali dobro protitumorsko učinkovitost. V teh študijah so uporabili plazmid, ki je nosil zapis za humani ali mačji IL-12, ki nima popolne homolognosti s pasjim IL-12. Poleg tega je nosil zapis za gen za odpornost proti ampicilinu, katerega pristojne organizacije ne odobravajo. Zato je priprava plazmida, ki bi nosil zapis za pasji IL-12 z ali brez gena za odpornost proti antibiotiku, ključna za uporabo v nadaljnjih kliničnih študijah na psih. Metode: V prvem sklopu smo pripravili in ovrednotili plazmide, ki nosijo zapis za pasji IL-12 pod konstitutivnim ali tkivno specifičnim promotorjem in z ali brez gena za odpornost proti antibiotiku. Poskuse smo v in vitro pogojih opravili na humanih in pasjih normalnih in tumorskih celičnih linijah ter spremljali stopnjo izražanja IL-12 na nivoju mRNA in na proteinskem nivoju po genskem elektroprenosu. V in vivo pogojih smo na ksenograftih pasjih melanomskih celic na imunsko oslabljenih miših poleg protitumorskega učinka spremljali tudi imunski odziv po genskem prenosu IL-12. Nenazadnje smo prijavili klinično študijo na pristojne organe ter začeli z vključevanjem pacientov v klinično študijo, kjer smo s kombinacijo elektrokemoterapije in elektrogenske terapije zdravili spontane kožne in oralne tumorje pri psih. Tekom terapije smo merili velikosti tumorjev in jemali vzorce krvi, urina in blata, ter brise z mesta aplikacije plazmida za določanje varnosti in učinkovitosti terapije. Rezultati: Rezultati so pokazali, da ima novi plazmid, ki nosi zapis za pasji IL-12 podobno ali višjo zmožnost ekspresije v in vitro pogojih na nivoju mRNA in proteinov kot plazmid, ki nosi zapis za humani IL-12. V in vivo poskusu na imunsko komprimiranih miših smo ugotovili, da je terapevtska učinkovitost po elektrogenskem prenosu primerljiva med plazmidoma, ki nosita zapis za pasji ali humani IL-12. V primeru plazmidov, ki nosijo zapis za pasji IL-12 pod konstitutivnim in tkivno specifičnim promotorjem ter nimajo gena za odpornost proti antibiotiku smo ugotovili, da odstranitev gena za odpornost proti antibiotiku ne zmanjša citotoksičnosti ter sposobnosti ekspresije transgena. V in vivo študiji smo ugotovili, da ima plazmid s konstitutivnim promotorjem višjo ekspresijo IL-12 kot plazmid s tkivno specifičnim promotorjem. Poleg tega je po genskem elektroprenosu plazmid s konstitutivnim promotorjem povzročil povišano vrednost IL-12 v serumu medtem, ko je bilo izražanje IL-12 v primeru tkivno specifičnega promotorja omejeno na tumor. Nenazadnje je bilo v primeru konstitutivnega promotorja zaznana povišana infiltracija grancim B pozitivnih celic v tumorju in vranici, kar kaže na sistemski učinek terapije. Po genskem elektroprenosu IL-12 smo opazili tudi povišano infiltracijo M1 makrofagov ter dendritičnih celic v tumorju na imunsko nezavrtih miših. V klinično študijo smo vključili 55 psov, 42 s kožnimi tumorji in 13 z oralnimi tumorji. V primeru kožnih tumorjev smo dosegli popolni odgovor pri 84 % pacientov. Odgovor je bil slabši pri oralnih tumorjih, kjer je delež popolnih odgovorov znašal 15 %. Pri preiskovanju zbranih vzorcev urina, blata in seruma, nismo zaznali prisotnosti plazmidne DNA. V primeru brisov kože smo pri nekaterih pacientih zaznali prisotnost plazmidne DNA takoj po aplikaciji (najvišja je bila 100 ng/mL), ki je v 4 tednih padla na ničelno vrednost. Poleg tega smo v krvi merili deleže imunskih celic po terapiji, kjer smo ugotovili statistično značilen upad celic T zaviralk ob koncu opazovanega obdobja, glede na delež celic T zaviralk ob terapiji. Zaključek: Novo pripravljeni plazmid, ki kodira pasji IL-12 in ima gen za odpornost proti kanamicinu, ima enako učinkovito stopnjo izražanja IL-12 ter enak protitumorski učinek kot plazmid, ki nosi zapis za humani IL-12. Uporaba novega plazmida v kombinaciji z elektrokemoterapijo za zdravljenje spontanih kožnih in oralnih tumorjev pri psih se je izkazala za varno ter učinkovito. Poleg tega novo pripravljeni plazmidi, ki nosijo zapis za pasji IL-12 pod tkivno specifičnim ali konstitutivnim promotorjem brez gena za odpornost proti antibiotikom, predstavljajo učinkovito orodje za nadaljnje klinične študije, ki so nujno potrebne za uspešno translacijo te terapije v humano medicino.

Jezik:Slovenski jezik
Ključne besede:genski elektroprenos interlevkin 12 veterinarska onkologija pasje maligne melanomske celične linije klinična študija
Vrsta gradiva:Doktorsko delo/naloga (mb31)
Organizacija:MF - Medicinska fakulteta
Leto izida:2018
Število ogledov:304
Število prenosov:152
Metapodatki:XML RDF-CHPDL DC-XML DC-RDF
 
Skupna ocena:(0 glasov)
Vaša ocena:Ocenjevanje je dovoljeno samo prijavljenim uporabnikom.
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Sekundarni jezik

Jezik:Angleški jezik
Naslov:Electrogenetherapy with interleukin 12 for the treatment of skin and oral tumors in dogs
Izvleček:
Scientific background: Spontaneous tumors in dogs represent an excellent model for testing new therapies and for their translation into human oncology. One such (new/novel) therapy is electrochemotherapy, which has proved to be effective in treating tumors in dogs and humans. Electrochemotherapy is a treatment with great success locally, but not systemically. Interleukin 12 (IL-12) electrogenetherapy has proved highly effective in inducing systemic effect in preclinical and clinical studies. IL-12 is an inflammatory cytokine that increases the activity of antitumor immune response through interferon γ (IFNɣ). In clinical studies of a combined electrochemotherapy and IL-12 electrogenetherapy treatment of spontaneous tumors in dogs, good antitumor efficacy has been demonstrated. In these studies plasmids encoding human or feline IL-12, which do not have a complete homology to canine IL-12, were used. In addition, these plasmids carried an ampicillin resistance gene which is disapproved by regulatory agencies. Therefore, the preparation of plasmids encoding canine IL-12 with or without an antibiotic resistance gene is essential for use in further clinical studies on dogs. Methods: Firstly, plasmids encoding canine IL-12 under a constitutive or a tissue specific promotor with or without the gene for antibiotic resistance were prepared and evaluated. The experiments were carried out in in vitro conditions on human and canine normal and tumor cell lines. The IL-12 expression on mRNA and protein levels was monitored following gene electrotransfer. In in vivo conditions, the antitumor effect of IL-12 gene electrotransfer of canine melanoma cell line xenografts on immunocompromised mice was evaluated. Also we observed the induced immune response after IL-12 gene electrotransfer. Lastly, we registered a clinical study to the competent authorities and began to enroll dogs with spontaneous tumors. Dogs were treated with a combination of electrochemotherapy and IL-12 electrogenetherapy. During the course of the therapy we measured the size of the tumors and took blood, swabs, urine and stool samples to determine the safety and efficacy of the therapy. Results: The new plasmid encoding canine IL-12 has a similar or a higher level IL-12 expression on mRNA and protein levels in in vitro conditions. In in vivo conditions on immunocompromised mice the therapeutic efficacy after IL-12 gene electrotransfer was found to be comparable between plasmids encoding canine and human IL-12. In the case of plasmids encoding canine IL-12 under the constitutive and the tissue-specific promoter without the antibiotic resistance gene, it has been found that the removal of the antibiotic resistance gene does not reduce the cytotoxicity or the expression level of the transgene. In in vivo conditions, it was found that the plasmid with the constitutive promoter had a higher expression of the transgene than a plasmid with a tissue-specific promoter. In addition, gene electrotransfer of the plasmid with the constitutive promoter caused an increase of serum IL-12, while in the case of the tissue specific promotor, IL-12 expression was limited to the tumor. In the case of the constitutive promoter, an increased infiltration of granzyme B positive cells was observed in the tumor and spleen, indicating a systemic effect of the treatment. Also, an increased infiltration of M1 macrophages and dendritic cells in the tumor was observed after the IL-12 gene electrotransfer. In our clinical study 55 dogs were enrolled, 42 dogs with skin tumors and 13 with oral tumors. In the case of skin tumors 84 % complete responses were achieved, whereas in oral tumors only 15 % complete responses were achieved. When screening for the presence of plasmid DNA, no plasmid DNA was detected in the stool, urine and serum samples. Plasmid DNA was present in skin swabs in some patients. The highest concentration of plasmid DNA (100 ng/mL) was detected immediately after the therapy, but after 4 weeks no plasmid DNA was detected. Lastly, the percentage of immune cells was measured after the therapy. We observed a statistically significant decline in the percentage of regulatory T cells at the end of the observation period compared to the time of the treatment. Conclusion: The new plasmid encoding canine IL-12 with kanamycin resistance gene has the same or a higher expression level of IL-12 and comparable antitumor effect than the plasmid encoding human IL-12. The use of a new plasmid in combination with electrochemotherapy for the treatment of spontaneous skin and oral tumors in dogs has proved to be safe and efficient. Additionally, newly prepared plasmids encoding canine IL-12 under the constitutive or the tissue-specific promoter without antibiotic resistance genes serves as an effective tool for further clinical studies, which are indispensable for translating this therapy into human medicine.

Ključne besede:gene electrotransfer interleukin 12 veterinary oncology canine malignant melanoma cell lines clinical study

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