TOLL BENZERİ ALMAÇLAR: ENFLAMASYONU YÖNLENDİREN MOLEKÜLLER İHSAN GÜRSEL Bilkent Üniversitesi, Moleküler Biyoloji ve Genetik Bölümü, UNAM, Malzeme Bilimi ve Nanoteknoloji Enstitüsü, Nanobiyoteknoloji Araştırma Birimi FDA VRC NCI NIAID NIH Clinical Center 1
KARŞILAŞMA: TÜRKİYE NİN BİLİM VE TEKNOLOJİDEKİ YERİ 1996-2005 (10 yıl) arasında uluslararası dergilerinde yayınlanan makalelerin tümü h-indeks: Bir araştırmanın uluslararası camiada ne kadar etkili olduğunu gösteren değer Stanford Üniversitesi, Türkiyeden 50 kattan daha fazla etkili araştırma yapıyor. Türkiye nin h-indisinin 80 i, uluslararası ortak çalışmaların sonucunda elde edilmiştir. Neden yüksek katma değer? İhraç Ürünleri $ Fındık (1 Ton) ~ 1,800 Tofaş Araba (1 Ton) ~ 12,000 Uzaktan Algılayan Lazer (1 Ton) ~ 2,500,000 AKM Mikroskopu (1 Ton) ~ 18,000,000 rifn* ve repo (15 grams, ithal!) ~ 450,000,000 Türkiye yılda ~ 15 gr rekombinant Eritropoetin kullanmaktadır. Bununla birlikte diğer interferonlar ve interlükinler de eklenince yılda harcanan rakam 0.8 Milyar $ seviyesine ulaşacağı tahmin ediliyor (2009 yılı için). *Bizim Amacımız: Bu biyolojik ajanları immün hücrelerine ürettirebilecek DNA kökenli nanoilaçlar tasarlamaktır. 2
The Danger Theory (Infectious non-self vs non-infectious self) Pathogen Associated Molecular Patterns (PAMPS) (Exist on NON SELF) Pathogen Recognition Receptors (PRR) ( Present on SELF) INNATE IMMUNE ACTIVATION Innate Immunity Inflammatory immune response Adaptive Immunity Antigen specific immune response Granulocytes, Macrophages T cells, B cells TLRs NLRs RLRs Dendritic cells (DC) Natural Killer cells (NK) Antigen presentation Cytokines NK killing Chemokines Interferons IgM Antigen-specific 1) Antibodies (IgG, A, E) 2) Cellular immune response (CTL, Th, Treg) minutes hours days months years Ishii KJ and Akira S. Innate Immunity Clinical Immunology 3 rd Ed. In press 3
TLRs sense both bacterial and viral invasion Bacteria Virus =Type-I IFN Peptidoglycan Lipopeptide Flagellin LPS Envelope protein (MMTV) F protein (RSV) Bacterial DNA Viral DNA Imidazoquinolines ssrna dsrna TLR2 TLR5 TLR4 TLR9 TLR3 TLR7 TLR (NLR, RLR) ligands derived from pathogens or damaged cells Innate Immunity 10 7 B 9 Proliferation IL-6 7 B 9 IgG Adaptive Immunity TLR (NLR, RLR)-for GOOD Mɸ Epithelial cells cytokines/chemokines 1/6 2/1 4 8 7 9 3 IFNα IFNα NK m(c)dc pdc IL-15 IFNα IFNα γδ T 10 2 IFNγ IL-12 Th1 Th2 2/6 1/2 4 CTL IFNγ + Antigens 2 4 RegT 7 4 8 5 3 TLR-(NLR, RLR)- Pathological effects Exaggerated inflammatory responses Septic shock Chronic inflammatory responses Atherosclerosis Transplant rejection Vaccine adjuvant Anti-allergy Anti-cancer Autoimmune diseases 4
Innate immune receptor; TLR, RLR and NLR CR, MR, SR, FcR TLR5 TLR11 NOD1 NOD2 CARD CARDCARD NOD NOD TLR2/1 TLR2/6 Extracellular Danger receptors MyD88 TRIF Intracellular AP Virus sensors Nucleic acid sensors MD-2 TLR4 TRAM Cytoplasm Endosome TLR3 TLR7, 8, 9 IL1/18 Pro-IL1/18 Cleavage NF-κB pro-inflammatory cytokines Caspase-1 IRF3/7 CARD Caspase IFNα, β Nucleus IPS-1 NALP3 IPAF NAIP5 PYD PYD CARD NOD NOD NOD BIRx3 Mitochondria Ishii KJ and Akira S. Innate Immunity Clinical Immunology 3 rd Ed. In press CARD CARDCARD CARDCARD? Helicase Helicase Helicase MyD88 RIG-I MDA5 LGP2 TLR Ulakları Biochem.. J. (2009) 422, 1-101 10 5
Multiple roles for MyD88 MyD88 is a central signal for many processes involving host defense to infection and inflammation and cancer Multiple roles for MyD88: mechanism for induction of EAE Signaling via MyD88 is essential for initiation of EAE and mediates mdc IL-6 and IL-23 production and T cell IL-23R production. MOG-specific Th17 cells are primed, differentiated and maintained in the presence of mdc IL-6 and IL- 23. The regulatory role of TLR4 or TLR9 on IL-17 production may act via downregulation of DC activation, and/or via dampened priming of encephalitogenic Th17 cells responses. Lines with bars represent inhibition 6
Dextran sülfat yollu kolon yaralanmalarında TLR4 e bağlı epitel doku rejenerasyonu Makrofajların, TLR4 ve MyD88 yollu COX2 yi etkinleşmesi sonucu Prostoglandin2 salımı artmaktadır. Bu olgu hücre prolierasyonu için gereklidir. TLR4 ün karaciğer stellat hücrelerini etkinleştirip fibrozisi artırmayı yönlendirme mekanizması 7
BASKILAYICI DNA: Memeli Genomunda Bulunur (TTAGGG)4 G tetrad oluşturabilen telomerik memeli DNA motifleri nanokompleks halde iken TLR antagonisti olarak davranmaktadırlar. Spontan nanoparçacık oluşturan DNA ODN lerin Atomik kuvvet mikroskop görüntüsü DNA nanoparçacığı Ave Size : 30 ± 10 nm 8
ARAŞMA AMAÇLI OLARAK SATILAN NANOPARÇACIK YAPISINDAKİ İMMÜN SİSTEMİ BASKILAYICI DNA (Invivogen, USA) BİZDE DAHA UCUZ!!! DNA BAZLI NANOİLAÇLAR İmmün Sistemi Baskılayıcı DNA motifleri- Immunosuppressive Motifs (Telomerik DNA) Otoimmün hastalıkların tedavisi Artrit Sepsis Lupus Diyabet Behçet Üveit FMF (sistemik oto-enflamatuvar) EAE 9
Baskılayıcı DNA fare de oluşturulan Adjuvant Artriti geriletmektedir PBS Bacteri DNA + Control ODN * * * Bacteri DNA + Sup ODN 0 5 10 15 0 1 2 0 5 10 JOINT HISTOLOGIC TNFa [SI] SWELLING SCORE -2 (mm x 10 ) Zeuner et.al, Arthrit Rheum, 2005 Aynı sonuçlar kollajenle indüklenmiş artrit modelinde de gösterilmiştir (Dong et al, Rheumatology, 2005) A % Survival 100 80 60 40 20 Baskılayıcı DNA Endotoksine Bağlı Septik Şoku Önlemektedir ** PBS Sup ODN Cont ODN 0 0 12 24 36 48 60 72 Hours post LPS challenge (200 μg) B 100 80 60 40 20 0 ** PBS Sup ODN ODN 2010 0 12 24 36 48 60 72 Hours post LPS challenge (200 μg) C % Survival 100 80 60 40 20 0 0 12 24 36 48 60 72 Hours post LPS challenge (100 μg) ** * PBS Sup ODN CpG ODN Shirota et.al,j.immunol, 2005 10
Yeni jenerasyon uyarıcı DNAlar Kendi kendine nanoparçacık oluşturabilen oligoların sentezi Nanoparçacık oluşturma şeması TCGACGTTGG TCGACGTTGG TCGACGTTGG TCGACGTTGG TCGACGTTGG TCGACGTTGG TCGACGTTGG ODN420: 5 -kısmına çift fonksiyonlu linker ekleyerek ikinci jenerasyonun sentezine olanak kılıyoruz. Sentez G=3 de durduruldu. Kontrol ODN (ODN421) CG nin GC ye dönüştürülmüş diziniyle hazırlandı) Patent pending 11
Nanoparçacık oluşturan ODN420 tipinin AKM görüntüsü Ave Size : 45 ± 10 nm (A) topographic (B) phase contrast Scan area is 5 µm x 5 µm Fare dalak hücrelerinin NP ODN420 uyarılmasından sonraki IL-6 salım profili 12
ODN420 induces TNFα secretion and ODN420- PO is a better candidate for type I IFN induction Following 5h incubation w/odns(1um) + Brefeldin A. pdcs are gated for TNFa. Tumor Xenograft Model 5 x 10 6 Huh7 cells, a human hepatocellular carcinoma cell line, is subcutaneously injected into T-cell deficient nude mice. Three doses of 100 µg of ODN-420 or ODN- A151 are injected intratumorally with one day-on and one day-off treatment starting from day 3 post-palpable tumor formation. Tumor dimensions are recorded daily as width, height and length by a caliper and calculated as mm 3. ODN Rx is initiated after observing ~75mm 3 tumor volume. 13
ODN-A151 cannot induce remission of the tumor but only delay the tumor growth (~d=30) Tumor volume in mm3 6000 5000 4000 3000 2000 1000 0 0 10 20 30 40 50 Days No Rx A151 (1) A151 (2) NP-ODN420 uygulaması tümör ilerlemesini durduruyr 6000 Tumor volume in mm3 5000 4000 3000 2000 1000 No Rx 420 (1) 420 (2) 0 0 10 20 30 40 50 Days 14
NP-ODN:anti-tümör immüniteyi başlatıyor 5580 mm3 550 mm3 Untreated @ d=35 NP-420 ODN Treated @ d=42 Tumor size reduction: >80% QUESTION? How can we follow the global gene expression changes mediating this immune activation? The Best Alternative APPROACH : MICROARRAY TECHNOLOGY 15
DNA Microarray: The Process Test sample Reference-commercial source Treatment Isolate RNA Control RNA RNA RT-reaction cdna cdna Label Cy5 Cy3 Mix Two Probes Wash Hybridize Scanning Representative image of the array after scanning 16
CpG DNA specific differentially up-regulated genes, and their common features over time. 2h (n=9) 4h (n=56) 1 3 21 5 29 164 8h (n=198) Explanation of the GO Database Contains 5 hierarchical levels (from global to specific). Example Biological Processes (BP): - Level 1: Physiological processes (GLOBAL) - Level 2: Response to external stimulus - Level 3: Response to biotic stimulus - Level 4: Defense response - Level 5: Immune response (SPECIFIC) Note: level 5 is a subset of level 4, which in turn is a subset of level 3 17
GO annotation chart (604 genes from 8h, CpG vs c-control group) 604 genes extracted at p<0.0001 by BRB (CpG vs c-cont at 8h) 521 genes are annotated by GO and divided into 3 sub-sections 1 2 3 Biological process 349 genes Molecular function 378 genes Cellular components 343 genes Extracting immune response genes by GO analysis (8 h 604 gene set) Biological process (BP) 349 genes Level 1 Physiological process 297 genes Level 2 Level 3 Response to stimulus Response to biotic stimulus 76 genes 62 genes Level 4 Defense response 60 genes Level 5 Immune response 53 genes 18
Fold change in the expression profile of selected gene groups over time Fold (log ratio) 9 8 7 6 5 4 3 2 1 0 1h cell surface signaling 2h receptor binding 4h cytokine activity 8h defense response 1h 2h 4h 8h Tüm Biyoterapötik ODN Lab üyelerine katkılarından dolayı teşekkür ederim 19
TEŞEKKÜRLER 20