GENE SILENCING USING
POLYPURINE REVERSE
HOOGSTEEN HAIRPINS
Carles J. Ciudad, Laura Rodríguez, Xenia Villalobos, Núria
Menc...
 
	
  
	
  
•  Double-­‐stranded	
  DNA	
  molecule:	
  
	
  
	
  
–  Reverse	
  Hoogsteen	
  bonds	
  between	
  an9paral...
INTRODUCTION	
  
PPRHS	
  
Binding	
  of	
  PPRH	
  causes	
  strand	
  displacement	
  
Watson-­‐Crick	
  bond	
   Revers...
•  Types:	
  
	
  	
  	
   	
  	
   	
   	
  Template-­‐PPRH 	
   	
  	
  	
  	
  	
  	
  	
  	
  	
   	
   	
  	
  	
   	...
1. Comparison	
   Coding-­‐	
   and	
   Template-­‐PPRHs	
   in	
   different	
   cell	
  
lines	
   in	
   terms	
   of	
 ...
•  Intracellular	
   protein	
   of	
   16.5-­‐
kDa	
  	
  
•  Belongs	
   to	
   IAP	
   family	
  
(inhibitor	
  of	
  a...
DISEÑO	
  PPRHs	
  
Survivin.	
  Survivin	
  gene	
  structure	
  and	
  localiza9on	
  of	
  designed	
  PPRHs	
  (arrows...
INTRODUCCIÓN	
  1.	
  CODING	
  VERSUS	
  TEMPLATE	
  
VIABILITY	
  
Most	
  effecDve	
  concentraDon	
  	
  
100	
  nM	
  ...
1.	
  CODING	
  VERSUS	
  TEMPLATE	
  
mRNA	
  and	
  protein	
  LEVELS	
  
Both	
  Template	
  and	
  Coding-­‐PPRHs	
  a...
INTRODUCCIÓN	
  1.	
  CODING	
  VERSUS	
  TEMPLATE	
  
APOPTOSIS	
  
ApoptoDc	
   assays.	
   Flow	
   cytometry	
   by	
 ...
1.  CODING	
  VERSUS	
  TEMPLATE	
  
NON-­‐TUMORAL	
  CELLS	
  
Survivin	
  mRNA	
  levels	
  in	
  HUVEC	
  	
  
(rela9ve...
2.	
  IN	
  VIVO	
  ASSAYS	
  
Intratumoral	
  versus	
  Intravenous	
  	
  administraDon	
  
Efficacy	
  Assay.	
  Administ...
3.	
  PROPERTIES:	
  IMMUNOGENICITY	
  siRNA	
  vs	
  PPRH	
  
Transcriptional induction of pro-inflammatory genes Inflamm...
RNA	
  
	
  
TLR-­‐3/7/8 	
  RIG1,	
  PKR	
  
	
  
	
  
DNA	
  
	
  
TLR-­‐9	
  	
  	
  	
  	
  DAI,	
  IFI16,	
  AIM2	
  ...
0.0	
  
0.5	
  
1.0	
  
1.5	
  
2.0	
  
CNT	
   PPRH	
   siRNA	
  
	
  Protein	
  levels	
  	
  
(relaDve	
  to	
  control...
0	
  
5	
  
10	
  
15	
  
20	
  
25	
  
30	
  
35	
  
40	
  
45	
  
50	
  
100	
  
nM	
  
100	
  
nM	
  
CNT	
   DTP	
   P...
siRNA	
  induces	
  Caspase-­‐1	
  cleavage	
  
and	
  IL-­‐1β	
  acDvaDon	
  
Caspase-­‐1	
  proteolyDc	
  acDvity.	
  De...
3.	
  STABILITY:	
  siRNA	
  vs	
  PPRH	
  
y	
  =	
  100e-­‐6E-­‐04x	
  
y	
  =	
  100e-­‐0.004x	
  
10	
  
100	
  
0	
  ...
3.	
  STABILITY:	
  siRNA	
  vs	
  PPRH	
  
PPRHs	
  are	
  more	
  stable	
  than	
  siRNAs	
  in	
  fetal,	
  mouse,	
  ...
CONCLUSIONS	
  
	
  
1.  Coding-­‐PPRHs	
   against	
   an9-­‐
apopto9c	
  genes	
  decrease	
  viability,	
  
at	
  least...
AGRADECIMIENTOS	
  
	
  
ACKNOWLEDGEMENTS	
  
	
  
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Gene Silencing using Polypurine Reverse Hoogsteen Hairpins

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Seminar led by Carles Ciudad, PhD

Recently, we developed an alternative type of molecules to decrease gene expression named Polypurine Reverse-Hoogsteen Hairpin (PPRH). PPRHs are DNA molecules formed by two antiparallel polypurine strands linked by a pentathymidine loop that allows the formation of intramolecularHoogsteen bonds between both strands. These hairpins bind polypyrimidine targets in the DNA via Watson-Crick bonds. Concretely, there are two types of PPRHs capable of decreasing gene expression, that differ in the location of the target sequence and their mechanism of action: Template-PPRHs, which bind to the template strand of the dsDNA (de Almagro et al., 2009), and Coding-PPRHs (de Almagro et al., 2011), which bind both to the template strand of the dsDNA and the mRNA. We analyzed important properties- stability and immunogenicity- of these molecules for their potential therapeutic approach. Stability experiments performed in different types of serum (human and murine) and in human prostate cells (PC3) revealed that PPRHs half-life is much longer than that of siRNAs, its main competitor. The activation of the innate immune response was evaluated analyzing the levels of the transcription factor IRF3, the cleavage of the proteolytic enzyme Caspase-1, and the expression levels of several pro-inflammatory cytokines: type-I interferons, TNFa, IL-6, IL-8, IL-1b, IL-18 and IL-33. These determinations indicate that PPRHs do not activate the immune response, unlike siRNAs, and therefore are suitable for in vivo administration. In this regard, we decided to further explore the in vitro and in vivo effect of PPRHs in cancer, choosing survivin as a target for its implication in apoptosis, mitosis and angiogenesis, and its overexpression in different tumors. We designed and tested several PPRHs against survivin. After an in vitro screening, including cytotoxicity, apoptosis, mRNA and protein levels, we chose the most effective one for in vivo studies. We conducted two types of administration, namely intratumoral and intravenous, in a xenografted model of prostate cancer cells (PC3). The results showed that the chosen Coding-PPRH proved to be effective in decreasing tumor volume and weight. These findings represent the proof of principle of PPRHs as a new silencing tool for cancer gene therapy.

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Gene Silencing using Polypurine Reverse Hoogsteen Hairpins

  1. 1. GENE SILENCING USING POLYPURINE REVERSE HOOGSTEEN HAIRPINS Carles J. Ciudad, Laura Rodríguez, Xenia Villalobos, Núria Mencia, Jeanne Prévot, Carlota Oleaga and Veronique Noé Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona
  2. 2.       •  Double-­‐stranded  DNA  molecule:       –  Reverse  Hoogsteen  bonds  between  an9parallel  purine  strands   –  Linked  by  5-­‐T  loop   –  Watson-­‐Crick  with  genomic  DNA   –  pH-­‐independent,  Salts  required   INTRODUCTION   PPRHs   PPRHs  =  PolyPurine  Reverse-­‐Hoogsteen  Hairpins   r-­‐H   r-­‐H   WC   WC  
  3. 3. INTRODUCTION   PPRHS   Binding  of  PPRH  causes  strand  displacement   Watson-­‐Crick  bond   Reverse-­‐Hoogsteen  bond   Coma et al. OLIGONUCLEOTIDES (2005) WC   WC   Decrease  in  gene  expression  
  4. 4. •  Types:                Template-­‐PPRH                                          Coding-­‐PPRH     INTRODUCTION   PPRHS   Watson-­‐Crick  bond   Reverse-­‐Hoogsteen  bond   5’     3’   3’   5’   3 ’   5 ’   5’   3’  mRNA   Ribosoma   Protein   3’   5’   3’     5’   5’     3’   3’     5’   De Almagro et al. THE JOURNAL OF BIOLOGICAL CHEMISTRY (2009) De Almagro et al. HUMAN GENE THERAPY (2011) Splicing  alteraDon   InhibiDon  of  transcripDon   New  gene  silencing  tool  
  5. 5. 1. Comparison   Coding-­‐   and   Template-­‐PPRHs   in   different   cell   lines   in   terms   of   decrease   in   viability,   mRNA   levels   and   apoptosis:   –  MiaPaCa  2  à  Pancrea9c  cancer   –  PC3  à  Prostate  cancer   –  HCT116  à  Colon  cancer   –  HUVEC  à  normal  cells         2.  In  vivo  administra9on  of  PPRHs:  Proof  of  principle         3.  PPRH’s  Proper9es:   –  Immunogenicity   –  Stability   GOALS    
  6. 6. •  Intracellular   protein   of   16.5-­‐ kDa     •  Belongs   to   IAP   family   (inhibitor  of  apoptosis)     •  Involved  in:   –  Cellular  division     –  Apoptosis  supression   –  Angiogenesis   –  Chemoresistance   INTRODUCCIÓN  1.  CODING  VERSUS  TEMPLATE   SURVIVIN   Altieri D.C. NATURE REVIEWS CANCER (2003; 2007) GOOD  TARGET   Human  survivin   structure   (1XOX)   Apopto9c  pathways   •  Overexpressed   in   cancer   cells,   undetectable  in  normal  9ssue  
  7. 7. DISEÑO  PPRHs   Survivin.  Survivin  gene  structure  and  localiza9on  of  designed  PPRHs  (arrows).   INTRODUCCIÓN  1.  CODING  VERSUS  TEMPLATE   PPRHs  DESIGN   NegaDve  controls.  Hps-­‐WC  has  intramolecular  Watson-­‐Crick  bonds  instead  of  reverse-­‐Hoogsteen   bonds.  Hps-­‐Sc  has  a  randon  polypurine  sequence  without  target  in  the  human  genome.      
  8. 8. INTRODUCCIÓN  1.  CODING  VERSUS  TEMPLATE   VIABILITY   Most  effecDve  concentraDon     100  nM     ≈  range  siRNA     HpsPr-­‐B  and  HpsPr-­‐C  efficient  in  all   lines   Viability  assays.  Comparison  between  coding-­‐  and  template-­‐PPRHs  designed  against   survivin  gene  in  three  different  cell  lines  :  PC3  (prostate  cancer),  MiaPaCa  2  (pancrea9c   cancer)  and  HCT116  (colon  cancer).    
  9. 9. 1.  CODING  VERSUS  TEMPLATE   mRNA  and  protein  LEVELS   Both  Template  and  Coding-­‐PPRHs  against  the  promoter  sequence  of  the   survivin  gene  decrease  mRNA  and  protein  levels  of  the  targeted  gene   mRNA  levels.  qRT-­‐PCR  of  survivin  levels  of   PC3   when   transfected   with   increasing   doses  of  HpsPr-­‐B  and  HpsPr-­‐C.       0.0   0.2   0.4   0.6   0.8   1.0   1.2   CONTROL   30   100   300   100   100   Hps-­‐SC    Hps-­‐WC     Survivin  mRNA  levels      (relaDve  to  CONTROL)   PPRHs  (nM)   HpsPr-­‐B   HpsPr-­‐C   0   20   40   60   80   100   Survivin  protein  levels     (relaDve  to  CONTROL)   PPRHs  (100nM)   HpsPr-­‐B   HpsPr-­‐C   Protein   levels.   WB   of   survivin   levels   of  PC3  when  transfected  with  100nM   of  HpsPr-­‐B  and  HpsPr-­‐C.      
  10. 10. INTRODUCCIÓN  1.  CODING  VERSUS  TEMPLATE   APOPTOSIS   ApoptoDc   assays.   Flow   cytometry   by   Rhodamine   method   or   Caspase-­‐3/7   Assay.   Comparison   between   coding-­‐   and   template-­‐PPRHs   against   survivin   in   3   different   cell   lines  :  PC3  (prostate  cancer),  MiaPaCa  2  (pancrea9c  cancer)  and  HCT116  (colon  cancer).     Coding-­‐PPRHs  cause  more  apoptosis  than  Template-­‐PPRHs  at  24h     0.8   0.9   1   1.1   1.2   1.3   1.4   1.5   1.6   CONTROL   DOTAP   HpsPr-­‐B   HpsPr-­‐C   HpsE3-­‐C   HpsI1-­‐C   Hps-­‐WC   Hps-­‐Sc   %  apoptosis    (relaDve  to  CONTROL)   PPRHs  (100nM)   Caspase-­‐3  acDvaDon  in  PC3  when  transfected  with   PPRHs  against  survivin  gene       0   10   20   30   40   50   60   70   CONTROL   DOTAP   HpsPr-­‐B   HpsPr-­‐C   HpsE3-­‐C   HpsI1-­‐C   HpsPr-­‐Sc   HpsPr-­‐WC   %  apoptoDc  cells   PPRHs  (100  nM)   Apoptosis  when  transfected  with  PPRHs  against   survivin   HCT116   MiaPaCa  2   PC3  
  11. 11. 1.  CODING  VERSUS  TEMPLATE   NON-­‐TUMORAL  CELLS   Survivin  mRNA  levels  in  HUVEC     (rela9ve  to  PC3  levels)   0.0   0.2   0.4   0.6   0.8   1.0   1.2   PC3   HUVEC   Survivin  mRNA  levels    (relaDve  to  PC3)   Cell  line   Survivin   19  KDa     AcDn    42  Kda                      PC3                      HUVEC   0" 20" 40" 60" 80" 100" 120" 140" DOTAP" HpsPr1B" HpsPr1C" Hps1Sc" %"viability" "(rela-ve"to"DOTAP)" PPRHs"(100nM)" Survivin  protein  levels  in  HUVEC   (rela9ve  to  PC3  levels)     Viability  assays.  Comparison   between  HpsPr-­‐B  and   HpsPr-­‐C  in  HUVEC The  most  cytotoxic  PPRHs  (HpsPr-­‐B  and  HpsPr-­‐C)  DO  NOT  cause   decrease  in  viability  in  HUVEC,  which  DO  NOT  express  survivin  
  12. 12. 2.  IN  VIVO  ASSAYS   Intratumoral  versus  Intravenous    administraDon   Efficacy  Assay.  Administra9on  of  HpsPr-­‐C  to  animals  with  a  xenograced   tumor  of  prostate  cancer  (PC3).  Tumor  volume  is  represented.     A.  Intratumoral  administra9on      (10µg/animal  twice  a  week)     B.  Intravenous  administra9on              (50µg/animal  twice  a  week)     Intratumoral  or  intravenous  of  the  Coding-­‐PPRH  against  survivin   administered    induces  a  significant  anD-­‐tumor  effect  without  effect  in   animal  body  weight  loss  
  13. 13. 3.  PROPERTIES:  IMMUNOGENICITY  siRNA  vs  PPRH   Transcriptional induction of pro-inflammatory genes Inflammasome-dependent caspase-1 activation dsRNA ssRNA CpG DNA Cytoplasm Adapted from Atianand MK, Fitzgerald KA. J Immunol. 2013
  14. 14. RNA     TLR-­‐3/7/8  RIG1,  PKR       DNA     TLR-­‐9          DAI,  IFI16,  AIM2     3.  PROPERTIES:  IMMUNOGENICITY   siRNA  vs  PPRH   IFN-­‐α,  TNF-­‐α,  IL-­‐6   IFN-­‐β,  IL-­‐6,  IL-­‐8   IFN  and   proinflammatory   cytokines       Inflammasome     ê   Caspase-­‐1     ê   IL-­‐1β,  IL-­‐18   IFN-­‐α,  IFN-­‐β,  TNF-­‐α   and  IL-­‐6   IFN-­‐β   Robbins et al. OLIGONUCLEOTIDES (2009) Barker B.R. et al. CURRENT OPINION IN IMMUNOLOGY (2011) Choubey D. CLINICAL IMMUNOLOGY (2012)
  15. 15. 0.0   0.5   1.0   1.5   2.0   CNT   PPRH   siRNA    Protein  levels     (relaDve  to  control)   NF-­‐kB  protein  levels  a)   b)   0   2   4   6   8   10   12   14   16   18   CNT   PPRH   sIRNA   Protein  levels     (relaDve  to  control)   IRF3  protein  levels   IRF3   Tubulin   NF-­‐kB   3.  PROPERTIES:  IMMUNOGENICITY  siRNA  vs  PPRH   siRNA  induces  an  increase  in  NF-­‐κβ  and  IRF3  
  16. 16. 0   5   10   15   20   25   30   35   40   45   50   100   nM   100   nM   CNT   DTP   PPRH   MTF   siRNA   LPS   mRNA  levels     (relaDve  to  control)   IFN-­‐ß  mRNA  levels  in  THP-­‐1  cells   0   0.5   1   1.5   2   2.5   100   nM   100   nM   CNT   DTP   PPRH   MTF   siRNA   LPS   mRNA  levels     (relaDve  to  control)   IFN-­‐α    mRNA  levels  in  THP-­‐1  cells   0   1   2   3   4   5   6   100   nM   100   nM   CNT   DTP   PPRH   MTF   siRNA   LPS   mRNA  levels     (relaDve  to  control)   IL-­‐6  mRNA  levels  in  THP-­‐1  cells   3.  PROPERTIES:  IMMUNOGENICITY  siRNA  vs  PPRH   siRNA  induces  an   increase  in  IL-­‐6,   TNF-­‐α  and  IFN-­‐β   levels   0   5   10   15   20   25   100   nM   100   nM   CNT   DTP   PPRH   MTF   siRNA   LPS   mRNA  levels      (relaDve  to  control)     TNF-­‐α  mRNA  levels  in  THP-­‐1  cells  
  17. 17. siRNA  induces  Caspase-­‐1  cleavage   and  IL-­‐1β  acDvaDon   Caspase-­‐1  proteolyDc  acDvity.  Determina9on  by  luciferase  assay.     3.  PROPERTIES:  IMMUNOGENICITY  siRNA  vs  PPRH   0   1   2   3   4   5   6   CNT   DTP   PPRH   MET  (1,5)   siRNA   (1,5)   LPS/ATP   F12   Caspase-­‐1  proteolyDc  acDvity     (relaDve  to  control)   Supernatant  
  18. 18. 3.  STABILITY:  siRNA  vs  PPRH   y  =  100e-­‐6E-­‐04x   y  =  100e-­‐0.004x   10   100   0   100   200   300   400   %  of  INPUT     IncubaDon  Dme  (min)   F-­‐PPRH  vs  F-­‐siRNA  stability  in  mouse  serum   y  =  100e-­‐4E-­‐04x   y  =  100e-­‐0.003x   10   100   0   100   200   300   400    %  of  INPUT   IncubaDon  Dme  (min)   F-­‐PPRH  vs  F-­‐siRNA  stability  in  human  serum   y  =  100e-­‐0.001x   y  =  100e-­‐0.011x   1   10   100   0   100   200   300   400   %  of  INPUT     IncubaDon  Dme  (min)   F-­‐PPRH  vs  F-­‐siRNA  stability  in  FCS  100%   y  =  100e-­‐0.01x   y  =  100e-­‐0.023x   10   100   0   20   40   60   80   Fluorescence  intensity     (%  relaDve  to  t  =  24h)   Decay  Dme  (h)   F-­‐PPRH  vs  F-­‐siRNA  stability  in  PC3  cells  
  19. 19. 3.  STABILITY:  siRNA  vs  PPRH   PPRHs  are  more  stable  than  siRNAs  in  fetal,  mouse,  human  serum  and  in  PC3  cells  
  20. 20. CONCLUSIONS     1.  Coding-­‐PPRHs   against   an9-­‐ apopto9c  genes  decrease  viability,   at  least,  as  efficiently  as  Template-­‐ PPRHs.   2.  Coding-­‐PPRHs   cause   a   higher   apopto9c   effect   than   Template-­‐ PPRHs  at  24h     3.  Administra9on   of   PPRHs   in   xenograced  tumors  is  effec9ve.   4.  PPRHs  are  less  immunogenic  than   siRNAs  in  THP-­‐1  cells.   5.  PPRHs  are  much  more  stable  than   siRNAs  in  FCS,  mouse  and  human   serum  and  inside  the  cells.       ü  Effec9ve  in  different  cell  lines   ü  Effec9ve  in  xenograced  tumors       ü  Low  immunogenicity     ü  High  stability  
  21. 21. AGRADECIMIENTOS     ACKNOWLEDGEMENTS    

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