Future  directions

The   administration   of   miR-­‐30   in   vivo   will   provide   significantly   deeper   insight   into   the   functions  of  the  described  target  genes  in  relation  to  DOX  injury.  Therefore,  the  imperative   study  in  our  future  work  section  is  the  establishment  of  a  relevant  model  to  prove  the  miR-­‐

30  mechanisms  discovered  in  vitro  and  return  to  the  original  in  vivo  setting  of  the  project.  

This   would   complete   the   full   circle   of   this   research   and   allow   us   to   draw   meaningful   conclusions  about  a  possible  translational  use  for  miR-­‐30.    

The   experimental   design   for   the   planned   in   vivo   model   comprises   pre-­‐treatment   with   our   rAAV  (AAV9.miR30e),  followed  by  the  reproduction  of  the  existing  DOX-­‐induced  HF  model   (Figure  61).  The  rationale  behind  the  order  of  the  treatment  combination  relies  on  the  start   of  chemotherapy  being  one  of  few  clinical  scenarios  where  the  date  of  initial  exposure  to  the   toxic  agent  is  known.  Also,  this  schedule  allows  sufficient  time  for  the  therapeutic  transgene   to  be  expressed  prior  to  the  insult.  The  high  toxicity  of  the  DOX  model  is  also  an  important   factor  that  contributed  to  the  timeline  design.  DOX-­‐induced  cardiotoxicity  develops  steadily   after  cumulative  dosing  and  once  the  heart  becomes  dysfunctional  the  window  to  examine   animals  prior  to  sudden  death  is  extremely  narrow.    

Figure  61.  Experimental  design  for  the  AAV9.miR30e/DOX  model.    

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The   phenotypic   characterization   at   the   endpoint   will   entail   echocardiography   to   assess   cardiac   function   across   treatment   groups,   histochemistry   to   check   cell   morphology   and   arrangement,  molecular  studies  (PCR  and  Western  Blot)  for  miRNA  and  target  levels,  as  well   as  cardiomyocyte  contractility  measures  ex  vivo.  Having  already  depicted  the  effects  of  high   miR-­‐30  expression  on  contractility  using  transfected  ARVCM,  it  is  intriguing  to  resolve  how   this   would   translate   to   AAV.miR30e-­‐treated   animals.   However,   considering   that   the   gene   transfer   will   be   performed   in   combination   with   DOX,   we   expect   opposing   effects   on   contractility   in   vivo   to   counterbalance   each   other.   Bearing   in   mind   the   recently   proposed   activity  of  miRNAs  as  paracrine/endocrine  signals,  as  well  as  their  potential  as  non-­‐invasive   biomarkers,   it   would   also   be   interesting   to   measure   circulating   miR-­‐30e.   In   addition,   measures   of   other   serum   biomarkers   that   are   currently   used   to   monitor   cardiac   function   (BNP,  TpnI)  could  be  incorporated  into  the  study  too.    

The  described  AAV9.miR30e+DOX  model  is  a  challenging  one,  with  numerous  variables  to  try   to  control.  Apart  from  the  on-­‐going  optimisation  of  the  gene  therapy,  troubleshooting  is  also   in  progress  with  reference  to  the  DOX-­‐induced  HF  model.  Even  though  the  total  DOX  dose   used   in   this   project   (15mg/kg)   is   widely   used   and   has   long   been   established   -­‐and   even   increased   up   to   18   or   20mg/kg-­‐  154,   319,   321,   421,   animal   welfare   issues   were   raised   deriving   from   the   high   toxicity   of   this   15mg/kg   model.   Admittedly,   high   mortality   rates   have   been   reported  by  other  authors  researching  the  DOX-­‐induced  cardiotoxicity  model,  ranging  from   30-­‐60%   when   applying   15mg/kg   cumulative   doses   or   higher  422,   423.   This   is   particularly   relevant  in  our  case,  since  a  second  potential  source  of  discomfort  (recovery  surgery  for  AAV   administration)  will  be  incorporated.  Aiming  to  mitigate  this  problem,  we  generated  another   cohort   where   animals   were   treated   with   a   cumulative   DOX   dose   of   10mg/kg.   After   four   weeks,   no   reduction   in   LVEF   was   observed   (not   shown)   and   miR-­‐30e   levels   were   not   dysregulated   (Figure   64).   As   a   result,   we   are   currently   investigating   an   intermediate   12.5mg/kg  dose,  as  it  has  been  proved  to  be  sufficient  to  trigger  cardiac  dysfunction  424,  425.     Eventually,  we  believe  that  it  would  be  interesting  to  combine  a  xenograft  model  with  the   evaluation   of   CO.   Given   the   present   and   published   evidence   showing   the   anti-­‐tumour   activity   of   miR-­‐30   and   the   dual   benefits   of   β-­‐blockers   in   a   cardioncology   context,   administration  of  both  DOX  and  miR-­‐30  mimics  could  potentially  have  synergic  anti-­‐cancer  

protocol   to   immunocompromised   mice,   and   also   an   alternative   formulation   for   miR-­‐30   delivery   that   is   not   exclusively   cardiotropic   in   order   to   target   the   tumour   as   well.   Still,   it   would   provide   the   most   accurate   indication   of   the   postulated   dual   benefits   of   miR-­‐30   achievable  in  a  pre-­‐clinical  setting.    

4.4 Conclusions  

This   research   has   unveiled   novel   mechanisms   that   seem   to   play   vital   roles   in   the   adverse   cardiac   effects   of   DOX.   The   discovery   of   aberrant   miR-­‐30   expression   in   the   myocardium   upon  DOX  treatment  has  proven  to  be  biologically  relevant,  given  its  sustained  alteration  in   a  model  of  late  stage  HF  as  well  as  its  demonstrated  impacts  on  the  targets  validated  here.    

Overall,  the  present  investigation  underlines  a  miR-­‐30-­‐induced  dampened  response  to  βAR   stimulation   and   a   protective   effect   against   DOX   insult   in   cardiomyocytes.   From   a   translational  perspective,  the  β-­‐blocker  activity  of  miR-­‐30  would  be  beneficial  as  it  replicates   the  effects  of  common  pharmacological  therapy  routinely  given  to  HF  patients.  The  fact  that   miR-­‐30   acts   as   a   β-­‐adrenergic   antagonist   is   relevant   beyond   DOX   cardiomyopathy,   considering  the  widespread  application  of  β-­‐blockers  in  cardiovascular  disease.  On  the  other   hand,  high  miR-­‐30  expression  seems  to  correlate  with  less  aggressive  tumours,  supporting  a   dual  therapeutic  use  for  miR-­‐30.  Importantly,  in  keeping  with  our  results,  published  research   indicates   anti-­‐cancer   effects   for   β-­‐blockers,   further   predicting   a   two-­‐pronged   therapeutic   role  for  miR-­‐30.  

Harnessing  miRNA  biology  could  bring  huge  benefits  to  the  clinic,  both  for  the  development   of   biomarker   assays   and   as   part   of   therapeutic   approaches.   The   findings   described   in   this   project  highlight  the  power  of  miRNA  as  master  gene  expression  regulators  and  propose  yet   another   scenario   in   the   growing   list   of   uses   of   miRNA   manipulation   in   disease.   From   a   holistic  or  systems  biology  point  of  view,  the  data  obtained  in  this  thesis  interestingly  links   cardiac  disease  and  cancer  biology  through  regulation  by  common  miRNAs.  Ultimately,  we   believe  that  the  improvement  of  DOX-­‐based  regimes  is  likely  to  come  from  two  directions:  

optimisation   of   the   drug   formulation   itself   in   conjunction   with   complementary   cardioprotective  strategies.  We  foresee  the  use  of  appropriately  formulated  miR-­‐30  mimics   as   an   attractive   option   for   adjuvant   treatment   to   achieve   the   second   goal,   as   well   as   pleiotropically  contributing  to  an  impairment  of  cancer  progression.  

5 APPENDICES