CLN2,  CLN3,  CLN6,  Adult,  Unknown  NCL  

Inves7ga7ng  the  molecular  basis  of  NCL:  a  path  to  improved  diagnosis  and  drug  development  

Susan  L.  Cotman,  Ph.D.  (Principal  Inves7gator),  Uma  Chandrachud,  Ph.D.,  Anna-­‐Lena  Hillje,  Ph.D.,  Ursula  Ilo,  M.Sci.,  Abigail  Nowell,  Hyejin  Oh,  Ph.D.,  Center  for  Human  Gene7c  Research,  Department  of  Neurology,  MassachuseQs  General  Hospital,  

Harvard  Medical  School  

Introduc)on  and  Laboratory  Objec)ves  

!   DNA  muta7ons  in  one  of  at  least  13  different  genes  lead  to  the  clinical  symptoms  of  BaQen  disease,  or  NCL  (for  neuronal  ceroid  lipofuscinosis).  In  some  cases,  iden7fying  the  gene7c  cause  of  disease  remains  a  significant  challenge.    

! In  many  forms  of  NCL,  how  the  DNA  muta7ons  lead  to  the  disrupted  cellular  processes  is  not  yet  completely  understood.  It  is  also  s7ll  not  well  understood  which  disrupted  processes  lead  to  the  disease  symptoms.  

!   Understanding  the  steps  in  the  disease  process,  from  gene7c  trigger  (DNA  muta7on)  to  clinical  onset  and  progression,  is  important  for  designing  therapies.    

!   Our  laboratory  uses  gene7c  model  organisms  as  well  as  human  cell  culture  systems  to  formulate  and  test  hypotheses  regarding  the  NCL  disease  process.    

! We  also  par7cipate  in  collabora7ve  efforts  to  improve  the  methods  for  iden7fying  the  DNA  muta7ons  and  to  further  improve  the  availability  of  pa7ent  samples.

 Conclusions  

 ! The  increasingly  well  characterized  disease  models  that  now  exist,  which  recapitulate  NCL  DNA  muta7ons,  are  contribu7ng  to  important  advances  in  our  understanding  of  the  molecular  basis  of  the  NCLs    ! Research  with  these  model  systems  is  leading  to  new  candidate  drug  targets  that  are  currently  being  studied  for  drug  development  

! Screening  of  drug  libraries  is  iden7fying  new  informa7on  and  new  candidate  drugs/drug  targets    ! Our  understanding  of  the  func7ons  of  the  NCL  proteins  is  increasing,  which  will  lead  to  beQer  targeted  therapies  and  biomarker  tools  for  monitoring  treatment    

! New  methods  for  determining  the  underlying  NCL  DNA  muta7ons  are  leading  to  an  increasing  awareness  of  shared  disease  biology  with  other  forms  of  human  disease  and  in  a  greater  apprecia7on  of  how  muta7ons  in  NCL  genes  affect  human  health  more  broadly.  This  knowledge  will  increase  awareness  and  correctly  iden7fy  more  pa7ents  and  the  underlying  genes  causing  their  disease  

! There  is  an  increasing  u7liza7on  of  pa7ent  samples  linked  to  gene7c  and  clinical  informa7on  and  a  greater  effort  to  deepen  this  important  resource      

Acknowledgements: We thank our numerous scientific and clinical collaborators and supporters, as well as the organizations who’ve provided funding to support our research. We would also like to expressly thank the families and patients who’ve donated samples and participated in our research studies. Recent funding sources include the Batten Disease Support and Research Association, the National Institutes of Health: National Institute for Neurological Diseases and Stroke, the MGH Executive Committee on Research, Catherine’s Hope for a Cure, Beyond Batten Disease Foundation and Beat Batten.

Drug  screening  in  a  CLN3  model  iden)fies  a  candidate  target  pathway  for  therapy  development    

   

Facilita)ng  the  gene)c  research  cycle  for  all  forms  of  NCL

   

Conceptualiza)on  of  the  NCL  disease  process  

 Model  systems  we  have  developed  and/or  use  for  NCL  research  

 

Gene7c  Studies  to  Iden7fy  ‘Unknowns’  and  Gene7c  Modifiers  • Next  Genera7on  Sequencing  of  Whole  Exomes/Genomes  • Candidate  Gene  Screening  • Adult  NCL  Gene  Discovery  Consor7um  • Analy7c  and  Transla7onal  Gene7cs  Unit  of  MGH  (Dr.  Mark  Daly,  Dr.  Daniel  MacArthur)  

Mouse  models  and  cell  culture  models      •  Useful  in  iden7fying    possible  early,  pre-­‐

clinical  symptoms  •  Biomarkers  development  •  Improved  descrip7on  of  the  disease  

process  

     Screening  for  drugs  using    mouse  and  human  neuronal  cells  • Unbiased  screen  of  a  large  drug  library    • Collabora7ng  partners  with  other  academic  labs  and  pharmaceu7cal/biotech  companies  to  test  candidate  treatments  

Systems  for  transla7on  of  findings  to  human  pa7ents  Fibroblasts  Lymphoblasts  **Human  induced  pluripotent  stem  cells  (hiPS  cells)—can  be  differen:ated  into  affected  cell  types,  like  neurons  and  glia  

MGH-­‐BaQen  Disease  Center  (Dr.  Kathryn  Swoboda,  Dr.  Winnie  Xin,)  • MGH  Neurogene7cs  DNA  Lab    • NCL  Registry  and  Biorepository  • Collabora7ve  efforts  with  Dr.  Jon  Mink  to  develop  merged,  searchable  clinical  database  linked  to  biorepository  samples  

Drug libraries (e.g. >2000 FDA-approved drugs)

Phenotypic brain cell-based assays

are developed Automated screen performed

Hits identified (e.g. potential CLN3 drugs)

Follow-up studies to validate and optimize leads

~2000 drugs screened

Candidate drugs that improve an abnormality One class of drugs identified as hits targeted certain Ca2+ channels, which prompted follow up studies on how CLN3 neurons handle Ca2+

Disease-modifying drugs; understanding these effects can lead to new information about target pathways

Elevated  lysosomal  Ca2+  in  cultured  CLN3  brain  cells  

Drugs  that  lower  the  elevated  lysosomal  Ca2+  in  cultured  CLN3  brain  cells  to  normal  levels  are  in  further  tes7ng  as  candidate  treatments  (collabora7on  with  other  groups  including  Dr.  Emyr  Lloyd-­‐Evans)

Cln3∆ex7/8 knock-in mice • Genetic replica of the ~1-kb

deletion mutation most frequently observed in CLN3

patients • Cln6nclf mice

CbCln3∆ex7/8 and CbCln6nclf mouse

neuronal precursor cells

Patient fibroblasts and reprogrammed human induced

pluripotent stem (hiPS) cells Can be turned into brain cells and

other relevant cell types

• Phenotyping (characterizing abnormalities at

the cellular and whole organism level)

• Disease modifier studies (cell-based screening and mouse

modifier studies)

• Molecular analysis (single gene and genomic level)

Potential modifiers: Mitochondrial pathways Intracellular Ca2+

Autophagy pathway modifiers

êAutophagy clearance êendocytosis êlysosomal protein trafficking Mitochondrial changes

Subunit c storage

Sensorimotor processing affected

Gliosis Motor function decline

Working chronology of the disease process in NCL genetic models

cln3 knockout Dictyostelium discoideum

•  Social amoeba, single cell stage to multicellular stage

developmental life cycle •  Expression of human

CLN3 in the cln3- Dicty cells rescues abnormalities

demonstrating conserved function across evolution

Conception NCL gene status

Lifeline of a person with two NCL mutations Clinical Diagnosis

End-stage disease

Conception NCL gene status

End-of-life

Lifeline of a person with at least one normal NCL gene

•  Different genetic or environmental modifiers could act at different stages and affect the progression towards end-stage disease.

•  Identifying these factors and then targeting them through

interventions/drugs (blue arrows) could slow or halt further advancement of disease progression.

CLN3