2016 BDSRA Cotman, Chandrachud, Hillje, Ilo, Nowell, Oh CLN2, CLN3, CLN6, Adult NCL
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Transcript of 2016 BDSRA Cotman, Chandrachud, Hillje, Ilo, Nowell, Oh CLN2, CLN3, CLN6, Adult NCL
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