APOPTOTIC DEFECTS IN NCL

1. Use of the oral antiapoptotic agent Flupirtine as a treatment strategy in human INCL, LINCL, JNCL and CLN6-deficientvLINCL

2. APOPTOTIC DEFECTS IN NCL • Anti-apoptotic effects of intact CLN3 is firmly established in humans: 1. By EM and TUNEL of human JNCL brain (Lane et al,. 96). 2. By JC-1, propidium iodide, TUNEL and LDH release of CLN3-deficient human neurons (Dhar et al., 2002). 3. Resistance of stable CLN3-overexpressing NT2 cells to apoptotic death resulting from serum starvation and chemotherapeutic agents. Inhibition of etoposide–induced PARP cleavage, and therefore caspase-3 activation in these cells (Puranam et al,. 99). 4. Increased apoptotic death of CLN3-deficient, naturally occurring patient derived fibroblasts and lymphoblasts resulting in net slower growth (Persaud-Sawin et al, 02). 5. Upregulation of CLN3 protein and RNA in a number of human cancer cell lines and solid colon cancer, as well as mouse breast cancer cells. Inhibition of cancer cell growth and initiation of apoptosis after blunting of CLN3 protein expression using antisense strategies (Rylova et al, 02). 6. Indirect evidence: developmental regulation of CLN3, upregulation of Bcl-2 in surviving neurons by immunocytochemistry, Western blot, RT-PCR and Affymetrix Genechip analysis. Upregulation of many other neuroprotective genes by Affy Genechip analysis of human, CLN3-deficient fibroblasts (Pane et al, 99; Puranam et al, 97, unpublished). 7. Increased ceramide in JNCL brain and JNCL fibroblasts and downregulation of ceramide levels in stable CLN3 overexpressing cells (Puranam et al, 97, Rylova et al, 02). 8. Recognition of anti-apoptotic motifs within the CLN3 protein (Persaud-Sawin et al, 02).

3. APOPTOTIC DEFECTS IN NCL • Established in LINCL: 1. By EM and TUNEL of human LINCL brain (Lane et al, 96). 2. By JC-1, propidium iodide and TUNEL and LDH release of CLN2-deficient human neurons (Dhar et al, 02). 3. Resistance of stable CLN2-overexpressing NT2 cells to proapoptotic insults such as vincristine, staurosporine and etoposide (ibid). 4. Increased apoptotic death of CLN2-deficient, naturally occurring patient derived fibroblasts and lymphoblasts resulting in net slower growth (ibid). 5. Restoration of growth by transfection of CLN2-deficient cells with a CLN2 bearing plasmid.

4. Apoptosis in NCL • In INCL: • Blocking of CLN1 expression results in apoptosis, and CLN1-overexpression protects from ceramide-induced cell death (Glyn Dawson’s group) • Slowed growth of CLN1-deficient patient lymphoblasts and fibroblasts (unpublished) • Growth restored by transfection of CLN1-deficient fibroblasts with a CLN1-bearing plasmid (unpublished).

6. APOPTOSIS IN NCL • In CLN6 deficient vLINCL: • By EM and TUNEL of affected human brain (Lane et al. 96). • Increased ceramide in vLINCL CLN6-deficient human brain and fibroblasts. • By flow cytometry (sub-diploid peak), DNA fragmentation by gel electrophoresis, TUNEL staining of affected sheep brain and retina observed at 14 months of age (provided by Bob Jolly, Lane et al,).

7. 8 200 V2 S1A2 160 S1C4 6 120 4 cell number * 10,000 of CLN2 RNA 80 Fold increase in expression 40 2 0 0 0 20 40 60 80 V2 S1A1 S1A2 S1C4 Time (hours ) 100 100 no etoposide no etoposide plus etoposide plus etoposide 80 80 60 60 40 40 cell number * 10,000 cell number *10,000 20 20 0 0 Control-AAV2 Sense-CLN2-AAV2 Control-AAV2 AS-CLN2-AAV2 Overexpression of CLN2 causes resistance to apoptosis

8. JC-1 Staining d V2 without etoposide V2 with etoposide S1C4 without etoposide S1C4 with etoposide

9. e PI Staining V2 without etoposide V2 with etoposide S1C4 without etoposide S1C4 with etoposide

10. Transduction of post-mitotic human neurons with AS-CLN2 causes spontaneous neuronal apoptotic death and LDH release Control-AAV2 AS-CLN2 AAV2

11. Transduction of NT2 precursor cells with antisense CLN3 adenovirus causes cell death and ↑LDH release

12. Transduction of hNT neurons with AS-Ad-CLN3 results in spontaneous apoptotic death by TUNEL staining Ad-control (without etoposide) Ad-control (with etoposide) Ad-AS-CLN3 (without etoposide) Ad-AS-CLN3 (with etoposide)

13. Flupirtine • It is a triaminopyridine analogue. • It was developed and is used as a non-opioid centrally acting analgesic. • It has powerful muscle-relaxant properties not mediated through the benzodiazepine receptor (ideal for treatment of back pain with spasms, orthopedic and cancer pain). • It has been shown to protect postmitotic neurons and photoreceptors from apoptotic death induced by a variety of insults: NMDA, amyloid, HIV and prionic proteins. • It has been shown to reduce the area of infarction in rats, and to improve their motor function following carotid artery ligation. • It has been shown to elevate Bcl-2 levels 5-7 fold, and glutathione levels. • There is an indirect functional antagonism to NMDA-induced cell death. • It activates a G-regulated, inwardly rectifying K+ channel. • It stabilizes retention of Ca++ at the mitochondrial membrane level.

14. Beneficial Clinical Characteristics of Flupirtine • Can be given orally/per gastrostomy tube • Proven safety after prolonged use in adults and children (for periods > 2years). Has been in use for 18 years. • *Pain-killer, less sedating than opioids. • * Muscle relaxant properties. • Centrally acting (ideal, as this is where you want it to act). • * Weak anti-convulsant action, and very little interaction with other anticonvulsants. • Cheap. • *Pain, spasticity, and seizures are key target symptoms in NCL

15. Normal Control m n o

16. Flupirtine Protects CLN1-deficient lymphoblasts from apoptosis b a c

17. Flupirtine protects CLN2-deficient lymphoblasts from etoposide-induced apoptosis d e f

18. Flupirtineprotects CLN3-deficient lymphoblasts from etoposide induced apoptosis g h i

19. Flupirtine protects CLN6-deficient lymphoblasts from etoposide-induced apoptosis j k l

20. Flupirtine protects healthy hNT neurons from etoposide induced apoptosis b a c

21. Flupirtine protects hNT neurons transduced with AS-CLN2-AAV2 from apoptosis g h + Flup Flupirtine protects hNT neurons transduced with Ad-AS-CLN3 from apoptosis i j i + Flup

23. Flupirtine • In Vitro Experimentation: Done • Pre-IND Application and FDA meeting : Done • Phase I/II Pilot Clinical Trial in LINCL NIH application to be resubmitted. • Why LINCL first? Can determine within shorter period than for JNCL whether effective. INCL fewer patients, and other oral therapies available. • Once FDA approved can be used off-label for other indications. • IND Application: To be submitted as soon as grant resubmitted. • COLLABORATORS: VIATRIS (drug); GENZYME (regulatory support); BDSRA (financial and logistical support); NINDS: G. Spinella, Dan Tagle and D. Hirtz (advice on design and strategy); DCRI (statistics and clinical trial expertise), A. Kohlschütter (clinical endpoints) IBR: K. Wisniewski & N. Zhong; MGH: Katherine Sims; Toronto Sick Kids: B. Minassian; BDSRA: Lance Johnston (diagnosis and patient referral).

24. Co-Workers (Batten Brigade) Present: Dixie-Ann Persaud Sawin Angela Schulz Alisa Ray Gabreal St.Claire Previous: Sumeer Dhar Svetlana Rylova Rhonda Bitting Paul Jansen Ellen Lockhart Carla Andreia Teixeira Collaborators Andrea Amalfitano Dwight Koeberl Alfried Kohlschütter Co-workers and Collaborators ACKNOWLEDGEMENTS Families and affected children, BDSRA, NINDS, Battin’ for Betsy Charities, Inc., Serbian Orthodox Church, McGee Family Sandy Hoffman (CLN1 cells),P. Lobel and D. Sleat (CLN2 cDNA), Nan Zhong, VIATRIS, and Genzyme