Batten Disease: Basic Biology and Therapy

Executive Summary

Batten Disease: Basic Biology and Therapy

A workshop sponsored by NINDS, Children's Brain Disease Foundation, and the NIH Office of Rare Diseases (ORD)

April 1-2, 1999

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Organizers:

C hris Campbell, William Mobley, Rose-Mary Boustany, and Giovanna Spinella

Attendees from NINDS and ORD:

Audrey Penn, Giovanna Spinella, Robert Baughman, Steven C. Groft, and Gerald Fischbach

Participants:

Raymond T. Bartus, Rose-Mary Boustany, Roscoe Brady, Jonathan Cooper, Beverly Davidson, Gregory Grabowski, Yusuf Hannun, Sandra Hoffman, Anu Jalanko, Terry Lerner, Peter Lobel., Vivek Malhotra, Hannah Mitchison, William Mobley, Robert Nussbaum, Leena Peltonen, Allen D. Roses, Mark Sands, David Sleat, and Kunihiko Suzuki

Parents/Guests:

Liz Aurelio, Ricky Bennett, Chris Campbell, Andrew Henery, Lance Johnston, Michael Joyce, Russelle Rankin, Alfred Rider, Andrea Schneider, and Caroline Wright

Introduction

This conference was conceived with the goals of: 1) generating novel ideas about pathogenesis, 2) attracting talented new scientists to the field, and 3) spurring on development of effective therapies based on current knowledge. The workshop sought to harness the power of existing knowledge, available resources, and know-how generated by academia, drug companies, private foundations, private biotech companies, and the National Institutes of Health to achieve these goals.

The conference was organized around three working groups:

• Cell death, Neuroprotection, and Signaling.
• Lysosomal Biology and Protein Trafficking.
• Gene, Protein, and/or Drug Delivery to the CNS.

On the first day of the workshop, following a brief overview, members of each of the three working groups addressed the following questions:

Which elements of pathogenesis are relevant?

What are potential therapeutic targets in these pathways?

How can these targets be best evaluated?

What resources are needed to accomplish this work?

Highlights of the Conference Overview presented by Drs. Leena Peltonen, Peter Lobel, and Terry Lerner:

We learned from Dr. Peltonen that protein palmitoyl thiotransferase (PPT1) colocalizes with synaptophysin in vesicles in axonal extensions of neurons and that it most likely represents a membrane protein. There is expression in the embryo beginning at E8-E11 in the neural tube region with subsequent spread to the forebrain and hindbrain. PPT was expressed in all neuronal cells. Neuron enriched regions stain highly positively for PPT1 mRNA between P10-P20. Protein distribution closely followed mRNA distribution. A PPT2 protein has been identified which can use palmitoylated esters as substrates, similar to PPT1, but only PPT1 recognizes thiol esters as substrates. PPT2 did not correct the PPT1 defect. There is a heavy clustering of PPT1 mutations in exon 2. Some cases of what appears to be LINCL have been reported with PPT1 mutations. The present mouse knockout for PPT1 is heterozygous lethal.

Dr. Peter Lobel discussed the CLN2 pepinase gene and protein. It is 562 amino acids long and has a 25 kDa propiece. The mature protein is 46 kDa. This pepinase is similar to a bacterial protein known as tripeptidyl protease 1. Both enzymes share an ability to degrade fluorescent artificial substrates in a pepstatin nonsensitive manner. It is not yet known what the natural substrates for these enzymes are. Dr. Lobel also noted that the defect in ovine congenital neuronal ceroid lipofuscinosis consisted of a mutation in cathepsin D, resulting in a single amino acid substitution (aspartate to asparagine).

Dr. Lerner reviewed what is known about the localization of the CLN3 protein. Different researchers have localized CLN3 to ER, golgi, mitochondrial and/or lysosomal membranes. She also discussed Dr. David Pierce's findings: A yeast knockout for the gene homologous to CLN3 (BTN1 or YCH3) had an altered vacuolar pH of 5.8 as opposed to the wild type yeast vacuolar pH which was 6.2. In addition the yeast knockout had an upregulation of HSP 30 and BTN2 (similar to HOOK 1). Dr. Lerner also noted that a mouse knockout model with exon 7 and 8 of the CLN3 gene deleted is now available. Other mouse models (mnd and nclf) showed neurodegeneration and gliosis by 8-9 months of age.

Dr. Allen Roses reviewed for the group the principles of exploratory discovery that drug companies can undertake for diseases where the genes are 1) known or 2) not known. This pharmacogenic process aims to identify effective drugs. The differences between differential display using a "closed" versus an "open" system were discussed. Comparing differential display of pre symptomatic affected versus normal tissues can reveal information about perturbed biochemical pathways. Once biochemical pathways are identified, drugs known to affect these pathways are systemically tried. Effective and safe drugs can then be identified by high through-put screening.

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Highlights of the discussion on Cell Death, Neuroprotection, and Signaling led by Dr. Yusuf Hannun:

It was agreed that investigation of apoptotic pathways was important for the understanding of CLN3, CLN2 and CLN1 defects as well as other variants, as neuronal and photoreceptor death is common to all. Corroboration that apoptotic pathways are engaged and investigation of mechanisms would be important. On a broader level, investigation of the link between lysosomal function/proteins and apoptosis may yield interesting approaches to therapy. It was recommended by the working group that a concerted effort be undertaken to: 1) study appropriate organisms such as yeast, C. elegans, and the now available mouse knockouts; 2) attract young investigators from different fields with different ideas and expertise in new technologies as this would allow productive cross-fertilization at many levels; and 3) encourage scientists to undertake collaborative efforts. The NIH and/or private foundations should collaborate on multiple scientist/multiple organization projects. It was suggested that an annual meeting/workshop perhaps concurrent with the Neuroscience meetings could be one way to keep the momentum going.

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Highlights of the discussion on Lysosomal Biology and Protein Trafficking led by Dr. Peter Lobel:

The neuronal ceroid lipofuscinoses, particularly CLN1 and CLN2, could represent a new form of "lysosomal disease" in which the "storage material" was less significant, as there is accumulation of material, but no ballooning of neurons. The enzyme deficiencies in both these disorders did not directly result in accumulation of an identified substrate, but were capable of producing neuronal loss or death. The delineation of the exact function of the defective proteins, their in vivo substrates and their associated metabolic pathways should be top priority items for investigation. The degree of selective CNS involvement vis a vis neuronal death could result from toxic metabolites immediately upstream of the biochemical block, decreased production of downstream metabolites vital for survival, or the more general model of accretion of storage material and corresponding general lysosomal dysfunction. Unexplained would remain why neurons are preferentially killed. The idea was suggested that in the brain and eye (two immune privileged sites), loss of function of an antiapoptotic element would be devastating to neurons and photoreceptors. Neuronal substrate specificity could also account for differences. Exact cellular localization of all the known proteins (CLN1, CLN2, CLN3, and CLN5) may help elucidate function as well. Dr. Peltonen and others suggested that studying whether there is an interaction between any of the known proteins may shed some light on function as well and this could be achieved using yeast two-hybrid systems or novel chemical cross-linking technologies.

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Highlights of the discussion on Gene, Protein, and/or Drug Delivery to the CNS led by Dr. Mark Sands:

Various difficulties associated with protein delivery were discussed. 1) The need for the protein to be soluble (CLN1 and CLN2) and preferably not membrane bound (CLN3 and CLN5) was addressed. Modes of direct delivery or carrier-mediated (e.g.., transferrin receptor antibody conjugated) of protein to the brain were discussed. Dr. Roscoe Brady shared with us his plans for direct injection of beta glucocerebrosidase into brain of patients affected with type III Gaucher disease. Rather widespread travel of the enzyme was expected. 2) The use of neural stem cells was mentioned as a future therapeutic modality, with the goals of repopulating neurons and as a source of enzyme. 3) The use of growth factors to both slow progression and to stimulate stem cells was also briefly discussed. 4) The different gene therapy vectors (Adenovirus, AAV, and others) were discussed. With respect to gene therapy the major challenges are a) identifying the best route of delivery of the gene; b) achieving long term source and persistence of expression over time, c) determining site and frequency of delivery, d) choosing the most efficient tissue-specific promoters; e) circumventing shutdown of expression, f) avoiding deleterious immunological consequences, and g) finally, but most importantly, determining the ultimate biologic availability and function of the transcribed protein. These are all hurdles scientists involved with gene therapy are working hard to overcome. It was concluded that the availability of mouse models would greatly facilitate research into gene therapy for the various Batten variants for which the gene defects were known. Ultimately, carefully planned human trials would be necessary. Additionally, combination therapies using enzyme and/or gene replacement, bone marrow transplantation and growth factors should also be discussed.

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On the second day of the workshop, the working groups addressed issues specific to CLN1, CLN2, CLN3, and variants and came up with specific recommendations for the different types.

Dr. Sandra Hoffman summarized the discussion and recommendations for CLN1:

• Define the natural history of cases and enter information for cases in an accessible database.
• Look into genetic screening for this form and other forms of Batten disease.
• Optimize enzyme and gene therapy using a mouse model for the disease.
• Continue studying the pathogenesis/biology of neurodegeneration.
• Identify thioesterase substrates and perhaps neural cell specific substrates.
• Attempt inhibition of substrate accumulation. Optimize a screening assay for substrate accumulation.
• Attempt to enhance substrate degradation by development of small molecules.

Dr. David Sleat summarized the discussion and recommendations for CLN2:

1. Develop a transgenic knockout mouse model for the CLN2 protease. A mouse model is essential for evaluation of enzyme replacement and gene therapies as well determining the biologic role of the CLN2 protein.
2. Establish collaborations to facilitate large-scale synthesis of the CLN2 protease for enzyme replacement. The problem of CNS delivery remains an obstacle to be overcome.
3. Develop effective viral based gene-delivery systems.
4. Identify the natural substrate of the CLN2 protease. Determine if accumulation of this substrate is deleterious to cells and attempt to do pharmacologic depletion as a therapeutic option.
5. Identify other lysosomal proteins in catabolic pathways involving the CLN2 protease.
6. Use differential display technologies to identify other proteins impacted by absence of the CLN2 protease, and conduct yeast two-hybrid and co-immunoprecipitation studies to fish out other interacting proteins.
7. Generate antibodies to the CLN2 protease.

Dr. Beverly Davidson summarized the discussion and recommendations for CLN3:

1. Investigate interaction of other proteins including CLN2, CLN1, and CLN5 with CLN3.
2. Determine upstream regulators and downstream targets of the CLN3 gene.
3. Encourage specific investigations of the mechanism of action of CLN3 in apoptosis and its role in neurodegeneration by more than one laboratory.
4. Share resources available to scientists, as for example mouse models and antibodies. This would be particularly useful in preparation for clinical trials of any kind.
5. Achieve more refined immuno-EM localization of the CLN3 protein as an aid to understand function.
6. Investigate protein/gene therapy in animal models, even if CLN3 is most likely a membrane-bound protein, particularly if neuronal targeting of the CLN3p is achievable.
7. Encourage collaborative efforts between scientists.

Dr. Hannah Mitchison summarized existing knowledge and recommendations regarding the Batten variants, including some existing animal models:

1. Finnish CLN5 (gene known)
2. CLN6/nclf/ovine NCL. In humans the defect is localized to chromosome 15. This variant LINCL has been described in Costa Rican, Romany gypsies, Portugese and British patients. The gene is not yet identified.
3. Turkish CLN7 variant.
4. CLN8 identifies the cloned gene (EPMR) responsible for Northern epilepsy.
5. CLN4 or the adult Kuf variant (gene unknown).
6. Congenital ovine NCL (CONCL) due to the recently identified cathepsin D defect
7. In addition many animal models were mentioned: English Setter, Collie, Schnauzer, Australian Blue Heeler and others.

• mnd mouse is a good model for CLN8.
• The researchers were finding difficulties identifying sufficient numbers of cases, and obtaining funding for the study of extremely rare diseases.
• Pooling of resources were recommended as well as the use of novel techniques to overcome these difficulties listed in 9). Those methods include the technique developed by Dr. Lobel and his group to identify additional lysosomal proteins, and differential display techniques.

RECOMMENDATIONS FOR NIH, SCIENTISTS, PRIVATE FOUNDATIONS AND PARENTS

Expectations for NINDS

The conference participants strongly urge the NINDS to 1) fund research into basic pathophysiology and development of therapies for Batten disease; 2) encourage collaboration between scientists by fostering collaborative proposals; 3) work to attract scientists new to the field, especially those with different expertise; 4) continue to support workshops and conferences on Batten disease; and 5) advise and guide investigators interested in creating a consortium for carrying out clinical trials.

Expectations for Families and Private Foundations

Families and private foundations should be encouraged to 1) continue having courage, optimism, hope, and patience; 2) allow scientists and physician-scientists a significant degree of freedom and independence in their work by allowing them to develop and test novel hypotheses; 3) continue fundraising for family support and research; and 4) educate their own extended families, co-workers, and friends about Batten disease.

Expectations for Scientists

Scientists studying Batten disease should 1) continue to be persistent, creative, and hard working; 2) share resources including patient material, animal models (mouse models in particular), antibodies, cell lines, vectors, and tissues; 3) cooperate and share ideas through conferences, workshops, publications, and collaborative proposals; 4) demonstrate collegiality and professionalism; 5) show sensitivity towards the parents and patients with Batten disease; and 6) create working groups to address or tackle specific problems such as gene therapy, protein replacement, and transfer of technologies to allow availability of quality-controlled testing for the known gene defects/defective enzymes.

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Conclusion

The meeting was intensely satisfying and productive for the participants. New ideas developed and new alliances were forged. More importantly, the workshop fueled everyone attending with energy and renewed hope and commitment. The concerted efforts of the scientific community, private Batten foundations, NIH, and drug and biotechnology companies will surely be needed to expand our knowledge base in Batten disease, transcend gene discovery and move on to targeted gene and protein therapies and pharmaceutical approaches. The organizers believe that the format of the meeting created a uniquely positive environment for discussing how to translate basic science advances into approaches for treatment.