The CLN Genes – When Children get Dementia
When it comes to dementia – the progressive loss of nerve cells and thus also of cognitive performance – most people immediately think of older people, perhaps their grandparents. Not many people know that dementia can affect the youngest. The diagnosis of “childhood dementia”, Batten disease, or neural ceroid lipofuscinosis, NCL for short, means a severe shock for around 15-20 parents a year in Germany. The prognosis is difficult to accept: Batten disease usually causes blindness and progressive loss of motor and cognitive functions and results in death in early adulthood.
The disease is based on mutations in one of the CLN genes, numbered from CLN1 to CLN14. The proteins encoded by these genes are all related in some way to an important part of all of our cells, the lysosome, since NCL belongs to a special subgroup of rare genetic diseases, the lysosomal storage diseases. There is no cure, but new therapeutic approaches might give reason for some hope.
The Lysosome and Lysosomal Storage Diseases
To understand why NCL causes such severe symptoms, one should first look at the normal function of the lysosomes. In essence, the lysosomes are our cells’ garbage disposal sites; or better: the recycling plants. Here the substances that are currently not needed by the cell are broken down into their individual parts, from which other substances can then be built up. If the function of the lysosomes is disturbed, more and more “garbage” accumulates in the cells. In the long run, this damages the cells so much that they eventually die. Since not just one but many proteins are important for the function of the lysosome, mutations in various genes can lead to a lysosomal storage diseases. The proteins are distributed differently in the lysosomes of the various tissues in our body, so that depending on which gene carries a mutation, the various tissues are affected to different degrees.
The CLN Genes and their Products
Even though NCL diseases were first described in the 19th century, it was not until 1995 that a first NCL gene, CLN1, and – still in the same year – a second NCL gene, CLN3, were identified. Two years later, in 1997, the CLN2 gene followed. Mutations in any of these three genes are the most common causes of NCL disease. The analysis of their specific functions helps to reveal characteristics of the different forms of NCL, as well as to explain some difficulties in NCL research.
The CLN1 gene represents the blueprint for an enzyme called palmitoyl protein thioesterase 1, or PPT1 for short. The ending –ase, as my cell biology professor once said during my undergraduate studies, usually indicates an “ugly enzyme that destroys something”. PPT1 breaks down long-chain fatty acids that are attached to some proteins. CLN2 also codes for a soluble enzyme, which, to keep everyone maximally confused, also has a similar abbreviation: TPP1, tripeptidyl peptidase 1. TPP1 cleaves proteins into small parts. The most important workplace of PPT1 and TPP1 is – you guessed it – the lysosome. But not all NCL genes swim around in the lysosome; others, such as CLN3, are located on its surface, in the membrane of the lysosome. The exact role that the healthy CLN3 protein plays in the lysosome membrane has not yet been fully clarified.
Symptoms, Diagnosis and Prognosis
The age at which children first show symptoms varies between the different forms of childhood dementia. Most patients with CLN1 mutations develop symptoms in their first year of life: they often never learn to stand, walk or speak properly. Many of them develop epilepsy and die in their early childhood. Children with CLN2 mutations usually learn to walk and speak, which, however, often becomes increasingly difficult at the age of 3-4 and they soon depend on intensive care. They usually die between 6 and 12 years of age. The CLN3 disease manifests mostly as progressive blindness, which begins at the age of about 4-7 years. Affected children develop increasing learning difficulties and lose their motor and language skills. They usually die in young adulthood. Other forms of the disease, which can be traced back to very rare mutations in the CLN4 or CLN6 gene, for example, only show up in adulthood.
Mostly, however, it is children who are affected and their parents usually consult a number of doctors before a genetic diagnosis for this rare disease is available. This represents an immense shock due to the prognosis of a relentless loss of skills and the inevitable fatal outcome. No wonder parents try everything they can to help their affected children. But is there any hope of slowing down or even stopping the course of the disease?
Enzyme Replacement Therapy for CLN2 Patients
For the CLN2 form, there is an approved therapy since 2017; the first ever to treat the disease itself and not just relieve symptoms. This is a so-called enzyme replacement therapy, which means that affected children are given an enzyme that takes over the missing function of the TPP1 enzyme. The recombinantly produced precursor to the active enzyme, Cerliponase Alfa, is sold by the company BioMarin under the trade name Brineura. It is given directly into the ventricle of the brain through a catheter every two weeks. Despite approval by EU authorities, it is still difficult to get such a therapy in some European countries, which not at least is due to the costs of over 500,000 Euros per year. The first results from the ongoing treatments indicate that the course of the disease can be slowed down considerably by the administration of Cerliponase Alfa. However, we will only be able to assess the longer-term effects of the therapy in a few years.
Gene Therapies using AAVs
As diseases that are caused by mutations in individual genes, the NCL diseases represent prime candidates for the use of gene therapies. Classic forms of gene therapy are based on giving the patient the “healthy” copy of the gene which, in a mutated form, causes the disease. One of the big problems with gene therapies is the delivery: how do you get the gene into the cells? Usually viruses are used for this, traditionally retroviruses. If you equip retroviruses with the desired gene and infect cells with it, this gene is integrated somewhere into the genome of the host cell, mainly by chance. However, this poses a risk: the gene can cause damage by integrating into critical areas of the genome. In the worst case, this cell can get out of control and form a tumor. That is why so-called adeno-associated viruses, or AAVs, have been used recently. AAVs have the peculiarity that – if they integrate into the genome at all – they always do so at a certain point; so they are much more predictable. Even better: most of the time, the AAVs do not integrate their genetic material into the genome at all; they bring it into the cell where it just lies around next to the other chromosomes; we call this an episome. AAVs can only be equipped with a DNA fragment of up to 4,700 base pairs. For some of the CLN genes, however, this is enough, so preclinical and clinical studies, in which the effectiveness and safety of gene therapies are determined, are currently being carried out for a number of the CLN genes.
Hollywood director Gordon Gray and his wife, for example, have two daughters who carry mutations in both copies of the CLN6 gene. When the disease was diagnosed in the then 4-year-old Charlotte Gray, her 2-year-old sister Gwenyth was not showing any symptoms. After an astonishingly rapid development initiated by their parents with a lot of commitment and money, one year after the diagnosis, in 2016, both sisters were treated with an AAV-based CLN6 gene therapy as part of a clinical study. By then Charlotte had already lost a lot of her motor and cognitive abilities, which unfortunately cannot be restored. Nevertheless, it remains exciting how the therapy will influence the course of the disease of the other participants and especially that of the younger sister.
Antisense Oligonucleotides as New Pharmacological Agents
A relatively new approach pursues a different strategy than classic gene therapies. For some of the CLN diseases it is possible to intervene in the splicing process in order to restore parts of the normal gene function. As a reminder: during splicing, the so-called introns are removed from a primary RNA copy of the gene, leaving only the joined exons (see also my article on ARHGAP11B). The mostfrequent mutation underlying the CLN3 disease, is based on a deletion that includes exons 7 and 8. The loss of these two exons alone would probably not be so bad, but the fact that exon 6 is added to exon 9 in this case results in a frameshift. This means that the reading frame, which results from the fact that all genes are coded in triplets, is shifted.
This initially leads to a short missense, i.e. “senseless“, code on exon 9 (orange in the figure), which then leads relatively quickly to a STOP codon. All sections of the gene behind this are no longer translated into protein (red in the figure). However, if a short piece of nucleic acid (purple in the figure) is introduced into the cells that is complementary to a splice site of exon 5 and therefore accumulates there, then the splice site of exon 5 is blocked. Exon 5 is therefore cut out together with the introns and exon 4 is split directly to exon 6. As a result, you not only lose what was coded on exon 5, but you also shift the reading frame so that exon 6 becomes “senseless“. The reading frame that makes sense in exon 4 is the same as the one that makes sense on exon 9. This intervention thus regains the gene function of exon 9 and all subsequent exons. The resulting protein is not identical to the normal CLN3 protein, but it is much more functional than the greatly shortened protein product of the mutated CLN3 gene. Because even if we still don’t exactly understand the functional role of CLN3, we know two so-called lysosomal targeting sequences (LTS) on exon 9 and exon 15, without which CLN3 cannot be transported to its site of operation. The effectiveness of this treatment in principle has recently shown promising effects in a preclinical study on mice and has slowed the course of the disease.
But is this approach suitable for treating patients? As a high-profile case has shown, it is already being tried. This was not a treatment of the CLN3 form but about a mutation of the CLN7 gene that is unique in the world. Little Mila inherited mutations in CLN7 from both of her parents, but they differ from one another. The mutation inherited from the mother is an insertion that has never been described before and was caused by a transposon. This transposon had its own splice site and integrated behind exon 6 of the CLN7 gene. As a result, exon 6 is not spliced to exon 7 but to the transposon integration, which quickly leads to a STOP codon. If one could block this splice site of the transposon, then that of exon 7 would be used again and the gene function would be completely restored. So an antisense nucleotide was designed, tested, manufactured for precisely this transposon splice site, approved for this experimental use in unprecedented speed and administered directly into Mila’s brain. So this therapy was developed for a single person; according to our current knowledge, there is no other person in the world who could benefit from this very oligonucleotide. With Mila, however, it has at least led to a reduction in the epileptic seizures that her illness brought with it. The drug was named milasen.
Since such new approaches are still very experimental and risky, for now they should only be used in cases where the prognosis is very poor and no other therapies are available. But they demonstrate where – in the opinion of many experts – the field could move: towards “personalized medicine“. This refers to the vision that treatments in the future could increasingly be tailored precisely to the patient’s individual illness situation. Patients who suffer from rare genetic diseases, such as child dementia, could benefit from these developments in the future.
Hamburg as a Center for Childhood Dementia
Anyone who knows me beyond my blog already learned the news: I’ll be moving to Hamburg soon! And to be honest, I only found out about childhood dementia when I researched Hamburg as a science location. The only enzyme replacement therapies in Germany take place here, which means that a number of children who suffer from the CLN2 form of the disease travel to the Universitätsklinikum Eppendorf, UKE for short, every two weeks. The NCL Foundation, which raises funds for research into child dementia and brings the disease to public awareness, is also based in Hamburg. So if you want to find out more about child dementia or want to get active yourself, take a look at the NCL Foundation’s homepage.