ULD is a catastrophic epilepsy with subtle onset that evolves into explosive intractable continuous myoclonus during the second and third decades of life. This is accompanied by convulsive seizures and rapidly worsening ataxia leading to wheelchair dependence and dementia. Symptoms somewhat diminish in intensity after the third decade but persist throughout the length of a reduced lifespan. 18 Given the severity, along with the neurodegenerative nature of the disease, the need for a treatment is acute. Lack of a treatment is also a high concern because relative to similarly severe conditions, ULD prevalence is high in certain countries (Finland), reaching 2/100,000. 18, 19 This is not merely due to a genetic founder effect. The causative mutation on at least one allele in all patients with ULD is a dodecamer repeat expansion that arises from a human-specific two or three-copy dodecamer repeat in the CSTB gene promoter. 3 The particularity of the location of this repeat sequence, and its predisposition to expand, suggest that ULD will continue to recur at this relatively high incidence. As a comparison, the closest differential diagnosis, the Lafora PME, only has an incidence of ~ 2/1,000,000, even in societies with high rates of consanguinity and despite the fact that it can be caused by recessively inherited mutations in two independent genes. 20
Numerous functions have been attributed to cystatin B, the first, where it derived its name, is as an inhibitor of lysosomal cysteine proteases. 21 Other functions include regulation of microglial activation and (neuro) inflammation, organizing synaptic transmission, interneuron migration, and chromatin structure during neural stem cell renewal and differentiation, prevention of oxidative stress induced cell death, and alteration of malignant characteristics of cancers. 14, 22–27 Consistent with its functional plurality, the CSTB protein has been documented in multiple subcellular locations, including at the lysosomal surface, in the lysosome, nucleus, and cytosol, and most recently, secreted extracellularly. 6, 22, 25, 28 The specific function of CSTB that contributes most prominently to ULD is unknown, which has been an impediment to developing disease-modifying therapies. A gene-based approach is therefore favored. Recent evidence that the product can be secreted, 24 and thus is potentially able to impact cells beyond those transduced by the gene therapy vector, offers further support for pursuing this modality.
Another favorable circumstance is that affected children are completely healthy prior to onset, and the disease is initially insidious. Early clinical or pre-symptomatic gene replacement could potentially protect the brain while it is still minimally affected, and neurons remain spared. Finally, the fact that all patients have a small amount of CSTB still produced, diminishes concerns of immune intolerance of the delivered gene product.
The Cstb−/− mice are not a genetic model of ULD. They are a genetic model of a disease caused by a complete absence of CSTB that is characterized by very severe early infantile encephalopathy and death in the rare cases where such children are even born. 4, 5 As such, phenotypic corrections in this mouse model point to a likely even greater impact in the less severe hypomorphic ULD phenotype. In the present work, to replicate the ULD clinical situation (late-childhood onset) we treated the mice at the relatively advanced ages of p21 and p60. At these ages, CSF-interstitial space barriers are mature and viral distribution is more restricted compared to gene therapies delivered in the neonatal period. 29 As such, our experimental design had a two-fold high bar, namely treating both later during disease progression and in a model of a much earlier-onset and much more severe disease than the target condition.
Despite this high bar for success, we were able to show that CSTB gene replacement reduces neuronal apoptosis, neuroinflammation and ataxia. Brain weight was not rescued. One explanation for the lack of brain weight rescue could be that a large portion of brain weight loss (likely reflecting neurodegeneration) had already occurred in these mice by 2 months of age (Figs. 4a and b; see also references). 10, 17 Our earliest gene therapy treatment in mice was administered at p21, and maximal and stable protein production requires 3–4 weeks, 30–32 i.e., in this case mice would subsequently be 6–8 weeks of age, when substantial neurodegeneration has already occurred. Related to this was our curious observation that CSTB protein expression was substantially higher in the brains of mice sacrificed at 9 versus 2 months (Fig. 2a, c, and e). It appears that maximal expression required even more time than documented with other gene therapies, suggesting an additional reason to aim for early intervention in future studies.
The relative resistance of mice to epilepsy is well documented, as is the impact of genetic background. This was previously formally studied in the Cstb−/− mice, where mice on a pure 129SvJ background had seizures, but this phenotype was lost on the C57BL/6 background. 7 Approximately one third of the genome of our mice was not 129SvJ, of which 10% was C57BL/6, which could explain why the epileptic phenotypes, at least the seizures and EEG abnormalities, were absent and could thus not be rescued with our treatment.
In addition to the epilepsy, ataxia is a cardinal feature of ULD and is a highly disabling symptom. The cerebellum is the most affected region of the brain with neurodegeneration noted in both the Cstb−/− mice and human patients. 33, 34 Historically, prior to the availability of more modern anti-seizure drugs, the use of phenytoin to control seizures would result in accelerated cerebellar neurodegeneration and rapid loss of ambulation. 35, 36 Remarkably, in the present work, despite treatment at time points after late-into-neurodegeneration intervention, sufficient cerebellar cells were rescued and murine ataxia improved, suggesting that gene therapy will likely benefit patients even once the disease has already progressed beyond the earliest symptoms.
Subsequent work should include experiments in pure 129SvJ mice to determine therapeutic impact on the epilepsy, and earlier time points of vector administration. However, existing results already suggest that gene therapy in ULD is likely to be effective, possibly even under the challenging circumstances of later intervention. Of course, newly diagnosed patients and genetically confirmed still asymptomatic siblings will likely benefit the most, as will cases detected very early through new-born screening in a hoped-for not too distant future.