Genetic prion disease: opportunities for early therapeutic intervention with rigorous pre-symptomatic trials

1. Introduction

Human prion diseases are associated with misfolded prion protein (Prion Protein Scrapie, PrP^Sc) causing rapidly and irreversibly progressive encephalopathic syndromes. PrP^Sc builds insoluble aggregates, spreads throughout the brain, and is potentially transmissible [1]. The incidence of human prion diseases is about two cases per million person-years and sporadic Creutzfeldt-Jakob disease (sCJD) is the most common form (about 85–90%). About 10–15% of prion disease cases are associated with point mutations or base pair insertions on the PrP gene (PRNP) on chromosome 20, mostly following an autosomal-dominant pattern [2]. Some gPD are associated with a ‘CJD-like’ phenotype (gCJD, e.g. E200K) whereas others may cause distinctive clinical syndromes such as Fatal Familial Insomnia (FFI, D178N-129 MM) or Gerstman-Sträussler-Scheinker syndrome (GSS, e.g. P102L).

Evidence for a pathogenic PRNP mutation gives the unique opportunity for a pre-clinical intervention that might be the only way to verify clinical effectivity of an investigational drug for human prion diseases. Nonetheless, known difficulties include the low prevalence of gPD, the need for an accurate pre-clinical diagnosis, and ethical questions concerning potentially invasive treatment of asymptomatic patients.

2. Current therapeutic concepts

No causal therapeutic strategies for human prion diseases have been established to date. Some characteristic symptoms, such as myoclonus in gCJD or insomnia in FFI, may respond to antiepileptic drugs, antipsychotics, antidepressants, or hypnagogues, respectively [3]. Thorough patient care, rigorous treatment of concomitant and secondary diseases, tube feeding, and artificial respiration may extend the patient’s survival time, but none of these measures is able to slow down the disease progression in pre-terminal stages substantially. Thus, best palliative care is the commonly accepted treatment concept in all human prion diseases.

3. Clinical trials: previous drawbacks and future opportunities

3.1. Previous investigational drugs and clinical trials

Over the last two decades, prion disease animal models identified several compounds and procedures that have potential to inhibit disease progression and to prolong the survival time [4]. Despite promising results from some observational studies, only very few clinical trials were performed and showed disappointing results, indicating no significant or at best marginal therapeutic benefits of Flupirtine, Quinacrine, and Doxycycline in humans. The reasons for these failures are manifold and include compound-specific problems such as lack of effectivity against human prion strains or insufficient brain distribution [5] but also specific problems in the execution of clinical trials (Table 1).

Table 1. Difficulties in the development of therapeutics for human prion diseases

Stages of drug development	Known and potential difficulties in prion diseases
Development or identification of compounds	low financial interest of potential sponsors (e.g. public agencies, foundations, pharmacological companies) due to low disease prevalence little wide-spread experience from previous research (compared to well-known pathogenic agents such as microbiotics) identification of potentially beneficial effects is challenging in in vitro models of proteinopathies
Translation of results from animal models into clinical trials	insufficient distribution of compounds in human brains no effectivity against human prion strains different sCJD subtypes may respond differently to interventions but can only be determined through neuropathological investigation individuals with clinical symptoms are already in a late disease stage (from a neuropathological point of view), in a phase of rapid disease progression, possibly non-responsive to any available disease-modifying approaches
Identification of eligible individuals, trial execution, and data analyses	accurate diagnosis is challenging, especially in early disease stages rapid disease progression and poor clinical condition may cause legal representatives either to reject any treatment or to assure medication (in case of easily available drugs, e.g. Doxycycline) low case numbers require alternative and innovative study designs as well as statistical methods noninvasive pre-clinical, stage-reflecting, and predictive biomarkers were not available in the past

The information in Table 1 reflect the authors’ clinical and research experience, as well as data from the literature [4–6].

In recent years, animal models of new therapeutic approaches such as immunotherapy or the lowering of PrP expression by the application of antisense oligonucleotides (ASOs) showed promising results [7,8], but clinical data are not available, yet. Interestingly, a trial on Doxycycline in 2014 showed no significant benefit, whereas an analysis of a subgroup of patients in early disease stages showed a slight increase of survival time in the doxycycline treatment group [9]. These clinical data, as well as data from animal models [8], indicate that an early intervention may be an important factor for future trials.

3.2. Genetic prion disease as an opportunity for pre-clinical interventions

Except for one ongoing intervention using Doxycycline in FFI [10], patients with gPD have not been enrolled in specific clinical trials. The most important reason is that gPD are very rare and have an up to 10-fold lower incidence than sCJD. On the other hand, diseases caused by autosomal-dominant mutations provide the unique opportunity to identify eligible individuals before the clinical onset, maybe even before the neuropathological onset [5]. Positivity for a pathogenic mutation in the PRNP gene in an individual implies a very high probability to develop a prion disease [2]. Nonetheless, the starting point of PrP^Sc seeding activity in the brain, of the loss of neuronal integrity, and the clinical onset cannot be determined precisely. Observation of iatrogenic CJD has shown that the incubation time in prion diseases may take up to four decades [11]. A recent multinational study provided data on the age of onset in the three most common mutations and observed median ages of 53 years (D178N), 56 years (P102L), and 62 (E200K) years with a huge variability ranging from 12 to 92 years [6]. These data may help to decide when advanced biomarker analyses to investigate study eligibility should be performed.

3.3. The role of advanced biomarker analyses

The course of an individual with a PRNP mutation may be divided into three phases. In the first ‘healthy’ phase, PrP^Sc seeding activity in the brain and loss of neuronal integrity have not started, yet (at best). In this phase, prophylactic therapeutic interventions might be possible if the procedure was not too invasive and well tolerated. In the second (pre-clinical) phase, PrP^Sc-related pathologic changes are present in the brain, but clinical symptoms have not become manifest. Unfortunately, the starting point and the duration of this phase cannot be predicted precisely. On the other hand, recent advances in biomarker research provide minimally invasive tools to detect pathologic changes in the brain. Neurofilament Light Chain (NfL), a marker of neuro-axonal damage, was shown to be elevated in the pre-clinical phase of gPD [12,13]. A not yet peer-reviewed study from 2020 analyzed plasma samples from 377 individuals with mostly sporadic prion diseases and followed nine healthy mutation carriers. The data indicate that NfL has potential to predict onset in at-risk individuals [13]. Imaging techniques like MRI in gCJD [14], Fluordesoxyglucose – Positron Emission Tomography in FFI [15] and neurophysiological markers [16] have shown diagnostic potential in pre-clinical phases as well. Early and accurate diagnosis is crucial for an effective intervention in the third phase (symptomatic patients). The Real-time Quaking Induced Conversion (RT-QuIC) in cerebrospinal fluid (CSF) has been a major improvement in the diagnosis of sCJD. It allows accurate diagnosis of early disease stages [17] and shows a very good sensitivity in gCJD. In other gPD, CSF RT-QUIC seems to be less sensitive, especially in FFI [18]. Unfortunately, case numbers of biomarker studies on gPD are rather low and data on RT-QuIC based on less invasive tissue sampling (for pre-clinical diagnosis) are not available. Figure 1 summarizes the clinical stages, implications for interventions, and biomarker analyses.

Figure 1. Timeline of interventions and biomarkers in genetic prion diseases. The course of gPD is characterized by subsequent stages and clinical milestones that are displayed in this flow chart. In addition, information on disease duration, age of onset, and gPD biomarkers is given. CSF biomarkers are mostly diagnostic, except for PrP that may also have potential as a monitor for therapeutic PrP lowering. Imaging as well as fluid markers may as well reflect disease stage or response to intervention. The figure is based on the authors’ expertise and on references [2,5,6,12–15]. It was created using Microsoft Office Power Point™

Abbreviations: gCJD (genetic Creutzfeldt-Jakob disease), FFI (Fatal Familial Insomnia), GSS (Gerstmann-Sträussler-Scheinker syndrome), RT-QuIC (Real-Time Quaking Induced Conversion), PrP (Prion Protein), MRI (Magnetic Resonance Imaging), FDG-PET (Fluordesoxyglucose – Positron Emission Tomography), NfL, (Neurofilament Light chain), gPD (genetic Prion Disease).

3.4. Study design of early interventional trials

To address the known difficulties (Table 1), early interventions may have to follow alternative study designs. Due to conventional power calculations, the needed number of participants is not realistic in gPD but the use of biomarkers as surrogate endpoints might increase the statistical power and reduce trial duration [5]. Most of the aforementioned diagnostic biomarkers were shown to be associated with disease stage or severity of symptoms, respectively. Besides plasma levels of NfL and Tau [18], the quantification of total PrP in the CSF may also be a promising tool, especially as monitor in trials on potentially PrP-reducing drugs like ASOs [19]. Previous experience with ASOs (Tofersen) in amyotrophic lateral sclerosis indicates that CSF protein concentrations (in this case, super-oxide dismutase 1) may be suitable surrogate trial endpoints [20].

The unavailability of established therapies that could be applied in a control group and the refusal of potential participants to be assigned to a placebo group [9] are major problems in the execution of placebo-controlled trials. In addition, invasive procedures such as intrathecal drug applications complicate the enrollment of healthy individuals and may preclude sufficient sham controls. For pre-clinical trials in gPD, the use of historical control data from prion disease reference centers may be an adequate alternative [5]. The ongoing Italian trial on Doxycycline uses historical data to detect possible effects of preventive Doxycycline application by determination of expected disease onset. In addition, it took advantage of recruiting a large and compliant kindred [10] and from the fact that Doxycycline is a well-known drug. Concerning experimental compounds, an open-labeled and single-armed pre-symptomatic trial on ASOs in spinal muscular atrophy (Nusinersen) [21] has demonstrated a study design possibly suitable for future trials in gPD.

4. Conclusion

Clinical data on therapeutic interventions in prion diseases are scarce and past clinical trials in sCJD showed no or only poor beneficial effects. These observations and data from animal models have shown that early, at best pre-clinical intervention may be crucial in future trials. High penetrance gPD give the opportunity to identify individuals at high-risk and recent advances in biomarker research may provide new tools for early diagnosis and trial monitoring. Nonetheless, low case numbers of distinctive clinical syndromes associated with various PRNP mutations, as well as a huge variability in the age of pre-clinical and clinical onset will challenge researchers to develop innovative study designs and to seek for international cooperation.

5. Expert opinion

The history of drug development and clinical trials in human prion diseases is characterized by specific difficulties (Table 1) and failures. Similar to experiences from other proteinopathies/neurodegenerative diseases, several promising compounds had been identified but clinical effectivity in symptomatic patients could not be verified. There is a high probability that symptomatic patients may have already passed a ‘point of no return’ and entered a condition that is irreversibly resistant to all known therapeutic concepts aiming at PrP or PrP^Sc stabilization, cleavage, and elimination, respectively.

Recently, the idea of pre-clinical or even prophylactic intervention in gPD has come into focus and the identification of minimal-invasive biomarkers for neuronal damage (e.g. plasma NfL) may pave the way for clinical trials. The reduction of PRNP gene expression by ASOs is an exciting new approach that may be particularly effective in gPD. However, early intervention will still be crucial and future research has to address several points:

• Evidence for all blood-based pre-clinical biomarkers of gPD is based on very small case numbers and has to be enhanced.

• Low prevalence of gPD requires global cooperation of prion disease research centers.

• Due to invariably fatal outcome and low prevalence of gPD, placebo-controlled trials may only be possible to a limited extend.

• Probably, existing data on natural history of gPD patients have to function as control group. Thus, study endpoints have to be chosen accordingly.

• High variability of age of onset may require very long trial durations.

• General and country-specific legal and ethical concerns have to be considered when healthy mutation carriers receive new compounds or invasive interventions.

Pre-clinical trials in gPD will be an exciting opportunity and may function as a role model for trials in numerous diseases associated with protein aggregation in the central nervous system. Registers of sCJD and gPD cases have been set up during surveillance activities in many countries because of the potentially infectious character of PrP^Sc. Nonetheless, design and execution of trials will be challenging and will require joint efforts of researchers in drug development, biomarkers, and epidemiology.

Article highlights

• Previous clinical trials in human prion diseases included only sporadic CJD cases and showed disappointing results.

• New therapeutic approaches such as immunotherapy and antisense oligonucleotides are promising but have to be evaluated through clinical trials.

• Genetic prion diseases may provide chances for effective early or even pre-clinical interventions.

• Genetic prion diseases are very rare and innovative study designs are needed to overcome the known difficulties.

• Recent advances in biomarker research provide new opportunities for the identification of eligible patients and trial monitoring.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

The authors receive funding from German federal ministry of health through grants from the Robert-Koch-Institute, grant no. 139-341.