Review Article

Opening New Horizons in the Treatment of Childhood Onset Leukodystrophies

Stina Schiller1 Marco Henneke1 Jutta Gärtner1

1 Department of Paediatrics and Adolescent Medicine, University Medical Address for correspondence Prof. Dr. med. Jutta Gärtner, Centre Göttingen, Georg August University Göttingen, Germany Department of Paediatrics and Adolescent Medicine, University Medical Centre Göttingen, Robert-Koch-Strasse 40, 37075, Neuropediatrics Göttingen, Germany (e-mail: [email protected]).

Abstract Leukodystrophies (LDs) predominantly affect the white matter of the central nervous system and its main component, the myelin. The majority of LDs manifests in infancy with progressive neurodegeneration. Main clinical signs are intellectual and motor function losses of already attained developmental skills. Classical LDs include lysosomal storage disorders like metachromatic leukodystrophy (MLD), peroxisomal disorders like X-linked adrenoleukodystrophy (X-ALD), disorders of mitochondrial dysfunction, and myelin defects like Pelizaeus-Merzbacher disease. So far, there are only single LD disorders with effective treatment options in an early stage of disease. The increasing number of patients Keywords diagnosed with LDs emphasizes the need for novel therapeutic options. Impressive ► leukodystrophy advances in biotechnology have not only led to the continuous identification of new ► myelin disease for so far unknown LDs but also led to new effective neuroprotective and ► neurometabolism disease-modifying therapeutic approaches. This review summarizes ongoing and novel ► replacement innovative treatment options for LD patients and their challenges. It includes in vitro and in ► therapy vivo approaches with focus on stem cell and gene therapies, intrathecal substrate or ► novel therapies enzyme replacement, and genome editing.

Introduction oxisomal disorders like X-linked adrenoleukodystrophy (X- ALD), or defects in myelin protein like Pelizaeus-Merzbacher Leukodystrophies (LDs) comprise a group of rare monogenetic disease (PMD). Over the past decade, the impressive advances neurological disorders that primarilyaffect thewhite matter of in next-generation sequencing technologies led to continuous 4 the central nervous system (CNS). Single LDs are associated expansion of the spectrum of defined white matter disorders. Downloaded by: King's College London. Copyrighted material. with an additional involvement of the peripheral nervous The knowledge of the underlying disease gene, the corre- system (PNS). Besides myelin, the major component of white sponding protein, and/or the affected metabolic pathways matter, other structural components, and metabolic pathways facilitates the establishment of new therapeutic approaches. – can be affected.1 3 LDs can manifest at all ages, which means as Thus, an early diagnosis is crucial, especially in those disorders early as in the fetal and neonatal period or as late as in where treatment or treatment approaches are possible. adolescence and adulthood. The clinical manifestations are highly variable. Besides progressive loss of motor and cognitive Innovative Treatment Options for function, LDs often go along with visual or hearing impairment Leukodystrophies and epilepsy. Brain imaging, especially magnetic resonance imaging (MRI) pattern recognition, plays a pivotal role in the In general, the treatment of LDs is hampered by the restricted diagnostic process and classification of LDs.3 Classical LDs have access of substances to the CNS. Therapeutic approaches have defects in neurometabolism including lysosomal storage dis- to implicate the need for overcoming the blood–brain barrier. orders (LSDs) like metachromatic leukodystrophy (MLD), per- Thus, potential therapies are based on small molecules with

received © Georg Thieme Verlag KG DOI https://doi.org/ August 16, 2018 Stuttgart · New York 10.1055/s-0039-1685529. accepted after revision ISSN 0174-304X. March 4, 2019 Novel Therapies for Leukodystrophies Schiller et al. high lipophilicity and low molecular weight or on specific cells allogeneic HSCT shows low or no efficiency in the treatment like macrophages or microglia. Another therapeutic approach of patients with late stages of disease or rapidly progressing is bypassing the blood–brain barrier by applying lacking disease courses.12 Furthermore, allogeneic HSCT can be substances including directly into the cerebrospinal complicated by an impeded and delayed identification of fluid (CSF). This mode of CNS application is extensively used in matched donors and potential transplantation-related risks the treatment of brain cancers, in cerebral folate deficiencyand like treatment-related toxicity or graft-versus-host disease. also with only limited success in the treatment of MLD. The transplantation of unrelated donor umbilical cord The increasing number of patients diagnosed with LDs blood (UCB) represents a more accessible source of hema- emphasizes the need for novel therapeutic options. Besides topoietic stem cells. Some cautiously optimistic results have widespread and complex problems that have to be solved in been reported in presymptomatic patients with late-infan- the development and application of new compounds, the tile MLD and patients with juvenile MLD with only minimal proof of treatment efficacy is further hindered by the rarity symptoms.13 Currently, HSCT should be considered as the of LDs and their highly variable clinical onset and course. therapy of choice for early stages of cerebral X-ALD and Wide-ranging insights into the pathogenic mechanisms of juvenile or adult onset MLD. Furthermore, HSCT is a possible neurodegeneration are of uppermost importance and set the treatment approach to attenuate the disease course in early basis for developing effective future neuroprotective and stages of early onset GLD.11 Accordingly, HSCT is a suitable disease modifying therapies. Furthermore, monitoring dis- treatment option for selected LDs but is, even then, limited to ease progression and treatment efficacy is crucial for eval- certain subtypes and early onset diseases. Although the uating novel therapies. Thus, searching for white matter general risk of HSCT including mortality significantly biomarkers is needed. MRI as the follow-up examination of declined within the last decade, one has still to take into choice mainly detects macroscopic lesions. Neurofilament consideration that HSCT can be accompanied by severe side light chain (NfL) as an unspecific marker of neuroaxonal effects such as graft versus host disease and severe life- injury is a potential biomarker for disease activity and axonal threatening infections due to immunosuppression. damage.5 Elevated NfL levels have been shown in children with white matter abnormalities as in newborns with Gene Therapy hypoxic–ischemic encephalopathy6 and in children with The general aim of gene therapy is to restore the function of acquired demyelinating syndromes7 the deficient protein, especially the enzymatic activity of a protein through the insertion of the wild-type form of the Intrathecal Enzyme Replacement Therapy disease gene. Possible techniques comprise in vivo methods The beginning of enzyme replacement therapy (ERT) goes being directly applied to the patient or ex vivo methods back to the early 1960s.8 In 1991, ERT was first applied as a isolating, modifying, and reinfusing target cells, which have pharmaceutical for the treatment of patients with Gaucher the capability to migrate into the CNS.14 An important disease, a LSD.9 At present, ERT is efficiently used for several obstacle for in vivo methods is the blood–brain barrier. metabolic diseases, which are characterized by mostly non- Moreover, immune responses to vectors or transgene pro- cerebral manifestations. The impermeability of the blood– ducts represent major problems. However, the direct and brain barrier for enzymes mandates to circumvent the fast transfer of the wild-type gene into the CSF constitutes a blood–brain barrier and to administer the lacking enzyme beneficial option. “After promising results in MLD mice2 and 15 directly into the CSF. non-human primates an open label study of direct intra- Downloaded by: King's College London. Copyrighted material. This procedure is currently being tested in clinical studies, cerebral administration of a replication deficient adeno- for example, in children with MLD using recombinant human associated virus vector expressing ARSA (ClinicalTrials.gov A via a surgically placed intrathecal drug delivery Identifier: NCT01801709) has been discontinued as without device. A phase ½ study was completed in 2017 (Clinical Trials. effect (personal communication).” Another in vivo clinical gov: NCT01510028, a long-term extension study started trial evaluates the self-inactivating lentiviral vector TYF- recently (Clinical Trials.gov: NCT01887938). Thisfirst attempts ABCD1 in X-ALD patients via intracerebral injection (Clin- to bypass the blood–brain barrier appear promising,10 but icalTrials.gov Identifier: NCT03727555). further long-term efficacy and safety data are needed. Examples for ex vivo methods are the reinfusion of bone marrow-derived CD34þ hematopoietic stem cells transduced Stem Cells with lentiviral vectors carrying human wild-type ARSA com- Allogeneic hematopoietic stem cell transplantation (HSCT) plementary deoxyribonucleic acid (cDNA) in MLD16 (Clinical- has shown to be beneficial for single LDs such as late infantile Trials.gov Identifier: NCT01560182) or wild-type ABCD1 cDNA and juvenile MLD, cerebral form of X-ALD, and globoid cell in cerebral X-ALD17 (ClinicalTrials.gov Identifier: NCT018 leukodystrophy (GLD/Krabbe disease). The ability of donor- 96102). In particular, children with early-onset, late-infantile, derived microglial cells to circumvent the blood–brain bar- or early juvenile MLD or cerebral X-ALD seems to benefitfrom rier and settle in the CNS enables the production and lentiviral HSCTs gene therapy.18,19 secretion of functional and thereby the replacement Gene therapy seems to be an especially beneficial thera- of nonfunctional or lacking enzymes by “cross-correction” peutic option for patients without matching donors for HSCT. similar to a local ERT.11 However, the engraftment and In adult X-ALD patients, one also has to take into account that functioning of the transplanted cells need months. Therefore, these patients have higher risks for fatal complications in

Neuropediatrics Novel Therapies for Leukodystrophies Schiller et al. allogeneic HSCT when compared with children and adoles- administration (ClinicalTrials.gov Identifier: NCT01510028, cents.19 However, further studies to evaluate the long-term ClinicalTrials.gov Identifier: NCT01887938) are ongoing. The efficacy and safety for each LD are still needed for general treatment results for conventional HSCT are controversial.27,28 recommendations. Other approaches refer to the use of UCB13 or HSCT gene therapy16,18 (ClinicalTrials.gov Identifier: NCT01560182, Clin- Drug Repurposing icalTrials.gov Identifier: NCT03392987). Currently, HSCT is an In view of LDs being rare diseases, the costs of developing new available therapy and has proven beneficial for patients with drugs are disproportionately high with only few patients to be early stage juvenile MLD.27 However, the results of the gene treated after approval. Moreover, there are only small numbers therapy study with lentivirally modified stem cells are pro- of patients that can be recruited for a clinical trial in a given mising and should be available soon as a regular therapy. time range to demonstrate efficacy and safety. Therefore, presymptomatic early juvenile MLD patients should The search for overlaps between common and rare diseases at least be considered to undergo gene therapy before treating might be a solution for these problems. Single gene mutations them with allogenic HCST. in rare diseases may disturb well-studied cellular proteins or metabolic pathways overlapping with common diseases for Krabbe Disease (Globoid Cell Leukodystrophy) which effective therapies and drugs with proven safety and GLD represents another characteristic example of an LSD. This pharmacokinetic properties have already been established. autosomal recessive disorder is caused by a deficiency of Such situations open the possibility of identifying new indica- galactocerebrosidase, which catalyzes the hydrolytic degrada- tions for approved drugs and significantly shorten the risky tion of galactocerebroside accumulating in cerebral macro- and costly process of drug development in rare LDs. phages that transform to multinucleated globoid cells. An Examples for successful drug repurposing for patients alternative pathway leads to the formation of toxic psychosine with LDs are the substitution of folate in cerebral folate (galactosphingosine) in oligodendrocytes destructing the mye- deficiency20 and the administration of luteolin and N-acetyl lin sheaths.29 Galactocerebrosidase is encoded by the GALC cysteine in NRF2 activation.21 For both examples, the success gene. In extremely rare cases, GLD is caused by mutations in the is evident. It underlines that drug repurposing is disease saposin A gene encoding for a cerebrosidase activator protein.30 specific and depends largely on the availability of safe and The clinical symptoms of GLD depend on the clinical active substances as well as their accessibility to the brain. course. A classification can be made according to the age at onset as infantile, late infantile, juvenile, and adult form with Innovative Treatment Options—Examples of the latter two being often combined as late forms. Severe Diseases phenotypes with early onset show progressive psychomotor impairment with blindness, deafness, ataxia, spastic tetra- Metachromatic Leukodystrophy paresis, and seizures followed by death within few years. Late LSDs are rare, genetically determined diseases that are pro- onset GLD shows more variable symptoms with progressive voked by deficiencies or malfunctions of lysosomal enzymes neurological disturbances and longer survival.31 or transporters. MLD, a characteristic example for an LSD, is an HSCT is considered in later32 or infantile forms33,34 but has autosomal recessive disorder caused by a deficiency of aryl- proven beneficial for patients with early disease stages. There- A due to mutation in the ARSA gene or, in extremely fore, some States in the United States implemented newborn

rare cases, mutations in the PSAP gene encoding for the ARSA screens for GLD. First results indicate that the prompt initiation Downloaded by: King's College London. Copyrighted material. activator protein saposin B.22 MLD is characterized by an of HSCT in specialized centers shortly after diagnosis is a accumulation of sulfatides in microglia, oligodendrocytes, particular challenge.35 There is also a clinical trial comprising and Schwann cells leading to inflammatory demyelination in the transplantation of human placental-derived stem cells the CNS and PNS.23 The clinical course is heterogeneous (ClinicalTrials.gov Identifier: NCT01586455). depending on the age at onset and rate of progression. Currently, HSCT is the only available therapy. Neverthe- The spectrum of clinical symptoms comprises progressive less, approximately only 10% of enzyme activity is needed to psychomotor retardation, spastic tetraparesis, hearing loss, prevent the neurological implications of GLD. Therefore, and blindness.24 There is some evidence for an at least partial interventions leading to a small increase of residual activity genotype–phenotype correlation with different residual could attenuate GLD symptoms.36 Experimental therapies enzyme activities, leading to distinct clinical courses.25 A with this focus will be discussed further below. late-infantile form (onset before 3 years of age), a juvenile form (onset before 16 years of age), and an adult form with Pelizaeus-Merzbacher Disease mainly cognitive and behavioral problems are known.26 The PMD is an X-linked LD that is caused by duplications, point incidence is estimated around 1/100.000 live births in the mutations, or deletions in the proteolipid protein gene 1 (PLP1) European population.26 encoding the major protein component of the myelinating MLD is one of the more common LDs, for which several oligodendroglia.37 PLP1-related disorders show a genotypic novel therapeutic approaches are ongoing. Clinical trials and phenotypic heterogeneity with a clinical continuum ran- regarding ERTs with recombinant human either ging from severe forms without psychomotor development to by intravenous (ClinicalTrials.gov Identifier: NCT00418561, pure spastic paraparesis. A connatal, a classic, and a transitional ClinicalTrials.gov Identifier: NCT00633139) or intrathecal subtype can be distinguished. Spastic paraplegia type 2 (SPG2)

Neuropediatrics Novel Therapies for Leukodystrophies Schiller et al. and PLP1 null disease are mild variants.38 Additionally, auto- underlining the significance of early diagnoses and immediate somal-recessive mutations in further genes can lead to com- identifications of appropriated allogenic stem cell donors. Best parable phenotypes called PMD-like disease (PMLD), of which outcomes can be expected with cells from related unaffected PMLD-1 due to mutations of the gap junction protein gamma 2 and HLA (human leukocyte antigen)-identical donors, who are (GJC2) gene is the most common.37 not always available.45 Thus, alternative approaches are on the The complete lack of PLP1 leads to milder forms of PMD/ way, for example, using autologous hematopoietic stem cells SPG2 than misfolded PLP1 proteins due to certain missense transduced ex vivo with a lentiviral vector including ABCD1 mutations. Accumulation of misfolded protein in the endo- cDNA. After individual applications19 a phase ⅔ study showed plasmic reticulum and the subsequent triggering of unfolded first promising results17 (Clinical Trials.gov: NCT01896102). protein response enforce apoptosis and play a decisive role in As with MLD, the only therapy currently available is HSCT. the pathogenic mechanisms of PMD.39,40 The results of the ongoing gene therapy trial with lentivirally For PMD there is currently no available efficient therapy. modified stem cells are promising and should also be avail- Transplantation of UCB in two affected children showed slow able as a regular therapy soon. neurocognitive improvements and stable or improved mye- lination,41 but the effect is controversially discussed.42 Allo- Canavan Disease geneic hematopoietic stem cell and umbilical cord-derived Canavan disease is a classical autosomal recessive LD. Pri- cell transplantation can be beneficial in myelin disorders mary cause is mutations in the ASPA gene encoding aspar- with single enzyme defects. Nevertheless, a convincing toacylase that hydrolyzes N-acetyl-L-aspartic acid to L- pathomechanical basis for their benefit in myelin disorders aspartic acid and acetate. The resulting highly elevated with structural protein defects like PMD is still missing. A urinary level of N-acetyl-L-aspartic acid is the diagnostic clinical trial with adjacent intrathecal administration of UCB biomarker.46 Canavan disease is characterized by diffuse cells in patients undergoing standard transplantation of spongiform white matter degeneration and intramyelinic unrelated UCB (ClinicalTrials.gov Identifier: NCT02254863) edema. The severity of the disease course seems to correlate is underway. Another trial is the intracerebral transplanta- with the type of mutation and the resulting residual enzyme tion of neuronal stem cells (ClinicalTrials.gov Identifier: activity.47 The spectrum of symptoms includes macroce- NCT01005004). Even though a clinical benefit could not be phaly, severe developmental delay, hypotonia, blindness, shown yet,43 this innovative treatment proved to be safe and and seizures. So far, there is no treatment. The lack of the further course after transplantation will be explored in available successful therapies reflects the lack of knowledge these patients (ClinicalTrials.gov Identifier: NCT01391637). of the exact disease mechanisms.46 Therapeutic approaches Another approach refers to a cholesterol-enriched diet in comprise gene therapies and substances to reduce the pro- a mouse model for PMD, which improved the clinical phe- duction of L-aspartic acid and acetate to prevent N-acetyl-L- notype and prevented disease progression.44 Unfortunately, aspartic acid accumulation46 (ClinicalTrials.gov Identifier: compassionate use in single patients showed no effect (per- NCT00278707, ClinicalTrials.gov Identifier: NCT00724802). sonal communication). Leukodystrophies Due to Mitochondrial Defects X-Linked Adrenoleukodystrophy Mitochondria have a key function in the energy metabolism X-ALD is characterized by an impaired peroxisomal β-oxida- of cells. A wide variety of defects in either mitochondrial- or

tion of very long-chain fatty acids (VLCFAs; C22) accumu- nuclear-encoded proteins can cause mitochondrial LDs. Downloaded by: King's College London. Copyrighted material. lating in all body fluids and tissues, especially in the cerebral Examples are Kearns–Sayre syndrome, mitochondrial ence- white matter and the adrenal cortex. Primary cause is phalomyopathy with lactic acidosis, and stroke-like episodes mutations in the ABCD1 gene encoding a peroxisomal pro- and Leigh’s disease.48 Mitochondrial disorders show a highly tein, which is involved in the transport of VLCFAs. heterogeneous spectrum of clinical phenotypes and parti- There are at least six distinct clinical phenotypes ranging cularly affect tissues with high energy demand like the CNS from the severe childhood cerebral form to asymptomatic or skeletal and heart muscles. In severe courses, mitochon- individuals, which can vary among affected members within drial disorders show congenital manifestations but disease the same family. Most common phenotypes are the child- onset in middle age is also possible.49 The underlying com- hood onset cerebral form and the adult onset adrenomyelo- plex etiopathology of mitochondrial disorders complicates neuropathy. The clinical symptoms for the childhood onset targeted therapies. Currently a multitude of different ther- cerebral form include behavioral changes, visual and hearing apeutic strategies is under development. A detailed review impairment, dementia, polyneuropathy, spastic paraplegia, has been published recently.50 and Addison disease. Due to the X-linked mode of inheri- tance severe courses predominantly affect males even Aicardi-Goutières Syndrome though a majority of heterozygous females develop symp- Aicardi-Goutières syndrome (AGS) is the prototype of a novel toms later in life. The individual course of disease is unpre- group of monogenetic autoinflammatory and autoimmune dictable; there is no genotype–phenotype correlation. disorders termed type I interferonopathies comprising all Cerebral X-ALD shows good responses to treatment with phenotypes associated with a pathological upregulation of HSCT when administered at first signs of cerebral demyelina- type I signaling. AGS is caused by autosomal tion. In later disease stages, the success of HSCT is limited45 recessive or, in rare cases, autosomal dominant/de novo

Neuropediatrics Novel Therapies for Leukodystrophies Schiller et al. arising heterozygous mutations in seven distinct genes encod- read-through drugs seem to have great potential, especially ing proteins involved in nucleotide metabolism or sensing in LSDs and other LDs in which relative low amounts of (TREX1, SAMHD1, ADAR1, RNASEH2A, RNASEH2B, RNASEH2C, enzyme can lead to an alleviation of clinical symptoms or IFIH1).51 The clinical phenotype of AGS resembles conge- (►Fig. 1).56 Accordingly, in vitro experiments with read- nital viral infection and is highly variable even among affected through drugs show first promising results in LSDs.57 members within the same family. AGS mainly presents as progressive encephalopathy with basal ganglia calcifications Nucleic Acids/Gene Editing Therapies and white matter destruction, concomitant with lymphocy- tosis and increase in type 1 interferon levels in CSF and blood. CRISPR/Cas9-Based Genome Editing Onset ranges from the neonatal period (particularly due to In nearly all cases, current therapies for the treatment of LD TREX1 mutations) to later-onset forms after normal initial can at best only attenuate disease progression. In contrast, psychomotor development (commonly due to RNASEH2B CRISPR/Cas9-based technologies keep the promise to correct mutations).52 There is currently no available efficient therapy. permanently disease-causing mutations by germline and Recent advances in the understanding of the cellular and postnatal genome editing. Studies concerning the mono- molecular pathogenesis of type I interferonopathies have genic disorder Duchenne muscular dystrophy show first enabled novel treatment strategies directed toward reducing success in human cell lines and mouse models.58 However, type I interferon production/activity or blocking type I inter- CRISPR/Cas9 is still in an experimental approach stage. feron-induced signaling.53 First clinical trials with different Before starting human clinical trials possible off-target therapeutic approaches are under way. In AGS patients, effects have to be excluded. Apart from this, the CRISPR/ clinical trials with reverse transcriptase inhibitors are in Cas9 system facilitates the fast and cost-efficient generation progress (ClinicalTrials.gov Identifier: NCT03304717, Clinical- of animal models mimicking human monogenetic diseases. Trials.gov Identifier: NCT02363452). Exon Skipping Future Perspectives Antisense oligonucleotides can modulate the splicing process of immature pre-mRNA by so-called exon skipping. Thus, it is Technologies like MRI and next-generation sequencing possible to restore the reading frame of a transcript to generate result in the discovery of more and more so far unknown a truncated but still functional form of the mutated protein. LDs and expand the phenotypic spectrum of already defined This approach is in ongoing clinical trials for Duchenne mus- disorders. The poor outcome of most of the current clinical cular dystrophy.59 A variety of neurogenetic diseases are treatment approaches emphasizes the need for novel ther- caused by mutations that interfere with the splicing process apeutic options. Furthermore, there is growing awareness making exon skipping to an interesting tool for the treatment that indeed many people are affected by rare diseases even if of LDs, too (►Fig. 1).60 For example, the normal PLP1 splicing a single rare disease affects only a small population. In this pattern could be restored in both a cell and a mouse model for context, increasing public interest led to first national and PMD using an antisense-based approach61,62 international research funding programs in many countries. Monogenetic diseases like LDs several advantages for Gene Silencing the research on innovative therapeutic options: A disease due Gene silencing can be used to reduce the expression of a

to a single gene defect implicates that distinct human cell and disease-causing gene to decrease the level of the mutated Downloaded by: King's College London. Copyrighted material. animal models are available to learn about pathogenic protein under a nontoxic threshold, for example, by using mechanisms. Investigations of single components of extensive antisense oligonucleotides. There are first promising results metabolic pathways allow conclusions concerning cellular from a mouse model for Alexander disease63 and a zebrafish networks with wide implications toward rare and common model for GLD (►Fig. 1).64 diseases. Defects of single genes also offer opportunities to Another approach refers to gene silencing by . Demye- correct the cause of a disease on the primary genetic level. New lination, a characteristic feature of LDs, is counteracted by promising and challenging attempts are as follows. remyelination, a process that is often insufficient in neurode- generative diseases. Remyelination and the associated prolif- Stop Codon Read-Through eration, migration, and differentiation of oligodendrocyte More than 10% of people suffering from monogenetic dis- progenitor cells are modulated by miRNAs (microRNA), which eases carry premature termination codons (PTCs) that result are endogenous, small, noncoding RNAs regulating gene in exon skipping, unstable messenger ribonucleic acids expression.65 Specific miRNAs might serve as therapeutics (mRNAs), or in the synthesis of truncated proteins.54 Some in neurodegenerative diseases like LDs.66 small molecule drugs allow the read-through of PTCs by RNA interference (RNAi) represents another promising incorporation of an amino acid at the mutated site and thus RNA-based therapeutic approach—a post-transcriptional enable the production of longer proteins with improved gene silencing process by using double-stranded RNAs function. So far, the only read-through compound that with one strand being complementary to a specificmRNA. reached clinical trials and drug approval is ataluren for The RNAi can be introduced as synthetic oligonucleotides or Duchenne muscular dystrophy, a genetic disease character- DNA vector templates.67 To date, this approach has not been ized by progressive muscle degeneration.55 Nevertheless, applied for LDs.

Neuropediatrics Novel Therapies for Leukodystrophies Schiller et al.

Fig. 1 Treatment approaches for leukodystrophies. Molecular pathomechanisms and therapeutic approaches for childhood onset leukody- strophies. AGS, Aicardi-Goutières syndrome; Alexander, Alexander disease; Canavan, Canavan disease; CFD, cerebral folate deficiency; GLD, globoid cell leukodystrophy (Krabbe disease); MLD, metachromatic leukodystrophy; NRF2, inborn activating mutations in NFE2L2; PMD, Pelizaeus-Merzbacher disease; X-ALD, X-linked adrenoleukodystrophy.

Pharmacological Chaperons lipids, and micronutrients like folate.71 The potential ability Missense mutations can lead to misfolded proteins and of EVs to serve as nanocarriers for site-specificdrugdeliv- subsequent early protein degradation. Natural chaperons ery72 is of particular interest, and their capability to cross the are proteins that stabilize newly synthesized proteins and blood–brain barrier safely turns them into a promising promote their correct folding. Pharmacological chaperons supply tool for the treatment of CNS diseases.73 belong to the class of so-called small molecules including Until now, the use of EVs is still limited by the insufficient

therapeutic agents with a low molecular weight, which are production quantities and unsatisfactory loading protocols. Downloaded by: King's College London. Copyrighted material. able to regulate biological processes by modifying the However, EVs loaded with specific drugs are currently being function of their target molecules. Accordingly, pharma- tested in animal models and clinical trials, especially for cological chaperons are small molecules facilitating the cancer therapies.74 folding process of a protein under physiologically accep- 68 table concentrations. Another group of compounds with Conclusion similar effects are proteostasis regulators, which are small molecules influencing signaling pathways to manipulate The study of long-term known and recently identified LD the cellular protein homeostasis. Both strategies, sepa- provides wide-ranging insight into the pathogenetic rately or in combination, aim to result in at least partially mechanisms of neurodegeneration in the CNS. It sets the active proteins with correct subcellular localizations.69 The basis for developing effective future neuroprotective and potential effectiveness of these compounds is now studied disease-modifying therapies, which might not be restricted and discussed for numerous LDs like GLD70 and PMD to LDs but also applicable to other common neurodegenera- (►Fig. 1).39,40 tive diseases. Already ongoing and new challenging attempts include stem cell and gene therapies, intrathecal substrate/ Extracellular Vesicles enzyme replacement, genome editing, and others. Extracellular vesicles (EVs) are a group of nanosized natural These innovative single approaches have great potential membrane vesicles released by probably all cell types to their to be the way for future cure, especially combined as indi- extracellular environment. Exosomes, one class of EVs that vidual multimodal therapies. Long-standing comparable and arise from the inward budding of multivesicular bodies, partially successful concepts are already established in can- serve, inter alia, as carriers for the transfer of RNAs, proteins, cer therapies.75,76

Neuropediatrics Novel Therapies for Leukodystrophies Schiller et al.

Conflict of Interest an ad-hoc analysis of a non-randomised, open-label, phase 1/2 Dr. Gärtner reports grants from Deutsche Forschungsge- trial. Lancet 2016;388(10043):476–487 meinschaft GA354/14–1, during the conduct of the study; 17 Eichler F, Duncan C, Musolino PL, et al. Hematopoietic stem-cell gene therapy for cerebral adrenoleukodystrophy. N Engl J Med personal fees from Bayer, personal fees from Biogen, 2017;377(17):1630–1638 personal fees from Novartis, outside the submitted work. 18 Biffi A, Montini E, Lorioli L, et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science 2013;341(6148):1233158 19 Cartier N, Aubourg P. Hematopoietic stem cell transplantation and References hematopoietic stem cell gene therapy in X-linked adrenoleuko- 1 Kevelam SH, Steenweg ME, Srivastava S, et al. Update on leuko- dystrophy. 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