Motor neuron disorders
Spinal muscular atrophy (SMA) is caused by homozygous LoF mutations in the
SMN1 gene that is ubiquitously expressed in the body. The age of onset and the severity of the phenotype (SMA type 1–4) depend on the copy number of the homologous
SMN2 gene. The mature
SMN2 mRNA differs from the
SMA1 mRNA by exclusion of exon 7 following alterative splicing, which results in instability and reduced amounts of the protein product. Two gene specific therapies have been developed that mediate the inclusion of exon 7 into the mature
SMN2 mRNA: the ASO Nusinersen (Fig.
1B) and the small molecule Risdiplam (Fig.
1C). Nusinersen (NCT02193074 [
11], NCT02292537 [
12]) is administered intrathecally every 4 months after a 1-year dosing phase. Since its approval it has revolutionized the treatment of infantile- and later-onset SMA [
13]. Meanwhile there is robust evidence that adult patients with SMA also benefit from a therapy with Nusinersen [
14]. Contrary to Nusinersen, Risdiplam is a small molecule and is given orally. It could in principle reach all tissues of the body. In a phase 3 study it has shown a similar clinical effectiveness as Nusinersen for SMA type 1 (i.e. infantile SMA) and is also effective in the other SMA types (NCT02908685, NCT02913482, NCT03032172, NCT03779334). Its approval for all forms of SMA is expected soon. With Branaplam another small molecule inducing exon 7 inclusion is being tested in a phase 1/2 trial (NCT02268552). The third gene specific therapy approach against SMA aims at restoring SMN1 expression through AAV9-mediated delivery of a functional SMN1 gene copy under control of a CMV promoter (Onasemnogene abeparvovec-xioi [Zolgensma]) (Fig.
1E). Onasemnogene abeparvovec-xioi, which is administered i.v. as a single dose during the first 2 years of life, has shown an excellent therapeutic effect and a sufficient safety profile in infants with SMA type 1 (NCT03306277) [
15]. However, a trial with intrathecal administration of a single dose of Onasemnogene abeparvovec-xioi was stopped by the FDA after a preclinical study had revealed dorsal root ganglia (DRG) mononuclear cell inflammation in non-human primates. The drug has meanwhile been approved for i.v. administration by the FDA and EMA.
Nucleocytoplasmic translocation of TDP-43 with formation of cytosolic pTDP-43 inclusions that might cause a nuclear LoF and cytoplasmic GoF toxicity is the prevailing neuropathology in
sporadic ALS. The propagation of TDP-43 pathology in the CNS correlates with the spreading of the clinical symptoms [
16]. Corroborating the significance of TDP-43 in ALS, mutations in its gene (
TARDBP) cause genetic forms of ALS [
17,
18]. Consequently, the TDP-43 pathology appears a reasoned pharmacological target. However, the reduction of TDP-43 expression itself is not a suitable therapeutic approach, since TDP-43 is physiologically essential. Mice with homozygous
Tardbp deletion are not viable and those with heterozygous KO develop an ALS-like phenotype [
19]. Therefore, approaches must aim at reducing the pTDP-43 inclusions or compensate for the loss of physiological function of TDP-43. Following this thought, an ASO approach has been developed to downregulate the
XPO1 mRNA (BIIB100), which codes for the protein Exportin 1. Exportin 1mediates the nuclear export of many proteins containing nuclear export signals including TDP-43 [
20] (Fig.
1A). Thus,
XPO1 inhibition is supposed to reduce nucleocytoplasmic translocation and cytosolic aggregation of TDP-43 [
21]. The recruitment of sporadic ALS patients for a respective phase 1/2 study has just started at the end of 2019 (NCT03945279).
Intermediate-length polyglutamine expansions in the
ATXN2 gene are associated with an increased risk of ALS. TDP-43 mutant mice with
Atxn2-KO or treated with ASOs downregulating
Atxn2 show a reduction of phosphorylated TDP-43 inclusions and a dramatically increased lifespan [
22] (Fig.
1A). Consequently, an ASO downregulating human
ATXN2 is being developed towards a clinical trial in sporadic ALS patients.
An intronic hexanucleotide repeat expansion in
C9Orf72 and missense mutations in
SOD1 are the most frequent causes of genetic ALS
(C9-ALS and
SOD1-ALS) in Europe [
23]. It is supposed that their pathogenicity is predominantly based on a GoF toxicity, although a LoF aspect is also discussed for
C9Orf72 mutations. After most promising results in mutant disease models, ASOs downregulating the expression of
SOD1 (Tofersen/BIIB067/IONIS-SOD1
Rx; allele unselective) and
C9Orf72 (BIIB078/IONIS-C9
Rx; allele selective) through RNase H mediated mRNA degradation are now being tested in clinical trials (Fig.
1A). The trial testing IONIS-SOD1
Rx is presently in phase 3 (NCT02623699); an interim analysis of phase 2 has raised hope for a positive study outcome [
24]. The IONIS-C9
Rx trial is currently in phase 2 (NCT03626012), there are no interim results available yet. Further, another consortium is developing an allele-selective silencing ASO towards clinical translation for C9-ALS and -FTD. AAV mediated gene delivery of DNA coding miRNA or shRNA that downregulate
SOD1 mRNA showed excellent efficacy in mutant hSOD1 rodent or primate models [
25] (Fig.
1F). Consequently, an AAV9-
SOD1-shRNA candidate is being developed towards clinical translation by another consortium.
Movement disorders
The current drug therapy of
idiopathic Parkinson’s disease (iPD) aims at restoring CNS dopamine levels. Two sponsors are currently testing MR-guided intraputaminal AAV2-delivered
AADC gene transfer in iPD patient in phase 1/2 trials (NCT02418598, NCT01973543). The
AADC gene encodes the aromatic L-amino acid decarboxylase responsible for converting Levodopa to Dopamine. An interim analysis of NCT01973543 has shown an increase in enzyme expression and dose dependent clinical improvements [
26]. While this approach might prolong the time of symptom control it is probably – like current dopaminergic drugs – incapable of influencing disease progression. Alpha-synuclein (a-syn) aggregations are the predominant neuropathology of iPD; its propagation in the CNS correlates with the spreading of the clinical symptoms [
27]. The significance of a-syn in iPS is further supported by the findings that multiplications, mutations, and single nucleotide polymorphisms in the
SNCA gene, encoding the alpha-synuclein protein, either cause or increase the risk for iPS. Cole and colleagues developed an ASO that downregulates the
SCNA mRNA after intraventricular application. It efficiently reduced seeded a-syn inclusion load in wildtype mice and rats treated with exogenous preformed a-syn fibrils [
28] (Fig.
1A). This therapy has not yet reached the clinical trial phase. Another approach targeting the a-syn pathology consists in the downregulation of
LRRK2. Missense mutations in
LRRK2 that lead to a GoF are a frequent cause of genetic PD [
29,
30] and certain polymorphisms in
LRRK2 locus modulate the risk for iPD [
31]. In addition, increased LRRK2 protein activity in dopaminergic neurons in post-mortem tissue of iPD patients seems to drive the α-syn pathology [
32]. Thus, downregulation of LRRK2 appears an interesting therapy strategy to alleviate α-syn pathology. Indeed, ASOs reducing expression of the
LRRK2 mRNA diminished fibril-induced seeding of a-syn inclusions in wildtype mice [
28] (Fig.
1A). Consequently, a pharma consortium is testing an ASO targeting the human
LRRK2 mRNA (BIIB094/ION859) in a phase 2 trial in PD patients with or without
LRRK2 GoF mutations (NCT03976349). In case the strategies reducing a-syn pathology are clinically effective they might also be tested in other synucleinopathies, such as multiple system atrophy (MSA) and Lewy body dementia (LBD).
Mutations in the
GBA1 gene, which presumably lead to a LOF, are the most frequent genetic contributor to PD pathogenesis (
GBA-PD) [
32].
GBA1 encodes the enzyme beta-glucocerebrosidase, which is required for the disposal and recycling of glycolipids. Accumulation of glycolipids leads to lysosomal dysfunction that in turn exacerbates lysosomal accumulation of a-syn. Recently, a phase 1/2 trial has been launched in patients with a pathogenic
GBA1 mutation (PD-GBA) testing a gene reconstitution therapy where a
GBA1 gene copy is delivered by an AAV9 (NCT04127578) (Fig.
1E). The drug is administered intracisternally as a single dose. A phase 1/2 trial testing the same therapeutic in patients with neuropathic Gaucher disease, that is caused by biallelic LoF mutations in the same gene, is expected to start soon. Further, the same sponsor announces the development of an approach combining
GBA1 gene transfer and
SCNA knockdown for treatment of synucleinopathies in general.
A CAG repeat expansion in the
HTT gene that encodes the Huntingtin protein causes
Huntington’s disease (HD). After most promising results in preclinical rodent studies [
33], several gene-specific therapeutic strategies are being tested in clinical trials in HD patients. In NCT03761849, currently in phase 3, an allele unselective ASO that downregulates pan-
HTT mRNA (Tominersen/RG6042/IONIS-HTT
Rx) is administered intrathecally (Fig.
1A). An interim analysis has demonstrated successful target engagement in that the HTT protein was reduced by 40% on average in the CSF of HD patients [
34]. In NCT03225833 and NCT03225846, currently in phase 1/2, allele specific ASOs (targeting a prevalent SNP) selectively downregulate the mutant
HTT mRNA after intrathecal adminstration (Fig.
1A). Third, in NCT04120493, currently in phase 1/2, an AAV5 vector is used to deliver a gene encoding a miRNA that blocks the HTT mRNA (
miHTT). This vector is administered by intrastriatal injection (Fig.
1F).
Dementias
Tau and beta-amyloid aggregates are the neuropathological hallmarks of
sporadicAlzheimer’s disease (AD). Mutations in the amyloid precursor protein gene
APP cause genetic AD through a GoF mechanism. However, beta-amyloid immunotherapy has been largely disappointing in clinical trials so far, questioning beta-amyloid as the ideal drug target, at least in progressed AD cases. According to autopsy and Tau-PET imaging studies spreading of Tau pathology seems to be highly correlated with disease progression [
35,
36], which raises hopes for a successful clinical translation of Tau targeted therapies. Genetic deletion or ASO-mediated downregulation of the
MAPT mRNA (encoding Tau protein) alleviated neuropathology and clinical symptoms in genetic AD mouse models. Consequently, an ASO designed to downregulate the
MAPT mRNA is being tested in AD patients in a phase 1/2 clinical trial (NCT03186989) (Fig.
1A). If this strategy proves successful it could be also tested in other tauopathies, such as the atypical Parkinson syndromes supranuclear palsy (PSP) and corticobasal syndrome (CBS).
Sporadic frontotemporal dementia (FTD) is neuropathologically associated with TDP-43, Tau or FUS protein pathology that each is considered the cause of the clinical symptoms. Therapies aiming at alleviating these proteinopathies are currently being tested in other indications (BIIB100 for TDP-43 pathology in ALS; BIIB080 for Tau pathology in AD, see respective paragraphs above). They might also be tested for the treatment of FTD patients, if the according ALS or AD studies are positive. Another prerequisite would be the availability of PET imaging and respective tracers that allow to reliably identify the underlying proteinopathy. The three most frequently mutated genes in
genetic FTD are
C9Orf72,
GRN, and
MAPT (with an autosomal dominant inheritance in all three cases). As described before, ASOs downregulating
C9Orf72 or
MAPT (both predominantly causing a GoF effect when mutated) are currently being tested in C9-ALS and sporadic AD patients and may be tested in C9-FTD cohorts next (see above). Mutations in
GRN cause FTD by a LOF and aggravate TDP-43 inclusion pathology. An AAV9-based gene reconstitution therapy for GRN-FTD is currently developed towards a phase 1/2 trial (Fig.
1E).
Polyneuropathies
Hereditary amyloidosis is caused by GoF mutations in the
ATTR gene leading to an abnormal, aggregation-prone TTR protein that is deposited in amyloid aggregates. The amyloidosis causes cardiomyopathy and/or PNP, also called
familial amyloid PNP (FAP). Two gene specific therapies based on RNA interference, Inotersen and Patisiran, have been developed and approved for the treatment of PNP caused by
hATTR amyloidosis. Inotersen is an ASO that is administered s.c. once per week, while Patisiran is a first-in-class siRNA therapeutic that is given i.v. every 3 weeks. They lead to the degradation of the mutant and the wildtype
hATTR mRNA through RNAse H (Inoteresen) (Fig.
1A) and RISC (Patisiran) (Fig.
1D), respectively. Longer lasting compounds based on the same therapeutic principles (AKCEA-TTR-LRx and Vutrisiran) are currently being tested by the same sponsors in phase 3 trials (NCT04136184 and NCT04153149).
Giant axonal neuropathy (GAN) is a very rare, autosomal recessive childhood onset disease owing to LoF mutations in the
GAN gene. It encodes the protein Gigaxonin. The mutations cause a progressive accumulation of neuronal intermediate filaments in axons. After a successful preclinical study [
37] a phase 1 trial is recruiting patients to test the intrathecal administration of an AAV9 vector to deliver a functional copy of the
GAN gene (NCT02362438) (Fig.
1E).
Duplications of the
PMP22 gene cause the most prevalent subtype of CMT,
CMT1A. Zhao et al. have developed an ASO to downregulate
PMP22 mRNA. After s.c. administration the ASO results in restoration of myelination and improvement of electroneurographic parameters in a mouse model based on overexpression of he human
PMP22 gene [
38] (Fig.
1A). A clinical trial has not yet been announced. Further, the upregulation of neurotrophin-3 (NT-3) has been shown to lead to a remyelination in CMT1A mouse models and patients [
39]. A gene transfer using an AAV1 vector has shown good efficacy in
PMP22 mutant mice [
40]. A clinical trial in CMT1A patients is underway (NCT03520751) (Fig.
1E). Preclinical studies testing gene reconstitution therapies have also been successful in mouse models of other CMT types [
41].
Muscle diseases
Muscle dystrophy is a X-linked genetic myopathy caused by mutations in the dystrophin gene
DMD. Depending on the residual function of the protein product, mutations either lead to the more severe phenotype of Duchenne muscular dystrophy (DMD) (complete LoF) or the milder form of Becker muscular dystrophy (partial LoF). Two ASO therapeutics (Eteplirsen and Golodirsen) have been approved for the treatment of DMD in 2016 and 2010, respectively, by the FDA, while the European authority EMA rejected their approval for reasons discussed below [
42]. Eteplirsen and Golodirsen, given s.c. or i.v. weekly, cause a skipping of exon 51 or 53, respectively, of the Dystrophin pre-mRNA. This strategy results in a shortened instead of an otherwise unfunctional protein (Fig.
1B). Both ASOs are restricted to DMD patients with mutations in exon 51 or 53, respectively. However, severe shortcomings of the clinical studies that led to their approval and serious SAEs have raised significant doubts about their efficacy and safety [
43,
44]. The FDA has instructed the responsible company to provide more robust evidence for the clinical effectiveness of both ASO therapeutics in post marketing studies by 2021 (Eteplirsen) and 2023 (Golodirsen), respectively. Multiple other ASOs that lead to skipping of various
DMD exons are being tested in clinical trials [
45]. Beyond the aforementioned ASO therapeutics, the small molecule Ataluren has been approved for treatment of patients with nonsense
DMD mutations, which produce a premature stop codon, after showing some clinical benefit [
46]. Ataluren causes the ribosomal readthrough of mRNAs with a premature stop codon and consequent translation of the complete protein. Further, multiple AAV-based i.v. gene transfer therapies have been developed and are being tested in phase 1/2 trials in DMD patients (see Table
1) (Fig.
1E). Since the
Dystrophin gene exceeds the packaging capacity of AAVs it has been shortened to the essential domains in these cases, called micro- or mini-dystrophin-genes.
Limb-girdle muscular dystrophy (LGMD) is a slowly progressive, symmetric, proximal myopathy with onset in childhood or adolescence. It is caused by mono- or biallelic LoF mutations in various genes encoding sarcoglycans, which tie the intracellular cytoskeleton to the extracellular matrix in muscle tissue. According to the mode of inheritance it is classified into LGMD1 (autosomal dominant) and LGMD2 (autosomal recessive). After successful preclinical studies in mice [
47,
48] AAV gene reconstitution therapies that supply healthy copies of the mutated genes have been developed and are being tested in phase 1/2 trials for the autosomal recessive LGMD2 forms D and E (NCT01976091 and NCT03652259 (Fig.
1E).
Pompe disease is a progressive myopathy and autosomal recessively inherited disorder caused by biallelic LoF mutations in the
GAA gene.
GAA encodes the lysosomal acidic alpha-glucosidase. Respective mutations lead to lysosomal accumulation of glycogen. After promising results from preclinical mouse studies [
49], AAV-mediated expression of
GAA in hepatocytes by a single i.v. infusion of the viral vector is now tested in Pompe disease patients in phase 1/2 trials (NCT04093349 and NCT03533673) (Fig.
1E). Perspectively, ASOs reducing glycogen synthesis, which are currently being developed for the treatment of Lafora disease, might be a general therapeutic option for glycogenoses.
Centronuclear Myopathy (CNM) is a group of congenital myopathies characterized by abnormal localization of the nucleus in the center of muscles cells. Mutations in several genes have been made responsible for CNM. The most severe from is X-linked CNM (XL-CNM; syn. Myotubular myopathy) is caused by LoF mutations in the
MTM1 gene, while autosomal dominant CNM (AD-CNM) is mostly caused by GoF mutations in the
DNM2 gene.
Mtm1-KO causes an overexpression of DNM2 and systemic administration of an ASO downregulating
Dnm2 mRNA prevented and reverted myotubular myopathy in
Mtm1-KO mice [
50]. Consequently, a consortium is testing its ASO candidate IONIS-DNM2–2.5
Rx (DYN101) that is administered i.v. in patients with centronuclear myopathies caused by mutations in either
DNM2 or
MTM1 (NCT04033159). The study is currently in phase 2. Further, Audentes Therapeutics is testing an AAV8-delivered replacement of the
MTM1 gene (AT132) by single dose i.v. administration in patients with XL-CNM in a phase 1/2 study (NCT03199469). Therapeutic efficacy has already been shown in
Mtm1-KO mice and XLMTM dogs before. An interim analysis has yielded promising results [
51].
Other neurological indications
Leber hereditary optic neuropathy (LHON) is a maternally inherited mitochondrial disease characterized by the degeneration of retinal ganglion cells and their axons leading to vision loss. It is caused by LoF mutations in the genes
ND4,
ND1 and
ND6 encoding the mitochondrial NADH dehydrogenase proteins. AAV transfer of a healthy
ND4 gene copy after intraocular administration prevented retinal ganglion cell degeneration and preserved visual function in a LHON rat model [
52]. GenSight Biologics is testing an AAV2 gene therapy delivering a
ND4 gene copy in a phase 3 trial in LHON patients with
ND4 mutation (NCT02652780, NCT02652767, NCT03293524) (Fig.
1E).
Neuronal ceroid lipofuscinoses (CLN) a group of rare rare, childhood-onset and fatal generally autosomal recessive genetic neurodegenerative lysosomal storage diseases caused by mutations in various genes (CLN1–7). The disease is characterized by symptomatic epilepsy and progressive decline of cognitive and motor functions. After successful preclinical studies in animals [
53] trials for the different CLN types testing intrathecal or intracranial administration of single-dose AAV-based gene delivery of the
CLN2,
CLN3 or
CLN6 gene have been launched by various sponsors and are currently ongoing (CLN2: NCT01414985, NCT01161576; CLN3: NCT03770572; CLN6: NCT02725580).
Fabry Disease is a X-linked genetic lysosomal storage disorder caused by mono- or biallelic LoF mutations in the
GLA gene and affects men and women. The mutations lead to a deficiency of the alpha-galactosidase, which causes ubiquitous accumulation of glycosphingolipids in lysosomes. This leads to multi-organ dysfunction and polymorphic symptoms, amongst others neuropathy and strokes. After successful preclinical studies in mouse models of Fabry disease [
54,
55], Freeline Therapeutics and Sangamo Therapeutics are already testing i.v. single dose AAV gene replacement therapies in phase 1/2 trials in Fabry disease patients (NCT04046224 and NCT04040049) (Fig.
1E).