Gene Therapy for Neurodegenerative Diseases: Slowing Down the Ticking Clock.

Gene therapy is an emerging and powerful therapeutic tool to deliver functional genetic material to cells in order to correct a defective gene. During the past decades, several studies have demonstrated the potential of AAV-based gene therapies for the treatment of neurodegenerative diseases. While some clinical studies have failed to demonstrate therapeutic efficacy, the use of AAV as a delivery tool has demonstrated to be safe. Here, we discuss the past, current and future perspectives of gene therapies for neurodegenerative diseases. We also discuss the current advances on the newly emerging RNAi-based gene therapies which has been widely studied in preclinical model and recently also made it to the clinic.

Development of an AAV-Based MicroRNA Gene Therapy to Treat Machado-Joseph Disease.

Spinocerebellar ataxia type 3 (SCA3), or Machado-Joseph disease (MJD), is a progressive neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene. The expanded CAG repeat is translated into a prolonged polyglutamine repeat in the ataxin-3 protein and accumulates within inclusions, acquiring toxic properties, which results in degeneration of the cerebellum and brain stem. In the current study, a non-allele-specific ATXN3 silencing approach was investigated using artificial microRNAs engineered to target various regions of the ATXN3 gene (miATXN3). The miATXN3 candidates were screened in vitro based on their silencing efficacy on a luciferase (Luc) reporter co-expressing ATXN3. The three best miATXN3 candidates were further tested for target engagement and potential off-target activity in induced pluripotent stem cells (iPSCs) differentiated into frontal brain-like neurons and in a SCA3 knockin mouse model. Besides a strong reduction of ATXN3 mRNA and protein, small RNA sequencing revealed efficient guide strand processing without passenger strands being produced. We used different methods to predict alteration of off-target genes upon AAV5-miATXN3 treatment and found no evidence for unwanted effects. Furthermore, we demonstrated in a large animal model, the minipig, that intrathecal delivery of AAV5 can transduce the main areas affected in SCA3 patients. These results proved a strong basis to move forward to investigate distribution, efficacy, and safety of AAV5-miATXN3 in large animals.

AAV5-miHTT Lowers Huntingtin mRNA and Protein without Off-Target Effects in Patient-Derived Neuronal Cultures and Astrocytes.

Huntington disease (HD) is a fatal neurodegenerative genetic disorder, thought to reflect a toxic gain of function in huntingtin (Htt) protein. Adeno-associated viral vector serotype 5 (AAV5)- microRNA targeting huntingtin (miHTT) is a HD gene-therapy candidate that efficiently lowers HTT using RNAi. This study analyzed the efficacy and potential for off-target effects with AAV5-miHTT in neuronal and astrocyte cell cultures differentiated from induced pluripotent stem cells (iPSCs) from two individuals with HD (HD71 and HD180). One-time AAV5-miHTT treatment significantly reduced human HTT mRNA by 57% and Htt protein by 68% in neurons. Small RNA sequencing showed that mature miHTT was processed correctly without off-target passenger strand. No cellular microRNAs were dysregulated, indicating that endogenous RNAi machinery was unaffected by miHTT overexpression. qPCR validation of in silico-predicted off-target transcripts, next-generation sequencing, and pathway analysis confirmed absence of dysregulated genes due to sequence homology or seed-sequence activity of miHTT. Minor effects on gene expression were observed in both AAV5-miHTT and AAV5-GFP-treated samples, suggesting that they were due to viral transduction rather than miHTT. This study confirms the efficacy of AAV5-miHTT in HD patient iPSC-derived neuronal cultures and lack of off-target effects in gene expression and regulation in neuronal cells and astrocytes.

In-Depth Characterization of a Mifepristone-Regulated Expression System for AAV5-Mediated Gene Therapy in the Liver.

Gene therapy is being developed for the treatment of inherited diseases, whereby a therapeutic gene is continuously expressed in patients after delivery via viral vectors such as adeno-associated virus (AAV). Depending on the transgene, there could be a limited therapeutic window, and regulating timing and levels of transgene expression is advantageous. To control transgene transcription, the regulatory system GeneSwitch (GS) was evaluated in detail both in vitro and in vivo. The classical two-plasmid mifepristone (MFP)-inducible GS system was put into one plasmid or a single AAV5 vector. Our data demonstrate the inducibility of multiple transgenes and the importance of promoter and regulatory elements within the GS system. Mice injected with AAV5 containing the GS system transiently expressed mRNA and protein after MFP induction. The inducer MFP could be measured in plasma and liver tissue, and assessment of MFP and its metabolites showed rapid clearance from murine plasma. In a head-to-head comparison, our single vector outclassed the classical two-vector GS system. Finally, we show repeated inducibility of the transgene that also translated into a dynamic phenotypic change in mice. Taken together, this in-depth analysis of the GS system shows its applicability for regulated gene therapy.

Targeting RNA-Mediated Toxicity in C9orf72 ALS and/or FTD by RNAi-Based Gene Therapy.

A hexanucleotide GGGGCC expansion in intron 1 of chromosome 9 open reading frame 72 (C9orf72) gene is the most frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The corresponding repeat-containing sense and antisense transcripts cause a gain of toxicity through the accumulation of RNA foci in the nucleus and deposition of dipeptide-repeat (DPR) proteins in the cytoplasm of the affected cells. We have previously reported on the potential of engineered artificial anti-C9orf72-targeting miRNAs (miC) targeting C9orf72 to reduce the gain of toxicity caused by the repeat-containing transcripts. In the current study, we tested the silencing efficacy of adeno-associated virus (AAV)5-miC in human-derived induced pluripotent stem cell (iPSC) neurons and in an ALS mouse model. We demonstrated that AAV5-miC transduces different types of neuronal cells and can reduce the accumulation of repeat-containing C9orf72 transcripts. Additionally, we demonstrated silencing of C9orf72 in both the nucleus and cytoplasm, which has an added value for the treatment of ALS and/or FTD patients. A proof of concept in an ALS mouse model demonstrated the significant reduction in repeat-containing C9orf72 transcripts and RNA foci after treatment. Taken together, these findings support the feasibility of a gene therapy for ALS and FTD based on the reduction in toxicity caused by the repeat-containing C9orf72 transcripts.

Artificial MicroRNAs Targeting C9orf72 Can Reduce Accumulation of Intra-nuclear Transcripts in ALS and FTD Patients.

The most common pathogenic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic GGGGCC (G4C2) repeat in the chromosome 9 open reading frame 72 (C9orf72) gene. Cellular toxicity due to RNA foci and dipeptide repeat (DPR) proteins produced by the sense and antisense repeat-containing transcripts is thought to underlie the pathogenesis of both diseases. RNA sequencing (RNA-seq) data of C9orf72-ALS patients and controls were analyzed to better understand the sequence conservation of C9orf72 in patients. MicroRNAs were developed in conserved regions to silence C9orf72 (miC), and the feasibility of different silencing approaches was demonstrated in reporter overexpression systems. In addition, we demonstrated the feasibility of a bidirectional targeting approach by expressing two concatenated miC hairpins. The efficacy of miC was confirmed by the reduction of endogenously expressed C9orf72 mRNA, in both nucleus and cytoplasm, and an ∼50% reduction of nuclear RNA foci in (G4C2)44-expressing cells. Ultimately, two miC candidates were incorporated in adeno-associated virus vector serotype 5 (AAV5), and silencing of C9orf72 was demonstrated in HEK293T cells and induced pluripotent stem cell (iPSC)-derived neurons. These data support the feasibility of microRNA (miRNA)-based and AAV-delivered gene therapy that could alleviate the gain of toxicity seen in ALS and FTD patients.

Design, Characterization, and Lead Selection of Therapeutic miRNAs Targeting Huntingtin for Development of Gene Therapy for Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by accumulation of CAG expansions in the huntingtin (HTT) gene. Hence, decreasing the expression of mutated HTT (mtHTT) is the most upstream approach for treatment of HD. We have developed HTT gene-silencing approaches based on expression cassette-optimized artificial miRNAs (miHTTs). In the first approach, total silencing of wild-type and mtHTT was achieved by targeting exon 1. In the second approach, allele-specific silencing was induced by targeting the heterozygous single-nucleotide polymorphism (SNP) rs362331 in exon 50 or rs362307 in exon 67 linked to mtHTT. The miHTT expression cassette was optimized by embedding anti-HTT target sequences in ten pri-miRNA scaffolds and their HTT knockdown efficacy, allele selectivity, passenger strand activity, and processing patterns were analyzed in vitro. Furthermore, three scaffolds expressing miH12 targeting exon 1 were incorporated in an adeno-associated viral serotype 5 (AAV5) vector and their HTT knock-down efficiency and pre-miHTT processing were compared in the humanized transgenic Hu128/21 HD mouse model. Our data demonstrate strong allele-selective silencing of mtHTT by miSNP50 targeting rs362331 and total HTT silencing by miH12 both in vitro and in vivo. Ultimately, we show that HTT knock-down efficiency and guide strand processing can be enhanced by using different cellular pri-miRNA scaffolds.

Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig Loci in activated B cells.

After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen-binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading to antibody class switch recombination (CSR). We asked if, during B cell activation, AID also induces DNA breaks at genes other than IgH genes. Using a nonbiased genome-wide approach, we have identified hundreds of reproducible, AID-dependent DSBs in mouse splenic B cells shortly after induction of CSR in culture. Most interestingly, AID induces DSBs at sites syntenic with sites of translocations, deletions, and amplifications found in human B cell lymphomas, including within the oncogene B cell lymphoma11a (bcl11a)/evi9. Unlike AID-induced DSBs in Ig genes, genome-wide AID-dependent DSBs are not restricted to transcribed regions and frequently occur within repeated sequence elements, including CA repeats, non-CA tandem repeats, and SINEs.