Revisiting the outcome of adult wild-type Htt inactivation in the context of HTT-lowering strategies for Huntington's disease.

Huntingtin-lowering strategies are central to therapeutic approaches for Huntington's disease. Recent studies reported the induction of age- and cell type-specific phenotypes by conditional huntingtin knockout, but these experimental conditions did not precisely mimic huntingtin-lowering or gene-editing conditions in terms of the cells targeted and brain distribution, and no transcriptional profiles were provided. Here, we used the adeno-associated delivery system commonly used in CNS gene therapy programmes and the self-inactivating KamiCas9 gene-editing system to investigate the long-term consequences of wild-type mouse huntingtin inactivation in adult neurons and, thus, the feasibility and safety of huntingtin inactivation in these cells. Behavioural and neuropathological analyses and single-nuclei RNA sequencing indicated that huntingtin editing in 77% of striatal neurons and 16% of cortical projecting neurons in adult mice induced no behavioural deficits or cellular toxicity. Single-nuclei RNA sequencing in 11.5-month-old animals showed that huntingtin inactivation did not alter striatal-cell profiles or proportions. Few differentially expressed genes were identified and Augur analysis confirmed an extremely limited response to huntingtin inactivation in all cell types. Our results therefore indicate that wild-type huntingtin inactivation in adult striatal and projection neurons is well tolerated in the long term.

Limitations of Dual-Single Guide RNA CRISPR Strategies for the Treatment of Central Nervous System Genetic Disorders.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a toxic gain-of-function CAG expansion in the first exon of the huntingtin (HTT) gene. The monogenic nature of HD makes mutant HTT (mHTT) inactivation a promising therapeutic strategy. Single nucleotide polymorphisms frequently associated with CAG expansion have been explored to selectively inactivate mHTT allele using the CRISPR/Cas9 system. One of such allele-selective approaches consists of excising a region flanking the first exon of mHTT by inducing simultaneous double-strand breaks at upstream and downstream positions of the mHTT exon 1. The removal of the first exon of mHTT deletes the CAG expansion and important transcription regulatory sites, leading to mHTT inactivation. However, the frequency of deletion events is yet to be quantified either in vitro or in vivo. Here, we developed accurate quantitative digital polymerase chain reaction-based assays to assess HTT exon 1 deletion in vitro and in fully humanized HU97/18 mice. Our results demonstrate that dual-single guide RNA (sgRNA) strategies are efficient and that 67% of HTT editing events are leading to exon 1 deletion in HEK293T cells. In contrast, these sgRNA actively cleaved HTT in HU97/18 mice, but most editing events do not lead to exon 1 deletion (10% exon 1 deletion). We also showed that the in vivo editing pattern is not affected by CAG expansion but may potentially be due to the presence of multiple copies of wildtype (wt)/mHTT genes HU97/18 mice as well as the slow kinetics of AAV-mediated CRISPR/Cas9 delivery.

Scalable Production of AAV Vectors in Orbitally Shaken HEK293 Cells.

Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo. Following upstream processing, we purified vectors via gradient centrifugation and immunoaffinity chromatography. The in vitro characterization revealed differences due to the purification method, as well as the transfection protocol and the corresponding HEK293 cell line. The purification method and cell line used also affected in vivo transduction efficiency after bilateral injection of AAV2/9 vectors expressing a GFP reporter fused with a nuclear localization signal (AAV2/9-CBA-nlsGFP) into the striatum of adult mice. These results show that AAV vectors deriving from suspension HEKExpress cells are bioequivalent and may exhibit higher potency than vectors produced with adherent HEK293 cells.

Therapeutic efficacy of regulable GDNF expression for Huntington's and Parkinson's disease by a high-induction, background-free "GeneSwitch" vector.

Gene therapy is currently an irreversible approach, without possibilities to fine-tune or halt the expression of a therapeutic gene product. Especially when expressing neurotrophic factors to treat neurodegenerative disorders, options to regulate transgene expression levels might be beneficial. We thus developed an advanced single-genome inducible AAV vector for expression of GDNF, under control of the approved small molecule drug mifepristone. In the rat brain, GDNF expression can be induced over a wide range up to three hundred-fold over endogenous background, and completely returns to baseline within 3-4 weeks. When applied with appropriate serotype and titre, the vector is absolutely free of any non-induced background expression. In the BACHD model of Huntington's disease we demonstrate that the vector can be kept in a continuous ON-state for extended periods of time. In a model of Parkinson's disease we demonstrate that repeated short-term expression of GDNF restores motor capabilities in 6-OHDA-lesioned rats. We also report on sex-dependent pharmacodynamics of mifepristone in the rodent brain. Taken together, we show that wide-range and high-level induction, background-free, fully reversible and therapeutically active GDNF expression can be achieved under tight pharmacological control by this novel AAV - "Gene Switch" vector.

CRISPR/Cas9-Mediated Genome Editing for Huntington's Disease.

This chapter describes the potential use of viral-mediated gene transfer in the central nervous system for genome editing in the context of Huntington's disease. Here, we provide protocols that cover the design of various genome editing strategies, the cloning of CRISPR/Cas9 elements into lentiviral vectors, and the assessment of cleavage efficiency, as well as potential unwanted effects.

The Self-Inactivating KamiCas9 System for the Editing of CNS Disease Genes.

Neurodegenerative disorders are a major public health problem because of the high frequency of these diseases. Genome editing with the CRISPR/Cas9 system is making it possible to modify the sequence of genes linked to these disorders. We designed the KamiCas9 self-inactivating editing system to achieve transient expression of the Cas9 protein and high editing efficiency. In the first application, the gene responsible for Huntington's disease (HD) was targeted in adult mouse neuronal and glial cells. Mutant huntingtin (HTT) was efficiently inactivated in mouse models of HD, leading to an improvement in key markers of the disease. Sequencing of potential off-targets with the constitutive Cas9 system in differentiated human iPSC revealed a very low incidence with only one site above background level. This off-target frequency was significantly reduced with the KamiCas9 system. These results demonstrate the potential of the self-inactivating CRISPR/Cas9 editing for applications in the context of neurodegenerative diseases.