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.

A randomized, blinded study of photobiomodulation in a mouse model of Alzheimer's disease showed no preventive effect.

Photobiomodulation (PBM), the process of exposing tissue to red or near-infrared light, has become a topic of great interest as a therapy for diverse pathologies, including neurodegenerative disorders. Here, we aimed to evaluate the potential beneficial effect of PBM on Alzheimer's disease (AD) using behavioral and histological readouts from a well-established transgenic murine AD model (5xFAD mice) in a randomized and fully blinded long-term in-vivo study following GLP (Good Laboratory Practices) guidelines. The heads of the mice were illuminated with no (sham), low or high power 810 nm light, three times a week for 5 months from the first to the sixth month of life corresponding to the prodromal phase of the pathology. The results showed that there were no significant differences between the groups in behavioral tests, including the Morris water maze, novel object recognition, and Y-maze. Similarly, histological analyses showed no differences in amyloid load, neuronal loss or microglial response. In conclusion, under the conditions of our experiment, we were unable to demonstrate any therapeutic effect of PBM for AD. This study calls for further evidence and caution when considering PBM as an effective treatment for AD.

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.

Semi-automated workflows to quantify AAV transduction in various brain areas and predict gene editing outcome for neurological disorders.

One obstacle to the development of gene therapies for the central nervous system is the lack of workflows for quantifying transduction efficiency in affected neural networks and ultimately predicting therapeutic potential. We integrated data from a brain cell atlas with 3D or 2D semi-automated quantification of transduced cells in segmented images to predict AAV transduction efficiency in multiple brain regions. We used this workflow to estimate the transduction efficiency of AAV2/rh.10 and AAV2.retro co-injection in the corticostriatal network affected in Huntington's disease. We then validated our pipeline in gene editing experiments targeting both human and mouse huntingtin genes in transgenic and wild-type mice, respectively. Our analysis predicted that 54% of striatal cells and 7% of cortical cells would be edited in highly transduced areas. Remarkably, in the treated animals, huntingtin gene inactivation reached 54.5% and 9.6%, respectively. These results demonstrate the power of this workflow to predict transduction efficiency and the therapeutic potential of gene therapies in the central nervous system.

Maximizing lentiviral vector gene transfer in the CNS.

Gene transfer is a widely developed technique for studying and treating genetic diseases. However, the development of therapeutic strategies is challenging, due to the cellular and functional complexity of the central nervous system (CNS), its large size and restricted access. We explored two parameters for improving gene transfer efficacy and capacity for the selective targeting of subpopulations of cells with lentiviral vectors (LVs). We first developed a second-generation LV specifically targeting astrocytes for the efficient expression or silencing of genes of interest, and to better study the importance of cell subpopulations in neurological disorders. We then made use of the retrograde transport properties of a chimeric envelope to target brain circuits affected in CNS diseases and achieve a broad distribution. The combination of retrograde transport and specific tropism displayed by this LV provides opportunities for delivering therapeutic genes to specific cell populations and ensuring high levels of transduction in interconnected brain areas following local administration. This new LV and delivery strategy should be of greater therapeutic benefit and opens up new possibilities for the preclinical development of gene therapy for neurodegenerative diseases.