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Rescuing Cognitive Deficits in Neurodevelopmental Disorders by Gene Editing in Brain Development: the Case of Down Syndrome

Periodic Reporting for period 2 - GenEdiDS (Rescuing Cognitive Deficits in Neurodevelopmental Disorders by Gene Editing in Brain Development: the Case of Down Syndrome)

Reporting period: 2019-04-01 to 2020-09-30

Neurodevelopmental disorders (ND) are chronic psychiatric conditions with different etiologies, but most share a strong genetic component, defective brain development, and cognitive impairment. Currently, treatment options are very limited, and early educational intervention is the cornerstone for the management of cognitive impairment in most ND, indicating the positive effect of early actions during brain development. Among ND, Down syndrome (DS) is caused by the presence of an extra chromosome 21, and it represents the leading cause of genetically-defined intellectual disability. Different pharmacological treatments targeting one of the many pathways downstream of the triplicated genes have been shown to rescue cognitive impairment in DS animal models. Nevertheless, most of these preclinical studies have been performed postnatally and often in adults, possibly because of concerns of unwanted drug side effects that may have long-lasting noxious sequelae on a developing brain at embryonic stages. On the other hand, viral (but also non-viral) gene therapy approaches in animal models of ND have been mostly neglected because of technical and ethical issues, when considered in the light of future translational applications. Yet, DS is mostly diagnosed prenatally, when many of its brain developmental abnormalities originate. Here, we will investigate whether in utero manipulation of specific and possibly converging gene networks in neuronal progenitors of DS mice by CRISPR-Cas9 gene-editing technology, may recover brain development and cognitive deficits later in life. Specifically targeting neuronal progenitors will allow us to act at early stages of brain development, while avoiding the involvement of genetic editing of germline cells and all related ethical issues. In parallel, we will also develop safer (viral-free) technological approaches for genetic manipulations in utero to minimize technical issues in the view of potential future translational applications.
The project entails three aims:
1) Gene editing technology to restore the expression of triplicated genes key for cognitive impairment in DS mice in utero.
So far, we have set up and validated the most appropriate molecular biology tools for two key genes. For one, we have found that our tool is able to rescue genesis of DS neurons form progenitors in an in vitro cell culture model. For the second one, we have performed experiments aimed at downregulating its expression (or inhibiting its activation) in vitro and in vivo. We have applied these tools to early development of DS mice and we were able to rescue cognitive impairment, hyperactivity and seizure susceptibility. The same tools utilized in adult animals only rescued cognition in DS mice. We have also setup new protocols to assess developmental mile stones and social behaviors in rodent pups (Naskar et al., Nature Communications, 2019). We are currently evaluating the mechanism underlying the positive effects of rescueing one of these genes at electrophysiological and molecular levels.
2) Comparison of the expression of genes in DS animals and DS human samples to understand what genes among the triplicated and non-triplicated may be the more responsible for cognitive impairment.
So far, we have collected a lot of data that we are still analyzing. Nevertheless, we have found a specific gene that is dysregulated both in DS mice (two different mouse models) and DS people. This same gene has been implicated already in other brain disorders. Moreover, we found that inflammatory pathways play a big role in cognitive impairment in DS mice. Accordingly, we showed that treatment with a commonly used anti-inflammatory drug can rescue learning and memory in DS mice (Savardi et al., Chem, 2020). We are currently evaluating whether the same approach can be extendend also to other neurodevelopmental disorders.
3) Development of new tools for implementing safe and reliable gene editing in vivo.
To perform these experiments we had to ask for a special animal license, which took more than expected. We eventually got authorized. In the meanwhile, we have written an extensive review on the already existing methods to allow the utilization of gene editing tools in the brain in vivo. We are currently performing experiments to setup the best parameters to achieve successful in utero sonoporation in rodents.
The project has already produced a lot of knowledge that we have conveyed in 10 manuscripts, 2 patents, and 1 sponsored research agreement

(1) We have opened to molecular biology to design new approaches to knock out specific genes in one only chromosomal allele. Moreover, we have designed gene-therapy approaches with potentials to be translated in humans. This have generated 1 patent and an important sponsored research agreement with a company interested in gene therapy.
(2) Based on our results, we have used classical pharmacological approaches to assess cognitive deficits by treatment with an FDA-approved anti-inflammatory drug. Currently, we are trying to find funding to perform a phase II clinical trial for the repurposing of this drug in DS people.
(3) Stemming from our results highlighting a causal link between the dysregulation of one of the gene focus of our study and the cognitive impairment in DS, we have also setup a drug-discovery program that has lead to the synthesis and testing of potential new class of compounds to be tested as possible drugs to treat cognitive impairment in Down syndrome. This has contributed to the generation of another patent.
(3) We have innovative protocols for testing developmental milestones and social behavior in rodent pups. This will promote the cross-talk between laboratories acquainted to study behavior in pups by possibly recruiting laboratories interested in (i) basic neurodevelopmental biology (ii) molecular mechanisms and potential therapeutic approaches to treat neurodevelopmental diseases (most of the studies on neurodevelopmental diseases are performed on adult animals for the lack of appropriate behavioral tools in developing pups). (ii) large scaling behavioral phenotyping of laboratory rodents to evaluate the efficacy of particular hit compounds in neurodevelopmental disorders (e.g. pharmaceutical companies and contract research organizations (CROs)).