Periodic Reporting for period 4 - GenEdiDS (Rescuing Cognitive Deficits in Neurodevelopmental Disorders by Gene Editing in Brain Development: the Case of Down Syndrome)
Período documentado: 2022-04-01 hasta 2023-09-30
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 investigated whether early in life manipulation by gene-therapy and pharmacological approaches on known, but also newly discovered converging gene-networks (relevant for the human condition) in neurons of DS mice may recover brain development, cognitive deficits, but also other comorbidities later in life. This approach avoided the involvement of genetic editing of germline cells and all related ethical issues. In parallel, we improved (viral-free) technological approaches for genetic manipulations in utero to minimize technical issues in the view of potential future translational applications.
Our investigation highlighted common pathways dysregulated in people with DS and mouse models, indicating a prevalent role for inflammation, early developmental processes such as axonogenesis, and possibly new targets. Early-in-life gene therapy/pharmacological interventions had long-lasting effects in the absence of any drug and led to rescue of behavioral phenotypes and molecular signatures that therapeutic interventions during adulthood could not rescue. In this context, although viral-free strategies lead to significant results in animal models (mostly in utero electroporation, which has low translational value), their potential applications in patients still seem to be far away in the future, due to low efficiency of sonoporation.
Our investigation highlighted common pathways dysregulated in people with DS and mouse models, indicating a prevalent role for inflammation, early developmental processes such as axonogenesis, and possibly new targets. Early-in-life gene therapy/pharmacological interventions had long-lasting effects in the absence of any drug and led to rescue of behavioral phenotypes and molecular signatures that therapeutic interventions during adulthood could not rescue. In this context, although viral-free strategies lead to significant results in animal models (mostly in utero electroporation, which has low translational value), their potential applications in patients still seem to be far away in the future, due to low efficiency of sonoporation.
The project entailed 3 aims:
1) Gene editing to restore the expression of triplicated genes key for cognitive impairment in DS mice in utero.
We set up and the most appropriate molecular biology tools for two key genes. For one, we found that our tool was able to rescue neurogenesis in DS in an in vitro cell-culture model. For the second one, we performed experiments for downregulating its expression (or inhibiting its activation) in vitro and in vivo. We applied these tools to early development of DS mice. We were able to rescue cognitive impairment, hyperactivity and seizure susceptibility. The same tools utilized in adult animals only rescued cognition in DS mice.
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.
We collected a lot of data that we are still analyzing. On the analyzed data, we 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 extended also to other neurodevelopmental disorders. We are also validating one completely new potential therapeutic target that was highlighted by our proteomic approach.
3) Development of new tools for implementing safe and reliable gene editing in vivo.
We strongly increased efficacy of in utero electroporation in the prefrontal cortex, and optimized the protocol for CRISPR-Cas9 gene editing by electroporation in the retina. We are currently performing experiments to setup the best parameters to achieve successful in utero sonoporation in rodents. We also cured a special issue in Frontiers in Neuroscience on gene therapies, and wrote an extensive review on already existing methods for in vivo manipulations for the utilization of gene-editing tools in the brain in vivo.
1) Gene editing to restore the expression of triplicated genes key for cognitive impairment in DS mice in utero.
We set up and the most appropriate molecular biology tools for two key genes. For one, we found that our tool was able to rescue neurogenesis in DS in an in vitro cell-culture model. For the second one, we performed experiments for downregulating its expression (or inhibiting its activation) in vitro and in vivo. We applied these tools to early development of DS mice. We were able to rescue cognitive impairment, hyperactivity and seizure susceptibility. The same tools utilized in adult animals only rescued cognition in DS mice.
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.
We collected a lot of data that we are still analyzing. On the analyzed data, we 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 extended also to other neurodevelopmental disorders. We are also validating one completely new potential therapeutic target that was highlighted by our proteomic approach.
3) Development of new tools for implementing safe and reliable gene editing in vivo.
We strongly increased efficacy of in utero electroporation in the prefrontal cortex, and optimized the protocol for CRISPR-Cas9 gene editing by electroporation in the retina. We are currently performing experiments to setup the best parameters to achieve successful in utero sonoporation in rodents. We also cured a special issue in Frontiers in Neuroscience on gene therapies, and wrote an extensive review on already existing methods for in vivo manipulations for the utilization of gene-editing tools in the brain in vivo.
The project 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, there is a phase II clinical trial for the repurposing of this drug to treat cognitive impairment 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 classes of compounds to be tested as possible drugs to treat cognitive impairment in Down syndrome. This has contributed to the generation of another patent and the launch of an innovative startup company.
(4) We have innovative protocols for testing developmental milestones and social behavior in rodent pups. This is promoting the cross-talk between laboratories acquainted to study behavior in pups by 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)).
(5) We set the ground to performing parallel studies in mouse models and children with cognitive impairments in future clinical trials, with possibly more chances of success.
(6) We identified a new molecular target relevant for DS, but possibly other neurodevelopmental disorders.
(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, there is a phase II clinical trial for the repurposing of this drug to treat cognitive impairment 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 classes of compounds to be tested as possible drugs to treat cognitive impairment in Down syndrome. This has contributed to the generation of another patent and the launch of an innovative startup company.
(4) We have innovative protocols for testing developmental milestones and social behavior in rodent pups. This is promoting the cross-talk between laboratories acquainted to study behavior in pups by 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)).
(5) We set the ground to performing parallel studies in mouse models and children with cognitive impairments in future clinical trials, with possibly more chances of success.
(6) We identified a new molecular target relevant for DS, but possibly other neurodevelopmental disorders.