Periodic Reporting for period 2 - BIND (Brain Involvement iN Dystrophinopathies)
Periodo di rendicontazione: 2021-07-01 al 2022-12-31
The DMD clinical care and translational research are focused on the skeletal and cardiac muscle involvement. However there is poor appreciation, assessment and management of the brain comorbidities, with negative outcomes for the affected individuals. Moreover, experimental therapies are currently not focused on this comorbidity. A deeper understanding of these difficulties will lead to better and globally adopted standards of care guidelines and support strategies for these patients.
DMD provides us with the unique opportunity to assess the direct correlation between the deficiency of a specific protein and its interacting partners in brain and a range of brain comorbidities, ultimately providing better understanding on how the brain works. This is likely to have implication for other conditions as well. Moreover, our ongoing genetic therapy studies focused on restoration of postnatal dystrophin gene expression in the dystrophic mdx mice will provide the unique opportunity to assess the degree of reversibility of these brain comorbidities in the different dystrophic animal models lacking specific dystrophin subunits, and recapitulating the different scenarios encountered in clinic. Our work will not only provide the foundation for considering future dystrophin restoration therapies in DMD patients, but will also facilitate the development of brain targeted therapies for other neurological disorders.
In the human, we are providing the more comprehensive assessment of the frequency and severity of brain involvement in the largest group of DMD and BMD patients ever studied.
In our human and mouse pathology studies, we are characterising for the first time the structure of the different protein complexes associated with the different dystrophin isoforms involved in brain function.
In the dystrophic animal studies, we are assessing the extent of improvement of brain comorbidities by genetic therapies aimed at restoring the production of different missing dystrophin proteins, paving the way to future clinical studies in the human.
DMD provides us with the unique opportunity to assess the direct correlation between the deficiency of a specific protein and its interacting partners in brain and a range of brain comorbidities, ultimately providing better understanding on how the brain works. This is likely to have implication for other conditions as well. Moreover, our ongoing genetic therapy studies focused on restoration of postnatal dystrophin gene expression in the dystrophic mdx mice will provide the unique opportunity to assess the degree of reversibility of these brain comorbidities in the different dystrophic animal models lacking specific dystrophin subunits, and recapitulating the different scenarios encountered in clinic. Our work will not only provide the foundation for considering future dystrophin restoration therapies in DMD patients, but will also facilitate the development of brain targeted therapies for other neurological disorders.
In the human, we are providing the more comprehensive assessment of the frequency and severity of brain involvement in the largest group of DMD and BMD patients ever studied.
In our human and mouse pathology studies, we are characterising for the first time the structure of the different protein complexes associated with the different dystrophin isoforms involved in brain function.
In the dystrophic animal studies, we are assessing the extent of improvement of brain comorbidities by genetic therapies aimed at restoring the production of different missing dystrophin proteins, paving the way to future clinical studies in the human.
We have now optimised protein and RNA detection techniques between the various laboratories to detect and quantify precisely the various dystrophin isoforms in both human and mouse brains, ranging from different techniques on homogenates (western, Jess, Wes) to quantitative transcriptomic studies in both human and mouse; to single cell transcriptomic studies (in mouse) in-situ RNA techniques (RNAScope and BaseScope focusing on the cellular sublocalisation of the different DMD transcripts). Our results provide a comprehensive atlas of DMD isoforms expression both in both species, which highlight some species differences, and the complex pattern of isoforms expression in different cell types (vascular; astrocytes; neurons; oligodendrocytes), raising the possibility that multiple isoforms could be co-expressed in the same cells. We have now optimised methodologies for immunoprecipitation of the complexes belonging dystrophin isoforms, and will confirm our preliminary studies in the 3rd reporting period.
We have firstly established the same mouse strains at each of the participating sites, deficient for the different dmd isoforms. We subsequently performed a deep phenotyping of the mdx mice deficient for either Dp427; or Dp427+Dp140. The deep phenotyping of the dmd null, lacking all isoforms is underway.
Our results have established that some of the previously associated pathological behaviour in the mdx23 are likely related to the mouse strain (C57BL/10) as not recapitulated on the C57BL/6J background. This is very relevant as most of the previously published literature is on the C57BL/10 background. Furthermore we have identified robust behavioural parametres allowing us to assign a function to Dp427; and to the combined absence of both Dp427 and Dp140. These observations are of fundamental importance both to better understand the role of the different isoforms on brain function; and as they provide robust outcome measures for the ongoing dystrophin restoration studies.
We have also compared a number of different techniques focused on the restoration of dystrophin in the mdx52. We have compared different antisense backbones and modalities of their administrations, which provide us both information on their biodistribution and also informing us on which phenotypes can be improved, and with which levels of dystrophin protein expression, and where, and which are phenotypes that are less responsive to postnatal dystrophin restoration.
Our work is currently progressing on 2 fronts, one a novel on-line assessment tool which allowed us to reach out to families also during the previous COVID-19 lockdown period (Protocol part 1); and since summer 2022 also the face to face assessments in clinic (part 2). These studies are currently underway, we expect to have the first interim results towards the summer 2023. The interim analysis will also allow us to compare the on-line assessments to the face-to-face more in depth assessment performed in clinic in a subset of DMD and BMD individuals, hence providing validation for the new on-line instrument
We have firstly established the same mouse strains at each of the participating sites, deficient for the different dmd isoforms. We subsequently performed a deep phenotyping of the mdx mice deficient for either Dp427; or Dp427+Dp140. The deep phenotyping of the dmd null, lacking all isoforms is underway.
Our results have established that some of the previously associated pathological behaviour in the mdx23 are likely related to the mouse strain (C57BL/10) as not recapitulated on the C57BL/6J background. This is very relevant as most of the previously published literature is on the C57BL/10 background. Furthermore we have identified robust behavioural parametres allowing us to assign a function to Dp427; and to the combined absence of both Dp427 and Dp140. These observations are of fundamental importance both to better understand the role of the different isoforms on brain function; and as they provide robust outcome measures for the ongoing dystrophin restoration studies.
We have also compared a number of different techniques focused on the restoration of dystrophin in the mdx52. We have compared different antisense backbones and modalities of their administrations, which provide us both information on their biodistribution and also informing us on which phenotypes can be improved, and with which levels of dystrophin protein expression, and where, and which are phenotypes that are less responsive to postnatal dystrophin restoration.
Our work is currently progressing on 2 fronts, one a novel on-line assessment tool which allowed us to reach out to families also during the previous COVID-19 lockdown period (Protocol part 1); and since summer 2022 also the face to face assessments in clinic (part 2). These studies are currently underway, we expect to have the first interim results towards the summer 2023. The interim analysis will also allow us to compare the on-line assessments to the face-to-face more in depth assessment performed in clinic in a subset of DMD and BMD individuals, hence providing validation for the new on-line instrument
Our protein and transcript localisation studies provide for the first time insight on the richness and complexity of the pattern of dystrophin expression in the human and the mouse; our ongoing immunoprecipitation studies are allowing us to have insight on dystrophin complexes in the brain that will shed light on how the deficiency of the different isoforms is associated- at the cellular level- with the DMD brain comorbidities.
The deep phenotyping of mdx mice deficient for different isoforms has allowed us to identify a robust set of outcome measures that we expect will be used in the future by any investigator interested in assessing the mdx mice. We have been able to conclude that some of the previously assigned “typical” mdx23 phenotypes are not related to brain dystrophin deficiency but to a particular strain; in addition we have assigned specific and novel phenotypes to the brain deficiency of Dp140 (for example cognitive flexibility and hyperactivity following frustrating conditions) that will now be assessed in mice following brain dystrophin restoration. We have also encountered that mutations located towards the 3’ end of the gene (the mdx23 has a mutation in exon 23, the mdx52 in exon 52) have significant lower levels of dystrophin transcription than assumed. This is related to transcript imbalance that uniquely affect DMD transcription in mouse and, at least in muscle, also in DMD boys. While this observation raises the bar of finding an efficacious method to restore dystrophin in the brain of boys with DMD, this observation is very helpful as the majority of the DMD boys carry mutations towards the 3’ end of the gene and not in the regions affected by the mdx23 mutation. This observation provides new information of what can be realistically rescued in terms of brain dystrophin restoration in the context of future clinical trials, and what is the lowest level of dystrophin to provide a measurable clinical benefit to the mdx mice. Our results have identified already a range of behavioural phenotypes that can be improved by restoring Dp427; the restoration of Dp140 is- in mice (but not in the human) more complex but this has also been accomplished using different techniques, and provide exciting and novel understanding on postnatal dystrophin brain restoration.
The deep phenotyping of mdx mice deficient for different isoforms has allowed us to identify a robust set of outcome measures that we expect will be used in the future by any investigator interested in assessing the mdx mice. We have been able to conclude that some of the previously assigned “typical” mdx23 phenotypes are not related to brain dystrophin deficiency but to a particular strain; in addition we have assigned specific and novel phenotypes to the brain deficiency of Dp140 (for example cognitive flexibility and hyperactivity following frustrating conditions) that will now be assessed in mice following brain dystrophin restoration. We have also encountered that mutations located towards the 3’ end of the gene (the mdx23 has a mutation in exon 23, the mdx52 in exon 52) have significant lower levels of dystrophin transcription than assumed. This is related to transcript imbalance that uniquely affect DMD transcription in mouse and, at least in muscle, also in DMD boys. While this observation raises the bar of finding an efficacious method to restore dystrophin in the brain of boys with DMD, this observation is very helpful as the majority of the DMD boys carry mutations towards the 3’ end of the gene and not in the regions affected by the mdx23 mutation. This observation provides new information of what can be realistically rescued in terms of brain dystrophin restoration in the context of future clinical trials, and what is the lowest level of dystrophin to provide a measurable clinical benefit to the mdx mice. Our results have identified already a range of behavioural phenotypes that can be improved by restoring Dp427; the restoration of Dp140 is- in mice (but not in the human) more complex but this has also been accomplished using different techniques, and provide exciting and novel understanding on postnatal dystrophin brain restoration.