Artificial escape from XCI by DNA methylation editing of the CDKL5 gene

Halmai, Julian; Deng, Peter; Gonzalez, Casiana E; Coggins, Nicole B.; Cameron, David L; Carter, Jasmine L; Buchanan, Fiona K B; Waldo, Jennifer J; Lock, Samantha; Anderson, Johnathon D.; O’Geen, Henriette; Segal, David J.; Nolta, Jan A.; Fink, Kyle D.

doi:10.1093/nar/gkz1214

Cited by 36 publications

(23 citation statements)

References 66 publications

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“…A splicing correction strategy has been applied to CDKL5, developing engineered U1snRNA variants that were able to rescue CDKL5 mRNA splicing, protein synthesis, and function [44]. Other researchers induced escape of CDKL5 from X chromosome inactivation by editing DNA methylation on the promoter of CDKL5 using a dCas9-TET1 fusion protein [45]. Recently, the first proof-of-concept study on gene replacement as a potential approach for the treatment of CDD was published, using adeno-associated virus (AAV)-CDKL5 vectors in in vivo and in vitro models of CDD [46].…”

Section: Review Of Emerging Therapiesmentioning

confidence: 99%

Current neurologic treatment and emerging therapies in CDKL5 deficiency disorder

Olson

Daniels

Haviland

et al. 2021

J Neurodevelop Disord

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Background CDKL5 deficiency disorder (CDD) is associated with refractory infantile onset epilepsy, global developmental delay, and variable features that include sleep, behavioral disturbances, and movement disorders. Current treatment is primarily symptom-based and informed by experience in caring for this population. Methods We describe medication and non-medication approaches to treatment of epilepsy and additional key neurologic symptoms (sleep disturbances, behavioral issues, movement disorders, and swallowing dysfunction) in a cohort of 177 individuals meeting criteria for CDD, 154 evaluated at 4 CDKL5 Centers of Excellence in the USA and 40 identified through the NIH Natural History Study of Rett and Related Disorders. Results The four most frequently prescribed anti-seizure medications were broad spectrum, prescribed in over 50% of individuals. While the goal was not to ascertain efficacy, we obtained data from 86 individuals regarding response to treatment, with 2-week response achieved in 14–48% and sustained 3-month response in 5–36%, of those with known response. Additional treatments for seizures included cannabis derivatives, tried in over one-third of individuals, and clinical trial medications. In combination with pharmacological treatment, 50% of individuals were treated with ketogenic diet for attempted seizure control. Surgical approaches included vagus nerve stimulators, functional hemispherectomy, and corpus callosotomy, but numbers were too limited to assess response. Nearly one-third of individuals received pharmacologic treatment for sleep disturbances, 13% for behavioral dysregulation and movement disorders, and 43% had gastrostomy tubes. Conclusions Treatment for neurologic features of CDD is currently symptom-based and empiric rather than CDD-specific, though clinical trials for CDD are emerging. Epilepsy in this population is highly refractory, and no specific anti-seizure medication was associated with improved seizure control. Ketogenic diet is commonly used in patients with CDD. While behavioral interventions are commonly instituted, information on the use of medications for sleep, behavioral management, and movement disorders is sparse and would benefit from further characterization and optimization of treatment approaches. The heterogeneity in treatment approaches highlights the need for systematic review and guidelines for CDD. Additional disease-specific and disease-modifying treatments are in development.

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Section: Review Of Emerging Therapiesmentioning

confidence: 99%

Current neurologic treatment and emerging therapies in CDKL5 deficiency disorder

Olson

Daniels

Haviland

et al. 2021

J Neurodevelop Disord

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“…Although this co-targeting was not under XIST downregulation, SadCas9-VPR-mediated activation of FOXP3 was enhanced by SadCas9-TET1, suggesting an XIST -independent reactivation of FOXP3 by TET1. Likewise, co-targeting of dCas9-TET1 and dCas9-VP64 synergistically reactivates the X-linked CDKL5 from XCI (25). Thus, XIST is likely to be dispensable during the reactivation of X-linked genes from XCI by TET1 with transactivators such as VPR or VP64.…”

Section: Discussionmentioning

confidence: 99%

“…These data indicate that, for females with cancer, it may be possible to reactivate the non-mutated, inactivated allele for therapeutic purposes. Recent research has shown that X-linked genes can be reactivated from XCI by targeted editing of DNA methylation (25), offering a possibility for targeted reactivation of X-linked genes from XCI.…”

Section: Introductionmentioning

confidence: 99%

Targeted Reactivation of X-linked Endogenous FOXP3 Gene from X-chromosome Inactivation in Human Female Breast Cancer Cells

Cui

Wang

et al. 2021

Preprint

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Unlike autosomal tumor suppressors, which must be inactivated by a two-hit Knudson mechanism, X-linked tumor suppressors can be inactivated by a single hit due to X-chromosome inactivation (XCI). Here, we argue that targeted reactivation of the non-mutated allele from XCI offers a potential therapy for female breast cancers. Towards this goal, we developed a dual CRISPR interference and activation (CRISPRi/a) approach for simultaneously silencing and reactivating multiple X-linked genes using two orthogonal, nuclease-deficient CRISPR/Cas9 (dCas9) proteins. We verified the efficacy for use of Streptococcus pyogenes (Sp) dCas9-KRAB for silencing XIST and Staphylococcus aureus (Sa) dCas9-VPR for activating FOXP3 in various cell lines of human female breast cancers. We confirmed CRISPR reactivation of the non-mutated copy of FOXP3 from XCI in human breast cancer CRL2316 cells, which express a synonymous heterozygous mutation (p.L266L; c.798G>C) in the coding region of FOXP3. Further, simultaneous silencing of XIST from XCI led to enhanced and prolonged FOXP3 reactivation. We optimized CRISPRa by fusing SadCas9 to the demethylase TET1 and observed enhanced FOXP3 activation. Analysis of the conserved CpG-rich region of FOXP3 intron 1 confirmed that CRISPRi/a-mediated simultaneous FOXP3 activation and XIST silencing were accompanied by elevated H4 acetylation, including H4K5ac, H4K8ac, and H4K16ac, and lower DNA methylation and H3K9me3. This indicates that CRISPRi/a targeting to XIST and FOXP3 loci alters their transcription and their nearby epigenetic modifications. The simultaneous activation and repression of the X-linked, endogenous FOXP3 and XIST from XCI offers a useful research tool and a potential therapeutic for female breast cancers. This approach also provides new routes of targeted therapy for other X-chromosome-linked genetic disorders.

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“…For example, rescue of X-linked mutations in CDKL5 has been recently demonstrated using dCas9 fused to TET1, a demethylation factor. Demethylation of the CDKL5 promoter by dCas9-TET1 targeting was able to reactive the silenced WT X-allele in vitro [47].…”

Section: Crispr For the Treatment Of Haploinsufficiencymentioning

confidence: 97%

Gene Editing and Modulation: the Holy Grail for the Genetic Epilepsies?

Carpenter

Lignani

2021

Neurotherapeutics

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Epilepsy is a complex neurological disorder for which there are a large number of monogenic subtypes. Monogenic epilepsies are often severe and disabling, featuring drug-resistant seizures and significant developmental comorbidities. These disorders are potentially amenable to a precision medicine approach, of which genome editing using CRISPR/Cas represents the holy grail. Here we consider mutations in some of the most ‘common’ rare epilepsy genes and discuss the different CRISPR/Cas approaches that could be taken to cure these disorders. We consider scenarios where CRISPR-mediated gene modulation could serve as an effective therapeutic strategy and discuss whether a single gene corrective approach could hold therapeutic potential in the context of homeostatic compensation in the developing, highly dynamic brain. Despite an incomplete understanding of the mechanisms of the genetic epilepsies and current limitations of gene editing tools, CRISPR-mediated approaches have game-changing potential in the treatment of genetic epilepsy over the next decade.

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Artificial escape from XCI by DNA methylation editing of the CDKL5 gene

Cited by 36 publications

References 66 publications

Current neurologic treatment and emerging therapies in CDKL5 deficiency disorder

Current neurologic treatment and emerging therapies in CDKL5 deficiency disorder

Targeted Reactivation of X-linked Endogenous FOXP3 Gene from X-chromosome Inactivation in Human Female Breast Cancer Cells

Gene Editing and Modulation: the Holy Grail for the Genetic Epilepsies?

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