Rett Syndrome Plasmid Collection and Resource Center

The Rett Syndrome Research Trust (RSRT) was launched in 2008 to drive the development of treatments and cures for Rett Syndrome and related MECP2 disorders. The RSRT has a comprehensive, strategic and aggressive plan to use gene editing, MECP2 reactivation, RNA editing, gene replacement therapy, RNA trans-splicing, and protein replacement to reverse the impacts of this disease. Addgene is working with the RSRT along with individual laboratories to assemble a Rett Syndrome plasmid resource for the scientific research community.

Background

Rett Syndrome

Rett syndrome is a neurodevelopmental disorder that presents in early toddlerhood primarily affecting females at an incidence of approximately 1:10,000. It is characterized by apparently normal development through the first 6 months of age, followed by developmental delay between 6 and 18 months of age. A defining feature, with onset typically between 18 to 30 months, is regression of previously acquired skills, notably loss of acquired purposeful hand movements and regression of speech. In contrast to the sustained loss of skills in neurodegenerative conditions, the regressive period in Rett syndrome is self-limited, after which a period of developmental stabilization or even limited recovery of skills occurs.

Diagnosis of Rett syndrome is currently based on specific clinical diagnostic criteria, defined as the presence of relatively normal early development, regression with a loss of spoken language and hand skills, development of repetitive hand stereotypies, and gait dysfunction or absence of gait. Greater than 95% of individuals who meet diagnostic criteria have disease-causing mutations in the gene methyl-CpG binding protein 2 (MECP2).

MECP2

Rett syndrome is an X-linked disorder caused from loss-of-function mutations in the MECP2 gene. Causative mutations for Rett syndrome typically arise spontaneously, although in rare cases Rett syndrome can be inherited. In females, random X inactivation results in approximately half of cells expressing wild-type MECP2 and half expressing mutant MECP2. Skewing of these X inactivation ratios can affect disease severity.

The MECP2 protein is a global transcriptional regulator of thousands of genes and studies have suggested roles in transcriptional repression, activity dependent de-repression, chromatin remodeling, gene activation, and more. How loss of MECP2 protein function results in Rett syndrome is not clear, however, the ability to bind to methylated DNA and recruit known co-repressors or other transcriptional modifiers suggests the major function of MECP2 is to regulate gene expression either locally or globally.

The MECP2 protein contains several functional domains, including:

• the N-terminal Domain (NTD)
• the Methyl Binding Domain (MBD)
• a Transcriptional Repressor Domain (TRD)
• the TRD contains a Nuclear receptor Co-Repressor 1/Silencing Mediator of Retinoic acid and Thyroid hormone receptor (NCoR/SMRT) interacting domain (NID)
• a nuclear localization signal (not essential for localization)
• the C-terminal Domain (CTD)

The most common missense mutations cluster in the MBD and NID demonstrating the importance of binding to methylated DNA and the recruitment of the NCoR complex for proper nervous system function.

While there is wide variability in symptoms even within patients with the same mutation, the location and type of MECP2 mutation can be a strong predictor of symptom severity. Broadly, early gene disruptions, missense mutations located in the MBD, and truncations prior to the NID are more severe than mutations further downstream in the NID and C-terminus. A large number of mutations are known to cause Rett syndrome, however, eight recurrent missense and nonsense mutations (R106W, R133C, T158M, R168X, R255X, R270X, R294X, and R306C) account for almost 46% of all Rett syndrome cases.

Research Milestones

• 1992 - MECP2 is discovered by Adrian Bird's lab. PMID: 1606614
• 1999 - Huda Zoghbi's lab identifies MECP2 as the genetic cause of RTT. PMID: 10508514
• 2007 - Adrian Bird's lab demonstrates that Rett syndrome symptoms are reversible in mice if MECP2 gene expression is switched on regardless of age or symptom severity, suggesting that restoring MECP2 expression in humans could be curative. PMID: 17289941
• 2010 - Allyson Muotri's lab develops the first iPSC model for Rett syndrome. PMID: 21074045
• 2017 - the Bird lab determines a minimally functional MECP2 that rescues neurological defects of Rett syndrome. PMID: 29019980

Animal Models

Studies of the MECP2 protein in mouse and rat models have accelerated our understanding of Rett syndrome at the molecular level. They are also an important tool for developing therapeutic strategies to treat Rett syndrome and restore functional MECP2 in cells.

• Search JAX Mice for mouse models that include MECP2 null alleles or common mutations that recapitulate Rett syndrome in humans.
• MECP2 knockout rats are available from Envigo.

The following cell lines are available directly from the labs in which they were created. Find their contact information by following the link to their laboratory website.

Mouse Line	Mutation (DNA)	Background Strain	Purpose	Publication	PI
Mecp2 NLucTom	Knock-in of NLuc-tdTomato at endogenous MECP2 locus	Castaneus	MECP2-NLuc-tdTomato mouse reporter cell line	PMID: 35148843	Joost Gribnau
Xist 2lox/2lox	Conditional Xist, Lox sites flanking exon 1,2,3	C57BL/6	Mouse line with conditional deletion of Xist	Unpublished	Joost Gribnau

Human Cell Line Models

Induced pluripotent stem cells (iPSCs) are a powerful system for generating Rett syndrome-specific differentiated cells that can be used to understand the mechanisms of the disease and for the development of successful therapeutics.

• ATCC - LUHMES cell line, an immortalized pre-neuronal cell line that can be differentiated into neurons
• Coriell Institute:
  • Patient-specific lines
  • RSRT iPSC lines and isogenic controls, patient-specific
• Infinity BiologiX - RSRT cell line collection (186.8 KB). To order, please email

The following cell lines are available directly from the labs in which they were created. Find their contact information by following the link to their laboratory website.

Cell Line	Mutation (DNA)	Mutation (protein)	Sex	Source of Material	Publication	PI
N126I	A377T	N126I	M	Fibroblasts & iPSC	PMID: 26944080	Alysson Muotri
Q83X	C247T	Q83X	M	Fibroblasts & iPSC	PMID: 26944080	Alysson Muotri
c.806delG	806delG	G269Afs*20	M	Fibroblasts & iPSC	Unpublished	Wendy Gold
A140V	C419T	A140V	M	Fibroblasts	Unpublished	Wendy Gold
AN_BU	808delC	Arg270Glufs*19	F	Fibroblasts & iPSC	Unpublished	Wendy Gold
BO_DI	806delG	G269Afs*20	F	Fibroblasts	Unpublished	Wendy Gold
HO_AN	G917A	R306H	F	Fibroblasts	Unpublished	Wendy Gold

For more information on generating iPSCs using plasmids, please see Addgene's Plasmids for Stem Cell Research page.

CRISPR Tools

CRISPR technology is a promising therapeutic approach for precise editing of mutations in the MECP2 gene. Please refer to Addgene's CRISPR Guide for a general introduction to CRISPR technology or the mammalian CRISPR resources for a full selection of plasmids expressing Cas9 and empty gRNA backbones. A few examples of additional CRISPR resources can be found below:

• Base Editing
• RNA Editing
• CRISPR-based DNA methylation editing system - Shawn Liu and Rudolf Jaenisch labs

Plasmids

MECP2 Plasmids

The table below highlights plasmids that express or target the MECP2 gene. Use the search bar or sort buttons to find plasmids based on:

• Expression system (mammalian or bacterial)
• Insert species (mouse or human)
• Mutation

External Resources

Research Databases

• InterRett - international online database of clinical features and genetic characteristics of Rett syndrome
• RettBase - MECP2 mutation database
• OMIM Rett Syndrome - Rett syndrome curated literature information
• OMIM MECP2 - MECP2 curated literature information
• UniProt MECP2 - MECP2 curated protein and functional information

Mouse and Cell Line Repositories

• Coriell Institute - cell line repository, including patient-derived iPSCs
• Jackson Labs - mouse line developer and repository, and CRO for centralized behavior scoring
• MMRRC Repository - NIH-supported mouse and cell line repository
• Sampled - one of the world's largest biorepositories
• ATCC - the world's largest cell line repository

Tissue Banks

• Harvard Brain Bank
• Autism Brain Bank
• University of Maryland Brain & Tissue Bank

Antibodies

• N-terminal Anti-MECP2 (M7443) mouse antibody from Sigma
• C-terminal Anti-MECP2 (M6818) mouse antibody from Sigma
• C-terminal Anti-MECP2 (D4F3) rabbit antibody from Cell Signalling Technology

Additional Resources

• BrainSpan Atlas of the Developing Human Brain - includes developmental transcriptome information, prenatal LMD microarray data, ISH image data, and anatomic reference atlases of prenatal and adult human brain
• ClinicalTrials.gov - a list of Rett syndrome clinical trials
• HRV Metrics Toolbox - algorithms for data analysis to calculate heart rate variability
• Protocols.io - protocol sharing site

Funding Opportunities

• Rett Syndrome Research Trust
• Rettsyndrome.org

References

Amir et al. 1999. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet. 23, 185–188. PMID: 10508514

Archer et al. 2007. Correlation between clinical severity in patients with Rett syndrome with a p.R168X or p.T158M MECP2 mutation, and the direction and degree of skewing of X-chromosome inactivation. J Med Genet. 44, 148–152. PMID: 16905679

Cuddapah et al. 2014. Methyl-CpG-binding protein 2 (MECP2) mutation type is associated with disease severity in Rett syndrome. J Med Genet. 51, 152–158. PMID: 24399845

Guy et al. 2007. Reversal of Neurological Defects in a Mouse Model of Rett Syndrome. Science. 315, 1143–1147. PMID: 17289941

Hagberg et al. 1983. A progressive syndrome of autism, dementia, ataxia, and loss of purposeful hand use in girls: Rett’s syndrome: report of 35 cases. Ann Neurol. 14, 471–479. PMID: 6638958

Kankirawatana et al. 2006. Early progressive encephalopathy in boys and MECP2 mutations. Neurology. 67, 164–166. PMID: 16832102

Krishnaraj et al. 2017. RettBASE: Rett syndrome database update. Hum Mutat. 38, 922–931. PMID: 28544139

Laurvick et al. 2006. Rett syndrome in Australia: a review of the epidemiology. J Pediatric. 148(3):347-352. PMID: 16615965

Leonard et al. 2017. Clinical and biological progress over 50 years in Rett syndrome. Nat Rev Neurol. 13, 37–51. PMID: 27934853

Neul et al. 2008. Specific mutations in methyl-CpG-binding protein 2 confer different severity in Rett syndrome. Neurology. 70, 1313–1321. PMID: 18337588

Neul et al. 2010. Rett syndrome: revised diagnostic criteria and nomenclature. Ann Neurol. 68, 944–950. PMID: 21154482

Neul et al. 2019. The array of clinical phenotypes of males with mutations in Methyl-CpG binding protein 2. Am J Med Genet B Neuropsychiatr Genet. 180, 55–67. PMID: 30536762

Shahbazian et al. 2002. Insight into Rett syndrome: MeCP2 levels display tissue- and cell-specific differences and correlate with neuronal maturation. Hum Mol Genet. 11, 115–124. PMID: 11809720

Tarquinio et al. 2015. Age of diagnosis in Rett syndrome: patterns of recognition among diagnosticians and risk factors for late diagnosis. Pediatr Neurol. 52, 585-591.e2. PMID: 25801175

Tillotson and Bird. 2019. The Molecular Basis of MeCP2 Function in the Brain. J Mol Biol. 17;S0022-2836(19)30595-9. PMID: 31629770