Generation and characterization of a preclinical models to study DDX3X disorder

Biochemistry; Genetics; Neuroscience and Neurobiology

DDX3X mutation cause intellectual disabilities, seizures, autism, poor muscle tone, and slower physical developments. We propose to generate a female mouse model carrying germline DDX3X mutations.

Research Interests

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List ofcollaborative team/co-principal investigators:

Dr. SanchitaBhatnagar, Ph.D.,  Dr.Howrad Goodkin, MD,Ph.D., Dr. Michael Wormington

Project Description:

Significance: DDX3X syndrome is a recently discovered disorder withdevelopmental delay and/or intellectual disability. It is characterized byserious limitations in intellectual functioning and autism-like behavior, suchas, epilepsy, tremors, hypotonia, and corpus callosum hypoplasia.     Currently,there is no cure for DDX3X syndrome. Typically, DDX3X patients areheterozygous for DEAD-box RNA helicase family protein (DDX3X)deficiency which is located on human Xchromosome p11.3 to p11.23 (1, 2).Some of the functions attributed to DDX3X, include transcriptional regulation,RNA unwinding, splicing, RNA nuclear export, ribosomal biogenesis, and mRNAtranslation.

Typically, this pediatric disease ischaracterized by its almost exclusive occurrence in girls, as boys are moreseverely affected and do not survive beyond infancy. The severity of thephenotype in males is due to the single copy of X chromosome. In females, the mutant DDX3X allele is diluted due to random X chromosome inactivation (XCI), an epigenetic process essentialfor dosage compensation between sexes. Because of the XCI, ~50% of the cells inDDX3X girls express non-functional DDX3X. However, everyDDX3X mutant female cell also carries a wild type but epigenetically dormant DDX3X on inactive X, which ifreactivated can compensate for DDX3X deficiency. To be clear, even thoughmutant genes on the active X chromosome are the cause of disease, eachdefective cell holds a possible key to a cure, if the healthy genes on the Xican be reactivated. This modality is clinically relevant as conditional expression of wild-type X-linked genes, suchas Mecp2 in a mouse model of Rettsyndrome, another neurodevelopmental disorder, is sufficient to rescue multipledisease features (3). Additionally, the results from genetically engineered mouse modelshave shown that wild-type Mecp2 isdominant in cells also expressing an Mecp2mutant (4). Most importantly, we have recently provided animportant proof-of-concept for the feasibility, tolerability and safetyof pharmacological reactivation of X-linked MECP2in post-mitotic neurons derived from induced pluripotent stem cells (iPSC) offemale Rett syndrome patient and cerebral cortical neurons of adult living mice(5, 6).

A major goal of this application is to determine whetherreactivation of the epigenetically silenced DDX3X gene is a feasibletherapeutic approach for treating DDX3X syndrome. Unfortunately, there is noDDX3X mouse model or human induced pluripotent stem cell model to directlyanalyze the therapeutic benefit of DDX3X reactivation. Therefore, we willundertake two complementary approaches to generate preclinical models for DDX3Xsyndrome.

The scientific goalsof the project are as follows:

Goal 1: Generate and characterize a mouse model carryingclinically relevant mutation in DDX3X gene.

Goal 2: Derivation of a series of clonal femaleDDX3X neuronal cell lines expressing mutant DDX3X from patient skin fibroblast.

Reference:

1.         ParkSH, Lee SG, Kim Y, Song K. Assignment of a human putative RNA helicase gene,DDX3, to human X chromosome bands p11.3-->p11.23. Cytogenet Cell Genet.1998;81(3-4):178-9. doi: 10.1159/000015022. PubMed PMID: 9730595.

2.         Owsianka AM, Patel AH.Hepatitis C virus core protein interacts with a human DEAD box protein DDX3.Virology. 1999;257(2):330-40. doi: 10.1006/viro.1999.9659. PubMed PMID:10329544.

3.         Cobb S, Guy J, Bird A.Reversibility of functional deficits in experimental models of Rett syndrome.Biochemical Society transactions. 2010;38(2):498-506. Epub 2010/03/20. doi:10.1042/bst0380498. PubMed PMID: 20298210.

4.         Heckman LD, Chahrour MH,Zoghbi HY. Rett-causing mutations reveal two domains critical for MeCP2function and for toxicity in MECP2 duplication syndrome mice. Elife.2014;3:e02676. Epub 2014/06/28. doi: 10.7554/eLife.02676. PubMed PMID:24970834.

5.         Bhatnagar S, Zhu X, Ou J,Lin L, Chamberlain L, Zhu LJ, Wajapeyee N, Green MR. Genetic andpharmacological reactivation of the mammalian inactive X chromosome. Proc NatlAcad Sci U S A. 2014;111(35):12591-8. Epub 2014/08/20. doi:10.1073/pnas.1413620111. PubMed PMID: 25136103; PMCID: Pmc4156765.

6.         Przanowski P, Wasko U,Zheng Z, Yu J, Sherman R, Zhu LJ, McConnell MJ, Tushir-Singh J, Green MR,Bhatnagar S. Pharmacological reactivation of inactive X-linked Mecp2 incerebral cortical neurons of living mice. Proc Natl Acad Sci U S A. 2018. doi:10.1073/pnas.1803792115. PubMed PMID: 30012595.

Desired outcomes

1)We hope to successfully generate a mouse model to study DDX3X syndrome. Our goal will be to characterize the various symptoms of the DDX3X syndrome in this model. Ultimately, we want to test our previously identified small molecule inhibitors of PDPK1 and ACVR1 pathway to study the reactivation of DDX3X from the inactive X chromosome.

2) We anticipate from the proposed investigations to generate the isogenic female neuronal cell line, a cell type that is most clinically relevant to DDX3X syndrome. We will perform a series ofcell culture-based and electrophysiological experiments to test if DDX3X expression could rescue characteristic phenotypic defects, reverse activity dependent calcium transients and correctsynaptic currents in DDX3X neurons.