A mixed modality approach towards Xi reactivation for PNAS PLUS Rett syndrome and other X-linked disorders Lieselot L. G. Carrettea,b,c,d, Chen-Yu Wanga,b,c, Chunyao Weia,b,c, William Pressa,b,c, Weiyuan Mae,f, Raymond J. Kelleher IIIe,f, and Jeannie T. Leea,b,c,1 aHoward Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114; bDepartment of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114; cDepartment of Genetics, Harvard Medical School, Boston, MA 02115; dCenter for Medical Genetics, Ghent University, 9000 Ghent, Belgium; eCenter for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114; and fDepartment of Neurology, Massachusetts General Hospital, Boston, MA 02114 Contributed by Jeannie T. Lee, December 1, 2017 (sent for review August 28, 2017; reviewed by Ingolf Bach and Gyorgyi Csankovszki) The X-chromosome harbors hundreds of disease genes whose however, two obstacles to an Xi-reactivation strategy. First, sex associated diseases predominantly affect males. However, a sub- chromosomal dosage compensation is known to be important set, including neurodevelopmental disorders, Rett syndrome (RTT), throughout development and life: Perturbing XCI by a germline fragile X syndrome, and CDKL5 syndrome, also affects females. deletion of the master regulator Xist resulted in inviable female These disorders lack disease-specific treatment. Because female embryos (8), an epiblast-specific deletion of Xist caused severely cells carry two X chromosomes, an emerging treatment strategy reduced female fitness (9), and a conditional deletion of Xist in has been to reawaken the healthy allele on the inactive X (Xi). blood caused fully penetrant hematological cancers (10). Per- Here, we focus on methyl-CpG binding protein 2 (MECP2) resto- turbing dosage balance via Xi reactivation could therefore have ration for RTT and combinatorially target factors in the interac- untoward physiological consequences. On the other hand, loss of tome of Xist, the noncoding RNA responsible for X inactivation. Xist and partial reactivation occur naturally in lymphocytes (11), We identify a mixed modality approach combining an Xist antisense and may therefore be tolerated in vivo under controlled cir- oligonucleotide and a small-molecule inhibitor of DNA methylation, cumstances. A second challenge is that the Xi has been difficult which, together, achieve 30,000-fold MECP2 up-regulation from Xist to reactivate via pharmacological means due to multiple parallel GENETICS the Xi in cultured cells. Combining a brain-specific genetic – ablation with short-term 5-aza-2′-deoxycytidine (Aza) treatment mechanisms of epigenetic silencing (1 3, 12). Progress has been models the synergy in vivo without evident toxicity. The Xi is made in recent years, however. Several siRNA screens identified selectively reactivated. These experiments provide proof of con- several factors regulating Xi stability, but no overlap of candi- cept for a mixed modality approach for treating X-linked disorders dates was observed between them (13, 14), perhaps because the in females. screens were not saturating. Others have identified the TGF-β pathway (15), a synergism between Aurora kinase and DNA X reactivation | antisense oligonucleotides | LNA | Rett syndrome | Xist methylation in a primed small-molecule screen (16), as well as a iseases caused by a mutation on the mammalian X chro- Significance Dmosome affect males and females differently as males have only one X chromosome and females have two. Female X The X-chromosome harbors hundreds of disease genes, a sub- chromosomes are, however, subject to a dosage compensation set of which gives rise to neurodevelopmental disorders mechanism in which one X chromosome is inactivated. Because such as Rett syndrome (RTT), fragile X syndrome, and of “X-chromosome inactivation” (XCI), the female mammal is a CDKL5 syndrome. There is presently no disease-specific treat- mosaic of cells that expresses either the maternal or paternal X ment. Here, we work toward a therapeutic program based on chromosome (1–3). Thus, heterozygous X-linked mutations reactivation of the silent X chromosome to restore expression would affect approximately half of a female’s somatic cells. For of the missing protein. We develop a mixed modality approach gene products with a non–cell-autonomous function, healthy that combines a small-molecule inhibitor of DNA methylation cells can usually compensate for those expressing the mutation and an antisense oligonucleotide against Xist RNA. This com- (e.g., factor VIII for hemophilia). With mutations in gene bination achieves up to 30,000-fold methyl-CpG binding pro- products that fulfill a critical role within the cells that produce tein 2 upregulation in cultured cells. In vivo modeling using a Xist ′ them on the other hand, deficits in just half of the body’s somatic conditional knockout and 5-aza-2 -deoxycytidine recapit- cells can result in a severe disorder. One well-known example is ulates inactive X reactivation. These findings provide proof of Rett syndrome (RTT), a human neurodevelopmental disorder concept for the mixed modality approach to treat X-linked caused by a mutation in the methyl-CpG binding protein 2 disorders, including RTT. (MECP2) (4), a chromatin-associated gene product that is cru- Author contributions: L.L.G.C. and J.T.L. designed research; L.L.G.C. performed research; cial for neuronal development. Whereas males do not survive, W.P., W.M., and R.J.K. contributed new mouse lines/analytic tools; L.L.G.C. and J.T.L. females are typically born and remain symptom-free until the analyzed data; C.-Y. W. and C.W. performed bioinformatic analysis; and L.L.G.C. and first or second year of life. Then, symptoms arise that include J.T.L. wrote the paper. motor abnormalities, severe seizures, absent speech, and autism Reviewers: I.B., UMass Medical School; and G.C., University of Michigan. (5). To date, no disease-specific therapy is available for this Conflict of interest statement: J.T.L. is a founder and scientific advisory board member of disorder, which affects one in ∼10,000 girls throughout Translate Bio and Fulcrum Therapeutics. Patents have also been filed on the subject matter. the world. Published under the PNAS license. Notably, females carry a potential cure within their own cells. Data deposition: The data reported in this paper have been deposited in the Gene Ex- Every affected cell harbors a normal but dormant copy of pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. MECP2 on the inactive X (Xi) chromosome, which may, in GSE97077). principle, be reactivated to alleviate disease burden. Intriguingly, 1To whom correspondence should be addressed. Email: [email protected]. in male RTT mouse models, restoring normal Mecp2 expression This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. can reverse disease after the onset of symptoms (6, 7). There are, 1073/pnas.1715124115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1715124115 PNAS Early Edition | 1of8 Downloaded by guest on September 29, 2021 synergism between a ribonucleotide reductase subunit (RRM2) A qB qD qF1 qE1 qC1 qA5 qA2 qA6 qE2 qE3 qF2 qF3 qF4 qA4 qF5 qC2 ′ qC3 and 5-aza-2 -deoxycytidine (17). In a more direct approach, an qA1.1 qA3.1 qA7.1 qA3.2 qA1.2 qA7.2 qA7.3 a Xist RNA proteomic screen identified more than 100 interacting X Xist proteins and demonstrated that de-repression of the Xi could be Mecp2 s p r l n achieved robustly only when two to three interactors were tar- L1cam Naa10 Irak1 Opn1mw Flna Dnase1l1 Plxna3 Ubl4 geted simultaneously (18). In all studies to date, MECP2 resto- k Avpr2 Hcfc1 Mecp2 Tktl1 Emd Gdi1 Lage3 << o m ration has been extremely limited ( 1% of normal levels). Here, q g we integrate the existing knowledge and explore new methods of hij d b Xi reactivation. We arrive at a mixed modality approach, in- e f c Mecp2 cluding an antisense oligonucleotide (ASO) against Xist and an inhibitor of DNA methylation, the combination of which achieves B 1.5 a 30,000-fold reactivation of MECP2 from the Xi. 1 0.5 Results 0.02 0.015 Pharmacological Synergy Through a Mixed Modality Approach. While 0.01 the pharmaceutical industry has focused almost exclusively on 0.005 0 i j l r f k s e g n o p q b d h c targeting proteins, long noncoding RNAs (lncRNAs) have be- m Aza come increasingly attractive as pharmacological targets (19). blank control Xist = a Xa active Improving ASO technology makes lncRNAs pharmacologically untreated relative luciferase counts / cell accessible. ASOs are high-molecular-weight compounds that reference Aza + ASO have been optimized over the past 50 y through chemical mod- ifications to acquire greater stability, selectivity, and bio- availability (20, 21). Since ASOs bind their target through C Function Protein Class Inhibitor DNA modification DNMT Aza (DNMT1) Watson–Crick base-pairing interactions, they can be rationally DNA topology TOP topotecan (TOP1), designed and hit previously “undruggable” targets. Notably, ASO etoposide (TOP2) DNA repair PARP olaparib technology has achieved success in treating hypercholesterolemia Histone modification Histone PRT4165 (RING1 of PRC1), (Kynamro) and spinal muscular atrophy (Spinraza). ubiquitination We asked whether an ASO could also be developed for Xi HMT EPZ6438 (EZH2 of PRC2), A366 (G9A) reactivation and screened a small ASO library against various AURK VX680 (AURK), barasertib (AURKB) targets of potential interest, including Xist RNA and an anti- HDAC vorinostat & vorinostat (HDAC), romidepsin (HDAC1&2), sense transcript to Mecp2 (Mecp2-as) (Fig. 1A, Fig. S1, and entinostat (HDAC1&3), Table S1). In designing the ASOs, we chose phosphorothioate rocilinostat & tubastatin a (HDAC6) backbone and locked nucleic acid (LNA) chemistry (22) for its in HAT SGC-CBP30 (CREBBP) Chromatin remodeling SWI/SNF PFI-3 (SMARCA4/BRG1) vivo and in vitro stability, and increased affinity and selectivity Epigenetic reader BET JQ1, I-BRD9 (BRD9) for RNA targets.