CA2832097C - Mecp2 isoform-specific antibody for detection of endogenous expression of mecp2e2 - Google Patents

Abstract

An antibody that binds a MeCP2E2 isoform of MeCP2 protein, wherein the antibody comprises a region comprising the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:11. A method for detecting and/or monitoring a disease or a disorder caused by an over-expression or an under-expression of a MeCP2E2 isoform of MeCP2 protein, comprising: (i) obtaining a first sample from a mammalian subject; (ii) contacting the first sample with the anti-MeCP2E2 antibody; (iii) removing unbound antibody from the sample; (iv) conducting an immunoassay on the first sample to determine a first value for expression of the MeCP2E2 isoform; (v) comparing the first value to a reference value for expression of the MeCP2E2 isoform in healthy mammalian subjects; wherein a deviation of the first value from the reference value indicates the presence of a disease or a disorder caused by an over-expression or an under- expression of the MeCP2E2 isoform.

Description

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TITLE: MeCP2 ISOFORM-SPECIFIC ANTIBODY FOR DETECTION OF
ENDOGENOUS EXPRESSION OF MeCP2E2 TECHNICAL FIELD
The present disclosure relates generally to compositions and methods for detecting and/or monitoring abnormalities associated with over-expression and under-expression of Methyl CpG
Binding Protein 2. More particularly, the present disclosure relates to antibodies or antigen-binding fragments thereof which specifically bind to the MeCP2E2 isoform of Methyl CpG
Binding Protein 2, to methods for preparing the antibodies, to compositions containing such antibodies, and to use of the antibodies and/or compositions for detecting the MeCP2E2 isoform of Methyl CpG Binding Protein 2.
BACKGROUND
Mutation or altered expression of the X-linked Methyl CpG Binding Protein 2 (MECP2) gene leads to a wide spectrum of neurodevelopmental disorders including Rett Syndrome.
MeCP2 is a multifunctional epigenetic factor that is involved in multiple nuclear events including transcriptional repression, transcriptional activation, RNA
splicing, and chromatin compaction. MeCP2 was first discovered as a repressor protein that binds to methylated DNA at the 5-methylcytosine (5mC) residues. However, recent studies have shown that MeCP2 also binds to 5-hydroxymethylcytosine (5hmC), presumably as an activator (Mellen et al., 2012, MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system. Cell 151:1417-1430). While 5mC is a hallmark of inactive genes (Delcuve et al., 2009, Epigenetic control. J. Cell. Physiol. 219:243-250), 5hmC is generally associated with active genes (Mellen et al., 2012). Currently, it is unclear how, as a single protein, MeCP2 provides so many different nuclear functions.
In mice and humans, alternative splicing of the Mecp2/MECP2 gene leads to the generation of two protein isoforms, MeCP2E1 and MeCP2E2. MeCP2E1 contains a unique 21 amino acid sequence at its N-terminus, whereas the N-terminus of MeCP2E2 includes 9 exclusive amino acids (Kriaucionis et al., 2004, The major form of MeCP2 has a novel N-V AN_LAW\ 1320991\4 terminus generated by alternative splicing. Nucleic Acids Res. 32:1818-1823).
Other than their N-terminal regions, MeCP2 isoforms are similar and share the same functional domains, including the Methyl Binding Domain (MBD) and the Transcriptional Repression Domain (TRD) (Zachariah et al., 2012, Linking epigenetics to human disease and Rett syndrome: the emerging novel and challenging concepts in MeCP2 research. Neural Plasticity 2012:415825).
Previous studies indicate differential properties of MeCP2E1 and MeCP2E2 regarding their interacting protein partners, impact on neuronal survival, role during embryonic development, and sensitivity to different drugs.
Distinct transcript expression patterns have been reported for Mecp2/MECP2 isoforms in brain, with higher expression of Mecp2e1 than of Mecp2e2 (Dragich et al., 2007, DOerential distribution of the MeCP2 splice variants in the postnatal mouse brain. J.
Com.p Neurol.
501:526-542). However, comparative analysis of MeCP2 isoforms at the protein levels in any system has not been reported to date, due to the lack of specific anti-MeCP2E2 antibodies.
SUMMARY
The exemplary embodiments of the present disclosure relate to antibodies that selectively bind to the MeCP2E2 isoform of the MeCP2 protein, to compositions comprising the anti-MeCP2E2 antibodies, to methods for producing the anti-MeCP2E2 antibodies and compositions comprising the anti-MeCP2E2 antibodies, and to use of the anti-MeCP2E2 antibodies and compositions for detection of and monitoring of the over-expression and/or under-expression of MeCP2E2.
One exemplary embodiment of the present disclosure pertains to methods of preparing anti-MeCP2E2 antibodies or antigen-binding fragments that do not bind to or otherwise engage the MeCP2E1 isoform of the MeCP2 protein. The anti-MeCP2E2 antibodies are generated by a synthetic peptide that consists of a sequence of twelve amino acids selected from the N-terminus of the anti-MeCP2E2 isoform. Alternatively, the anti-MeCP2E2 antibodies are generated by a synthetic peptide that consists of a sequence of eleven amino acids selected from the N-terminus of the anti-MeCP2E2 isoform.
VAN_LAW \ 1320991\4 Another exemplary embodiment of the present disclosure pertains to compositions that include the foregoing antibodies or antigen-binding fragments thereof.
Another exemplary embodiment of the present disclosure pertains to foregoing isolated anti-MeCP2E2 antibodies or antigen-binding fragments thereof packaged in lyophilized form, or packaged in an aqueous medium.
Another exemplary embodiment of the present disclosure pertains to kits for detecting over-expression of MeCP2E2 or under-expression of MeCP2E2 for diagnosis, prognosis or monitoring. The kits include the foregoing isolated anti-MeCP2E2 antibody or antigen-binding fragment thereof labelled with a selected compound, and one or more compounds for detecting the label. Preferably the label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.
Another exemplary embodiment of the present disclosure pertains to methods for detecting an over-expression of MeCP2E2 or an under-expression of MeCP2E2, in a sample from a mammalian subject. The methods include contacting the sample with any of the foregoing antibodies or antigen-binding fragments thereof which specifically bind to an extracellular or a N-terminal domain of MeCP2E2, for a time sufficient to allow the formation of a complex between the antibody or antigen-binding fragment thereof and MeCP2E2, and detecting the MeCP2E2-antibody complex or MeCP2E2-antigen-binding fragment complex. The presence of a complex in the sample is indicative of the presence in the sample of MeCP2E2 or a cell expressing MeCP2E2.
In another aspect, the invention provides other methods for diagnosing a MeCP2E2-mediated disease or disorder in a mammalian subject. The methods include administering to a subject suspected of having or previously diagnosed with MeCP2E2-mediated disease an amount of any of the foregoing antibodies or antigen-binding fragments thereof which specifically bind to an extracellular or a N-terminal domain of MeCP2E2 antigen. The method also includes allowing the formation of a complex between the antibody or antigen-binding fragment thereof and MeCP2E2, and detecting the formation of the MeCP2E2-antibody complex or MeCP2E2-VAN_LAW\ 1320991\4 antigen-binding fragment antibody complex to the target epitope. The presence of a complex in the subject is indicative of the presence of a MeCP2E2-mediated disease or disorder.
BRIEF DESCRIPTION OF THE DRAWINGS
The present disclosure will be described in conjunction with reference to the following drawings, in which:
Fig. 1 shows the alignment of MeCP2E1 Isoform 1 amino acid sequences between human, mouse, and rat;
Fig. 2 is a Western blot gel to detect MeCP2E1 expression in control non-transfected (NT), MECP2E1 transfected (El -T), MECP2E2 transfected (E2-T), and MECP2E1 pre-incubated with the antigenic peptide. Anti-MYC labelling was used as a positive control while GAPDH labelling was used as a loading control;
Figs. 3(A)-3(D) are micrographs showing detection of MeCP2E1 by immunofluorescence in NIH3T3 cells transduced with MECP2E1 (Figs 3(A), 3(C)), NI113T3 cells transduced with MECP2E2 (Fig. 3(B)), and non-transduced NI113T3 cells (Fig. 3(D)) (Scale bars represent 10 lm);
Fig. 4 shows the alignment of MeCP2E2 Isoform 2 amino acid sequences between human, mouse, and rat;
Figs. 5(A) and 5(B) show negative controls for immunofluorescence detection of 5(A) MeCP2E2 and C-MYC in non-transduced NIH3T3 cells, and 5(B) absence of signals in primary omission controls with Rhodamine Red X (RRDX) and FITC in MECP2E2 transduced cells (scale bars represent 10 p.m);
Fig. 6 is a Western blot analysis for detection of MeCP2E2 expression in control non-transfected (NT), MECP2E1 transfected (El -T), MECP2E2 transfected (E2-T), and E2-T pre-incubated with E2 antigenic peptide. Anti-MYC labelling was used as a positive control;
Fig. 7 shows a Western blot analysis with Phoenix cell extracts from non-transfected cells (NT), and MECP2E2 transfected cells (E2-T), probed with the anti-MeCP2E2 antibody after pre-VAN_LAVA 1320991\4 incubation with increasing concentrations of peptide (0%, 0.1%, 1%, and 5%, of peptide compared to the amount of antibody used);
Figs. 8(A), 8(C), 8(E), 8(G) are micrographs showing detection by immunofluorescence staining of MeCP2E2 in NI113T3 cells transduced with a MECP2E1 retroviral vector, while Figs. 8(B), 8(D), 8(F), 8(11) are micrographs showing detection by immunofluorescence staining of MeCP2E2 in NIH3T3 cells transduced with a MECP2E2 retroviral vector (scale bars represent 20 m);
Fig. 9(A) is a Western blot gel showing Detection of MeCP2E1 in the nuclear extracts from adult mouse brain but not in the cytoplasmic extracts, while Fig. 9(B) is a Western blot gel showing Detection of MeCP2E2 in the nuclear extracts from adult mouse brain but not in the cytoplasmic extracts. Increasing amounts of nuclear and cytoplasmic protein extracts were used.
and the membranes were re-probed with GAPDH as a loading control;
Fig. 10 shows a Western blot analysis of MeCP2E1 in the isolated nuclear extracts from the whole brain at the indicated developmental time points. Nuclear extracts from null brain were used as negative controls while ACTIN was used as a loading control (E =
embryonic days; P =
postnatal days; null = mecp2tm1.1Bird y/-brain tissue; N = 3 SEM);
Fig. 11 is a chart showing quantitative RT-PCR with specific primers to detect Mecp2e1 and Mecp2e2 transcripts. Total RNA from null brain was used as the negative control (E ¨
embryonic days; P = postnatal days; null = mecp2tmLiBird Y/ brain tissue; N =
3 SEM; significant differences are indicated at P<0.001).;
Fig. 12 shows the results of a Pearson's correlation analysis for the indicated Mecp2 transcripts and MeCP2 protein levels;
Fig. 13 is a schematic representation of the MECP2E1 retroviral vector with a C-MYC
tag (Retro-EF 1 a-E1) and the MECP2E2 retroviral vector with a C-MYC tag (Retro-EF 1 a-E2) retroviral vectors with C-MYC tag that were used for transfection of Phoenix cells (shown in Figs. 2, 6) and transduction of NIH3T3 cells (shown in Figs. 3, 8);
VAN_LAW\ 1320991\4 Figs. 14(A), 14(C), 14(E) are micrographs showing detection by immunohistochemistry of endogenous MeCP2E2 in the CA1 region of adult mouse hippocampus from wild type mecp2tm1.1Bird y/+ mice, while Figs. 14(B), 14(D), 14(F) are micrographs showing absence of detection of endogenous MeCP2E2 in the CA1 region of adult mouse hippocampus from null (mecp2tm 1Bird y/-) Mecp2 mice (scale bars represent 20 [im);
Figs. 15(A)-15(E) are micrographs of controls to verify the specificity of anti-MeCP2E2 detection by immunohistochemistry in the adult mouse brain wherein 15(A) are views of primary omission, 15(B) are views of anti-MeCP2E2 incubation with IgY and pre-incubation of the newly generated anti-MeCP2E2 antibody with the antigenic peptide MeCP2E2 shown in 15(C), 15(D) are views of pre-incubation of the newly generated anti-MeCP2E2 antibody with the antigenic peptide MeCP2E1, 15(E) are views of anti-MeCP2E2 incubation with a peptide against the C-terminus of MeCP2 (scale bars represent 10 [im);
Fig. 16 is a Western blot analysis to detect endogenous MeCP2E2 expression in the WT
adult mouse brain and its absence in Mecp2 null mice brain, GAPDH was used as a loading control;
Figs. 17(A)-17(C) are micrographs of confocal images of MeCP2E1 in WT adult mouse brain hippocampus CA1 region, while 17(D) is a chart showing the signal intensity profile of MeCP2E1 and DAPI co-localization indicating MeCP2E1 detection at the DAPI-rich heterochromatin regions of nuclei (scale bar represents 2 gm);
Fig. 18(A)-18(C) are micrographs of confocal images of MeCP2E2 in WT adult mouse brain hippocampus CAI region, while 18(D) is a chart showing the signal intensity profile of MeCP2E2 and DAPI co-localization indicating MeCP2E2 detection at the DAPI-rich heterochromatin regions of nuclei (scale bar represents 2 pm);
Fig. 19 shows a Western blot analysis of MeCP2E2 in the isolated nuclear extracts from the whole brain at the indicated developmental time points. Nuclear extracts from null brain were used as negative controls while ACTIN was used as a loading control (E =
embryonic days; P =
tm1.-postnatal days; null = Mecp2 1Bird y/
brain tissue; N = 3 SEM);
VAN_LAW\ 1320991\4 Figs. 20(A) are micrographs of immunofluorescence expression of the MeCP2E1 isoform in neurons from the CA1 hippocamus region of adult male mouse brain, while Figs.
20(B) are micrographs of immunofluorescence expression of the MeCP2E2 isoform (scale bar represents 20 gm);
Figs. 21(A) are micrographs of immunofluorescence expression of the MeCP2E1 isoform in a single neuron nucleus from the CA1 hippocamus region of adult male mouse brain, while Figs. 21(B) are micrographs of immunofluorescence expression of the MeCP2E2 isoform in a single neuron nucleus from the CA1 hippocamus region of adult male mouse brain (scale bar represents 2 p.m);
Figs. 22(A) are micrographs of immunofluorescence expression of the MeCP2E1 isoform in GFAP-positive astrocytes from the CA1 hippocamus region of adult male mouse brain, while Figs. 22(B) are micrographs of immunofluorescence expression of the MeCP2E2 isoform in GFAP-positive astrocytes from the CA1 hippocamus region of adult male mouse brain (scale bar represents 20 gm);
Figs. 23(A) are micrographs of immunofluorescence expression of the MeCP2E1 isoform in CNPase-positive oligodendrocytes from the CA1 hippocamus region of adult male mouse brain, while Figs. 23(B) are micrographs of immunofluorescence expression of the MeCP2E2 isoform in CNPase-positive oligodendrocytes from the CA1 hippocamus region of adult male mouse brain (scale bar represents 20 p.m);
Figs. 24(A) are micrographs of immunofluorescence expression of the MeCP2E1 isoform in a single astrocyte nucleus from the CA1 hippocamus region of adult male mouse brain, while Figs. 24(B) are micrographs of immunofluorescence expression of the MeCP2E2 isoform in a single astrocyte nucleus from the CA1 hippocamus region of adult male mouse brain (scale bar represents 2 gm);
Figs. 25(A) are micrographs of immunofluorescence expression of the MeCP2E1 isoform in a single oligodendrocyte nucleus from the CA1 hippocamus region of adult male mouse brain, while Figs. 25(B) are micrographs of immunofluorescence expression of the MeCP2E2 isoform VAN_LAW\ 1320991\4 in a single oligodendrocyte nucleus from the CA1 hippocamus region of adult male mouse brain (scale bar represents 2 um);
Figs. 26(A) are micrographs of immunofluorescence expression of the MeCP2E1 isoform in neurons from the CA1 hippocamus region of adult female mouse brain, while Figs. 26(B) are micrographs of immunofluorescence expression of the MeCP2E2 isoform (scale bar represents 20 um);
Figs. 27(A) are micrographs of immunofluorescence expression of the MeCP2E1 isoform in a single neuron nucleus from the CA1 hippocamus region of adult female mouse brain, while Figs. 27(B) are micrographs of immunofluorescence expression of the MeCP2E2 isoform in a single neuron nucleus from the CA1 hippocamus region of adult female mouse brain (scale bar represents 2 um);
Figs. 28(A) are micrographs of immunofluorescence expression of the MeCP2E1 isoform in GFAP-positive astrocytes from the CA1 hippocamus region of adult female mouse brain, while Figs. 28(B) are micrographs of immunofluorescence expression of the MeCP2E2 isoform (scale bar represents 20 um);
Figs. 29(A) are micrographs of immunofluorescence expression of the MeCP2E1 isoform in CNPase-positive oligodendrocytes from the CA1 hippocamus region of adult female mouse brain, while Figs. 29(B) are micrographs of immunofluorescence expression of the MeCP2E2 isoform in CNPase-positive oligodendrocytes from the CA1 hippocamus region of adult female mouse brain (scale bar represents 20 pm);
Figs. 30(A) are micrographs of immunofluorescence expression of the MeCP2E1 isoform in a single astrocyte nucleus from the CA1 hippocamus region of adult female mouse brain, while Figs. 30(B) are micrographs of immunofluorescence expression of the MeCP2E2 isoform in a single astrocyte nucleus from the CA1 hippocamus region of adult female mouse brain (scale bar represents 2 um);
Figs. 31(A) are micrographs of immunofluorescence expression of the MeCP2E1 isoform in a single oligodendrocyte nucleus from the CA1 hippocamus region of adult mouse brain, while Figs. 31(B) are micrographs of immunofluorescence expression of the MeCP2E2 VAN_LAW\ 1320991\4 isoform in a single oligodendrocyte nucleus from the CA1 hippocamus region of adult mouse brain (scale bar represents 2 pm);
Figs. 32(A, A) - 32(A, D) are micrographs of immunohistochemical detection of the expression of the MeCP2E1 isoform in the whole hippocampus (32(A, A)), in the CA2 region of the hippocampus (32(A, B)), in the CA3 region of the hippocampus (32(A, C)), and the dentate gyms region of the hippocampus (32(A, D)), while Figs. 32(B, Al) - 32(B, D1) are micrographs of immunohistochemical detection of the expression of the MeCP2E2 isoform in the whole hippocampus (32(B, Al)), in the CA2 region of the hippocampus (32(B, B1)), in the CA3 region of the hippocampus (32(B, Cl)), and the dentate gyrus region of the hippocampus (32(B, D1)) (scale bars represent 200 pin in A, Al; scale bars represent 20 pm in B, Bl, C, Cl, D, D1);
Figs. 33(A, A) - 33(A, C) are micrographs of immunohistochemical detection of the expression of the MeCP2E1 isoform in the olfactory bulb region of the brain (33(A, A)), in the striatum region of the brain (33(A, B)), and in the cortex region of the brain (33(A, C)), while Figs. 33(B, Al) - 33(B, Cl) are micrographs of immunohistochemical detection of the expression of the MeCP2E2 isoform in the olfactory bulb region of the brain (33(B, Al)), in the striatum region the brain (33(B, B1)), and in the cortex region of the brain (33(B, Cl)) (scale bars represent 20 pm);
Figs. 34(A, A) - 34(A, C) are micrographs of immunohistochemical detection of the expression of the MeCP2E1 isoform in the thalamus region of the brain (34(A, A)), in the brain stem region of the brain (34(A B)), and in the cerebellum region of the brain (34(A, C)), while Figs. 34(B,A1) - 34(B,C1) are micrographs of immunohistochemical detection of the expression of the MeCP2E2 isoform in the thalamus region of the brain (34(B, Al)), in the brain stem region the hippocampus (34(B, B1)), and in the cerebellum region of the brain (34(B, Cl)) (scale bars represent 20 p.m);
Figs. 35(A)-35(D) are micrographs of immunohistochemical detection of the expression of the MeCP2E1 isoform and the MeCP2E2 isoform in the molecular layer (m1), the Purkinje cell layer (pep, and granule cell layer (gcl) of the cerebellum (the scale bar represents 20 m);
VAN_LAW\ 1320991\4 Figs. 36(A)-36(C) are micrographs showing colocalization of the expression of the MeCP2E1 isoform and the MeCP2E2 isoform in the molecular layer of the cerebellum (36(A)), in the Purkinje cell layer of the cerebellum (36(B)), and in the granule cell layer of the cerebellum (36(C)) (the scale bar represents 2 }im);
Fig. 37 is a chart and a Western gel showing the expression of the MeCP2E1 isoform in adult mouse brain regions. Whole WT Mecp2 and null Mecp2 adult brains were used as controls, while ACTIN used as the loading control (OB = olfactory bulb; STR = striatum;
CTX = cortex;
HIPP = hippocampus; THAL = thalamus; BS = brain stem; CERE = cerebellum; N = 3 SEM);
Fig. 38 is a chart showing the results of quantitative RT-PCR to detect transcript levels of -- Mecp2 isoforms in adult mouse brain regions (OB = olfactory bulb; STR =
striatum; CTX =
cortex; HIPP = hippocampus; THAL = thalamus; BS = brain stem; CERE =
cerebellum; N =
3 SEM; significant differences between the two isoforms are indicated with P<0.0001****, P<0.001***, P<0.01** or P<0.05*);
Fig. 39 shows the results of a Pearson's correlation analysis for indicated Mecp2 -- transcripts and MeCP2 protein levels;
Fig. 40 is a chart and a Western gel showing the expression of the MeCP2E2 isoform in adult mouse brain regions. Whole WT Mecp2 and null Mecp2 adult brains were used as controls, while ACTIN used as the loading control (OB = olfactory bulb; STR = striatum;
CTX = cortex;
HIPP = hippocampus; THAL = thalamus; BS = brain stem; CERE = cerebellum; N = 3 SEM);
and Fig. 41(A) is a chart showing a semi-quantitative representation of MeCP2E I
and MeCP2E2 levels in WT Mecp2 and null Mecp2 adult brain (N = 3 SEM; significant differences between the two isoforms are indicated with P<0.001***), while Fig. 41(B) is a chart showing a Quantitative RT-PCR to detect transcript levels of Mecp2 isoforms in WT Mecp2 and null -- Mecp2 adult mouse whole brains. (N = 3 SEM; significant differences between the two isoforms are indicated with P<0.0001****).
VAN LAW\ 1320991\4 DETAILED DESCRIPTION
The exemplary embodiments of the present disclosure pertain to antibodies that selectively bind to the MeCP2E2 isoform of the MeCP2 protein, to compositions comprising the anti-MeCP2E2 antibodies, to methods for producing the anti-MeCP2E2 antibodies and compositions comprising the anti-MeCP2E2 antibodies, and to use of the anti-MeCP2E2 antibodies and compositions for detection of and monitoring of the over-expression and/or under-expression of MeCP2E2.
Accordingly, one exemplary embodiment of the present disclosure pertains to methods of preparing anti-MeCP2E2 antibodies or antigen-binding fragments that do not bind to or otherwise engage the MeCP2E1 isoform of the MeCP2 protein. The anti-MeCP2E2 antibodies can be generated by a peptide that consists of the sequence of twelve amino acids set forth in SEQ ID NO:10. Alternatively, the anti-MeCP2E2 antibodies can be generated by a peptide that consists of the sequence of eleven amino acids set forth in SEQ ID NO:11.
Another exemplary embodiment of the present disclosure pertains to compositions that include the foregoing antibodies or antigen-binding fragments thereof.
Another exemplary embodiment of the present disclosure pertains to foregoing isolated anti-MeCP2E2 antibodies or antigen-binding fragments thereof packaged in lyophilized form, or packaged in an aqueous medium.
Another exemplary embodiment of the present disclosure pertains to kits for detecting over-expression of MeCP2E2 or under-expression of MeCP2E2 or relative protein expression to MeCP2E1 for diagnosis, prognosis or monitoring a disease or a disorder in a subject. The kits include the foregoing isolated anti-MeCP2E2 antibody or antigen-binding fragment thereof labelled with a selected compound, and one or more compounds for detecting the label.
Preferably the label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.
Another exemplary embodiment of the present disclosure pertains to methods for detecting an over-expression of MeCP2E2 or an under-expression of MeCP2E2 or relative VAN JAW\ 1320991\4 protein expression to MeCP2E1, in a sample collected from a mammalian subject.
The methods include contacting the sample with any of the foregoing antibodies or antigen-binding fragments thereof which specifically bind to an extracellular or a N-terminal domain of MeCP2E2, for a time sufficient to allow the formation of a complex between the antibody or antigen-binding -- fragment thereof and MeCP2E2, and detecting the MeCP2E2-antibody complex or MeCP2E2-antigen-binding fragment complex. The presence of a complex in the sample is indicative of the presence in the sample of MeCP2E2 or a cell expressing MeCP2E2.
Another exemplary embodiment pertains to methods for diagnosing a MeCP2E2-mediated disease or disorder in a mammalian subject. The methods include administering to a -- subject suspected of having or previously diagnosed with MeCP2E2-mediated disease an amount of any of the foregoing antibodies or antigen-binding fragments thereof which specifically bind to an extracellular or a N-terminal domain of MeCP2E2 antigen. The method also includes allowing the formation of a complex between the antibody or antigen-binding fragment thereof and MeCP2E2, and detecting the formation of the MeCP2E2-antibody complex or MeCP2E2--- antigen-binding fragment antibody complex to the target epitope. The presence of a complex in the subject is indicative of the presence of a MeCP2E2-mediated disease or disorder.
Another exemplary embodiment pertains to use of the anti-MeCP2E2antibodies or antigen-binding fragments thereof, and/or use of the compositions that include the foregoing antibodies or antigen-binding fragments thereof, and/or use of the kits, and/or use methods for -- detecting and/or diagnosing Rett's syndrome in a mammalian subject.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Certain terms are discussed in the specification to provide additional guidance to the practitioner in describing the methods, uses and the like of embodiments of the disclosure, and -- how to make or use them. It will be appreciated that the same thing may be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein. No significance is to be placed upon whether or not a term is elaborated or discussed herein. Recital of one or a few synonyms or equivalents does not exclude use of other synonyms or equivalents, unless it is explicitly stated. Use of examples in the specification, VAN_LAW\ 1320991 \ 4 including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of the embodiments of the disclosure herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
To facilitate understanding of the disclosure, the following definitions are provided.
The word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers.
As used herein, the word "complexed" means attached together by one or more linkages.
The term "a cell" includes a single cell as well as a plurality or population of cells.
Administering an agent to a cell includes both in vitro administrations and in vivo administrations.
The term "subject" as used herein includes all members of the animal kingdom, and specifically includes humans.
The term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.
The term "homologous" in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin,"
including homologous proteins from different species. Such proteins (and their encoding genes) have sequence homology, as reflected by their high degree of sequence similarity.
This homology is greater than about 75%, greater than about 80%, greater than about 85%. In some cases the homology will be greater than about 90% to 95% or 98%.
"Amino acid sequence homology" is understood to include both amino acid sequence identity and similarity. Homologous sequences share identical and/or similar amino acid residues, where similar residues are conservative substitutions for, or "allowed point mutations"
of, corresponding amino acid residues in an aligned reference sequence. Thus, a candidate polypeptide sequence that shares 70% amino acid homology with a reference sequence is one in VAN_LAW\ 1320991\4 which any 70% of the aligned residues are either identical to, or are conservative substitutions of, the corresponding residues in a reference sequence.
The term "polypeptide" refers to a polymeric compound comprised of covalently linked amino acid residues. Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group. A polypeptide of the disclosure preferably comprises at least about 14 amino acids.
The term "protein" refers to a polypeptide which plays a structural or functional role in a living cell.
The term "corresponding to" is used herein to refer to similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured. A nucleic acid or amino acid sequence alignment may include spaces.
Thus, the term "corresponding to" refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.
The term "derivative" refers to a product comprising, for example, modifications at the level of the primary structure, such as deletions of one or more residues, substitutions of one or more residues, and/or modifications at the level of one or more residues. The number of residues affected by the modifications may be, for example, from 1, 2 or 3 to 10, 20, or 30 residues. The term derivative also comprises the molecules comprising additional internal or terminal parts, of a peptide nature or otherwise. They may be in particular active parts, markers, amino acids, such as methionine at position ¨1. The term derivative also comprises the molecules comprising modifications at the level of the tertiary structure (N-terminal end, and the like). The term derivative also comprises sequences homologous to the sequence considered, derived from other cellular sources, and in particular from cells of human origin, or from other organisms, and possessing activity of the same type or of substantially similar type. Such homologous sequences may be obtained by hybridization experiments. The hybridizations may be performed based on VAN_LAWN 1320991\4 nucleic acid libraries, using, as probe, the native sequence or a fragment thereof, under conventional stringency conditions or preferably under high stringency conditions.
The term "antibody" as used herein refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Cl q) of the classical complement system.
The term "antigen-binding fragment" of an antibody as used herein, refers to one or more portions of an antibody that retain the ability to specifically bind to an antigen (e.g., MeCP2E2 isoform of the MeCP2 protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI
domains; (ii) a F(ab1)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains;
(iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546) which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, V and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which VAN JLAW\ 1320991\4 the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.
NatL Acad. Sci. USA
85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional procedures, such as proteolytic fragmentation procedures, as described in J.
Goding, Monoclonal Antibodies: Principles and Practice, pp 98-118 (N.Y.
Academic Press 1983). The fragments are screened for utility in the same manner as are intact antibodies.
An "isolated antibody", as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to MeCP2E2 isoform of the MeCP2 protein and is substantially free of antibodies that specifically bind antigens other than the MeCP2E2 isoform). As used herein, "specific binding" refers to antibody binding to a predetermined antigen. Typically, the antibody binds with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.
The term "complementarity determining regions" as used herein refers to the regions within antibodies where these proteins complement an antigen's shape. The acronym CDR is used herein to mean "complementarity determining region".
The antibodies of the present disclosure may be polyclonal antibodies and can be produced by a variety of techniques well known in the art. Procedures for raising polyclonal antibodies are well known. For example anti-MeCP2E2 polyclonal antibodies may be raised by administering a synthetic peptide (e.g., SEQ ID NO:10, or alternatively, SEQ
ID NO:11) subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The synthetic peptide can be injected at a total volume of 100 IA per site at six different sites, typically with one or more adjustments. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is collected 10 days after each boost. Polyclonal antibodies are recovered from the serum, preferably by affinity chromatography using the synthetic peptide to capture the antibody.
VAN_LAW\ 1320991\4 4 = . V86625CA 17 This and other procedures for raising polyclonal antibodies are disclosed in E. Harlow, et. al., editors, Antibodies: A Laboratory Manual (1988).
An embodiment of the present disclosure relates to a method of detecting cells or portions thereof in a biological sample (e.g., histological or cytological specimens, biopsies, whole blood samples, separated blood cells, and the like) wherein the MeCP2E2 is overexpressed. This method involves providing an antibody or an antigen-binding binding fragment thereof, probe, or ligand, which specifically binds to an a peptide having a sequence with at least 90% to SEQ ID
NO:10, preferably at least about 95% identical, more preferably at least about 97% identical, still more preferably at least about 98% identical, and most preferably is at least about 99% identical.
This method also involves providing an antibody or an antigen-binding binding fragment thereof, probe, or ligand, which specifically binds to an a peptide having a sequence with at least 90% to SEQ ID NO:11, preferably at least about 95% identical, more preferably at least about 97%
identical, still more preferably at least about 98% identical, and most preferably is at least about 99% identical. The anti-MeCP2E2 antibody is bound to a label that permits the detection of the cells or portions thereof upon binding of the anti-MeCP2E2 antibody to the cells or portions thereof. The biological sample is contacted with the labeled anti-MeCP2E2 antibody under conditions effective to permit binding of the anti-MeCP2E2 antibody to the extracellular domain or N-terminal domain of MeCP2E2 of any of the cells or portions thereof in the biological sample. The presence of any cells or portions thereof in the biological sample is detected by detection of the label. In one preferred form, the contact between the anti-MeCP2E2 antibody and the biological sample is carried out in a living mammal and involves administering the anti-MeCP2E2 antibody to the mammal under conditions that permit binding of the anti-MeCP2E2 antibody to MeCP2E2 of any of the cells or portions thereof in the biological sample. Again, such administration can be carried out by any suitable method known to one of ordinary skill in the art.
In addition, the anti-MeCP2E2 antibodies of the present disclosure can be used in immunofluorescence techniques to examine human tissue, cell and bodily fluid specimens. In a typical protocol, slides containing cryostat sections of frozen, unfixed tissue biopsy samples or cytological smears are air dried, formalin or acetone fixed, and incubated with the monoclonal antibody preparation in a humidified chamber at room temperature. The staining pattern and REPLACEMENT SHEET

intensities within the sample are then determined by fluorescent light microscopy and optionally photographically recorded.
As yet another alternative, computer enhanced fluorescence image analysis or flow cytometry can be used to examine tissue specimens or exfoliated cells, i.e., single cell preparations from aspiration of tissues or organs using the anti-MeCP2E2 antibodies of this disclosure. The percent MeCP2E2 positive cell population, alone or in conjunction with determination of other attributes of the cells (e.g., DNA ploidy of these cells), may, additionally, provide very useful prognostic information by providing an early indicator of disease progression.
The method of the present disclosure can be used to screen patients for diseases or disorders associated with the over-expression of MeCP2E2 or under-expression of MeCP2E2 or changes in MeCP2E2 protein expression relative to MeCP2E1. Alternatively, it can be used to identify the recurrence of such diseases or disorders, particularly when the disease or disorder is localized in a particular biological material of the patient.
Also within the scope of the disclosure are kits comprising the compositions of the disclosure and instructions for use. Kits containing the antibodies or antigen-binding fragments thereof of the present disclosure can be prepared for in vitro diagnosis, prognosis and/or monitoring of the over-expression of MeCP2E2 or the under-expression of MeCP2E2 or its protein expression relative to MeCP2E1 by the immunohistological, immunocytological and immuno serological methods described above. The components of the kits can be packaged either in aqueous medium or in lyophilized form. When the antibodies or antigen-binding fragments thereof are used in the kits in the form of conjugates in which a label moiety is attached, such as an enzyme or a radioactive metal ion, the components of such conjugates can be supplied either in fully conjugated form, in the form of intermediates or as separate moieties to be conjugated by the user or the kit.
A kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more container means or series of container means such as test tubes, vials, flasks, bottles, syringes, or the like. A first of said container means or series of container means may contain one or more anti-MeCP2E2 antibodies or antigen-binding fragments thereof or MeCP2E2. A second container means or series of container means may contain a label or linker-VAN_LAW\ 1320991\4 label intermediate capable of binding to the primary anti-MeCP2E2 antibodies (or fragment thereof).
The present disclosure will be further elaborated in the following examples.
However, it is to be understood that these examples are for illustrative purposes only, and should not be used to limit the scope of the present disclosure in any manner.
EXAMPLES
Example 1: Generation of polyclonal and monoclonal MeCP2E1 isoform specific antibodies An isoform-specific antibody that was generated in a different species was required to enable double-labelling of the MeCP2E1 and MeCP2E2 isoforms in a single organism..
Accordingly, the strategy disclosed by Zachariah et al. (2012, Novel MeCP2 isoform-specific antibody reveals the endogenous MeCP2E1 expression in murine brain, primary neurons and astrocytes. PloS ONE 7, e49763) was followed as taught for the production of polyclonal MeCP2E1 antibodies in rabbits.
Fig. 1 shows the alignment of MeCP2E1 Isoform 1 amino acid sequences between human, mouse, and rat, and a peptide sequence "GGGEEERLEEKS" (SEQ ID NO:4;
shaded section in Fig. 1) selected from the N-terminus for use as an antigen for generating polyclonal MeCP2E1 antibodies in rabbits. The IgY molecules were purified from rabbit blood and anti-MeCP2E1-specific immunoglobulins were isolated by peptide affinity purification.
An additional "C" residue was inserted at the N-terminal end of the sequence "GGGEEERLEEKSC" (SEQ ID NO:6). The additional C (underlined) was used for conjugation with BSA (Bovine Serum Albumin) and KLH (Keyhole limpet hemocyanin) for antibody purification. The generated antibodies were tested against MeCP2E1 peptide by ELISA during production, and were also tested by WB (Western Blot) and immunofluorescence (IF) studies in transfected Phoenix cells and transduced NIH3T3 cells with MECP2E1 retroviral vectors carrying human MECP2E1 cDNA. The overexpressed MeCP2E1 has a MYC tag and we confirmed the detection of similar signals with C-MYC antibody. Importantly, C-MYC also detects MeCP2E2 (the other MeCP2 isoform that has a MYC tag). However, the anti-MeCP2E1 VAN_LAW\ 1320991\4 antibody disclosed herein does not cross-react with the MeCP2E2 isoform.
The specificity and sensitivity of the anti-MeCP2E1 antibody was initially verified by Western blotting. Probing protein extracts from non-transfected, MeCP2E1-transfected and MeCP2E2-transfected phoenix cells, the affinity purified anti-MeCP2E1 detected specific bands at approximately 75 kDa in MeCP2E1-transfected extracts (Fig. 2, lane 2). No signal was detected in non-transfected cells (Fig. 2, lane 1), nor in transfected cells with MECP2E2 (Fig. 2, lane 3). The presence of exogenous MeCP2 in the transfected cells with either Retro-EF la-El or Retro-EF 1 a-E2 was verified by immunolabelling with an anti-C-MYC antibody (Fig. 2, lanes 5-6), with no detectable signal in non-transfected cells (Fig. 2, lane 4).
Furthermore, pre-incubation of the anti-MeCP2E1 antibody with the antigenic peptide before probing the membranes with MECP2E1 transfected cell lysate (Fig. 2, lane 7) completely abrogated the detection of exogenous MeCP2E1. Immunofluorescence staining with the anti-MeCP2E1 antibody revealed the expression of MeCP2 in the DAPI-rich heterochromatic foci within the NIH3T3 cells transduced with MECP2E1 (Fig. 3(A)), but no signal was detected in the MECP2E2 transduced cells (Fig. 3(B)) confirming that the anti-MeCP2E1 antibody does not cross-react with the overexpressed MECP2E2. In both MECP2E1 and MECP2E2 overexpressed cells, incubation with an anti-C-MYC antibody resulted in detectable signals indicating that the transduced protein is properly expressed in both cases. As expected, no signals were detected in primary omission experiments using Retro-EF 1 a-E1 transduced cells with the same secondary antibody (Fig. 3(C).
The absence of endogenous MECP2E2 expression was confirmed in the non-transduced NIH3T3 cells by using the anti-MeCP2E2 antibody (Fig. 3(D)), thus confirming that the anti-MeCP2E1 antibody disclosed herein does not cross-react with the MeCP2E2 isoform.
Additionally, mouse monoclonal antibodies were generated against MeCP2E1 with the same strategy and the corresponding clones were selected based on positive ELISA readings. It was confirmed that these monoclonal antibodies detect exogenous MeCP2E1 by WB
and IF.
The data generated in this study shows that inclusion or exclusion of an extra amino acid at the N-terminal part of the peptide does not affect the specificity of the generated antibody and does not cause any cross-reactivity with the other MeCP2E2 isoform. Therefore, both peptides:
"GGGEEERLEEKS" (SEQ ID NO:4) or "GGGEEERLEEK" (SEQ ID NO:5) can be VAN_LAW\ 1320991\4 successfully used for generating polyclonal or monoclonal antibodies against MeCP2E1 protein isoform.
Example 2: Generation of polyclonal and monoclonal MeCP2E2 isoform specific antibodies Fig. 4 shows the alignment of MeCP2E2 Isoform 2 amino acid sequences between human, mouse, and rat, and a peptide sequence "VAGMLGLREEKS" (SEQ ID NO:10;
shaded section in Fig. 4) selected from the N-terminus for use as an antigen for generating polyclonal MeCP2E1 antibodies in chickens. The IgY molecules were purified from chicken egg yolks and anti-MeCP2E1-specific immunoglobulins were isolated by peptide affinity purification.
Equivalent anti-MeCP2E2 antibodies have also been generated in rabbit and show similar specificity for detecting MeCP2E2 protein isoform.
An additional "C" residue was inserted at the N-terminal end of the sequence "VAGMLGLREEKSC" (SEQ ID NO:12). The additional C (underlined) was used for conjugation with BSA (Bovine Serum Albumin) and KLH (Keyhole limpet hemocyanin) for antibody purification. The generated antibodies were tested against MeCP2E2 peptide by ELISA
during production, and were also tested by WB (Western Blot) and immunofluorescence (IF) studies in transfected Phoenix cells and transduced NIH3T3 cells with MECP2E2 retroviral vectors carrying human MECP2E2 cDNA. The overexpressed MeCP2E2 has a MYC tag and we confirmed the detection of similar signals with C-MYC antibody. Importantly, C-MYC also detects MeCP2E1 (the other MeCP2 isoform that has a MYC tag). However, the anti-MeCP2E2 antibody disclosed herein does not cross-react with the MeCP2E1 isoform.
The specificity of the anti-MeCP2E2 antibody by Western blot (WB) and immunofluorescence (IF) experiments at various stages of the antibody production and after IgY
purification. For validations by WB, the affinity purified antibody was tested using protein extracts from Phoenix cells transfected with either Retro-EF1a-E1 or Retro-EF
1 a-E2 (Fig. 6), in parallel to non-transfected control cells following the method taught by Rastegar et al. (2009, MECP2 isoform-specific vectors with regulated expression for Rett syndrome gene therapy. PloS
ONE 4:e6810).
Western blot analysis with the anti-MeCP2E2 antibody yielded a specific band at the VAN_LAW1 1320991\4 expected molecular weight (approximately75 kDa) in MECP2E2-transfected cells (Fig. 6, lane 3). In contrast, no signal was detected in non-transfected cells (Fig. 6, lane 1), nor in transfected cells with MECP2E1 (Fig. 6, lane 2). Importantly, pre-incubation of the anti-MeCP2E2 antibody with the antigenic peptide used to generate the antibody (peptide competition) eliminated the detection of signal in the MECP2E2 transfected cells (Fig. 6, lane 7). The specificity and sensitivity of this newly developed anti-MeCP2E2 antibody was verified by pre-incubation of antibody with increasing concentrations of the antigenic peptide before probing the membranes with MECP2E2 transfected cell lysates (Fig. 7, lanes 2-4). The presence of exogenous MeCP2 in the transfected cells with either Retro-EF 1 a-E1 or Retro-EF 1 a-E2 was verified by immunolabelling with an anti-C-MYC antibody (Fig. 6, lanes 5-6), with no detectable signal in non-transfected cells (Fig. 6, lane 4).
Further verification of the specificity of the custom-made anti-MeCP2E2 antibody using IF, revealed the localization of MeCP2E2 in the DAPI-rich heterochromatic foci within the NIH3T3 cells transduced with MECP2E2 (Fig. 8(B)). No signal was detected in the MECP2EI
transduced cells (Fig. 8(A)). C-MYC labelling confirmed the successful transduction of both MeCP2E1 and MeCP2E2 within the tested samples (Figs, 8(C), 8(D)). The absence of endogenous MECP2E2 expression was verified in non-transduced NIH3T3 cells using the anti-MeCP2E2 antibody (Fig. 5(A)). No signal could be observed in primary omission experiments using Retro-EF 1 a-E1 transduced cells with the same secondary antibodies (Fig. 5(B)).
Additionally, mouse monoclonal antibodies were generated against MeCP2E2 with the same strategy using the amino acid sequence set forth in SEQ ID NO:11 and the corresponding clones were selected based on positive ELISA readings. It was confirmed that these monoclonal antibodies detect exogenous MeCP2E2 by WB and IF.
The data shows that inclusion or exclusion of an extra amino acid at the N-terminal part of the peptide does not affect the specificity of the generated antibody and does not cause any cross-reactivity with the other MeCP2E1 isoform. Therefore, both peptides:
"VAGMLGLREEKS" (SEQ ID NO:10) or "VAGMLGLREEK" (SEQ ID NO: 11) can be successfully used for generating polyclonal or monoclonal antibodies against MeCP2E2 protein isoform.
VANJAW\ 1320991\4 Example 3: Generation of MECP2E1/E2 transfected/transduced cells Retro-EF 1 a-E1 (expressing MECP2E1) and Retro-EF 1 a-E2 (expressing MECP2E2) vectors were transfected into Phoenix retroviral packaging cells (Kinsella et al., 1996, Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum.
Gene. Ther .7:1405-1413.) following the method taught by Rastegar et al. (2009, MECP2 isoform-specific vectors with regulated expression for Rett syndrome gene therapy. PLoS One 4: e6810) to generate (i) infectious retroviral MECP2E1 vectors with a C-terminal C-Myc tag particles, and (ii) retroviral MECP2E2 vectors with a C-terminal C-Myc tag particles. Culture supernatants containing viral particles were harvested at 48 hours post-transfections. The transfected phoenix cells were collected and lysed for protein extraction, and the retroviral particles were used to transduce NIH3T3 mouse fibroblasts following the method taught by Rastegar et al.
(2009). The transduced cells were fixed with 4% paraformaldehyde for imrnunofluorescent studies, 48 hours after transduction. NIH2T3 cells, Phoenix cells, and MECP2 vectors were obtained from The Hospital for Sick Children (Toronto, ON, CA).
Example 4: Quantitative Real Time PCR (qRT-PCR) Total RNA from brain regions and brains at developmental stages were extracted using RNEASY Mini Kit (RNEASY is a registered trademark of Qiagen GmbH, Hilden, Fed. Rep.
Ger.) and converted to cDNA using SUPERSCRIPT III Reverse Transcriptase (SUPERSCRIPT
is a registered trademark of Life Technologies Corp., Carlsbad, CA, USA) following the methods taught by Barber et al. (2013, Dynamic expression of MEIS1 homeoprotein in E14.5 forebrain and differentiated forebrain-derived neural stem cells. Annals of Anatomy /
Anatomischer Anzeiger: official organ of the Anatomische Gesellschaft), Kobrossy et al. (2006, Interplay between chromatin and trans-acting factors regulating the Hoxd4 promoter during neural differentiation. J. Biol. Chem. 281:25926-25939), and Nolte et al. (2006, Stereospecificity and PAX6 function direct Hoxd4 neural enhancer activity along the antero-posterior axis. Devel. Biol. 99:582-593). Quantitative RT-PCR was carried out using SYBR
Green-based RT2 qPCR Master Mix (SYBR is a registered trademark of Molecular Probes Inc., Eugene, OR, USA) in a Fast 7500 Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA) following the method taught by Barber et al. (2013, Dynamic expression of MEIS1 VAN_LAW\ 1320991\4 homeoprotein in E14.5 forebrain and differentiated forebrain-derived neural stem cells. Ann. of Anat. Available on line). Transcript levels of Mecp2e1 and Mecp2e2 were examined using gene specific primers (Table 1). PCR program for Mecp2 consisted of initial denaturation at 95 C for 3 mm followed by 35 cycles of 1 mm at 95 C, 30 sec at 60 C, and 45 sec at 72 C, with a final extension step at 72 C for 10 mm. The threshold cycle values (Ct) for each gene was normalized against the housekeeping gene Gapdh to obtain ACt values for each sample.
Relative quantification of gene expression was carried out by calculating 2-Act of each sample. Analysis was performed using MICROSOFT EXCEL 2010 and GraphPad Prism 6.0 (MICROSOFT
and EXCEL are registered trademarks of Microsoft Corp., Redmond, WA, USA). Two-Way ANOVA was used to calculate significant differences between different brain regions.
Table 1: List of primers used for qRT-PCR
Gene SEQ ID NO: Direction Sequence (5' to 3') SEQ ID NO:13 Forward AGG AGA GAC TGG AGG AAA AGT
Mecp2 e 1 SEQ ID NO:14 Reverse CTT AAA CTT CAG TGG CTT GTC TCT G
SEQ ID NO:15 Forward CTC ACC AGT TCC TGC TTT GAT GT
Mecp2e2 SEQ ID NO:16 Reverse CTT AAA CTT CAG TGG CTT GTC TCT G
SEQ ID NO:17 Forward AAC GAC CCC TTC ATT GAC
Gapdh SEQ ID NO:18 Reverse TCC ACG ACA TAC TCA GCA C
Example 5: Immunofluorescence and immunohistochemistry Detection of immunofluorescence (IF) antibodies in cultured NI113T3 cells was carried out following the method taught by Zachariah et al. (2012, Novel MeCP2 isoform-specific antibody reveals the endogenous MeCP2E1 expression in murine brain, primary neurons and astrocytes. PloS ONE 7, e497630) using the primary antibodies listed in Table

2 and the secondary antibodies listed in Table 3. Briefly, cultured NIH3T3 cells on coverslips were washed with phosphate buffered saline (PBS, GIBCO) and fixed in 4% formaldehyde.
Fixed cells were then permeabilized with 2% NP40 in PBS for 10 min, followed by preblocking with 10% normal goat serum (NGS, Jackson Immunoresearch Laboratories Inc.) in PBS for lb.
Primary antibodies were diluted in PBS with 10% NGS and the cells were incubated in primary antibodies overnight at 4 C followed by three washes with PBS. Secondary antibodies diluted in 10%
NGS were added to the cells for 1 h, followed by three washes with PBS. Coverslips were mounted on glass VAN_LAW\ 1320991\4 slides with PROLONG Gold antifade (PROLONG is a registered trademark of Molecular Probes Inc., Eugene, OR, USA) containing 2 jig/m1 4',6-diamidino-2-phenylindole (DAPI) (Calbiochem, EMD Millipore) counter-stain.
Table 2: Primary antibodies Primary Antibody Application & dilution Description Source MeCP2 (C-terminal) IHC 1:300 Rabbit polyclonal Millipore, 07-MeCP2 (C-terminal) WB 1:100 Mouse monoclonal Abcam, Ab50005 MeCP2E1 WB 1:100, IHC: 3mg/m1 Chicken polyclonal Custom-made 3 WB 1:100, IF 1:200, MeCP2E2 Chicken polyclonal Custom-made IHC: 1mg/m1 WB 1:1000, MeCP2E1 Rabbit polyclonal Custom-made IHC: 3mg/m1 GAPDH WB 1:500 Rabbit polyclonal Santa Cruz, Sc Beta-ACTIN WB 1:2000 Mouse monoclonal Sigma Aldrich, C-MYC WB 1:1500, IF 1:200 Rabbit polyclonal Santa Cruz, Sc789 C-MYC IF 1:200 Mouse monoclonal Invitrogen, GFAP IHC 1:500 Mouse monoclonal Invitrogen, NEUN IHC 1:400 Mouse monoclonal Millipore, Mab377 CNPase IHC 1:5000 Mouse monoclonal Covance, SMI-Table 3: Secondary antibodies Application Secondary AntibodySource and dilution Rhodamine Red-X conjugated goat anti mouse IgG IF 1:400 Jackson Immunoresearch, 115-259-146 Dylight 649 conjugated goat anti chicken IgY IF 1:400 Jackson Immunoresearch, 103-485-155 FITC-Conjugated Affinipure goat anti rabbit IgG IF 1:400 Jackson Immunoresearch, 111-095-144 Rhodamine Red-X conjugated goat anti chicken IgY IHC 1:400 Jackson Immunoresearch, 103-295-155 Alexa 488 goat anti rabbit IHC 1:1000 Invitrogen, A11034 Alexa 488 goat anti mouse IHC 1:1000 Invitrogen, A11017 Alexa 488 goat anti chicken IHC 1:1000 Invitrogen, A11042 Peroxidase-Affinipure sheep anti-mouse IgG WB 1:7500 Jackson ImmunoResearch 115-035-174 Peroxidase-AffiniPure donkey anti-rabbit IgG WB 1:7500 Jackson ImmunoResearch 711-036-152 Peroxidase-AffiniPure goat anti-chicken IgY WB 1:5000 Jackson ImmunoResearch 103-035-155 VAN_LAW\ 1320991\4 Immunohistochemistry (IHC) experiments for adult murine brain were carried out following the method taught by Zachariah et al. (2012). Briefly, brain tissues were fixed in ice-cold freshly de-polymerized paraformaldehyde (PFA) (0.16 M sodium phosphate buffer, pH 7.4 with PFA). Subsequently, tissue blocks were incubated in cryoprotectant (25 mM
sodium phosphate buffer, pH 7.4, 10% sucrose, 0.04% NaN3) at 4 C for approximately 24 h.
Cryosections were processed onto gelatinized slides and stored at -20 C.
Prior to IHC
experiments, tissues were permeabilized with 0.3% Triton X-100 Iris-buffered saline (TBS-Tr) (50 mM Tris-HC1, pH 7.6, containing 1.5% NaC1) solution. The slides were then pre-blocked with normal goat serum (NGS) in TBS-Tr and incubated with appropriate primary antibodies diluted inTBS-Tr/serum overnight at 4 C. Secondary antibodies were diluted in TBS-Tr/serum and applied, followed by washes using Tris-HC1 buffer (50 mM, pH 7.4).
Coverslips were prepared after incubation with 0.2 g/m1 DAPI counterstain, washes with Tris-HC1, and application of PROLONG Gold antifade (Life Technologies Inc.).
Immunolabelled signals were detected using a ZEISS AXIO Observer Z1 inverted microscope and LSM710 Confocal microscope (Carl Zeiss Canada Ltd, Toronto, ON, CA;
ZEISS and AXIO are registered trademarks of Carl Zeiss AG Corp., Oberkochen, Fed. Rep.
Ger.). Images were obtained using AXIO VISION 4.8 (AXIO VISION is a registered trademark of Carl Zeiss AG Corp.), Zen Blue 2011, Zen Black 2009 and Zen Black 2011 softwares (Carl Zeiss Canada Ltd) and assembled using ADOBE PHOTOSHOP CS5 and ADOBE
ILLUSTRATOR CS5 (ADOBE, PHOTOSHOP, and ILLUSTRATOR are registered trademarks of Adobe Systems Inc., San Jose, CA, USA).
Example 6: Western blotting Total cell extracts were prepared and Western blotting (WB) was done following the methods taught by Rastegar et al. (2009). 2 jig of total protein extracts from transfected cells or 20-80 lig of nuclear or cytoplasmic proteins were loaded into each lane and were subjected to WB analysis. Nuclear extracts were prepared with the NE-PER (NE-PER is a registered trademark of Pierce Biotechnology Inc., Rockford, IL, USA) Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific Inc., Waltham, MA, USA)) according to the manufacturer's instructions. All probed membranes were subjected to a second WB with an anti-ACTIN
VAN LAW\ 1320991\4 antibody as a loading control. Quantification of detected MeCP2 or MeCP2E1 bands was done with ADOBE PHOTOSHOP CS5 software and all bands were normalized to ACTIN
signals.
Student's t-test was used to analyze the significance of MeCP2 protein levels between samples.
For peptide incubation experiments, increasing amounts of peptide antigen (as compared to the antibody concentration) was pre-incubated with the antibody for 3-5 hours at 4 C before probing the membrane. Primary antibodies and secondary antibodies used in these experiments are listed in Tables 2 and 3, respectively.
Example 7: MeCP2E2 expression displays a later onset than MeCP2E1 during brain development It is known that the temporal expression profile of MeCP2 increases during the course of mouse brain development and follows neuronal maturation (Jung et at., 2003, The expression of methyl CpG binding factor MeCP2 correlates with cellular differentiation in the developing rat brain and in cultured cells. J. Neurobiol. 55:86-96; Shahbazian et al., 2002, Insight into Rett syndrome: MeCP2 levels display tissue- and cell-specific differences and correlate with neuronal maturation. Hum. Molec. Gen. 11, 115-124). Accordingly, anti-MeCP2E1 antibodies produced as disclosed in Example 1 and anti-MeCP2E2 antibodies produced as disclosed in Example 2 were used to detect and assess the expression of MeCP2E1 and MeCP2E2 during brain development before and after birth. The studies included brain tissues from embryonic day (E) 14, E18, postnatal day (P) 1, P7, P21, and P28. Expression of both Mecp2/MeCP2 isoforms at the transcript and protein levels was studied by qRT-PCR and WB
experiments, respectively.
MeCP2E1 expression was assessed using the anti-MeCP2E1 antibody. Nuclear extracts were prepared from dissected whole brain tissues at the aforementioned developmental time points for WB experiments. Because MeCP2 is a nuclear protein, the first step was to confirm whether MeCP2E1 could be detected specifically in the nuclear fractions of the samples. Therefore, increasing concentrations of brain nuclear and cytoplasmic extracts were subjected to WB
analysis with anti-MeCP2E1 antibody.
As expected, MeCP2E1 was only detected in the nuclear fractions, and not in cytoplasmic fractions (Fig. 9(A)) confirming that MeCP2E1 is a nuclear protein in the adult brain. Further VAN_LAWN 1320991\4 WB experiments using developmental brain samples indicated that MeCP2E1 is detected as early as E14.
A gradual increase was observed in MeCP2E1 expression levels, which reached a plateau between P7 to P21, and subsequent decline at P28 that was not statistically significant (Fig. 11;
Table 4). Surprisingly, Mecp2e1 transcripts were significantly higher at E14 and E18, with noticeable decline after birth (Fig. 11). In all of the protein and transcript analysis studies disclosed herein, Mecp2 null mouse brain (Mecp2"11Bird YID were used as controls because this mouse model is reported to lack Mecp2/MeCP2 transcript and protein (Guy et al., 2001, A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome.
Nature Genetics 27:322-326).
Pearson's correlation analysis (r) was performed between MeCP2E1 protein and Mecp2e1 transcript levels, and indicated no significant relation between Mecp2e1/MeCP2E1 at the analyzed time points (r=0.44) (Fig. 12).
Next, a similar analysis of Mecp2e2/MeCP2E2 expression was performed at the same selected developmental time points using the chicken polyclonal anti-MeCP2E2 antibody disclosed in Example 2. The specificity of this anti-MeCP2E2 antibody was validated by multiple techniques including WB and IF in transfected Phoenix cells (for WB) and transduced NIH3T3 cells (for IF) with Retro-EF 1 a-E1 or Retro-EF 1 a-E22 (Fig. 13).
Control studies included non-transfected Phoenix and non-transduced NIH3T3 cells (Figs. 5(A), 5(B), 7, 8(A), 8(B)). The anti-MeCP2E2 antibody produced as disclosed in Example 2 showed positive signals in WT adult mouse hippocampus CA1 region in brain by IHC experiments (Figs.
14(A), 14(C), 14(E)), but as expected, MeCP2E2 signals were not detected in the null mice brain (me cp2m1.113irdy/-) (Figs. 14(B), 14(D), 14(F)). Further control experiments showed that MeCP2E2 signals were eliminated by MeCP2E2-antigenic peptide, but not when the antibody was pre-incubated with MeCP2E1-specific or a MeCP2 C-terminal-specific peptide (Abeam PLC, San Francisco, CA, USA) (Figs 15(A), 15(B), 15(C)). Additional controls with primary omission and IgY incubation did not result in any detectable signal, as expected (Figs. 15(D), 15(E)). WB analysis with the anti-MeCP2E2 antibody produced a clear band at about 75kDa in WT brain nuclear extracts that was absent in the null mice brain (Fig. 16).
VAN_LAW \ 1320991 \ 4 Table 4: Differences in expression of Mecp2/MeCP2 isoforms during brain development MeCP2E1 MeCP2E2 Mecp2e1 Mecp2e2 AGE MD SIG P MD SIG P MD SIG P
MD SIG
E14 vs. E18 -0.2647 **** <0.0001 -0.1089 ns 0.2642 -0.00262 ns 0.7446 -0.00158 ns 0.9979 E14 vs. P1 -0.3697 **** <00001 -0.4495 ****
<00001 -0.00192 ns 0.9787 -0.0051 * 0.0162 E14 vs. P7 -0.5155 **** < 0.0001 -0.7871 **** <
0.0001 0.004596 * 0.0424 -0.00472 * 0.0335 E14 vs. P21 -0.4578 **** < 0.0001 -0.7829 **** < 0.0001 0.009475 **** < 0.0001 -0.00075 ns >
0.9999 E14 vs. P28 -0.3498 **** <0.0001 -0.4899 **** <0.0001 0.009832 **** <0.0001 1.57E-05 ns > 0.9999 E14 vs. Null 0.5253 **** <0.0001 0.00697 ns >
0.9999 0.01573 **** <0.0001 0.004131 **** <0.0001 E18 vs. P1 -0.105 ns 0.3166 -0.3407 **'""`
<0.0001 0.000703 ns >0.9999 -0.00353 * 0.0347 E18 vs. P7 -0.2508 **** < 0.0001 -0.6782 ****
< 0.0001 0.007218 *** 0.0002 -0.00315 * 0.0254 E18 vs. P21 -0.1931 ** 0.0016 -0.6741 ****
<0.0001 0.0121 **** <0.0001 0.000828 ns > 0.9999 E18 vs. P28 -0.0851 ns 0.668 -0.3811 **** <0.0001 0.01245 **** <0.0001 0.001591 ns 0.9976 1.) E18 vs. NULL 0.79 **** <00001 0.1158 ns 0.1862 0.01835 **** <00001 0.005706 **** <00001 1.) P1 vs. P7 -0.1458 * 0.0333 -0.3375 ****
<0.0001 0.006515 *** 0.001 0.000379 ns > 0.9999 P1 vs. P21 -0.0881 ns 0.6105 -0.3334 ****
<0.0001 0.01139 **** <0.0001 0.004354 *** 0.001 1.) P1 vs. P28 0.0199 ns >0.9999 -0.0404 ns 0.9998 0.01175 **** <0.0001 0.005117 *** 0.0005 P1 vs. NULL 0.895 **** <0.0001 0.4565 ****
<0.0001 0.01765 **** <0.0001 0.009232 **** <0.0001 P7 vs. P21 0.0577 ns 0.9837 0.00413 ns >
0.9999 0.004878 * 0.0249 0.003975 ** 0.0086 P7 vs. P28 0.1657 ** 0.0096 0.2971 **** <
0.0001 0.005235 * 0.0125 0.004737 ** 0.0031 P7 vs. NULL 1.041 **** <0.0001 0.794 ****
<0.0001 0.01113 **** <0.0001 0.008853 **** <0.0001 P21 vs. P28 0.108 ns 0.2748 0.293 **** <0.0001 0.000357 ns > 0.9999 0.000763 ns > 0.9999 P21 vs. NULL 0.9831 **** < 0.0001 0.7899 **** <
0.0001 0.006255 *** 0.0009 0.004878 **** < 0.0001 P28 vs. NULL 0.8751 **** <0.0001 0.4969 ****
<0.0001 0.005898 *** 0.00034 0.004116 **** <0.0001 vs = versus MD = mean difference SIG = significance P = P value Bonferroni's multiple comparisons test (P<0.05 was considered to be statistically significant) (N=3) VAN_LAW\ 1320991\4 The MeCP2E2 signal from the WT mouse brain was only present in the nuclear extracts and not cytoplasmic extracts (Fig. 11(B)), indicating that similar to MeCP2 (total) and MeCP2E1, MeCP2E2 is also a nuclear protein. Similar to MeCP2E1 (Figs. 17(A)-17(D)), confocal analysis of a single nucleus from adult mouse hippocampus showed that MeCP2E2 is also enriched in the DAPI-rich chromocenters (Figs. 18(A)-18(D)).
After confirming that the newly developed anti-MeCP2E2 antibody specifically detects endogenous MeCP2E2, MeCP2E2 expression levels during mouse brain development were assessed. WB analysis of nuclear extracts from selected developmental time points indicated that MeCP2E2 has a delayed onset of protein expression compared to MeCP2E1, with the earliest detection at E18 (Fig. 19). From El 8 onwards, MeCP2E2 showed a similar expression pattern when compared to MeCP2E1, albeit at lower levels (Fig. 19; Table 5). On the other hand, Mecp2e2 transcript levels increased prenatally until birth with highest levels at P1 -P7, and significantly declined by P28 (Fig. 10; Table 5). Comparison of Mecp2ellMecp2e2 transcript levels indicated significantly higher Mecp2e1 levels from E14 until birth;
however no significant difference in transcript expression was detected between P7-P28 (Fig. 11).
Pearson's correlation analysis showed no significant relation between Mecp2e2/MeCP2E2 transcript and protein (r=0.58) at these selected developmental time points (Fig. 12).
Taken together, the results disclosed herein confirm that in the adult mouse brain, MeCP2E2 is a nuclear protein that is enriched in the chromocenters of the hippocampus CA1 nuclei, and that while the onset of expression of MeCP2E1 and MeCP2E2 significantly differs prenatally during mouse brain development, their expression overlap after birth. Additionally, these data indicate there is no significant correlation between Mecp2/MeCP2 isoform-specific transcript and protein expression levels during mouse brain development.
Example 8: MeCP2 protein isoforms show similar cell type-specific patterns in brain hippocampus of both male and female adult mice.
It is known that MeCP2 is expressed in the three main brain cell types including neurons, astrocytes and oligodendrocytes (Rastegar et at., 2009, MECP2 isoform-specific vectors with regulated expression for Rett syndrome gene therapy. PloS ONE 4:e6810; Ballas et al., 2009, VAN_LAW \ 1320991\4 Non-cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology. Nat.
Neurosci. 12:311-317). Because consistent and uniform expression patterns were detected for both MeCP2E1 and MeCP2E2 in the nuclei of hippocampal cells in the CA1 region (Figs.
17(A)-17(D), 18(A)-18(D)), further assessments were made of MeCP2E1 and MeCP2E2 -- expression in neurons, astrocytes and oligodendrocytes in this region.
In male mouse brain, IF co-labelling of MeCP2E1 with a neuronal nuclei marker NeuN
showed that the majority of MeCP2E1-labelling was localized to NeuN + positive cells in the hippocampus CA1 layer (Fig. 20(A)). Similarly to MeCP2E1, MeCP2E2 was also detected in the majority of neuronal nuclei in the hippocampus CA1 layer of male mice (Fig.
20(B)). Confocal -- analysis showed similar nuclear patterns for MeCP2E1 and MeCP2E2 in NeuN+
nuclei (Fig.
21(A), 21(B)). Immunofluorescence detection of MeCP2E1 and MeCP2E2 occurred less frequently in DAPI counterstained NeuN- cells indicating the possibility for expression of MeCP2 isoforms in non-neuronal cells. Therefore, IF co-labelling experiments were performed with anti-MeCP2E1 or anti-MeCP2E2 antibodies in combination with an astrocyte marker -- (GFAP; Figs. 22(A), 22(B)), or an oligodendrocyte marker (CNPase; Figs.
23(A), 23(B)).
Abundant labelling for each cell type-specific marker was detected in the CA1 of male brain.
However, co-localization of MeCP2 isoforms with glial cell markers was not easily determined by regular microscopy. Therefore, confocal microscopy imaging analysis was performed, and MeCP2E1 and MeCP2E2 signals were detected in both GFAP+ astrocytes (Figs.
24(A), 24(B)) -- and CNPase+ oligodendrocytes (Figs. 25(A), 25(B)) in the hippocampus CA1 layer of adult male brain.
Immunofluorescence labelling similar to that of male for MeCP2E1 and MeCP2E2 was detected in the hippocampus CA1 layer of female mouse brain. IF co-labelling of MeCP2E1 with a neuronal nuclei marker NeuN also showed the majority of MeCP2E1 and MeCP2E2 signals to -- be localized to NeuN + positive cells in the hippocampus CA1 layer (Figs.
26(A), 26(B)).
Confocal analysis showed similar nuclear patterns for MeCP2E1 and MeCP2E2 in NeuN+ nuclei (Figs. 27(A), 27(B)). Also detected was abundant labelling for each cell type-specific marker in the CA1 of female brain (Figs. 28(A), 28(B), 29(A), 29(B)). Confocal microscopy imaging analysis was performed and MeCP2E1 and MeCP2E2 signals were detected in GFAP+
astrocytes -- (Figs. 30(A), 30(B)) and CNPase+ oligodendrocytes (Figs.31(A), 31(B)) in the hippocampus VAN LA W\ 1320991\4 CA1 layer of adult female brain. Also compared were distributions of MeCP2 isoforms in the CA2, CA3 and dentate gyrus regions (DG) of male hippocampus (Figs. 32(A-A), 32(A-A1).
Higher magnification of CA2, CA3 and DG in mouse hippocampus sections showed no obvious differences for MeCP2E1 and MeCP2E2 labelling in these regions (Figs. 32(A-B,C,D), 32(A1-B1,C1,D1)) similar to what was observed in the CA1 region. Nuclear labelling was evident in other hippocampus layers surrounding the pyramidal and dentate regions as seen in the low magnification tiled images (Figs. 32(A), 32(B)).
It is known that Mecp2e1 and Mecp2e2 transcripts are differentially distributed throughout different mouse brain regions (Dragich et al., 2007, Differential distribution of the MeCP2 splice variants in the postnatal mouse brain. J. Comp. Neurol. 501:526-542)., and that the pattern of distribution of MeCP2E1 in the adult mouse brain is similar to the distribution pattern total MeCP2 (Zachariah et al., 2012). However, comparative analysis of MeCP2E1 and MeCP2E2 endogenous protein expression and localization in different brain regions has not been reported to date. IHC experiments in different regions of the adult mouse brain, i.e. the olfactory bulb, striatum, cortex, hippocampus, thalamus, brain stem and cerebellum were performed to assess the spatial expression of the two MeCP2 isoforms. As was also observed in the hippocampus, labelling for both MeCP2 isoforms in the other brain regions was abundant with no obvious differences in staining patterns in the majority of studied regions (Figs. 33(A), 33(B), 34(A), 34(B)). The most intense signals detected in the olfactory bulb by anti-MeCP2 isoform-specific antibodies were in the mitral cell layer, presumably in mitral cells (Figs. 33(A-A), 33(B-Al). Similar staining intensities were also observed in the nuclei localized within the inner and outer plexiform layers. Lower levels of staining intensities were observed for both MeCP2 isoforms in the granule cell layer. Interestingly, while MeCP2E1 and MeCP2E2 labelling was observed in the same juxtaglomerular nuclei of the olfactory bulb, some nuclei were devoid of labelling for both isoforms.
Similar IF labelling patterns of MeCP2 isoforms were observed in dorsal/ventral and medial regions of the striatum (Figs. 33(A-B), 33(B-B1)). IF labelling of MeCP2E1 and MeCP2E2 was detected in all layers of rostral to caudal cerebral cortex as shown in layers 5-6 (Figs. 33(A-C), 33(B-C1)). Likewise, positive labelling for both MeCP2 isoforms was observed throughout the thalamus of mouse brain, including medial areas (Figs. 34(A-A), 34(B-A)) and VAN JAW\ 1320991\4 the dorsal region underlying the ventral hippocampus (Figs. 34(A-A), 34(B-A1)), as well as throughout regions of the brain stem, including the medial vestibular nucleus (Figs. 34(A-B), 34(B-B1)).
Interestingly, distribution of MeCP2E1 was different from the distribution of MeCP2E2 in the cerebellum (Figs.34(A-C), 34(B-C1)), where under low magnification;
detection of MeCP2E2 was the greater of the two isoforms in the granule cell layer of the cerebellum. To further confirm the differential levels of MeCP2E1 and MeCP2E2 signals, IHC
double labelling was performed for both MeCP2E1 using rabbit polyclonal anti-MeCP2E1 antibodies and anti-MeCP2E2 antibodies in sections of male mouse cerebellum. Confocal microscopy of double IF-labelling with rabbit anti-MeCP2E1 and chicken anti-MeCP2E2 antibodies in mouse cerebellum supported the single-labelling data and confirmed differential detection levels of MeCP2E1 and MeCP2E2 in the granule cell layer of mouse cerebellum (Figs. 35(A)-35(D)).
Using confocal microscopy, it was observed that in the cerebellum sub-regions of molecular layer, Purkinje cell and granule cell layer, MeCP2E1 and MeCP2E2 signals were co-localized with each other at the chromocenters (Figs. 36(A), 36(B), 36(C)).
Taken together, these data demonstrate an overall similar cell type-specific distribution of MeCP2 isoforms between CA1 region of mouse male and female brain. Although MeCP2E1 and MeCP2E2 signals are mostly identical throughout brain regions, differential abundance of MeCP2E1 and MeCP2E2 exists at least in the granule cell layer of the cerebellum as seen in male brain. Furthermore, both MeCP2 isoforms were detected in all three neural cell types examined in the present study.
Example 9: Mecp2/MeCP2 isoforms show differential abundance in adult murine brain regions Since highly similar distribution and localization of MeCP2 isoforms were observed by IHC within all the brain regions except for the cerebellum, the next step was to quantify the abundance of MeCP2 isoforms in different brain regions by WB. Nuclear extracts were for these experiments because the previous WB, IHC and IF studies disclosed herein showed the nuclear localization of both MeCP2 isoforms. Expression analysis of MeCP2E1 protein levels showed VAINLLAW\ 1320991\4 uniform expression levels across different brain regions that were analyzed (Fig. 37; Table 5).
Similar expression profile was seen with Mecp2e1 transcripts in all the studied brain regions (Fig. 38; Table 5). Pearson's correlation analysis revealed a statistically significant correlation between Mecp2e//MeCP2E1 transcripts and protein (r---0.91, P<0.01) (Fig. 39).
In contrast to the results obtained with MeCP2E1, MeCP2E2 showed a differential expression pattern across different brain regions with significantly higher expression in the olfactory bulb and the cerebellum compared to other regions (Fig. 40; Table 5). Brain stem showed the lowest expression of MeCP2E2 compared to other examined regions. Mecp2e2 transcript levels were also differentially expressed in different brain regions with significant differences between the cortex and thalamus, and cortex and brain stem (Fig. 40; Table 5).
Correlation analysis between MeCP2E2 protein and Mecp2e2 transcript levels revealed a statistically significant correlation between Mecp2e2/MeCP2E2 P<0.05). As positive and negative controls for the aforementioned analysis, Whole Mecp2 WT and null adult brains (adult mice at 6 weeks of age) were used as the positive and negative controls respectively for analysis of Mecp2/MeCP2 isoform-specific expression. As expected, the expression levels of MeCP2E1 were significantly higher (2.8-fold) than that of MeCP2E2 in the WT
whole brain, whereas neither isoform was detected in the nuclear extracts of null mouse brain (Fig. 41(A)).
Similarly, higher Mecp2e1 transcript levels were detected in the WT brain (2.6-fold), relative to lower Mecp2e2 levels, while no transcripts were detected in the null brain (Fig. 41(B)). These observations further confirm that MeCP2E1 is the major isoform in the adult mouse brain.
Taken together, these results demonstrate that Mecp2/MeCP2 isoforms are different with respect to their distribution and expression levels in the adult mouse brain regions.
VAN_LAW\ 1320991\4 Table 5:
Differences in expression of Mecp2/MeCP2 isoforms in different regions of the brain MeCP2E1 I MeCP2E2 Mecp2e1 Mecp2e2 REGION MD SIG P MD SIG P MD
SIG P MD SIG P
WB vs. NULL 0.9703 **** <0.0001 0,3291 ****
<0.0001 0.00785 **** <0.0001 0.002907 * 0.0422 WB vs. OB -0.07358 ns 0.997 -0.5935 ****
<0.0001 0.001652 ns 0.8598 -0.00198 ns 0.5525 WB vs. STR -0.08335 ns 0.9804 -0.3884 ****
<0.0001 -0.000085 ns > 0.9999 -0.0022 ns 0.3449 WB vs. CTX -0,04608 ns > 0.9999 -0.1679 ns 0.0638 0.000652 ns > 0.9999 -0.00397 *** 0.001 WB vs. HIPPO -0.03852 ns >0.9999 -0.3517 ****
<0.0001 0.001999 ns 0.5294 -0.00112 ns 0.9993 WB vs. THAL -0.05912 ns > 0.9999 -0.0257 ns >
0.9999 -0.00083 ns > 0.9999 -0.00084 ns >0.9999 WB vs. BS -0.08505 ns 0.9745 0.1816 * 0.0302 0.001533 ns 0.9312 0.00059 ns > 0.9999 WB vs. CERE -0.07208 ns 0.9979 -0.6069 ****
<0.0001 0.001866 ns 0.6671 -0.00212 ns 0.4079 OB vs. STR -0.00977 ns > 0.9999 0.2051 ** 0.0078 -0.001738 ns 0.7902 -0.00022 ns > 0.9999 OB vs. CTX 0.0275 ns > 0.9999 0.4256 ****
<0.0001 -0.001 ns > 0.9999 -0.00199 ns 0.5356 OB vs. HIPPO 0.03507 ns > 0.9999 0.2418 *** 0.0009 0.00034 ns > 0.9999 0.000854 ns > 0.9999 0 4) OB vs. THAL 0.01447 ns > 0.9999 0.5678 ****
<0.0001 -0.002482 ns 0.1596 0.001139 ns 0.9991 OB vs. BS -0.01147 ns > 0.9999 0.7751 ****
<0.0001 -0.00011 ns > 0.9999 0.002566 ns 0.1244 N.) co OB vs. CERE 0.0015 ns > 0.9999 -0.0134 ns >
0.9999 0.000213 ns > 0.9999 -0.00015 ns >0.9999 w N.) STR vs. CTX 0.03727 ns > 0.9999 0.2205 **
0.0031 0.000737 ns > 0.9999 -0.00177 ns 0.7573 0 ko STR vs. HIPPO 0.04483 ns > 0.9999 0.0367 ns > 0.9999 0.002085 ns 0.4441 0.001073 ns 0.9997 ....I
STR vs. THAL 0.02423 ns > 0.9999 0.3627 ****
<0.0001 -0.000744 ns > 0.9999 0.001358 ns 0.9843 N.) STR vs. BS -0.0017 ns > 0.9999 0.57 **** <0.0001 0.001619 ns 0.8827 0.002785 ns 0.0628 w ' STR vs. CERE 0.01127 ns >0.9999 -0.2185 ** 0,0035 0.001951 ns 0.5783 7.19E-05 ns >0.9999 CTX vs. HIPPO 0.00756 ns >0.9999 -0.1838 * 0.0267 0.001347 ns 0.986 0.002847 ns 0.0514 0 CTX vs. THAL -0.01303 ns >0.9999 0.1422 ns 0.2286 -0.001482 ns 0.9526 0.003132 * 0.0197 w 1-, CTX vs. BS -0.03897 ns > 0.9999 0.3495 ****
<0.0001 0.000881 ns > 0.9999 0.004559 *** 0.0008 CTX vs. CERE -0.026 ns > 0.9999 -0.439 ****
<0.0001 0.001214 ns 0.9972 0.001846 ns 0.6871 HIPPO vs. THAL -0.0206 ns > 0.9999 0.326 ****
<0.0001 -0.002829 ns 0.0544 0.000285 ns > 0.9999 HIPPO vs. BS -0.04653 ns > 0.9999 0.5333 ****
<0.0001 -0.000465 ns > 0.9999 0.001713 ns 0.8122 HIPPO vs. CERE -0.03357 ns >0.9999 -0.2552 ***
0.0004 -0.000133 ns > 0.9999 -0.001 ns > 0.9999 THAL vs. BS -0.02593 ns > 0.9999 0.2073 ** 0.0069 0.002363 ns 0.2233 0.001427 ns 0.9698 THAL vs. CERE -0.01297 ns >0.9999 -0.5812 ****
<0.0001 0.002696 ns 0.0835 -0.00129 ns 0.9929 BS vs. CERE 0.01297 ns > 0.9999 -0.7885 ****
<0.0001 0.000332 ns > 0.9999 -0.00271 ns 0.079 vs = versus MD = mean differences SIG = significance P = P value Bonferroni's multiple comparison's test (P<0.05 was considered to be statistically significiant) N = 3 VAN_LAW\ 1320991\4 SUMMARY:
The studies and related data disclosed herein report a number of comparative analyses of Mecp2/MeCP2 isoform-specific expression during mouse brain development and in different brain regions of young adult mice at 6 weeks of age. MeCP2 isoforms show significant increase at the protein levels during the early postnatal mouse development (P1 -P7).
This time period has been reported to coincide with the onset of neuronal maturation and synaptogenesis in several brain regions (Jung et al., 2003, The expression of methyl CpG binding factor MeCP2 correlates with cellular differentiation in the developing rat brain and in cultured cells. J. Neurobiol.
55:86-96; Shahbazian et al., 2002, Insight into Rett syndrome: MeCP2 levels display tissue- and cell-spec(ic differences and correlate with neuronal maturation. Hum. Mol.
Gen. 11:115-124).
Thus, the possibility of both MeCP2 isoforms contributing to these processes cannot be ruled out. It is noteworthy that the later onset of MeCP2E2 protein expression, as compared to the onset of MeCP2E1 expression, might reflect the developmental pattern of a regional, neuronal or cellular subtype in the brain. This is important in light of the knowledge that MeCP2 dysfunction affects different regions of the brain to different extents, suggesting that MeCP2E2 may contribute to normal function of specific types of neurons or other brain cell types. Moreover, the absence of a significant correlation between Mecp2/MeCP2 transcript and protein expression of the two isoforms during brain development suggest possible post-transcriptional regulation of Mecp2 isoforms during development.
Recent studies have shown that MeCP2 expression levels are critical to maintain, and higher or lower levels than normal in different brain regions correlate with specific behavioural impairments (Wither et al., 2013, Regional MeCP2 expression levels in the female MeCP2-deficient mouse brain correlate with specific behavioral impairments. Exp.
Neurol. 239:9-59).
Moreover, deletion of Mecp2 in neurons in specific brain-regions is associated with RTT
phenotypes (Adachi et al., 2009, MeCP2-mediated transcription repression in the basolateral amygdala may underlie heightened anxiety in a mouse model of Rett syndrome. J.
Neurosci.
29:4218-4227; Wu et al., 2009, MeCP2 function in the basolateral amygdala in Rett syndrome.
J. Neurosci. 29:9941-9942; Gemelli et al., 2006, Postnatal loss of methyl-CpG
binding protein 2 in the forebrain is sufficient to mediate behavioral aspects of Rett syndrome in mice. Biol. Psych.
VAN_LAW\ 1320991\4 59:468-476). This reinforces the requirement for precise levels of MeCP2 expression for normal brain function, as both higher or lower levels of MeCP2 expression (compared to normal) results in neurological dysfunction.
The WB data disclosed herein show that both MeCP2 isoforms are present in the adult mouse brain, with MeCP2E1 showing more uniform expression levels in different brain regions compared to MeCP2E2 and confirm that, similar to MeCP2, MeCP2E1 is also a nuclear protein.
Therefore, the influence of cellular size and also nuclear to cytoplasmic ratio which might not be the same in different cell types or regions of the brain, was eliminated by the use of nuclear extracts. Although the significance of the uniform nuclear distribution of MeCP2E1 in brain remains to be elucidated, it may be hypothesized to relate to a specialized MeCP2E1 nuclei structural function in different brain regions. The data disclosed herein regarding the MeCP2E2 expression patterns suggest that it may contribute to MeCP2 brain region-specific functions or target genes.
Interestingly, semi-quantitative WB analysis showed similar overall protein expression levels of MeCP2 isoforms in the adult mouse cerebellum, but further IHC
characterization revealed differential localization of MeCP2 isoforms in sub regions of the cerebellum. The data disclosed herein indicate that MeCP2E2 is the more abundant isoform in the granule cell layer of the cerebellum compared to MeCP2E1. Thus, these data confirm that at the protein levels, MeCP2 isoforms are also differentially localized in this part of the brain.
The detected differentiation distribution of MeCP2 isoforms in the cerebellum might be helpful to understand the contribution of individual MeCP2 isoforms in cerebellar functions and gene expression, The anti-MeCP2E2 antibody produced with an antigen comprising a peptide with a sequence of twelve amino acids (e.g., SEQ ID NO:10) or alternatively with an antigen comprising a peptide with a sequence of eleven amino acids (e.g., SEQ ID
NO:11) from the N-terminus of MeCP2E2 as disclosed herein, provides novel avenues for understanding brain region and/or cell type-specific expression of MeCP2E2 that will provide vital insights for the efficient design of future gene therapy approaches. The data further indicate that in adult mice brain, MeCP2E2 signals overlap with DAPI-rich heterochromatin regions in the nucleus. The data disclosed herein show that both MeCP2 isoforms are expressed in three major brain cell VAN_LAW\ 1320991\4 types; neurons, astrocytes and oligodendrocytes of both male and female adult mouse.
Furthermore, it is noted that transcript and protein expression of Mecp2/MeCP2 isoforms significantly correlate with each other in different regions of the adult brain, while such correlations do not exist in the developing brain. The generated and validated anti-MeCP2E2 antibody will have important applications for future diagnosis, prognosis or understanding the mechanism of MeCP2-associated diseases.
VAN_LAW\ 1320991\4

Claims (24)

39

1. An antibody that specifically binds a MeCP2E2 isoform of Methyl CpG
Binding Protein 2, wherein said antibody is produced with an antigen with a region comprising the amino acid sequence set forth in SEQ ID NO:10 or the amino acid sequence set forth in SEQ
ID NO:11.

2. The antibody of claim 1, wherein said antibody does not bind a MeCP2E1 isoform of Methyl CpG Binding Protein 2.

3. The antibody of claim 1, wherein said antibody is a polyclonal antibody.

4. The antibody of claim 1, wherein said antibody is a monoclonal antibody.

5. The antibody of claim 1, wherein the antibody is conjugated to a detectable label.

6. The antibody of claim 5, wherein the label is a fluorescent label, an enzymatic label, a luminescent label, or a chromophore label.

7. A composition comprising:
an antibody that specifically binds a MeCP2E2 isoform of Methyl CpG Binding Protein 2; and a carrier therefor;
wherein said antibody is produced with an antigen with a region comprising the amino acid sequence set forth in SEQ ID NO:10 or the amino acid sequence set forth in SEQ
ID NO:11.

8. The composition of claim 7, wherein said antibody does not bind a MeCP2E1 isoform of Methyl CpG Binding Protein 2.

9. The composition of claim 7, wherein said antibody is a polyclonal antibody.

10. The composition of claim 7, wherein the antibody is conjugated to a detectable label.

11. The composition of claim 10, wherein the label is a fluorescent label, an enzymatic label, a luminescent label, or a chromophore label.

12. The composition of claim 7, wherein the antibody is packaged in a lyophilized form.

13. The composition of claim 7, wherein the antibody is packaged in an aqueous medium.

14. A peptide for use as an antigen to prepare the antibody of claim 1, wherein said peptide comprises a region comprising the amino acid sequence set forth in SEQ ID
NO:10 or the amino acid sequence set forth in SEQ ID NO:11.

15. A kit for detecting or monitoring an over-expression or an under-expression of a MeCP2E2 isoform of Methyl CpG Binding Protein 2 or expression of the MeCP2E2 isoform relative to an expression of MeCP2E1, the kit comprising;
the antibody of claim 1; and a carrier therefore.

16. The kit of claim 15, wherein the kit additionally comprises a detectable label.

17. The kit of claim 16, wherein the detectable label is a fluorescent label, an enzymatic label, a luminescent label, or a chromophore label.

18. The kit of claim 16, additionally comprising one or more compounds for detecting the label.

19. Use of any of the antibody of claim 1, the composition of claim 7, and the kit of claim 15 for detecting and/or monitoring an over-expression or an under-expression of a MeCP2E2 isoform of Methyl CpG Binding Protein 2 or expression of the MeCP2E2 isoform relative to an expression of MeCP2E1.

20. A method for detecting and/or monitoring an over-expression or an under-expression of a MeCP2E2 isoform of Methyl CpG Binding Protein 2, comprising the steps of:
contacting a first sample collected from a mammalian subject with an antibody that specifically binds a MeCP2E2 isoform of Methyl CpG Binding Protein 2, wherein said antibody is produced with an antigen with a region comprising the amino acid sequence set forth in SEQ
ID NO:10 or the amino acid sequence set forth in SEQ ID NO:11;
removing unbound antibody from the sample;
conducting an immunoassay on the first sample to determine a first value for expression of the MeCP2E2 isoform;
comparing the first value to a reference value for expression of the MeCP2E2 isoform in healthy mammalian subjects;
wherein a deviation of the first value from the reference value indicates the presence of a disease or a disorder caused by an over-expression or an under-expression of the MeCP2E2 isoform.

21. The method of claim 20, additionally comprising:
contacting a second sample collected from the mammalian subject with the antibody;
conducting an immunoassay on the second sample to determine a second value for expression of the MeCP2E2 isoform;
comparing the second value to one or both of the first value and the reference value;
wherein a deviation of the second value from one or both of the first value and the reference value indicates the over-expression or the under-expression of the MeCP2E2 isoform.

22. The method of claim 20, wherein the antibody is conjugated to a detectable label.

23. The method of claim 22, wherein the label is a fluorescent label, an enzymatic label, a luminescent label, or a chromophore label.

24. The method of claim 20, wherein said antibody does not bind a MeCP2E1 isoform of Methyl CpG Binding Protein 2.