WO2019241410A1 - Compositions et procédés d'activation synergique de p53 par modulation de l'épissage de mdm2 et mdm4 - Google Patents

Compositions et procédés d'activation synergique de p53 par modulation de l'épissage de mdm2 et mdm4 Download PDF

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WO2019241410A1
WO2019241410A1 PCT/US2019/036806 US2019036806W WO2019241410A1 WO 2019241410 A1 WO2019241410 A1 WO 2019241410A1 US 2019036806 W US2019036806 W US 2019036806W WO 2019241410 A1 WO2019241410 A1 WO 2019241410A1
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mdm2
mdm4
sma
exon
smn
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Livio Pellizzoni
Meaghan VAN ALSTYNE
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The Trustees Of Columbia University In The City Of New York
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3233—Morpholino-type ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • the present disclosure provides, inter alia, compositions, kits and methods for treating cancers and other diseases by modulating Mdm2 and Mdm4 alternative splicing.
  • sequence listing text file “000607-seq.txt” file size of 10 KB, created on June 11 , 2019.
  • sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. ⁇ 1.52(e)(5).
  • RNA homeostasis is a common theme of many neurodegenerative disorders in which genetic mutations are associated with the direct or indirect impairment of RNA-binding proteins or factors controlling RNA processing at multiple levels (Conlon and Manley 2017; Li et al. 2014; Cooper et al. 2009).
  • disease-linked proteins are ubiquitously expressed and carry out essential biological processes, pathology is characterized by selective degeneration of specific neuronal populations.
  • these proteins are often multifunctional and involved in the regulation of diverse RNA pathways, making it difficult to discern specific, disease-relevant events among many transcriptome abnormalities.
  • These complexities have made it remarkably challenging in most instances to establish causal links between RNA dysregulation and disease etiology. Nonetheless, dissecting the RNA-mediated mechanisms underlying the dysfunction and death of select neurons is necessary to uncover the molecular basis of human disease and to identify drivers of neurodegeneration, which may also represent key targets for therapeutic intervention.
  • RNA dysfunction spinal muscular atrophy
  • SMA spinal muscular atrophy
  • SmRNPs small nuclear ribonucleoproteins
  • U7 snRNPs U7 snRNPs that carry out histone mRNA 3’ end processing
  • SMN has also been implicated in the assembly of other RNA-binding proteins with a variety of coding and noncoding RNAs (Li et al. 2014), the most characterized of which is the formation of specific mRNPs for axonal transport and localized translation in neurons (Donlin-Asp et al. 2017).
  • RNA-binding proteins Li et al. 2014
  • mRNPs coding and noncoding RNAs
  • the present disclosure relates to the molecular mechanisms of motor neuron death in SMA.
  • SMA motor neuron death occurs cell-autonomously (Fletcher et al. 2017; Gogliotti et al. 2012; Martinez et al. 2012; McGovern et al. 2015) and distinct motor neuron pools display differential vulnerability to SMN deficiency (Fletcher et al. 2017; Mentis et al. 2011 ; Simon et al.
  • Mdm2 mainly serves as an E3 ubiquitin ligase that targets p53 for degradation, while Mdm4 inhibits p53 transcriptional activity in addition to enhancing Mdm2 function (Shadfan et al. 2012; Marine et al. 2006).
  • Skipping of exon 7 in Mdm4 introduces an early stop codon that induces nonsense-mediated decay or production of an inactive protein leading to p53 activation through loss of function (Bardot et al. 2015; Bezzi et al. 2013; Dewaele et al. 2016).
  • Mdm2 exon 3 and Mdm4 exon 7 are identified as critical downstream targets whose coordinated alternative splicing is regulated by SMN through its function in snRNP biogenesis. Importantly, it has been shown that defective splicing of these key regulatory exons induced by SMN deficiency - which occurs early and is most pronounced in vulnerable motor neurons - acts as a key biological switch governing initiation of the p53 response in SMA mice.
  • one embodiment of the present disclosure is an anti-sense morpholino oligonucleotide (MO) targeting a gene in a subject, wherein the gene is selected from mdm2 and mdm4.
  • MO anti-sense morpholino oligonucleotide
  • composition comprising at least one anti-sense morpholino oligonucleotide (MO) as disclosed herein and a pharmaceutically acceptable carrier.
  • MO anti-sense morpholino oligonucleotide
  • Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a cancer in a subject, comprising administering to the subject an effective amount of an anti-sense MO as disclosed herein or a composition comprising the same as disclosed herein.
  • Another embodiment of the present disclosure is a vector comprising a full-length cDNA of a gene in a subject, wherein the gene is selected from mdm2 and mdm4.
  • composition comprising at least one vector as disclosed herein and a pharmaceutically acceptable carrier.
  • Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a neurodegenerative disease in a subject, comprising administering to the subject an effective amount of a vector as disclosed herein or a composition comprising the same as disclosed herein.
  • kits comprising a composition as disclosed herein, and instructions for use thereof.
  • Fig. 1A is an RT-PCR analysis of Mdm2 exon 3 splicing in NIFI3T3- Smn RNAi and NIFI3T3-SMN/Smn RNAi cells cultured with or without Dox using primers in exon 2 (E2) and exon 5 (E5).
  • E2 exon 2
  • E5 exon 5
  • a schematic of the alternatively spliced exon is shown at the top. Arrowheads indicate the location of primers.
  • Fig. 1 B is an RT-PCR analysis of Mdm4 exon 7 splicing in NIH3T3- Smn RNAi and NIH3T3-SMN/Smn RNAi cells cultured with or without Dox using primers in exon 5 (E5) and exon 5 (E8).
  • a schematic of the alternatively spliced exon is shown at the top. Arrowheads indicate the location of primers.
  • Fig. 1 E is an RT-PCR of Mdm2 exon 3 splicing in WT and SMA spinal cord at P1 , P6 and P11 using E2 and E5 primers.
  • Fig. 1 F is an RT-PCR of Mdm4 exon 7 splicing in WT and SMA spinal cord at P1 , P6 and P11 using E5 and E8 primers.
  • Fig. 1 J is a schematic of the experimental approach used to isolate retrogradely labeled, muscle-identified motor neurons from WT and SMA mice.
  • Fig. 1 L is an RT-PCR analysis of Mdm2 exon 3 and Mdm4 exon 7 splicing in L1 -L3 motor neurons from WT and SMA mice at P6. E2 and E5 primers were used for Mdm2. E5 and E8 primers were used for Mdm4.
  • Figs. 2A - 2J show that defective snRNP biogenesis dysregulates Mdm2 and Mdm4 alternative splicing and induces p53 activation.
  • Statistics were performed with two-tailed unpaired Student’s f-test (Figs. 2C, 2D, 2E and 2H) or one- way ANOVA with Tukey’s post hoc test (J). * P ⁇ 0.05; ** P ⁇ 0.01 ; *** P ⁇ 0.001.
  • Fig. 2A is an RT-PCR analysis of Mdm2 exon 3 splicing in NIFI3T3- Sm B RNAi cells cultured with or without Dox using E2 and E5 primers.
  • Fig. 2B is an RT-PCR analysis of Mdm4 exon 7 splicing in NIFI3T3- SmB RNAi cells cultured with or without Dox using E5 and E8 primers.
  • Fig. 2F is a Western blot analysis of NIFI3T3-SmB RNAj cells cultured with and without Dox.
  • Fig. 2J shows normalized SmB fluorescence intensity in WT as well as p53 + and p53 SMA L2 motor neurons at P6 from experiments as in Fig. 2I. Each point represents SmB fluorescent intensity in a single L2 motor neuron and data are collected from 3 mice.
  • Figs. 3A - 3I show that skipping of SMN-regulated Mdm2 and Mdm4 exons synergistically trigger p53 activation.
  • Fig. 3A is a schematic of splice-switching MOs targeting the 5’ splice- sites of Mdm2 exon 3 and Mdm4 exon 7.
  • Fig. 3B is an RT-PCR analysis of Mdm2 exon 3 and Mdm4 exon 7 splicing in the spinal cord of WT mice at P11 following injection of 400pg of control MO or 200pg of each splice-switching MO individually or together at P0.
  • E2 and E5 primers were used for Mdm2.
  • E5 and E8 primers were used for Mdm4.
  • Fig. 3F shows ChAT and p53 immunostaining of L5 spinal segments at P11 from WT mice injected with 400pg of control or 200pg of each splice-switching MO at P0.
  • Fig. 3H shows ChAT and phospho-p53 Ser18 immunostaining of L5 spinal segments from experiments as in Fig. 3F.
  • Figs. 5A - 5D show that full length Mdm2 and Mdm4 overexpression prevents motor neuron degeneration in SMA mice. All statistics were performed with one-way ANOVA with Tukey’s post hoc test. * P ⁇ 0.05; ** P ⁇ 0.01 ; *** P ⁇ 0.001.
  • Fig. 5B shows the total number of L2 motor neurons from the same groups as in Fig. 5A at P11. Data represent mean and SEM (n>3).
  • Fig. 5D shows the total number of L5 MMC motor neurons number from the same groups as in Fig. 5A at P1 1. Data represent mean and SEM (n>4).
  • Figs. 6A - 6K show that full length Mdm2 and Mdm4 overexpression moderately improves spinal reflexes and motor behavior in SMA mice.
  • Fig. 6B shows the spinal reflex amplitudes from the same groups as in Fig. 6A. Data represent mean and SEM (n>3).
  • Fig. 6D shows the total number of VGIuTI + synapses on L2 motor neuron somata from the same groups as in Fig. 6A at P11. Data represent mean and SEM (n>22 neurons from 3 mice per group).
  • Fig. 6F shows the percentage of fully denervated NMJs in the QL muscle from the same groups as in Fig. 6A at P11. Data represent mean and SEM (n>3).
  • Fig. 7 shows splicing mechanisms of p53 anti-repression and motor neuron death in SMA.
  • Dysregulation of both splicing events is required to synergistically induce p53 stabilization, which is the initiating step in the death pathway leading to motor neuron loss in SMA mice. See text for additional details.
  • Figs. 8A - 8F show effects of SMN deficiency on splicing of Mdm2 and Mdm4 exons in NIH3T3 cells.
  • Fig. 8B is an RT-PCR analysis of Mdm2 mRNA in NIFI3T3-Smn RNAi cells cultured with or without Dox using primers located in exon 2 (E2) and at the junction between exon 6 and 7 (E6/7). DNA size in base pairs is shown on the left.
  • Fig. 8C is an RT-PCR analysis of Mdm2 mRNA in NIFI3T3-Smn RNAi cells cultured with or without Dox using primers located in exon 12 (E12) and at the junction between exon 6 and 7 (E6/7). DNA size in base pairs is shown on the left.
  • Fig. 8D shows the Gene structure of mouse Mdm4 depicting protein- coding exons in blue and non-coding portions in grey, except for the SMN-regulated exon 7 that is shown in red. Arrowheads point to the location of primers and brackets indicate the portion of the mRNA analyzed by RT-PCR in Figs. 8E and 8F.
  • Fig. 8E is an RT-PCR analysis of Mdm4 mRNA in NIFI3T3-Smn RNAi cells cultured with or without Dox using primers located in exon 1 (E1 ) and exon 5 (E5). DNA size in base pairs is shown on the left.
  • Fig. 8F is an RT-PCR analysis of Mdm4 mRNA in NIFI3T3-Smn RNAi cells cultured with or without Dox using primers located in exon 5 (E5) and exon 11 (E11 ). DNA size in base pairs is shown on the left.
  • Figs. 9A - 9C show dose-response analysis of p53 induction in the spinal cord of WT mice by MO-mediated skipping of Mdm2 exon 3 and Mdm4 exon 7.
  • Fig. 9A is an RT-PCR analysis of Mdm2 and Mdm4 splicing in the spinal cord at P11 from WT mice injected at P0 with increasing doses of equivalent amounts of splice-switching MOs (25, 50, 100, 200, 400 pg total) or with control MO (400 pg).
  • E2 and E5 primers were used for Mdm2.
  • E5 and E8 primers were used for Mdm4.
  • Fig. 9C is an RT-qPCR analysis of p53 transcriptional targets in the spinal cord at P11 from the same groups as in Fig. 9B. Data represent mean and 95% Cl from three technical replicates.
  • Figs. 10A - 10E show that full-length Mdm2 and Mdm4 overexpression does not increase SMN levels in the spinal cord of SMA mice.
  • Fig. 10A is a schematic of the AAV9 vectors used to express full-length Mdm2 or Mdm4 cDNAs in vivo.
  • Fig. 10E shows a Western blot analysis of spinal cords isolated at P11 from uninjected WT mice and SMA mice injected at P0 with AAV9-GFP or AAV9- Mdm2 and AAV9-Mdm4 together. Three independent mice per group were analyzed.
  • one embodiment of the present disclosure is an anti-sense morpholino oligonucleotide (MO) targeting a gene in a subject, wherein the gene is selected from mdm2 and mdm4.
  • MO anti-sense morpholino oligonucleotide
  • the anti-sense morpholino oligonucleotide (MO) is complementary to the 5’ splice site of an exon of the gene, and thereby blocks U1 snRNP binding and promotes exon skipping without altering the endogenous mRNA level.
  • the exon of the gene is selected from mouse mdm2 exon 3 or its orthologues, and mouse mdm4 exon 7 or its orthologues.
  • exon refers to any part of a gene that will encode a part of the final mature RNA produced by that gene after RNA splicing.
  • An exon refers to both the DNA sequence within a gene and to the corresponding sequence in RNA transcripts.
  • Another embodiment of the present disclosure is a composition comprising at least one anti-sense morpholino oligonucleotide (MO) as disclosed herein and a pharmaceutically acceptable carrier.
  • MO anti-sense morpholino oligonucleotide
  • the composition comprises both an anti-sense MO targeting mdm2 and an anti-sense MO targeting mdm4.
  • compositions of the present disclosure are pharmaceutically acceptable and may comprise one or more active ingredients in admixture with one or more pharmaceutically-acceptable carriers and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the agents/compounds of the present disclosure are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.). More generally, "pharmaceutically acceptable” means that which is useful in preparing a composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use
  • Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a cancer in a subject, comprising administering to the subject an effective amount of an anti-sense MO as disclosed herein or a composition comprising the same as disclosed herein.
  • the terms "treat,” “treating,” “treatment” and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient.
  • the methods and compositions of the present disclosure may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development.
  • every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population, may fail to respond or respond inadequately to treatment.
  • ameliorate means to decrease the severity of the symptoms of a disease in a subject.
  • the administration of the composition synergistically induces p53 anti-repression.
  • the cancer retains a functional p53 allele.
  • the cancer is selected from the group consisting of glioblastoma, liposarcoma, breast cancer, oesophagus cancer, osteosarcoma, colorectal cancer, melanoma and diffuse large B cell lymphoma.
  • an "effective amount” or “therapeutically effective amount” of a compound or pharmaceutical composition is an amount of such a compound or composition that is sufficient to affect beneficial or desired results as described herein when administered to a subject.
  • Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of the subject, and like factors well known in the arts of, e.g., medicine and veterinary medicine.
  • a suitable dose of a compound or pharmaceutical composition according to the disclosure will be that amount of the compound or composition, which is the lowest dose effective to produce the desired effect with no or minimal side effects.
  • the effective dose of a compound or pharmaceutical composition according to the present disclosure may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
  • Another embodiment of the present disclosure is a vector comprising a full-length cDNA of a gene in a subject, wherein the gene is selected from mdm2 and mdm4.
  • the present disclosure includes use of unctional equivalents to the full-length cDNA.
  • the vector is an adeno-associated virus serotype 9 (AAV9) vector.
  • AAV9 vectors suitable for human use are also within the scope of the present disclosure.
  • Non-limiting examples of such vectors include adenovirus vectors, e.g., Ad5; adeno-associated virus vectors, e.g., AAV2, AAV3, AAV5, AAV6, AAV8, AAV9; herpes simplex virus vectors, e.g., HSV1 , HSV; retrovirus vectors, e.g.,MMSV, MSCV; Lentivirus vectors, e.g., HIV-1 , HIV2; alphavirus vectors, e.g., SFV, SIN, VEE, M1 ; flavivirus vectors, e.g., Kunjin, West Nile, Dengue virus; rhabdovirus vectors, e.g., rabies, VSV; measles virus vectors, e.g.,
  • Another embodiment of the present disclosure is a composition comprising at least one vector as disclosed herein and a pharmaceutically acceptable carrier.
  • Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a neurodegenerative disease in a subject, comprising administering to the subject an effective amount of a vector as disclosed herein or a composition comprising the same as disclosed herein.
  • Non-limiting examples of neurodegenerative disease include spinal muscular atrophy (SMA), Alzheimer’s disease, Parkinson’s disease, and ischemic stroke and traumatic brain injury.
  • the neurodegenerative disease is spinal muscular atrophy (SMA).
  • a “subject” is a mammal, preferably, a human.
  • categories of mammals within the scope of the present disclosure include, for example, agricultural animals, veterinary animals, laboratory animals, etc.
  • agricultural animals include cows, pigs, horses, goats, etc.
  • veterinary animals include dogs, cats, etc.
  • laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc.
  • the subject is a human.
  • administering means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, intracerebroventricular or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject.
  • Administration is by any route including parenteral, and transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal).
  • Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, intracerebroventricular and intracranial.
  • Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.
  • the administration is carried out by intracerebroventricular (ICV) injection.
  • ICV intracerebroventricular
  • kits comprising a composition disclosed herein, and instructions for use thereof.
  • Plasmids containing cDNAs for full-length mouse Mdm2 and Mdm4 were purchased from DharmaconTM (Mdm2 Clone ID: 6415937 Accession: BC050902; Mdm4 Clone ID: 3599906 Accession: BC003750). DNA fragments corresponding to the open reading frames of GFP and full-length Mdm2 and Mdm4 generated by PCR using these plasmids as templates were cloned downstream of the GUSB promoter of vectors harboring AAV2 ITRs for the production of self- complementary AAV9. All constructs were verified by DNA sequencing.
  • DNA for production of AAV9 vectors was purified using endotoxin free Mega prep kit (Qiagen) according to the manufacturer’s instructions.
  • the recombinant plasmids were each packaged into AAV serotype-9 capsid by triple- plasmid transfection of human HEK-293 cells, and virions were purified by CsCI gradient centrifugation as previously described (Mueller et al. 2012).
  • the resulting vectors were concentrated to final titers of about 2x10 13 genome copies/ml using Amicon Ultracel centrifugal filter devices with a 30,000 nominal molecular weight limit (Millipore).
  • NIH3T3 cell lines used in this study were described previously (Lotti et al. 2012; Ruggiu et al. 2012) and were treated with doxycycline (100ng/ml) for 5 days (Smn RNAi and SMN/Smn RNAi ) or 60 hours (SmB RNAi ) prior to analysis.
  • mice All mouse work was performed in accordance with the National Institutes of Health Guidelines on the Care and Use of Animals and approved by the IACUC committee of Columbia University. Equal proportions of mice of both sexes were used and aggregated data are presented because gender-specific differences were not found.
  • the SMNA7 mouse line ( Smn +/ ⁇ /SMN2 +/+ /SMNA7 +/+ ) used in this study to generate SMA mice was on a pure FVB background and was obtained from Jackson Mice (Jax stock #005025). Genotyping of the Smn knockout allele was performed using tail DNA PCR and the primers listed in Table 1 as previously described (Fletcher et al. 2017).
  • mice were sacrificed and tissue collection was performed in a dissection chamber under continuous oxygenation (95%0 2 /5%C0 2 ) in the presence of cold ( ⁇ 12°C) artificial cerebrospinal fluid (aCSF) containing 128.35mM NaCI, 4mM KCI, 0.58mM NaH 2 P0 4 , 21 mM NaHC0 3 , 30mM D-Glucose, 1.5mM CaCI 2 , and 1 mM MgS0 4.
  • aCSF artificial cerebrospinal fluid
  • LCM of motor neurons was carried out essentially as previously described (Lotti et al. 2012). Briefly, to retrogradely label motor neurons, the IL and QL muscles of P2 WT and SMA mice were exposed, and ⁇ 1 pl of cholera toxin B subunit (CTb) conjugated to Alexa 488 was delivered by intramuscular injection using a finely pulled glass microelectrode. At P6, the spinal cord was dissected, and the L1 through L3 segments were embedded in OCT and flash frozen. Cryosections of 14pm were mounted on PEN-Membrane Slides 2.0 (Zeiss) and fixed in 100% ethanol for 15 seconds prior to laser capture microdissection.
  • CTb cholera toxin B subunit
  • motor neurons were microdissected using a DM6000B microscope equipped with a LMD6000 laser capture unit (Leica). Approximately 200 motor neurons were collected bilaterally from L1 -L3 spinal segments of one or more mice for each sample group and biological replicate.
  • RNA analysis For RNA analysis, purification of total RNA from mouse spinal cords and NIH3T3 cells was carried out using TRIzol reagent (Invitrogen) as per manufacturer’s instructions followed by treatment with RNAse-free DNasel (Ambion). cDNA was generated using RevertAid RT Reverse Transcription Kit (ThermoFisher) with random hexamer and oligo dT primers. RT-PCRs were first performed at increasing cycle numbers to ensure analysis was performed within the linear range of amplification using AmpliTaq Gold DNA Polymerase (Thermo Fisher). The identity of all the RT-PCR products was confirmed by DNA sequencing.
  • RT-qPCR analysis was done using SYBR Green (Applied Biosystems) in technical triplicates.
  • Total RNA was purified from LCM motor neurons using the Absolutely RNA Nanoprep Kit (Agilent).
  • Amplified cDNA was prepared from total RNA using the Ovation PicoSL WTA System V2 Kit (Nugen) and purified with the MinElute Reaction Cleanup Kit (Qiagen). RNA quality and quantity was assessed using the 2100 Bioanalyzer (Agilent).
  • the primers used for RT-PCR and RT-qPCR experiments are listed in Table 1.
  • proteins from spinal cord tissue or NIFI3T3 cells were homogenized in SDS PAGE sample buffer and quantified using RC DCTM protein assay (BioRad). Protein extracts were run on 12% polyacrylamide gel and transferred to nitrocellulose membranes for probing. Blocking was done in 5% milk in PBS/0.1 % Tween and primary and secondary antibodies were diluted in PBS/0.1 % Tween. The antibodies used for these experiments are listed in Table 2. Immunohistochemistry and immunofluorescence analysis
  • NIFI3T3 fibroblasts plated on coverslips were fixed in 4% PFA for 15 minutes then permeabilized with 0.5% Triton- X 100 in PBS for 10 minutes at room temperature. Samples were blocked in 3% BSA/0.05% sodium azide in PBS for 1 hour, and incubated with primary antibodies in blocking buffer for 2 hours. Following three 5 minutes washes, coverslips were incubated with secondary antibodies (Jackson ImmunoResearch) and DAPI diluted in blocking buffer for 1 hour, washed three times, and mounted using ProLongTM Gold Antifade Mountant (Thermo Fisher).
  • the stimulus threshold was defined as the current at which the minimal evoked response was recorded in three out of five trials. Recordings were fed to an A/D interface (Digidata 1440A, Molecular Devices) and acquired with Clampex (v10.2, Molecular Devices) at a sampling rate of lOkFIz. Data were analyzed off-line using Clampfit (v10.2, Molecular Devices). Measurements were taken from averaged traces of five trials elicited at 0.1 Flz. The temperature of the physiological solution ranged between 21 - 25°C.
  • the stimulus threshold was defined as the current at which the minimal evoked response was recorded in three out of five trials.
  • the nerve was stimulated at 1 , 2, 5 and 10X threshold to ensure a supramaximal stimulation of the muscle.
  • the maximum CMAP amplitude was determined as the average from five measurements.
  • Results are expressed as mean + standard error of the mean (SEM) from at least three independent experiments and biological replicates unless otherwise indicated. For statistical analysis, differences between two groups were analyzed by two-tailed unpaired Student's f-test and differences among three or more groups were analyzed by one-way or two-way ANOVA followed by Tukey’s post hoc test as appropriate. GraphPad Prism 5 was used for all statistical analyses and P values are indicated as follows: * P ⁇ 0.05; ** P ⁇ 0.01 ; *** P ⁇ 0.001.
  • RT-PCR analysis revealed that the inclusion of both Mdm2 exon 3 and Mdm4 exon 7 was significantly reduced upon Smn deficiency (Figs. 1A - 1 D), while splicing of other Mdm2 and Mdm4 exons was not affected (Figs. 8A - 8F). Moreover, these splicing changes were reversed by expression of RNAi-resistant human SMN in NIFI3T3-SMN/Smn RNAi cells (Figs. 1A - 1 D) (Ruggiu et al. 2012; Lotti et al. 2012; Tisdale et al. 2013), highlighting the specificity of the effects for SMN depletion. These results demonstrate that SMN regulates the alternative splicing of specific exons of Mdm2 and Mdm4 mRNAs in vitro.
  • SMN deficiency induces time-dependent, progressive accumulation of Mdm2A3 and Mdm4A7 mRNAs in the spinal cord of SMA mice relative to control mice (Figs. 1 E - 1 H), revealing SMN-dependent dysregulation of Mdm2 and Mdm4 alternative splicing in vivo.
  • CTb fluorescently conjugated cholera toxin B subunit
  • RT-PCR analysis showed a severe reduction in the inclusion of Mdm2 exon 3 and Mdm4 exon 7 in vulnerable SMA motor neurons relative to WT motor neurons (Figs. 1 L, 1 M).
  • SMN deficiency strongly affects the alternative splicing of Mdm2 and Mdm4 mRNAs in vulnerable SMA motor neurons.
  • splicing dysregulation of these mRNAs occurs earlier and to a much greater extent in these disease-relevant neurons than in whole spinal cord of SMA mice.
  • Defective snRNP biogenesis induces Mdm2 and Mdm4 splicing dysregulation and p53 activation
  • RT-PCR analysis showed that SmB knockdown strongly reduced inclusion of Mdm2 exon 3 and Mdm4 exon 7 in Dox- treated NIH3T3-SmB RNAi cells relative to untreated controls (Figs. 2A - 2D), mimicking effects of SMN deficiency on the splicing of these mRNAs.
  • NIH3T3-SmB RNAi cells Further characterization of NIH3T3-SmB RNAi cells revealed that SmB deficiency also led to marked upregulation of p53 protein levels (Figs. 2E, 2F), nuclear accumulation of p53 (Fig. 2G), and robust mRNA upregulation of p53 transcriptional targets (Fig. 2H).
  • defective snRNP biogenesis dysregulates Mdm2 and Mdm4 alternative splicing and activates p53 in mammalian cells.
  • p53 + SMA motor neurons had significantly lower levels of nuclear SmB than either SMA or WT motor neurons that are p53 (Figs. 2I, 2 J), indicating a correlation between snRNP reduction and p53 activation in SMA motor neurons.
  • the Mdm2 and Mdm4 MOs were delivered by intracerebroventricular (ICV) injection in WT mice at P0 either alone or in combination in order to define the individual contribution of these splicing events to p53 activation.
  • RT-PCR analysis of Mdm2 exon 3 and Mdm4 exon 7 splicing in the spinal cord of injected mice at P11 showed that Mdm2 and Mdm4 MOs acted selectively to induce skipping of their targeted exons in the spinal cord of WT mice at P11 (Fig. 3B).
  • phosphorylation of serine 18 of mouse p53 is a specific marker of degenerating SMA motor neurons, and overexpression of unphosphorylated p53 is not sufficient to induce degeneration of WT motor neurons in vivo (Simon et al. 2017).
  • We did not observe phospho-p53 S18 expression in motor neurons or other spinal cells Fig. 3H
  • Fig. 3I skipping of SMN-regulated Mdm2 and Mdm4 exons can act as an initiating step for p53 stabilization, but not for phosphorylation of serine 18 in vivo.
  • Mdm2 and Mdm4 gene delivery inhibits p53 activation in SMA mice
  • AAV9-Mdm2 and AAV9-Mdm4 did not alter the low levels of SMN produced by the SMN2 gene at the mRNA or protein level (Figs. 10D, 10E).
  • AAV9-Mdm2 or AAV9- Mdm4 affected the expression of p53 in vulnerable motor neurons of SMA mice. In agreement with our previous study (Simon et al.
  • Mdm2 and Mdm4 restoration inhibits motor neuron degeneration and improves motor function in SMA mice
  • Mdm2 and Mdm4 are negative regulators of p53 that prevent its undue activation under normal conditions (Vousden and Prives 2009). However, they are also oncogenes as their overexpression promotes uncontrolled cell proliferation through excess inhibition of the tumor suppressor activity of p53 in certain cancers (Wade et al. 2013). Moreover, enhanced skipping of Mdm4 exon 7 (exon 6 in human MDM4) has been proposed as a candidate therapeutic approach to activate the p53 response in specific cancers such as melanomas and diffuse large B cell lymphoma that retain a functional p53 allele (Dewaele et al.
  • hypoxia is a modifier of SMN2 splicing and disease severity in a severe SMA mouse model.
  • Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model. Nature 478: 123-126.
  • SMN is essential for the biogenesis of U7 small nuclear ribonucleoprotein and 3’-end formation of histone mRNAs. Cell Rep 5: 1187-95.
  • SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing. Cell 133: 585-600.

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Abstract

La présente invention concerne, entre autres, des compositions et des procédés destinés à traiter ou à atténuer les effets de cancers tels que le mélanome et d'autres maladies telles que l'amyotrophie spinale (SMA) chez un sujet par modulation de l'épissage alternatif de Mdm2 et Mdm4. L'invention concerne également des kits contenant de telles compositions ou destinés à mettre en œuvre de tels procédés.
PCT/US2019/036806 2018-06-13 2019-06-12 Compositions et procédés d'activation synergique de p53 par modulation de l'épissage de mdm2 et mdm4 WO2019241410A1 (fr)

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