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Definitions

• This disclosure is directed to therapeutic strategies for the treatment of Charcot-Marie- Tooth disease (CMT) via targeting PMP22 mRNA with antisense oligonucleotides (ASOs), including methods and compositions for the same.
• CMT Charcot-Marie- Tooth disease
• ASOs antisense oligonucleotides
• CMT1A the most common form of CMT, is a demyelinating neuropathy caused by genetic duplication of the peripheral myelin protein-22 (PMP-22) gene (SEQ ID NO: 1) (Patel et al., 1992; Timmerman et al., 1992; Valentijn et al., 1992; Matsunami et al., 1992).
• PMP22 is an essential structural component of the myelin sheath that surrounds axons (Lee et al., 2014; Mittendorf et al., 2017; Snipes et al., 1992). Functionally, myelin acts as a biological insulator that facilitates the efficient transmission of electrical impulses along an axon. While myelin is produced in the CNS by glial cells, Schwann cells (SC) are responsible for the production of PMP22 and myelin in the peripheral nervous system (PNS).
• SC Schwann cells
• PMP22 While too much PMP22 results in the development of CMT1A, an autosomal dominant disease, it is equally important to maintain sufficient PMP22 expression as the loss of PMP22 results in a distinct neuropathy called hereditary neuropathy with predisposition to pressure palsy (HNPP). Taken together, PMP22 is an essential component of the myelin sheath and the delicate homeostatic balance of this gene should be paramount in the development of effective therapeutics.
• HNPP hereditary neuropathy with predisposition to pressure palsy
• PMP22 peripheral myelin protein 22
• the peripheral myelin protein 22 (PMP22) gene which encodes the major myelin protein, peripheral myelin protein 22, resides within the 1.4-Mb duplicated interval.
• PMP22 is an intrinsic membrane protein of myelin that alters lipid organization/ distribution and is developmentally induced within Schwann cells as they initiate myelination of peripheral nerves.
• PMP22 overexpression in rodent models can be reduced by high-dose ascorbic acid (Cortese et al., 2020); however, in clinical trials, ascorbic acid didnot reduce the level ofPMP22 mRNA in skin biopsies from treated CMTIApatients (Eichinger et al., 2018; Gautier et al., 2021; Kagiava et al., 2018).
• Progesterone antagonists and GABAB agonists have also been shown to reduce PMP22 mRNA expression (Lee et al., 2020; Massade and Charbel, 2020), but their potential is hampered by diverse effects on the gene -regulation program of Schwann cells and possibly other cell types, which may complicate a chronic treatment of an inherited disease.
• CMT1A is monogenic; the disease gene has been identified; and inhibition of PMP22 expression can be accomplished through a variety a molecular mechanisms.
• a panel of 2 ’-O-2-m ethoxy ethyl phosphorothioate based backbone (2’ MOE) ASOs were developed and analyzed as a means to interfere with PMP22 expression (Zhao et al., 2018).
• Zhao et al. ASOs were identified that decreased PMP22 expression in several important cellular and in vivo contexts, including K-562 cells and the C22 transgenic mouse model.
• each of these experimental contexts is predicated upon the presence of the human PMP22 gene.
• ASO treatment decreased PMP22 expression and significantly improved the CMT phenotype, including neuronal pathology, degree of myelination, and CMAP/MNCV.
• This disclosure relates to molecules (small molecules, antisense oligonucleotides, antibodies, etc) that bind to the pre-mRNA region of human PMP22 related to exon splicing.
• the molecules bind to one (or more) of these key regions of the pre-mRNA prior to splicing to induce an exon skipping event during translation/transcription that results in mRNA being produced that is similar to the full-length version of the natural mRNA, but missing one or more exons or a portion thereof.
• the resulting exon-skipped mRNA is stable and measurable, either in vitro, in vivo, or in situ.
• composition comprising an antisense oligonucleotide (ASO) that comprises or consists of a complementary region that is complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to a target region of a PMP22 pre- mRNA.
• ASO antisense oligonucleotide
• binding in a cell of the complementary region of the ASO to the target region of the PMP22 pre-mRNA induces exon skipping during RNA transcription.
• the induced exon skipping reduces full-length PMP22 mRNA production.
• the induced exon skipping produces an exon-skipped PMP22 mRNA.
• Also provided for herein is a method of decreasing the amount of full-length PMP22 mRNA expression in a cell.
• Such method comprises administering to the cell an exon skipping inducing composition comprising an antisense oligonucleotide (ASO) of this disclosure.
• ASO antisense oligonucleotide
• an PMP22 exon-skipped mRNA is produced.
• the amount of PMP22 protein produced in the cell is decreased.
• Also provided for herein is a method of producing an exon-skipped PMP22 pre-mRNA.
• Such method comprises administering to a cell an exon-skipping inducing composition comprising an antisense oligonucleotide (ASO) of any this disclosure.
• ASO antisense oligonucleotide
• the amount of full-length PMP22 mRNA expression in the cell is decreased.
• the amount of functional PMP22 protein produced in the cell is decreased.
• Also provided for herein is a method of treating Charcot-Marie-Tooth disease.
• Such method comprises administering to a subject in need thereof an exon-skipping inducing composition comprising an antisense oligonucleotide (ASO) of any this disclosure.
• ASO antisense oligonucleotide
• composition comprising an antisense oligonucleotide (ASO) comprising or consisting of a complementary region that is complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to a target region of a PMP22 pre-mRNA.
• ASO antisense oligonucleotide
• the target region of the PMP22 pre-mRNA comprises an intron/exon junction of one of the coding exons.
• Figure 1 shows PMP22 pre-mRNA (top), resulting in full length mRNA that contains all of the amino acid coding Exons (middle) and functioning protein (bottom). Introns between each exon are shown as double horizontal lines and the base pair sequences are removed during splicing.
• Figure ! shows the PMP22 gene process when an anti-sense oligonucleotide (ASO) is added that hybridizes to a portion of the premRNA and induces a skipping event when the pre-mRNA is converted to mRNA.
• ASO anti-sense oligonucleotide
• Figure 3A,B,C is a schematic of the changes in structure to a pre-mRNA during splicing.
• Figure 4A-D Figure 4 shows a schematic of a number of strategies for biding to the pre-mRNA to induce exon skipping.
• Figure 5A,B Figure 5 shows the sequence of PMP22 gene around the 5’- and 3 ’-regions of Exon 3 (all caps) and associated upstream and downstream introns.
• 5A shows the 5’-end of the exon and 5B shows the 3 ’-end.
• a number of different representative 25-mer oligonucleotides ASO sequences of the present disclosure are shown (below the sequence at the top) that may produce a skipping of Exon 3 during conversion from pre-mRNA to mRNA.
• 5A SEQ ID NOs: 2-34; SHC-006 25-mer (SEQ ID NO: 71); SHC-001 24-mer (SEQ ID NO: 72); SHC-005 25-mer (SEQ ID NO: 73).
• 5B SEQ ID NOs: 35-70; SCH-010 21-mer (SEQ ID NO: 74); SCH-012 20-mer (SEQ ID NO: 75).
• Figure 6 shows the results from PCR amplification followed by gel electrophoresis analysis of the selected Exon 3 skipping ASOs from Figure 5.
• a scramble injection control (that does not effect PMP22 pre-mRNA or mRNA) is shown as well.
• Figure 7 shows RT-PCR from C3 mice (liver) treated with SHC-012 (SEQ ID NO: 75), a scramble (2x) ASO, or a water control. Human full-length PMP22 mRNA is detectable in addition to the GAPDH control. SHC-012-induced exon 3 skipping (confirmed by sequence) is present as marked.
• Figure 8 shows the PCR results (and corresponding change in amount of full-length PMP22 mRNA) for a number of tissues after a single subcutaneous injection of SHC- 012 (SEQ ID NO: 75) was given and the animals sacrificed 2 days later.
• the data in Figure 8 represents percent remaining. Specifically, for each tissue type we quantitated the total amount of PMP22 full-length mRNA per tissue type and compared to the SHC-012 animals. 20% remaining indicates the 80% of the pre-mRNA was blocked from making full-length mRNA and made exonskipped mRNA instead.
• Figure 9 shows the amount of time taken to walk across the dowel apparatus described for different treatment groups (using SHC-012; SEQ ID NO: 75), scramble animals, and wild-type mice. For all groups, 3 animals were in each group and the histogram shows the average values and standard deviations. P values were calculated (to determine confidence levels against the scramble control) for each treatment group. For each group, p ⁇ 0.05. All data is for average values of all animals that are 12 weeks old.
• Figure 10 shows the amount of time spent on a rotarod apparatus for the scramble, wild-type, and treatment groups first injected at 5-weeks of age. For all groups, 3 animals were in each group and the histogram shows the average values and standard deviations. P values were calculated (to determine confidence levels against the scramble control) for each treatment group and are shown. All data is for average values of all animals that are 12 weeks old.
• Figure 11A,B Figure 11 shows the sequence of PMP22 gene around the 5’- and 3’- regions of Exon 4 (all caps) and associated upstream and downstream introns.
• 11A is the 5’-end of the exon and 11B is the 3’-end.
• a number of different 25-mer oligonucleotides ASO sequences of this disclosure are shown (below the sequence at the top) that may produce a skipping of Exon 4 during conversion from pre-mRNA to mRNA.
• Six representative morpholino anti-sense oligo sequences that were designed (and synthesized) to bind to the intro/ exon junctions are shown where data is presented.
• 11A SEQ ID NOs: 76-110; SHC-029 21-mer (SEQ ID NO: 146); SHC-028 20- mer (SEQ ID NO: 147); SHC-027 20-mer (SEQ ID NO: 148).
• 11B SEQ ID NOs: 111-145; SCH- 031 21-mer (SEQ ID NO: 149); SCH-030 20-mer (SEQ ID NO: 150); SCH-032 20-mer (SEQ ID NO: 151).
• Figure 12 shows the results from PCR amplification followed by gel electrophoresis analysis of the selected Exon 4 skipping ASOs from Figure 11.
• Figure 13 shows quantitation results using two different methodologies from the gel results for 3 of the Exon 4 skipping compounds.
• Figure 14 shows the 5’- and 3’-ends of Exon 3 (top) and 3 different ASO compounds that were designed and tested that bridge the target regions (shown for each). These ASO target regions of the Exon that are non-continuous and will have a different effect on the three dimensional structure of the pre-mRNA than those described above.
• Figure 15 shows the results from PCR amplification followed by gel electrophoresis analysis of the selected Exon 3 skipping ASOs from Figure 14.
• Figure 16A,B Figure 16 shows the sequence of PMP22 gene around the 5’- and 3’- regions of Exon 2 (all caps) and associated upstream and downstream introns. 15A is the 5’-end of the exon and 15B is the 3 ’-end. 16A: SEQ ID NOs: 163-197. 16B: SEQ ID NOs: 198-232. [0033] Figure 17. Figure 17 shows images of the sciatic nerve for WT, untreated, and treated animals (top) and images from the peroneal portion of the nerve (bottom).
• Figure 18 shows a TEM image at higher magnification of portions of a Peroneal nerve.
• Figure 19 shows electrophysiology plots from the sciatic gastric section of sedated mice (top).
• Figure 19 (bottom) shows the average values of MUNE and CMAP from 3 measured animals per group.
• Figure 20 shows results for treatment groups (with the scramble animals’ group and wild-type animals shown for comparison at 4 months of age). Each data set of the histogram is an average of 4 days dowel travers time at the end of each month.
• Figure 21 shows the results of the dowel traverse time experiment (with 3 month old wild-type animals also plotted for comparison).
• identity refers to a relationship between two or more nucleotide sequences or between two or more amino acid sequences.
• sequences are said to be “identical” at that position.
• the percentage “sequence identity” is calculated by determining the number of positions at which the identical nucleic acid base or amino acid occurs in both sequences to yield the number of “identical” positions.
• the number of “identical” positions is then divided by the total number of positions in the comparison window and multiplied by 100 to yield the percentage of “sequence identity.” Percentage of “sequence identity” is determined by comparing two optimally aligned sequences over a comparison window.
• the portion of a nucleotide or amino acid sequence in the comparison window can comprise additions or deletions termed gaps while the reference sequence is kept constant.
• An optimal alignment is that alignment which, even with gaps, produces the greatest possible number of “identical” positions between the reference and comparator sequences.
• sequence identity between two sequences can be determined using, e.g., the program “BLAST” which is available from the National Center for Biotechnology Information, and which program incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for amino acid sequence comparison), which programs are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993).
• BLAST Altschul
• polypeptide is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
• polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
• peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms.
• polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-standard amino acids.
• a polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
• a “protein” as used herein can refer to a single polypeptide, i.e., a single amino acid chain as defined above, but can also refer to two or more polypeptides that are associated, e.g., by disulfide bonds, hydrogen bonds, or hydrophobic interactions, to produce a multimeric protein.
• an "isolated" polypeptide or a fragment, variant, or derivative thereof or the like is intended a polypeptide that is not in its natural milieu. No particular level of purification is required.
• an isolated polypeptide can be removed from its native or natural environment.
• Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.
• polynucleotide is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., premessenger RNA (pre-mRNA), messenger RNA (mRNA), or plasmid DNA (pDNA).
• pre-mRNA premessenger RNA
• mRNA messenger RNA
• pDNA plasmid DNA
• a polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
• PNA peptide nucleic acids
• nucleic acid refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.
• isolated nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
• a recombinant polynucleotide encoding a polypeptide subunit contained in a vector is considered isolated as disclosed herein.
• Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
• Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically.
• polynucleotide or a nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
• a "coding region” is a portion of nucleic acid comprising codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors.
• any vector can contain a single coding region, or can comprise two or more coding regions, e.g., a single vector can separately encode a selection marker gene and a gene of interest.
• a vector, polynucleotide, or nucleic acid can encode heterologous coding regions, either fused or unfused to a nucleic acid encoding a polypeptide subunit or fusion protein as provided herein.
• Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.
• an "exon” refers to the portion of a DNA or RNA sequence that results in the synthesis of an amino acid sequence.
• an "intron” refers to the portion of a DNA or RNA sequence that does not result in the synthesis of an amino acid sequence.
• the polynucleotide or nucleic acid is DNA.
• a polynucleotide comprising a nucleic acid that encodes a polypeptide normally can include a promoter and/or other transcription or translation regulatory elements operably associated with one or more coding regions.
• An operable association or linkage can be when a coding region for a gene product, e.g., a polypeptide, can be associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s).
• Two DNA fragments can be "operably associated” or “operably linked” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
• a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid.
• the promoter can be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells.
• Other transcription regulatory elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.
• transcription regulatory regions are known to those skilled in the art. These include, without limitation, transcription regulatory regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription regulatory regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit beta-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription regulatory regions include tissue-specific promoters and enhancers.
• translation regulatory elements include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picomaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
• a polynucleotide can be RNA, for example, in the form of a pre-mRNA or messenger RNA (mRNA).
• mRNA messenger RNA
• Polynucleotide and nucleic acid coding regions can be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide as disclosed herein.
• proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
• polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature” form of the polypeptide.
• the native signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it.
• a heterologous mammalian signal peptide, or a functional derivative thereof can be used.
• the wild-type leader sequence can be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse B-glucuronidase.
• a "vector” is nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
• a vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
• a vector can also include one or more selectable marker gene and other genetic elements known in the art. Illustrative types of vectors include plasmids, phages, viruses and retroviruses.
• a "transformed” cell, or a "host” cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques.
• transformation encompasses those techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
• a transformed cell or a host cell can be a bacterial cell or a eukaryotic cell.
• expression refers to a process by which a gene produces a biochemical, for example, a polypeptide.
• the process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into pre-mRNA and messenger RNA (mRNA), and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors.
• a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide that is translated from a transcript.
• Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.
• engineered includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g. by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these techniques).
• composition refers to a preparation or mixture of substances suitable for administering to a subject, i.e., that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the composition would be administered.
• Such composition can be sterile.
• a pharmaceutical composition may comprise an oligomeric compound and a sterile aqueous solution.
• a pharmaceutically acceptable carrier or diluent is suitable for administration. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension, and lozenges for the oral ingestion by a subject.
• a pharmaceutically acceptable carrier or diluent is sterile water, sterile saline, sterile buffer solution, or sterile artificial cerebrospinal fluid.
• pharmaceutically acceptable salts are physiologically and pharmaceutically acceptable salts of compounds. Pharmaceutically acceptable salts retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
• an “antisense compound” is a compound capable of achieving at least one antisense activity.
• an antisense compound comprises an antisense oligonucleotide (ASO) and optionally one or more additional features, such as a conjugate group or terminal group.
• ASO antisense oligonucleotide
• additional features such as a conjugate group or terminal group.
• an antisense compound has been engineered and synthesized to contain non-naturally occurring backbone structures (such as changes in the sugars and/or phosphate backbone).
• the antisense compounds have a morpholino backbone.
• antisense activity means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid.
• antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.
• the entire human PMP22 gene was downloaded from the University of California Santa Cruz genome.ucsc.edu database. Consistent with standard nomenclature, as shown in the Figures, regions of sequence in introns or non-coding portions of the genome appear in lower case letters. Regions of sequence encoding amino acids appear in upper case letters.
• the UCSC database above was used and the option to download the sequences from the Human Assembly Dec. 2013 (GRCh38/hg38) with the protein coding option was selected. If other databases are available and acceptable or become available and acceptable with slight sequence variations, one skilled in the art would understand this description to also cover those variants.
• complementary in reference to an oligonucleotide refers to two nucleic acid singles strands or portions of a single strand capable of hybridizing into a doublestranded sequence via hydrogen bonding of complementary bases.
• Complementary bases include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5-methyl cytosine (mC) and guanine (G).
• Complementary oligonucleotides and/or nucleic acids do not need to be complementarity at each positions. Some mismatches can be tolerated.
• oligonucleotides As used herein, “fully complementary” or “100% complementary” in reference to oligonucleotides means that the two oligo-strands have complementary bases at each corresponding position. In certain embodiments, complementary oligonucleotides have only at least 70% complementary bases at each corresponding position.
• hybridization means the pairing or annealing of complementary oligonucleotides and/or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleotides.
• oligomeric compound means an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group.
• An oligomeric compound may be paired with a second oligomeric compound that is complementary to the first oligomeric compound or may be unpaired.
• a “singled-stranded oligomeric compound” is an unpaired oligomeric compound.
• oligomeric duplex means a duplex formed by two oligomeric compounds having complementary nucleobase sequences. Each oligomeric compound of an oligomeric duplex may be referred to as a “duplexed oligomeric compound.”
• exon skipping can involve skipping an entire exon or a portion of an exon.
• an exon-skipping compound relates to any compound that binds to a pre-mRNA species and induces transcribed mRNA that is stable and measurable, but not full- length (i.e., exon-skipped mRNA).
• a gene or genetic locus may be identified by a particular reference sequence, e.g., the human PMP22 gene (SEQ ID NO: 1), it is understood that the gene corresponding to the reference sequence can comprise various allelic forms or variations in sequence and still be considered by one of ordinary skill in the art to be the same gene.
• nucleic acid sequences set forth herein and in each corresponding sequence ID Number are independent of any modification to a sugar moiety, an inter-nucleoside linkage, or a nucleobase.
• any nucleic acid of this disclosure including oligonucleotides, may comprise, independently, one or more modifications to a sugar moiety, an inter-nucleoside linkage, or a nucleobase.
• ASOs antisense oligonucleotides
• a direct therapeutic strategy targeting PMP22 RNA with antisense oligonucleotides comprising a method of using ASOs that bind to and interact with the pre-mRNA of the PMP22 gene cascade and effectively “splice out” certain portions of the mRNA by binding to junctions between the spacer region in the pre-mRNA and the exon regions that convert into full mRNA.
• the ASOs do not prevent the rest of the PMP22 gene exons from being expressed into amino acids.
• the resulting altered amino acid sequence is shorter or different than the naturally occurring PMP22 amino acid sequence, causing loss of function of the resulting protein and/or enhanced degradation compared to the naturally occurring protein (i.e., a reduction/decrease of functional PMP22 protein).
• the splicing event causes the resulting alternatively spliced mRNA (exon-skipped mRNA) to be degraded within the cell at a faster rate than the full-length mRNA, thus lowering protein production.
• exon-skipped mRNA is produced using an ASO of this disclosure
• the molecular activity of a drug can be specifically measured in subjects (e.g., laboratory animals and/or patients including human patients over time).
• the amount of exon-skipped mRNA can be correlated to various downstream effects of the drug that can have positive outcomes on patients, such as to lower the effect of functional protein levels and the phenotypic response of the patient or laboratory animal to the drug.
• embodiments of the present disclosure can be used as a diagnostic method to confirm the activity of a drug in a specific subject, alter the amount of the drug given to a patient to achieve a desirable response, and/or alter the dosing frequency of the drug to achieve a desirable response, etc.
• Figure 2 One embodiment of this disclosure is shown in Figure 2 and relates to any compounds that interact with the pre-mRNA shown in Figure 2 to eliminate one (or more) of the exons during translation from pre-mRNA to mRNA and allow the other exons to be included, creating a stable, measurable new exon-skipped mRNA that is similar to full-length mRNA but does not code for fully functioning PMP22 protein.
• Figure 2 schematically shows a compound that would eliminate Exon 3, but other examples are described below for Exon 2 and Exon 4.
• a method where the amount of full-length PMP22 mRNA, which will lead to effective protein, and the presence of exon-skipped mRNA, that will lead to non- function protein, can be compared within a given subject, making this monitoring possible with the practical natural variation of background mRNA and protein levels.
• this can be done in animals studies, both simplifying these studies and providing more information within a given study. And, in certain embodiments, this can be done in human patients receiving treatment.
• the approach of this disclosure leads to two measurable markers within the patient sample and the ratio of those two markers is a defining factor of the drugs activity.
• the amount of exon-skipped mRNA in an animal or patient sample is used to track therapeutic activity.
• the amount of exon-skipped mRNA is compared to the amount of full-length mRNA and that ratio is used to track the therapeutic activity.
• CMT1A For CMT1A, it is desirable to target PMP22 protein production. Further, it would be highly desirable to do so by altering the patient’s dosing schedule. Dosing requirements may be highly dependent on a number of patient factors including weight, age, disease progression, lifestyle, etc. Previously described approaches for treating CMT1A (and other monogenic and multi-genetic) diseases do not allow an accurate methodology for tracking the mRNA production and thus total (or partial) protein production from that mRNA.
• the overall strategy is to study drug effects versus concentration delivered over a large number of cellular and animal experiments to correlate the drugs effect at altering phenotypic responses.
• Drugs can then be studies in humans (typically at multiple dosing strategies mainly focuses on safety and toxicity profiling) in order to come up with a single recommended dose across a target patient population. This often leads to drugs that show a modest effect in clinical setting, but often leads to showing no measurable phenotypic effect whatsoever in a subset of the patient population. Often, these unresponsive patients are simply being dosed ineffectively (either not enough or too much of the drug) based on semi-correlated previous studies.
• ASOs of this disclosure When ASOs of this disclosure are added to cells (or subject, e.g., animals or humans for in vivo treatment), a portion of the ASOs interact with the pre-mRNA, forcing that exon (or more exons) to be skipped during transcription.
• An mRNA with the “skipped” exon (“exonskipped mRNA”) is still formed but produces non-functioning protein.
• exon-skipped mRNA molecule can be quantitated as a measurement of drug activity at a molecular level and therefore is not dependent on certain other environmental and experimental parameters (that are always present) which can confound the results obtained [0087]
• a new class of therapeutics that elicit a molecular response in subjects wherein both the native unaffected, full-length mRNA and the exon-skipped mRNA can be monitored during the course of treatment and related to downstream protein production.
• samples can be taken from target tissues of interest.
• samples can be taken from other (more accessible) portions of the body, such as blood plasma. This approach allows monitoring of the effect of the therapeutic compositions of this disclosure, both during animals studies and in actual patients during clinical deployment.
• PCR is an elegantly sensitive and specific method for analyzing the amount of RNA present - for example in both the target tissues of interest and in corresponding plasma levels - since RNA is not stable and the amount of RNA present in the blood may be significantly lower than the amount and nature of the RNA in the tissue, PCR monitoring can become problematic. Monitoring protein production in the target tissue and corresponding plasma levels can be even more problematic, since the protein levels may be even more variable based on environmental factors including patient activity and inherent protein stability differences in the target tissues versus other portion of the body during biodistribution.
• An important aspect of this disclosure is the unique approach of targeting the junction between the introns and exons of the pre-mRNA prior to transcription.
• the junction points between introns and exons are highly susceptible for ASO targeting (compared to the non-overlapping regions of only intro or only exon) for a number of reasons.
• Second, the binding of any ASO to a genetic target will be in “competition” with the proteins and enzymes (and other molecules) for those binding sites and can be displaced by said proteins.
• Figure 3 and Figure 4 describe an overall targeting approach.
• Figure 3A shows a schematic of Exon 1, the intron region, then Exon 2 (referred to as the pre-mRNA).
• the pre-mRNA Prior to the elimination of introns to join to sequential exons to one another during the conversion of pre- mRNA to mRNA , the pre-mRNA forms a three dimensional structure in the presence of snRNPs to create an intermediate structure shown in Figure 3B where the 3 ’-end of one Exonl is brought into close proximity to the 5’-end of Exon 2.
• biological molecules such as enzyme
• This disclosure relates to binding molecules to selective regions within this genome to disrupt a specific exon-intron-exon junction and allow the remainder of the transcription to occur (either in part or whole). This region is shown (Iz, dotted circle in Figure 3B) and the 3D structure is critical in this region in order for the splicing process to occur efficiently.
• This region is critical due to a number of factors including the enzymes recognizing the 3D structure, the chemical nature of the 3D structure, the close proximity of the ends of the two exons which will be joined, etc. Once splicing occurs, the two ends of Exon 1 and 2 are joined ( Figure 3C) forming the mRNA.
• an anti-sense oligonucleotide is introduced that specifically binds or hybridizes to a region of the intron and/or exon itself that is the target of the exon skipping.
• the ASO added comprises a sequence that is complimentary to the pre-mRNA in a region close to the proximal site described (Iz in Figure 3) in order to disrupt exon inclusion and produce a final mRNA product that does not include the exon targeted but does include other exons within the gene targeted where no drug has bound.
• RNA translation occurs The presence of the ASO within the nucleus of the cell (where RNA translation occurs) will form an equilibrium with the pre-mRNA that will be directly affected by the amount of the drug present, the amount of pre- mRNA present, binding efficiency to the region targeted, strength of the attachment once bound, etc.
• an amount of drug is introduced that does not force exon skipping of all the pre-mRNA present in the cell.
• both full- length PMP22 mRNA and exon-skipped mRNA will be produced and the amount of skipping that occurs will be dependent on factors such as the chemical composition of the drug and the amount of drug added. Other factors may affect the ratio of the exon skipping.
• certain embodiments of the disclosure relate to molecules that do not completely eliminate all full-length mRNA production.
• ASOs that are complimentary to continuous regions of the pre-mRNA can target a 3 ’-end of an exon, a 5’-end of an exon, or other regions of the intron or exon, provided that they induce a measurable exon skipping event where shortened, non-full length exon-skipped mRNA is produced.
• the length of the ASO shown in Figure 4B can vary depending on the desired effect.
• ASOs are disclosed that specifically target the PMP22 pre-mRNA, thus a minimum length of ASO bases is preferable that is specific to the PMP22 mRNA but does not also specifically bind to other areas of the human genome.
• very long ASOs can be problematic in their use, since long strands of DNA, RNA, etc. may fold onto themselves and not be available for binding to the target pre-mRNA. Manufacturing concerns also come under consideration with very long ASOs.
• an ASO is between any of about
• an ASO is between about 12 and 30 nucleotides long. In certain embodiments, an ASO is between about 15 and 25 nucleotides long. In certain embodiments, an ASO is between about 18 and 25 nucleotides long.
• the ASOs of this disclosure do not necessarily have to perfectly match (i.e., be 100% complementary to) all of the target pre-mRNA bases for hybridization.
• an 18-mer ASO maybe designed and synthesized where one or one or more of the bases is not complimentary to the target pre-mRNA, but will still hybridize/possess antisense activity.
• two or two or more of the ASO/target region base pairs can be considered “mis-matches” where they are not complimentary.
• an ASO comprises a complementary region that is complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to a target region of the pre-mRNA.
• an ASO comprises a complementary region that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% complementary to a target region of the pre-mRNA.
• FIG. 4B Another embodiment of this disclosure is shown in Figure 4B, where an ASO is used that does not bind to a continuous portion of the pre-mRNA, but rather spans two different portions of the pre-mRNA.
• the ASO shown can have bases that are complimentary to the 3’-end of Exon 1 and bases that are complimentary to the 5’-end of Exon 2.
• This ASO could bind once the 3D structure is formed (as is shown in Figure 4B) and disrupt the actual splicing event.
• the ASO could also bind and effect the entire formation of the 3D structure and its corresponding chemical nature. Numerous other ASO configurations are also envisioned, provided that they induce exon skipping in one more of the exons present.
• the ASO overlaps both a portion of the intro and exon both. In other embodiments, the ASO only overlaps an exon portion of the target region. In other embodiments, the ASO only overlaps a portion of the intro near the target region.
• FIG. 4C Another embodiment is shown in Figure 4C, where an antibody specific to a given intron/ exon junction is added that produces a exon skipping event. Still another embodiment is shown in Figure 4D, where a small molecule is used to induce the exon skipping event.
• the molecules described in this patent must be specific to certain portions of a genome and not randomly bind to intro/ exon junction regions, which would cause undesirable side effects and potentially disrupt normal biological processes not targeted.
• the molecules act by preventing the formation and stabilization of the 3D structure shown.
• the molecules bind to the region and physically or chemically block the cofactors and enzymes from completing the splicing event.
• compositions disclosed herein will allow ASOs to be more fully studied in animals to understand the various effects of drug dosing in order to further define safe and effective treatment strategy for human patients. It is also contemplated that other factors related to drug dosing (such as weight or disease progression) can be studied in animals, allowing patients to be “stratified” or “categorized” during clinical trials and receive a different dose depending on the particular description of different patients. It is also contemplated that mRNA levels can be monitored in patients as a companion diagnostic during treatment and their dosing can be altered on a patient-by-patient basis based on their molecular response to the drug treatment.
• the use of morpholino anti-sense oligonucleotides enhances the strength of the bonding of an ASO once it has hybridized to the target region of the pre-mRNA. Since non-charged backbone structures have no ionic repulsion competing with base pair binding (such as with 2’Me-O ASOs or miRNA drugs), once the ASO is bound, it resists displacement by the transcription proteins (and other cofactors) and leads to significant improvement. Morpholino backbones are well studied in the literature and used by Sarepta Therapeutics for certain FDA approved ASO products for other uses (such as Eteplirsen).
• non-natural amino acid backbones for the synthesis of ASOs of this disclosure are also contemplated.
• 2’Me-O modifications to the ASO sugar and backbone can be made in order to stabilize ASOs from enzymatic degradation.
• Numerous of backbone chemistries, sugar modification, terminal modifications, etc. can also be used in the methods of this disclosure to form a stable ASO having antisense activity (see all of the examples listed in US 2019/0062741 Al).
• Certain embodiments encompass any ASO structure that hybridizes to the complimentary pre-mRNA to enable exon skipping.
• compositions comprising an antisense oligonucleotide (ASO) that comprises or consists of a complementary region that is complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to a target region of a PMP22 pre-mRNA.
• ASO antisense oligonucleotide
• the complementary region is 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% complementary to a target region of the pre-mRNA.
• the ASO need not be complementary to the full length of the target region but is complementary to a sufficient portion of the target region to hybridize, i.e., the defined target region can be longer than the ASO complementary region and longer than the entire ASO, i.e., the complementary region of the ASO is complementary to a subset of the target region sequence. In certain embodiments, the complementary region and the target region are the same length.
• binding in a cell of the complementary region of the ASO to the target region of the PMP22 pre-mRNA induces exon skipping during RNA transcription. Binding of the ASO to the target region can reduce full-length PMP22 mRNA production.
• binding of the ASO to the target region can lead to a reduction in functional PMP22 protein and/or production of nonfunctional PMP22 protein. Both the reduction in full-length mRNA and the production of exon- skipped mRNA can be detected and measured. The reduction in functional protein and/or the production of non-functional protein can also be detected and measured. Further the correlation and/or ratio between full-length and exon-skipped mRNAs and functional and non-functional proteins can be determined and calculated for purposes such as disclosed in detail elsewhere herein.
• the ASO comprises or consists of a complementary region of at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 contiguous nucleotides that are complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to the PMP22 pre-mRNA target region.
• the length of the ASO’s complementary region can vary depending on, for example, the PMP22 pre-mRNA target region sequence and/or the particular application or conditions of administration.
• Illustrative examples of ASOs with a complementary region of “contiguous nucleotides” are disclosed in Example 1 and Example 4 that follow (e.g., Figure 5A,B and Figure 11A,B).
• the PMP22 pre-mRNA target region comprises two separate segments of the PMP22 pre-mRNA such as described in Example 5.
• the ASO comprises or consists of a complementary region of at least about 6, 8, 9, 10, 11, or 12 nucleotides that are complementary, or complementary except for one, two, or three mismatched nucleotides, to a first segment of contiguous sequence of the PMP22 pre-mRNA target region and the ASO also comprises or consists of a complementary region of at least about 6, 8, 9, 10, 11, or 12 nucleotides that are complementary, or complementary except for one, two, or three mismatched nucleotides, to a second segment of contiguous sequence of the PMP22 pre-mRNA target region.
• the complementary region of the ASO hybridizes to the first and second segments of the PMP22 pre-mRNA and also spans a region of the PMP22 pre-mRNA that it is not complementary/ does not hybridize to.
• the ASO comprises or consists of a complementary region between any of about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, or 45 and any of about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleotides that are complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to the PMP22 pre-mRNA target region.
• the ASO comprises or consists of a complementary region of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides that are complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to the PMP22 pre-mRNA target region.
• the ASO has a length of any of about
• the ASO is a modified and/or synthetic oligonucleotide as known in the art and/or defined elsewhere herein.
• the ASO can be a phosphorodiamidate morpholino oligomer (PMO).
• the ASO causes PMP22 pre-mRNA exon skipping, in certain embodiments this can be done in a manner in which downstream exons are still expressed as they would be from full-length PMP22 pre-mRNA, absent the portion from skipped exon. In certain embodiments, however, the exon skipping forces early termination of protein translation and/or downstream exons to be out of frame.
• the target region of the PMP22 pre-mRNA spans an intron/exon junction of at least one of the coding exons (e.g., PMP22 Exon 2, Exon 3, Exon 4, and Exon 5).
• the targeted intron/exon junction can be at the 3’-end and/or the 5’-end of an exon.
• the target region of the PMP22 pre-mRNA comprises the 3 ’-end of an exon.
• the target region of the PMP22 pre-mRNA comprises the 5 ’-end of an exon.
• the target region of the PMP22 pre- mRNA spans an intron/exon junction comprising or consisting of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the intron and a portion of the exon (e.g., Figure 5A,B and Figure 11A,B).
• the target region of the PMP22 pre-mRNA consists of 2, 3, 4, 5, 6, 7, 8,
• the target region of the PMP22 pre-mRNA spans an intron/exon junction comprising or consisting of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the exon and a portion of the intron (e.g., Figure 5A,B and Figure 11A,B).
• the target region of the PMP22 pre-mRNA consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the exon and a portion of the intron.
• the exon portion of the intron/exon junction comprises PMP22 Exon 3 ( Figure 5A,B).
• the exon portion of the intron/ exon junction comprises PMP22 Exon 4 ( Figure 11A,B).
• the PMP22 pre-mRNA target region comprises the 5 ’-end of Exon 3.
• the PMP22 pre-mRNA target region comprises or consists of SEQ ID NO: 2 or a portion or subset/fragment thereof.
• the PMP22 pre-mRNA target region comprises the 3 ’-end of Exon 3.
• the PMP22 pre-mRNA target region comprises or consists of SEQ ID NO: 35 or a portion or subset/fragment thereof.
• the PMP22 pre-mRNA target region comprises the 5 ’-end of Exon 4.
• the PMP22 pre-mRNA target region comprises or consists of SEQ ID NO: 76 or a portion or subset/fragment thereof.
• the PMP22 pre-mRNA target region comprises the 3 ’-end of Exon 4.
• the PMP22 pre-mRNA target region comprises or consists of SEQ ID NO: 111 or a portion or subset/fragment thereof.
• the PMP22 pre-mRNA target region comprises the 5 ’-end of Exon 2.
• the PMP22 pre-mRNA target region comprises or consists of SEQ ID NO: 163 or a portion or subset/fragment thereof.
• the PMP22 pre-mRNA target region comprises the 3 ’-end of Exon 2.
• the PMP22 pre-mRNA target region comprises or consists of SEQ ID NO: 198 or a portion or subset/fragment thereof.
• the ASO comprises or consists of a complementary region that is complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to SEQ ID NO: 2 (Exon 3, 5’-end), SEQ ID NO: 35 (Exon 3, 3’-end), SEQ ID NO: 76 (Exon 4, 5’-end), SEQ ID NO: 111 (Exon 4, 3’-end), SEQ ID NO: 163 (Exon 2, 5’-end), and/or SEQ ID NO: 198 (Exon 2, 3 ’-end).
• the ASO comprises or consists of a nucleotide sequence of SEQ ID NOs: 3-34 (Exon 3, 5’-end), SEQ ID NOs: 37-70 (Exon 3, 3’-end), SEQ ID NOs: 77-110 (Exon 4, 5’-end), SEQ ID NOs: 112-145 (Exon 4, 3’-end), SEQ ID NOs: 164-197 (Exon 2, 5’-end), or SEQ ID NOs: 199-232 (Exon 2, 3’-end), or a subset/fragment thereof sufficient to hybridize to PMP22 pre-mRNA.
• the ASO comprises or consists of a nucleotide sequence of SEQ ID NOs: 3-34 (Exon 3, 5’-end), SEQ ID NOs: 37-70 (Exon 3, 3’-end), SEQ ID NOs: 77-110 (Exon 4, 5’-end), SEQ ID NOs: 112-145 (Exon 4, 3’-end), SEQ ID NOs: 164-197 (Exon 2, 5’-end), or SEQ ID NOs: 199-232 (Exon 2, 3’-end), except for having one, two, or three nucleotide substitutions, or a subset/fragment thereof sufficient to hybridize to PMP22 pre-mRNA.
• the ASO comprises or consists of the nucleic acid sequence of: SEQ ID NO: 71 (SHC-006 25-mer), SEQ ID NO: 72 (SHC-001 24-mer), SEQ ID NO: 73 (SHC-005 25-mer), SEQ ID NO: 74 (SHC-010 21-mer), SEQ ID NO: 75 (SHC-012 20-mer), SEQ ID NO: 146 (SHC-029 21-mer), SEQ ID NO: 147 (SHC-028 20-mer), SEQ ID NO: 148 (SHC- 027 20-mer), SEQ ID NO: 149 (SHC-031 21-mer), SEQ ID NO: 150 (SHC-030 20-mer), SEQ ID NO: 151 (SHC-032 20-mer), SEQ ID NO: 156, SEQ ID NO: 159, SEQ ID NO: 162, SEQ ID NO: 235, or SEQ ID NO: 238.
• the ASO comprises or consists of the nucleic acid sequence of: SEQ ID NO: 71 (SHC-006 25-mer), SEQ ID NO: 72 (SHC-001 24-mer), SEQ ID NO: 73 (SHC-005 25-mer), SEQ ID NO: 74 (SHC-010 21-mer), SEQ ID NO: 75 (SHC-012 20-mer), SEQ ID NO: 146 (SHC-029 21-mer), SEQ ID NO: 147 (SHC-028 20-mer), SEQ ID NO: 148 (SHC-027 20-mer), SEQ ID NO: 149 (SHC-031 21-mer), SEQ ID NO: 150 (SHC-030 20- mer), SEQ ID NO: 151 (SHC-032 20-mer), SEQ ID NO: 156, SEQ ID NO: 159, SEQ ID NO: 162, SEQ ID NO: 235, or SEQ ID NO: 238, except for having one, two, or three nucleotide
• a method of decreasing the amount of full-length PMP22 mRNA expression in a cell comprising administering to the cell a composition comprising an antisense oligonucleotide (ASO) of this disclosure.
• decreasing the amount of full- length PMP22 mRNA comprises targeting a junction between an intron and an exon within a PMP22 pre-mRNA as described in greater detail elsewhere herein.
• administering to the cell is understood to cover all situations where the ASO is placed in contact with the cell in a manner that the cell may take-up the ASO so that the ASO may exert its antisense activity.
• administering to the cell includes exposing cells in an in vitro experiment to the ASO, such as to cells grown in tissue culture.
• Administering to the cell also includes providing the ASO to a subject, such as a research animal in an in vivo experiment, such that at least one cell of the subject, through administration locally, systemically, etc., is contacted with the ASO.
• Administering to the cell also includes providing the ASO to a patient, such as treating a human patient, such that at least one cell of the patient, through administration locally, systemically, etc., is contacted with the ASO.
• the subject cell may reside in a tissue, organ, body part, biological fluid, whole organism, and the like.
• the amount of full-length PMP22 mRNA in the cell is decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 95% in response to the ASO.
• the amount of full-length PMP22 mRNA and/or its decrease in any method of this disclosure can be compared against the amount of full-length PMP22 mRNA in an untreated cell, subject, patient, etc., to which the ASO composition has not been administered, such as described in the Examples that follow.
• the amount of full-length PMP22 mRNA in the cell is decreased by not more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in response to the ASO. In certain embodiments, the amount of full-length PMP22 mRNA in the cell is decreased by from any of about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% to any of about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in response to the ASO.
• a PMP22 exon-skipped mRNA is produced.
• the amount/reduction of full-length PMP22 mRNA can be compared against the amount of PMP22 exon-skipped mRNA to determine a correlation, calculate a ratio, etc.
• the amount of functional PMP22 protein produced in the cell is decreased. This decrease in functional protein in any method of this disclosure can be determined against an untreated control cell, subject, patient, etc. to which the ASO composition has not been administered.
• the amount of functional PMP22 protein in the cell is decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 95% in response to the ASO. As noted, in some embodiments it is not contemplated or desired to completely eliminate PMP22 protein. Thus, in some embodiments, the amount of functional PMP22 protein in the cell is decreased by not more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in response to the ASO.
• the amount of functional PMP22 protein in the cell is decreased by from any of about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% to any of about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in response to the ASO.
• a method of producing an exon-skipped PMP22 pre-mRNA comprising administering to a cell a composition comprising an antisense oligonucleotide (ASO) of this disclosure.
• the method comprises targeting a junction between an intron and an exon within a PMP22 pre-mRNA as described in greater detail elsewhere herein.
• the amount of full-length PMP22 mRNA expression in the cell is decreased.
• the amount of full-length PMP22 mRNA in the cell is decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 95% in response to the ASO.
• the amount of full-length PMP22 mRNA in the cell is decreased by not more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in response to the ASO. In certain embodiments, the amount of full-length PMP22 mRNA in the cell is decreased by from any of about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% to any of about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in response to the ASO.
• the amount/reduction of full-length PMP22 mRNA can be compared against the amount of PMP22 exon-skipped mRNA produced to determine a correlation, calculate a ratio, etc. Further, in certain embodiments, the amount of functional PMP22 protein produced in the cell is decreased. In certain embodiments, the amount of functional PMP22 protein in the cell is decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 95% in response to the ASO. As noted, in some embodiments it is not contemplated or desired to completely eliminate PMP22 protein.
• the amount of functional PMP22 protein in the cell is decreased by not more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in response to the ASO. In certain embodiments, the amount of functional PMP22 protein in the cell is decreased by from any of about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% to any of about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in response to the ASO.
• Charcot-Marie-Tooth disease type 1A a method of treating Charcot-Marie-Tooth disease, such as Charcot-Marie-Tooth disease type 1A, comprising administering to a subject in need thereof a composition comprising an antisense oligonucleotide (ASO) of this disclosure.
• the subject is a model system for Charcot-Marie-Tooth disease such as a research animal.
• the subject is a human patient.
• the composition is administered orally, locally, systemically, e.g., subcutaneously, perineurally, etc.
• the method comprises targeting a junction between an intron and an exon within a PMP22 pre-mRNA as described in greater detail elsewhere herein.
• a PMP22 exon-skipped mRNA is produced and in certain embodiments can be compared to the amount/decrease in full-length PMP22 mRNA as described in detail elsewhere herein. In certain embodiments, the amount of full-length PMP22 mRNA in the cell is decreased. In certain embodiments, the amount of full-length PMP22 mRNA in the cell is decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 95% in response to the ASO.
• the amount of full-length PMP22 mRNA in the cell is decreased by not more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in response to the ASO.
• the amount of full-length PMP22 mRNA in the cell is decreased by from any of about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% to any of about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in response to the ASO.
• the amount of functional PMP22 protein in the cell is decreased.
• the amount of functional PMP22 protein in the cell is decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 95% in response to the ASO. As noted, in some embodiments it is not contemplated or desired to completely eliminate PMP22 protein. Thus, in some embodiments, the amount of functional PMP22 protein in the cell is decreased by not more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in response to the ASO.
• the amount of functional PMP22 protein in the cell is decreased by from any of about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75% to any of about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in response to the ASO.
• the ASO is administered as a pharmaceutically acceptable salt. In certain embodiments, the ASO is administered in a pharmaceutically acceptable carrier or diluent.
• At least one symptom of the disease is alleviated.
• the rate of progression of at least one symptom of the disease is decreased.
• the method of treatment results in no side-effects or fewer or less severe side-effects in comparison to other CMT treatments.
• the correlation or ratio between the amount of exon-skipped PMP22 mRNA produced and the amount/reduction of full-length PMP22 mRNA can be used to adjust the dosage of the ASO treatment to increase its effectiveness and/or to decrease side-effects.
• composition comprising an antisense oligonucleotide (ASO) comprising or consisting of a complementary region that is complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to a target region of a PMP22 pre-mRNA, wherein the target region of the PMP22 pre-mRNA comprises an intron/ exon junction of one of the coding exons (i.e., PMP22 Exon 2, Exon 3, Exon 4, or Exon 5).
• ASO antisense oligonucleotide
• the ASO need not be complementary to the full length of the target region but is complementary to a sufficient portion of the target region to hybridize, i.e., the defined target region can be longer than the ASO complementary region and longer than the entire ASO.
• the ASO comprises or consists of a complementary region of at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 contiguous nucleotides that are complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to the PMP22 pre-mRNA target region.
• the ASO comprises or consists of a complementary region between any of about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, or 45 and any of about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 contiguous nucleotides that are complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to the PMP22 pre-mRNA target region.
• the ASO comprises or consists of a complementary region of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides that are complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to the PMP22 pre-mRNA target region.
• the target region of the PMP22 pre-mRNA comprises the 3 ’-end of an exon.
• the target region of the PMP22 pre-mRNA comprises the 5’-end of an exon.
• the target region of the PMP22 pre-mRNA comprises an intron/ exon junction comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the intron and a portion of the exon.
• the target region of the PMP22 pre-mRNA comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the intron and a portion of the exon.
• the target region of the PMP22 pre-mRNA comprises an intron/ exon junction comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the exon and a portion of the intron.
• the target region of the PMP22 pre-mRNA comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the exon and a portion of the intron.
• the exon portion of the intron/exon junction comprises PMP22 Exon 3.
• the exon portion of the intron/exon junction comprises PMP22 Exon 4.
• the ASO is a modified and/or synthetic oligonucleotide as known to those of ordinary skill in the art and/or as disclosed herein.
• the ASO is a phosphorodiamidate morpholino oligomer (PMO).
• the PMP22 pre-mRNA target region comprises or consists of SEQ ID NO: 2 (Exon 3, 5’-end), SEQ ID NO: 35 (Exon 3, 3’-end), SEQ ID NO: 76 (Exon 4, 5’-end), SEQ ID NO: 11 1 (Exon 4, 3’-end), SEQ ID NO: 163 (Exon 2, 5’-end), and/or SEQ ID NO: 198 (Exon 2, 3 ’-end).
• a tailored method of treatment can be used for patients that depends on the age of the patient and severity of the symptoms (e.g., Example 9).
• small doses of the compositions of this disclosure can have a positive and complete impact on axon improvement.
• the 5 mg/kg group showed only a small amount of improvement at the 30 day mark compared to the 50 mg/kg groups (2 and 4) that were tested at day 30.
• the dowel walking time was greatly improved.
• certain embodiments provide for prescribing a dosing regimen on a subject- to-subject basis based on their symptom progression or age.
• the treatment amount of a composition of this disclosure administered to a subject is based both on their disease progression and overall body weight.
• the disease progression is based on age of the patient, or the physical performance of the patient, or both.
• subjects with greater symptom severity and/or older subjects are given a loading dose of the treatment described for a period of time which is then later changed to a lower dose.
• the loading period can be the same amount of drug given to the subject simply more often, or it can be a larger dose of the composition initially administered.
• the dosing amount and/or dosing frequency can be lowered.
• the length of the complementary region of the ASO can vary and be either longer or shorter and still be effective as long as the ASO is able to specifically hybridize to the target region of PMP22 pre-mRNA. For example, some of the ASOs tested were of shorter length.
• the sequence of PMO SHC-001 (SEQ ID NO: 72) is ctaagagagatcGTTACCTAGCAC, which is 22 base pairs long and a subset of the 25-mer (SEQ ID NO: 15) shown in Figure 5.
• Schwann cells are the primary producer of PMP22 and are therefore the primary target in any therapeutic envisioned for CMT1A.
• Schwann cells are heavily myelinated, and their biochemistry is greatly affected by their surrounding tissues, making cellular studies (while feasible) quite challenging as compared to some other target cells.
• disclosed herein is an optimized method to do this and developed a robust assay for inducing PMP22 exon-skipping in the pre-mRNA.
• C3 mice (3 copies of human PMP22).
• C3 mice were bred and genotyped according to the published protocol from Jackson Labs. At 5 weeks, animals were injected subcutaneously with a single 6.7 mg of SHC-012 (SEQ ID NO: 75) or a scramble control PMO. Untreated C3 mice were included as a control. Animals were sacrificed after 24 hours after the injection and tissues were collected (sciatic nerve, kidney, liver, brain, spinal cord, etc.) and analyzed for PMP22 mRNA reduction/ expression and the exon skipped version of the PMP22 mRNA. RT-PCR was used to evaluate PMP22 expression (3% agarose gel).
• C3 mice (3x human PMP22 gene) were selected as the animal model since they have been shown to exhibit CMT like behaviors, have been well studied, and most importantly for this project, express the human genomic PMP22 gene (Huxley C, Passage E, Manson A, Putzu G, Figarella-Branger D, Pellissier JF, Fontes M. Construction of a mouse model of Charcot-Marie- Tooth disease type 1A by pronuclear injection of human YAC DNA. Human molecular genetics. 1996;5(5):563-9. Epub 1996/201701. doi: 10. 1093/hmg/5.5.563. PubMed PMID: 8733121). This is essential since the ASOs rely on sequence identity within the exon and intronic regions.
• Figure 17 shows images of the sciatic nerve for WT, untreated and treated animals;
• Figure 17 (bottom) shows images from the peroneal portion of the nerve.
• Figure 18 is a TEM image at higher magnification of portions of a Peroneal nerve from each animal.
• FIG 19 (top), electrophysiology plots from the sciatic gastric section of sedated mice are shown.
• the table at the bottom of Figure 19 shows the average values of MUNE and CMAP from 3 measured animals per group.
• the “dowel time” which is a measure of general fitness, balance and mobility (described below) is included and illustrates that improved dowel performance tracks with improved electrophysiological measures.
• a key specification of the envisioned CMT1A treatment is that treatment will only be required 2-4 times per year for each patient. This is potentially possible because PMO ASOs are resistant to nucleases and are very stable once cellular uptake has occurred.
• animals from the 1 mg/kg treatment and 10 mg/kg treatment groups above were monitored for 5 months after their last treatment ( Figure 20).
• the half-life of PMO molecules once they reach cells such as Schwann cells) is ⁇ 3-4 months for most tissue; thus, our hypothesis was that treatment benefit would persist for extended periods of time.
• the C3 animals at 5 months post-treatment have continued to perform well on the dowel test, with little or no reduction in activity after 4 months from last treatment (extended testing is still ongoing).
• Figure 20 shows the results for both treatment groups (with the scramble animals’ group and wild-type animals shown for comparison at 4 months of age).
• Each data set of the histogram is an average of 4 days dowel travers time at the end of each month.
• the last data set plotted is for animals that are 7 months old.
• a number of ASOs were designed for exon skipping of Exon 4 in the PMP22 RNA (Figure 11A and Figure 11B). 25 base pair long ASOs are shown but shorter subsets are also envisioned and disclosed. Both 5’-end and 3 ’-end region ASOs were designed. Referring to Figure 3, these ASOs were designed at the 3’- and 5’-end of Exon 4.
• PMOs SHC-043 SEQ ID NO: 156
• SHC-044 SEQ ID NO: 159
• SHC-045 SEQ ID NO: 162
• a number of ASOs were designed for exon skipping of Exon 2 in the PMP22 pre- mRNA (Figure 16). As disclosed elsewhere herein, shorter length and mis-matched ASOs are also contemplated. These ASOs were designed at the 3’- and 5’-end of Exon 2.
• CMT1A it is desirable to monitor the activity at a molecular level of a given drug over time without having to sacrifice the animal (during research studies) or perform a major biopsy on a human patient, in order to access the tissue of interest and determine if the pre-mRNA, mRNA, or protein for PMP22 has been effected.
• CMT1A is a disease where the peripheral nerve tissue is the desired tissue to affect the level of PMP22 and within this tissue the myelinated sheets around the tissue and the PMP22 protein is largely produced within the Schwann cells within this region.
• a major tissue biopsy must be performed and/or the animal must be sacrificed in order to assess these tissues.
• This method gives an internal control to the measurement which simplifies that assay measurement and increases accuracy. As that value goes down over time (indicative of new cells being created that do not contain the drug, loss of activity of the drug, etc.), an additional dose of the drug chosen can be given at that time to re-equilibrated exon skipping to the desired value.
• the method of this disclosure of measuring the exon-skipped mRNA in the blood can also be used to adjust the initial dose on a patient-by-patient basis or during dose escalation studies during clinical trials.
• CMT1A and other diseases
• the variance in actual measurements could be disastrous for certain patients.
• Incorrect results could lead to underdosing of patients (thus missing the drug activity window) or significant overdosing of patients (leading to potentially dangerous side effects).
• Due to a number of factors two different patients may require different amounts of drug to achieve desired levels of PMP22 reduction. For instance, if it is desirable to block 50% of PMP22 protein production, a physically smaller patient may require less drug than a heavier patient.
• Patients with different metabolisms or lifestyles may also require different amounts of drug per treatment to achiever optimal molecular changes.
• a key consideration for the treatment of CMT is to determine how the treatment may be effective for older patients that have progressed in the disease for a longer period of time and may have more axon damage than other patients.
• C3 animals were allowed to progress until 3 months of age and monitored for their walking ability across a suspended dowel as described above. The amount of time to cross the dowel was monitored prior to their treatment. The animals where then treated with 17 mg/kg of SCH-012 weekly for 6 weeks then allowed to progress (untreated) for an additional 6 weeks and again monitored for walking capability.
• Figure 21 shows the results of this experiment (with 3 month old wild-type animals also plotted for comparison). As can be seen, the group of animals were performing very poorly at 3 months of age (average dowel travers time of 18.1 seconds with a standard deviation of 7.1 seconds). After treatment and a recovery period, the same animals significantly improved to and average walking time of 7.1 seconds with a standard deviation of 2.4 seconds). For each bar in the graph, animals were measured for 4 days (2 days apart) and the averages of those time points are shown.
• C3 animals were allowed to progress untreated until 12 months of age. At 12 months of age, wild-type animals will also begin to show some degradation in walking and balance ability, so wild-type animals were also studied. At 12 months of age, C3 animals were treated (Table 1). For treatment groups 1, 2, 3, 5, 6, and 7, five animals were studied in each group and for groups 4 and 5, four animals were studied.
• SUBSTITUTE SHEET ( RULE 26 ) data set is the front paw grip strength (in grams for a 25 Newton pull setting) for the entire group tested on one day.
• Table 3 shows the data from Table 2 where the average percent improvement (compared to the first day for each group separately) is shown.
• the animals in the group showed on average a 26% improvement in walking performance from before treatment and after treatment (since a reduction in time is an improvement for walking).
• both time point percent improvements are compared to the original pre-testing data.
• the animals in groups 4 and 5 that had two performance testing days (30 days and 60 days).
• both time point percent improvements are compared to the original pre-testing data.
• For example, for Group 4 grip strength the animals showed a 90% improvement after 1 treatment (Time 1-2) and a 256% improvement at the 60 day mark compared to the pre-treatment value. Positive increases in grip strength demonstrate improvement.
• a composition comprising an antisense oligonucleotide (ASO), wherein the ASO comprises or consists of a complementary region that is complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to a target region of a PMP22 pre-mRNA; wherein binding in a cell of the complementary region of the ASO to the target region of the PMP22 pre-mRNA induces exon skipping during RNA transcription, thereby reducing full- length PMP22 mRNA production and producing exon-skipped PMP22 mRNA.
• ASO antisense oligonucleotide
• the ASO comprises or consists of a complementary region of at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 contiguous nucleotides that are complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to the PMP22 pre- mRNA target region; or wherein the PMP22 pre-mRNA target region comprises two separate segments of the PMP22 pre-mRNA, and optionally, wherein the ASO comprises or consists of a complementary region of at least about 6, 8, 9, 10, 11, or 12 nucleotides that are complementary, or complementary except for one, two, or three mismatched nucleotides, to a first segment of contiguous sequence of the PMP22 pre-mRNA target region and wherein the ASO comprises or consists of a complementary region of at least about 6, 8, 9, 10, 11, or 12 nucleotides that are complementary, or complementary except for one, two, or three mismatched nucleotides, to a second segment of con
• composition of paragraph 1 or 2 wherein the ASO comprises or consists of a complementary region between any of about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, or 45 and any of about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleotides that are complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to the PMP22 pre-mRNA target region; optionally, wherein the ASO comprises or consists of a complementary region of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides that are complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to the PMP22 pre- mRNA target region.
• the ASO is a modified and/or synthetic oligonucleotide; optionally, wherein the ASO is a phosphorodiamidate morpholino oligomer (PMO).
• PMP22 pre-mRNA spans an intron/exon junction of one of the coding exons; optionally, wherein the exon portion of the intron/exon junction comprises PMP22 Exon 3; and/or optionally, wherein the exon portion of the intron/exon junction comprises PMP22 Exon 4.
• PMP22 pre-mRNA comprises the 5 ’-end of an exon.
• PMP22 pre-mRNA comprises the 3 ’-end of an exon.
• PMP22 pre-mRNA spans an intron/exon junction comprising or consisting of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the intron and a portion of the exon; optionally, where the target region of the PMP22 pre-mRNA consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 nucleotides of the intron and a portion of the exon.
• PMP22 pre-mRNA spans an intron/exon junction comprising or consisting of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the exon and a portion of the intron; optionally, where the target region of the PMP22 pre-mRNA consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 nucleotides of the exon and a portion of the intron.
• composition of any one of paragraphs 1 to 11, wherein the PMP22 pre-mRNA target region comprises or consists of SEQ ID NO: 2 (Exon 3, 5’-end), SEQ ID NO: 35 (Exon 3, 3’-end), SEQ ID NO: 76 (Exon 4, 5’-end), SEQ ID NO: 111 (Exon 4, 3’-end), SEQ ID NO: 163 (Exon 2, 5’-end), and/or SEQ ID NO: 198 (Exon 2, 3 ’-end), or a portion or subset/fragment thereof.
• SEQ ID NO: 2 Exon 3, 5’-end
• SEQ ID NO: 35 Exon 3, 3’-end
• SEQ ID NO: 76 Exon 4, 5’-end
• SEQ ID NO: 111 Exon 4, 3’-end
• SEQ ID NO: 163 Exon 2, 5’-end
• SEQ ID NO: 198 Exon 2, 3 ’-end
• composition of paragraph 1 wherein the ASO comprises or consists of the nucleic acid sequence of:
• SEQ ID NO 146 (SHC-029 21-mer), SEQ ID NO 147 (SHC-028 20-mer), SEQ ID NO 148 (SHC-027 20-mer), SEQ ID NO 149 (SHC-031 21-mer),
• SEQ ID NO 150 (SHC-030 20-mer), SEQ ID NO 151 (SHC-032 20-mer),
• SEQ ID NO: 71 (SHC-006 25-mer), SEQ ID NO: 72 (SHC-001 24-mer), SEQ ID NO: 73 (SHC-005 25-mer), SEQ ID NO: 74 (SHC-010 21-mer),
• SEQ ID NO 146 (SHC-029 21-mer), SEQ ID NO 147 (SHC-028 20-mer), SEQ ID NO 148 (SHC-027 20-mer), SEQ ID NO 149 (SHC-031 21-mer),
• SEQ ID NO 150 (SHC-030 20-mer), SEQ ID NO 151 (SHC-032 20-mer),
• SEQ ID NO: 2308 except for having one, two, or three nucleotide substitutions.
• a method of decreasing the amount of full-length PMP22 mRNA expression in a cell comprising administering to the cell a composition comprising an antisense oligonucleotide (ASO) of any one of paragraphs 1 to 14; optionally, wherein an PMP22 exon-skipped mRNA is produced; and/or optionally, wherein the amount of functional PMP22 protein produced in the cell is decreased.
• ASO antisense oligonucleotide
• a method of producing an exon-skipped PMP22 pre-mRNA comprising administering to a cell a composition comprising an antisense oligonucleotide (ASO) of any one of paragraphs 1 to 14.
• ASO antisense oligonucleotide
• a method of treating Charcot-Marie-Tooth disease comprising administering to a subject in need thereof a composition comprising an antisense oligonucleotide (ASO) of any one of paragraphs 1 to 14.
• ASO antisense oligonucleotide
• a composition comprising an antisense oligonucleotide (ASO), wherein the ASO comprises or consists of a complementary region that is complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to a target region of a PMP22 pre-mRNA; wherein the target region of the PMP22 pre-mRNA comprises an intron/exon junction of one of the coding exons.
• ASO antisense oligonucleotide
• the ASO comprises or consists of a complementary region of at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 contiguous nucleotides that are complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to the PMP22 pre- mRNA target region; or wherein the PMP22 pre-mRNA target region comprises two separate segments of the PMP22 pre-mRNA, and optionally, wherein the ASO comprises or consists of a complementary region of at least about 6, 8, 9, 10, 11, or 12 nucleotides that are complementary, or complementary except for one, two, or three mismatched nucleotides, to a first segment of contiguous sequence of the PMP22 pre-mRNA target region and wherein the ASO comprises or consists of a complementary region of at least about 6, 8, 9, 10, 11, or 12 nucleotides that are complementary, or complementary except for one, two, or three mismatched nucleotides, to a second segment of con
• composition of paragraph 34 or 35 wherein the ASO comprises or consists of a complementary region between any of about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, or 45 and any of about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 contiguous nucleotides that are complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to the PMP22 pre-mRNA target region; optionally, wherein the ASO comprises or consists of a complementary region of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides that are complementary, or complementary except for one, two, three, four, or five mismatched nucleotides, to the PMP22 pre-mRNA target region.
• PMP22 pre-mRNA comprises the 3 ’-end of an exon.
• PMP22 pre-mRNA comprises the 5 ’-end of an exon.
• PMP22 pre-mRNA comprises an intron/exon junction comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the intron and a portion of the exon; optionally, where the target region of the PMP22 pre-mRNA comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the intron and a portion of the exon.
• PMP22 pre-mRNA comprises an intron/exon junction comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the exon and a portion of the intron; optionally, where the target region of the PMP22 pre-mRNA comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides of the exon and a portion of the intron.
• ASO is a modified and/or synthetic oligonucleotide; optionally, wherein the ASO is a phosphorodiamidate morpholino oligomer (PMO).
• composition of any one of paragraphs 34 to 42, wherein the PMP22 pre-mRNA target region comprises or consists of SEQ ID NO: 2 (Exon 3, 5’-end), SEQ ID NO: 35 (Exon 3, 3’-end), SEQ ID NO: 76 (Exon 4, 5’-end), SEQ ID NO: 111 (Exon 4, 3’-end), SEQ ID NO: 163 (Exon 2, 5’-end), and/or SEQ ID NO: 198 (Exon 2, 3 ’-end).
• peripheral myelin protein PMP-22 is a candidate for Charcot-Marie-Tooth disease type 1A. Nature genetics. 1992; 1(3): 159-65. Epub 1992/06/01. doi: 10.1038/ng0692-159. PubMed PMID: 1303228.
• peripheral myelin protein gene PMP-22 is contained within the Charcot- Marie-Tooth disease type 1A duplication. Nature genetics. 1992; 1 (3) : 171 -5. Epub 1992/06/01. doi: 10.1038/ng0692-171. PubMed PMID: 1303230.
• Landis SC Amara SG, Asadullah K, Austin CP, Blumenstein R, Bradley EW, Crystal RG, Darnell RB, Ferrante RJ, Fillit H, Finkelstein R, Fisher M, Gendelman HE, Golub RM, Goudreau JL, Gross RA, Gubitz AK, Hesterlee SE, Howells DW, Huguenard J, Kelner K, Koroshetz W, Krainc D, Lazic SE, Levine MS, Macleod MR, McCall JM, Moxley RT, 3rd, Narasimhan K, Noble LJ, Perrin S, Porter JD, Steward O, Unger E, Utz U, Silberberg SD.

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