Patent Application
20250114464
The present invention relates generally to pre-mRNA splice modulating peptide-conjugated antisense therapeutics for treatment of diseases and in particular, peptide-conjugated antisense therapeutics for the treatment of Duchenne Muscular Dystrophy (DMD) and Spinal Muscular Atrophy (SMA).
Antisense therapeutics that modulate pre-messenger RNA splicing by either inducing exon splicing or inhibiting exon splicing function by binding to the pre-messenger RNA and have been approved for the treatment of various diseases including Duchenne's Muscular Dystrophy and Spinal Muscular Atrophy.
Duchenne Muscular Dystrophy (DMD) is a fatal, X-linked recessive disorder caused by mutations in the DMD gene that lead to absence of dystrophin in muscle. Dystrophin stabilizes the sarcolemma by bridging cytoskeletal actin to the extracellular matrix, via forming a membrane-associated glycoprotein complex [1, 2]. Dystrophin loss results in progressive body-wide muscle degeneration, loss of ambulation before the teens, and cardiorespiratory malfunction during the twenties that typically leads to death [3]. DMD is present in 19.8 per 100,000 male births and is the most common inherited neuromuscular disorder worldwide [4].
Most patients (˜70%) have large out-of-frame deletions in DMD [5]. Exon skipping based therapeutics are based on the observation that, at least ˜90% of the time, in-frame mutations in DMD give rise to milder phenotypes, as found in Becker Muscular Dystrophy (BMD) [5, 6]. By excluding out-of-frame exons from the DMD transcript using antisense oligonucleotides, exon skipping converts out-of-frame into in-frame mutations, producing truncated but partially functional dystrophin. Four exon skipping therapies have been approved by the U.S. FDA: eteplirsen/Exondys 51 (Sarepta) [7], golodirsen/Vyondys 53 (Sarepta) [8], viltolarsen/Viltepso (NS Pharma) [9], and casimersen/Amondys45 (Sarepta) [10]. All are phosphorodiamidate morpholino oligomer (PMO) antisense oligonucleotides.
The applicability of single-exon skipping is, however, limited due to its mutation-specific nature of DMD. The therapies above could each treat at most only 8-13% of all patients [11]. Multi-exon skipping overcomes this issue, particularly skipping exons 45-55. Exons 45-55 is a mutation hotspot in the DMD gene, harboring 66% of large (≥1 exon) deletions and 15% of large duplications in patients (5). Exons 45-55 skipping could theoretically treat more than 40% of all DMD patients [12].
Deletion of exons 45-55 is also commonly associated with asymptomatic to mild phenotypes. In the Leiden DMD database, 90% of patients with the exons 45-55 deletion have BMD; in other databases, including UMD-TREAT-NMD, and clinical studies, the deletion is associated with BMD or asymptomatic individuals in all examined cases [12-15].
Exons 45-55 skipping cocktails that restored dystrophin synthesis in the muscles of dystrophic mice have been developed [16]. A PMO cocktail that skipped human DMD exons 45-55 in immortalized patient myotubes and humanized DMD mice have been developed [13, 17]. Average skipping efficacies of 27-61% and 15-22% were observed, respectively, and treatment produced up to 14% dystrophin of normal levels in vitro [13, 17]. However, this cocktail used one PMO per exon (except exon 48, which required two) to skip exons 45-55. Efficacy is reduced and off-target effects increased as all PMOs have to be present in the same nuclei to induce skipping of exons 45-55, PMO based exon skipping therapies efficacy is impacted by their rapid clearance from the bloodstream, poor uptake into muscle, and inability to escape from endosomes [19, 20]. Eteplirsen for instance, only restored 0.93% dystrophin of normal levels in patients after 180 weeks of treatment with a 30 or 50 mg/kg/week dose [7].
Accordingly, there exists a need for improved exon-skipping therapies for the treatment of Duchene Muscular Dystrophy.
Spinal muscular atrophy (SMA) is a devastating neurodegenerative disorder affecting motor neurons in the anterior horn of the spinal cord [45]. SMA can result in progressive muscular weakness, respiratory distress, or even paralysis, and is one of the leading genetic causes of infant mortality [46]. SMA is characterized by mutations in the survival of the telomeric motor neuron 1 (SMN1) gene leading to its homozygous deletion [47]. The complete loss of SMN protein is embryonically lethal [48,49]. Humans possess a paralog of SMN1 called SMN2, enabling patients to be born [47,50]. However, SMN2 cannot fully compensate for SMN1 loss because of a C-to-T transition in exon 7, leading to its exclusion.
While the full-length SMN2 (FL-SMN2) transcripts can produce a stable functional protein (˜10%), the transcripts without exon 7 (A7 SMN2) are unstable, and the protein produced is rapidly degraded (˜90%) [51]. Exclusion of SMN2 exon 7 is due to ISS-N1 (intronic splicing silencer N1) that binds to a repressor protein hnRNP A1 (heterogeneous ribonucleoprotein A1) [52-54].
All SMA patients lack a functional SMN1 gene and therefore, are dependent on SMN2 gene to produce SMN protein necessary for survival. The number of copies of SMN2 is therefore a potent genetic modifier of SMA, with the copy number correlating inversely with the severity of SMA [55,56]. The inability of SMN2 to compensate for SMN1 loss is responsible for the preferential degeneration of motor neurons [51,57]. Although the pathological hallmark of SMA is motor neuron degeneration, recent studies highlight defects in peripheral tissues [58-60]. Additionally, in vivo mouse studies have reported bradycardia and dilated cardiomyopathy, which improved with restoration of SMN levels [61-63]. While the reason remains unclear, motor neurons are particularly susceptible to the loss of SMN [64].
Currently, three SMA-modifying treatments have been approved by the U.S. Food and Drug Administration (FDA): nusinersen (brand name Spinraza), onasemnogene abeparvovec (brand name Zolgensma), and risdiplam (brand name Evrysdi). However, these three drugs have several associated concerns. Onasemnogene abeparvovec is gene-therapy drug and is only approved for patients less than 2 years of age [65]. Risdiplam is an orally deliverable small molecule drug. However, there are safety concerns due to its associated off-target effects on pre-mRNA splicing [66].
Nusinersen is a splice-switching antisense oligonucleotide (AO) with a 2′-O-methoxyethyl (MOE) phosphorothioate chemistry. It modulates SMN2 splicing by targeting ISS-N1 and promotes the inclusion of exon 7. Since nusinersen does not cross the blood-brain barrier (BBB), it requires repeated intrathecal administration that ensures a robust restoration of SMN protein mainly to the central nervous system (CNS) tissues [67]. Despite providing a safe profile, nearly one-third of the treated patients suffer from scoliosis, persistent lumbar-fluid leakage, thrombocytopenia, and other coagulation defects [68,69].
To overcome issues associated with intrathecal injections, several AO chemistries and modifications have been studied such as phosphorodiamidate morpholino oligomers (PMOs), locked nucleic acid (LNAs), and constrained ethyl (c-Et) [70]. The uncharged backbone, low protein binding affinity, and nuclease stability make PMO chemistry an ideal candidate as several studies have shown that the administration of multiple high doses can be achieved with minimal toxicity 70,71]. However, the main pitfall associated with PMO is low efficacy which is tied to its rapid clearance from the bloodstream, poor uptake in tissues such as the skeletal muscle, and endosome-trapping [72,73]. To improve delivery of PMOs to the nuclei in target tissues and reduce the administered dose, numerous cell-penetrating peptides (CPPs) conjugated PMOs are being studied. Some of these peptides facilitate the transport of PMOs across the BBB and thus ensure the possibility of non-invasive administration for the treatment of neuronal disorders [74]. Hammond et al. showed that the systemic administration of Pip6a, a peptide from the family of PMO internalizing peptide (Pip) into adult mice, increased the FL-SMN2 expression in the peripheral and CNS tissues [71]. However, the localization and distribution of AOs in the CNS remain unknown. Additionally, Pip peptides are known to be highly toxic [74].
Accordingly, there exists a need for improved splice modulating therapies for the treatment of Spinal Muscular Atrophy.
The present invention provides pre-mRNA splice modulating peptide-conjugated antisense therapeutics for treatment of diseases.
In one aspect of the invention, there is provided a conjugate comprising an antisense oligonucleotide capable of modulating exon splicing of pre-mRNA attached to a cell penetrating peptide (CPP) comprising the amino acid sequence:
uppercase: L-amino acids, lowercase: D-amino acids.
In another aspect of the invention, there is provided a conjugate comprising an antisense oligonucleotide capable of inducing exon skipping in human dystrophin covalently attached to a cell penetrating peptide (CPP) comprising the amino acid sequence:
uppercase: L-amino acids, lowercase: D-amino acids.
In some embodiments, the conjugate capable of inducing exon skipping in human dystrophin has an antisense oligonucleotide that binds to a target in exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54 and/or exon 55 of human dystrophin pre-mRNA.
In some embodiments, the conjugate capable of inducing exon skipping in human dystrophin has an antisense oligonucleotide that comprises or consists of any one of the following oligonucleotides:
In accordance with another aspect of the invention, there is provided a composition comprising a first conjugate comprising a first antisense oligonucleotide capable of inducing exon skipping in human dystrophin covalently attached to a cell penetrating peptide (CPP) comprising the amino acid sequence:
uppercase: L-amino acids, lowercase: D-amino acids and a second conjugate comprising a second antisense oligonucleotide capable of inducing exon skipping in human dystrophin covalently attached to the cell penetrating peptide (CPP).
In some embodiments, the composition, further comprises at least one other conjugate comprising another antisense oligonucleotide capable of inducing exon skipping in human dystrophin covalently attached to the cell penetrating peptide (CPP).
In some embodiments, the composition capable of inducing multi-exon skipping in human dystrophin comprises a peptide-conjugated antisense oligonucleotide targeting exon 45, a peptide-conjugated antisense oligonucleotide targeting exon 46, a peptide-conjugated antisense oligonucleotide targeting exon 47, a peptide-conjugated antisense oligonucleotide targeting exon 48, a peptide-conjugated antisense oligonucleotide targeting exon 49, a peptide-conjugated antisense oligonucleotide targeting exon 50, a peptide-conjugated antisense oligonucleotide targeting exon 51, a peptide-conjugated antisense oligonucleotide targeting exon 52, a peptide-conjugated antisense oligonucleotide targeting exon 53, a peptide-conjugated antisense oligonucleotide targeting exon 54 and a peptide-conjugated antisense oligonucleotide targeting exon 55.
In some embodiments, the composition capable of inducing multi-exon skipping in human dystrophin comprises a peptide-conjugated antisense oligonucleotide targeting exon 45, a peptide-conjugated antisense oligonucleotide targeting exon 47, a peptide-conjugated antisense oligonucleotide targeting exon 49, a peptide-conjugated antisense oligonucleotide targeting exon 51, a peptide-conjugated antisense oligonucleotide targeting exon 53 and a peptide-conjugated antisense oligonucleotide targeting exon 55.
In some embodiments, the composition capable of inducing multi-exon skipping in human dystrophin comprises a peptide-conjugated antisense oligonucleotide targeting exon 45, a peptide-conjugated antisense oligonucleotide targeting exon 47 and a peptide-conjugated antisense oligonucleotide targeting exon 53.
In accordance with an aspect of the invention, there is provided a method of treating a subject having DMD, comprising administering a therapeutically effective amount one or more peptide conjugates of the invention capable of inducing exon skipping in human dystrophin.
In accordance with another aspect of the invention, there is provided a conjugate comprising an antisense oligonucleotide capable of inducing exon inclusion in human SMN2 gene covalently attached to a cell penetrating peptide (CPP) comprising the amino acid sequence:
uppercase: L-amino acids, lowercase: D-amino acids.
In some embodiments, the conjugate comprising an antisense oligonucleotide capable of inducing exon inclusion in human SMN2 gene binds to intronic splicing silencer N1 of SMN2 pre-nIRNA.
In some embodiments, the conjugate comprising an antisense oligonucleotide capable of inducing exon inclusion in human SMN2 gene includes the antisense oligonucleotide comprises or consists of the sequence 5′-TCACTTTCATAATGCTGG-3′ and wherein the thymines are optionally replaced with uracil, optionally wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer.
In accordance with another aspect of the invention, there is provided a method of treating spinal muscular atrophy (SMA) in a subject, comprising administering a therapeutically effective amount of the conjugate comprising an antisense oligonucleotide capable of inducing exon inclusion in human SMN2 gene of the invention.
In accordance with another aspect of the invention, there is provided a pharmaceutical composition comprising one or more peptide conjugates of the invention, and a pharmaceutically acceptable excipient.
Embodiments of the present invention will now be described, by way of example only, with reference to the attached Figures.
The invention provides pre-mRNA splicing modulating therapeutics optionally suitable for systematic delivery. The therapeutics comprise antisense oligonucleotides conjugated to a cell penetrating peptide derived from the protein transduction domain (PTD) of the human Hph-1 transcription factor.
The therapeutics are designed to either promote exon skipping or exon inclusion. The therapeutics includes peptide-conjugated antisense therapeutics for the treatment of Duchenne Muscular Dystrophy (DMD) and Spinal Muscular Atrophy (SMA).
DMD therapeutics promote exon skipping. Optionally such therapeutics comprise more than one conjugate and are configured to promote multi-exon skipping. Optionally, the therapeutic comprises two, three, four, five, six, seven or more different conjugates wherein each conjugate comprises antisense oligonucleotides conjugated to a cell penetrating peptide derived from the protein transduction domain (PTD) of the human Hph-1 transcription factor. In some embodiments, each different conjugate targets a different exon. In some embodiments, different conjugates target the same exon.
SMA therapeutics promote exon inclusion.
Conjugates of the invention comprise an antisense oligonucleotide capable of modulating exon splicing of pre-mRNA attached to a cell penetrating peptide (CPP). The cell penetrating peptide of the invention, also referred to as DG9, facilitates the delivery of the conjugated antisense oligonucleotide and has improved activity over the R6G cell penetrating peptide. In some embodiments, conjugates of the invention are for intravenous delivery.
The cell penetrating peptide of the invention is derived from the protein transduction domain of human HPH-1 transcription factor and comprises the amino acid sequence:
Optionally, in some embodiments, one or two amino are deleted or substituted. Optionally, amino acids are substituted with non-naturally occurring amino acids.
In some embodiments, to increase serum stability one or more L-amino acids are replaced with D-amino acids. In some embodiments, two, three, four, five or six L-amino acids are replaced with D-amino acids. Optionally, two, three, four, five or six L-arginines are replaced with D-arginines. In some embodiments, the cell penetrating peptide (CPP) has the amino acid sequence:
wherein uppercase represent L-amino acids and lowercase represent D-amino acids.
In some embodiments, the CPP is conjugated to the 5′ end of the oligonucleotide. In other embodiments, the OPP is conjugated at the 3′ of the oligonucleotide.
As used herein interchangeably, “antisense oligonucleotides”, “antisense therapeutics”, “AOs”, “oligos”, “oligomers” refer to a sequence of subunits, each having a base carried on a backbone subunit, and where the backbone groups are linked by intersubunit linkages that allow the bases in the compound to hybridize to a target sequence in a nucleic acid by Watson-Crick base pairing, to form a nucleic acid:oligonucleotide heteroduplex within the target sequence. The oligonucleotides may have exact sequence complementarity to the target sequence or sufficient complementarity to selectively bind the target sequence.
The antisense oligonucleotide are between about 20 to about 50 nucleotides in length, including at least 10, 12, 15, 17, or 20 consecutive nucleotides of complementary to the target sequence.
In one embodiment, the antisense oligonucleotide is 20 to 30 or 24 to 28 nucleotides in length.
In some embodiments, the antisense oligonucleotide is an antisense oligonucleotide analogue.
The term ‘oligonucleotide analogue’ and ‘nucleotide analogue’ refers to any modified synthetic analogues of oligonucleotides or nucleotides respectively that are known in the art.
Examples of oligonucleotide analogues include, but are not limited to, peptide nucleic acids (PNAs), morpholino oligonucleotides, phosphorothioate oligonucleotides, phosphorodithioate oligonucleotides, alkylphosphonate oligonucleotides, acylphosphonate oligonucleotides, phosphoramidite oligonucleotides, tricyclo-DNA, and 2′methoxyethyl oligonucleogides.
In some embodiments, the antisense oligonucleotide comprises morpholino subunits.
In some embodiments, the antisense oligonucleotide is a morpholino antisense oligonucleotide.
In some embodiments, the antisense oligonucleotide comprises morpholino subunits linked together by phosphorus-containing linkages. In a specific example, the antisense oligonucleotide is a phosphoramidate or phosphorodiamidate morpholino antisense oligonucleotide.
The terms ‘morpholino antisense oligonucleotide’ or ‘PMO’ (phosphoramidate or phosphorodiamidate morpholino oligonucleotide) refer to an antisense oligonucleotide analog composed of morpholino subunit structures, where (i) the structures are linked together by phosphorus-containing linkages, for example one to three atoms long, for example two atoms long, and for example uncharged or cationic, joining the morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, and (ii) each morpholino ring bears a purine or pyrimidine base-pairing moiety effective to bind, by base specific hydrogen bonding, to a base in a polynucleotide.
In some embodiments, the antisense oligonucleotide comprises phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.
Optionally, variations can be made to the intersubunit linkage as long as the variations do not interfere with binding or activity. For example, the oxygen attached to phosphorus may be substituted with sulfur (thiophosphorodiamidate). The 5′ oxygen may be substituted with amino or lower alkyl substituted amino. The pendant nitrogen attached to the phosphorus may be unsubstituted, monosubstituted, or disubstituted with (optionally substituted) lower alkyl.
Antisense oligonucleotide may be made through the well-known technique of solid phase synthesis.
In some embodiments, the conjugate further comprises a label. The label provides a direct or indirect detectable signal. Appropriate labels are known in the art and include fluorescent or radioactive labels.
Methods of conjugating the CPP to the oligonucleotide are known in the art or the conjugate can be prepared commercially.
There are provided exon skipping peptide-conjugated antisense therapeutics for the treatment of Duchenne Muscular Dystrophy. Treatment of DMD with peptide-conjugated antisense therapeutics restores partially functional dystrophin to the DMD patient.
A ‘functional’ dystrophin protein refers to a dystrophin protein having sufficient biological activity to reduce the progressive degradation of muscle tissue that is otherwise characteristic of muscular dystrophy when compared to the defective form of dystrophin protein that is present in subjects with a muscular disorder such as DMD.
In some embodiments, a functional dystrophin protein may have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the in vitro or in vivo biological activity of wild-type dystrophin. The activity of dystrophin in muscle cultures in vitro can be measured according to myotube size, myofibril organization, contractile activity, and spontaneous clustering of acetylcholine receptors.
Accordingly, in some embodiments, the peptide-conjugated antisense therapeutics of the invention are for use in the treatment of DMD by inducing exon skipping in the human dystrophin pre-mRNA to restore functional dystrophin protein expression.
In one embodiment, the therapeutic comprises one peptide-conjugated antisense oligonucleotide. In alternative embodiments, the therapeutic comprises more than one peptide conjugated antisense oligonucleotide, wherein each peptide conjugated antisense oligonucleotide targets a different sequence in the dystrophin pre-mRNA.
As described herein, there is provided peptide-conjugated antisense therapeutics that may be therapeutically effective for exon skipping therapy skipping in the human dystrophin (DMD) gene, of one or more of exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54 and/or exon 55.
In some embodiments, the peptide-conjugate antisense therapeutic comprises a plurality of peptide-conjugated antisense oligonucleotides and promotes skipping of exons 45-55.
In some embodiments, the peptide-conjugate antisense therapeutic comprises a peptide-conjugated antisense oligonucleotide targeting exon 45, a peptide-conjugated antisense oligonucleotide targeting exon 46, a peptide-conjugated antisense oligonucleotide targeting exon 47, a peptide-conjugated antisense oligonucleotide targeting exon 48, a peptide-conjugated antisense oligonucleotide targeting exon 49, a peptide-conjugated antisense oligonucleotide targeting exon 50, a peptide-conjugated antisense oligonucleotide targeting exon 51, a peptide-conjugated antisense oligonucleotide targeting exon 52, a peptide-conjugated antisense oligonucleotide targeting exon 53, a peptide-conjugated antisense oligonucleotide targeting exon 54 and a peptide-conjugated antisense oligonucleotide targeting exon 55.
In other embodiments, the peptide-conjugate antisense therapeutic comprises a peptide-conjugated antisense oligonucleotide targeting exon 45, a peptide-conjugated antisense oligonucleotide targeting exon 47, a peptide-conjugated antisense oligonucleotide targeting exon 49, a peptide-conjugated antisense oligonucleotide targeting exon 51, a peptide-conjugated antisense oligonucleotide targeting exon 53 and a peptide-conjugated antisense oligonucleotide targeting exon 55.
In still other embodiments, the peptide-conjugate antisense therapeutic comprises a peptide-conjugated antisense oligonucleotide targeting exon 45, a peptide-conjugated antisense oligonucleotide targeting exon 47 and a peptide-conjugated antisense oligonucleotide targeting exon 53.
In some embodiments, the antisense oligonucleotide of the peptide-conjugated antisense therapeutics binds to a target sequence in exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54 or exon 55 of the human dystrophin (DMD) gene. Optionally, the target sequence is proximal to the exon acceptor splice site in the pre-mRNA. In some embodiments, the target sequence is at the exon acceptor splice site, within 10 bases of the exon splice site, within 15 bases of the exon splice site, within 20 bases of the exon splice site, within 30 bases of the exon splice site, within 40 bases of the axon splice site, within 50 bases of the exon splice site, within 75 bases of the axon splice site or within 100 bases of the exon splice site
In some embodiments, the anti-sense oligonucleotide of the conjugate comprises or consists of at least 10, 12, 14, 16, 18 or 20 consecutive nucleotides of any one of the following sequences wherein, thymine bases in the sequences are optionally uracil:
Optionally, the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).
In some embodiments, the conjugates have increased delivery to, uptake or retention by cardiac cells.
There are provided exon inclusion peptide-conjugated antisense therapeutics for the treatment of Spinal Muscular Atrophy. Treatment of SMA with the peptide-conjugated antisense therapeutics results in the production of stable SMN2 to compensate for the loss of SMN1.
The peptide-conjugated antisense therapeutics comprises DG-9 conjugated to an antisense oligonucleotide that binds to intronic splicing silencer N1 of SMN2 pre-mRNA. Optionally, the antisense oligonucleotide comprises or consists of the sequence 5′-TCACTTTCATAATGCTGG-3′ and wherein the thymines are optionally replaced with uracil.
In some embodiments, the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer.
In one aspect there is described a pharmaceutical composition comprising the antisense oligonucleotide(s) of the invention or a conjugate thereof, further comprising one or more pharmaceutically acceptable excipients.
In some embodiments, the pharmaceutical composition is prepared in a manner known in the art, with pharmaceutically inert inorganic and/or organic excipients being used.
The term ‘pharmaceutically acceptable’ refers to molecules and compositions that are physiologically tolerable and do not typically produce an allergic or similarly untoward reaction when administered to a patient.
In some embodiments, the pharmaceutical composition may be formulated as a pill, tablet, coated tablet, hard gelatin capsule, soft gelatin capsule and/or suppository, solution and/or syrup, injection solution, microcapsule, implant and/or rod, and the like.
In some embodiments, the pharmaceutical composition may be formulated as an injection solution.
In some embodiments, pharmaceutically acceptable excipients for preparing pills, tablets, coated tablets and hard gelatin capsules may be selected from any of: Lactose, corn starch and/or derivatives thereof, talc, stearic acid and/or its salts, etc.
In some embodiments, pharmaceutically acceptable excipients for preparing soft gelatin capsules and/or suppositories may be selected from fats, waxes, semisolid and liquid polyols, natural and/or hardened oils, etc.
In some embodiments, pharmaceutically acceptable excipients for preparing solutions and/or syrups may be selected from water, sucrose, invert sugar, glucose, polyols, etc.
In some embodiments, pharmaceutically acceptable excipients for preparing injection solutions may be selected from water, saline, alcohols, glycerol, polyols, vegetable oils, etc.
In some embodiments, pharmaceutically acceptable excipients for preparing microcapsules, implants and/or rods may be selected from mixed polymers such as glycolic acid and lactic acid or the like.
In some embodiments, the pharmaceutical composition may comprise a liposome formulation.
In some embodiments, optionally, the pharmaceutical composition may comprise two or more different antisense oligonucleotides or conjugates thereof.
In some embodiments, optionally, the antisense oligonucleotide and/or conjugate may be present in the pharmaceutical composition as a physiologically tolerated salt.
Suitably, physiologically tolerated salts retain the desired biological activity of the antisense oligonucleotide and/or conjugate thereof and do not impart undesired toxicological effects. For antisense oligonucleotides, suitable examples of pharmaceutically acceptable salts include (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.
In some embodiments, in addition to the active ingredients and excipients, a pharmaceutical composition may also comprise additives, such as fillers, extenders, disintegrants, binders, lubricants, wetting agents, stabilizing agents, emulsifiers, preservatives, sweeteners, dyes, flavorings or aromatizing agents, thickeners, diluents or buffering substances, and, in addition, solvents and/or solubilizing agents and/or agents for achieving a slow release effect, and also salts for altering the osmotic pressure, coating agents and/or antioxidants. Suitable additives may include Tris-HCl, acetate, phosphate, Tween 80, Polysorbate 80, ascorbic acid, sodium metabisulfite, Thimersol, benzyl alcohol, lactose, mannitol, or the like.
In some embodiments, the peptide conjugated antisense oligonucleotide(s) and/or pharmaceutical composition is for topical, enteral or parenteral administration.
In some embodiments, the peptide conjugated antisense oligonucleotide(s) and/or pharmaceutical composition may be for administration orally, transdermally, intravenously, intrathecally, intramuscularly, subcutaneously, nasally, transmucosally or the like.
In some embodiments, the antisense oligonucleotide(s) and/or pharmaceutical composition is for intramuscular administration.
In some embodiments, the antisense oligonucleotide(s) and/or pharmaceutical composition is for intramuscular administration by injection.
An ‘effective amount’ or ‘therapeutically effective amount’ refers to an amount of the antisense oligonucleotide, administered to a subject, either as a single dose or as part of a series of doses, which is effective to produce a desired physiological response or therapeutic effect in the subject.
In some embodiments, the antisense oligonucleotide(s) or conjugate thereof are administered daily, once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months.
In some embodiments, the antisense oligonucleotide or conjugate thereof may be administered as two, three, four, five, six or more sub-doses separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway.
Duchenne muscular dystrophy (DMD) is primarily caused by out-of-frame deletions in the dystrophin gene. Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) converts out-of-frame to in-frame mutations, producing partially functional dystrophin. Skipping of exons 45-55 could treat 40-47% of all patients and is associated with improved clinical outcomes. The development of peptide-conjugated PMOs for exons 45-55 skipping is reported. Experiments with immortalized patient myotubes revealed that exons 45-55 could be skipped by targeting as few as 5 exons. Conjugating DG9, a cell-penetrating peptide to PMOs improved single-exon 51 skipping, dystrophin restoration, and muscle function in hDMDdel52;mdx mice. Local administration of a minimized exons 45-55-skipping DG9-PMO cocktail restored dystrophin production.
The number of PMOs necessary to skip DMD exons 45-55 was minimized using two strategies.
First, a PMO (or PMOs) that targeted an exon in the region were removed from the previously developed exons 45-55 skipping cocktail [13) (Table 1). The full cocktail, with PMOs targeting all exons within exons 45-55, is referred to as the “all” cocktail (
For the second strategy, minimized cocktails based on the endogenous splicing of DMD exons 45-55 were prepared. A model proposes that exons 45-49, 50-52, and 53-55 are spliced first, after which these groups are spliced together to complete the exons 45-55 region (25). Derivative cocktails from the “all” cocktail with PMOs targeting the terminal exons of these three groups were designed. Transfection into healthy KM155 myotubes revealed three derivative cocktails to skip exons 45-55 significantly higher than the mock (p<0.05) (
The “all”, “base” “base-51”, “block”, and “3-PMO” exons 45-55 skipping cocktails were subsequently tested in the following immortalized patient-derived muscle cell lines: KMI55 (healthy), KM571 (Δex52), 6594 (Δex48-50), and 6311 (Δex45-52) (
The dystrophin restoration capabilities of the “block” and “3-PMO” cocktails in KM571 myotubes were evaluated, since these cocktails induced the highest levels of exons 45-55 skipping in this line. Western blot using antibodies against the rod (DYS1, exons 26-30) and C-terminal domains (ab15277) of dystrophin detected dystrophin upon treatment with the “all” and “block” cocktails, but not with the “3-PMO” cocktail (
Single Intravenous Treatment with DG9-PMO Induces Higher Dystrophin Production than Unconjugated PMO in the Skeletal Muscles and the Heart
To evaluate DG9 as a conjugate for the minimized exons 45-55 skipping cocktail, its efficacy in vivo as applied to single-exon skipping was evaluated. DG9 was conjugated to Ex51_Ac0, an exon 51-skipping PMO from the “all” cocktail that was up to 7 times more effective than eteplirsen in restoring dystrophin production in vitro [13, 26). hDMDdel52;mdx mice at 3 months were given a single retro-orbital injection of either saline, 50 mg/kg unconjugated PMO, or 64 mg/kg DG9-PMO (equimolar to the PMO dose) and assessed a week later (
Widespread dystrophin-positive fibers were observed in the tibialis anterior, diaphragm, and heart of DG9-PMO-treated mice, and very few to none in saline- or PMO-treated mice (
Repeated Intravenous Treatment with DG9-PMO Improves Dystrophin Production, Muscle Function, and Fiber Size in Dystrophic Mice
A repeated-dose treatment study was performed to provide more insight into the efficacy of DG9-PMO exon skipping therapy with regard to ameliorating dystrophic symptoms. hDMDdel52;mdx mice at 2 months were retro-orbitally injected with saline or 30 mg/kg of DG9-PMO, once weekly for 3 weeks. Functional assessments were done at baseline and at 2 weeks following the last injection, after which tissues were collected (
Body weights between groups did not significantly differ over the course of treatment (
Most impressively, repeated DG9-PMO treatment significantly improved forelimb (p<0.05) (
Histological analysis still did not show any significant reductions in CNFs in the tibialis anterior and diaphragm (
Local Treatment with the DG9-Conjugated Minimized Exons 45-55 Skipping Cocktail Induces Successful Skipping and Dystrophin Production
Finally, DG9 was conjugated to each PMO of the minimized “block” exons 45-55 skipping cocktail for in vivo testing. The “block” cocktail was chosen as it induced high levels of exons 45-55 skipping and dystrophin protein restoration in KM571 myotubes (
A minimized exons 45-55 skipping cocktail that induces dystrophin restoration in immortalized patient cells and dystrophic hDMDdel52;mdx mice was developed. For exon 52-deleted DMD transcripts, the number of PMOs used for exons 45-55 skipping was reduced from 11 in the “all” to 5 in the “block” cocktail (
The efficacy of conjugating DG9 to PMOs in single- and multi-exon skipping applications was demonstrated. Treatment with DG9-PMO resulted in higher single-axon skipping and dystrophin restoration levels in vivo compared to unconjugated PMO (
The advantage of DG9 is in its potentially better toxicity profile compared to other peptides. Peptide-conjugated PMOs have induced dose-dependent toxic effects in pre-clinical studies, including lethargy, weight loss, and kidney damage (20). This is linked to the membrane-disruptive properties of cell-penetrating peptides, which are largely influenced by their amino acid compositions (20, 29). Certain L-arginine residues in DG9 were converted to D-arginina, as this improves the viability of peptide-conjugated PMO-treated cells in vitro (30). DG9 also does not contain any 6-aminohexanoic acid residues, which have been associated with increased toxicity (30). Compared to DG9, previous peptides mostly used L-arginine and contained multiple 6-aminohaxanoic acid residues, but were of similar length and overall arginina content (20). Even though the above modifications decrease the antisense activity of peptide-conjugated PMOs, it comes with the benefit of increased safety. A balance must be struck between efficacy and safety for peptide-conjugated PMOs, as too frequent or too high doses increase toxicity (20, 29). No evidence of toxicity caused by DG9-PMO on the liver and kidney (
On the topic of efficacy, even though high exon 51 skipping was induced by our DG9-PMO, the dystrophin restoration observed were generally lower than that achieved by other peptide-conjugated PMOs (20, 29). This may be due to a difference in strategy, as the majority of peptide-conjugated PMO studies performed exon 23 skipping (20). Similar discrepancies between exon skipping and dystrophin restoration levels in vivo were observed by Aoki et al. (2010), who skipped exon 51 with PMOs (31), and Aupy et al. (2020), who also skipped exon 51 but with adeno-associated virus-delivered U7 small nuclear RNAs (32). Exon 51-skipped transcripts or proteins may be less stable than their axon 23-skipped counterparts, leading to the reduced dystrophin levels observed. An in vitro study using various internally truncated dystrophin proteins revealed that exon 51-skipped dystrophins were mostly as stable as full-length dystrophin (33), favoring a hypothesis of decreased transcript stability. While previous work has shown that DMD transcript stability has an impact on dystrophin protein production (34, 35), it remains to be determined if this explains the differences in the case of exon 23- and exon 51-skipped transcripts. Another factor would be the animal model used for testing. As humanized dystrophic mice such as hDMDdel52;mdx have only recently been developed, it would be important to characterize the stabilities of the human DMD transcript and protein in these models, to determine if this would have an impact on the efficacy of tested exon skipping therapies.
Despite its relatively reduced activity, functional improvement in mice given repeated doses of DG9-PMO (
In terms of multi-exon skipping, it was promising that the DG9-conjugated version of the minimized “block” cocktail successfully skipped axons 45-55 at 9.5% efficiency and restored dystrophin production in hDMDdal52;mdx mice at nearly 0.8% of wild-type levels, on average (
Cell culture. All immortalized human muscle cells were kindly provided by the MRC Center for Neuromuscular Diseases Biobank (NHS Research Ethics Committee reference 06/Q0406/33, HTA license number 12198), through Dr. Francesco Muntoni. The following immortalized patient-derived myoblast cell lines were used: KM155 (healthy), KM571 (DMD Δex52), 6594 (DMD Δex48-50), and 6311 (DMD Δex45-52). Information on these cell lines are summarized in Table 2. Myoblasts were grown in DMEM/F12 medium (with HEPES; Gibco) containing 20% fetal bovine serum (Sigma), 1 vial of skeletal muscle growth supplement mix (Promocell), 50 U/mL penicillin, and 50 μg/mL streptomycin. Myoblasts were then seeded into collagen type 1-coated 12-well plates, at a density of 0.53×105 cells/cm2. Once 90% confluent, myoblasts were differentiated into myotubes by replacing the growth medium with differentiation medium (DMEM/F12 containing 2% horse serum [GE Healthcare], 1×ITS solution [Sigma], 50 U/mL penicillin, and 50 μg/mL streptomycin). All cells were incubated at 37° C., 5% CO2.
PMO transfection. The PMOs used are summarized in Table 1 and were derived from cocktail set no. 3 in our previous publication [1]. Prior to transfection, PMOs (Gene Tools) were heated at 65° C. for 15 min to remove aggregates. PMOs were then transfected into muscle fibers at 3 days post-differentiation using 6 μM Endoporter reagent (Gene Tools) in differentiation medium. Each PMO in a cocktail was transfected at a final 5 μM concentration for all experiments. Cells were incubated in PMOs for 2 days, after which they were harvested for RNA and protein (
Animal treatments. Only male hDMDdel52;mdx mice (C57BL/6J background) (2, 3) heterozygous for the hDMDdel52 transgene were used. For single-exon skipping studies, DG9 (sequence N-YArVRRrGPRGYArVRRrGPRr-C: uppercase: L-amino acids, lowercase: D-amino acids) was conjugated to the 3′ end of Ex51_Ac0 (Table 1), a human DMD exon 51-skipping PMO we previously developed [1, 4]. We performed two experiments: single-dose and repeated-dose treatment. For single-dose treatment, 3-month-old hDMDdel52;mdx mice were retro-orbitally injected with either phosphate-buffered saline (PBS), PMO (50 mg/kg, unconjugated Ex51_Ac0), or DG9-PMO (64 mg/kg, equimolar to 50 mg/kg PMO) and then euthanized a week later for tissue collection. For repeated-dose treatment, 2-month-old hDMDdel52;mdx mice were given three retro-orbital injections of either PBS or DG9-PMO (30 mg/kg) once a week for 3 weeks. Body weights were recorded throughout the course of treatment, and mice were subjected to muscle function tests before the first injection and 2 weeks after the last injection as described in Functional testing. Mice were euthanized after the post-function tests for tissue collection. Upon dissection, tissues were mounted in tragacanth gum on corks and snap-frozen in liquid nitrogen-cooled isopentane.
For multi-exon skipping, DG9 was conjugated to each PMO of the minimized “block” exons 45-55 skipping cocktail (
Functional testing. Forelimb and total limb grip tests were conducted by blinded personnel according to TREAT-NMD SOP DMD_M.2.2.001 using the Chatillon DFE II grip strength meter (Columbus Instruments). The average of the three most consistent readings was used per mouse, and results were normalized to body weight. The rotarod test was performed using the AccuRotor 4-channel rotarod (Omnitech Electronics, Inc.). Mice were first placed on a rod rotating at a steady speed of 5 rpm. Once all mice were in place, rotation was accelerated from 5 to 45 rpm over a span of 300 s [5]. Fall times were automatically recorded by the software. Three trials were conducted, spaced 15 min apart, and the average or peak fall time from these trials was used for analysis. The run-to-exhaustion testwas performed using the Exer 3/6 animal treadmill (Columbus Instruments), according to TREAT-NMD SOP DMD_M.2.1.003. Mice were run on the treadmill with the following program: 5 m/min for 5 min, and then speed is increased by 1 m/min every minute until exhaustion. Exhaustion is considered as the point when the mouse does not get back on the treadmill within 10 s following repeated, gentle nudges. The maximum test duration was set at 15 min. All tests were performed at baseline (prior to receiving treatment) and at 2 weeks after receiving final treatment. Age-matched wild-type male C57BL/6J mice were used as controls.
RT-PCR and exon skipping evaluation. Total RNA was extracted from cells and 20-μm tissue sections using Trizol (Invitrogen), following the manufacturer's instructions.
For exons 45-55 skipping analysis, the SuperScript™ III One-Step RT-PCR system with Platinum™ Taq (Invitrogen) was used. Briefly, 200 ng of total RNA was used as template in a 25-μL solution containing 1× reaction mix, 0.2 μM each of forward and reverse primers for DMD or GAPDH/Gapdh (Table 3), and 1 μL of SuperScript III RT/Platinum Taq. The reaction was run under the following conditions: 1) 50° C., 5 min, 2) 94° C., 2 min, 3) 35 cycles of 94° C., 15 s; 60° C., 30 s; 680° C., 33-118 s, 4) 680° C., 5 min, 5) 4° C., hold.
For exon 51 skipping analysis, cDNA was synthesized from 750-1000 ng of total RNA using SuperScrip™ IV Reverse Transcriptase (Invitrogen) with 2.5 μM random hexamers (Invitrogen) in a 20-μL reaction following manufacturer's instructions. From this, 8 μL of cDNA was used for PCR with 1× GoTaq® Green Master Mix (Promega) and 0.3 μM each of forward and reverse primers for DMD or Gapdh (Table 3) in a 25-μL reaction. The reaction was run as follows: 1) 95° C., 2 min, 2) 40 cycles of 95° C., 30 s; 60° C., 30 s; 72° C., 35 s, 3) 72° C., 5 min, and 4) 4° C., hold. All PCR products (exons 45-55 or exon 51 skipping) were run in 1.5% agarose gels in 1× tris-borate-EDTA buffer, and band intensities were quantified by Image J (NIH). The % of successful exon skipping was calculated using the following formula: (intensity of desired skipped band/total intensity of unskipped, intermediate, and desired skipped bands)×100.
Western blot. Total protein was extracted from cells using RIPA buffer (Sigma) supplemented with cOmplete, Mini, EDTA-free protease inhibitor cocktail (Roche), according to our previously published protocol (6). On the other hand, total protein was extracted from 20-μm tissue sections using a high-SDS lysis buffer containing 10% SDS, 70 mM Tris-HCl (pH 6.7), 5 mM EDTA (pH 8.0), 5% β-mercaptoethanol, and cOmplete protease inhibitor cocktail in water, also according to a previous protocol (7). Proteins were quantified using the Pierce™ BCA kit (Thermo Scientific) or the Pierce™ Coomassie (Bradford) kit (Thermo Fisher), respectively.
In preparation for Western blot, proteins were mixed with NuPAGE™ LDS Sample Buffer (Invitrogen; 1× final concentration) and NuPAGE™ Sample Reducing Agent (Invitrogen; 1× final concentration), then heated at 70° C. for 10 min. SDS-PAGE was performed using pre-cast NuPAGE™ 3-8% Tris-Acetate Midi gels (Invitrogen), run at 150 V for 75 min. Proteins were then transferred onto a PVDF membrane (Millipore) using a semi-dry blotting system at 20 V for 70 min. Membranes were blocked overnight in 2% ECL Prime Blocking Agent (GE Healthcare) while shaking at 4° C.; the post-transfer gel was stained with PageBlue Protein Staining solution (Thermo Scientific) for 1 hr at room temperature to detect myosin heavy chain bands. After blocking, membranes were cut and incubated in one of the following primary antibodies for 1 hr at room temperature: 1:200 NCL-DYS1 (Leica), 1:2,500 anti-dystrophin C-terminal (Abcam, ab15277), 1:100 MANEX45A (Developmental Studies Hybridoma Bank; DSHB), (8) 1:100 MANEX4850E (MDA Monoclonal Antibody Resource, Wolfson Centre for Inherited Neuromuscular Disease) (8), or 1:4,000 anti-desmin (Abcam, ab8592) in blocking agent. The membranes were then washed thrice for 10 min each with PBS containing 0.05% Tween 20 (PBST), before incubating in either anti-mouse IgG2a, anti-mouse IgG1, or anti-rabbit IgG (H+L) horseradish peroxidase-conjugated secondary antibodies (Invitrogen) as appropriate, all 1:10,000 in PBST. Membranes were then similarly washed in PBST, and detected with ECL Select Detection Reagent (GE Healthcare). DYS1 band intensities were quantified using Image Lab™ software, v.6.0.1 (Bio-Rad), and dystrophin levels were expressed relative to the intensity of the least concentrated wild-type sample in a series.
Dystrophin immunofluorescence. Frozen muscle and heart samples were sectioned at 7-μm thickness and placed on poly-L-lysine-coated slides. After thawing at room temperature for 30 min, sections were blocked for 2 hr in PBS with 10% goat serum and 0.1% Triton X-100 at room temperature. Sections were then incubated with 1:50 NCL-DYS1 in the blocking agent overnight at 4° C. The following day, sections were washed thrice with PBS for 5 min each and subsequently incubated with Alexa 488-conjugated goat anti-mouse IgG2a secondary antibody (Life Technologies) for 30 min at room temperature. Sections were washed again with PBS, and mounted with Vectashield HardSet Antifade Mounting Medium with DAPI (Vector Laboratories). Samples were visualized for dystrophin and DAPI using a Zeiss LSM 710 confocal microscope at 200× magnification, by personnel blinded to the treatment condition.
Histology. Frozen muscles were sectioned at 7-μm and placed on poly-L-lysine-coated slides. After thawing at room temperature for 30 min, slides were stained with Mayer's hematoxylin (Electron Microscopy Sciences) for 15 min, washed with running tap water for 15 min, and then stained with eosin Y (Electron Microscopy Sciences) for 10 min. Sections were then dehydrated with an ethanol series (70%-90%-99%), cleared with a xylene substitute, and mounted with Permount™ (Fisher Scientific). Blinded personnel visualized the samples under brightfield using the Optika B-290 TB microscope at 200× magnification, taking three randomly chosen fields of view per sample. CNF percentage was calculated by (#CNFs/total #fibers)×100, with fibers counted manually using Image J. The average CNF percentage from all fields of view was taken per sample. Minimal Feret's diameters were quantified by blinded personnel in two steps. First, images were semi-automatically measured using an in-house developed Image J macro based on Open-CSAM (9). As Open-CSAM was initially developed for immunofluorescence images, we had to extensively modify it for compatibility with hematoxylin and eosin-stained images, which required the use of the Colour Deconvolution 2 plugin [10, 11]. Second, images that passed semi-automatic measurement were manually curated to correct fiber boundaries. Individual fiber measurements across samples were considered for analysis. For both CNF and minimal Feret's diameter quantification, fibers that touched the edges of an image were not considered.
Statistical Analysis. All statistical tests were performed using Prism v.9.0.1 (GraphPad Software). Unpaired two-tailed t-test, or one-way ANOVA with post-hoc Tukey's or Dunnett's multiple comparisons test were conducted as appropriate. P-values<0.05 were considered statistically significant.
Spinal muscular atrophy (SMA) is the most common genetic cause of infant mortality resulting from mutations in the survival motor neuron 1 (SMN1) gene. Humans have an SMN1 paralog SMN2. However, SMN2 cannot rescue the SMA phenotype because 90% of exon 7 in SMN2 mRNA is spliced out due to a single C-to-T nucleotide transition. The currently approved antisense oligonucleotide (AO) nusinersen (brand name Spinraza) targets SMN2 and induces SMN expression by promoting exon 7 inclusion. However, delivering AOs to both the central nervous system (CNS) and bodywide tissues is difficult. Although current approaches have been restricted to using highly invasive intrathecal injections and treats mainly motor neurons, recent studies indicate that SMA is a multi-organ disorder, calling for a body-wide treatment approach. To bypass intrathecal injections a novel cell-penetrating peptide called DG9 that increases the uptake of AO significantly following a single subcutaneous administration. It was demonstrated that DG9-conjugated AO (DG9-PMO) extends the lifespan of SMA mice and improves breathing dysfunction, which is the most common cause of mortality in SMA. DG9-PMO treatment leads to a significant increase in full-length SMN2expression and SMN protein levels in both peripheral and central nervous system tissues. Additionally, the treatment also improved the pathology of skeletal muscles and neuromuscular junctions. Lastly, we show that DG9-PMO penetrates a fully-developed blood brain barrier, and can distribute in the CNS following a systemic injection.
To examine whether DG9 peptide can increase the efficacy of PMO, DGS peptide was conjugated to an 18-mer PMO with a sequence identical to nusinersen. The DG9-conjugated PMO and unconjugated-PMO and 2′-O-methoxyethyl-RNA (MOE) were injected into SMA model mice. We used the Taiwanese SMA mouse model (Smn−/− SMN2Tg/−) that exhibits a severe phenotype and has a median survival of 8 days (d) [77,78].
Mice were subcutanteously injected with the AOs or saline (NT) administered at postnatal day 0 (PD0) at two different doses-40 mg/kg or 80 mg/kg. The survival was recorded until a humane endpoint was reached. For the 40 mg/kg doses, the median survival was 12 d (unconjugated-PMO), 15.5 d (MOE), and increased to 58 d for DG9-PMO treatment (
Next, the FL-SMN2 expression was evaluated relative to Δ7 SMN2 transcripts using real-time quantitative PCR (RT-qPCR) at PD7. In both doses, DG9-PMO treatment led to a 4-to-30-fold higher FL-SMN2 expression than NT control in both peripheral and CNS tissues (
To evaluate the effects of systemic DG9-PMO treatment on muscle strength and the function of motor neurons, several functional tests were used to compare motor function in the treated and NT mice at various time-points. The hindlimb suspension (HLS) assay and the righting-reflex test were performed during the early weeks of life. After PD6, NT mice had a decreasing HLS score as they could not extend their hindlimbs when suspended by the tail (
Majority of the SMA patients suffer from compromised breathing functions and rely on an external source of breathing support. To evaluate the effects of DG9-PMO treatment on breathing function, we performed whole-body plethysmography recordings at PD7 under normoxic (21% 02) and hypoxic (11% 02) conditions (
When switched to a hypoxic environment, the Hets, DG9-PMO, and MOE-treated mice exhibited an increase in the respiratory parameters—fR VE, and VT relative to normoxia (
Atrophic musculature is a classical characteristic feature of SMA with diminished skeletal muscle fiber size. To determine the treatment effects on muscle pathology, we assessed the physiology and architecture of the myofibers of two affected muscle groups− the quadriceps and intercostal muscles, as well as the sparingly affected diaphragm at PD7 and PD30 [79,80]. We quantified the cross-sectional area (CSA), the minimal feret's diameter and centrally nucleated fibers for at least 500 myofibers per muscle for each treatment using hematoxylin & eosin (H & E) staining at PD7 and PD30 (
At PD7, the feret's diameter and CSA of the myofibers were significantly larger in all three muscle groups following DG9-PMO treatment when compared to the NT control (
SMA mice typically begin to exhibit neuropathological deficits around PD4-PD5, with denervated and collapsed neuromuscular junctions (NMJs) as the disease progresses [37]. The NMJ architecture of the quadriceps and the intercostal muscles at PD30 was assessed to understand the phenotypic rescue following our injections at PD0 (
To determine the effects of DG9 peptide in increasing the PMO uptake, we evaluated the biodistribution of unconjugated-PMO and DG9-PMO in the peripheral and CNS tissues [82]. We used a well-established hybridization-based ELISA assay designed specifically to detect the PMO to compare the concentration of DG9-PMO and unconjugated-PMO in the tissues of treated PD7 mice [82]. Conjugation of DG9 to the PMO led to a significantly higher uptake in most tissues except for the liver, with an average AO detection of 12202 μM (quadriceps muscle, (n=5)), 2×108 pM (liver (n=5)), 8359 μM (brain (n=5)), 13907 μM (spinal cord (n=4)), 8886 μM (heart (n=4)), and 2×107 μM (kidney (n=6)) (
PMO uptake is mediated by the caveolin-dependent pathway in myotubes [83]. Some CPP-PMOs often get trapped in endosomes, limiting the efficiency of splicing correction [84]. To demonstrate intracellular localization of DG9-PMO, we attached a fluorescent tag to the DG9-PMO. SMA mice were subcutaneously injected at PD0 (40 mg/kg) and performed immunohistochemistry (IHC) to examine the localization of DG9-PMOs in frozen tissue sections collected at PD7. Some DG9-PMOs localized in nuclei of cells in the hearts and quadriceps muscle, and to a lesser extent in the CNS tissues (
To investigate whether the DG9-PMO can cross the fully developed BBB, a milder SMA model (F0) (Smn−/− SMN2+/+) was injected at PD5 with DG9-PMO or fluorescently tagged DG9-PMO. These mice have a normal life span and usually only exhibit necrosis of the tail and ears around 8-12 weeks of age. Tissues were harvested at PD7. First, we observed localization of fluorescently tagged DG9-PMO inside the nuclei of the cells in both peripheral and CNS tissues (
DG9-PMO Treatment Rescues the SMA Phenotype without Apparent Toxicity
Peptides can typically pose as antigens, leading to immune reactions. Therefore, we examined the susceptibility of DG9-PMO to cause immune activation at an early neonatal stage, by looking at CD68 positive cells (CD68+), indicative of circulating and tissue macrophages in the quadriceps muscle sections at PD7 (
The unconjugated-PMO and MOE-treated muscle had a significantly higher number of CD68+ macrophages when compared to the Hets, while NT and DG9-PMO mice had no significant difference (
To further elucidate the possibility of long-term toxicity, serum from the Hets and treated mice at PD 30-35 was collected. A toxicological evaluation was performed on the levels of alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), creatinine, total bilirubin, total protein, albumin, globulin, and gamma-glutamyl transferase (GOT). All examined indicators were comparable between the groups, suggesting no apparent toxic effects (
Duplications and mutations in the telomeric SMN1 gene led to the formation of its paralog centromeric SMN2 gene with a difference in only a few nucleotides [85]. Since SMN2 can produce a small amount of functional SMN, it is a viable target for therapy in SMA patients. 95% of SMA patients have mutations in the SMN1 gene, making SMN2 gene a candidate for AO therapy to treat almost all the SMA patients.
We deployed a CPP conjugated to an AO (PMO) because some of the studied CPPs can deliver biological cargos into the cell and can also cross the BBB [86,73]. Several peptides also have demonstrated neuroprotective effects, minimized the risk of toxicity and adverse effects, making them suitable for clinical applications [87]. With several concerns still associated with nusinersen, a therapy addressing the invasive administrations, immune reactions, toxicity, and other adverse events is still needed for SMA treatment.
In this study, we evaluated the efficacy of a novel peptide DG9, identified from screening several peptides in the zebrafish model system [75]. To overcome invasive intrathecal injections and to ensure body-wide restoration of the SMN protein, we subcutaneously injected neonatal SMA mice with 40 mg/kg of DG9-PMO, unconjugated-PMO, MOE, or saline at PD0. DG9-PMO treatment significantly prolonged survival, increased bodyweights, FL-SMN2 expression, and SMN levels in both the peripheral and CNS tissues.
DG9-PMO treated neonatal and adult mice showed robust improvement in muscle strength and coordination, with significant improvement in the functional tests. We also observed a dose-dependent increase in survival and in the FL-SMN2 expression when mice were administered a higher dose of 80 mg/kg of the AOs, with DGY-PMO treated mice maintaining an improvement in motor function even at PD60.
The DG9 peptide led to sustained delivery of PMO to the skeletal and respiratory muscles that, in turn, reduced the number of myofibers with central nuclei and increased the myofiber size. At PD30, the unconjugated-PMO mice had significantly smaller myofibers in both the intercostal and quadriceps muscles, hinting at a possibility of compromised breathing, and atrophic myofibers being the cause of early mortality. Though born normally, SMA mice begin to demonstrate decreased respiratory rates and an increase in apnea frequencies by PD7, typically due to weakening of the inspiratory and expiratory muscles [88]. Our plethysmography recordings at PD7 under normoxic and hypoxic conditions demonstrate the weak, slow, and irregular breathing in NT SMA mice. We did not perform statistics to compare treatments alone, but when compared to the Hets, half the PMO mice exhibited a severe respiratory phenotype with irregular breathing and apneas, while most of the MOE mice exhibited a normal breathing function. The variability in the results, especially in unconjugated-PMO mice points towards a deeper understanding of respiratory dysfunction in SMA, leading to their abrupt mortality. Under both normoxic and hypoxic conditions, DG9-PMO mice were similar to the Hets, with significantly better respiratory outcomes than NT PD7 mice. These findings provide evidence for the efficiency of DG9-PMO in rescuing early respiratory dysfunction in SMA mice.
At PD30, there was an improvement in the NMJ architecture with fewer collapsed structures and increased innervation patterns in the DG9-PMO mice. The unconjugated-PMO mice, on the other hand, had a higher number of denervated endplates and collapsed NMJs, suggesting an overt SMA phenotype. NMJ and motor neuron development go hand-in-hand, even though the link is not strongly established. Defects in NMJs are an early pathology in motor neuron diseases and require SMN for proper innervation [81,89,90]. Our treatment improved NMJ innervation, which correlates inversely with muscle atrophy [94], thereby leading to better performance in the functional tests.
Both peripheral and CNS tissues demonstrated a significantly higher uptake of PMO when conjugated to the DG9 peptide compared to unconjugated-PMO alone, which serves as evidence for the peptide to ensure widespread distribution of the AOs even to the brain and spinal cord.
Since our mouse model has a median survival of only 8 d, the precise efficiency of treatment at a late-onset symptomatic phase cannot be completely elucidated. To demonstrate the ability of DG9 to penetrate the fully developed BBB in mice, we used a milder model (Smn−/−; SMN2Tg/Tg) with a normal lifespan. We observed robust uptake of PMO and localization of the DG9 peptide in the CNS tissues following a single subcutaneous injection at PD5. However, from a therapeutic standpoint, it is necessary to corroborate our findings in detail in other mild SMA models with multiple copies of SMN2 that could serve as models to treat patients with type III-IV late-onset SMA or in type I-II at a symptomatic clinical stage.
Toxicity associated with cationic peptide conjugated PMOs is typically seen within 24 hours of administration. No apparent physiological differences in the neonates receiving subcutaneous injections of the AOs. They were active, similar to their healthy counterparts. We also quantified the number of CD68+ cells in quadriceps muscle at PD7, that are indicative of macrophages activated during an immune response in the body upon administration of AOs. SMA mice suffer from immune dysregulation and have an elevated number of immune cells. While DG9-PMO and NT mice had similar numbers of CD68+ cells, we believe that although DG9-PMO treatment might evoke an immune response, the simultaneous improvement in muscle pathology and amelioration of the atrophic muscles reduced the total number of circulating macrophages. No apparent toxicity in the liver or kidney as per the serum and histology analyses at PD30 was observed.
DG9 (sequence N-YArVRRrGPRGYArVRRrGPRr-C; uppercase: L-amino acids, lowercase: D-amino acids) was synthesized and covalently conjugated to the 3′ end of the PMO. The PMO targeting ISS-N1 intron 7 (5′-TCACTTTCATAATGCTGG-3′) was purchased from Gene Tools LLC. The 2′MOE was purchased from Eurogentec North America, USA.
All animal experiments were conducted in University of Alberta and approved by the Animal Care and Use Committee, University of Alberta Research Ethics Office. SMA transgenic mice (JAX stock #005058 FVB.Cg-Tg(SMN2)2HungSmn1tm1Hung/J (Smn−/−; SMN2Tg/Tg), also known as the Taiwanese mice) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Heterozygous mice for Smn1 (Smn1+/−; SMN2−/−) were crossed with mice homozygous for Smn1 Smn−/−; SMN2Tg/Tg to obtain the SMA mice (Smn−/−; SMN2Tg/−) or the heterozygous healthy control (Smn1+/−; SMN2Tg/−). The SMA mice display a severe overt phenotype similar to SMA type I patients. The mice were genotyped using a PCR assay on genomic DNA isolated from tail biopsies using the Phire Tissue Direct PCR kit (ThermoFisher) as per the manufacturer's instructions. The DNA was amplified using the primers described in Table 5 using the condition Mouse Smn: 98° C./5 min→98° C./5 s_58° C./10_72° C./10 s×35 cycles=72° C./1 min.
Injections were carried out using a 30-gauge Hamilton syringe. SMA neonates were subcutaneously injected with 40 or 80 mg/kg of AOs at P0, while NT and heterozygous control mice were injected with saline (n=10-25) per group. Tissues including the quadricep muscle, liver, kidney, spleen, diaphragm, intercostal muscle, heart, brain and spinal cord were collected and snap frozen in dry-ice cooled isopentane, and then subsequently stored at −80° C.
Frozen tissues harvested at P7 were sectioned using a cryostat (Leica C M 1950, Leica) into 20 μm sections. RNA was extracted from these sections using TRizol 10 Reagent (Invitrogen). cDNA was synthesized from 50 ng/μl RNA using superscript IV Reverse Transcriptase (ThermoFisher) and oligo(dT) primers (ThermoFisher) following the manufacturer's instructions. qPCR reaction was performed using the SsoAdvanced Universal SYBR Green Supermix (BioRad), and QuantStudio3 real-time PCR system (Applied Biosystems). The relative gene expression for full-lengthSMN2 over the deletion SMN2 transcripts without exon 7 was normalized to the NT control samples and analyzed using the ΔΔCt method.
Total protein was extracted from frozen tissues harvested at P7 or P30 by using RIPA buffer (Sigma) with cOmplete, Mini, EDTA-free protease inhibitor cocktail (Sigma). The protein concentration was quantified using the Pierce BCA Protein Assay Kit (ThermoFisher). For SDS-PAGE, 5-10 μg of protein per well was run per well in NuPAGE Novex 4-12% Bis-Tris Midi protein gels (Life Technologies) at 150 V for 60 minutes, followed by semi-dry transfer at 20 V for 30 minutes. The polyvinylidene fluoride (PVDF) membrane was blocked overnight with 5% skim milk and 0.05% Tween20 in PBS (PBST). The membrane was incubated with a mouse purified anti-SMN antibody (BD Biosciences) (1:10,000) for one hour at room temperature (RT) under agitation. The membrane was washed three times with PBST (10 minutes each wash) and incubated with HRP conjugated goat anti-mouse (IgG H+L) at RT for 1 hour under agitation. The bands were detected using the Amersham ECL Select Western Blotting Detection Kit (GE Healthcare) and visualized by ChemiDoc Touch Imaging system (BioRad). For β-tubulin, the membrane was incubated with the stripping buffer (15 g glycine, 1 g SDS, 10 nil Tween20 at pH 2.2) for 10 minutes at RT, followed by two washes in PBS (10 minutes each) and two washes in tris-buffered saline and 0.05% Tween20 (TBST) (5 minutes each). Similar to the primary antibody protocol, the membrane was subsequently blocked overnight and incubated with β-tubulin rabbit antibody (Abcam ab6046, 1:5000) at room temperature for 1 hour under agitation the next day. The secondary antibody used was HRP conjugated goat anti-rabbit (IgG H+L) for Tubulin (BioRad), 1:10,000). The bands were visualized as mentioned previously.
ELISA was performed as described previously [83,96]. In brief, protein was extracted from frozen tissue sections (˜20 μm) using RIPA buffer (Sigma) with cOmplete, Mini, EDTA-free protease inhibitor cocktail (Sigma). The probes (Integrated DNA Technologies) were designed complementary to the PMO sequence with phosphorothioated backbones at the 5′ and 3′ ends. The 5′ and 3′ ends were labelled with digoxigenin and biotin, respectively. The tissue lysates (0.02 mg/ml protein concentration) were pre-treated with 2.5 mg/mL trypsin containing 10 mM CaCl2 at 37° C. overnight to digest the DG9 peptide. The probes were added to the samples and allowed to hybridize at 37° C. for 30 minutes. Following the probe-PMO hybridization, the hybridized samples were added to Pierce NeutrAvidin Coated 96-Well Plates, Black (Thermo Fisher Scientific) to allow avidin-biotin interaction between the plate and the probes. The unhybridized probes were digested using micrococcal nuclease enzyme at 0.1 gel unit/pi (New England Biolabs). This was followed by the addition of anti-digoxigenin antibody conjugated with alkaline phosphatase (1:5000, Roche Applied Sciences). Attophos AP Fluorescent Substrate (Promega) was added to the PMO/DG9-PMO probes and fluorescence was detected at 444 nm excitation and 555 nm emission by using a rnonochromator SpectraMax M3 plate reader (Molecular Devices).
7 μm cryosections of the quadricep muscle, diaphragm and intercostal muscle were stained with Meyer's H&E reagents (Electron Microscopy Reagents) [97]. For the centrally nucleated fibers and myofiber size, 500-800 were randomly selected per muscle per treatment. The cross-sectional area and minimal Feter's diameter were quantified using Image J. All H and E analyses were performed in a blinded fashion.
NMJ staining was carried out as previously described [98]. Briefly, the quadricep and the intercostal muscle were fixed in 4% paraformaldehyde (PFA) for 2 hours. The muscles were washed quicky with PBS and blocked with 5% goat serum and 2% Triton-X in PBS for 1 hour at RT. This was followed by an overnight incubation at 4 with the primary mouse monoclonal antibodies anti-neurofilament 2H3 (1:100) and anti-synaptophysin (1:500) (DSHB, Iowa). Subsequently, the muscles were incubated in the dark with Alexafluor 488-goat anti-mouse IgG1 (1:1000) (Life Technologies) and Alexafluor 594-bungarotoxin (1:5000) (ThermoFisher Scientific). At least 300 NMJs were z-stacked and visualized for the analysis of denervation and cross-sectional area using a confocal microscope (Zeiss LSM 710) and Zeiss Zen software. The synaptic area was quantified using image J. All analyses were performed in a blinded fashion.
7 μm cryosections of the quadricep muscle were fixed with 4% PFA. The sections were incubated with rat anti-mouse CD68 antibody (Bio-Rad, MCA1957T). The number of CD68+ cells were counted and averaged from at least 5 sections per sample at random intervals at 20× magnification.
This spontaneous ability of the mice to right themselves up was tested from P2 until the weaning stage P20. The neonates were placed on their backs and the time taken to reposition and place all four paws on the ground was noted. The recording time was a maximum of 60 s. Each neonate underwent three trials, with a resting period of at least 10 minutes between trials. The data is expressed as the mean time to complete the righting reflex test ±SEM.
This test was performed on neonatal mice from P2-P12 as described in Treat NMD protocol SMA_M.2.2.001. Briefly neonatal mice were suspended on their hindlimbs from a tube. They were scored based on the position of the hindlimbs and their latency to fall was recorded with a 30 second cut-off. Each neonatal pup underwent three trials, with a 15-minute break between each trial. The average score was noted down by the observer who was blinded to this test.
This assay was conducted as described in Treat NMD protocol SMA_M.2.1.002. In short, the mouse is placed on the wire mesh of an automated grip strength meter (Columbus Instruments) such that only the front paws are allowed to grip the metal grid. The mouse is steadily pulled with the help of its tail, such that it lets go of the grid completely. P30 and P60 mice were used. Each mouse underwent three trials.
The rotarod test (AccuScan Instruments) was performed on mice between P30-P40, using an acceleration profile of 300s as previously described [99].
Blood was collected from the mice around P30-P40 (n=4-10 per group) during the dissection procedure. The blood was left at RT for 30 minutes and centrifuged at 2000 rcf. The resulting supernatant (serum) was transferred to a new 1.5 ml tube and stored at −20° C. The serum samples were analyzed by Idexxx Bioanalytics. The evaluation was performed on a standard set of toxicity markers: glucose, total bilirubin, blood urea nitrogen (BUN), creatine kinase (CK), creatinine, alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), globulin, albumin, and total protein.
Measurements were performed in whole body, cylindrical transparent plexiglass plethysmographs that had one inflow and two outflow ports for the continuous delivery of fresh room air and removal of expired carbon dioxide [100,101]. The plethysmograph of volumes were 10 ml (inner diameter: 1.9 cm, length: 3.5 cm for P7 mice with body weight less than 2.5 g), 30 ml (inner diameter: 2.6 cm, length: 5.6 cm for P7 mice with body weight more than 2.5 g) and 80 ml (inner diameter: 3.8 cm, length: 7 cm for P30) for measures of respiratory parameters with a flow rate of 15, 45, 120 ml/min, respectively. The gas was mixed with gas mixer (GSM-3, CWE InC, USA), delivered from compressed pure oxygen and pure nitrogen cannisters, being monitored using 0-200 ml/min gas regulators (Porter Instrument Company, USA).
Hypoxic challenge (11% of oxygen for 5 min) was performed with continuous monitoring of plethysmographic recordings without physical handling of the animal by switching inflow gas from normoxia (21% of oxygen, balanced by nitrogen) to hypoxia (11% of oxygen, balanced by nitrogen). It took about 1 min to finish gas exchange, confirmed with gas analyzer (Model: ML206, ADlnstruments). For P7, the plethysmograph was contained within an infant incubator (Isolette, model C-86: Air-Shields/Drager Medical, USA) to maintain the ambient temperature at the approximate nest temperature of 32° C. For P30, the plethysmograph was recorded at room temperature of around 22° C. Pressure changes were detected with a pressure transducer (model DP 103; Validyne, USA), signal conditioner (CD-15; Validyne), recorded with data acquisition software (Axoscope) via analog-digital board (Digidata 1322A). Signals were high pass filtered (0.01 kHz), with a sampling rate at 1 kHz. Respiratory frequency and tidal volume (VT) was measured with blood pressure settings using Labchart 8 (AD Instruments Inc., USA). Threshold levels for bursts were set, and bursts were then automatically detected so that frequency (calculated from cycle duration) and tidal volume (calculated from maximal pressure minus minimal pressure).
It should be noted that our plethysmograph is effective for studying respiratory frequency (fR) and detection of apneas. An apnea is defined as the absence of airflow (pressure changes) for a period equivalent or greater than two complete respiratory cycles. Our whole-body plethysmographic system provided semiquantitative measurements of tidal volume (VT, mL/g) and minute ventilation, from which we report changes relative to the wildtype normoxia (Ren et al., 2009, 2015). The coefficient of variation of frequency, a measure of relative variability, is the ratio of the standard deviation to the mean (average). The smaller the ratio, the more regular the breathing. The experiments were conducted between 10 am and 5 pm. Animals were sent back to the animal facility after experiments.
Respiratory parameters were calculated over an average of 1 min of continuous plethysmography recordings. The respiratory parameters VT and VE were reported relative to the mean of heterozygotes in normoxia (100%). The nature of the hypothesis testing is two-tailed. We first ran the normality test (Shapiro-Wilk) and equal variance test (Brown-Forsythe).
For those data (
All statistical analyses for all data (except respiratory analysis) were performed with GraphPad Prism 9 software. One-way ANOVA with Tukey's test for mUltiple comparisons, or long-rank Mantel-Cox test for survival analysis were used as needed. No statistical power calculation was conducted before the study. The sample size was based on our previous experience with these experimental protocols.
Nat Genet 24 (1):66-70. doi:10.1038/71709
Neurobiol Dis 3 (2):97-110. doi:10.1006/nbdi,1996.0010
The embodiments described herein are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.
All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.
The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.
This application claims priority to U.S. Provisional Application Ser. No. 63/303,577 filed Jan. 27, 2022 which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2023/050096 | 1/26/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63303577 | Jan 2022 | US |
