Methods for treating spinal muscular atrophy

Abstract

The present invention provides a method for treating spinal muscular atrophy (SMA) in a fetal subject in need thereof, the method comprising administering to a carrier of the fetal subject an amount of lisethopram. Administration of the amount of lisethopram to the carrier of the fetal subject causes placental delivery to the fetal subject, thereby causing an increase in SMN protein production.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application S / N 63 / 416,416 filed on October 14, 2022, U.S. Provisional Patent Application S / N 63 / 428,348 filed on November 28, 2022, and U.S. Provisional Patent Application S / N 63 / 434,915 filed on December 22, 2022, the entire contents of which are incorporated herein by reference. Background Art

[0003] Spinal muscular atrophy (SMA) is described in its broadest sense as a collection of inherited and acquired central nervous system (CNS) diseases characterized by progressive motor neuron loss in the spinal cord and brainstem, causing muscle weakness and atrophy. The most common form of SMA is caused by mutations in the survival motor neuron (SMN) gene and exhibits varying degrees of severity, affecting infants to adults. See Crawford and Pardo (1996) Neurobiol. Dis., 3:97.

[0004] The SMN gene has been mapped to a complex region of chromosome 5q by linkage analysis. In humans, this region contains approximately 500,000 base pairs (kb) of inverted repeats, resulting in two nearly identical copies of the SMN gene. The telomeric copies of the gene (SMN1) in the two copies (alleles) of chromosome 5 are inactivated by mutation or deletion, resulting in loss of function of the SMN1 gene and causing SMA. However, all patients retain a centromeric copy of the gene (SMN2), and the number of copies of the SMN2 gene in SMA patients is generally negatively correlated with disease severity; that is, patients with less severe SMA have more copies of SMN2. Approximately 73% of individuals with type 1 SMA have two copies of the SMN2 gene ("SMN2X2"). See Calucho (2018) Neuromuscul.Disord.28:208-215.

[0005] However, SMN2 cannot fully compensate for the loss of SMN1 function due to alternative splicing of exon 7 caused by a translationally silent C to T mutation in exon 7. Therefore, the majority of transcripts produced by SMN2 lack exon 7 (Δ7SMN2) and encode truncated SMN proteins that are functionally impaired and rapidly degraded.

[0006] The SMN protein is thought to play a systemic role in RNA processing and metabolism, with a well-characterized function of mediating the assembly of a specific class of RNA-protein complexes, known as snRNPs. Specifically, the SMN protein plays a role in RNA processing and metabolism in motor neurons. Reduced levels of functional SMN protein affect this RNA processing and metabolism, leading to clinical symptoms.

[0007] In most cases, SMA is diagnosed based on clinical symptoms and testing for the presence of any functional SMN1 gene copies. In some cases, when SMN1 gene testing is not feasible or does not show any abnormalities, other tests such as electromyography (EMG) or muscle biopsy may be necessary.

[0008] The clinical spectrum of SMA disease has been divided into the following five groups.

[0009] (a) Type 0 SMA (e.g., intrauterine SMA) is the most severe form of the disease and begins before birth. Typically, the first symptom of type 0 SMA is decreased fetal movement in the uterus, which can be first observed between 30 and 36 weeks of pregnancy. After birth, these newborns have little movement, difficulty swallowing and breathing, and often die after birth.

[0010] (b) Type 1 SMA (infantile SMA or Wernicke-Hoffmann disease) presents with SMA symptoms between 0 and 6 months of age and is also very severe. Patients are unable to achieve independent sitting and usually die within the first 2 years without ventilatory support.

[0011] (c) Type 2 SMA (intermediate SMA) has an age of onset of 7 to 18 months. Patients are able to sit without support but are unable to stand or walk independently. The prognosis of this group depends largely on the extent of respiratory involvement.

[0012] (d) Type 3 SMA (juvenile SMA or Kugelberg-Weiland disease) is generally diagnosed after 18 months of age. Individuals with Type 3 SMA are able to walk independently at some point during their lives, but typically become wheelchair-dependent in adolescence or adulthood.

[0013] (e) Type 4 SMA (adult-onset SMA) causes weakness in the legs or hands and feet that develops in the late teens to adulthood and then spreads to other areas of the body. Adult-onset SMA progresses much more slowly and has little or no effect on life expectancy.

[0014] Infantile SMA is the most severe form of this neurodegenerative disorder. Symptoms include muscle weakness, poor muscle tone, a weak cry, limp or a tendency to fall, difficulty sucking or swallowing, secretions in the lungs or throat, feeding difficulties, and increased susceptibility to respiratory infections. The legs are often weaker than the arms, and developmental milestones such as lifting the head or sitting up cannot be reached. In general, the earlier the symptoms appear, the shorter the life span. As the motor neuron cells deteriorate, symptoms will appear soon after. Severe forms of the disease are fatal, and there is no known cure for all forms. The course of SMA is directly related to the rate at which the motor neuron cells degenerate and the severity of the resulting weakness. Infants with severe forms of SMA often die of respiratory diseases due to weakness of the muscles that support breathing. Children with milder forms of SMA survive much longer, although they may require extensive medical support, especially those at the more severe end of the symptom spectrum.

[0015] Neuropathological studies have confirmed that for fetuses with SMA, until and including the third trimester of pregnancy, motor neurons in the brainstem and spinal cord are lost in large quantities and there are motor neuron lesions, and this loss increases rapidly in the first six months of life. Most infants with SMA and with two copies of the SMN2 gene show early symptoms of SMA (including muscle weakness and abnormal reflexes) at the first assessment of one week to two weeks of age, and score below the tenth percentile in the Hammersmith neonatal neurological examination test. See Pane (2022) Eur. J. Pediatr. 181 (7): 2821-2829. Electrophysiological testing of these infants with SMA shows that compared with healthy newborns of the same age, the amplitude of the response induced in the motor nerve conduction study test (such as the ulnar nerve compound muscle action potential (CMAP) amplitude) is reduced, indicating that the loss of motor neurons and the reduction of functional motor nerve fiber capacity have occurred before birth. See Finkel (2022) Brain. 145(7):2247-2249; Strauss (2022) Nat. Med. 28:1381-1389; Baranello (2021) N. Engl. J. Med. 384(10):915-923; Alves (2021) Mol. Ther. Methods Clin. Dev. 23:524-538; Kolb (2016) Ann. Clin. Transl. Neurol. 3:132-145) and De Vivo (2019) Neuromuscul. Disord. 29(11):842-856. In addition, plasma and cerebrospinal fluid neurofilament levels are elevated in neonates with SMA compared with healthy newborns, indicating the presence of active axonal disease and neurodegenerative processes in infants who appear clinically normal. See Darras (2019) Ann. Clin. Transl. Neurol. 6(5):932-944 and De Vivo (2019) Neuromuscul. Disord. 29:842-856.

[0016] These studies show that infants with SMA and carrying at least one copy of the SMN2 gene have lost a large number of motor neurons before full-term birth. In fact, the need for SMN in developing motor neurons is most urgent during the third trimester of fetal development to the first three months of postnatal life. Restoring SMN protein as early as possible during the development of tissues including motor neurons is believed to have the greatest benefit for subjects with SMA. See Kong (2021) Sci. Transl. Med. 13 (578): eabb6871; Ramos (2019) J. Clin. Invest. 129 (11): 4817-4831; Iwatani (2017) Front. Pediatr. 5: 194; Martínez-Hernández (2013) J. Pathol. 229 (1): 49-61; and Soler-Botija (2002) Brain. 125 (Chapter 7): 1624-34. Therefore, identifying newborn subjects with SMA via newborn screening may miss the opportunity for complete rescue, even if SMA therapy is initiated soon after birth.

[0017] Most SMA subjects with two copies of the SMN2 gene have type 1 SMA, the most severe form of the disease. See Calucho et al. (2018) Neuromuscul. Disord. 38: 208-15. Even if these patients are diagnosed early through newborn screening, they already have obvious early neurological signs during their first visit (3-13 days after birth). See Pane et al. (2022) Eur. J. Pediat. 181: 2821-9. Data from studies of presymptomatic SMA patients treated with current disease-modifying therapies (DMTs) indicate that even subjects who remain asymptomatic at the start of treatment may not follow a normal developmental trajectory (e.g., will still experience muscle weakness and / or will not be able to stand or walk independently). See De Vivo et al. (2019) Neuromuscl. Disord. 29:842-56; Strauss et al. (2022) Nat. Med. 28:1381-9; and Strauss et al. (2022) Nat. Med. 28:1390-7.

[0018] There are multiple SMA treatments that aim to increase SMN levels in patients. SMN2 splicing modification therapy is a treatment option for SMA. Sold) is an antisense oligonucleotide directed against SMN2 that modulates SMN2 splicing to increase SMN protein levels. See Ando (2020) Sci. Rep. 10(1):17472. It was approved by the U.S. Food and Drug Administration (FDA) in December 2016 for the treatment of children and adults with SMA.

[0019] Another SMN2 splicing-modifying therapy (i.e., the first oral SMA therapy) is lisporam, which is used to treat SMA in pediatric and adult patients. Lisporam is chemically described as 7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one. Lisporam is commercially available as an oral solution containing 60 mg of lisporam as a powder for formulation to provide a 0.75 mg / mL solution, as sell. Indicated for the treatment of SMA in children and adults.

[0020] Administer orally once daily using an oral syringe. Specifically, for patients younger than two months of age, The recommended daily dose is 0.15 mg / kg. For patients aged between two months and two years, The recommended daily dose is 0.2 mg / kg. For patients aged two years and above and weighing less than 20 kg, The recommended daily dose is 0.25 mg / kg. In addition, for patients two years and older weighing 20 kg or more, The recommended daily dose is 5mg.

[0021] SMN1 expression gene therapy is another treatment option for SMA. Onasemnogene abeparvovec (also known as "apeonoxi" or "AVXS-101") is a Apione (sold under the brand name Apione) is a gene therapy based on an adeno-associated virus vector, which is suitable for treating pediatric patients under the age of 2 (US FDA approval) or weighing less than 21 kg (EU EMA approval) with SMA and biallelic mutations in the SMN1 gene. Apione restores the production of SMN protein via a one-time systemic administration. See Thomsen (2021) Nat. Med. 27(10): 1701-1711; and Hoy (2019) Drugs. 79(11): 1255-1262.

[0022] Current SMA therapies have improved survival and motor function in presymptomatic individuals with type 1 SMA, particularly when treated shortly after birth while they are somewhat presymptomatic, see Mercuri (2022) Nat. Rev. Dis. Primers 8(1):52, it is not known whether these SMA treatment options (e.g., nusinersen and AVXS-101) can cross the human placenta and therefore will not effectively address SMA. Treatment of individuals with SMA and two copies of the SMN2 gene (who are highly likely to develop type 1 SMA) initiated after birth may provide only a partial response in terms of saving vulnerable motor neurons, and clinical outcomes are generally suboptimal. Therefore, new therapies for SMA treatment are needed to treat the fetus during a critical period when these motor neurons particularly require adequate SMN protein levels to prevent premature degeneration before birth and optimize the response of subjects with SMA to treatment. Specifically, new therapies for SMA treatment are needed for subjects diagnosed with SMA before birth. Therefore, in the case of prenatal diagnosis of SMA with 2 copies of SMN2, intervention with an SMN protein enhancer that can cross the placenta orally administered to carriers during the fetal stage may help support the child's prenatal development and lead to better postnatal outcomes. Summary of the invention

[0023] Provided herein is a method for treating SMA in a fetal subject in need thereof, comprising administering an amount of lisporam to a carrier of the fetal subject. An amount of lisporam is administered to a carrier of the fetal subject such that a therapeutically effective amount of lisporam is delivered to the fetal subject via the placenta, thereby further leading to an increase in the production of SMN protein in certain tissues, including the central nervous system (CNS) and spinal motor neurons therein. In some embodiments, lisporam is administered orally to a carrier of the fetal subject. Oral administration can take any suitable form, including a solution or a tablet.

[0024] Various embodiments are contemplated herein. For example, in Embodiment 1, a method of treating SMA Type 0 or Type 1 in a fetal subject in need thereof is provided, the method comprising administering a first amount of lisapram to a carrier of the fetal subject.

[0025] Embodiment 2: The method according to Embodiment 1, wherein administering the first amount of lisipril to the carrier of the fetal subject results in administering a therapeutically effective amount to the fetal subject.

[0026] Embodiment 3: The method according to any one of embodiments 1 to 2, wherein the fetal subject is in the third trimester of its development.

[0027] Embodiment 4: The method according to embodiment 3, wherein the fetal subject is at about twenty-eight weeks to about thirty-eight weeks of gestation.

[0028] Embodiment 5: The method according to any one of embodiments 1 to 4, wherein the fetal subject has no detectable functional survival motor neuron 1 (SMN1) gene.

[0029] Embodiment 6: The method according to any one of embodiments 1 to 5, wherein the fetal subject has at least one survival motor neuron 2 (SMN2) gene.

[0030] Embodiment 7: The method according to any one of embodiments 1 to 5, wherein the fetal subject has two or fewer copies of the survival motor neuron 2 (SMN2) gene.

[0031] Embodiment 8: The method according to any one of Embodiments 1 to 7, wherein administering the first amount of lisipril to the carrier of the fetal subject occurs orally.

[0032] Embodiment 9: The method according to any one of Embodiments 1 to 8, wherein administering the first amount of lisporamin to the carrier of the fetal subject occurs daily.

[0033] Embodiment 10: The method according to any one of embodiments 1 to 9, wherein the first amount of lisapram is administered at a dose of about 5 mg.

[0034] Embodiment 11: The method of Embodiment 1, further comprising terminating administration of the first amount of lisapram to the carrier of the fetal subject when the fetal subject is delivered from the carrier as a newborn subject.

[0035] Embodiment 12: The method according to embodiment 11, wherein the level of neurofilaments is elevated in at least the umbilical cord blood of the neonatal subject.

[0036] Embodiment 13: The method of any one of Embodiments 11-12, further comprising administering a second SMA therapy to the neonatal subject.

[0037] Embodiment 14: The method of Embodiment 13, wherein the second SMA therapy is administered to the neonatal subject beginning within a time period from birth of the neonatal subject to about sixty days after birth of the neonatal subject.

[0038] Embodiment 15: The method according to any one of embodiments 13 to 14, wherein the second SMA therapy is SMN expression gene therapy or SMN2 splicing modification therapy.

[0039] Embodiment 16: The method according to Embodiment 15, wherein the second SMA therapy is an SMN2 splicing modification therapy comprising administering liximab to the neonatal subject.

[0040] Embodiment 17: The method according to Embodiment 16, wherein the amount of lisiporam administered to the neonatal subject is an amount equal to or less than 0.15 mg / kg.

[0041] Embodiment 18: The method of any one of Embodiments 16-17, wherein lisiporam is administered to the neonatal subject if the level of lisiporam in the blood of the neonatal subject is less than about 100 ng / mL.

[0042] Embodiment 19: The method according to embodiment 18, wherein the level of lisapram in the blood of the neonatal subject is the level in the umbilical cord blood of the neonatal subject at the time of delivery.

[0043] Embodiment 20: The method according to embodiment 19, wherein the mean steady-state concentration-time area under the curve (AUC) of the lisapram in the umbilical cord blood of the neonatal subject at the time of delivery ss ) level corresponds to a mean steady-state area under the concentration-time curve (AUC) of less than about 2000 ng·h / mL ss ).

[0044] Embodiment 21: The method according to any one of embodiments 14 to 20, wherein administering the second amount of lisipril to the neonatal subject occurs daily.

[0045] Embodiment 22: The method according to any one of embodiments 14 to 21, wherein administering the second amount of lisporamin to the neonatal subject occurs orally.

[0046] Embodiment 23: The method of any one of Embodiments 1 to 22, wherein lisapram is administered as a pharmaceutical composition further comprising ascorbic acid, edetate disodium dihydrate, isomalt, mannitol, polyethylene glycol 6000, sodium benzoate, strawberry flavor, sucralose and tartaric acid.

[0047] Embodiment 24: A method of treating spinal muscular atrophy (SMA) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an SMA therapy, wherein the subject has received lisapram in utero as a fetal subject by a carrier of the subject. In some such embodiments, the subject is a newborn infant or older. In some such embodiments, the subject is a neonatal subject. In embodiments, the subject has SMA type 0, type 1, type 2, type 3, or type 4. In embodiments, the SMA therapy is an SMN expression gene therapy or an SMN2 splicing modification therapy.

[0048] Embodiment 25: A method of treating spinal muscular atrophy (SMA) in a subject in need thereof, the method comprising: administering a first amount of lisporam to a carrier of a fetal subject in a carrier dosing cycle to administer a therapeutically effective amount of lisporam to the fetal subject; terminating the carrier dosing cycle when the fetal subject is delivered from the carrier to become a neonatal subject; and administering a second amount of lisporam to the neonatal subject in a neonatal dosing cycle. In an embodiment, if the level of lisporam in the blood of the neonatal subject is less than about 100 ng / mL, the second amount of lisporam is administered to the neonatal subject in the neonatal dosing cycle.

[0049] Example 26: A use of lisapram for treating SMA in a fetal subject in need thereof.

[0050] Embodiment 27: Lisapram according to Embodiment 26, wherein the fetal subject is in the third trimester of its development.

[0051] Embodiment 28: Lisapram according to any one of Embodiments 26 to 27, wherein the fetal subject is at about twenty-eight to about thirty-eight weeks of gestation.

[0052] Embodiment 29: Lisapram according to any one of Embodiments 26 to 27, wherein the fetal subject is at least about thirty-two weeks of gestation.

[0053] Embodiment 30: Lisapram according to any one of embodiments 26 to 29, wherein the fetal subject has no detectable functional survival motor neuron 1 (SMN1) gene.

[0054] Embodiment 31: Lisapram according to any one of Embodiments 26 to 30, wherein the fetal subject has at least one survival motor neuron 2 (SMN2) gene.

[0055] Embodiment 32: Lisapram according to any one of embodiments 26 to 30, wherein the fetal subject has two or fewer copies of the survival motor neuron 2 (SMN2) gene.

[0056] Embodiment 33: Lisporam according to any one of Embodiments 26 to 32, wherein the lisporamin is administered to the carrier of the fetal subject.

[0057] Embodiment 34: Lisporam according to Embodiment 33, wherein administration of the lisporam to the carrier of the fetal subject occurs orally.

[0058] Embodiment 35: Lisporam according to any one of Embodiments 33 to 34, wherein administering the lisporam to the carrier of the fetal subject occurs daily.

[0059] Embodiment 36: Lisporam according to any one of Embodiments 33 to 35, wherein the lisiporam is administered to the carrier of the fetal subject at a dose of about 5 mg.

[0060] Embodiment 37: Lisporam according to any one of Embodiments 33 to 36, wherein administration of the lisporam to the carrier of the fetal subject is terminated when the fetal subject is delivered as a neonatal subject.

[0061] Embodiment 38: Lisapram according to Embodiment 37, wherein the level of neurofilaments is elevated in at least the umbilical cord blood of the neonatal subject.

[0062] Embodiment 39: Lisapram according to any one of Embodiments 33 to 38, further comprising administering a second SMA therapy to the neonatal subject.

[0063] Embodiment 40: Lisapram according to Embodiment 39, wherein the second SMA therapy is administered to the neonatal subject during a period from delivery of the fetal subject to the neonatal subject to about sixty days after delivery.

[0064] Embodiment 41: Lisapram according to any one of Embodiments 39 to 40, wherein the second SMA therapy is SMN expression gene therapy or SMN2 splicing modification therapy.

[0065] Embodiment 42: Lisporam according to any one of Embodiments 39 to 41, wherein the second SMA therapy is an SMN2 splicing modification therapy comprising Lisporam.

[0066] Embodiment 43: Lisporam according to Embodiment 42, wherein the first administration of the SMN2 splicing-modifying therapy comprising Lisporam to the neonatal subject occurs via oral administration daily in an amount equal to or less than 0.15 mg / kg.

[0067] Embodiment 44: Lisporam according to Embodiment 43, wherein the first administration of lisporam to the neonatal subject occurs if the level of lisporam in the blood of the neonatal subject is less than about 100 ng / mL.

[0068] Embodiment 45: Lisporam according to Embodiment 44, wherein the level of Lisporam in the blood of the neonatal subject is the level in the umbilical cord blood of the neonatal subject at the time when the fetal subject is delivered to become the neonatal subject.

[0069] Embodiment 46: Lisporam according to any one of Embodiments 41 to 45, wherein the second administration of the SMN2 splicing modification therapy comprising lisporam to the neonatal subject occurs via oral administration daily in an amount equal to or less than 0.20 mg / kg, and wherein the second administration of the lisporam occurs during a time period of about 2 months to about 2 years after delivery of the neonatal subject.

[0070] Embodiment 47: The lisporam of any one of Embodiments 26 to 46, wherein the lisporam is administered in a pharmaceutical composition further comprising ascorbic acid, edetate disodium dihydrate, isomalt, mannitol, polyethylene glycol 6000, sodium benzoate, strawberry flavor, sucralose and tartaric acid.

[0071] Embodiment 48: Lisapram according to any one of embodiments 26 to 47, wherein the SMA is type 0 SMA or type 1 SMA. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Included are diagrams related to administering an amount of lisapram to a carrier of a fetal subject and monitoring the carrier and the fetal subject in accordance with at least some embodiments disclosed herein.

[0073] Figure 2 is a graph comparing whole blood SMN protein levels in Example 1 with a reference study according to at least some embodiments disclosed herein.

[0074] Figure 3 is a graph depicting average neurofilament protein levels in the blood of carriers (eg, carriers) on different days according to at least some embodiments disclosed herein.

[0075] Figure 4 is a graph depicting average neurofilament levels in the blood of neonatal subjects (eg, infants) on different days, according to at least some embodiments disclosed herein.

[0076] Figure 5is a graph of a reference study measuring plasma phosphorylated neurofilament heavy chain subunit (pNF-H) concentration (pg / mL) levels at baseline in infants < 1 year old without SMA, according to at least some embodiments disclosed herein.

[0077] Figure 6 is a schematic diagram of a research scheme related to Example 2 according to at least some embodiments disclosed herein. DETAILED DESCRIPTION

[0078] Unless specifically defined otherwise, all technical and scientific terms used herein shall be construed to have the same meanings as commonly understood by one of ordinary skill in the art.

[0079] definition

[0080] "Treatment" or similar phrases refer to obtaining a beneficial or desired outcome, such as a clinical outcome, in a subject, for example, genetically diagnosed as having SMA. Beneficial or desired outcomes include any one or more of the following: alleviating one or more symptoms of SMA, reducing the extent of SMA, delaying or slowing disease progression, improving quality of life, and improving the disease state.

[0081] In some embodiments, treatment results in a subject having milder symptoms than would be expected based on their genetic analysis and / or the amount of SMN1 protein in circulation during a pre-treatment period.

[0082] The term "treating spinal muscular atrophy (SMA)" or "treatment of spinal muscular atrophy (SMA)" includes one or more of the following effects: (i) reducing or improving the severity of SMA; (ii) delaying the onset of SMA; (iii) inhibiting the progression of SMA; (iv) reducing the number of hospitalizations of the subject; (v) shortening the length of hospital stay of the subject; (vi) increasing the survival rate of the subject; (vii) improving the quality of life of the subject; (viii) reducing the number of symptoms associated with SMA; (ix) reducing or improving the severity of one or more symptoms associated with SMA; (x) shortening the duration of symptoms associated with SMA; (xi) preventing the recurrence of symptoms associated with SMA; (xii) inhibiting the development or onset of SMA symptoms; and / or (xiii) inhibiting the progression of symptoms associated with SMA. More specifically, "treating SMA" means one or more of the following beneficial effects: (i) reduced loss of muscle strength; (ii) increased muscle strength; (iii) reduced muscle atrophy; (iv) reduced loss of motor function; (v) increased motor neurons; (vii) reduced loss of motor neurons; (viii) protection of SMN-deficient motor neurons from degeneration; (ix) increased motor function; (x) increased bulbar function; and / or (xi) reduced loss of lung function. "Treating SMA" may further result in or help maintain the functional ability of a human infant or human toddler (e.g., a newborn subject) to perform certain physical activities, such as independently standing, independently walking, independently running, independently breathing, independently coughing, independently turning over during sleep, or independently swallowing in the case of a human infant, human toddler, human child, or human adult.

[0083] "Survival motor neuron" or "SMN" refers to the protein encoded by the human SMN1 gene and SMN2 gene.

[0084] "Survival motor neuron 1" or "SMN1" refers to the telomeric copy of the gene encoding the SMN protein.

[0085] "Survival motor neuron 2" or "SMN2" refers to the centromeric copy of the gene encoding the SMN protein. Mutations in the SMN2 gene alone do not cause SMA. Mutations in both the SMN1 gene and the SMN2 gene can cause embryonic lethality.

[0086] A "treatment cycle" refers to the period of time during which a set of SMA therapy doses is administered to a subject, such as to a carrier of a fetal subject or to a neonatal subject.

[0087] "Carrier" refers to a pregnant human subject who is carrying a fetal subject.

[0088] A "fetal subject" refers to a human subject prior to delivery from a carrier. Specifically, a "fetal subject" has been diagnosed with SMA prior to delivery from a carrier. A fetal subject may be referred to herein as a human subject.

[0089] "Neonatal subject" refers to a human subject after delivery from a carrier. A neonatal subject is a fetal subject that has been delivered. For the purposes of this application, a "neonatal subject" refers to a human subject whose age is, for example, 30 to 60 days, and may also refer to a subject whose age is infancy up to 2 years old. A neonatal subject may be referred to as a human subject herein.

[0090] "Gestation" refers to the period of time during which a fetus develops within a carrier.

[0091] The "First trimester" of development refers to the period of time between conception and about 13 weeks of gestation of a fetal subject.

[0092] The "Second trimester" of development refers to a fetal subject between 14 weeks and about 27 weeks of gestation.

[0093] The "third trimester" of development refers to the period of gestation of a fetal subject between about 28 weeks and until delivery at about 42 weeks.

[0094] In some embodiments, the "third trimester" of development refers to a fetal subject's gestation between about 28 weeks until delivery, which may occur before about 42 weeks or after about 42 weeks. In some embodiments, the "third trimester" may also refer to a fetal subject's gestation from about 28 weeks until delivery.

[0095] "Delivery" and "birth" are used interchangeably herein to refer to the removal of a fetal subject from a carrier to become a newborn subject.

[0096] "Lisporam" is a SMN2-directed RNA splicing modifier used as a drug for the treatment of SMA. The chemical name of Lisporam is 7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazine-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one, and the structural formula is:

[0097]

[0098] Lisporam is disclosed in U.S. Published Patent Application No. 2017 / 1979901A1 and International Publication No. WO 201 / 5173181 (titled "Compounds for the Treatment of Spinal Muscular Atrophy"). Certain crystalline forms of lisiporam are disclosed in U.S. Published Patent Application No. 2021 / 403487A1 and International Publication No. WO 2020 / 079203 (titled "New Forms of Pyrido[1,2-a]Pyrimidin-4-one Derivatives, Formulations Thereof, and Methods of Making Them"). Lisporam is primarily metabolized by flavin monooxygenases 1 and 3 (FMO1 and FMO3) and also by CYP 1A1, 2J2, 3A4, and 3A7. Metabolites of lisiporam are considered inactive. Lisporam is believed to cross the placenta of rodents and rabbits and not interfere with other placental transport mechanisms at doses that produce fetal exposures proportional to the 5 mg daily dose approved for humans weighing more than 20 kg (e.g., pregnant carriers). In preclinical studies in which pregnant animals were dosed, increasing doses of lisporam were administered to rats and rabbits until overt carrier toxicity was observed to determine the NOAEL. Weight loss and slight differences in skeletal ossification were noted in rat fetuses treated with dams at about 1 mg / kg / day to about 7.5 mg / kg / day. Fetal weight gain was reduced and skull or brain variations were present in rabbit fetuses treated with dams at about 1 mg / kg / day to about 12 mg / kg / day, and at about 12 mg / kg / day, some rabbit fetuses developed hydrocephalus, abnormally small gallbladders, and lung variations. Humans weighing up to about 20 kg are treated at 0.15 mg / kg / day to 0.25 mg / kg / day, and children and adults weighing greater than 20 kg are treated at a dose of 5 mg / day.

[0099] The term "mg / kg" refers to the dose (in milligrams) of lisporam administered, for example, per kilogram of body weight of the subject to be treated. For example, 0.15 mg / kg lisporam means a dose of 0.15 milligrams of lisporam per kilogram of body weight of a neonatal subject to be treated.

[0100] "Adverse event" means any undesirable clinical incident that occurs in a fetal subject (compared to the fetal subject's baseline health status) or a neonatal subject (compared to the neonatal subject's baseline health status), and is any undesired medical incident, defined as an unexpected disease or injury or an undesired clinical symptom (including abnormal laboratory results) in the fetal subject or neonatal subject. More specifically, the grades of adverse events include those published in the "Common Terminology Criteria for Adverse Events" (or "CTCAE") (version 4.03 released on June 14, 2010) published by the National Cancer Institute of the United States. These include mild (grade 1) adverse events, manifested as minor symptoms that do not require medical intervention; moderate (grade 2) adverse events, which require minimally invasive, local, or non-invasive intervention; severe or medically significant (grade 3) adverse events, which do not immediately endanger life but may require hospitalization or an extended hospital stay; life-threatening (grade 4) adverse events, which require urgent intervention; and death related to the adverse event (grade 5).

[0101] "No observed adverse effect level" or "NOAEL" means the level of exposure of an organism found by experiment or observation at which there is no biologically or statistically significant increase in the frequency or severity of any adverse reaction to the test protocol.

[0102] References herein to a "about" value or parameter include (and describe) variations that involve the value or parameter itself. In the examples, the term "about" means + / - 10%, + / - 5%, or + / - 1% of the specified value.

[0103] Unless the context clearly dictates otherwise, the singular forms "a / an", "or", and "the" include plural referents.

[0104] "Comprise" or variations such as "comprises" or "comprising" should be understood to imply the inclusion of the stated elements, integers, or steps or groups of elements, integers, or steps, but not the exclusion of any other elements, integers, or groups of elements, integers, or steps. The embodiments described herein also include aspects of "consisting of" and / or "consisting essentially of".

[0105] method

[0106] The present disclosure describes methods for treating SMA, particularly type 0 and type 1 SMA, as well as methods for treating neonates and infants who have received SMA treatment while in utero, thus addressing the unmet needs of this important patient population.

[0107] Importantly, the methods provided herein offer treatment options during a critical developmental period, which may provide long-term and durable benefits to the subject.

[0108] If there is a risk that the carrier may give birth to a child with SMA, the carrier can undergo chorionic villus sampling (CVS) testing, which is usually performed during the 11th to 14th week of pregnancy to extract a cell sample from the placenta for testing, and / or the carrier can undergo amniocentesis during the 15th to 20th week of pregnancy to determine the number of copies of the SMN gene mutation inherited by the fetal subject. The parents of the fetal subject can also undergo genetic testing (such as blood testing) to detect the most common mutations of SMN. After the fetal subject is born as a newborn subject, the newborn subject can undergo a genetic blood test to confirm the SMA condition. Other tests, such as physical examination, electromyography / neurophysiology and / or muscle biopsy, can also be performed. Electrophysical testing of newborn subjects can also be used as prognostic biomarkers.

[0109] In an embodiment, a fetal subject diagnosed with SMA by genetic testing is, for example, a homozygous deletion or a heterozygous deletion (predicted to be a loss of function of the SMN1 gene and one or two SMN2 gene copies). In an embodiment, the fetal subject has no detectable functional copy of the SMN1 gene and has at least one SMN2 gene copy. In an embodiment, a fetal subject diagnosed with SMA by genetic testing has no detectable functional copy of the SMN1 gene and three or fewer SMN2 gene copies. In some embodiments, the fetal subject has at least one SMN2 gene.

[0110] Described herein are methods for treating SMA in a fetal subject in need thereof, wherein the method comprises a carrier treatment cycle. In embodiments, the carrier treatment cycle comprises administering a first SMA therapy to a carrier of the fetal subject, the therapy comprising an SMN2 splicing modification therapy. For example, in embodiments, a method for treating type 0 or type 1 SMA in a fetal subject in need thereof is provided, wherein the method comprises administering a first amount of lisporam to the fetal subject by a carrier of the fetal subject during the carrier treatment cycle. In embodiments, administering a first amount of lisporam to a carrier of the fetal subject results in administering a therapeutically effective amount of lisporam to the fetal subject.

[0111] In an embodiment, prior to starting a carrier treatment cycle, a fetal subject is diagnosed as having SMA by genetic testing. In an embodiment, a carrier treatment cycle is started when the basic embryogenesis of a fetal subject is substantially complete. In an embodiment, a carrier treatment cycle is started when a fetal subject is in the third trimester of its development. In an embodiment, a carrier treatment cycle is started when the pregnancy of a fetal subject is at about twenty-eight weeks to about thirty-eight weeks of pregnancy. In an embodiment, a carrier treatment cycle is started when the pregnancy of a fetal subject is at least about thirty-two weeks of pregnancy. In an embodiment, a carrier treatment cycle is started when the pregnancy of a fetal subject is at least about twenty-eight weeks. In an embodiment, a carrier treatment cycle is started when the pregnancy of a fetal subject is at least about twenty-eight weeks. In an embodiment, a carrier treatment cycle is started when the pregnancy of a fetal subject is at about twenty-eight weeks to about thirty weeks of pregnancy.

[0112] In embodiments, administering the first amount of lisporam to a fetal subject comprises orally administering to a carrier of the fetal subject. In embodiments, the first amount of lisporam is administered to a carrier of the fetal subject daily. In embodiments, administering the first amount of lisporam to a carrier of the fetal subject comprises orally administering lisporam to a carrier of the fetal subject daily. In embodiments, the first amount of lisporam is a dose of about 5 mg.

[0113] In an embodiment, the method further includes terminating the carrier treatment cycle when an event occurs. In an embodiment, the event includes the delivery of a fetal subject from a carrier. In other embodiments, if an adverse change is detected in the biophysical profile of the fetal subject and / or if an uncontrollable medical problem occurs on the carrier side, the carrier treatment cycle is terminated. In an embodiment, a baseline pre-treatment comprehensive anatomical ultrasound study and / or a growth scan of the fetal subject can be performed and can be repeated during the carrier's pregnancy. Anatomical ultrasound studies monitor fetal growth and any signs of organ maldevelopment, especially in the brain, heart, kidneys and liver. If any adverse results are identified, these results will be evaluated for possible relevance to lisporam, and if it is considered to be possibly drug-related, the administration of the first amount of lisporam to the carrier may be interrupted or terminated. In an embodiment, after the fetal subject is delivered from the carrier, the carrier treatment cycle continues so that the newborn subject can continue to receive lisporam through the carrier's breast milk.

[0114] In an embodiment, the method further includes a neonatal treatment cycle. In an embodiment, during the carrier treatment cycle or after the carrier treatment cycle is terminated, the neonatal treatment cycle may be started or not started. In an embodiment, a neonatal subject blood (venous and / or arterial) sample including umbilical cord blood and amniotic fluid is collected at delivery to assess the intrauterine exposure of lisproram to the neonatal subject. In an embodiment, the start of the neonatal treatment cycle occurs within a certain period of time from the delivery of the neonatal subject. In an embodiment, the time period is within the range of from birth to about sixty days or two months. In an embodiment, the start of the neonatal treatment cycle occurs within thirty days of the delivery of the neonatal subject. In an embodiment, the start of the neonatal treatment cycle occurs within 7-10 days of the delivery of the neonatal subject. In an embodiment, the start of the neonatal treatment cycle occurs within two days of the delivery of the neonatal subject. In an embodiment, the start of the neonatal treatment cycle occurs within twenty-four hours of the delivery of the neonatal subject.

[0115] In embodiments, the SMN protein level in a neonatal subject is measured during a first moment and during a second moment. In embodiments, the first moment is before the start of the neonatal dosing cycle, and the second moment is after the start of the neonatal dosing cycle. In some embodiments, when the second moment is compared to the first moment, the SMN protein level of the neonatal subject increases by at least about 20%, at least about 30%, at least about 40%, or at least about 50%, or between about 20% and about 50%, or between about 30% and about 40%. In some embodiments, the time period between the first moment and the second moment can be several days, several weeks, two weeks, one month, or two months. In embodiments, the SMN protein level of the neonatal subject increases by about 30-40% within one month of administering the second amount of lisproram.

[0116] In embodiments, the neonatal treatment cycle comprises administering the second SMA therapy directly to the neonatal subject without passing through a carrier. In embodiments, the second SMA therapy is administered to the neonatal subject if the level of lisporam in the neonatal subject's blood is less than about 100 ng / mL. In embodiments, the second SMA therapy is administered to the neonatal subject within a certain period of time of delivery of the neonatal subject (e.g., within about 30 days after birth) if the level of lisporam in the neonatal subject's cord blood is less than about 100 ng / mL before and after delivery.

[0117] In an embodiment, the second SMA therapy is an SMN expression gene therapy or an SMN2 splicing modification therapy. In an embodiment, the neonatal treatment cycle includes the administration of SMN2 splicing modification therapy as a second SMA therapy. SMN2 splicing modification therapy can be administered to a neonatal subject within a certain period of time after the delivery of the neonatal subject / the delivery of the neonatal subject. In an embodiment, the time period is within the range from birth to about sixty days. In an embodiment, the start of administration of SMN2 splicing modification therapy to a neonatal subject occurs within thirty days of the delivery of a neonatal subject. In an embodiment, the start of administration of SMN2 splicing modification therapy to a neonatal subject occurs within 7-10 days of the delivery of a neonatal subject. In an embodiment, the start of administration of SMN2 splicing modification therapy to a neonatal subject occurs within 1-2 days of the delivery of a neonatal subject.

[0118] In embodiments, the SMN2 splicing modification therapy comprises administering a second amount of lisporam to a neonatal subject (the first amount being the amount of lisporam administered to a subject who is a fetal subject by a carrier). In embodiments, the second amount of lisporam is administered to a neonatal subject if the level of lisporam in the blood of the neonatal subject is less than about 100 ng / mL. In embodiments, if the level of lisporam in the cord blood of the neonatal subject around the time of delivery corresponds to a safe exposure average defined for humans, e.g. The area under the curve [AUC ss ] is 2000 ng / h / mL, a second dose of lisproram is administered to the neonatal subject within the time period of delivery of the neonatal subject (e.g., within 30 days after birth).

[0119] In an embodiment, the second amount of lisporam is a therapeutically effective amount of lisporam. In an embodiment, the therapeutically effective amount of lisporam is 0.15 mg / kg equal to or less than for a neonatal subject. In an embodiment, the therapeutically effective amount of lisporam is 0.15 mg / kg for a neonatal subject. In an embodiment, the administration of a second amount of lisporam to a neonatal subject occurs daily. In an embodiment, the administration of a second amount of lisporam to a neonatal subject occurs orally. In an embodiment, a second amount of lisporam is administered to a neonatal subject at an oral dose of up to 0.15 mg / kg / day. In an embodiment, lisporam is administered to a neonatal subject at a daily oral dose of up to 0.15 mg / kg during a period of about 2 months from the delivery of a neonatal subject to the delivery of a neonatal subject.

[0120] In embodiments, the second amount of lisapram is administered to the neonatal subject at a daily oral dose of 0.20 mg / kg over a period of about 2 months to about 2 years after delivery of the neonatal subject.

[0121] In embodiments, the neonatal treatment cycle comprises a first administration of lisiporam to a neonatal subject at a daily oral dose of 0.15 mg / kg during a period of time from birth of the neonatal subject to about 2 months after birth of the neonatal subject, followed by a second administration of lisiporam to the neonatal subject at a daily oral dose of 0.20 mg / kg during a period of time from about 2 months to about 2 years after birth of the neonatal subject. In embodiments, the first administration of lisiporam at a daily oral dose of 0.15 mg / kg to the second administration of lisiporam at a daily oral dose of 0.20 mg / kg is sequential. In embodiments, the first administration of lisiporam at a daily oral dose of 0.15 mg / kg and the second administration of lisiporam at a daily oral dose of 0.20 mg / kg are sequential.

[0122] In an embodiment, the SMN2 splicing modification therapy comprises administering to a neonatal subject an amount of nusinersen or In an embodiment, the amount of nusinersen is about 12 mg / 5 mL (2.4 mg / mL). In an embodiment, the amount of nusinersen is administered to a neonatal subject via a single dose by intrathecal administration. In an embodiment, the administration of SMN2 splicing modification therapy (such as nusinersen) to a neonatal subject as a second SMA therapy occurs with at least one loading dose. In such an embodiment, such SMN2 splicing modification therapy is initiated with up to four loading doses, wherein the first, second, and third loading doses are regularly administered to neonatal subjects at certain time intervals. In an embodiment, the time interval is a fourteen-day time interval. In an embodiment, the fourth loading dose is administered within a period of time after the first three loading doses. In an embodiment, the time period is about 30 days after the third loading dose. In an embodiment, the administration of SMN2 splicing modification therapy includes administering a maintenance dose within a period of time after the loading dose. In an embodiment, the time period is once every four months after the fourth loading dose.

[0123] In embodiments, the neonatal treatment cycle comprises administering a second SMA therapy to the neonatal subject, wherein the second SMA therapy is an SMN expression gene therapy. In embodiments, the SMN expression gene therapy is an adeno-associated viral vector-based gene therapy. In embodiments, the adeno-associated viral vector-based gene therapy is apionein-xioi or apionein-xioi or In an embodiment, the SMN expression gene therapy comprises administering to a neonatal subject an amount of apioneinol. In an embodiment, the amount of apioneinol is 1.1×10 14A therapeutically effective amount of a vector genome (vg). In an embodiment, the amount of apione base is administered via a single intravenous infusion that occurs once per time period. In an embodiment, the time period includes about sixty minutes. In an embodiment, a certain amount of systemic corticosteroids is administered within a period of time before the start of administration of the certain amount of apione base. In an embodiment, the time period is one day. In an embodiment, the amount of systemic corticosteroids is equivalent to oral prednisolone at about 1 mg / kg body weight. In an embodiment, one day before the start of administration of the certain amount of apione base, the administration of the certain amount of systemic corticosteroids occurs once a day. In an embodiment, after a period of time, the administration of the certain amount of systemic corticosteroids is terminated. In an embodiment, the time period is thirty days.

[0124] In embodiments, the second SMA therapy is combined with other therapies, such as muscle enhancement therapies. As defined herein, a "muscle enhancement therapy" is any therapy that is capable of increasing the size (muscle growth) and / or strength of a muscle, such as a skeletal muscle. In embodiments, the muscle enhancement therapy is a myostatin inhibitor. In embodiments, the myostatin inhibitor is an anti-myostatin antibody designed to target skeletal muscle, and in particular the myostatin pathway. Without being bound by any particular theory, it is believed that inhibiting myostatin may contribute to increases in muscle size and strength and / or to improvements in motor function. In embodiments, the myostatin inhibitor is apitegromab (SRK-015), a monoclonal antibody to the antigen myostatin. In embodiments, the myostatin inhibitor is Taldefgrobep alfa (BHV2000), a monoclonal antibody to the antigen myostatin. In embodiments, the myostatin inhibitor is GYM329 (Roche), a circulating and antigen-clearing monoclonal anti-myostatin antibody. In an embodiment, the myostatin inhibitor is BIIB110 (formerly ALG801) (Biogen), a recombinant protein that acts as an ActRIIB (type 2B activin receptor) ligand trap. See WO2016098357, WO2017104783, WO2023057404, and WO2017049011, the entire contents of which are hereby incorporated by reference in their entirety. In an embodiment, the administration of the myostatin inhibitor to the neonatal subject begins at least six months from the date of delivery of the fetal subject to become a neonatal subject. The myostatin inhibitor may be administered within at least the first day, first week, or first month after the fetal subject is delivered to become a neonatal subject. In an embodiment, the myostatin inhibitor is administered within about 3 to 6 months after delivery from a carrier. In an embodiment, the myostatin inhibitor is administered after the start of administration of a second SMA therapy. In an embodiment, the myostatin inhibitor is administered at the same time as the start of administration of a second SMA therapy.

[0125] In an embodiment, the method includes determining when a neonatal subject who receives a carrier treatment cycle as a fetal subject needs further treatment (such as a neonatal treatment cycle) and / or evaluating the clinical response of a neonatal subject to the disclosed therapy. The disclosed detection and the parameters measured for evaluating clinical response (such as biomarkers, electrophysiology, and neurophysical parameters) can be measured in association with the natural history of SMA or the natural history of SMA in human subjects. Therefore, a conclusion of complete or partial clinical response can be drawn based on the measured parameters, which confirms that the natural course is improved, the condition is maintained, or the natural course of deterioration is stopped, or the non-inferiority deterioration in the natural disease process. In an embodiment, the natural history of SMA is consistent with the data of the reference study. In an embodiment, the reference study is the NURTURE clinical trial (ClinicalTrials.gov ID NCT02386553), a study of delivering multiple doses of nusinersen (ISIS 396443) to infants with genetically diagnosed and presymptomatic spinal muscular atrophy. In the Examples, the reference study is the RAINBOWFISH clinical trial (ClinicalTrials.gov ID NCT03779334), a study of lisapram in infants with genetically diagnosed and presymptomatic spinal muscular atrophy.

[0126] The treatment eligibility and / or clinical response of the neonatal subject to the carrier treatment cycle can be evaluated at the time of delivery of the fetal subject and / or when the carrier treatment cycle is stopped. Whether the neonatal subject is eligible for the neonatal treatment cycle can be determined by the attending physician using clinical response evaluation tools, neurophysiological assessments, biomarker evaluations, histological clearance evaluations and / or according to the judgment of the physician. The clinical response can include a complete response, a partial response, a stable disease or a progressive disease. The clinical response can be evaluated based on a variety of criteria, including exposure to lisapram to fetal subjects based on amniotic fluid or fetal umbilical cord blood samples, complete blood count (CBC) screening, comprehensive metabolic panel (CMP), urine analysis (UA) and detected neurological problems or lisapram-specific adverse events (e.g., based on a physical examination of the neonatal subject). After completing the carrier treatment cycle, a complete response is not achieved but a partial remission is achieved, or a neonatal subject who exhibits stable or progressive disease can continue to receive further treatment, including the neonatal treatment cycle, according to the judgment of the treating physician.

[0127] In some embodiments, SMA biomarkers, such as the SMN protein level, CMAP amplitude, and neurofilament level of a neonatal subject, can be used to evaluate the efficacy of risdiplam in a neonatal subject treated in utero. CMAP is an objective measure of the electrophysiological output that is the total output of all motor units supplying a particular muscle following supramaximal stimulation of the innervating nerve. CMAP can be used to monitor disease progression in SMA because it is considered a surrogate for motor neuron loss and is associated with disease severity, functional status, SMN2 copy number, and age. Neonatal subjects with type 1 SMA show a significant decline in CMAP amplitude in the first few months of life. See Kolb (2016) Ann Clin Transl Neurol 3(2):132-45; Finkel (2013) Neuromuscul Disord 23(2):112-5; Lewelt (2010) Muscle Nerve 42(5):703-8; and Swoboda (2005) Ann Neurol 57(5):704-12. Additionally, CMAP amplitude can be used to determine the onset of disease in pre-symptomatic neonatal subjects. See Lee (2022) Neurology 99(14):e1527-37; Weng (2021) Genet Med. 23(2):415-20; and Vill (2019) J Neuromuscul Dis 6(4):503-15. CMAP amplitude is correlated with functional motor scores (see Kolb (2017) Ann Neurol 82(6):883-91) and can predict walking status at 2 years of age (see Kariyawasam (2023) Lancet Child Adolesc Health 7(3):159-70).

[0128] In some embodiments, when a fetal subject is delivered as a neonatal subject, the CMAP amplitude of the neonatal subject can be evaluated within a period of time after delivery from the carrier, such as one week after delivery from the carrier, two weeks after delivery from the carrier, one month after delivery from the carrier, two months after delivery from the carrier, six months after delivery from the carrier, and / or twelve months after delivery from the carrier, etc., to predict the efficacy of in utero treatment with risdiplam.

[0129] In some embodiments, fetal subjects diagnosed with SMA by genetic testing have elevated neurofilament levels in at least one body fluid, such as in plasma or cerebrospinal fluid (CSF). When the fetal subject is born as a neonatal subject, it can be determined that the neonatal subject has elevated neurofilament levels at least in cord blood. Neurofilaments are neuron-specific and consist of four subunits including neurofilament light chain (NfL) and heavy chain (NfH). After axonal injury occurs in SMA, neurofilaments are released into interstitial fluid, CSF, and peripheral blood. Elevated levels of NfL and pNfH associated with motor neuron injury have been observed in subjects with SMA compared to healthy controls. See Paris (2023) CPT Pharmacometrics Syst Pharmacol 12(2):196 - 206; Alves (2021) Mol Ther Methods ClinDev. 23:524 - 38; and Darras (2019) Ann Clin Transl Neurol 6(5):932 - 44. Neurofilaments serve as potential blood biomarkers for SMA, and the serum neurofilament light chain level (sNfL) is higher in treatment-naive SMA patients with 2 SMN2 copies than in those with >2 SMN2 copies. See Nitz (2021) Ann.Clinc.Transl.Neurol. 8(10):2013 - 2024. Additionally, neonatal subjects with SMA have elevated NfL and pNfH levels in the first few months of life compared to healthy controls. These levels are inversely correlated with the maximum ulnar CMAP negative peak amplitude value. See Alves (2021) Mol Ther Methods Clin Dev. 23:524 - 38. Furthermore, compared to sham control groups, the plasma concentration of pNfH decreases more rapidly and to a greater extent in neonatal subjects with SMA less than seven months of age treated with SMA disease-modifying therapy (DMT). See Darras (2019) Ann Clin Transl Neurol 6(5):932 - 44. In some embodiments, the pNfH level of a neonatal subject can be evaluated within a period of time after delivery from the carrier, such as after delivery from the carrier, in the first month after delivery from the carrier, and / or in the second month after delivery from the carrier, etc., to predict the efficacy of in utero treatment with risdiplam.

[0130] As described herein, a complete response to various assessments can include, for example, achieving certain developmental milestones as evaluated according to the metrics described in Table 1.

[0131] Table 1.

[0132]

[0133]

[0134] *MFM32 is a thirty-two clinician-reported outcome measure used to assess functional ability in subjects with neuromuscular disease, including subjects with SMA.

[0135] **Duration of permanent ventilation includes tracheostomy or ventilation [bilevel positive airway pressure] for >3 weeks or continuous intubation for >3 weeks for ≥16 hours per day in the absence of an acute reversible event or after resolution of an acute reversible event as assessed using the Kaplan-Meier method.

[0136] Complete response can include evaluating a neonatal subject according to one or more of the above results, and the resulting evaluation falls within the expected range of a neonatal subject without SMA or is considered normal. For example, if a neonatal subject is at or below the third percentile of the normal range of weight for age, height for age, and height for age / visit time, and head circumference for age / visit time based on the World Health Organization (WHO) child growth standard, it is considered that its pregnancy is small. Therefore, a complete response can include evaluating one or more of weight for age, height for age, and height for age / visit time, and head circumference for age / visit time based on the World Health Organization (WHO) child growth standard, and finding that the neonatal subject is higher than the third percentile or normal range. As another example, if a neonatal subject has a scale score of less than 1.5 standard deviations of the time reference standard as measured by the third edition of the Bailey Scales of Infant Development (BSID-III) cognitive scale, it is considered that it has cognitive impairment. Thus, a complete response may include evaluating a neonatal subject for cognitive deficits, as measured by the Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III) cognitive scale; and finding that the scale score is no less than 1.5 standard deviations of the time reference. A complete response may include demonstrating normal, age-appropriate tone, posture, strength, activity, reflexes, eating, and breathing patterns based on a neurological examination. Based on a birth weight table, a complete response may include a normal birth weight for its pregnancy. A complete response may include a head circumference for age / visit time that is above the third percentile of the normal range based on the WHO Child Growth Standards (WHO 2019). In some embodiments, administering lisapram to a carrier during pregnancy can increase the chance of being evaluated as normal based on one or more of the criteria in Table 1, compared to not administering lisapram to the carrier during pregnancy. Such an increase in the chance of achieving the evaluation (up to and including a normal response) can be evaluated or demonstrated by comparing newborn subjects with SMA who were administered lisapram by their carriers during pregnancy to newborn subjects with SMA who were not administered lisapram by their carriers during pregnancy.

[0137] The clinical response can be assessed at any suitable time or times during or after the disclosed treatment. In an embodiment, the clinical response is determined after the carrier treatment cycle is completed. In an embodiment, the clinical response is determined after the neonatal treatment cycle is completed. In an embodiment, the clinical response is determined after the carrier and neonatal treatment cycles are completed. In an embodiment, the clinical response is determined after the carrier treatment cycle is completed but before the neonatal treatment cycle begins.

[0138] In some embodiments, a neonatal treatment cycle is not required and treatment ends after the carrier treatment cycle. In some embodiments, a neonatal treatment cycle is required. For example, for neonatal subjects who do not achieve a complete response like fetal subjects after the carrier treatment cycle, or for neonatal subjects who may benefit from more than one treatment cycle, the neonatal treatment cycle can provide effective treatment. In some embodiments, neonatal subjects receiving a neonatal treatment cycle achieve a complete response after the carrier treatment cycle, like fetal subjects. In some embodiments, neonatal subjects receiving a neonatal treatment cycle do not achieve a complete response after the carrier treatment cycle, like fetal subjects (e.g., achieve only a partial response or exhibit stable or progressive disease). In embodiments, neonatal subjects eligible for additional treatment cycles show a clinical response after the carrier treatment cycle but before the neonatal treatment cycle. In embodiments, eligible neonatal subjects show a partial clinical response. In embodiments, eligible neonatal subjects show stable disease. In embodiments, eligible neonatal subjects show progressive disease. In embodiments, eligible neonatal subjects may be asymptomatic, but due to the progressive nature of SMA, may still require a second SMA therapy (e.g., to prevent disease manifestation or maintain the benefits of fetal therapy).

[0139] In embodiments, the treatment achieves a beneficial clinical response (e.g., partial response or stable disease) for at least one of the symptoms after the carrier treatment cycle but before the neonatal treatment cycle. In embodiments, the disclosed treatment does not achieve complete remission for at least one of the symptoms after the carrier treatment cycle (e.g., achieves partial remission or stable disease), but achieves complete remission for at least one of the symptoms after the neonatal treatment cycle. In embodiments, the disclosed treatment achieves a beneficial clinical response (e.g., complete response, partial response, or stable disease) for at least one of the symptoms.

[0140] Examples

[0141] The following examples are set forth to provide a more complete understanding of the disclosure. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting the disclosure in any way.

[0142] Example 1. A single patient study protocol designed to evaluate the safety and efficacy of risdiplam in the treatment of SMA in fetal patients

[0143] Fetal subjects were considered eligible for inclusion in the study if they met all of the following criteria: (1) the fetal subject was diagnosed with SMA by genetic testing, (2) the fetal subject was in the third trimester of development, (3) the fetal subject's gestation was within the range of about twenty-eight weeks to about thirty-eight weeks of gestation, (4) the fetal subject had no detectable functional copies of the SMN1 gene, and (5) the fetal subject had at least one copy of the SMN2 gene. Specifically, genetic testing of fetal subjects was highly predictive of type 1 SMA, which genetic testing combined with a family history of SMA confirmed no copies of the SMN1 gene and two copies of the SMN2 gene. See Jones (2020) J. Neuromuscul. Dis. 7:33-40; and Glascock (2018) J. Neuromuscul. Dis. 5(2):145-158. Fetal subjects will be excluded from this study if: (1) the fetal subject is in the first or second trimester of development, (2) the fetal subject has at least one copy of the SMN1 gene, or (3) the fetal subject has less than one copy of the SMN2 gene.

[0144] Fetal subjects assigned to this treatment plan received lisapram via the carrier during the third trimester of development, i.e., within the range of about thirty-two and a half weeks to their delivery from the carrier at about thirty-eight and a half weeks of gestation, after all essential organ systems of the fetal subject had substantially completed basal embryogenesis. During this period, lisapram was administered to the fetal subject via a carrier treatment cycle, wherein a single daily dose of 5 mg of lisapram was orally administered to the carrier of the fetal subject.

[0145] The carrier treatment cycle is terminated when the carrier of the fetal subject delivers the fetal subject as a neonatal subject. The carrier treatment cycle may also be terminated if an adverse change in the biophysical score of the fetal subject is detected. The carrier treatment cycle may also be terminated if the expected growth rate of fetal growth decreases by more than 10% compared to baseline. Ten percent was selected to confirm the variability of fetal weight in fetal ultrasound predictions (using the Hadlock A formula, the generally accepted error rate is 5%). See Milner (2018) Ultrasound 26(1):32-41. The carrier treatment cycle may also be terminated if fetal organs change in ventricular size, cardiac anatomy, kidneys, or liver. In addition, the carrier treatment cycle may also be terminated if unmanageable medical problems occur (including, for example, pregnancy-induced hypertension / preeclampsia in the case of carriers). None of these events occurred.

[0146] Trough drug levels of lisapram were obtained from carriers and subjects who were neonatal subjects at the time points presented in Tables 2A and 2B, i.e., from 32.5 weeks of gestation before administration of lisapram to carriers to 43 days after delivery. Lisapram drug levels (ng / mL) detected in plasma of carriers and neonatal subjects were determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Table 2B is a continuation of Table 2A.

[0147] Table 2A.

[0148]

[0149] *Below the limit of quantification

[0150] Table 2B.

[0151]

[0152] As set forth in Table 2A, steady-state plasma levels of lisporam in carriers were approximately 14 ng / mL. Steady-state plasma lisporam levels in carriers were achieved by 3 weeks and remained roughly stable despite changes in amniotic fluid volume and volume of distribution. Since actual drug levels in fetal subjects cannot be determined, but can be predicted using cord blood levels at delivery as a surrogate measure, it is still possible that steady-state may be achieved more quickly.

[0153] According to Table 2B, the level of lisporam in amniotic fluid (AF) was about 33% of the carrier plasma venous (V) lisporam level, while the level of lisporam in the umbilical cord venous (V) and arterial (A) plasma of fetal subjects was about 69% of the carrier plasma lisporam drug level. The drug elimination half-life in the umbilical cord blood and postpartum day 8 (d8) samples of neonatal subjects was 46 hours.

[0154] Safety considerations need to be prioritized to avoid excessively exceeding the mean ssAUC 0-24 The following considerations may be given: the liver of neonates may be relatively immature and therefore the rate of hepatic metabolism of lisapram may be reduced compared with older subjects, and any transient exposure of lactating neonates via breast milk.

[0155] The elimination half-life calculated for the neonatal subjects indicated that there was no need to delay the initiation of lisporam in the neonatal subjects, and the neonatal subjects were fed with formula milk, thereby avoiding additional drug administration via breast milk. Since it was determined that the level of lisporam in the umbilical cord blood of the neonatal subjects was less than about 100 ng / mL or about 0.1 mg / L at birth, the neonatal treatment cycle was started. The neonatal treatment cycle started on day 8 (d8) after birth and included administering lisporam to the neonatal subjects at a single daily dose of 0.15 mg / kg.

[0156] After 35 days of oral treatment of neonatal subjects at a dose of 0.15 mg / kg / day, the drug trough level at d43 (62 ng / ml) was similar to the reference data. See Baranello (2021) N. Engl. J. Med. 384: 915-923. In the reference study, the first group of four infants were each treated with a final dose of 0.08 mg / kg body weight / day of lisporam at 12 months of treatment, and the second group of seventeen infants were each treated with a final dose of 0.2 mg / kg body weight / day of lisporam at 12 months of treatment. See Baranello (2021) N. Engl. J. Med. 384 (10): 915-923. It is estimated that the lisporam drug level in the plasma of neonatal subjects is similar to the exposure observed in the reference study.

[0157] The antenatal study schedule and administration schedule for lisapram are summarized in Table 3.

[0158] Table 3.

[0159]

[0160] *Lisapram dosing occurred once daily throughout the study.

[0161] As shown in Table 3, "X1" occurs once a week, or more frequently as may be determined by the physician. X1 includes routine monitoring of carriers during the third trimester using relevant blood and urine tests, particularly for the development of hypertension and features of pre-eclampsia or HELLP syndrome. X1 also includes monitoring of carriers for nausea, vomiting, or dehydration, as well as placental integrity and amniotic fluid volume. X1 further includes fetal monitoring of fetal growth, organ system development, and fetal health, as well as any signs of organ maldevelopment, particularly in the brain, heart, kidneys, and liver. A baseline full anatomical ultrasound study and growth scan were performed prior to treatment, and follow-up ultrasound examinations were performed three weeks and six weeks prior to delivery of the fetal subject to become a neonatal subject.

[0162] In addition, in Table 3, "X2" includes safety laboratory data of carriers, such as CBC screening, CMP screening, and UA to assess the potential toxicity of lisapram, which are collected before dosing and then before delivery. Safety laboratory data are collected at baseline before dosing, and then every 3-4 weeks until delivery, including preoperative data before planned cesarean section. More frequent testing may be performed if clinically indicated.

[0163] In addition, in Table 3, blood samples for lisiporam PK and PD were collected from carriers before the first dose of lisiporam and 24 hours after the dose of lisiporam. PK / PD samples were collected from carriers every two weeks for up to six weeks after delivery to assess lisiporam clearance. At delivery, umbilical cord blood and amniotic fluid samples were collected to assess the exposure of lisiporam to fetal subjects in utero. If amniocentesis is deemed necessary, the collection of umbilical cord blood and amniotic fluid may also include the collection of umbilical cord blood and amniotic fluid during the carrier's pregnancy.

[0164] The neonatal study plan and dosing schedule for lisapram are summarized in Table 4.

[0165] Table 4.

[0166]

[0167] In Table 4, the “X5” post-delivery PK / PD collection and safety experimental data for neonatal subjects include CBC screening, CMP screening, and UA, and such safety experimental data can be repeated if the results are considered abnormal compared with healthy individuals.

[0168] Table 5 summarizes the results of clinical evaluations in carrier and fetal subjects when exposed to lisprolam during carrier pregnancy, and in neonatal subjects 6 weeks after delivery, a minimum of 30 days after the last dose of lisprolam.

[0169] Table 5 describes the treatment-emergent adverse events detected from this example, the severity of the event, the intensity of the adverse event, the relevance of the adverse event to the administration of lisapram, the study duration, and the start and end dates of the adverse event.

[0170] Each adverse event term is associated with a Medical Dictionary for Regulatory Activities (or MedDRA) term and the intensity is classified based on the CTCAE grade. In Table 5, "Y" means yes and "N" means no, and the intensity is measured from the lowest intensity level or "I" to the highest intensity level or "V." "VSD" is ventricular septal defect, "GERD" is gastroesophageal reflux disease, and "NICU" refers to neonatal intensive care unit.

[0171] Table 5.

[0172]

[0173]

[0174] *The neonatal subject was born 43 days after the start of lisapram administration to the carrier, and lisapram administration to the neonatal subject was started 8 days after birth.

[0175] Figure 1 Included are diagrams related to administering an amount of lisapram to a carrier of a fetal subject and monitoring the carrier and the fetal subject. Specifically, Figure 1 A depicts events associated with carrier and fetal subjects, Figure 1 B depicts assessments relevant to carrier and fetal subjects, Figure 1 C depicts significant adverse events associated with carrier and fetal subjects, and Figure 1 D depicts interventions associated with carrier and fetal subjects during administration of the amount of lisapram.

[0176] The neonatal subject's postpartum course was complicated by fetal fluid retention in the lungs, which manifested as mild tachypnea and hypoxemia in the delivery room. Hypoglycemia was not identified. The neonatal subject received 2 days of care in the neonatal intensive care unit (NICU), including intubation and mechanical ventilation for 3 hours, was quickly restored to room air, and no longer required support on the third day after delivery. As shown in Table 5, the neonatal subject's initial transition difficulty (attributable to retained fetal fluid in the neonatal subject's lungs) and VSD were unlikely to be related to lisprolam. Minor congenital heart defects were reported in 11% of 56 untreated individuals with SMA-1 and SMN2X2. See Rudnik- (2008) J. Med. Genet. 45: 635-638. Intermittent lethargy and slow weight gain during the first 3 weeks were not considered excessive and were unlikely to be drug related. Thus, carriers and subjects (as fetal subjects and neonatal subjects) tolerated each procedure and treatment cycle without adverse events related to the drug.

[0177] Table 6 describes further clinical evaluations relevant to this example.

[0178] Table 6.

[0179]

[0180] *The average for a typically developing infant at 3 months of age is 5.5 mV) and # Mean score for typically developing infants at 3.3 months = 50 (SD 10, range 32-62).

[0181] Despite the administration of lisapram, surveillance laboratory data during the carrier pregnancy were normal until proteinuria was detected at 36 WG, prompting a recommendation for a change to bed rest until a scheduled cesarean section at 396 / 7 WG. There were no signs of infection or gestational diabetes. Ultrasound evaluation identified an increase in amniotic fluid volume at 37 WG.

[0182] Fetal ultrasound examination was unremarkable and growth parameters followed a normal trajectory. Biophysical scores were normal at each time point. The experimental data on transient hypercapnia presented by the neonatal subject in the absence of acidosis or hypoxemia despite being in the NICU is noteworthy. CBC, CMP and U / A were normal, as shown in Table 6.

[0183] The postpartum course was complicated by transient hypertension, for which the carrier took antihypertensive medication for 11 days. A planned repeat cesarean section delivery at 38 weeks 6 days was uncomplicated.

[0184] After birth, neonatal subjects were assessed at approximately one week of age and again at approximately six weeks of age using the Hammersmith Infant Neurological Examination (HINE), the Bayley Scales of Infant and Toddler Development, 4th edition (“Bayley Scales of Infant and Toddler Development”), and the TM -4”), Children’s Hospital of Philadelphia Infant Neuromuscular Disorders Test (CHOP INTEND), Oral and Swallowing Ability Tool (OrSAT), Respiratory Inductance Plethysmography (RIP), complete physical examination, anthropometric measurements (including vital signs, length, weight, head circumference, and chest circumference), nerve conduction studies (specifically compound muscle action potential (CMAP) and motor unit number estimate (MUNE)), brain and muscle ultrasound, blood and urine collection for CBC, CMP, U / A, and lisapram PK / PD. If the neonatal subject has not started any SMN-directed therapy, the CBC, CMP, and U / A may be repeated at six weeks if clinically indicated at the time of the physical examination or if the one-week laboratory data have clinically significant abnormal results.

[0185] The maximum score of HINE-2 is 26 points, wherein higher scores reflect higher functions, respectively. The score of a neonatal subject is 8 points, which may indicate the motor ability of a neonatal subject.

[0186] Bayley TM-4 is a comprehensive assessment tool for determining high-risk newborns with developmental delay. See Bayley & Aylward (2019) Bayley Scales of Infant and Toddler Development (4th Edition) Technical Manual. Bloomington, MN: NCS Pearson. Bayley TM -4 scores come from an assessment of early childhood development. Specifically, Bayley TM -4 tests the neonatal subjects' developmental skills such as cognition, language, and motor skills. The scores indicate how the neonatal subjects perform in these areas compared to a group of neonatal subjects of the same age. Bayley TM -4 The mean standard score is 100 and the standard deviation is 15. As shown in Table 6, Bayley TM -4 was performed at 18 weeks of age, and the standard scores of motor composite, global communication, cognition, social, and adaptive behavior of the neonatal subjects were within normal ranges.

[0187] In Table 6, "CHOP INTEND" refers to such a test, which provides information about the strength of the muscles of the newborn subject and the ability of the newborn subject to control its muscles. During the CHOP INTEND inspection, the health care provider will measure 16 types of muscle actions, including head control (keeping the head upright), elbow flexion and knee extension (bending joints), arm and leg mobility and handshake. Each of the 16 motor skills is given a score from 0 to 4, wherein the score is 0 indicating that the subject cannot complete the action, and the intermediate score of 1,2 or 3 means that the subject can partially perform the motor skill, and 4 points means that the subject can completely complete the action by himself without assistance. All CHOP INTEND scores add up to a total score, and the highest possible score of the CHOP INTEND test is 64. If the newborn is scored as 50 at 6 weeks and scored as 55 at 18 weeks, it is considered that the newborn subject is normal.

[0188] The "OrSAT" or "Oral and Swallowing Ability Tool" is one such tool that is specifically designed to record structured information about different aspects of oral, swallowing and feeding abilities of patients with SMA type 1. See Berti (2022) Arch. Dis. Child. archdischild-2022-323899. Specifically, the OrSAT includes several questions in which responses are graded using a scoring system: each item is scored as 0 or 1 depending on whether the patient is able or unable to perform a given activity. Since some items are age-dependent, the number of items and the maximum score increase with age. If the neonatal subject is less than 6 months of age and swallowing of semi-solids or solids cannot be assessed, the maximum score is 7, while if the neonatal subject is between 6 and 9 months of age and this criterion can be assessed, the maximum score is 10. If the neonatal subject is 10 months of age or older and the consistency of all foods, including solids, can be assessed, the maximum total score is 12. See Berti (2021) J. Neuromuscul. Dis. 8(4):589-601. As shown in Table 6, an OrSAT score of 7 / 7 is considered normal.

[0189] The electrophysiological detection described in this example can serve as a prognostic biomarker. See Pino (2021) Biomark. Insights. 16: 11772719211035643. Electrophysiological studies were performed on a Natus UltraPro S100 instrument using standard techniques, with Natus pre-gelled disposable surface electrodes (#9013S0242) and standard settings for motor nerve conduction studies (LFF 10Hz and HFF 10MHz). The right ulnar nerve was stimulated at the elbow and recorded from the abductor digiti minimi (ADM), and the peroneal (fibular) nerve was stimulated at the knee and recorded from the tibialis anterior (TA).

[0190] Electrophysiological measurements, such as the compound muscle action potential (CMAP) and motor unit number estimate (MUNE) measured in millivolts (mV), can monitor the functional status of the motor unit pool and are particularly relevant to motor neuron disorders. See Arnold (2014) Ann. Clin. Transl. Neurol. 1(1):34:44. Cross-sectional studies have shown that CMAP and MUNE are associated with other measures of motor function, clinical severity, and overall function. See Arnold (2014) Ann. Clin. Transl. Neurol. 1(1):34:44.

[0191] The CMAP response measures the output of the motor units supplying a specific muscle or muscle group. The size of the CMAP depends on the size and number of muscle fibers that depolarize after supramaximal nerve stimulation. Three active electrodes were placed for each test nerve, and the maximum amplitude of the CMAP negative peak was used for analysis. As a reference study for the CMAP data provided in Table 6, an ongoing Phase 2 open-label study (NURTURE) is designed to evaluate the safety and efficacy of nusinersen in preventing or significantly reducing the severity of SMA when started before symptom onset. See De Vivo (2019) Neuromuscul. Disord. 29(11): 842-856. According to this reference study, the mean (SD) of the CMAP amplitude was 2.7 (1.5) and the median (range) was 2.3 (1.0-6.7), while the mean (SD) of NeuroNEXT (typically developing infants) was 5.5±2.0. See Kolb (2016) Ann. Clin. Transl. Neurol. 3(2): 132-45. The conventional ulnar CMAP score is 5.5 mV (+ / - 2) as reported in the NeuroNEXT study, which compared infants with SMA to typically developing infants. See Kolb (2016) Ann. Clin. Transl. Neurol. 3(2): 132-45. According to this referenced study of SMA involving neonatal subjects with 2 copies of SMN2 (SMN2X2), the median peroneal CMAP was 3.20 mV, with a range of 1.1-9.7 mV, a mean of 2.69 mV, and a standard deviation of 1.516 mV. See De Vivo (2019) Neuromuscul. Disord. 29(11): 842-856. Compared to the ulnar nerve CMAP (median 2.3 mV) of participants with SMN2XS in the NURTURE study, the data for neonatal subjects were 4.6 mV (2-fold higher) at 1 week and 5.8 mV (2.5-fold higher) at 6 weeks, indicating relative preservation of motor neurons in neonatal subjects. See De Vivo (2019) Neuromuscl. Discord. 29:842-856. These values ​​are similar to the 5.5 mV mean reported for typically developing neonatal subjects at an average age of 3.3 months. See Kolb (2016) Ann. Clin. Transl. Neurol. 3:132-145.

[0192] MUNE is an electrophysiological method that measures the number of motor units supplying a specific muscle. It should be understood that the "N" in MUNE (ulnar nerve) in Table 6 refers to "nerve". For presymptomatic SMA neonates, MUNE (ulnar N) ranges from 70-270. See Bromberg (2002) Muscle Nerve. 25(3):445-7; and Swoboda (2005) Ann Neurol. 57(5):704-12. In this example, MUNE was performed on the right ulnar nerve using a multipoint technique. At least ten unique single motor unit potentials were captured, the average of which was used to calculate MUNE. Published data on MUNE for five presymptomatic neonates with SMA range from 70 to 270, indicating that the neonatal subjects still maintained a large pool of motor neurons at at least 6 weeks of age. See Swoboda et al. (2005) Ann. Neurol. 57:704-712; and Bromberg (2002) Muscle Nerve 25:445-447.

[0193] Muscle ultrasound was performed using an 11 mHz linear array transducer (60 mm wide) in B mode (GE Vivid E95; GE, Chicago, IL, USA) to evaluate fasciculations in the right and left biceps brachii, abductor digiti minimi, quadriceps femoris, transverse abdominis, gastrocnemius-soleus, lumbar paraspinal muscles, and tongue. The thickness of the quadriceps femoris was measured. These examinations were performed by two experienced reviewers.

[0194] Tables 7A and 7B show the SMN protein levels (in ng / mL) in whole blood of the carriers and the subjects who were fetal subjects and neonatal subjects of this example, and the reference values ​​are shown in Table 7C below. Table 7B is a continuation of Table 7A from 32.5 weeks of gestation before administration of lisapram to the carriers to 43 days after delivery. SMN protein levels in whole blood were measured on the platform.

[0195] Table 7A.

[0196]

[0197] Table 7B.

[0198]

[0199] As shown in Table 7A and Table 7B, the SMN protein level of carriers decreased by 48% from prenatal treatment until delivery, and then increased by 15-28% in the postpartum period. The SMN protein level of the newborn subjects decreased by 52% from the umbilical cord blood level to d8, during which no drug was administered, and then increased by 36% on d43 starting with medication from d8.

[0200] SMN protein levels decrease with age, and theoretically, the decrease in SMN protein levels in carriers may reflect a similar situation during pregnancy. SMN protein levels at birth in newborn subjects suggest a pharmacodynamic effect of prenatal treatment. The target blood levels of SMN in fetal subjects required to ensure optimal survival and function of motor neurons during fetal development are unclear. Unlike the theoretical risks of SMN overexpression from gene therapy (e.g., apione therapy) (see Van Alstyne (2021) Nat. Neurosci. 24(7): 930-940), there is no such concern for lisprolam because its action is limited to the maximum possible shift in SMN2 pre-mRNA splicing to fully include exon 7 in the transcript.

[0201] Figure 2 Depicted is a graph comparing SMN protein levels in whole blood in Example 1 with a reference study having reference SMN protein levels provided in Table 7C.

[0202] Table 7C.

[0203]

[0204] *See Baranello (2021) N. Engl. J. Med. 384(10):915-923.

[0205] exist Figure 2 In Table 7C, the x-axis is associated with the study day and the y-axis is associated with the median SMN protein level in whole blood (measured in ng / mL). Lines D1, D2, and D3 are depicted. In Table 7C associated with the reference study, line D1 refers to the low dose of 0.08 mg / kg / day and line D2 refers to the high dose of 0.2 mg / kg / day. Line D3 depicts the data points associated with this example. As in Figure 2 As can be seen in the figure, at d0, the prenatal SMN protein level in whole blood was about twice the reference amount. At d8, after discontinuation of lisproram, the prenatal SMN protein level in whole blood decreased by about 52%, and at d43, the prenatal SMN protein level in whole blood was the median of the reference amount.

[0206] Plasma and serum samples were assayed for neurofilament light chain and phosphorylated heavy chain using ELISA assays according to the manufacturer's instructions (ProteinSimple, San Jose, CA, USA). Two rounds were performed, with three aliquots run on the same plate in each round and the average value used. The average of the two rounds was used for interpretation and construction Figure 3 and Figure 4 .

[0207] Figure 3 providing a graph depicting average neurofilament levels in the blood of carriers on different days according to at least some embodiments disclosed herein, and Figure 4 A graph is provided depicting average neurofilament levels in the blood of neonatal subjects on different days according to at least some embodiments disclosed herein.

[0208] Figure 3 and Figure 4 The x-axis in is related to the date. Figure 3 The x-axis in includes both antenatal dates (eg, 32.5 WG or gestational weeks – 37.6 WG) and postnatal dates or “PNDs” (eg, d0-d43, referring to days after the fetal subject was born as a neonatal subject). Figure 3 and Figure 4 The y-axis in FIG. 5 is related to the mean neurofilament level in blood (measured in pg / mL).

[0209] Further, in Figure 3 and Figure 4 In the text, "NF-L" refers to "neurofilament light chain", which is a neuronal protein highly expressed in large-caliber myelinated axons. In a variety of neurological disorders (including inflammatory diseases, neurodegenerative diseases, traumatic diseases, and cerebrovascular diseases), NF-L levels in cerebrospinal fluid and blood increase in direct proportion to the degree of axonal damage. See Gaetani (2019) J. Neurol. Neurosurg. Psychiatry. 90(8): 870-881. In addition, "pNF-H" refers to the phosphorylated neurofilament heavy chain subunit (or phosphorylated NF-H). In addition, as Figure 3 and Figure 4 As shown in the Figures, "Ser" refers to serum, and "Pla" refers to plasma.

[0210] like Figure 3 and Figure 4As shown, NF-L levels were low, did not vary much, and were similar between carriers and neonatal subjects. pNF-H levels in neonatal subjects at delivery were approximately 10 times higher in neonatal subjects compared to carriers. During the 6-week observation period, postpartum pNF-H levels in carriers increased approximately 2-fold, with relatively small changes in NF-L levels. NF-L and serum pNF-H levels in neonatal subjects increased slightly from cord blood samples to d8 and then stabilized, while plasma pNF-H showed a transient decrease from birth to d8 and then increased to d43.

[0211] Figure 5 Depicts a graph of a reference study measuring baseline plasma pNF-H concentration (pg / mL) levels in NURTURE infants and infants without SMA aged <1 year according to at least some embodiments disclosed herein. See De Vivo (2019) Neuromuscul. Disord. 29(11):842-856 Figure 6 A. Specifically, Figure 5 pNF-H levels in NURTURE were assessed using the ProteinSimple pNF-H ELLA, and if pNF-H concentrations were below the limit of quantification, 7.46 pg / mL was used as an estimate. Baseline pNF-H values ​​for NURTURE infants were obtained prior to or four hours after nusinersen administration on Study Visit Day 1. See De Vivo (2019) Neuromuscul. Disord. 29(11): 842-856. Figure 6 A. Levels in neonatal subjects were similar to those in typically developing infants and were Figure 5 The NURTURE study participants were 35% of their nadir before lisapram administration. Figure 4 The data point with pNF-H of 293 pg / mL shown in Figure 5 The data points shown are related.

[0212] The neurofilament levels in this example can also be compared with limited published data on individuals with type 1 SMA, individuals untreated or treated with nusinersen or apioronic acid, and typically developing infants. See Alves (2021) Mol. Ther. Methods Clin. Dev. 23:524 - 538; De Vivo (2019) Neuromuscl. Discord. 29:842 - 856; Darras (2019) Ann. Clin. Transl. Neurol. 6:932 - 944; and Pironkova (2017) Exp. Ther. Med. 14:228 - 238. Table 8 presents the neurofilament levels (mean, pg / mL) in plasma reported in the literature.

[0213] Table 8.

[0214]

[0215] See 1 Pironkova (2017) Exp. Th. Med. 14:228 - 238; 2 Darras (2019) ACTN 6:932 - 44; 3 Alves (2021) Molec. Ther.: Methods and Clin. Dev. 524 - 38; and 4 De Vivo (2019) Neuromusc. Dis. 29:842–856.

[0216] Results from this example indicate that the pNF - H level is more informative than the relatively low and less reactive NF - L level. The cord blood pNF - H levels (mean 137 ± 54 pg / ml, measured using a different assay) reported for full - term neonate subjects in the NURTURE study were approximately half those of the neonate subjects in this example, indicating a higher level of active axonal lesion in the index cases. See Pironkova (2017) Exp. Ther. Med. 14:228 - 238. In contrast, the median (range) value for "pre - symptomatic" neonate subjects in the NURTURE study who were less than 6 weeks old and had SMN2X2 was 20881 (845 - 52,900), e.g., 1.4% of the median in this example, indicating that fetal subjects exposed to risdiplam may have a favorable response to prenatal stabilization of axonal lesions. See De Vivo (2019) Neuromuscl. Discord. 29:842 - 856.

[0217] According to this example, it is safe and well tolerated to treat carriers with oral lisporam. No treatment-related adverse events were identified by the investigator or treating physician in carriers or subjects who were fetal subjects or neonatal subjects. Lisporam reaches fetal subjects from carriers through the placenta, reaching therapeutically effective steady-state drug levels in fetal subjects, which are about two-thirds of the carrier levels. Compared with symptomatic infants or after oral treatment started postpartum, neonatal subjects had higher SMN protein levels and lower neurofilament levels at birth, demonstrating favorable pharmacodynamic effects, indicating that even moderate exposure to drugs in utero can produce SMN protein increases that will be clinically significant. Clinical observations were good, including normal examinations, high CHOP INTEND scores, and strong CMAP and MUNE.

[0218] Example 2. A Phase II, Open-label, Single-group, Multicenter Study to Evaluate the Safety, Pharmacokinetics, and Efficacy of Lisaprolamine Administered to Carriers for the Treatment of Fetal Subjects Genetically Diagnosed with SMA

[0219] This is an open-label, single-arm, multicenter Phase II study designed to evaluate the safety, pharmacokinetics, pharmacodynamics, and efficacy of lisapram administered to carriers in the third trimester of pregnancy for the treatment of fetal subjects genetically diagnosed with SMA. As stated in Table 9, the primary objective of this study is to determine the safety of lisapram in subjects with SMA treated in utero during the third trimester of pregnancy and in carriers after oral administration of lisapram to them, and to evaluate the pharmacokinetics of lisapram in carriers, fetal subjects, and neonatal subjects with SMA and to assess the pharmacodynamics (SMN protein) of subjects with SMA. The secondary objectives of this study are to evaluate the efficacy of lisapram in subjects with SMA on SMA biomarkers, motor function achievement, survival and permanent ventilation, and feeding ability.

[0220] Table 9.

[0221]

[0222]

[0223]

[0224] The study will enroll carriers aged 18 to 40 years who will begin oral lisapram between gestational weeks 28 and 30 after all eligibility criteria are confirmed to be met. The population for this study is carriers in the third trimester of pregnancy carrying a fetal subject confirmed by genetic diagnosis of SMA with either a homozygous deletion or a heterozygous deletion (predicted as loss of function of both SMN1 and 2 SMN2 gene copies).

[0225] Inclusion criteria

[0226] [1] Potential subjects are eligible for inclusion in the study only if all of the following criteria are met:

[0227] Carriers are pregnant women aged ≥18 and ≤40 years old

[0228] The carrier is apparently healthy and the fetal subject is developing normally, as determined by a medical evaluation performed by the attending obstetrician according to applicable local pregnancy care guidelines. The carrier has undergone antenatal follow-up (prior to enrollment) according to local guidelines and as determined by the attending obstetrician, which may include medical / obstetric / family history, current medication history, nutrition, smoking and drug use history, mental health issues, infectious disease screening, Rh D blood typing, fetal malformation screening, and other risk factor assessments (e.g., gestational diabetes, preeclampsia, venous thromboembolism)

[0229] The carrier's body mass index before pregnancy is ≤ 32 kg / m2 and the weight gain during pregnancy is within the recommended limits based on the pre-pregnancy weight as determined by the attending obstetrician

[0230] Prior to screening, fetal subjects with SMA confirmed by prenatal diagnosis (and documented in the carrier history) as previously determined, tested for cells with homozygous or heterozygous deletions (predicted loss of function of the SMN1 gene and both copies of the SMN2 gene) by amniocentesis or chorionic villus sampling at a local laboratory

[0231] Exclusion criteria

[0232] [2] Potential subjects were excluded from the study if any of the following criteria were met:

[0233] The fetus suffers from severe congenital malformations or other fetal abnormalities that are clinically significant as determined by the attending obstetrician

[0234] Multiple pregnancy (twins or other multiple births)

[0235] Conditions considered high-risk pregnancy factors, such as high blood pressure, diabetes, placental abnormalities, blood clotting disorders, or other problems that the attending obstetrician believes have a serious impact on the outcome of the ongoing pregnancy

[0236] Exposure to genotoxic substances, including drugs and / or environmental factors, within 6 months before or during pregnancy, plus 5 elimination half-lives from the last exposure

[0237] Treatment with any non-genotoxic teratogenic drug within 1 month or 5 elimination half-lives (whichever is longer) before or during pregnancy

[0238] Treatment with investigational therapy within 1 month or 5 elimination half-lives (whichever is longer) before or during pregnancy

[0239] The carrier takes any of the following:

[0240] o Any CYP3A4 inhibitor taken within 2 weeks (or 5 times the elimination half-life, whichever is longer) prior to the first dose of lisprolam, including but not limited to: ketoconazole, miconazole, itraconazole, fluconazole, erythromycin, clarithromycin, ranitidine, cimetidine, and including foods or beverages known to modulate CYP3A activity (e.g., Seville oranges, grapefruit or grapefruit juice, pomegranates, exotic citrus fruits, grapefruit hybrids or juices)

[0241] o Any CYP3A4 inducer taken within 4 weeks (or within 5 times the elimination half-life, whichever is longer) prior to the first dose of lisprolam, including but not limited to: rifampicin, rifabutin, glucocorticoids, carbamazepine, phenytoin, phenobarbital, or St. John's wort

[0242] o Any multiple drug and toxin extrusion (MATE) substrate taken within 2 weeks (or within 5 elimination half-lives, whichever is longer) prior to the first dose of lisprolam

[0243] Any serious medical condition or abnormality in clinical laboratory testing that would prevent safe participation in and completion of the study

[0244] Carriers will receive lisporam from the 28th to 30th week of pregnancy until the fetal subject is delivered to become a newborn subject. Pregnancy should be estimated by the most accurate method as determined by the attending obstetrician. Lisporam will be administered orally at a dose of 5 mg once a day. Newborn subjects will receive lisporam orally at a dose of 0.15 mg / kg body weight once a day after birth for the first two months since birth, and after two months of age until two years of age at a dose of 0.2 mg / kg body weight.

[0245] For carriers, lisapram treatment will be started at an oral dose of 5 mg, once daily. Blood samples will be obtained from carriers during pregnancy to assess lisapram levels. If the carrier requires amniocentesis during the third trimester for clinical reasons, samples of amniotic fluid and fetal cord blood may be collected to monitor fetal subject lisapram exposure. If any of the stopping rules are met, treatment may be stopped.

[0246] Safety monitoring of fetal subjects will consist of the standard of care (SOC) for the pregnancy risk level and will include at least biophysical scoring and comprehensive anatomical and growth ultrasounds. Assessment of carrier safety will include the collection of adverse events or serious adverse events, including selected abnormal pregnancy outcomes (e.g., spontaneous abortion, stillbirth, elective abortion, preterm and postterm birth) and pregnancy complications. Evaluation of the safety of lisapram in subjects with SMA treated in utero will include the collection of adverse events or serious adverse events (including major and minor congenital disorders, small gestational age, prenatal and postnatal growth disorders, and cognitive and neurodevelopmental disorders) until the first two years of life of newborn subjects.

[0247] Once the fetal subject is delivered to a neonatal subject, the carrier will stop treatment with lisapram. At delivery, samples of cord blood (venous and arterial) and amniotic fluid will be collected, if possible, to assess the fetal subject's in utero exposure to lisapram.

[0248] On the second day after birth, neonatal subjects will begin oral treatment with lisapram. Blood samples will be obtained from neonatal subjects to assess lisapram levels and SMN protein.

[0249] After delivery, monitoring of newborn subjects will include study assessments, which include brain ultrasound scans, safety laboratory data (hematology, chemistry panel), vital signs, and ECG. In addition, children will undergo the following assessments: Hammersmith Infant Neurological Examination Module 2 (HINE-2), Children's Hospital of Philadelphia Infant Neuromuscular Disorders Test (CHOP-INTEND), Bayley Scales of Infant and Toddler Development-Third Edition (BSID-III), Oral and Swallowing Ability Tool (OrSAT), Compound Muscle Action Potential (CMAP) amplitude, and phosphorylated neurofilament heavy chain (pNfH).

[0250] Neonatal subjects were considered to have small gestation if they were at or below the third percentile of the normal range for weight-for-age, length / height-for-age, and weight-for-length / height at age / visit time, and head circumference at age / visit time based on the World Health Organization (WHO) Child Growth Standards. Neonatal subjects were considered to have cognitive deficits if they had a scale score below 1.5 standard deviations of the time-referenced standard as measured by the Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III) cognitive scale.

[0251] Pharmacokinetic and pharmacodynamic evaluations of both carriers and neonatal subjects can include measurement of plasma concentrations of lisapram (and its metabolite M1) at designated time points during pregnancy (carriers) and up to 8 weeks after birth (neonatal subjects), and measurement of SMN protein in neonatal subjects with SMA up to 8 weeks after birth.

[0252] Evaluation of the efficacy of lisapram in neonatal subjects treated in utero will be based on SMA biomarkers as early predictors of benefit (CMAP amplitude and neurofilaments) and clinical outcomes assessing motor function and milestones. These outcomes have been extensively used in previous SMA clinical trials and can be objectively assessed. Results of efficacy assessments will be correlated with corresponding data from subjects treated presymptomatically and with normative data from healthy neonatal subjects when available.

[0253] Safety and efficacy in neonatal subjects will continue to be observed for two years, and safety in carriers will be observed until two months after the fetal subjects are delivered as neonatal subjects. Figure 6 is a schematic diagram of the study protocol associated with Example 2. The primary analysis will be performed once the last neonatal subject in the study completes the 2 month study visit.

[0254] Other embodiments

[0255] This application cites various published patent applications, journal articles, and other publications, each of which is incorporated herein by reference.

[0256] The foregoing has described certain non-limiting embodiments of the present disclosure. It will be appreciated by those skilled in the art that various changes and modifications may be made to the specification without departing from the spirit or scope of the present disclosure as defined in the following claims.

Claims

1. A method of treating spinal muscular atrophy (SMA) in a fetal subject in need thereof, the method comprising administering a first amount of lisapram to a carrier of the fetal subject.

2. The method of claim 1, wherein administering the first amount of lisipril to the carrier of the fetal subject results in administering a therapeutically effective amount to the fetal subject.

3. The method according to any one of claims 1 to 2, wherein the fetal subject is in the third trimester of its development.

4. The method of claim 3, wherein the fetal subject is at about twenty-eight to about thirty-eight weeks of gestation.

5. The method of any one of claims 1 to 2, wherein initiation of administering the first amount of liximab to a carrier of the fetal subject occurs when the fetal subject is at least about twenty-eight weeks of gestation.

6. The method of claim 5, wherein initiation of administering the first amount of liximab to a carrier of the fetal subject occurs when the fetal subject is between about twenty-eight weeks and about thirty weeks of gestation.

7. The method of any one of claims 1 to 4, wherein the fetal subject has no detectable functional survival motor neuron 1 (SMN1) gene.

8. The method of any one of claims 1 to 7, wherein the fetal subject has at least one survival motor neuron 2 (SMN2) gene.

9. The method of any one of claims 1 to 7, wherein the fetal subject has two or fewer copies of the survival motor neuron 2 (SMN2) gene.

10. The method of any one of claims 1 to 7, wherein the fetal subject has two copies of the survival motor neuron 2 (SMN2) gene.

11. The method of any one of claims 1 to 10, wherein administering the first amount of lisipriram to the carrier of the fetal subject occurs orally.

12. The method of any one of claims 1 to 11, wherein administering the first amount of lisipriram to the carrier of the fetal subject occurs daily.

13. The method of any one of claims 1 to 12, wherein the first amount of lisipriram is administered at a dose of about 5 mg.

14. The method of any one of claims 1 to 13, further comprising measuring at least one of the plasma concentration of the lisapram and the plasma concentration of a metabolite of the lisapram in the carrier of the fetal subject.

15. The method of claim 1, further comprising terminating administration of the first amount of liximab to the carrier of the fetal subject when the fetal subject is delivered from the carrier as a newborn subject.

16. The method of claim 15, further comprising measuring at least one of the following of the neonatal subject at least once over a period of time: the plasma concentration of the lisporam, the plasma concentration of a metabolite of the lisporam, SMN protein levels, ulnar nerve compound muscle action potential (CMAP) amplitude, phosphorylated neurofilament heavy chain subunit (pNfH) levels, and the Children's Hospital of Philadelphia Infant Neuromuscular Disorders Test (CHOP INTEND) score.

17. The method of claim 16, further comprising measuring neurofilament levels in at least umbilical cord blood in the neonatal subject at least once within the time period.

18. The method of claim 16 or 17, wherein the time period is the day the fetal subject is delivered from the carrier to become the newborn subject.

19. The method of claim 16, wherein the time period is up to six months after the fetal subject becomes the newborn subject from delivery by the carrier.

20. The method of claim 16, wherein the time period is up to three months after the fetal subject becomes the newborn subject from delivery by the carrier.

21. The method of claim 17 or 18, wherein the neonatal subject has elevated neurofilament levels in at least the umbilical cord blood relative to baseline neurofilament levels.

22. The method according to claim 16, in: measuring the SMN protein level in the neonatal subject during a first moment in time and during a second moment in time in the time period, and The first moment occurs before the second amount of lisapram is administered.

23. The method of claim 22, wherein the SMN protein level increases by at least about 20% to about 50% between the first time and the second time.

24. The method of claim 22, wherein the SMN protein level increases by at least about 30% to about 40% between the first time and the second time.

25. The method of claim 15, further comprising administering a second SMA therapy to the neonatal subject.

26. The method of claim 25, wherein initiation of administering the second SMA therapy to the neonatal subject occurs within a time period from birth of the neonatal subject to about sixty days after birth of the neonatal subject.

27. The method of any one of claims 25 to 26, wherein the second SMA therapy is SMN expression gene therapy or SMN2 splicing modification therapy.

28. The method of claim 27, wherein the second SMA therapy is an SMN2 splicing modification therapy comprising administering a second amount of lisapram to the neonatal subject.

29. The method of claim 28, wherein the second amount of lisapram administered to the neonatal subject is in an amount of 0.15 mg / kg.

30. The method of claim 28, wherein the second amount of lisapram administered to the neonatal subject is in an amount of 0.20 mg / kg when the neonatal subject is between about two months and about two years old.

31. The method of claim 28, further comprising: include: After administering the second amount of lisapram to the neonatal subject, the level of SMN protein in the neonatal subject is measured at least once.

32. The method of claim 31, wherein the SMN protein level increases by at least about 20% to about 50% after administering the second amount of lisipril to the neonatal subject.

33. The method of claim 31, wherein the SMN protein level increases by at least about 30% to about 40% after administering the second amount of lisipriram to the neonatal subject.

34. The method of claim 27, wherein the second SMA therapy is an SMN2 splicing modification therapy comprising administering an amount of nusinersen to the neonatal subject.

35. The method of claim 34, wherein the amount of nusinersen administered to the neonatal subject is in an amount equal to or less than 5 mg / mL.

36. The method of claim 34, wherein the amount of nusinersen administered to the neonatal subject is in an amount equal to or less than 2.5 mg / mL.

37. The method of claim 25, wherein the second SMA therapy is an SMN-expressing gene therapy.

38. The method of claim 37, wherein the SMN expression gene therapy comprises an adeno-associated viral vector-based gene therapy.

39. The method of claim 38, wherein the adeno-associated viral vector-based gene therapy comprises administering an amount of apionein-xioi to the neonatal subject.

40. The method of claim 38, wherein the adeno-associated viral vector-based gene therapy comprises administering an amount of apione to the neonatal subject.

41. The method of claim 40, wherein the amount of apioneinol administered to the neonatal subject is equal to or less than 2×10 14 The amount of vector genome (vg).

42. The method of claim 40, wherein the amount of apioneinol administered to the neonatal subject is equal to or less than 1.5 x 10 14 The amount of vector genome (vg).

43. The method of claim 16, wherein lisiporam is administered to the neonatal subject if the level of lisiporam in the blood of the neonatal subject is less than about 100 ng / mL.

44. The method of claim 17, wherein the level of lisapram in the blood of the neonatal subject is the level in the umbilical cord blood of the neonatal subject at the time of delivery.

45. The method of claim 44, wherein at the time of delivery, the mean steady-state area under the concentration-time curve (AUC) of lisapram in the cord blood of the neonatal subject is ss ) is less than about 2000 ng·h / mL.

46. ​​The method of any one of claims 25 to 30, wherein administering the second amount of lisipriram to the neonatal subject occurs daily.

47. The method of any one of claims 25 to 30, wherein administering the second amount of lisipriram to the neonatal subject occurs orally.

48. The method of any one of claims 1 to 47, wherein lisapram is administered in a pharmaceutical composition further comprising ascorbic acid, edetate disodium dihydrate, isomalt, mannitol, polyethylene glycol 6000, sodium benzoate, strawberry flavor, sucralose, and tartaric acid.

49. A method of treating spinal muscular atrophy (SMA) in a neonatal subject in need thereof, the method comprising administering to the neonatal subject a therapeutically effective amount of lisapram, wherein the subject received lisapram in utero as a fetal subject from a carrier of the subject.

50. A method of treating spinal muscular atrophy (SMA) in a fetal subject in need thereof, the method comprising administering to the fetal subject a therapeutically effective amount of lisapram, wherein the fetal subject receives the lisapram in utero via a carrier of the fetal subject.

51. A method of treating spinal muscular atrophy (SMA) in a subject in need thereof, the method include: administering a first amount of lisiporam to a carrier of a fetal subject in a carrier dosing cycle to administer a therapeutically effective amount of lisiporam to the fetal subject; When the fetal subject is delivered from the carrier to become a newborn subject, terminating the carrier's dosing cycle; as well as A second amount of lisapram is administered to the neonatal subject during a neonatal dosing cycle.

52. Lisapram for use in treating SMA Type 0 or Type 1 in a fetal subject in need thereof.

53. Lisapram according to claim 52, wherein the fetal subject is in the third trimester of its development.

54. Lisapram according to any one of claims 52 to 53, wherein the fetal subject is at about thirty-two to about thirty-eight weeks of gestation.

55. Lisapram according to any one of claims 52 to 54, wherein the fetal subject has no detectable functional survival motor neuron 1 (SMN1) gene.

56. Lisapram according to any one of claims 52 to 55, wherein the fetal subject has at least one survival motor neuron 2 (SMN2) gene.

57. Lisapram according to any one of claims 52 to 55, wherein the fetal subject has two or fewer copies of the survival motor neuron 2 (SMN2) gene.

58. Lisapram according to any one of claims 52 to 55, wherein the fetal subject has two copies of the survival motor neuron 2 (SMN2) gene.

59. Lisporam according to any one of claims 52 to 58, wherein the lisiporam is administered to the carrier of the fetal subject.

60. Lisporam according to claim 59, wherein administration of said lisporam to said carrier of said fetal subject occurs orally.

61. Lisporam according to any one of claims 59 to 60, wherein administration of the lisporam to the carrier of the fetal subject occurs daily.

62. Lisporam according to any one of claims 59 to 61, wherein the lisporam is administered to the carrier of the fetal subject at a dose of about 5 mg.

63. The lisporadic acid of any one of claims 59 to 62, wherein administration of the lisporadic acid to the carrier of the fetal subject is terminated when the fetal subject is delivered as a neonatal subject.

64. Lisapram according to claim 63, wherein the neonatal subject has elevated neurofilament levels in at least umbilical cord blood.

65. The liximab of any one of claims 63 to 64, further comprising administering a second SMA therapy to the neonatal subject.

66. The method of claim 65, wherein the second SMA therapy is administered to the neonatal subject during a period from delivery of the fetal subject to the neonatal subject to about sixty days after the delivery.

67. Lisapram according to any one of claims 65 to 66, wherein the second SMA therapy is SMN expression gene therapy or SMN2 splicing modification therapy.

68. The method of claim 67, wherein the second SMA therapy is a SMN2 splice-modifying therapy, and wherein the SMN2 splice-modifying therapy comprises lisiporam.

69. The lisporam of claim 68, wherein the first administration of the SMN2 splicing-modifying therapy comprising lisporam to the neonatal subject occurs via oral administration daily in an amount of 0.15 mg / kg.

70. The lisiporam of claim 68, wherein the first administration of lisiporam to the neonatal subject occurs if the level of lisiporam in the blood of the neonatal subject is less than about 100 ng / mL.

71. The method of claim 68, wherein the level of lisiporam in the blood of the neonatal subject is the level in the umbilical cord blood of the neonatal subject at the time of delivery of the fetal subject to become the neonatal subject.

72. The method of any one of claims 68 to 71, wherein a second administration of the SMN2 splicing-modifying therapy comprising lisiporam to the neonatal subject occurs via daily oral administration in an amount equal to or less than 0.20 mg / kg, and wherein the second administration of lisiporam occurs during a time period of about 2 months to about 2 years after delivery of the neonatal subject.

73. The lisporam of any one of claims 52 to 72, wherein the lisporam is administered in a pharmaceutical composition further comprising ascorbic acid, edetate disodium dihydrate, isomalt, mannitol, polyethylene glycol 6000, sodium benzoate, strawberry flavor, sucralose, and tartaric acid.

74. Lisapram according to any one of claims 52 to 73, wherein the SMA is type 0 SMA or type 1 SMA.

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