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KR-20260066056-A - Composition and method for treating spinal muscular atrophy

KR20260066056AKR 20260066056 AKR20260066056 AKR 20260066056AKR-20260066056-A

Abstract

An expression cassette for the delivery of a nucleotide sequence encoding hSMN1 and a recombinant AAV vector containing the same are provided. The provided composition is useful for a method for treating spinal muscular atrophy (SMA) in subjects requiring treatment.

Inventors

  • 오르도 줄리에트

Assignees

  • 더 트러스티스 오브 더 유니버시티 오브 펜실베니아

Dates

Publication Date
20260512
Application Date
20240810
Priority Date
20230810

Claims (20)

  1. A recombinant adeno-associated virus (AAV) comprising an AAV capsid containing a vector genome, wherein the vector genome is (a) UbC promoter sequence, (b) Encoding sequence of the survival of motor neuron 1 (SMN1) protein, (c) at least four miR182 target sequences, and (d) polyA sequence It includes an expression cassette containing, The above-mentioned coding sequence, the above-mentioned at least four miR182 target sequences, and the above-mentioned polyA sequence are recombinant AAVs operably linked to the above-mentioned UbC promoter sequence.
  2. In claim 1, the SMN1 protein is a recombinant AAV, which is an SMN1 isoform D protein.
  3. A recombinant AAV according to claim 1 or 2, wherein the encoding sequence comprises the nucleotide sequence of SEQ ID NO. 7 or a nucleotide sequence that is at least 95% identical to SEQ ID NO. 7.
  4. In any one of paragraphs 1 to 3, the AAV capsid is a recombinant AAV, wherein the AAV capsid is a clad F AAV capsid.
  5. In any one of paragraphs 1 to 4, the recombinant AAV, wherein the AAV capsid is an AAVhu68 capsid.
  6. In claim 5, the AAVhu68 capsid comprises recombinant AAV, wherein the VP1, VP2, and VP3 proteins are expressed from a nucleic acid sequence encoding the amino acid sequence (amino acids 1 to 736) of SEQ ID NO. 9.
  7. A recombinant AAV according to any one of claims 1 to 3, wherein the AAV capsid is an AAVhu95 capsid, an AAVhu96 capsid, or an AAV9 capsid.
  8. In any one of claims 1 to 7, the at least four miR182 target sequences are recombinant AAVs located at the 3' of the SMN1 encoding sequence and the 5' of the polyA sequence.
  9. A recombinant AAV according to any one of claims 1 to 7, wherein the at least four miR182 target sequences comprise at least one target sequence located at the 5' of the UbC promoter and at least one target sequence located at the 3' of the SMN1 encoding sequence and the 5' of the polyA sequence.
  10. In any one of claims 1 to 9, each of the miR182 target sequence comprises the nucleotide sequence of SEQ ID NO. 12, recombinant AAV.
  11. In any one of claims 1 to 10, the recombinant AAV wherein the UbC promoter comprises the nucleotide sequence of SEQ ID NO. 10.
  12. A recombinant AAV according to any one of claims 1 to 11, wherein the polyA sequence is a rabbit beta-globin (rBG) polyA sequence.
  13. In claim 12, the rBG polyA sequence comprises the nucleotide sequence of sequence no. 13, recombinant AAV.
  14. In any one of claims 1 to 13, the expression cassette further comprises an intron, recombinant AAV.
  15. In any one of claims 1 to 14, the recombinant AAV wherein the intron comprises the nucleotide sequence of SEQ ID NO. 11.
  16. A recombinant AAV according to any one of claims 1 to 15, wherein the vector genome further comprises an AAV 5' ITR sequence and an AAV 3' ITR sequence, and optionally, the AAV 5' ITR sequence and the AAV 3' ITR sequence are derived from AAV2.
  17. In any one of claims 1 to 16, the expression cassette comprises a nucleic acid sequence of SEQ ID NO. 16 or a nucleic acid sequence that is at least 99% identical to SEQ ID NO. 16, a recombinant AAV.
  18. In any one of claims 1 to 17, the recombinant AAV comprising the nucleic acid sequence of SEQ ID NO. 2 or a sequence that is at least 99% identical to SEQ ID NO. 2.
  19. In any one of claims 1 to 18, the recombinant AAV is formulated in the form of an aqueous suspension for systemic delivery and/or delivery to the central nervous system.
  20. A composition comprising a recombinant AAV stock and an aqueous suspension medium according to any one of claims 1 to 18.

Description

Composition and method for treating spinal muscular atrophy Mention of electronic sequence list The electronic sequence list submitted by this invention under the title "21-10509.PCT.xml" (53,945 bytes, created August 8, 2024) is incorporated herein by reference in its entirety. Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by a mutation in the SMN1 gene, resulting in reduced expression of survival motor neuron (SMN) proteins. It is the most common genetic cause of infant mortality, and the global incidence of SMA is approximately 1 in 10,000 births. SMN proteins play a crucial role in spliciosome assembly, protein translation, cytoskeletal mechanics, and mitochondrial function. The destruction of SMA motor units (motor neurons and the muscle fibers through which they innervate) underlies the most evident clinical symptoms of SMA. Infants with Type 1 SMA exhibit severe symptoms accompanied by severe hypotonia, progressive respiratory distress, and dysphagia. Patient management typically includes physiotherapy, orthopedic support, respiratory management, and management of medullary paralysis. Several treatments for SMA have been developed, including the antisense oligonucleotide nusinersen (Spinraza) that modifies SMN2 protein splicing, the onasemnogene aveparvovec (Zolgensma), and the adeno-associated virus (AAV)-mediated gene therapy risdiplam (Evrysdi), which modifies SMN2 protein splicing. Despite significant progress in SMA treatment, these therapies (in the case of nusinersen and risdiplam) are expensive and may require repeated administration. While AAV-mediated gene therapies are effective with a single dose, the current FDA-approved product for Type I SMA treatment (Zolgensma) is expensive and associated with several serious toxicity cases, including acute liver failure. Additionally, the safety profile of Zolgensma prohibits its extended use in patients with less severe and older Type II/III SMA. What is needed is an improved SMA treatment that is effective in alleviating the symptoms of the disorder while simultaneously reducing potential toxic side effects. In one embodiment, a recombinant adeno-associated virus (AAV) comprising an AAV capsid containing a vector genome is provided herein, wherein the vector genome comprises an expression cassette comprising (a) a UbC promoter sequence, (b) a sequence encoding the functional human motor neuron survival 1 (SMN1) protein, (c) at least four miR182 target sequences, and (d) a polyA sequence, wherein the encoding sequence, at least four miR182 target sequences, and the polyA sequence are operably linked to the UbC promoter sequence. In a specific embodiment, the recombinant AAV of claim 1, wherein the SMN1 protein is the SMN1 isoform D protein. In a specific embodiment, the encoding sequence comprises the nucleotide sequence of SEQ ID NO. 7 or a nucleotide sequence that is at least 95% identical to SEQ ID NO. 7. In a specific embodiment, the recombinant AAV is not a self-complementary AAV vector. In another aspect, a composition comprising a recombinant AAV stock in an aqueous suspension medium is provided herein. In a given embodiment, the suspension is formulated for intrathecal delivery, optionally intrathecal delivery is an intraventricular (ICV) injection or an intracerebellar (ICM) injection. In another aspect, a pharmaceutical composition comprising recombinant AAV and an aqueous formulation buffer is provided herein. In a specific embodiment, the composition is formulated for intrathecal delivery. In a specific embodiment, the composition is formulated for intraventricular (ICV) injection or intracerebellar (ICM) injection. In another embodiment, a recombinant nucleic acid molecule is provided herein, comprising an expression cassette comprising (a) a 5' AAV ITR sequence, (b) a UbC promoter sequence, (c) a coding sequence of a functional human motor neuron survival 1 (SMN1) protein, (d) at least four miR182 target sequences, (e) a polyA sequence, and (f) a 3' AAV ITR sequence, wherein the coding sequence, at least four miR182 target sequences, and the polyA sequence are operably linked to the UbC promoter sequence. In a specific embodiment, the SMN1 protein is an SMN1 isoform D protein. In a specific embodiment, the coding sequence comprises the nucleotide sequence of SEQ ID NO. 7 or a nucleotide sequence that is at least 95% identical to SEQ ID NO. 7. In a specific embodiment, a plasmid comprising the nucleic acid is provided. In another embodiment, a packaged host cell comprising the recombinant nucleic acid molecule or the plasmid is provided herein. In another aspect, a recombinant AAV production system useful for recombinant AAV production is provided herein. In another aspect, a method for treating spinal muscular atrophy (SMA) in a subject requiring treatment is provided herein, said method comprising the step of administering to the subject an aqueous suspension containing recombinant AAV as described herein. In