Search

KR-20260064747-A - Anti-TfR:GAA and anti-CD63:GAA insertion for the treatment of Pompe disease

KR20260064747AKR 20260064747 AKR20260064747 AKR 20260064747AKR-20260064747-A

Abstract

Nucleic acid constructs and compositions are also provided such that the coding sequence of a multi-domain therapeutic protein (e.g., GAA fusion protein) is inserted into a target genomic locus, e.g., an endogenous ALB locus, and/or the coding sequence of a multi-domain therapeutic protein (e.g., GAA fusion protein) is expressed. The nucleic acid constructs and compositions may be used for methods of incorporating the nucleic acid of a multi-domain therapeutic protein (e.g., GAA fusion protein) into a target genomic locus, methods of expressing the multi-domain therapeutic protein (e.g., GAA fusion protein) in cells, methods of reducing glycogen accumulation, methods of treating Pompe disease or GAA deficiency in subjects, and methods of preventing or reducing the onset of signs or symptoms of Pompe disease in subjects including neonatal cells and subjects.

Inventors

  • 백, 앤드류
  • 린, 엘렌
  • 프라가스티스, 마리아
  • 시그나, 캐서린
  • 페파니스, 에반젤로스
  • 사빈, 레아

Assignees

  • 리제너론 파마슈티칼스 인코포레이티드

Dates

Publication Date
20260507
Application Date
20240726
Priority Date
20230728

Claims (20)

  1. A composition comprising a nucleic acid construct comprising a coding sequence of a multidomain therapeutic protein including a delivery domain fused to a lysosomal alpha-glucosidase polypeptide, wherein the lysosomal alpha-glucosidase coding sequence is CpG-depleted compared to the coding sequence of wild-type lysosomal alpha-glucosidase, and optionally, the delivery domain is a TfR-binding delivery domain or a CD63-binding delivery domain.
  2. A composition according to claim 1, wherein the nucleic acid construct comprises a polyadenylation signal or sequence downstream of the coding sequence of a multidomain therapeutic protein.
  3. A composition according to claim 2, wherein the polyadenylation signal comprises a bovine growth hormone (BGH) polyadenylation signal, a Simian virus 40 (SV40) polyadenylation signal, or a combination of a bovine growth hormone polyadenylation signal and an SV40 polyadenylation signal.
  4. A composition according to claim 3, wherein the SV40 polyadenylation signal is a unidirectional SV40 late polyadenylation signal, and each case of the sequence AATAAA in the reverse strand is mutated from the unidirectional SV40 late polyadenylation signal, optionally, the SV40 polyadenylation signal is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence presented in SEQ ID NO. 752, and optionally, the SV40 polyadenylation signal comprises the sequence presented in SEQ ID NO. 752.
  5. A composition according to claim 3 or 4, wherein the polyadenylation signal comprises a BGH polyadenylation signal, optionally, the BGH polyadenylation signal is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence presented in SEQ ID NO. 751, and optionally, the BGH polyadenylation signal comprises the sequence presented in SEQ ID NO. 751.
  6. A composition according to any one of claims 3 to 5, wherein the polyadenylation signal comprises a BGH polyadenylation signal and an SV40 polyadenylation signal, optionally the BGH polyadenylation signal comprises the sequence presented in SEQ ID NO. 751 and the SV40 polyadenylation signal comprises the sequence presented in SEQ ID NO. 752, the polyadenylation signal comprising the BGH polyadenylation signal and the SV40 polyadenylation signal is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence presented in SEQ ID NO. 795, and optionally the polyadenylation signal comprising the BGH polyadenylation signal and the SV40 polyadenylation signal comprises the sequence presented in SEQ ID NO. 795.
  7. A composition according to any one of claims 1 to 6, wherein the nucleic acid construct is a unidirectional nucleic acid construct.
  8. A composition according to any one of claims 1 to 7, wherein the coding sequence of the delivery domain is modified to remove one or more latent splice sites, the coding sequence of the lysosomal alpha-glucosidase polypeptide is modified to remove one or more latent splice sites, or the coding sequence of the multi-domain therapeutic protein is modified to remove one or more latent splice sites.
  9. A composition according to any one of claims 1 to 8, wherein the coding sequence of the delivery domain is CpG-depleted or the coding sequence of the multi-domain therapeutic protein is CpG-depleted.
  10. A composition according to any one of claims 1 to 9, wherein the coding sequence of the delivery domain is codon-optimized and CpG-depleted, the coding sequence of the lysosomal alpha-glucosidase polypeptide is codon-optimized and CpG-depleted, and the coding sequence of the multi-domain therapeutic protein is codon-optimized and CpG-depleted.
  11. A composition according to any one of claims 1 to 10, wherein the nucleic acid composition comprises a splice receptor upstream of the coding sequence of a multi-domain therapeutic protein.
  12. A composition according to any one of claims 1 to 11, wherein the nucleic acid composition does not include a homologous arm.
  13. In any one of claims 1 to 12, the nucleic acid construct comprises a 5' to 3' splice receptor, a coding sequence of a multidomain therapeutic protein, and a polyadenylation signal or sequence, and The nucleic acid construct does not contain a promoter that induces the expression of a multi-domain therapeutic protein, and A nucleic acid composition that does not contain homologous arms.
  14. A composition according to any one of claims 1 to 11, wherein the nucleic acid composition comprises a homologous arm.
  15. A composition according to any one of claims 1 to 14, wherein the nucleic acid composition does not include a promoter that induces the expression of a multi-domain therapeutic protein.
  16. A composition according to any one of claims 1 to 14, wherein the coding sequence of a multi-domain therapeutic protein is operably linked to a promoter, and optionally, the promoter is a liver-specific promoter.
  17. A composition according to any one of claims 1 to 16, wherein the C-terminus of the delivery domain is fused to the N-terminus of the lysosome alpha-glucosidase polypeptide.
  18. A composition according to any one of claims 1 to 17, wherein the transfer domain is fused to a lysosome alpha-glycosidase protein through a peptide linker.
  19. A composition according to any one of claims 1 to 18, wherein the lysosomal alpha-glucosidase lacks a lysosomal alpha-glucosidase signal peptide and a propeptide.
  20. A composition according to any one of claims 1 to 19, wherein the lysosome alpha-glucosidase is essentially composed of the sequence presented in SEQ ID NO. 727 or is composed thereof.

Description

Anti-TfR:GAA and anti-CD63:GAA insertion for the treatment of Pompe disease Cross-reference of related applications This application claims the benefit of U.S. Application No. 63/516,395 filed July 28, 2023, the entirety of which is incorporated herein by reference for all purposes. Reference to the list of sequences submitted as an XML file The sequence list written in file 616967SEQLIST.xml is 1,213,989 bytes in size, was created on July 24, 2024, and is incorporated herein by reference. Pompe disease (PD), or Type II glycogen storage disease, is a monogenic lysosomal disease caused by a deficiency in the activity of the enzyme lysosomal acid alpha-glucosidase (GAA). GAA deficiency leads to the accumulation of its substrate, glycogen, in the lysosomes of cells within tissues, including skeletal muscle and cardiac muscle. This abnormal accumulation of glycogen within muscle fibers results in progressive damage to muscle tissue, with symptoms that may include cardiac hypertrophy, mild to severe muscle weakness, and eventually death due to cardiac or respiratory failure. Infantile-onset PD (IOPD) is associated with less than 1% of normal GAA activity. It is severe and affects visceral organs, muscles, and the central nervous system (CNS). Late-onset PD (LOPD) is associated with 2 to 40% of GAA activity. It is less severe and primarily involves the respiratory system and skeletal muscle. The only approved therapy for PD is enzyme replacement therapy (ERT). Recombinant human (rh) GAAs are delivered to patients by intravenous infusion every two weeks. While ERT has been highly successful in treating the cardiac manifestations of PD, skeletal muscle and the CNS are still minimally treated by ERT. The primary mechanism by which rhGAA reaches the lysosome is absorption via the cation-independent mannose 6-phosphate (M6P) receptor (CIMPR), which binds to M6P on rhGAA. However, CI-MPR expression in skeletal muscle is very low, and rhGAA is insufficiently mannose 6-phosphorylated. Furthermore, CI-MPR can be misdelivered to the autophagic vacuoles of affected cells rather than the lysosome, while a large amount of the drug can also be absorbed by the liver, an organ that does not exhibit the major pathology of PD. ERT does not cross the blood-brain barrier. Furthermore, PD requires early life treatment, which presents additional obstacles due to the unique circumstances of neonatal and adolescent patients. Nucleic acid constructs and compositions are provided such that the coding sequence of a multi-domain therapeutic protein (e.g., GAA fusion protein) is inserted into a target genomic locus, e.g., an endogenous ALB locus, and/or the coding sequence of a multi-domain therapeutic protein (e.g., GAA fusion protein) is expressed. The nucleic acid constructs and compositions may be used in a method of incorporating or inserting the multi-domain therapeutic protein (e.g., GAA fusion protein) nucleic acid into a target genomic locus of a cell or cell population or subject, a method of expressing the multi-domain therapeutic protein (e.g., GAA fusion protein) in a cell or cell population or subject, a method of reducing glycogen accumulation in a cell or cell population or subject, a method of treating Pompe disease or GAA deficiency in a subject, and a method of preventing or reducing the onset of signs or symptoms of Pompe disease in subjects such as subjects having reduced GAA activity or expression, including neonatal subjects, and subjects diagnosed with Pompe disease. In some embodiments, the cell, cell population, or subject is a neonatal cell, a neonatal cell population, or a neonatal subject. In one embodiment, a composition is provided comprising a nucleic acid construct comprising a coding sequence of a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha-glucosidase polypeptide, wherein the lysosomal alpha-glucosidase coding sequence is CpG-depleted relative to the wild-type lysosomal alpha-glucosidase coding sequence, and optionally, the delivery domain is a CD63-binding delivery domain or a TfR-binding delivery domain. In some of these compositions, the nucleic acid construct comprises a polyadenylation signal or sequence downstream of the coding sequence of the multidomain therapeutic protein. In some of these compositions, the polyadenylation signal comprises a bovine growth hormone (BGH) polyadenylation signal, a Simian virus 40 (SV40) polyadenylation signal, or a combination of a bovine growth hormone polyadenylation signal and an SV40 polyadenylation signal. In some of these compositions, the SV40 polyadenylation signal is a unidirectional SV40 late polyadenylation signal, and each case of the sequence AATAAA in the reverse strand is mutated from the unidirectional SV40 late polyadenylation signal, and the SV40 polyadenylation signal is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence presented in SEQ ID NO. 752, and option