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US-20260124227-A1 - BIOLOGIC AGENTS AND METHODS OF USE

US20260124227A1US 20260124227 A1US20260124227 A1US 20260124227A1US-20260124227-A1

Abstract

Disclosed herein are nucleic acid compositions and methods of use. The nucleic acid compositions may have a therapeutic nucleic acid sequence operably linked to a nuclear targeting sequence that increases expression of the therapeutic nucleic acid in a cell by at least 1.25 fold; at least 80% sequence identity to SEQ ID NO: 6; or a first regulatory element comprising a promoter sequence operably linked to a hemoglobin subunit gamma intron (hBGi) sequence, and a second regulatory element comprising a woodchuck hepatitis posttranscriptional regulatory element (WPRE) sequence. The nucleic acid compositions with these features may enhance therapeutic nucleic acid transfection and expression. Also disclosed herein are transgenes optimized for gene therapy applications, including novel FVIII transgene sequences, in which the B domain may be non-naturally occurring the A1 and/or A3 domain may include at least one amino acid substitution.

Inventors

  • Ivan Krivega
  • Margarita KRIVEGA
  • Kenneth Greenberg
  • Steven B. Feinstein

Assignees

  • SONOTHERA, INC.

Dates

Publication Date
20260507
Application Date
20251008

Claims (20)

  1. 1 . A nucleic acid composition comprising a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 6.
  2. 2 . The nucleic acid composition of claim 1 , wherein the nucleic acid sequence is SEQ ID NO: 6.
  3. 3 . The nucleic acid composition of claim 1 , further comprising a therapeutic nucleic acid sequence, wherein the nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 6 increases expression of the therapeutic nucleic acid sequence in a cell by at least 1.25-fold as compared to an otherwise identical control composition lacking the nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 6.
  4. 4 . The nucleic acid composition of claim 1 , wherein the nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 6 is a nuclear targeting sequence.
  5. 5 . The nucleic acid composition of claim 1 , wherein the nucleic acid composition is an isolated nucleic acid molecule.
  6. 6 . The nucleic acid composition of claim 1 , wherein the nucleic acid composition further comprises one or more Inverted Terminal Repeat (ITR) sequences.
  7. 7 . The nucleic acid composition of claim 1 , wherein the nucleic acid composition is a non-viral vector.
  8. 8 . A kit comprising a sonoactive agent and a nucleic acid composition, wherein the nucleic acid composition comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 6.
  9. 9 . The kit of claim 8 , wherein the nucleic acid sequence is SEQ ID NO: 6.
  10. 10 . The kit of claim 8 , nucleic acid composition further comprises a therapeutic nucleic acid sequence, wherein the nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 6 increases expression of the therapeutic nucleic acid sequence in a cell by at least 1.25-fold as compared to an otherwise identical control composition lacking the nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 6.
  11. 11 . The kit of claim 8 , wherein the nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 6 is a nuclear targeting sequence.
  12. 12 . The kit of claim 8 , wherein the nucleic acid composition is an isolated nucleic acid molecule.
  13. 13 . The kit of claim 8 , wherein the nucleic acid composition further comprises one or more Inverted Terminal Repeat (ITR) sequences.
  14. 14 . The kit of claim 8 , wherein the nucleic acid composition is a non-viral vector.
  15. 15 . The kit of claim 8 , wherein the sonoactive agent comprises a shell filled with a perfluorinated gas.
  16. 16 . The kit of claim 8 , wherein the sonoactive agent comprises protein-stabilized microstructures.
  17. 17 . The kit of claim 8 , wherein the sonoactive agent comprises lipid-stabilized microstructures.
  18. 18 . The kit of claim 17 , wherein the lipid-stabilized microstructures comprise a lipid stabilized shell surrounding a perfluorinated gas core.
  19. 19 . The kit of claim 18 , wherein the lipid stabilized shell comprises a monomolecular membrane of hydrogenated egg yolk phosphatidyl serine, wherein the perfluorinated gas core comprises perfluorobutane gas.
  20. 20 . The kit of claim 8 , further comprising instructions for administering ultrasound acoustic energy to a subject to facilitate delivery of the nucleic acid composition to a subject.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of International Application No. PCT/US2025/024558, filed Apr. 14, 2025, which claims the benefit of U.S. Provisional Patent Application No. 63/634,905, filed Apr. 16, 2024, and U.S. Provisional Patent Application No. 63/709,966, filed Oct. 21, 2024, each of which is incorporated herein by reference in its entirety and for all purposes. INCORPORATION BY REFERENCE OF SEQUENCE LISTING The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file 62668-732301_Sequence_Listing.XML, created Oct. 8, 2025, which is 147,357 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. BACKGROUND Gene therapy, in which a functional copy of a gene is transfected or otherwise delivered into a cell, has been proposed as a possible method of treating genetic diseases, however many existing gene therapies suffer from significant shortcomings when used to treat subjects due to failure to achieve or maintain therapeutic levels of transgene expression, which often leaves subjects without an effective treatment due to inability to re-dose many such gene therapies. Vectors for transgene delivery which improve gene transfection, expression, or durability could significantly improve gene therapies and patient outcomes. Similarly, transgenes which provide for improved gene expression or durability, or which otherwise provide for an improved therapeutic effect in a gene therapy treatment, could significantly improve gene therapies and patient outcomes. SUMMARY Vectors for transgene delivery can be improved by the incorporation of regulatory elements which improve gene delivery and transfection. However, the identification of genetic regulatory elements which tend to have a positive therapeutic effect in improving transgene expression remains a challenge. Disclosed herein are nucleic acid compositions comprising genetic regulatory elements operably linked to therapeutic nucleic acid sequences which improve gene transfection and expression. In some cases, the genetic regulatory elements may include novel nuclear targeting sequences which increase nuclear localization of the nucleic acid composition and resulting gene expression. In some cases, the genetic regulatory elements may also include novel combinations of nuclear targeting sequences, promoters, post-transcriptional elements, nuclear targeting sequences, and other genetic regulatory elements, which increase gene transfection and expression. In addition to improved vectors, transgenes themselves can be modified to improve their transfection and expression in a subject, and can otherwise be modified to provide an improved therapeutic effect to a subject. However, the identification of transgene elements which tend to have a positive therapeutic effect in improving transgene expression or function remains a challenge, especially in cases of very large transgenes where modifications at any point may disrupt gene expression or degrade transgene performance. Aspects disclosed herein provide a Factor VIII polypeptide comprising: an A1 domain, and A2 domain, a non-naturally occurring B domain (B), an A3 domain, a C1 domain, and a C2 domain, positioned from N- to C-terminus as A1-A2-B-A3-C1-C2, wherein the B domain comprises SEQ ID NO: 27; and wherein one or both of: the A1 domain comprises at least one amino acid substitution relative to SEQ ID NO: 36; and the A3 domain comprises at least two amino acid substitutions as compared to SEQ ID NO: 37. Aspects disclosed herein provide a Factor VIII polypeptide comprising an amino acid sequence having up to 3 amino acid substitutions relative to SEQ ID NO: 24. In some embodiments, the FVIII polypeptide disclosed herein may improve over prior FVIII transgenes by providing for increased gene expression, secretion, and coagulation activity of the FVIII polypeptide, among other benefits described herein. Aspects disclosed herein provide a nucleic acid composition comprising a therapeutic nucleic acid sequence, wherein the therapeutic nucleic acid sequence encodes a FVIII polypeptide having an A1 domain, and A2 domain, a non-naturally occurring B domain (B), an A3 domain, a C1 domain, and a C2 domain, positioned from N- to C-terminus as A1-A2-B-A3-C1-C2, wherein the B domain comprises SEQ ID NO: 27; and wherein: the A1 domain comprises substitution of phenylalanine at position 328 to serine when the Factor VIII polypeptide is aligned with SEQ ID NO: 35; and the A3 domain comprises substitution of cysteine at position 1249 to glycine when the Factor VIII polypeptide is aligned with SEQ ID NO: 35 and substitution of cysteine at position 1253 to glycine when the Factor VIII polypeptide is aligned with SEQ ID NO: 35. In some embodiments, the therapeutic nucleic acid sequence encodes a Factor VIII (FVIII) polypeptide comprising an amino acid