Nucleotide sequence and pharmaceutical composition based thereon with prolonged VEGF transgene expression

• US 9,896,493 B2
• Filed: 03/15/2016
• Issued: 02/20/2018
• Est. Priority Date: 05/26/2015
• Status: Active Grant

• Current Assignee: NextGen Company Limited (Human Stem Cells Institute OJSC)
• Original Assignee: NextGen Company Limited (Human Stem Cells Institute OJSC)
• Inventors: Kiselev, Sergej L'vovich
• Primary Examiner(s): GALISTEO GONZALEZ, ANTONIO
• Application Number:
  US15/070,239
• Publication Number:
  US 20160347803A1
• Time in Patent Office: 707 Days
• Field of Search: None
• CPC Class Codes: A61K 31/00 Medicinal preparations containing organic active ingredients
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 2830/50 regulating RNA stability, not being an intron, e.g. poly A signal

Abstract

A nucleic acid or transgene comprising a modified VEGF 3′-untranslated region (3′-UTR) polynucleotide sequence and a polynucleotide sequence encoding a Vascular Endothelial Growth Factor (VEGF). When transformed into a host cell, the nucleic acid or transgene exhibits a high stability and provides prolonged and reliable expression of VEGF. A method for extending the lifetime of transgene mRNA encoding VEGF in a mammalian host cell. A method for treating a subject in need of increased or modified expression of VEGF using this nucleic acid or transgene.

Claims

1. An engineered polynucleotide comprising:

a modified 3′

-UTR sequence from a vascular endothelial growth factor (VEGF) gene that comprises at least one nucleotide sequence modification to a native 3′

-UTR sequence of a VEGF gene, and

a structural gene encoding VEGF,

wherein when said engineered polynucleotide is transformed into and transcribed by a cell, its transcription product has an extended lifetime compared to the VEGF transcription product of the polynucleotide described by SEQ ID NO;

1 when transformed into an otherwise identical cell,

wherein the engineered polynucleotide comprises at least one nucleotide sequence modification selected from the group consisting of;

C-deletion at position 1071,

C-deletion at position 1079,

T-deletion at position 1111,

A/C-substitution at position 1144,

A-deletion at position 1148,

C-deletion at position 1155,

A-deletion at position 1173,

G-deletion at position 1083,

A-deletion at position 1185, and

G/C-substitution at position 1536;

wherein said positions correspond to those described by SEQ ID NO;

1, or

the engineered polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO;

2 to SEQ ID NO;

10 and SEQ ID NO;

11.

2. The polynucleotide of claim 1, comprising SEQ ID NO:

11.

3. The engineered polynucleotide of claim 1, wherein when said engineered polynucleotide is transformed into and transcribed by a HEK293 cell, its transcription product has an extended lifetime compared to the VEGF transcription product of the polynucleotide described by SEQ ID NO:

1 when transformed into an otherwise identical HEK293 cell.

4. The engineered polynucleotide of claim 1, wherein the transcription product encoding VEGF is from isoform VEGF-121, VEGF-145, VEGF-148, VEGF-165, VEGF-183, VEGF-189, and VEGF-206.

5. A vector or polynucleotide construct comprising the engineered polynucleotide according to claim 1.

6. The vector or polynucleotide of claim 5 that is a plasmid.

7. The vector or polynucleotide construct of claim 5 that comprises the polynucleotide sequence of SEQ ID NO:

11.

8. A pharmaceutical composition comprising the vector or polynucleotide construct according to claim 5 in a form suitable for administration to a mammalian subject, and, optionally, comprising at least one cryoprotectant and/or pH stabilizer.

9. A cell that has been transformed or transfected with the vector or polynucleotide construct according to claim 5.

10. The cell according to claim 9 that is autologous to a subject receiving said cell.

11. A method for treating a subject in need of VEGF comprising administering the vector or polynucleotide construction of claim 5 to said subject.

12. The method according to claim 11, wherein said subject is in need of regeneration of a connective, neural, muscular, osseous or cardiovascular tissue.