Gene therapy using nucleic acid constructs comprising methyl CPG binding protein 2 (MECP2) promoter sequences
By using engineered MeCP2 promoters and introns in AAV vectors, the problems of unstable transgene expression and insufficient CNS targeting in PGRN gene therapy with AAV vectors were solved, achieving efficient PGRN expression and disease correction effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- UCB BIOPHARMA SPRL
- Filing Date
- 2021-08-11
- Publication Date
- 2026-06-09
AI Technical Summary
Existing AAV vectors have difficulty achieving robust transgene expression and CNS targeting when delivering PGRN gene therapy, and traditional methods may affect transgene expression or increase immune response, failing to effectively correct FTD-related behavioral defects.
An engineered MeCP2 promoter, containing a minimal promoter sequence and introns, was linked to a PGRN coding sequence to construct an AAV vector to improve expression levels and transduction efficiency in CNS cells.
It achieved efficient PGRN expression and transduction in CNS cells, corrected symptoms of neurological diseases associated with GRN deficiency, and provided robust transgene expression and CNS targeting.
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Figure CN116113441B_ABST
Abstract
Claims
1. A nucleic acid construct comprising a methyl CpG-binding protein 2 (MeCP2) promoter operatively linked to a nucleotide sequence encoding a granulosome protein precursor (PGRN) protein, wherein the MeCP2 promoter is an engineered MeCP2 promoter composed of the nucleotide sequence of SEQ ID NO:
3.
2. The nucleic acid construct as described in claim 1, wherein: The nucleotide sequence encoding the PGRN protein comprises the nucleotide sequence of SEQ ID NO: 12; and / or The PGRN protein contains the amino acid sequence of SEQ ID NO:
13.
3. The nucleic acid construct according to any one of claims 1-2, further comprising: (a) A post-transcriptional regulatory element (WPRE) sequence of marmot hepatitis virus (WHP), wherein the WPRE is located at the 3' of the nucleotide sequence encoding the PGRN protein, and / or the WPRE comprises the nucleotide sequence of SEQ ID NO: 15; (b) a polyadenylation signal sequence, wherein the polyadenylation signal sequence is located at the 3' of the nucleotide sequence encoding the PGRN protein, and / or the polyadenylation signal sequence comprises the nucleotide sequence of SEQ ID NO: 16; or (c) (a) and (b) above.
4. The nucleic acid construct of claim 3, wherein, in the 5' to 3' direction, the nucleic acid construct comprises the MeCP2 promoter, the nucleotide sequence encoding the PGRN protein, the WPRE, and the polyadenylation signal sequence.
5. The nucleic acid construct according to any one of claims 1-2 and 4, wherein the length is 3700 to 4800 bp.
6. A vector comprising a nucleic acid construct as defined in any one of claims 1-5.
7. The vector as described in claim 6, wherein it is a plasmid or a viral vector.
8. The vector of claim 6, wherein the viral vector comprises the nucleotide sequence of SEQ ID NO:
11.
9. The vector according to any one of claims 6-8, wherein it is a viral vector selected from: (a) an adeno-associated virus (AAV) vector or a vector containing an AAV genome; or (b) a lentiviral vector or a vector containing a lentiviral genome.
10. The vector of claim 9, wherein the AAV genome is chimeric, recombinant, or capsid-modified.
11. The viral vector of claim 9, wherein the AAV vector comprises a genome derived from AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), or AAV serotype rh10 (AAVrh10), or wherein the AAV comprises a genome derived from AAV2, AAV9, or AAVrH10.
12. The AAV vector of claim 11, wherein the AAV vector comprises a genome derived from AAV2, or wherein the AAV is AAV-TT.
13. The AAV vector of claim 11, wherein the AAV vector comprises a nucleotide sequence, the nucleotide sequence comprising one or more of the following in the 5' to 3' direction: (a) 5'ITR; (b) 5' adjacent segments; (c) Engineered MeCP2 promoter sequence; (d) At least one synthetic intron; (e) Kozak sequence; (f) The polynucleotide sequence encoding the PGRN protein; (g)SV40 poly(A) sequence; (h)3' adjacent segments; and (i)3'ITR.
14. The AAV carrier as claimed in claim 13, wherein: (a) The 5'ITR consists of the following: the nucleotide sequence of SEQ ID NO: 20; (b) The 5' adjacent fragment consists of the following: the nucleotide sequence of SEQ ID NO: 21; (c) The engineered MeCP2 promoter sequence consists of the following: the nucleotide sequence of SEQ ID NO: 3; (d) The Kozak sequence consists of the following: the nucleotide sequence of SEQ ID NO: 24; (e) The polynucleotide sequence encoding the PGRN protein consists of the following: the nucleotide sequence of SEQ ID NO: 12; (f) The SV40 poly(A) sequence consists of the following: the nucleotide sequence of SEQ ID NO: 16; (g) The 3' adjacent fragment consists of the following: the nucleotide sequence of SEQ ID NO: 22; and / or (h) The 3'ITR consists of the following: the nucleotide sequence of SEQ ID NO:
23.
15. The AAV vector according to any one of claims 12-14, wherein the AAV vector comprises or is composed of the following nucleotide sequences: SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO:
19.
16. A host cell comprising a nucleic acid construct according to any one of claims 1-5 and / or a vector according to any one of claims 6-15, and / or the host cell producing a viral vector according to any one of claims 8-15.
17. The host cell of claim 16, wherein the host cell is an HEK293 cell or an HEK293T cell.
18. A pharmaceutical composition comprising a nucleic acid construct according to any one of claims 1-5, a vector according to claim 6 or 7 and / or a viral vector according to any one of claims 8-15, and a pharmaceutically acceptable carrier, excipient or diluent.
19. Use of a nucleic acid construct as defined in any one of claims 1-5, a vector as defined in claim 6 or 7, or a viral vector as defined in any one of claims 8-15 in the preparation of a medicament for treating, in patients in need, a disease characterized by a deficiency of granulosome protein precursor (PGRN), wherein the disease characterized by a PGRN deficiency is frontotemporal dementia (FTD) or neuronal cerebrolipofuscinosis type 11 (NCL11).
20. The use as claimed in claim 19, wherein the nucleic acid construct, vector, or viral vector is administered to the patient by delivery to the patient's brain and / or cerebrospinal fluid (CSF).
21. The use as claimed in claim 20, wherein the delivery is performed by injection into: (i) the patient's brain, wherein the injection into the brain is selected from intracerebral injection, intraparenchymal injection, intranuclear-shell injection, and combinations thereof; and / or (ii) the patient’s CSF, wherein the injection into the CSF is selected from intracerebellomedullary cistern injection, intrathecal injection, intraventricular (ICV) injection, and combinations thereof.