Granted Patent B2

US 12,491,267 · App. 17/779,980 · Granted Dec 9, 2025

Inventors: Kimberley S. Gannon (Boston, MA); Martin Goulet (Boston, MA); Neil R. Hackett (Boston, MA)

Assignee: The Brigham and Women's Hospital, Inc.

A61K48/0066C07K14/4711C12N15/86C12N2750/14143C12N2830/50C12N2840/10

Abstract

Claims (54)

1 . An isolated polynucleotide set forth in SEQ ID NO: 37 or SEQ ID NO: 39, wherein the polynucleotide is not naturally occurring.

2 . The isolated polynucleotide of claim 1 , having the nucleotide sequence set forth in SEQ ID NO: 37.

3 . The isolated polynucleotide of claim 1 , having the nucleotide sequence set forth in SEQ ID NO: 39.

4 . A nucleic acid expression cassette comprising:

(I) a polynucleotide encoding presenilin 1, wherein the polynucleotide comprises:

(a) a polynucleotide set forth in SEQ ID NO: 37 or SEQ ID NO: 39; and

(II) one or more regulatory elements operably linked to the polynucleotide encoding presenilin 1.

5 . The nucleic acid expression cassette of claim 4 , wherein one of the regulatory elements is a Kozak translation initiation signal.

6 . The nucleic acid expression cassette of claim 5 , wherein the Kozak translation initiation signal comprises a polynucleotide set forth in SEQ ID NO: 5.

7 . The nucleic acid expression cassette of claim 4 , wherein one of the regulatory elements is a chromatin insulator sequence.

8 . The nucleic acid expression cassette of claim 7 , wherein the chromatin insulator sequence comprises a polynucleotide set forth in SEQ ID NO: 4.

9 . The nucleic acid expression cassette of claim 4 , wherein one of the regulatory elements is a neuron-specific promoter.

10 . The nucleic acid expression cassette of claim 9 , wherein the neuron-specific promoter comprises (i) a polynucleotide set forth in SEQ ID NO: 2; or (ii) a polynucleotide set forth in SEQ ID NO: 3.

11 . The nucleic acid expression cassette of claim 4 , wherein one or more of the regulatory elements is independently selected from a mRNA stability element.

12 . The nucleic acid expression cassette of claim 11 , wherein the mRNA stability element comprises a polynucleotide set forth in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

13 . The nucleic acid expression cassette of claim 11 , wherein the mRNA stability element is located 3′ of an open reading frame of the polynucleotide encoding presenilin 1 or 5′ of a polyadenylation signal.

14 . The nucleic acid expression cassette of claim 13 comprising a first and a second mRNA stability element, wherein the first mRNA stability element is located 3′ of an open reading frame of the polynucleotide encoding presenilin 1; and the second mRNA stability element is located 5′ of a polyadenylation signal.

15 . A vector comprising the nucleic acid expression cassette of claim 4 .

16 . The vector of claim 15 , wherein the vector is a viral vector.

17 . The vector of claim 16 , wherein the viral vector is an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, or an adenoviral vector.

18 . The vector of claim 17 , wherein the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVDJ, AAVrh10, AAV11, AAV12, AAV2/1, AAV2/5, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV2/rh10, AAV2/11, or AAV2/12.

19 . The vector of claim 17 , wherein the vector comprises:

a. a promoter selected from a CAG promoter, a presenilin-1 promoter, a ubiquitin C promoter, a CBA promoter, a synapsin-1 promoter, a PGK promoter, and an EF1α promoter, operatively linked to:

b. a presenilin-1 coding sequence selected from SEQ ID NO: 37 or SEQ ID NO: 39; and

c. a polyadenylation sequence selected from a human growth hormone polyadenylation sequence and a rabbit β-globin polyadenylation sequence.

20 . The vector of claim 19 additionally comprising an intron selected from a human beta globin intron or a minute virus of mice intron, wherein the intron is located in between the promoter and the presenilin-1 coding sequence.

21 . The vector of claim 20 , wherein the vector comprises:

a. nucleotides 1-141 of SEQ ID NO: 41, nucleotides 237-1200 of SEQ ID NO: 41, nucleotides 1221-1786 of SEQ ID NO: 41, nucleotides 1899-3299 of SEQ ID NO: 41, nucleotides 3330-3806 of SEQ ID NO: 41, and nucleotides 4553-4693 of SEQ ID NO: 41;

b. Nucleotides 1-141 of SEQ ID NO: 42, nucleotides 237-1323 of SEQ ID NO: 42, nucleotides 1344-1909 of SEQ ID NO: 42, nucleotides 1983-3416 of SEQ ID NO: 42, nucleotides 3447-3923 of SEQ ID NO: 42, and nucleotides 4554-4694 of SEQ ID NO: 42;

c. Nucleotides 1-141 of SEQ ID NO: 43, nucleotides 237-890 of SEQ ID NO: 43, nucleotides 911-1476 of SEQ ID NO: 43, nucleotides 1550-2983 of SEQ ID NO: 43, nucleotides 3014-3490 of SEQ ID NO: 43, and nucleotides 4553-4694 of SEQ ID NO: 43;

d. Nucleotides 1-141, nucleotides 237-1415 of SEQ ID NO: 44, nucleotides 1436-2001 of SEQ ID NO: 44, nucleotides 2075-3508 of SEQ ID NO: 44, nucleotides 3539-4015 of SEQ ID NO: 44, and nucleotides 4500-4640 of SEQ ID NO: 44;

e. Nucleotides 1-141 of SEQ ID NO: 45, nucleotides 237-664 of SEQ ID NO: 45, nucleotides 684-1249 of SEQ ID NO: 45, nucleotides 1323-2756 of SEQ ID NO: 45, 2787-3263 of SEQ ID NO: 45, and nucleotides 4533-4673 of SEQ ID NO: 45;

f. Nucleotides 1-141 of SEQ ID NO: 46, nucleotides 237-684 of SEQ ID NO: 46, nucleotides 705-1270 of SEQ ID NO: 46, nucleotides 1344-2777 of SEQ ID NO: 46, nucleotides 2808-3284 of SEQ ID NO: 46, and nucleotides 4554-4695 of SEQ ID NO: 46; or

g. Nucleotides 1-105 of SEQ ID NO: 47, nucleotides 113-766 of SEQ ID NO: 47, nucleotides 776-867 of SEQ ID NO: 47, nucleotides 881-2311 of SEQ ID NO: 47, nucleotides 2319-2367 of SEQ ID NO: 47, and nucleotides 2386-2526 of SEQ ID NO: 47.

22 . The vector of claim 21 , wherein the vector comprises a nucleotide sequence of any one of SEQ ID NOs: 41-47.

23 . The nucleic acid expression cassette of claim 4 , wherein the regulatory elements operably linked to the polynucleotide encoding presenilin 1 comprise:

(i) a Kozak translation initiation signal;

(ii) a neuron-specific promoter;

(iii) a chromatin insulator sequence;

(iv) a mRNA stability element located 3′ of an open reading frame of the polynucleotide encoding presenilin 1; and

(v) a mRNA stability element located 5′ of a polyadenylation signal.

24 . The nucleic acid expression cassette of claim 23 , wherein:

the Kozak translation initiation signal comprises a polynucleotide set forth in SEQ ID NO: 5;

the chromatin insulator sequence comprises a polynucleotide set forth in SEQ ID NO: 4;

the mRNA stability element located 3′ of the open reading frame of the polynucleotide encoding presenilin 1 comprises a polynucleotide set forth in SEQ ID NO: 9, or SEQ ID NO: 10;

the at least one mRNA stability element located 5′ of a polyadenylation signal comprises a polynucleotide set forth in SEQ ID NO: 11; and

the neuron-specific promoter comprises a polynucleotide set forth in SEQ ID NO: 2 or SEQ ID NO: 3.

25 . The nucleic acid expression cassette of claim 4 , wherein the promoter operably linked to the polynucleotide encoding presenilin 1 is a ubiquitous promoter.

26 . A method of treating a neurodegenerative disease, disorder, or condition comprising administering to a subject in need thereof the vector of claim 15 .

27 . The method of claim 26 , wherein the neurodegenerative disease, disorder, or condition is Alzheimer's disease, Familial Alzheimer's Disease (FAD), presenilin 1 (PSEN-1) mediated FAD, frontotemporal dementia, frontotemporal lobar degeneration, Pick's disease, Lewy body dementia, memory loss, cognitive impairment, or mild cognitive impairment.

28 . A method of treating Familial Alzheimer's Disease (FAD) or presenilin 1 (PSEN-1) mediated FAD comprising administering to a subject in need thereof the vector of claim 15 .

29 . A method of producing presenilin 1 protein comprising:

transforming a host cell with a vector comprising an optimized polynucleotide set forth in SEQ ID NO: 37 or SEQ ID NO: 39; and

culturing the cell under conditions and for a time that allow expression of the presenilin 1 protein, wherein the expression of the presenilin 1 protein encoded by the optimized polynucleotide in the host cell is greater than a level of expression of presenilin 1 protein encoded by a wilt-type polynucleotide in a host cell, thereby producing presenilin 1 protein.

Assignments (2)

Continuity (3)

References Cited (295)