Granted Patent B2

US 12,571,002 · App. 17/369,168 · Granted Mar 10, 2026

Inventors: Asa Abeliovich (New York, NY); Laura Heckman (Yardley, PA); Herve Rhinn (New York, NY)

Assignee: PREVAIL THERAPEUTICS, INC

C12N15/861A61K48/0058C07K14/435C07K14/70596C12N7/00C12N9/2402C12N15/113C12N15/86C12Y302/01045A61K9/0019A61K48/00C12N2310/14C12N2310/141C12N2710/14043C12N2750/14143

Abstract

Claims (44)

1 . An isolated nucleic acid comprising: (i) an expression construct comprising a promoter operably linked to a transgene insert encoding a beta-glucocerebrosidase (Gcase) protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; and (ii) two adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences flanking the expression construct, wherein the first ITR sequence is a 5′ ITR, and the second ITR sequence is a 3′ ITR.

2 . The isolated nucleic acid of claim 1 , wherein the promoter is a chicken beta actin (CBA) promoter.

3 . The isolated nucleic acid of claim 1 , further comprising a cytomegalovirus (CMV) enhancer (CMVe).

4 . The isolated nucleic acid of claim 1 , further comprising a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).

5 . The isolated nucleic acid of claim 1 , further comprising a Bovine Growth Hormone polyA signal tail.

6 . The isolated nucleic acid of claim 1 , wherein each of the two ITR sequences is a wild-type AAV2 ITR sequence.

7 . The isolated nucleic acid of claim 1 , wherein each of the two ITR sequences comprises a “D” region (SEQ ID NO: 27) that is proximal to the expression construct.

8 . The isolated nucleic acid of claim 1 , wherein at least one of the two ITR sequences comprises a “D” region (SEQ ID NO: 27) positioned on the outside of the ITR sequence relative to the expression construct.

9 . The isolated nucleic acid of claim 1 , wherein the 5′ ITR sequence comprises a “D” region (SEQ ID NO: 27) that is proximal to the expression construct, and the 3′ ITR sequence comprises a “D” region (SEQ ID NO: 27) positioned on the outside of the ITR sequence relative to the expression construct.

10 . The isolated nucleic acid of claim 9 , wherein the nucleic acid sequence of the 5′ ITR is nucleotides 1-145 of SEQ ID NO: 1 and the nucleic acid sequence of the 3′ ITR is nucleotides 3867-4011 of SEQ ID NO: 1.

11 . The isolated nucleic acid of claim 9 , further comprising a TRY region between the 5′ ITR and the expression construct, wherein the TRY region has the sequence set forth in SEQ ID NO: 28.

12 . An isolated nucleic acid comprising, in 5′ to 3′ order:

(a) a 5′ AAV ITR;

(b) a CMVe;

(c) a CBA promoter;

(d) a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15;

(e) a WPRE;

(f) a Bovine Growth Hormone polyA signal tail; and

(g) a 3′ AAV ITR.

13 . A plasmid comprising a nucleic acid comprising:

(i) an expression construct comprising a promoter operably linked to a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; and

(ii) two AAV ITR sequences flanking the expression construct.

14 . A Baculovirus vector comprising a nucleic acid comprising:

(i) an expression construct comprising a promoter operably linked to a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; and

(ii) two AAV ITR sequences flanking the expression construct.

15 . An isolated cell comprising:

(i) a first vector encoding one or more AAV rep proteins and/or one or more AAV cap proteins; and

(ii) a second vector comprising a nucleic acid comprising:

(a) an expression construct comprising a promoter operably linked to a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; and

(b) two AAV ITR sequences flanking the expression construct.

16 . The cell of claim 15 , wherein

(i) the first vector is a plasmid and the second vector is a plasmid; or

(ii) the first vector is a Baculovirus vector and the second vector is a Baculovirus vector.

17 . The cell of claim 15 , wherein the cell is a mammalian cell or an insect cell.

18 . The cell of claim 17 , wherein the mammalian cell is a HEK293 cell.

19 . A method of producing a recombinant adeno-associated virus (rAAV), the method comprising:

(i) culturing the cell of claim 15 under conditions allowing for packaging the rAAV; and

(ii) harvesting the cultured host cell or a culture medium for collection of the rAAV.

20 . The method of claim 19 , wherein the rAAV comprises an AAV9 capsid protein.

21 . A rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a Gcase protein, wherein the transgene insert is SEQ ID NO: 15.

22 . A rAAV comprising:

(i) an AAV capsid protein; and

the rAAV vector of claim 21 .

23 . The rAAV of claim 22 , wherein the AAV capsid protein is AAV9 capsid protein.

Assignments (1)

Continuity (5)

References Cited (123)