JPWO2020186150A5

JPWO2020186150A5 -

Info

Publication number: JPWO2020186150A5
Application number: JP2021554695A
Authority: JP
Publication date: 2023-03-22

Claims

A capsid-free closed- end DNA (ceDNA) vector comprising:
at least one nucleic acid sequence positioned between adjacent inverted terminal repeats (ITRs), said at least one nucleic acid sequence encoding at least one phenylalanine hydroxylase ( PAH) protein, said at least one PAH protein wherein said at least one nucleic acid sequence encoding has at least 90% identity with a sequence selected from the group consisting of the sequences shown in Table 1.

2. The ceDNA vector of claim 1, wherein said ceDNA vector comprises a promoter operably linked to said at least one nucleic acid sequence encoding said at least one PAH protein.

3. The ceDNA vector of claim 2, wherein said promoter comprises a nucleic acid sequence having at least 85% identity to SEQ ID NO:191.

The ceDNA vector is
enhancer ;
5'UTR and/or intron sequences;
3'UTR; and/or
at least one poly A sequence
The ceDNA vector according to any one of claims 1 to 3, comprising

12. The ceDNA vector of any one of the preceding claims , wherein said at least one nucleic acid sequence is cDNA.

4. The ceDNA vector of any one of the preceding claims, wherein at least one of said flanking ITRs comprises a functional terminal resolution site (TRS) and a replication ( Rep ) protein binding site.

4. The ceDNA vector of any one of the preceding claims, wherein one or both of said flanking ITRs are derived from a virus selected from the group consisting of parvovirus, dependovirus and adeno-associated virus (AAV).

said adjacent ITRs are symmetrical or asymmetrical ;
said adjacent ITRs are symmetrical or substantially symmetrical;
said adjacent ITRs are asymmetric; and/or
one of said flanking ITRs is wild-type, or both said flanking ITRs are wild-type;
ceDNA vector according to any one of the preceding claims.

The ceDNA vector of any one of the preceding claims , wherein said flanking ITRs are derived from different viral serotypes.

4. The ceDNA vector of any one of the preceding claims, wherein the flanking ITRs are from a viral serotype pair selected from the group consisting of the viral serotype pairs shown in Table 2.

one or both of said flanking ITRs comprises a sequence selected from the group consisting of the sequences of Table 3, Table 5A, Table 5B, and Table 6 ;
at least one of said flanking ITRs is altered from a wild-type AAV ITR sequence by a deletion, addition, or substitution that affects the overall three-dimensional conformation of said ITR;
one or both of said flanking ITRs are from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12;
one or both of said flanking ITRs are synthetic; and/or
one of said flanking ITRs is not a wild-type ITR, or both of said flanking ITRs are not wild-type;
ceDNA vector according to any one of the preceding claims.

deletion, insertion in at least one of the ITR regions, wherein one or both of said flanking ITRs are selected from the group consisting of A, A', B, B', C, C', D, and D' , and/or modified by substitutions .

4. Said deletion, insertion and/or substitution results in the deletion of all or part of the stem-loop structure formed by said A, A', B, B', C or C' regions. 12. The ceDNA vector according to 12 .

one or both of said flanking ITRs are modified by deletion, insertion and/ or substitution resulting in the deletion of all or part of the stem-loop structure formed by said B and B' regions ;
one or both of said flanking ITRs have been modified by deletion, insertion and/or substitution resulting in deletion of all or part of the stem-loop structure formed by said C and C'regions;
One or both of the adjacent ITRs lack a portion of the stem-loop structure formed by the B and B' regions and/or a portion of the stem-loop structure formed by the C and C' regions. modified by deletions, insertions, and/or substitutions resulting in;
in a region in which one or both of the adjacent ITRs contain a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions, contains a single stem-loop structure;
in a region in which one or both of the adjacent ITRs contain a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions, comprising a single stem and two loops; and/or
in a region in which one or both of the adjacent ITRs contain a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions, containing a single stem and a single loop,
ceDNA vector according to any one of the preceding claims.

13. The ceDNA of any one of the preceding claims, wherein both of said adjacent ITRs are altered in such a manner that when said ITRs are flipped with respect to each other they result in an overall three-dimensional symmetry. vector.

said at least one nucleic acid sequence is SEQ ID NO: 381, SEQ ID NO: 382, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 390 391, SEQ ID NO : 392, SEQ ID NO : 393, and SEQ ID NO:394, comprising a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO:394.

wherein said at least one nucleic acid sequence comprises a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO:382, SEQ ID NO:384, SEQ ID NO:394, SEQ ID NO:385, and SEQ ID NO:386 The ceDNA vector according to any one of claims 1 to 3 .

19. The ceDNA of any one of the preceding claims , wherein the ceDNA vector comprises a nucleic acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 192, SEQ ID NO: 193 and SEQ ID NO: 194. vector.

The ceDNA vector of any one of the preceding claims , wherein the ceDNA vector comprises at least one regulatory switch.

said at least one regulatory switch is a binary regulatory switch, a small molecule regulatory switch, a passcode regulatory switch, a nucleic acid-based regulatory switch, a post-transcriptional regulatory switch, a radiation-controlled or ultrasound-controlled regulatory switch, a hypoxia-mediated regulatory switch, an inflammatory response 20. The ceDNA vector of claim 19 , which is selected from the group consisting of regulatory switches, shear-activated regulatory switches, and kill switches.

A pharmaceutical composition comprising a ceDNA vector according to any one of the preceding claims for use in a method of expressing PAH proteins in cells.

22. The pharmaceutical composition of claim 21 , wherein said cells are hepatocytes .

23. The pharmaceutical composition of claim 21 or 22 , wherein said cells are in vitro or in vivo.

The pharmaceutical composition according to any one of claims 21-23 , wherein said at least one nucleic acid sequence is codon-optimized for expression in a cell .

25. The pharmaceutical composition according to any one of claims 21-24 , wherein said at least one nucleic acid sequence is selected from the group consisting of the sequences shown in Table 1.

A pharmaceutical composition comprising a ceDNA vector according to any one of the preceding claims for use in a method of treating a subject with phenylketonuria (PKU).

27. The pharmaceutical of claim 26 , wherein said at least one nucleic acid sequence encoding said at least one PAH protein has at least 90% identity with a sequence selected from the group consisting of sequences shown in Table 1. composition .

28. The pharmaceutical composition according to claim 26 or 27 , wherein said ceDNA vector is administered to hepatocytes .

The pharmaceutical composition according to any one of claims 21-28 , wherein said ceDNA vector expresses said PAH protein in hepatocytes .

The pharmaceutical composition of any one of claims 26-29 , wherein the ceDNA vector is administered by intravenous injection .

said subject exhibits at least about a 50% reduction in the level of serum phenylalanine compared to the level of serum phenylalanine in said subject prior to administration ;
said subject has a serum phenylalanine level of less than about 1500 uM after administration; and/or
said subject exhibits at least about a 10% increase in PAH activity after administration compared to the level of PAH activity before administration;
The pharmaceutical composition according to any one of claims 26-30 .

A pharmaceutical composition comprising a ceDNA vector according to any one of the preceding claims .

A cell comprising a ceDNA vector according to any one of the preceding claims .

34. The cell of claim 33 , wherein said cell is a hepatocyte .

A composition comprising the ceDNA vector of any one of the preceding claims and a lipid.

36. The composition of claim 35 , wherein said lipid is a lipid nanoparticle (LNP).

A ceDNA vector according to any one of the preceding claims , a pharmaceutical composition according to claim 32 , a cell according to claim 33 or claim 34 , or a composition according to claim 35 or 36. Kits containing objects and instructions for use .