Granted Patent B2

US 12,502,438 · App. 17/415,643 · Granted Dec 23, 2025

Inventors: Stylianos Michalakis (Munich, DE); Martin Biel (Starnberg, DE); Elvir Becirovic (Munich, DE)

Assignee: Vigeneron GmbH

A61K48/0066A61K35/76A61P27/02C12N15/102C12N15/113C12N15/86C12N2320/33C12N2750/14143

Abstract

Claims (134)

1 . A pre-mRNA trans-splicing molecule comprising,

(i) an acceptor splice region, comprising a sequence of at least 80% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4, further comprising

(ia) a pyrimidine tract, wherein the pyrimidine tract comprises

(iaa) 5 to 25 nucleotides;

(iab) wherein at least 60% of the nucleotides within these 5 to 25 nucleotides are cytosine (C), thymine (T) and/or uracil (U);

(iac) wherein the 5 to 25 nucleotides of the pyrimidine tract comprise the sequence TTTTTT or TCTTTT; and

(ib) an acceptor splice site,

(iba) wherein the acceptor splice site is located 3′ to the pyrimidine tract; and

(ibb) wherein the acceptor splice site comprises from 5′ to 3′ a sequence of CAGG;

(ii) a nucleotide sequence of interest or a portion thereof, wherein the acceptor splice region is localized 3′ or 5′ to the nucleotide sequence of interest or the portion thereof;

(iii) a binding domain targeting pre-mRNA, which is localized 3′ or 5′ to the nucleotide sequence of interest or the portion thereof, and

(iv) optionally a spacer sequence, wherein the spacer sequence is localized between the binding domain and the acceptor splice region.

2 . The pre-mRNA trans-splicing molecule of claim 1 comprising,

(i) the nucleotide sequence of interest or the portion thereof, wherein the acceptor splice region is localized 5′ to the nucleotide sequence of interest or the portion thereof;

(ii) a donor splice site, wherein the donor splice site is localized 3′ to the nucleotide sequence of interest or the portion thereof;

(iii) a first binding domain targeting pre-mRNA located 5′ to the nucleotide sequence of interest or the portion thereof,

(iv) a second binding domain targeting pre-mRNA located 3′ to the nucleotide sequence of interest or the portion thereof;

(v) optionally a first spacer sequence, wherein the first spacer is localized between the first binding domain and the acceptor splice region; and

(vi) optionally a second spacer sequence, wherein the second spacer is localized between the second binding domain and the donor splice site.

3 . The pre-mRNA trans-splicing molecule of claim 1 , wherein the acceptor splice region is localized 5′ to the nucleotide sequence of interest or the portion thereof and the binding domain is localized 5′ to acceptor splice region.

4 . The pre-mRNA trans-splicing molecule of claim 1 , further comprising a termination sequence.

5 . The pre-mRNA trans-splicing molecule of claim 1 , wherein

(a) the acceptor splice region comprises the sequence of SEQ ID NOs: 3 or 4; and/or

(b) the sequence between the last pyrimidine of the pyrimidine tract and the acceptor splice site has less than 10 bases.

6 . The pre-mRNA trans-splicing molecule of claim 1 , wherein the acceptor splice region further comprises

(a) 7 nucleotides 5′ to the pyrimidine tract having at least 4 nucleotides of the sequence CAACGAG, wherein the first 5′ nucleotide is C;

(b) 7 nucleotides 5′ to the pyrimidine tract having at least 5 nucleotides of the sequence CAACGAG, wherein the first 5′ nucleotide is C;

(c) 7 nucleotides 5′ to the pyrimidine tract having at least 4 nucleotides of the sequence CAACGAG, wherein the first 5′ nucleotides are CAA; or

(d) 7 nucleotides 5′ to the pyrimidine tract having at least 5 nucleotides of the sequence CAACGAG, wherein the first 5′ nucleotides are CAAC.

7 . A DNA molecule comprising a promoter and a sequence encoding the pre-mRNA trans-splicing molecule according to claim 1 .

8 . An in vitro method for producing a nucleic acid sequence, the method comprising

(A) providing a first nucleic acid sequence comprising one or more donor splice site sequences;

(B) providing a second nucleic acid sequence, wherein the second nucleic acid sequence comprises

(i) an acceptor splice region sequence comprising a sequence of at least 80% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4, further comprising;

(ia) a pyrimidine tract, wherein the pyrimidine tract comprises

(iaa) 5 to 25 nucleotides;

(iab) wherein at least 60% of the nucleotides within these 5 to 25 nucleotides are cytosine (C), thymine (T) and/or uracil (U);

(iac) wherein the 5 to 25 nucleotides of the pyrimidine tract comprise the sequence TTTTTT or TCTTTT; and

(ib) an acceptor splice site,

(iba) wherein the acceptor splice site is located 3′ to the pyrimidine tract; and

(ibb) wherein the acceptor splice site comprises from 5′ to 3′ a sequence of CAGG;

wherein the second nucleic acid sequence further comprises a nucleotide sequence of interest or a portion thereof, wherein the acceptor splice region is localized 3′ or 5′ to the nucleotide sequence of interest or the portion thereof and a binding domain targeting pre-mRNA, which is localized 3′ or 5′ to the nucleotide sequence of interest or the portion thereof, and, optionally, a spacer sequence, wherein the spacer sequence is localized between the binding domain and the acceptor splice region;

(C) cleaving the first nucleic acid sequence at the one or more donor splice site sequence(s) and cleaving the second nucleic acid sequence in the acceptor splice site,

(D) ligating the first cleaved nucleic acid sequence to the second cleaved nucleic acid sequence,

thereby obtaining a nucleic acid sequence.

9 . The in vitro method of claim 8 , wherein the first nucleic acid sequence further comprises a nucleotide sequence of interest or a portion thereof, wherein at least a portion of the nucleotide sequence of interest is located 5′ to the donor splice site and wherein at least a portion of the nucleotide sequence of interest is located 3′ to the splice acceptor splice region in the second nucleic acid sequence.

10 . The in vitro method of claim 8 , wherein the first and the second nucleic acid sequence are introduced into a host cell.

11 . The in vitro method of claim 8 , comprising

(A) introducing into a host cell a first nucleic acid sequence comprising a pre-mRNA trans-splicing molecule sequence or a nucleic acid sequence encoding said pre-mRNA trans-splicing molecule, wherein the first pre-mRNA trans-splicing molecule comprises from 5′ to 3′

(a) a 5′ portion of a nucleotide acid sequence of interest;

(b) a donor splice site;

(c) optionally a spacer sequence;

(d) a first binding domain; and

(e) optionally a termination sequence, or a polyA sequence, and

(B) introducing into the host cell a second nucleic acid sequence comprising a pre-mRNA trans-splicing molecule sequence or a nucleic acid sequence encoding said pre-mRNA trans-splicing molecule, wherein the second pre-mRNA trans-splicing molecule comprises from 5′ to 3′;

(i) a second binding domain, which is complementary to the first binding domain of the first nucleic acid sequence;

(ii) an acceptor splice region sequence comprising a sequence of at least 80% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4, further comprising;

(iia) a pyrimidine tract, wherein the pyrimidine tract comprises

(iiaa) 5 to 25 nucleotides;

(iiab) wherein at least 60% of the nucleotides within these 5 to 25 nucleotides are cytosine (C), thymine (T) and/or uracil (U);

(iiac) wherein the 5 to 25 nucleotides of the pyrimidine tract comprise the sequence TTTTTT or TCTTTT; and

(iib) an acceptor splice site,

(iiba) wherein the acceptor splice site is located 3′ to the pyrimidine tract; and

(iibb) wherein the acceptor splice site comprises from 5′ to 3′ a sequence of CAGG;

(iii) a 3′ portion of the nucleotide sequence of interest, and

(iv) a termination sequence, or a polyA sequence,

(C) cleaving the first nucleic acid sequence at the donor splice site sequence and cleaving the second nucleic acid sequence in the acceptor splice site; and

(D) ligating the first cleaved nucleic acid sequence comprising the 5′ portion of the nucleotide sequence of interest to the second cleaved nucleic acid sequence comprising the 3′ portion of the nucleotide sequence of interest, thereby obtaining the nucleic acid sequence of interest.

12 . An adeno-associated virus (AAV) vector comprising at least two inverted terminal repeats comprising a nucleic acid sequence between these two inverted terminal repeats, wherein said nucleic acid sequence comprises from 5′ to 3′

(i) a promoter;

(ii) a binding domain;

(iii) optionally a spacer sequence;

(iv) an acceptor splice region sequence comprising a sequence of at least 80% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4, further comprising

(a) a pyrimidine tract, wherein the pyrimidine tract comprises

(aa) 5 to 25 nucleotides;

(ab) wherein at least 60% of the nucleotides within these 5 to 25 nucleotides are cytosine (C), thymine (T) and/or uracil (U);

(ac) wherein the 5 to 25 nucleotides of the pyrimidine tract comprise the sequence TTTTTT or TCTTTT; and

(b) an acceptor splice site,

ba) wherein the acceptor splice site is located 3′ to the pyrimidine tract; and

bb) wherein the acceptor splice site comprises from 5′ to 3′ a sequence of CAGG;

(v) a nucleotide sequence of interest or a portion thereof, and

(vi) optionally a poly A sequence.

13 . An adeno-associated virus (AAV) vector system comprising

(I) a first AAV vector comprising at least two inverted terminal repeats comprising a nucleic acid sequence between these two inverted terminal repeats, wherein said nucleic acid sequence between these two inverted terminal repeats comprises from 5′ to 3′

(a) a promoter,

(b) a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest;

(c) a donor splice site;

(d) optionally a spacer sequence;

(e) a first binding domain; and

(f) optionally a termination sequence, or a polyA sequence;

(II) a second AAV vector comprising a nucleic acid sequence comprising at least two inverted terminal repeats comprising a nucleic acid sequence between these two inverted terminal repeats, wherein said nucleic acid sequence between these two inverted terminal repeats comprises from 5′ to 3′

(i) a promoter;

(ii) a second binding domain, which is complementary to the first binding domain of the first AAV vector;

(ii) optionally a spacer sequence

(iii) an acceptor splice region sequence comprising a sequence of at least 80% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4, further comprising

(a) a pyrimidine tract, wherein the pyrimidine tract comprises

(aa) 5 to 25 nucleotides;

(ab) wherein at least 60% of the nucleotides within these 5 to 25 nucleotides are cytosine (C), thymine (T) and/or uracil (U); and

(ac) wherein the 5 to 25 nucleotides of the pyrimidine tract comprise the sequence TTTTTT or TCTTTT; and

(b) an acceptor splice site,

(ba) wherein the acceptor splice site is located 3′ to the pyrimidine tract; and

(bb) wherein the acceptor splice site comprises from 5′ to 3′ a sequence of CAGG;

(iv) a nucleotide sequence encoding a C-terminal portion of the polypeptide of interest;

(iva) wherein the C-terminal portion of the polypeptide of interest and the N-terminal portion of the polypeptide of interest reconstitute the polypeptide of interest; and

(v) a termination sequence.

14 . The AAV vector system of claim 13 , wherein the polypeptide of interest is a full-length polypeptide of interest and the first AAV vector comprises an N-terminal portion of the full-length polypeptide of interest and the second AAV vector comprises a C-terminal portion of the full-length polypeptide of interest.

15 . A nucleic acid sequence comprising

(i) an acceptor splice region sequence comprising a sequence of at least 90% sequence identity to SEQ ID No: 3 or SEQ ID No: 4, further comprising

(ia) a pyrimidine tract, wherein the pyrimidine tract comprises

(aa) 5 to 25 nucleotides;

(ab) wherein at least 60% of the nucleotides within these 5 to 25 nucleotides are cytosine (C), thymine (T) and/or uracil (U);

(ac) wherein the 5 to 25 nucleotides of the pyrimidine tract comprise the sequence TTTTTT or TCTTTT; and;

(ib) an acceptor splice site,

ba) wherein the acceptor splice site is located 3′ to the pyrimidine tract; and

bb) wherein the acceptor splice site comprises from 5′ to 3′ a sequence of CAGG; and

(ii) a nucleotide sequence of interest, wherein the nucleotide sequence of interest

(iia) is located 3′ or 5′ to the acceptor splice region.

16 . The in vitro method of claim 8 , wherein

(a) the sequence between the last pyrimidine of the pyrimidine tract and the acceptor splice site has less than 10 bases;

(b) the acceptor splice region further comprises 7 nucleotides 5′ to the pyrimidine tract having at least 4 nucleotides of the sequence CAACGAG, wherein the first 5′ nucleotide is C; and/or

(c) the acceptor splice region comprises the sequence of SEQ ID NO: 3 or 4.

17 . The AAV vector of claim 12 , wherein

(a) the sequence between the last pyrimidine of the pyrimidine tract and the acceptor splice site has less than 10 bases;

(b) the acceptor splice region further comprises 7 nucleotides 5′ to the pyrimidine tract having at least 4 nucleotides of the sequence CAACGAG, wherein the first 5′ nucleotide is C; and/or

(c) the acceptor splice region comprises the sequence of SEQ ID NO: 3 or 4.

18 . The nucleic acid sequence of claim 15 , wherein

(a) the sequence between the last pyrimidine of the pyrimidine tract and the acceptor splice site has less than 10 bases;

(b) the acceptor splice region further comprises 7 nucleotides 5′ to the pyrimidine tract having at least 4 nucleotides of the sequence CAACGAG, wherein the first 5′ nucleotide is C; and/or

(c) the acceptor splice region comprises the sequence of SEQ ID NO: 3 or 4.

19 . A method of treating an ocular disorder in a patient in need thereof, comprising administering to the patient the adeno-associated virus vector system of claim 13 by subretinal or intravitreal injection.

20 . A method of treating an ocular disorder a patient in need thereof, comprising administering to the patient the AAV vector of claim 12 by subretinal or intravitreal injection.

21 . The pre-mRNA trans-splicing molecule of claim 1 , wherein the acceptor splice region further comprises 7 nucleotides 5′ to the pyrimidine tract having the sequence CAACGAG.

22 . The AAV vector of claim 12 , wherein the acceptor splice region further comprises 7 nucleotides 5′ to the pyrimidine tract having the sequence CAACGAG.

23 . The AAV vector system of claim 13 , wherein the acceptor splice region further comprises 7 nucleotides 5′ to the pyrimidine tract having the sequence CAACGAG.

Assignments (2)

Priority Claims (1)

Continuity (1)

References Cited (22)