Optimized acceptor splice site module for biological and biotechnological applications

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.

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.

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.

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.