Methods and compositions for use in spliceosome mediated RNA trans-splicing

A cell comprising a nucleic acid molecule wherein said nucleic acid molecule comprises:

• a) one or more target binding domains that target binding of the nucleic acid molecule to a factor VIII pre-mRNA expressed within the cell;
• b) a 3′ splice acceptor site;
• c) a spacer region that separates the 3′ splice region from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A cell comprising a nucleic acid molecule wherein said nucleic acid molecule comprises:

• a) one or more target binding domains that target binding of the nucleic acid molecule to a factor VIII pre-mRNA expressed within the cell;
• b) a 5′ splice site;
• c) a spacer region that separates the 5′ splice site from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A cell comprising a recombinant vector wherein said vector expresses a nucleic acid molecule comprising:

• a) one or more target binding domains that target binding of the nucleic acid molecule to a factor VIII pre-mRNA expressed within the cell;
• b) a 3′ splice region comprising a branch point, a pyrimidine tract and a 3′ splice acceptor site;
• c) a spacer region that separates the 3′ splice region from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A cell comprising a recombinant vector wherein said vector expresses a nucleic acid molecule comprising:

• a) one or more target binding domains that target binding of the nucleic acid molecule to a factor VIII pre-mRNA expressed within the cell;
• b) a 3′ splice acceptor site;
• c) a spacer region that separates the 3′ splice region from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A cell comprising a recombinant vector wherein said vector expresses a nucleic acid molecule comprising:

• a) one or more target binding domains that target binding of the nucleic acid molecule to a factor VIII pre-mRNA expressed within the cell;
• b) a 5′ splice site;
• c) a spacer region that separates the 5′ splice site from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A method of producing a chimeric RNA molecule in a cell comprising:

• contacting a target pre-mRNA expressed in the cell with a nucleic acid molecule recognized by nuclear splicing components wherein said nucleic acid molecule comprises:
• a) one or more target binding domains that target binding of the nucleic acid molecule to a factor VIII pre-mRNA expressed within the cell;
• b) a 3′ splice region comprising a branch point, a pyrimidine tract and a 3′ splice acceptor site;
• c) a spacer region that separates the 3′ splice region from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• under conditions in which a portion of the nucleic acid molecule is trans-spliced to a portion of the target pre-mRNA to form a chimeric RNA within the cell.

A method of producing a chimeric RNA molecule in a cell comprising:

• contacting a target pre-mRNA expressed in the cell with a nucleic acid molecule recognized by nuclear splicing components wherein said nucleic acid molecule comprises:
• a) one or more target binding domains that target binding of the nucleic acid molecule to a factor VIII pre-mRNA expressed within the cell;
• b) a 3′ splice acceptor site;
• c) a spacer region that separates the 3′ splice region from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• under conditions in which a portion of the nucleic acid molecule is trans-spliced to a portion of the target pre-mRNA to form a chimeric RNA within the cell.

A method of producing a chimeric RNA molecule in a cell comprising:

• contacting a target pre-mRNA expressed within the cell with a nucleic acid molecule recognized by nuclear splicing components wherein said nucleic acid molecule comprises:
• a) one or more target binding domains that target binding of the nucleic acid molecule to a factor VIII pre-mRNA expressed within the cell;
• b) a 5′ splice site;
• c) a spacer region that separates the 5′ splice site from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A nucleic acid molecule comprising:

• a) one or more target binding domains that target binding of the nucleic acid molecule to a factor VIII pre-mRNA expressed within the cell;
• b) a 3′ splice region comprising a branch point, a pyrimidine tract and a 3′ splice acceptor site;
• c) a spacer region that separates the 3′ splice region from the target binding domain;
• d) a safety sequence comprising one or more complementary sequences that bind to one or both sides of the 3′ splice site;
• and
• e) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A nucleic acid molecule comprising:

• a) one or more target binding domains that target binding of the nucleic acid molecule to a factor VIII pre-mRNA expressed within the cell;
• b) a 3′ splice acceptor site;
• c) a spacer region that separates the 3′ splice region from the target binding domain;
• d) a safety sequence comprising one or more complementary sequences that bind to one or both sides of the 3′ splice site;
• and
• e) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A nucleic acid molecule comprising:

• a) one or more target binding domains that target binding of the nucleic acid molecule to a factor VIII pre-mRNA expressed within the cell;
• b) a 5′ splice site;
• c) a spacer region that separates the 5′ splice site from the target binding domain;
• d) a safety sequence comprising one or more complementary sequences that bind to one or both sides of the 5′ splice site;
• and
• e) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A eukaryotic expression vector wherein said vector expresses a nucleic acid molecule comprising:

• a) one or more target binding domains that target binding of the nucleic acid molecule to a factor VIII protein pre-mRNA expressed within the cell;
• b) a 3′ splice region comprising a branch point, a pyrimidine tract and a 3′ splice acceptor site;
• c) a spacer region that separates the 3′ splice region from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A eukaryotic expression vector wherein said vector expresses a nucleic acid molecule comprising:

• a) one or more target binding domains that target binding of the nucleic acid molecule to factor VIII protein pre-mRNA expressed within the cell;
• b) a 3′ splice acceptor site;
• c) a spacer region that separates the 3′ splice region from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A eukaryotic expression vector wherein said vector expresses a nucleic acid molecule comprising:

• a) one or more target binding domains that target binding of the nucleic acid molecule to a factor VIII protein pre-mRNA expressed within the cell;
• b) a 5′ splice site;
• c) a spacer region that separates the 5′ splice site from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.