Chimeric RNA molecules generated by trans-splicing
OwnerLloyd G. Mitchellinventor
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Abstract
The molecules and methods of the present invention provide a means for in vivo production of a therapeutic molecule in a selected subset of cells. The pre-therapeutic molecules of the invention are substrates for a trans-splicing reaction between the pre-therapeutic molecules and a pre-mRNA which is uniquely expressed in the specific target cells. The in vivo trans-splicing reaction provides an active therapeutic RNA which is functional as RNA or encodes a protein to be expressed in the target cells. The expression product of the mRNA is a protein of therapeutic value to the cell or a toxin which causes killing of the specific cells.
Claims34
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 target 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 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 target 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 target 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 target 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 target 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 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 target 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 target 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 .sub.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 target 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 target pre-mRNA expressed within the cell;
- b) a 3' splice region comprising a branch point, a pyrimidine tact 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 a target 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.
Dependencies are inferred from the claim text.