Trans-splicing mediated photodynamic therapy

Abstract

The present invention provides methods and compositions for conferring selective death on cells expressing a specific target precursor messenger RNA (selective target pre-mRNA). The compositions of the invention include pre-trans-splicing molecules (PTMs) designed to interact with a target precursor messenger RNA molecule (target pre-mRNA) expressed within a cell and mediate a trans-splicing reaction resulting in the generation of a novel chimeric mRNA molecule (chimeric mRNA) capable of encoding a light producing protein or enzyme. Cell death is further mediated by the presence of a photosensitizer which upon photoactivation produces cytotoxicity.

1 . 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 3′ splice acceptor site;   (c) a spacer region that separates the 3′ splice region from the target binding domain; and   (d) a nucleotide sequence encoding a light producing protein or enzyme to be trans-spliced to the target pre-mRNA;   
       wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell and wherein the light producing protein or enzyme activates a cytotoxic photosensitizer that causes cell death. 
     
     
         2 . The cell of  claim 1  wherein the 3′ splice region further comprises a branch point and a pyrimidine tract. 
     
     
         3 . 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 encoding a light producing protein or enzyme to be trans-spliced to the target pre-mRNA;   
       wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell and wherein the light producing protein or enzyme activates a cytotoxic photosensitizer that causes cell death. 
     
     
         4 . The nucleic acid of  claim 1  or  2  wherein the nucleic acid molecule further comprises a 5′ donor site. 
     
     
         5 . An isolated cell comprising nucleic acid molecule wherein said nucleic acid molecule comprises:
 (a) one or more target binding domains, wherein at least one target binding domain is more than about 100 nucleotides in length, that target binding of the nucleic acid molecule to a target pre-mRNA expressed within the cell;   (b) a 3′ splice region comprising 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 encoding a light producing protein or enzyme to be trans-spliced to the target pre-mRNA;   
       wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell and wherein the light producing protein or enzyme activates a cytotoxic photosensitizer that causes cell death. 
     
     
         6 . The cell of  claim 5  wherein the 3′ splice region further comprises a branch point and a pyrimidine tract. 
     
     
         7 . An isolated cell comprising a nucleic acid molecule wherein said nucleic acid molecule comprises:
 (a) one or more target binding domains, wherein at least one target binding domain is more than about 100 nucleotides in length, 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 encoding a light producing protein or enzyme to be trans-spliced to the target pre-mRNA;   
       wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell and wherein the light producing protein or enzyme activates a cytotoxic photosensitizer that causes cell death. 
     
     
         8 . The cell of  claim 5  or  6  wherein the nucleic acid molecule further comprises a 5′ donor site. 
     
     
         9 . A method of producing a chimeric mRNA molecule in a cell wherein said chimeric molecule expresses a light producing protein or enzyme 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 more than about 100 nucleotides in length that target binding of the nucleic acid molecule to a target pa e-mRNA expressed within the cell;   (b) a 3′ splice region comprising 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 encoding a light producing protein or enzyme 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-m RNA to form a chimeric mRNA within the cell wherein the light producing protein or enzyme activates a cytotoxic photosensitizer that causes cell death. 
     
     
         10 . The method of  claim 5  wherein said 3′ splice region further comprises a branch point and a pyrimidine tract. 
     
     
         11 . A method of producing a chimeric mRNA molecule in a cell wherein said chimeric molecule expresses a light producing protein or enzyme 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, wherein at least one target binding domain is more than about 100 nucleotides in length, 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 encoding a light producing protein or enzyme 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 mRNA within the cell wherein the light producing protein or enzyme activates a cytotoxic photosensitizer that causes cell death. 
     
     
         12 . The method of  claim 9  or  10  wherein the nucleic acid molecule further comprises a 5′ donor site. 
     
     
         13 . A method for targeting cell death comprising:
 (i) contacting said cell with a nucleic acid molecule wherein said nucleic acid molecule comprises:
 a) one or more target binding domains, wherein at least one target binding domain is more than about 100 nucleotides in length, that target binding of the nucleic acid molecule to a target pre-mRNA expressed within the cell; 
 b) a 3′ region comprising 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 encoding a light producing protein enzyme to be trans-spliced to the target pre-mRNA; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell; and 
   (ii) placing a photosensitizer in close enough proximity to the cell to permit activation of the photosensitizer by the light producing enzyme, wherein said activation results in cell death.   
     
     
         14 . The method of  claim 13  wherein said 3′ splice region further comprises a branch point and a pyrimidine tract. 
     
     
         15 . A method for targeting cell death comprising
 (i) contacting said cell with a nucleic acid molecule wherein said nucleic acid molecule comprises:
 a) one or more target binding domains, wherein at least one target binding domain is more than about 100 nucleotides in length, 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 3′ splice region from the target binding domain; and 
 d) a nucleotide sequence encoding a light producing protein enzyme to be trans-spliced to the target pre-mRNA; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell; and 
   (ii) placing a photosensitizer in close enough proximity to the cell to permit activation of the photosensitizer by the light producing enzyme, wherein said activation results in cell death.   
     
     
         16 . The method of  claim 13  or  14  wherein the nucleic acid molecule further comprises a 5′ donor site. 
     
     
         17 . The method of  claim 13 ,  14  or  15  further comprising contacting said cell with a substrate specific for the light producing protein or enzyme. 
     
     
         18 . The method of  claim 17  further comprising contacting said cell with a substrate specific for the light producing protein or enzyme.