Self-cleaving ribozymes and uses thereof

Abstract

In certain embodiments, the disclosure relates to compositions and methods relating to a ribozyme-based gene regulation system that functions in mammalian cells. In certain specific embodiments, the disclosure relates to schistosome self-cleaving RNA mutant motifs.

1 . A self-cleaving ribozyme, which efficiently cleaves an RNA molecule which comprises the self-cleaving ribozyme in a mammalian cell. 
     
     
         2 . A schistosome ribozyme mutant, comprising a loop on stem III. 
     
     
         3 . The ribozyme mutant of  claim 2 :
 a) wherein the loop comprises at least three nucleotides;   b) wherein the loop is selected from the group consisting of: 5′-UUCG-3′, 5′-CUUCGG-3′, and 5′-GCUUCGGU-3′;   c) wherein the ribozyme mutant further comprises at least one nucleotide substitution in the ribozyme core sequence;   d) wherein the nucleotide C at position 7 is substituted with a nucleotide selected from the group consisting of: U, A and G; or   e) wherein the ribozyme mutant comprises a schistosome ribozyme mutant having a nucleotide sequence selected from SEQ ID NOs: 1-63.   
     
     
         4 - 7 . (canceled) 
     
     
         8 . A self-cleaving RNA motif having the nucleotide sequence as set forth in SEQ ID NO: 64, wherein,
 each N is independently any nucleotide;   each A, U, G and C, is adenosine, uridine, guanosine and cytidine, respectively;   n is 3-40; and   X at the 5′ and Y at the 3′ end of said self-cleaving RNA motif are meant to indicate additional nucleotides which may be present or absent.   
     
     
         9 . A self-cleaving RNA motif of  claim 8 , having the nucleotide sequence as set forth in SEQ ID NO: 65, wherein,
 N is any nucleotide;   each A, U, G and C, is adenosine, uridine, guanosine and cytidine, respectively; and   X at the 5′ and Y at the 3′ end of said self-cleaving RNA motif are meant to indicate additional nucleotides which may be present or absent; and optionally wherein N is nucleotide U.   
     
     
         10 . (canceled) 
     
     
         11 . A self-cleaving RNA motif comprising a schistosome ribozyme mutant having a nucleotide sequence selected from SEQ ID NOs: 1-63. 
     
     
         12 . A recombinant nucleic acid encoding:
 a) the schistosome ribozyme mutant of  claim 2 ; or   b) the schistosome ribozyme mutant of  claim 2  and an aptamer in a position such that the cleaving activity of said schistosome ribozyme mutant can be modulated by binding of an effector to the aptamer.   
     
     
         13 - 14 . (canceled) 
     
     
         15 . A host cell comprising or transformed with the recombinant nucleic acid of  claim 12 . 
     
     
         16 . A recombinant polynucleotide molecule comprising:
 (a) a promoter;   (b) a nucleic acid encoding a nucleic acid product; and   (c) a nucleic acid encoding a schistosome ribozyme mutant motif,   wherein the nucleic acid of (b) and the nucleic acid of (c) are operably linked to the promoter and transcription of the nucleic acid of (b) and the nucleic acid of (c) produces a RNA molecule comprising said schistosome ribozyme mutant motif and a mRNA encoding said nucleic acid product, wherein said schistosome ribozyme mutant motif is capable of cleaving said RNA molecule intramolecularly.   
     
     
         17 . The polynucleotide molecule of  claim 16 :
 i) wherein the nucleic acid of (c) further comprises a nucleic acid encoding an aptamer which is at a position such that the cleaving activity of the schistosome ribozyme mutant motif is regulatable by binding of an effector to the aptamer; or   ii) wherein the nucleic acid of (c) encodes at least two schistosome ribozyme mutant motifs.   
     
     
         18 . (canceled) 
     
     
         19 . The polynucleotide molecule of  claim 16 , wherein the self-cleaving RNA mutant motif includes a loop on stem III. 
     
     
         20 . The polynucleotide molecule of  claim 19 :
 a) wherein the loop comprises at least three nucleotides;   b) wherein the loop is selected from the group consisting of: 5′-UUCG-3′,5′-CWCGG-3′, and 5′-GCUUCGGU-3′;   c) wherein the nucleotide C at position 7 is substituted with a nucleotide selected from the group consisting of: U, A and G;   d) wherein the polynucleotide molecule is present in the genome of a cell; or   e) wherein the polynucleotide molecule is present on a vector.   
     
     
         21 - 24 . (canceled) 
     
     
         25 . A host cell comprising the polynucleotide molecule of  claim 16 . 
     
     
         26 . The host cell of  claim 25 , wherein the cell is a mammalian cell, or wherein the cell further comprises an inhibitor of the self-cleaving RNA mutant motif. 
     
     
         27 . (canceled) 
     
     
         28 . A virus or viral vector comprising the recombinant polynucleotide molecule of  claim 16 . 
     
     
         29 . The virus or viral vector of  claim 28 , wherein the nucleic acid of (c) further comprises a nucleic acid encoding an aptamer which is at a position such that the cleaving activity of said schistosome ribozyme mutant motif is regulatable by binding of an effector to said aptamer. 
     
     
         30 - 31 . (canceled) 
     
     
         32 . A method of modulating expression of a nucleic acid product in the host cell of  claim 25 , comprising contacting said host cell with an agent which modulates cleavage of a self-cleaving schistosome RNA mutant motif. 
     
     
         33 . The method of  claim 32 , wherein the expression of the nucleic acid product is induced and the agent inhibits cleavage of said self-cleaving schistosome RNA mutant motif. 
     
     
         34 . The method of  claim 33 :
 a) wherein the agent is an antibiotic;   b) wherein the agent is selected from the group consisting of toyocamycin, 8-azaadenosine, sangivamycin, tubercidin, tubercidin-cyclic monophosphate, tubercidin-monophosphate, tubercidin-triphosphate, nebularine, tricyclic nucleoside, 5-fluorouridine, 5-bromouridine, 5-fluorouracil, Syto-83, homidium bromide, and acridine orange; or   c) wherein the nucleic acid of (c) encodes at least two self-cleaving RNA mutant motifs.   
     
     
         35 - 36 . (canceled) 
     
     
         37 . The method of  claim 33 , wherein the self-cleaving schistosome RNA mutant motif includes a loop on stem III. 
     
     
         38 . The method of  claim 37 :
 a) wherein the loop comprises at least three nucleotides;   b) wherein the loop is selected from the group consisting of: 5′-UUCG-3′,5′-CUUCGG-3′, and 5′-GCLTUCGGU-3′; or   c) wherein the nucleotide C at position 7 is substituted with a nucleotide selected from the group consisting of: U, A and G.   
     
     
         39 - 40 . (canceled) 
     
     
         41 . A method for modulating expression of a nucleic acid product in an individual, comprising:
 (a) obtaining cells from the individual under conditions appropriate for cell growth and cell division;   (b) introducing into said cells a DNA construct which comprises the recombinant polynucleotide molecule of  claim 16 ;   (c) returning said cell produced in step (b) to the individual; and   (d) administering to the individual an agent which modulates cleavage of said self-cleaving schistosome RNA mutant motif such that expression of the nucleic acid product is modulated.   
     
     
         42 . The method of  claim 41 , wherein the expression of the nucleic acid product is induced and the agent administered to the individual is capable of inhibiting cleavage of said self-cleaving schistosome RNA mutant motif. 
     
     
         43 . The method of  claim 42 , wherein the agent is an antibiotic. 
     
     
         44 . A method of inducing expression of a nucleic acid product in the host cell of  claim 25  comprising contacting said host cell with an antisense oligonucleotide of a self-cleaving schistosome RNA mutant motif. 
     
     
         45 . The method of  claim 44 :
 a) wherein the self-cleaving schistosome RNA mutant motif includes a loop on stem III;   b) wherein the antisense oligonucleotide base pairs with a region of the self-cleaving schistosome RNA mutant motif as set forth in SEQ ID NO: 67; or   c) wherein the antisense oligonucleotide is a modified oligonucleotide selected from the group consisting of: morpholino, phosphorothioate RNA, 2′-O-methyl RNA, and phosphorothioate 2′-O-methoxyethyl RNA.   
     
     
         46 - 47 . (canceled) 
     
     
         48 . A modified antisense oligonucleotide of a self-cleaving schistosome RNA mutant motif. 
     
     
         49 . The modified antisense oligonucleotide of  claim 48 :
 a) selected from the group consisting of: morpholino, phosphorothioate RNA, 2′-O-methyl RNA, and phosphorothioate 2′-O-methoxyethyl RNA; or   b) which oligonucleotide base pairs with a region of the self-cleaving schistosome RNA mutant motif as set forth in SEQ ID NO: 67.   
     
     
         50 . (canceled) 
     
     
         51 . A method of screening for an agent which is capable of modulating the catalytic activity of a schistosome ribozyme mutant comprising:
 (a) introducing into host cells a DNA construct which comprises:
 (1) a promoter; 
 (2) a nucleic acid encoding a reporter; and 
 (3) a nucleic acid encoding the schistosome ribozyme mutant of  claim 2 , 
 wherein the nucleic acid of (2) and the nucleic acid of (3) are downstream of the promoter and operably linked to said promoter; 
   (b) contacting said host cells with an agent to be assessed for its ability to modulate catalytic activity of said schistosome ribozyme mutant under conditions appropriate for expression of said reporter; and   (c) assaying reporter activity, wherein detection of reporter activity in the presence of said agent greater than or less than reporter activity in the absence of the said agent identifies the agent as one which modulates the catalytic activity of said schistosome ribozyme mutant.   
     
     
         52 . A method for producing a transgenic nonhuman animal comprising introducing a DNA construct into a germ cell of a nonhuman animal or a germ cell of an ancestor of said animal, wherein said DNA construct comprises:
 (a) a promoter;   (b) a nucleic acid encoding a desired nucleic acid product; and   (c) a nucleic acid encoding the schistosome ribozyme mutant of  claim 2 ,   wherein the nucleic acid of (b) and the nucleic acid of (c) are operably linked to said promoter and transcription of the nucleic acid of (b) and the nucleic acid of (c) produces a RNA molecule comprising said schistosome ribozyme mutant and a mRNA encoding said nucleic acid product, wherein said schistosome ribozyme mutant is capable of cleaving said RNA molecule intramolecularly.   
     
     
         53 . The method of  claim 52 , wherein the nucleic acid of (3) further comprises nucleic acid encoding an aptamer which is at a position such that the cleaving activity of said schistosome ribozyme mutant is regulatable by binding of an effector to said aptamer. 
     
     
         54 . A method for determining the level of a modulator of a schistosome ribozyme mutant in a cell, comprising:
 (a) introducing into a cell a DNA construct which comprises:
 (1) a promoter; 
 (2) a nucleic acid encoding a reporter; and 
 (3) a nucleic acid encoding the schistosome ribozyme mutant of  claim 2 , 
 wherein the nucleic acid of (2) and the nucleic acid of (3) are downstream of the promoter and operably linked to said promoter, under conditions which result in modulation of the ribozyme mutant and expression of the reporter; and 
   (b) assaying reporter activity in the cell produced by (a), wherein the level of said modulator in the cell is identified by comparing the reporter activity with an appropriate control.   
     
     
         55 . The method of  claim 54 :
 a) wherein the modulator is an inhibitor selected from the group consisting of: 5-fluorouracil and 5-fluorouridine; or   b) wherein the cell is a cancer cell.   
     
     
         56 . (canceled) 
     
     
         57 . A method for determining the level of a modulator of a schistosome ribozyme mutant in a biological sample, comprising:
 (a) contacting a cell with the biological sample, wherein the cell expresses a DNA construct which comprises:
 (1) a promoter; 
 (2) a nucleic acid encoding a reporter; and 
 (3) a nucleic acid encoding the schistosome ribozyme mutant of  claim 2 , 
 wherein the nucleic acid of (2) and the nucleic acid of (3) are downstream of the promoter and operably linked to said promoter, under conditions which result in modulation of the ribozyme mutant and expression of the reporter; and 
   (b) assaying reporter activity in the presence of the biological sample, wherein the level of said modulator in the biological sample is identified by comparing the reporter activity with an appropriate control.   
     
     
         58 . A method of modulating activity of a catalytic RNA in a cell, comprising contacting a cell with a modulator of the schistosome ribozyme mutant of  claim 2 . 
     
     
         59 . The method of  claim 58 :
 a) wherein the cell is infected with a virus or a pathogenic microorganism;   b) wherein the modulator is an inhibitor selected from the group consisting of toyocamycin, 8-azaadenosine, sangivamycin, tubercidin, tubercidin-cyclic monophosphate, tubercidin-monophosphate, tubercidin-triphosphate, nebularine, tricyclic nucleoside, 5-fluorouridine, 5-bromouridine, 5-fluorouracil, Syto-83, homidium bromide, and acridine orange; or   c) wherein the modulator is an antisense oligonucleotide.   
     
     
         60 - 61 . (canceled) 
     
     
         62 . A method of inhibiting infection by a virus or a pathogenic microorganism in a cell, comprising contacting a cell with an inhibitor of the schistosome ribozyme mutant of  claim 2 . 
     
     
         63 . The method of  claim 62 :
 a) wherein the infection is caused by a virus selected from the group consisting of a human immunodeficiency virus, a herpes virus, a hepatitis virus, and a human papillomavirus;   b) wherein the infection is caused by a pathogenic microorganism selected from the group consisting of  Notophthalmus viddescens, Ambystoma talpoideum, Amphiuma tridactylum , and  Schistosoma mansoni;      c) wherein the cell is a mammalian cell; or   d) wherein the cell is a plant cell.   
     
     
         64 - 66 . (canceled) 
     
     
         67 . A kit for regulating gene expression, comprising a nucleic acid comprising:
 (a) a schistosome ribozyme mutant sequence; and   (b) a cloning site for introduction of a target nucleotide sequence to be transcribed operatively linked to the schistosome ribozyme mutant sequence.   
     
     
         68 . The kit of  claim 67 :
 a) further comprising an inhibitor of the schistosome ribozyme mutant, wherein transcription of the target gene is inhibited in the absence of the inhibitor;   b) wherein the schistosome ribozyme mutant sequence comprises a nucleotide sequence selected from SEQ ID NOs: 1-63; or   c) wherein the schistosome ribozyme mutant comprises a loop on stem III; or   d) wherein the nucleic acid comprises at least two self-cleaving RNA mutant motifs.   
     
     
         69 - 71 . (canceled)