Use of ribozymes in the detection of adventitious agents

Abstract

The present invention provides a method of detecting adventitious agents in a composition comprising a microorganism by using ribozyme-expressing indicator cells, as well as indicator cells useful in such detection.

We claim:  
     
         1 . A method of detecting the presence of an adventitious agent in a composition comprising a reovirus, comprising: 
 (a) providing a population of indicator cells that expresses a ribozyme, wherein the ribozyme is capable of specifically cleaving the genome of the reovirus;    (b) contacting the indicator cells with the composition under conditions that allow for cleavage of the genome of the reovirus by the ribozyme; and    (c) determining the effect of the composition on the indicator cells, wherein any pathogenic effect indicates the presence of an adventitious agent in the composition in addition to the reovirus.    
     
     
         2 . The method of  claim 1  wherein the indicator cells express the ribozyme from a gene that is integrated into the genome of the cells.  
     
     
         3 . The method of  claim 1  wherein the ribozyme is capable of specifically cleaving the s1 RNA of the reovirus.  
     
     
         4 . The method of  claim 1  wherein the ribozyme is Rz-553 or Rz-984.  
     
     
         5 . The method of  claim 1  wherein the reovirus is a mammalian reovirus.  
     
     
         6 . The method of  claim 1  wherein the reovirus is a human reovirus.  
     
     
         7 . The method of  claim 1  wherein the reovirus is a Dearing strain reovirus.  
     
     
         8 . The method of  claim 1  wherein the reovirus is a recombinant reovirus.  
     
     
         9 . The method of  claim 8  wherein the recombinant reovirus is generated by co-infection of mammalian cells with different subtypes of reovirus.  
     
     
         10 . The method of  claim 8  wherein the recombinant reovirus is naturally-occurring.  
     
     
         11 . The method of  claim 8  wherein the recombinant reovirus is non-naturally-occurring.  
     
     
         12 . The method of  claim 8  wherein the recombinant reovirus is from two or more strains of reovirus.  
     
     
         13 . The method of  claim 12  wherein the two or more strains of reovirus are selected from the group consisting of strain Dearing, strain Abney, strain Jones, and strain Lang.  
     
     
         14 . The method of  claim 8 , wherein the recombinant reovirus results from reassortment of reoviruses selected from the group consisting of serotype 1 reovirus, serotype 2 reovirus and serotype 3 reovirus.  
     
     
         15 . The method of  claim 8  wherein the recombinant reovirus comprises naturally-occurring variant coat protein coding sequences.  
     
     
         16 . The method of  claim 8  wherein the recombinant reovirus comprises mutated coat protein coding sequences.  
     
     
         17 . A method of detecting the presence of an adventitious agent in a composition comprising a virus wherein the virus contains an RNA genome or utilizes an RNA transcript to replicate, comprising: 
 (a) providing a population of indicator cells that expresses a ribozyme, wherein the ribozyme is capable of specifically cleaving the RNA genome or RNA transcript of the virus to inhibit replication or infection of the virus;    (b) contacting the indicator cells with the composition under conditions that allow for cleavage of the RNA genome or RNA transcript of the virus by the ribozyme; and    (c) determining the effect of the composition on the indicator cells, wherein any pathogenic effect indicates the presence of an adventitious agent in the composition in addition to the virus.    
     
     
         18 . The method of  claim 17  wherein the indicator cells express the ribozyme from a gene that is integrated into the genome of the cells.  
     
     
         19 . The method of  claim 17  wherein the virus is a DNA virus.  
     
     
         20 . The method of  claim 17  wherein the virus is capable of selectively infecting neoplastic cells.  
     
     
         21 . The method of  claim 17  wherein the virus is selected from the group consisting of modified adenovirus, modified HSV, modified vaccinia virus, modified parapoxvirus orf virus, modified influenza virus, p53-expressing viruses, the ONYX-015 virus, the Delta24 virus, and vesicular stomatitis virus.  
     
     
         22 . A method of validating a composition comprising a microorganism, comprising: 
 (a) providing a population of indicator cells that expresses a ribozyme, wherein the ribozyme is capable of specifically cleaving the RNA genome or RNA transcript of the microorganism to inhibit replication or infection of the microorganism;    (b) contacting the indicator cells with the composition under conditions that allow for cleavage of the RNA genome or RNA transcript of the microorganism by the ribozyme; and    (c) determining the effect of the composition on the indicator cells, wherein the absence of any pathogenic effect validates the composition as having no detectable adventitious agent.    
     
     
         23 . The method of  claim 13  wherein the microorganism is a virus.  
     
     
         24 . The method of  claim 13  wherein the microorganism is a virus capable of selectively infecting neoplastic cells.  
     
     
         25 . The method of  claim 13  wherein the microorganism is a reovirus.  
     
     
         26 . The method of  claim 13  wherein the microorganism is selected from the group consisting of modified adenovirus, modified HSV, modified vaccinia virus, modified parapoxvirus orf virus, modified influenza virus, p53-expressing viruses, the ONYX-015 virus, the Delta24 virus, and vesicular stomatitis virus.  
     
     
         27 . An indicator cell useful for detecting an adventitious agent in a composition of microorganism wherein the indicator cell permanently expresses a ribozyme that is capable of cleaving the genome or RNA transcript of the microorganism.  
     
     
         28 . The indicator cell of  claim 27  wherein a gene coding for the ribozyme is integrated into the genome of the cell.  
     
     
         29 . The indicator cell of  claim 27  wherein the microorganism is a virus.  
     
     
         30 . The indicator cell of  claim 27  wherein the microorganism is reovirus.  
     
     
         31 . The indicator cell of  claim 27  wherein the cell is derived from human embryonic kidney 293 cells.