Selective killing of cells by activation of double-stranded rna dependent protein kniase-pkr

Abstract

Novel methods and compositions for selective killing of cells by activation of PKR are disclosed. In a preferred embodiment, a method is provided for causing cell death in a targeted population of cells that includes the steps of: selecting a nucleotide sequence at a single genetic locus in the targeted population that is absent from the equivalent locus in a population of non-targeted cells; obtaining one or more anti-sense RNA having sequence homology with the locus in the targeted population; permitting the anti-sense RNA to hybridize with an RNA transcribed from the locus in the targeted population so as to form a contiguous double stranded RNA for interacting with PKR. The activation of PKR gives rise to selective cell death in the targeted population.

1 . A method for selectively activating double stranded RNA dependent protein kinase in a targeted population of cells, comprising: 
 (a) selecting a nucleotide sequence at a single genetic locus in the targeted population that is absent from the equivalent locus in a population of non-targeted cells;    (b) obtaining one or more anti-sense RNA having sequence homology with the locus in the targeted population;    (c) permitting the anti-sense RNA to hybridize with an RNA transcribed from the locus in the targeted population so as to form a contiguous double stranded RNA for interacting with double stranded RNA dependent protein kinase, thereby selectively activating double stranded RNA dependent protein kinase.    
     
     
         2 . A method according to  claim 1 , wherein the step of obtaining an anti-sense RNA in step (b) further comprises selecting a DNA sequence encoding the anti-sense RNA, wherein the DNA sequence is transcribed in the targeted population to provide the anti-sense RNA.  
     
     
         3 . A method according to ( 2 ) wherein the DNA sequence is contained within any of a plasmid, a virus or a liposome or is delivered to the cell as naked DNA or is transcribed from an RNA sequence contained within a retrovirus  
     
     
         4 . A method according to ( 2 ). wherein the DNA sequence is contained within a virus selected from the group consisting of an adenovirus, vaccinia virus and a herpes virus.  
     
     
         5 . A method according to ( 3 ), wherein the DNA sequence is naked DNA, the naked being attached to a carrier.  
     
     
         6 . A method according to ( 1 ). wherein obtaining an antisense RNA in step (b) further comprises: selecting an RNA sequence encoding anti-sense RNA wherein the RNA sequence is replicated in the target population to form double stranded RNA.  
     
     
         7 . A method according to ( 6 ), wherein the RNA sequence is contained within a virus.  
     
     
         8 . A method according to ( 7 ),wherein the virus is a lentivirus.  
     
     
         9 . A method according to ( 6 ), wherein the RNA sequence includes a U6 small nuclear RNA promoter.  
     
     
         10 . A method according to ( 1 ),wherein the target population of cells is selected from the group consisting of: neoplastic cells, autoimmune T-lymphocytes, infectious agents, smooth muscle cells in blood vessel plaque, cells in abnormal blood vessel growth, cells in psoriasis and keloid scarring.  
     
     
         11 . A method according to ( 1 ),wherein the targeted population of cells are neoplastic cells.  
     
     
         12 . A method according to ( 11 ),wherein the neoplastic cells are glioblastoma cells.  
     
     
         13 . A method according to ( 11 ), wherein the neoplastic cells are lymphomas  
     
     
         14 . A method according to ( 1 ), wherein the neoplastic cells are located in vivo in a subject  
     
     
         15 . A method according to  1 , wherein the neoplastic cells are located ex vivo in a culture vessel,  
     
     
         16 . A method according to  1 , wherein step (c) further comprises administering to the population of cells, an effective amount of interferon.  
     
     
         17 . A method according to  1 , wherein the anti-sense RNA has a sequence selected from SEQ 1-4 .  
     
     
         18 . A method according to  1 , wherein the double stranded RNA is at least 30 nucleotides in length.  
     
     
         19 . A method according to  17 , wherein the double stranded RNA is lee than 100 nucleotides in length.  
     
     
         20 . A method for treating a subject having a disorder characterized by proliferating cells, comprising: 
 (a) obtaining a nucleotide sequence at a single genetic locus in the proliferating cells that differs from the equivalent locus in a population of non-proliferating cells;    (b) administering an effective amount of a nucleic acid so as to provide anti-sense RNA having sequence homology with the locus in the proliferating cells. such that the antisense RNA hybridizes in situ with messenger RNA transcribed from the locus to form a single contiguous double stranded RNA, the double stranded RNA having a length suited for activating double stranded RNA dependent protein kinase; and    (c) treating the disorder in the subject by selectively killing the proliferating cells.    
     
     
         21 . A method according to  20 , wherein the genetic locus is characterized by a mutation.  
     
     
         22 . A method according to  20 , where the proliferative disorder is neoplastic disease.  
     
     
         23 . A method according to  20 , wherein the proliferative disorder is psoriasis.  
     
     
         24 . A method according to  20 , wherein the proliferative disorder results from abnormal autoimmune T-lymphocytes.  
     
     
         25 . A method according to  20 , wherein the proliferative disorder includes vasculogenesis or angiogenesis  
     
     
         26 . A pharmaceutical composition for treating proliferative disorders, comprising: an effective tumor cell growth inhibiting amount of anti-sense RNA, the antisense RNA having a sequence that is complementary to a contiguous mRNA sequence present in a neoplastic cell and absent in non-neoplastic cells, so as to form a double stranded RNA for activating a double stranded RNA dependent protein kinase and inhibiting tumor cell growth  
     
     
         27 . A composition according to  26 , wherein the anti-sense RNA has a sequence selected from the group consisting of SEQ ID Nos 1,3, 7, 9, 11, and 13 .  
     
     
         28 . A composition according to  26 , wherein the pharmaceutical composition. further comprises an interferon.  
     
     
         29 . An anti-sense RNA, comprising: 
 a natural or synthetic molecule having a length of less than 100 nucleotides, and having a sequence that hybridizes in situ or under stringent conditions in vitro with a contiguous nucleotide sequence that spans a site-specific mutation on a mammalian chromosome    
     
     
         30 . An anti-sense mRNA according to  29 , comprising: a sequence selected from the group consisting of SEQ ID Nos 1, 3, 5, 7, 9, 11 and 13.  
     
     
         31 . A nucleic acid comprising: a sequence that is a template for an anti-sense RNA, wherein the anti-sense RNA hybridizing under stringent conditions to a messenger RNA, the messenger RNA being transcribed from a contiguous nucleotide sequence that spans a site specific mutation on a mammalian chromosome, the mutation occurring in a neoplastic cell and absent in a non-neoplastic cell, the nucleic acid having a length of less than 100 nucleotides  
     
     
         32 . A vector comprising: a sequence that is a template for an anti-sense RNA of greater than 30 nucleotides and less than 100 nucleotides in length, wherein the anti-sense RNA hybridizes to an RNA, the RNA being identical to a transcription product from a contiguous nucleotide sequence that spans a site specific mutation on a mammalian chromosome, the mutation occurring in a neoplastic cell, being absent in a non-neoplastic cell at that site.  
     
     
         33 . A vector according to  32 , consisting of a retroviral vector  
     
     
         34 . A vector according to  33  wherein the retroviral vector is a lentivirus  
     
     
         35 . A cell population having transfected therein a vector according to  32   
     
     
         36 . A cell population according to  35 , wherein the transfection is a stable and the cell population is maintained in a cell culture medium,