Engineered viral vector reduces induction of inflammatory and immune responses

Abstract

Modified viral genomes are able to reduce induction of inflammatory and immune antiviral responses. This manifests itself in reduced NF-kB activity, increased viral transduction rates, and increased expression of transgenes. Viral genomes are modified by incorporating one or more oligonucleotide sequences which are able to bind to TLR9 but not induce activation of it. The oligonucleotide sequences may be synthetic, bacterial, human, or from any other source.

1 . A nucleic acid molecule comprising:
 a viral genome covalently linked to an inhibitory nucleic acid sequence which binds to TLR9 but does not trigger TLR9 activation; or   an inverted terminal repeat and a nucleic acid sequence that inhibits TLR9-mediated inflammation.   
     
     
         2 . A recombinant virus for delivery of a desired function to a mammalian cell, comprising:
 the nucleic acid molecule of  claim 1 .   
     
     
         3 .- 44 . (canceled) 
     
     
         45 . A method of treating a mammal, comprising:
 administering the recombinant virus of  claim 2  to a mammal in need thereof.   
     
     
         46 .- 58 . (canceled) 
     
     
         59 . A method of making a viral genome of a recombinant virus, comprising:
 inserting an inhibitory nucleic acid sequence into a viral genome, wherein the inhibitory nucleic acid sequence binds to TLR9 but does not trigger TLR9 activation; or   inserting a nucleic acid sequence into a viral genome, wherein the nucleic acid sequence inhibits TLR9-mediated inflammation.   
     
     
         60 .- 80 . (canceled) 
     
     
         81 . A method of increasing expression in a host cell of a virally introduced transgene, the method comprising introducing into a host the recombinant virus of  claim 2 , whereby the modified virus results in higher transgene expression in a host cell as compared to a virus that does not contain the nucleic acid sequence. 
     
     
         82 . A composition comprising a viral capsid encapsidating the nucleic acid molecule of  claim 1 . 
     
     
         83 .- 90 . (canceled) 
     
     
         91 . A method of reducing an inflammatory response in a subject in need thereof, the method comprising:
 administering to the subject a recombinant adeno-associated virus (AAV) comprising a viral genome, wherein the viral genome comprises a therapeutic transgene and a nucleic acid molecule comprising three tandem repeat monomers of SEQ ID NO: 6 or SEQ ID NO: 9, thereby reducing an inflammatory response in the subject, relative to a control.   
     
     
         92 . The method of  claim 91 , wherein the AAV is a self-complementary AAV (scAAV). 
     
     
         93 . The method of  claim 91 , wherein the AAV is an AAV2 or AAV8. 
     
     
         94 . The method of  claim 91 , wherein the viral genome further comprises a 5′ untranslated region, and wherein the nucleic acid molecule is in the 5′ untranslated region. 
     
     
         95 . The method of  claim 91 , wherein the viral genome further comprises a 3′ untranslated region, and wherein the nucleic acid molecule is in the 3′ untranslated region. 
     
     
         96 . The method of  claim 91 , wherein the therapeutic gene is a human gene. 
     
     
         97 . The method of  claim 91 , wherein the recombinant scAAV is administered systemically the subject. 
     
     
         98 . The method of  claim 91 , wherein the recombinant scAAV is administered subretinally to the subject. 
     
     
         99 . The method of  claim 91 , wherein the recombinant AAV is administered in an amount effective to prevent TLR-mediated inflammation in the subject. 
     
     
         100 . The method of  claim 91 , wherein the recombinant AAV is administered in an amount effective to reduce induction of pro-inflammatory cytokines in the subject, relative to a control. 
     
     
         101 . The method of  claim 91 , wherein the recombinant AAV is administered in an amount effective to increase expression of the therapeutic transgene in the subject, relative to a control.