Use of novel mirna-binding site cassettes for antigen-presenting cell detargeting of transgene expression by raav gene therapy

Abstract

The disclosure, in some aspects, relates to nucleic acids, compositions and kits useful for gene therapy with reduced immune response to transgene products.

1 . A method of delivering a transgene to target cells of a subject, the method comprising administering to the subject a recombinant Adeno-Associated Virus (rAAV) comprising a transgene, wherein the transgene comprises a promoter operably linked to a nucleic acid sequence that encodes an RNA transcript that comprises at least one immune-associated miRNA binding site, wherein the rAAV infects target cells of the subject thereby delivering the transgene to the target cells, wherein the at least one immune-associated miRNA binding site is a miR-652-5pBS, alone or in combination with one or more of the following miRNA binding sites: a miR-33-5pBS, a miR-223BS, or a miR142BS. 
     
     
         2 - 3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein the at least one immune-associated miRNA is expressed in dendritic cells, macrophages, T-lymphocytes, B-lymphocytes, monocytes, myeloid cells, and/or MF. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , further comprising a binding site for one of the following miRNAs: miR-106, miR-125a, miR-125b, miR-126a, miR-142, miR-146a, miR-15, miR-150, miR-155, miR-16, miR-17, miR-18, miR-181a, miR-19a, miR-19b, miR-20, miR-21a, miR-223, miR-24-3p, miR-29a, miR-29b, miR-29c, miR-302a-3p, miR-30b, miR-33-5p, miR-34a, miR-424, miR-652-3p, miR-652-5p, miR-9-3p, miR-9-5p, miR-92a, and miR-99b-5p (SEQ ID NOs: 1-33). 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the at least one miRNA binding site is miR-652-5p BS, optionally wherein the binding site is encoded by a nucleic acid having the sequence set forth in SEQ ID NO: 39. 
     
     
         9 . The method of  claim 1 , wherein the at least one miRNA binding site is a combination of miR-142BS and miR-652BS, optionally wherein the binding site is encoded by a nucleic acid having the sequence set forth in SEQ ID NO: 40. 
     
     
         10 - 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the RNA transcript is a messenger RNA (mRNA) and at least one of the miRNA binding sites is present in the 3′-UTR of the messenger RNA, optionally wherein the RNA transcript is a mRNA and each of the miRNA binding sites is present in the 3′-UTR of the mRNA. protein. 
     
     
         13 . The method of  claim 1 , wherein the RNA transcript encodes a therapeutic 
     
     
         14 . The method of  claim 1 , wherein the administration is intramuscular. 
     
     
         15 . The method of  claim 1 , wherein the subject is a human. 
     
     
         16 . A recombinant Adeno-Associated Virus (rAAV) comprising a transgene encoding an RNA transcript that comprises at least one immune-associated miRNA binding sites, wherein the at least one immune-associated miRNA binding site is a miR-652-5pBS, alone or in combination with one or more of the following miRNA binding sites: a miR-33-5pBS, a miR-223BS, or a miR142BS. 
     
     
         17 . (canceled) 
     
     
         18 . The rAAV of  claim 16 , further comprising a binding site for one of the following miRNAs: miR-106, miR-125a, miR-125b, miR-126a, miR-142, miR-146a, miR-15, miR-150, miR-155, miR-16, miR-17, miR-18, miR-181a, miR-19a, miR-19b, miR-20, miR-21a, miR-223, miR-24-3p, miR-29a, miR-29b, miR-29c, miR-302a-3p, miR-30b, miR-33-5p, miR-34a, miR-424, miR-652-3p, miR-652-5p, miR-9-3p, miR-9-5p, miR-92a, and miR-99b-5p (SEQ ID NOs: 1-33). 
     
     
         19 . (canceled) 
     
     
         20 . The rAAV of  claim 16  comprising miR-652-5pBS, wherein the rAAV comprise a sequence corresponding to SEQ ID NO: 43, optionally wherein miR-652-5p BS is encoded by a nucleic acid having the sequence set forth in SEQ ID NO: 39. 
     
     
         21 . The rAAV of  claim 16 , wherein the rAAV comprises a combination of miR-142BS and miR-652BS having a sequence corresponding to SEQ ID NO: 44, optionally wherein the combination of miR-142BS and miR-652BS is encoded by a nucleic acid having the sequence set forth in SEQ ID NO: 40. 
     
     
         22 - 23 . (canceled) 
     
     
         24 . The rAAV of  claim 16 , wherein the RNA transcript is a messenger RNA (mRNA) and at least one of the miRNA binding sites is present in the 3′-UTR of the messenger RNA, optionally wherein the RNA transcript is a mRNA and each of the miRNA binding sites is present in the 3′-UTR of the messenger RNA. 
     
     
         25 . The rAAV of  claim 16 , wherein the RNA transcript encodes a therapeutic protein, optionally wherein the RNA transcript is an inhibitory RNA. 
     
     
         26 . (canceled) 
     
     
         27 . The rAAV of  claim 16 , wherein the rAAV comprises a capsid of a serotype selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and variants thereof. 
     
     
         28 . The rAAV of  claim 16 , wherein the transgene is flanked by inverted terminal repeat (ITR) sequences. 
     
     
         29 . A host cell comprising the nucleic acid rAAV of  claim 16 . 
     
     
         30 . The host cell of  claim 29 , wherein the host cell is a mammalian cell.