Multicistronic expression constructs

Abstract

Some aspects of this invention provide nucleic acid constructs for transgene expression. Some aspects of this invention provide multicistronic nucleic acid constructs, for example, comprising an expression cassette encoding a hairpin RNA and a reporter expression cassette. Some aspects of this invention provide nucleic acid constructs comprising two or more self-complementary nucleic acid sequences, for example, hairpin RNA encoding nucleic acid sequences and AAV inverse terminal repeats. Methods for the use of the constructs in therapy and research are also provided.

1 .- 80 . (canceled) 
     
     
         81 . A recombinant Adeno-associated Virus (rAAV) comprising:
 (a) a nucleic acid construct, comprising:
 (i) Adeno-associated Virus (AAV) inverted terminal repeats (ITRs); 
 (ii) a first expression cassette, comprising a nucleic acid encoding a heterologous gene product under the control of a first promoter positioned between the ITRs, and an intron, wherein the intron is positioned between the transcriptional start site of the first expression cassette and the nucleic acid encoding the gene product; and 
 (iii) a second expression cassette, comprising a self-complementary nucleic acid sequence under the control of a second promoter, wherein the second expression cassette is positioned within the intron of the first expression cassette in proximity with the first promoter; and 
   (b) an AAV capsid protein.   
     
     
         82 . The rAAV of  claim 81 , wherein the first promoter is an RNA polymerase II promoter. 
     
     
         83 . The rAAV of  claim 81 , wherein the second promoter is an RNA polymerase III promoter or a U6 or an H1 promoter. 
     
     
         84 . A composition comprising the rAAV of  claim 81 . 
     
     
         85 . A kit, comprising a container housing the rAAV of  claim 81 . 
     
     
         86 . The rAAV of  claim 81 , wherein the nucleic acid construct is configured such that the direction of transcription of the first expression cassette and the direction of transcription of the second expression cassette are opposed. 
     
     
         87 . The rAAV of  claim 81 , wherein the nucleic acid construct is configured such that the direction of transcription of the first expression cassette and the direction of transcription of the second expression cassette are the same. 
     
     
         88 . The rAAV of  claim 81 , wherein the gene product is a reporter protein. 
     
     
         89 . The rAAV of  claim 88 , wherein the reporter protein is a fluorescent protein, enzyme or surface antigen. 
     
     
         90 . The rAAV of  claim 81 , wherein the gene product is a therapeutic protein. 
     
     
         91 . The rAAV of  claim 81 , wherein the self-complementary nucleic acid encodes self-complementary RNA. 
     
     
         92 . The rAAV of  claim 91 , wherein the self-complementary RNA is short hairpin RNA (shRNA) or micro RNA (miRNA). 
     
     
         93 . The rAAV of  claim 92 , wherein the shRNA or miRNA is complementary to a messenger RNA (mRNA). 
     
     
         94 . The rAAV construct of  claim 93 , wherein the mRNA is human mRNA. 
     
     
         95 . The rAAV of  claim 81 , wherein at least one of the ITRs is an AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, or AAV6 ITR. 
     
     
         96 . The rAAV of  claim 81 , wherein one of the ITRs lacks a functional terminal resolution site. 
     
     
         97 . The rAAV of  claim 96 , wherein the ITR lacking a functional terminal resolution site is a ΔTRS ITR. 
     
     
         98 . A method, comprising contacting a cell expressing a target gene with the rAAV of  claim 81 . 
     
     
         99 . The method of  claim 98 , wherein the target gene is an oncogene, a tumor suppressor gene, or a viral gene. 
     
     
         100 . The method of  claim 98 , wherein the gene product is a therapeutic protein.