Genome-modified recombinant adeno-associated virus vectors

Abstract

Provided herein are recombinant adeno-associated virus (rAAV) nucleic acid vectors that comprise one or more modifications within at least one inverted terminal repeat (ITR) region. Exemplary modifications include ITR sequences comprising a glucocorticoid responsive element and/or a transcription factor binding site. Also provided are plasmids, libraries, rAAV particles, compositions, kits, and methods related to such vectors.

1 - 20 . (canceled) 
     
     
         21 . A library comprising a plurality of recombinant adeno-associated virus (rAAV) particles, each rAAV particle in the plurality comprising a viral capsid encapsidating a nucleic acid vector, wherein the nucleic acid vector comprises a glucocorticoid receptor responsive element and/or a transcription factor binding site inserted within an inverted terminal repeat (ITR). 
     
     
         22 . The library of  claim 21 , wherein the viral capsid of each rAAV particle in the plurality comprises a modified capsid protein comprising at least one mutation. 
     
     
         23 . A library comprising a plurality of plasmids, each plasmid comprising a glucocorticoid receptor responsive element and/or a transcription factor binding site inserted within an inverted terminal repeat (ITR). 
     
     
         24 . (canceled) 
     
     
         25 . The library of  claim 21 , wherein the transcription factor binding site is a transcription factor binding site for POU3F2, FOXJ2, NR2E3, MEF-2, AP-1, Sp-1, KLF-4, GATA-6, HNF-1, MyOD, C/EBP, POU, MRF, NKX2-5, GATA-4, GCM2, OTX-2, Pax-2, or GATA-3. 
     
     
         26 . The library of  claim 21 , wherein the glucocorticoid receptor responsive element comprises the sequence GGTACANNNTGT(T/C)CT (SEQ ID NO: 1), AGAACANNNTGTTCT (SEQ ID NO: 8), or GGCACAGTGTGGTCT (SEQ ID NO:9), wherein each N is independently a T, C, G or A and wherein (T/C) is either a T or a C nucleotide. 
     
     
         27 . The library of  claim 21 , further comprising a multiple cloning site, a promoter, and/or a heterologous nucleic acid region comprising a sequence encoding a protein, polypeptide, or RNA of interest. 
     
     
         28 . The library of  claim 27 , wherein the protein or polypeptide of interest is beta-catenin (CTNNB 1), pyruvate dehydrogenase (PDH), parvovirus B19 non-structural protein (B19 NS1), Trichosanthin (TCS), beta-globin (HBB), acid alpha-glucosidase (GAA), Methyl CpG binding protein (MECP2), Aromatic L-amino acid decarboxylase (AADC), Glial cell-derived neurotrophic factor (GDNF), Cystic fibrosis transmembrane conductance regulator (CFTR), Tumor necrosis factor receptor fused to an antibody Fe (TNFR:Fc), HIV-1 gag-proΔrt (tgAAC09), Sarcoglycan, Alpha-1-antitrypsin (AAT), Glutamate decarboxylase 1(GAD1), Glutamate decarboxylase 2 (GAD2), Aspartoacylase (ASPA), Nerve growth factor (NGF), Granulocyte-macrophage colonystimulating factory (GM-CSF), Cluster of Differentiation 86 (CD86 or B7-2), IL-12, neuropeptide Y, ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 (SERCA2), Dystrophin or Minidystrophin, Ceroid lipofuscinosis neuronal 2, Neurturin (NR TN), N-acetylglucosaminidase, alpha (NAGLU), Iduronidase, alpha-1 (IDUA), Iduronate 2-sulfatase (IDS), Glucuronidase, beta (GUSB), Hexosaminidase A, Hexosarninidase B, RPE65, Factor IX, Adenine nucleotide translocator, ApaLI, NADH ubiquinone oxidoreductase subunit 4 (ND4), very long-acyl-CoA dehydrogenase, medium-chain acyl-CoA dehydrogenase (MCAD), Myotubularin 1 (MTM 1), Myophosphorylase (PYGM), Lipoprotein lipase (LPL), sFLTOl, Glucocerebrosidase (GC), UDP glucuronosyltransferase 1 family, polypeptide A1 (UGTlA1), Glucose 6-phosphatase (G6Pase), Ornithine carbamoyltransferase (OTC), Cystathionine-beta-synthase (CBS), Factor VIII (F8), Hemochromatosis (HFE), Low density lipoprotein receptor, Galactosidase, alpha (AGA), Phenylalanine hydroxy lase (P AH), Propionyl CoA carboxylase, alpha polypeptide (PCCA), or myosin 7A (MY07A). 
     
     
         29 . The library of  claim 21 , wherein the nucleic acid vector is single-stranded. 
     
     
         30 . The library of  claim 21 , wherein the nucleic acid vector is self-complementary. 
     
     
         31 . The library of  claim 27 , wherein the protein, polypeptide, or RNA of interest is a human protein, polypeptide, or RNA. 
     
     
         32 . The library of  claim 21 , wherein each rAAV particle is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. 
     
     
         33 . The library of  claim 21 , wherein the ITR is of serotype AAV2. 
     
     
         34 . The library of  claim 22 , wherein the modified capsid protein comprises at least one of the following mutations and combinations of mutations:
 (a) Y705F, Y731F, or T492V numbered with reference to AAV1;   (b) Y444F+Y500F+Y730F, Y272F+Y444F+Y500F+Y730F+T491V, or Y252F+Y272F+Y444F+Y500F+Y704F+Y730F numbered with reference to AAV2;   (c) Y705F+Y731F, or S663V+T492V numbered with reference to AAV3;   (d) Y719F numbered with reference to AAV5;   (e) Y705F+Y731F, or T492V numbered with reference to AAV6;   (f) Y733F numbered with reference to AAV8; and   (g) Y731F numbered with reference to AAV9.   
     
     
         35 . The library of  claim 21 , wherein the glucocorticoid receptor responsive element and/or the transcription factor binding site replaces a D-sequence or a portion thereof within the ITR.