High Transgene Expression Of A Pseudotyped Adeno-Associated Virus Type

Abstract

The present invention related to methods and compositions comprising recombinant vectors comprising chimeric capsids and recombinant pseudotyped vectors with non-native capsid protein(s). The recombinant vectors of the invention confer an altered tropism that permits selective targeting of desired cells.

1 .- 31 . (canceled) 
     
     
         32 . A recombinant pseudotyped adeno-associated virion for use in neural cells comprising:
 a transgene flanked 5′ and 3′ by inverted terminal repeat (ITR) sequences, wherein at least one ITR sequence is derived from a first adeno-associated virus (AAV);   a Rep expression product from the rep coding region of a second adeno-associated virus (AAV), wherein the second AAV is different from the first AAV; and   a non-native capsid derived from the first adeno-associated virus (AAV), such that the transgene is packaged within the non-native capsid, and wherein the non-native capsid provides a modified tropism and can bind to an attachment site present on a cell surface of a neural cell with a higher affinity than a corresponding adeno-associated virion with a wild type capsid.   
     
     
         33 . The recombinant virion of  claim 32 , wherein at least one of the ITR sequences is derived from AAV2 and the other ITR sequence is derived from an adeno-associated virus (AAV) selected from the group consisting of AAV1, AAV3, AAV4, AAV5, and AAV6. 
     
     
         34 . The recombinant virion of  claim 32 , wherein the first adeno-associated virus type is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6. 
     
     
         35 . The recombinant virion of  claim 32 , wherein the second adeno-associated virus type is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6. 
     
     
         36 . The recombinant virion of  claim 32 , wherein the first AAV is AAV5. 
     
     
         37 . The recombinant virion of  claim 32 , wherein second AAV is AAV2. 
     
     
         38 . The recombinant virion of  claim 32 , wherein the Rep expression product and capsid are encoded by the nucleic acid of SEQ ID NO:14. 
     
     
         39 . The recombinant virion of  claim 32 , wherein the transgene is operably linked to a promoter in a transgene expression cassette. 
     
     
         40 . The recombinant virion of  claim 39 , wherein the promoter is a tissue-specific promoter. 
     
     
         41 . The recombinant virion of  claim 39 , wherein the promoter is operable in a brain or spinal cord neural cell. 
     
     
         42 . The recombinant virion of  claim 39 , wherein the promoter is operable in a stratium neural cell. 
     
     
         43 . The recombinant virion of  claim 39 , wherein the promoter is operable in a hippocampal neural cell. 
     
     
         44 . The recombinant virion of  claim 32 , wherein the recombinant virion has a cell transduction rate that is about 2-fold higher than the transduction rate of the corresponding wild type adeno-associated virion. 
     
     
         45 . A recombinant pseudotyped adeno-associated virus type-5 virion for use in a neural cell comprising:
 a transgene flanked 5′ and 3′ by inverted terminal repeat sequences derived from adeno-associated virus-5 (AAV5);   a Rep expression product from the rep coding region of adeno-associated virus-2 (AAV2); and   a non-native capsid derived from adeno-associated virus-5 (AAV5), such that the transgene is packaged within the AAV5 capsid, wherein the AAV5 capsid has a higher affinity to an attachment site present on a cell surface of the neural cell than a corresponding adeno-associated virion with a wild type capsid.   
     
     
         46 . The recombinant virion of  claim 45 , wherein the Rep expression product and capsid are encoded by the nucleic acid of SEQ ID NO:14. 
     
     
         47 . The recombinant virion of  claim 45 , wherein the transgene is operably linked to a promoter in a transgene expression cassette. 
     
     
         48 . The recombinant virion of  claim 47 , wherein the promoter is operable in a brain or spinal cord neural cell. 
     
     
         49 . The recombinant virion of  claim 47 , wherein the promoter is operable in a stratium neural cell. 
     
     
         50 . The recombinant virion of  claim 47 , wherein the promoter is operable in a hippocampal neural cell. 
     
     
         51 . A method of making a recombinant pseudotyped adeno-associated virion with a modified tropism comprising:
 providing a first construct comprising a transgene flanked 5′ and 3′ with inverted terminal repeat sequences derived from a first adeno-associated virus type, wherein at least one inverted terminal repeat sequence comprises a packaging signal, and a second helper construct comprising a rep coding region derived from a second adeno-associated virus type and a cap coding region derived from the first adeno-associated virus type, wherein the cap coding region encodes for a non-native capsid, and wherein the first adeno-associated virus type is different from the second adeno-associated virus type; and   contacting a population of cells with the first and second constructs, such that the population of cells allows assembly of a recombinant virion comprising a non-native capsid, to thereby produce a recombinant pseudotyped virion with a modified tropism, wherein the recombinant pseudotyped virion can bind to an attachment site present on a cell surface of a neural cell, with a higher affinity than a corresponding adeno-associated virion with a wild type capsid.   
     
     
         52 . The method of  claim 51 , wherein the first adeno-associated virus type is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6. 
     
     
         53 . The method of  claim 51 , wherein the second adeno-associated virus type is adeno-associated virus type-2 (AAV2). 
     
     
         54 . The method of  claim 51 , wherein the step of contacting the population of cells further comprises contacting a population of 293 cells. 
     
     
         55 . The method of  claim 51 , wherein the step of contacting the population of cells comprises the recombinant pseudotyped virion capable of transducing the population of cells at about 2-fold to about 30-fold higher transduction rate than the corresponding wild type adeno-associated virion.