Utilization of non-viral sequences for minus-strand DNA transfer and gene reconstitution

Abstract

A retroviral vector for gene reconstitution is provided that includes a 3′ portion of a heterologous nucleic acid sequence 5′ of a first att site of the retroviral vector, a 5′ portion of the heterologous nucleic acid sequence 3′ of a second att site of the retroviral vector. In this vector, a sub-portion of the 3′ portion of the heterologous nucleic acid sequence and the 5′ region of the heterologous nucleic acid sequence are direct repeats. Transformation of a eukaryotic cell with the retroviral vector results in reconstitution and duplication of the heterologous nucleic acid sequence. A retroviral vector for gene reconstitution is also provided that includes a 3′ portion of a heterologous nucleic acid sequence inserted into or adjacent to a 5′ retroviral terminal repeat of the retroviral vector, and a 5′ portion of the heterologous nucleic acid sequence inserted into or adjacent to a 3′ retroviral terminal repeat of the retroviral vector, wherein the 5′ retroviral terminal repeat and the 3′ retroviral terminal repeat each comprise an att site. A sub-portion of the 3′ portion of the heterologous nucleic acid sequence and the 5′ region of the heterologous nucleic acid sequence are direct repeats. Transformation of a eukaryotic cell with this retroviral vector results in reconstitution and duplication of the heterologous nucleic acid sequence. Method and kits are provided for reconstituting and duplicating a nucleic acid molecule in a host cell using these retroviral vectors to introduce a heterologous nucleic acid in the cell. A method is provided for treating a subject that includes contacting a cell of the subject with a therapeutically effective amount of a retroviral vector for gene reconstitution and duplication.

1 . A retroviral vector for gene reconstitution, comprising: 
 a 3′ portion of a heterologous nucleic acid sequence 5′ of a first att site of the retroviral vector;    a 5′ portion of the heterologous nucleic acid sequence 3′ of a second att site of the retroviral vector;    wherein a sub-portion of the 3′ portion of the heterologous nucleic acid sequence and the 5′ portion of the heterologous nucleic acid sequence are direct repeats, and transformation of a eukaryotic cell with the retroviral vector results in reconstitution and duplication of the heterologous nucleic acid sequence.    
     
     
         2 - 45 . (canceled)  
     
     
         46 . A method for reconstituting and duplicating a nucleic acid molecule in a host cell, comprising 
 transforming the host cell with a retroviral vector comprising a 3′ portion of a heterologous nucleic acid sequence inserted into or adjacent to a 5′ retroviral terminal repeat of the retroviral vector, and a 5′ portion of the heterologous nucleic acid sequence inserted into or adjacent to a 3′ retroviral terminal repeat of the retroviral vector, wherein a sub-portion of the 3′ portion of the heterologous nucleic acid sequence and the 5′ region of the heterologous nucleic acid sequence are direct repeats,    wherein the transformation results in viral integration and production of a 5′ long terminal repeat and a 3′ long terminal repeat, thereby reconstituting and duplicating the nucleic acid sequence within the 5′ and the 3′ long terminal repeats.    
     
     
         47 . The method of  claim 46 , wherein the cell is a eukaryotic cell.  
     
     
         48 . The method of  claim 46 , wherein the transformation is in vitro.  
     
     
         49 . The method of  claim 46 , wherein the transformation is in vivo.  
     
     
         50 . The method of  claim 46 , wherein the nucleic acid sequence encodes a polypeptide, and wherein the viral integration results in expression of the nucleic acid sequence encoding the polypeptide.  
     
     
         51 - 52 . (canceled)  
     
     
         53 . The method of claim  51 , wherein the introduction is in vivo.  
     
     
         54 - 55 . (canceled)  
     
     
         56 . A method for treating a subject, comprising contacting a cell of the subject with a therapeutically effective amount of a retroviral vector for gene reconstitution, comprising a 3′ portion of a heterologous nucleic acid sequence 5′ of a first att site of the retroviral vector, a 5′ portion of the heterologous nucleic acid sequence 3′ of a second att site of the retroviral vector, wherein a sub-portion of the 3′ portion of the heterologous nucleic acid sequence and the 5′ region of the heterologous nucleic acid sequence are direct repeats, wherein the contact of the cell with the retroviral vector results in integration of the retroviral vector in a genome of the cell, and treatment of the subject.  
     
     
         57 . The method of  claim 56 , wherein the heterologous nucleic acid sequence is a therapeutic polypeptide or an antisense sequence.  
     
     
         58 . The method of  claim 56 , wherein the subject has a disorder, and wherein expression of the heterologous nucleic acid sequence results in alleviating of a symptom of the disorder.  
     
     
         59 . The method of  claim 56 , wherein the retroviral vector is introduced into the subject's cells ex vivo and the cells are then reintroduced into the subject.  
     
     
         60 . The method of  claim 56 , wherein the subject is a mammal.  
     
     
         61 . The method of  claim 60 , wherein the subject is a human.  
     
     
         62 - 84 . (canceled)