Methods and compositions relating to restricted expression lentiviral vectors and their applications

Abstract

The present invention provides HIV-derived lentivectors which are safe, highly efficient, and very potent for expressing transgenes for human gene therapy, especially, in human hematopoietic progenitor cells as well as in all other blood cell derivatives. The lentiviral vectors comprise promoters active to promote expression specific to cell types or tissues. Further, promoters are providing that are amenable to control by activators, enhancers, or repressors. These vectors are in a self-inactivating configuration for biosaftey. Additional promoters are also described. The vectors can also comprise additional transcription enhancing elements such as the wood chuck hepatitis virus post-transcriptional regulatory element, without any decrease in the specificity or control exerted by the promoters. These vectors therefore provide useful tools for genetic treatments such as inherited and acquired lympho-hematological disorders, gene-therapies for cancers especially the hematological cancers, as well as for the study of hematopoiesis via lentivector-mediated modification of human HSCs.

1 .- 68 . (canceled) 
     
     
         69 . A lymphocyte transduced in vitro with a lentivirus comprising a transgene positioned under the control of a promoter that is active to support detectable transcription of the transgene in the lymphocyte, and capable of promoting expression of the transgene in the lymphocyte at a signal-to-noise ratio of between about 10 and about 200. 
     
     
         70 . The lymphocyte of  claim 69 , wherein the lymphocyte is a T lymphocyte. 
     
     
         71 . The lymphocyte of  claim 69 , wherein the lentivirus comprises a central polypurine tract (cPPT) positioned upstream of the transgene. 
     
     
         72 . The lymphocyte of  claim 71 , wherein the central polypurine tract (cPPT) comprises the nucleotide sequence of SEQ ID NO: 1. 
     
     
         73 . The lymphocyte of  claim 71 , wherein the lentivirus comprises multiple unique cloning sites positioned adjacent to the central polypurine tract (cPPT). 
     
     
         74 . The lymphocyte of  claim 73 , wherein the multiple unique cloning sites are positioned upstream of the central polypurine tract (cPPT). 
     
     
         75 . The lymphocyte of  claim 73 , wherein the multiple unique cloning sites are positioned downstream of the central polypurine tract (cPPT). 
     
     
         76 . The lymphocyte of  claim 73 , wherein multiple unique cloning sites are positioned both upstream and downstream of the central polypurine tract (cPPT). 
     
     
         77 . The lymphocyte of  claim 69 , wherein the lentivirus is a self-inactivating lentivirus (SIN). 
     
     
         78 . The lymphocyte of  claim 77 , wherein the LTR region of said lentivirus has reduced transcriptional activity by virtue of deletions in the U3 region of a 3′ LTR. 
     
     
         79 . The lymphocyte of  claim 78 , wherein the deletions are of nucleotides at positions −418 through −18 relative to the U3-R region boundary. 
     
     
         80 . The lymphocyte of  claim 69 , wherein the lentivirus comprises at least one enhancer sequence. 
     
     
         81 . The lymphocyte of  claim 69 , wherein the promoter is an EF1-α promoter, a PGK promoter, a gp91-phox promoter, a WIC class II promoter, a CD11 promoter, a CD4 promoter, a CD2 promoter or a gp47-phox promoter. 
     
     
         82 . The lymphocyte of  claim 81 , wherein the promoter is an EF1-α promoter. 
     
     
         83 . The lymphocyte of  claim 81 , wherein the promoter is a PGK promoter. 
     
     
         84 . The lymphocyte of  claim 81 , wherein the promoter is a gp91-phox promoter. 
     
     
         85 . The lymphocyte of  claim 81 , wherein the promoter is a WIC class II promoter. 
     
     
         86 . The lymphocyte of  claim 81 , wherein the promoter is a CD11 promoter. 
     
     
         87 . The lymphocyte of  claim 81 , wherein the promoter is a CD4 promoter. 
     
     
         88 . The lymphocyte of  claim 81 , wherein the promoter is a CD2 promoter. 
     
     
         89 . The lymphocyte of  claim 81 , wherein the promoter is a gp47-phox promoter. 
     
     
         90 . The lymphocyte of  claim 69 , wherein the promoter is capable of promoting expression of the transgene at a signal-to-noise ratio of between about 40 and about 200. 
     
     
         91 . The lymphocyte of  claim 69 , wherein the promoter is capable of promoting expression of the transgene at a signal-to-noise ratio of between about 150 and about 200. 
     
     
         92 . The lymphocyte of  claim 69 , wherein the promoter is capable of promoting expression of the transgene in response to a transcriptional activator. 
     
     
         93 . The lymphocyte of  claim 92 , wherein the transcriptional activator is INF-gamma. 
     
     
         94 . The lymphocyte of  claim 69 , wherein the transgene comprises erythropoietin, an interleukin, a colony-stimulating factor, integrin αIIbβ, a multidrug resistance gene, gp91-phox, gp47-phox, an antiviral gene, a gene coding for blood coagulation factor VIII, a gene coding for blood coagulation factor IX, a T cell antigen receptor, a B cell antigen receptor, a T cell antigen receptor in combination with a single chain antibodies (scFv), a B cell antigen receptor in combination with an ScFv, an ScFv, TNF, gamma interferon, CTLA4, B7, Melana, MAGE, or GFP. 
     
     
         95 . The lymphocyte of  claim 94 , wherein the transgene comprises an interleukin. 
     
     
         96 . The lymphocyte of  claim 94 , wherein the transgene comprises interleukin-2. 
     
     
         97 . The lymphocyte of  claim 94 , wherein the transgene comprises a T cell antigen receptor. 
     
     
         98 . The lymphocyte of  claim 94 , wherein the transgene comprises a T cell antigen receptor in combination with an scFv. 
     
     
         99 . The lymphocyte of  claim 94 , wherein the transgene comprises an scFv. 
     
     
         100 . The lymphocyte of  claim 69 , wherein the lentivirus comprises a posttranscriptional regulatory sequence positioned to promote the expression of the transgene. 
     
     
         101 . The lymphocyte of  claim 100 , wherein the posttranscriptional regulatory sequence is an intron. 
     
     
         102 . The lymphocyte of  claim 101 , wherein the intron is positioned in an orientation opposite that of the lentivirus genomic transcript. 
     
     
         103 . The lymphocyte of  claim 100 , wherein the posttranscriptional regulatory sequence is a posttranscriptional regulatory element. 
     
     
         104 . The lymphocyte of  claim 103 , wherein the posttranscriptional regulatory element comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). 
     
     
         105 . The lymphocyte of  claim 103 , wherein the posttranscriptional regulatory element comprises a hepatitis B virus posttranscriptional regulatory element (HPRE). 
     
     
         106 . A method for transducing a lymphocyte comprising contacting a population of human cells that include lymphocytes with a lentivirus comprising a transgene positioned under the control of a promoter that is active to support detectable transcription of the transgene in the lymphocyte at a signal-to-noise ratio of between about 10 and about 200, under conditions to effect the transduction of a human lymphocyte in said population by said vector.