Recombinant vectors for use in position-independent transgene expression within chromatin

Abstract

The embodiments of the present invention are directed to discrete, cis-acting regulatory elements that include a barrier element, an insulating element, a silencing element, and matrix attachment regions (“MARs”). Additional embodiments of the present invention are directed to nucleic acid molecules that are useful for facilitating stable transgene expression within a chromatin environment. Additional embodiments of the present invention are directed to recombinant expression vectors including nucleic acid molecules of the present invention that can be incorporated into artificial chromosomes, eukaryotic cell-lines, non-human transgenic animals, and transgenic plants, to improve recombinant protein production in a broad range of eukaryotic hosts, and pharmaceutical compositions including nucleic acid molecules of the present invention that are also useful for gene therapy in the treatment of various genetic diseases.

1 . An isolated nucleic acid molecule having one or more activities including an enhancer-blocking activity, an insulating activity, a silencing activity, and a barrier activity, the isolated nucleic acid derived from a c-myc locus of a mammal, and having at least 50% nucleotide identity with a corresponding subsequence of the c-myc locus, the nucleic acid molecule comprising one of: 
 a MINE (c-myc Insulator Element) sequence;    a MINE sequence fragment;    a variant sequence; and    a variant sequence fragment.    
     
     
         2 . The nucleic acid molecule of  claim 1  wherein the mammal is a human or a rodent.  
     
     
         3 . The nucleic acid molecule of  claim 1  wherein the MINE sequence is SEQ ID NO:1, a variant of SEQ ID NO:1, SEQ ID NO:9, or a variant of SEQ ID NO:9.  
     
     
         4 . The nucleic acid molecule of  claim 1  wherein the fragment of the MINE comprises a BE (barrier element).  
     
     
         5 . The nucleic acid molecule of  claim 4  wherein the BE has the sequence of: SEQ ID NO:3; a variant of SEQ ID NO:3; SEQ ID NO:10; or a variant of SEQ ID NO:10.  
     
     
         6 . The nucleic acid molecule of  claim 1  wherein the fragment of the MINE comprises a 5′CTCF-binding site.  
     
     
         7 . The nucleic acid molecule of  claim 6  wherein the 5′CTCF-binding site has the sequence of: SEQ ID NO: 15; a variant of SEQ ID NO: 15; SEQ ID NO:16; or a variant of SEQ ID NO: 16.  
     
     
         8 . The nucleic acid molecule of  claim 1  wherein the fragment comprises a first silencing element that suppresses an expression of an exogenous gene.  
     
     
         9 . The nucleic acid molecule of  claim 1  wherein the fragment comprises a second silencing element that suppresses an expression of an exogenous gene.  
     
     
         10 . The nucleic acid molecule of  claim 8  wherein the first silencing element has the sequence of: SEQ ID NO:4; a variant of SEQ ID NO:4; SEQ ID NO:207; or a variant of SEQ ID NO:207.  
     
     
         11 . The nucleic acid molecule of  claim 9  wherein the second silencing element has the sequence of: SEQ ID NO:5; a variant of SEQ ID NO:5; SEQ ID NO:208; or a variant of SEQ ID NO:208.  
     
     
         12 . An isolated nucleic acid molecule that functions as a nuclear-matrix attachment region (“MAR”) when inserted into a host chromosome, the isolated nucleic acid derived from a c-myc locus of a mammal, and having at least 50% nucleotide identity with a corresponding subsequence of the c-myc locus, the nucleic acid molecule comprising one of: 
 a MAR sequence;    a MAR fragment;    a variant sequence of MAR; and    a variant fragment.    
     
     
         13 . The nucleic acid molecule of  claim 12  wherein the MAR is derived from a region 5′ of a human c-myc gene (MYC-5′MAR), and has the sequence of SEQ ID NO:6.  
     
     
         14 . The nucleic acid molecule of  claim 12  wherein the MAR is derived from a region 3′ of a human c-myc gene (MYC-3′MAR), and has the sequence of SEQ ID NO:7.  
     
     
         15 . The nucleic acid molecule of  claim 14  that comprises a sub-region of the MYC-3′MAR that has the sequence of SEQ ID NO:8, or that has at least 50% identity to SEQ ID NO:8.  
     
     
         16 . The nucleic acid molecule of  claim 12  wherein the MAR is derived from a region 5′ of a mouse c-myc gene (MYC-5′MAR), and has the sequence of SEQ ID NO:11.  
     
     
         17 . The nucleic acid molecule of  claim 12  wherein the MAR is derived from a region 3′ of a mouse c-myc gene (MYC-3′MAR), and has the sequence of SEQ ID NO:12.  
     
     
         18 . The nucleic acid molecule of  claim 17  that has at least 50% identity to SEQ ID NO: 12.  
     
     
         19 . An expression vector comprising: 
 a transgene that is operably linked to a promoter and an enhancer; and    a MINE (c-Myc Insulator Element), or a MINE fragment, that is operably linked to the gene, the promoter, and the enhancer.    
     
     
         20 . The expression vector of  claim 19  wherein the components are positioned in a 5′-to-3′ direction in the following order: the MINE sequence or the MINE fragment, the enhancer, the promoter, and the gene of interest.  
     
     
         21 . The expression vector of  claim 19  wherein the MINE, or the MINE fragment, is positioned relative to the enhancer, the promoter, and the gene of interest, in an orientation that is either 5′-to-3′ direction, or 3′-to-5′ direction.  
     
     
         22 . The expression vector of  claim 19  wherein the MINE fragment comprises a BE.  
     
     
         23 . The expression vector of  claim 19  wherein the MINE fragment comprises a 5′CTCF-binding site.  
     
     
         24 . The expression vector of  claim 19  wherein vector includes a selectable marker.  
     
     
         25 . The expression vector of  claim 19  that further comprises a matrix attachment region (MAR) derived from any gene locus of any eukaryotic organism, and wherein the MAR is positioned 5′ (upstream) of a MINE or positioned 3′ (downstream) of the gene of interest.  
     
     
         26 . The expression vector of  claim 25  wherein the MAR is derived from the 5′ region of a c-myc gene (MYC-5′MAR), or the 3′ region of a c-myc gene (MYC-3′MAR).  
     
     
         27 . An artificial chromosome comprising the vector of  claim 19 .  
     
     
         28 . An eukaryotic host cell comprising the vector of  claim 19 .  
     
     
         29 . An eukaryotic host cell comprising the artificial chromosome of  claim 27 .  
     
     
         30 . A non-human transgenic animal comprising the vector of  claim 19 .  
     
     
         31 . A transgenic plant comprising the vector of  claim 19 .  
     
     
         32 . A pharmaceutical composition comprising: 
 the vector of  claim 19;  and    a pharmaceutical carrier.    
     
     
         33 . A method for decreasing a position-effect variegation effect on an expression level of an exogenous gene, the method comprising: 
 introducing the vector of  claim 19  into a eukaryotic host cell; and    permitting the vector to randomly integrate into heterochromatin of the host cell, wherein a MINE, a MINE fragment, or a MINE variant within the vector inhibits the progression of the heterochromatin at an integration site.    
     
     
         34 . The method of  33  wherein the sequence comprises the one or more of the following sequences, or variants of the sequences: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; and SEQ ID NO:12.  
     
     
         35 . A method for decreasing a position-effect variegation effect on an expression level of an exogenous gene, which is expressed to treat a diseased eukaryotic host animal, the method comprising: 
 administering a pharmaceutical composition comprising the vector of  claim 19  into the diseased host;    permitting the vector to enter a eukaryotic cell of the diseased host;    permitting the vector to randomly integrate into an heterochromatin genome of the host cell; and    enabling a sequence within the vector to inhibit the progression of the heterochromatin at an integration site.    
     
     
         36 . The method of  claim 35  wherein the eukaryotic host contains a mutant allele the expression of which causes a diseased phenotype that is amenable to treatment by expression of a transgene comprising a wildtype allele.