Genetically modified msc and therapeutic methods

Abstract

This disclosure relates to vectors, isolated cells, compositions, and methods for the treatment of critical limb ischemia and associated disorders. One aspect of the disclosure relates to a vector comprising a nucleic acid encoding a 165A isoform VEGF protein and a promoter that regulates expression of the nucleic acid encoding the VEGF.

1 . A vector comprising:
 (a) a nucleic, acid encoding a 165A isoform VEGF protein or an equivalent thereof, and   (b) a promoter that regulates expression of e nucleic acid encoding the 165A isoform VEGF or an equivalent thereof.   
     
     
         2 . The vector of  claim 1 , further comprising an enhancer element. 
     
     
         3 - 5 . (canceled) 
     
     
         6 . The vector of  claim 1 , further comprising
 (c) a nucleic acid encoding a tetracycline activator protein; and   (d) a promoter that regulates expression of the tetracycline activator protein.   
     
     
         7 . The vector of  claim 6 , wherein the promoter that regulates expression of the tetracycline activator protein is a constitutive promoter. 
     
     
         8 . (canceled) 
     
     
         9 . The vector of  claim 1 , further comprising a suicide gene and a promoter that regulates expression of the suicide gene 
     
     
         10 . The vector of  claim 9 , wherein the nucleic acid encoding the tetracycline activator protein and the suicide gene are regulated by one promoter. 
     
     
         11 . The vector of  claim 10 , further comprising a protease cleavage site between the suicide gene and the nucleic acid encoding the tetracycline activator protein. 
     
     
         12 - 18 . (canceled) 
     
     
         19 . An isolated polynucleotide corresponding to a vector, the polynucleotide comprising nucleotides 4654-8071 of SEQ ID NO: 2 or nucleotides 4667-8160 of SEQ ID NO. 23, or an equivalent of each thereof,
 wherein an equivalent of nucleotides 4654-8071 of SEQ ID NO: 2 comprises a polynucleotide encoding a peptide having VEGF biological activity and having at least 80% sequence identity to nucleotides 4654 to 8071 of SEQ ID NO. 2 or a polynucleotide encoding a peptide having VEGF biological activity and that hybridizes under conditions of high stringency to nucleotides 4654 to 8071 of SEQ ID NO. 2 or its complement, wherein conditions of high stringency comprise incubation temperatures of about 55° C. to about 68° C.; buffer concentrations of about 1×SSC to about 0.1×SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1×SSC, 0.1×SSC, or deionized water, and   wherein an equivalent of nucleotides 4667-8160 of SEQ ID NO. 23 comprises a polynucleotide encoding a peptide having VEGF biological activity and having at least 80% sequence identity to nucleotides 4667-8160 of SEQ ID NO. 23 or a polynucleotide that hybridizes under conditions of high stringency to nucleotides 4667-8071 of SEQ ID NO. 23 or its complement, wherein conditions of high stringency comprise incubation temperatures of about 55° C. to about 68° C.; buffer concentrations of about 1×SSC to about 0.1×SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1×SSC, 0.1×SSC, or deionized water.   
     
     
         20 . A vector comprising the following operatively linked to each other: a first promoter, a nucleic acid encoding a 165A isoform VEGF protein or an equivalent thereof, a second promoter and a TK gene. 
     
     
         21 . The vector of  claim 20 , further comprising an WPRE enhancer. 
     
     
         22 - 23 . (canceled) 
     
     
         24 . A vector comprising the following operatively linked to each other: a 5′LTR, a MNDU3 promoter, a nucleic acid encoding a 165A isoform VEGF protein or an equivalent thereof, a phosphoglycerate kinase 1 (PGK) constitutive promoter, a TK gene, an enhancer, and a 3′ LTR. 
     
     
         25 - 27 . (canceled) 
     
     
         28 . A viral packaging system comprising:
 (a) the vector of  claim 1 ;   (b) a packaging plasmid; and   (c) an envelope plasmid.   
     
     
         29 - 31 . (canceled) 
     
     
         32 . A method for producing a pseudotyped viral particle, comprising transducing a packaging cell line with the system of  claim 28  under conditions suitable to package the viral vector. 
     
     
         33 - 34 . (canceled) 
     
     
         35 . A pseudotyped viral particle comprising a viral vector comprising a polynucleotide encoding a 165A isoform VEGF protein or an equivalent thereof and an envelope protein comprising a ZZ  S. aureus  domain. 
     
     
         36 . An isolated cell comprising the vector of  claim 20 . 
     
     
         37 - 50 . (canceled) 
     
     
         51 . A method for treating peripheral artery disease and/or critical limb ischemia in a patient in need thereof comprising administering an effective amount of the isolated cell of  claim 36 . 
     
     
         52 - 53 . (canceled) 
     
     
         54 . A method for promoting wound healing, promoting or increasing the rate of angiogenesis or wound healing, decreasing the size of a wound, or decreasing the time to wound healing in a patient in need thereof comprising administering an effective amount of the isolated cell of  claim 36 . 
     
     
         55 - 56 . (canceled) 
     
     
         57 . A method for salvaging a limb in a patient with peripheral artery disease or critical limb ischemia comprising administering an effective amount the isolated cell of  claim 36 . 
     
     
         58 - 59 . (canceled) 
     
     
         60 . A method for increasing vascularization in a patient in need thereof comprising administering an effective amount of the isolated cell of  claim 36 . 
     
     
         61 - 74 . (canceled) 
     
     
         75 . An isolated marrow stromal cell expressing the phenotype CD34−/CD45−/CD105+/CD90+/CD73+ and comprising a 165A isoform VEGF polynucleotide or protein or an equivalent of each thereof. 76-82. (canceled)