Vectors and methods to treat ischemia

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.

What is claimed is: 
     
         1 . A method for treating peripheral artery disease and/or critical limb ischemia in a patient in need thereof comprising administering to the patient an effective amount of an isolated marrow stromal cell comprising a lentiviral vector, wherein the vector comprises:
 (a) a nucleic acid encoding a 165A isoform VEGF protein or an equivalent thereof, and   (b) a promoter that regulates expression of the nucleic acid encoding the 165A isoform VEGF or an equivalent thereof.   
     
     
         2 . 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 to the patient an effective amount of an isolated marrow stromal cell comprising a lentiviral vector, wherein the vector comprises:
 (a) a nucleic acid encoding a 165A isoform VEGF protein or an equivalent thereof, and   (b) a promoter that regulates expression of the nucleic acid encoding the 165A isoform VEGF or an equivalent thereof.   
     
     
         3 . A method for salvaging a limb in a patient with peripheral artery disease or critical limb ischemia comprising administering to the patient an effective amount an isolated marrow stromal cell comprising a lentiviral vector to a patient in need thereof, wherein the vector comprises:
 (a) a nucleic acid encoding a 165A isoform VEGF protein or an equivalent thereof, and   (b) a promoter that regulates expression of the nucleic acid encoding the 165A isoform VEGF or an equivalent thereof.   
     
     
         4 . A method for increasing vascularization in a patient in need thereof comprising administering to the patient an effective amount of an isolated marrow stromal cell comprising a lentiviral vector, wherein the vector comprises:
 (a) a nucleic acid encoding a 165A isoform VEGF protein or an equivalent thereof, and   (b) a promoter that regulates expression of the nucleic acid encoding the 165A isoform VEGF or an equivalent thereof.   
     
     
         5 . The method of any one of  claims 1 - 4 , wherein the promoter comprises a constitutive promoter. 
     
     
         6 . The method of claims any one of  claims 1 - 4 , wherein the cell further comprises a nucleic acid sequence encoding thymidine kinase (TK). 
     
     
         7 . The method of any one of  claims 1 - 4 , wherein the isolated marrow stromal cell expresses the marker phenotype CD34−/CD45−/CD105+/CD90+/CD73+, or the marker phenotype CD105+/CD90+/CD73+. 
     
     
         8 . The method of any one of  claims 1 - 4 , wherein the vector comprises at least nucleotides 4661-6466 of SEQ ID NO: 26, or an equivalent thereof, wherein an equivalent has at least 80% sequence identity thereto or its complement; or a polynucleotide that hybridizes under conditions of high stringency to nucleotides 4661-6466 of SEQ ID NO: 26, or its complement, wherein the 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 
     
     
         9 . The method of any one of  claims 1 - 4 , wherein the promoter comprises a MNDU3 promoter. 
     
     
         10 . The method of any one of  claims 1 - 4 , wherein the patient is a human and the effective amount is a dosage of between about 50 million cells to about 100 million cells in total. 
     
     
         11 . An isolated marrow stromal cell expresses the marker phenotype CD34−/CD45−/CD105+/CD90+/CD73+, or the marker phenotype CD105+/CD90+/CD73+, wherein the cell comprises a lentiviral vector, and wherein the vector comprises:
 (a) a nucleic acid encoding a 165A isoform VEGF protein or an equivalent thereof, and 
 (b) a promoter that regulates expression of the nucleic acid encoding the 165A isoform VEGF or an equivalent thereof. 
 
     
     
         12 . The cell of  claim 11 , wherein the promoter comprises a constitutive promoter. 
     
     
         13 . The cell of  claim 11  or  12 , wherein the cell further comprises a nucleic acid sequence encoding thymidine kinase (TK). 
     
     
         14 . The cell of  claim 11 , wherein the vector comprises at least nucleotides 4661-6466 of SEQ ID NO: 26, or an equivalent thereof, wherein an equivalent has at least 80% sequence identity thereto or its complement; or a polynucleotide that hybridizes under conditions of high stringency to nucleotides 4661-6466 of SEQ ID NO: 26, or its complement, wherein the 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. 
     
     
         15 . The cell of  claim 11 , wherein the promoter comprises a MNDU3 promoter.