Lectin-derived progenitor cell preservation factors and methods of use

Abstract

The invention relates to an isolated nucleic acid molecule that encodes a protein that is effective to preserve progenitor cells, such as hematopoietic progenitor cells. The nucleic acid comprises a sequence defined by SEQ ID NO:1, a homolog thereof, or a fragment thereof. The encoded protein has an amino acid sequence that comprises a sequence defined by SEQ ID NO:2, a homolog thereof, or a fragment thereof that contains an amino acid sequence TNNVLQVT. Methods of using the encoded protein for preserving progenitor cells in vitro, ex vivo, and in vivo are also described. The invention, therefore, include methods such as myeloablation therapies for cancer treatment wherein myeloid reconstitution is facilitated by means of the specified protein. Other therapeutic utilities are also enabled through the invention, for example, expanding progenitor cell populations ex vivo to increase chances of engraftation, improving conditions for transporting and storing progenitor cells, and facilitating gene therapy to treat and cure a broad range of life-threatening hematologic diseases.

1 . An isolated nucleic acid molecule comprising a sequence that encodes a protein comprising an amino acid sequence as defined by SEQ ID NO:2, a homolog thereof, or a fragment thereof that contains the sequence TNNVLQVT.  
     
     
         2 . A isolated nucleic acid molecule according to  claim 1 , comprising a nucleotide sequence as defined by SEQ ID NO:1, a homolog thereof, or a unique fragment thereof.  
     
     
         3 . An isolated nucleic acid molecule that encodes a protein that preserves progenitor cells, wherein the nucleic acid is isolated from a legume.  
     
     
         4 . An isolated nucleic acid according to  claim 3 , wherein the encoded protein comprises an amino acid sequence as defined by SEQ ID NO:2 or a fragment thereof that contains the sequence TNNVLQVT.  
     
     
         5 . An isolated nucleic acid according to  claim 3 , wherein the nucleic acid encodes a mannose/glucose-specific legume lectin.  
     
     
         6 . An isolated nucleic acid according to  claim 3 , wherein the nucleic acid is isolated from a legume from the tribe Phaseoleae.  
     
     
         7 . An isolated nucleic acid according to  claim 7 , wherein the nucleic acid is isolated from red kidney beans, white kidney beans, hyacinth beans, or black-eyed peas.  
     
     
         8 . An isolated nucleic acid according to  claim 3 , wherein the nucleic acid comprises a nucleotide sequence as defined by SEQ ID NO:1 or a unique fragment thereof.  
     
     
         9 . An isolated nucleic acid according to  claim 3 , wherein the encoded protein preserves progenitor cells that are at least unipotent progenitor cells.  
     
     
         10 . An isolated nucleic acid according to  claim 3 , wherein the encoded protein preserves progenitor cells that are pluripotent progenitor cells.  
     
     
         11 . An isolated nucleic acid according to  claim 3 , wherein the encoded protein preserves progenitor cells that are totipotent progenitor cells.  
     
     
         12 . An isolated nucleic acid according to  claim 3 , wherein the encoded protein preserves progenitor cells that are hematopoietic progenitor cells.  
     
     
         13 . An isolated nucleic acid according to  claim 3 , wherein the encoded protein preserves progenitor cells that comprise nerve, muscle, skin, gut, bone, kidney, liver, pancreas, or thymus progenitor cells.  
     
     
         14 . An isolated nucleic acid according to  claim 9 , wherein the encoded protein preserves progenitor cells that express the CD34 antigen.  
     
     
         15 . An isolated nucleic acid according to  claim 9 , wherein the encoded protein preserves progenitor cells that express the FLK2/FLT3 receptor.  
     
     
         16 . An isolated nucleic acid according to  claim 15 , wherein the encoded protein preserves progenitor cells that are cells modified to express FLK2/FLT3 receptors on their surface.  
     
     
         17 . An isolated nucleic acid according to  claim 16 , wherein the encoded protein has significant binding affinity for FLK2/FLT3 receptor on the cells, wherein binding of the encoded protein with the FLK2/FLT3 receptor mediates the inhibition of differentiation of the cells.  
     
     
         18 . A method for preserving progenitor cells, comprising contacting progenitor cells with a protein encoded by an isolated nucleic acid comprising a nucleotide sequence defined by SEQ ID NO:1, a homolog thereof, or a fragment thereof that encodes an amino acid sequence TNNVLQVT, in an amount sufficient to preserve the progenitor cells.  
     
     
         19 . A method according to  claim 18 , wherein the progenitor cells comprise hematopoietic progenitor cells.  
     
     
         20 . A method according to  claim 19 , wherein the progenitor cells comprise human cells that express the CD34 antigen and/or the FLK2/FLT3 receptor.  
     
     
         21 . A method according to  claim 19 , wherein the progenitor cells comprise murine cells that express the Sca antigen but that do not express mature blood cell lineage antigens.  
     
     
         22 . A method according to  claim 18 , wherein the method comprises contacting the progenitor cells with the protein in vitro, ex vivo, or in vivo.  
     
     
         23 . A method according to  claim 18 , wherein the method further comprises contacting the progenitor cells with FLK2/FLT3 ligand in an amount sufficient to selectively expand the number of progenitor cells without inducing differentiation thereof.  
     
     
         24 . A method of treating a mammal in need of hematopoietic therapy, comprising: 
 a) obtaining a tissue sample from the mammal, the tissue sample comprising hematopoietic progenitor cells;    b) culturing the progenitor cells in the presence of a protein that preserves the progenitor cells, to provide cultured cells enriched in the progenitor cells, wherein the protein is encoded by an isolated nucleic acid comprising a nucleotide sequence defined by SEQ ID NO:1, a homolog thereof, or a fragment thereof that encodes an amino acid sequence TNNVLQVT;    c) subjecting the mammal to conditions sufficient to effect myeloablation; and    d) administering the cultured cells to the mammal following the myeloablation to reconstitute the hematopoietic system of the mammal.    
     
     
         25 . A method according to  claim 24 , wherein the myeloablation conditions comprise bone marrow irradiation, whole body irradiation, or chemically-induced myeloablation.  
     
     
         26 . A method of enriching progenitor cells, comprising culturing progenitor cells in a progenitor-preserving amount of a protein encoded by an isolated nucleic acid comprising a nucleotide sequence defined by SEQ ID NO:1, a homolog thereof, or a fragment thereof that encodes an amino acid sequence TNNVLQVT, wherein the protein specifically preserves the progenitor cells, and wherein the culturing is performed under conditions permitting preservation of progenitor cells while permitting the number of differentiated cells to decrease.  
     
     
         27 . A method according to  claim 26 , wherein the progenitor cells include primitive progenitor cells.  
     
     
         28 . A method according to  claim 26 , wherein the progenitor cells include mature progenitor cells.  
     
     
         29 . A method according to  claim 26 , wherein the progenitor cells are at least substantially free of stromal cells.  
     
     
         30 . A method according to  claim 26 , wherein the culturing conditions include culturing in a medium containing a cytotoxic agent that exhibits selective toxicity for proliferating cells.  
     
     
         31 . A method according to  claim 30 , wherein the cytotoxic agent is adriamycin, cyclophosphamide, taxol or other taxane, cisplatin, or 5-fluorouracil.  
     
     
         32 . A method of improving hematopoietic competence in a mammal, comprising: 
 a) culturing a tissue sample comprising hematopoietic progenitor cells in a growth medium containing a protein encoded by an isolated nucleic acid comprising a nucleotide sequence defined by SEQ ID NO:1, a homolog thereof, or a fragment thereof that encodes an amino acid sequence TNNVLQVT, in an amount sufficient to preserve the progenitor cells and to provide cultured cells enriched in the progenitor cells; and    b) transfusing the enriched cultured cells to the mammal to provide progenitor cells for generating blood cellular components in the mammal.    
     
     
         33 . A method according to  claim 32 , wherein the tissue sample comprises peripheral blood, umbilical cord blood, placental blood, or bone marrow.  
     
     
         34 . A method according to  claim 33 , wherein the tissue sample is autologous to the mammal.  
     
     
         35 . A method according to  claim 32 , wherein the tissue sample is at least substantially free of stromal cells.  
     
     
         36 . A method according to  claim 32 , further comprising ablating hematopoietic tissues in the mammal prior to the transfusing.  
     
     
         37 . In a method of transfecting an exogenous DNA sequence into somatic cells, 
 the improvement comprising transfecting progenitor cells selectively preserved by a protein encoded by an isolated nucleic acid comprising a nucleotide sequence defined by SEQ ID NO:1, a homolog thereof, or a fragment thereof that encodes an amino acid sequence TNNVLQVT.    
     
     
         38 . A composition for preserving viability of progenitor cells ex vivo, comprising a cell growth medium and a protein that preserves progenitor cells, wherein the protein is encoded by a nucleic acid comprising a nucleotide sequence defined by SEQ ID NO:1, a homolog thereof, or a fragment thereof that encodes an amino acid sequence TNNVLQVT.  
     
     
         39 . A method for preserving progenitor cells in a mammal, comprising: 
 a) administering to the mammal a protein that specifically preserves progenitor cells, the protein being encoded by an isolated nucleic acid comprising a nucleotide sequence defined by SEQ ID NO:1, a homolog thereof, or a fragment thereof that encodes an amino acid sequence TNNVLQVT, in an amount sufficient to preserve progenitor cells of the mammal in a substantially non-proliferative state;    b) exposing the mammal to myeloablative conditions sufficient to effect ablation of proliferating myeloid cells but sparing non-proliferating progenitor cells; and    c) following the exposing, reestablishing proliferation or differentiation of the preserved progenitor cells.    
     
     
         40 . A method according to  claim 39 , wherein the reestablishing comprises administering to the mammal a cytokine in an amount sufficient to improve the viability of the progenitor cells  
     
     
         41 . A method according to  claim 40 , wherein the viability-improving cytokine is IL-1, IL-3, IL-6, IL-11, KL, or a combination thereof.  
     
     
         42 . A method according to  claim 39 , wherein the reestablishing comprises administering to the mammal a proliferation-stimulating amount of the FLK2/FLT3 ligand.