Treatment of cone cell degeneration with transfected lineage negative hematopoietic stem cells

Abstract

Transfected, mammalian, adult bone marrow-derived, lineage negative hematopoietic stem cell populations (Lin − HSCs) contain endothelial progenitor cells (EPCs) capable of rescuing retinal blood vessels and neuronal networks in the eye. Preferably at least about 20% of the cells express the cell surface antigen CD31. The transfected stem cells express an antiangiogenic fragment of tryptophanyl tRNA synthetase (TrpRS). The transfected Lin − HSC populations are useful for treatment of ocular vascular diseases and to ameliorate cone cell degeneration in the retina. In a preferred embodiment, the Lin − HSCs are isolated by extracting bone marrow from an adult mammal; separating a plurality of monocytes from the bone marrow; labeling the monocytes with biotin-conjugated lineage panel antibodies to one or more lineage surface antigens; removing of monocytes that are positive for the lineage surface antigens from the plurality of monocytes, and recovering a Lin − HSC population containing EPCs. The treatment may be enhanced by stimulating proliferation of activated astrocytes in the retina using a laser.

1 . A method of ameliorating cone cell degeneration in the retina of a mammal comprising the step of administering to the retina of a mammal that suffers from an ocular disease a transfected, mammalian bone marrow-derived, lineage negative hematopoietic stem cell population, which comprises hematopoietic stem cells and endothelial progenitor cells, the transfected stem cell population expressing an antiangiogenic fragment of human tryptophanyl tRNA synthetase (TrpRS), the transfected stem cell population being administered in an amount sufficient to retard cone cell degeneration in the retina.  
     
     
         2 . The method of  claim 1  wherein the fragment of TrpRS is selected from the group consisting of T2-TrpRS (SEQ ID NO: 3) and T2-TrpRS-GD (SEQ ID NO: 4).  
     
     
         3 . The method of  claim 1  wherein at least about 20% of the cells in the transfected, lineage negative, hematopoietic stem cell population express the surface antigen CD31.  
     
     
         4 . The method of  claim 1  wherein at least about 50% of the cells in the transfected, lineage negative, hematopoietic stem cell population express the surface antigen CD31.  
     
     
         5 . The method of  claim 1  wherein at least about 75% of the cells in the transfected, lineage negative, hematopoietic stem cell population express the surface antigen CD31.  
     
     
         6 . The method of  claim 1  wherein at least about 50% of the cells in the transfected, lineage negative, hematopoietic stem cell population express the surface antigen integrin α 6 .  
     
     
         7 . The method of  claim 1  wherein the transfected lineage negative, hematopoietic stem cell population comprises transfected adult bone marrow cells.  
     
     
         8 . The method of  claim 1  wherein the transfected, lineage negative, hematopoietic stem cell population comprises transfected murine cells.  
     
     
         9 . The method of  claim 8  wherein at least about 50% of the cells in the transfected, lineage negative, hematopoietic stem cell population express the surface antigen CD31 and at least about 50% of the cells express the surface antigen CD117.  
     
     
         10 . The method of  claim 8  wherein at least about 65% of the cells in the transfected, lineage negative, hematopoietic stem cell population express the surface antigen CD117.  
     
     
         11 . The method of  claim 8  wherein at least about 80% of the cells in the transfected, lineage negative, hematopoietic stem cell population express the surface antigen CD31 and at least about 70% of the cells express the surface antigen CD117.  
     
     
         12 . The method of  claim 1  wherein the transfected, lineage negative, hematopoietic stem cell population comprises transfected human cells.  
     
     
         13 . The method of  claim 12  wherein the cells in the transfected, lineage negative, hematopoietic stem cell population are CD133 negative, at least about 50% of the cells express the surface antigen integrin α 6 , and at least about 50% of the cells express the surface antigen CD31.  
     
     
         14 . The method of  claim 12  wherein the cells in the transfected, lineage negative, hematopoietic stem cell population are CD133 positive, less than about 30% of the cells express the surface antigen integrin α 6 , and less than about 30% of the cells express the surface antigen CD31.  
     
     
         15 . The method of  claim 1  including the additional steps of isolating a lineagen negative, hematopoietic stem cell population from the mammal that suffers from the ocular disease, and transfecting the cells with a gene that operably encodes an antiangiogenic fragment of TrpRS, prior to administering the cells to the retina.  
     
     
         16 . The method of  claim 15  wherein the lineage negative, hematopoietic stem cell population is isolated by: 
 (a) extracting bone marrow from the mammal to be treated;    (b) separating a plurality of monocytes from the bone marrow;    (c) labeling the monocytes with biotin-conjugated lineage panel antibodies to one or more lineage surface antigens selected from the group consisting of CD2, CD3, CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, and CD235a; and    (d) removing monocytes that are positive for said one or more lineage surface antigens from the plurality of monocytes and recovering a population of lineage negative hematopoietic stem cells containing endothelial progenitor cells.    
     
     
         17 . The method of  claim 16  wherein the mammal is a mouse.  
     
     
         18 . The method of  claim 16  wherein the mammal is a mouse and the monocytes are labeled in step (c) with biotin-conjugated lineage panel antibodies to CD3, CD11, CD45, Ly-6G, and TER-119.  
     
     
         19 . The method of  claim 16  wherein the mammal is a human.  
     
     
         20 . The method of  claim 19  wherein the monocytes are labeled in step (c) with biotin-conjugated lineage panel antibodies to CD2, CD3, CD4, CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86, and CD235a.  
     
     
         21 . The method of  claim 19  wherein the method includes the additional steps of labeling the monocytes with a biotin-conjugated CD133 antibody and recovering a population of CD133 positive, lineage negative hematopoietic stem cells.  
     
     
         22 . The method of  claim 19  wherein the method includes the additional steps of labeling the monocytes with a biotin-conjugated CD133 antibody, removing CD133 positive cells, and recovering a population of CD133 negative, lineage negative hematopoietic stem cells.  
     
     
         23 . The method of  claim 1  wherein the transfected, lineage negative, hematopoietic stem cell population is administered by intraocular injection.  
     
     
         24 . The method of  claim 1  wherein the ocular disease is selected from the group consisting of a retinal degenerative disease, an ischemic retinopathy, a vascular hemorrhage, a vascular leakage, a choroidopathy, age related macular degeneration, diabetic retinopathy, presumed ocular histoplasmosis, retinopathy of prematurity, sickle cell anemia, and retinitis pigmentosa.  
     
     
         25 . The method of  claim 1  wherein the lineage negative hematopoietic stem cell population is transfected with a gene that operably encodes a therapeutically useful peptide, in addition to a TrpRS fragment, prior to administering the stem cells to the retina of the mammal.  
     
     
         26 . The method of  claim 25  wherein the therapeutically useful peptide is an anti-angiogenic peptide.  
     
     
         27 . The method of  claim 25  wherein the therapeutically useful peptide is a neurotrophic agent.  
     
     
         28 . The method of  claim 27  wherein the neurotrophic agent is selected form the group consisting of nerve growth factor, neurotrophin-3, neurotrophin-4, neurotrophin-5, ciliary neurotrophic factor, retinal pigmented epithelium-derived neurotrophic factor, insulin-like growth factor, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor.  
     
     
         29 . The method of  claim 1  wherein the transfected, lineage negative, hematopoietic stem cell population is prepared by: 
 (a) extracting bone marrow from an adult mammal;    (b) separating a plurality of monocytes from the bone marrow;    (c) labeling the plurality of monocytes with biotin-conjugated lineage panel antibodies to CD2, CD3, CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, and CD235a;    (d) separating monocytes that are positive for said one or more lineage surface antigens from the plurality of monocytes and recovering a population of lineage negative hematopoietic stem cells containing endothelial progenitor cells; and    (e) transfecting the lineage negative hematopoietic stem cells recovered in step (d) with a polynucleotide that operably encodes an antiangiogenic TrpRS fragment.    
     
     
         30 . The method of  claim 29  wherein the TrpRS fragment is selected from the group consisting of T2-TrpRS (SEQ ID NO: 3) and T2-TrpRS-GD (SEQ ID NO: 4).  
     
     
         31 . The method of  claim 1  wherein astrocytes in the retina of the mammal are stimulated using a laser prior to administering the cells.  
     
     
         32 . A method of preserving cone cells in the retina of a mammal suffering from and an ocular disease comprising isolating from the bone marrow of the mammal a transfected, lineage-negative hematopoietic stem cell population that includes endothelial progenitor cells and expresses an antiangiogenic fragment of human tryptophanyl tRNA synthetase (TprRS), and subsequently intravitreally injecting cells from the transfected stem cell population into an eye of the mammal in an amount sufficient to ameliorate the degeneration of cone cells in the retina.  
     
     
         33 . The method of  claim 32  wherein the amount of injected stem cells is effective for repairing retinal damage of the mammal's eye.  
     
     
         34 . The method of  claim 32  wherein the amount of injected stem cells is effective for stabilizing retinal neovasculature of the mammal's eye.  
     
     
         35 . The method of  claim 32  wherein the amount of injected stem cells is effective for maturing retinal neovasculature of the mammal's eye.  
     
     
         36 . The method of  claim 32  wherein astrocytes in the retina of the mammal are stimulated using a laser prior to administering the cells.  
     
     
         37 . The method of  claim 32  wherein the fragment of TrpRS is selected from the group consisting of T2-TrpRS (SEQ ID NO: 3) and T2-TrpRS-GD (SEQ ID NO: 4).  
     
     
         38 . A method of preserving cone cells in the retina of a mammal suffering from and an ocular disease comprising isolating from the bone marrow of the mammal a lineage negative hematopoietic stem cell population that includes endothelial progenitor cells, transfecting the isolated cells with a gene that operably encodes an antiangiogenic fragment of human tryptophanyl tRNA synthetase, treating the retina with a laser to stimulate local proliferation of activated astrocytes in the retina, and subsequently intravitreally injecting the transfected stem cells into the eye of the mammal in a number sufficient to ameliorate the degeneration of cone cells in the retina.  
     
     
         39 . The method of  claim 38  wherein the fragment of TrpRS is selected from the group consisting of T2-TrpRS (SEQ ID NO: 3) and T2-TrpRS-GD (SEQ ID NO: 4).