Hematopoietic stem cells and methods of treatment of neovascular eye diseases therewith

a technology of hematopoietic stem cells and eye diseases, which is applied in the direction of biocide, drug composition, extracellular fluid disorder, etc., can solve the problems of insufficient epc targeting of neovasculature, no treatment is currently available to specifically treat ocular vascular disease, and significant loss of visual function, so as to stabilize the degeneration of retinal vasculature, and stabilize the effect o

Inactive Publication Date: 2007-05-24
THE SCRIPPS RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007] The EPC's in the lineage negative HSC populations of the present invention extensively incorporate into developing retinal vessels and remain stably incorporated into neovasculature of the eye. The isolated, lineage negative HSC populations of the present invention can be used to rescue and stabilize degenerating retinal vasculature in mammals. In one embodiment of the isolated Lin− HSC populations of the present invention, the cells are transfected with a therapeutically useful gene. The transfected cells can selectively target neovasculature and inhibit new vessel formation without affecting already established vessels through a form of cell-based gene therapy. Cells from isolated, lineage negative HSC population of the present invention that have been transfected with a gene encoding angiogenesis inhibiting peptides are useful for modulating abnormal blood vessel growth in diseases such as ARMD, DR and certain retinal degenerations associated with abnormal vasculature.
[0008] A particular advantage of ocular treatments with the isolated Lin− HSC population of the present invention is a vasculotrophic and neurotrophic rescue effect observed in eyes intravitreally treated with the Lin− HSC. Retinal neurons and photoreceptors are preserved and visual function is maintained in eyes treated with the isolated Lin− HSC of the invention.

Problems solved by technology

Since the retina consists of well-defined layers of neuronal, glial, and vascular elements, relatively small disturbances such as those seen in vascular proliferation or edema can lead to significant loss of visual function.
While significant progress has been made in identifying factors that promote and inhibit angiogenesis, no treatment is currently available to specifically treat ocular vascular disease.
Although these cells have been used in several experimental models of angiogenesis, the mechanism of EPC targeting to neovasculature is not known and no strategy has been identified that will effectively increase the number of cells that contribute to a particular vasculature.

Method used

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  • Hematopoietic stem cells and methods of treatment of neovascular eye diseases therewith
  • Hematopoietic stem cells and methods of treatment of neovascular eye diseases therewith
  • Hematopoietic stem cells and methods of treatment of neovascular eye diseases therewith

Examples

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example 1

Cell Isolation and Enrichment; Preparation of a Lin− HSC Populations A and B

[0041] General Procedure. All in vivo evaluations were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and all evaluation procedures were approved by The Scripps Research Institute (TSRI, La Jolla, Calif.) Animal Care and Use Committee. Bone marrow cells were extracted from B6.129S7-Gtrosa26, Tie-2GFP, ACTbEGFP, FVB / NJ (rd / rd mice) or Balb / cBYJ adult mice (The Jackson Laboratory, Me.).

[0042] Monocytes were then separated by density gradient separation using HISTOPAQUE® polysucrose gradient (Sigma, St. Louis, Mo.) and labeled with biotin conjugated lineage panel antibodies (CD45, CD3, Ly-6G, CD 11, TER-119, Pharmingen, San Diego, Calif.) for Lin− selection. Lineage positive (Lin+) cells were separated and removed from Lin− HSC using a magnetic separation device (AUTOMACS™ sorter, Miltenyi Biotech, Auburn, Calif.). The resulting Lin− HSC population, containing endotheli...

example 2

Intravitreal Administration of Cells

[0046] An eyelid fissure was created with a fine blade to expose the P2 to P6 eyeball. Lineage negative HSC Population A of the present invention (approximately 105 cells in about 0.5 μl to about 1 μl of cell culture medium) was then injected intravitreally using a 33-gauge (Hamilton, Reno, Nev.) needled-syringe.

example 3

EPC Transfection

[0047] Lin− HSC (Population A) were transfected with DNA encoding the T2 fragment of TrpRS also enclosing a His6 tag (SEQ ID NO: 1, FIG. 7) using FUGENE™6 Transfection Reagent (Roche, Indianapolis, Ind.) according to manufacturer's protocol. Cells from a Lin− HSC composition (about 106 cell per ml) were suspended in opti-MEM® medium (Invitrogen, Carlsbad, Calif.) containing stem cell factor (PeproTech, Rocky Hill, N.J.). DNA (about 1 μg) and FuGENE reagent (about 3 μl) mixture was then added, and the mixtures were incubated at about 37° C. for about 18 hours. After incubation, cells were washed and collected. The transfection rate of this system was approximately 17% that was confirmed by FACS analysis. T2 production was confirmed by western blotting. The amino acid sequence of His6-tagged T2-TrpRS is shown as SEQ ID NO: 2, FIG. 8.

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Abstract

Isolated, mammalian, bone marrow-derived, hematopoietic stem cell populations contain endothelial progenitor cells (EPC) capable of forming retinal blood vessels. At least about 50% of the cells in the isolated cell population express CD31 and c-kit. Up to about 8% of the cells can express Sca-1, and up to about 4% of the cells can express Flk-1 / KDR. The cells can incorporate into the vasculature of the retina when intravitreally injected into the eye, and can thereby stabilize or rebuilt diseased or abnormal retinal blood vessels.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a division of U.S. Application for patent Ser. No. 10 / 628,783, which was filed on Jul. 25, 2003, now U.S. Pat. No. 7,153,501, which claims the benefit of Provisional Application for Patent Ser. No. 60 / 398,522, filed on Jul. 25, 2002, and Provisional Application for Patent Ser. No. 60 / 467,051, filed on May 2, 2003, each of which is incorporated herein by reference.STATEMENT OF GOVERNMENT INTEREST [0002] This invention was made with U.S. Government support under grant number CA92577 from the National Cancer Institute and under grants number EY11254, EY12598 and EY12599 from the National Institutes of Health. The United States Government has certain rights in this invention.FIELD OF THE INVENTION [0003] This invention relates to isolated, mammalian, lineage negative hematopoietic stem cells (Lin− HSC) derived from bone marrow. The invention also relates to treatment of vascular diseases of the eye by administering Lin− ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/14C12N5/06C12N5/08A01N63/00C12N15/09A61K35/12A61K35/28A61K48/00A61P9/10A61P27/02C12N5/00C12N5/02C12N5/071C12N5/078C12N5/0789C12N5/10
CPCA61K48/00A61K2035/124C12N5/0647C12N5/0692A61P27/02A61P7/06A61P9/10A61P9/14A61K35/12C12N5/0696
Inventor FRIEDLANDER, MARTINOTANI, ATSUSHISILVA, KAREN DA
Owner THE SCRIPPS RES INST
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