Methods and compositions for modulating angiogenesis and vasculogenesis
Inventors
Dao, Monique • Tate, Ciara • Case, Casey C.
Assignees
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Publication Number
US-10245286-B2
Publication Date
2019-04-02
Expiration Date
Abstract
Disclosed herein are methods and compositions for stimulating angiogenesis, using cells descended from marrow adherent stromal cells that have been transfected with sequences encoding a Notch intracellular domain. Applications of these methods and compositions include treatment of ischemic disorders such as stroke.
Core Innovation
The disclosure describes methods and compositions for modulating angiogenesis and vasculogenesis using SB623 cells. SB623 cells are derived from marrow adherent mesenchymal stem cells (MSCs) genetically modified to transiently express a Notch intracellular domain (NICD) from a polynucleotide that does not encode full-length Notch protein. The disclosed cell preparation is described as SB623-conditioned medium and SB623 cell transplantation.
The disclosure states that SB623-conditioned medium and SB623 cell transplantation promote endothelial survival and angiogenic behaviors. Endothelial survival is described using reduced PI uptake and increased Bcl-2, and endothelial proliferation is described using increased Ki67. Angiogenic behaviors are described using tube formation in HUVECs and increased vessel sprouting and branching in a rodent aortic ring assay.
The disclosure further states that conditioned medium from MSCs versus SB623 cells differentially expresses trophic and angiogenic factors. The disclosed factors include higher angiogenin, ANG-2, and HB-EGF in SB623, while VEGF is reported as high in both MSC and SB623 conditioned media. The disclosure reports partial reversal of SB623-mediated effects by a VEGFR2 inhibitor (SU5416), supporting a role for VEGF signaling.
Embodiments are described for ischemic disorders occurring outside the central nervous system, including stroke and other ischemic indications. The disclosure describes the relationship between the NICD-expressing MSC derivatives producing angiogenin, angiopoietin-2 (ANG-2), and HB-EGF at higher levels than the starting MSCs, and endothelial and vessel outcomes including endothelial cell death prevention/survival enhancement, endothelial cell proliferation, blood vessel sprouting, and vessel branching. The disclosure also describes combinational use with a second pro-angiogenic agent such as a VEGF-activating transcription factor, including an engineered zinc finger transcription factor associated with VEGF gene transcription.
Claims Coverage
The provided independent claims cover treatment of an ischemic disorder outside the central nervous system using NICD-expressing mesenchymal stem cells cultured without selection and without trophic factors, and a combination regimen including such cells plus a second pro-angiogenic agent. Across the independent claims, four core inventive elements are emphasized: target location outside the central nervous system, NICD polynucleotide modification of MSCs, culture conditions excluding selection and trophic factors, and increased production of angiogenin, ANG-2, and HB-EGF relative to the starting MSCs.
Treating ischemic disorder outside the central nervous system with NICD-expressing MSCs
Administering, to a site of ischemic disorder in a subject that is outside the central nervous system, a therapeutically effective amount of cells.
NICD polynucleotide not encoding full-length Notch protein in MSCs
Making the cells by providing a culture of mesenchymal stem cells and contacting the cell culture with a polynucleotide comprising sequences encoding a Notch intracellular domain (NICD) wherein the polynucleotide does not encode a full-length Notch protein, selecting cells that comprise the polynucleotide.
Culture without selection and without trophic factors
Further culturing the selected cells in the absence of selection and in the absence of trophic factors.
Cells produce angiogenin, ANG-2 and HB-EGF at higher levels
The cells produce angiogenin, angiopoietin-2 (ANG-2) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) at higher levels than do the mesenchymal stem cells.
Combination including first NICD-expressing pro-angiogenic cells and a second pro-angiogenic agent
Providing a combination comprising a first pro-angiogenic agent comprising cells made by culturing NICD polynucleotide-contacted mesenchymal stem cells in the absence of selection and in the absence of trophic factors so that the cells produce angiogenin, ANG-2 and HB-EGF at higher levels than the mesenchymal stem cells, and a second pro-angiogenic agent.
Second pro-angiogenic agent as polypeptide or nucleic acid, including VEGF-activating transcription factor
Defining the second pro-angiogenic agent as a polypeptide or a nucleic acid, and specifying the transcription factor as a non-naturally-occurring zinc finger protein that activates transcription of the VEGF gene.
Claim coverage centers on ischemic treatment outside the central nervous system using mesenchymal stem cell derivatives transiently expressing NICD from a polynucleotide that does not encode full-length Notch, cultured without selection and without trophic factors, and resulting in higher production of angiogenin, ANG-2, and HB-EGF versus the starting MSCs. A related independent claim expands to a combination including a second pro-angiogenic agent, with dependent refinements specifying modulation of VEGF gene transcription via a non-naturally-occurring zinc finger transcription factor.
Stated Advantages
Promotes endothelial survival (reduced PI uptake and increased Bcl-2).
Promotes endothelial cell proliferation (increased Ki67).
Enhances angiogenic behaviors including tube formation and increased vessel sprouting and branching.
Produces angiogenin, angiopoietin-2 (ANG-2), and HB-EGF at higher levels than do the mesenchymal stem cells.
Partial reversal of SB623-mediated effects by a VEGFR2 inhibitor (SU5416) supports a role for VEGF signaling.
Documented Applications
Treating an ischemic disorder in a subject where the ischemic disorder occurs outside the central nervous system, including stroke as an ischemic indication described in the disclosure.
Treating specific ischemic disorders selected from cardiac ischemia, ischemia of the bowel, ischemia of the limb, cutaneous ischemia, and retinal ischemia, and cerebral palsy.
Augmenting angiogenesis by prevention of endothelial cell death, enhancement of endothelial cell survival, stimulation of endothelial cell proliferation, and enhancement of blood vessel sprouting and vessel branching.
Combination use with a second pro-angiogenic agent, including a VEGF-activating transcription factor such as an engineered zinc finger protein associated with VEGF gene transcription.
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