Compositions and methods for osteogenic gene therapy
Inventors
Lau, Kin-Hing William • Baylink, David J. • Hall, Susan L. • Chen, Shin-Tai • Mohan, Subburaman
Assignees
Loma Linda University • US Department of Veterans Affairs
Publication Number
US-8772571-B2
Publication Date
2014-07-08
Expiration Date
2026-06-13
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Abstract
The present disclosure provides compositions and methods for increasing bone growth and/or enhancing wound healing, for example, fracture repair. The disclosure provides recombinant nucleic acids useful for promoting bone growth. For example, the disclosure provides recombinant nucleic acids that encode a fibroblast growth factor-2 (FGF-2) analog. The disclosure also provides vectors and cells incorporating these nucleic acids, as well as FGF-2 analogs encode by them. The disclosure also provides a mouse system of bone marrow transplantation and methods for producing as well as methods for using the system. Methods for inducing division and/or inducing differentiation of a hematopoietic stem cell are also provided, as are methods for enhancing bone growth and/or wound repair (for example, fracture repair).
Core Innovation
The present disclosure provides compositions and methods for increasing bone growth and/or enhancing wound healing, such as fracture repair, by using recombinant nucleic acids encoding osteogenic growth factors, including fibroblast growth factor-2 (FGF-2) analogs. It also describes vectors and cells incorporating these nucleic acids and methods for using a mouse system of bone marrow transplantation to induce division and/or differentiation of hematopoietic stem cells, thereby enhancing bone growth and wound repair.
The background describes the role of growth factors like FGF-2 in regulating cellular proliferation, differentiation, and angiogenesis, with known benefits in wound healing and tissue repair. However, prior gene transfer approaches with FGF-2 yielded inconsistent results. The problem being solved is the need for compositions and methods that increase the efficacy of osteogenic growth factor therapies, such as FGF-2, to enhance bone growth and repair for treating skeletal disorders.
The disclosure addresses this need by delivering osteogenic growth factors via hematopoietic stem cells or progenitor cells that home to and engraft in bone marrow. These cells can be modified ex vivo with vectors encoding growth factors that promote self-renewal and bone formation, such as FGF-2 analogs with increased stability and secretion, including cysteine to serine or asparagine mutations and BMP2/4 secretion signal sequences. The methods utilize constitutive, tissue-specific, or regulatable promoters to control gene expression, enabling targeted and controllable therapy to increase bone growth, assist fracture repair, and enhance wound healing.
Claims Coverage
The claims present one independent claim that describes a multi-step method for identifying agents that modulate bone growth using genetically modified hematopoietic stem cells in a mouse transplantation model.
Use of hematopoietic stem cells expressing secretable FGF-2 analog
Harvesting at least one hematopoietic stem cell and transducing it with heterologous nucleic acid encoding a secretable analog of human fibroblast growth factor-2 comprising a BMP2/4 hybrid secretion signal peptide and mutations substituting alanine for cysteine at amino acid position 70 and asparagine for cysteine at amino acid position 88.
Engraftment in immunologically compatible mouse and increased serum FGF-2 levels
Introduction of the modified hematopoietic stem cells or progeny into an immunologically compatible mouse such that expression of the secretable analog of human FGF-2 results in increased serum FGF-2 levels compared to mice engrafted with cells transduced with BMP2/4 secretion signal peptide and wild-type FGF-2.
Screening for modulation of FGF-2 secretion to identify bone growth agents
Administering an agent to the mouse and screening for modulation of FGF-2 secretion, where an increase in FGF-2 secretion identifies an agent that increases bone growth.
Stem cell characteristics
The hematopoietic stem cell is pluripotent and expresses CXCR4, including being a Sca-1+ cell.
Methods of introducing heterologous nucleic acid
Introducing the nucleic acid into hematopoietic stem cells via electroporation, biolistic transformation, lipid mediated transformation, or viral vector transfection using adenovirus, adeno-associated virus, murine leukemia virus, or lentivirus vectors that are replication defective.
Use of regulatable transcription control sequences
The polynucleotide encoding the osteogenic growth factor is operably linked to a regulatable promoter, for instance, a tetracycline regulatable promoter.
The independent claim covers a method employing genetically modified pluripotent hematopoietic stem cells expressing a secretable FGF-2 analog with specific cysteine substitutions and BMP2/4 secretion signals, engrafting these cells into immunologically compatible mice, administering agents, and screening for modulation of FGF-2 secretion as a proxy for bone growth, with additional dependent claims reciting specific features related to cell types, methods of nucleic acid introduction, and regulatable promoters.
Stated Advantages
Increased stability and secretion of FGF-2 protein through inclusion of a BMP2/4 secretion signal and cysteine-to-serine or cysteine-to-asparagine mutations.
More effective and consistent systemic delivery of osteogenic growth factors via hematopoietic stem cells that home to and engraft in bone marrow.
Targeted bone formation focused primarily on endosteal and trabecular bone, enhancing bone strength and fracture healing.
Use of regulatable promoters allows controlled therapeutic protein expression to mitigate side effects such as excessive bone formation or calcium deficiency.
Improved murine bone marrow transplantation model yields consistent, high-level long-term engraftment with reduced host morbidity and mortality.
Systemic gene therapy using transduced Sca-1+ hematopoietic progenitor cells is effective in old as well as young adult animals.
Documented Applications
Treatment of skeletal disorders including osteoporosis, osteogenesis imperfecta, osteomyelitis, cancer-induced bone defects, and bone fractures through enhanced bone growth and repair.
Systemic delivery of osteogenic growth factors to promote localized and systemic bone formation.
Methods for enhancing wound healing and fracture repair applications.
Use of a mouse bone marrow transplantation model to identify agents that modulate bone growth.
Gene therapy applications using hematopoietic stem cells to express osteogenic growth factors for therapeutic bone formation.
Use in veterinary and human medicine for treating bone degradation and promoting bone health in various mammals including domestic animals and humans.
Potential use of regulated gene expression systems (e.g., tetracycline inducible promoters) to control therapeutic levels of osteogenic factors.
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