Compositions and methods for osteogenic gene therapy
Inventors
Lau, Kin-Hing William • Hall, Susan L. • Chen, Shin-Tai • Mohan, Subburaman • Baylink, David J.
Assignees
Loma Linda University • US Department of Veterans Affairs
Publication Number
US-9458215-B2
Publication Date
2016-10-04
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, including fracture repair, through the use of recombinant nucleic acids that encode fibroblast growth factor-2 (FGF-2) analogs. These recombinant nucleic acids are designed to promote bone growth by encoding modified FGF-2 proteins, and the disclosure also provides for vectors and cells incorporating these nucleic acids as well as the FGF-2 analogs themselves.
A key aspect of the invention is the engineering of hematopoietic stem cells or progenitor cells to express osteogenic growth factors such as FGF-2 analogs, specifically designed to promote both stem cell self-renewal and bone formation in vivo. The nucleic acid constructs used in this method typically include a bone morphogenetic protein 2/4 (BMP2/4) hybrid secretion signal fused to a modified FGF-2 cDNA (with specific amino acid substitutions to increase protein stability), and are delivered into suitable cells using appropriate vectors and promoters.
The problem addressed by the invention is the need for more effective compositions and methods for increasing the efficacy of treatment using osteogenic growth factors like FGF-2 to enhance bone growth and repair, particularly for treating skeletal disorders such as osteoporosis and fractures. Prior approaches using FGF-2 in gene transfer yielded inconsistent or insufficient results; the modifications and delivery systems described aim to overcome these limitations and produce reliable therapeutic outcomes.
Claims Coverage
There are two independent claims, each defining unique inventive features related to recombinant nucleic acids and modified FGF-2 analogs.
Recombinant nucleic acid encoding a modified FGF-2 analog with BMP2/4 signal and dual cysteine substitution
A recombinant nucleic acid is described which, in a 5′ to 3′ direction: - Contains a polynucleotide sequence encoding a bone morphogenetic protein 2/4 (BMP2/4) hybrid secretion signal sequence. - Contains a polynucleotide sequence encoding a mature human FGF-2 polypeptide with: - A mutation resulting in the substitution of an alanine for a cysteine at amino acid position 70 (C2) of FGF-2. - A mutation resulting in the substitution of an asparagine for a cysteine at amino acid position 88 (C3) of FGF-2. These features are directed specifically at the nucleotide composition and amino acid sequence modifications of the FGF-2 analog, including the secretion signal for improved stability and delivery.
Modified FGF-2 analog encoded by nucleic acid with BMP2/4 secretion signal and dual cysteine substitution
The invention also covers a modified fibroblast growth factor-2 (FGF-2) analog that is: - Encoded by a nucleic acid comprising, in a 5′ to 3′ direction: - A polynucleotide sequence encoding a BMP2/4 hybrid secretion signal sequence. - A polynucleotide sequence encoding a mature human FGF-2 polypeptide, where: - An alanine is substituted for cysteine at position 70 (C2). - An asparagine is substituted for cysteine at position 88 (C3). This claim focuses on the structure of the encoded protein product resulting from these specific substitutions and inclusion of the BMP2/4 signal sequence.
The claims cover both nucleic acid constructs encoding FGF-2 analogs with a BMP2/4 hybrid secretion signal and dual cysteine substitutions (C2A, C3N), as well as the resulting modified FGF-2 analog itself incorporating these features.
Stated Advantages
The disclosed compositions and methods allow for efficient and consistent secretion of FGF-2 from mammalian cells by combining a potent secretion signal sequence and specific amino acid mutations.
The modified FGF-2 analogs exhibit increased stability and biological activity compared to wild-type FGF-2, enabling robust production and secretion suitable for therapeutic applications.
Enhanced systemic or local bone formation is achieved, providing improved outcomes for treating bone defects, injuries, fractures, and diseases such as osteoporosis.
The delivery system using hematopoietic stem or progenitor cells enables targeted production of therapeutic proteins at bone tissue sites, promoting bone growth and repair.
Regulatable promoters (such as tet-on systems) permit controlled expression of the therapeutic protein, allowing adjustment of therapy as needed.
The system provides a reliable animal model for bone marrow transplantation and for screening agents that modulate bone growth.
Documented Applications
Increasing bone growth and enhancing wound healing, including fracture repair, by expressing osteogenic growth factors such as modified FGF-2 in stem or progenitor cells.
Treating systemic or local bone defects, including bone injuries, fractures, osteoporosis, osteomyelitis, and cancer-related bone conditions.
Targeted therapeutic protein delivery to bone tissue using hematopoietic stem or progenitor cells transduced with osteogenic growth factor genes.
Use in animal models (such as mouse bone marrow transplantation) for screening and identifying agents that modulate bone growth and promote tissue regeneration.
Gene therapy applications for skeletal disorders by transplantation of modified human or animal stem/progenitor cells.
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