Production and delivery of a stable collagen
Inventors
Marinkovich, M. Peter • Lane, Alfred T. • Rajadas, Jayakumar
Assignees
US Department of Veterans Affairs • Leland Stanford Junior University
Publication Number
US-9040484-B2
Publication Date
2015-05-26
Expiration Date
2032-04-26
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Abstract
Improved methods are provided for the recombinant synthesis of collagen, particularly collagen VII, in host cell, and for therapeutic delivery of the same. The recombinant collagen is produced in a host cell that has increased levels of prolyl-4-hydroxylase, relative to basal cell levels. The collagen produced by the methods of the invention has increased numbers of modified proline residues, relative to a recombinant collagen produced in a host cell having basal levels of prolyl-4-hydroxylase. The increased proline modification provides for a collagen having increased stability, including increased in vivo stability.
Core Innovation
The invention provides improved methods for the recombinant synthesis of collagen, particularly collagen VII, within host cells that have been genetically modified to express increased levels of prolyl-4-hydroxylase compared to basal levels. This genetic modification leads to collagen having a higher number of modified proline residues as a result of enhanced prolyl hydroxylation, which confers increased stability, including increased in vivo stability such as a longer half-life after injection.
The recombinant collagen is produced in host cells which may be mammalian or other suitable cell types, where increased expression of prolyl-4-hydroxylase is achieved either by introduction of exogenous sequences encoding the enzyme or by modification of endogenous gene expression. The methods further include delivery devices comprising microneedle arrays made of biocompatible or biodegradable polymers incorporating the stable collagen, designed to penetrate the skin's stratum corneum to deliver therapeutic collagen to the viable epidermis and epidermal/dermal junction.
Claims Coverage
The patent contains one independent method claim relating to synthesizing collagen VII with increased prolyl-4-hydroxylase expression in a host cell, supported by dependent claims detailing embodiments involving vectors, host cells, and therapeutic administration.
Genetic modification of host cells to increase prolyl-4-hydroxylase expression
The host cell is genetically modified to increase expression of prolyl-4-hydroxylase via introduction of an episomal vector encoding prolyl-4-hydroxylase operably linked to a promoter, or by modifying the endogenous prolyl-4-hydroxylase gene to increase expression.
Recombinant synthesis of collagen VII with increased stability
Recombinant collagen VII is synthesized in genetically modified host cells with increased prolyl-4-hydroxylase expression, resulting in collagen with increased stability compared to collagen produced in cells without such modification.
The claims principally cover a method to produce collagen VII with enhanced stability by genetically modifying host cells to overexpress prolyl-4-hydroxylase, leading to collagen with improved post-translational modifications and stability, and further include aspects of isolating and administering the collagen for therapeutic uses such as treating epidermolysis bullosa.
Stated Advantages
Increased hydroxylation of proline residues in recombinant collagen results in enhanced stability of the collagen triple helix, including increased in vivo half-life.
The genetic modification ensures stable, sustained production of collagen VII with improved quality suited for therapeutic applications.
Microneedle-based delivery allows targeted, efficient, and less painful administration of therapeutic collagen to the skin layers where it is needed.
Documented Applications
Treatment of acquired or genetic epidermolysis bullosa, including recessive and dominant dystrophic forms, by administration of recombinant collagen VII.
Topical use in wound healing applications utilizing collagen VII formulations.
Use of microneedle arrays for delivering recombinant collagen VII or other therapeutic proteins to the epidermal/dermal junction in skin.
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