Background: Heterotopic ossification (HO) is a profound problem for wounded warriors with polytrauma and amputations resulting from high energy blast explosions. The molecular pathogenesis of HO remains unclear, but overexpression of bone morphogenetic protein (BMP) and possibly noncollagenous proteins plays a significant role. Delivery of short interfering RNA (siRNA) has been shown to regulate proteins in vitro and in vivo.Objective/Hypothesis: A subset of overexpressed proteins wound tissue can be uniquely identified as causative factors in HO by proteomic analysis, and these proteins can then be modulated by siRNA therapy to understand the etiology of HO and eventually prevent or reverse the progression of this syndrome.Specific Aims: 1) Identify protein targets through proteomic analysis of tissue, tissue exudates, and serum from human HO patients.2) Develop siRNA's to down-regulate the targeted proteins. 3) Develop bisphosphonated Graplon-siRNA nanocomplexes targeted to bone cells; 4) Screen the Graplon-siRNA delivery efficacy in osteoblasts in vitro. 5) Evaluate selected Graplon-siRNA complexes in vivo in a mouse HO model produced by BMP.6) Evaluate the best Graplon-siRNA's from the mouse study in a rat trauma model of HO.Study Design: Proteomic analysis will be performed on samples taken from trauma patients statistically likely to develop HO who will be followed for 6 months to determine if HO is present. Protein analyses from known HO patients and healthy controls will be compared to select proteins for gene silencing. SiRNA's will be developed against both established (BMP) and newly discovered genes implicated in HO. Graplon-siRNA nanocomplex delivery vectors with bisphosphonate ligands will be prepared by established self-assembly methods and evaluated in vitro in murine preosteoblastic cells. Efficacy testing in rodent HO models will be performed by i.v. injection of selected Graplon-siRNA complexes and the extent of HO characterized by micro-CT and histological analyses.Innovation: Integration of proteomic analysis of wound tissue to identify causative proteins for HO with the design and delivery of siRNAs that downregulate those proteins has not been performed. While the Graplon delivery vector for siRNA uniquely permits effective transport and intracellular activity of the siRNA, targeting of the Graplons with bisphosphonate ligands to bone tissue has not yet been demonstrated. The proposed research therefore constitutes a major new direction in seeking an effective treatment for HO.Military Benefit: Studies of combat-related extremity traumas requiring orthopaedic interventions, including fixation or amputation, reveal that HO occurs in over 60% of these patients. While prophylaxis with nonsteroidal anti-inflammatory drugs and local radiation therapy can be effective, many patients (ca. 7%) require one or more surgical excisions of ectopic bone. The proposed siRNA therapy may eliminate the need for such secondary remediation, halting ectopic bone formation and preventing chronic pain associated with HO.Focus Area: This proposal is focused on the enhancement of the tissue environment for healing and preventing the onset of HO using optimal indicators of viability of bone and surrounding soft tissue.
|Effective start/end date||9/30/10 → 10/29/14|
- Congressionally Directed Medical Research Programs (CDMRP)