The Delivery and Evaluation of RNAi Therapeutics for Heterotopic Ossification Pathologies

Shrivats, Arun R.; Hollinger, Jeffrey O.

doi:10.1007/7651_2013_34

Cited by 9 publications

(11 citation statements)

References 26 publications

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“…Runx2 and Osx are key transcriptional molecules of the cascade [63]. Thus, dosing, temporal, and spatial parameters of RNAi must be finely tuned.…”

Section: Discussionmentioning

confidence: 99%

“…To compensate, additional osteogenic signaling factors should be targeted. Mid-and late-stage osteogenic markers including OPN, bone sialoprotein (BSP), and OCN are temporal successors to RUNX2 and OSX expression after rhBMP-2 stimulation [63]. The complex temporal expression profiles of OPN, BSP, and OCN have been elucidated [15,16,23,25,26,30,53,59,66]; therefore, a coordinated, temporal RNAi treatment cycle may enhance the duration and intensity of gene silencing and prevent HA deposition in vitro.…”

Section: Discussionmentioning

confidence: 99%

“…Lipofectamine RNAiMAX was used as a positive control for siRNA delivery, defining the positive control as a treatment producing an expected reduction of ALP activity. ALP activity was assessed as previously detailed [63]. Briefly, at experimental endpoints, cells were rinsed with 1 9 PBS and lysed with 1 9 cell lysis buffer.…”

Section: Alkaline Phosphatase Activity and Cell Viabilitymentioning

confidence: 99%

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Cationic Nanogel-mediated Runx2 and Osterix siRNA Delivery Decreases Mineralization in MC3T3 Cells

Shrivats

Hsu

Averick

et al. 2015

Clinical Orthopaedics &Amp; Related Research

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Background Heterotopic ossification (HO) may occur after musculoskeletal trauma, traumatic brain injury, and total joint arthroplasty. As such, HO is a compelling clinical concern in both military and civilian medicine. A possible etiology of HO involves dysregulated signals in the bone morphogenetic protein osteogenic cascade. Contemporary treatment options for HO (ie, nonsteroidal antiinflammatory drugs and radiation therapy) have adverse effects associated with their use and are not biologically engineered to abrogate the molecular mechanisms that govern osteogenic differentiation. Questions/purposes We hypothesized that (1) nanogelmediated short interfering RNA (siRNA) delivery against Runt-related transcription factor 2 (Runx2) and osterix (Osx) genes will decrease messenger RNA expression; (2) inhibit activity of the osteogenic marker alkaline phosphatase (ALP); and (3) inhibit hydroxyapatite (HA) deposition in osteoblast cell cultures. Methods Nanogel nanostructured polymers delivered siRNA in 48-hour treatment cycles against master osteogenic regulators, Runx2 and Osx, in murine calvarial preosteoblasts (MC3T3-E1.4) stimulated for osteogenic differentiation by recombinant human bone morphogenetic protein (rhBMP-2). The efficacy of RNA interference (RNAi) therapeutics was determined by quantitation of messenger RNA knockdown (by quantitative reverse transcription-polymerase chain reaction), downstream protein knockdown (determined ALP enzymatic activity assay), and HA deposition (determined by OsteoImage TM assay). Results Gene expression assays demonstrated that nanogelbased RNAi treatments at 1:1 and 5:1 nanogel:short interfering RNA weight ratios reduced Runx2 expression by 48.59% ± 19.53% (p \ 0.001) and 43.22% ± 18.01% (both p \ 0.001). The same 1:1 and 5:1 treatments against both Runx2 and Osx reduced expression of Osx by 51.65% ± 10.85% and 47.65% ± 9.80% (both p \ 0.001). Moreover, repeated 48-hour RNAi treatment cycles against Runx2 and Osx rhBMP-2 administration reduced ALP activity after 4 and 7 days. ALP reductions after 4 days in culture by nanogel 5:1 and 10:1 RNAi treatments were 32.4% ± 12.0% and 33.6% ± 13.8% (both p \ 0.001). After 7 days in culture, nanogel 1:1 and 5:1 RNAi treatments produced 35.9% ± 14.0% and 47.7% ± 3.2% reductions in ALP activity. Osteoblast mineralization data after 21 days suggested that Two of the authors (ARS, EH) contributed equally.

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“…Runx2 and Osx are key transcriptional molecules of the cascade [63]. Thus, dosing, temporal, and spatial parameters of RNAi must be finely tuned.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Alkaline Phosphatase Activity and Cell Viabilitymentioning

confidence: 99%

See 1 more Smart Citation

Cationic Nanogel-mediated Runx2 and Osterix siRNA Delivery Decreases Mineralization in MC3T3 Cells

Shrivats

Hsu

Averick

et al. 2015

Clinical Orthopaedics &Amp; Related Research

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“…As previously reported, 36 cells were fixed in ice-cold 70% ethanol for 1 h and rinsed three times with DI water. 40 mM Alizarin Red S stain was added (pH adjusted to 4.2) for 10 min under rotation.…”

Section: Methodsmentioning

confidence: 99%

Nanogel-Mediated RNAi Against Runx2 and Osx Inhibits Osteogenic Differentiation in Constitutively Active BMPR1A Osteoblasts

Shrivats

McDermott

Klimak

et al. 2015

ACS Biomater. Sci. Eng.

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Trauma-induced heterotopic ossification (HO) and fibrodysplasia ossificans progressiva (FOP) are acquired and genetic variants of pathological bone formation occurring in soft tissues. Conventional treatment modalities target the inflammatory processes preceding bone formation. We investigated the development of a prophylaxis for heterotopic bone formation by addressing the biological basis for HO – dysregulation in the bone morphogenetic protein (BMP) signaling pathway. We previously reported the synthesis of cationic nanogel nanostructured polymers (NSPs) for efficient delivery of short interfering ribonucleic acids (siRNAs) and targeted gene silencing. Results suggested that nanogel:siRNA weight ratios of 1:1 and 5:1 silenced Runx2 and Osx gene expression in primary mouse osteoblasts with a constitutively active (ca) BMP Receptor 1A (BMPR1A) by the Q233D mutation. Repeated RNAi treatments over 14 days significantly inhibited alkaline phosphatase activity in caBMPR1A osteoblasts. Hydroxyapatite (HA) deposition was diminished over 28 days in culture, though complete suppression of HA deposition was not achieved. Outcome data suggested minimal cytotoxicity of nanogel-based RNAi therapeutics, and the multistage disruption of BMP-induced bone formation processes. This RNAi based approach to impeding osteoblastic differentiation and subsequent bone formation may form the basis of a clinical therapy for heterotopic bone formation.

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“…Finally, novel therapeutic methods such as small interfering RNAs (siRNA) have been tested in other diseases. This approach shows promise in FOP SHED cells transfected with allele‐specific siRNA; however, it remains to be seen whether sufficiently specific targeting of ACVR1 and a pharmacological response can be achieved in vivo .…”

Section: Emerging Therapeutic Strategies For the Treatment Of Fopmentioning

confidence: 99%

Therapeutic advances for blocking heterotopic ossification in fibrodysplasia ossificans progressiva

Wentworth

Masharani

Hsiao

2019

Brit J Clinical Pharma

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Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease in which heterotopic bone forms in muscle and soft tissue, leading to joint dysfunction and significant disability. FOP is progressive and many patients are wheelchair‐bound by the 3rd decade of life. FOP is caused by an activating mutation in the ACVR1 gene, which encodes the activin A Type 1 receptor. Aberrant signalling through this receptor leads to abnormal activation of the pSMAD 1/5/8 pathway and triggers the formation of bone outside of the skeleton. There is no curative therapy for FOP; however, exciting advances in novel therapies have developed recently. Here, we review the clinical and translational pharmacology of three drugs that are currently in clinical trials (palovarotene, REGN 2477 and rapamycin) as well as other emerging treatment strategies for FOP.

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The Delivery and Evaluation of RNAi Therapeutics for Heterotopic Ossification Pathologies

Cited by 9 publications

References 26 publications

Cationic Nanogel-mediated Runx2 and Osterix siRNA Delivery Decreases Mineralization in MC3T3 Cells

Cationic Nanogel-mediated Runx2 and Osterix siRNA Delivery Decreases Mineralization in MC3T3 Cells

Nanogel-Mediated RNAi Against Runx2 and Osx Inhibits Osteogenic Differentiation in Constitutively Active BMPR1A Osteoblasts

Therapeutic advances for blocking heterotopic ossification in fibrodysplasia ossificans progressiva

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