Evaluation of BMP2/miRNA co-expression systems for potent therapeutic efficacy in bone-tissue regeneration

Abstract

Reconstruction of bone defects and compensation of deficient repair mechanisms represent important goals within the field of regenerative medicine and require novel safe strategies for translation into the clinic. A non-viral osteogenic gene therapeutic vector system (‘hybrid vectors’) was generated, combining an improved bone morphogenetic protein 2 (BMP2) gene cassette and single pro-osteogenic microRNAs (miR-148b-3p, miR-20-5p, miR-590b-5p), driven by the U6 promoter. The vectors were tested in vitro for their osteogenic differentiation potential in C2C12 and C3H/10T1/2 cell lines, using BMP2 alone as control. After confirming BMP2 expression and miRNA transcription, increased osteogenic differentiation was observed by all hybrid vectors, but most consistently by BMP2/miR-590-5p, using alkaline phosphatase enzyme activity assays and osteogenic marker mRNA quantitation, including runt-related transcription factor 2 (Runx2), collagen type 1 (Col1a1) and osteocalcin. To visualise target mRNAs of the respective miRNAs, next generation sequencing was performed, confirming down-regulation of mRNA targets of the hybrid vectors. Since the hybrid vector consisting of BMP2 and miR-590-5p showed the largest increase in osteogenic differentiation in vitro, this was tested in a mouse ectopic-bone model. Mineralisation was more than with BMP2 alone. The present study showed hybrid vectors as a novel non-viral gene therapeutic plasmid system for combining therapeutic effects of recombinant protein expression and miRNA transcription that did not add to the burden of the translation machinery, while improving the therapeutic efficacies. In vivo proof-of-principle in the context of bone regeneration suggested that such hybrid vectors will be applicable in a wide array of gene therapeutic strategies.

Effect of LSD1 on osteogenic differentiation of human periodontal ligament stem cells in periodontitis

“…miR‐590‐3p discouraged TNF‐α level to relieve inflammatory symptom and strengthen tissue damage repair (Deng et al, 2021). On the other hand, miR‐590‐5p brought a host of benefits to cell osteogenic differentiation potential (Brenner et al, 2021). Srinaath et al pointed out that miR‐590 was positively related to ALP and Runx2 and upregulated upon osteogenic induction to defense inflammatory reaction, modulate bone metabolism, and alleviate osteal diseases (Srinaath et al, 2019).…”

Functions of the bone morphogenetic protein signaling pathway through non-coding RNAs

“…BMPs signaling pathway cascades are post-transcriptionally modulated by different miRNAs that stimulate or inhibit osteogenesis, angiogenesis or neurogenesis. Specifically, several miRNAs can directly increase or reduce the expression of the genes coding for components of the BMPs signaling pathways or the transcription factors involved in cells differentiation, ultimately exerting both stimulatory and inhibitory effects on osteogenesis, angiogenesis or neurogenesis [ 13 , 14 ].…”

Regulatory of miRNAs in tri-lineage differentiation of C3H10T1/2

“…Sun et al [ 41 ] discovered that the down-regulation of miR-138 could promote the osteogenetic differentiation of C3H10T1/2, and that the quantity of ALP-positive cells increased significantly, and that the expression levels of osteogenetic associated genes, like ALP, COL-1, BMP-2, OCN and Runx2, were the most remarkable in cells. Brenner et al [ 42 ] constructed a non-viral osteogenetic genetic therapy vector system, namely BMP-2/miR-590-5p, which displayed the largest elevation in osteogenetic differentiation and more severe mineralization in vitro compared with BMP-2 alone. This hybrid vector provides a new gene therapy strategy for the reconstruction and repair of bone defects.…”