Gene-Enhanced Personalized Regenerative Medicine for Bone

It is said that RM replaces or regenerates human cells, tissue or organs using different cell types and strategies.1 Stem cells, as an important source of cells used in regenerative medicine (RM), have the ability to renew themselves for long periods and can be differentiated into various cell types.2,3 Gene therapy can modify embryonic/adult stem cells or induced somatic cells into pluripotent stem cells (iPSCs) to further use in RM. Personalized medicine is a novel field of medicine which refers to the employing of specific therapeutics procedure for an individual. This approach has been established based on genomic information and data. Nowadays personalized medicine covers many fields of healthcare such as RM.4,5 Accordingly, RM and cell-based therapy uses individual cell-based products in order to develop personalized treatments and regeneration. Science the use of gene therapy and personalized medicine can affect the efficiency of the regeneration strategies, it is suggested to apply genomic data of an individual in RM as personalized regenerative medicine or PRM. In this article, different strategies such as cell therapy, gene therapy, RNA based therapy etc used in bone regeneration according to the use in bone PRM is presented.


Introduction
It is said that RM replaces or regenerates human cells, tissue or organs using different cell types and strategies. 1 Stem cells, as an important source of cells used in regenerative medicine (RM), have the ability to renew themselves for long periods and can be differentiated into various cell types. 2,3ene therapy can modify embryonic/adult stem cells or induced somatic cells into pluripotent stem cells (iPSCs) to further use in RM.Personalized medicine is a novel field of medicine which refers to the employing of specific therapeutics procedure for an individual.This approach has been established based on genomic information and data.Nowadays personalized medicine covers many fields of healthcare such as RM. 4,5ccordingly, RM and cell-based therapy uses individual cell-based products in order to develop personalized treatments and regeneration.Science the use of gene therapy and personalized medicine can affect the efficiency of the regeneration strategies, it is suggested to apply genomic data of an individual in RM as personalized regenerative medicine or PRM.
In this article, different strategies such as cell therapy, gene therapy, RNA based therapy etc used in bone regeneration according to the use in bone PRM is presented.
one patient, the use of gene therapy for a special growth factor may be more effective than other growth factors due to its genetics.In one patient, using siRNA against corticosteroids receptors may be more effective than other strategies. 6

Personalized Cell Therapy in Bone Regeneration
There are some limitations in the clinical utility of stem cells.For example the immune rejection of heterologous stem cells, limit their applications. 7Also, the low quantity of reachable autologous cells requires in vitro expansion in xeno-derived media and may result in genomic alterations and unwanted cell differentiation and functions.This is why some appropriate tests should be performed for analyzing cells before transplantation.][10] Banking and the use of human cord blood stem cells or iPSCs) in order to achieve personalized cell source will be an approach in personalized cell therapy in bone regeneration. 11

Gene Therapy in Bone Regeneration
Bone regeneration can be enhanced through the delivery of genes or other regulators of genes that stimulate osteogenesis and angiogenesis. 12Gene therapy is transferring DNA or RNA into cells for some proposes. 13In vivo, ex vivo and in situ approaches are the most popular local gene therapy in RM.The in vivo approach is simple, fast, and less expensive but the ex vivo gene therapy is safer and more helpful; because genetic manipulations get done outside the body.In this method, the progenitor cells can be used as a primer place for bone development.Both methods are presently used for bone regeneration. 14Viral and non-viral vectors can be used in RM. 14,15 Viral and non-viral deliveries have some advantages and disadvantages.Ultimately, clinical trial for gene therapy in bone regeneration is essential for the follow-up study of side effects. 13nes and Factors in Bone Gene Therapy In general, genes or factors that are used in bone regeneration can be divided into the following.The delivery of growth factor genes and the expression of these genes is the original item for modifying the bone matrix during bone curing for regeneration purposes. 16The GF regulate vascularization and induce proliferation and differentiation of osteoprogenitors cells and neighboring tissues in the periodontal and gingival structures.Hence, they can be valuable for improving the repairing processes and to stimulate bone regeneration.
Transforming growth factor-b1 (TGFb1) is a growth factor that has an essential role in MSC stimulate to osteoblast precursors and chondrocyte proliferation.Also, TGFb1 has some functions in some extracellular bone matrix protein such as collagen, osteopontin, and alkaline phosphatase.On the other hand, biomaterials were advancely industrialized to combine GFs for synergistic effects on bone regeneration 16 .Finally, vascular endothelial growth factor (VEGF) is an important GF in bone formation and angiogenesis. 17,18.The GF transcription factors, such as Runx2, Sox9, and Osterix are also necessary for osteoblastogenesis and regulators for osteogenesis of chondrocytes and osteoblasts and have been used in gene therapy for bone regeneration (Figure 1). 19

Epigenetic Therapy in Bone Regeneration
Epigenetic modifications lead to functional relevant alterations in the genome without any changes in the nucleotide sequence. 21Epigenetic mechanisms play essential roles in stem cell maintenance, differentiation and expression pattern during bone regeneration processes. 22In osteogenic differentiation of stem cells, DNA methylation, histone modifications, and microRNAs (miRs) regulation are involved in bone regeneration. 23For example, CpG methylation of the osteocalcin promoter considerably decreases during in vitro osteoblast differentiation of MSCs.
In addition, hypermethylation of the LIN28 promoter reduces LIN28 expression during osteoblast differentiation.It has Figure 1.Some of the Factors and Genes Are Shown in Osteogenesis. 20een demonstrated that the activation of CDK1 promotes osteogenic differentiation of MSCs through phosphorylation of EZH2, the catalytic subunit of PRC2, that in turn catalyzes the methylation of histone H3 on lysine 27 (H3K27). 24owever, epigenetic mechanisms are not limited to the following.

MiRs in Bone Regeneration
The MiRs acts as the main regulators of bone formation, regeneration, and degeneration. 25The delivery of miRs is the most common epigenetics control that was used in bone regeneration.They regulate expression of many genes in various vital processes; including growth and differentiation.Also, MiRs play an essential role in any step of bone formation.
MiRs regulate expression of genes by targeting mRNAs. 26p-regulate or down-regulate occur in miRs expression throughout the development and differentiation process for therapeutic special effects. 27Ex vivo manipulation of the miRs levels is an important approach in the differentiation of various stem cells in RM. 28 The use of miR levels could be an attractive approach for bone regeneration. 29MiRs that induced osteogenic had several fundamental functions in the stimulation of bone formation at different stages such as growth and development.Also, miRs contribute to the endochondral ossification process. 30So, particular miRs have essential roles in numerous pathways for promoting osteoblast differentiation. 31MiRs regulate osteogenic, adipogenic, and chondrogenic differentiation.There have been a number of the significant role of miRs in osteoblast proliferation and differentiation.For example, in positive osteogenesis, miRs can target Runx2 co-repressors in MSCs.This is while during negative osteogenesis, Runx2 or its co-activators can be targeted by miRs. 32For example, miR-29 is one type of miRs which its role in bone formation has been determined.Actually, miR-29 can stimulate osteogenic differentiation by targeting negative regulators on the related signaling pathway.It has also been shown that miR-34c with other signaling pathway are involved in osteogenic differentiation in mice. 16tudies have shown that miRNA-26a can promote bone regeneration through the positive regulation osteogenesis. 33

RNA-Based Therapy
Coding RNAs (mRNAs) code proteins in translation inside ribosome.Non-coding RNA are rRNA, tRNA, snRNA and other regulatory RNAs like short interference RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Piwi-interacting RNA (piRNA) and Long noncoding RNA (lncRNA).5][36] the RNA-based therapeutics such as the use of siRNAs, miRNAs, antisense oligonucleotides, aptamers, synthetic mRNAs and clustered regularly interspaced short palindromic repeats (CRISPR).][38][39] It is also considered as a new method for tissue repair and regeneration (Advances in the delivery of RNA therapeutics: from concept to clinical reality, overcoming cellular barriers for RNA therapeutics, and RNA therapeutics for tissue engineering).RNA therapy because of its cytoplasmic function and degradation reduce the risk and have some advantages. 40,41n the context of tissue engineering and RM, the probable turnover of the exogenous RNA is a further advantage. 42As conventional RNAs have strong immunogenicity and low stability, chemical modifications are needed to facilitate. 25,43

CRISPR/Cas9 System
The CRISPR/CRISPR-Cas9 pathway is revolutionizing biological research. 44,45Modifications to this primitive prokaryotic immune system now enable scientists to efficiently edit DNA or modulate gene expression in living eukaryotic cells and organisms. 46,47Thus, many laboratories can now perform important experiments that were previously considered as scientifically risky or too costly.

Delivery Approachs for Gene Enhanced Regeneration
Sustainable local delivery, regulated gene expression, safety and failed clinical trial, are subjects which should be considered in gene therapy for RM. 48,49Viral transduction of target cells is a possible approach for regenerative applications.
As mentioned, gene therapy by delivery through viral and non-viral vectors, lipid-based delivery systems and polymerbased delivery systems are general approaches for RM. 50For gene and often for miRs delivery, systemic or local delivery systems could be used. 51Transfection for primary stem cells is more difficult than non-stem cell lines. 52Thus more efficient systems for miRs delivery are required for RM.Some regeneration methods are a prolonged process.Depending on the expression levels of the targets, miRs replacement or inhibition therapy that up-regulates miRs expression and down-regulates targets of miRs expression can be done. 53everal viral and non-viral vectors with high transfection efficiency, excellent biocompatibility, and high-level targeting efficiencies have been developed. 51ral Delivery Systems Gene transfer mediated by viral vectors represents the most common approach in gene therapy studies. 54The different types of viral vectors whether integrated or nonintegrated form scan used for RM. 55The integrated viruses are important because they are stable and their expressions are continual.Under different conditions, such as bone regeneration, transient transgene expression is useful.For this aim, recombinant adenoviral vectors can be the most attractive.Also adenoviral vectors have the highest level of transgene efficiency.So, the use of adenovirus vectors in bone regeneration is common.Retroviral vectors have been also used in a small amount. 14n-viral Delivery Systems Delivery of naked DNA/plasmids contains direct injection, liposome, particle or polymer mediated transfection, electroporation, etc.On the other hand, the use of non-viral delivery systems is limited by their efficiency of transfer and the level of transgene expression.Lipid or polymerbased delivery systems can be used in the various opinion of stem cell differentiation.Non-viral transmission systems are not appropriate for clinical applications in some cases.
For some purposes such as a systemic anti-miR delivery was recommended nanoparticles and liposomes for delivery. 56,57affold-Based Delivery Systems The scaffold-based delivery systems were planned to be a successful, stable, low cytotoxicity and efficient non-viral delivery system. 58Scaffold-mediated delivery via encapsulating the genes or miRs in or onto the tissue engineering scaffold can be used for more controlled and localized transfection in RM applications.The findings show some of the mechanisms of miRs regulation by biomaterials and their development.
These delivery systems would have probable curative values in bone regeneration. 59Localized and efficient gene transfer can be promoted by exploiting the interaction between the vector or miRs and biomaterial. 60Different materials can be designed in order to use as gene delivery systems for bones.Biodegradable natural or synthetic materials or polymers with the potential of attachment and carrying vectors or other nucleic acids and analogs can be used.These systems in addition to carrying and delivering the desired construct can be effective in bone regeneration by itself. 61For example, chitosan-based hydrogel have been used in bone regeneration as gene delivery systems to transfer the BMP2 gene. 62

Conclusions
Personalized medicine is an important feature in RM.Since bone formation and regeneration processes take place using the cells and the co-expression of many molecules, including GFs, specific transcription factors, and morphogenic bone proteins, the understanding of which of the genes, factors or epigenetic modifications can lead to more success in a patient requires a related genomic analysis of the patient.Application of personalized bone regeneration approaches can greatly alter the outcomes in the near future.