“Genetic scissors” CRISPR/Cas9 genome editing cutting-edge biocarrier technology for bone and cartilage repair

Li, Chao; Du, Yawei; Zhang, Tongtong; Wang, Haoran; Hou, Zhiyong; Zhang, Yingze; Cui, Wenguo; Chen, Wei

doi:10.1016/j.bioactmat.2022.09.026

Cited by 10 publications

(8 citation statements)

References 161 publications

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“…The CRISPR-Cas9 system has revolutionized the field of biomedical research, enabling the development of gene therapy-based clinical strategies, and the in vitro modeling of cellular pathways to unravel genetic determinants of diseases. For cartilage regeneration, a multitude of CRISPR-Cas9-based studies have been reported [ 44 ]. However, they all involve the use of cellular models which do not recapitulate the physiological phenotype of human cartilage (e.g., iPSCs, cell lines [ 17 – 20 ]) and they employ viral vectors [ 9 – 11 ].…”

Section: Discussionmentioning

confidence: 99%

Streamlined, single-step non-viral CRISPR-Cas9 knockout strategy enhances gene editing efficiency in primary human chondrocyte populations

Ponta,

Bonato,

Neidenbach

et al. 2024

Arthritis Res Ther

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Background CRISPR-Cas9-based genome engineering represents a powerful therapeutic tool for cartilage tissue engineering and for understanding molecular pathways driving cartilage diseases. However, primary chondrocytes are difficult to transfect and rapidly dedifferentiate during monolayer (2D) cell culture, making the lengthy expansion of a single-cell-derived edited clonal population not feasible. For this reason, functional genetics studies focused on cartilage and rheumatic diseases have long been carried out in cellular models that poorly recapitulate the native molecular properties of human cartilaginous tissue (e.g., cell lines, induced pluripotent stem cells). Here, we set out to develop a non-viral CRISPR-Cas9, bulk-gene editing method suitable for chondrocyte populations from different cartilaginous sources. Methods We screened electroporation and lipid nanoparticles for ribonucleoprotein (RNP) delivery in primary polydactyly chondrocytes, and optimized RNP reagents assembly. We knocked out RELA (also known as p65), a subunit of the nuclear factor kappa B (NF-κB), in polydactyly chondrocytes and further characterized knockout (KO) cells with RT-qPCR and Western Blot. We tested RELA KO in chondrocytes from diverse cartilaginous sources and characterized their phenotype with RT-qPCR. We examined the chondrogenic potential of wild-type (WT) and KO cell pellets in presence and absence of interleukin-1β (IL-1β). Results We established electroporation as the optimal transfection technique for chondrocytes enhancing transfection and editing efficiency, while preserving high cell viability. We knocked out RELA with an unprecedented efficiency of ~90%, confirming lower inflammatory pathways activation upon IL-1β stimulation compared to unedited cells. Our protocol could be easily transferred to primary human chondrocytes harvested from osteoarthritis (OA) patients, human FE002 chondroprogenitor cells, bovine chondrocytes, and a human chondrocyte cell line, achieving comparable mean RELA KO editing levels using the same protocol. All KO pellets from primary human chondrocytes retained chondrogenic ability equivalent to WT cells, and additionally displayed enhanced matrix retention under inflamed conditions. Conclusions We showcased the applicability of our bulk gene editing method to develop effective autologous and allogeneic off-the-shelf gene therapies strategies and to enable functional genetics studies in human chondrocytes to unravel molecular mechanisms of cartilage diseases.

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Section: Discussionmentioning

confidence: 99%

Streamlined, single-step non-viral CRISPR-Cas9 knockout strategy enhances gene editing efficiency in primary human chondrocyte populations

Ponta,

Bonato,

Neidenbach

et al. 2024

Arthritis Res Ther

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“…Collagen-based delivery methods have also demonstrated potential in lowering CRISPR/Cas9 off-target effects, a significant problem in gene therapy. The development of collagen-based delivery methods for the effective and targeted administration of CRISPR/Cas9 to target cells still faces obstacles ( Maeder and Gersbach, 2016 ; Li et al, 2022a ).…”

Section: Need For Biomaterials: Advantages Over Other Delivery Methodsmentioning

confidence: 99%

Biomaterials-mediated CRISPR/Cas9 delivery: recent challenges and opportunities in gene therapy

Dubey,

Mostafavi

2023

Front. Chem.

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The use of biomaterials in delivering CRISPR/Cas9 for gene therapy in infectious diseases holds tremendous potential. This innovative approach combines the advantages of CRISPR/Cas9 with the protective properties of biomaterials, enabling accurate and efficient gene editing while enhancing safety. Biomaterials play a vital role in shielding CRISPR/Cas9 components, such as lipid nanoparticles or viral vectors, from immunological processes and degradation, extending their effectiveness. By utilizing the flexibility of biomaterials, tailored systems can be designed to address specific genetic diseases, paving the way for personalized therapeutics. Furthermore, this delivery method offers promising avenues in combating viral illnesses by precisely modifying pathogen genomes, and reducing their pathogenicity. Biomaterials facilitate site-specific gene modifications, ensuring effective delivery to infected cells while minimizing off-target effects. However, challenges remain, including optimizing delivery efficiency, reducing off-target effects, ensuring long-term safety, and establishing scalable production techniques. Thorough research, pre-clinical investigations, and rigorous safety evaluations are imperative for successful translation from the laboratory to clinical applications. In this review, we discussed how CRISPR/Cas9 delivery using biomaterials revolutionizes gene therapy and infectious disease treatment, offering precise and safe editing capabilities with the potential to significantly improve human health and quality of life.

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“…Callus remodeling, often regarded as the final stage of fracture repair, involves the degradation of initially formed woven bone and its replacement with mature lamellar bone. Towards the end of the healing process, osteoclasts are activated; inhibiting them during the remodeling phase leads to increased callus density 63 . Differentiated osteoclasts, attaching to bone surfaces, express biomarkers such as translocon‐associated protein (TRAP), MMP‐9, and Cathepsin K (CTSK), facilitating their polarization and maturation.…”

Section: Effect Of Mechanical Strain On Cellsmentioning

confidence: 99%

“…Towards the end of the healing process, osteoclasts are activated; inhibiting them during the remodeling phase leads to increased callus density. 63 Differentiated osteoclasts, attaching to bone surfaces, express biomarkers such as translocon‐associated protein (TRAP), MMP‐9, and Cathepsin K (CTSK), facilitating their polarization and maturation. The RANK/RANKL pathway is essential for the differentiation of precursors into mature osteoclasts.…”

Section: Effect Of Mechanical Strain On Cellsmentioning

confidence: 99%

Biomechanical Effects of Mechanical Stress on Cells Involved in Fracture Healing

Wu,

Zhao,

Wang

et al. 2024

Orthopaedic Surgery

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Fracture healing is a complex staged repair process in which the mechanical environment plays a key role. Bone tissue is very sensitive to mechanical stress stimuli, and the literature suggests that appropriate stress can promote fracture healing by altering cellular function. However, fracture healing is a coupled process involving multiple cell types that balance and limit each other to ensure proper fracture healing. The main cells that function during different stages of fracture healing are different, and the types and molecular mechanisms of stress required are also different. Most previous studies have used a single mechanical stimulus on individual mechanosensitive cells, and there is no relatively uniform standard for the size and frequency of the mechanical stress. Analyzing the mechanisms underlying the effects of mechanical stimulation on the metabolic regulation of signaling pathways in cells such as in bone marrow mesenchymal stem cells (BMSCs), osteoblasts, chondrocytes, and osteoclasts is currently a challenging research hotspot. Grasping how stress affects the function of different cells at the molecular biology level can contribute to the refined management of fracture healing. Therefore, in this review, we summarize the relevant literature and describe the effects of mechanical stress on cells associated with fracture healing, and their possible signaling pathways, for the treatment of fractures and the further development of regenerative medicine.

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“Genetic scissors” CRISPR/Cas9 genome editing cutting-edge biocarrier technology for bone and cartilage repair

Cited by 10 publications

References 161 publications

Streamlined, single-step non-viral CRISPR-Cas9 knockout strategy enhances gene editing efficiency in primary human chondrocyte populations

Streamlined, single-step non-viral CRISPR-Cas9 knockout strategy enhances gene editing efficiency in primary human chondrocyte populations

Biomaterials-mediated CRISPR/Cas9 delivery: recent challenges and opportunities in gene therapy

Biomechanical Effects of Mechanical Stress on Cells Involved in Fracture Healing

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