RAW264.7 is a monocyte/macrophage-like cell line, which is a transformed cell line of Abelson leukemia virus derived from BALB/c microRNA. RAW 264.7 is one of the most commonly used in vitro models for osteoclasts and inflammation.
1.？ Study on the formation of osteoclasts;
It has been proved that RAW 264.7 can easily differentiate into osteoclasts induced by RANKL. Unlike the precursor of primary osteoclasts, the differentiation of RAW 264.7 does not require the addition of macrophage colony stimulating factor (M-CSF).
2.？ Inflammation research:
RAW264.7 is the most commonly used in vitro research model for screening anti-inflammatory active substances and studying inflammation. Under the action of inducers (such as lipopolysaccharide LPS), RAW264.7 cells can simulate inflammatory reaction, releasing or up-regulating various inflammatory mediators such as nitric oxide (NO), cyclooxygenase -2(COX-2), tumor necrosis factor α(TNF-α), interleukin 6(IL-6) and so on.
It is reported that rheumatoid arthritis (RA) affects more than 2,654.38+million people all over the world. Rheumatoid arthritis is an autoimmune inflammatory disease that affects joints. It is characterized by infiltration of macrophages and lymphocytes, proliferation of synovial fibroblasts and ultimate destruction of joints. Macrophages play an important role in the pathogenesis of rheumatoid arthritis. The number of macrophages in RA inflammatory synovium is higher than that in normal joints, which is positively correlated with the severity of joint pain and inflammation. Many drugs have been approved for the treatment of rheumatoid arthritis, gene or cell therapy.
MicroRNA155 (mir-155) was found in the BIC gene of mouse chromosome 16 and human chromosome 2 1. In clinical and experimental models, miR- 155 is related to the pathogenesis of RA, because it is up-regulated in synovial membrane and synovial macrophages of RA patients. SiRNA interfering with miR- 155 (KD) can inhibit the production of proinflammatory cytokines. MiR- 155 may participate in the formation of RA in many ways. One way is that MiR- 155 targets three untranslated regions of Src homologous -2, including inositol phospholipase 1 (SHIP 1), which is a negative regulator of inflammation. Therefore, the increase of miR- 155 in RA leads to the decrease of SHIP 1 level and the increase of pro-inflammatory cytokines.
Researchers successfully mutated the endogenous miR- 155 gene of mouse macrophage RAW264.7 by using CRISPR/CAS9 technology, and obtained the miR- 155 genome knockout (GKO) clone. Further analysis showed that miR- 155 GKO clone expressed higher levels of SHIP 1 under LPS stimulation, but produced less pro-inflammatory cytokines.
By using the miR- 155 GKO clone, the removal of miR- 155 will lead to the decrease of pro-inflammatory cytokines produced by macrophages, thus confirming the previous observation that the increased miR- 155 contributes to the sustained level of cytokine production in RA patients. By transfecting the miR- 155 mimetic back to GKO clone, researchers can reintroduce the miR- 155 effect. In a word, these results indicate that the mutation of endogenous miR- 155 gene may cause the pre-miR- 155 product to be truncated and unable to mature into a shorter but stable miR- 155.
NLR family protein NLRP3 is a cytoplasmic sensor for exogenous pathogens and endogenous damage-related molecular patterns (DAMPs). After activation, NLRP3 assembles with adaptor protein ASC and cysteine protease caspase- 1 to form an inflammatory body of NLRP3, which leads to the cleavage and activation of caspase- 1. Activated capase- 1 cleaves the cytokine of IL- 1 and the precursor of IL- 18, making them mature and leading to the release of several pro-inflammatory cytokines, including IL- 1 and IL- 183. It is reported that NLRP3 inflammatory corpuscles play an important role in the occurrence and development of various inflammatory diseases. Inhibition of NLRP3 inflammatory corpuscle signal has been proved to be effective in relieving septic shock, peritoneum, Alzheimer's disease, atherosclerosis, T2D, multiple sclerosis, gout and other diseases. Therefore, NLRP3 inflammatory corpuscles are excellent targets for the treatment of various inflammatory diseases.
Using CRISPR/Cas9 to directly destroy the key molecule-nlrp 3 at the genome level can not only completely inhibit the activation of NLRP 3 inflammatory bodies, but also avoid the potential risk of inhibiting the off-target pathway of anti-inflammatory biological agents and inhibitors. Studying the strategy of knocking out NLRP3 by CRISPR/Cas9 is expected to be a more effective method to treat a variety of inflammatory diseases.
In this study, the researchers reported a systematic delivery system CRISPR/cas9? Encapsulate mCas9 and gNLRP3 into CLAN. CLAN is a kind of nanoparticles based on PEG -b- PLGA supplemented with cationic lipid BHEM-Chol, which is used to deliver nucleic acid therapy. In previous work, researchers have introduced small interfering RNA, RNA aptamer and hepatitis B virus CpG into tumor cells, cardiomyocytes, macrophages or plasma-like dendritic cell families.
However, mCas9/gNLRP3 is different from other nucleic acid therapies, and the properties of nanoparticles affect the drug delivery efficiency. In order to test whether CLAN42 can effectively transmit mCas9/ gRNA, the researchers encapsulated Cas9, enhanced green fluorescent protein (EGFP) * * mRNA (CAS9-EGFP mRNA, or mCas9-EGFP) and negative control gRNA (gNC) into the selected clan (CLANmCas9-EGFP/gNC). Bone marrow-derived macrophages were transfected with different CLANmCas9- EGFP/gNC. The positive rate of EGFP in bone marrow stromal cells in transfection group was the highest. Next, the researchers tested the gene knockout efficiency by transfecting Raw264.7 cells (macrophage cell line) which stably expressed GFP (Raw264.7-GFP) and CLAN (CLAN mCas9/GGFP) which encapsulated MCAS9 and gRNA-targeted GFP. The percentage of gfp gene knockout Raw264.7-GFP cells in transfection group was the highest, reaching 53.9%. By injecting different CLANmCas9- EGFP /gNC into mice, the researchers further confirmed the transmission efficiency of CLAN42 in mCas9/gRNA in vivo. The positive rate of EGFP in peritoneal macrophages in injection group was the highest (48.4%). To sum up, CLAN42 is the most effective in mCas9/gRNA transmission because of its strongest macrophage uptake capacity, and CLAN42 is more suitable for wrapping mCas9/gNLRP3 (named CLAN mCas9/gNLRP3) for treating various inflammatory diseases.
Therefore, by adjusting the weight of cationic lipid BHEM-Chol and the mass fraction of PEG5K-b-PLGA 1 1K in the polymer, the researchers established a group library with different surface charges and PEG densities. Researchers screened families in vitro and in vivo, and selected a better family, and introduced mCas9/gNLRP3 into macrophages, so as to improve septic shock, peritonitis and T2D induced by HFD by destroying NLRP3 in macrophages. This study provides a promising strategy for CRISPR/Cas9 to enter macrophages and treat various inflammatory diseases.
The results show that CLANmCas9/ gNLRP3 is a promising method to treat NLRP3-dependent inflammatory diseases. This study also provides an example for the treatment of immune-related diseases through gene editing of immune cells mediated by nanoparticles.