**5. Extracellular Vesicles**

Genome-editing factors, packaged in engineered CRISPR/Cas9 complexes, can be enclosed in extracellular vesicles (EVs), for delivery to specific target cells [86,87]. EVs are composed of cellular constituents such as lipids, proteins, RNA, and DNA [86,88]. EVs may cross the blood–brain barrier, target cells in vivo, and protect their components from degradation in the circulatory system [87,89]. Their function is dependent upon their origin, and EVs derived from MSCs could have the potential to deliver contents to OA cells [90–93], as shown in Figure 3 [85,94,95]. The use of exosomes as a nonselective cell system may have limitations, including the delivery of contents to unintended cell targets. Possible solutions to this challenge include the design of targeted exosomes, as suggested by Bellavia and colleagues [96].

**Figure 3.** Extracellular vesicle delivery of CRISPR/Cas9 in the treatment of OA. Inside a producer cell (**left**), engineered OA-targeted sgRNA transcription may occur. SgRNA combines with Cas9 to form CRISPR/Cas9 sgRNA complexes. CRISPR/Cas9 complexes load into extracellular vesicles with fluorescent tags containing a dimerization domain compatible with a dimerization domain in engineered CRISPR/Cas9 complexes. These fluorescent tags also contain targets for the target osteoarthritic cell (**right**). Loaded EVs attach to target cells and unload the CRISPR/Cas9 complexes, which are then transported to the nucleus to perform gene modification.

Exogenous-cell-based therapy is gaining traction for the regeneration of articular cartilage over stimulation therapy such as electric stimulation of endogenous cartilage growth factors [97]. Exogenous cell therapy can be done through the delivery of chondrocytes (either autologous or allogeneic), mesenchymal stem cells (MSCs), or extracellular vesicles [98]. MSCs are used for regenerative medicine due to their ability to promote regeneration based on environmental signals at sites of injury. As inhibitors of the immune system and multilineage differentiators, MSCs are an important alternative

cell source for articular cartilage repair and regeneration [97]. MSCs derived from the synovial membranes of joints have been shown to be more effective in terms of articular cartilage formation in in vitro studies when compared to MSCs from other tissues, joint and nonjoint [99,100].

In vitro studies performed in mice showed that microparticles and exosomes, which are EVs derived fromMSCs, exerted similar chondroprotective and anti-inflammatory functions, protecting mice from developing osteoarthritis and reproducing the main therapeutic effect of reducing symptoms [101, 102]. Thus, a combinatorial approach to the treatment of OA may be feasible.

#### **6. Potential CRISPR**/**Cas9 Molecular Targets for the Treatment of Osteoarthritis**

Cell therapy has great potential to help treat OA, but inflammation can prevent new articular cartilage from forming after the introduction of stem cells. Inflammation and inflammatory modulators must be addressed in the treatment of OA, and these inflammatory modulators may serve as targets for CRISPR/Cas9 strategies [103]. Table 1 identifies potential targets for CRISPR/Cas9 editing and the laboratories that are making progress in the use of CRISPR/Cas9 techniques in OA treatment.

IL-1β is a pro-inflammatory cytokine secreted primarily by neutrophils. IL-1β induces the expression of many OA-related genes and other cytokines, including tumor necrosis factor-alpha (TNFα) [104]. Current OA therapies target TNFα, however, deleterious side effects occur due to TNFα's role in facilitating many other functions [105,106]. Human articular cartilage (hAC) exposed to TNFα displays increased levels of expression of interleukin IL-1β [105,106]. Karlsen and colleagues, in their 2016 study, were able to silence the IL-1β cytokine receptor (IL1-R1) in hACs to determine its effect on inflammation and the redifferentiation potential of the hACs after exposure to the interleukin IL-1β. The hACs were isolated from cartilage, and CRISPR/Cas9 was used to knock out the IL1-R1 receptor and insert a puromycin-resistance gene to allow the selection of the knockout cells. The colonies of knockout cells were expanded and exposed to recombinant IL-1β and TNFα to assess their response. The results showed that the addition of recombinant IL-1β increased inflammation to high levels in the control group, as expected. However, in the knockout group, exposure to recombinant IL-1β did not cause measurable inflammation. Therefore, the therapeutic knockdown of IL1-R1 in articular cartilage cells in vitro prior to re-injection into the body may improve cell-therapy results [106].


**Table 1.** Potential CRISPR/Cas9 molecular targets for CRISPR/Cas treatment of osteoarthritis.

Recent gene-editing efforts have targeted cellular senescence. Ren and colleagues found that by targeting CBX4, cellular senescence could be alleviated, with positive outcomes for OA [116]. FOXD1 is a transcription factor that can be regulated by YAP. Recent research indicates that the upregulation of FOXD1 by YAP may hold promise for OA treatment by alleviating senescence [117]. Further studies that focused on connexin 43 modulation were able to demonstrate that the attenuation of cellular senescence could promote the regenerative capacity of cells and improve tissue quality in OA [115].

Degradative enzymes such as matrix metalloproteinases (MMP) play important roles in joint health. Seidl and colleagues utilized CRISPR/Cas9 to modify the MMP13 levels in human chondrocytes and found that by reducing the level of MMP activity, cells were able to accumulate higher levels of the beneficial type II collagen to strengthen the extracellular matrix of the articular cartilage [114].
