Therapeutic potential of extracellular vesicle‐associated long noncoding RNA

Abstract Both extracellular vesicles (EVs) and long noncoding RNAs (lncRNAs) have been increasingly investigated as biomarkers, pathophysiological mediators, and potential therapeutics. While these two entities have often been studied separately, there are increasing reports of EV‐associated lncRNA activity in processes such as oncogenesis as well as tissue repair and regeneration. Given the powerful nature and emerging translational impact of other noncoding RNAs such as microRNA (miRNA) and small interfering RNA, lncRNA therapeutics may represent a new frontier. While EVs are natural vehicles that transport and protect lncRNAs physiologically, they can also be engineered for enhanced cargo loading and therapeutic properties. In this review, we will summarize the activity of lncRNAs relevant to both tissue repair and cancer treatment and discuss the role of EVs in enabling the potential of lncRNA therapeutics.


INTRODUCTION
Extracellular vesicles (EVs), including exosomes, microvesicles, and other subtypes, have emerged as a novel class of cell-derived therapeutics with vast potential. EVs are released from virtually every cell type and are capable of transferring lipids, proteins, and nucleic acids to recipient cells in paracrine or endocrine fashion. 1,2 A majority of studies to date have specified proteins and/or microRNAs (miRNAs) as the primary therapeutic components of EVs; however, long noncoding RNAs (lncRNAs) are increasingly being recognized as important mediators of EV biological effects. lncRNAs are defined as any RNA over 200 bps with no apparent coding function, [3][4][5] and their abilities to interact with cellular miRNAs via both complementary sequence binding and secondary structure effects have been linked to therapeutic outcomes. 6,7 Intercellular transfer of lncRNAs is naturally accomplished by EVs, and thus there is particular interest in and potential for harnessing this mechanism for therapeutic benefit. This review summarizes research on the therapeutic activity of lncRNA and EV-associated lncRNA to date relevant to both tissue repair and cancer treatment. It further discusses current knowledge and expected challenges of EV-mediated lncRNA delivery as well as issues for future consideration to enable translation of EV-associated lncRNA therapeutics.

THERAPEUTIC ACTIVITY OF lncRNA FOR TISSUE REPAIR AND REGENERATION
of diseases and injuries across all organ systems. In this section, therapeutic effects of lncRNAs relevant to the integumentary, musculoskeletal, cardiovascular, nervous, and gastrointestinal systems are discussed.

Integumentary system
The integumentary system consists of the skin, hair, and nails, as well as exocrine glands, and is responsible for providing protection from the environment. Wound healing is an essential process in dealing with insults that breach this protective barrier. Endothelial cells play a major role in wound healing, as the formation of new blood vessels is crucial for tissue repair and regeneration. 8 Increasing the proliferative and migratory properties of these cells is required for new blood vessels to form and facilitate healing. Aimed at this goal, Tao et al. utilized extracellular mimics to deliver lncRNA H19, resulting in improved proliferation, migration, and tube formation of human dermal microvascular endothelial cells. Further, using a streptozocin-induced diabetic mouse model, they showed that delivery of H19-containing vesicles improved blood supply to the wound and helped aid healing overall.
The angiogenic effects observed were attributed to potential H19 involvement in the insulin-PI3K-Akt pathway. 9 Endothelial cells are also a potential source for EVs with therapeutic lncRNAs. Shyu et al. altered the environmental conditions of human coronary artery endothelial cells to augment levels of lncRNA MALAT1 in their secreted exosomes. They delivered these exosomes to ischemic hindlimbs of rats and saw improved neovascularization via a potential mechanism of action of sequestration of miR-92a and upregulation of KLF2. 10 In a separate study, Lamichhane et al. altered environmental conditions of human umbilical vein endothelial cells (HUVECs) by supplementing media with low doses of ethanol. EVs collected from these preconditioned cells were found to have increased levels of lncRNAs HOTAIR and MALAT1 that produced enhanced vascularization responses in vitro and in vivo ( Figure 1). 11 This phenomenon was replicated in a scalable bioreactor culture system, demonstrating the potential to produce EVs with specifically enhanced therapeutic lncRNA content at large scale. 12 In addition to endothelial cells, fibroblasts are also crucial players in wound healing as they deposit extracellular matrix in newly formed skin and release a variety of growth factors that help orchestrate the angiogenic component of wound repair. 13 Copper et al. assessed the effects of exosomes from human adipose-derived mesenchymal stem cells (hAD-MSCs) on human dermal fibroblasts (HDFs). Unmodified hAD-MSC exosomes increased HDF migration in vitro, but this effect was reduced when the hAD-MSCs were transfected with an antisense oligonucleotide to MALAT1 prior to exosome collection. When hAD-MSC-conditioned media was applied to an ischemic excisional wound healing model, wound closure improved compared to control media treatment. 14 Additionally, a study employing a skin fibroblast in vitro thermal injury model demonstrated that fibroblast proliferation, migration, and extracellular matrix synthesis was enhanced through the upregulation of lncRNA XIST by downregulating miR-29a and upregulating Lin28a. 15 Full keratinocyte coverage (i.e., epithelization) is the defining clinical criteria of a closed wound. 16 18 -that both increase keratinocyte migration in wound F I G U R E 1 Role of long noncoding RNA (lncRNA) in endothelial cell (EV)-derived bioactivity. (a) Expression levels of the indicated lncRNAs were assessed by qPCR in EVs from human umbilical vein endothelial cells (HUVECs) cultured in the presence versus absence of 100 mM ethanol (EtOH) for 24 hr (n = 3; *p < .05). (b-d) HUVEC gap closure was assessed following 24 hr stimulation by 100 μg/ml EVs from HUVECs cultured in the absence (−EtOH) or presence (+EtOH) of 100 mM EtOH for 24 hr following transfection with a scrambled small interfering RNA (siRNA) (scr) or siRNA specific to (b) HOTAIR, (c) MALAT1, or (d) both HOTAIR and MALAT1 (double transfection) (n = 4; ##p < .01 vs. − EtOH + scr; **p < .01, ***p < .001 vs. + EtOH + scr). HUVECs incubated in basal medium (EBM2, without growth factors) were used as the negative control (−) and HUVECs incubated in growth medium (EGM2, with growth factors) were used as positive controls (+). Data reproduced from Reference 11 (open access) with no changes beds. Another study by Sawaya et al. found that mevastatin upregulated lncRNA GAS5, which led to the inhibition of c-myc, a transcription factor that inhibits keratinocyte migration and whose overexpression has also been shown to be a hallmark of chronic wounds. 19 Beyond the cell types mentioned above, hair follicles contain a number of stem cells at their base that play a role in wound healing. [20][21][22] Si et al. found that transfecting hair follicle stem cells with the lncRNA PlncRNA-1 increased their proliferation and differentiation. Treatment of these transfected cells with the TGF-β1 inhibitor LY2109761 decreased these effects, suggesting a role for PlncRNA-1 in the TGF-β1-mediated Wnt/β-catenin signaling cascade. 23 In a similar vein, Cai et al. found that transfection of hair follicle stem cells with IncRNA5322 also promoted their proliferation and differentiation due to the activation of a miR-21-mediated PI3K-AKT signaling pathway. 24 Finally, transfusions of autologous blood and blood-derived products have been developed to address chronic wounds. [25][26][27] Guo et al.
utilized autologous blood transfusions in a streptozocin-induced diabetic mouse model in which lncRNA H19 was upregulated, improving wound healing. Mechanistically, they showed that H19 increased the expression of the angiogenic protein HIF-1α in fibroblasts by methylating histones via H3K4me3. 28 Another transfusion study by Liu et al. investigated the effects of autologous blood transfusions on wound healing in a similar diabetic mouse model. Mice treated with autologous blood efficacious in speeding wound healing showed increased levels of lncRNA MALAT1, which was found to activate fibroblasts through the initiation of the HIF-1α signaling pathway. 29 Key data are summarized in Table 1.

Musculoskeletal system
Osteoarthritis is the most common joint disorder in western populations and is characterized by chronic degradation of articular cartilage as well as osteophyte formation. 30,31 With regard to cartilage, Liu et al. showed that exosomes derived from human MSCs contained lncRNA KLF3-AS1and promoted chondrocyte proliferation in vitro. In an in vivo collagenase-induced model of osteoarthritis, the same MSC exosomes improved cartilage repair. 32 The same group explored the molecular action of KLF3-AS1 and found that it sponges miR-206 leading to the upregulation of G-protein-coupled receptor kinase interacting protein-1 and resulting in chondrocyte proliferation and apoptosis inhibition. 33 34 Interestingly, in a study by Pan et al., lncRNA MALAT1 was able to upregulate miR-19b, contributing to the inactivation of Wnt/β-catenin and NF-κB pathways. This ultimately led to a reduction of LPS-induced inflammatory injury in murine ATDC5 cells. 35 Osteoporosis is characterized by skeletal fragility and microarchitectural deterioration, 36 and differentiating bone marrow stromal cells to osteoblasts as a treatment has been an active area of research. 37 Rheumatoid arthritis is characterized by invasive fibroblast-like synoviocytes that cause joint destruction. 36 Li et al. showed that the phytochemical Tanshinone IIA was able to promote the apoptosis of these fibroblast-like synoviocytes by upregulating lncRNA GAS5. 41 Another study by Zhang et al. utilized LPS-induced chondrocytes and showed that lncRNA HOTAIR inactivated NF-κB signaling by downregulating miR-138. In rheumatoid arthritis animal models, it was found that chondrocyte proliferation was upregulated and inflammatory markers IL-17 AND IL-23 were downregulated by HOTAIR. 42 Bone fractures primarily heal by formation of a callus, which is eventually vascularized and calcified. Ciu et al. showed that endothelial progenitor cells cocultured with bone marrow-derived macrophages released exosomes enriched with lncRNA MALAT1. MALAT1 was found to sequester miR-124 and subsequently upregulate integrin subunit β1 as well as promote neovascularization at the bone fracture site in an in vivo mouse bone fracture model. Healing was improved in mice treated with these MALAT1-containing exosomes compared to those administered from bone marrow-derived macrophages. 43 In addition, Tang et al. found that lncRNA OG interacts with heterogeneous nuclear ribonucleoprotein K to regulate the expression of BMP family proteins to promote osteogenic differentiation of bone marrow-derived MSCs. 44 Key data are summarized in Table 2.

Cardiovascular system
Common cardiovascular ailments include ischemic heart disease, ischemia-reperfusion injury, arrhythmias, and inflammation, among  Table 3.

Nervous system
Ischemic strokes are a common cause of brain injury, and promoting neuronal survival after ischemia is essential when developing a treatment. Ruan  Another form of brain injury occurs by direct insult. Patel et al.
found that exosomes derived from human adipose MSCs exhibited therapeutic properties when administered intravenously in a murine traumatic brain injury model. These exosomes were found to aid in recovery of motor function and reduction in cortical brain lesions via delivery of MALAT1, as exosomes depleted of MALAT1 were not efficacious ( Figure 2). 56 Key data are summarized in Table 4.

Gastrointestinal system
Liver failure is a major threat to human health, and although liver transplant is a viable option, it is limited by cost and donor availability.
Increasing the proliferation of hepatocytes is one means of a treatment. induced inflammatory injured intestine. H19 was found to inhibit the expression of p53, miR-34a, and let-7, enabling cell growth and proliferation. 59 Key data are summarized in Table 5.

Breast cancer
Breast cancer is the second most common type of cancer in women in the United States. 60 Zhang et al. showed that overexpression of lncRNA MEG3 in breast cancer cells acted as a tumor suppressor in vitro and in an in vivo xenograft model. This was found to be due in part to MEG3 enhancing ER-stress-related genes and inducing NF-kB and p53 signaling pathways. 61 Another lncRNA, NKILA, was shown to activate similar pathways. 62 The sensitivity of breast cancer cells to paclitaxel was enhanced by the antisense intronic lncRNA EGOT due to the upregulation ITPR1, as reported by Xu et al. 63 Key data are summarized in Table 6.  83 Key data are summarized in Table 7.

Neurological cancer
Tumors that develop in the brain and spinal cord are classified as gliomas. Xia et al. found that lncRNA PTCSC3 suppressed proliferation, migration, and invasion of glioma cell lines, and further found that these functional effects were due to PTCSC3's downregulation of LRP6 and subsequent suppression of the Wnt/βcatenin signaling pathway. 84 Huo et al. found that lncRNA GAS5 was downregulated in glioma cell lines with low sensitivity to cisplatin. By overexpressing GAS5, they found that glioma cells were sensitized to cisplatin by restoring cisplatin-inhibited mammalian target of rapamycin activation. 85 A Stage IV glioma is classified as a glioblastoma and is the most aggressive type of cancer that develops in the brain. Xu et al. found that lncRNA AC003092.1 was decreased in glioblastoma cells resistant to temozolomide. When the lncRNA was overexpressed, sensitivity of glioblastoma cell lines to temozolomide was enhanced resulting in increased apoptosis through a TFPI-2 mediated pathway. 86 Key data are summarized in Table 8.

Lung cancer
Lung cancer is the leading cause of cancer-related deaths in both men and women. 87  advanced non-small cell lung cancer decreased proliferation of such cells by upregulating lncRNA MEG3, which increased the expression of p53. 91 Key data are summarized in Table 9.

Bone cancer
Osteosarcoma is the most common form of bone cancer. Guo et al.
found that lncRNA SRA1 was able to inhibit proliferation, migration, and invasion as well as facilitate apoptosis by sponging miR-208a in osteosarcoma cells. 92 Another study found that lncRNA FER1L4 sequestered miR-18a-5p in order to modulate the expression of PTEN in osteosarcoma cells. 93 Additionally, Wang et al. showed that lncRNA CTA was downregulated in doxorubicin-resistant osteosarcoma cells.
When CTA was overexpressed, doxorubicin resistance was overcome in vitro and in vivo. CTA was found to promote apoptosis of osteosarcoma cells via sponging miR-210. 94 Key data are summarized in Table 10.

Skin cancer
There are three types of cancers that can develop in the skin. Basal cell carcinoma and squamous cell carcinoma arise from keratinocytes, while melanoma arises from melanocytes. Mei et al. found that lncRNA LINC00520 targeted EGFR inhibition and resulted in the inactivation of the PI3K/Akt pathway leading to inhibition of cutaneous squamous cell carcinoma development. 95 104 Key data are summarized in Table 13.

Summary of therapeutic activity of lncRNA in cancer
The therapeutic potential of lncRNAs in cancer is still a nascent area of study, as lncRNAs are most commonly studied as possible cancer biomarkers or drug targets. These studies reveal a confluence of signaling pathways regulated by lncRNAs in cancer, and not surprisingly many of the most studied pathways are listed (e.g., PI3K, KRAS). As more dedicated studies of lncRNA activity in cancer are conducted, it is likely that a greater diversity of pathway interactions will be reported. It is also notable that MEG3 appears to be the most versatile lncRNA with regard to therapeutic potential in cancer, and thus this lncRNA may be a good candidate for future EV delivery studies.

EV DELIVERY OF lncRNA
As with other RNA therapeutics, there is a presumed need to protect therapeutic lncRNAs from circulating nucleases to enable efficacy. As synthetic lncRNAs are currently not widely available, utilization of EVs as natural lncRNA delivery vehicles is of high interest. Within this paradigm, several approaches have been taken (Figure 4). One method is collecting exosomes from a cell type known to secrete EVs enriched with an lncRNA of interest. (Figure 4,   Exogenous loading techniques have also been developed for EVs and could be applied to lncRNA loading once synthetic production of these RNAs is achieved. A common technique for loading EVs with nucleic acids is electroporation, as seminally reported by Wood et al. 109 More recently, Kao et al. utilized electroporation to load large (~7 kb) DNA plasmids into human megakaryocytic microparticles at thousands of copies per particle. 110 This result suggests that some level of lncRNA loading may be achievable using this process. Beyond electroporation, sonoporation has also been used successfully for EV loading of both nucleic acids 111 and large proteins such as catalase, 112 a~240 kDa enzyme. Additional methods, such as the use of pH-gradient-mediated loading 113 or cellular nanoporation, 114 among others, may also eventually be useful for loading synthetic lncRNAs into EVs.

FUTURE CONSIDERATIONS
In addition to the need for better mechanistic understanding of lncRNA activity, there are several open questions whose answers will be critical in defining appropriate dose and scheduling considerations for EV delivery of lncRNA. For example, it is not clear how many copies of a particular lncRNA per cell are needed to achieve an effect or how long lncRNA effects last. This issue is especially critical as the relatively large size of lncRNAs should limit their loading capacity in EVs (or any other delivery vehicle) relative to other ncRNAs such as small interfering RNA and miRNA. Identifying off-target effects of lncRNAs, especially for therapies requiring systemic administration, is also necessary to ensure safety. To this end, knowledge about potential toxicity of lncRNAs, both intracellularly and extracellularly, must be established. Further, while EV-associated lncRNAs are thought to act primarily in the cytoplasm as miRNA sponges, they could perform other functions, such as serving as scaffolds for transcriptional complexes following transport to the nucleus. Understanding these phenomena is paramount to developing effective EV-lncRNA therapeutics.

CONCLUSIONS
lncRNAs are an exciting class of regulatory RNAs with increasing rates of newly discovered therapeutic properties. They are naturally contained within EVs in the body, promoting optimism for the development of a novel class of EV-based therapies. However, a number of hurdles must be overcome for EV-lncRNA-based treatments to be realized, including improved understanding of mechanisms of action, pharmacokinetics, and toxicity. Addressing these issues, combined with further developments in generation of synthetic lncRNAs and improved biomanufacturing of EVs, will be required to enable the full potential of EV-lncRNA therapeutics.

ACKNOWLEDGMENT
This work was supported by NIH HL141611.