Genetic tools for the stable overexpression of circular RNAs

Abstract

Circular RNAs (circRNAs) are a class of non-coding RNAs featuring a covalently closed ring structure formed through backsplicing. circRNAs are broadly expressed and contribute to biological processes through a variety of functions. Standard gain-of-function and loss-of-function approaches to study gene functions have significant limitations when studying circRNAs. Overexpression studies in particular suffer from the lack of efficient genetic tools. While mammalian expression plasmids enable transient circRNA overexpression in cultured cells, most cell biological studies require long-term ectopic expression. Here we report the development and characterization of genetic tools enabling stable circRNA overexpression in vitro and in vivo . We demonstrated that circRNA expression constructs can be delivered to cultured cells via transposons, whereas lentiviral vectors have limited utility for the delivery of circRNA constructs due to viral RNA splicing in virus-producing cells. We further demonstrated ectopic circRNA expression in a hepatocellular carcinoma mouse model upon circRNA transposon delivery via hydrodynamic tail vein injection. Furthermore, we generated genetically engineered mice harbouring circRNA expression constructs. We demonstrated that this approach enables constitutive, global circRNA overexpression as well as inducible circRNA expression directed specifically to melanocytes in a melanoma mouse model. These tools expand the genetic toolkit available for the functional characterization of circRNAs.

Exosomal circular RNAs: New player in breast cancer progression and therapeutic targets

“…In term of circRNAs regulating circRNA expression in vivo, Researchers proposed to use transposon to deliver a circRNA expression cassette (Mecozzi et al, 2022). In addition, it is possible to produce circRNAs artificially and enhance their capabilities (Rossbach, 2019).…”

Hydrodynamic Delivery: Characteristics, Applications, and Technological Advances

“…Hepatocellular carcinoma is the second most frequently explored liver disease; it is addressed in approximately 10% of the total publications employing hydrodynamic delivery in the last 5 years. Malignant diseases, including hepatoblastoma, cholangiocellular carcinoma, colorectal cancer [23,24,[120][121][122], lung cancer [123], brain tumors [53], lymphoma [124], melanoma [24,121,125,126], and metastasis of renal cell carcinoma to the lungs, liver, and kidneys [118], have been investigated using hydrodynamic delivery. More than half of the reports on malignant diseases focus on hepatocellular carcinoma.…”

“…Although no protective ingredients are included in its formulation, hydrodynamic delivery can act as an effective carrier, not only of DNA [33,114,124,132,136,137,143,171] but also of other materials, including RNA and proteins, because rapid injection of a large volume of solution physically pushes away preexisting blood components, including nucleases, and brings nucleic acids or proteins directly into the cell interior within a short period of time. Since the establishment of the concept of RNA interference to suppress gene expression, there have been reports of the effective hydrodynamic delivery of various types of RNA, including microRNA [47,150,152,[188][189][190][191], circular RNA [126], short hairpin RNA [94,192,193], and small interfering RNA (siRNA) [87,[194][195][196]. Hydrodynamic delivery can also extrude cells from blood vessels and, thus, offers a simple and easy way to develop animal models for metastatic diseases [118] (Table 3).…”

“…Melanoma [24,125,126] Leber congenital amaurosis [141] Atopic skin & cutaneous diseases [114,117,164,165] Sepsis [81] Metastasis (melanoma, breast cancer, RCC * (lungs, liver, kidneys)) [118,119,121] Sickle cell disease [142] Cardiovascular & ischemic diseases [115,[166][167][168][169][170][171] Trypanosome [163] *, renal cell carcinoma Cystathionine β-synthase deficiency [143] Kidney diseases & hyperparathyroidism [172][173][174][175] Fabry disease [144] Diabetes mellitus & obesity [176][177][178][179] α-1 antitrypsin deficiency [69] Hypertriglyceridemia [83] Growth hormone deficiency [145] Inflammatory diseases [180][181][182][183][184] Metachromatic leukodystrophy [146] Osteoporosis [185] Short-chain acyl-CoA dehydrogen. def.…”

“…Minicircle DNA [33,114,124,132,136,137,143,171] US-targeted microbubble destruction [178] PhiC31 Integrase [197] microRNA [47,150,152,[188][189][190][191] Computer-assisted hydrodynamic delivery [12,198] Sleeping Beauty [30,96,111,142,191,[199][200][201][202] Circular RNA [126] Bioluminescence imaging [47,[203][204][205][206] piggyBac [126,207] shRNA [94,192,193] Tissue clearing [206] Cre-loxP [208][209][210] siRNA [87,[194][195][196] Repopulation [189,211] CreER…”