Lipid nanomaterials-based RNA therapy and cancer treatment

We summarize the most important advances in RNA delivery and nanomedicine.We describe lipid nanoparticle-based RNA therapeutics and the impacts on the development of novel drugs.The fundamental properties of the key RNA members are described.We introduced recent advances in the nanoparticles to deliver RNA to defined targets,with a focus on lipid nanoparticles(LNPs).We review recent advances in biomedical therapy based on RNA drug delivery and state-of-the-art RNA application platforms,including the treatment of different types of cancer.This review presents an overview of current LNPs based RNA therapies in cancer treatment and provides deep insight into the development of future nanomedicines sophisticatedly combining the unparalleled functions of RNA therapeutics and nanotechnology.

The tumor therapeutic potential of long non-coding RNA delivery and targeting

Long non-coding RNAs(lncRNAs)is a type of RNA over 200 nt long without any protein coding ability,which has been investigated relating to crucial biological function in cells.There are many key lncRNAs in tumor/normal cells that serve as a biological marker or a new target for tumor treatment.However,compared to some small non-coding RNA,lncRNA-based drugs are limited in clinical application.Different from other non-coding RNA,like microRNAs,most lncRNAs have a high molecular weight and conserved secondary structure,making the delivery of lncRNAs more complex than the small non-coding RNAs.Considering that lncRNAs constitute the most abundant part of the mammalian genome,it is critical to further explore lncRNA delivery and the subsequent functional studies for potential clinical application.In this review,we will discuss the function and mechanism of lncRNAs in diseases,especially cancer,and different approaches for lncRNA transfection using multiple biomaterials.

Nucleic acid nanoassembly-enhanced RNA therapeutics and diagnosis

RNAs are involved in the crucial processes of disease progression and have emerged as powerful therapeutic targets and diagnostic biomarkers.However,efficient delivery of therapeutic RNA to the targeted location and precise detection of RNA markers remains challenging.Recently,more and more attention has been paid to applying nucleic acid nanoassemblies in diagnosing and treating.Due to the flexibility and deformability of nucleic acids,the nanoassemblies could be fabricated with different shapes and structures.With hybridization,nucleic acid nanoassemblies,including DNA and RNA nanostructures,can be applied to enhance RNA therapeutics and diagnosis.This review briefly introduces the construction and properties of different nucleic acid nanoassemblies and their applications for RNA therapy and diagnosis and makes further prospects for their development.

Use of recombinant microRNAs as antimetabolites to inhibit human non-small cell lung cancer

During the development of therapeutic microRNAs(miRNAs or miRs),it is essential to define their pharmacological actions.Rather,miRNA research and therapy mainly use miRNA mimics synthesized in vitro.After experimental screening of unique recombinant miRNAs produced in vivo,three lead antiproliferative miRNAs against human NSCLC cells,miR-22-3p,miR-9-5p,and miR-218-5p,were revealed to target folate metabolism by bioinformatic analyses.Recombinant miR-22-3p,miR-9-5p,and miR-218-5p were shown to regulate key folate metabolic enzymes to inhibit folate metabolism and subsequently alter amino acid metabolome in NSCLC A549 and H1975 cells.Isotope tracing studies further confirmed the disruption of one-carbon transfer from serine to folate metabolites by all three miRNAs,inhibition of glucose uptake by miR-22-3p,and reduction of serine biosynthesis from glucose by miR-9-5p and-218-5p in NSCLC cells.With greater activities to interrupt NSCLC cell respiration,glycolysis,and colony formation than miR-9-5p and-218-5p,recombinant miR-22-3p was effective to reduce tumor growth in two NSCLC patient-derived xenograft mouse models without causing any toxicity.These results establish a common antifolate mechanism and differential actions on glucose uptake and metabolism for three lead anticancer miRNAs as well as antitumor efficacy for miR-22-3p nanomedicine,which shall provide insight into developing antimetabolite RNA therapies.

Clinical progress of nanomedicine-based RNA therapies

The clinical application of nanoparticles(NPs)to deliver RNA for therapy has progressed rapidly since the FDA approval of Onpattro®in 2018 for the treatment of polyneuropathy associated with hereditary transthyretin amyloidosis.The emergency use authorization or approval and widespread global use of two mRNA-NP based vaccines developed by Moderna Therapeutics Inc.and Pfizer-BioNTech in 2021 has highlighted the translatability of NP technology for RNA delivery.Furthermore,in clinical trials,a wide variety of NP formulations have been found to extend the half-life of RNA molecules such as microRNA,small interfering RNA,and messenger RNA,with limited safety issues.In this review,we discuss the NP formulations that are already used in the clinic to deliver therapeutic RNA and highlight examples of RNA-NPs which are currently under evaluation for human use.We also detail NP formulations that failed to progress through clinical trials,in hopes of guiding future successful translation of nanomedicine-based RNA therapeutics into the clinic.

Simultaneous elimination of cancer stem cells and bulk cancer cells by cationic-lipid-assisted nanoparticles for cancer therapy

Convincing evidence indicates that the existence of cancer stem cells （CSCs） within malignant tumors is mostly responsible for the failure of chemotherapy. Therefore, instead of merely targeting bulk cancer cells, simultaneous elimin- ation of both CSCs and bulk cancer cells is necessary to improve therapeutic outcomes. Herein, we designed cationic-lipid-assisted nanopartides DTXLNPsRNA for simultaneous encapsulation of the conventional chemotherapeutic agent docetaxel （DTXL） and small interfering RNA （siRNA） targeting BMI-1 （siBMI-1）. We confirmed that nanopartides vrxLNPsiBMI-l effectively deliver both therapeutic agents into CSCs and bulk cancer cells. The bulk cancer cells were effectively killed by the DTXL encapsulated in DVXL NPsiBMI-1. In breast CSCs, BMI-1 expression was significantly downregulated by DVXLNpsiBMI-1; consequently, the sternness was reduced and chemosensitivity of CSCs to DTXL was enhanced, resulting in the elimination of CSCs. Therefore, via DTXLNPsiBMI-1, the combination of siBMI-1 and DTXL completely inhibited tumor growth and prevented a relapse by synergistic kiUing of CSCs and bulk cancer cells in a murine model of an MDA-MB-231 orthotropic tumor.

Specific anti-viral effects of RNA interference on replication and expression of hepatitis B virus in mice

Background RNA interference （RNAi） is a powerful tool to silence gene expression post-transcriptionally. Our previous study has demonstrated that small interfering RNAs （siRNAs） have sufficiently inhibited hepatitis B virus （HBV） replication and expression in vitro. In this study we observed the RNAi-mediated inhibitory effects on HBV replication in mice models and accessed the specificity of these effects. Methods A mutant RNAi vector （pSI-C mut） with two base pairs different from the original target gene sequence at the RNAi vector （pSI-C） was constructed according to the method described in this study, A mouse model of acute hepatitis B virus infection was established by injecting naked plasmid pHBV1.3 via the tail vein with acute circulatory overload, pSI-C, pSI-C mut and the irrelevant RNAi control plasmid for green fluorescent protein （GFP） gene, pSIGFP were respectively delivered with pHBV1.3 by tail vein injection method. Six days post injection, enzyme-linked immunosorbent assay （ELISA） assay was used to measure the concentration of HBV surface antigen （HBsAg） in mouse serum, immunohistochemical straining method was used to visualize the expressin of HBV core protein （HBcAg） in liver tissues, and the transcriptional level of HBV C mRNA in liver tissues was detectedd by reverse transcriptase PCR （RT-PCR） analysis. Results Injection of pSI-C exerted magnificent and specific inhibitory effects on the replication and expression of HBV in the murine model. After 6-day post-injection （ p. i. ）, the OD values were shown to be 5.07 ± 1.07 in infecting group and 0.62 ± 0. 59 in pSI-C group. The concentration of HBsAg in pSI-C group was significantly lower than that in infecting group （ P 〈 0. 01 ）. Liver intracellular synthesis of viral core protein was sharply reduced to 0. 9% ±0. 1%, compared with 5.4% ± 1.2% of positive hepatocytes in infecting group （P 〈0. 01 ）, and the transcriptional level of HBV C mRNA was greatly reduced by 84. 7%. However, the irrelevant RNAi control plasmid （pSIGFP）, and the pSI-C mut did not show the same robust inhibitory effects as pSI-C. Conclusion pSI-C exert efficient and specific inhibitory effects on HBV replication and expression in mice models.

Advances in quantum dot-mediated siRNA delivery

Nano-sized quantum dots（QDs） exhibit uniquely optical properties that are tunable with different sizes and shapes.QDs can emit narrow symmetric bands under a wide excitation range,possess antiphotobleaching stability,and be bio-functionalized on the large surface area.Therefore,QDs are attractive vectors for imaging-guided therapy.Small-interfering RNA（siRNAs）-based therapeutics hold great potential to target a large part of the currently undruggable genes,but overcoming the lipid bilayer to deliver siRNA into cells has remained a major challenge to solve for widespread development of siRNA therapeutics.In this mini-review,we focus on theranostic QD/siRNA assembles for enhancing delivery of siRNA and facilitating evaluation of therapeutic efficacy via imaging of QDs,with special attention to carbonaceous QDs for delivery of siRNA.

Potent and specific inhibition of SARS-CoV antigen expression by RNA interference

Background Severe acute respiratory syndrome (SARS) is an infectious disease caused by SARS-CoV. There are no effective antiviral drugs for SARS although the epidemic of SARS was controlled. The aim of this study was to develop an RNAi (RNA interference) approach that specifically targeted the N gene sequence of severe acute respiratory syndrome associated coronavirus (SARS-CoV) by synthesizing short hairpin RNA (shRNA) in vivo , and to assess the inhibitory effect of this shRNA on SARS-CoV N antigen expression. Methods The eukaryotic expression plasmid pEGFP-C1-N, containing SARS-CoV N gene, was co-transfected into 293 cells with either the RNAi plasmid pshRNA-N or unrelated control plasmid pshRNA-HBV-C4. At 24, 48 and 72 hours post transfection, the green fluorescence was observed through a fluorescence microscope. The RNA levels of SARS-CoV N were determined by reverse transcription polymerase chain reaction (RT-PCR). The expression of Green Fluorescent Protein (GFP) and protein N were detected using Western blot.Results The vector, pshRNA-N expressing shRNA which targeted the N gene of SARS-CoV, was successfully constructed. The introduction of RNAi plasmid efficiently and specifically inhibited the synthesis of protein N. RT-PCR showed that RNAs of N gene were clearly reduced when the pEGFP-C1-N was cotransfected with pshRNA-N, whereas the control vector did not exhibit inhibitory effect on N gene transcription.Conclusions Our results demonstrate that RNAi mediated silencing of SARS-CoV gene could effectively inhibit expression of SARS-CoV antigen, hence RNAi based strategy should be further explored as a more efficacious antiviral therapy of SARS-CoV infection.

Bioengineered miR-124-3p prodrug selectively alters the proteome of human carcinoma cells to control multiple cellular components and lung metastasis in vivo

With the understanding of microRNA(miRNA or miR) functions in tumor initiation,progression,and metastasis,efforts are underway to develop new miRNA-based therapies.Very recently,we demonstrated effectiveness of a novel humanized bioengineered miR-124-3 p prodrug in controlling spontaneous lung metastasis in mouse models.This study was to investigate the molecular and cellular mechanisms by which miR-124-3 p controls tumor metastasis.Proteomics study identified a set of proteins selectively and significantly downregulated by bioengineered miR-124-3 p in A549 cells,which were assembled into multiple cellular components critical for metastatic potential.Among them,plectin(PLEC) was verified as a new direct target for miR-124-3 p that links cytoskeleton components and junctions.In miR-124-3 p-treated lung cancer and osteosarcoma cells,protein levels of vimentin,talin 1(TLN1),integrin beta-1(ITGB1),IQ motif containing GTPase activating protein 1(IQGAP1),cadherin2 or N-cadherin(CDH2),and junctional adhesion molecule A(F11 R or JAMA or JAM1) decreased,causing remodeling of cytoskeletons and disruption of cell-cell junctions.Furthermore,miR-124-3 p sharply suppressed the formation of focal adhesion plaques,leading to reduced cell adhesion capacity.Additionally,efficacy and safety of biologic miR-124-3 p therapy was established in an aggressive experimental metastasis mouse model in vivo.These results connect miR-124-3 p-PLEC signaling to other elements in the control of cytoskeleton,cell junctions,and adhesion essential for cancer cell invasion and extravasation towards metastasis,and support the promise of miR-124 therapy.