DMD transcript. However, the treatment efficacy of the already conditionally approved AONs remains low. Aiming to optimize AON efficiency, we assessed exon 53 skipping of the DMD transcript with different chemically modified AONs, all with a phosphorothioate backbone: 2′-O-methyl (2′OMe), locked nucleic acid (LNA)-2′OMe, 2′-fluoro (FRNA), LNA-FRNA, αLNA-FRNA, and FANA-LNA-FRNA. Efficient exon 53 skipping was observed with the FRNA, LNA-FRNA, and LNA-2′OMe AONs in human control myoblast cultures. Weekly subcutaneous injections (50 mg/kg AON) for a duration of 6 weeks were well tolerated by hDMDdel52/mdx males. Treatment with the LNA-FRNA and LNA-2′OMe AONs resulted in pronounced exon 53 skip levels in skeletal muscles and heart up to 90%, but no dystrophin restoration was observed. This discrepancy was mainly ascribed to the strong binding nature of LNA modifications to RNA, thereby interfering with the amplification of the unskipped product resulting in artificial overamplification of the exon 53 skip product. Our study highlights that treatment effect on RNA and protein level should both be considered when assessing AON efficiency.Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy (DMD) patients to restore dystrophin expression by reframing the disrupted open reading frame of the  DMD gene encoding for the protein dystrophin . Samples were heated at 95°C for 5 min to ensure denaturation of the single strands. Sample collection was performed between 95°C and 5°C and the sample temperature was lowered at an interval of 0.5°C/min. Nitrogen was used when sample temperature fell below 15°C. Absorbance was measured at 260 nm and the thermal melting temperature (mT) were determined by calculating the midpoint of dissociation curves by first derivatives.A Varian CARY 100 UV-visible spectrophotometer equipped with a Peltier temperature controller was used to perform the UV thermal melting studies. RNA complement and AON (2 nmol each) were added and dissolved in the appropriate buffer  in zirconium bead prefilled tubes with the MagNA Lyser. Protein lysates were denatured at 95°C, after which the protein concentration was determined with the Pierce BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Protein lysates from the corresponding muscles of a hDMD/Protein samples  were prepared in Laemmli buffer  and supplemented with 2% β-mercaptoethanol and 0.01% bromophenol blue and incubated for 5 min at 95°C. Thereafter, protein samples were loaded on a Criterion XT 3%–8% Tris-Acetate gel in XT Tricine running buffer  and were run for 1 h at 75 V and 1 h at 150 V. Proteins were transferred onto a 0.2 μm Nitrocellulose membrane with the Trans-Blot Turbo Transfer system (Bio-Rad).Membranes were blocked in 5% nonfat dried milk powder (Sigma-Aldrich) in Tris-buffered saline for 1 h. Next, blots were incubated with the primary antibodies Ab154168  to detect human dystrophin, and 66895-1-Ig  and to detect α-actinin as a loading control, in Takara Western BLoT Immunobooster . The following day, blots were washed to remove unbound primary antibodies and then incubated with the secondary antibodies donkey-α-Rabbit IRDye-800CW and donkey-α-Mouse IRDye-680RD . The membrane was visualized with the Odyssey CLx infrared imaging system and software (Li-Cor).AON levels were detected in the tibialis anterior using the peptide nucleic acid (PNA) assay as previously described . In brieP < 0.05 was considered statistically significant.Data analyses were performed with GraphPad Prism  or SPSS . Values are presented as means ± standard deviation. Q = 1%) in GraphPad Prism. Outliers are visualized in the graphs but were removed for further analysis or descriptive statistics. Bodyweight was analyzed with a mixed model for linear regression in SPSS. A Shapiro-Wilk test was performed to test the normality of the plasma markers data. A Kruskal-Wallis test and the Dunn's multiple comparison test were performed to compare between the different treatment groups for the CK, GOT, and GPT data. The ALP and urea data were analyzed with a one-way analysis of variance followed by a Tukey's multiple comparison test.Outliers in plasma markers and exon 53 skip percentages were determined with the ROUT method . The bodyweight was recorded once a week for the duration of the experiment but did not statistically differ between the treatment groups over time  and heart . We obseAON accumulation was determined for the tibialis anterior. The 2′OMe and LNA-2′OMe AONs were most abundantly present after systemic treatment, while the concentration of FRNA AONs was much lower. Notably, LNA-FRNA AONs could not be detected in the tibialis anterior , despiteSince high levels of exon 53 skip were observed after treatment with the LNA-2′OMe and the LNA-FRNA AONs compared to the 2′OMe AON, we assessed whether this resulted in dystrophin restoration by western blot analysis. We observed very low levels of dystrophin protein (<1%) in the saline-treated group. Unexpectedly, we did not observe a visual increase in the dystrophin protein amounts in the AON-treated groups in the gastrocnemius, tibialis, and heart .To investigate potential causes for the discrepancies observed between high exon skip levels after treatment with LNA-2′OMe and LNA-FRNA AONs and the absence of treatment effects on dystrophin restoration, we assessed whether the increased binding affinity of the LNA-modified AONs to the target exon interfered with the amplification of the hDMDdel52 product in the RT-PCR. To do so, we added 10 or 20 ng of LNA-2′OMe, LNA-FRNA, or 2′OMe, as a control, in either the first or both PCR steps of the nested RT-PCR of four saline-treated gastrocnemius samples, in which we observe low levels of spontaneous exon 53 skip product . The addWhen 20 ng LNA-FRNA AON was added to the reaction, the effect on the amplification of both products was even more pronounced, resulting in the absence of the hDMDdel52 unskipped and exon 53 skip product when added to both RT-PCR rounds . The addAlthough we confirmed the effect of AON interference in the RT-PCR, it is more likely that the presence of LNA-modified AONs itself already influences cDNA synthesis. Therefore, we aimed to remove RNA structures before cDNA synthesis and optimize the RT-PCR. To melt out RNA secondary structures, it is recommended to incubate RNA samples at 70°C in the presence of primers and dNTPs before cDNA synthesis. Since the LNA modification increases the affinity of the AON for the target RNA, we evaluated the thermal affinities of the AONs to the complementary RNA. Indeed, we measured that the thermal affinity of the LNA-FRNA or LNA-2′OMe AONs was >95°C .Therefore, we increased the RNA incubation temperature to 95°C to melt the LNA-modified AONs from the mRNA before cDNA synthesis. In addition, we optimized our RT-PCR to a single PCR since nested RT-PCR is known to overamplify smaller fragments, in this case, the exon 53 skip products. To ensure that the RNA integrity was not impacted, we ran an RNA Nano chip, which confirmed that the RNA integrity number was sufficient for RT-PCR analysis before and after incubation at higher temperatures .We observed that in a single RT-PCR with cDNA made from the gastrocnemius RNA samples, which were incubated at 70°C before cDNA synthesis, the amount of exon skip levels was drastically decreased for the LNA-2′OMe and LNA-FRNA AONs to 19.0% ± 4.4% and 13.9% ± 3.3%, respectively . When weA similar reduction in exon 53 skip level was observed in the triceps, tibialis anterior, diaphragm, and heart with RNA incubated at 95°C before cDNA synthesis. We did not observe an effect of RT-PCR type or RNA incubation temperature on exon 53 skip levels in skeletal muscles or heart of mice treated with the 2′OMe AON, and only a slight decrease in FRNA AON-treated samples .DMD transcript levels, we performed a RT-qPCR at different exon-exon junctions in the gastrocnemius of hDMDdel52/mdx mice. We observed a trend toward increase in DMD transcript levels at the exon 3–4 junction in all AON-treated groups compared to the saline-treated group. At the exon 46–47 and exon 78-UTR junction, all treatment groups had comparable levels. We also assessed the DMD transcript levels with cDNA generated from RNA incubated at 95°C before cDNA synthesis. We observed that the 95°C RNA pretreatment resulted in overall lower exon 3–4 DMD transcript levels compared to the 70°C pretreatment although the pattern in which AON-treatment affected DMD transcript levels remained the same. At the exon 46–47 and 78-UTR junctions, no decrease was observed  interfered with cDNA synthesis and could be  prevented by incubating the RNA samples at 95°C instead of the standard 70°C incubation to remove RNA secondary structures. Furthermore, the addition of LNA-FRNA AON to the RT-PCR almost completely prevented amplification of the unskipped product and resulted in artificial overamplification of the exon 53 skip product while the effect of the LNA-2′OMe AON on the amplification of the unskipped product was smaller but still detectable.Since the LNA modifications greatly increase the binding affinity of the AONs to the target RNA and some of the AONs might remain bound to the RNA during RNA isolation, we hypothesized that the LNA-2′OMe and the LNA-FRNA AONs were hybridizing to the unskipped fragments and therefore interfering during cDNA synthesis and PCR amplification. Indeed, the high hybridization affinity of the LNA modified AONs . DM1 is caused by a CTG repeat expansion in the SLR mice . While tMbln1, which raises the question whether these AONs were able to induce exon 7 skipping of Mbnl1 or if there was a discrepancy between the treatment effect on alternative splicing of Mbln1 and Serca1 [However, while they show that the LNA-2′OMe AONs accumulate in skeletal muscles and heart, they do not mention the effect of AON treatment on exon 7 skipping of d Serca1 .Notably, in the first two studies, the LNA modifications were primarily located around the 5′ and 3′ end of the AON, while in the last study, the LNA modifications were more evenly spread across the AONs, whereas in our study the LNA modifications are located in the core of the AONs. The position of the LNA modification within the AON might greatly influence the ability to which the LNA modified AON are able to interfere during PCR analysis. Interestingly, LNA/DNA mixmers have been used in assays to block amplification of a wild-type DNA fragment, allowing for the amplification and detection of single-point mutant DNA fragments ,28 and, in vitro to block viral reverse transcription of viral RNA either by physically blocking initiation or elongation of cDNA synthesis, by inducing RNase H-mediated cleavage or by direct binding to the RT [Furthermore, different chemically modified DNA oligos have been utilized in cell-free systems as well as o the RT .mdx mice with the LNA-FRNA and 2′OMe AONs resulted in higher exon skip levels in skeletal muscles and heart compared to the 2′OMe AON, even after optimization of the cDNA synthesis and RT-PCR analysis to reduce the LNA-modified AON interference, further long-term studies are necessary to evaluate whether these AONs or other permutations of these chimeric AONs can result in dystrophin restoration. Furthermore, based on the study identifying an alternative target size for exon 51 skipping in hDMDdel52/mdx [Given that treatment of hDMDdel52/el52/mdx , alternaIn conclusion, our results show that 2′OMePS-LNA- and LNA-FRNA-modified AONs may have the potential to increase exon-skipping efficacy in a splice modulating approach in skeletal muscles and heart. However, care must be taken during RNA analysis as binding of these and other high-affinity AONs to unskipped transcripts can result in an artificial amplification of efficacy. This likely also applies to applications using RNase H AONs where qPCR is used to assess efficacy.


Lymph node (LN) status is vital to evaluate the curative potential of relatively early gastric cancer  treatment (endoscopic or surgery). Currently, there is a lack of robust and convenient methods to identify LN metastasis before therapeutic decision-making.Genome-wide expression profiles of long noncoding RNA (lncRNA) in primary T1 gastric cancer data from The Cancer Genome Atlas (TCGA) was used to identify lncRNA expression signature capable of detecting LN metastasis of GC and establish a 10-lncRNA risk-prediction model based on deep learning. The performance of the lncRNA panel in diagnosing LN metastasis was evaluated both in silico and clinical validation methods. In silico validation was conducted using TCGA and Asian Cancer Research Group (ACRG) datasets. Clinical validation was performed on T1 and T2 patients, and the panel’s efficacy was compared with that of traditional tumor markers and computed tomography (CT) scans.Profiling of genome-wide RNA expression identified a panel of lncRNA to predict LN metastasis in T1 stage gastric cancer (AUC = 0.961). A 10-lncRNA risk-prediction model was then constructed, which was validated successfully in T1 and T2 datasets . Thereafter, the clinical performance of the lncRNA panel was validated in clinical cohorts . Notably, the panel demonstrated significantly better performance compared with CT and traditional tumor markers. The novel 10-lncRNA could diagnose LN metastasis robustly in relatively early gastric cancer (T1–T2), with promising clinical potential.The online version contains supplementary material available at 10.1007/s10120-023-01428-8. Globally, gastric cancer (GC) is fifth in the list of most common cancers and ranks fourth as the most common cause of cancer death . Lymph nGastric cancer is still treated using surgery and endoscopic resection. Currently, Asian and European guidelines identify endoscopic submucosal dissection (ESD) and endoscopic mucosal resection (EMR) as the first choice treatments for most cases of early GC (cT1a) and are considered to be safe and definitive treatments , 7. HoweIt is highlighted by genome-wide association studies in cancer that single-nucleotide polymorphisms (SNPs) are related to cancer risk and more than 80% of cancer-associated SNPs occur in noncoding regions of the genome . In addiHerein, transcriptome-wide expression profiles of long noncoding RNA (lncRNA) were analyzed comprehensively and systematically, and a 10-lncRNA panel was established to identify GC LN metastasis (T1 and T2). We verified the effectiveness of the panel in independent databases and clinical tissue samples. The performance of the lncRNA panel was also compared with that of CEA, CA19-9, and CT, highlighting the value of this panel in predicting LN metastasis of T1 and T2 GC. The lncRNA panel could function as the basis for clinical decision-making for patients with GC.6, and then carrying out log2-transformation. We filtered all lncRNA expression levels from the TCGA and ACRG processed data according to the human gene annotation file [https://ftp.ensembl.org/pub/release97/gtf/homo_sapiens/Homo_sapiens.GRCh38.97.chr.gtf.gz). Then, logistic regression analysis was performed using the Logistic Regression (LR) function from Pytorch [https://arxiv.org/abs/1912.01703). Feature importance was estimated using coefficients from the LR model. To assess the lncRNA panel's diagnostic accuracy, the selected lncRNA features were used to construct a multivariate LR model, followed by calculation of the area under the receiver operator characteristic (ROC) curve (AUC) values. Ultimately, the probability of each patient being identified as LNP was used as the basis to calculate the risk scores. The flowchart of this study is shown in Supplementary Fig. 1.To identify an lncRNA expression signature for the detection of lymph node (LN) metastasis in gastric cancer (GC), the study used genome-wide expression profiles of lncRNAs from primary tumors with and without LN metastasis, which were obtained from The Cancer Genome Atlas (TCGA) database. Only pathological T1 and T2 RNA-sequencing (RNA-Seq) data were used for further analysis. The T1 data were from 15 LN metastasis negative (LNN) and 5 LN metastasis positive (LNP) samples, and the T2 data were from 34 LNN and 48 LNP samples. The processed TCGA level 3 RNA-Seq data for GC were obtained from the Firehose Broad GDAC portal . Indepenion file  . Cohort 3 included 127 patients . Patients in the clinical cohorts were treated at the Lihuili Hospital affiliated to Ningbo University (China). These patients had biopsy-proven primary GC and underwent curative surgery between December 2017 and January 2022. During surgery, we obtained tissues samples from a representative malignant lesion located in the surgically excised stomach specimen. The tissue samples were added with RNAstore , frozen rapidly in liquid nitrogen, and stored at − 80 ºC. The summarized characteristics of the patients in the clinical cohorts are shown in Table ACTB (encoding beta actin) was used to determine and correct the relative expression of target genes, employing the 2Ct–Δ method. In this method, ΔCt is the difference in cycle threshold (Ct) values between ACTB and the gene of interest. The data were then log2 transformed. Supplementary Table 1 details the PCR primers used.An RNeasy mini kit  was used to isolate total RNA from frozen surgical tissues, following the supplier's guidelines. The RT-PCR step of the qRT-PCR protocol was carried out using a SensiFAST probe Lo-ROX Kit  and the qPCR step used the QuantStudio 6 Flex Real-Time PCR System . Assay reproducibility was ensured via multiple techniques, such as including appropriate controls, excluding specimens with poor RNA quality, and the analysis of multiple replicates carried out at various time points. The QuantStudio 6 Flex Real-Time PCR System Software (Applied Biosystems) was used to assess gene expression. The expression level of DeLong [https://www.python.org/) was used to carry out the statistical analyses. Two-tailed t test-determined p values less than 0.05 indicated statistical significance.The method of DeLong  was usedhttps://doi.org/10.1093/nar/gkaa806). The data were visualized on a histogram and bubble chart using ggplot2 [For lncRNA enrichment analysis, we used the website application constructed by Chen et al.   and five lncRNAs with relatively low expression  in the LNP versus LNN comparison. In addition, tumor functional enrichment analysis was carried out for this panel. The results revealed that this 10-lncRNA panel was closely related to tumor prognosis, epithelial–mesenchyme transition (EMT), and metastasis, and was specific for gastrointestinal system cancer  and a cohort of 186 T2 patients from the ACRG  curves revealing the diagnostic performance of the model to distinguish lymph node-positive (LNP) and lymph node-negative (LNN) patients in The Cancer Genome Atlas (TCGA) T1 samples. B. Histogram and bubble diagrams showing the enrichment analysis of cancer hallmark and disease in the 10-lncRNA panel. C. The lymph node (LN) risk scores divided by LN status in the T1 and T2 cohorts from the TCGA, shown as a waterfall diagram, and a receiver operating characteristic (ROC) curve showing how the 10-lncRNA risk-prediction model performed in diagnosing patients with T1 and T2 stage disease in the TCGA data. D. The LN risk scores divided by LN status in the T2 cohort from Asian Cancer Research Group (ACRG), shown as a waterfall diagram, and a ROC curve showing how the 10-lncRNA risk-prediction model performed in diagnosing patients T2 stage disease in the ACRG data.The accuracy of diagnosis using the 10-lncRNA panel was assessed using RNA-seq in validation clinical cohort 1 and by qRT-PCR in validation clinical cohorts 2 and 3 (Table The patients in cohort 2 and 3 had T1 (19 LNP and 79 LNN) stage and T2 (38 LNP and 89 LNN) stage GC, respectively. Multivariate LR analysis was used to assess the effectiveness of the 10-lncRNA panel in T1–T2 tumors. In cohort 2, the panel showed an AUC of 0.812 (Table p < 0.05) and clinical cohort 3 (C) (p < 0.05), respectively. D. ROC curves illustrating the diagnostic value for identification of LN metastasis of the 10-lncRNA panel compared with that of CEA, CA19-9, and CT in clinical cohort 2 and cohort 3 (p < 0.05). E. ROC curves illustrating the diagnostic accuracy of the combinatorial model integrating the 10-lncRNA panel and clinicopathological features in clinical cohort 2 + 3 compared with that of the 10-lncRNA panel alone (p < 0.05).A. The risk score curve and heatmap of the lncRNAs expressed between lymph node-positive (LNP) and lymph node-negative (LNN) patients in clinical cohort 1. B and C. Receiver operating characteristic (ROC) curves showing how the 10-lncRNA risk-prediction model performed in identifying lymph node (LN) metastasis compared with that of the carcinoembryonic antigen (CEA), carbohydrate antigen 19–9 (CA19-9), and computed tomography (CT) in clinical cohort 2 (B) . In addition, we combined the 10-lncRNA panel with clinicopathological features  in cohorts 2 + 3. The results were also encouraging: this combination further improved the diagnostic accuracy of our panel (AUC = 0.813) compared with the 10-lncRNA panel alone  and CT in clinical cohort. Our 10-lncRNA panel showed significant superiority over preoperative clinical factors, CEA, CA19-9, and CT . Pathological diagnosis following radical gastrectomy remains the optimal way to evaluate a patient's GC's LN status, considering our lack of effective molecular markers that can robustly detect LN metastasis before therapeutic decision-making. Moreover, only patients with GC in situ (Tis stage) and T1a GC without LN metastasis can be treated successfully using endoscopic mucosal or submucosal resection. However, the actual LN metastasis rate of early GC (T1) is only around 20%. In addition, the incidence of regional LN metastasis is limited in patients with T2 GC, in which D2 gastrectomy might be an excessively invasive surgery, involving in a significant waste of medical resources , 10, 12.LncRNAs are mRNA-like transcripts of > 200 nucleotides with no capacity to encode proteins . A varieIn this article, we used RNA-sequencing to gain insights into the molecular biology of tumor heterogeneity and disease processes to identify LN metastasis. A systematic and comprehensive analysis of transcriptome-wide expression profiles of patients with T1–T2 GC, with and without LN metastasis, was used to establish an optimized 10-lncRNA panel to identify LN metastasis using logistic regression analysis. Subsequently, the panel was validated in three independent validation cohorts based on RNA-seq and qRT-PCR, achieving encouraging results. Our study is based on the concept of minimally invasive and non-invasive, devoted to the prediction of lymph node metastasis in early gastric cancer and clinical decision support. At the initial stage of the study, we also verified the predictive value of our panel in patients with T3 and T4 stage GC by TCGA and ACRG databases, but the results were not as expected , which may related to the heterogeneity of GC with different T stages.H19 is considered a carcinogenic factor in GC, and its upregulation is related to tumor cell proliferation, invasion, migration, and EMT [HOTAIR has been reported to be related to the expression of HER2  and facilitates GC lymph node metastasis [HAR1A is a tumor suppressor involved in tumor progression via EMT regulation and is negatively associated with prognosis [HAR1A also acted as a negative factor for early lymph node metastasis in GC. Similarly, TP53TG1 and TTTY15 have been confirmed to be differentially expressed in GC tissues compared with that in normal gastric mucosa [Carcinoembryonic antigen (CEA) and carbohydrate antigen 19–9 (CA19-9) are the most commonly used clinical monitoring serum indicators of digestive system tumors. It has been widely reported that elevated serum CEA and CA19-9 levels correlated well with lymph node metastasis, lymphatic invasion, stage grouping, and depth of invasion , 34–36.  and EMT . HOTAIR tastasis . A studyrognosis , 40. In c mucosa , 41. FinThis study has certain limitations. First, this was a retrospective study, and its design means that although we validated our findings in multiple clinical cohorts, prospective studies are still required. Second, the main aim of this study was to find early-stage GC biomarkers; therefore, the samples were concentrated in the T1 and T2 GC stages, which limited the sample size to discover biomarkers and had a certain impact on obtaining the panel with maximum efficiency. To overcome these limitations, larger cohorts comprising patients with GC and T1 and T2 LN metastasis are required, which might involve the participation of multiple medical institutions.Our panel provided and validated a class of biomarkers that could robustly categorize patients with relatively early GC according to their LN status prior to therapeutic decision-making, thus permitting individualized treatment. Our panel offers promising diagnostic potential to identify patients with GC with or without LN metastasis; however, our findings should be validated prospectively using clinical cohorts.Supplementary file1 (DOCX 353 KB)Below is the link to the electronic supplementary material.


The accumulation of toxic proteins and RNA molecules with repetitive sequences, and/or sequestration of RNA-binding proteins by RNA molecules containing expanded repeats are thought to be important contributors to disease aetiology9. Here we reveal that the adenosine in CAG repeat RNA can be methylated to N1-methyladenosine (m1A) by TRMT61A, and that m1A can be demethylated by ALKBH3. We also observed that the m1A/adenosine ratio in CAG repeat RNA increases with repeat length, which is attributed to diminished expression of ALKBH3 elicited by the repeat RNA. Additionally, TDP-43 binds directly and strongly with m1A in RNA, which stimulates the cytoplasmic mis-localization and formation of gel-like aggregates of TDP-43, resembling the observations made for the protein in neurological diseases. Moreover, m1A in CAG repeat RNA contributes to CAG repeat expansion-induced neurodegeneration in Caenorhabditis elegans and Drosophila. In sum, our study offers a new paradigm of the mechanism through which nucleotide repeat expansion contributes to neurological diseases and reveals a novel pathological function of m1A in RNA. These findings may provide an important mechanistic basis for therapeutic intervention in neurodegenerative diseases emanating from CAG repeat expansion.Microsatellite repeat expansions within genes contribute to a number of neurological diseases N1-methyladenosine on CAG repeat RNA, resulting in the formation of gel-like TDP-43 aggregates in the cytoplasm that resemble those observed in neurological disease pathology.TDP-43 binds to  For instance, expansion of a GGGGCC hexanucleotide repeat in the C9ORF72 gene is the major genetic cause of amyotrophic lateral sclerosis10 (ALS), and CAG repeat expansions contribute to the development of multiple neurodegenerative disorders including Huntington’s disease and various forms of spinocerebellar ataxia2 (SCA). Several mechanisms have been proposed for neurological diseases elicited by repeat expansions; these include neurotoxicity arising from formation of neuronal intranuclear inclusions and amyloid-like aggregates of proteins translated from repeat RNAs4, and the ensuing impairment of the ubiquitin–proteasome system11. Additionally, accumulation and aberrant phase separation of toxic RNAs, and their sequestration of RNA-binding proteins are also important contributors to disease aetiology9.Nucleotide repeat expansions contribute to a number of neurological diseases13 (FTLD), and CAG repeat expansion disorders, such as Huntington’s disease14 and SCA15. Additionally, intermediate-length CAG repeat expansions in the ATXN2 gene are significantly associated with ALS16, in which genetic depletion of ATXN2 extends lifespan and mitigates pathological aggregates in TDP-43 transgenic mice15. TDP-43 contains two RNA-recognition motifs (RRMs) and a C-terminal low-complexity domain13 (LCD), through which the protein can undergo intermolecular interactions and assemble into phase-separated liquid condensates17. Furthermore, phase-separated droplets of RNA–protein complexes can become less dynamic and aggregate over time, suggesting that aberrant phase transition contributes to the aggregation of RNA-binding proteins18.Cytoplasmic mis-localization and aggregation of TDP-43 in the degenerated regions of the brain are clinical hallmarks of many neurological diseases, including ALS, frontotemporal lobar degenerationN6 position of adenosine, constitute an important mechanism regulating the stability and translation efficiency of mRNAs20, and dysregulation of this methylation in mRNAs are linked with human diseases21. Here we show that CAG repeat expansions lead to increased levels of m1A, which binds to TDP-43, alters its subcellular distribution and phase separation behaviour, and contributes to neurodegeneration.Dynamic post-transcriptional modifications in RNA, especially methylation at the 2. We set out to test whether methylated adenosines—especially N6-methyladenosine (m6A) and m1A—are present in CAG repeat RNA and if so, how their modification frequencies may be influenced by repeat length and how they modulate RNA–protein interactions.Previous studies documented that the age of onset of neurological disorders arising from CAG repeat expansion is negatively correlated with repeat length, whereas disease severity is positively associated with repeat length1A and m6A in the resulting RNA samples using liquid chromatography–tandem mass spectrometry22 (LC–MS/MS). The results revealed a progressive increase in the frequency of m1A (relative to adenosine (rA)) with repeat length, which is accompanied with a gradual diminution in m6A level 38 RNA was even higher than that of m6A 38 repeat than in flanking sequences 38 RNAs were expressed at similar levels  in the levels of m1A in (CAG)38 after ectopic expression of ALKBH3, but not ALKBH1, FTO or ALKBH5 38 could be demethylated by FTO 38 RNA  to knock down TRMT61A expression in human cells 38 RNA. TRMT61A depletion led to a significant decrease in m1A level in (CAG)38 RNA, whereas we observed no change in m6A level 16 RNA in vitro, whereas the heat-inactivated enzyme did not 38 RNA  or transgenic mice expressing one wild-type endogenous Htt allele and a second Htt allele with knock-in of human mutant HTT (mHTT) exon 1, which contains 140 CAG repeats31 (Q140). LC–MS/MS analysis showed that the m1A level was significantly higher in Q140 mRNA than in Q7 mRNA isolated from striatal tissues , than those with a short Q27 CAG repeat6 40 and (CAG)67 mRNA (Q40 and Q67) than in worms expressing (CAG)19 mRNA32 (Q19), although no difference was detected in the levels of m6A in these mRNA samples  targeting the W02A11.1 gene, which is the nematode orthologue of the human TRMT61A gene. Knockdown of W02A11.1 led to a significantly diminished level of m1A in CAG repeat RNA extracted from the nematodes, whereas there was no significant difference in m6A level 38, with a more pronounced diminution being observed for the latter -based affinity screening led to the identification of several candidate m1A-containing RNA. Electrophoretic mobility shift assay (EMSA) showed that recombinant TDP-43 binds more strongly to the m1A-carrying RNA substrate used in the quantitative proteomic experiment than its unmethylated or m6A-containing counterpart, with the dissociation constants  of 18, 38 and 46 nM, respectively 38 RNA was significantly attenuated in cells with ectopic expression of wild-type ALKBH3, but not with a catalytically inactive mutant 38 RNA was significantly decreased in HEK293T cells upon ectopic co-expression of wild-type ALKBH3, but not its catalytically inactive mutant H257A34 22, and the amount of TDP-43 in the pull-down mixture was diminished upon ectopic co-expression of ALKBH3 38 13, which, along with our observations of the interaction between this protein and m1A-containing RNA, prompted us to examine the effect of CAG repeat RNA on the cleavage and subcellular distribution of TDP-43.Truncation, cytoplasmic redistribution and aggregation of TDP-43 are histopathological hallmarks of ALS and FTLD22 and (CAG)38 RNA, with (CAG)38 RNA being more effective in eliciting truncated TDP-43 protein 7 and (CAG)16 RNA carrying zero or three m1A residues. Our results showed more truncated TDP-43 in cells transfected with (CAG)16-3m1A RNA than with the corresponding unmodified RNA, though no apparent difference was detected for transfections with (CAG)7-0m1A and (CAG)7-3m1A RNAs ues Fig. , suggest22 or (CAG)38 resulted in cytoplasmic redistribution of TDP-43 22, where the foci size was decreased upon ectopic expression of ALKBH3 -43 Fig. . In addiBH3 Fig.  or upon BH3 Fig. . Consist38 RNA than with (CAG)22 RNA  and immunofluorescence assays to monitor subcellular localizations of CAG repeat RNA and TDP-43 protein, respectively. The results showed more extensive co-localization between endogenous TDP-43 and (CAG)RNA Fig. . AdditioRNA Fig.  or upon RNA Fig. . SimilarRNA Fig. ; howeverRNA Fig. . This isRNA Fig. .1A in the repeat RNA and requires the functional RRM domains of TDP-43. Additionally, CAG repeat RNAs promote the cytoplasmic redistribution and truncation of TDP-43, which again require m1A in CAG repeat RNA and its interaction with TDP-43.Together, these results substantiate that CAG repeat RNA binds to TDP-43 in cells, which is driven by m36; we next examined whether CAG repeat RNA and endogenous TDP-43 are localized to stress granules. We observed cytoplasmic foci for endogenous G3BP1 and its co-localization with endogenous TDP-43 in U2OS cells upon expression of (CAG)22 and (CAG)38, with the extent of co-localization being more pronounced for cells expressing (CAG)38 than (CAG)22 22 Fig. . Further)22 Fig. .Fig. 3m11A-marked RNA was also sequestered into stress granules. Our RNA-FISH and immunofluorescence assay results showed that endogenous G3BP1 granules were co-localized with (CAG)38 RNA 7 and (CAG)16 RNA carrying zero or three m1A residues. Transfection of cells with the synthetic CAG repeat RNAs led to cytoplasmic redistribution of TDP-43 in a sub-population of cells, and transfection with (CAG)7-3m1A or (CAG)16-3m1A RNA conferred larger TDP-43 foci in the cytosol than with the same amount of the corresponding unmodified RNAs RNA Fig. . Togethe1A in CAG repeat RNA. We first examined the physical properties of GFP–TDP-43 in cells expressing RNA with expanded CAG repeats. Our results showed that after photobleaching, the fluorescence of GFP–TDP-43 was rapidly recovered in cells with ectopic expression of (CAG)22 RNA 38 RNA, indicating that TDP-43 protein is immobile in these cells 22 with 1,6-hexanediol , which disrupts liquid-like condensates37. Upon treatment with 1,6-HD, there were fewer GFP–TDP-43 granules in cells expressing (CAG)22 1A regulates phase separation of TDP-43 protein in vitro using synthetic (CAG)7 RNA containing 0, 1 or 3 m1A residues. Although the unmodified (CAG)7 RNA did not alter the phase separation of TDP-43 7 RNA with 0 or 1 m1A, indicating that TDP-43 protein exhibits LLPS  and demethylase (ALKBH3) for m−1 penicillin and 100 μg ml−1 streptomycin under standard cell culture conditions . The cell lines were authenticated by ATCC using short tandem repeat (STR) analysis as described in 2012 in ANSI Standard (ASN-0002) Authentication of Human Cell Lines. The cells were confirmed to be free of mycoplasma contamination by using LookOut Mycoplasma PCR Detection Kit .HEK293T cells and U2OS cells were obtained from ATCC. The cells were maintained in DMEM  with 10% FBS, 100 U mlTRMT61A, TRMT61B and TRMT10C were designed and their sequences are listed in Supplementary Table shRNAs against Total RNA was extracted from HEK293T cells using TRI reagent (Sigma). The RNA sample (~20 μg) was subsequently incubated with 200 μM of 5′-biotinylated 5×CTG oligodeoxyribonucleotides in a 2-volume hybridization buffer  at room temperature for 4 h with gentle mixing. To the resulting mixture were then added streptavidin-conjugated agarose beads (Thermo Scientific) and the suspension was incubated at 4 °C for 2 h. After the incubation, the oligodeoxyribonucleotide-bound beads were washed for four times with 2× SSC buffer  and the beads was resuspended in RNase-free water for digestion.−1 RNase inhibitor. The supernatant was incubated with anti-Flag M2 beads (Sigma) at 4 °C overnight. The beads were washed for three times with a buffer containing 50 mM HEPES (pH 7.5), 200 mM NaCl, 2 mM EDTA, 0.05% IGEPAL CA-630, and 0.5 mM DTT. The TDP-43-bound RNA was extracted from the beads using TRI reagent, and mRNA was isolated and purified by using PolyATtract mRNA Isolation System IV (Promega) according to the manufacturer’s instructions.For the cellular pull-down experiment, HEK293T cells were transfected with Flag-tagged TDP-43 or 5FL mutant plasmid. After a 24-h incubation, the cells were washed with PBS and lysed in a buffer containing 10 mM HEPES (pH 7.5), 150 mM KCl, 2 mM EDTA, 0.5% IGEPAL CA-630, 0.5 mM DTT, protease inhibitor (Sigma), and 40 units ml2. The mixture was then incubated at 37 °C for 2 h, and to the mixture were added 0.5 unit of Antarctic phosphatase and 3 μl of 1.0 M NH4HCO3. After incubation at 37 °C for an additional 2 h, the digestion mixture was dried and reconstituted in 100 μl ddH2O. [13C5]-adenosine, [D3]-m6A, and [D3]-m1A were used as internal standards for the quantifications of rA, m6A, and m1A, respectively. In parallel, total RNA (400 ng as input control RNA) was digested in the same way. The enzymes in the digestion mixture were removed by extraction using chloroform:isoamyl alcohol (24:1), and salt in the samples was removed by acetonitrile precipitation. The resulting supernatant was dried, and the dried residues were reconstituted in 10 μl ddH2O and injected for LC–MS/MS analysis on a TSQ Altis triple-quadrupole mass spectrometer (Thermo). For m1A and m6A quantifications, the pre-column and analytical column were packed with porous graphitic carbon and Zorbax SB-C18 stationary phase materials, respectively, where a gradient of 0–15% B in 10 min, 15–95% B in 30 min, and 95% B in 10 min was used. The mass spectrometer was operated in the multiple-reaction monitoring (MRM) mode, where the loss of a ribose from the [M + H]+ ions of rA, m6A, m1A and their stable isotope-labelled counterparts were monitored.The above affinity-purified RNA (400 ng) was digested with 1 unit of nuclease P1 in 25 μl buffer containing 25 mM NaCl and 2.5 mM ZnCl38 repeat. In particular, the isolated RNA was annealed in the RNase H reaction buffer (New England Biolabs) by heating to 95 °C and cooling slowly to 70 °C. The temperature was held at 70 °C for 10 min to melt nonspecific RNA structures, before cooling slowly to 37 °C. RNase H was added to the mixture, incubated at 37 °C for 2 h, and the reaction was terminated with 0.5 M EDTA. (CAG)38 RNA was subsequently isolated by affinity purification, digested with enzymes, and analysed by LC–MS/MS, as described above. Unless specifically noted, the reported levels for m1A and m6A in CAG repeat RNA were without RNase H treatment.RNase H was used to remove the 5′ and 3′ flanking RNA sequences of the (CAG)mHTT exon 1 containing 140 CAG repeats were described previously40. All mice were maintained and bred under standard conditions consistent with National Institutes of Health guidelines and approved by the University of California, Los Angeles Institutional Animal Care and Use Committee. The cages were maintained on a 12:12 light:dark cycle, with food and water ad libitum. Striatum and cortex tissues were harvested from wild-type and heterozygous HD knock-in Q140 mice at six months of age and immediately frozen on dry ice, where tissues from both male and female mice were used. Q19 , Q40  and Q67 worm strains  were purchased from Caenorhabditis Genetics Center (CGC). Drosophila expressing a SCA3 transgene with 78 repeats of CAG (UAS-MJDtrQ78) and those with a short (UAS-HA-MJDtrQ27 line c19.1) CAG repeat (Bloomington Drosophila Stock Center line 8149) have been described41, where the transgene was expressed in neurons using ElavGal4 (Bloomington line 458). Coding sequences of wild-type and catalytically inactive mutant human ALKBH3 were subcloned into a pUAST transformation vector, and transgenic fly lines were subsequently generated . CAG repeat RNA was isolated from total RNA extracted from mouse striatum and cortex tissues, Drosophila heads and C. elegans, digested, and analysed in a similar way as described above for cellular RNA.C57BL/6 mice expressing one wild-type endogenous Htt allele and a second Htt allele with knock-in of human 6A and m1A were quantified by employing calibration curves obtained from pure nucleoside standards analysed under the same instrument conditions 7-0m1A with (CAG)7-1m1A and (CAG)7-1m6A at defined m1A/rA and m6A/rA molar ratios, and quantified the levels of m1A and m6A by following the same procedures as described above for CAG repeat RNA isolated from cells and tissues. The recoveries of m1A and m6A were determined to be approximately 85% and 87%, respectively . The lysates were centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was pre-cleared by incubating with streptavidin-conjugated agarose beads (Thermo Scientific) at 4 °C for 1 h, and 20 μl samples were taken as inputs. Biotinylated RNA baits (3 µg) were incubated, at room temperature for 4 h, with pre-cleared cell lysates in a hybridization buffer containing protease and RNase inhibitors. Streptavidin-conjugated agarose beads were then added to the mixture, and the mixture was kept in a shaker at 4 °C for 2 h. The beads were washed extensively and boiled for 10 min, and the supernatant collected. The samples were subsequently resolved by 10% SDS–PAGE and transferred to a PVDF membrane . Membranes were blocked in 5% milk at room temperature for 1 h, and then incubated with primary antibodies in 5% BSA at 4 °C overnight. Primary antibodies for the following proteins or epitope were used: TDP-43 , G3BP1 , TRMT61A , Flag epitope , ALKBH3 , GAPDH , and α-tubulin . After washing with PBS containing 0.05% Tween (PBS-T), the membranes were incubated with secondary antibodies at room temperature for 1 h. Secondary antibodies used for western blotting included anti-rabbit IgG (whole molecule)-Peroxidase antibody produced in goat  and m-IgGκ BP-HRP . Membranes were then washed with PBS-T and bands were visualized using a LI-COR imaging system and quantified by using ImageJ. All western blots shown are representative results obtained from at least three biological replicates.42. In brief, cells were washed once with ice-cold PBS, lysed with CelLytic M cell lysis reagent (Sigma), and incubated on ice for 10 min. Following a brief sonication on ice, lysates were centrifuged for 40 min at 15,000g at 4 °C. Supernatants were collected as the detergent-soluble fraction. The pellets were subsequently resuspended in a urea buffer , briefly sonicated on ice, and lysates were centrifuged at 15,000g at room temperature for 1 h, where supernatant was collected as the detergent-insoluble, urea-soluble fraction. Proteins in the detergent-soluble and insoluble fractions were then resolved by SDS–PAGE and analysed by western blot.Detergent solubility fractionation of TDP-43 was performed as described43. For purification of full-length TDP-43–MBP–6×His, TDP-43-5FL–MBP–His, TDP-43-5FL–eGFP–MBP–His and TDP-43-ΔLCD–eGFP–MBP–His proteins, BL21-DE3 Escherichia coli cells were cultured at 37 °C to an OD at 600 nm of 0.6–0.9, and induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 18 °C for 16 h. The cells were subsequently collected, pelleted and resuspended in a binding buffer containing 20 mM Tris-HCl (pH 8.0), 1 M NaCl, 10% (v/v) glycerol, 1 mM DTT, and EDTA-free protease inhibitor cocktail according to the manufacturer’s instructions. The cells were lysed by sonication and centrifuged at 10,000g for 20 min. The proteins were purified over pre-packed Dextrin Sepharose High Performance MBPTrap HP  and eluted by using the binding buffer containing 10 mM maltose. Proteins were further purified over pre-packed Ni Sepharose High Performance HisTrap HP (GE) and eluted with a buffer containing 50 mM Tris (pH 7.4), 500 mM NaCl and 400 mM imidazole. Protein concentration was determined by Bradford assay (Bio-Rad). The eluted fractions were analysed by using denaturing SDS–PAGE, and the purities of the recombinant proteins were assessed by SDS–PAGE analysis.Recombinant TDP-43 proteins were purified by following previously described procedures1A, A or m6A; Supplementary Table 44. In brief, [5′-32P]cytidine-3′,5′-bis(phosphate) (pCp) was prepared by incubating 0.5 μl 0.5 mM cytidine-3′-monophosphate  with 1.0 μl 10× T4 PNK buffer (NEB), 1 μl T4 PNK , and 8.25 μl [γ-32P]ATP at 37 °C for 1 h. The mixture was incubated at 65 °C for 10 min. RNA probe was 3′-end labelled in a 30-μl buffer containing 3 μl 10× T4 RNA ligase buffer (NEB), 3 μl 10 mM ATP (NEB), 1 μl 100 mM DTT (Promega), 3 μl DMSO (Sigma), 9.1 μl [5′-32P]pCp, and 3 μl T4 RNA ligase I (NEB) at 4 °C for 24 h. The probes were then purified by using micro bio-spin P-30 columns (Bio-Rad). In addition, synthetic 21-mer (CAG)7 RNA with or without m1A  was incubated with increasing concentrations of TDP-43 or its truncated or mutated variants in a binding buffer  at 4 °C for 1 h. The entire 20-µl sample was subjected to electrophoresis on an 8% native polyacrylamide gel and the gel band intensities were quantified using phosphorimager analysis with a Typhoon 9410 Variable Mode Imager. The dissociation constant (Kd) was calculated using Prism software (version 8.0.1).RNA probe, 5′-biotin-CCGUUCCGCCCXGGCCGCGCCCAGCUGGAAUGCA-3′  were calculated. The intensity and area of co-localized granules were calculated using plot profile tool in Fiji.2-controlled top-stage incubator for live-cell imaging. Photobleaching was conducted by a 488-nm line from an Argon laser at 50% power. Three regions of interest (ROIs) were defined for these experiments: ROI-1 was the indicated circular region in the droplet; ROI-2 was a circular, un-photobleached region of similar size in the same droplet; and ROI-3 was defined as background and drawn outside of the droplet, where its signal was subtracted from those of ROI-1 and ROI-2. Raw data were processed and plotted using Prism.FRAP experiments were performed using an LSM 880 laser scanning confocal microscope (Zeiss 880 Inverted Airyscan Fast) coupled with a temperature-, humidity- and COU2OS cells expressing the indicated RNA were fixed at 24 h following transfection and permeabilized by incubation for 10 min in methanol containing 10% (v/v) acetic acid. RNA was detected using a Cy3-labelled DNA probe (5′-/5Cy3/CTGCTGCTGCTGCTGCTGCTGCTG-3′). Hybridization and washing buffers were obtained from Biosearch Technologies and used following the manufacturer’s protocol. For immunofluorescence detection of proteins following RNA-FISH, methanol-fixed cells were stained using antibodies against TDP-43  and G3BP1 , and Alexa Fluor 488 and 647-labelled secondary antibodies . The samples were subsequently stained with DAPI and imaged using confocal microscopy as described above. The data were analysed using Zen 2 Blue software (version 2.3).2, 0.02% NP-40, and 50% glycerol. Dextran (10%) was also added as a crowding agent to induce phase separation of TDP-43. The droplets on a coverslip were detected using a 100× oil immersion objective by confocal microscopy. After TDP-43 droplet formation following the addition of m1A-RNA, partition coefficients were calculated for stably formed TDP-43-containing droplets based on the ratio of intensity of TDP-43 in phase-separated droplets over that located in the immediately adjacent region.In vitro TDP-43 droplet formation was induced in a buffer containing 20 mM HEPES (pH 7.4), 300 mM KCl, 6 mM MgCl45 and was modified to contain a 3C protease cleavage sequence C-terminal to the 6×His tag. Recombinant TRMT6–TRMT61A was purified following previously described procedures45. For the methyltransferase assay, synthetic (CAG)16 RNA was annealed by first heating to 95 °C for 5 min and then cooling to 25 °C. The reactions were performed at room temperature overnight in 20 μl buffer containing 50 mM ammonium acetate, 3 mM MgCl2, 50 mM Tris-HCl (pH 8.0), 1 mM DTT, 1 mM S-adenosylmethionine, 2.5 μM RNA substrate and 12.5 μM TRMT6–TRMT61A complex. After the reaction, proteins were removed by chloroform extraction, and the RNA was subsequently digested by nuclease P1 and Antarctic phosphatase, and the levels of rA and m1A were measured by LC–MS/MS, as described above.The expression plasmid, pET17b-6×HisTrm6-Trm61, was isolated from a strain provided by J. Finer-MooreC. elegans strains were maintained at 20 °C on standard nematode growth medium agar plates seeded with E. coli OP50 bacteria unless otherwise stated46. A list of strains used in this study is provided in Supplementary Table C. elegans strains  was fused to the C-terminus of ALKBH3 to monitor the expression of ALKBH3 protein in worms. All fragments with TBB2 3′-UTR were ligated by employing PCR walking technique and inserted into pCR2.1-TOPO (Thermo Fisher). The primer sequences are listed in Supplementary Table rol-6 (su1006) dominant marker plasmid pRF4 into the germline cells of early-adult hermaphrodites, where the injection mixtures contained 50 μg ml−1 each of pRF4 and ALKBH3 plasmids47. The F1 progenies of the transgenic strains generated in this manner carried heritable extrachromosomal multi-copy arrays of both the experimental and marker plasmids and were used for microscopy assessment. The gaps between neurons were quantified using ImageJ.To generate transgenic W02A11.1 RNAi constructs were obtained from the Vidal RNAi library. Hermaphrodite worms were fed E. coli OP50 containing an empty control vector (L4440) or expressing double-stranded RNA when they reached adulthood48. Plates for RNAi analysis were prepared by supplementing agar with 100 μg ml−1 carbenicillin and 1 mM IPTG after autoclave. Worms were synchronized and grown from hatching on RNAi-feeding plates unless otherwise stated. RNAi efficiency was verified by quantitative PCR with reverse transcription and western blot.For RNAi experiments, P values and number of samples are included in the figure panels or the corresponding figure legends. Significance was defined as any statistical outcome that resulted in a P value of less than 0.05, unless otherwise indicated49.All statistical analyses were performed in GraphPad Prism (version 8.0.1) or Microsoft Excel 2016. The outcomes of all statistical tests including Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-023-06701-5.Supplementary InformationSupplementary Tables 1–4 and Supplementary Figs 1–12.Reporting SummarySupplementary Video 17 RNAs containing 0, 1 or 3 m1A.Fusion events of TDP-43–eGFP with or without synthetic (CAG)Supplementary Video 27 RNAs containing 0, 1 or 3 m1A.Fusion events of TDP-43–eGFP with or without synthetic (CAG)Supplementary Video 37 RNAs containing 0, 1 or 3 m1A.Fusion events of TDP-43–eGFP with or without synthetic (CAG)Supplementary Video 47 RNAs containing 0, 1 or 3 m1A.Fusion events of TDP-43–eGFP with or without synthetic (CAG)Supplementary Video 57 RNAs containing 0, 1 or 3 m1A.Fluorescence recovery after photobleaching (FRAP) of TDP-43–eGFP in the presence or absence of synthetic (CAG)Supplementary Video 67 RNAs containing 0, 1 or 3 m1A.Fluorescence recovery after photobleaching (FRAP) of TDP-43–eGFP in the presence or absence of synthetic (CAG)Supplementary Video 77 RNAs containing 0, 1 or 3 m1A.Fluorescence recovery after photobleaching (FRAP) of TDP-43–eGFP in the presence or absence of synthetic (CAG)Supplementary Video 87 RNAs containing 0, 1 or 3 m1A.Fluorescence recovery after photobleaching (FRAP) of TDP-43–eGFP in the presence or absence of synthetic (CAG)Source DataSource data for Supplementary Figs. 4–8 and 11.Source Data Fig. 1Source Data Fig. 2Source Data Fig. 3Source Data Fig. 4Source Data Extended Data Fig. 1Source Data Extended Data Fig. 2Source Data Extended Data Fig. 3Source Data Extended Data Fig. 4Source Data Extended Data Fig. 5Source Data Extended Data Fig. 6Source Data Extended Data Fig. 7


Transposable elements (TEs) are DNA sequences that can transpose and replicate within the genome, leading to genetic changes that affect various aspects of host biology. Evolutionarily, hosts have also developed molecular mechanisms to suppress TEs at the transcriptional and post-transcriptional levels. Recent studies suggest that stress-induced formation of ribonucleoprotein (RNP) granules, including stress granule (SG) and processing body (P-body), can play a role in the sequestration of TEs to prevent transposition, suggesting an additional layer of the regulatory mechanism for TEs. RNP granules have been shown to contain factors involved in RNA regulation, including mRNA decay enzymes, RNA-binding proteins, and noncoding RNAs, which could potentially contribute to the regulation of TEs. Therefore, understanding the interplay between TEs and RNP granules is crucial for elucidating the mechanisms for maintaining genomic stability and controlling gene expression. In this review, we provide a brief overview of the current knowledge regarding the interplay between TEs and RNP granules, proposing RNP granules as a novel layer of the regulatory mechanism for TEs during stress. Alu and SVA elements can be mobilized in trans by hijacking the L1 enzymatic machinery in new genomic sites [Sequences of mobile elements, namely transposable elements (TEs), account for an astonishing portion of the host genome in virtually all organisms. In humans, approximately half of the genome is composed of TEs, whereas 1–2% of the genome is composed of protein-coding sequences [ic sites ,4.Once considered as junk DNA or selfish elements that reside in the host genome, an enormous effort, together with the advent of technical advances, has allowed us to appreciate the impact of TEs on the host genome, not only in terms of local mutations and genome size, but also in terms of deleterious and adaptive outcomes for individuals . Even TEDrosophila melanogaster, other organisms, including humans, have employed this type of small noncoding RNA to silence TEs as well [One of the well-known mechanisms to repress TEs is to utilize small noncoding RNAs that are grouped by their size and association with the type of Argonaute proteins. In particular, the PIWI-clade Argonaute proteins predominantly present in the germline associate with a class of small RNAs known as PIWI-interacting RNAs (piRNAs). Initially discovered in the germline of  as well ,17,18,19 as well . Exporte as well . The RNP as well . In addi as well ,23,24. A as well ,26,27,28 as well , ZFP57/K as well . Especia as well ,32. Appr as well . Epitope as well ,35,36. F as well ,38.Recent studies have shed light on new aspects of RNP granules involved in TE regulation that could serve as an additional layer of the host defense mechanisms beyond transcriptional and post-transcriptional silencing of TEs. RNP granules are non-membrane-bound structures consisting of protein and RNA components, which are believed to serve as centers for the regulation of RNA metabolism, including mRNA storage, transport, translation, and degradation. TEs have been found to interact with RNP granules, suggesting that these granules may modulate TE activity through various mechanisms such as TE mRNA sequestration, translational control, and degradation. Therefore, this review aims to discuss the newly discovered regulatory mechanisms associated with RNP granules and their potential implications in the context of TE regulation.The control of gene expression in eukaryotic cells takes place at the transcriptional to translational levels. As translation occurs in eukaryotes, mature mRNAs must be exported from the nucleus to encounter the translation machinery present in the cytoplasm. In other words, the localization of mRNAs to subcellular compartments can influence their interaction with various protein complexes, thereby providing a mechanism for the regulation of gene expression at the post-transcriptional level. Cytoplasmic RNP granules are membraneless subcellular compartments in the cytoplasm of eukaryotic cells, which serve as important sites for RNA regulation such as RNA storage and decay ,40. AlthDuring physiological stress, cells employ various defense mechanisms to counteract the continuous threats to their functionality and survival. SG is a prominent cytoplasmic RNP granule that plays an important role in coping with physiological stress . Once ceAlthough various stress stimuli can induce SG, the assembly of SG is typically associated with translational suppression . SG targTraditionally, the primary function of SG was thought to inhibit mRNA translation. However, emerging evidence increasingly challenges this understanding, suggesting that SG may not necessarily interfere with mRNA translation. Recent research using single-molecule imaging of translating RNAs has shown that many RNA molecules undergo translation in SG . AdditioRBPs containing intrinsically disordered regions (IDRs), such as PABP1, TIA1, and G3BP1, play an essential role in SG formation ,51. IDRsRecent studies suggest that stress-induced RNA granulation is not solely directed by the translational arrest of bulk mRNA species ,50. RNA SG actively participates in a variety of cellular processes such as metabolism, cellular signaling, apoptosis, and cellular senescence. SG harbors signaling proteins to modulate their function. For instance, the RACK1 protein that activates stress-responsive MTK1 is located in SG under severe stress, leading to the suppression of apoptosis . SG alsoDuring viral infection, host cells display a dynamic relationship with SG . SG formOn the other hand, some viruses have evolved mechanisms to either inhibit or utilize SG to avoid antiviral activity and evade their replication. For instance, viruses can inhibit SG formation by sequestering or modifying SG components such as G3BP1, which has been shown to facilitate the replication of many viruses, including the hepatitis C virus (HCV) ,78. The Processing body (P-body) is a cytoplasmic RNP granule that exists in all eukaryotic cells. The structure is characterized by its heterogeneity and dynamic nature with variable sizes and compositions, depending on the specific cellular conditions. Although present in nonstressed cells, P-body increases in both number and size in response to glucose starvation, osmotic stress, growth to the stationary phase, and DNA replication stress . Like otP-body consists of a variety of RNA and protein components that play important roles in post-transcriptional gene regulation. The molecular composition of P-body has been extensively studied, leading to the identification of numerous key components that are essential for P-body function. The RNA components of P-body include untranslated mRNAs, microRNAs, and long noncoding RNAs, which can modulate gene expression and post-transcriptional gene silencing. P-body also contains a diverse array of proteins involved in regulating RNA degradation ,84. The Although extensive studies have revealed that P-body contains molecules involved in RNA metabolism, the precise functions and mechanisms of P-body remain unclear. For instance, it is uncertain whether mRNA decay primarily occurs within P-body or elsewhere in the cytoplasm. While P-body contains various factors involved in RNA degradation, accumulating evidence suggests that P-body may function more as a site for mRNA storage and sequestration rather than degradation, and that mRNA decay can occur efficiently in the absence of P-body ,94. HoweLINE1 or L1). Although L1 makes up about 17% of the human genome and most L1 copies in the genome are considered to be incompetent to mobilize autonomously, about 100 L1 copies are known to propagate in the genome, with the estimation of a new insertion in every 20–200 births [L1 elements is about 6 kb, containing a bidirectional promoter in a 5′ UTR region, a 40 kDa ORF1 protein, a 150 kDa ORF2 protein, and a 3′ UTR region with a poly(A) tract. The process of L1 retrotransposition requires both the ORF1 protein, which encodes an RNA-binding protein with nucleic acid chaperone activity, and ORF2 protein, which has endonuclease and reverse transcriptase activities [Among class I retrotransposons, one of the heavily studied TEs is long interspersed nuclear element 1 , are enriched in cytoplasmic foci with a diffuse cytoplasmic expression, which have a similar pattern to cytoplasmic aggregates of endogenous mouse L1 ORF1p in embryonic carcinoma (EC) cells [L1 constructs containing tagged ORF1/2 as well as L1 mRNAs are also co-localized with SG markers, further suggesting a role of SG in the life cycle of L1s [In addition to the defense mechanism against C) cells ,99. These of L1s ,101.L1s in SG appears to negatively impact on their retrotransposition events , another interferon-stimulated gene, also restricts L1 retrotransposition by enhancing the sequestration of ORF1p to G3BP1-containing cytoplasmic granules [L1 RNPs into SG might prevent the accession of L1 RNPs to the nucleus for their reverse transcription. In addition, SAM domain and HD domain containing protein 1 (SAMHD1), involved in Aicardi–Goutières syndrome (AGS) as an improper immune activation [L1 retrotransposons [L1 RNPs into SG, subsequently resulting in the restriction of L1 retrotransposition. Lastly, the formation of SG induced by hepatitis C virus (HCV) infection shows a reduction in human L1 retrotransposition in hepatoma cells where L1 ORF1p is trapped within the SG. Collectively, these reports suggest that the sequestration of L1 components into SG inversely correlates with retrotransposition [Mounting evidence indicates that localization of n events . The zinL2 in SG ,104. Givgranules . As MxB granules , the seqtivation , is an ansposons ,109. Besposition .LINEs, Alu elements belong to the class of retrotransposons termed as short interspersed elements (SINEs). There are approximately 1 million copies of Alu elements in the human genome, which are known to be nonautonomous retrotransposons. Despite the requirement of transacting factors for their propagation, Alu elements have successfully continued to mobilize in the genome. They undoubtedly contribute various aspects to their host such as genetic diversity and diseases [Alu elements to some extent. The expression of the GFP reporter containing inverted Alu repeats at its 3′ UTR was found to be suppressed but prominently localized in cytoplasmic SG [Alu-containing mRNAs can be inhibited in SG. Similarly, even though exogenously expressed Alu RNA alone is typically found in the nucleus, it has been demonstrated that when Alu-like 7SL RNA containing MS2 stem loops and GFP-tagged L1 ORF1p are co-expressed, they co-localize in cytoplasmic granules [In addition to diseases . Interesasmic SG , indicatgranules .HERV) consists of 5′- and 3′-long terminal repeats (LTRs) flanking a Gag-Pro-Pol protein-coding sequence [HERVK mRNAs and proteins are expressed for a short time period during development, and cis-acting elements in the ERVs can act as an enormous reservoir of gene regulatory modules under various conditions [L1 localization in SG, ERV also appears to be localized in SG to some extent. Staufen-1 is required for the development of the anteroposterior axis during embryogenesis [HERVK (HML-2), localizes HERVK Rec proteins in SG induced by sodium arsenite treatment [ERVK proteins aggregated in the cytoplasm, and their localization was within G3BP1-positive SG [ERVKs via autophagy, suggesting a potential interplay between SG and autophagy.Comprising approximately 8% of the human genome, a complete human endogenous retrovirus , homologous to MOV10-like-1 (MOV10L1), which interacts with the piRNA pathway proteins MILI and MIWI [L1s, Alu, and SVAs in cell culture. This is unlike other RNA helicases that are associated with L1 ribonucleoprotein particles (RNPs) [L1 RNPs to SG to silence/degrade L1 via RNA interference (RNAi).Moreover, genetic factors related to TE silencing have been linked to SG as a potent site for TE regulation. In various cell culture systems, members of the cytidine deaminases, APOBEC3 family , have been shown to play an inhibitory role in the retrotransposition activity of L1  as well MusD TEs ,125,126.MusD TEs ,129,130.MusD TEs ,132,133,and MIWI ,135. MOVs (RNPs) ,137. Dues (RNPs) ,139, it Ty elements are extensively studied retrotransposons in the budding yeast Saccharomyces cerevisiae [Ty1 and Ty3 are exported to the cytoplasm where they assemble into nucleocapsids, known as virus- like particles (VLPs). Genetic evidence has suggested that Ty1 and Ty3 require host factors related to P-body to complete their replication during their life cycle  are endogenous retroviruses that actively mobilize in the mouse genome [Ty3 element in yeast [IAPs [IAP mRNAs and the Gag protein and causes a shift in IAP mRNA from a nonpolysomal to a polysomal fraction, suggesting that P-body plays a role in inhibiting the retrotranposition of IAPs. Additionally, the same group also reported that siRNA treatment against MOV10, a component of P-body, inhibits the retrotranposition of IAPs [Comprising ~12% of the mouse genome, intracisternal A-type particles  or chromatoid body from male germ cells ,155, hasor decay ,159.biomRecent studies have suggested that RNP granules such as SG and P-body may be potential candidates for TE silencing. Although the localization of transposable elements (TEs) does not necessarily imply their suppression, growing evidence indicates that the localization of TE ribonucleoproteins (RNPs) in SG can reduce the retrotransposition rate. Furthermore, certain factors within SG and P-body have been shown to suppress TEs. This evidence collectively suggests that RNP granules may regulate TEs, in addition to their role in translational attenuation. However, numerous questions remain regarding the mechanisms of TE regulation within RNP granules. For instance, it is still unclear which core factor(s) is (are) responsible for recruiting TE RNPs into SG, and what the consequences of TE RNPs within SG are. Further research is required to fully elucidate the interaction between RNP granules and TEs during stress and the potential regulatory mechanisms involved.


AluJb RNA/polymer complexes  were engineered to form colloidally stable nanoparticles that exhibited immunostimulatory activity in vitro and in vivo. Finally, the therapeutic potential of Alu-NPs for the treatment of cancer was demonstrated by attenuated tumor growth and prolonged survival in the B16.F10 murine melanoma tumor model. Thus, these data collectively implicate intratumoral Alu RNA as a potentiator of antitumor innate immunity and identify AluJb RNA as a novel nucleic acid immunotherapeutic for cancer.It was recently found that patients with relapsing remitting multiple sclerosis exhibit widespread loss of adenosine-to-inosine (A-to-I) RNA editing, which contributes to the accumulation of immunostimulatory double-stranded Alu RNA in circulating leukocytes and an attendant increase in levels of proinflammatory cytokines . A specific Alu RNA  was implicated in activating multiple RNA-sensing pathways and found to be a potent innate immune agonist. Here, we have performed a bioinformatic analysis of A-to-I RNA editing in human melanoma samples and determined that pre-therapy levels of A-to-I RNA editing negatively correlate with survival times, suggesting that an accumulation of endogenous double-stranded Alu RNA might contribute to cancer patient survival. Furthermore, we demonstrated that immunostimulatory Alu RNA can be leveraged pharmacologically for cancer immunotherapy. AluJb RNA was Loss of A-to-I editing leads to accumulation of unedited Alu RNAs that activate innate immunity via RNA-sensing pattern recognition receptors. When packaged into endosome-releasing polymer nanoparticles, AluJB RNA becomes highly immunostimulatory and can be used pharmacologically to inhibit tumor growth in mouse melanoma models. These findings identify Alu RNAs as a new class of nucleic acid innate immune agonists for cancer immunotherapy. RNA sensing is a prominent defense mechanism employed by mammalian cells to detect intracellular pathogens and cellular distress . The abnUnder homeostatic conditions, cellular RNAs tend to be immunologically inert and avoid spurious innate immune activation, because RNA-sensing PRRs can largely discriminate against them based on their subcellular localization, composition, and local concentration. Furthermore, cellular RNAs are naturally processed in a variety of ways to avoid innate immune recognition . However, in certain diseases and pathologic states, mechanisms of RNA processing are impaired, resulting in the activation of RNA-sensing PRRs in the absence of pathogen invasion  was found to be highly immunostimulatory as determined in a reporter cell screen for both IFN-I and NFκB activity . Using vin vitro than poly(IC) RNA in terms of both IFN-I and NFκB activity ] (D-PDB), previously described by our group and others  genomic location, chromosome, and nucleotides from the p terminus, (ii) type of edit; either A-to-G or T-to-C, strand (+ or −), and (iii) number of edits per site and total number of reads per nucleotide site. Mathematica programs were developed to synthesize data including determination of numbers of editing sites within groups unique or shared by groups, mean numbers of total reads and edits for each editing sites, and mean numbers of total reads and edits per total reads across individual gene regions. This information was tied to an Alu database to annotate each A-to-I editing site: location relative to annotated genes and if intronic, noncoding or RNA gene, 3′ or 5′ untranslated regions, or intergenic and if sites were within Alu or non-Alu elements. For these analyses, we defined three categories of A-to-I editing sites: (i) sites with greater than or equal to 1 edit/per read and greater than or equal to 1 read, “all”, (ii) sites with edit/read ratios greater than or equal to 0.05 and greater than or equal to 5 total reads, and (iii) sites with edit/read ratios greater than or equal to 0.25 and greater than or equal to 10 total reads. To create genome-wide A-to-I–editing indices, we identified all A-to-I–editing sites present in one sample and summed edit/read ratios for all editing sites across the genome.D-PDB and DB were synthesized via reversible addition-fragmentation chain transfer polymerization as described previously . Alu DNANanoparticle formulation was performed as described previously . SpecifiNanoparticle physical characterization was conducted as described previously . Dynamicl-glutamine, 10% heat-inactivated FBS (Gibco), 100 U mL−1 penicillin/100 µg mL−1 streptomycin (Gibco), and 100 µg/mL Normocin. To maintain selection pressure of the RAW-Dual cells, 200 µg/mL Zeocin was added every other passage. THP1-Dual cells (InvivoGen) were cultured in RPMI1640 Medium (Gibco) supplemented with 25 mmol/L HEPES, 2 mmol/L l-glutamine, 10% heat-inactivated FBS (Gibco), 100 U mL−1 penicillin/100 µg mL−1 streptomycin (Gibco), and 100 µg/mL Normocin. To maintain selection pressure of the THP1-Dual cells, 10 µg/mL Blasticidin and 100 µg/mL Zeocin were added every other passage. B16.F10 cells (ATCC) and B16.F10 IFN-LUC cells were cultured in DMEM (Gibco) supplemented with 4.5 g/L glucose, 2 mmol/L l-glutamine, 10% heat-inactivated FBS (Gibco), and 100 U mL−1 penicillin/100 µg mL−1 streptomycin (Gibco). To maintain selection pressure of the B16.F10 IFN-LUC cells, 10 µg/mL puromycin was added every passage. All of the cell lines used in this work were cultured in a humidified environment  and routinely tested for Mycoplasma contamination.All of the cell lines used in this work were maintained according to supplier specifications. RAW-Dual cells (InvivoGen) were cultured in DMEM (Gibco) supplemented with 4.5 g/L glucose, 2 mmol/L 50 values for the various treatments.The RNA/polymer complexes were screened in 96-well plates . The wells were seeded with 50,000 reporter cells in 100 µL media. Treatments were diluted to 100 µL in PBS and added to the existing 100 µL media when the wells were approximately 80% confluent. Approximately 24 hours after treatment, cell supernatants were evaluated via the Quanti-Luc (InvivoGen) assay, following manufacturer's instructions. A microplate reader  was used to quantify luminescence and/or absorbance. White, opaque-bottom 96-well plates  were employed for luminescence measurements. For each sample, signal was calculated from three biological replicates, each with three technical replicates. Signal intensity was normalized by subtracting the average value of a negative control group. For presentation purposes, bell-shaped dose–response curves were truncated at their plateau. Curve fitting analysis (GraphPad Prism software) was employed to estimate ECin vivo IFN activity experiment. Approximately 1 × 106 B16.F10 IFN-LUC cells suspended in 100 µL of PBS were inoculated into the rear right flank of 6–8 weeks old C57BL/6 mice (The Jackson Laboratory) via a subcutaneous injection. When tumors reached a volume of approximately 100 mm3, mice were given a single 100 µL intratumoral injection of PBS or Alu-NPs  at a 2 µg RNA dose. Bioluminescence was recorded longitudinally . At each predetermined timepoint, the mice were given a dorsal subcutaneous injection of 4.5 mg Pierce d-Luciferin  solublized in 150 µL PBS, and 15 minutes thereafter, a luminescence image was taken. Mice were shaved and anesthetized via isoflurane gas when necessary.An IVIS Lumina III (PerkinElmer) was employed to evaluate the 6 B16.F10 cells suspended in 100 µL of PBS were inoculated into the rear right flank of 6–8 weeks old C57BL/6 mice (The Jackson Laboratory) via a subcutaneous injection. When tumors reached a volume of approximately 200 mm3, mice were given two 100 µL intratumoral injections administered every 3 days with treatments of either PBS or Alu-NPs  at a 2 µg RNA dose. Mice were euthanized 6 hours after the second intratumoral treatment, and tumors were harvested. A TissueLyser II (Qiagen) was employed to homogenize the tumors, and a RNeasy Plus Mini Kit (Qiagen) was used to isolate tumor RNA.Approximately 1 × 10Ppib (Mm00478295_m1), mouse Ifnb1 (Mm00439552_s1), mouse Cxcl10 (Mm00445235_ m1), mouse Il6 (Mm00446190_m1), and mouse Tnf (Mm00443258_m1). For each treatment group, at least four biological samples were evaluated with technical duplicates for the each gene. The threshold cycle numbers of each technical duplicate were averaged. Employing the 2−ddCt method of analysis, gene expression was normalized to the house-keeping gene  and subsequently normalized to the negative control  treatment values.For the qPCR analysis of proinflammatory gene expression, an iScript cDNA synthesis kit (Bio-Rad) was used according to manufacturer's instructions to reverse transcribe 1 µg of tumor RNA. A CFX Connect Real-time System (Bio-Rad) was employed for qPCR on the resultant cDNA, with the threshold cycle number automatically determined by Bio-Rad CFX manager software V.3.0. The TaqMan gene expression kits (Thermo Fisher Scientific) utilized in this work include mouse 6 B16.F10 cells suspended in 100 µL of PBS were inoculated into the rear right flank of 6–8 weeks old C57BL/6 mice (The Jackson Laboratory) via a subcutaneous injection. When tumors reached a volume of approximately 50 mm3, mice were given three 100 µL intratumoral injections administered every 3 days with treatments of either PBS, D-PDB polymer NPs, Edited Alu-NPs, or Alu-NPs  at a 2 µg RNA dose or polymer equivalent. Edited Alu-NPs utilized an inactive form of AluJB RNA (Left Arm). Tumor volume, total murine mass, and murine well-being were recorded every other day. A maximum tumor volume of approximately 1,500 mm3 was the predetermined study endpoint and used as the basis for “survival” analysis. A T-cell depletion study was employed to evaluate the T-cell dependence of treatment efficacy. Mice were inoculated and treated as described above. However, T-cell depletion via intraperitoneal injection of α-CD8 mAb  was initiated 24 hours prior to NP treatment. To this end, 100 µg of α-CD8 mAb was injected on days −1, 2, 5, 8, and 11  reviewed and approved all of the animal experiments performed in this work, and all surgical and experimental procedures were performed in accordance with the regulations and guidelines imposed by the Vanderbilt University IACUC. All mice were maintained under pathogen-free conditions at the animal facilities of Vanderbilt University .P < 0.0001; ***, P < 0.005; **, P < 0.01; *, P < 0.05; ns, not significant.For each experiment, statistical significance was determined as indicated in the corresponding figure caption. GraphPad Prism software (Version 7.0c) was employed for all statistical analyses of this work. Plotted values represent experimental means, and error bars represent one SD, with the exception of those in tumor growth plots, which instead represent one SEM for improved visual presentation. ****, The data generated in this study are available upon request from the corresponding author.N = 23); whole-genome RNA-seq and whole-exome sequencing were performed [European Genome Phenome Archive Study ID: EGAS00001004545 (19)].We employed the SPRINT software package  to measut test = 0.25, paired t test = 0.14). Overall, we interpret these results to indicate that initial levels of tumor AEI may contribute to overall survival time in melanoma.We next investigated whether there was a statistically significant correlation between the AEI determined in each melanoma sample both prior and after onset of therapy and survival times . We founADAR (adenosine deaminase acting on RNA) family of genes encodes for ADAR enzymes that convert adenosine to inosine  have recently been reported . Becauseactivity . It was activity . As a rein vitro transcribed, and the endosomolytic polymer, D-PDB was employed to mediate intracellular delivery of the RNA  was  the RNA . While w the RNA , and com the RNA . At an N the RNA  with sim the RNA . The lac the RNA , which iin vitro activity assays in reporter cell lines. Alu-NP complexes were administered to IFN-I reporter cells that express a secreted luciferase downstream of IFN-stimulated response elements, and 24 hours after treatment, the relative IFN-I production was measured via luminescence  or NPs complexed with edited, inactive AluJB (Left Arm). D-PDB NPs without RNA  failed to elicit a response in RAW-Dual macrophages and induced only a modest response in THP1-Dual monocytes. Furthermore, while AluJB (Left Arm) complexed with non-endosomolytic DMAEMA-block-BMA (D-B) polymer slightly stimulated type-I IFN production in RAW-Dual macrophages, it failed to activate a response in THP1-Dual monocytes. Thus, D-PDB was necessary for effective intracellular delivery of AluJb RNA, and only the unedited form of AluJb RNA was able to potently activate RNA sensing pathways.The immunostimulatory activity of Alu-NPs was then investigated using nescence  and E. Iin vivo, they were injected intratumorally twice, spaced 3 days apart, in mice bearing subcutaneous B16.F10 tumors, and 6 hours after the second treatment, tumor RNA was collected for qPCR analysis . Intratumorally administered Alu-NPs elicited significantly increased gene expression of Ifnb1, Cxcl10, and Tnf in the tumor microenvironment (TME). Il6 expression also tended to increase relative to PBS-treated tumors, but in a statistically insignificant manner (P = 0.086).To determine whether the Alu-NPs conserved their immunostimulatory activity when administered analysis . Gene exSubsequently, B16.F10 IFN-LUC tumors, which express luciferase in response to IFN signaling , were us+ T cells were antibody depleted with the initiation of CD8+ T-cell depletion one day prior to the onset of treatment. When Alu-NPs were administered in conjunction with CD8+ T-cell depletion, no inhibition of tumor growth was observed, demonstrating that CD8+ T cells are required for efficacy  was formulated with the endosomolytic polymer, D-PDB to form Alu-NP, which enables the intracellular delivery of RNA and targeted activation of RNA-sensing PRRs. At an N/P charge ratio of 4, Alu-NP complexes exhibited high RNA loading and formed uniform nanoparticles of approximately 100 nm in diameter that could potently activate innate immune signaling pathways as determined with onocytes . Upon inin vitro  and in v exist and have potential to further enhance delivery of Alu RNA, including advanced polymeric carriers recently described by our group (Regarding future work and applications of Alu-NPs, further characterization of the pharmacokinetic properties of the nanoparticle may be necessary to fully understand the kinetics of signaling and its associated effects on innate immune activation. Moreover, comprehensive cellular and molecular immunophenotyping of the TME in response to Alu-NP remains necessary to establish the major cellular effectors responsible for mediating antitumor responses. However, the lack of antitumor efficacy of Alu-NPs when CD8ur group , and theSupplementary Table 1Clinical Characteristics from Anagnostou et al.Click here for additional data file.