m6A hypomethylation of DNMT3B regulated by ALKBH5 promotes intervertebral disc degeneration via E4F1 deficiency

Clinical and translational medicine

2022 Mar 01

Li, G;Luo, R;Zhang, W;He, S;Wang, B;Liang, H;Song, Y;Ke, W;Shi, Y;Feng, X;Zhao, K;Wu, X;Zhang, Y;Wang, K;Yang, C;
PMID: 35340126 | DOI: 10.1002/ctm2.765

The intervertebral disc (IVD) degeneration is the leading cause of low back pain, which accounts for a main cause of disability. N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNAs and is involved in various diseases and cellular processes by modulating mRNA fate. However, the critical role of m6A regulation in IVD degeneration remains unclear. Nucleus pulposus cell (NPC) senescence is critical for the progression of IVD degeneration. Here, we uncovered the role and explored the regulatory mechanism of m6A in NPC senescence during IVD degeneration.Identification of NPC senescence during IVD degeneration was based on the analysis of tissue samples and the cellular model. ALKBH5 upregulation inducing cellular senescence was confirmed by functional experiments in vivo and in vitro. ChIP-qPCR and DNA-Pulldown were used to reveal increased ALKBH5 was regulated by KDM4A-mediated H3K9me3. Furthermore, Me-RIP-seq was performed to identify m6A hypomethylation of DNMT3B transcripts in senescent NPCs. Stability analysis showed that DNMT3B expression was enhanced for less YTHDF2 recognition and increased DNMT3B promoted NPC senescence and IVD degeneration via E4F1 methylation by in vivo and in vitro analyses.Expression of ALKBH5 is enhanced during IVD degeneration and NPC senescence, due to decreased KDM4A-mediated H3K9me3 modification. Functionally, ALKBH5 causes NPC senescence by demethylating DNMT3B transcripts and in turn promoting its expression via less YTHDF2 recognition and following degradation due to transcript hypomethylation in vitro and in vivo. Increased DNMT3B promotes the development of IVD degeneration and NPC senescence, mechanistically by methylating CpG islands of E4F1 at the promoter region and thus restraining its transcription and expression.Collectively, our findings reveal an epigenetic interplay mechanism in NPC senescence and IVD degeneration, presenting a critical pro-senescence role of ALKBH5 and m6A hypomethylation, highlighting the therapeutic potential of targeting the m6A/DNMT3B/E4F1 axis for treating IVD degeneration.

Biodistribution and Tolerability of AAV-PHP.B-CBh-SMN1 in Wistar Han Rats and Cynomolgus Macaques Reveal Different Toxicologic Profiles

Human gene therapy

2021 Dec 21

Palazzi, X;Pardo, I;Sirivelu, M;Newman, L;Kumpf, S;Qian, J;Franks, T;Lopes, S;Liu, J;Monarski, L;Casinghino, S;Ritenour, C;Ritenour, H;Dubois, C;Olson, J;Graves, J;Alexander, K;Coskran, T;Lanz, TA;Brady, J;McCarty, D;Somanathan, S;Whiteley, L;
PMID: 34931542 | DOI: 10.1089/hum.2021.116

Recombinant adeno-associated viruses (AAVs) have emerged as promising vectors for human gene therapy, but some variants have induced severe toxicity in Rhesus monkeys and piglets following high dose intravenous (IV) administration. To characterize biodistribution, transduction and toxicity amongst common preclinical species, an AAV9 neurotropic variant expressing the survival motor neuron-1 (SMN-1) transgene (AAV-PHP.B-CBh-SMN1) was administered by IV bolus injection to Wistar Han rats and cynomolgus monkeys at doses of 2x1013, 5x1013, or 1x1014 vg/kg. A dose-dependent degeneration/necrosis of neurons without clinical manifestations occurred in dorsal root ganglia (DRGs) and sympathetic thoracic ganglia in rats, while liver injury was not observed in rats. In monkeys, one male at 5x1013 vg/kg was found dead on Day 4. Clinical pathology data on Days 3 and/or 4 at all doses suggested liver dysfunction and coagulation disorders, which led to study termination. Histologic evaluation of the liver in monkeys showed hepatocyte degeneration and necrosis without inflammatory cell infiltrates or intravascular thrombi suggesting that hepatocyte injury is a direct effect of the vector following hepatocyte transduction. In situ hybridization (ISH) demonstrated a dose-dependent expression of SMN1 transgene mRNA in the cytoplasm and DNA in the nucleus of periportal to panlobular hepatocytes, while qPCR confirmed the dose-dependent presence of SMN1 transgene mRNA and DNA in monkeys. Monkeys produced a much greater amount of transgene mRNA compared with rats. In DRGs, neuronal degeneration/necrosis and accompanying findings were observed in monkeys as early as 4 days after test article administration. The present results show sensory neuron toxicity following IV delivery of AAV vectors at high doses with an early onset in Macaca fascicularis and after one month in rats, and suggest adding the autonomic system in the watch-list for preclinical and clinical studies. Our data also suggest that the rat may be useful for evaluating the potential DRG toxicity of AAV vectors, while acute hepatic toxicity associated with coagulation disorders appears to be highly species-dependent.

Traumatic-noise-induced hair cell death and hearing loss is mediated by activation of CaMKKβ

Cellular and molecular life sciences : CMLS

2022 Apr 19

Wu, F;Hill, K;Fang, Q;He, Z;Zheng, H;Wang, X;Xiong, H;Sha, SH;
PMID: 35438341 | DOI: 10.1007/s00018-022-04268-4

The Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) are serine/threonine-directed protein kinases that are activated following increases in intracellular calcium, playing a critical role in neuronal signaling. Inner-ear-trauma-induced calcium overload in sensory hair cells has been well documented in the pathogenesis of traumatic noise-induced hair cell death and hearing loss, but there are no established pharmaceutical therapies available due to a lack of specific therapeutic targets. In this study, we investigated the activation of CaMKKβ in the inner ear after traumatic noise exposure and assessed the prevention of noise-induced hearing loss (NIHL) with RNA silencing.Treatment with short hairpin RNA of CaMKKβ (shCaMKKβ) via adeno-associated virus transduction significantly knocked down CaMKKβ expression in the inner ear. Knockdown of CaMKKβ significantly attenuated noise-induced hair cell loss and hearing loss (NIHL). Additionally, pretreatment with naked CaMKKβ small interfering RNA (siCaMKKβ) attenuated noise-induced losses of inner hair cell synapses and OHCs and NIHL. Furthermore, traumatic noise exposure activates CaMKKβ in OHCs as demonstrated by immunolabeling for p-CaMKI. CaMKKβ mRNA assessed by fluorescence in-situ hybridization and immunolabeling for CaMKKβ in OHCs also increased after the exposure. Finally, pretreatment with siCaMKKβ diminished noise-induced activation of AMPKα in OHCs.These findings demonstrate that traumatic-noise-induced OHC loss and hearing loss occur primarily via activation of CaMKKβ. Targeting CaMKKβ is a key strategy for prevention of noise-induced hearing loss. Furthermore, our data suggest that noise-induced activation of AMPKα in OHCs occurs via the CaMKKβ pathway.

A novel protein encoded by circHNRNPU promotes multiple myeloma progression by regulating the bone marrow microenvironment and alternative splicing

Journal of experimental & clinical cancer research : CR

2022 Mar 08

Tang, X;Deng, Z;Ding, P;Qiang, W;Lu, Y;Gao, S;Hu, Y;Yang, Y;Du, J;Gu, C;
PMID: 35260179 | DOI: 10.1186/s13046-022-02276-7

Multiple myeloma (MM) is an incurable plasma cell malignancy in the bone marrow (BM), while immunoglobulin D type of MM (IgD MM) is a very rare but most severe subtype in all MM cases. Therefore, systemic study on IgD MM is purposeful to disclose the recurrent and refractory features in both IgD and other types of MM, and beneficial to the development of potent therapeutic strategy on MM.Agilent SBC-ceRNA microarray chips were employed to examine 3 normal plasma cell samples (NPCs), 5 lgD MM samples and 5 lgG MM samples, respectively. Sanger sequencing, RNase R digestion and qPCR assays were used to detect the existence and expression of circHNRNPU. BaseScope RNA ISH assay was performed to test circHNRNPU levels in paraffin-embedded MM tissues. The protein encoded by circHNRNPU was identified by LC-MS/MS, which was named as circHNRNPU_603aa. The function of circHNRNPU_603aa on cellular proliferation and cell cycle was assessed by MTT test, colony formation assay, flow cytometry and MM xenograft mouse model in vivo. RIP-seq, RIP-PCR and WB analysis for ubiquitination were performed to explore the potential mechanism of circHNRNPU_603aa in MM. Exosomes were isolated from the culture supernatant of MM cells by ultracentrifugation and characterized by Transmission Electron Microscope and WB confirmation of exosomes markers Alix and CD9.CircHNRNPU was one of the top most abundant and differentially expressed circRNA in IgD MM relative to lgG and NPCs samples. Increased circHNRNPU was associated with poor outcomes in four independent MM patient cohorts. Intriguingly, MM cells secreted circHNRNPU, which encoded a protein named as circHNRNPU_603aa. Overexpressed circHNRNPU_603aa promoted MM cell proliferation in vitro and in vivo, in contrast knockdown of circHNRNPU_603aa by siRNA abrogated these effects. Due to circHNRNPU_603aa including RNA-binding RGG-box region, it regulated SKP2 exon skipping, thereby competitively inhibited c-Myc ubiquitin so as to stabilize c-Myc in MM. MM cells secreted circHNRNPU through exosomes to interfere with various cells in the BM microenvironment.Our findings demonstrate that circHNRNPU_603aa is a promising diagnostic and therapeutic marker in both MM cells and BM niche.

hnRNPL-activated circANKRD42 back-splicing and circANKRD42-mediated crosstalk of mechanical stiffness and biochemical signal in lung fibrosis

Molecular therapy : the journal of the American Society of Gene Therapy

2022 Mar 10

Xu, P;Zhang, J;Wang, M;Liu, B;Li, R;Li, H;Zhai, N;Liu, W;Lv, C;Song, X;
PMID: 35278674 | DOI: 10.1016/j.ymthe.2022.01.045

Increasing circular RNAs (circRNAs) are involved in the progression of idiopathic pulmonary fibrosis (IPF). However, circRNA biogenesis and circRNA-mediated crosstalk between mechanical stiffness and biochemical signals in IPF remain obscure. In this study, a novel circRNA-ankyrin repeat domain 42 (ANKRD42) from peripheral blood of patients with IPF, which participated in pulmonary fibrosis through the close communication of mechanical stiffness and biochemical signals, was identified. Mechanistic studies revealed that the heterogeneous nuclear ribonucleoprotein L (hnRNP L) activated the circANKRD42 reverse splicing biogenesis. The biogenetic circANKRD42 sponged miR-324-5p to promote the AJUBA expression, which blocked the binding between phosphorylated yes-associated protein 1 (YAP1) and large tumor suppressor kinase 1/2 (LATS1/2), leading to increased YAP1 entering the nucleus. circANKRD42 also sponged miR-136-5p to promote the YAP1 translation. Accumulating YAP1 in nucleus bound to TEAD, which initiated the transcription of genes related to mechanical stiffness. Finally, the therapeutic effect of circANKRD42 was evaluated in mice and the association between circANKRD42 and clinicopathological features was analyzed in IPF patients. Our findings supported that circANKRD42 is a promising biomarker and a potential therapeutic target related to cytoskeleton tension for IPF treatment.

Intermittent Lipid Nanoparticle mRNA Administration Prevents Cortical Dysmyelination Associated with Arginase Deficiency

Molecular Therapy - Nucleic Acids

2022 Apr 01

Khoja, S;Liu, X;Truong, B;Nitzahn, M;Lambert, J;Eliav, A;Nasser, E;Randolph, E;Burke, K;White, R;Zhu, X;Martini, P;Nissim, I;Cederbaum, S;Lipshutz, G;
| DOI: 10.1016/j.omtn.2022.04.012

Arginase deficiency is associated with prominent neuromotor features including spastic diplegia, clonus, and hyperreflexia; intellectual disability and progressive neurological decline are other signs. In a constitutive murine model, we recently described leukodystrophy as a significant component of the central nervous system features of arginase deficiency. In the present studies, we sought to examine if the administration of a lipid nanoparticle carrying human ARG1 mRNA to constitutive knockout mice could prevent abnormalities in myelination associated with arginase deficiency. Imaging of the cingulum, striatum, and cervical segments of the corticospinal tract, revealed a drastic reduction of myelinated axons; signs of degenerating axons were also present with thin myelin layers. Lipid nanoparticle/ARG1 mRNA administration resulted in both light and electron microscopic evidence of a dramatic recovery of myelin density compared with age-matched controls; oligodendrocytes were seen to be extending processes to wrap many axons. Abnormally thin myelin layers, when myelination was present, was resolved with intermittent mRNA administration, indicative of not only a greater density of myelinated axons but also an increase in the thickness of the myelin sheath. In conclusion, lipid nanoparticle/ARG1 mRNA administration in arginase deficiency prevents the associated leukodystrophy and restores normal oligodendrocyte function.

The gRNA Vector Level Determines the Outcome of Systemic AAV CRISPR Therapy for Duchenne Muscular Dystrophy

Human gene therapy

2022 May 04

Wasala, NB;Million, ED;Watkins, TB;Wasala, LP;Han, J;Yue, Y;Lu, B;Chen, SJ;Hakim, CH;Duan, D;
PMID: 35350865 | DOI: 10.1089/hum.2021.130

Adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR) editing holds promise to restore missing dystrophin in Duchenne muscular dystrophy (DMD). Intramuscular coinjection of CRISPR-associated protein 9 (Cas9) and guide RNA (gRNA) vectors resulted in robust dystrophin restoration in short-term studies in the mdx mouse model of DMD. Intriguingly, this strategy failed to yield efficient dystrophin rescue in muscle in a long-term (18-month) systemic injection study. In-depth analyses revealed a selective loss of the gRNA vector after long-term systemic, but not short-term local injection. To determine whether preferential gRNA vector depletion is due to the mode of delivery (local vs. systemic) or the duration of the study (short term vs. long term), we conducted a short-term systemic injection study. The gRNA (4e12 vg/mouse in the 1:1 group or 1.2e13 vg/mouse in the 3:1 group) and Cas9 (4e12 vg/mouse) vectors were coinjected intravenously into 4-week-old mdx mice. The ratio of the gRNA to Cas9 vector genome copy dropped from 1:1 and 3:1 at injection to 0.4:1 and 1:1 at harvest 3 months later, suggesting that the route of administration, rather than the experimental duration, determines preferential gRNA vector loss. Consistent with our long-term systemic injection study, the vector ratio did not influence Cas9 expression. However, the 3:1 group showed significantly higher dystrophin expression and genome editing, better myofiber size distribution, and a more pronounced improvement in muscle function and electrocardiography. Our data suggest that the gRNA vector dose determines the outcome of systemic AAV CRISPR therapy for DMD.

Targeted clearance of p21- but not p16-positive senescent cells prevents radiation-induced osteoporosis and increased marrow adiposity

Aging cell

2022 Apr 01

Chandra, A;Lagnado, AB;Farr, JN;Doolittle, M;Tchkonia, T;Kirkland, JL;LeBrasseur, NK;Robbins, PD;Niedernhofer, LJ;Ikeno, Y;Passos, JF;Monroe, DG;Pignolo, RJ;Khosla, S;
PMID: 35363946 | DOI: 10.1111/acel.13602

Cellular senescence, which is a major cause of tissue dysfunction with aging and multiple other conditions, is known to be triggered by p16Ink4a or p21Cip1 , but the relative contributions of each pathway toward inducing senescence are unclear. Here, we directly addressed this issue by first developing and validating a p21-ATTAC mouse with the p21Cip1 promoter driving a "suicide" transgene encoding an inducible caspase-8 which, upon induction, selectively kills p21Cip1 -expressing senescent cells. Next, we used the p21-ATTAC mouse and the established p16-INK-ATTAC mouse to directly compare the contributions of p21Cip1 versus p16Ink4a in driving cellular senescence in a condition where a tissue phenotype (bone loss and increased marrow adiposity) is clearly driven by cellular senescence-specifically, radiation-induced osteoporosis. Using RNA in situ hybridization, we confirmed the reduction in radiation-induced p21Cip1 - or p16Ink4a -driven transcripts following senescent cell clearance in both models. However, only clearance of p21Cip1 +, but not p16Ink4a +, senescent cells prevented both radiation-induced osteoporosis and increased marrow adiposity. Reduction in senescent cells with dysfunctional telomeres following clearance of p21Cip1 +, but not p16Ink4a +, senescent cells also reduced several of the radiation-induced pro-inflammatory senescence-associated secretory phenotype factors. Thus, by directly comparing senescent cell clearance using two parallel genetic models, we demonstrate that radiation-induced osteoporosis is driven predominantly by p21Cip1 - rather than p16Ink4a -mediated cellular senescence. Further, this approach can be used to dissect the contributions of these pathways in other senescence-associated conditions, including aging across tissues.

Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A

Nature medicine

2022 Apr 01

Fong, S;Yates, B;Sihn, CR;Mattis, AN;Mitchell, N;Liu, S;Russell, CB;Kim, B;Lawal, A;Rangarajan, S;Lester, W;Bunting, S;Pierce, GF;Pasi, KJ;Wong, WY;
PMID: 35411075 | DOI: 10.1038/s41591-022-01751-0

Factor VIII gene transfer with a single intravenous infusion of valoctocogene roxaparvovec (AAV5-hFVIII-SQ) has demonstrated clinical benefits lasting 5 years to date in people with severe hemophilia A. Molecular mechanisms underlying sustained AAV5-hFVIII-SQ-derived FVIII expression have not been studied in humans. In a substudy of the phase 1/2 clinical trial ( NCT02576795 ), liver biopsy samples were collected 2.6-4.1 years after gene transfer from five participants. Primary objectives were to examine effects on liver histopathology, determine the transduction pattern and percentage of hepatocytes transduced with AAV5-hFVIII-SQ genomes, characterize and quantify episomal forms of vector DNA and quantify transgene expression (hFVIII-SQ RNA and hFVIII-SQ protein). Histopathology revealed no dysplasia, architectural distortion, fibrosis or chronic inflammation, and no endoplasmic reticulum stress was detected in hepatocytes expressing hFVIII-SQ protein. Hepatocytes stained positive for vector genomes, showing a trend for more cells transduced with higher doses. Molecular analysis demonstrated the presence of full-length, inverted terminal repeat-fused, circular episomal genomes, which are associated with long-term expression. Interindividual differences in transgene expression were noted despite similar successful transduction, possibly influenced by host-mediated post-transduction mechanisms of vector transcription, hFVIII-SQ protein translation and secretion. Overall, these results demonstrate persistent episomal vector structures following AAV5-hFVIII-SQ administration and begin to elucidate potential mechanisms mediating interindividual variability.

Time 2EVOLVE: predicting efficacy of engineered T-cells- how far is the bench from the bedside?

Journal for immunotherapy of cancer

2022 May 01

Guedan, S;Luu, M;Ammar, D;Barbao, P;Bonini, C;Bousso, P;Buchholz, CJ;Casucci, M;De Angelis, B;Donnadieu, E;Espie, D;Greco, B;Groen, R;Huppa, JB;Kantari-Mimoun, C;Laugel, B;Mantock, M;Markman, JL;Morris, E;Quintarelli, C;Rade, M;Reiche, K;Rodriguez-Garcia, A;Rodriguez-Madoz, JR;Ruggiero, E;Themeli, M;Hudecek, M;Marchiq, I;
PMID: 35577501 | DOI: 10.1136/jitc-2021-003487

Immunotherapy with gene engineered CAR and TCR transgenic T-cells is a transformative treatment in cancer medicine. There is a rich pipeline with target antigens and sophisticated technologies that will enable establishing this novel treatment not only in rare hematological malignancies, but also in common solid tumors. The T2EVOLVE consortium is a public private partnership directed at accelerating the preclinical development of and increasing access to engineered T-cell immunotherapies for cancer patients. A key ambition in T2EVOLVE is to assess the currently available preclinical models for evaluating safety and efficacy of engineered T cell therapy and developing new models and test parameters with higher predictive value for clinical safety and efficacy in order to improve and accelerate the selection of lead T-cell products for clinical translation. Here, we review existing and emerging preclinical models that permit assessing CAR and TCR signaling and antigen binding, the access and function of engineered T-cells to primary and metastatic tumor ligands, as well as the impact of endogenous factors such as the host immune system and microbiome. Collectively, this review article presents a perspective on an accelerated translational development path that is based on innovative standardized preclinical test systems for CAR and TCR transgenic T-cell products.

A Chimeric Antigen Receptor Targeting Malonaldehyde-modified Low-density lipoprotein Cholesterol Activates Regulatory T Cells in the Presence of Human Atherosclerotic Plaque

JVS-Vascular Science

2022 Jan 01

Shaligram, S;Lopez, JL;Lin, PY;Ho, P;Huang, A;
| DOI: 10.1016/j.jvssci.2022.05.021

Background: Regulatory T cells (Tregs) suppress inflammation in atherosclerosis, and therefore have the therapeutic potential to decrease the risk of myocardial infarction and stroke. However, there is currently no method to generate antigen specific Tregs that target atherosclerosis. We therefore engineered Tregs that express a chimeric antigen receptor (CAR) targeting malonaldehyde-modified low-density lipoprotein cholesterol (MDA-LDL), the most common form of oxidized LDL and a key molecular component of atherosclerosis. Methods: Novel single chain variable fragments (scFv) were synthesized using sequences from antibodies targeting human MDA-LDL. OxidizedLDL specific CARs (ox-CARs) were subsequently engineered by fusing each scFv to an IgG4 hinge, CD28 transmembrane, and CD28/CD3z cytoplasmic domains. CD4+ CD25+ CD127low/e Tregs were purified from human blood via fluorescent activated cell sorting and lentivirally transduced to express the novel ox-CARs (ox-CAR-Tregs). Human atherosclerotic plaques were obtained from patients undergoing carotid endarterectomy. Autologous ox-CAR-Tregs were analyzed for activation after ex vivo coculture with carotid endarterectomy samples. Results: A rationally designed panel of 42 ox-CARs were engineered using scFv derived from 12 antibodies targeting MDA-LDL. We first assessed CAR expression and activation in Jurkat T cells to identify promising oxCAR variants for further evaluation in human Tregs. After culture in the presence of MDA-LDL, six ox-CAR-Treg variants consistently showed significant activation, compared with controls, based on CD71 expression, cytokine expression, and proliferation in the absence of CD3/28 stimulation. Human atherosclerotic samples were identified to have substantial amounts of MDA-LDL epitopes using immunohistochemistry. Autologous ox-CAR-Tregs showed a dose-dependent increase in CD71 expression after ex vivo co-culture with atherosclerotic plaque. Conclusions: An optimized CAR targeting MDA-LDL activates Tregs Q10 when cultured with human atherosclerotic plaque ex vivo.

Antisense oligonucleotides ameliorate kidney dysfunction in podocyte specific APOL1 risk variant mice

Molecular therapy : the journal of the American Society of Gene Therapy

2022 Apr 20

Yang, YW;Poudel, B;Frederick, J;Dhillon, P;Shrestha, R;Ma, Z;Wu, J;Okamoto, K;Kopp, JB;Booten, SL;Gattis, D;Watt, AT;Palmer, M;Aghajan, M;Susztak, K;
PMID: 35450819 | DOI: 10.1016/j.ymthe.2022.04.007

Coding variants (named G1 and G2) in Apolipoprotein L1 (APOL1) can explain the most excess risk of kidney disease observed in African Americans. It has been proposed that risk variant APOL1 dose, such as increased risk variant APOL1 level serves as a trigger (second hit) for disease development. The goal of this study was to determine whether lowering risk variant APOL1 levels protects from disease development in podocyte specific transgenic mouse disease model. We administered antisense oligonucleotides (ASO) targeting APOL1 to podocyte specific G2APOL1 mice and observed efficient reduction of APOL1 levels. APOL1 ASO1, which more efficiently lowered APOL1 transcript levels, protected mice from albuminuria, glomerulosclerosis, tubulointerstitial fibrosis, and renal failure. The administration of APOL1 ASO1 was effective even for established disease in the NEFTA-rtTA/TRE-G2APOL1 (NEFTA/G2APOL1) mice. We observed a strong correlation between APOL1 transcript level and disease severity. We concluded that an APOL1 ASO1 may be an effective therapeutic approach for APOL1-associated glomerular disease.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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