Second-generation (Gen 2) Antisense oligonucleotides (ASOs) show increased nuclease stability and affinity for their RNA targets, which has translated to improved potency and therapeutic index in the clinic. Gen 2 ASOs are typically modified using the phosphorothioate (PS) backbone modification, which enhances ASO interactions with plasma, cell surface, and intracellular proteins. This facilitates ASO distribution to peripheral tissues and also promotes cellular uptake after injection into animals. Previous work identified that Stabilin receptors specifically internalize PS-ASOs in the sinusoidal endothelial cells of the liver and the spleen. By modulating expression of specific proteins involved in the trafficking and maturation of the endolysosomal compartments, we show that Rab5C and EEA1 in the early endosomal pathway, and Rab7A and lysobisphosphatidic acid in the late endosomal pathway, are important for trafficking of PS-ASOs and facilitate their escape from endolysosomal compartments after Stabilin-mediated internalization. In conclusion, this work identifies key rate-limiting proteins in the pathway for PS-ASO translocation and escape from the endosome.
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References
1.
MillerCM and HarrisEN. (2016). Antisense oligonucleotides: treatment strategies and cellular internalization. RNA Dis, 3:1393.
2.
CrookeST, WangS, VickersTA, ShenW and LiangXH. (2017). Cellular uptake and trafficking of antisense oligonucleotides. Nat Biotechnol, 35:230–237.
3.
AltmannK-H, DeanNM, FabbroD, FreierSM, GeigerT, HaneraR, HiiskenDA, MartinaP, MoniaBPB, MiillerM, et al. (1996). Second generation of antisense oligonucleotides: from nuclease resistance to biological efficacy in animals. CHIMIA Int J Chem, 50:168–176.
4.
KoleR, KrainerAR and AltmanS. (2012). RNA therapeutics: beyond RNA interference and antisense oligonucleotides. Nat Rev Drug Discov, 11:125–140.
5.
WangS, SunH, TanowitzM, LiangXH and CrookeST. (2016). Annexin A2 facilitates endocytic trafficking of antisense oligonucleotides. Nucleic Acids Res, 44:7314–7330.
6.
MillerCM, DonnerAJ, BlankEE, EggerAW, KellarBM, OstergaardME, SethPP and HarrisEN. (2016). Stabilin-1 and Stabilin-2 are specific receptors for the cellular internalization of phosphorothioate-modified antisense oligonucleotides (ASOs) in the liver. Nucleic Acids Res, 44:2782–2794.
7.
HarrisEN, WeigelJA and WeigelPH. (2008). The human hyaluronan receptor for endocytosis (HARE/Stabilin-2) is a systemic clearance receptor for heparin. J Biol Chem, 283:17341–17350.
8.
PandeyMS, BaggenstossBA, WashburnJ, HarrisEN and WeigelPH. (2013). The hyaluronan receptor for endocytosis (HARE) activates NF-kappaB-mediated gene expression in response to 40-400-kDa, but not smaller or larger, hyaluronans. J Biol Chem, 288:14068–14079.
9.
HarrisEN, KyossevaSV, WeigelJA and WeigelPH. (2007). Expression, processing, and glycosaminoglycan binding activity of the recombinant human 315-kDa hyaluronic acid receptor for endocytosis (HARE). J Biol Chem, 282:2785–2797.
10.
HarrisEN, WeigelJA and WeigelPH. (2004). Endocytic function, glycosaminoglycan specificity, and antibody sensitivity of the recombinant human 190-kDa hyaluronan receptor for endocytosis (HARE). J Biol Chem, 279:36201–36209.
11.
ZhouB, WeigelJA, FaussL and WeigelPH. (2000). Identification of the hyaluronan receptor for endocytosis (HARE). J Biol Chem, 275:37733–37741.
12.
MuFT, CallaghanJM, Steele-MortimerO, StenmarkH, PartonRG, CampbellPL, McCluskeyJ, YeoJP, TockEP and TohBH. (1995). EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine “fingers” and contains a calmodulin-binding IQ motif. J Biol Chem, 270:13503–13511.
13.
SimonsenA, LippeR, ChristoforidisS, GaullierJM, BrechA, CallaghanJ, TohBH, MurphyC, ZerialM and StenmarkH. (1998). EEA1 links PI(3)K function to Rab5 regulation of endosome fusion. Nature, 394:494–498.
14.
ChenPI, KongC, SuX, and StahlPD. (2009). Rab5 isoforms differentially regulate the trafficking and degradation of epidermal growth factor receptors. J Biol Chem, 284:30328–30338.
15.
JovicM, SharmaM, RahajengJ and CaplanS. (2010). The early endosome: a busy sorting station for proteins at the crossroads. Histol Histopathol, 25:99–112.
16.
VonderheitA and Helenius.A (2005). Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes. PLoS Biol, 3:e233.
17.
BucciC, ThomsenP, NicozianiP, McCarthyJ, and van DeursB. (2000). Rab7: a key to lysosome biogenesis. Mol Biol Cell, 11:467–480.
18.
VitelliR, SantilloM, LatteroD, ChiarielloM, BifulcoM, BruniCB and BucciC. (1997). Role of the small GTPase Rab7 in the late endocytic pathway. J Biol Chem, 272:4391–4397.
19.
LebrandC, CortiM, GoodsonH, CossonP, CavalliV, MayranN, FaureJ and GruenbergJ. (2002). Late endosome motility depends on lipids via the small GTPase Rab7. EMBO J, 21:1289–1300.
WangS, SunH, TanowitzM, LiangXH and CrookeST. (2017). Intra-endosomal trafficking mediated by lysobisphosphatidic acid contributes to intracellular release of phosphorothioate-modified antisense oligonucleotides. Nucleic Acids Res, 45:5309–5322.
22.
RajaRH, LeBoeufRD, StoneGW and WeigelPH. (1984). Preparation of alkylamine and 125I-radiolabeled derivatives of hyaluronic acid uniquely modified at the reducing end. Anal Biochem, 139:168–177.
23.
BitterT and MuirHM. (1962). A modified uronic acid carbazole reaction. Anal Biochem, 4:330–334.
24.
WheelerTM, LegerAJ, PandeySK, MacLeodAR, NakamoriM, ChengSH, WentworthBM, BennettCF and ThorntonCA. (2012). Targeting nuclear RNA for in vivo correction of myotonic dystrophy. Nature, 488:111–115.
25.
GutschnerT, HammerleM, EissmannM, HsuJ, KimY, HungG, RevenkoA, ArunG, StentrupM, et al. (2013). The noncoding RNA MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells. Cancer Res, 73:1180–1189.
26.
LaweDC, ChawlaA, MerithewE, DumasJ, CarringtonW, FogartyK, LifshitzL, TuftR, LambrightD and CorveraS. (2002). Sequential roles for phosphatidylinositol 3-phosphate and Rab5 in tethering and fusion of early endosomes via their interaction with EEA1. J Biol Chem, 277:8611–8617.
27.
WilsonJM, de HoopM, ZorziN, TohBH, DottiCG and PartonRG. (2000). EEA1, a tethering protein of the early sorting endosome, shows a polarized distribution in hippocampal neurons, epithelial cells, and fibroblasts. Mol Biol Cell, 11:2657–2671.
28.
JohanssonM, LehtoM, TanhuanpaaK, CoverTL and OlkkonenVM. (2005). The oxysterol-binding protein homologue ORP1 L interacts with Rab7 and alters functional properties of late endocytic compartments. Mol Biol Cell, 16:5480–5492.
29.
ChenPI, SchauerK, KongC, HardingAR, GoudB and StahlPD. (2014). Rab5 isoforms orchestrate a “division of labor” in the endocytic network; Rab5C modulates Rac-mediated cell motility. PLoS One, 9:e90384.
30.
GuerraF and BucciC. (2016). Multiple Roles of the Small GTPase Rab7. Cells, 5:pii: E34.
31.
BessonN, Hullin-MatsudaF, MakinoA, MurateM, LagardeM, PageauxJF, KobayashiT and Delton-VandenbrouckeI. (2006). Selective incorporation of docosahexaenoic acid into lysobisphosphatidic acid in cultured THP-1 macrophages. Lipids, 41:189–196.
32.
PoteryaevD, DattaS, AckemaK, ZerialM and SpangA. (2010). Identification of the switch in early-to-late endosome transition. Cell, 141:497–508.
33.
SonnichsenB, De RenzisS, NielsenE, RietdorfJ and ZerialM. (2000). Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11. J Cell Biol, 149:901–914.
34.
ZerialM and McBrideH. (2001). Rab proteins as membrane organizers. Nat Rev Mol Cell Biol, 2:107–117.
35.
MishraA, EathirajS, CorveraS and LambrightDG. (2010). Structural basis for Rab GTPase recognition and endosome tethering by the C2H2 zinc finger of Early Endosomal Autoantigen 1 (EEA1). Proc Natl Acad Sci U S A, 107:10866–10871.
36.
RothCM. (2005). Molecular and cellular barriers limiting the effectiveness of antisense oligonucleotides. Biophys J, 89:2286–2295.
37.
JensenKD, KopeckovaP and KopecekJ. (2002). Antisense oligonucleotides delivered to the lysosome escape and actively inhibit the hepatitis B virus. Bioconjug Chem, 13:975–984.
38.
KobayashiT, StangE, FangKS, de MoerlooseP, PartonRG and GruenbergJ. (1998). A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature, 392:193–197.
39.
KobayashiT, BeuchatMH, LindsayM, FriasS, PalmiterRD, SakurabaH, PartonRG and GruenbergJ. (1999). Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nat Cell Biol, 1:113–118.
40.
van der GootFG and GruenbergJ. (2006). Intra-endosomal membrane traffic. Trends Cell Biol, 16:514–521.
41.
RussellMR, NickersonDP and OdorizziG. (2006). Molecular mechanisms of late endosome morphology, identity and sorting. Curr Opin Cell Biol, 18:422–428.
42.
TrioulierY, TorchS, BlotB, CristinaN, Chatellard-CausseC, VernaJM and SadoulR. (2004). Alix, a protein regulating endosomal trafficking, is involved in neuronal death. J Biol Chem, 279:2046–2052.
43.
PempeEH, XuY, GopalakrishnanS, LiuJ and HarrisEN. (2012). Probing structural selectivity of synthetic heparin binding to stabilin protein receptors. J Biol Chem, 287:20774–20783.
44.
KyossevaSV, HarrisEN and WeigelPH. (2008). The hyaluronan receptor for endocytosis mediates hyaluronan-dependent signal transduction via extracellular signal-regulated kinases. J Biol Chem, 283:15047–15055.
45.
GearyRS, NorrisD, YuR, and BennettCF. (2015). Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Adv Drug Deliv Rev, 87:46–51.
46.
HareAK and HarrisEN. (2015). Tissue-specific splice variants of HARE/Stabilin-2 are expressed in bone marrow, lymph node, and spleen. Biochem Biophys Res Commun, 456:257–261.
47.
ParkSY, YunY, LimJS, KimMJ, KimSY, KimJE and KimIS. (2016). Stabilin-2 modulates the efficiency of myoblast fusion during myogenic differentiation and muscle regeneration. Nat Commun, 7:10871.
48.
KimGW, ParkSY and KimIS. (2016). Novel function of stabilin-2 in myoblast fusion: the recognition of extracellular phosphatidylserine as a “fuse-me” signal. BMB Rep, 49:303–304.
49.
HansenB, LongatiP, ElvevoldK, NedredalGI, SchledzewskiK, OlsenR, FalkowskiM, KzhyshkowskaJ, CarlssonF, et al. (2005). Stabilin-1 and stabilin-2 are both directed into the early endocytic pathway in hepatic sinusoidal endothelium via interactions with clathrin/AP-2, independent of ligand binding. Exp Cell Res, 303:160–173.
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