Letters pubs.acs.org/acschemicalbiology
siRNA Conjugates Carrying Sequentially Assembled Trivalent NAcetylgalactosamine Linked Through Nucleosides Elicit Robust Gene Silencing In Vivo in Hepatocytes Shigeo Matsuda, Kristofer Keiser, Jayaprakash K. Nair, Klaus Charisse, Rajar M. Manoharan, Philip Kretschmer, Chang G. Peng, Alexander V. Kel’in, Pachamuthu Kandasamy, Jennifer L.S. Willoughby, Abigail Liebow, William Querbes, Kristina Yucius, Tuyen Nguyen, Stuart Milstein, Martin A. Maier, Kallanthottathil G. Rajeev,* and Muthiah Manoharan* Alnylam Pharmaceuticals, 300 Third Street, Cambridge, Massachusetts 02142, United States S Supporting Information *
ABSTRACT: Asialoglycoprotein receptor (ASGPR) mediated delivery of triantennary N-acetylgalactosamine (GalNAc) conjugated short interfering RNAs (siRNAs) to hepatocytes is a promising paradigm for RNAi therapeutics. Robust and durable gene silencing upon subcutaneous administration at therapeutically acceptable dose levels resulted in the advancement of GalNAc-conjugated oligonucleotide-based drugs into preclinical and clinical developments. To systematically evaluate the effect of display and positioning of the GalNAc moiety within the siRNA duplex on ASGPR binding and RNAi activity, nucleotides carrying monovalent GalNAc were designed. Evaluation of clustered and dispersed incorporation of GalNAc units to the sense (S) strand indicated that sugar proximity is critical for ASGPR recognition, and location of the clustered ligand impacts the intrinsic potency of the siRNA. An array of nucleosidic GalNAc monomers resembling a trivalent ligand at or near the 3′ end of the S strand retained in vitro and in vivo siRNA activity, similar to the parent conjugate design. This work demonstrates the utility of simple, nucleotide-based, cost-effective siRNA−GalNAc conjugation strategies.
R
neutrals (siRNNs)17 in mice, and anti-microRNA therapeutics18 in humans, confirming the value of the parent trivalent design. The objective of the present work was to further characterize the GalNAc−ASGPR system and to refine the ligand design using nucleosidic linkers within the siRNA duplex to explore the effects of ligand positioning, proximity, and steric factors on ligand−receptor interaction and silencing activity. Here, we evaluated various sites of attachment including the 2′- and 3′positions of the ribosugar and a particular site in the nucleobase (Figure 1, structures II−V). Furthermore, successive placement of a trinucleotide motif, where each nucleotide contains a monovalent GalNAc, results in a trivalent GalNAc cluster II−V that resembles the well optimized triantennary GalNAc ligand design I. The postsynthetic “click” conjugation strategy reported earlier from our laboratory,19 using the copper-assisted azide−alkyne cycloaddition (CuAAC) to link oligonucleotides containing 2′-O-propargylated nucleotides with a corresponding azido-GalNAc derivative, was employed to introduce
NA interference (RNAi) is a naturally occurring biological pathway of gene silencing. In this pathway, long doublestranded RNAs are processed to short interfering RNAs (siRNAs) that suppress expression of homologous genes via mRNA cleavage or translation repression.1−3 The mechanism can be harnessed for therapeutic applications using synthetic siRNAs.4−6 The success of RNAi-based therapeutics relies on the ability to efficiently deliver siRNAs to target tissues of interest and then to the cytoplasm where gene silencing occurs.7,8 We have recently demonstrated that a synthetic triantennary N-acetylgalactosamine (GalNAc) ligand conjugated to siRNA (Figure 1, structure I) binds to the asialoglycoprotein receptor9−11 (ASGPR) present on the surface of hepatocytes and facilitates tissue-specific gene silencing.12 Recent reviews have summarized the evaluation of several oligonucleotidecarbohydrate conjugates for targeted delivery.13,14 Successful translation of this delivery approach into human clinical trials has laid the foundation for GalNAc-conjugated siRNAs (siRNA−GalNAc) as a new class of therapeutics for the treatment of diseases involving liver-expressed genes such as transthyretin (TTR) amyloidosis.15 The triantennary GalNAc ligand was subsequently used for hepatocyte-specific delivery of antisense oligonucleotides16 and short interfering ribonucleic © XXXX American Chemical Society
Received: December 17, 2014 Accepted: February 18, 2015
A
DOI: 10.1021/cb501028c ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
Figure 1. Representative nucleotidic siRNA−GalNAc conjugate designs used in this study: non-nucleosidic parent triantennary GalNAc (I); three successive nucleotidic monomers with GalNAc conjugated to 2′- (II, III) and 3′- (IV) of the ribosugar and to nucleobase (V), separated by phosphodiester or phosphorothioate linkages.
To illustrate possible GalNAc conjugation through the nucleobase as an alternative mode of attachment, the N − 1 position of pseudouridine,21 an isomer of uridine, was explored. The GalNAc ligand was linked to the N − 1 position of pseudouridine through an aminolinker22,23 to yield the monovalent GalNAc-containing pseudouridine monomers 21 and 23 (Scheme 2). Three contiguous pseudouridine units with monovalent GalNAc moiety (Figure 1, V) were introduced at the 3′ terminus of the S strand, followed by annealing with the AS strand to yield the conjugate 56. Insertion of the phosphoramidite 21 at alternating positions in the S strand gave the siRNA−GalNAc conjugate 58 (Table 1). The corresponding non-GalNAc containing pseudouridine modified siRNA duplexes 57 and 59 (Table 1) were also synthesized (Scheme S2, in the SI) and evaluated as controls for both ASGPR binding and siRNA activity in vitro. The ASGPR binding affinities of the conjugated GalNAc moieties to siRNA were determined using a fluorescence based assay24 (Table 1). Incorporation of three contiguous GalNAc units on the S strand of the siRNAs 42−48 resulted in high ASGPR binding with affinity similar to that of the parent design 41, in which the siRNA was conjugated to the original triantennary ligand I (Figure 1). A 4- to 6-fold decrease in binding affinity was observed when the distance between the GalNAc moieties was increased through dispersed placement across the S strand (49−51, Table 1). We then evaluated gene silencing activity of these GalNAc conjugated siRNAs either in the presence or in the absence of a transfection agent (free uptake) in the appropriate cell type expressing the receptor. While the silencing activity with a transfection agent measures intrinsic RNAi activity of the duplex, the free uptake mediated gene silencing assesses the
clustered and dispersed monovalent GalNAc to the 2′ position of the ribosugar moiety (Scheme S1 in the SI). Successive incorporation of three 2′-O-propargylated nucleosides into the sense (S) strand, followed by the CuAAC reaction with an azido-GalNAc derivative and subsequent annealing with the corresponding antisense (AS) strand afforded trivalent GalNAc conjugated siRNAs 42−48 (Table 1). Sense strands were synthesized with GalNAc moieties placed at adjacent trinucleotides to evaluate the impact of positioning of the GalNAc trivalent cluster. The dispersed GalNAc conjugate designs 49−51 in which the monovalent GalNAc moieties were separated by two or more nucleotides were also synthesized. The sequential trinucleotide walk (42−48) and dispersed GalNAc (49−51) placement across the S strand allowed evaluation of the proximity effect and spacing between the GalNAc moieties on ASGPR binding and internalization. The CuAAC postsynthetic GalNAc conjugation approach was very useful for rapid conjugate synthesis and initial screening; however, it was important to evaluate alternate synthetic strategies utilizing established phosphoramidite chemistry to avoid possible copper ion contamination19 and potential difficulties associated with the scale-up of “click” reactions. The carboxylic acid 3 containing a monovalent GalNAc moiety was covalently attached to the 2′ or to the 3′ position of the ribose of 5-methyluridine through a 2′/3′-Ohexylamino linker20 and then converted to the corresponding phosphoramidites and solid supports for incorporation into oligonucleotides (Scheme 1) to obtain conjugate designs III and IV (Figure 1). Phosphorothioate (PS) linkages were introduced between GalNAc-bearing nucleotides at the 3′ end of the S strands of conjugates 54 and 55 to enhance stability against nucleases. B
DOI: 10.1021/cb501028c ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology Table 1. siRNA-GalNAc Conjugates Used in This Study
a
siRNA targeting mRNA encoding mouse transthyretin (mTTR).12 bS and AS indicate sense and antisense strands. cItalicized upper case and normal lower case letters indicate 2′-deoxy-2′-fluoro (2′-F) and 2′-O-methyl (2′-OMe) sugar modifications, respectively; • indicates phosphorothioate (PS) linkage; letters in parentheses represent structures shown below, N is one of the four bases with identity given in the sequence.
d Binding affinity to ASGPR (on mouse primary hepatocytes, fluorescent based assay).24 eHalf maximal inhibitory concentration (IC50) for in vitro gene silencing under transfection (Lipofectamine RNAiMAX) or free uptake conditions in primary mouse hepatocytes. fNot determined (lack of cell uptake due to poor recognition and binding by the receptor). gDue to the absence of ligand to bind to the ASGPR.
(IC50 = 8.0 pM) under similar in vitro transfection conditions. Importantly, siRNAs 42−44 and 48 (IC50 = 1.093, 0.725, 0.785, and 1.208 nM respectively) with clustered ligands near the 3′ and 5′ ends of the S strand had in vitro potency similar to or slightly better than that of the parent design 41 (IC50 = 1.542 nM) under receptor-mediated free uptake conditions, whereas the dispersed design 49 (IC50 = 14.104 nM) showed a significant loss in potency (Table 1, IC50, free uptake). The IC50 of 50 was above the concentration level tested under the same conditions. These data indicate that efficient receptor binding
actual receptor-mediated intracellular transport enabled by the GalNAc ligand. Placement of the GalNAc units at certain positions on the S strand had a negative impact on the intrinsic potency of the siRNAs as gene silencing agents, as reflected in their in vitro half maximal inhibitory concentrations (IC50), measured after transfection with lipid-based transfection agents (Table 1, IC50, transfection). siRNAs 45 and 46 (IC50 = 53.0 and 96.0 pM respectively) with three GalNAc units in the center of the S strand were significantly less potent than the parent siRNA 41 C
DOI: 10.1021/cb501028c ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology Scheme 1. Synthesis of 2′- and 3′-Ribonucleoside-GalNAc Monomersa
a
(i) Bz2O, DMAP/Py, 75%. (ii) RuCl2, NaIO4, H2O/MeCN/CH2Cl2, 86%. (iii) EDC, DIEA/CH2Cl2, 96%. (iv) Et3N/CH2Cl2 (7, 83%; 11, 66%). (v) 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite, 4,5-dicyanoimidazole/CH2Cl2 (8, 95%; 12, 83%). (vi) a. succinic anhydride/DMAP/ CH2Cl2. b. lcaa CPG, DIEA, HBTU/CH3CN (loading, 9, 73 μmol/g; 13, 56 μmol/g).
efficacy relative to the parent conjugate 41 in wild-type C57BL/ 6 mice. Levels of circulating mTTR protein were analyzed 72 h after single dose subcutaneous administration of 5 mg/kg (Figure 2). The results generally correlated well with the in vitro data. The conjugates that had in vitro potencies similar to the parent, such as 42−44, showed similar in vivo activities, whereas compounds that did not bind tightly to the receptor or did not have similar intrinsic potency exhibited reduced in vivo activities. The exception was conjugate 48 with three GalNAc units at the 5′ end of the S strand, which had in vitro activity similar to the parent under free uptake conditions but showed only moderate activity in vivo. This loss of in vivo activity may be due to reduced metabolic stability due to the absence of 3′GalNAc, which hinders 3′-exonulcease attack on the S strand. Subcutaneous administration of 54, 55, and 56 in mice resulted in the robust reduction of circulating mTTR protein levels similar to that observed with the parent trivalent conjugate 53 (Figure 3a,b). These findings further confirmed that three monovalent GalNAc units placed in close proximity to each other at the 3′ end of the S strand results in highaffinity binding of the siRNA conjugate to ASGPR. The efficient ASGPR-mediated delivery thus translates in robust RNAi-mediated silencing in vivo irrespective of the type of GalNAc attachment and nature of the linker.
and hepatocellular uptake require multiple GalNAc units in close proximity to each other. The binding affinities and in vitro potencies under transfection and free uptake conditions of conjugates 54 and 55 with 3′-5′ and 2′-5′ phosphate linkages between the GalNAcnucleosides, respectively, were comparable to the parent triantennary design 53 (Table 1, IC50). Conjugate 56 that showed a slight loss in binding affinity to ASGPR had potencies (both in the presence and in the absence of transfection agent) comparable to the parent conjugate. As observed with the other designs, dispersed placement of the pseudouridine-GalNAc units in conjugate 58 again resulted in significant loss in binding affinity (Table 1) and in vitro silencing activity, compared to the control 53 (free uptake IC50 = 14.456 and 1.966 nM, respectively). The corresponding control siRNAs 57 and 59 that contained the N − 1 modified pseudouridine moiety (Table 1, Scheme S2 in the SI) without GalNAc at the identical position in the S strand showed no binding to the ASGPR and no in vitro activity under free uptake conditions. However, in the presence of a transfection reagent, these siRNAs were as potent as the parent siRNA-GalNAc, confirming the importance of the ligand−receptor interaction for productive hepatocyte uptake. We next evaluated siRNA−GalNAc conjugates 42−52, designed to target mouse transthyretin mRNA (mTTR),12 for D
DOI: 10.1021/cb501028c ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology Scheme 2. Synthesis of Nucleobase-GalNAc Monomersa
a
(i) Methyl acrylate, triethylammonium bicarbonate 1 M, pH 8.5/EtOH, 98%. (ii) DMTr-Cl, DMAP/Py, 88%. (iii) ethylenediamine, 80%. (iv) HBTU, DIEA/DMF, 61%. (v) TBDMS-Cl, imidazole/Py, 20, 23%; 22, 32%. (vi) 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite, 4,5dicyanoimidazole/CH2Cl2, 83%. (vii) a. succinic anhydride, DMAP/CH2Cl2. b. lcaa CPG, HBTU, DIEA/DMF, loading: 47 μmol/g.
Figure 2. Gene silencing of conjugates 41−52 in wild-type C57BL/6 mice (n = 3), Results are presented as % mTTR protein remaining in circulation after single dose SC administration at 5 mg/kg dose, relative to PBS-treated mice. Blood samples were drawn 72 h postdose for mTTR protein evaluation by ELISA. Serum TTR protein levels from individual animals were normalized to a PBS treated control group and are expressed as the mean ± standard error.
exhibited in vitro and in vivo potencies similar to the compounds containing the parent triantennary ligand as long as the ligand placement did not interfere with the RNA induced silencing complex (RISC) machinery. Such simple trivalent ligand designs will support the advancement of robust ASGPRmediated delivery of RNAi therapeutics to hepatocytes, a platform with tremendous potential for the treatment of liverassociated diseases. Finally, the results reported herein reaffirm the importance of proximity and clustering of carbohydrate moieties for recognition by and binding to the receptor and corroborate with our own recent results using an alternative
In summary, nucleoside−GalNAc monomers were designed, synthesized, and incorporated site-specifically into siRNAs via postsynthetic conjugation using “click” chemistry or during solid-phase synthesis using phosphoramidite chemistry. The receptor binding affinity, and in vitro and in vivo gene silencing activities of the novel siRNA−GalNAc conjugates, were compared with those of a previously reported triantennary conjugate.12 The results indicate that three clustered monomeric GalNAc units attached to siRNAs, either through the ribosugar or through the nucleobase, in a fashion that resembles a trivalent ligand design resulted in siRNAs with receptor binding affinities similar to the parent. These novel conjugates E
DOI: 10.1021/cb501028c ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
Figure 3. Comparison of gene silencing by conjugates 54, 55, and 56 with 53 in wild-type C57BL/6 mice. Results are presented as % mTTR protein remaining in circulation after single dose subcutaneous administration. (a) 53, 54, and 55 at 5 mg/kg dose (n = 5) and (b) 53 and 56 at 15 mg/kg dose (n = 3). Blood samples were drawn 48 h postdose for mTTR protein evaluation by ELISA. Serum TTR protein levels from individual animals were normalized to the PBS treated control group and are expressed as the mean ± standard error. RNA using simple bioconjugates. Expert Opin. Drug Delivery 11, 791− 822. (8) Soni, R. S., Gohil, A., and Avachat, A. M. (2013) Delivering small interfering RNA for novel therapeutics. Int. Pharm. Sci. 3, 11−19 19 pp.. (9) Meier, M., Bider, M. D., Malashkevich, V. N., Spiess, M., and Burkhard, P. (2000) Crystal Structure of the Carbohydrate Recognition Domain of the H1 Subunit of the Asialoglycoprotein Receptor. J. Mol. Biol. 300, 857−865. (10) Grewal, P. K. (2010) The Ashwell-Morell receptor. Methods Enzymol. 479, 223−241. (11) Spiess, M. (1990) The asialoglycoprotein receptor: a model for endocytic transport receptors. Biochemistry 29, 10009−10018. (12) Nair, J. K., Willoughby, J. L. S., Chan, A., Charisse, K., Alam, M. R., Wang, Q., Hoekstra, M., Kandasamy, P., Kel’in, A. V., Milstein, S., Taneja, N., O’Shea, J., Shaikh, S., Zhang, L., van der Sluis, R. J., Jung, M. E., Akinc, A., Hutabarat, R., Kuchimanchi, S., Fitzgerald, K., Zimmermann, T., van Berkel, T. J. C., Maier, M. A., Rajeev, K. G., and Manoharan, M. (2014) Multivalent N-Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAiMediated Gene Silencing. J. Am. Chem. Soc. 136, 16958−16961. (13) Lonnberg, H. (2009) Solid-Phase Synthesis of Oligonucleotide Conjugates Useful for Delivery and Targeting of Potential Nucleic Acid Therapeutics. Bioconjugate Chem. 20, 1065−1094. (14) Spinelli, N., Defrancq, E., and Morvan, F. (2013) Glycoclusters on oligonucleotide and PNA scaffolds: synthesis and applications. Chem. Soc. Rev. 42, 4557−4573. (15) http://www.alnylam.com/capella/presentations/positive-initialrevusiran-phase-2-data/. (16) Prakash, T. P., Graham, M. J., Yu, J., Carty, R., Low, A., Chappell, A., Schmidt, K., Zhao, C., Aghajan, M., Murray, H. F., Riney, S., Booten, S. L., Murray, S. F., Gaus, H., Crosby, J., Lima, W. F., Guo, S., Monia, B. P., Swayze, E. E., and Seth, P. P. (2014) Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice. Nucleic Acids Res. 42, 8796−8807. (17) Meade, B. R., Gogoi, K., Hamil, A. S., Palm-Apergi, C., Berg, A. v. d., Hagopian, J. C., Springer, A. D., Eguchi, A., Kacsinta, A. D., Dowdy, C. F., Presente, A., Lonn, P., Kaulich, M., Yoshioka, N., Gros, E., Cui, X.-S., and Dowdy, S. F. (2014) Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications. Nat. Biotechnol. 32, 1256−1261. (18) http://ir.regulusrx.com/releasedetail.cfm?ReleaseID=877462. (19) Yamada, T., Peng, C. G., Matsuda, S., Addepalli, H., Jayaprakash, K. N., Alam, M. R., Mills, K., Maier, M. A., Charisse, K., Sekine, M., Manoharan, M., and Rajeev, K. G. (2011) Versatile Site-Specific
sequential design, which utilizes a non-nucleosidic linker-based trivalent siRNA−GalNAc conjugate.25
■
ASSOCIATED CONTENT
S Supporting Information *
Experimental, compound characterization, and assays. This material is available free of charge via the Internet at http:// pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Author Contributions
The manuscript was written through contributions of all authors. Notes
The authors declare the following competing financial interest(s): All authors except K.K., R.J.M. and P.K. are current employees of Alnylam Pharmaceuticals.
■
REFERENCES
(1) Bumcrot, D., Manoharan, M., Koteliansky, V., and Sah, D. W. Y. (2006) RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat. Chem. Biol. 2, 711−719. (2) Deleavey, G. F., and Damha, M. J. (2012) Designing Chemically Modified Oligonucleotides for Targeted Gene Silencing. Chem. Biol. 19, 937−954. (3) Novina, C. D., and Sharp, P. A. (2004) The RNAi revolution. Nature 430, 161−164. (4) Joshi, B. H., and Pachchigar, K. P. (2014) siRNA: novel therapeutics from functional genomics. Biotechnol. Genet. Eng. Rev. 30, 1−30. (5) Karunapriyachitra, Ramadevibhimavarapu, Srinath, N., Sabasu, N., Mahesh, B., and Balavamsikrishna (2013) siRNA - an excellent technique for downregulation of gene expression. Int. J. Invent. Pharm. Sci. 1, 6−14. (6) Li, T., Wu, M., Zhu, Y. Y., Chen, J., and Chen, L. (2014) Development of RNA Interference-Based Therapeutics and Application of Multi-Target Small Interfering RNAs. Nucleic Acid Ther. 24, 302−312. (7) Nielsen, C., Kjems, J., Sorensen, K. R., Engelholm, L. H., and Behrendt, N. (2014) Advances in targeted delivery of small interfering F
DOI: 10.1021/cb501028c ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology Conjugation of Small Molecules to siRNA Using Click Chemistry. J. Org. Chem. 76, 1198−1211. (20) Manoharan, M., Tivel, K. L., Andrade, L. K., and Cook, P. D. (1995) 2′-O- and 3′-O-pyrimidine amino-tether-containing oligodeoxyribonucleotides: synthesis and conjugation chemistry. Tetrahedron Lett. 36, 3647−3650. (21) Carlile, T. M., Rojas-Duran, M. F., Zinshteyn, B., Shin, H., Bartoli, K. M., and Gilbert, W. V. (2014) Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature 515, 143−146. (22) Manoharan, M.; Rajeev, K. G.; Kelin, A. V.; Takkellapati, S. R.; Matsuda, S. Nucleotides modified at the 2′-position of the sugar moiety for use in nucleic acids for therapeutic use. PCT Int. Appl. WO 2010101951, 2010. (23) Ramzaeva, N., Rosemeyer, H., Leonard, P., Muhlegger, K., Bergmann, F., Von der Eltz, H., and Seela, F. (2000) Oligonucleotides functionalized by fluorescein and rhodamine dyes: Michael addition of methyl acrylate to 2′-deoxypseudouridine. Helv. Chim. Acta 83, 1108− 1126. (24) Severgnini, M., Sherman, J., Sehgal, A., Jayaprakash, N. K., Aubin, J., Wang, G., Zhang, L., Peng, C. G., Yucius, K., Butler, J., and Fitzgerald, K. (2012) A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development. Cytotechnology 64, 187−195. (25) Rajeev, K. G., Nair, J. K., Jayaraman, M., Charisse, K., Taneja, N., O’Shea, J., Willoughby, J. L. S., Yucius, K., Nguyen, T., ShulgaMorskaya, S., Milstein, S., Liebow, A., Querbes, W., Borodovsky, A., Fitzgerald, K., Maier, M. A., and Manoharan, M. (2015) HepatocyteSpecific Delivery of siRNA Conjugated to Novel Non-Nucleosidic Trivalent N-Acetylgalactosamine Elicits Robust Gene Silencing In Vivo. ChemBioChem, DOI: 10.1002/cbic.201500023.
G
DOI: 10.1021/cb501028c ACS Chem. Biol. XXXX, XXX, XXX−XXX
