Intrinsically cell-penetrating multivalent andmultitargeting ligands for myotonic dystrophy type 1JuYeon Leea,1, Yugang Baib,c,d,1, Ullas V. Chembazhie, Shaohong Pengf, Kevin Yume, Long M. Luua, Lauren D. Haglera,Julio F. Serranoa, H. Y. Edwin Chanf, Auinash Kalsotrae,2, and Steven C. Zimmermana,2

aDepartment of Chemistry, University of Illinois at Urbana–Champaign, Urbana, IL 61801; bInstitute of Chemical Biology and Nanomedicine, HunanUniversity, Changsha, 410082 Hunan, China; cState Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, 410082 Hunan,China; dDepartment of Chemistry, Hunan University, Changsha, 410082 Hunan, China; eDepartment of Biochemistry, University of Illinois at Urbana–Champaign,Urbana, IL 61801; and fLaboratory of Drosophila Research, School of Life Sciences, The Chinese University of Hong Kong, Shatin N.T., Hong Kong SpecialAdministrative Region, People’s Republic of China

Edited by Jennifer M. Heemstra, Emory University, and accepted by Editorial Board Member Thomas E. Mallouk March 18, 2019 (received for reviewDecember 6, 2018)

Developing highly active, multivalent ligands as therapeutic agents ischallenging because of delivery issues, limited cell permeability, andtoxicity. Here, we report intrinsically cell-penetrating multivalentligands that target the trinucleotide repeat DNA and RNA inmyotonic dystrophy type 1 (DM1), interrupting the disease progres-sion in two ways. The oligomeric ligands are designed based on therepetitive structure of the target with recognition moieties alternat-ing with bisamidinium groove binders to provide an amphiphilic andpolycationic structure, mimicking cell-penetrating peptides. Multiplebiological studies suggested the success of our multivalency strategy.The designed oligomers maintained cell permeability and exhibitedno apparent toxicity both in cells and in mice at working concentra-tions. Furthermore, the oligomers showed important activities inDM1 cells and in a DM1 liver mouse model, reducing or eliminatingprominent DM1 features. Phenotypic recovery of the climbing defectin adult DM1 Drosophila was also observed. This design strategyshould be applicable to other repeat expansion diseases and moregenerally to DNA/RNA-targeted therapeutics.

multivalent ligand | DNA/RNA-targeting therapeutics |cell-penetrating peptide mimic | myotonic dystrophy type 1 |trinucleotide repeat expansion diseases

Multivalency is an effective strategy for the development ofpotent and selective ligands, especially at cell surfaces. The

thermodynamic and kinetic advantage of multiple binding moi-eties interacting with many receptor sites is attributed to severalpossible factors, including steric stabilization, the chelation ef-fect, and concentration effects (1–3). For the maximum benefitof multivalency to accrue, there must be a suitable spatial ar-rangement of the ligands and a strain-free, ligand-receptor “fit”upon complexation. Dimeric ligands can be viewed as simplifiedversions of multivalent ligands, and there is ample evidence thatoptimization of the linkage between two binding motifs is nec-essary to obtain significant affinity improvement (4–6). However,rather than using an optimized dimeric linkage, multivalent ligandsare often prepared by covalent attachment of ligands to readilyavailable extrinsic scaffolds, such as a PNA or dendrimer. In thesecases, the ease of synthesis often comes with loss of tunability andspatial optimization (7–9). A separate consideration comes intoplay when the target is intracellular. Namely, the increase in masscan reduce cell permeability, although the use of a cell-penetratingpeptide (CPP) as the scaffold can help (9).Trinucleotide repeat expansion diseases (TREDs) are outstanding

targets for exploring intracellular multivalent approaches, becausethe pathogenic targets involve regular and repeating sequencesof DNA and RNA. For example, myotonic dystrophy type 1(DM1), an incurable TRED, originates in expanded CTG re-peats [d(CTG)exp] in the 3′-UTR of the DMPK gene on chro-mosome 19 (Fig. 1). An RNA gain-of-function model for DM1involves r(CUG)exp transcripts altering the level of splicing reg-ulators, such as muscleblind-like 1 (MBNL1) and CUG binding

protein 1 (CUGBP1), leading to misregulation of pre-mRNAsplicing (10–13).Recent reports shed light on additional pathobiology of DM1,

such as microRNA dysregulation (14) and repeat-associatednon-ATG (RAN) translation (15, 16). Nonetheless, the seques-tration of MBNL1 by r(CUG)exp, and their coaggregation intodistinctive nuclear foci is considered as a hallmark of DM1 (17).Importantly, MBNL1 knockout mouse model studies havereported that >80% of DM1 missplicing defects and relatedsymptoms are directly associated with the loss of MBNL1 byr(CUG)exp (18, 19). Thus, therapeutic efforts focus on inhibitingthe transcription of d(CTG)exp to reduce r(CUG)exp levels (20–22)or on inhibiting the interaction of r(CUG)exp with MBNL1 (23–27). Here, we report an oligomeric ligand that targets bothd(CTG)exp and r(CUG)exp in a multivalent fashion and containsa structure designed to mimic CPPs for inherent cell perme-ability (Fig. 1). The mixture of oligomers reduces the diseasefoci and toxic r(CUG)exp level in cellular assay and showspromising in vivo activity using a known adult DM1 Drosophilaphenotypic assay and a liver-specific DM1 mouse model.

Significance

Multivalent ligands have tremendous potential as therapeuticagents; however, their efficacy is limited by delivery issues, poorcell permeability, and toxicity. We report here a strategy whereinmultivalent ligands are designed to be intrinsically cell-penetrating,allowing them to target the expanded trinucleotide repeat se-quences of DNA and RNA that cause myotonic dystrophy type 1(DM1). The multivalent ligand studied shows cell permeability andlow toxicity both in cells and in mice. Importantly, the ligand re-duced or eliminated DM1 defects in DM1 cells and in vivo, vali-dating the multivalent strategy. The approach should be broadlyapplicable to other repeat expansion diseases and to any multi-valent oligomeric therapeutic agent whose activity can accom-modate structural elements that mimic cell-penetrating peptides.

Author contributions: J.L., Y.B., U.V.C., S.P., K.Y., L.M.L., L.D.H., H.Y.E.C., A.K., and S.C.Z.designed research; J.L., Y.B., U.V.C., S.P., K.Y., L.M.L., L.D.H., and J.F.S. performed research;J.L., U.V.C., S.P., L.D.H., J.F.S., H.Y.E.C., and A.K. analyzed data; and J.L., Y.B., L.D.H.,H.Y.E.C., A.K., and S.C.Z. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. J.M.H. is a guest editor invited by theEditorial Board.

Published under the PNAS license.1J.L. and Y.B. contributed equally to this work.2To whom correspondence may be addressed. Email: sczimmer@illinois.edu or kalsotra@illinois.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1820827116/-/DCSupplemental.

Published online April 11, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1820827116 PNAS | April 30, 2019 | vol. 116 | no. 18 | 8709–8714

CHEM

ISTR

YBIOCH

EMISTR

Y

Dow

nloa

ded

by g

uest

on

Oct

ober

27,

202

0

ResultsRational Design of a Multivalent Ligand. We recently reported asmall-molecule inhibitor of the r(CUG)exp–MBNL1 interaction(26) whose structure (1 in Fig. 2) features a bisamidinium core asa general RNA groove binder and two triaminotriazine unitsdesigned for selective recognition of U–U mismatches. Althoughthe synthesis was not trivial, we were able to prepare dimericligand 2 using the alkyne-azide copper-assisted (CuAAC) clickreaction and thus a triazole linker between two monomer units(Fig. 2A) (5). Dimer 2 inhibited formation of the MBNL1–r(CUG)16 complex with a Ki = 25 ± 8 nM that was nearly 1,000-fold lower than that measured for monomer 1 and r(CUG)12(Ki = 16 ± 6 μM). However, 2 did not perform well in DM1 cellularassays compared with 1,000-fold enhanced activity in vitro.Several lines of evidence suggest that the structure of 2 is

particularly unsuitable for cell-membrane penetration. First, itslarge size (molecular mass = 1,166.4 Da) (5) places it well out-side of the Lipinski rule of 5 (Ro5), where compounds withmolecular mass < 500 are most likely to show excellent passivediffusion across the cellular membrane. Although some clinicallyuseful and orally bioavailable drugs have molecular masses be-tween 500 and 1,000, a range known as “beyond the Ro5”(bRo5), these agents are usually cyclic peptides, often with N-methyl groups. Invariably, a steep drop-off in membrane per-meability occurs at molecular mass > 1,000, and an upper sizelimit appears to be molecular mass ∼ 1,000 for passive diffusionof small molecules across cell membranes (28–30). Beyond thehigh molecular mass, dimer 2 violates at least two other Ro5s

with a large number of N–H and hydrogen-bond acceptor groups.The final challenge for 2 to enter the cell comes from the need tomove four cations (amidinium groups) across the membrane. In-terestingly, previous studies with oligoarginines and aromatic amidefoldamers found that four positive charges were not enough toefficiently translocate into cells, whereas eight cationic groups werefound to be the most efficient (31, 32). These CPPs and their mimicsfrequently have molecular masses >1,000, yet readily enter cellsthrough either energy-dependent or -independent mechanisms.Given the considerations above, it appears that ligand 2 is too

large and too charged to passively penetrate the cell membraneas a small molecule and yet too small and insufficiently cationicto enter the cells through CPP-type mechanisms. Thus, wesought to connect four to six monomeric units of 1 to create aCPP mimic with 8–12 cationic groups. Ligand 1 is designed torecognize (CUG)3 with the bisamidinium linker, designatedlinker A in Fig. 2A, binding the groove of the central CUG andthe triaminotriazine units recognizing the terminal U–U mis-matches. In linking monomeric units of 1, it can be critical toselect the appropriate linker. The monomeric units of dimer 2are linked by a triazole group (linker B, Fig. 2A). In this study, weconsidered using the optimized linker A, as seen in ligand 3. Thecentral bisamidinium in 3 provides two more positive charges, aswell as additional groove binding, potentially improving cellpermeability and affinity for the target r(CUG)exp. The regularalternating structure of 3 further suggested oligomeric or poly-meric ligand 4 with multivalent binding capability (Fig. 2A).To validate our molecular design, we performed a preliminary

molecular modeling study using molecular dynamics (MD) sim-ulations. Following the same approach we reported (26) for theMD simulation of ligand 1 bound to [r(CUG)6]2, the oligomericligand 4 was manually docked into [r(CUG)15]2. The ligand usedhad three bisamidinium moieties and four triaminotriazine units,corresponding to a degree of polymerization (DP; i.e., number ofrepeat units) of 3.5. A 10-ns MD simulation suggested stablemajor groove binding with the triaminotriazine units recognizingevery other U–U mismatch along the sequence (SI Appendix, Fig.S1). The triaminotriazine units formed multiple hydrogen bonds toand were positioned roughly coplanar with one of the uracil groups,whereas the other was flipped out. This simple computational studysupported our rational design wherein oligomeric ligand 4 can se-lectively recognize r(CUG)exp in a multivalent fashion.

Preparation of Oligomer 4 by Polycondensation and Interaction withr(CUG)16. The alternation of triaminotriazine and bisamidiniummoieties in 4 suggested a single-step synthesis involving thepolycondensation of diamine 5 and bisimidate 6 (Fig. 2B), pre-cursors that can be prepared in one or two steps from commerciallyavailable compounds (SI Appendix, SI Materials and Methods). Thepolycondensation reaction was performed in DMF at 35 °C, reliably

Oligomeric inhibitor

GUC

CUG

MBNL1

GACGAC

Transcription Splicing misregulation

and DM1 defects

Recovery of DM1 defects

Small molecule inhibitor

CTG CTG

DMPK

Fig. 1. DM1 pathogenesis and therapeutic strategy. CTG expanded repeatsin 3′-UTR of the DMPK gene is transcribed to r(CUG)exp, forming a hairpinsecondary structure with U–U mismatches. The structured RNA sequestersMBNL1 and causes splicing misregulation and other DM1 defects. A designedmultivalent ligand has a multitargeting ability toward d(CTG)exp andr(CUG)exp. The oligomeric inhibitor blocks transcription of d(CTG·CAG)exp byd(CTG)exp binding and intervenes the r(CUG)exp–MBNL1 interaction. Thesestrategies are expected to decrease toxic r(CUG)exp levels and restore MBNL1levels, respectively, thereby recovering DM1 defects.

A

B

C

Fig. 2. Rational design and synthesis of multivalentligands. (A) Structure of ligand 1 with two triaminotriazineU–U or T–T recognition moieties (green hexagons)connected through a groove-binding bisamidiniumlinker (optimized linker A). Dimeric ligand 2 was pre-viously prepared by click chemistry (triazole linker B).Ligand 3 shows an alternative design for a dimer,linking two monomers using a groove binder linker A.This design provides a more regular structure, en-abling further elongation to an oligomeric ligand 4.(B) Synthesis of oligomeric ligand 4 by polycondensa-tion reaction. (C) MALDI-TOF characterization of oligomer4 shows the formation of an oligomeric mixture rangingfrom 4 to 8 mer (average molecular mass of ∼2 kDa).See text for explanation of R end groups. DHB wasused as the matrix. The spectrumwas collected by usingreflective mode. Intens., intensity.

8710 | www.pnas.org/cgi/doi/10.1073/pnas.1820827116 Lee et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

27,

202

0

producing low-molecular-mass oligomeric products. Tempera-ture control was important to restrict the DP.The small-molecule by-products and the smallest oligomers

were removed by dialysis, giving a mixture of oligomers thatexhibited a single HPLC peak (SI Appendix). The MALDI-MSindicated formation of the desired oligomeric products rangingfrom 4 to 8 mers with an average molecular mass of ∼2 kDa (DP∼ 5; Fig. 2C). Thus, two prominent mass series are seen in theMALDI-MS, each separated by m/z 396, the molecular mass ofthe repeat unit. The cluster of peaks in each mass series in theMALDI-MS spectrum suggests that different end groups arepresent on the oligomers. 1H NMR indicates the presence ofboth ester groups from hydrolysis of imidate ester 6 and ami-nobutyl groups expected for 5 as terminal groups (SI Appendix).The more prominent mass series (green arrows, Fig. 2C) can beassigned to the oligomeric mixture 4 with ester groups at bothends. The second mass series (magenta arrows, Fig. 2C) is con-sistent with two possible structures. The first involves 5 as oneterminal group and a 4-cyanobenzamidinium as the other. Thecyano group may arise from incomplete conversion of 1,4-dicyanobenzene into bisimidate 6. The second possibility in-volves dual MALDI-MS fragmentation at the benzamidiniumunit, consistent with the known fragmentation of arginine. Themass series with blue arrows likely involves one ester end groupand fragmentation at the benzamidinium unit (additional detailson the MALDI-MS can be found in SI Appendix, Characterization)(33). The presence of amine end groups in 4 derived from 5 issupported by the ability of the oligomer to be fluorescently labeled(see below).Given that 4 is an oligomeric mixture, in vitro binding studies

with a discrete r(CUG)n will lead to a complex mixture of boundstructures. Indeed, the binding of 4 with r(CUG)16 was studiedby using isothermal titration calorimetry (ITC) (SI Appendix, Fig.S2A), which showed a strongly enthalpic interaction and a U-shaped curve consistent with positive cooperativity. In compari-son, the ITC curve for monomer 1 was consistent with a 1:1complex and significantly weaker binding (SI Appendix, Fig.S2B). Dynamic light scattering (DLS) studies were also per-formed at similar concentrations to examine the nature of thecomplexation. The data suggested large polyplex formation withincreased concentration of oligomer 4 and r(CUG)16 (SI Ap-pendix, Fig. S3). The DLS also indicated that oligomer 4 ismonomeric at 20 μM and below. It needs to be noted that theITC and DLS experiments require a concentration considerablyhigher than the expected working concentration in biologicalenvironments.

Cell Permeability of Oligomer 4. To investigate the cell perme-ability of oligomer 4, we used confocal microscopy to visualizeoligomer 4 inside live cells. The oligomer was fluorescently la-beled with fluorescein (FAM) or Rhodamine B (RhB) by usingisothiocyanate-amine conjugation chemistry. Either RhB– orFAM–oligomer 4 was incubated with HeLa cells. A significantamount of fluorescence was observed inside the cells and nucleus,confirming the cell permeability of oligomer 4 (SI Appendix, Fig.S4A). Preliminary studies were undertaken with Lysotracker Red toshed light on the mechanism of the cell penetration. The Lyso-tracker Red fluorescence was observed to overlap significantly withthe FAM fluorescence from oligomer 4, consistent with an endo-cytotic uptake mechanism (SI Appendix, Fig. S4 B and C). However,nonoverlapping FAM fluorescence was also observed, indicatingeither escape from the endosome or a direct penetration mecha-nism. No disruption of the cell surface was observed in any of theexperiments.

rCUG-Targeting in DM1 Model Cell Culture and Patient-DerivedMyoblasts. Given that the key cellular feature of DM1 is the pres-ence of ribonuclear foci, formed between r(CUG)exp and MBNL1,

we examined whether oligomer 4 could inhibit their formation.Initial studies were performed with a standard DM1 model cellculture produced by transfecting HeLa cells with a plasmid con-taining GFP and DMPK exons 11−15, with 960 CTG repeats inexon 15 (DT960–GFP) (34). The r(CUG)960 and MBNL1 in thefoci were separately visualized, the former by using fluorescence insitu hybridization (FISH) with Cy3-(CAG)10 and the latter by usingimmunofluorescence with an anti-MBNL antibody (17). Untreatedcells showed significant areas of nuclear foci observed as yellowpunctate within the nucleus, demonstratingMBNL1 protein (green)–r(CUG)960 RNA (red) colocalization (SI Appendix, Fig. S5A, row1 merge). Treatment with oligomer 4 at various concentrations for48 h significantly suppressed the foci formation (SI Appendix, Fig.S5A). Quantitative analysis indicated a small dose-dependent re-sponse from 100 to 500 nM of oligomer 4, with 68–76% reductionin the foci area (SI Appendix, Fig. S5B). It is noteworthy thatmonomeric ligand 1 showed similar activity at ∼1,000-foldhigher concentration (100 μM) (26), consistent with an oligovalentadvantage conferred on 4.More interestingly, we used FAM-conjugated oligomer 4 in an

effort to track its location within the DM1 model cells. At aconcentration of 200 nM, colocalization of the FAM signal andr(CUG)960 were observed within the nucleus. This observationprovides evidence for 4 penetrating both the nuclear and cellmembranes and directly engaging r(CUG)960 (SI Appendix, Fig.S6). At a higher concentration of 500 nM, the FAM–oligomer 4appeared as cytoplasmic punctate, suggesting lack of endosomalrelease, limited nuclear membrane penetration, or saturation ofr(CUG)960 binding sites at the higher concentration (SI Appen-dix, Fig. S6). This would explain the modest dose dependence forfoci disruption upon increasing the concentration of 4 from 100to 500 nM and the inability to fully dissolve all foci at the highestconcentrations (SI Appendix, Fig. S5B).For a more disease-relevant cellular assay, we used DM1

patient-derived myoblasts, which were prepared by successfuldifferentiation of DM1 fibroblasts (GM03987; Coriell Institute)usingMyoD-containing retrovirus (SI Appendix) (35, 36). Whereasthe DM1 model cell culture used above produces r(CUG)960 witha doxycycline (Dox)-inducing expression system (22, 34), the DM1myoblasts spontaneously generate heterogeneous transcripts up tor(CUG)500. Treatment of the patient cells with 200 nM 4 for 48 hled to a 30% reduction in foci area (Fig. 3A and SI Appendix, Fig.S5C). The smaller reduction in the DM1 myoblasts may indicate alower level of uptake in DM1 myoblasts compared with in HeLacells. It is important to note that no sign of cytotoxicity was ob-served throughout the working concentration ranges both in HeLacells and DM1 patient-derived fibroblasts (GM03987) (SI Ap-pendix, Fig. S7).Inspired by enhanced activity of oligomer 4 in foci disruption

relative to monomer 1, we further evaluated an importantdownstream feature, the misregulation of alternative splicing ofpre-mRNAs. IR pre-mRNA was chosen for this study because itsmissplicing is relatively difficult to correct (37). The IR pre-mRNA undergoes two possible splicing pathways dependent onthe exclusion or inclusion of exon 11, which produces isoform Aor B, respectively (Fig. 3B). The DM1 model cell culture pro-duced by transfecting HeLa cells with plasmids containingDT960 and IR minigenes is able to reproduce the IR splicingpattern observed in normal and disease states (13, 36). Oligomer4 showed full rescue of IR missplicing at a concentration of 200nM, although the recovery rate diminished slightly and leveledoff at higher concentrations, similar to our observation with focidisruption. (Fig. 3B). Again the strength of oligovalent approachwas evident when comparing to monomeric ligand 1, which un-der similar conditions provided only 43% missplicing rescue at a500-fold higher concentration (100 μM) (26).

Lee et al. PNAS | April 30, 2019 | vol. 116 | no. 18 | 8711

CHEM

ISTR

YBIOCH

EMISTR

Y

Dow

nloa

ded

by g

uest

on

Oct

ober

27,

202

0

dCTG-Targeting by Oligomer 4 and Inhibition of Toxic RNA Synthesis.As explained, recent studies showed that r(CUG)exp can produceadditional toxicity, which adds to the complexity of the DM1disease pathobiology. For example, the r(CUG)exp transcript candisrupt the translation of MEF2 protein, leading to microRNAdysregulation in DM1 heart tissue (14). Additionally, r(CUG)exp

undergoes RAN translation, producing homopeptides that maybe toxic (15, 16). For these reasons, we tested the potential ofoligomer 4 to regulate the levels of toxic r(CUG)exp. Although inour previous studies, we did not see d(CTG)exp binding by mo-nomeric ligand 1, N-substituted analogs that add additionalcationic groups are able to target both the DM1 DNA and RNAexpansions (22).We first checked whether oligomer 4 can inhibit the transcription

of d(CTG)exp in vitro using a (CTG)74-containing DNA template.A strong dose-dependent inhibition effect of 4 was observed towardthe (CTG)74 template (IC50 = 100 nM), whereas no inhibition wasapparent for the transcription of a non-repeat-containing controltemplate (Fig. 3C and SI Appendix, Fig. S8A). This finding is con-sistent with oligomer 4 selectively and potently targeting d(CTG)exp

transcription, possibly by stabilizing an analogous hairpin secondarystructure formed in transcription bubble (38). The potential of 4 toact as a transcription inhibitor and down-regulate the toxic RNAlevels in cells was tested. After treatment of the DM1 model cellculture with 4 for 72 h, the level of r(CUG)960 was found to bereduced by 20–50%, depending on the oligomer concentration.These results indicate that oligomer 4, by inhibiting d(CTG)exp

transcription, can suppress the level of r(CUG)exp, the DM1 caus-ative agent (SI Appendix, Fig. S8B).

Recovery of Phenotypic Defect in DM1 Drosophila Adult ClimbingAssay. The promising cell-permeability and multitargeting fea-tures of 4 produced significant improvement of cellular DM1phenotypes, which motivated us to further study its activityin vivo. We used a phenotypic assay with adult DM1 Drosophilathat takes advantage of their innate climbing behavior (39).Because DM1 is a neuromuscular disease affecting both thecentral nervous system and the motor system, DM1 flies showage-dependent progressive climbing defects (39, 40). By agingDM1 adult flies for several days or even months, more r(CUG)exp

is accumulated, allowing for disease symptoms that range from amild to a severe climbing defect. In turn, this tunability allowsscreening of therapeutic ligands with widely different activities.Moreover, the climbing assay allows fast screening using a largernumber of adult flies, generating statistically important data fasterand more conveniently.With the aged adult DM1 flies created with (CTG)480 as

reported, but with an Elav–GAL4-inducing system (40), we

evaluated head to head the ability of monomeric ligand 1 andoligomer 4 to reverse the climbing failure (SI Appendix, Fig. S9).For normal flies with CTG60, treatment with 4 (80 μM) andmonomer 1 (400 μM) did not affect their climbing ability, sug-gesting minimal toxicity of compounds. When it came to DM1flies (CTG480), almost 80% of the flies failed to climb, buttreatment with oligomer 4 at a concentration of 20–80 μM res-cued their climbing ability in a dose-dependent fashion. At thehighest dose of 80 μM, the failure rate was dropped to 37%,representing a 54% recovery. In contrast, monomer 1 decreasedthe failure rate to 55%, a 31% recovery, at a fivefold higherconcentration (400 μM). The phenotypic improvement in DM1adult flies demonstrated the in vivo activity of 4, although themolecular mechanism of action was not investigated.

In Vivo Activities of Multivalent Ligands in a Liver-Specific DM1Mouse Model. As a result of the multisystemic nature of DM1,symptoms other than muscle wasting and myotonia are oftenexhibited by patients. These symptoms include cardiac conductiondefects, cataracts, testicular atrophies, dysphagic symptoms, andother gastrointestinal-tract-related symptoms (41, 42). Abnormalelevation of liver enzymes such as aspartate aminotransferase, al-anine aminotransferase, alkaline phosphatase, and lactate dehy-drogenase along with nonalcoholic fatty liver disease and gallbladderdysfunction are also common in DM1 patients (43–45), and eventhough such liver-associated pathologies may be the first symptomobserved, they remain understudied. Therefore, we developed aliver-specific DM1 mouse model by expressing 960 CUG repeat-containing DMPK RNAs in a tetracycline-inducible (46) andhepatocyte-specific manner (TRE-960I;ApoE-rtTA mouse model;SI Appendix, Fig. S10A). The liver-specific CUG960 mouse modelserved as an attractive model for screening in vivo efficacy ofoligomer 4, considering the ease of delivery of compounds to theliver via i.p. injections (47). An 8-d pathogenic r(CUG)960 RNAinduction and treatment scheme was used in this study (Fig. 4A).Efficient r(CUG)960 RNA expression was observed in the mouselivers upon feeding a 6 g/kg Dox-containing diet, as seen byquantitative RT-PCR (qRT-PCR) (Fig. 4B). Continued injectionswith oligomer 4 led to a significant reduction in the level ofr(CUG)960 RNA, likely due to its inhibitory effect on d(CTG)exp

transcription, as observed with in vitro and DM1 cell studies. Doxdiet and oligomer 4 treatment as mentioned above had no signifi-cant impact on the body weight or liver-to-body-weight ratios (SIAppendix, Fig. S11 A and B). Histological analysis showed no overtsigns of liver toxicity—such as inflammation, injury, or fibrosis—inoligomer 4-treated mice (SI Appendix, Fig. S11F). Furthermore, theeffects of oligomer 4 in reducing r(CUG)960 RNA load were spe-cific, as the mRNA levels of three other nontargeted genes,

10 11 12

10 11 12

10 12

Isoform B

Isoform A

Normal: A < BDiseased: A> B

10

40

60

2030

50

70

% Is

ofor

m B

(%)

DiseasedNormal 0.1 0.2 1 2 3 4

Oligomer 4 (μM)

Hoechst33342 Cy3-(CAG)10 MBNL1 Merge

Unt

reat

ed20

0 nM

** * * * *

400 300 200 100 50 20 10150 0

20406080

100120140

Nor

mal

ized

RN

A le

vel (

%)

[Oligomer 4 / nM]

(CUG)74

Control RNA

A B C

Fig. 3. Ligand 4 as an inhibitor of r(CUG)exp–MBNL1 and d(CTG·CAG)exp transcription. (A) Reduction of disease foci in DM1 patient-derived myoblasts uponthe treatment of oligomer at 200 nM. Foci areas are indicated with white arrows. (Scale bars, 5 μm.) (B) Schematic description of splicing pattern of IR pre-mRNA and the result of splicing assay in a presence of oligomer 4. Full recovery of the IR splicing defect was obtained at 200 nM. P values were calculatedagainst data points of the diseased sample. *P < 0.05. No significant differences between normal cells and each of all the treated samples (P > 0.05, Student’st test, two-tailed). (C) A plot of normalized RNA transcript level against the oligomer 4 concentration. RNA transcript level was normalized to the level of aloading reference RNA, HIV fs RNA, for quantitative comparison between the loading samples. Oligomer 4 inhibited d(CTG)exp transcription in a dose-dependent manner (IC50 = 100 nM), but no significant inhibition was detected with the control DNA template throughout all the tested concentrations. Errorbars indicate standard error of the mean from three independent experiments.

8712 | www.pnas.org/cgi/doi/10.1073/pnas.1820827116 Lee et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

27,

202

0

Mapkap1, Pcolce, and Mllt3—which contain 26, 12, and 8 CUGrepeats, respectively—were not reduced after treatment (SIAppendix, Fig. S11C).We then studied the most prominent molecular features of

DM1 to understand the effects of the oligomer. To analyze thecharacteristic disease ribonucleic foci, FISH and immunofluo-rescence were performed on frozen liver-tissue sections (17).Upon Dox induction, RNA foci were clearly visible in the he-patocyte nuclei, which were significantly reduced after treatmentwith oligomer 4 (Fig. 4D and SI Appendix, Fig. S11D). Colocal-ization of MBNL1 and CUG RNA also exhibited a marked re-duction, demonstrating reduced sequestration of MBNL1 (Fig. 4C and D). In addition, we identified several developmentallyregulated splicing events that are sensitive to MBNL1 levels inthe liver by screening their splicing patterns in wild-type fetal andadult as well as MBNL1-knockout mice (SI Appendix, Fig. S10 Band C) (18, 48).When induced with Dox, TRE-960I;ApoE-rtTA mouse livers

consistently reproduced the alternative splicing defects evidentin the livers of MBNL1-knockout mice, which were significantlyreversed with oligomer 4 treatment (Fig. 4E and SI Appendix,Fig. S11E). As expected, the developmentally regulated splicingevents (Add1_34 and Usp4_141) that are insensitive to MBNL1levels in the liver showed no change upon r(CUG)exp inductionor after oligomer 4 treatment (Fig. 4E and SI Appendix, Fig.S11E). Together, these results demonstrated that systemic admin-istration of oligomer 4 to a liver-specific transgenic r(CUG)960mouse model can reduce the burden of r(CUG)exp RNA levels,disperse the ribonuclear foci-releasing MBNL1 proteins, and cor-rect the splicing defects for a number of pre-mRNAs that areregulated by MBNL1 in the liver.

DiscussionDM1 serves as an excellent disease platform for testing intracellularmultivalent approaches because the pathogenic agents d(CTG)exp

and r(CUG)exp involve regularly repeating sequences. Current ap-proaches for multivalency heavily depend on linker optimizationbetween monomeric units or incorporation of binding moietiesonto a delivery vehicle such as CPPs or their mimics (4, 5, 9, 24).The former strategy often requires laborious in vitro optimization,yet the increased size and low cell permeability frequently limits thepotency in cellular assays or in vivo. In the latter approaches, it isoften difficult to control the positions and spatial arrangement ofbinding ligands on the carrier scaffold. In this study, we introducedmultivalent ligands that are inherently cell-permeable. The alter-nating structure of triaminotriazines and bisamidinium recognitionunits contains the CPP features of cationic charges andamphipathicity.Despite its large molecular mass, oligomer 4 significantly

outperformed monomer 1 in each cellular and in vivo assay. Theoligomer exhibited excellent multitargeting activity binding bothd(CTG)exp and r(CUG)exp, thereby inhibiting the DM1 patho-genesis. These activities include reducing the toxic r(CUG)exp

level by inhibiting transcription and dispersing disease foci(rCUGexp-MBNL1 coaggregates) in both DM1 model cells andDM1 patient myoblasts. The difference in the foci-dissolvingactivity observed in DM1 model cell culture and differentiatedDM1 myoblasts may reflect a different extent of uptake. Mem-brane permeability can be modified by abnormal protein–membraneinteractions, loss of integral proteins, etc., which are possibledifferences between DM1 model cells and differentiated DM1myoblasts (49, 50).More interestingly, the same activities of correcting DM1

molecular defects were also observed in vivo in a liver-specificCUG960 mouse model, fully rescuing multiple MBNL1-regulatedsplicing defects. Our liver-specific r(CUG)960 mouse model isexpected to be a useful model to understand pathogenic mech-anisms of DM1 in the liver and related symptoms, which havenot been examined before. Also, considering a facile delivery ofcompounds to the liver via i.p. injections, our mouse model can

Mice fed with Dox (6g/kg)

IP injection ends

Daily injections & continued Dox diet

IP injection starts;experimental group w/ Oligomer 4 (2mg/kg)

control group w/ H O

Mice sacked

DAPI Cy3-(CAG) MBNL1 Merge

r(C

UG

)960 m

ouse

+ H

O

2

0

20000

40000

60000

80000

100000

CU

G R

NA

expr

essi

on(R

Q)

*

n=4

n=4n=3

ApoE-rtTA+/- +DoxTRE-960I +/-; ApoE-rtTA+/- + Dox + H2OTRE-960I +/-; ApoE-rtTA+/- + Dox + Oligomer 4

Nor

mal

ized

col

oca l

izatio

n a

rea

/ cel

l (%

)

2

n=4

Sorbs1_168

Arhgef10l_15

Synj2_135

Inf2_157

Git2_90

H2afy_100

Add1_34

Usp4_141

0204040

60

80

100

Perc

ent s

plic

ed in

(PSI

, %) *

ns

*

*ns

*

*ns

*

*ns

* *ns

* nsns ns

nsns ns

nsns ns

**

100806040200

120

r(C

UG

)960 m

ouse

+ O

ligom

er 4

A

DE

B C

Fig. 4. In vivo activities of oligomer 4 in a liver-specific CUG960 transgenic mouse model. (A) Schematic of disease induction and 4 administration in the livers ofTRE-960I;ApoE-rtTAmice. (B) qRT-PCR result shows efficient expression of r(CUG)exp in TRE-960I;ApoE-rtTAmice upon Dox treatment. Oligomer 4 efficiently reducesr(CUG)exp expression. Data are normalized to β-actin. (C) Quantitative analysis of colocalized disease foci area per cell. Significant amount of disease foci withMBNL1 was released with 4 treatment. (D) Foci disruption upon 4 treatment in the liver tissue. Colocalization of r(CUG)exp and MBNL1 was detected by FISHcombined with immunofluorescence. (Scale bars, 5 μm.) (E) Rescue of splicing defects caused by expression of r(CUG)exp. Mean (±SD) of percent spliced in (PSI) isplotted for different events from n = 3, 4, and 4 mice for control, untreated, and oligomer 4-treated mice, respectively. *P < 0.05; ns, not significant.

Lee et al. PNAS | April 30, 2019 | vol. 116 | no. 18 | 8713

CHEM

ISTR

YBIOCH

EMISTR

Y

Dow

nloa

ded

by g

uest

on

Oct

ober

27,

202

0

serve as an initial in vivo platform for potential DM1 therapeuticagents. Importantly, oligomer 4 showed no sign of toxicity at theworking concentrations in HeLa cells, DM1 patient cells (GM03987),and both in vivo models. The maximum tolerated dose (MTD) wasdetermined to be 40 mg/kg in C57BL/6 mice, which is significantlyhigher than that for dimeric ligand 2 (3 mg/kg) (5).Our oligomeric ligand inherently mimics the structure of CPPs

such as oligoarginines or Tat. In this regard, we believe that thisapproach represents a paradigm in multivalent ligand design, wherethe direct assembly of binding moieties serves as their own deliveryagent. The ability to prepare oligomer 4 by a condensation poly-merization streamlines the synthesis, but moving forward it willbe important to prepare homogenous oligomers for testing.Nonetheless, our general design principle should be applicable toother TREDs, such as Huntington’s disease and spinocerebellarataxias, considering the similarities in their pathogenesis.

Materials and MethodsAll reagents were purchased from standard suppliers and used withoutfurther purification unless otherwise noted. r(CUG)16 was purchased from

Integrated DNA Technologies or synthesized on an Applied Biosystems 3400DNA Synthesizer, followed by HPLC purification. NMR spectra and massspectral analyses were performed by using instrumentation available in theSchool of Chemical Sciences, University of Illinois. Details of the synthesis,compound, and oligomer characterization, as well as additional experi-mental details and additional figures, can be found in SI Appendix. Westrictly followed the National Institutes of Health guidelines for use and careof laboratory animals, and all experimental protocols were approved by theInstitutional Animal Care and Use Committee at the University of Illinois,Urbana–Champaign.

ACKNOWLEDGMENTS. We thank Maurice Swanson (University of Florida,Gainesville) for the (CTG)74 plasmid; Nicholas Webster (University of California,San Diego) for the IR minigene plasmid; A. Dusty Miller (Fred HutchinsonCancer Research Center) for MyoD-containing retrovirus; and Dr. HyangYeon Lee and Prof. Paul J. Hergenrother for the MTD. This work was sup-ported by National Institutes of Health Grants R01AR058361 (to S.C.Z.) andR01HL126845 (to A.K.); Muscular Dystrophy Association Grants 295229 and514335 (to A.K.); and the Fundamental Research Funds for the Central Uni-versities from Hunan University (Y.B.). L.D.H. is a member of NIH Chemistry-Biology Interface Training Grant NRSA 1-T-32-GM070421.

1. Kiessling LL, Gestwicki JE, Strong LE (2000) Synthetic multivalent ligands in the ex-ploration of cell-surface interactions. Curr Opin Chem Biol 4:696–703.

2. Lundquist JJ, Toone EJ (2002) The cluster glycoside effect. Chem Rev 102:555–578.3. Mammen M, Choi SK, Whitesides GM (1998) Polyvalent interactions in biological

systems: Implications for design and use of multivalent ligands and inhibitors. AngewChem Int Ed Engl 37:2754–2794.

4. Jahromi AH, et al. (2013) Developing bivalent ligands to target CUG triplet repeats,the causative agent of myotonic dystrophy type 1. J Med Chem 56:9471–9481.

5. Luu LM, et al. (2016) A potent inhibitor of protein sequestration by expanded triplet(CUG) repeats that shows phenotypic improvements in a Drosophila model of myo-tonic dystrophy. ChemMedChem 11:1428–1435.

6. Mahajan SS, et al. (2006) Optimization of bivalent glutathione S-transferase inhibitorsby combinatorial linker design. J Am Chem Soc 128:8615–8625.

7. Englund EA, et al. (2012) Programmable multivalent display of receptor ligands usingpeptide nucleic acid nanoscaffolds. Nat Commun 3:614.

8. Yang L, et al. (2011) Engineering polymeric aptamers for selective cytotoxicity. J AmChem Soc 133:13380–13386.

9. Bai Y, et al. (2016) Integrating display and delivery functionality with a cell pene-trating peptide mimic as a scaffold for intracellular multivalent multitargeting. J AmChem Soc 138:9498–9507.

10. Fu YH, et al. (1992) An unstable triplet repeat in a gene related to myotonic musculardystrophy. Science 255:1256–1258.

11. Brook JD, et al. (1992) Molecular basis of myotonic dystrophy: Expansion of a tri-nucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase familymember. Cell 68:799–808.

12. Yum K, Wang ET, Kalsotra A (2017) Myotonic dystrophy: Disease repeat range,penetrance, age of onset, and relationship between repeat size and phenotypes. CurrOpin Genet Dev 44:30–37.

13. Philips AV, Timchenko LT, Cooper TA (1998) Disruption of splicing regulated by aCUG-binding protein in myotonic dystrophy. Science 280:737–741.

14. Kalsotra A, et al. (2014) TheMef2 transcription network is disrupted in myotonic dystrophyheart tissue, dramatically altering miRNA and mRNA expression. Cell Rep 6:336–345.

15. Pearson CE (2011) Repeat associated non-ATG translation initiation: One DNA, twotranscripts, seven reading frames, potentially nine toxic entities! PLoS Genet 7:e1002018.

16. Cleary JD, Ranum LPW (2013) Repeat-associated non-ATG (RAN) translation in neu-rological disease. Hum Mol Genet 22:R45–R51.

17. Mankodi A, et al. (2001) Muscleblind localizes to nuclear foci of aberrant RNA inmyotonic dystrophy types 1 and 2. Hum Mol Genet 10:2165–2170.

18. Kanadia RN, et al. (2006) Reversal of RNA missplicing and myotonia after muscleblindoverexpression in a mouse poly(CUG) model for myotonic dystrophy. Proc Natl AcadSci USA 103:11748–11753.

19. Kanadia RN, et al. (2003) A muscleblind knockout model for myotonic dystrophy.Science 302:1978–1980.

20. Siboni RB, et al. (2015) Actinomycin D specifically reduces expanded CUG repeat RNAin myotonic dystrophy models. Cell Rep 13:2386–2394.

21. Coonrod LA, et al. (2013) Reducing levels of toxic RNA with small molecules. ACSChem Biol 8:2528–2537.

22. Nguyen L, et al. (2015) Rationally designed small molecules that target both the DNAand RNA causing myotonic dystrophy type 1. J Am Chem Soc 137:14180–14189.

23. Gareiss PC, et al. (2008) Dynamic combinatorial selection of molecules capable ofinhibiting the (CUG) repeat RNA-MBNL1 interaction in vitro: Discovery of lead com-pounds targeting myotonic dystrophy (DM1). J Am Chem Soc 130:16254–16261.

24. Childs-Disney JL, Hoskins J, Rzuczek SG, Thornton CA, Disney MD (2012) Rationallydesigned small molecules targeting the RNA that causes myotonic dystrophy type 1are potently bioactive. ACS Chem Biol 7:856–862.

25. Parkesh R, et al. (2012) Design of a bioactive small molecule that targets the myotonicdystrophy type 1 RNA via an RNA motif-ligand database and chemical similaritysearching. J Am Chem Soc 134:4731–4742.

26. Wong CH, et al. (2014) Targeting toxic RNAs that cause myotonic dystrophy type 1(DM1) with a bisamidinium inhibitor. J Am Chem Soc 136:6355–6361.

27. Arambula JF, Ramisetty SR, Baranger AM, Zimmerman SC (2009) A simple ligand thatselectively targets CUG trinucleotide repeats and inhibits MBNL protein binding. ProcNatl Acad Sci USA 106:16068–16073.

28. Doak BC, Over B, Giordanetto F, Kihlberg J (2014) Oral druggable space beyond therule of 5: Insights from drugs and clinical candidates. Chem Biol 21:1115–1142.

29. Pye CR, et al. (2017) Nonclassical size dependence of permeation defines bounds forpassive adsorption of large drug molecules. J Med Chem 60:1665–1672.

30. Matsson P, Kihlberg J (2017) How big is too big for cell permeability? J Med Chem 60:1662–1664.

31. Futaki S, et al. (2001) Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery.J Biol Chem 276:5836–5840.

32. Iriondo-Alberdi J, Laxmi-Reddy K, Bouguerne B, Staedel C, Huc I (2010) Cellular in-ternalization of water-soluble helical aromatic amide foldamers. ChemBioChem 11:1679–1685.

33. Gogichaeva NV, Williams T, Alterman MA (2007) MALDI TOF/TOF tandem mass spec-trometry as a new tool for amino acid analysis. J Am Soc Mass Spectrom 18:279–284.

34. Lee JE, Bennett CF, Cooper TA (2012) RNase H-mediated degradation of toxic RNA inmyotonic dystrophy type 1. Proc Natl Acad Sci USA 109:4221–4226.

35. Weintraub H, et al. (1989) Activation of muscle-specific genes in pigment, nerve, fat,liver, and fibroblast cell lines by forced expression of MyoD. Proc Natl Acad Sci USA86:5434–5438.

36. Savkur RS, Philips AV, Cooper TA (2001) Aberrant regulation of insulin receptor al-ternative splicing is associated with insulin resistance in myotonic dystrophy. NatGenet 29:40–47.

37. Jog SP, et al. (2012) RNA splicing is responsive to MBNL1 dose. PLoS One 7:e48825.38. Mirkin SM (2007) Expandable DNA repeats and human disease. Nature 447:932–940.39. González ÀL, et al. (2017) In silico discovery of substituted pyrido[2,3-d]pyrimidines

and pentamidine-like compounds with biological activity in myotonic dystrophymodels. PLoS One 12:e0178931.

40. Bargiela A, et al. (2015) Increased autophagy and apoptosis contribute tomuscle atrophyin a myotonic dystrophy type 1 Drosophila model. Dis Model Mech 8:679–690.

41. Machuca-Tzili L, Brook D, Hilton-Jones D (2005) Clinical and molecular aspects of themyotonic dystrophies: A review. Muscle Nerve 32:1–18.

42. Harper PS (2004) Myotonic Dystrophy (Oxford Univ Press, Oxford).43. Heatwole CR, Miller J, Martens B, Moxley RT, 3rd (2006) Laboratory abnormalities in

ambulatory patients with myotonic dystrophy type 1. Arch Neurol 63:1149–1153.44. Achiron A, et al. (1998) Abnormal liver test results in myotonic dystrophy. J Clin

Gastroenterol 26:292–295.45. Shieh K, Gilchrist JM, Promrat K (2010) Frequency and predictors of nonalcoholic fatty

liver disease in myotonic dystrophy. Muscle Nerve 41:197–201.46. Morriss GR, Rajapakshe K, Huang S, Coarfa C, Cooper TA (2018) Mechanisms of

skeletal muscle wasting in a mouse model for myotonic dystrophy type 1. Hum MolGenet 27:2789–2804.

47. Lukas G, Brindle SD, Greengard P (1971) The route of absorption of intraperitoneallyadministered compounds. J Pharmacol Exp Ther 178:562–564.

48. Bhate A, et al. (2015) ESRP2 controls an adult splicing programme in hepatocytes tosupport postnatal liver maturation. Nat Commun 6:8768.

49. Frandsen SK, McNeil AK, Novak I, McNeil PL, Gehl J (2016) Difference in membranerepair capacity between cancer cell lines and a normal cell line. J Membr Biol 249:569–576.

50. González-Barriga A, et al. (2015) Cell membrane integrity in myotonic dystrophy type1: Implications for therapy. PLoS One 10:e0121556.

8714 | www.pnas.org/cgi/doi/10.1073/pnas.1820827116 Lee et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

27,

202

0