BNANC Gapmers Revert Splicing and Reduce RNA Foci with Low

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Letter NC

BNA gapmers revert splicing and reduce RNA foci with low toxicity in myotonic dystrophy cells Kassie S. Manning, Ashish N. Rao, Miguel Castro, and Thomas A. Cooper ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00416 • Publication Date (Web): 30 Aug 2017 Downloaded from http://pubs.acs.org on September 3, 2017

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ACS Chemical Biology

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A

CTG(300) GFP CMV

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n.s. (p=0.15)

Control 30nM gapmer 30nM mixmer

***

200000

100000 0

CNPY3

GAPDH GAPDH

1.2 1.0 0.8 0.6 0.4 0.2 0.0 ***



A

B Polymorphic BpmI site

Allele 1: 152 bp Unexpanded in DM1

RT-PCR and BpmI digestion

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Nuclear Cytoplasmic

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30nM BNANC gapmers


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A 40 Percentage of nuclei

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BNANC gapmers revert splicing and reduce RNA foci with low toxicity in myotonic dystrophy cells Kassie S. Manning

1, 2

3

5

, Ashish N. Rao , Miguel Castro , Thomas A. Cooper

1

1, 2, 3, 4 *

2

Departments of Integrative Molecular and Biomedical Sciences Program, Pathology and 3

4

Immunology, Molecular and Cellular Biology, Molecular Physiology and Biophysics, Baylor 5

College of Medicine, Houston, TX 77030, Bio-Synthesis, Inc., 612 East Main Street, Lewisville, TX 75057 *

corresponding author: [email protected]


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ABSTRACT Myotonic dystrophy type 1 (DM1) is a multisystemic disease caused by an expanded CTG repeat in the 3’ UTR of the dystrophia myotonica protein kinase (DMPK) gene. Short, DNA-based antisense oligonucleotides termed gapmers are a promising strategy to degrade toxic CUG expanded repeat (CUGexp) RNA. Nucleoside analogs are incorporated to increase gapmer affinity and stability, however some analogs also exhibit toxicity. In this study, we demonstrate that the NC

NC

2’,4’-BNA [NMe] (BNA ) modification is a promising nucleoside analog with high potency NC

similar to 2’,4’-LNA (LNA). BNA

gapmers targeting a non-repetitive region of the DMPK 3’ UTR

show allele-specific knockdown of CUGexp RNA and revert characteristic DM1 molecular defects NC

including mis-splicing and accumulation of RNA foci. Notably, the BNA

gapmers tested in this

study did not induce caspase activation, in contrast to a sequence matched LNA gapmer. This NC

study indicates BNA

gapmers warrant further study as a promising RNA targeting therapeutic.

INTRODUCTION Myotonic dystrophy is a multisystemic disease affecting approximately 1 in 8,000 individuals.(1) Myotonic dystrophy type I (DM1) is caused by a CTG repeat expansion within the 3’ UTR of the DMPK gene.(2,3) Expanded CUG repeat (CUGexp) RNA is retained in the nucleus and forms RNA foci that sequester the MBNL family of splicing factors and induces upregulation of CELF1 through PKC-mediated phosphorylation and altered microRNA regulation.(4-6) Altered MBNL and CELF1 activity in DM1 leads to defects in developmentally regulated alternative splicing.(1) Antisense oligonucleotides (ASOs) are a promising therapeutic approach for diseases caused by an RNA gain of function, including DM1. Addition of chemical modifications greatly stabilizes the affinity and stability of ASOs. These modifications include altered backbone chemistry, such as phosphorothioate (PS), and altered ribose chemistry, such as the 2’-Omethoxyethyl (MOE) and 2’-O,4’-C-methylene-bridged nucleic acid (LNA).(7) However, the addition of 2’ ribose modifications abolishes RNase H1-mediated target degradation.(8) Chimeric ASOs called gapmers contain 2’ ribose modifications at the 5’ and 3’ ends, leaving an internal 6– 10 nucleotide gap that maintains RNase H1 activity.(8-10) This is in contrast to mixmers, which contain modified analogs throughout the ASO and are unable to recruit RNase H1 for target cleavage (Figure S1A).(8) A recent clinical trial using a MOE gapmer (IONIS-DMPKRx) for the treatment of myotonic dystrophy demonstrated promise with the approach with current work focused on improving tissue delivery (Ionis Pharmaceuticals and Biogen press release, January 2017). Comparison of LNA and MOE gapmers indicates that LNA gapmers are 5–10 fold more potent, eliciting initial interest as a more potent alternative to MOE gapmers.(11,12) However, multiple studies have



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indicated that LNA gapmers induce RNase H1-dependent hepatotoxicity and apoptosis in both mice and cell models, reducing suitability for translational studies.(12-15) NC

NC

The 2’4’-BNA [NMe] (BNA ) modification was developed following similar principles as the LNA modification, with a bridged structure that greatly increases its affinity (Figure S1B).(16,17) NC

BNA

ASOs are more stable than LNA ASOs, and have been shown to be well tolerated in NC

mice.(16,18) However, BNA

gapmers have not been systematically tested in repeat expansion NC

models and it is unknown whether BNA NC

gapmers. We demonstrate that BNA

gapmers have similar toxicity concerns as LNA

gapmers have comparable potency to LNA gapmers,

display potentially lower propensity to induce caspase activity, and functionally rescue NC

characteristic DM1 defects, making BNA

gapmers a promising alternative chemistry for

therapeutic development.

RESULTS AND DISCUSSION NC

We first sought to compare the efficiency of BNA

and LNA gapmers at targeting DMPK

transcripts containing CUGexp. COSM6 cells were transfected with two plasmids: an rtTA expression plasmid and the pBitetDT700ctgGFP (Bi700CTG) plasmid, which contains a bidirectional tet-inducible promoter to express GFP and exons 11–15 of DMPK, including ~300 CUG repeats (Figure 1A). Twenty-four hours after plasmid transfection, cells were treated with NC

increasing concentrations of LNA or BNA

gapmers targeting either within the CUG repeat (CAG

gapmers) or in a non-repetitive region of DMPK (DMPK gapmers) (Figure 1; Table S1).(19) DMPK mRNA levels were quantified by RT-PCR relative to the GFP mRNA to standardize for NC

transfection efficiency. Compared to the CTG negative control gapmers, both LNA and BNA

CAG gapmers gave significant knockdown of CUGexp RNA at the lowest gapmer dose (0.3nM), and maximum knockdown to 65–80% of baseline at doses above 10nM (Figure 1C-D). We next NC

tested the efficiency of LNA and BNA

DMPK gapmers that target upstream of the expanded

repeat. While neither DMPK gapmer showed significant knockdown at the lowest dose, they both induced over 70% knockdown at 30nM. Interestingly, the DMPK gapmers displayed more potent knockdown than the CAG gapmers at the highest dose (100nM), with only 2–14% of the target NC

remaining (Figure 1C-D). The LNA DMPK gapmer was also more effective than the BNA

DMPK

gapmer at the low dosage levels. It is likely that the CAG gapmer is more effective than the DMPK gapmer at low concentrations because there are more binding sites per molecule. At higher concentrations, the residual amount of DMPK RNA that cannot be degraded by the CAG gapmer suggests the CUG repeat is partially protected, potentially by MBNL proteins or RNA NC

structure. Taken together, these results indicate that both LNA and BNA

gapmers are potent

and effective for knockdown of CUGexp RNA. NC

After establishing comparable potency between LNA and BNA NC

toxicity of BNA

gapmers, we compared the

and LNA gapmers. Non-DM1 TeloMyoD fibroblasts were treated with 30nM of


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NC

LNA or BNA

gapmers or mixmers and were assayed for membrane integrity, viability, and

caspase-3/7 activity 32 hours after transfection. We show a sequence-, chemistry- and RNase H1-dependent increased caspase response specifically caused by the LNA CAG gapmer and not NC

the BNA

CAG gapmer or the LNA CAG mixmer (Figure 2A, Figure S2A). Increased caspase

was not due to differences in cell number between samples because only the staurosporine treated cells showed a statistically significant decrease in viability (Figure 2B). Cytotoxicity was only elevated in cells treated with the positive control digitonin, indicating the ASOs tested do not display cytotoxic effects at 30nM (Figure S2B). Given that the LNA CAG mixmer did not elicit caspase activity, we hypothesized that LNA CAG gapmers induce caspase activity through on-target but non-desirable knockdown of nonNC

DMPK transcripts that contain CUG6+. However, we demonstrate that both LNA and BNA

CAG

gapmers induce RNase H1-mediated knockdown of all 11 tested CUG6+-containing mRNAs (Figure 2C, Figure S3). The question remains why LNA CAG gapmers induce caspase activity NC

but the sequence-matched BNA

CAG gapmers do not. Gapmer-mediated RNase H1 activity

includes off-target effects within intronic or exonic regions of long pre-mRNA transcripts.(14,20) Additionally, a variety of proteins bind ASOs and modulate their activity, and ASO chemistry can either promote or inhibit protein binding.(21,22) Therefore, differences in off-target effects or in the ASO-protein interactome could explain the differential caspase induction seen between the NC

LNA and BNA

CAG gapmers.

Previous work indicates that gapmers targeting within the repetitive region of DMPK preferentially degrade the mutant allele, and this region is an attractive target given the higher number of potential binding sites across the repeat tract.(23) However, given our results that nonDMPK transcripts containing short CUG repeat tracts were degraded by CAG gapmers, we NC

analyzed endogenous allele-specific RNA expression in DM1 cells after treatment with BNA gapmers. To distinguish between the expanded and non-expanded alleles we utilized a polymorphic site within DMPK and confirmed RNA transcripts from the expanded allele are NC

retained in the nucleus in these DM1 cells (Figure 3A-C).(24) Treatment with 30nM BNA

DMPK

gapmers induces preferential knockdown of the nuclear-retained CUGexp RNA without affecting the nuclear or cytoplasmic expression of the nonexpanded allele (Figure 3D-E). Unexpectedly, this preferential knockdown was not seen with the BNA

NC

CAG gapmer, which produced over

60% knockdown of total DMPK, but at the expense of targeting both the expanded and nonexpanded alleles (Figure 3E). In contrast, the BNA

NC

DMPK gapmer produced only 20%

knockdown of total DMPK, but this was largely accounted for by knockdown of the nuclearNC

retained, expanded allele. It is possible that the high potency of the BNA

gapmer saturated

knockdown of the expanded allele, leading to secondary degradation of the non-expanded DMPK mRNA. A recent study demonstrated that DMPK knockout mice do not have muscle or cardiac dysfunctions, making it possible that the CAG gapmer would still be viable for clinical



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translation.(25) However, the relative low specificity of this gapmer and higher toxicity seen for the LNA gapmer of this sequence indicate that targeting this repetitive region confers higher risk. NC

We next sought to determine whether BNA

gapmers could functionally rescue

characteristic defects in DM1 cells. First, we used fluorescent in situ hybridization (FISH) and NC

immunofluorescence (IF)-FISH to determine whether BNA

gapmers can reduce foci load and

release sequestered MBNL1. DM1 and non-DM1 TeloMyoD fibroblasts were treated with 30nM of NC

BNA

CAG or DMPK gapmers and differentiated for 6 days to induce the myogenic

program.(26,27) CUGexp RNA foci were visualized by RNA FISH (Figure S4A) and the number of foci per nucleus was quantified across three biological replicates (Figure 4A). Nearly all (97% ± 2.3%) of mock treated DM1 cells had nuclear RNA foci (Figure 4A, Figure S4C), while none of the 150 analyzed non-DM1 cells had nuclear foci. The total number of foci in 150 cells decreased NC

from 738 in DM1 mock treated cells, to 334 and 383 after treatment with BNA

CAG or DMPK

gapmers, respectively. This is reflected as a shift in the distribution of foci number per cell (Figure 4A). While over 30% of mock treated cells had more than 6 foci, only 5–7% of cells treated with NC

the BNA

CAG or DMPK gapmers had more than 6 foci. This effect was even more pronounced

in undifferentiated cells assayed 24 hours after gapmer treatment (Figure S4B-D). Partial foci knockdown, despite significant transcript knockdown by RT-PCR, may be a result of foci nucleation by a low absolute number of transcripts..(28) It is also possible that the MBNL-bound CUGexp RNA region is partially protected from exonuclease activity following RNase H1 cleavage. Given that the majority of cells still had at least one RNA focus after gapmer treatment, we were interested to determine the degree to which MBNL1 was released in cells that had decreased levels of RNA foci. We demonstrated that diffuse MBNL1 levels increased in the NC

nucleoplasm for cells treated with BNA NC

the BNA

CAG gapmers, however this result was not significant in

DMPK gapmers (Figure 4B-C and Figure S5). The increase in free MBNL1 is visible

even in cells that contain foci, indicating the presence of RNA foci is not necessarily a direct measure of MBNL1 availability. Our results in both differentiated and undifferentiated DM1 cells indicate that RNA foci are reduced and MBNL1 activity may be increased after treatment with NC

30nM of BNA

gapmers.

As RNA foci can be indicative of MBNL1 sequestration in DM1, we next analyzed whether NC

characteristic DM1 splicing abnormalities are rescued by BNA

gapmers. Differentiated DM1

TeloMyoD fibroblasts recapitulate splicing defects observed in DM1 skeletal muscle. Treatment of NC

these cells with 30nM of BNA

CAG or DMPK gapmers significantly reverted splicing of ALPK3,

MBNL1, and KIF13A towards the pattern in non-DM1 cells (Figure 4D). The splicing rescue for MBNL1 and KIF13A was robust while rescue was more moderate for the less severely affected event ALPK3. The level of splicing rescue for these events is notable, especially given the residual number of about 1–3 foci per cell after gapmer treatment. This suggests that decreased


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foci load can be sufficient to revert splicing without complete degradation of all RNA foci and NC

demonstrates that BNA

gapmers potently revert splicing defects in DM1 cells. NC

In conclusion, we have shown that BNA

gapmers are a promising therapeutic option to NC

target toxic RNA in myotonic dystrophy. The allele-specific knockdown achieved with the BNA

DMPK, but not CAG, gapmers indicates targeting a non-repetitive region improves preferential knockdown of the expanded allele. Determining the mechanism responsible for differences in NC

caspase induction between LNA and BNA

CAG gapmers is a key area of interest and will aid in

overcoming toxicity concerns for future gapmer-based therapies.

METHODS NC

Antisense oligonucleotides. LNA and BNA

ASOs were purchased from Exiqon and Bio-

Synthesis, respectively.

Cell models and assays SB TeloMyoD (control, non-DM1) and KB TeloMyoD (DM1, 400 CTG repeats) immortalized human fibroblast cell lines express telomerase and contain a tetracycline-inducible MyoD to promote the myogenic program in response to growth for 6 days in low serum media (1% FBS) supplemented with 1 µg/ml doxycycline.(26,27) Toxicity analyses were conducted using the ApoTox-Glo Triplex Assay (Promega). Additional assay details and RT-PCR primers are listed in Supplementary Data and Table S2.

Microscopy TeloMyoD fibroblasts were electroporated with 30nM of gapmers, differentiated for 6 days and visualized by RNA FISH using the (CAG)5 TYE 563 LNA probe (CAGCAGCAGCAGCAG) or by IF/FISH using MBNL1 (Santa Cruz, sc-47740). For further details see Supplementary Data.

Statistical analysis Data is expressed as mean ± standard deviation. All data shown is the summary of three or more biological replicates and statistical analyses were completed in Prism 7. For datasets where three or more groups were analyzed simultaneously, one-way ANOVA was used with ungrouped data and two-way ANOVA was used with grouped data and were corrected for multiple comparisons. Statistical values used: *P

BNANC Gapmers Revert Splicing and Reduce RNA Foci with Low

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