Fluorescence Imaging of Huntingtin mRNA Knockdown - American

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Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

Fluorescence Imaging of Huntingtin mRNA Knockdown Eunseon Oh,¶,# Yuhong Liu,# Mahesh V. Sonar,§ Diane E. Merry, and Eric Wickstrom* Department of Biochemistry & Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, United States ABSTRACT: Huntington’s disease (HD) is an autosomal-dominant neurodegenerative genetic disorder caused by CAG repeat expansion in exon 1 of the HTT gene. Expression of the mutant gene results in the production of a neurotoxic polyglutamine (polyQ)-expanded huntingtin (Htt) protein. Clinical trials of knockdown therapy of mutant polyglutamine-encoding HTT mRNA in Huntington’s disease (HD) have begun. To measure HTT mRNA knockdown effectiveness in human cells, we utilized a fluorescent hybridization imaging agent specific to the region encompassing the human HTT mRNA initiation codon. We designed, synthesized, purified, and characterized Cal560-spacer-peptide nucleic acid (PNA)-spacer-IGF1 tetrapeptides. The human HTT PNA 12mer complement was CATGGCGGTCTC, while the rat htt equivalent 12mer contained the sequence CATGaCGGcCTC, with two bases differing from the human sequence. The cyclized IGF1 tetrapeptide fragment D(CSKC) that promotes IGF1 receptor-mediated endocytosis was bonded to the C-terminus. We tested the reliability of HTT mRNA imaging with Cal560-spacer-peptide nucleic acid (PNA)-spacer-IGF1 tetrapeptides in human embryonic kidney (HEK) 293T cells that express endogenous HTT and IGF1 receptor. By qPCR, we quantitated HTT mRNA in HEK293T cells with and without HTT mRNA knockdown by three different siRNAs. By confocal fluorescence imaging, we quantitated the accumulation of fluorescent HTT hybridization agent in the same cells. A rat homologue differing from the human sequence by two bases showed negligible fluorescence. qPCR indicated 86 ± 5% knockdown of HTT mRNA by the most effective siRNA. Similarly, Cal560-HTT PNA-peptide fluorescence intensity indicated 69 ± 6% reduction in HTT mRNA. We concluded that the fluorescence hybridization method correlates with the established qPCR method for quantitating HTT mRNA knockdown by siRNA in HEK293T cells, with a Pearson correlation coefficient of 0.865 for all three siRNA sequences. These results will enable real time imaging and quantitation of HTT mRNA in animal models of HD.

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INTRODUCTION Huntington’s disease (HD) is an autosomal-dominant neurodegenerative genetic disorder that is caused by the expansion of a polyglutamine-encoding CAG repeat within exon 1 of the huntingtin HTT gene.1 HD is a fatal disorder that gradually damages brain cells and causes muscle uncoordination, cognitive decline, and psychiatric problems.2 Striatal medium spiny neurons and cortical neurons are the primary neuronal substrates for disease symptoms, although other cell types, both neuronal and non-neuronal, contribute to the disease phenotype.3,4 Htt protein is expressed in all human and mammalian cells, including cells of the central nervous system and peripheral tissues, and occurs at particularly high levels in the brain.5−7 The HTT CAG repeat is polymorphic in normal individuals, ranging in number from 6 to 35, while in affected individuals, the number of CAG repeats exceeds 37, with expansions as long as 200 observed in some early onset cases.2 The CAG expansion leads to production of a mutant, polyglutamineexpanded Htt protein, which is characterized by misfolding and aggregation.8 Currently, therapies for HD include antidepressants and antipsychotics for psychiatric symptoms, and tetrabenazine or its deuterated form to treat choreic movements,9,10 but there is no effective therapy to prevent or slow © XXXX American Chemical Society

the progression of HD. Finding therapeutic agents to slow down or stabilize HD is thus a critical unmet need. In recent years, investigators have applied various strategies to reduce HTT mRNA. Striatal injections of adeno-associated virus (AAV) expressing either a short hairpin RNA (shRNA)11 or an artificial microRNA (miRNA) expression scaffold, mi2.4,12 as well as intraventricular delivery of antisense oligonucleotides (ASOs) that target cellular mRNA transcripts via complementary base pairing13,14 have resulted in Htt protein knockdown efficacy in animal models of HD. Furthermore, the striatal infusion of synthetic small interfering RNA (siRNA) duplexes with cholesterol-conjugates attenuated neuronal pathology, and delayed the abnormal behavioral phenotypes in transgenic mouse models.15 The development of therapeutic strategies that utilize HTT silencing requires appropriate, proximate biomarkers to ensure target engagement. The levels of mutant Htt protein or mutant HTT mRNA represent important, proximate biomarkers to determine the efficacy of experimental knockdown therapies. Encouraging results from HTT knockdown studies in mice13,14 Received: January 18, 2018 Revised: February 13, 2018 Published: February 16, 2018 A

DOI: 10.1021/acs.bioconjchem.8b00048 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 1. Structure of human Cal560-AEEA-HTT PNA-AEEA-IGF1 tetrapeptide, HsHTT.

established a basis for a human trial with the same ASO.16 Thus, the current human trial carries a requirement for a surrogate marker to determine efficacy of the therapeutic HTT knockdown agent in live patients in real time during therapy. An immunoassay of mutant Htt protein in cerebrospinal fluid withdrawn by spinal tap17 has been utilized in the current human trial. In an effort to develop a noninvasive assay to determine the efficacy of HTT mRNA knockdown, we designed a fluorescent HTT mRNA hybridization agent (Figure 1) to enable imaging and quantitation of HTT mRNA in Htt-expressing18 HEK293T cells. We conceived and pioneered PET imaging of mRNA levels in tumors19 and fluorescence imaging of mRNA levels in cells, which showed equivalent results in both live and fixed cells.20,21 The objective of the present study was to develop an HTT mRNA imaging agent composed of a complementary peptide nucleic acid (PNA) labeled on the N-terminus with a fluorescent dye for imaging, and conjugated on the C-terminus to a peptide moiety for receptor-mediated intracellular delivery (Figure 1). A PNA is an oligonucleotide analog for which the sugarphophodiester backbone is replaced with a peptide-like aminoethylglycine backbone; PNAs have great potential for biomedical applications.22 Owing to their achiral, uncharged, flexible backbone, PNAs hybridize with mRNA more strongly and specifically than do normal RNAs or DNAs with mRNA.23 They are resistant to enzymatic degradation and are stable over a wide range of pH. However, due to their uncharged nature, naked PNAs are poorly taken up by mammalian cells.24 As a result, the challenge of delivering PNA imaging agents into targeted cells must be overcome. One approach to the delivery of PNA imaging agents to cells utilizes the incorporation of a cell penetrating peptide.25 An alternative approach to target imaging agents specifically to HTT-expressing neuronal cells, and to enable efflux of unbound agents, is to incorporate a peptide ligand that enables receptormediated cellular uptake. We previously showed that inclusion of a cyclized tetrapeptide fragment of insulin-like growth factor (IGF1) (Figure 1) enabled IGF1 receptor-mediated cellular uptake20 and cytoplasmic release from endosomes,21 which was blocked in mice by excess IGF1.26 Radiolabeled or fluorescent agents are taken up by cells overexpressing high levels of the target receptor at a million or more copies per cell. In contrast, the mRNA targets are overexpressed at thousands of copies per cell. As a result, the target mRNAs are saturated with reporter agents. After excess reporter agents have effluxed, the remaining hybridized radiolabeled or fluorescent agents report the level of

the target mRNAs.19 Moreover, we previously found that reporter-PNA 12mers with a C-terminal IGF1 tetrapeptide stay in circulation by complexing with IGF1 binding proteins.27 Here we report the synthesis, characterization, and cellular uptake of human Cal560-HTT PNA-IGF1 tetrapeptide in tumorigenic human embryonic kidney HEK293T cells. In addition, we evaluate the ability of this imaging moiety to report on altered HTT mRNA levels in response to HTT siRNA knockdown. The initiation codon target region of the HTT mRNA is the same for both wild type and excess CAG mutants. Therapeutic agents in the clinic similarly knock down both wild type and mutant HTT mRNA.

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RESULTS AND DISCUSSION Cal560-HTT PNA-Peptide Synthesis. Fluorescent hybridization agents (Table 1) were synthesized at the 10 μmol scale

Table 1. Fluorophore-PNA-Peptide Sequencesa name HsHTT Rrhtt

sequence Cal560-AEEACATGGCGGTCTC-AEEAD(CSKC) Cal560-AEEACATGaCGGcCTC-AEEAD(CSKC)

calc. mass

exp. mass

4463.62 Da

4463.39 Da

4432.61 Da

4432.38 Da

a

HsHTT complementary to initiation codon domain, nt 308−319 from NM_002111.7, 13669 nt. Rat. RrHTT differences from the human HTT sequence are shown in bold lowercase.

by solid phase synthesis. Representative reversed phase HPLC (Figure 2) and MALDI-TOF MS (Figure 3) profiles are shown for the human HTT mRNA agent, HsHTT. Measured masses of each agent are shown in Table 1. Validation of Cal560-HTT PNA-D(CSKC) as a Specific Imaging Agent for HTT mRNA. Preliminary experiments with a fluorophore-PNA-HIV Tat cell-penetrating peptide revealed excellent cellular uptake, regardless of cell type or PNA sequence (not shown). However, although internalization via the Tat cell penetrating peptide was efficient, little or no efflux of unbound agent occurred. Thus, the use of the Tat cellpenetrating peptide precluded mRNA-specific imaging. Therefore, in order to direct cytoplasmic uptake by human IGF1 receptor, as described,19 an IGF1 tetrapeptide was included at the C-terminus of the PNA sequence. IGF1R is expressed in the cortex and striatum,28−30 and in HEK293 cells.31 For visualization, a Cal560 fluorophore was included at the Nterminus (Figure 1). HEK293T cells were incubated with 100 B

DOI: 10.1021/acs.bioconjchem.8b00048 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 2. Representative analytical HPLC of H. sapiens Cal560-HTT PNA-D(CSKC) fluorescent imaging agent on a 10 × 250 mm Microsorb C18 column, eluted with a 35 min gradient from 0% to 60% CH3CN in aqueous 0.1% CF3CO2H, at 1 mL/min, λ = 260 nm, at 50 °C.

Figure 3. Representative MALDI-ToF mass spectrum of H. sapiens Cal560-HTT PNA-D(CSKC) fluorescent imaging agent. Calculated exact mass: 4463.62 Da; Experimental mass: 4463.39 Da (M + H).

Figure 4. Fluorescent HTT imaging agent cellular uptake. HEK293T cells were incubated for 4 h at 37 °C (Left) with 100 nM HsHTT (H. sapiens Cal560-HTT PNA-D(CSKC) or (Right) 100 nM RrHTT (R. rattus Cal560-htt PNA-D(CSKC). Cells were fixed and imaged by an individual blinded to the treatment condition using a Leica DMR fluorescence microscope. Fields used for imaging were selected based on viewing DAPI-stained nuclei with a blue filter and all images within an experiment were collected at the same exposure.

vealed specific uptake of the human HTT agent into the cytoplasm, but not the nucleus (Figure 4). In contrast, incubation with 100 nM Cal560-Rrhtt PNA-IGF1 tetrapeptide

nM Cal560-HsHTT PNA-IGF1 tetrapeptide in culture medium for 2 or 4 h at 37 °C to allow uptake while enabling efflux of unbound hybridization agent. Fluorescence microscopy reC

DOI: 10.1021/acs.bioconjchem.8b00048 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Table 2. Unique to Human HTT siRNA 27mer Duplexes type

27mer siRNA guide strands

location, nta

A B C NTC

rGGAUAGUAGACAGCAAUAACUCGGT rGGGAUGUAGAGAGGCGUUAGUGGGC rCCUGUUACAACAAGUAAAUCCUCAT rAUACGCGUAUUAUACGCGAUUAACGAC

3′UTR, 12836−12860 3′UTR, 10534−10558 Coding, exon 28, 3974−3998 Origene #SR302082

a

From NM_002111.7, 13669 nt; A = adenosine-3′-phosphate, 3′-adenylic acid; C = cytidine-3′-phosphate, 3′-cytidylic acid; G = guanosine-3′phosphate, 3′-guanylic acid; T = ribothymidine-3′-phosphate, 3′-ribothymidylic acid; U = uridine-3′-phosphate, 3′-uridylic acid.

Figure 5. siRNA knockdown of HTT mRNA in HEK293T cells. Each bar represents the average of three replicates in each of 3 experiments. The two-tailed unpaired t test results are indicated by the asterisks (*for p < 0.05; ** for p < 0.01). Error bars represent SEM.

displayed a weak fluorescent signal (Figure 4). The weak fluorescence data imply that the two base difference between the Rrhtt rat probe and the human HTT mRNA resulted in poor hybridization of the Rrhtt rat probe to the human HTT mRNA. qPCR of HTT mRNA Following siRNA Knockdown. The trisilencer-27 siRNA kit containing three Dicer-Substrate duplexes (A, B, C) and a scrambled nontargeting control (NTC) (Table 2) were used to determine the most effective HTT mRNA knockdown reagent. No additional HTT gene expression was needed in the HEK293T cells because HTT is constitutively expressed in these cells. siRNA transfection efficiency was determined using the cotransfected plasmid CMV-EGFP-C1. Following siRNA transfection, HEK293T cells were incubated for an additional 48 h, at which time, a portion of the cells were harvested to determine HTT mRNA levels. The combined results of three independent siRNA knockdown experiments are shown in Figure 5. The percent knockdown of HTT mRNA for the type A siRNA was 85.7 ± 4.5% (Table 3). Three independent experiments confirmed that the type A siRNA duplex most effectively reduced the level of HTT mRNA in HEK293T cells (p < 0.01). Fluorescence Imaging of HTT mRNA Following siRNA Knockdown. After cells were transfected with siRNA knockdown sequences (Table 2) for 48 h, a portion of the

cells was replated and incubated with match (HsHTT) and mismatch (Rrhtt) PNA agents (Table 1), in order to determine if changes in the PNA fluorescence intensity correlated with HTT RNA levels determined by qPCR. Fluorescence images of HEK293T cells transfected with control (NTC) or specific (A, B, C) siRNAs (Table 2) were acquired after incubation with the human match hybridization agent, HsHTT, or the rat mismatch agent, Rrhtt. Five images were collected from each of three replicate slides in each of three experiments for image analysis and quantification. Six representative images of cells transfected with NTC or type A siRNA and incubated with HsHTT or Rrhtt are shown in Figure 6. These results illustrate both the efficacy of type A siRNA and the specificity of the HsHTT imaging agent. Quantification of these and additional images was carried out using ImageJ and the results are shown in Figure 7 and Table 4. Analysis of the qPCR results (Figure 5) revealed that the type A siRNA knockdown duplex produced the lowest HTT mRNA level out of the three duplexes. Similarly, quantification (Figure 7) of fluorescence images, acquired following treatment with HsHTT PNA imaging agents, revealed that, while all siRNA types produced a statistically significant reduction in PNA intensity, the type A siRNA resulted in the most substantial reduction, similar to what was observed by qPCR (Figures 6, 7). Finally, in order to determine if Cal560-PNA-IGF1 tetrapeptide fluorescence imaging could be used as a proxy for direct RNA knockdown analysis by all three siRNA types, we determined the Pearson correlation between the fluorescence and qPCR data sets. This analysis revealed a strong correlation between PNA fluorescence intensity and HTT mRNA levels for all three siRNAs (0.865), further supporting the development of this approach to quantify human HTT mRNA levels.

Table 3. Average Knockdown Extent of Three HTT siRNAsa type

%knockdown

A B C

85.7 ± 4.5 72.5 ± 9.5 57.6 ± 3.9

a

Results of three independent siRNA knockdown experiments. Each sample out of 24 was loaded in triplicate. D

DOI: 10.1021/acs.bioconjchem.8b00048 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 6. Fluorescence images of HEK293T cells transfected with siRNA, then incubated with Cal560-PNA-peptide. Left: NTC siRNA and human HsHTT. Center: NTC siRNA and rat Rrhtt differing from human by two bases. Right: HTT siRNA A and human HsHTT.

and remain long enough to be detected by fluorescence microscopy. Elevated fluorescence intensity was expected to reflect elevated HTT mRNA copy number in the cytoplasm to bind with the introduced fluorescent agents, as we previously observed for oncogene mRNAs19 and MAOA mRNA.21 We found that the strongest siRNA sequence, type A, knocked down HTT mRNA in HEK293T cells by 86 ± 5%. In parallel, we observed 69 ± 6% reduction in cellular fluorescence intensity in type A siRNA-treated cells. These results suggest that quantitative fluorescence imaging of HTT mRNA in therapeutic animal models of HD before treatment and longitudinally during treatment can likely be achieved. Therapeutically, siRNA knockdown shows its limitations in these results, compared with the clinical success by singlestranded RNA analogs.16 Ultimately, these fluorescence hybridization results suggest that PET imaging of HTT mRNA in HD patients undergoing HTT knockdown therapy could provide a noninvasive approach to quantitating this important proximate biomarker.

Figure 7. Average cell fluorescence intensities in HEK293T cells transfected with siRNAs, then incubated with human fluorescent agent HsHTT. Image analysis of gray value data for fluorescence intensity was performed by using a one-way ANOVA, indicated by asterisks (* for p < 0.05; **for p < 0.01). Error bars represent SEM.

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Table 4. Percent reduction of cellular fluorescence intensity in siRNA-treated cells siRNA type

A

B

C

Average

69 ± 6%

42 ± 20%

54 ± 10%

EXPERIMENTAL PROCEDURES Fluorescent Hybridization Agent Synthesis. Cal560HTT PNA-peptides (Figure 1) (Table 1) were synthesized, purified, and analyzed as described.32 Briefly, Fmoc-D-amino acids (Novabiochem, San Diego, CA) composing the Nterminal peptide, Fmoc-AEEA linkers, and Fmoc-PNA monomers (Link Technologies, Bellshill, Scotland), and were first activated by O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (Applied Biosystems, Foster City, CA), followed by coupling onto 0.2−0.3 mmol/g 9-Fmoc-amino-xanthen-3-yloxy NovaSyn TG Sieber resin (Novabiochem) on a PS3 peptide synthesizer (Protein Technologies, Tucson, AZ). Then, the D-cysteine residues (where present) were deprotected, followed by cyclization with 0.1 M I2 in (CH3)2NCHO for 4 h on solid phase. After thorough rinsing of I2 from the resin, Cal560 carboxylic acid (Biosearch Technologies, Petaluma, CA) was activated with HATU and coupled to the N-terminal AEEA amine in the synthesizer. Following assembly, the Cal560-HTT PNA-peptides were deprotected and cleaved at room temperature with 85%

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CONCLUSIONS We quantitated HTT mRNA in HEK293T cells by qPCR with and without HTT siRNA knockdown, and also quantitated the accumulation of fluorescent human HTT hybridization agent in the same cells by fluorescence microscopic imaging. We asked whether the fluorescence intensity of treated cells was significantly different between the human HTT agent and the rat htt agent that differed by two bases, and whether the agents were stable in the cells. First, the rat htt agent that differed by two bases should not hybridize stably enough to maintain its localization in the cytoplasm, even if the agent penetrates into the cells. Cells use passive and active transport mechanisms to import and export materials between cells and their environment. The unbound agents likely efflux by active transport activities.21 The HsHTT PNA agent that hybridizes with HTT mRNA should be stable E

DOI: 10.1021/acs.bioconjchem.8b00048 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry CF3CO2H, 5% CH2Cl2, 9.5% m-cresol, and 0.5% Et3SiH. Finally, the deprotected conjugates were purified by liquid chromatography on an Alltima reversed phase C18 column (Alltech, Grace, Deerfield, IL) and analyzed on a 4800 matrixassisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) (Applied Biosystems, Foster City, CA) from an α-cyano-hydroxycinnamic acid matrix. Cell Culture. We used HEK293T cells (American Type Culture Collection, Manassas, VA), which express both HTT mRNA18 and IGF1R,31 for in vitro studies of agent uptake. Thus, no further HTT DNA construction, transfection, or transformation was necessary for the cell culture model. HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 50 U/mL penicillin, 5 μg/mL streptomycin, and 2 mM glutamine, all from Thermo Fisher (Springfield Township, NJ) incubated at 37 °C in humidified air containing 10% CO2, according to the ATCC protocol. siRNA Knockdown of HTT mRNA. HEK293T cells grown in six-well plates (Thermo Fisher) were transfected with 2 μg/ well of three unique 27mer siRNAs (#SR302082, OriGene Technologies, Rockville, MD) targeting human HTT mRNA, NM_002111.7 (Table 2), or nontargeting scrambled control siRNA (NTC, #SR30004) using Lipofectamine 2000 (Life Technologies, Carlsbad, CA). Two μg/well of CMV-EGFP-C1 plasmid (Genentech, South San Francisco, CA) were included with the siRNAs to monitor the transfection rate. Cells were incubated at 37 °C for 48 h after transfection, then trypsinized, pelleted, washed with PBS, resuspended, and titered. Five × 104 cells were transferred to 24-well plates with poly(D-lysine) coated cover glasses (Thermo Fisher) for fluorescent hybridization uptake. The rest of the cells were left in the wells for RNA isolation. HTT Fluorescent Hybridization Agent Uptake. 48 h after transfection with siRNAs and indicator plasmids, the cells grown on coverslips were treated with 100 nM Cal560-PNApeptides (Table 1) and incubated at 37 °C for 4 h. Treated cells were washed twice for 15 min with culture medium at 37 °C, then twice for 5 min with DPBS at room temperature. The cells were then fixed with 4% paraformaldehyde followed by three washes in DPBS. Nuclei were stained with Hoechst 22242 fluorescent stain (#28491-52-3, Thermo Fisher) for 10 min, followed by three 5 min washes with DPBS. Coverslips were mounted with Vectashield (Thermo Fisher) onto the glass slides. Three replicate treatments were carried out with both 100 nM and 200 nM Cal560-PNA-peptides. Quantitative Polymerase Chain Reaction for HTT mRNA. RNA was isolated from treated cells as described.19 Briefly, 48 h post-transfection, cells from each well were homogenized with 1 mL Trizol reagent (#15596-026, Life Technologies) per well, then stabilized against RNase activity by addition of 0.2 mL CHCl3, homogenized again, and sedimented. RNA in the aqueous phase was precipitated by addition of 0.5 mL of 100% isopropanol, then sedimented. The RNA pellet was washed with 1 mL of 75% EtOH, 25% water, then redissolved in 50 μL RNase-free water (Ambion, Austin, TX) at 55 °C for 15 min, and quantitated by A260. Single stranded cDNA was synthesized from 1 μg total RNA using a Taqman High Capacity cDNA Reverse Transcription Kit (#4368814, Applied Biosystems) by combining 2× reverse transcription (RT) master mix and total RNA with random primers in a volume of 20 μL reaction. Reverse transcription was performed for 2 h at 37 °C in a thermocycler. To

determine relative gene expression, 5′-FAM, 3′-TAMRA probebased fluorescence qPCR was performed on the cDNA using Gene Expression Assays probes for human HTT (#Hs00169273_m1, Applied Biosystems). Duplication cycle thresholds were normalized to a human GAPDH primer/probe set for determination of HTT mRNA knockdown, vs the experimental control probe. Fluorescence Imaging of HTT mRNA. Slides were mounted with Vectashield after PNA treatment and visualized by fluorescence microscopy (Leica DMRXA, #C8484-03). To define the nuclear or cytoplasmic localization of PNA:mRNA complexes, images were collected using an oil 60× objective lens. Images were captured with a Hamamatsu digital camera. The images that revealed an intact plasma membrane and cytosol free of bright puncta were used for quantification using Ivision software (Biovision Technologies, Exton, PA). Five images were acquired from each of 3 replicates for each treatment. The NTC sample was imaged first in each experiment in order to set the exposure times for rest of the siRNA treated samples. All images within a treatment were acquired with identical exposure times. Fluorescence intensities of imaged cells were measured and quantified using ImageJ (National Institutes of Health, Bethesda, MD) by the gray mean value (red images) obtained with the freehand selection tool. Ten background measurements were taken from each image to subtract an average background. Statistical Analysis. qPCR data were normalized to HTT mRNA levels in samples treated with the nontargeting control. Comparative levels were statistically evaluated using two-tailed unpaired Student’s t test. Analysis of fluorescent image intensity was performed by using one-way ANOVA. Post-hoc analyses were performed to assess for significant differences between individual groups. Pearson’s correlation coefficient was determined from the qPCR and fluorescence quantification data obtained in 3 independent experiments, for all three siRNAs.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Eric Wickstrom: 0000-0001-7729-640X Present Addresses ¶

Spark Therapeutics, Philadelphia, PA 19104 Innovassynth Technologies, Khopoli, India 410203

§

Author Contributions #

Eunseon Oh and Yuhong Liu contributed equally to the work.

Notes

The authors declare the following competing financial interest(s): We wish to inform the Editor of potential conflicts of interest. Prof. Eric Wickstrom holds shares in GeneSeen LLC, which might ultimately benefit from the results of this investigation, but did not support the work.

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ACKNOWLEDGMENTS This work was supported by funding from the Hereditary Disease Foundation to E.W. E.W. holds shares in GeneSeen LLC, which might ultimately benefit from the results of this investigation; however, it did not support the work. F

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ABBREVIATIONS AEEA, aminoethoxyethoxyacetyl; DAPI, 4′,6-diamidino-2phenylindole; HTT, huntingtin; PBS, phosphate buffered saline; PNA, peptide nucleic acid

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REFERENCES

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DOI: 10.1021/acs.bioconjchem.8b00048 Bioconjugate Chem. XXXX, XXX, XXX−XXX