Fluorescence Imaging of Huntingtin mRNA Knockdown

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Fluorescence Imaging of Huntingtin mRNA Knockdown Eunseon Oh, Yuhong Liu, Mahesh V. Sonar, Diane Merry, and Eric Wickstrom Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00048 • Publication Date (Web): 16 Feb 2018 Downloaded from http://pubs.acs.org on February 17, 2018

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Bioconjugate Chemistry

Fluorescence Imaging of Huntingtin mRNA Knockdown

Eunseon Oh¶*, Yuhong Liu*., Mahesh V. Sonar§, Diane E. Merry, and Eric Wickstrom1

Department of Biochemistry & Molecular Biology Thomas Jefferson University, Philadelphia, PA 19107

* These authors contributed equally to the work. 1

Corresponding author. Email: [email protected]

¶

Current address: Spark Therapeutics, Philadelphia, PA 19104

§

Current address: Innovassynth Technologies, Khopoli, India 410203

Keywords: fluorescence, Huntington’s disease, hybridization, IGF1 receptor, peptide nucleic acid Running Title: Fluorescence Imaging of HTT mRNA Knockdown Abstract: 288 words

Body text: 3097 wd

References: 32

Tables/Figures: 11

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Fluorescent HTT mRNA Imaging

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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 homolog 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, polyglutamine-expanded 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 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


Comment [DM1]: I had asked previously about the text that I deleted below. In lieu of sending it back to you, I’ve deleted it. If you would like to keep it in, perhaps you can edit it in some way. Its inclusion does not seem fully justified to me.

Bioconjugate Chemistry 1 2 3 4 5 6 7 Fluorescent HTT mRNA Imaging 4 Oh, Liu, et al. 8 results from HTT knockdown studies in mice13, 14 established a basis for a human trial with the same ASO.16 9 10 11 Thus, the current human trial carries a requirement for a surrogate marker to determine efficacy of the 12 therapeutic HTT knockdown agent in live patients in real time during therapy. An immunoassay of mutant Htt 13 14 protein in cerebrospinal fluid withdrawn by spinal tap17 has been utilized in the current human trial. 15 In an effort to develop a noninvasive assay to determine the efficacy of HTT mRNA knockdown, we 16 17 18 designed a fluorescent HTT mRNA hybridization agent (Fig. 1) to enable imaging and quantitation of HTT 19 mRNA in Htt-expressing18 HEK293T cells. We conceived and pioneered PET imaging of mRNA levels in 20 21 tumors19 and fluorescence imaging of mRNA levels in cells, which showed equivalent results in both live and 22 23 fixed cells.20, 21 The objective of the present study was to develop an HTT mRNA imaging agent composed of a 24 25 complementary peptide nucleic acid (PNA) labeled on the N-terminus with a fluorescent dye for imaging, and 26 conjugated on the C-terminus to a peptide moiety for receptor-mediated intracellular delivery (Fig. 1). 27 28 29 30 31 32 33 34 35 36 37 38 39 Fig. 1. Structure of human Cal560-AEEA-HTT PNA-AEEA-IGF1 tetrapeptide, HsHTT. 40 41 A PNA is an oligonucleotide analog for which the sugar-phophodiester backbone is replaced with a 42 43 peptide-like aminoethylglycine backbone; PNAs have great potential for biomedical applications.22 Owing to 44 45 their achiral, uncharged, flexible backbone, PNAs hybridize with mRNA more strongly and specifically than do 46 23 47 normal RNAs or DNAs with mRNA. They are resistant to enzymatic degradation and are stable over a wide 48 range of pH. However, due to their uncharged nature, naked PNAs are poorly taken up by mammalian cells.24 49 50 As a result, the challenge of delivering PNA imaging agents into targeted cells must be overcome. 51 52 53 54 55 56 57 58 59 ACS Paragon Plus Environment 60

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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 receptor-mediated cellular uptake. We previously showed that inclusion of a cyclized tetrapeptide fragment of insulin-like growth factor (IGF1) (Fig. 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 by solid phase synthesis. Representative reversed phase HPLC (Fig. 2) and MALDI-TOF MS (Fig. 3) profiles are shown for the human HTT mRNA agent, HsHTT. Measured masses of each agent are shown in Table 1.

Table 1. Fluorophore-PNA-peptide sequences Name

Sequence

Calc. Mass

Exp. Mass

HsHTT

Cal560-AEEA-CATGGCGGTCTC-AEEA-D(CSKC)

4463.62 Da

4463.39 Da

Rrhtt

Cal560-AEEA-CATGaCGGcCTC-AEEA-D(CSKC)

4432.61 Da

4432.38 Da

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.

Fig. 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 50oC.


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Validation of Cal560-HTT PNAD(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,

Fig. 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).

little or no efflux of unbound agent occurred. Thus, the use of the Tat cell-penetrating peptide precluded

Fig. 4. Fluorescent HTT imaging agent cellular uptake. HEK293T cells were incubated for 4 h at 37oC (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.




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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 N-terminus (Fig. 1). HEK293T cells were incubated with 100 nM Cal560-HsHTT PNA-IGF1 tetrapeptide in culture medium for 2 hr or 4 hr at 37°C to allow uptake while enabling efflux of unbound hybridization agent. Fluorescence microscopy revealed specific uptake of the human HTT agent into the cytoplasm, but not the nucleus (Fig. 4). In contrast, incubation with 100 nM Cal560-Rrhtt PNA-IGF1 tetrapeptide displayed a weak fluorescent signal (Fig. 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 tri-silencer-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. Table 2: Unique to human HTT siRNA 27mer duplexes Type

27mer siRNA guide strands

Location, nt*

A

rGGAUAGUAGACAGCAAUAACUCGGT

3’UTR, 12836-12860

B

rGGGAUGUAGAGAGGCGUUAGUGGGC

3’UTR, 10534-10558

C

rCCUGUUACAACAAGUAAAUCCUCAT

Coding, exon 28, 3974-3998

NTC

rAUACGCGUAUUAUACGCGAUUAACGAC

Origene #SR302082

* 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.


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Fig. 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

Fluorescence Imaging of Huntingtin mRNA Knockdown

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