Delivery of antisense oligonucleotides for splice‐correction of androgen receptor pre‐mRNA in castration‐resistant prostate cancer models using cell‐penetrating peptides

Abstract Background Cell‐penetrating peptides (CPPs) are a promising approach for delivering antisense oligonucleotides (AONs) as they form nanosized complexes through noncovalent interactions that show efficient cellular uptake. Previously, we have designed an AON system to correct splicing of the androgen receptor (AR) pre‐mRNA, thereby preventing the generation of the splice variant AR‐V7 mRNA. AON‐mediated knockdown of AR‐V7 resulted in inhibition of androgen‐independent cell proliferation. In this study, we evaluated the CPP‐mediated delivery of this AON into castration‐resistant prostate cancer cell line models 22Rv1, DuCaP (dura mater cancer of the prostate), and VCaP (vertebral cancer of the prostate). Methods Nanoparticles (polyplexes) of AONs and CPPs were formed through rapid mixing. The impact of the peptide carrier, the formulation parameters, and cell incubation conditions on cellular uptake of fluorescently labeled AONs were assessed through flow cytometry. The cytotoxic activity of these formulations was measured using the CellTiter‐Glo cell viability assay. The effectivity of CPP‐mediated delivery of the splice‐correcting AON‐intronic splicing enhancer (ISE) targeting the ISE in the castration‐resistant prostate cancer (CRPC)‐derived 22Rv1, DuCaP, and VCaP cells was determined by measuring levels of AR‐V7 mRNA normalized to those of the human heterochromatin protein 1 binding protein 3 (HP1BP3). Western blot analysis was used to confirm AR‐V7 downregulation at a protein level. The cellular distribution of fluorescently labeled AON delivered by a CPP or a transfection reagent was determined through confocal laser scanning microscopy. Results The amphipathic and stearylated CPP PepFect 14 (PF14) showed higher uptake efficiency than arginine‐rich CPPs. Through adjustment of formulation parameters, concentration and incubation time, an optimal balance between carrier‐associated toxicity and delivery efficiency was found with a formulation consisting of an amino/phosphate ratio of 3, 0.35 μM AON concentration and 30 min incubation time of the cells with polyplexes. Cellular delivery of AON‐ISE directed against AR pre‐mRNA achieved significant downregulation of AR‐V7 by 50%, 37%, and 59% for 22Rv1, DuCaP, and VCaP cells, respectively, and reduced androgen‐independent cell proliferation of DuCaP and VCaP cells. Conclusions This proof‐of‐principle study constitutes the basis for further development of CPP‐mediated delivery of AONs for targeted therapy in prostate cancer.


| INTRODUCTION
Patients with advanced prostate cancer have a 5-year relative survival rate of 30%. 1 Current therapy, directed to inhibit the androgen/androgen receptor signaling axis, only achieve modest survival benefits as the disease develops soon into a hormonerefractory state known as castration-resistant prostate cancer (CRPC). The elevated expression of C-terminally truncated androgen receptor splice variants has been described as a mechanism of CRPC progression. Variants such as AR-V7 can act as constitutively active transcription factors, promoting androgenindependent tumor growth. 2,3 Previously, we designed two antisense oligonucleotides (AONs) to target splicing enhancers within the AR pre-mRNA, responsible for the generation of an AR-V7 transcript. AON-mediated inhibition of AR-V7 generation resensitized cells to androgen depletion, inducing apoptosis of diverse CRPC cell line models. 4 Translation of AON-based therapeutic approaches into an in vivo application requires additional tailoring to ensure specific cellular targeting, sufficient cellular uptake and endosomal release, and to prevent a rapid clearance by the body. 5 Cationic cell-penetrating peptides (CPPs) spontaneously associate with AONs into polyplexes that yield cellular uptake, both in vitro and in vivo. 6,7 The amphipathic CPP PepFect 14 (PF14) has shown superior activity in the cellular delivery of diverse oligonucleotides due to its capacity to induce endosomal release. 8,9 This study first demonstrates that PF14 outperforms the two stereoisomers of nona-arginine and the human lactoferrin-derived peptide (hLF) 10,11 concerning uptake efficiency. We next defined the optimal conditions to increase uptake efficiency while minimizing CPP-associated toxicity. Polyplexes of PF14 and the splicingcorrecting AON-intronic splicing enhancer (ISE) resulted in a significant downregulation of AR-V7 mRNA levels in three different CRPC cell lines, which was accompanied by a decrease in cell viability under castrate androgen conditions. These results demonstrate the feasibility of using CPPs such as PF14 to complement AON technology for targeting prostate cancer cells.

| AONs
An RNA AON of 22 nucleotides (AON-ISE) and a control sense oligonucleotide (SON-ISE) were previously described. 4 Both oligonucleotides were modified with a phosphorothioate backbone and 2′-O-methyl groups at the ribose (Biolegio). The oligonucleotides were dissolved in ultrapure water. The 5' Cy3-or Alexa Fluor 568 (AF568)labeled AON Luc-S-oligo, previously described, were used for microscopy and flow cytometry. 12

| CPPs and polyplex formulations
The peptides PF14 (Stearyl-AGYLLGKLL-Orn-Orn-LAAAAL-Orn-Orn-L-L-NH 2 , Orn corresponding to ornithine), hLF derived from human lactoferrin (Ac-KCFQWQRNMRKVRGPPVSCIKR-NH 2 ) and nona-arginine (L)-R9 (Ac-RRRRRRRRR-NH 2 ) and the D-enantiomer (D)-r9 (Ac-rrrrrrrrr-NH 2 ) were purchased from EMC microcollections. The noncovalent peptide complexes (or polyplexes) of CPPs and AONs were formed based on the amino/ phosphate (N/P) ratio, representing the ratio between the positively charged peptide side-chain amino groups (N = nitrogen) and the negatively charged phosphorothioate (P) groups in the backbone of the AON. Polyplexes were generated by diluting the peptide and AON with ultrapure water at 10x their final concentration and pipetting equal volumes of both solutions simultaneously against the wall of a polypropylene 1.5 ml centrifuge tube. Before cell incubation, the polyplexes were incubated at room temperature for 1 h.

| Dynamic light scattering
Size measurements of polyplexes were performed on a Zetasizer Nano S, using a HeNe laser with 4 mW, 633 nm. Polyplexes were diluted to a concentration of 0.2 µM with respect to the oligonucleotide in ultrapure water. For the determination of size, three technical replicates were performed per sample with an automatic selection for the number of runs. For size measurements, the backward scatter was used. The data analysis was carried out with Ze-taSizer software 7.03.

| RNA isolation and RT-PCR
For gene expression, 140,000 22Rv1, DuCaP, or VCaP cells were seeded per well of a 24-well plate 1 day before the experiments and cultured in CSS-containing medium. Four days after treatment with polyplexes or X-tremeGENE 9 transfection, the cell culture medium was removed, and total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The concentration and purity of the RNA were determined on a Nanodrop-1000 spectrophotometer (Thermo Scientific). Subsequently, 2 μg of total RNA was treated with DNaseI and used to synthesize complementary DNA (cDNA) using random hexamer primers and Su-perScript II Reverse Transcriptase (Invitrogen). Real-time PCR Results were reproduced in two independent experiments.

| Statistical analysis
For cell viability assays with 22Rv1, DuCaP, and VCaP cells, data are presented as means ± SEM from at least three independent experiments with three technical replicates. Two-tailed unpaired t tests were performed using GraphPad Prism 7 (GraphPad Software).
Pearson correlation coefficients were used to determine the relationships between relative gene expression profiles, considering a 95% confidence interval. A p-value of <0.05 was considered statistically significant and p < 0.05 is represented by one star (*) and p < 0.01 is represented by two stars (**).

| Optimal parameters for PF14-mediated delivery
To determine the optimal conditions for the delivery of AONs by PF14, three different parameters were evaluated, which were the formulation N/P ratio, the concentration of oligonucleotide and the for all formulation conditions. An N/P ratio of 1 was the least efficient in promoting uptake and an N/P ratio of 3 the most efficient, outperforming even X-tremeGENE 9-mediated transfection ( Figure 2B). Of note, AON/PF14 polyplex size was hardly affected when using different N/P ratios but was surprisingly different between AON and SON ( Figure 2C). Altogether, to ensure targeting of a high number of cells using a minimal quantity of peptide and oligonucleotide and to minimize toxicity, an N/P ratio of 3, an oligonucleotide concentration of 0.35 μM, and an incubation time of 30 min were determined as the most optimal parameters for testing of oligonucleotide activity.

| Intracellular distribution of PF14 polyplexes
By using an AF568-labeled AON, we evaluated the cellular uptake and intracellular distribution of the delivered AON. In line with our flow cytometry data, both X-tremeGENE 9 and PF14 yielded high intracellular fluorescence. In both cases, fluorescence was mostly present in large punctate structures, most likely corresponding to endosomes. For X-tremeGENE 9 the major part of cells also showed a clearly discernible nuclear staining. By comparison, for PF14, although in fewer cells, a homogenous fluorescence inside the nuclei was also present (Figure 3).
Nuclear localization of the oligonucleotides is essential for splicecorrecting AONs as they target pre-mRNA molecules.

| Splice-correcting activity of PF14delivered AONs
Following a demonstration of uptake, next we were interested in assessing the activity of delivered AONs. It is well-established that cellular uptake as such is no predictor of activity as AONs need to be released into the cytosol. 9,16,17 The antisense oligonucleotide AON-ISE, designed to target an ISE present in the AR pre-mRNA and to prevent the synthesis of an AR-V7 transcript, was used to form polyplexes with PF14 using the optimal parameters described earlier.  Figure 4C). Consistent with our previous study, 4 treatment with AON-ISE did not reduce full-length AR levels, highlighting the specificity of our AON system ( Figure 4D).
Lastly, AR-V7 is able to promote androgen-independent cell proliferation, 18 which is inhibited upon knockdown of this variant. 4 Therefore, effective delivery of this AON into the nucleus should reduce the cell viability under androgen-depleted conditions. For PF14-mediated delivery of AON-ISE in DuCaP and VCaP cells this was indeed the case ( Figure 4E). However, the effect on 22Rv1 cells did not reach statistical significance ( Figure 4E). The degree of reduction in cell viability for all PF14 polyplex conditions was lower than for X-tremeGENE 9-AON formulations, which may indicate that PF14 by itself exerts some degree of cell stress, even at concentrations at which no acute toxicity was observed.

| DISCUSSION
CRPC is a late-stage disease with no curative treatment. CRPC tumors often develop several mechanisms to reactivate the androgen/ androgen receptor signaling axis, which make them irresponsive to AR-targeted therapy. One of these mechanisms is the elevated expression of constitutively active androgen receptor splice variants.
Variants such as AR-V7 support androgen-independent tumor growth and its expression can be predictive of therapy failure. 19 Previously, we designed an AON approach to correct splicing of AR pre-mRNA preventing the synthesis of an AR-V7 transcript. 4 Here, we demonstrate that CPP-mediated delivery provides a suitable approach to deliver this AON into prostate cancer cells. of oligoarginine and PF14-mediated AON uptake in myoblasts. In this system, PF14 also yielded more efficient uptake. 9 In addition, only PF14 polyplexes achieved delivery of the AON to the nucleus. Interestingly, this nuclear localization was more pronounced than the one observed in this study.
On average, PF14 and X-tremeGENE 9 yielded the same AON uptake efficiency. However, for X-tremeGENE 9 two cell populations that differed in uptake efficiency were observed, while for PF14 uptake was uniform across the entire cell population with an uptake efficiency that was intermediate with respect to the two populations observed for X-tremeGENE 9. At this point, we cannot conclude whether the higher activity observed for delivery through the latter may be attributed to the cell population showing higher uptake efficiency. Nevertheless, the presence of two populations for the lipidbased delivery agent X-tremeGENE 9 is in line with what we had observed for mRNA delivery. 22 Uptake of naked (gymnos in Greek) phosphorothioate oligonucleotides by cells without the use of a delivery agent is a process known as gymnosis. 23 Gymnosis has been described in different cell types in culture and offered as an alternative approach for difficult-to-transfect cells. In our experiments, treatment with naked AONs and SONs resulted in poor uptake and no effect on AR-V7 mRNA levels or cell viability. Gymnosis has been described for oligonucleotide concentrations from 2.5 to 10 µM, whereas in our experiments CPP formulations and transfection with X-tremeGENE 9 used a maximum concentration of 0.5 µM. These findings suggests that our low oligonucleotide doses may not be sufficient for gymnotic delivery to take place and demonstrating the gain in activity that can be obtained with delivery agents.
With respect to reduction of AR-V7 levels and cell viability, PF14-mediated delivery was less efficient than lipid-based delivery.
Remarkably, the difference in activity was larger for reduction in cell viability than for transcript reduction. This difference may be attributed to the fact that in spite of the absence of acute toxicity, the PF14 nanoparticles by themselves also reduced cell viability to some extent. Another possibility is that for X-tremeGENE 9-mediated delivery, AON activity could mostly be attributed to the subpopulation of cells showing high uptake, while PF14 achieved a lower albeit uniform effect across the entire cell population. Importantly, we showed that the higher activity of a lipid-based formulation such as X-tremeGENE 9 in comparison to PF14 was only present in vitro but not in vivo. 24 Lastly, PF14 has been reported to be amenable to alterations in the charge and fatty acid moiety for augmentation of in vivo gene delivery, 25 as well as to the addition of targeting moieties to improve tissue specificity in vivo. [26][27][28] For a peptide-based delivery agent, extension with peptide-based targeting ligands and also modification with small molecule targeting ligands is straightforward. The prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein expressed in normal prostate epithelial cells and overexpressed on nearly all prostate cancer cells, [29][30][31] with the highest expression levels found in advanced stages like CRPC. 29,[32][33][34] Interestingly, ligand binding to PSMA results in internalization. This principle has been exploited in prostate cancer diagnosis and therapy, and several PSMA ligands have been developed with some of them currently used in the clinics. 35,36 The addition of a PSMA ligand to PF14 could increase targeting specificity to the prostate and prostate cancer cells, aiding delivery of therapeutic AONs and is, therefore, a highly interesting next step.

| CONCLUSIONS
Translation of therapeutic AONs from in vitro to in vivo requires cellular targeting, sufficient cellular penetration and endosomal release, and avoidance of rapid clearance by the body. In this study, we have assessed key parameters on AON formulation and cellular delivery that serve as the basis for further development of CPP-mediated delivery of splicecorrecting AONs for targeted therapy in prostate cancer.