Therapeutic Potential of Chemically Modified, Synthetic, Triplex Peptide Nucleic Acid–Based Oncomir Inhibitors for Cancer Therapy

miRNA (miR) is a class of noncoding RNAs that control gene expression at the post-transcription level (1). miRNAs play key roles in maintaining physiological processes by controlling gene expression through regulating messenger RNA (mRNA) stability and translation. Hence, aberrant expression of miRNAs causes several devastating diseases. In cancer, atypical miRNA levels lead to altered processes, including differentiation, proliferation, and apoptosis (2). Hence, miRNAs have been explored as promising molecular targets for the development of precision medicine in cancer (3). Synthetic nucleic acid–based antimiRs have been evaluated in conjunction with delivery systems to repress miRNAs upregulated in multiple tumors (also called oncomiRs) for potential cancer therapeutics (4).

Several antimiRs have been developed to specifically target full-length miRNAs by Watson–Crick recognition to prevent their interaction with target mRNAs. In particular, peptide nucleic acids (PNA) have gained attention as potential antimiR agents. PNAs are synthetic nucleic acid analogs that possess a neutral backbone and are resistant to enzymatic degradation (5). It is well known that PNAs can target the full-length miRNAs by Watson–Crick base pairing and thus control gene expression (6, 7). Targeting full-length miRNAs provides numerous advantages, especially the sequence-specific targeting of preferred miRNA sites minimizes the off-target toxicity (8). A few studies reported using shorter antimiRs targeting the miRNA seed region to inhibit its function (9, 10). Still, clinical translation of targeting seed region-based strategies could be hampered due to off-target effects because of nonspecific binding with other coding and noncoding RNAs. Though promising results have been shown, increasing the binding affinity of antimiRs without compromising their specificity remains a continuous goal.

Herein, we demonstrated that compared with full-length PNA, another novel class of PNAs called tail-clamp PNA (tcPNA) possesses superior binding properties to target miRNAs and inhibit their activity. In tcPNA, one strand binds via Watson–Crick base pairing, and the other strand binds via Hoogsteen base pairing (11). Hence, tcPNAs possess a higher binding affinity compared with single-stranded PNAs. tcPNAs have been designed to target double-strand DNA containing homopurine stretches (12). A few biophysical studies have shown that tcPNAs containing regular PNA units can bind to short double-stranded RNA targets (13). However, most of these studies, as mentioned above, were focused only on biophysical studies and not much progress has been made in evaluating their biological activity in cell culture as well as in vivo.

To our knowledge, this is the first study centered on the evaluation of chemically modified tcPNAs for targeting biologically relevant miRNAs for therapeutic application. To boost the binding affinity, we used gamma (γ) PNA-containing tcPNA-based antimiRs. Gamma PNAs are next-generation PNAs that possess superior binding properties due to their preorganized locked structure (14). Because of their high binding affinity, gamma PNAs have been used in gene editing (15–17) and gene barcoding applications (18).

We established that anti–miR-155 gamma tcPNAs show superior miR-155 inhibition activity compared with conventional single-stranded anti–miR-155 design both in cell culture and in vivo. miR-155 has been upregulated in various cancers, especially in lymphoma and leukemia (19, 20). Recent studies have demonstrated that miR-155 is a clinically relevant target for treatment of various lymphomas (6). The drug cobomarsen (MRG-106) is in a phase 2 clinical trial targeting miR-155 for lymphoma therapy (21). Here, we performed comprehensive studies where anti–miR-155 gamma tcPNAs inhibit miR-155 in a lymphoma cell line, and its downstream targets both in the cell culture and in in vivo studies. In a parallel comparison, we demonstrate that anti–miR-155 gamma tcPNAs effectively decrease tumor growth in a lymphoma cell line–derived xenograft mouse model compared with the regular PNA. Overall, herein we established that gamma tcPNA–based synthetic oncomiR inhibitors could be explored for the next-generation miRNA therapeutics.

Boc-protected regular monomers (for PNA-155) and serine gamma monomers used for gamma tcPNA-155, gamma PNA-155 and Scr-gamma tcPNA-155 synthesis were purchased from ASM Research Chemicals GmbH. The monomers were vacuum-dried before start of solid-phase synthesis. Around 100 mg arginine-loaded resin was soaked in dichloromethane for 5 hours in a reaction vessel. The dichloromethane was drained and the resin was deprotected using trifluoroacetic acid-m-cresol (95:5) mixture for 5 minutes. This deprotection step was repeated two additional times followed by washing the resin with dichloromethane and N,N-Dimethylformamide. The monomer was dissolved in a coupling solution comprising of a mixture of 0.2 mol/L N-Methyl pyrrolidone (NMP), 0.52 mol/L Di-isopropylethylamine, and 0.39 mol/L O-Benzotriazole-N,N,N′,N′-tetramethyl–uronium–hexafluoro–phosphate. The coupling solution was added to the reaction vessel and rocked for 2 hours. The resin was capped using a capping solution (a mixture of NMP, Pyridine, and acetic anhydride). The resin was washed with dichloromethane (8X). The entire process was repeated until the last monomer was added. 5-Carboxytetramethylrhodamine (TAMRA) was conjugated to N-terminus of gamma tcPNA-155. The PNA was cleaved from the resin using a cleavage cocktail (thioanisole, m-cresol, TMFSA, TFA(1:1:2:6), and the vessel was rocked for 1.5 hours. The PNA was collected and precipitated using diethyl ether and centrifuged at 3,500 rpm for 5 minutes. The PNA was washed with ether twice and vacuum dried. The PNA was purified by RP-HPLC (reverse-phase high-performance liquid chromatography) and absorbance of the PNA was measured using Nanodrop One (Thermo Fisher Scientific). The extinction coefficient of the individual monomers used for calculating PNA concentration [6,600 M⁻¹ cm⁻¹ (C), 13,700 M⁻¹ cm⁻¹ (A), 8,600 M⁻¹ cm⁻¹ (T), 11,700 M⁻¹ cm⁻¹ (G)].

Different concentrations of PNA-155 or gamma tcPNA-155 were made in physiological buffer (2 mmol/L MgCl₂, 150 mmol/L KCl, 10 mmol/L NaPi) and incubated overnight at 37°C with 1 μmol/L DNA-155 target. The samples were run through 8% PAGE gel in IX TBE buffer at 120 volts for 35 minutes. The gel was stained with SyBr Gold for 2 minutes and visualized using Gel Doc EZ Imager (Bio-Rad).

U2932 are suspended cell lines and were purchased from Leibniz Institute (DSMZ, Germany). The cells were regularly tested for Mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza). The authenticity of the cell lines was confirmed by Human cell STR profiling service by the ATCC. All the cells used in the experiment were passaged less than 8 times. 50,000 U2932 cells were seeded in 24-well plate (37°C and 5% CO₂). The cells were treated with gamma tcPNA-155 TAMRA (500 nmol/L concentration). After 48 hours, the cells were washed twice with PBS and then fixed using 4% paraformaldehyde for 10 minutes at room temperature. The cells were washed with PBS and then permeablized using 0.1% triton X for 10 minutes at room temperature. The cells were washed with PBS and the cells were finally resuspended in 50 μL PBS. A drop of mounting media with DAPI (Life Technologies) was placed on the slide. 10 μL of cells were mixed with the drop of DAPI on the slide and coverslip was placed on the slide. The slide was allowed to dry overnight and imaged using Keyence digital microscope.

For evaluating cellular uptake by flow cytometry—400,000 U2932 cells were collected in a 12-well plate (37°C, 5% CO₂). Three wells were untreated and three other cells were treated with 500 nmol/L gamma tcPNA-155 TAMRA. After 48 hours, the cells were washed twice with PBS and then fixed using 4% paraformaldehyde for 10 minutes at room temperature. The cells were passed through the FACS tube. Furthermore, analysis was performed using LSR Fortessa X-20 Cell analyzer (BD Biosciences). The FACS data were plotted using FlowJo software.

400,000 U2932 cells were seeded in a 12-well plate. The cells were treated with 500 nmol/L PNA-155, gamma tcPNA-155, gamma PNA-155, Scr gamma tcPNA-155 or were PBS treated (control) for 48 hours in an incubator (37°C and 5% CO₂). The cells were centrifuged at 2,000 rpm for 4 minutes at 4°C. The total RNA from the cell pellet was extracted using the RNeasy Mini Kit (Qiagen). The cDNA was prepared in thermal cycler (Bio-Rad) using reverse transcriptase, RNase inhibitor, dNTPs, nuclease-free water, and RT primers specific for miR-155 and U6. Random primers were used for the preparation of cDNA for downstream targets. The cDNA was amplified using miR-155 assay, U6 assay, or specific downstream target assays in CFX Connect Real-time PCR detection system (Bio-Rad). The samples were subjected to polymerase activation (95°C for 10 minutes), followed by 40 cycles of denaturation (95°C for 15 seconds) and annealing (60°C for 1 minute). The 2 ^−ΔΔC_t method was used to calculate the fold change in target genes.

Similarly, 400,000 SUDHL-2 cells were seeded in a 12-well plate and treated with 500 nmol/L Scr gamma tcPNA-155, PNA-155 and gamma tcPNA-155 for 48 hours in an incubator (37°C and 5% CO₂). The RNA was extracted and the samples for gene expression were prepared in the same manner as described above.

Diffused large B-cell lymphoma (DLBCL) cell lines like SUDHL-2 (ATCC CRL-2956) and SUDHL-5 (ATCC CRL-2958) cells were purchased from the ATCC. The cells were regularly tested for Mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza). All the cells used in the experiment were passaged less than 8 times. 10,000 U2932, SUDHL-5, or SUDHL-2 cells were plated in a 96-well plate. The cells were treated with different doses (500, 1,000, 2,000, and 4,000 nmol/L) of PNA-155, gamma tcPNA-155 or Scr-gamma tcPNA-155 for 48 hours in an incubator (37°C and 5% CO₂). The dead cells were marked with trypan blue. Further counting was performed using an automated cell counter (Bio-Rad).

A total of 400,000 U2932 cells were seeded in a 12-well plate. The cells were treated with 500 nmol/L PNA-155, gamma PNA-155, gamma tcPNA-155, Scr-gamma tcPNA-155 or were PBS treated (control) for 48 hours in an incubator (37°C, 5% CO₂). The cells were washed with PBS twice. The cells were centrifuged at 2,000 rpm for 4 minutes at 4°C. The cell pellet was suspended in IX Annexin V–binding buffer. The cells were then counted and 100 μL of cell suspension (containing 2.5 × 10⁵ cells) was passed through the FACS tube. The cells were stained with 12.5 μL phycoerythrin (PE) Annexin dye and 12.5 μL 7-amino-actinomycin (7AAD) and kept in dark for 15 minutes. 400 μL of IX Annexin V binding buffer was added to the cells and the cells were then analyzed using LSR Fortessa X-20 Cell analyzer as indicated above.

For Annexin V FITC–stained fluorescent imaging method, 10,000 U2932 cells were seeded in 96-well plate (37°C and 5% CO₂). The cells were treated with Scr-γtcPNA-155, PNA-155, γPNA-155, γtcPNA-155 (500 nmol/L concentration) for 48 hours. Annexin V FITC diluted 1:10 in 1X Annexin binding buffer was supplemented to each well. The plate was kept at room temperature for 15 minutes. The cells were imaged using ×10 lens on Keyence digital microscope.

400,000 U2932 cells were collected in a 12-well plate and treated with 500 nmol/L PNA-155, gamma PNA-155, gamma tcPNA-155 or Scr-gamma tcPNA-155 for 48 hours in an incubator. The cell pellet was collected by centrifuging at 2,000 rpm for 4 minutes at 4°C. 1X RIPA buffer and 1X protease inhibitor were added to the cell pellet and subjected to intermittent vortexing after 10 minutes (3X) to extract the proteins from the cell pellet. The protein was collected after centrifuging the tube at 10,000 rpm for 10 minutes at 4°C. The protein concentration was measured by Lowry protein assay. About 25-μg protein was loaded on SDS PAGE gel (4%–20% MP TGX stain-free gels, Bio-Rad) and separated at 101 Volts for 90 minutes. The proteins were transferred from the gel to a PVDF (polyvinylidene difluoride) membrane at 110 Volts for 90 minutes. The PVDF membrane was blocked using 5% milk in 1X tris buffered saline for 1 hour. The Western blotting was performed using the following antibodies: Mcl1 (39224), CASP3 (9622S), vinculin (13901), antirabbit IgG horseradish peroxidase–linked antibody (7074; Cell Signaling Technology). The blots were imaged using ChemiDoc Imaging System (Bio-Rad). Protein expression intensities were determined using ImageJ software and normalized relative to loading control and treatment control.

Primary blood mononuclear cells (PBMC; ATCC PCS-800–011) were purchased from the ATCC. The cells were regularly tested for Mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza). All the cells used in the experiment were passaged less than 2 times. 10,000 PBMC cells were seeded in a 96-well plate. The cells were treated with 500, 1,000, 2,000, and 4,000 nmol/L PNA-155 and γtcPNA-155 for 48 hours in an incubator. The dead cells were examined with trypan blue and further counted using an automated cell counter (Bio-Rad).

All the animals' experiments were performed at the University of Connecticut, Storrs campus, in compliance and approved by the Institutional Animal Care and Use Committee (IACUC). The authorization number for approved IACUC protocol is A21–041.

Female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, strain 005557) weighing 20–23 g were procured from The Jackson Laboratory. 1 × 10⁷ U2932 cells suspended in RPMI-1640 medium were injected subcutaneously in the right and left flank of 6-week-old NSG mice. The mice developed a small bump in 2 weeks. When the tumor volume reached 100–200 mm³, the mice were randomly assigned to the treatment group (n = 6 per group).

For SUDHL-2 xenografts, 1 × 10⁷ SUDHL-2 cells suspended in RPMI-1640 medium were injected subcutaneously in the right and left flank of 6-week-old NSG mice. The mice developed a small bump in 2–3 weeks. When the tumor volume reached 100–200 mm³, the mice were randomly allocated to the treatment group (n = 5 per group).

The γtcPNA-155 TAMRA PNA was intratumorally injected in the NSG mice (n = 3) bearing 200 mm³ tumors at the dose of 3 mg kg^–1 at an interval of 1 week. When the tumor volume reached 2,000 mm³, the mice were euthanized by CO₂ inhalation. The tumors were harvested and imaged using IVIS Spectrum system. The tumors were embedded in OCT compound. The tumors were cryosectioned at 10-mm thickness using Leica cryostat. The tumor sections were fixed using 4% formaldehyde for 10 minutes, followed by washing with PBS for 10 minutes. The tumor sections were then permeabilized using 0.2% triton X for 20 minutes followed by washing with PBS. A drop of mounting media with DAPI (Life Technologies) was placed on the tumor section and a coverslip was placed on it. The tumor sections were imaged using Keyence digital microscope.

The mice were injected intratumorally with 1 mg kg^–1 dose of PNA-155, gamma tcPNA-155, gamma PNA-155, Scr gamma tcPNA-155 or were untreated. The injections were repeated two additional times after 1 week each. The length, breadth, and depth of the tumors were measured daily using a vernier caliper. The mice were euthanized when the tumor volumes reached 2,000 mm³. The resected tumor sections were finely minced using a sterile blade and suspended in dissociation media (4 mL) comprising of RPMI-1640, 1.2 mg/mL dispase, and 0.5 mg/mL collagenase for 90 minutes at 500 rpm at 37°C. The dissociated tumors were washed with buffer saline at 2,500 rpm (4 minutes) at 4°C and then suspended in 0.25% trypsin for 4 minutes at room temperature. RPMI-1640 media were added to the trypsinized tumor mass and the cells were passed through a 70-μm filter. The cells were centrifuged at 2,500 rpm for 4 minutes at 4°C. The cell pellet was then suspended in 1X RBC lysis buffer (Sigma) and incubated on ice for 10 minutes. PBS was added to the cells and the cells were passed through a 40-μm filter. The cells were centrifuged at 2,500 rpm for 4 minutes at 4°C. The cell pellet was resuspended in 0.5% BSA in PBS. The mouse cells were removed from the tumor cells using a mouse cell depletion kit (Miltenyi Biotec) according to the manufacturer's protocol. The enriched tumor cells from each tumor sample were divided into two fractions. RNA for gene expression analysis was extracted from one tumor fraction by the same procedure as mentioned earlier. The protein for Western blot analysis was extracted from the second fraction using the method described above.

The mice were sacrificed by CO₂ inhalation when tumor volume reached 2,000 mm³. The tumor and vital organs (e.g., liver, kidney, spleen, lungs, and heart) were carefully isolated, weighed, and fixed in the 10% NBF solution. The sections (5 μmol/L) of formalin-fixed paraffin-embedded liver and kidney were stained by hematoxylin and eosin for the histological analysis. The sections of 5 μmol/L of the formalin-fixed paraffin-embedded tumor were heated (95°C, 20 minutes) in citrate buffer (10 mmol/L) for antigen recovery. Further incubation was performed with primary antibodies. The concentrations of rabbit anti–Ki-67 (D2H10) and rabbit anti-caspase-3 (9962) were 1:100. The antigen-primary antibody complexes were examined by fluorescent-tagged secondary antibodies. Images were taken using a Zeiss confocal microscope (LSM 510).

Graphpad Prism 9 software (Version 9.2) was used for all statistical analyses. The data are reported as means SEM of triplicates, and the numbers and replicates are included in the Figure captions. Unpaired two-tailed and multiple Student t test was performed for experiments. For in vivo studies, animals were randomly assigned in groups to minimize the bias, and based on prior experience with animal models, sample size was selected (9, 22). The statistical comparisons are significant when *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.

We designed and synthesized anti–miR-155 gamma tcPNAs (γtcPNA) and control PNA oligomers to test in a series of lymphoma cell lines and a xenograft mouse model (Fig. 1A). It has been established that tcPNAs can only bind to target RNA sequences containing homopurine stretches (12). Homopurine stretches are present at the 3′ end of the miR-155 sequence (Fig. 1B). Hence, we designed an anti–miR-155 gamma tcPNA containing Watson–Crick and Hoogsteen domain joined by flexible trioxo (-OOO-) linker to target the miR-155 sequence (Fig. 1B and C, γtcPNA-155). In prior studies, it has been noted that gamma-modified PNAs exhibit superior binding and biocompatibility features compared with the conventional PNAs due to their right-handed helical pre-organization properties (23). γtcPNA-155 contains pseudoisocytosine (J) units on the Hoogsteen end as J can form hydrogen bonding at physiological pH (24). We also conjugated a TAMRA (5-Carboxytetramethylrhodamine) fluorophore to the 5′ terminal of γtcPNA-155 to study cellular uptake in cell lines and intratumoral biodistribution in the xenograft mice (Fig. 1C, γtcPNA-155-Tam). For comparison, we synthesized full-length regular PNA (23mer long) as it has been demonstrated to inhibit miR-155 in prior studies (Fig. 1C, PNA-155; ref. 6). We synthesized single-stranded full-length serine gamma PNAs that can bind to target miR-155 by Watson–Crick base pairing (Fig. 1C, γPNA-155) and a scrambled γtcPNA-155 (Fig. 1C, Scr-γtcPNA-155) as control. For cell permeability, we appended two arginine residues to the C- and N-terminus of each PNA (25). We selected four arginine residues to minimize the cytotoxicity associated with cationic domains (9). The PNAs, γPNAs, and γtcPNAs were synthesized by established solid-phase synthesis-based protocols (26), and quality control analyses were performed by RP-HPLC (Supplementary Fig. S1).

Next, we evaluated the binding affinity of γtcPNA-155 and PNA-155 with the miR-155 target by PAGE-based protocols (Fig. 1D). We incubated PNA-155 and γtcPNA-155 with miR-155 at indicated concentrations in physiological buffer and temperature and assessed the binding by PAGE followed by SYBR gold staining. As expected, we noticed an increase in the formation of a retarded band (PNA-155–miR-155 bound fraction) with the increase in concentration of PNA-155. We also noted the complete disappearance of the unbound miR-155 target at a miR-155:PNA-155 stoichiometry ratio of 1:2.0 (Fig. 1D, left). Whereas in the case of γtcPNA-155, we noticed the complete disappearance of unbound miR-155 target at miR-155:γtcPNA-155 stoichiometry ratio of 1:1.0 (Fig. 1D, right). These results confirmed that γtcPNA-155 possesses a stronger binding affinity compared with regular PNA-155. We also observed three distinct retarded band patterns in the case of γtcPNA-155–incubated with target miR-155. Three separate retarded bands are consistent with tcPNA-binding modes with the complementary target strands. The top retarded band corresponds to the presence of γtcPNA-155 Watson–Crick, and the Hoogsteen domain bonded to miR-155 target. The other two retarded bands indicate binding with either Watson–Crick or Hoogsteen domain of γtcPNA-155 with the miR-155 target.

Various studies established that miR-155 is upregulated in B-cell malignancies and is recognized as a therapeutically bona fide molecular target for treating lymphoma (6). Hence, we performed cellular uptake studies of γtcPNA-155-Tam in lymphoma cell lines (U2932 and SUDHL-2) by flow cytometry analysis. Significant cellular uptake of the TAMRA was observed in the U2932 (Fig. 2A) and SUDHL-2 cells (Supplementary Fig. S2A) 48 hours post incubation with γtcPNA-155-TAMRA without using any transfection agent. Furthermore, we also confirmed the cellular uptake of γtcPNA-155-TAMRA in U2932 cells by confocal microscopy (Supplementary Fig. S2B).

Next, we assessed the miR-155 inhibitory activity of γtcPNA-155, γPNA-155, and PNA-155 in the U2932 cell line. U2932 cells were treated with a 500 nmol/L dose of Scr-γtcPNA-155, PNA-155, γPNA-155, and γtcPNA-155 for 48 hours. Our RT-PCR results confirmed approximately 80%, 83%, and 90% miR-155 inhibition with PNA-155, γPNA-155, and γtcPNA-155 treatments, respectively, relative to scrambled control (Fig. 2B).

To verify miR-155 inhibition by the antimiRs, we evaluated the gene expression of known miR-155 downstream genes by RT-PCR–based gene expression analysis. High miR-155 gene expression results in repression of tumor suppressor genes via activation of the PI3-AKT signaling pathway, thereby promoting tumor cell survival, proliferation, and progression (27). Therefore, downregulating miR-155 will result in the derepression of tumor suppressor genes. We examined the gene expression of panel of known miR-155–associated tumor suppressor genes by RT-PCR analysis after treatment with γtcPNA-155, γPNA-155, and PNA-155 (Fig. 2C; ref. 22). We noted significant derepression of miR-155 target tumor suppressor genes in the U2932 cells treated with γPNA-155 and γtcPNA-155. In particular, CSF1R, CUX1, and SHIP1 negatively regulate PI3-AKT signaling and are a direct target of miR-155 (28–30). We noted a 1.8-fold increase in CSF1R levels, a 1.6-fold increase in CUX1 levels, and a 1.4-fold increase in the SHIP1 levels in γtcPNA-155–treated groups in comparison with PNA-155. Overall, we noted that γtcPNA-155 followed by γPNA-155 shows optimal derepression of tumor suppressor genes in comparison with PNA-155. One plausible explanation of these findings is that γtcPNA-155 has a higher binding affinity with the miR-155 target.

In addition to the tumor suppressors, miR-155 is also known to impact expression of oncogenes. MCL1 is a known miR-155 downstream oncogene (20, 31). Our gene expression results confirmed a 40% reduction in MCL1 gene expression levels after pretreatment with γtcPNA-155 (Fig. 2D). γPNA-155 and PNA-155 pretreatment results in 19% and 10% decline in MCL1 gene expression, respectively. We confirmed the effect of γtcPNA-155 on the validated miR-155 downstream genes MCL1 and caspase-3 (CASP3) by Western blot analysis. Caspase-3 has also been identified as a one of the direct targets of miR-155 (32). Our results indicated that U2932 pretreatment with γtcPNA-155 at 500 nmol/L concentration led to significant downregulation of Mcl1 (50%; Fig. 2E and Supplementary Fig. S3A) and upregulation of caspase-3 (80%; Fig. 2F and Supplementary Fig. S3B).

Next, we assessed the efficacy of γtcPNA-155 in SUDHL-2 cell lines that exhibit overexpression of miR-155. Consistent with our aforementioned findings, RT-PCR results show that pretreatment of SUDHL-2 cell lines with γtcPNA-155 results in a 63% decrease in miR-155 gene expression (Supplementary Fig. S4). We also investigated that the pretreatment of SUDHL-2 cell lines with γtcPNA-155 results in significant upregulation of miR-155–associated tumor suppressor genes in comparison with Scr-γtcPNA-155 and PNA-155 (Supplementary Fig. S5).

Prior studies indicated that miR-155 inhibition results in a decrease in cell proliferation and viability (22). Therefore, we assessed the dose-dependent cell viability in multiple lymphoma cell lines, U2932, SUDHL-5, and SUDHL-2 by trypan blue assay. As expected, we noticed a dose-dependent reduction in cell viability in U2932, SUDHL-2, and SUDHL-5 cell lines. We observed approximately 80% decrease in cell viability in three cell lines at a 4 μmol/L concentration (Fig. 3). We also noted that γtcPNA-155 pretreatment causes a significant reduction in cell viability in U2932, SUDHL-5, and SUDHL-2 cell lines at a lesser concentration of 500 nmol/L followed by γPNA-155 compared with the PNA-155–treated group (Fig. 3 and Supplementary Fig. S6). These results are consistent with prior results signifying that γtcPNA-155 has a superior binding affinity followed by γPNA-155, and thus anti–miR-155 activity compared with regular full-length PNA-155. We did not notice any effect on cell viability with Scr-γtcPNA-155 in comparison with PBS-treated control cells (Supplementary Fig. S7).

Furthermore, to test whether the decrease in cell viability is due to apoptosis, we treated the U2932 cells with the same amount of γtcPNA-155 and PNA-155 and performed an annexin V–based apoptosis assay. Our results indicated that treatment with γtcPNA-155 results in increased apoptosis in U2932 cells than the PNA-155 and γPNA-155–treated groups (Fig. 4). We also performed confocal imaging on U2932 cells treated with indicated PNAs and assessed the apoptosis by Annexin-V FITC–stained methods. Consistent with our flow cytometry–based results, we noted higher apoptosis in the γtcPNA-155–treated cells (Supplementary Fig. S8).

Prior studies successfully confirmed that pre-treatment of cobomarsen at a dose of 10 μmol/L results in 4-fold increase in apoptosis in the U2932 lymphoma cell line compared with control (22). In a parallel comparison, we also performed an apoptosis assay after pretreatment of U2932 cells with γtcPNA-155 at a dose of 10 μmol/L. Our results indicated that the γtcPNA-155 result in a 4.5-fold increase in apoptosis in comparison with control (Supplementary Fig. S9). Hence, we suggest that γtcPNA-155 efficacy is comparable with cobomarsen in a cell culture–based analysis.

Overall, these results demonstrate that γtcPNA-155, followed by γPNA-155, led to robust inhibition of miR-155, decreased cell viability, and increased apoptosis in U2932 cell lines. We also evaluated the safety of PNA-155, and γtcPNA-155 in PBMC cell lines in a dose-dependent manner using trypan blue assay. We did not notice a significant reduction in viability of PBMC cell lines, indicating that the γtcPNA-155 and Scr-γtcPNA-155 do not cause any nonspecific effects in the primary cells (Supplementary Fig. S10).

To evaluate whether γtcPNA-155 can inhibit tumor growth more effectively than regular PNA-155 in vivo, we performed studies in xenografts derived from DLBCL cells. DLBCL U2932 cells were selected for implants as they show maximum miR-155 gene expression levels (33). Hence, they provide a robust xenograft model to study miR-155 inhibitory effects of γtcPNA-155. For our in vivo study, we assessed the anti–miR-155 impact of γtcPNA-155 and γPNA-155 compared with PNA-155 via intratumoral delivery. Cobomarsen, an investigative drug to inhibit miR-155 has also received orphan drug designation to treat lymphoma by intratumoral delivery. Because we selected the intratumoral route of delivery, we injected the U2932 cells in the right and left flank of mice (Supplementary Fig. S11). After 10–14 days, when the tumor volume reached 100–200 mm³, the mice were divided into the five treatment groups. We also tested scrambled PNA (Fig. 1C, Scr-γtcPNA-155) as a control for in vivo study. The mice were randomized into groups based on the tumor volumes for uniform distribution of tumor volumes in each group. Before the efficacy study, we performed a biodistribution analysis of TAMRA-conjugated γtcPNA-155 (Fig. 1C, γtcPNA-155-Tam) at a dose of 3 mg kg^–1 intratumoral in the xenograft. We noticed a significant biodistribution in tumor after intratumoral injection by IVIS imaging (Supplementary Fig. S12). We confirmed the biodistribution by confocal imaging of cryosections from the TAMRA-treated group and control mice. Significant biodistribution of TAMRA fluorescence was noted in the tumor as compared with the control group (Fig. 5A).

For the efficacy study, each mouse received three intratumoral injections of either γtcPNA-155, γPNA-155 or PNA-155 on days 0, 7, and 14. We noticed that the control and scrambled treated tumors reached 2,000 mm³ much faster during our study, so we could not inject a third time in these mice. We did not observe: A significant decrease in the body weight (Supplementary Fig. S13); histological damage to the kidney and liver (Supplementary Fig. S14); general behavioral change in mice treated with indicated PNAs. Intratumoral administration reduced the tumor growth in the γtcPNA-155–treated group, followed by γPNA-155 and PNA-155 (Fig. 5B and Supplementary Fig. S15). In contrast, as stated earlier, there was no effect on the control and scrambled PNA control on the tumor volume.

To ascertain the efficacy of γtcPNA-155, the histological sections of tumors isolated from γtcPNA-155–treated mice were immunostained for apoptosis marker caspase-3 (Fig. 5C and D) and cell proliferation marker Ki67 (Supplementary Fig. S16A and S16B). We noted a significant decrease in the Ki67-positive cells and an increase in the caspase-3–positive cells in the tumor sections isolated from γtcPNA-155–treated groups, which further corroborates the antitumor efficacy of γtcPNA-155.

We next assessed the levels of miR-155 and its downstream genes in the tumors of xenograft mice to assess the mechanism of tumor reduction. After tumor harvest, mouse and DLBCL cells were separated by a bead-separation method to remove the false background from mice tissues. Subsequently, we investigated the level of miR-155 gene expression. Our results indicated that the γtcPNA-155–treated group shows a 90% decrease in miR-155 gene expression, whereas PNA-155 and γPNA-155–treated tumors indicated an 75% and 80% decline in the miR-155 expression, respectively (Fig. 6A). As anticipated, we did not observe a change in the miR-155 expression level in PBS-treated cells and scrambled groups.

Furthermore, we measured the expression of miR-155 downstream genes, both tumor suppressor and oncogenes, as described previously in the in vitro result section. Upon γtcPNA-155 treatment, there was a significant reduction in the oncogenes and derepression of tumor suppressor genes compared with the PNA-155–treated group. Compared with the PNA-155–treated group, we observed about a 50% decrease in MCL1 mRNA levels in the γtcPNA-155–treated group (Fig. 6B). Furthermore, we confirmed these results by measuring reduction in the protein levels of Mcl1 in γtcPNA-155–treated tumors (Fig. 6C and Supplementary Fig. S17). Consistent with tumor growth studies, we did not notice any alteration in the gene expression and Mcl1 protein level for the Scr-γtcPNA-155–treated groups (Supplementary Fig. S18). We also examined the in vivo derepression of tumor suppressor genes as mentioned in the cell culture studies. Consistent with our cell culture–based results, we noted significant upregulation of tumor suppressor genes in the tumors of mice that received γtcPNA-155 intratumorally (Fig. 6D). We also measured caspase-3 protein levels in the tumors and observed a 23% increase in the in vivo γtcPNA-155–treated tumors than Scr-γtcPNA-155–treated tumors (Supplementary Fig. S19). These results collectively indicated that γtcPNA-155 exhibits a superior anti–miR-155 effect, followed by γPNA-155, decreasing tumor growth compared with its scrambled control and classical PNA-155.

Furthermore, we corroborated our results in the SUDHL-2–derived xenograft mouse model. We injected the SUDHL-2 cells in the right and left flank of mice. After 14–21 days, when the tumor volume reached 100–200 mm³, the mice were randomized into three treatment groups based on uniform tumor volume distribution in each group. Consistent with prior in vivo results, we noted that γtcPNA-155 reduced the tumor growth as compared with the Scr-γtcPNA-155 and PNA-155 control (Supplementary Fig. S20). We further confirmed that PNA-155 significantly reduces miR-155 expression level in the SUDHL-2 cell line–based xenograft after in vivo treatment (Supplementary Fig. S21).

The antisense field has seen a rapid surge of FDA approvals of various nucleic acid-based drugs; Nusinersen, Onpattro, Gilvari, and Milasen to name a few, targeting coding mRNA for diverse therapeutic applications (34). However, targeting noncoding RNAs like miRNAs for clinical applications lags behind and still needs further optimization as a broader platform. miRNAs have been established for their key roles in cancer progression and transformation (35). Especially, miR-155 is considered an important biomarker and highly expressed in B-cell lymphoma and DLBCL (36). Hence, in recent years, interest and research in this area has grown. miRagen Therapeutics (now Viridian Therapeutics) has made strides in developing cobomarsen as a drug candidate to target miR-155 for cutaneous T-cell lymphoma treatment. Cobomarsen has shown promise in decreasing miR-155 levels followed by reduced tumor burden. Recent studies also indicated that systemic delivery of cobomarsen shows favorable outcomes in patients that developed resistance to CHOP and CAR–T-cell therapy (22). Overall, these studies underpin the significance of miR-155 as an important molecular target for developing precision medicine for lymphoma therapy.

However, practicing miRNA therapeutics has been challenging due to delivery, plasma stability, and moderate efficacy of nucleic acid analogs-based antimiRs (37). Novel chemical modifications have been performed to increase the efficacy and enzymatic stability of next-generation antimiRs with minimal off-target effects. Though numerous synthetic nucleic acid analogs have been developed as potential antimiRs, LNA and PNA have gained enormous attention due to their enzymatic stability and superior physicobiochemical properties (38, 39). Both full-length and seed-targeting LNAs have been explored as potential anti–miR-155–based therapeutics (10, 40). Though LNA-based cobomarsen has shown promise, the field still needs to increase the antimiR-based repertoire that can be used for broader applications and targeting of diverse miRNAs with increased efficacy.

PNA has been widely used as antimiR for targeting full-length miRNA. Apart from its binding properties, PNA-based technology has been amenable to several delivery platforms like nanoparticles (41), liposomes (42), and peptide conjugations (43), to target the tumor microenvironment and inhibit the target miRNA selectively. Conventional regular PNA targets full-length miRNAs and inhibits their target mRNA interaction by steric hindrance (44). Herein, we tested novel gamma-modified tcPNA antimiRs that can bind with both Watson–Crick and Hoogsteen base pairing with a miR-155 target containing a homopurine stretch and inhibit its activity. In prior studies, it has been well established that gamma tcPNAs can induce a higher percentage of gene editing than the regular PNA due to their high binding affinity (15, 17, 45). We report that gamma tcPNAs can inhibit the miR-155 at a higher level than conventional full-length PNAs.

Herein, we also noted that full-length serine gamma PNA causes increased miR-155 inhibition in both cell culture and in vivo analysis compared with conventional PNA-155. Prior studies indicated that poly-lactic-co-glycolic acid (PLGA) nanoparticles loaded with diethylene-glycol units containing gamma anti–miR-210 PNA results in significant miR-210 inhibition in a HeLa cell line–derived xenograft (23). Diethylene-glycol containing gamma PNAs have enhanced loading and release in the PLGA NPs as compared with the regular PNA-210, resulting in increased antimiR efficacy. To the best of our knowledge, our study here is the first where a parallel comparison of the antimiR efficacy of cationic serine gamma PNAs and conventional PNAs based on their binding affinity have been performed. In addition, serine gamma PNAs have not been explored before for in vitro and in vivo antimiR efficacy. Though we noted that gamma tcPNAs show superior inhibition due to the gamma substitutions and the presence of Hoogsteen-binding domains, it would also be interesting to study the full-length gamma PNAs for targeting mixed sequence containing target miRNAs.

We performed an extensive analysis of gamma tcPNA's anti–miR-155 activity in both cell culture and in vivo analysis. We validated our anti–miR-155 results by gene expression and protein level analysis of direct and indirect miR-155 targets in U2932 lymphoma cell lines. We demonstrated that repressing miR-155 levels decreases cell viability in multiple lymphoma cell lines SUDHL-2, SUDHL-5, and U2932. We also found that gamma tcPNA-155–induced apoptosis in vitro. Our in vivo results in the xenograft mice indicated that gamma tcPNA design could inhibit miR-155 and further affect the miR-155 downstream targets in vivo more efficiently compared with regular PNA-155. Furthermore, a decrease in cellular proliferation and an increase in apoptosis markers mechanistically support the retardation of tumor growth in mice receiving intratumoral injections of gamma tcPNA-155. Prior studies successfully demonstrated that pretreatment of cobomarsen at a dose of 10 μmol/L results in 4-fold increase in apoptosis in the U2932 lymphoma cell line compared with control. Our results indicate that treatment of U2932 cell lines with gamma tcPNAs at a dose of 10 μmol/L causes a 4.5-fold increase in apoptosis compared with control. Hence, we examined that gamma tcPNA results are comparable with cobomarsen based on in-vitro studies. Various important miR-155 targets are involved in lymphoma cell proliferation. In a prior study, it has been noted that CUX1 and WEE1 are established miR-155–predicted targets and play an essential role in tumor proliferation (22, 46). In both in vitro and in vivo analysis, we noted that gene expression of multiple miR-155 targets, including CUX1 and WEE1, increased after treatment with gamma tcPNA-155.

We have presented a novel antimiR strategy that could target multiple miRNA-based molecular targets for diverse therapeutic applications. One possible limitation of gamma tcPNA design is that it can only target miRNA-containing homopurine stretches. Still, numerous miRNAs containing homopurine stretches play a crucial role in various cancers and other metabolic diseases. It would be noteworthy to target other miRNAs that contain more than five homopurine stretches. It has been demonstrated that targeting longer homopurine stretches results in stronger binding affinity and increases the PNA-mediated gene-editing efficacy (47). Hence, we would anticipate that preorganized features of gamma PNAs and targeting the extended Hoogsteen domain will enhance antimiR efficacy. In addition, it would be interesting to study the direct systemic delivery of gamma tcPNAs in conjunction with nanoparticles or pH low insertion peptide (pHLIP)–based technology that directly targets these novel antimiRs in the acidic tumor microenvironment.

In conclusion, an efficient antimiR-based repertoire is required to target the miRNAs that are involved in disease pathogenesis. Our results demonstrate that gamma-modified tcPNA-based anti–miR-155 not only improves its affinity toward the target miR-155 but also increases its efficacy in retarding tumor growth. Furthermore, gamma-modified tcPNA-based antimiRs appeared safe in our mice throughout treatment. Hence, gamma tcPNA-based molecules hold the potential to be used in combination with other drugs like R-CHOP and radiotherapy as adjuvant therapy to improve the drug response of patients with DLBCL. Finally, the gamma tcPNA technology can also be used to target mRNAs and other noncoding RNAs involved in the development of other health disorders.

No disclosures were reported.

K. Dhuri: Conceptualization, formal analysis, validation, investigation, methodology, writing–original draft, writing–review and editing. R.R. Gaddam: Formal analysis, supervision, validation, investigation, writing–review and editing. A. Vikram: Formal analysis, supervision, validation, investigation, writing–review and editing. F.J. Slack: Formal analysis, funding acquisition, validation, writing–review and editing. R. Bahal: Conceptualization, formal analysis, supervision, funding acquisition, validation, investigation, methodology, writing–original draft, writing–review and editing.

This work was supported by Charles H. Hood Foundation Grant (to R. Bahal) and National Institutes of Health (CA241194 to R. Bahal and F.J. Slack).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.