MicroRNA silencing for cancer therapy targeted to the tumour microenvironment.

MicroRNAs are short non-coding RNAs expressed in different tissue and cell types that suppress the expression of target genes. As such, microRNAs are critical cogs in numerous biological processes, and dysregulated microRNA expression is correlated with many human diseases. Certain microRNAs, called oncomiRs, play a causal role in the onset and maintenance of cancer when overexpressed. Tumours that depend on these microRNAs are said to display oncomiR addiction. Some of the most effective anticancer therapies target oncogenes such as EGFR and HER2; similarly, inhibition of oncomiRs using antisense oligomers (that is, antimiRs) is an evolving therapeutic strategy. However, the in vivo efficacy of current antimiR technologies is hindered by physiological and cellular barriers to delivery into targeted cells. Here we introduce a novel antimiR delivery platform that targets the acidic tumour microenvironment, evades systemic clearance by the liver, and facilitates cell entry via a non-endocytic pathway. We find that the attachment of peptide nucleic acid antimiRs to a peptide with a low pH-induced transmembrane structure (pHLIP) produces a novel construct that could target the tumour microenvironment, transport antimiRs across plasma membranes under acidic conditions such as those found in solid tumours (pH approximately 6), and effectively inhibit the miR-155 oncomiR in a mouse model of lymphoma. This study introduces a new model for using antimiRs as anti-cancer drugs, which can have broad impacts on the field of targeted drug delivery.

Tumor-Targeted, Cytoplasmic Delivery of Large, Polar Molecules Using a pH-Low Insertion Peptide.

Tumor-targeted drug delivery systems offer not only the advantage of an enhanced therapeutic index, but also the possibility of overcoming the limitations that have largely restricted drug design to small, hydrophobic, "drug-like" molecules. Here, we explore the ability of a tumor-targeted delivery system centered on the use of a pH-low insertion peptide (pHLIP) to directly deliver moderately polar, multi-kDa molecules into tumor cells. A pHLIP is a short, pH-responsive peptide capable of inserting across a cell membrane to form a transmembrane helix at acidic pH. pHLIPs target the acidic tumor microenvironment with high specificity, and a drug attached to the inserting end of a pHLIP can be translocated across the cell membrane during the insertion process. We investigate the ability of wildtype pHLIP to deliver peptide nucleic acid (PNA) cargoes of varying sizes across lipid membranes. We find that pHLIP effectively delivers PNAs up to ∼7 kDa into cells in a pH-dependent manner. In addition, pHLIP retains its tumor-targeting capabilities when linked to cargoes of this size, although the amount delivered is reduced for PNA cargoes greater than ∼6 kDa. As drug-like molecules are traditionally restricted to sizes of ∼500 Da, this constitutes an order-of-magnitude expansion in the size range of deliverable drug candidates.

Effective Cellular Delivery of Antisense Peptide Nucleic Acid by Conjugation to Guanidinylated Diaminobutanoic Acid-Based Peptide Dendrons.

A series of amino- and guanidino-terminating 3- and 4-generation 2,4-diaminobutanoic acid (Dab) dendrons have been robustly synthesized on a solid phase and characterized as cellular delivery agents in antisense peptide nucleic acid (PNA) conjugates in the pLuc705 HeLa cell splice switching system. The dendron-PNA conjugates exhibited splice correction activity at one digit micromolar concentrations, and guanidino-terminating dendrons were significantly more effective than analogous amine terminating ones. Furthermore, introduction of lipophilic groups such as phenyl, alkyl, or fatty acids increased efficacy, but also increased cellular toxicity. Fluorescence microscopy analyses supported an endosomal uptake mechanism and furthermore predominantly showed colocalization with late endosomes and lysosomes. The robust solid phase synthesis should make such Dab-dendrons a useful platform for further in vitro as well as in vivo optimization.

Antibacterial Peptide Nucleic Acids-Facts and Perspectives.

Antibiotic resistance is an escalating, worldwide problem. Due to excessive use of antibiotics, multidrug-resistant bacteria have become a serious threat and a major global healthcare problem of the 21st century. This fact creates an urgent need for new and effective antimicrobials. The common strategies for antibiotic discovery are based on either modifying existing antibiotics or screening compound libraries, but these strategies have not been successful in recent decades. An alternative approach could be to use gene-specific oligonucleotides, such as peptide nucleic acid (PNA) oligomers, that can specifically target any single pathogen. This approach broadens the range of potential targets to any gene with a known sequence in any bacterium, and could significantly reduce the time required to discover new antimicrobials or their redesign, if resistance arises. We review the potential of PNA as an antibacterial molecule. First, we describe the physicochemical properties of PNA and modifications of the PNA backbone and nucleobases. Second, we review the carriers used to transport PNA to bacterial cells. Furthermore, we discuss the PNA targets in antibacterial studies focusing on antisense PNA targeting bacterial mRNA and rRNA.

Peptide Nucleic Acids and Gene Editing: Perspectives on Structure and Repair.

Unusual nucleic acid structures are salient triggers of endogenous repair and can occur in sequence-specific contexts. Peptide nucleic acids (PNAs) rely on these principles to achieve non-enzymatic gene editing. By forming high-affinity heterotriplex structures within the genome, PNAs have been used to correct multiple human disease-relevant mutations with low off-target effects. Advances in molecular design, chemical modification, and delivery have enabled systemic in vivo application of PNAs resulting in detectable editing in preclinical mouse models. In a model of ß-thalassemia, treated animals demonstrated clinically relevant protein restoration and disease phenotype amelioration, suggesting a potential for curative therapeutic application of PNAs to monogenic disorders. This review discusses the rationale and advances of PNA technologies and their application to gene editing with an emphasis on structural biochemistry and repair.

Peptide Nucleic Acid-Functionalized Adenoviral Vectors Targeting G-Quadruplexes in the P1 Promoter of Bcl-2 Proto-Oncogene: A New Tool for Gene Modulation in Anticancer Therapy.

The B-cell lymphoma 2 (Bcl-2) gene encodes for an antiapoptotic protein associated with the onset of many human tumors. Several oligonucleotides (ONs) and ON analogues are under study as potential tools to counteract the Bcl-2 expression. Among these are Peptide Nucleic Acids (PNAs). The absence of charges on PNA backbones allows the formation of PNA/DNA complexes provided with higher stability than the corresponding natural DNA/DNA counterparts. To date, the use of PNAs in antigene or antisense strategies is strongly limited by their inability to efficiently cross the cellular membranes. With the aim of downregulating the expression of Bcl-2, we propose here a novel antigene approach which uses oncolytic adenoviral vectors (OAds) as a new cancer cell-targeted PNA delivery system. The ability of oncolytic Ad5D24 vectors to selectively infect and kill cancer cells was exploited to transfect with high efficiency and selectivity a short cytosine-rich PNA complementary to the longest loop of the main G-quadruplex formed by the 23-base-long bcl2midG4 sequence located 52-30 bp upstream of the P1 promoter of Bcl-2 gene. Physico-chemical and biological investigations confirmed the ability of the PNA-conjugated Ad5D24 vectors to load and transfect their PNA cargo into human A549 and MDA-MB-436 cancer cell lines, as well as the synergistic (OAd+PNA) cytotoxic effect against the same cell lines. This approach holds promise for safer chemotherapy because of reduced toxicity to healthy tissues and organs.

Inhibiting Methicillin-Resistant Staphylococcus aureus by Tetrahedral DNA Nanostructure-Enabled Antisense Peptide Nucleic Acid Delivery.

One of the biggest obstacles for the use of antisense oligonucleotides as antibacterial therapeutics is their limited uptake by bacterial cells without a suitable carrier, especially in multi-drug-resistant bacteria with a drug efflux mechanism. Existing vectors, such as cell-penetrating peptides, are inefficient and nontargeting, and accordingly are not ideal carriers. A noncytotoxic tetrahedral DNA nanostructure (TDN) with a controllable conformation has been developed as a delivery vehicle for antisense oligonucleotides. In this study, antisense peptide nucleic acids (asPNAs) targeting a specific gene ( ftsZ) were efficiently transported into methicillin-resistant Staphylococcus aureus cells by TDNs, and the expression of ftsZ was successfully inhibited in an asPNA-concentration-dependent manner. The delivery system specifically targeted the intended gene. This novel delivery system provides a better platform for future applications of antisense antibacterial therapeutics and provides a basis for the development of a new type of antibacterial drug for multi-drug-resistant bacterial infections.

Polyethylene Glycol-Engrafted Graphene Oxide as Biocompatible Materials for Peptide Nucleic Acid Delivery into Cells.

Graphene oxide (GO) is known to strongly bind single-stranded nucleic acids with fluorescence quenching near the GO surface. However, GO exhibits weak biocompatibility characteristics, such as low dispersibility in cell culture media and significant cytotoxicity. To improve dispersibility in cell culture media and cell viability of GO, we prepared nanosized GO (nGO) constructs and modified the nGO surface using polyethylene glycol (PEG-nGO). Single-stranded peptide nucleic acid (PNA) was adsorbed onto the PEG-nGO and was readily desorbed by adding complementary RNA or under low pH conditions. PNA adsorbed on the PEG-nGO was efficiently delivered into lung cancer cells via endocytosis without affecting cell viability. Furthermore, antisense PNA delivered using PEG-nGO effectively downregulated the expression of the target gene in cancer cells. Our results suggest that PEG-nGO is a biocompatible carrier useful for PNA delivery into cells and serves as a promising gene delivery tool.

Self-assembled nanoparticles for cellular delivery of peptide nucleic acid using amphiphilic N,N,N-trimethyl-O-alkyl chitosan derivatives.

Peptide nucleic acid (PNA) holds enormous potentials as antisense/antigenic drug due to its specific binding ability and biostability with DNA or RNA. However, the poor cellular delivery is the key obstacle in development of PNA therapy. To overcome this difficulty, we developed self-assembled nanoparticles (NPs) for delivery of PNA to living cells using amphiphilic CS derivatives. A series of N,N,N-trimethyl-O-alkyl chitosans (TMACs) with different lengths of alkyl chains were synthesized. The structures of these synthesized chemicals were characterized with FT-IR and 1H NMR. We found that the TMACs were all able to self-assemble in aqueous condition to form nano-size NPs. These nano-size NPs are spherical shape with a size range of around 100 nm and a zeta potential above +30 mV. PNA was easily encapsulated into chitosan derivative NPs by an ultrasonic method with entrapment efficiency up to 75%. The PNA-loaded TMAC NPs released the drug in a sustained manner in PBS (pH 7.4) at 37 °C. N,N,N-trimethyl-O-cetyl chitosan (TMCC) showed the best in vitro hemocompatibility and cell viability. These TMCC based NPs were able to dramatically increase the cellular uptake of PNA, specifically, 66-fold higher compared to without using these nanoparticles. The results suggest that the designed TMCC NPs might be a promising solution for improving cellular delivery of PNA.

Self-Assembling Lipid-Peptide Hybrid Nanoparticles of Phospholipid-Nonaarginine Conjugates for Enhanced Delivery of Nucleic Acid Therapeutics.

Despite potential applications of nucleic acid therapeutics, the lack of effective delivery systems hinders their clinical application. To overcome the barriers to nucleic acid delivery, we previously reported nanoparticles using phospholipid-polyethylenimine conjugates. However, toxicity of polyethylenimine remains as a problematic issue. Herein, we proposed to substitute the polyethylenimine with arginine-rich peptide to obtain a less-toxic carrier system. Nonaarginine was conjugated to the distal end of phospholipid hydrocarbon chains leading to phospholipid-nonaarginine conjugates (PL9R) and then lipid-peptide hybrid nanoparticles carrying oligonucleotide therapeutics (hNP) were constructed by self-assembly process. The hNP were further modified with cell penetrating Tat peptide (T-hNP) to enhance cellular uptake. The PL9R was less cytotoxic, and the hNP showed high loading capacity and colloidal stability. The T-hNP showed higher cellular uptake and transfection efficiency and effective accumulation to tumor tissue and silencing effect in tumor bearing mice. Altogether, T-hNP could provide a promising nanocarrier for nucleic acid therapeutics.

Therapeutic Peptide Nucleic Acids: Principles, Limitations, and Opportunities.

Since their invention in 1991, peptide nucleic acids (PNAs) have been used in a myriad of chemical and biological assays. More recently, peptide nucleic acids have also been demonstrated to hold great potential as therapeutic agents because of their physiological stability, affinity for target nucleic acids, and versatility. While recent modifications in their design have further improved their potency, their preclinical development has reached new heights due to their combination with recent advancements in drug delivery. This review focuses on recent advances in PNA therapeutic applications, in which chemical modifications are made to improve PNA function and nanoparticles are used to enhance PNA delivery.

Combined Delivery of Temozolomide and Anti-miR221 PNA Using Mesoporous Silica Nanoparticles Induces Apoptosis in Resistant Glioma Cells.

Mesoporous silica nanoparticles (MSNPs), 100 nm in size, incorporating a Cy5 fluorophore within the silica framework, are synthesized and loaded with the anti-cancer drug temozolomide (TMZ), used in the treatment of gliomas. The surface of the particles is then decorated, using electrostatic interactions, with a polyarginine-peptide nucleic acid (R8-PNA) conjugate targeting the miR221 microRNA. The multi-functional nanosystem thus obtained is rapidly internalized into glioma C6 or T98G cells. The anti-miR activity of the PNA is retained, as confirmed by reverse transcription polymerase chain reaction (RT-PCR) measurements and induction of apoptosis is observed in temozolomide-resistant cell lines. The TMZ-loaded MSNPs show an enhanced pro-apoptotic effect, and the combined effect of TMZ and R8-PNA in the MSNPs shows the most effective induction of apoptosis (70.9% of apoptotic cells) thus far achieved in the temozolomide-resistant T98G cell line.

Incorporation of Naked Peptide Nucleic Acids into Liposomes Leads to Fast and Efficient Delivery.

The delivery of peptide nucleic acids (PNAs) to cells is a very challenging task. We report here that a liposomal formulation composed of egg PC/cholesterol/DSPE-PEG2000 can be loaded, according to different encapsulation techniques, with PNA or fluorescent PNA oligomers. PNA loaded liposomes efficiently and quickly promote the uptake of a PNA targeting the microRNA miR-210 in human erythroleukemic K562 cells. By using this innovative delivery system for PNA, down-regulation of miR-210 is achieved at a low PNA concentration.

Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B.

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers, such as chitosan (CS), appear of particular interest as nonviral vectors due to their capacity to facilitate cellular delivery of bioactive cargoes including peptide nucleic acids (PNAs) or DNA vaccines. We have investigated the ability of a PNA conjugated to different CPPs to inhibit the replication of duck hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its PNA cargo, was able to drastically inhibit late stages of DHBV replication. In the mouse model, conjugation of HBV DNA vaccine to modified CS (Man-CS-Phe) improved cellular and humoral responses to plasmid-encoded antigen. Moreover, other systems for gene delivery were investigated including CPP-modified CS and cationic nanoparticles. The results showed that these nonviral vectors considerably increased plasmid DNA uptake and expression. Collectively promising results obtained in preclinical studies suggest the usefulness of these safe delivery systems for the development of novel therapeutics against chronic hepatitis B.

Activatable cell penetrating peptide-peptide nucleic acid conjugate via reduction of azobenzene PEG chains.

The use of stimuli-responsive bioactive molecules is an attractive strategy to circumvent selectivity issues in vivo. Here, we report an activatable cell penetrating peptide (CPP) strategy ultimately aimed at delivering nucleic acid drugs to the colon mucosa using bacterial azoreductase as the local reconversion trigger. Through screening of a panel of CPPs, we identified a sequence (M918) capable of carrying a nucleic acid analogue payload. A modified M918 peptide conjugated to a peptide nucleic acid (PNA) was shown to silence luciferase in colon adenocarcinoma cells (HT-29-luc). Reversible functionalization of the conjugate's lysine residues via an azobenzene self-immolative linkage abolished transfection activity, and the free CPP-PNA was recovered after reduction of the azobenzene bond. This activatable CPP conjugate platform could find applications in the selective delivery of nucleic acid drugs to the colon mucosa, opening therapeutic avenues in colon diseases.

Preclinical Pharmacokinetics in Tumors and Normal Tissues of the Antigene PNA Oligonucleotide MYCN-Inhibitor BGA002.

Although MYCN has been considered an undruggable target, MYCN alterations confer poor prognosis in many pediatric and adult cancers. The novel MYCN-specific inhibitor BGA002 is an antigene peptide nucleic acid oligonucleotide covalently bound to a nuclear localization signal peptide. In the present study, we characterized the pharmacokinetics (PK) of BGA002 after single and repeated administration to mice using a novel specific enzyme-linked immunosorbent assay. BGA002 concentrations in plasma showed linear PK, with dose proportional increase across the tested dose levels and similar exposure between male and female and between intravenous and subcutaneous route of administration. Repeated dosing resulted in no accumulation in plasma. Biodistribution up to 7 days after single subcutaneous administration of [14C]-radiolabeled BGA002 showed broad tissues and organ distribution (suggesting a potential capability to reach primary tumor and metastasis in several body sites), with high concentrations in kidney, liver, spleen, lymph nodes, adrenals, and bone marrow. Remarkably, we demonstrated that BGA002 concentrates in tumors after repeated systemic administrations in three mouse models with MYCN amplification (neuroblastoma, rhabdomyosarcoma, and small-cell lung cancer), leading to a significant reduction in tumor weight. Taking into account the available safety profile of BGA002, these data support further evaluation of BGA002 in patients with MYCN-positive tumors.

Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo.

In vivo delivery is a major barrier to the use of molecular tools for gene modification. Here we demonstrate site-specific gene editing of human cells in vivo in hematopoietic stem cell-engrafted NOD.Cg-Prkdc(scid)IL2rγ(tm1Wjl) (abbreviated NOD-scid IL2rγ(null)) mice, using biodegradable nanoparticles loaded with triplex-forming peptide nucleic acids (PNAs) and single-stranded donor DNA molecules. In vitro screening showed greater efficacy of nanoparticles containing PNAs/DNAs together over PNA-alone or DNA-alone. Intravenous injection of particles containing PNAs/DNAs produced modification of the human CCR5 gene in hematolymphoid cells in the mice, with modification confirmed at the genomic DNA, mRNA and functional levels. Deep sequencing revealed in vivo modification of the CCR5 gene at frequencies of 0.43% in hematopoietic cells in the spleen and 0.05% in the bone marrow: off-target modification in the partially homologous CCR2 gene was two orders of magnitude lower. We also induced specific modification in the ß-globin gene using nanoparticles carrying ß-globin-targeted PNAs/DNAs, demonstrating this method's versatility. In vivo testing in an enhanced green fluorescent protein-ß-globin reporter mouse showed greater activity of nanoparticles containing PNAs/DNAs together over DNA only. Direct in vivo gene modification, such as we demonstrate here, would allow for gene therapy in systemic diseases or in cells that cannot be manipulated ex vivo.

Targeted gene modification of hematopoietic progenitor cells in mice following systemic administration of a PNA-peptide conjugate.

Hematopoietic stem cell (HSC) gene therapy offers promise for the development of new treatments for a variety of hematologic disorders. However, efficient in vivo modification of HSCs has proved challenging, thus imposing constraints on the therapeutic potential of this approach. Herein, we provide a gene-targeting strategy that allows site-specific in vivo gene modification in the HSCs of mice. Through conjugation of a triplex-forming peptide nucleic acid (PNA) to the transport peptide, antennapedia (Antp), we achieved successful in vivo chromosomal genomic modification of hematopoietic progenitor cells, while still retaining intact differentiation capabilities. Following systemic administration of PNA-Antp conjugates, sequence-specific gene modification was observed in multiple somatic tissues as well as within multiple compartments of the hematopoietic system, including erythroid, myeloid, and lymphoid cell lineages. As a true functional measure of gene targeting in a long-term renewable HSC, we also demonstrate preserved genomic modification in the bone marrow and spleen of primary recipient mice following transplantation of bone marrow from PNA-Antp-treated donor mice. Our approach offers a minimally invasive alternative to ex vivo gene therapy, by eliminating the need for the complex steps of stem cell mobilization and harvesting, ex vivo manipulation, and transplantation of stem cells. Therefore, our approach may provide new options for individualized therapies in the treatment of monogenic hematologic diseases such as sickle cell anemia and thalassemia.

Safety and Biodistribution of Nanoligomers Targeting the SARS-CoV-2 Genome for the Treatment of COVID-19.

As the world braces to enter its fourth year of the coronavirus disease 2019 (COVID-19) pandemic, the need for accessible and effective antiviral therapeutics continues to be felt globally. The recent surge of Omicron variant cases has demonstrated that vaccination and prevention alone cannot quell the spread of highly transmissible variants. A safe and nontoxic therapeutic with an adaptable design to respond to the emergence of new variants is critical for transitioning to the treatment of COVID-19 as an endemic disease. Here, we present a novel compound, called SBCoV202, that specifically and tightly binds the translation initiation site of RNA-dependent RNA polymerase within the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome, inhibiting viral replication. SBCoV202 is a Nanoligomer, a molecule that includes peptide nucleic acid sequences capable of binding viral RNA with single-base-pair specificity to accurately target the viral genome. The compound has been shown to be safe and nontoxic in mice, with favorable biodistribution, and has shown efficacy against SARS-CoV-2 in vitro. Safety and biodistribution were assessed using three separate administration methods, namely, intranasal, intravenous, and intraperitoneal. Safety studies showed the Nanoligomer caused no outward distress, immunogenicity, or organ tissue damage, measured through observation of behavior and body weight, serum levels of cytokines, and histopathology of fixed tissue, respectively. SBCoV202 was evenly biodistributed throughout the body, with most tissues measuring Nanoligomer concentrations well above the compound KD of 3.37 nM. In addition to favorable availability to organs such as the lungs, lymph nodes, liver, and spleen, the compound circulated through the blood and was rapidly cleared through the renal and urinary systems. The favorable biodistribution and lack of immunogenicity and toxicity set Nanoligomers apart from other antisense therapies, while the adaptability of the nucleic acid sequence of Nanoligomers provides a defense against future emergence of drug resistance, making these molecules an attractive potential treatment for COVID-19.

Nanoparticles deliver triplex-forming PNAs for site-specific genomic recombination in CD34+ human hematopoietic progenitors.

Triplex-forming peptide nucleic acids (PNAs) are powerful gene therapy agents that can enhance recombination of short donor DNAs with genomic DNA, leading to targeted and specific correction of disease-causing genetic mutations. Therapeutic use of PNAs is severely limited, however, by challenges in intracellular delivery, particularly in clinically relevant targets such as hematopoietic stem and progenitor cells. Here, we demonstrate efficient and nontoxic PNA-mediated recombination in human CD34(+) cells using poly(lactic-co-glycolic acid) (PLGA) nanoparticles for intracellular oligonucleotide delivery. Treatment of progenitor cells with nanoparticles loaded with PNAs and DNAs targeting the ß-globin locus led to levels of site-specific modification in the range of 0.5-1% in a single treatment, without detectable loss in cell viability, resulting in a 60-fold increase in modified and viable cells as compared to nucleofection. As well, the differentiation capacity of the progenitor cells treated with nanoparticles did not change relative to untreated progenitor cells, indicating that nanoparticles are safe and minimally disruptive delivery vectors for PNAs and DNAs to mediate gene modification in human primary cells. This is the first demonstration of the use of biodegradable nanoparticles to deliver genome-editing agents to human primary cells, and provides a strong rationale for systemic delivery of complex nucleic acid mixtures designed for gene correction.