Phosphorodiamidate Morpholino Oligonucleotides Research Articles

Characterization of Nonclinical Drug Metabolism and Pharmacokinetic Properties of Phosphorodiamidate Morpholino Oligonucleotides, a Novel Drug Class for Duchenne Muscular Dystrophy.

Eteplirsen, golodirsen, and casimersen are phosphorodiamidate morpholino oligomers (PMOs) that are approved in the United States for the treatment of patients with Duchenne muscular dystrophy (DMD) with mutations in the DMD gene that are amenable to exon 51, 53, and 45 skipping, respectively. Here we report a series of in vivo and in vitro studies characterizing the drug metabolism and pharmacokinetic (DMPK) properties of these three PMOs. Following a single intravenous dose, plasma exposure was consistent for all three PMOs in mouse, rat, and nonhuman primate (NHP), and plasma half-lives were similar for eteplirsen (2.0-4.1 h) and golodirsen (2.1-8.7 h) across species and more variable for casimersen (3.2-18.1 h). Plasma protein binding was low (<40%) for all three PMOs in mouse, rat, NHP, and human and was largely concentration independent. In the mdx mouse model of DMD, following a single intravenous injection, extensive biodistribution was observed in the target skeletal muscle tissues and the kidney for all three PMOs; consistent with the latter finding, the predominant route of elimination was renal. In vitro studies using liver microsomes showed no evidence of hepatic metabolism, and none of the PMOs were identified as inhibitors or inducers of the human cytochrome P450 enzymes or membrane drug transporters tested at clinically relevant concentrations. These findings suggest that key DMPK features are consistent for eteplirsen, golodirsen, and casimersen and provide evidence for the concept of a PMO drug class with potential application to novel exon-skipping drug candidates. SIGNIFICANCE STATEMENT: The PMOs eteplirsen, golodirsen, and casimersen share similar absorption, distribution, metabolism, and excretion and DMPK properties, which provides evidence for the concept of a PMO treatment class. A PMO drug class may support a platform approach to enhance understanding of the pharmacokinetic and pharmacodynamic behavior of these molecules. The grouping of novel agent series into platforms could be beneficial in the development of drug candidates for populations in which traditional clinical trials are not feasible.

Synthesis of Phosphorodiamidate Morpholino Oligonucleotides (PMOs) Using Staudinger Reduction as a Deblocking Condition and Its Usefulness for Orthogonal Conjugation in Bi- and Trifunctionalized PMOs

AbstractThe synthesis of short phosphorodiamidate morpholino oligonucleotides (PMOs) has been successfully achieved using azidoaryl carbamate protected chlorophosphoramidate monomers. The deprotection step carried out in a neutral medium with triphenylphosphine-based reagents avoids the need for chlorinated solvents. This method uses a meticulously tailored combination of resin support, solvents, deblocking agents, and coupling reagents to ensure efficient synthesis. Additionally, the azidoaryl carbamate protecting group has been adapted as an orthogonal protection, enabling the development of bi- and trifunctionalized PMOs for bioconjugation. These advancements are expected to broaden the potential applications of PMOs in biomedical research.

The Usefulness of Determining Plasma and Tissue Concentrations of Phosphorodiamidate Morpholino Oligonucleotides to Estimate Their Efficacy in Duchenne Muscular Dystrophy Patients.

Currently, four kinds of phosphorodiamidate morpholino oligomers (PMOs), such as viltolarsen, have been approved for the treatment of Duchenne muscular dystrophy (DMD); however, it is unclear whether human efficacy can be estimated using plasma concentrations. This study summarizes the tissue distribution of viltolarsen in mice and cynomolgus monkeys and evaluates the relationship between exposure and efficacy based on exon skipping. In the tissue distribution studies, all muscles in DMD-model mice showed higher concentrations of viltolarsen than those in wild-type mice and cynomolgus monkeys, and the concentrations in skeletal muscle were correlated with the exon-skipping efficiency in mice and cynomolgus monkeys. In addition, a highly sensitive bioanalytical method using liquid chromatography with tandem mass spectrometry shows promise for determining plasma concentrations up to a later time point, and the tissue (muscle)/plasma concentration ratio (Kp) in DMD-model mice was shown to be useful for predicting changes in pharmacodynamic (PD) markers in humans. Our results suggest that pharmacokinetic (PK)/PD analysis can be conducted by using the human PK profile or Kp values and skipping efficiency in DMD-model mice. This information will be useful for the efficient and effective development of PMOs as therapeutic agents. SIGNIFICANCE STATEMENT: We evaluated the relationship between the plasma or tissue concentrations and the efficiency of exon skipping for viltolarsen as an example phosphorodiamidate morpholino oligomers in the skeletal and cardiac muscle of mice and cynomolgus monkeys for pharmacokinetic/pharmacodynamic (PK/PD) analysis. The results suggest that PK/PD analysis can be conducted by using the human PK profile or tissue (muscle)/plasma concentration ratios and skipping efficiency in DMD-model mice.

Evaluation of Transiently O-6-Protected Guanosine Morpholino Nucleosides in Phosphorodiamidate Morpholino Oligonucleotide Synthesis

AbstractA novel strategy is presented for the synthesis of morpholino guanosine monomers protected at O-6 with 1-(4-azidophenyl)ethan-1-ol, p-methoxybenzyl alcohol and trimethylsilylethyl groups. The introduction of these protecting groups increases the solubility of the morpholino nucleosides which is crucial during the synthesis of phosphorodiamidate morpholino oligonucleotides (PMOs). HPLC analysis shows that the trimethylsilylethyl-protected monomer gives better coupling efficiency in PMO synthesis compared to the regular monomer. Moreover the nonpolar nature of the O-6-protected monomer facilitates the preparation of guanosine-rich oligomer in solution.

Understanding Antisense Oligonucleotide Efficiency in Inhibiting Prokaryotic Gene Expression.

Oligonucleotides offer a unique opportunity for sequence specific regulation of gene expression in bacteria. A fundamental question to address is the choice of oligonucleotide, given the large number of options available. Different modifications varying in RNA binding affinities and cellular uptake are available but no comprehensive comparisons have been performed. Herein, the efficiency of blocking expression of β-galactosidase (β-Gal) in E. coli was evaluated utilizing different antisense oligomers (ASOs). Fluorescein (FAM)-labeled oligomers were used to understand their differences in bacterial uptake. Flow cytometry analysis revealed significant differences in uptake, with high fluorescence seen in cells treated with FAM-labeled peptidic nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO) and phosphorothioate (PS) oligomers, and low fluorescence observed in cells treated with phosphodiester (PO) oligomers. Thermal denaturation (Tm) of oligomer:RNA duplexes and isothermal titration calorimetry (ITC) studies reveal that ASO binding to target RNA demonstrates a good correlation between Tm and Kd values. There was no correlation between Kd values and reduction of β-Gal activity in bacterial cells. However, cell-free translation assays demonstrated a direct relationship between Kd values and inhibition of gene expression by antisense oligomers, with tight binding oligomers such as LNA being the most efficient. Membrane active compounds such as polymyxin B and A22 further improved the cellular uptake of FAM-PNA and FAM-PS oligomers in wild-type E. coli cells. PNA and PMO were most effective in cellular uptake and reducing β-Gal activity as compared to oligomers with PS or those with PO linkages. Overall, cell uptake of the oligomers is shown as the key determinant in predicting their differences in bacterial antisense inhibition, and the RNA affinity is the key determinant in inhibition of gene expression in cell free systems.

Synthesis and Biophysical Properties of Triazole-Incorporated PMOs (TzPMOs): A Convergent, Click Ligation Approach.

The synthesis of phosphorodiamidate morpholino oligonucleotides (PMOs) incorporating single or double triazole rings in the backbone has been achieved via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The synthetic approach implemented is fundamentally convergent, involving the ligation of a 5'-azide PMO fragment to a 3'-alkyne fragment both in solution and on solid support. To access the 3'-alkyne PMO fragment, we synthesized 3'-N-propargyl chlorophosphoramidate morpholino monomers for all four nucleobases. The resulting triazole-incorporated PMOs (TzPMOs) have exhibited comparable or improved binding affinity toward complementary deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) strands compared to its regular analogues. Finally, a full-length TzPMO was designed to target the Nanog gene, demonstrating almost identical hybridization properties when compared to its regular version. Circular dichroism studies revealed a B-type helical conformation for the duplexes formed by TzPMOs.

Synthesis of Self Permeable Antisense PMO Using C5-Guanidino-Functionalized Pyrimidines at the 5'-End Enables Sox2 Downregulation in Triple Negative Breast Cancer Cells.

Delivery of macromolecular drugs inside cells has been a huge challenge in the field of oligonucleotide therapeutics for the past few decades. Earliest natural inspirations included the arginine rich stretch of cell permeable HIV-TAT peptide, which led to the design of several molecular transporters with varying numbers of rigid or flexible guanidinium units with different tethering groups. These transporters have been shown to efficiently deliver phosphorodiamidate morpholino oligonucleotides, which have a neutral backbone and cannot form lipoplexes. In this report, PMO based delivery agents having 3 or 4 guanidinium groups at the C5 position of the nucleobases of cytosine and uracil have been explored, which can be assimilated within the desired stretch of the antisense oligonucleotide. Guanidinium units have been connected by varying the flexibility with either a saturated (propyl) or an unsaturated (propargyl) spacer, which showed different serum dependency along with varied cytoplasmic distribution. The effect of cholesterol conjugation in the delivery agent as well as at the 5'-end of full length PMO in cellular delivery has also been studied. Finally, the efficacy of the delivery has been studied by the PMO mediated downregulation of the stemness marker Sox2 in the triple-negative breast cancer cell line MDA-MB 231. These results have validated the use of this class of delivery agents, which permit at a stretch PMO synthesis where the modified bases can also participate in Watson-Crick-Franklin base pairing for enhanced mRNA binding and protein downregulation and could solve the delivery problem of PMO.

Peptide-conjugated antimiRs improve myotonic dystrophy type 1 phenotypes by promoting endogenous MBNL1 expression

Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by a CTG repeat expansion in the DMPK gene that generates toxic RNA with a myriad of downstream alterations in RNA metabolism. A key consequence is the sequestration of alternative splicing regulatory proteins MBNL1/2 by expanded transcripts in the affected tissues. MBNL1/2 depletion interferes with a developmental alternative splicing switch that causes the expression of fetal isoforms in adults. Boosting the endogenous expression of MBNL proteins by inhibiting the natural translational repressors miR-23b and miR-218 has previously been shown to be a promising therapeutic approach. We designed antimiRs against both miRNAs with a phosphorodiamidate morpholino oligonucleotide (PMO) chemistry conjugated to cell-penetrating peptides (CPPs) to improve delivery to affected tissues. In DM1 cells, CPP-PMOs significantly increased MBNL1 levels. In some candidates, this was achieved using concentrations less than two orders of magnitude below the median toxic concentration, with up to 5.38-fold better therapeutic window than previous antagomiRs. In HSALR mice, intravenous injections of CPP-PMOs improve molecular, histopathological, and functional phenotypes, without signs of toxicity. Our findings place CPP-PMOs as promising antimiR candidates to overcome the treatment delivery challenge in DM1 therapy.

Convergent synthesis of phosphorodiamidate morpholino oligonucleotides (PMOs) by the H-phosphonate approach

Phosphorodiamidate morpholino oligonucleotides (PMOs) are a promising type of antisense oligonucleotides, but their challenging synthesis makes them difficult to access. This research presents an efficient synthetic approach for PMOs using the H-phosphonate approach. The use of phosphonium-type condensing reagents significantly reduced coupling times compared with the current synthetic approach. Furthermore, phosphonium-type condensing reagents facilitated the fragment condensation of PMO, synthesizing up to 8-mer containing all four nucleobases with remarkable coupling efficacy. This is the first report on the convergent synthesis of PMOs. This approach would facilitate the large-scale synthesis of PMOs and accelerate their popularity and accessibility as a next-generation therapy.

Self-transfecting GMO-PMO chimera targeting Nanog enable gene silencing in vitro and suppresses tumor growth in 4T1 allografts in mouse

Phosphorodiamidate morpholino oligonucleotide (PMO)-based antisense reagents cannot enter cells without the help of a delivery technique, which limits their clinical applications. To overcome this problem, self-transfecting guanidinium-linked morpholino (GMO)-PMO or PMO-GMO chimeras have been explored as antisense agents. GMO facilitates cellular internalization and participates in Watson-Crick base pairing. Targeting NANOG in MCF7 cells resulted in decline of the whole epithelial to mesenchymal transition (EMT) and stemness pathway, evident through its phenotypic manifestations, all of which were promulgated in combination with Taxol due to downregulation of MDR1 and ABCG2. GMO-PMO-mediated knockdown of no tail gene resulted in desired phenotypes in zebrafish even upon delivery after 16-cell stages. In BALB/c mice, 4T1 allografts were found to regress via intra-tumoral administration of NANOG GMO-PMO antisense oligonucleotides (ASOs), which was associated with occurrence of necrotic regions. GMO-PMO-mediated tumor regression restored histopathological damage in liver, kidney, and spleen caused by 4T1 mammary carcinoma. Serum parameters of systemic toxicity indicated that GMO-PMO chimeras are safe. To the best of our knowledge, self-transfecting antisense reagent is the first report since the discovery of guanidinium-linked DNA (DNG), which could be useful as a combination cancer therapy and, in principle, can render inhibition of any target gene without using any delivery vehicle.

Therapeutic phosphorodiamidate morpholino oligonucleotides: Physical properties, solution structures, and folding thermodynamics

Elucidating the structure-function relationships for therapeutic RNA mimicking phosphorodiamidate morpholino oligonucleotides (PMOs) is challenging due to the lack of information about their structures. While PMOs have been approved by the US Food and Drug Administration for treatment of Duchenne muscular dystrophy, no structural information on these unique, charge-neutral, and stable molecules is available. We performed circular dichroism and solution viscosity measurements combined with molecular dynamics simulations and machine learning to resolve solution structures of 22-mer, 25-mer, and 30-mer length PMOs. The PMO conformational dynamics are defined by the competition between non-polar nucleobases and uncharged phosphorodiamidate groups for shielding from solvent exposure. PMO molecules form non-canonical, partially helical, stable folded structures with a small 1.4- to 1.7-nm radius of gyration, low count of three to six base pairs and six to nine base stacks, characterized by -34 to -51kcal/mol free energy, -57 to -103kcal/mol enthalpy, and -23 to -53kcal/mol entropy for folding. The 4.5- to 6.2-cm3/g intrinsic viscosity and Huggins constant of 4.5-9.9 are indicative of extended and aggregating systems. The results obtained highlight the importance of the conformational ensemble view of PMO solution structures, thermodynamic stability of their non-canonical structures, and concentration-dependent viscosity properties. These principles form a paradigm to understand the structure-properties-function relationship for therapeutic PMOs to advance the design of new RNA-mimic-based drugs.

Synthesis of Chlorophosphoramidate Monomer Morpholinos and PMOs.

Phosphorodiamidate morpholino oligonucleotides (PMOs) are a successful class of antisense reagents that efficiently modulate gene expression. Because PMOs do not follow standard phosphoramidite chemistry, optimized synthetic protocols for these compounds are relatively scarce in the literature. This paper presents detailed protocols for synthesizing full-length PMOs using chlorophosphoramidate chemistry by manual solid-phase synthesis. We first describe the synthesis of Fmoc-protected morpholino hydroxyl monomers, and the corresponding chlorophosphoramidate monomers, from commercially available protected ribonucleosides. The new Fmoc chemistry necessitates the use of a milder base, such as N-ethylmorpholine (NEM), and coupling reagent, such as 5-(ethylthio)-1H-tetrazole (ETT), which are also tolerated for acid-sensitive trityl chemistry. These chlorophosphoramidate monomers are then employed for PMO synthesis in a manual solid-phase procedure using four sequential steps. The synthetic cycle for each nucleotide incorporation consists of (a) deblocking of the 3'-N protecting group using an acidic deblocking cocktail for trityl and base deblocking for Fmoc, (b) neutralization, (c) coupling in the presence of ETT and NEM, and (d) capping of the unreacted morpholine ring-amine. The method uses safe, stable, and inexpensive reagents, and the process is expected to be scalable. After full-length PMO synthesis and ammonia-mediated cleavage from the solid support and deprotection, a range of PMOs with different lengths can be obtained conveniently and efficiently with reproducible good yields. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of the novel Fmoc-protected morpholino monomers Basic Protocol 2: Synthesis of the phosphorylating reagent (N,N-dimethylphosphoramic dichloride) required for chlorophosphoramidate monomer synthesis Basic Protocol 3: Synthesis of chlorophosphoramidate monomers of Fmoc-protected morpholino monomers Basic Protocol 4: Solution-phase standardization of dimer and trimer PMO synthesis using Fmoc chemistry Basic Protocol 5: Solid-phase synthesis, purification, and characterization of full-length (25-mer) no-tail PMO using both trityl and Fmoc chemistry.

C5-pyrimidine-functionalized morpholino oligonucleotides exhibit differential binding affinity, target specificity and lipophilicity.

C5-substituted uridine and cytidine morpholino chlorophosphoramidate monomers were synthesized and incorporated into a 12-mer Phosphorodiamidate Morpholino Oligonucleotide (PMO) using semi-automated solid phase synthesis. PMOs with most of the tested pyrimidine C5-substitutions have significantly increased thermal stability when bound to the complementary RNA strand relative to the PMO. They exhibit higher binding with RNA than DNA. CD-spectra show B-type helical conformation of duplexes. HPLC analysis indicates their greater lipophilicity compared to regular PMOs. These chemical modifications have significant potential towards the development of better antisense technologies.

Synthesis of 5'-Thiol Functionalized Morpholino Oligo-Nucleotide and Subsequent Conjugation with IGT to Improve Delivery and Antisense Efficacy In Vitro.

Thiol functionalized oligonucleotides are useful intermediates for a wide range of applications including DNA nanobiotechnology field through conjugation with various types of probes and cargos. Due to the limitation of synthetic process, phosphorodiamidate morpholino oligonucleotides (PMOs) have not been explored like other oligonucleotides through SH conjugation as mentioned above. In this paper, we report the synthesis of 5'-SH functionalized PMO using a solid support synthesis protocol with an optimized cysteine derived linker so that loading and coupling efficiency of morpholino monomers were effective enough to get a 25-mer 5'-SH functionalized PMO against human Nanog. The PMO with SH functionality was subsequently conjugated with our previously reported Internal Oligo-guanidinium Transporter (IGT) in solution phase to obtain the IGT-PMO conjugate. Interestingly, 5'-conjugated PMO (IGT-PMO) showed 2.5 times better antisense efficacy than 3'-conjugated PMO with IGT (PMO-IGT). 5'-Conjugation enables us to use IGT-PMO for further conjugation at the 3'-N terminal of PMO which was not possible earlier with 5'-OH-PMO-IGT. PMO has become an important class of antisense reagents because four PMO-based drugs have been approved for the treatment of Duchenne muscular dystrophy; hence such an improved result with 5'-modified PMO could be useful for enhancing the therapeutic efficacy of DMD drugs. Similarly, thiol-modified PMO could also be explored like other thiol-containing oligonucleotides for various other applications.

Peptide-Conjugated PMOs for the Treatment of Myotonic Dystrophy.

Antisense oligonucleotides (ASOs) have shown great therapeutic potential in the treatment of many neuromuscular diseases including myotonic dystrophy 1 (DM1). However, systemically delivered ASOs display poor biodistribution and display limited penetration into skeletal muscle. The conjugation of cell-penetrating peptides (CPPs) to phosphorodiamidate morpholino oligonucleotides (PMOs), a class of ASOs with a modified backbone, can be used to enhance ASO skeletal muscle penetration. Peptide-PMOs (P-PMOs) have been shown to be highly effective in correcting the DM1 skeletal muscle phenotype in both murine and cellular models of DM1 and at a molecular and functional level. Here we describe the synthesis and conjugation of P-PMOs and methods for analyzing their biodistribution and toxicity in the HSA-LR DM1 mouse model and their efficacy both in vitro and in vivo using FISH and RT-PCR splicing analysis.

Synthesis of Phosphorodiamidate Morpholino Oligonucleotides Using Trityl and Fmoc Chemistry in an Automated Oligo Synthesizer.

Phosphorodiamidate morpholino oligonucleotides (PMOs) constitute 3 out of the 11 FDA-approved oligonucleotide-based drugs in the last 6 years. PMOs can effectively silence disease-causing genes and modify splicing. However, PMO synthesis has remained challenging for a variety of reasons: inefficient deprotection and coupling methods and instability of monomers. Here, we report the development of a suitable combination of resin supports, deblocking and coupling reagents for synthesizing PMOs using either trityl or Fmoc-protected chlorophosphoramidate monomers. The synthesized PMOs using both the methods on a solid support have been validated for gene silencing in a zebrafish model. The protocol was successfully transferred into an automated DNA synthesizer to make several sequences of PMOs, demonstrating for the first time the adaptation of regular PMOs in a commercial DNA synthesizer. Moreover, PMOs with longer than 20-mer sequences, including FDA-approved Eteplirsen (30-mer), were achieved in >20% overall yield that is superior to previous reports. Hybridization study shows that PMOs exhibit a higher binding affinity toward complementary DNA relative to the DNA/DNA duplex (>6 °C). Additionally, the introduction of Fmoc chemistry into PMOs opens up the possibility for PMO synthesis in commercial peptide synthesizers for future development.

Casimersen for the treatment of Duchenne muscular dystrophy

Casimersen is a single-stranded, antisense phosphorodiamidate morpholino oligonucleotide where six-membered morpholino rings substitute the ribofuranose rings of the natural DNA and RNA backbone. The morpholino is conjugated to the following monomer via a charge-neutral phosphorodiamidate instead of the phosphodiester linkage containing a negative charge. Phosphorodiamidate morpholino oligonucleotides are resistant to enzymatic degradation and their lack of charge facilitates cellular penetration.

Enhancement of Regnase-1 expression with stem loop-targeting antisense oligonucleotides alleviates inflammatory diseases.

Regnase-1 is an ribonuclease that plays essential roles in restricting inflammation through degrading messenger RNAs (mRNAs) involved in immune reactions via the recognition of stem-loop (SL) structures in the 3' untranslated regions (3'UTRs). Dysregulated expression of Regnase-1 is associated with the pathogenesis of inflammatory and autoimmune diseases in mice and humans. Here, we developed a therapeutic strategy to suppress inflammatory responses by blocking Regnase-1 self-regulation, which was mediated by the simultaneous use of two antisense phosphorodiamidate morpholino oligonucleotides (MOs) to alter the binding of Regnase-1 toward the SL structures in its 3'UTR. Regnase-1-targeting MOs not only enhanced Regnase-1 expression by stabilizing mRNAs but also effectively reduced the expression of multiple proinflammatory transcripts that were controlled by Regnase-1 in macrophages. Intratracheal administration of Regnase-1-targeting MOs ameliorated acute respiratory distress syndrome and chronic fibrosis through suppression of inflammatory cascades. In addition, intracranial treatment with Regnase-1-targeting MOs attenuated the development of experimental autoimmune encephalomyelitis by promoting the expansion of homeostatic microglia and regulatory T cell populations. Regnase-1 expression was inversely correlated with disease severity in patients with multiple sclerosis, and MOs targeting human Regnase-1 SL structures were effective in mitigating cytokine production in human immune cells. Collectively, MO-mediated disruption of the Regnase-1 self-regulation pathway is a potential therapeutic strategy to enhance Regnase-1 abundance, which, in turn, provides therapeutic benefits for treating inflammatory diseases by suppressing inflammation.

Structural Modifications to the Internal Oligoguanidinium Transporter Uncover Two Potent Analogues that Effectively Deliver the Proapoptotic Peptide in Multiple Cancer Cell Lines.

Efficient cytosolic delivery with serum-independent kinetics and low toxicity are the ultimate challenges towards the transformation of an antisense oligonucleotide or a therapeutic peptide to a suitable drug candidate for clinical trials. Most delivery vehicles falter on at least one of the above requirements, which hinders their potential in in vivo models as well. Our previous reports on internal guanidinium transporters (IGTs) have established the diversity of this particular class of molecule with the efficient delivery of antisense phosphorodiamidate morpholino oligonucleotides. In this paper, we report twenty IGTs with different types of evidence-backed structural modifications with different types of head-group linkage R, which significantly change the transfection, toxicity, and endosomal escape. Based on these three criteria, the analogues were sorted systematically to find the more promising IGTs, which were then further examined by LysoTracker studies. Finally, two analogues, with cholesteryl linkage (R = Chol) and pentafluorobenzyl linkage (R = PF Cbz), were selected for a proapoptotic peptide delivery as the final validation using a long-chain di-acid linker conjugation. Detailed mechanistic studies also revealed that the primary pathway of endocytosis is macropinocytosis, and that other pathways play different roles depending on the head group of the IGT. Since endocytosis pathways for entry depend on the nature of the cell line, we have shown the mechanistic variations in two cell lines for validation.

Modular Synthesis of Trifunctional Peptide-oligonucleotide Conjugates via Native Chemical Ligation.

Cell penetrating peptides (CPPs) are being increasingly used as efficient vectors for intracellular delivery of biologically active agents, such as therapeutic antisense oligonucleotides (ASOs). Unfortunately, ASOs have poor cell membrane permeability. The conjugation of ASOs to CPPs have been shown to significantly improve their cellular permeability and therapeutic efficacy. CPPs are often covalently conjugated to ASOs through a variety of chemical linkages. Most of the reported approaches for ligation of CPPs to ASOs relies on methodologies that forms non-native bond due to incompatibility with in-solution phase conjugation. These approaches have low efficiency and poor yields. Therefore, in this study, we have exploited native chemical ligation (NCL) as an efficient strategy for synthesizing CPP-ASO conjugates. A previously characterized CPP [ApoE(133–150)] was used to conjugate to a peptide nucleic acid (PNA) sequence targeting human survival motor neuron-2 (SMN2) mRNA which has been approved by the FDA for the treatment of spinal muscular atrophy. The synthesis of ApoE(133–150)-PNA conjugate using chemo-selective NCL was highly efficient and the conjugate was obtained in high yield. Toward synthesizing trifunctional CPP-ASO conjugates, we subsequently conjugated different functional moieties including a phosphorodiamidate morpholino oligonucleotide (PMO), an additional functional peptide or a fluorescent dye (Cy5) to the thiol that was generated after NCL. The in vitro analysis of the bifunctional CPP-PNA and trifunctional CPP-(PMO)-PNA, CPP-(peptide)-PNA and CPP-(Cy5)-PNA showed that all conjugates are cell-permeable and biologically active. Here we demonstrated chemo-selective NCL as a highly efficient and superior conjugation strategy to previously published methods for facile solution-phase synthesis of bi-/trifunctional CPP-ASO conjugates.