Amphiphilic lipopeptide significantly enhances uptake of charge-neutral splice switching morpholino oligonucleotide in spinal muscular atrophy patient-derived fibroblasts

Tajik-Ahmadabad, Behnoosh; Polyzos, Anastasios; Separovic, Frances; Shabanpoor, Fazel

doi:10.1016/j.ijpharm.2017.08.116

Cited by 15 publications

(12 citation statements)

References 40 publications

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“…The direct conjugation of siRNAs to CPP may result in neutralizing the charges on CPPs, which would lead to a reduced efficiency of membrane translocation [ 58 ]. To circumvent this problem, nucleic acid analogs with reduced charges, such as PNA and PMO, have been used in conjugation with CPPs [ 87 , 88 ]. In studies on cells in culture and on animals, such as mice and nonhuman primates, it was shown that conjugation to CPPs much improved the cellular uptake and efficiency of PMOs for the treatment of DMD [ 89 , 90 ].…”

Section: Delivery Issuesmentioning

confidence: 99%

Current Challenges in Delivery and Cytosolic Translocation of Therapeutic RNAs

Johannes

Lucchino

2018

Nucleic Acid Therapeutics

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RNA interference (RNAi) is a fundamental cellular process for the posttranscriptional regulation of gene expression. RNAi can exogenously be modulated by small RNA oligonucleotides, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), or by antisense oligonucleotides. These small oligonucleotides provided the scientific community with powerful and versatile tools to turn off the expression of genes of interest, and hold out the promise of new therapeutic solutions against a wide range of gene-associated pathologies. However, unmodified nucleic acids are highly instable in biological systems, and their weak interaction with plasma proteins confers an unfavorable pharmacokinetics. In this review, we first provide an overview of the most efficient chemical strategies that, over the past 30 years, have been used to significantly improve the therapeutic potential of oligonucleotides. Oligonucleotides targeting and delivery technologies are then presented, including covalent conjugates between oligonucleotides and targeting ligand, and noncovalent association with lipid or polymer nanoparticles. Finally, we specifically focus on the endosomal escape step, which represents a major stumbling block for the effective use of oligonucleotides as therapeutic agents. The need for approaches to quantitatively measure endosomal escape and cytosolic arrival of biomolecules is discussed in the context of the development of efficient oligonucleotide targeting and delivery vectors.

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Section: Delivery Issuesmentioning

confidence: 99%

Current Challenges in Delivery and Cytosolic Translocation of Therapeutic RNAs

Johannes

Lucchino

2018

Nucleic Acid Therapeutics

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“…Both peptides were purified to greater than 95% purity as determined by analytical HPLC and obtained in a high yield (Table 1, ESI Supplementary Figures S1, S2). In order to synthesize the trifunctional peptide-(PMO)-PNA conjugate, a 20-mer PMO with the same sequence as the PNA (Table 1) was functionalized at its 3′-end with maleimidopropionic acid, as previously described (Tajik-Ahmadabad et al, 2017). The purified Maleimide-PMO was obtained in excellent yield (78%) (ESI Supplementary Figures S1D, S2D).…”

Section: Resultsmentioning

confidence: 99%

“…The conjugates were tested at 1 and 2 µm concentrations to enable comparison of CPP efficiency in delivering the conjugated PNA, PMO and fluorophore into the cells. The maximum concentration for CPP-ASO conjugates was set at 2 µm, as higher concentrations would results in saturation of response based on our previous studies on cellular uptake of ApoE and similar peptides (Shabanpoor et al, 2017;Tajik-Ahmadabad et al, 2017). All conjugates significantly increased the level of full-length SMN2 compared to untreated cells, as measured by RT-qPCR (Figure 2A).…”

Section: Resultsmentioning

confidence: 99%

“…CPPs are relatively short cationic, amphipathic peptides (Kurrikoff et al, 2020) and are being widely used for intracellular drug delivery for a wide range of cell-impermeable cargoes, such as therapeutic antisense oligonucleotides (ASOs). The conjugation of CPPs to ASOs has been shown to enhance their cell permeability, intracellular bioavailability, and subsequently, an increase in their therapeutic efficacy (Tajik-Ahmadabad et al, 2017;Klein et al, 2019;Nikan et al, 2020). CPPs have also been utilized to cross the blood-brain barrier for delivery of therapeutic ASOs into the central nervous system (CNS) (Hammond et al, 2016;Shabanpoor et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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“…Some SMA patients carry the mutation(s) in the SMN2 gene and c.804T mutation resulted in the deletion of exon 7 in nearly 90% of the SMN2 transcripts (Δ7SMN2) [ 143 ]. Using a steric-blocking PMO to bind the pre-mRNA of the mutated SMN2 gene can successfully block the aberrant splicing event, produce full-length functional SMN2 [ 144 ], and compensate for the deficiency of SMN1. Thus, these steric-blocking PMOs provide a significant therapeutic benefit for SMA patients.…”

Section: Rna Therapies To Treat Single-gene Neurological Disordersmentioning

confidence: 99%

Recent Advances in RNA Therapy and Its Carriers to Treat the Single-Gene Neurological Disorders

Lee

Wang

2022

Biomedicines

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The development of new sequencing technologies in the post-genomic era has accelerated the identification of causative mutations of several single gene disorders. Advances in cell and animal models provide insights into the underlining pathogenesis, which facilitates the development and maturation of new treatment strategies. The progress in biochemistry and molecular biology has established a new class of therapeutics—the short RNAs and expressible long RNAs. The sequences of therapeutic RNAs can be optimized to enhance their stability and translatability with reduced immunogenicity. The chemically-modified RNAs can also increase their stability during intracellular trafficking. In addition, the development of safe and high efficiency carriers that preserves the integrity of therapeutic RNA molecules also accelerates the transition of RNA therapeutics into the clinic. For example, for diseases that are caused by genetic defects in a specific protein, an effective approach termed “protein replacement therapy” can provide treatment through the delivery of modified translatable mRNAs. Short interference RNAs can also be used to treat diseases caused by gain of function mutations or restore the splicing aberration defects. Here we review the applications of newly developed RNA-based therapeutics and its delivery and discuss the clinical evidence supporting the potential of RNA-based therapy in single-gene neurological disorders.

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Amphiphilic lipopeptide significantly enhances uptake of charge-neutral splice switching morpholino oligonucleotide in spinal muscular atrophy patient-derived fibroblasts

Cited by 15 publications

References 40 publications

Current Challenges in Delivery and Cytosolic Translocation of Therapeutic RNAs

Current Challenges in Delivery and Cytosolic Translocation of Therapeutic RNAs

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

Recent Advances in RNA Therapy and Its Carriers to Treat the Single-Gene Neurological Disorders

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