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(A) Nucleotide sequence (in 5 to 3 orientation) of HD-PNA is bordered on amino terminus by biotin (bio) residue and by tyrosine (Y) and lysine (K) residues at carboxyl terminus. There are 5 linkers (designated O) flanking nucleotide sequence. Complementary nucleotide sequence of HD target mRNA (in 5 to 3 orientation) is shown, and methionine initiation codon (ATG) is underlined. HD exon-1 sequence is downstream of T3 RNA polymerase promoter (left solid box denoted by arrow), which allows for in vitro transcription of HD exon-1 mRNA. (B) Combined in vitro transcription/translation assays resulted in formation of 3 H-labeled exon-1 huntingtin protein that was precipitated by TCA. Translation of HD exon-1 protein was inhibited in dose response by either PO-ODN (III) or by PNA. (C) RNase protection assay shows formation of HD mRNA protected fragment after complete nuclease digestion, because of hybridization of biotinylated HD PNA to huntingtin exon-1 mRNA (lane 2). Conjugation of antisense PNA to mAb-SA transport vector does not inhibit the hybridization of PNA to target mRNA, based on formation of RNase protected oligonucleotide shown in lane 4. Conversely, no protected fragment is observed after mixing of anti-luc PNA with HD RNA, either in unconjugated form (lane 3) or conjugated to mAb-SA vector (lane 5). BPB bromophenol blue.
Source publication
Disease-specific genes of unknown function can be imaged in vivo with antisense radiopharmaceuticals, providing the transcellular transport of these molecules is enabled with drug-targeting technology. The current studies describe the production of 16-mer peptide nucleic acid (PNA) that is antisense around the methionine initiation codon of the hun...
Contexts in source publication
Context 1
... PNA complementary to nucleotides -1 to 15 of the human HD exon 1 is designated HD-PNA ( Fig. 2A) and was synthesized at the University of Texas Southwestern Medical Center using automated synthesis described previously (14). The biotin at the amino terminus is followed by 5 linkers (designated -O-), a 16-mer PNA sequence, another 5 linkers, a tyrosine and lysine residue, and an amidated carboxyl terminus. Each of the 5 linkers is ...
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... to the firefly luc gene as previously described (10). The antiluciferase PNA, designated luc-PNA, was custom synthesized at PE Biosystems (Framingham, MA); had the follow- ing nucleic acid sequence: CTTCCATTTTACCAAC; and con- tained biotin, 5 linkers flanking the nucleotide sequence, tyrosine, and lysine in an order identical to HD-PNA ( Fig. ...
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... hybridization of the HD-PNA to the HD mRNA is sequence specific ( Fig. 2A) and was confirmed with both the transcription/translation assay (Fig. 2B) and the RPA (Fig. 2C). The PNA inhibits the cell-free translation of the exon-1 fragment of the HD mRNA in a dose response that is comparable with the dose response inhibition of translation caused by a PO-ODN of the same sequence, and designated PO-ODN-III ...
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... hybridization of the HD-PNA to the HD mRNA is sequence specific ( Fig. 2A) and was confirmed with both the transcription/translation assay (Fig. 2B) and the RPA (Fig. 2C). The PNA inhibits the cell-free translation of the exon-1 fragment of the HD mRNA in a dose response that is comparable with the dose response inhibition of translation caused by a PO-ODN of the same sequence, and designated PO-ODN-III (Fig. 2B). The hybridization of the PNA to the target HD transcript was ...
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... hybridization of the HD-PNA to the HD mRNA is sequence specific ( Fig. 2A) and was confirmed with both the transcription/translation assay (Fig. 2B) and the RPA (Fig. 2C). The PNA inhibits the cell-free translation of the exon-1 fragment of the HD mRNA in a dose response that is comparable with the dose response inhibition of translation caused by a PO-ODN of the same sequence, and designated PO-ODN-III (Fig. 2B). The hybridization of the PNA to the target HD transcript was verified with the RPA as ...
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... ( Fig. 2A) and was confirmed with both the transcription/translation assay (Fig. 2B) and the RPA (Fig. 2C). The PNA inhibits the cell-free translation of the exon-1 fragment of the HD mRNA in a dose response that is comparable with the dose response inhibition of translation caused by a PO-ODN of the same sequence, and designated PO-ODN-III (Fig. 2B). The hybridization of the PNA to the target HD transcript was verified with the RPA as shown in Figure 2C (lane 2). The RPA was performed with either the unconjugated PNA (lane 2), which has a molecular weight of 6,300 Da, or the PNA conjugated to the mAb-SA vector (lane 4), which has a molecular weight of 200,000 Da. The presence of ...
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... PNA inhibits the cell-free translation of the exon-1 fragment of the HD mRNA in a dose response that is comparable with the dose response inhibition of translation caused by a PO-ODN of the same sequence, and designated PO-ODN-III (Fig. 2B). The hybridization of the PNA to the target HD transcript was verified with the RPA as shown in Figure 2C (lane 2). The RPA was performed with either the unconjugated PNA (lane 2), which has a molecular weight of 6,300 Da, or the PNA conjugated to the mAb-SA vector (lane 4), which has a molecular weight of 200,000 Da. ...
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... presence of a PNA-protected RNA fragment after complete nuclease digestion of the HD mRNA is indicative of se- quence-specific hybridization of the PNA to the target mRNA molecule. The RPA studies in Figure 2C show that the biotinylated HD PNA specifically hybridizes to the target mRNA, and this hybridization is not altered after conjugation of the PNA to the mAb-SA vector. Conversely, mixing of an anti-luc PNA with the HD RNA did not protect the RNA from nuclease digestion, either in the unconju- gated form (lane 3) or as a conjugate with the mAb-SA vector (lane 5). ...
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... results of these studies are consistent with the fol- lowing conclusions. First, the pharmacokinetics and organ uptake of a PNA antisense radiopharmaceutical are pro- foundly altered by conjugation of the PNA to the mAb targeting vector (Tables 1 and 2). Second, the brain uptake of the PNA radiopharmaceutical by the mouse brain is increased by conjugation to the targeting vector, whereas there is no significant uptake of the unconjugated PNA by the mouse brain (Fig. 3). ...
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... targeting vector enables both the import of the PNA into the brain (Fig. 3) and the export of the PNA-mAb conjugate back to the blood over a 6-h period (Fig. 4). Fourth, conjugation of the PNA to the targeting vector does not affect hybridization of the PNA to the target HD mRNA, based on either cell-free translation or RNase protection assays (Fig. 2). Fifth, the expression of the HD exon-1 gene in the R6/2 HD transgenic mouse can be detected in vivo with the combined use of a sequence-specific PNA radio- pharmaceutical and brain drug-targeting technology. The brain uptake of the 125 I-HD-PNA-8D3 conjugate is in- creased in the transgenic mice compared with littermate controls (Fig. ...
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... that the hybridization of the PNA to the target mRNA is not sterically inhibited by conjugation of the PNA to the targeting 8D3-SA vector. The in vitro studies showed that conjugation of the PNA to the 8D3 mAb did not impair the ability of the PNA to hybridize to the target HD mRNA, based on either cell-free translation or RNase protection assays (Fig. ...
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Citations
... Thus, the conjugation of ASOs with antibodies targeting TfR1 may promote ASO transcytosis across the BBB by receptor-mediated endocytosis. It has been reported that biotinylated radiolabeled PNAs linked to a streptavidin-conjugated monoclonal antibody targeting the mouse transferrin receptor are distributed in the brain following intravenous injection, unlike the unconjugated ASO [129]. Other strategies, such as conjugation to CPPs, loading into extracellular vesicles, and encapsulation in glucose-coated polymeric nanocarriers, have been reported to deliver ASOs to the mouse brain following systemic administration [130][131][132]. ...
Myotonic dystrophy type 1 (DM1) is a dominant genetic disease in which the expansion of long CTG trinucleotides in the 3′ UTR of the myotonic dystrophy protein kinase (DMPK) gene results in toxic RNA gain-of-function and gene mis-splicing affecting mainly the muscles, the heart, and the brain. The CUG-expanded transcripts are a suitable target for the development of antisense oligonucleotide (ASO) therapies. Various chemical modifications of the sugar-phosphate backbone have been reported to significantly enhance the affinity of ASOs for RNA and their resistance to nucleases, making it possible to reverse DM1-like symptoms following systemic administration in different transgenic mouse models. However, specific tissue delivery remains to be improved to achieve significant clinical outcomes in humans. Several strategies, including ASO conjugation to cell-penetrating peptides, fatty acids, or monoclonal antibodies, have recently been shown to improve potency in muscle and cardiac tissues in mice. Moreover, intrathecal administration of ASOs may be an advantageous complementary administration route to bypass the blood-brain barrier and correct defects of the central nervous system in DM1. This review describes the evolution of the chemical design of antisense oligonucleotides targeting CUG-expanded mRNAs and how recent advances in the field may be game-changing by forwarding laboratory findings into clinical research and treatments for DM1 and other microsatellite diseases.
... Direct delivery to the eye using intravitreal injection or subretinal delivery is also well tolerated and intranasal delivery has also been achieved whereby the drug is transported into the brain along the rostral migratory stream [11,12]. Passage through the vascular BBB after systemic delivery can be achieved by exploiting existing receptor-mediated endocytosis pathways; for example, transferrin-targeting nanoparticles or antibodies complexed with AONs have been used in small animal model studies [13,14]. AONs conjugated to arginine-rich cell penetrating peptides (CPPs) are also known to cross the BBB in mice [15,16]. ...
RNA-based therapeutics have entered the mainstream with seemingly limitless possibilities to treat all categories of neurological disease. Here, common RNA-based drug modalities such as antisense oligonucleotides, small interfering RNAs, RNA aptamers, RNA-based vaccines and mRNA drugs are reviewed highlighting their current and potential applications. Rapid progress has been made across rare genetic diseases and neurodegenerative disorders, but safe and effective delivery to the brain remains a significant challenge for many applications. The advent of individualized RNA-based therapies for ultra-rare diseases is discussed against the backdrop of the emergence of this field into more common conditions such as Alzheimer’s disease and ischaemic stroke. There remains significant untapped potential in the use of RNA-based therapeutics for behavioural disorders and tumours of the central nervous system; coupled with the accelerated development expected over the next decade, the true potential of RNA-based therapeutics to transform the therapeutic landscape in neurology remains to be uncovered.
... According to the affinity and tight binding properties of streptavidin and biotinase, Lee and colleagues used biotinase to label antisense oligonucleotides, combining streptavidin with a radiolabeled mouse transferrin receptor monoclonal antibody. e tracing results showed that the antisense oligonucleotides combined with the transferrin receptor monoclonal antibody to form a conjugate, which reached the brain through receptor-mediated endocytosis in the transgenic mouse model [50]. e second mechanism is a cell-penetrating peptide-(CPP-) based delivery system. ...
Antisense nucleic acids are single-stranded oligonucleotides that have been specially chemically modified, which can bind to RNA expressed by target genes through base complementary pairing and affect protein synthesis at the level of posttranscriptional processing or protein translation. In recent years, the application of antisense nucleic acid technology in the treatment of neuromuscular diseases has made remarkable progress. In 2016, the US FDA approved two antisense nucleic acid drugs for the treatment of Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA), and the development to treat other neurodegenerative diseases has also entered the clinical stage. Therefore, ASO represents a treatment with great potential. The article will summarize ASO therapies in terms of mechanism of action, chemical modification, and administration methods and analyze their role in several common neurodegenerative diseases, such as SMA, DMD, and amyotrophic lateral sclerosis (ALS). This article systematically summarizes the great potential of antisense nucleic acid technology in the treatment of hereditary neurodegenerative diseases.
... For example, it has been shown that a radioactively labelled ASO conjugated to a monoclonal antibody against the transferrin receptor was able to reach the brain through the endogenous transferrin transport pathway in a preclinical model. This resulted in a~tenfold increase in brain uptake of the ASO, owing to an enhanced ability to permeate the BBB and brain cell membranes [82]. Another solution is the cellpenetrating peptide (CPP)-based delivery mechanism. ...
Epilepsy is a common and serious neurological disorder characterised by recurrent spontaneous seizures. Frontline pharmacotherapy includes small-molecule antiseizure drugs that typically target ion channels and neurotransmitter systems, but these fail in 30% of patients and do not prevent either the development or progression of epilepsy. An emerging therapeutic target is microRNA (miRNA), small noncoding RNAs that negatively regulate sets of proteins. Their multitargeting action offers unique advantages for certain forms of epilepsy with complex underlying pathophysiology, such as temporal lobe epilepsy (TLE). miRNA can be inhibited by designed antisense oligonucleotides (ASOs; e.g., antimiRs). Here, we outline the prospects for miRNA-based therapies. We review design considerations for nucleic acid-based approaches and the challenges and next steps in developing therapeutic miRNA-targeting molecules for epilepsy.
... Currently, there are several possible approaches, from IV injection to more direct delivery methods, including ICV or IT injection. The systemic route for delivery into the CNS includes diffusion [192] and RME [193,194] and requires the development of a siRNA nanodelivery system, which is able to cross the BBB, such as the RVG-targeted SNALPs we have been referring to. So far, however, the most widely used methods to bypass the BBB are the direct brain parenchyma injections, which allow for the use of non-targeted nanocarriers or chemically modified naked therapeutic molecules. ...
More than two thirds of Lysosomal Storage Diseases (LSDs) present central nervous system involvement. Nevertheless, only one of the currently approved therapies has an impact on neuropathology. Therefore, alternative approaches are under development, either addressing the underlying enzymatic defect or its downstream consequences. Also under study is the possibility to block substrate accumulation upstream, by promoting a decrease of its synthesis. This concept is known as substrate reduction therapy and may be triggered by several molecules, such as small interfering RNAs (siRNAs). siRNAs promote RNA interference, a naturally occurring sequence-specific post-transcriptional gene-silencing mechanism, and may target virtually any gene of interest, inhibiting its expression. Still, naked siRNAs have limited cellular uptake, low biological stability, and unfavorable pharmacokinetics. Thus, their translation into clinics requires proper delivery methods. One promising platform is a special class of liposomes called stable nucleic acid lipid particles (SNALPs), which are characterized by high cargo encapsulation efficiency and may be engineered to promote targeted delivery to specific receptors. Here, we review the concept of SNALPs, presenting a series of examples on their efficacy as siRNA nanodelivery systems. By doing so, we hope to unveil the therapeutic potential of these nanosystems for targeted brain delivery of siRNAs in LSDs.
... Derzeit konzentrieren sich wissenschaftliche Untersuchungen auf die Entwicklung geeigneter Applikationsformen und -systeme. Die Verbesserung der rezeptorvermittelten Endozytose von ASOs mittels monoklonaler Antikörper (15), zellpenetrierender Peptide (CPP) (16), Nanopartikelsysteme (17) oder (Adeno-) Virusvektoren (18) sind nur einige Beispiele hierfür und könnten zukünftig die zelluläre Aufnahme und systemische (intravenöse, subkutane, orale und nasale) Gabe ermöglichen. Derzeit müssen die meisten der zur Verfügung stehenden ASOs zur Behandlung neurologischer Er-krankungen jedoch noch intrathekal mittels Lumbalpunktion verabreicht werden. ...
Zusammenfassung
Molekulargenetische Untersuchungen konnten die Pathomechanismen vieler neurodegenerativer Erkrankungen aufklären, jedoch standen lange Zeit keine krankheitsmodifizierenden Therapieoptionen zur Verfügung. Dies änderte sich seit der Einführung von sogenannten Antisense-Oligonukleotiden in die klinische Praxis. Im Jahr 2016 wurden erstmals Antisense-Oligonukleotide zur Behandlung der spinalen Muskelatrophie und der Muskeldystrophie Duchenne zugelassen. In der Entwicklung von Antisense-Oligonukleotiden waren Hürden zu überwinden und chemische Modifikationen notwendig, die deren Einsatz erst möglich machten. In diesem Artikel stellen wir Prinzipien dieser chemischen Modifikationen und Wirkmechanismen von Antisense-Oligonkuleotiden vor. Darüber hinaus geben wir einen Überblick über den aktuellen Stand der Wissenschaft zu verschiedenen Antisense-Oligonukleotiden und fokussieren dabei auf neuromuskuläre und neurodegenerative Erkrankungen wie die spinale Muskelatrophie, die Muskeldystrophie Duchenne, die myotone Dystrophie, Chorea Huntington und die Amyotrophe Lateralsklerose.
... 41 A biotinylated ASO captured with a streptavidin-conjugated monoclonal antibody to the mouse transferrin receptor reached the brain by this mechanism. 42 The same receptor-mediated endocytosis pathway was used to deliver nanoparticles carrying ASOs into the brain parenchyma. 43 Another mechanism was demonstrated by cellpenetrating peptide (CPP)-based delivery systems. ...
The introduction of genetics revolutionized the field of neurodegenerative and neuromuscular diseases and has provided considerable insight into the underlying pathomechanisms. Nevertheless, effective treatment options have been limited. This changed recently when antisense oligonucleotides (ASOs) could be translated from in vitro and experimental animal studies into clinical practice. In 2016, two ASOs were approved by the United States US Food and Drug Administration (FDA) and demonstrated remarkable efficacy in Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). ASOs are synthetic single-stranded strings of nucleic acids. They selectively bind to specific premessenger ribonucleic acid (pre-mRNA)/mRNA sequences and alter protein synthesis by several mechanisms of action. Thus, apart from gene replacement, ASOs may therefore provide the most direct therapeutic strategy for influencing gene expression. In this review, we shall discuss basic mechanisms of ASO action, the role of chemical modifications needed to improve the pharmacodynamic and pharmacokinetic properties of ASOs, and we shall then focus on several ASOs developed for the treatment of neurodegenerative and neuromuscular disorders, including SMA, DMD, myotonic dystrophies, Huntington’s disease, amyotrophic lateral sclerosis and Alzheimer’s disease.
... A common strategy is to utilize receptor-mediated transcytosis, which allows macromolecules such as transferrin, insulin, leptin, and insulin-like growth factor 1 to cross the BBB. For example, a monoclonal antibody to transferrin receptor has been conjugated as a delivery ligand directly to ASO [40] and to nanoparticles carrying ASO [41], but no products utilizing receptor-mediated transcytosis have proceeded to clinical development. Tagging cell-penetrating peptides with ASO might also be possible because of the peptides' strong transmembrane capacity [42], but adverse effects such as lethargy, weight loss, and renal dysfunction are yet to be overcome [43]. ...
Within the field of RNA therapeutics, antisense oligonucleotide-based therapeutics are a potentially powerful means of treating intractable diseases. However, if these therapeutics are used for the treatment of neurological disorders, safe yet efficient methods of delivering antisense oligonucleotides across the blood-brain barrier to the central nervous system must be developed. Here, we examined the use of angubindin-1, a binder to the tricellular tight junction, to modulate paracellular transport between brain microvascular endothelial cells in the blood-brain barrier for the delivery of antisense oligonucleotides to the central nervous system. This proof-of-concept study demonstrated that intravenously injected angubindin-1 increased the permeability of the blood-brain barrier and enabled transient delivery of subsequently administered antisense oligonucleotides into the mouse brain and spinal cord, leading to silencing of a target RNA without any overt adverse effects. We also found that two bicellular tight junction modulators did not produce such a silencing effect, suggesting that the tricellular tight junction is likely a better target for the delivery of antisense oligonucleotides than the bicellular tight junction. Our delivery strategy of modulating the tricellular tight junction in the blood-brain barrier via angubindin-1 provides a novel avenue of research for the development of antisense oligonucleotide-based therapeutics for the treatment of neurological disorders.
... In research publications, targeted particles are often described as successful if the accumulation within the target organ is increased at least 1.5 or 2-fold compared to a nontargeted system, in the case of the brain or other tissues that are difficult to reach, that corresponds to well below 2% or even 1% of the total dose [23,76,77]. While such results may constitute an important progress in basic research, a direct translation to therapeutic use is questionable. ...
Although recent clinical successes of antisense, splice-switching, and siRNA oligonucleotides have established the therapeutic utility of this novel class of medicines, the efficient systemic application for non-liver targets remains elusive. Exploitation of active receptor-mediated targeting followed by efficient and productive cellular uptake is required for enabling the therapy of extrahepatic diseases on the expressional level. Evasion of liver accumulation and organ-specific targeting and also efficient cytosolic delivery after endosomal internalization are currently insufficiently solved issues. Lipid and polymer-based nanoparticles can be engineered for efficient cellular uptake and enhancement of endosomal escape, but are characterized by preferential liver accumulation based on biodistribution largely determined by particle size and biophysical properties. Oligonucleotide bioconjugates with receptor-binding ligands have been evolved for highly efficient targeting, but frequently result in a large extent of endosomal entrapment and consequently a lack of sufficient cytosolic concentrations. Non-immunoglobulin protein-based receptor recognition affords high cell-type selectivity and is promising for achieving nonhepatic oligonucleotide targeting. The use of such novel protein scaffolds, including designed ankyrin repeat proteins (DARPins), for oligonucleotide delivery is attractive for achieving effective tissue targeting. Issues for further development and optimization to advance approaches for extrahepatic oligonucleotide delivery by nanoparticles or bioconjugates are discussed.
... AON biodistribution using various delivery strategies. Schematic overview of in vivo biodistribution, based on quantitative data described in [33][34][35][36][37] . Colours indicate AON levels reached in various organs. ...
Antisense oligonucleotide (AON)-based therapies hold promise for a range of neurodegenerative and neuromuscular diseases and have shown benefit in animal models and patients. Success in the clinic is nevertheless still limited, due to unfavourable biodistribution and poor cellular uptake of AONs. Extensive research is currently being conducted into the formulation of AONs to improve delivery, but thus far there is no consensus on which of those strategies will be the most effective. This systematic review was designed to answer in an unbiased manner which delivery strategies most strongly enhance the efficacy of AONs in animal models of heritable neurodegenerative and neuromuscular diseases. In total, 95 primary studies met the predefined inclusion criteria. Study characteristics and data on biodistribution and toxicity were extracted and reporting quality and risk of bias were assessed. Twenty studies were eligible for meta-analysis. We found that even though the use of delivery systems provides an advantage over naked AONs, it is not yet possible to select the most promising strategies. Importantly, standardisation of experimental procedures is warranted in order to reach conclusions about the most efficient delivery strategies. Our best practice guidelines for future experiments serve as a step in that direction.
