This is our publication "General Principles for Yield Optimization of Nucleoside Phosphorylase-Catalyzed Transglycosylations" that described the prediction and optimization of yields in nucleoside transglycosylations mediated by nucleoside phosphorylases. As a continuation to previous reports, we highlight the varying effect of excess phosphate on product yield both theoretically and experimentally, as this is a crucially important feature of the reaction system previous undecribed. Furthermore, we provide a simplified equation for the estimation of product yield that allows for straightforward analytical solutions instead of the numerical solutions previously required. Herein, we provide the full text draft of our work along with the supplementary information. Externally hosted supplementary files can be accessed via the links cited in the main document.

Nucleoside-5’-triphosphates (NTPs) and their analogs are building blocks of DNA and are important compounds in both pharmaceutical and molecular biology applications. Currently, commercially available base or sugar modified NTPs are mainly synthesized chemically. Since the chemical production of NTPs is time-consuming and generally inefficient, alternative approaches are under development. Here we present a simple, efficient and generalizable enzymatic synthesis method for the conversion of nucleosides to NTPs. Our one-pot method is modular, applicable to a wide range of natural and modified nucleotide products and accesses NTPs directly from cheap nucleoside precursors. Nucleoside kinases, nucleoside monophosphate (NMP) kinases and a nucleoside diphosphate (NDP) kinase were applied as biocatalysts. Enzymes with different substrate specificities were combined to produce derivatives of adenosine and cytidine triphosphate with conversions of 4 to 26%. The implementation of a (deoxy)ATP recycling system resulted in a significant increase in the conversion to all NTP products, furnishing 4 different NTPs in quantitative conversion. Natural (deoxy)NTPs were synthesized with 60 to >99% conversion and sugar- and base-modified NTPs were produced with 69 to >99% and 27 to 75% conversion, respectively. The presented method is suitable for the efficient synthesis of a wide range of natural and modified NTPs in a sustainable one-pot process.

Nucleoside analogs represent a class of important drugs for cancer and antiviral treatments. Nucleoside phosphorylases (NPases) catalyze the phosphorolysis of nucleosides and are widely employed for the synthesis of pentose-1-phosphates and nucleoside analogs, which are difficult to access via conventional synthetic methods. However, for the vast majority of nucleosides, it has been observed that either no or incomplete conversion of the starting materials is achieved in NPase-catalyzed reactions. For some substrates, it has been shown that these reactions are reversible equilibrium reactions that adhere to the law of mass action. In this contribution, we broadly demonstrate that nucleoside phosphorolysis is a thermodynamically controlled endothermic reaction that proceeds to a reaction equilibrium dictated by the substrate-specific equilibrium constant of phosphorolysis, irrespective of the type or amount of NPase used, as shown by several examples. Furthermore, we explored the temperature-dependency of nucleoside phosphorolysis equilibrium states and provide the apparent transformed reaction enthalpy and apparent transformed reaction entropy for 24 nucleosides, confirming that these conversions are thermodynamically controlled endothermic reactions. This data allows calculation of the Gibbs free energy and, consequently, the equilibrium constant of phosphorolysis at any given reaction temperature. Overall, our investigations revealed that pyrimidine nucleosides are generally more susceptible to phosphorolysis than purine nucleosides. The data disclosed in this work allow the accurate prediction of phosphorolysis or transglycosylation yields for a range of pyrimidine and purine nucleosides and thus serve to empower further research in the field of nucleoside biocatalysis.

The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient alternative to chemical synthesis routes. In this study, dihalogenated nucleoside analogues were produced by thermostable nucleoside phosphorylases in transglycosylation reactions using uridine or thymidine as sugar donors. Prior to the enzymatic process, ideal maximum product yields were calculated after the determination of equilibrium constants through monitoring the equilibrium conversion in analytical-scale reactions. Equilibrium constants for dihalogenated nucleosides were comparable to known purine nucleosides, ranging between 0.071 and 0.081. To achieve 90% product yield in the enzymatic process, an approximately five-fold excess of sugar donor was needed. Nucleoside analogues were purified by semi-preparative HPLC, and yields of purified product were approximately 50% for all target compounds. To evaluate the impact of halogen atoms in positions 2 and 6 on the antiproliferative activity in leukemic cell lines, the cytotoxic potential of dihalogenated nucleoside analogues was studied in the leukemic cell line HL-60. Interestingly, the inhibition of HL-60 cells with dihalogenated nucleoside analogues was substantially lower than with monohalogenated cladribine, which is known to show high antiproliferative activity. Taken together, we demonstrate that thermodynamic calculations and small-scale experiments can be used to produce nucleoside analogues with high yields and purity on larger scales. The procedure can be used for the generation of new libraries of nucleoside analogues for screening experiments or to replace the chemical synthesis routes of marketed nucleoside drugs by enzymatic processes.

The increased interest in (enzymatic) transformations between nucleosides and nucleobases has demanded the development of efficient analytical tools. In this report, we present an update and extension of our recently described method for monitoring these reactions by spectral unmixing. The presented method uses differences in the UV absorption spectra of nucleosides and nucleobases after alkaline quenching to derive their ratio based on spectral shape by fitting normalized reference spectra. It is applicable to a broad compound spectrum comprising more than 35 examples, offers HPLC-like accuracy, ease of handling and significant reductions in both cost and data acquisition time compared to other methods. This contribution details the principle of monitoring reactions by spectral unmixing, gives recommendations regarding solutions to common problems and applications that necessitate special sample treatment. We provide software, workflows and reference spectra that facilitate the straightforward and versatile application of the method.

Introduction: Development of agonistic analgesic drugs requires proof of selectivity in vivo attainable by selective antagonists or several knockdown strategies. The Kv7.2 potassium channel encoded by the KCNQ2 gene regulates neuronal excitability and its activation inhibits nociceptive transmission. Although it is a potentially attractive target for analgesics, no clinically approved Kv7.2 agonists are currently available and selectivity of drug candidates is hard to demonstrate in vivo due to the expenditure to generate KCNQ2 knockout animals and the lack of Kv7.2 selective antagonists. The present study describes the set-up of an RNA interference-based model that allows studying the selectivity of Kv7.2 openers. Methods: Adeno-associated virus (AAV) vectors were used to deliver the expression cassette for a short hairpin RNA targeting KCNQ2. Heat nociception was tested in rats after intrathecal AAV treatment. Results: Surprisingly, screening of AAV serotypes revealed serotype 7, which has rarely been explored, to be best suited for transduction of dorsal root ganglia neurons following intrathecal injection. Knockdown of the target gene was confirmed by qRT-PCR and the anti-nociceptive effect of a Kv7.2 agonist was found to be completely abolished by the treatment. Discussion: We consider this approach not only to be suitable to study the selectivity of novel analgesic drugs targeting Kv7.2, but rather to serve as a general fast and simple method to generate functional and phenotypic knockdown animals during drug discovery for central and peripheral pain targets.

Efficient reaction monitoring is crucial for data acquisition in kinetic and mechanistic studies. However, for conversions of nucleosides to their corresponding nucleobases, as observed in enzymatically catalyzed nucleoside phosphorylation reactions, the current analytical arsenal does not meet modern requirements regarding cost, speed of analysis and high throughput. Herein, we present a UV/Vis spectroscopy-based assay employing an algorithm for spectral unmixing in a 96-well plate format. The algorithm relies on fitting of reference spectra of nucleosides and their bases to experimental spectra and allows determination of nucleoside/nucleobase ratios in solution with high precision. The experimental procedure includes appropriate dilution of a sample into aqueous alkaline solution, transfer to a multi-well plate, measurement of a UV/Vis spectrum and subsequent in silico spectral unmixing. This enables data collection in a high-throughput fashion and reduces costs compared to state-of-the-art HPLC analyses by approximately 5-fold while being 20-fold faster and offering comparable precision. Additionally, the method is robust regarding dilution and sample transfer errors as it only considers spectral form and not absolute intensity. It can be applied to all natural nucleosides and nucleobases and even unnatural ones as demonstrated by several examples.

Pyrimidine-nucleoside phosphorylases (Py-NPases) have a significant potential to contribute to the economic and ecological production of modified nucleosides. These can be produced via pentose-1-phosphates, an interesting but mostly labile and expensive precursor. Thus far, no dynamic model exists for the production process of pentose-1-phosphates, which involves the equilibrium state of the Py-NPase catalyzed reversible reaction. Previously developed enzymological models are based on the understanding of the structural principles of the enzyme and focus on the description of initial rates only. The model generation is further complicated, as Py-NPases accept two substrates which they convert to two products. To create a well-balanced model from accurate experimental data, we utilized an improved high-throughput spectroscopic assay to monitor reactions over the whole time course until equilibrium was reached. We examined the conversion of deoxythymidine and phosphate to deoxyribose-1-phosphate and thymine by a thermophilic Py-NPase from Geobacillus thermoglucosidasius. The developed process model described the reactant concentrations in excellent agreement with the experimental data. Our model is built from ordinary differential equations and structured in such a way that integration with other models is possible in the future. These could be the kinetics of other enzymes for enzymatic cascade reactions or reactor descriptions to generate integrated process models.

Natural ribonucleoside-5’-monophosphates are building blocks for nucleic acids which are used for a number of purposes, including food additives. Their analogues, additionally, are used in pharmaceutical applications. Fludarabine-5´-monophosphate, for example, is effective in treating hematological malignancies. To date, ribonucleoside-5’-monophosphates are mainly produced by chemical synthesis, but the inherent drawbacks of this approach have led to the development of enzymatic synthesis routes. In this study, we evaluated the potential of human deoxycytidine kinase (HsdCK) as suitable biocatalyst for the synthesis of natural and modified ribonucleoside-5’-monophosphates from their corresponding nucleosides. Human dCK was heterologously expressed in E. coli and immobilized onto Nickel-nitrilotriacetic acid (Ni-NTA) superflow. A screening of the substrate spectrum of soluble and immobilized biocatalyst revealed that HsdCK accepts a wide range of natural and modified nucleosides, except for thymidine and uridine derivatives. Upon optimization of the reaction conditions, HsdCK was used for the synthesis of fludarabine-5´-monophosphate using increasing substrate concentrations. While the soluble biocatalyst revealed highest product formation with the lowest substrate concentration of 0.3 mM, the product yield increased with increasing substrate concentrations in the presence of the immobilized HsdCK. Hence, the application of immobilized HsdCK is advantageous upon using high substrate concentration which is relevant in industrial applications.

Nucleoside phosphorylases catalyze the reversible phosphorolysis of pyrimidine and purine nucleosides in the presence of phosphate. They are valuable catalysts in the synthesis of nucleosides and their analogues, which are often used as pharmaceuticals or their precursors. Thermostable nucleoside phosphorylases are promising biocatalysts, as they withstand harsh reaction conditions such as high pH or the addition of organic solvents. In this review, the characteristics and properties of thermostable nucleoside phosphorylases are described. Differences in amino acid content and protein structure were compared to their mesophilic homologues to identify features involved in thermostability. Substrate spectra of thermostable nucleoside phosphorylases were analyzed, and it is shown that thermostable nucleoside phosphorylases have a wider substrate spectrum than their mesophilic counterparts. Thus, thermostable nucleoside phosphorylases are interesting biocatalysts for industrial applications.

With the world moving toward green chemistry approaches, the enzymatic synthesis of nucleoside analogues offers several advantages over chemical methods, which include higher total yields, a higher regio- and stereo-selectivity, and higher product purity. This chapter focuses on enzymatic approaches using nucleoside phosphorylases (NPs). NPs are of high interest as biocatalysts because of their wide substrate spectrum and abundance in almost all living organisms. Since NPs were first described by Kalckar, many research projects were conducted to test whether definite nucleoside analogues are used as substrates for NPs. NPs from different mesophiles, like Escherichia coli or Bacillus subtilis, were applied in the synthesis of pharmacologically active compounds. An interesting feature of purine NPs (PNPs) is their ability to accept pyrimidine nucleosides as substrate. Thus, they may be interesting catalysts for the synthesis of cytidine and deoxycytidine that are not utilized by many pyrimidine NPs.

alpha-D-pentofuranose-1-phosphates (Pentose-1Ps) are key intermediates in nucleoside metabolism and important precursors for the enzymatic synthesis of modified nucleosides. To date, Pentose-1Ps are mainly produced by chemical approaches which have numerous disadvantages. Therefore, several enzymatic methods employing mesophilic enzymes have been developed but are not widely applied due to their limited substrate spectrum. Here we report the use of thermostable nucleoside phosphorylases for the chemo-enzymatic synthesis of modified Pentose-1Ps (2-deoxy-2-fluoro-alpha-D-ribofuranose-1-phosphate, alpha-D-arabinofuranose-1-phosphate, and 2-deoxy-2-fluoro-alpha-D-arabinofuranose-1-phosphate), which are interesting building blocks for the synthesis of modified nucleosides. After optimizing the synthesis protocol using the natural substrates uridine and thymidine, grams of modified Pentose-1Ps were purified as their Ba-salts with over 95% purity. Their structures were confirmed by NMR spectroscopy and the temperature and pH stability of natural and modified Pentose-1Ps in aqueous solution was -evaluated. Four of the Pentose-1P-Ba salts were stable with no visible degradation up to 60 degrees C and pH above 5, while 2-deoxy-alpha-D-ribofuranose-1-phosphate was less stable. The presented protocol provides an easy, fast, and environmentally-friendly method to produce grams of modified Pentose-1P-Ba salts of high purity.

Pyrimidine and purine nucleoside phosphorylases catalyze the reversible phosphorolytic cleavage and formation of the glycosidic bond of purine and pyrimidine nucleosides, respectively, and are thus, key catalysts for the synthesis of new compounds. The selection of the best combination of enzyme and reaction conditions is not trivial, as each of the two enzymes can perform the reaction in two directions and thus, also competes with the other one for the reaction intermediates. A generic approach to the solution of this problem based on the formulation of a mixed integer dynamic optimization program using MOSAICmodeling is presented.

Activation of the neuronal potassium channel Kv7.2 encoded by the KCNQ2 gene has recently been shown to be an attractive mechanism to inhibit nociceptive transmission. However, potent, selective, and clinically proven activators of Kv7.2/Kv7.3 currents with analgesic properties are still lacking. An important prerequisite for the development of new drugs is a model to test the selectivity of novel agonists by abrogating Kv7.2/Kv7.3 function. Since constitutive knockout mice are not viable, we developed a model based on RNA interference-mediated silencing of KCNQ2. By delivery of a KCNQ2-specific short hairpin RNA with adeno-associated virus vectors, we completely abolished the activity of the specific Kv7.2/Kv7.3-opener ICA-27243 in rat sensory neurons. Results obtained in the silencing experiments were consistent between freshly prepared and cryopreserved dorsal root ganglion neurons, as well as in dorsal root ganglion neurons dissociated and cultured after in vivo administration of the silencing vector by intrathecal injections into rats. Interestingly, the tested associated virus serotypes substantially differed with respect to their transduction capability in cultured neuronal cell lines and primary dorsal root ganglion neurons and the in vivo transfer of transgenes by intrathecal injection of associated virus vectors. However, our study provides the proof-of-concept that RNA interference-mediated silencing of KCNQ2 is a suitable approach to create an ex vivo model for testing the specificity of novel Kv7.2/Kv7.3 agonists.

Background: Nucleoside phosphorylases catalyze the reversible phosphorolysis of pyrimidine and purine nucleosides in the presence of phosphate. They are relevant to the appropriate function of the immune system in mammals and interesting drug targets for cancer treatment. Next to their role as drug targets nucleoside phosphorylases are used as catalysts in the synthesis of nucleosides and their analogs that are widely applied as pharmaceuticals. Methods: Based on their substrates nucleoside phosphorylases are classified as pyrimidine and purine nucleoside phosphorylases. This article describes the substrate spectra of nucleoside phosphorylases and structural properties that influence their activity. Substrate ranges are summarized and relations between members of pyrimidine or purine nucleoside phosphorylases are elucidated. Results: Nucleoside phosphorylases accept a broad spectrum of substrates: they accept both base and sugar modified nucleosides. The most widely studied nucleoside phosphorylases are those of Escherichia coli, mammals and pathogens. However, recently the attention has been shifted to thermophilic nucleoside phosphorylases due to several advantages. Nucleoside phosphorylases have been applied to produce drugs like ribavirin or fludarabine. However, limitations were observed when drugs show an open ring structure. Site-directed mutagenesis approaches were shown to alter the substrate specificity of nucleoside phosphorylases. Conclusion: Nucleoside phosphorylases are valuable tools to produce modified nucleosides with therapeutic or diagnostic potential with high affinity and specificity. A wide variety of nucleoside phosphorylases are available in nature which differ in their protein sequence and show varying substrate spectra. To overcome limitations of the naturally occurring enzymes site-directed mutagenesis approaches can be used.

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models. In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects.

SummaryThe mobility and bioavailability of heavy metals in soils contaminated by irrigation with wastewater increase with increasing mineralization of accumulated organic substance and decreasing pH. In laboratory experiments addition of biochar reduced heavy metal availability to plants and enhanced plant growth. However, literature from field trials is scarce. Therefore, we conducted a 2‐year field experiment with orchard grass (Dactylis glomerata L.) to study the effects of miscanthus‐derived biochar applied to sewage field soil on biomass production and concentrations of zinc (Zn), copper (Cu), cadmium (Cd), lead (Pb) and various nutrients in plants and in the soil solution. Biochar was mixed into the contaminated topsoil (30 cm) with addition rates of 0, 1, 2.5 and 5% on a dry‐mass basis (g 100 g−1). The soil solution was collected with suction plates installed at a depth of 30 cm. Addition of biochar increased biomass production and reduced Zn and Cd concentrations in the soil solution. Zinc concentrations were also reduced in plants. This effect seems to be attributable to an increase in pH caused by biochar addition. In contrast, Pb and Cu concentrations in the soil solution generally increased and were related to the concentrations of dissolved organic carbon (DOC). Copper concentrations also increased in the plants; however, only at the beginning of the measurement period. Our data indicate that increased concentrations of DOC, Cu and Pb in the soil solution might be a transient effect. Therefore, further research is needed to determine the long‐term effect of biochar amendment on element immobilization and leaching into groundwater.

Future Medicinal ChemistryVol. 7, No. 13 CommentaryApplication of modified antisense oligonucleotides and siRNAs as antiviral drugsAnke Wagner, Claus-Thomas Bock, Henry Fechner & Jens KurreckAnke WagnerBioNukleo, Department of Bioprocess Engineering, Berlin University of Technology, Berlin, Germany, Claus-Thomas BockDepartment of Infectious Diseases, Robert Koch Institute, Berlin, Germany, Henry FechnerDepartment of Applied Biochemistry, Institute of Biotechnology, Berlin University of Technology, Berlin, Germany & Jens Kurreck*Author for correspondence: E-mail Address: jens.kurreck@tu-berlin.deDepartment of Applied Biochemistry, Institute of Biotechnology, Berlin University of Technology, Berlin, GermanyPublished Online:18 Sep 2015https://doi.org/10.4155/fmc.15.114AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: antisense oligonucleotideantiviral therapydelivery systemmodified nucleotidespost-transcriptional gene silencingsiRNAsReferences1 Yamamoto T, Nakatani M, Narukawa K, Obika S. Antisense drug discovery and development. Future Med. Chem. 3(3), 339–365 (2011).Link, CAS, Google Scholar2 Kurreck J. RNA interference: from basic research to therapeutic applications. Angew. Chem. Int. Ed. Engl. 48(8), 1378–1398 (2009).Crossref, Medline, CAS, Google Scholar3 Eckstein F. Phosphorothioates, essential components of therapeutic oligonucleotides. Nucleic Acid Ther. 24(6), 374–387 (2014).Crossref, Medline, CAS, Google Scholar4 Davis ME, Zuckerman JE, Choi CH et al. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464(7291), 1067–1070 (2010).Crossref, Medline, CAS, Google Scholar5 Song E, Zhu P, Lee SK et al. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat. Biotechnol. 23(6), 709–717 (2005).Crossref, Medline, CAS, Google Scholar6 Kastelein JJ, Ross CJ, Hayden MR. From mutation identification to therapy: discovery and origins of the first approved gene therapy in the western world. Hum. Gene Ther. 24(5), 472–478 (2013).Crossref, Medline, CAS, Google Scholar7 Morrissey DV, Lockridge JA, Shaw L et al. Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat. Biotechnol. 23(8), 1002–1007 (2005).Crossref, Medline, CAS, Google Scholar8 Wooddell CI, Rozema DB, Hossbach M et al. Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection. Mol. Ther. 21(5), 973–985 (2013).Crossref, Medline, CAS, Google Scholar9 De Clercq E. Curious discoveries in antiviral drug development: the role of serendipity. Med. Res. Rev. 35(4), 698–719 (2015).Crossref, Medline, CAS, Google Scholar10 Iversen PL, Warren TK, Wells JB et al. Discovery and early development of AVI-7537 and AVI-7288 for the treatment of Ebola virus and Marburg virus infections. Viruses 4(11), 2806–2830 (2012).Crossref, Medline, CAS, Google Scholar11 De Clercq E. Ebola virus (EBOV) infection: therapeutic strategies. Biochem. Pharmacol. 93(1), 1–10 (2015).Crossref, Medline, CAS, Google Scholar12 Thi EP, Mire CE, Lee AC et al. Lipid nanoparticle siRNA treatment of Ebola-virus-Makona-infected nonhuman primates. Nature 521(7552), 362–365 (2015).Crossref, Medline, CAS, Google Scholar13 Brake OT, Hooft K, Liu YP, Centlivre M, Jasmijn Von Eije K, Berkhout B. Lentiviral vector design for multiple shRNA expression and durable HIV-1 inhibition. Mol. Ther. 16(3), 557–564 (2008).Crossref, Medline, Google Scholar14 Stein EA, Pinkert S, Becher PM et al. Combination of RNA interference and virus receptor trap exerts additive antiviral activity in coxsackievirus B3-induced myocarditis in mice. J. Infect. Dis. 211(4), 613–622 (2015).Crossref, Medline, CAS, Google Scholar15 Grundhoff A, Sullivan CS. Virus-encoded microRNAs. Virology 411(2), 325–343 (2011).Crossref, Medline, CAS, Google Scholar16 Lindow M, Kauppinen S. Discovering the first microRNA-targeted drug. J. Cell Biol. 199(3), 407–412 (2012).Crossref, Medline, CAS, Google Scholar17 Lanford RE, Hildebrandt-Eriksen ES, Petri A et al. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 327(5962), 198–201 (2010).Crossref, Medline, CAS, Google Scholar18 Saayman S, Ali SA, Morris KV, Weinberg MS. The therapeutic application of CRISPR/Cas9 technologies for HIV. Expert Opin. Biol. Ther. 15(6), 819–830 (2015).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByChemistry of Peptide-Oligonucleotide Conjugates: A Review6 September 2021 | Molecules, Vol. 26, No. 17Silencing acpP gene via antisense oligonucleotide-niosome complex in clinical Pseudomonas aeruginosa isolatesResearch in Microbiology, Vol. 172, No. 4-5Mesyl Phosphoramidate Oligonucleotides as Potential Splice-Switching Agents: Impact of Backbone Structure on Activity and Intracellular LocalizationNucleic Acid Therapeutics, Vol. 31, No. 3Oligonucleotide‐Based Therapeutics: An Emerging Strategy for the Treatment of Chronic Liver Diseases16 March 2021 | Hepatology, Vol. 73, No. 4Allele-Specific RNA Knockdown with a Biologically Stable and Catalytically Efficient XNAzyme22 March 2021 | Journal of the American Chemical Society, Vol. 143, No. 12A perspective on RNA interference-based therapeutics for metabolic liver diseases29 January 2021 | Expert Opinion on Investigational Drugs, Vol. 30, No. 3Antiviral Activity of Nanocomplexes of Antisense Oligonucleotides Targeting VP72 Protein in Vero Cells Infected by African Swine Fever Virus26 April 2021 | Russian Journal of Bioorganic Chemistry, Vol. 47, No. 2Novel Lipid-Oligonucleotide Conjugates Containing Long-Chain Sulfonyl Phosphoramidate Groups: Synthesis and Biological Properties27 January 2021 | Applied Sciences, Vol. 11, No. 3New oligodeoxynucleotide derivatives containing N-(methanesulfonyl)-phosphoramidate (mesyl phosphoramidate) internucleotide group3 February 2018 | Russian Journal of Bioorganic Chemistry, Vol. 43, No. 6НОВЫЕ ПРОИЗВОДНЫЕ ОЛИГОДЕЗОКСИНУКЛЕОТИДОВ, СОДЕРЖАЩИЕ МЕЖНУКЛЕОТИДНУЮ N-(МЕТАНСУЛЬФОНИЛ)-ФОСФОРАМИДНУЮ (МЕЗИЛФОСФОРАМИДНУЮ) ГРУППУ1, "Биоорганическая химия"Биоорганическая химия, No. 6Frontiers in nucleic acid-based drug research and developmentLun Quan Sun & Jonathan P Wong14 October 2015 | Future Medicinal Chemistry, Vol. 7, No. 13 Vol. 7, No. 13 Follow us on social media for the latest updates Metrics Downloaded 154 times History Published online 18 September 2015 Published in print September 2015 Information© Future Science LtdKeywordsantisense oligonucleotideantiviral therapydelivery systemmodified nucleotidespost-transcriptional gene silencingsiRNAsFinancial & competing interests disclosureA Wagner is the CEO and CSO of the biotech startup BioNukleo UG and J Kurreck is the scientific board member of the same company. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

Reconstituted three-dimensional (3D) liver models obtained by engrafting hepatic cells into an extracellular matrix (ECM) are valuable tools to study tissue regeneration, drug action and toxicology ex vivo. The aim of the present study was to establish a system for the functional investigation of a viral vector in a 3D liver model composed of human HepG2 cells on a rat ECM. An adeno-associated viral (AAV) vector expressing the Emerald green fluorescent protein (EmGFP) and a short hairpin RNA (shRNA) directed against human cyclophilin b (hCycB) was injected into the portal vein of 3D liver models. Application of the vector did not exert toxic effects, as shown by analysis of metabolic parameters. Six days after transduction, fluorescence microscopy analysis of EmGFP production revealed widespread distribution of the AAV vectors. After optimization of the recellularization and transduction conditions, averages of 55 and 90 internalized vector genomes per cell in two replicates of the liver model were achieved, as determined by quantitative PCR analysis. Functionality of the AAV vector was confirmed by efficient shRNA-mediated knockdown of hCycB by 70-90%. Our study provides a proof-of-concept that a recellularized biological ECM provides a valuable model to study viral vectors ex vivo.