Investigation of Sequence-Penetration Relationships of Antisense Oligonucleotides

A major limitation for the development of more effective oligonucleotide therapeutics has been a lack of understanding of their penetration into the cytosol. While prior work has shown how backbone modifications affect cytosolic penetration, it is unclear how cytosolic penetration is affected by other features including base composition, base sequence, length, and degree of secondary structure. We have applied the chloroalkane penetration assay, which exclusively reports on material that reaches the cytosol, to investigate the effects of these characteristics on the cytosolic uptake of druglike oligonucleotides. We found that base composition and base sequence had moderate effects, while length did not correlate directly with the degree of cytosolic penetration. Investigating further, we found that the degree of secondary structure had the largest and most predictable correlations with cytosolic penetration. These methods and observations add a layer of design for maximizing the efficacy of new oligonucleotide therapeutics.     

Personalized Development of Antisense Oligonucleotides for Exon Skipping Restores Type XVII Collagen Expression in Junctional Epidermolysis Bullosa

Intermediate junctional epidermolysis bullosa caused by mutations in the COL17A1 gene is characterized by the frequent development of blisters and erosions on the skin and mucous membranes. The rarity of the disease and the heterogeneity of the underlying mutations renders therapy developments challenging. However, the high number of short in-frame exons facilitates the use of antisense oligonucleotides (AON) to restore collagen 17 (C17) expression by inducing exon skipping. In a personalized approach, we designed and tested three AONs in combination with a cationic liposomal carrier for their ability to induce skipping of COL17A1 exon 7 in 2D culture and in 3D skin equivalents. We show that AON-induced exon skipping excludes the targeted exon from pre-mRNA processing, which restores the reading frame, leading to the expression of a slightly truncated protein. Furthermore, the expression and correct deposition of C17 at the dermal–epidermal junction indicates its functionality. Thus, we assume AON-mediated exon skipping to be a promising tool for the treatment of junctional epidermolysis bullosa, particularly applicable in a personalized manner for rare genotypes.     

Oligonucleotides Containing C5-Propynyl Modified Arabinonucleic Acids: Synthesis,Biophysical and Antisense Properties

The introduction of chemical modifications on the nucleic acid scaffold has allowed for the progress of antisense oligonucleotides (ASOs) in the clinic for the treatment of a variety of disorders. In contribution to the repertoire of gene-silencing nucleic acid modifications, herein we report the synthesis and incorporation of C5-propynyl arabinouridine ( araU^P ) and arabinocytidine ( araC^P ) into mixed-base ASOs containing a pyrimidine core. Substitution of the core with araU ^P and araC^P resulted in stabilization of the duplex formed with RNA but not with DNA. Similar results were obtained with ASOs bearing phosphorothioate linkages or methoxyethyl (MOE) wings in a gapmer design. All modified ASOs were compatible with E. coli RNase H mediated degradation of target RNA. Substitution of DNA for araU^P and araC^P in the central portion of a gapmer with MOE wings demonstrated improved nuclease resistance. These results suggest C5-modified arabinonucleic acids may serve as a potential chemical modification for therapeutic ASOs.     

Boron Clusters as Enhancers of RNase H Activity in the Smart Strategy of Gene Silencing by Antisense Oligonucleotides

Boron cluster-conjugated antisense oligonucleotides (B-ASOs) have already been developed as therapeutic agents with “two faces”, namely as potential antisense inhibitors of gene expression and as boron carriers for boron neutron capture therapy (BNCT). The previously observed high antisense activity of some B-ASOs targeting the epidermal growth factor receptor (EGFR) could not be rationally assigned to the positioning of the boron cluster unit: 1,2-dicarba-closo-dodecaborane (0), [(3,3′-Iron-1,2,1′,2′-dicarbollide) (1-), FESAN], and dodecaborate (2-) in the ASO chain and its structure or charge. For further understanding of this observation, we performed systematic studies on the efficiency of RNase H against a series of B-ASOs models. The results of kinetic analysis showed that pyrimidine-enriched B-ASO oligomers activated RNase H more efficiently than non-modified ASO. The presence of a single FESAN unit at a specific position of the B-ASO increased the kinetics of enzymatic hydrolysis of complementary RNA more than 30-fold compared with unmodified duplex ASO/RNA. Moreover, the rate of RNA hydrolysis enhanced with the increase in the negative charge of the boron cluster in the B-ASO chain. In conclusion, a “smart” strategy using ASOs conjugated with boron clusters is a milestone for the development of more efficient antisense therapeutic nucleic acids as inhibitors of gene expression.     

Progress and Prospects of Anti-HBV Gene Therapy Development

Despite the availability of an effective vaccine against hepatitis B virus (HBV), chronic infection with the virus remains a major global health concern. Current drugs against HBV infection are limited by emergence of resistance and rarely achieve complete viral clearance. This has prompted vigorous research on developing better drugs against chronic HBV infection. Advances in understanding the life cycle of HBV and improvements in gene-disabling technologies have been impressive. This has led to development of better HBV infection models and discovery of new drug candidates. Ideally, a regimen against chronic HBV infection should completely eliminate all viral replicative intermediates, especially covalently closed circular DNA (cccDNA). For the past few decades, nucleic acid-based therapy has emerged as an attractive alternative that may result in complete clearance of HBV in infected patients. Several genetic anti-HBV strategies have been developed. The most studied approaches include the use of antisense oligonucleotides, ribozymes, RNA interference effectors and gene editing tools. This review will summarize recent developments and progress made in the use of gene therapy against HBV.     

A Versatile and Convenient Synthesis of 34S‐Labeled Phosphorothioate Oligonucleotides

A synthetic protocol for ³⁴S‐labeled phosphorothioate oligonucleotides (PS ONs) was developed to facilitate MS‐based assay analysis. This was enabled by a highly efficient, two‐step, one‐pot synthesis of ³⁴S‐labeled phenylacetyl disulfide (³⁴S‐PADS), starting from ³⁴S‐enriched elemental sulfur (³⁴S₈). ³⁴S‐PADS was subsequently used for stable isotope labeling (SIL) of oligonucleotides containing a phosphorothioate backbone. The ³⁴S‐SIL PS ONs are shown to retain the same melting temperature, antisense activity, and secondary structure as those of the corresponding unlabeled ³²S PS ONs.     

Trans-Splicing Improvement by the Combined Application of Antisense Strategies

Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.     

Antisense Oligonucleotides Conjugated with Lipophilic Compounds: Synthesis and In Vitro Evaluation of Exon Skipping in Duchenne Muscular Dystrophy

Our groups previously reported that conjugation at 3′-end with ursodeoxycholic acid (UDCA) significantly enhanced in vitro exon skipping properties of ASO 51 oligonucleotide targeting the human DMD exon 51. In this study, we designed a series of lipophilic conjugates of ASO 51, to explore the influence of the lipophilic moiety on exon skipping efficiency. To this end, three bile acids and two fatty acids have been derivatized and/or modified and conjugated to ASO 51 by automatized solid phase synthesis. We measured the melting temperature (T_m) of lipophilic conjugates to evaluate their ability to form a stable duplex with the target RNA. The exon skipping efficiency has been evaluated in myogenic cell lines first in presence of a transfection agent, then in gymnotic conditions on a selection of conjugated ASO 51. In the case of 5′-UDC-ASO 51, we also evaluated the influence of PS content on exon skipping efficiency; we found that it performed better exon skipping with full PS linkages. The more efficient compounds in terms of exon skipping were found to be 5′-UDC- and 5′,3′-bis-UDC-ASO 51.     

2基因片段的克隆和反义表达载体的构建及遗传转化初步研究

以我国重要的生物能源灌木——中间锦鸡儿枝叶和种子为材料,根据Genbank中已经发表fad2基因的同源序列,利用PCR技术克隆得到两个基因片段.在GenBank中Blast(GenBank登录号AY957393和AY957394),所得基因片段和同属豆科的Glycine max Gm fad2-2a,同源性高达88 %,属于fad2基因编码区中间片断.将所得片段经BamHI和SacI酶切后插入表达载体质粒pBI121,构建了反义表达载体pBI121 fad2,并利用农杆菌介导法转入烟草叶片,获得了抗卡那霉素和安苄青霉素的再生烟草植株.初步分析结果表明,与对照烟草相比较,转基因烟草种子脂肪酸含量没有明显差异,而亚油酸则减少了10.3 %.这为进行下一步柠条分子改良,获得高单不饱和脂肪酸的优质中间锦鸡儿新品种以及改造其它木本生物柴油用木本原料植物奠定了基础.     

Conjugation and Evaluation of Triazole‐Linked Single Guide RNA for CRISPR‐Cas9 Gene Editing

The CRISPR‐Cas9 gene editing system requires Cas9 endonuclease and guide RNAs (either the natural dual RNA consisting of crRNA and tracrRNA or a chimeric single guide RNA) that direct site‐specific double‐stranded DNA cleavage. This communication describes a click ligation approach that uses alkyne–azide cycloaddition to generate a triazole‐linked single guide RNA (sgRNA). The conjugated sgRNA shows efficient and comparable genome editing activity to natural dual RNA and unmodified sgRNA constructs.     

Variable Expression of GABAA Receptor Subunit Gamma 2 Mutation in a Nuclear Family Displaying Developmental and Encephalopathic Phenotype

Mutations in GABA_A receptor subunit genes (GABRs) are a major etiology for developmental and epileptic encephalopathies (DEEs). This article reports a case of a genetic abnormality in GABRG2 and updates the pathophysiology and treatment development for mutations in DEEs based on recent advances. Mutations in GABRs, especially in GABRA1, GABRB2, GABRB3, and GABRG2, impair GABAergic signaling and are frequently associated with DEEs such as Dravet syndrome and Lennox–Gastaut syndrome, as GABAergic signaling is critical for early brain development. We here present a novel association of a microdeletion of GABRG2 with a diagnosed DEE phenotype. We characterized the clinical phenotype and underlying mechanisms, including molecular genetics, EEGs, and MRI. We then compiled an update of molecular mechanisms of GABR mutations, especially the mutations in GABRB3 and GABRG2 attributed to DEEs. Genetic therapy is also discussed as a new avenue for treatment of DEEs through employing antisense oligonucleotide techniques. There is an urgent need to define treatment targets and explore new treatment paradigms for the DEEs, as early deployment could alleviate long-term disabilities and improve quality of life for patients. This study highlights biomolecular targets for future therapeutic interventions, including via both pharmacological and genetic approaches.     

Regulation of Gene Expression by Telomere Position Effect

Many diseases that involve malignant tumors in the elderly affect the quality of human life; therefore, the relationship between aging and pathogenesis in geriatric diseases must be under-stood to develop appropriate treatments for these diseases. Recent reports have shown that epigenetic regulation caused by changes in the local chromatin structure plays an essential role in aging. This review provides an overview of the roles of telomere shortening on genomic structural changes during an age-dependent shift in gene expression. Telomere shortening is one of the most prominent events that is involved in cellular aging and it affects global gene expression through genome rearrangement. This review provides novel insights into the roles of telomere shortening in disease-affected cells during pathogenesis and suggests novel therapeutic approaches.     

Evaluation of Orbital Lymphoproliferative and Inflammatory Disorders by Gene Expression Analysis

Non-specific orbital inflammation (NSOI) and IgG4-related orbital disease (IgG4-ROD) are often challenging to differentiate. Furthermore, it is still uncertain how chronic inflammation, such as IgG4-ROD, can lead to mucosa-associated lymphoid tissue (MALT) lymphoma. Therefore, we aimed to evaluate the diagnostic value of gene expression analysis to differentiate orbital autoimmune diseases and elucidate genetic overlaps. First, we established a database of NSOI, relapsing NSOI, IgG4-ROD and MALT lymphoma patients of our orbital center (2000–2019). In a consensus process, three typical patients of the above mentioned three groups (mean age 56.4 ± 17 years) at similar locations were selected. Afterwards, RNA was isolated using the RNeasy FFPE kit (Qiagen) from archived paraffin-embedded tissues. The RNA of these 12 patients were then subjected to gene expression analysis (NanoString nCounter^®), including a total of 1364 target genes. The most significantly upregulated and downregulated genes were used for a machine learning algorithm to distinguish entities. This was possible with a high probability (p < 0.0001). Interestingly, gene expression patterns showed a characteristic overlap of lymphoma with IgG4-ROD and NSOI. In contrast, IgG4-ROD shared only altered expression of one gene regarding NSOI. To validate our potential biomarker genes, we isolated the RNA of a further 48 patients (24 NSOI, 11 IgG4-ROD, 13 lymphoma patients). Then, gene expression pattern analysis of the 35 identified target genes was performed using a custom-designed CodeSet to assess the prediction accuracy of the multi-parameter scoring algorithms. They showed high accuracy and good performance (AUC ROC: IgG4-ROD 0.81, MALT 0.82, NSOI 0.67). To conclude, genetic expression analysis has the potential for faster and more secure differentiation between NSOI and IgG4-ROD. MALT-lymphoma and IgG4-ROD showed more genetic similarities, which points towards progression to lymphoma.     

Chemically Tuned Intracellular Gene Delivery by Core-Shell Nanoparticles: Effects of Proton Buffering,Acid Degradability,and Membrane Disruption

Nanoparticles consisting of a condensed nucleic acid core surrounded by protective layers which aid to overcome extracellular and intracellular hurdles to gene delivery (i. e., core-shell nanoparticles, CSNPs) synthetically mimic viruses. The outer shells shield the core and are particularly designed to enable facilitated release of the gene payload into the cytoplasm, the major limiting step in intracellular gene delivery. The hypothetical proton sponge effect and degradability in response to a stimulus (i. e., mildly acidic pH in the endosome) are two prevailing, although contested, principles in designing effective carriers for intracellular gene delivery via endosomal escape. Utilizing the highly flexible chemical-tuning of the polymeric shell via surface-initiated photo-polymerization of the various monomers at different molecular ratios, the effects of proton buffering capacity, acid-degradability, and endosomal membrane-lysis property on intracellular delivery of plasmid DNA by CSNPs were investigated. This study demonstrated the equivalently critical roles of proton buffering and acid-degradability in achieving efficient intracellular gene delivery, independent of cellular uptake. Extended proton buffering resulted in further improved transfection as long as the core structure was not compromised. The results of the study present a promising synthetic strategy to the development of an efficient, chemically-tunable gene delivery carrier.