Towards RNAi based therapy of liver diseases : diversity and complexity of shRNA and miRNA processing and functions

Familial hypercholesterolemia (FH) is a genetic disorder characterized by high levels of low density lipoprotein cholesterol (LDL-C) and increasing the risk of cardio vascular diseases. FH and many other liver diseases can possibly be treated with RNA interference (RNAi). RNAi is a natural process of regulation of gene expression by binding of small RNA molecules to complementary sequences in the mRNA of a gene and hence inducing its degradation or translational repression. In this thesis, we aimed at developing a safe and robust RNAi-based therapy for FH by inhibiting Apolipoprotein B100 (ApoB). ApoB is a primary component of the low density lipoprotein (LDL), a lipoprotein that transports __bad__ cholesterol LDL-C. We have shown that RNAi therapy using AAV-delivered artificial miRNA is a promising approach for treatment of FH and possibly other liver diseases. Our mechanistic studies revealed differences in shRNA and miRNA processing and functioning in vivo. Finally, our findings have significant impact on understanding and overcoming toxicity and off-targeting related problems of RNAi based gene therapy in the liver using AAV vectors.uniQure Biopharma, Macrogen, Leiden UniversityUBL - phd migration 201

New design of a lentiviral shRNA vector with inducible co-expression of ARGONAUTE 2 for enhancing gene silencing efficiency

Background: RNA interference (RNAi) is a robust tool for inhibiting specific gene expression, but it is limited by the uncertain efficiency of siRNA or shRNA constructs. It has been shown that the overexpression of ARGONAUTE 2 (AGO2) protein increases silencing efficiency. However, the key elements required for AGO2-mediated enhancement of gene silencing in lentiviral vector has not been well studied.Results: To explore the application of AGO2-based shRNA system in mammalian cells, we designed shRNA vectors targeting the EGFP reporter gene and evaluated the effects of various factors on silencing efficiency including stem length, loop sequence, antisense location as well as the ratio between AGO2 and shRNA. We found that 19 ~ 21-bp stem and 6- or 9-nt loop structure in the sense-loop-antisense (S-L-AS) orientation was an optimal design in the AGO2-shRNA system. Then, we constructed a single lentiviral vector co-expressing shRNA and AGO2 and demonstrated that the simultaneous expression of shRNA and AGO2 can achieve robust silencing of exogenous DsRed2 and endogenous ID1 and P65 genes. However, the titers of packaged lentivirus from constitutive expression of AGO2 vector were extremely low, severely limiting its broad application. For the first time, we demonstrated that the problem can be significantly improved by using the inducible expression of AGO2 lentiviral system.Conclusions: We reported a novel lentiviral vector with an optimal design of shRNA and inducible AGO2 overexpression which provides a new tool for RNAi research.Peer reviewedPhysiological Science

Towards RNAi based therapy of liver diseases : diversity and complexity of shRNA and miRNA processing and functions

Familial hypercholesterolemia (FH) is a genetic disorder characterized by high levels of low density lipoprotein cholesterol (LDL-C) and increasing the risk of cardio vascular diseases. FH and many other liver diseases can possibly be treated with RNA interference (RNAi). RNAi is a natural process of regulation of gene expression by binding of small RNA molecules to complementary sequences in the mRNA of a gene and hence inducing its degradation or translational repression. In this thesis, we aimed at developing a safe and robust RNAi-based therapy for FH by inhibiting Apolipoprotein B100 (ApoB). ApoB is a primary component of the low density lipoprotein (LDL), a lipoprotein that transports __bad__ cholesterol LDL-C. We have shown that RNAi therapy using AAV-delivered artificial miRNA is a promising approach for treatment of FH and possibly other liver diseases. Our mechanistic studies revealed differences in shRNA and miRNA processing and functioning in vivo. Finally, our findings have significant impact on understanding and overcoming toxicity and off-targeting related problems of RNAi based gene therapy in the liver using AAV vectors

Geological and geomorphological interpretation of Airborne Laser Scanning (ALS) data of the Kasprowy Wierch area (Tatra Mts.)

The paper presents the geological interpretation of Airborne Laser Scanning data of Kasprowy Wierch Mt. area. The analyzed points cloud data represent the ground surface after removal of the land cover (primarily vegetation) objects. The ALS data were characterized by very high density, presenting even minor terrain forms, very difficult to identify in forested and mountain dwarfpine areas as well. The resulting image of detailed geomorphology of the study area was compared with existing maps at scale of 1 : 10 000 and literature data. This allowed verifying the extent of geological units of basement rocks and shed new light on existing opinions on the genesis of glacial sediments. An extensive landslide has been identified on the eastern slopes of Sucha Czuba Mt. The landslide has not been reported in the literature so far. Application of laser scanning data was extremely useful and allowed making new observations that enrich the existing knowledge about the geology of this part of the Tatra Mts

Mir-142-3p target sequences reduce transgene-directed immunogenicity following intramuscular adeno-associated virus 1 vector-mediated gene delivery

Cellular mechanisms in basic and clinical gastroenterology and hepatolog

Embedding siRNA sequences targeting Apolipoprotein B100 in shRNA and miRNA scaffolds results in differential processing and in vivo efficacy

Cellular mechanisms in basic and clinical gastroenterology and hepatolog

Therapeutic expression of hairpins targeting apolipoprotein B100 induces phenotypic and transcriptome changes in murine liver

Constitutive expression of short hairpin RNAs (shRNAs) may cause cellular toxicity in vivo and using microRNA (miRNA) scaffolds can circumvent this problem. Previously, we have shown that embedding small interfering RNA sequences targeting apolipoprotein B100 (ApoB) in shRNA (shApoB) or miRNA (miApoB) scaffolds resulted in differential processing and long-term efficacy in vivo. Here we show that adeno-associated virus (AAV)-shApoB- or AAV-miApoB-mediated ApoB knockdown induced differential liver morphology and transcriptome expression changes. Our analyses indicate that ApoB knockdown with both shApoB and miApoB resulted in alterations of genes involved in lipid metabolism. In addition, in AAV-shApoB-injected animals, genes involved in immune system activation or cell growth and death were affected, which was associated with increased hepatocyte proliferation. Subsequently, in AAV-miApoB-injected animals, changes of genes involved in oxidoreductase activity, oxidative phosphorylation and nucleic bases biosynthetic processes were observed. Our results demonstrate that long-term knockdown of ApoB in vivo by shApoB or miApoB induces several transcriptome changes in murine liver. The increased hepatocyte profileration by AAV-shRNA may have severe long-term effects indicating that AAV-mediated RNA interference therapy using artificial miRNA may be a safer approach for familial hypercholesterolemia therap