Design and application of RNA therapeutics for splice site mutations

Precursor messenger RNA splicing is one of the most fundamental and complex mechanisms in eukaryotes. Over 90% of the human genes undergo alternative splicing, which is essential for the regulation of gene expression. A dynamic RNA-protein complex called the spliceosome catalyzes splicing. The spliceosome recognizes core splice site signals, and its function is furthermore regulated by other sequence elements that can either silence or enhance splicing in a cell-specific manner. These regulatory elements are recognized by trans-acting protein factors which modulate the function of the spliceosome. However, the fact that splicing is one of the most regulated mechanisms in the cell also makes it prone to dysfunctions caused by mutations. Mis-splicing diseases account for up to 30% of the inherited genetic diseases. They can be caused by mutations in the core splice sites as well as in the regulatory elements. Mutations that disrupt the splicing mechanism may result in RNA degradation, non-functional protein products, or toxic proteins that might alter the cellular environment. The understanding of mis-splicing disease mechanisms has been subject to various studies aiming at finding the appropriate therapeutics. In parallel with the development of gene therapy, where the classical aim is to introduce the corrected gene to cure a disease, another field has emerged; antisense oligonucleotide therapeutics. These RNA-DNA-based oligonucleotides can be designed to alter gene expression as well as to manipulate the splicing mechanism. Since their emergence during 1970s, antisense oligonucleotides have been extensively studied within the mis-splicing disease field, with several clinical trials ongoing. In this thesis, in paper I, we report a novel solid-phase synthesis method with a biological proof-of-concept. This synthesis method allows the conjugation of therapeutic oligonucleotides via a cleavable disulfide linker. The developed method can be applied to target several transcripts or different parts within the same transcript by allowing delivery of equimolar amounts of therapeutics. In paper II, we explore the possibility of using splice- correction approach for restoring the aberrant splicing of the gene BTK. Lack of BTK causes a primary immunodeficiency disease called XLA, which is a B cell developmental disorder. For the first time, we show splice-correction in B cells by modified oligonucleotide therapeutics both ex vivo and in vivo. In paper III, we aim at developing methods to rescue the core splice site mutations in BTK by using bifunctional oligonucleotide therapeutics. These oligonucleotides have the ability to recruit splice factor proteins, improving the splicing of mutated sites. We show that rescuing of core splice site mutations is possible yet it has its own challenges, which need to be taken into account in the design process. The results in this thesis have provided new therapeutics for a genetic disease, and more generally explores new methods for improving the function and delivery of oligonucleotide therapeutics

Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective. Keywords: CRISPR; FRGN; ex vivo; genome editing; hepatocyte transplantation; liver-humanized mouse; primary hepatocytes; urea cycle disorder

The viral protein corona directs viral pathogenesis and amyloid aggregation

Artificial nanoparticles accumulate a protein corona layer in biological fluids, which significantly influences their bioactivity. As nanosized obligate intracellular parasites, viruses share many biophysical properties with artificial nanoparticles in extracellular environments and here we show that respiratory syncytial virus (RSV) and herpes simplex virus type 1 (HSV-1) accumulate a rich and distinctive protein corona in different biological fluids. Moreover, we show that corona pre-coating differentially affects viral infectivity and immune cell activation. In addition, we demonstrate that viruses bind amyloidogenic peptides in their corona and catalyze amyloid formation via surface-assisted heterogeneous nucleation. Importantly, we show that HSV-1 catalyzes the aggregation of the amyloid beta-peptide (A beta(42)), a major constituent of amyloid plaques in Alzheimer's disease, in vitro and in animal models. Our results highlight the viral protein corona as an acquired structural layer that is critical for viral-host interactions and illustrate a mechanistic convergence between viral and amyloid pathologies.Peer reviewe

Optimizing anti-gene oligonucleotide ‘Zorro-LNA’ for improved strand invasion into duplex DNA

Zorro-LNA (Zorro) is a newly developed, oligonucleotide (ON)-based, Z-shaped construct with the potential of specific binding to each strand of duplex DNA. The first-generation Zorros are formed by two hybridized LNA/DNA mixmers (2-ON Zorros) and was hypothesized to strand invade. We have now established a method, which conclusively demonstrates that an LNA ON can strand invade into duplex DNA. To make Zorros smaller in size and easier to design, we synthesized 3′–5′–5′–3′ single-stranded Zorro-LNA (ssZorro) by using both 3′- and 5′-phosphoramidites. With ssZorro, a significantly greater extent and rate of double-strand invasion (DSI) was obtained than with conventional 2-ON Zorros. Introducing hydrophilic PEG-linkers connecting the two strands did not significantly change the rate or extent of DSI as compared to ssZorro with a nucleotide-based linker, while the longest alkyl-chain linker tested (36 carbons) resulted in a very slow DSI. The shortest alkyl-chain linker (3 carbons) did not reduce the extent of DSI of ssZorro, but significantly decreased the DSI rate. Collectively, ssZorro is smaller in size, easier to design and more efficient than conventional 2-ON Zorro in inducing DSI. Analysis of the chemical composition of the linker suggests that it could be of importance for future therapeutic considerations

Histopathological features of chronic hepatitis B reactivation

It was aimed to investigate histopathological changes in acute hepatic exacerbations. A total of 20 patients, who developed acute hepatic exacerbation on the base of chronic hepatitis B infection, were enrolled in the study and liver biopsy was performed. Histological activity index (HAI) and its components, fibrosis scores and total HAI score were calculated according to the Ishak scoring system. All of the patients enrolled in the study had portal inflammation and spotty necrosis. Fibrosis was present in 17 patients. The median grade, stage and total HAI scores of the patients were 7 (2-14), 2 (0-3) and 8.5 (4-16), respectively. Positive correlation was determined between necroinflammation and ALT. While grade and total HAI score did not show significant difference among genders, fibrosis was significantly higher in males (p=0.008). The findings of this study showed that there are significant correlation between histopathologic changes (grade and total HAI score) in liver biopsy and plasma ALT, AST and Total bilirubin levels during acute hepatic exacerbation in patients with chronic hepatitis B infection. [Med-Science 2020; 9(3.000): 721-6