Modification to the immunodominant loop of hepatitis B virus core protein to enhance vector properties of virus-like particles

Gene therapy has shown potential in alleviating a wide range of diseases, ranging from viral infections to autosomal diseases. One of the obstacles to gene therapy reaching its full potential is the inadequacy of methods to deliver therapeutic nucleic sequences. Current delivery of gene therapy entails use of viral and non-viral vectors. Viral vectors are however associated with drawbacks such as potential toxicity, high cost and labour-intensive production. Thus non-viral delivery alternatives are being developed in an attempt to overcome difficulties associated with nucleic acid delivery for gene therapy. Virus-like particles are potentially very useful delivery vehicles as their production is simple and cost effective, the particle surface is amenable to modification and the capsid interior can be altered to accommodate a variety of cargoes. One such particle is the recombinant HBV capsid, which comprises a single species of protein and is tolerant of amino acid substitutions on the surface exposed immunodominant loops. This study aimed to enhance the vector-like properties of the HBV virus-like particle by including amino acid substitutions on the particle surface. These substituted residues in turn provided a conjugation site for tropic and immuno evasive moieties. It was found that lysine substitutions resulted in poor conjugation to the capsid surface, whereas substituted cysteine residues resulted in almost all protein-moiety conjugates forming. In order to introduce lysine and cysteine substitutions, a novel method of cloning into the HBV was generated. In doing so, complicated procedures associated with cloning into the immunodominant loop of the HBV capsid have been alleviated. Ligands containing galactose were utilised on the surface of both the HBV capsid and liposomes to confer hepatotropism. The presence of the galactose moieties on the surface of the HBV capsid prevented indiscriminate cellular uptake in cultured cells, however did not improve hepatotropism. Galactose ligands on the surface of liposomes did improve transfection efficiency, however they required a short linker distance between liposome surface and galactose group. The inclusion of galactose in liposome formulations also provided a means to deliver siRNA to the liver of transgenic HBV mice. It is believed that with alterations to the ligand structure, it is possible to provide HBV capsids with hepatotropism in future experimentation. This study demonstrated that the exposed external surface of the HBV capsid is amenable to convenient conjugation, which potentially facilitates immune evasion and conferring of defined tropism

An improved ribozyme processing system that generates active RNA interference effector molecules from Pol II expression cassettes

RNA interference (RNAi) has been shown to be highly effective in targeted gene knockdown and has the potential to be applied for therapy by silencing pathology-causing genes. However, there remain several undesired properties associated with the utilisation of RNAi for therapeutic purposes. These include: innate immunostimulation, “off-target” cellular sequences and the possibility of saturating the endogenous RNAi pathway, which is required for microRNA biogenesis. RNA Polymerase III (Pol III) promoters have been used predominantly to generate exogenous expressed RNAi precursors. Pol III promoters possess constitutive activity in most tissue types and their transcripts can be easily tailored into microRNA-like cellular precursor structures. Regulation of Pol III promoters is however difficult to achieve, and their lack of tissue specificity and high activity are responsible for the toxic saturating effects on the RNAi pathway. In contrast, RNA polymerase II promoters express mRNAs which can be regulated and are differentially expressed in specific tissues. However, Pol II-transcripts have additional sequences such as the 5’ 7-methyl guanosine cap and 3’ polyadenylation sequence which make them unsuitable for the generation of important RNAi precursors such as short hairpin RNAs (shRNAs). This study aimed to produce a series of cytomegalovirus (CMV) promoter-controlled expression cassettes that would generate shRNAs lacking unwanted flanking sequences. The precise hairpin RNA strand was processed post-transcriptionally through the action of chimaeric ciscleaving hammerhead ribozymes that are incorporated up- and down-stream of the shRNA. The hammerhead ribozymes were restored from a minimal state by inserting additional extra-core elements allowing for intracellular activity. This design for producing active RNAi effector sequences was termed a Ribozyme Processing System (RyPS). To evaluate the inhibitory efficacy of RyPS, a previously characterised shRNA targeted against the X open reading frame of the hepatitis B virus was inserted into a RyPS expression cassette. In vitro co-transcription and cleavage experiments demonstrated the processing potential of RyPS. This resulted in the formation of 3 products; the upstream and downstream ribozymes and the shRNA. Northern blot analysis of in vitro transcription products revealed the shRNA and downstream ribozyme were smaller than anticipated. Using primer extension analysis the precise ribozyme cleavage sites of the up- and downstream ribozymes were mapped. The upstream ribozyme mapped the predicted site, however multiple cleavage sites were mapped for the downstream ribozyme. The aberrant cleavage of the downstream ribozyme resulted in an shRNA cleaved within the antisense region, a sequence which dictates the targeting and inhibitory potential of the shRNA. Intracellular transfection of RyPS resulted in little to no inhibition of both live virus and targeted reporter genes. It was noted however that the ribozymes maintained intracellular activity according to a luciferase-based knockdown assay, in which ribozyme activity resulted in luciferase mRNA destruction. By applying these results to RNA folding algorithms, a model can be developed where atypical cleavage by the flanking ribozymes is avoided, and further allow for the design of more stable RyPS and individual ribozyme. Although the current design of the RyPS cassette was shown to be ineffective at producing active shRNAs, possible optimisation would involve the substitution of the shRNA, or replacing the chimaeric hammerhead ribozyme species with a naturally occurring species. With these changes further developments of this post-transcriptional processing system may soon result in effective Pol II-generated sequences for therapeutic RNAi

Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.International Society for Advancement of Cytometry Marylou Ingram Scholar 2019-2023H2020 EXPERTSwedish foundation of Strategic Research (SSF-IRC; FormulaEx)ERC CoG (DELIVER)Swedish Medical Research CouncilAccepte

Effect of aliskiren on post-discharge outcomes among diabetic and non-diabetic patients hospitalized for heart failure: insights from the ASTRONAUT trial

Aims The objective of the Aliskiren Trial on Acute Heart Failure Outcomes (ASTRONAUT) was to determine whether aliskiren, a direct renin inhibitor, would improve post-discharge outcomes in patients with hospitalization for heart failure (HHF) with reduced ejection fraction. Pre-specified subgroup analyses suggested potential heterogeneity in post-discharge outcomes with aliskiren in patients with and without baseline diabetes mellitus (DM). Methods and results ASTRONAUT included 953 patients without DM (aliskiren 489; placebo 464) and 662 patients with DM (aliskiren 319; placebo 343) (as reported by study investigators). Study endpoints included the first occurrence of cardiovascular death or HHF within 6 and 12 months, all-cause death within 6 and 12 months, and change from baseline in N-terminal pro-B-type natriuretic peptide (NT-proBNP) at 1, 6, and 12 months. Data regarding risk of hyperkalaemia, renal impairment, and hypotension, and changes in additional serum biomarkers were collected. The effect of aliskiren on cardiovascular death or HHF within 6 months (primary endpoint) did not significantly differ by baseline DM status (P = 0.08 for interaction), but reached statistical significance at 12 months (non-DM: HR: 0.80, 95% CI: 0.64-0.99; DM: HR: 1.16, 95% CI: 0.91-1.47; P = 0.03 for interaction). Risk of 12-month all-cause death with aliskiren significantly differed by the presence of baseline DM (non-DM: HR: 0.69, 95% CI: 0.50-0.94; DM: HR: 1.64, 95% CI: 1.15-2.33; P < 0.01 for interaction). Among non-diabetics, aliskiren significantly reduced NT-proBNP through 6 months and plasma troponin I and aldosterone through 12 months, as compared to placebo. Among diabetic patients, aliskiren reduced plasma troponin I and aldosterone relative to placebo through 1 month only. There was a trend towards differing risk of post-baseline potassium ≥6 mmol/L with aliskiren by underlying DM status (non-DM: HR: 1.17, 95% CI: 0.71-1.93; DM: HR: 2.39, 95% CI: 1.30-4.42; P = 0.07 for interaction). Conclusion This pre-specified subgroup analysis from the ASTRONAUT trial generates the hypothesis that the addition of aliskiren to standard HHF therapy in non-diabetic patients is generally well-tolerated and improves post-discharge outcomes and biomarker profiles. In contrast, diabetic patients receiving aliskiren appear to have worse post-discharge outcomes. Future prospective investigations are needed to confirm potential benefits of renin inhibition in a large cohort of HHF patients without D

The rough endoplasmatic reticulum is the central site of siRNA-mediated RNA silencing

Despite the rapid advancement of our mechanistic understanding of the RNA interference (RNAi) pathways in the past years, the subcellular sites of RNA silencing still remain under debate. Here we show that a lion’s share of transfected small interfering RNA (siRNA) is cleared quickly and only few siRNA molecules are finally getting loaded into Ago2, with as little as 20 30 siRISC molecules per cell sufficient to promote 50 % mRNA knockdown. While the major RNAi pathway proteins are found in most subcellular compartments, the microRNA (miRNA)- and siRNA loaded Ago2 population as well as the RNAi mediated mRNA cleavage product co-sediment exclusively with the membranes of the rough endoplasmatic reticulum (rER) together with the RISC loading complex (RLC) factors Dicer, TRBP and PACT. Moreover, siRNA-loaded Ago2 associates with the cytosolic side of membranes through TRBP and PACT in an RNA-independent manner, potentially mediated through indirect interaction via Dicer. Our findings demonstrate that the outer membrane of the rER is the central site of RNA silencing, which explains the remarkable thermodynamic and kinetic efficiency of this mechanism

Exosomes surf on filopodia to enter cells at endocytic hot spots, and shuttle with endosomes to scan the endoplasmic reticulum – a highway to the cell.

Exosomes are nanovesicles released by virtually all cells which act as intercellular messengers by transfer of protein, lipid and RNA cargo. Their quantitative efficiency, routes of cell uptake and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake which saturates with dose and time and reaches near 100 % ‘transduction’ efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia, as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. Following internalization, exosomes shuttle with endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction and suggest unanticipated routes of subcellular cargo delivery. vonBueren S1, Graff-Meyer A4, Genoud C4, Martin K5, Voshol H1, Morrissey DV6, 7, EL Andaloussi S3,8, Wood MJ3, Meisner-Kober NC

Exosomes surf on filopodia to enter cells at endocytic hot spots and shuttle within endosomes to scan the ER

Exosomes are nanovesicles released by virtually all cells which act as intercellular messengers by transfer of protein, lipid and RNA cargo. Their quantitative efficiency, routes of cell uptake and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake which saturates with dose and time and reaches near 100 % ‘transduction’ efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia, as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. Following internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction and suggest unanticipated routes of subcellular cargo delivery

Inhibition of hepatitis B virus replication in vivo using lipoplexes containing altritol-modified antiviral siRNAs

Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the world's population and carriers of the virus are at risk for complicating hepatocellular carcinoma. Current treatment options have limited efficacy and chronic HBV infection is likely to remain a significant global medical problem for many years to come. Silencing HBV gene expression by harnessing RNA interference (RNAi) presents an attractive option for development of novel and effective anti HBV agents. However, despite significant and rapid progress, further refinement of existing technologies is necessary before clinical application of RNAi-based HBV therapies is realized. Limiting off target effects, improvement of delivery efficiency, dose regulation and preventing reactivation of viral replication are some of the hurdles that need to be overcome. To address this, we assessed the usefulness of the recently described class of altritol-containing synthetic siRNAs (ANA siRNAs), which were administered as lipoplexes and tested in vivo in a stringent HBV transgenic mouse model. Our observations show that ANA siRNAs are capable of silencing of HBV replication in vivo. Importantly, non specific immunostimulation was observed with unmodified siRNAs and this undesirable effect was significantly attenuated by ANA modification. Inhibition of HBV replication of approximately 50% was achieved without evidence for induction of toxicity. These results augur well for future application of ANA siRNA therapeutic lipoplexes