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The regulatory approval of Onpattro, a lipid nanoparticle-based short interfering RNA drug for the treatment of polyneuropathies induced by hereditary transthyretin amyloidosis, paves the way for clinical development of many nucleic acid-based therapies enabled by nanoparticle delivery.

The world raced to develop vaccines to protect against the rapid spread of SARS-CoV-2 infection upon the recognition of COVID-19 as a global pandemic. A broad spectrum of candidates was evaluated, with mRNA-based vaccines emerging as leaders due to how quickly they were available for emergency use while providing a high level of efficacy. As a modular technology, the mRNA-based vaccines benefitted from decades of advancements in both mRNA and delivery technology prior to the current global pandemic. The fundamental lessons of the utility of mRNA as a therapeutic were pioneered by Dr. Katalin Kariko and her colleagues, perhaps most notably in collaboration with Drew Weissman at University of Pennsylvania, and this foundational work paved the way for the development of the first ever mRNA-based therapeutic authorized for human use, COMIRNATY®. In this Special Issue of Pharmaceutics, we will be honoring Dr. Kariko for her great contributions to the mRNA technology to treat diseases with unmet needs. In this review article, we will focus on the delivery platform, the lipid nanoparticle (LNP) carrier, which allowed the potential of mRNA therapeutics to be realized. Similar to the mRNA technology, the development of LNP systems has been ongoing for decades before culminating in the success of the first clinically approved siRNA-LNP product, ONPATTRO®, a treatment for an otherwise fatal genetic disease called transthyretin amyloidosis. Lessons learned from the siRNA-LNP experience enabled the translation into the mRNA platform with the eventual authorization and approval of the mRNA-LNP vaccines against COVID-19. This marks the beginning of mRNA-LNP as a pharmaceutical option to treat genetic diseases.

Most known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle–based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9 , a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases. Base editors are effective and safe for cholesterol reduction in non-human primates.

Immunostimulatory oligodeoxynucleotides (ODN) are effective adjuvants in the induction of humoral and cellular immune responses when administered parenterally with antigen. The skin has recently become a target organ for the design of non-invasive vaccine technologies. Using ovalbumin (OVA) as a model antigen, we demonstrate that the application of ODN sequences to tape-stripped skin promotes the induction of potent cytotoxic T lymphocyte (CTL) responses to co-administered peptide. Induction of peptide-specific CTL required the presence of CpG motifs within the ODN. CTL afforded tumor protection against a tumor expressing an immunodominant OVA CTL epitope. CTL could also be induced to whole protein administered onto the skin. Differential CpG sequence activity was noted with respect to the induction of CTL to epicutaneous protein with an ODN sequence containing a poly-G motif having an optimal effect. Peptide-specific CTL could be detected in the peripheral blood as early as 6 d after a single immunization. These results highlight the potential of the bare skin as a route for vaccine development and indicate an important role for immunostimulatory ODN as adjuvants to generate functional CTL with the help of the skin immune system.

Efficient siRNA delivery remains a key challenge to realizing the full potential of RNAi-based therapeutics. Semple et al . accomplish unprecedented potency for liposome-mediated siRNA delivery by applying rational design to refine an empirically identified cationic lipid widely regarded as the benchmark for use in lipid nanoparticles. We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.

Phosphorothioate c-myc antisense oligodeoxynucleotides [S]ODNs (free INX-6295) were encapsulated in a new liposome formulation and the antitumor activity was compared to the unencapsulated antisense in a human melanoma xenograft. The systemic administration of INX-6295 encapsulated in stabilized antisense lipid particles (SALP INX-6295) improved plasma AUC (area under the plasma concentration-time curve) and initial half-life of free INX-6295, resulting in a significant enhancement in tumor accumulation and improvement in tumor distribution of antisense oligodeoxynucleotides. Animals treated with SALP INX-6295 exhibited a prolonged reduction of c-myc expression, reduced tumor growth and increased mice survival. When administered in combination with cisplatin (DDP), SALP INX-6295 produced a complete tumor regression in approximately 30% of treated mice, which persisted for at least 60 days following the first cycle of treatment. Finally, the median survival of mice treated with DDP/SALP INX-6295 increased by 105% compared to 84% for animals treated with the combination DDP/free INX-6295. These data indicate that the biological activity and the therapeutic efficacy of c-myc antisense therapy may be improved when these agents are administered in lipid-based delivery systems.

This study describes the effect of ethanol and the presence of poly(ethylene) glycol (PEG) lipids on the interaction of nucleotide-based polyelectrolytes with cationic liposomes. It is shown that preformed large unilamellar vesicles (LUVs) containing a cationic lipid and a PEG coating can be induced to entrap polynucleotides such as antisense oligonucleotides and plasmid DNA in the presence of ethanol. The interaction of the cationic liposomes with the polynucleotides leads to the formation of multilamellar liposomes ranging in size from 70 to 120 nm, only slightly bigger than the parent LUVs from which they originated. The degree of lamellarity as well as the size and polydispersity of the liposomes formed increases with increasing polynucleotide-to-lipid ratio. A direct correlation between the entrapment efficiency and the membrane-destabilizing effect of ethanol was observed. Although the morphology of the liposomes is still preserved at the ethanol concentrations used for entrapment (25–40%, v/v), entrapped low-molecular-weight solutes leak rapidly. In addition, lipids can flip-flop across the membrane and exchange rapidly between liposomes. Furthermore, there are indications that the interaction of the polynucleotides with the cationic liposomes in ethanol leads to formation of polynucleotide-cationic lipid domains, which act as adhesion points between liposomes. It is suggested that the spreading of this contact area leads to expulsion of PEG-ceramide and triggers processes that result in the formation of multilamellar systems with internalized polynucleotides. The high entrapment efficiencies achieved at high polyelectrolyte-to-lipid ratios and the small size and neutral character of these novel liposomal systems are of utility for liposomal delivery of macromolecular drugs.

BackgroundOver half the world is exposed daily to the smoke from combustion of solid fuels. Chronic obstructive pulmonary disease (COPD) is one of the main contributors to the global burden of disease and can be caused by biomass smoke exposure. However, studies of biomass exposure and COPD show a wide range of effect sizes. The aim of this systematic review was to quantify the impact of biomass smoke on the development of COPD and define reasons for differences in the reported effect sizes.MethodsA systematic review was conducted of studies with sufficient statistical power to calculate the health risk of COPD from the use of solid fuel, which followed standardised criteria for the diagnosis of COPD and which dealt with confounding factors. The results were pooled by fuel type and country to produce summary estimates using a random effects model. Publication bias was also estimated.ResultsThere were positive associations between the use of solid fuels and COPD (OR=2.80, 95% CI 1.85 to 4.0) and chronic bronchitis (OR=2.32, 95% CI 1.92 to 2.80). Pooled estimates for different types of fuel show that exposure to wood smoke while performing domestic work presents a greater risk of development of COPD and chronic bronchitis than other fuels.ConclusionDespite heterogeneity across the selected studies, exposure to solid fuel smoke is consistently associated with COPD and chronic bronchitis. Efforts should be made to reduce exposure to solid fuel by using either cleaner fuel or relatively cleaner technology while performing domestic work.

Mammalian promoters can be separated into two classes, conserved TATA box–enriched promoters, which initiate at a well-defined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3′ UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis -acting elements associated with them.