Influence of ionizable lipid tail length on lipid nanoparticle delivery of mRNA of varying length.

RNA-based therapeutics have gained traction for the prevention and treatment of a variety of diseases. However, their fragility and immunogenicity necessitate a drug carrier. Lipid nanoparticles (LNPs) have emerged as the predominant delivery vehicle for RNA therapeutics. An important component of LNPs is the ionizable lipid (IL), which is protonated in the acidic environment of the endosome, prompting cargo release into the cytosol. Currently, there is growing evidence that the structure of IL lipid tails significantly impacts the efficacy of LNP-mediated mRNA translation. Here, we optimized IL tail length for LNP-mediated delivery of three different mRNA cargos. Using C12-200, a gold standard IL, as a model, we designed a library of ILs with varying tail lengths and evaluated their potency in vivo. We demonstrated that small changes in lipophilicity can drastically increase or decrease mRNA translation. We identified that LNPs formulated with firefly luciferase mRNA (1929 base pairs) and C10-200, an IL with shorter tail lengths than C12-200, enhance liver transfection by over 10-fold. Furthermore, different IL tail lengths were found to be ideal for transfection of LNPs encapsulating mRNA cargos of varying sizes. LNPs formulated with erythropoietin (EPO), responsible for stimulating red blood cell production, mRNA (858 base pairs), and the C13-200 IL led to EPO translation at levels similar to the C12-200 LNP. The LNPs formulated with Cas9 mRNA (4521 base pairs) and the C9-200 IL induced over three times the quantity of indels compared with the C12-200 LNP. Our findings suggest that shorter IL tails may lead to higher transfection of LNPs encapsulating larger mRNAs, and that longer IL tails may be more efficacious for delivering smaller mRNA cargos. We envision that the results of this project can be utilized as future design criteria for the next generation of LNP delivery systems for RNA therapeutics.

In situ combinatorial synthesis of degradable branched lipidoids for systemic delivery of mRNA therapeutics and gene editors.

The ionizable lipidoid is a key component of lipid nanoparticles (LNPs). Degradable lipidoids containing extended alkyl branches have received tremendous attention, yet their optimization and investigation are underappreciated. Here, we devise an in situ construction method for the combinatorial synthesis of degradable branched (DB) lipidoids. We find that appending branch tails to inefficacious lipidoids via degradable linkers boosts mRNA delivery efficiency up to three orders of magnitude. Combinatorial screening and systematic investigation of two libraries of DB-lipidoids reveal important structural criteria that govern their in vivo potency. The lead DB-LNP demonstrates robust delivery of mRNA therapeutics and gene editors into the liver. In a diet-induced obese mouse model, we show that repeated administration of DB-LNP encapsulating mRNA encoding human fibroblast growth factor 21 alleviates obesity and fatty liver. Together, we offer a construction strategy for high-throughput and cost-efficient synthesis of DB-lipidoids. This study provides insights into branched lipidoids for efficient mRNA delivery.

Predictive High-Throughput Platform for Dual Screening of mRNA Lipid Nanoparticle Blood-Brain Barrier Transfection and Crossing.

Lipid nanoparticle (LNP)-mediated nucleic acid therapies, including mRNA protein replacement and gene editing therapies, hold great potential in treating neurological disorders including neurodegeneration, brain cancer, and stroke. However, delivering LNPs across the blood-brain barrier (BBB) after systemic administration remains underexplored. In this work, we engineered a high-throughput screening transwell platform for the BBB (HTS-BBB), specifically optimized for screening mRNA LNPs. Unlike most transwell assays, which only assess transport across an endothelial monolayer, HTS-BBB simultaneously measures LNP transport and mRNA transfection of the endothelial cells themselves. We then use HTS-BBB to screen a library of 14 LNPs made with structurally diverse ionizable lipids and demonstrate it is predictive of in vivo performance by validating lead candidates for mRNA delivery to the mouse brain after intravenous injection. Going forward, this platform could be used to screen large libraries of brain-targeted LNPs for a range of protein replacement and gene editing applications.

siRNA lipid nanoparticles for CXCL12 silencing modulate brain immune response during Zika infection.

CXCL12 is a key chemokine implicated in neuroinflammation, particularly during Zika virus (ZIKV) infection. Specifically, CXCL12 is upregulated in circulating cells of ZIKV infected patients. Here, we developed a lipid nanoparticle (LNP) to deliver siRNA in vivo to assess the impact of CXCL12 silencing in the context of ZIKV infection. The biodistribution of the LNP was assessed in vivo after intravenous injection using fluorescently tagged siRNA. Next, we investigated the ability of the developed LNP to silence CXCL12 in vivo and assessed the resulting effects in a murine model of ZIKV infection. The LNP encapsulating siRNA significantly inhibited CXCL12 levels in the spleen and induced microglial activation in the brain during ZIKV infection. This activation was evidenced by the enhanced expression of iNOS, TNF-α, and CD206 within microglial cells. Moreover, T cell subsets exhibited reduced secretion of IFN-É£ and IL-17 following LNP treatment. Despite no observable alteration in viral load, CXCL12 silencing led to a significant reduction in type-I interferon production compared to both ZIKV-infected and uninfected groups. Furthermore, we found grip strength deficits in the group treated with siRNA-LNP compared to the other groups. Our data suggest a correlation between the upregulated pro-inflammatory cytokines and the observed decrease in strength. Collectively, our results provide evidence that CXCL12 silencing exerts a regulatory influence on the immune response in the brain during ZIKV infection. In addition, the modulation of T-cell activation following CXCL12 silencing provides valuable insights into potential protective mechanisms against ZIKV, offering novel perspectives for combating this infection.

mRNA Lipid Nanoparticles for Ex Vivo Engineering of Immunosuppressive T Cells for Autoimmunity Therapies.

Cell-based therapies for autoimmune diseases have gained significant traction, with several approaches centered around the regulatory T (Treg) cell─a well-known immunosuppressive cell characterized by its expression of the transcription factor Foxp3. Unfortunately, due to low numbers of Treg cells available in circulation, harvesting and culturing Treg cells remains a challenge. It has been reported that engineering Foxp3 expression in CD4+ T cells can result in a Treg-like phenotype; however, current methods result in the inefficient engineering of these cells. Here, we develop an ionizable lipid nanoparticle (LNP) platform to effectively deliver Foxp3 mRNA to CD4+ T cells. We successfully engineer CD4+ T cells into Foxp3-T (FP3T) cells that transiently exhibit an immunosuppressive phenotype and functionally suppress the proliferation of effector T cells. These results demonstrate the promise of an LNP platform for engineering immunosuppressive T cells with potential applications in autoimmunity therapies.

Ionizable Lipid Nanoparticles for Therapeutic Base Editing of Congenital Brain Disease.

Delivery of mRNA-based therapeutics to the perinatal brain holds great potential in treating congenital brain diseases. However, nonviral delivery platforms that facilitate nucleic acid delivery in this environment have yet to be rigorously studied. Here, we screen a diverse library of ionizable lipid nanoparticles (LNPs) via intracerebroventricular (ICV) injection in both fetal and neonatal mice and identify an LNP formulation with greater functional mRNA delivery in the perinatal brain than an FDA-approved industry standard LNP. Following in vitro optimization of the top-performing LNP (C3 LNP) for codelivery of an adenine base editing platform, we improve the biochemical phenotype of a lysosomal storage disease in the neonatal mouse brain, exhibit proof-of-principle mRNA brain transfection in vivo in a fetal nonhuman primate model, and demonstrate the translational potential of C3 LNPs ex vivo in human patient-derived brain tissues. These LNPs may provide a clinically translatable platform for in utero and postnatal mRNA therapies including gene editing in the brain.

A (Controlled) Spill of IL2 for Localized Treatment of Mesothelioma.

A microencapsulated, cell-based IL2 cytokine factory was recently developed, and the safety and efficacy of this platform in a mouse model of mesothelioma were demonstrated. This platform has the potential to overcome current challenges in the delivery of therapeutic cytokines for cancer immunotherapy. See related article by Nash et al., p. 5121.

Amniotic fluid stabilized lipid nanoparticles for in utero intra-amniotic mRNA delivery.

Congenital disorders resulting in pathological protein deficiencies are most often treated postnatally with protein or enzyme replacement therapies. However, treatment of these disorders in utero before irreversible disease onset could significantly minimize disease burden, morbidity, and mortality. One possible strategy for the prenatal treatment of congenital disorders is in utero delivery of messenger RNA (mRNA). mRNA is a nucleic acid therapeutic that has previously been investigated as a platform for protein replacement therapies and gene editing technologies. While viral vectors have been explored to induce intracellular expression of mRNA, they are limited in their clinical application due to risks associated with immunogenicity and genomic integration. As an alternative to viral vectors, safe and efficient in utero mRNA delivery can be achieved using ionizable lipid nanoparticles (LNPs). While LNPs have demonstrated potent in vivo mRNA delivery to the liver following intravenous administration, intra-amniotic delivery has the potential to deliver mRNA to cells and tissues beyond those in the liver, such as in the skin, lung, and digestive tract. However, LNP stability in fetal amniotic fluid and how this stability affects mRNA delivery has not been previously investigated. Here, we engineered a library of LNPs using orthogonal design of experiments (DOE) to evaluate how LNP structure affects their stability in amniotic fluid ex utero and whether a lead candidate identified from these stability measurements enables intra-amniotic mRNA delivery in utero. We used a combination of techniques including dynamic light scattering (DLS), transmission electron microscopy (TEM), and chromatography followed by protein content quantification to screen LNP stability in amniotic fluids. These results identified multiple lead LNP formulations that are highly stable in amniotic fluids ranging from small animals to humans, including mouse, sheep, pig, and human amniotic fluid samples. We then demonstrate that stable LNPs from the ex utero screen in mouse amniotic fluid enabled potent mRNA delivery in primary fetal lung fibroblasts and in utero following intra-amniotic injection in a murine model. This exploration of ex utero stability in amniotic fluids demonstrates a means by which to identify novel LNP formulations for prenatal treatment of congenital disorders via in utero mRNA delivery.

Delivery technologies for in utero gene therapy.

Advances in prenatal imaging, molecular diagnostic tools, and genetic screening have unlocked the possibility to treat congenital diseases in utero prior to the onset of clinical symptoms. While fetal surgery and in utero stem cell transplantation can be harnessed to treat specific structural birth defects and congenital hematological disorders, respectively, in utero gene therapy allows for phenotype correction of a wide range of genetic disorders within the womb. However, key challenges to realizing the broad potential of in utero gene therapy are biocompatibility and efficiency of intracellular delivery of transgenes. In this review, we outline the unique considerations to delivery of in utero gene therapy components and highlight advances in viral and non-viral delivery platforms that meet these challenges. We also discuss specialized delivery technologies for in utero gene editing and provide future directions to engineer novel delivery modalities for clinical translation of this promising therapeutic approach.

Phosphatase activity tunes two-component system sensor detection threshold.

Two-component systems (TCSs) are the largest family of multi-step signal transduction pathways in biology, and a major source of sensors for biotechnology. However, the input concentrations to which biosensors respond are often mismatched with application requirements. Here, we utilize a mathematical model to show that TCS detection thresholds increase with the phosphatase activity of the sensor histidine kinase. We experimentally validate this result in engineered Bacillus subtilis nitrate and E. coli aspartate TCS sensors by tuning their detection threshold up to two orders of magnitude. We go on to apply our TCS tuning method to recently described tetrathionate and thiosulfate sensors by mutating a widely conserved residue previously shown to impact phosphatase activity. Finally, we apply TCS tuning to engineer B. subtilis to sense and report a wide range of fertilizer concentrations in soil. This work will enable the engineering of tailor-made biosensors for diverse synthetic biology applications.

Size is an essential parameter in governing the UVB-protective efficacy of silver nanoparticles in human keratinocytes.

BACKGROUND: Ultraviolet (UV) radiation from sun, particularly its UVB component (290-320 nm), is considered the major etiological cause of skin cancer that impacts over 2 million lives in the United States alone. Recently, we reported that polydisperse colloidal suspension of silver nanoparticles (AgNPs) protected the human keratinocytes (HaCaT) against UVB-induced damage, thus indicating their potential for prevention of skin carcinogenesis. Here we sought out to investigate if size controlled the chemopreventive efficacy of AgNPs against UVB-induced DNA damage and apoptosis. METHODS: Percent cell viability was examined by WST-1 assay after treating the cells with various doses (1-10 µg/mL) of AgNPs of different sizes (10, 20, 40, 60 and 100 nm) for 12 and 24 h. For protection studies, cells were treated with AgNPs of different sizes at a uniform concentration of 1 µg/mL. After 3 h, cells were irradiated with UVB (40 mJ/cm(2)) and dot-blot analysis was performed to detect cyclobutane pyrimidine dimers (CPDs) as an indication of DNA damage. Apoptosis was analyzed by flow cytometry after staining the cells with 7-Amino-Actinomycin (7-AAD) and PE Annexin V. Immunoblot analysis was accomplished by processing the cells for protein extraction and Western blotting using specific antibodies against various proteins. RESULTS: The data show that the pretreatment of HaCaT cells with AgNPs in the size range of 10-40 nm were effective in protecting the skin cells from UVB radiation-induced DNA damage as validated by reduced amounts of CPDs, whereas no protection was observed with AgNPs of larger sizes (60 and 100 nm). Similarly, only smaller size AgNPs (10-40 nm) were effective in protecting the skin cells from UV radiation-induced apoptosis. At the molecular level, UVB -irradiation of HaCaT cells led to marked increase in expression of pro-apoptotic protein (Bax) and decrease in anti-apoptotic proteins (Bcl-2 and Bcl-xL), while it remained largely unaffected in skin cells pretreated with smaller size AgNPs (10-40 nm). CONCLUSIONS: Altogether, these findings suggest that size is a critical determinant of the UVB-protective efficacy of AgNPs in human keratinocytes.

Silver nanoparticles protect human keratinocytes against UVB radiation-induced DNA damage and apoptosis: potential for prevention of skin carcinogenesis.

Ultraviolet (UV)-B radiation from the sun is an established etiological cause of skin cancer, which afflicts more than a million lives each year in the United States alone. Here, we tested the chemopreventive efficacy of silver-nanoparticles (AgNPs) against UVB-irradiation-induced DNA damage and apoptosis in human immortalized keratinocytes (HaCaT). AgNPs were synthesized by reduction-chemistry and characterized for their physicochemical properties. AgNPs were well tolerated by HaCaT cells and their pretreatment protected them from UVB-irradiation-induced apoptosis along with significant reduction in cyclobutane-pyrimidine-dimer formation. Moreover, AgNPs pre-treatment led to G1-phase cell-cycle arrest in UVB-irradiated HaCaT cells. AgNPs were efficiently internalized in UVB-irradiated cells and localized into cytoplasmic and nuclear compartments. Furthermore, we observed an altered expression of various genes involved in cell-cycle, apoptosis and nucleotide-excision repair in HaCaT cells treated with AgNPs prior to UVB-irradiation. Together, these findings provide support for potential utility of AgNPs as novel chemopreventive agents against UVB-irradiation-induced skin carcinogenesis. FROM THE CLINICAL EDITOR: Excessive exposure to the sun is known to increase the risk of skin cancer due to DNA damage. In this work, the authors tested the use of silver nanoparticles as protective agents against ultraviolet radiation. The positive results may open a door for the use of silver nanoparticle as novel agents in the future.