ABSTRACT
Ionizable amino lipids are a major constituent of the lipid nanoparticles for delivering nucleic acid therapeutics (e.g., DLin-MC3-DMA in ONPATTRO , ALC-0315 in Comirnaty , SM-102 in Spikevax ). Scarcity of lipids that are suitable for cell therapy, vaccination, and gene therapies continue to be a problem in advancing many potential diagnostic/therapeutic/vaccine candidates to the clinic. Herein, we describe the development of novel ionizable lipids to be used as functional excipients for designing vehicles for nucleic acid therapeutics/vaccines in vivo or ex vivo use in cell therapy applications. We first studied the transfection efficiency (TE) of LNP-based mRNA formulations of these ionizable lipid candidates in primary human T cells and established a workflow for engineering of primary immune T cells. We then adapted this workflow towards bioengineering of CAR constructs to T cells towards non-viral CAR T therapy. Lipids were also tested in rodents for vaccine applications using self-amplifying RNA (saRNA) encoding various antigens. We have then evaluated various ionizable lipid candidates and their biodistribution along with the mRNA/DNA translation exploration using various LNP compositions. Further, using ionizable lipids from the library, we have shown gene editing of various targets in rodents. We believe that these studies will pave the path to the advancement in nucleic acid based therapeutics and vaccines, or cell gene therapy agents for early diagnosis and detection of cancer, and for targeted genomic medicines towards cancer treatment and diagnosis.
ABSTRACT
Background & Aim: The new UCOE models we have recently developed, tested on many cell groups (including mouse ES and human iPS cells) and human mAb recombinant production studies as well, shows a powerful resistance to DNA methylation- mediated silencing and provides a higher and stable transfection profile. By the urgent need of vaccine development for COVID-19 during the pandemic, in this study we aimed to produce a potential recombinant vaccine by using the new generation UCOEs models of our own design. Methods, Results & Conclusion(s): Existing new-generation UCOE models and standard plasmid vectors to be used as control group were provided. Then, the sequences related to the PCR method were amplified for sufficient stock generation and cloning experiments. Verification in the plasmid vector was carried out in gel electrophoresis. Transfection of 293T cells was performed with clone plasmids carrying antigen genes and plasmids carrying genetic information of lentivirus units for the production of lentiviral vectors. Afterwards, 293T cells produced lentiviral vectors carrying antigen genes. Harvesting of these vectors was carried out during 48th and 72nd hours. Afterwards, CHO cells were transduced with appropriate quantity of lentiviral vectors. Isolation and purification of targeted proteins from the relevant medium were performed by HPLC and Q-TOF methods. A part of the spike and nucleocapsid gene sequences of COVID-19 were firstly cloned into our UCOE models. These UCOEs plasmids were then transferred into 293T cells along with plasmids carrying the genes that will form the lentivirus vectors (LVs). After harvesting and calculation of LV vector titers, the cloned vectors were then transfected into the CHO cells which the targeted recombinant production of the antigen proteins will be carried out. Antigenic structures were then isolated from the culture medium of CHO cells in following days for confirmation. Using HPLC and qTOF mass spectrometer methods, these structures in the medium were confirmed to be the units of spike and nucleocapsid proteins of the COVID-19 virus. In order to produce large amount of the recombinant antigens, the culture was then carried out with bioreactors in liters. At the final stage, these recombinantly produced antigen proteins were tested on rats to measure their immunogenic responses, and the study recently been completed successfully as a potential recombinant vaccine against COVID-19.Copyright © 2023 International Society for Cell & Gene Therapy
ABSTRACT
Background: BST2/Tetherin is an interferon-stimulated gene with antiviral activity against enveloped viruses. Particularly, BST2 tethers virions at their site of assembly, preventing their release and spread. In addition to this primary role, BST2 is as an important bridge between the innate and adaptive immune system, since (i) BST2 routes tethered particles to lysosomes, which generates viral breakdown products that engage pattern recognition receptors;and (ii) trapped virions facilitate antibody-dependent cell-mediated cytotoxicity (ADCC). In turn, viruses have evolved mechanisms to bypass BST2, either by targeting BST2 for proteasomal/lysosomal degradation or by removing BST2 from sites of virion assembly. However, the role of BST2 in SARS-CoV-2 replication, spread, evolution, and pathogenesis remains largely unknown. Method(s): The antiviral potential of BST2 against SARS-CoV-2 was investigated by infecting different SARS-CoV-2 isolates (Hong Kong, Alpha, Beta, Delta, and Omicron) in BST2+ and BST2- cells. Culture supernatants were collected to assess virion production by ELISA and infectivity by TCID50. Infected cells were analyzed by western blot and flow cytometry to examine viral and cellular protein levels, including BST2. Transfection of individual SARS-CoV-2 ORFs and mutagenesis studies allowed us to identify the genes that the virus uses to downregulate BST2. Immunoprecipitation assays revealed protein-protein interactions and changes in ubiquitination patterns. Experiments with proteasomal and lysosomal inhibitors furthered our mechanistic understanding of how SARS-CoV-2 counteracts BST2. Finally, fluorescence microscopy studies uncovered changes in the subcellular distribution of BST2 in SARS-CoV-2 infected cells. Result(s): While BST2 reduces virion release, SARS-CoV-2 has evolved to counteract this effect. Specifically, SARS-CoV-2 uses the Spike to interact with BST2, sequester the protein at perinuclear locations, and ultimately route it for lysosomal degradation. By surveying different SARS-CoV-2 variants of concern (Alpha-Omicron), we found that each variant is more efficient than the previously circulating strain at downregulating BST2 and facilitating virion production, and that mutations in the Spike account for their enhanced BST2 antagonism. Conclusion(s): As part of its adaptation to humans, SARS-CoV-2 is improving its capacity to counteract BST2, highlighting that BST2 antagonism is important for SARS-CoV-2 infectivity and transmission.
ABSTRACT
Background: Next-generation SARS-CoV-2 vaccines demonstrated that nanoparticle messenger ribonucleic acid (mRNA) delivery is effective and safe for in vivo delivery in humans. Current treatments for cystic fibrosis (CF) primarily focus on modulator drug therapies designed to correct malfunctioning CF transmembrane conductance regulator (CFTR) protein, but these modulators are ineffective for the 10% of people with CF with variants that do not allow protein production. Among these is the splice variant 3120 + 1G >A, the most common CF-causing mutation in native Africans. Gene editing would allow production of CFTR protein and enhancement of function using available CFTR modulators. We have demonstrated that electroporation of a modified CRISPR-Cas9 base editor to primary human bronchial epithelial cells carrying 3120 + 1G >A and F508del mutant alleles achieved 75% genome editing of the splice variant, resulting in approximately 40% wild-type (WT) CFTR function [1]. Here,we evaluate the effectiveness of several new nanoparticle formulations at delivering green fluorescent protein (GFP) mRNA to CF bronchial epithelial (CFBE41o-) cells. Using the optimal formulation,we then tested the efficacy correction of the 3120 + 1G >Avariant in a CFTR expression minigene (EMG) integrated into the genome of isogenic CFBE cells using mRNA and plasmid deoxyribonucleic acid (DNA) encoding adenine base editor (ABE) and guide (g)RNA. Method(s): GFP served as a reporter to evaluate transfection efficiency, cell viability, and mean fluorescence intensity (MFI) for three dosages (150, 75, 32.5 ng of mRNA), four polymer-to-mRNA to weight (w/w) ratios (60, 40, 30, 20), and four polymers (R, Y, G, B). 7-AAD served as a live/dead stain to quantify viability, with flow cytometry results analyzed using FlowJo software. CFBE cells stably expressing the 3120 + 1G >A EMG were transfected with the optimized nanoparticle formulation to deliver ABE and gRNA at two dosages (150, 75 ng) of mRNA and DNA. CFTR function in CFBE cellswas measured by short circuit current, forskolin stimulation, and inh-172 inhibition as a measure of editing efficiency. Result(s): Flow cytometry showed that polymer R achieved more than 85% GFP transfection, compared with a maximum of approximately 35% for the other three polymers at the maximum 150-ng dose, with approximately 80% viability normalized to untreated cells. In addition, polymer R achieved GFP MFI more than one order of magnitude as high as other formulations (~30 000 vs 2700 MFI) for the other three polymers at 150-ng dose and 40 w/w ratio. CFBE cells transfected with polymer R nanoparticles containing ABE and guide RNA at 75 ng and 150 ng showed mean CFTR function increase to 10 muA 6 (standard error of the mean [SEM] 1.1 muA) (~10% of WT) and 6.3 muA (SEM 0.9 muA) (~6% of WT), respectively. Greater toxicity at the higher dose could explain the larger increase in CFTR current at the lower dose. DNA-encoded ABE plasmid and gRNA showed a less robust increase in CFTR function (2.9 muA [SEM 0.4 muA] for 75-ng dose;3.0 muA [SEM 0.4 muA] for 150-ng dose), which was probably a result of the nanoparticle formulation being optimized for RNA instead of DNA cargo or the additional intracellular barriers that must be overcome for successful DNA delivery. Conclusion(s): We demonstrated that an optimized nanoparticle formulation containing ABE and gRNA can correct splicing of isogenic cells bearing the 3120 + 1G >A CFTR variant, resulting in recovery of CFTR function. In ongoing work, we are adapting these nanoparticles for RNA- and DNAencoded ABE and gRNA delivery to primary human bronchial epithelial cells.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved
ABSTRACT
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the current global pandemic of the COVID-19, which has persisted partly through the emergence of new variants. A non-infectious, convenient, and reproducible in vitro system is needed to assess drug susceptibility of new variants of concern and potential drug resistance mutations. Method(s): The SARS-CoV-2 replicon protocol was adapted and optimized based on {Zhang 2021}. The replicon RNA was produced by in vitro transcription of full-length replicon DNA assembled by ligation of plasmid fragments encoding for the SARS-CoV-2 non-structural proteins (Nsps), nucleoprotein and gaussia luciferase reporter protein. Wild-type and mutant replicon RNAs were transfected into Huh7-1CN cells by electroporation and treated with remdesivir (RDV). To determine EC50 values, luciferase activity was determined at 48 hours post transfection. A recombinant SARS-CoV-2 virus rescue system {Xie 2020} was used to generate matching Nsp mutants for comparison with the replicon system. Result(s): The selected substitutions reflective of Omicron BA.5 sub-lineage BF.7 variant: the triple mutants (Nsp12 (P323L) +Nsp13 (R392C) + Nsp14 (I42V), and a single Nsp12 L247F mutant as well as several specific Nsp12 mutations identified by in vitro resistance selection with RDV or RDV parent nucleoside analog GS-441524 were cloned into the replicon and tested for susceptibility to RDV. RDV inhibited the SARS-CoV-2 wild-type replicon with a mean EC50 value of 14.7 +/- 3.5 nM (N=9). The Nsp12 P323L substitution, a common polymorphism in all major variants of concern including Omicron, was fully susceptible to RDV with a 0.6-fold change in EC50 from the wild-type. The Omicron BF.7 triple mutants and L247F were also fully susceptible to RDV with 0.5- and 0.4-fold changes, respectively. Nsp12 substitutions F480L, V557L, V792I, S759A+V792I, and C799F resulting from in vitro resistance selections with RDV showed minimal to moderate levels of reduced susceptibility to RDV (1.8 to 18.3-fold change) (Table 1). The RDV EC50 fold changes correlated between the noninfectious replicon and recombinant infection virus system (Table 1). Conclusion(s): The replicon system is a convenient and reproducible model to test the susceptibility of SARS-CoV-2 mutant variants to RDV and potentially other antivirals. The common Nsp12 polymorphisms in all variants including the highly transmissible Omicron variant were fully susceptible to RDV.
ABSTRACT
The year 2020 was the most challenging period due to the havoc caused by the outbreak of novel coronavirus SARS-CoV-2. Scientists and researchers all around the world have endeav-ored every possible approach to find solutions in context to therapeutics and vaccines to control the spread of this life-threatening virus. The acceleration instigated by the outbreak of SARS-CoV-2 and its mutated strains has leveraged the use of numerous platform technologies for the development of vaccines against this unfathomable disease. Vaccines could play an important role in miti-gating the effects of COVID-19 and reducing the ongoing health crisis. Various innovative plat-forms like proteins, nucleic acids, viruses, and viral vectors have been exploited to fabricate vaccines depicting almost 90% of efficacy like BNT162b2, AZD1222, Ad5-nCoV, etc. Some of these vaccines are multipotent and have shown potent activity against newly emerged malicious strains of SARS-CoV-2 like B.1.351 and B.1.1.7. In this review article, we have gathered key findings from various sources of recently popularized vaccine candidates, which will provide an overview of potential vaccine candidates against this virus and will help the researchers to investi-gate possible ways to annihilate this menace and design new moieties.Copyright © 2022 Bentham Science Publishers.
ABSTRACT
During the COVID-19 pandemics we have all witnessed the clinical importance of mRNA as current vaccines and future therapeutics. mRNA therapies have a potential to revolutionize cancer treatment. Delivery of mRNA requires lipid nanoparticles (LNP) to protect the cargo from degradation. mRNA has a negative charge and depends on positively charged lipids to be encapsulated in LNP. These lipids can be either ionizable at certain pH or constantly cationic. Even though previous studies had evaluated the formulation properties of ionizable and cationic LNP systems, there is the need to understand their specificity in terms of mRNA delivery and protein expression in breast cancer tumor microenvironment. The objective of this work was to assess the kinetics of LNP cellular uptake and mRNA expression inv breast cancer (BC) cells and fibroblasts, the most frequent cell type in the tumor microenvironment cells, while studying the mechanisms involved in differential behaviors of LNP formulated with cationic and ionizable lipids. To achieve this goal mRNA-LNP containing ionizable lipids (LNP-A) and cationic lipids (LNP-B) were designed and formulated using Nanoassemblr Benchtop microfluidics mixer (Precision NanoSystems). mRNA-LNP were characterized for size, zeta potential using dynamic light scattering (DLS) and mRNA encapsulation efficiency using RiboGreen assay. LNP were tagged with rhodamine lipid to investigate the uptake kinetic and a reporter GFP mRNA to evaluate mRNA expression in murine 4T1 and human MCF7, MDA-231, SUM-159 and T47D breast cancer cells and BJ fibroblasts. Live fluorescence microscopy imaging, IncuCyte S3, was used to determine the LNP uptake and GFP mRNA expression. In vitro biocompatibility was assessed with WST-1 assay. Additionally, expression of mRNA delivered from LNP in tumor microenvironment was evaluated in vivo in a syngeneic 4T1 breast cancer model using mRNA luciferase and IVIS imaging. mRNA-LNPs possessed an average diameter of 77 - 107 nm, narrow size distribution, neutral zeta potential and high mRNA encapsulation efficiency (>94%). Our results demonstrated that mRNA expression was higher in breast cancer cells when delivered from LNP-A formulation and in BJ fibroblasts when delivered from LNP-B. LNP-A, the ionizable LNP, was tested in the breast cancer cells to confirm the efficacy of the delivery. The highest transfection efficacy, from high to low, T-47D, MCF7, SUM-159, 4T1 and MDA-231.We have further investigated the cellular uptake mechanisms of LNP using uptake pathway inhibitors for caveolae endocytosis, clathrin endocytosis, and phagocytosis. Our data confirm that there are differences in mechanisms that govern the uptake of mRNA LNP in breast cancer cells and fibroblasts. Clathrin-mediated endocytosis was active in 4T1 breast cancer cells for ionizable and cationic LNP. Interestingly, despite in vitro differences in uptake and mRNA expression, in vivo results show that both formulations efficiently delivered luciferasemRNA in the tumor microenvironment. Histology results demonstrated similar luciferase expression for both LNP in tumors. Additionally, we were able to confirm the prominent presence of fibroblast and similar distribution in the 4T1 subcutaneous model which could explain the similar efficacy of cationic and ionizable LNP. Understanding uptake and mRNA expression of different LNP formulations in the tumor microenvironment can help in achieving the necessary protein expression for breast cancer therapies. Furthermore, determining the most efficient carrier in early stages may reduce the time required for clinical translation. Acknowledgement: This research was supported in part by CPRIT Core for RNA Therapeutics and Research.
ABSTRACT
SARS-CoV-2 protease Nsp3 is a therapeutic target for developing anti-SARS-CoV-2 drugs. Nsp3 is a large multi-spanning membrane protein, and its characterization in vitro has been challenging. Here we describe an in vitro assay to characterize the biochemical activity of full-length Nsp3 isolated from cells. The assay can be used to evaluate Nsp3 inhibitors. © 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
ABSTRACT
SARS-CoV-2 remains a significant public health threat, causing severe respiratory illness in susceptible individuals. Several effective Covid-19 vaccines have been developed but novel SARS-CoV-2 variants continuously emerge that are more transmissible and have potential to evade vaccine immune responses. We are developing a novel therapy that does not depend on an immune response, based on siRNA-mediated silencing of Angiotensin-converting enzyme 2 (ACE2) receptor and Transmembrane Serine Protease 2 (TMPRSS2). SARS-CoV-2 requires these host proteins to enter respiratory epithelial cells at the cell surface, through binding and priming of its Spike protein. As a cell model for SARS-CoV-2 infection, we have utilised primary nasal epithelial cells (NHNE), as well as HEK293T cells overexpressing ACE2 and TMPRSS2. siRNA transfection in NHNE cells led to a 78%-88% knockdown of ACE2 and TMPRSS2, as determined by qRT-PCR and western blot data. TMPRSS2 knockdown in the overexpressing HEK293T cells resulted in an 87% reduction in infectivity from SARS-CoV-2 Spike-pseudotyped lentiviruses expressing a luciferase transgene, indicative of a significant reduction in virus entry (p < 0.0001 by one-way ANOVA). We are now working to confirm these results with live SARS-CoV-2 and to test lipid nanoparticle delivery of the siRNAs to air-liquid interface grown NHNEs to more accurately model the respiratory airway. This siRNA approach could provide a novel therapy for immunocompromised individuals who do not gain sufficient protection from SARS-CoV-2 vaccines. Additionally, by targeting host proteins rather than virus components, our therapy is likely to remain effective in spite of emerging SARS-CoV-2 variants that circumvent pre-existing immune responses.
ABSTRACT
The COVID-19 crisis and the rapid development of highly effective mRNA vaccines opened a new era for gene therapy. While viral vectors were for a long time the only tool for efficient delivery, new non-viral vectors have recently emerged, spawning new opportunities (indications, tissues, etc.). A new one is set to take off thanks to its safety profile, its specificity toward tissues, and its versatility toward both genetic materials and indications. Gas-filled microbubbles (MB), clinically used as ultrasound (US) contrast agents, have proven their benefits in various animal models and clinical applications for targeted delivery of drugs/genes. Herein, we disclose the development of new MB formulations allowing the delivery of various genetic materials at a specific location under the control of an ultrasound probe. We set forth a study to elicit the expression of a foreign enzyme in a liver mouse model. To this aim, MB were systemically co-injected with a Luciferase pDNA (6 to 65 mug) in the tail vein, then Ultrasound were delivered at MB arrival in the liver. The effective pDNA transfection was observed by bioluminescence 24 hours after treatment. Mice were divided into three groups: pDNA alone;pDNA with US;pDNA with US and MBs (n >= 5). The use of our MB allowed increasing the signal up to 5 folds in comparison to the US alone. These results highlight the potential of MB plus US to efficiently deliver locally genetic material without any safety concerns.
ABSTRACT
Introduction: RNA-based therapies of the myocardium offer a range of therapeutic opportunities for conditions in which gene transfer needs to be highly effective but transient, such as cardiac regeneration and cardiac gene editing. MicroRNA and mRNA gene transfer can be achieved by intramyocardial injection of lipofectamine-based products, but these are not amenable to clinical translation. In contrast, lipid nanoparticles (LNPs) have already reached clinical approval for siRNA administration with patisiran in 2018 and for mRNA delivery with the COVID-19 vaccines by Pfizer-BioNTech and Moderna in 2020. Objective(s): We wanted to develop Stable Nucleic Acid Lipid nanoParticles (SNALPs) for the effective myocardial delivery of microRNAs and mRNAs. Material(s) and Method(s): We generated various SNALP formulations via ethanol injection method, carrying the pro-regenerative microRNA-199a-3p or GFP mRNA and assessed their transfection efficiency by high-content microscopy (stimulation of EdU incorporation and GFP fluorescence respectively) in neonatal rat and mouse cardiomyocytes and upon intramyocardial injection in mice. Result(s): We started by investigating the inclusion of different ionisable lipids, helper lipids and PEG-lipids in the nanoparticle shell. Then, we systematically tested the effect of varying the molar ratios of each of the constituents to improve transfection. After optimising the formulations in vitro, we tested the same in mice through direct intramyocardial injection. Conclusion(s): The results obtained highlight the applicability of the LNP technology for efficient delivery of mRNA and microRNA into cardiomyocytes. Copyright © 2022
ABSTRACT
Background Messenger ribonucleic acid (mRNA) is a powerful tool for transferring genetic information. Its advantages include potent but transient gene expression without risk of genomic insertion, tailorable immunogenicity to match therapeutic application, and the potential for efficient, scalable manufacturing.1 The recent success of mRNA-based SARSCoV- 2 vaccines has inspired interest in mRNA as a cancer therapy to deliver immunostimulatory molecules and tumor antigens. However, clinical translation is limited by mRNA instability at physiological conditions and inefficient in vivo delivery.2 A reliable, non-toxic, and stabilizing in vivo delivery system for immunotherapeutic mRNA would help to advance mRNA as a viable cancer therapy. Here, we utilized calcium phosphate mineral-coated microparticles (MCMs) as a delivery system for mRNA-lipid complexes (lipoplexes) to transfect melanoma cells. Methods MCMs were prepared as previously described3 by suspending beta-tricalcium phosphate particles in modified simulated body fluid under rotation for 7 days at 37degreeC, refreshing the media daily. MCMs were then washed in deionized water and freeze dried. Custom-synthesized reporter or therapeutic mRNA constructs were complexed with a lipidic transfecting agent through mixing, then resulting lipoplexes were incubated briefly with MCMs to facilitate electrostatic binding to the porous CaP coating (figure 1a). Loaded MCMs or soluble lipoplexes were added to B16F10 murine melanoma cell culture, and transfection was measured through various assays, including fluorescence microscopy, bioluminescence, and enzymelinked immunosorbent assays. Results Scanning electron microscopy was used to verify platelike, porous coating morphology following MCM fabrication (figure 1b). MCMs enhanced transfection of B16F10 melanoma cells compared to soluble mRNA lipoplex delivery. This was demonstrated with reporter constructs encoding enhanced green fluorescent protein (eGFP, figure 1c) and Gaussia luciferase (G-Luc), as well as with a therapeutic construct encoding interleukin 15 (IL-15), a T cell growth factor. Timelapse imaging also revealed more rapid transfection with MCMs. A close proximity of cells to MCMs was observed as necessary for transfection. Conclusions We demonstrated that MCMs efficiently and locally deliver mRNA lipoplexes to melanoma cells and cause elevated levels of protein expression compared to soluble lipoplex delivery. This enhanced delivery profile makes MCMs a potential drug delivery platform for future in vivo tumor studies and clinical translation. (Figure Presented).
ABSTRACT
Background: Platelets are transfused therapeutically for hemostasis, and are an integral part of hemorrhage management. However, transfusions can be ineffective in the most severe cases of hemorrhage. Platelets are also a potential cell therapy in other applications, but development has been hindered by inadequate methods to control which proteins are expressed by platelets. Currently, there are no methods to express exogenous proteins in transfusable platelets, which would expand their use to help treat the diseases they modulate. A method is therefore needed to modify transfusable platelets, and thus enhance their protein composition for specific applications. Aim(s): To produce engineered, transfusable platelets to enhance their natural coagulability and functional repertoire by directly transfecting donor-derived platelets with mRNA via lipid nanoparticle (LNP)-mediated delivery. The recent advances through the COVID-19 mRNA vaccines demonstrates the clinical safety and efficacy of LNP-mediated gene therapy, and thus offers a promising strategy to effectively engineer modified platelets. Method(s): Donor-derived platelets were washed and subsequently incubated with a systematic array of LNPs encapsulating Cy5-labeled mRNA encoding for nanoluciferase in comparison to commercial transfection reagents. LNP uptake and platelet activation via CD62p levels was assessed following 4 hours by flow cytometry, while luciferase expression was assessed by normalizing the luminescence intensity to the total protein content. Result(s): Platelets took up the mRNA through all conditions tested;nanoluciferase was only expressed, however, in platelets treated with LNPs and not commercial reagents. Systematically optimizing LNPs increased nanoluciferase expression nine-fold relative to pre-optimized LNPs. Exogenous protein expression did not appear to correlate with mRNA uptake nor platelet activation. Conclusion(s): Platelets transfected with LNPs can express exogenous protein. Further optimization can eventually lead to the creation of a platform technology that in the long-term will allow platelets to deliver therapeutic proteins and yield more effective platelet products.
ABSTRACT
Introduction: Ischemic necrosis of dermal flaps is a devastating complication of reconstructive surgery. The increasing prevalence of diabetes, obesity, and an aging population adds to this concern. Hypoxia-inducible factor-1alpha (HIF-1alpha), a master regulator of the adaptive response to hypoxia, controls the expression of angiogenic growth factors. The development of biologically active, gene-specific mRNAs, especially in COVID-19 vaccines, has shown the ability for intracellular protein expression. We sought to express HIF-1alpha through mRNA transfection and determined its biological activity by measuring the upregulation of selected downstream targets. Method(s): 5'-methyl-capped poly-A tailed mRNA was generated using T7 RNA polymerase and verified by gel electrophoresis. Predominant and variant HIF-1alpha mRNA were transfected into primary human dermal fibroblasts via Lipofectamine in triplicate, and RNA levels were assessed using RT-qPCR. All gene expression levels were normalized to beta-actin expression levels Results: At one day after transfection, the levels of HIF-1alpha transcript were significantly higher in the cells transfected with predominant (p = 0.0104) and variant (p = 0.0007) HIF-1alpha transcripts relative to the control. Additionally, the expression of HIF-1alpha transcription product genes VEGF (p = 0.0274) and ANG-1 (p = 0.05) were significantly higher in the cells transfected with the HIF-1alpha transcripts than the control. Conclusion(s): Our approach led to the successful transfection of HIF-1alpha mRNA into human fibroblasts, resulting in upregulation of HIF-1alpha downstream angiogenic targets. Thus, the use of biologically active HIF-1alpha mRNA transfection offers a promising approach to inhibit ischemic necrosis.
ABSTRACT
Purpose/Objectives: The delivery of nucleic acids to cells has revolutionized medicine and enabled new technologies such as mRNA vaccines and stem cell therapies. These recent advances rely on delivery vehicles to stabilize the genetic payload and increase cellular transfection. While engineered viruses are efficient vectors for ex vivo cellular reprogramming, they are not ideal for in vivo gene therapies as repeated dosing leads to anti-vector immunity. Lipid nanoparticles have thus emerged as the best alternative to viral vectors for in vivo nucleic acid delivery. However, all FDA-approved lipid nanoparticles have been linked to inflammatory responses, undesirable for regenerative medicine applications that require precise immunomodulation. Thus, non-immunogenic delivery materials must be developed to fulfill the immense potential of gene therapy in regenerative medicine. Lipid nanoparticles typically comprise 4 different lipids, with the ionizable amino lipid being the main driver of potency and immunogenicity. A way to reduce immunogenicity is to develop lipid nanoparticles that minimize the amount of lipids per gram of nucleic acids. To do so, we developed a novel class of ionizable amino lipids with high charge density. Our primary objective is to design a lipid nanoparticle that maximizes RNA delivery and minimizes immunogenicity. Methodology: We designed a library of proprietary ionizable lipids based on the structure of a poly(amido amine) dendron. The structure is modular, which allowed us to systematically vary molecular motifs to optimize important physiochemical parameters: Lipid-to-RNA ratio;apparent pKa;surface zeta potential;size distribution;and RNA encapsulation These structures are also designed to include a higher number of amines compared to current ionizable lipids. This improves ionization charge density of the lipid and lowers the amount of lipid required to encapsulate RNA. In this study, lipid nanoparticles contain an ionizable lipid selected from our library, cholesterol, a phospholipid, and a PEG-lipid. The lipids and formulation conditions were selected to mimic Moderna's COVID-19 vaccine (SpikeVax), albeit with different lipid-to-RNA ratios. C57BL/6 mice were injected intramuscularly with nanoparticles co-formulated with a firefly luciferase mRNA and ovalbumin mRNA to simultaneously study transfection efficiency and antigen-specific immune responses. Nanoparticles that comprise SM-102, the ionizable lipid used in SpikeVax, were used as a comparative control due to their high potency and immunogenicity. Luciferase activity was detected using an IVIS Spectrum, and key organs were harvested for immune phenotyping. Results: We have so far determined the effect of hydrophobic motifs on apparent pKa and RNA encapsulation. Our best lipids with optimized tails did not induce IFN-I responses in vitro and demonstrated comparable in vivo efficacy to SM-102. We are currently in the process of collecting immunogenicity data which we expect to complete prior to the conference. Conclusion/Significance: We have produced a novel set of lipid nanoparticles that efficiently transfect cells in vivo. These new particles deliver RNA with half of the lipid mass used in SpikeVax, which can reduce the amount of material-induced immunogenicity. This result opens the door to developing mRNA vaccines with fewer side effects and equitable gene therapies for untreatable diseases such as inflammatory and autoimmune disorders.
ABSTRACT
Myocardial infarction is a global health burden for which there is no treatment available that aims to recover the damaged tissue after the ischemic event. Lipid nanoparticles (LNPs) represent a well characterized class of mRNA delivery systems, which were recently approved for clinical usage in their application for mRNA-based covid-19 vaccines. After myocardial infarction, endogenous mechanisms that enable repair of the functional damaged tissue can be triggered by modified mRNA (modRNA) delivery, locally in the infarcted area. As a first step, in order to optimize the LNP formulation for effective myocardial delivery and study cellular tropism of the LNPs in the heart, different LNPs formulations will be evaluated as delivery systems in a murine healthy heart model. Different LNP formulations varying in type and amount of helper lipid were used as delivery systems for modRNA encoding the reporter genes luciferase or eGFP. In vitro, LNPs were evaluated for modRNA delivery in a human endothelial cell line (HMEC-1), induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) and induced pluripotent stem cell -derived fibroblasts (iPS-FBs). In vivo, modRNA delivery was evaluated in C57BL-6 mice, undergoing open chest heart surgery under general anaesthesia in order to infuse LNPs into the left ventricular wall. For determination of luciferase expression levels, animals were infused with luciferin substrate intraperitoneally 24 hrs after injection. Heart, liver, lungs, spleen and kidneys were extracted for imaging in a bioluminescence imaging system. The organs were then stored in liquid nitrogen for further ex-vivo modRNA delivery analysis. For determining cellular tropism, histology was performed on mice treated with eGFP modRNA. Both bioluminescence imaging and luminescence analysis in tissue lysates showed that mRNA transfection is achieved in the myocardium 24 hours after LNP intramyocardial administration. However, all LNP formulations also resulted in high expression levels in other organs, including liver and spleen. Changes in type or amount of helper lipid in LNPs strongly affected transfection levels. Histology of the treated hearts revealed a distinct transfection pattern. The targeted, interstitial cells were negative for CD31 (marker for endothelial cells and monocytes) and Troponin I3 (marker for cardiomyocytes) (Figure 1). We show that, using an optimized LNP formulation, a significant degree of modRNA local transfection of the heart can be achieved. However, despite the local route of administration (into the left ventricular wall), the highest LNP transfection is shown in remote organs such as liver and spleen. More improvements of the LNP formulations must be done to increase their tropism towards the heart tissue for their optimization as cardiac delivery systems. Determining which cell types are being targeted is also important in order to establish a therapeutic target when applying the LNPs for cardiac therapy. (Figure Presented).
ABSTRACT
The current state of pulmonary vaccine delivery will be discussed in this review. The prospects for lung immunization using dry powder generation technologies and specialized medicinal formulations are discussed. In terms of vaccine durability and antigenicity, dry powder vaccine generation technologies may be advantageous. The non-invasive, reasonably safe, and low-cost nature of pulmonary delivery could help the public health vaccination significantly. The vaccines, which are all given intramuscularly, produce systemic antibodies in the blood but not antibodies in the pulmonary mucosal lining. Inhalation vaccines provide a number of potential benefits over injectable vaccines, including ease of delivery, and even self-administration. To create a dry powder inhalation formulation that is breathable and mediates robust transfection in the lung, a safe and effective mRNA delivery vector as well as a suitable particle engineering approach is needed.
ABSTRACT
Introduction: COVID-19 severe pneumonia has been associated to systemic inflammation and elevation of blood parameters and reminiscent of cytokine storm syndrome. Stimulation of PBMC from patients with severe COVID-19 have shown a high secretion of IL-1β, a pivotal cytokine driving inflammatory phenotypes, which maturation and secretion is regulated by NLRP3 inflammasome. Steroidal anti-inflammatory therapies have shown efficacy in reducing mortality in critically ill patients, however the mechanisms by which SARS-CoV2 virus triggers such an extensive inflammation remain unexplained. Objectives: The overall objective of this study was to investigate if SARS-CoV2 drives inflammation in COVID-19 patients through NLRP3 inflammasome activation and IL-1β secretion. Methods: Samples from SARS-CoV2 infected patients, were collected at day 0 and at 3 and 7 following treatment with anakinra. Fresh monocytes, purified through adherence, were cultured for 3, 6, 18 h in the presence or absence of LPS (100 ng/ml) and MCC950 (10μM). Release of IL-1β, IL-1Ra, IL-6, TNF-α, IL-18 was quantified by ELISA kit. Relative gene expression analysis of ORF3a gene was performed by RT-qPCR. THP-1 cells were transfected with a plasmid containing ORF3a sequence by nucleofection. NLRP3 inflammasome and ASC speck formation were detected by confocal microscopy and/or by FACS analysis. Results: In the present study we show that circulating monocytes from COVID-19 patients display ASC specks, index of NLRP3 activation, and spontaneously secrete IL-1β in vitro. This spontaneous activation reverts following patient's treatment with the IL-1 receptor antagonist anakinra. Transfection of a monocytic cell line with cDNA coding for the ORF3a SARS-CoV2 protein, resulted in NLRP3- dependent ASC speck formation. The involvement of ORF3a in inflammasome activation was further supported by the detection by RT-PCR of ORF3a in monocytes from COVID-19 patients. Conclusion: In summary, these results provide a mechanistic explanation for the strong inflammatory manifestations associated to COVID-19 and further evidence that NLRP3 and IL-1β targeting could represent an effective strategy in this disease.
ABSTRACT
In this research, the messenger ribonucleic acid vaccine has proven to be the best vaccine among all others against the Coronavirus disease of 2019, such that the Food and Drug Administration recently gave it full approval. This approval is made possible due to its clinical efficacy, the rare occasion of insertional mutagenesis associated with it, and the absence of anti-vector immunity. Also, its rapid production process gives it an added advantage over conventional DNA vaccines. A literature review was carried out based on available literature online. This review elaborates on the steps involved in the in vitro generation of the messenger ribonucleic acid vaccine. An example is made using a plasmid that contains both the receptor-binding domain found in the spike protein of the Coronavirus and the super folder green fluorescent protein. The use of a viral vector in making these vaccines is also added as a note for additional knowledge.