bims-novged 2024-04-28 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2024‒04‒28
twelve papers selected by
the Merkel lab, Ludwig-Maximilians University

Modulating the Nature of Ionizable Lipids and Number of Layers in Hyaluronan-Decorated Lipid Nanoparticles for In Vitro Delivery of RNAi.
Fine tuning of the net charge alternation of polyzwitterion surfaced lipid nanoparticles to enhance cellular uptake and membrane fusion potential.
Lipo-Xenopeptide Polyplexes for CRISPR/Cas9 based Gene editing at ultra-low dose.
Tumor-Tailored Ionizable Lipid Nanoparticles Facilitate IL-12 Circular RNA Delivery for Enhanced Lung Cancer Immunotherapy.
Data-balanced transformer for accelerated ionizable lipid nanoparticles screening in mRNA delivery.
Lipid Nanoparticle-Associated Inflammation is Triggered by Sensing of Endosomal Damage: Engineering Endosomal Escape Without Side Effects.
Engineering pH-sensitive dissolution of lipid-polymer nanoparticles by Eudragit integration impacts plasmid DNA (pDNA) transfection.
Superior COL7A1 and TGM1 gene expression in difficult-to-transfect skin cell mediated by highly branched poly(β-amino esters) through stepwise fractionation.
Codelivery of CPT and siPHB1 with GSH/ROS Dual-Responsive Hybrid Nanoparticles Based on a [12]aneN3-Derived Lipid for Synergistic Lung Cancer Therapy.
Design and Characterization of a New Formulation for the Delivery of COVID-19-mRNA Vaccine to the Nasal Mucosa.
Major Role of S-Glycoprotein in Providing Immunogenicity and Protective Immunity in mRNA Lipid Nanoparticle Vaccines Based on SARS-CoV-2 Structural Proteins.
Machine learning methods for predicting guide RNA effects in CRISPR epigenome editing experiments.

Pharmaceutics. 2024 Apr 20. pii: 563. [Epub ahead of print]16(4):

Modulating the Nature of Ionizable Lipids and Number of Layers in Hyaluronan-Decorated Lipid Nanoparticles for In Vitro Delivery of RNAi.

Victor Passos Gibson, Houda Tahiri, Claudia Gilbert, Chun Yang, Quoc Thang Phan, Xavier Banquy, Pierre Hardy.

  Lipid nanoparticles (LNPs) have established their position as nonviral vectors for gene therapy. Tremendous efforts have been made to modulate the properties of LNPs to unleash their full clinical potential. Among the strategies being pursued, the layer-by-layer (LbL) technique has gained considerable attention in the biomedical field. Illuminated by our previous work, here we investigate if the LbL approach could be used to modify the LNP cores formulated with three different ionizable lipids: DODMA, MC3, and DODAP. Additionally, we wondered if more than three layers could be loaded onto LNPs without disrupting their gene transfection ability. Taking advantage of physicochemical analysis, as well as uptake and gene silencing studies, we demonstrate the feasibility of modifying the surface of LNPs with the LbL assembly. Precisely, we successfully modified three different LNPs using the layer-by-layer strategy which abrogated luciferase activity in vitro. Additionally, we constructed a 5×-layered HA-LNP containing the MC3 ionizable lipid which outperformed the 3×-layered counterpart in transfecting miRNA-181-5p to the pediatric GBM cell line, as a proof-of-concept in vitro experiment. The method used herein has been proven reproducible, of easy modification to adapt to different ionizable lipid-containing LNPs, and holds great potential for the translation of RNA-based therapeutic strategies.

Keywords:  glioblastoma; layer-by-layer; lipid nanoparticles; siRNA; surface modification

DOI:  https://doi.org/10.3390/pharmaceutics16040563
Sci Technol Adv Mater. 2024 ;25(1): 2338785

Fine tuning of the net charge alternation of polyzwitterion surfaced lipid nanoparticles to enhance cellular uptake and membrane fusion potential.

Keitaro Homma, Yutaka Miura, Motoaki Kobayashi, Wanphiwat Chintrakulchai, Masahiro Toyoda, Koichi Ogi, Junya Michinishi, Tomoyuki Ohtake, Yuto Honda, Takahiro Nomoto, Hiroyasu Takemoto, Nobuhiro Nishiyama.

  Lipid nanoparticles (LNPs) coated with functional and biocompatible polymers have been widely used as carriers to deliver oligonucleotide and messenger RNA therapeutics to treat diseases. Poly(ethylene glycol) (PEG) is a representative material used for the surface coating, but the PEG surface-coated LNPs often have reduced cellular uptake efficiency and pharmacological activity. Here, we demonstrate the effect of pH-responsive ethylenediamine-based polycarboxybetaines with different molecular weights as an alternative structural component to PEG for the coating of LNPs. We found that appropriate tuning of the molecular weight around polycarboxybetaine-modified LNP, which incorporated small interfering RNA, could enhance the cellular uptake and membrane fusion potential in cancerous pH condition, thereby facilitating the gene silencing effect. This study demonstrates the importance of the design and molecular length of polymers on the LNP surface to provide effective drug delivery to cancer cells.

Keywords:  Lipid nanoparticles; membrane fusion; pH-responsiveness; polycarboxybetaine; siRNA

DOI:  https://doi.org/10.1080/14686996.2024.2338785
J Control Release. 2024 Apr 23. pii: S0168-3659(24)00266-9. [Epub ahead of print]

Lipo-Xenopeptide Polyplexes for CRISPR/Cas9 based Gene editing at ultra-low dose.

Janin Germer, Anna-Lina Lessl, Jana Pöhmerer, Melina Grau, Eric Weidinger, Miriam Höhn, Mina Yazdi, Martino Alfredo Cappelluti, Angelo Lombardo, Ulrich Lächelt, Ernst Wagner.

  Double pH-responsive xenopeptide carriers containing succinoyl tetraethylene pentamine (Stp) and lipo amino fatty acids (LAFs) were evaluated for CRISPR/Cas9 based genome editing. Different carrier topologies, variation of LAF/Stp ratios and LAF types as Cas9 mRNA/sgRNA polyplexes were screened in three different reporter cell lines using three different genomic targets (Pcsk9, eGFP, mdx exon 23). One U-shaped and three bundle (B2)-shaped lipo-xenopeptides exhibiting remarkable efficiencies were identified. Genome editing potency of top carriers were observed at sub-nanomolar EC50 concentrations of 0.4 nM sgRNA and 0.1 nM sgRNA for the top U-shape and top B2 carriers, respectively, even after incubation in full (≥ 90%) serum. Polyplexes co-delivering Cas9 mRNA/sgRNA with a single stranded DNA template for homology directed gene editing resulted in up to 38% conversion of eGFP to BFP in reporter cells. Top carriers were formulated as polyplexes or lipid nanoparticles (LNPs) for subsequent in vivo administration. Formulations displayed long-term physicochemical and functional stability upon storage at 4 °C. Importantly, intravenous administration of polyplexes or LNPs mediated in vivo editing of the dystrophin gene, triggering mRNA exon 23 splicing modulation in dystrophin-expressing cardiac muscle, skeletal muscle and brain tissue.

Keywords:  CRISPR Cas9; Delivery; Genome editing; Lipid nanoparticle; Polyplexes; Xenopeptide; mRNA

DOI:  https://doi.org/10.1016/j.jconrel.2024.04.037
Adv Mater. 2024 Apr 24. e2400307

Tumor-Tailored Ionizable Lipid Nanoparticles Facilitate IL-12 Circular RNA Delivery for Enhanced Lung Cancer Immunotherapy.

Shufen Xu, Yue Xu, Nicholas C Solek, Jingan Chen, Fanglin Gong, Andrew James Varley, Alex Golubovic, Anni Pan, Songtao Dong, Gang Zheng, Bowen Li.

  The advancement of mRNA-based immunotherapies for cancer is highly dependent on the effective delivery of RNA payloads using ionizable lipid nanoparticles (LNPs). However, the clinical application of these therapies is hindered by variable mRNA expression among different cancer types and the risk of systemic toxicity. The transient expression profile of mRNA further complicates this issue, necessitating frequent dosing and thus increasing the potential for adverse effects. Addressing these challenges, we utilized a high-throughput combinatorial method to synthesize and screen LNPs that efficiently deliver circular RNA (circRNA) to lung tumors. Our lead LNP, H1L1A1B3, demonstrated a fourfold increase in circRNA transfection efficiency in lung cancer cells over ALC-0315, the industry-standard LNPs, while providing potent immune activation. A single intratumoral injection of H1L1A1B3 LNPs, loaded with circRNA encoding interleukin-12 (IL-12), induced a robust immune response in a Lewis lung carcinoma model, leading to marked tumor regression. Immunological profiling of treated tumors revealed substantial increments in CD45+ leukocytes and enhanced infiltration of CD8+ T cells, underscoring the ability of H1L1A1B3 LNPs to modulate the tumor microenvironment favorably. These results highlight the potential of tailored LNP platforms to advance RNA drug delivery for cancer therapy, broadening the prospects for RNA immunotherapeutics. This article is protected by copyright. All rights reserved.

Keywords:  Ionizable lipid; cancer immunotherapy; lipid nanoparticles; rna delivery

DOI:  https://doi.org/10.1002/adma.202400307
Brief Bioinform. 2024 Mar 27. pii: bbae186. [Epub ahead of print]25(3):

Data-balanced transformer for accelerated ionizable lipid nanoparticles screening in mRNA delivery.

Kun Wu, Xiulong Yang, Zixu Wang, Na Li, Jialu Zhang, Lizhuang Liu.

  Despite the widespread use of ionizable lipid nanoparticles (LNPs) in clinical applications for messenger RNA (mRNA) delivery, the mRNA drug delivery system faces an efficient challenge in the screening of LNPs. Traditional screening methods often require a substantial amount of experimental time and incur high research and development costs. To accelerate the early development stage of LNPs, we propose TransLNP, a transformer-based transfection prediction model designed to aid in the selection of LNPs for mRNA drug delivery systems. TransLNP uses two types of molecular information to perceive the relationship between structure and transfection efficiency: coarse-grained atomic sequence information and fine-grained atomic spatial relationship information. Due to the scarcity of existing LNPs experimental data, we find that pretraining the molecular model is crucial for better understanding the task of predicting LNPs properties, which is achieved through reconstructing atomic 3D coordinates and masking atom predictions. In addition, the issue of data imbalance is particularly prominent in the real-world exploration of LNPs. We introduce the BalMol block to solve this problem by smoothing the distribution of labels and molecular features. Our approach outperforms state-of-the-art works in transfection property prediction under both random and scaffold data splitting. Additionally, we establish a relationship between molecular structural similarity and transfection differences, selecting 4267 pairs of molecular transfection cliffs, which are pairs of molecules that exhibit high structural similarity but significant differences in transfection efficiency, thereby revealing the primary source of prediction errors. The code, model and data are made publicly available at https://github.com/wklix/TransLNP.

Keywords:  data imbalance; ionizable lipid nanoparticles; transfection cliffs

DOI:  https://doi.org/10.1093/bib/bbae186
bioRxiv. 2024 Apr 18. pii: 2024.04.16.589801. [Epub ahead of print]

Lipid Nanoparticle-Associated Inflammation is Triggered by Sensing of Endosomal Damage: Engineering Endosomal Escape Without Side Effects.

Serena Omo-Lamai, Yufei Wang, Manthan N Patel, Eno-Obong Essien, Mengwen Shen, Aparajeeta Majumdar, Carolann Espy, Jichuan Wu, Breana Channer, Michael Tobin, Shruthi Murali, Tyler E Papp, Rhea Maheshwari, Liuqian Wang, Liam S Chase, Marco E Zamora, Mariah L Arral, Oscar A Marcos-Contreras, Jacob W Myerson, Christopher A Hunter, Andrew Tsourkas, Vladimir Muzykantov, Igor Brodsky, Sunny Shin, Kathryn A Whitehead, Peter Gaskill, Dennis Discher, Hamideh Parhiz, Jacob S Brenner.

Lipid nanoparticles (LNPs) have emerged as the dominant platform for RNA delivery, based on their success in the COVID-19 vaccines and late-stage clinical studies in other indications. However, we and others have shown that LNPs induce severe inflammation, and massively aggravate pre-existing inflammation. Here, using structure-function screening of lipids and analyses of signaling pathways, we elucidate the mechanisms of LNP-associated inflammation and demonstrate solutions. We show that LNPs' hallmark feature, endosomal escape, which is necessary for RNA expression, also directly triggers inflammation by causing endosomal membrane damage. Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which bind to sugars on the inner endosomal membrane and then regulate downstream inflammation. We find that inhibition of galectins abrogates LNP-associated inflammation, both in vitro and in vivo . We show that rapidly biodegradable ionizable lipids can preferentially create endosomal holes that are smaller in size and reparable by the endosomal sorting complex required for transport (ESCRT) pathway. Ionizable lipids producing such ESCRT-recruiting endosomal holes can produce high expression from cargo mRNA with minimal inflammation. Finally, we show that both routes to non-inflammatory LNPs, either galectin inhibition or ESCRT-recruiting ionizable lipids, are compatible with therapeutic mRNAs that ameliorate inflammation in disease models. LNPs without galectin inhibition or biodegradable ionizable lipids lead to severe exacerbation of inflammation in these models. In summary, endosomal escape induces endosomal membrane damage that can lead to inflammation. However, the inflammation can be controlled by inhibiting galectins (large hole detectors) or by using biodegradable lipids, which create smaller holes that are reparable by the ESCRT pathway. These strategies should lead to generally safer LNPs that can be used to treat inflammatory diseases.

DOI: https://doi.org/10.1101/2024.04.16.589801
Eur J Pharm Biopharm. 2024 Apr 19. pii: S0939-6411(24)00125-5. [Epub ahead of print] 114299

Engineering pH-sensitive dissolution of lipid-polymer nanoparticles by Eudragit integration impacts plasmid DNA (pDNA) transfection.

Diviya Santhanes, Huiming Zhang, Alex Wilkins, Robert John Aitken, Anne-Louise Gannon, Mingtao Liang.

  Lipid-polymer nanoparticles offer a promising strategy for improving gene nanomedicines by combining the benefits of biocompatibility and stability associated with the individual systems. However, research to date has focused on poly-lactic-co-glycolic acid (PLGA) and resulted in inefficient transfection. In this study, biocompatible Eudragit constructs E100 and RS100 were formulated as lipid-polymer nanoparticles loaded with pDNA expressing red fluorescent protein (RFP) as a model therapeutic. Using a facile nanoprecipitation technique, a core-shell structure stabilised by lipid-polyethylene glycol (PEG) surfactant was produced and displayed resistance to ultracentrifugation. Both cationic polymers E100 (pH-sensitive dissolution at 5) and RS100 (pH-insensitive dissolution) produced 150-200 nm sized particles with a small positive surface charge (+3-5 mV) and high pDNA encapsulation efficiencies (EE) of 75-90 %. The dissolution properties of the Eudragit polymers significantly impacted the biological performance in human embryonic kidney cells (HEK293T). Nanoparticles composed of polymer RS100 resulted in consistently high cell viability (80-100 %), whereas polymer E100 demonstrated dose-dependent behaviour (20-90 % cell viability). The low dissolution of polymer RS100 over the full pH range and the resulting nanoparticles failed to induce RFP expression in HEK293T cells. In contrast, polymer E100-constructed nanoparticles resulted in reproducible and gradually increasing RFP expression of 26-42 % at 48-72 h. Intraperitoneal (IP) injection of the polymer E100-based nanoparticles in C57BL/6 mice resulted in targeted RFP expression in mouse testes with favourable biocompatibility one-week post-administration. These findings predicate Eudragit based lipid-polymer nanoparticles as a novel and effective carrier for nucleic acids, which could facilitate pre-clinical evaluation and translation of gene nanomedicines.

Keywords:  Eudragit; Gene transfection; Lipid-polymer nanoparticles; Nanoprecipitation; plasmid DNA (pDNA)

DOI:  https://doi.org/10.1016/j.ejpb.2024.114299
J Control Release. 2024 Apr 23. pii: S0168-3659(24)00251-7. [Epub ahead of print]370 82-94

Superior COL7A1 and TGM1 gene expression in difficult-to-transfect skin cell mediated by highly branched poly(β-amino esters) through stepwise fractionation.

Chaolan Pan, Chenfei Wang, Yitong Zhao, Tao Bo, Liping Han, Dingjin Yao, Yumeng Wang, Xiaoxiao Wang, Linjing Shi, Anqi Zhao, Qiaoyu Cao, Fuying Chen, Wei He, Ying Ye, Si Zhang, Ming Li.

  Delivering functional gene into targeted skin cells or tissues to modulate the genes expression, has the potential to treat various hereditary cutaneous disorders. Nevertheless, the lack of safe and effective gene delivery vehicles greatly limits the clinical translation of gene therapy for inherited skin diseases. Herein, we developed a facile elution fractionation strategy to isolate eight HPAEs with Mw ranging from 7.6 to 131.8 kg/mol and Đ < 2.0 from the one crude HPAE23.7k, and investigated the expression efficiency for TGM1 and COL7A1 plasmids. Gene transfection results revealed that the intermediate MW HPAEs, HPAE20.6k, exhibited the highest gene transfection efficiency (46.4%) and the strongest mean fluorescence intensity (143,032 RLU), compared to other isolated components and the crude product. Importantly, best-performing isolated HPAE effectively delivered COL7A1 (15,974 bp) and TGM1 (7181 bp) plasmids, promoting the efficient expression of type VII collagen (C7) and transglutaminase-1 proteins in cutaneous cells. Our study establishes a straightforward step-by-step elution fractionation strategy for the development of HPAEs gene delivery vectors, expediting their clinical translation in inherited skin diseases.

Keywords:  Branched poly(β-amino ester)s; COL7A1 and TGM1 gene expression; Gene therapy; Gene transfection; Stepwise fractionation

DOI:  https://doi.org/10.1016/j.jconrel.2024.04.030
ACS Appl Bio Mater. 2024 Apr 23.

Codelivery of CPT and siPHB1 with GSH/ROS Dual-Responsive Hybrid Nanoparticles Based on a [12]aneN3-Derived Lipid for Synergistic Lung Cancer Therapy.

Ya-Xuan Liang, Xue-Yi Sun, De-Zhong Xu, Yi-Nan Gao, Quan Tang, Zhong-Lin Lu, Yang Liu.

  The combination of small-interfering RNA (siRNA)-mediated gene silencing and chemotherapeutic agents for lung cancer treatment has attracted widespread attention in terms of a greater therapeutic effect, minimization of systemic toxicity, and inhibition of multiple drug resistance (MDR). In this work, three amphiphiles, CBN1-CBN3, were first designed and synthesized as a camptothecin (CPT) conjugate and gene condensation agents by the combination of CPT prodrugs and di(triazole-[12]aneN3) through the ROS-responsive phenylborate ester and different lengths of alkyl chains (with 6, 9, 12 carbon chains for CBN1-CBN3, respectively). CBN1-CBN3 were able to be self-assembled into liposomes with an average diameter in the range of 320-240 nm, showing the ability to effectively condense siRNA. Among them, CBN2, with a nine-carbon alkyl chain, displayed the best anticancer efficiency in A549 cells. In order to give nanomedicines a stealth property and PEGylation/dePEGylation transition, a GSH-responsive PEGylated TPE derivative containing a disulfide linkage (TSP) was further designed and prepared. A combination of CBN2/siRNA complexes and DOPE with TSP resulted in GSH/ROS dual-responsive lipid-polymer hybrid nanoparticles (CBN2-DP/siRNA NPs). In present GSH and H2O2, CBN2-DP/siRNA NPs were decomposed, resulting in the controlled release of CPT drug and siRNA. In vitro, CBN2-DP/siPHB1 NPs showed the best anticancer activity for suppression of about 75% of A549 cell proliferation in a serum medium. The stability of CBN2-DP/siRNA NPs was significantly prolonged in blood circulation, and they showed effective accumulation in the A549 tumor site through an enhanced permeability and retention (EPR) effect. In vivo, CBN2-DP/siPHB1 NPs demonstrated enhanced synergistic cancer therapy efficacy and tumor inhibition as high as 71.2%. This work provided a strategy for preparing lipid-polymer hybrid NPs with GSH/ROS dual-responsive properties and an intriguing method for lung cancer therapy.

Keywords:  [12]aneN3; dual-responsive; gene/drug codelivery; lipid–polymer hybrid nanoparticles; synergistic therapy

DOI:  https://doi.org/10.1021/acsabm.4c00206
Vaccines (Basel). 2024 Apr 12. pii: 409. [Epub ahead of print]12(4):

Design and Characterization of a New Formulation for the Delivery of COVID-19-mRNA Vaccine to the Nasal Mucosa.

Ayça Altay Benetti, Eugene Yang Zhi Tan, Zi Wei Chang, Ki Hyun Bae, Ma Thinzar Thwin, Ram Pravin Kumar Muthuramalingam, Kuo-Chieh Liao, Yue Wan, Lisa F P Ng, Laurent Renia, Jianping Liu, Xiaoyuan Chen, Yi Yan Yang, Kevin P White, Giorgia Pastorin.

  Chitosan, a natural polysaccharide derived from chitin, possesses biocompatibility, biodegradability, and mucoadhesive characteristics, making it an attractive material for the delivery of mRNA payloads to the nasal mucosa and promoting their uptake by target cells such as epithelial and immune cells (e.g., dendritic cells and macrophages). In this project, we aimed at developing novel lipid-based nanoformulations for mRNA delivery to counteract the pandemic caused by SARS-CoV-2 virus. The formulations achieved a mRNA encapsulation efficiency of ~80.2% with chitosan-lipid nanoparticles, as measured by the RiboGreen assay. Furthermore, the evaluation of SARS-CoV-2 Spike (S) receptor-binding domain (RBD) expression via ELISA for our vaccine formulations showed transfection levels in human embryonic kidney cells (HEK 293), lung carcinoma cells (A549), and dendritic cells (DC 2.4) equal to 9.9 ± 0.1 ng/mL (174.7 ± 1.1 fold change from untreated cells (UT)), 7.0 ± 0.2 ng/mL (128.1 ± 4.9 fold change from UT), and 0.9 ± 0.0 ng/mL (18.0 ± 0.1 fold change from UT), respectively. Our most promising vaccine formulation was also demonstrated to be amenable to lyophilization with minimal degradation of loaded mRNA, paving the way towards a more accessible and stable vaccine. Preliminary in vivo studies in mice were performed to assess the systemic and local immune responses. Nasal bronchoalveolar lavage fluid (BALF) wash showed that utilizing the optimized formulation resulted in local antibody concentrations and did not trigger any systemic antibody response. However, if further improved and developed, it could potentially contribute to the management of COVID-19 through nasopharyngeal immunization strategies.

Keywords:  COVID-19; Vaccines; liposomes; mRNA; spike protein; stability

DOI:  https://doi.org/10.3390/vaccines12040409
Vaccines (Basel). 2024 Apr 02. pii: 379. [Epub ahead of print]12(4):

Major Role of S-Glycoprotein in Providing Immunogenicity and Protective Immunity in mRNA Lipid Nanoparticle Vaccines Based on SARS-CoV-2 Structural Proteins.

Evgeniia N Bykonia, Denis A Kleymenov, Vladimir A Gushchin, Andrei E Siniavin, Elena P Mazunina, Sofia R Kozlova, Anastasia N Zolotar, Evgeny V Usachev, Nadezhda A Kuznetsova, Elena V Shidlovskaya, Andrei A Pochtovyi, Daria D Kustova, Igor A Ivanov, Sergey E Dmitriev, Roman A Ivanov, Denis Y Logunov, Alexander L Gintsburg.

  SARS-CoV-2 variants have evolved over time in recent years, demonstrating immune evasion of vaccine-induced neutralizing antibodies directed against the original S protein. Updated S-targeted vaccines provide a high level of protection against circulating variants of SARS-CoV-2, but this protection declines over time due to ongoing virus evolution. To achieve a broader protection, novel vaccine candidates involving additional antigens with low mutation rates are currently needed. Based on our recently studied mRNA lipid nanoparticle (mRNA-LNP) platform, we have generated mRNA-LNP encoding SARS-CoV-2 structural proteins M, N, S from different virus variants and studied their immunogenicity separately or in combination in vivo. As a result, all mRNA-LNP vaccine compositions encoding the S and N proteins induced excellent titers of RBD- and N-specific binding antibodies. The T cell responses were mainly specific CD4+ T cell lymphocytes producing IL-2 and TNF-alpha. mRNA-LNP encoding the M protein did not show a high immunogenicity. High neutralizing activity was detected in the sera of mice vaccinated with mRNA-LNP encoding S protein (alone or in combinations) against closely related strains, but was undetectable or significantly lower against an evolutionarily distant variant. Our data showed that the addition of mRNAs encoding S and M antigens to mRNA-N in the vaccine composition enhanced the immunogenicity of mRNA-N and induced a more robust immune response to the N protein. Based on our results, we suggested that the S protein plays a key role in enhancing the immune response to the N protein when they are both encoded in the mRNA-LNP vaccine.

Keywords:  SARS-CoV-2; efficacy; immunogenicity; lipid nanoparticles; mRNA vaccine

DOI:  https://doi.org/10.3390/vaccines12040379
bioRxiv. 2024 Apr 19. pii: 2024.04.18.590188. [Epub ahead of print]

Machine learning methods for predicting guide RNA effects in CRISPR epigenome editing experiments.

Wancen Mu, Tianyou Luo, Alejandro Barrera, Lexi R Bounds, Tyler S Klann, Maria Ter Weele, Julien Bryois, Gregory E Crawford, Patrick F Sullivan, Charles A Gersbach, Michael I Love, Yun Li.

CRISPR epigenomic editing technologies enable functional interrogation of non-coding elements. However, current computational methods for guide RNA (gRNA) design do not effectively predict the power potential, molecular and cellular impact to optimize for efficient gRNAs, which are crucial for successful applications of these technologies. We present "launch-dCas9" (machine LeArning based UNified CompreHensive framework for CRISPR-dCas9) to predict gRNA impact from multiple perspectives, including cell fitness, wild-type abundance (gauging power potential), and gene expression in single cells. Our launch-dCas9, built and evaluated using experiments involving >1 million gRNAs targeted across the human genome, demonstrates relatively high prediction accuracy (AUC up to 0.81) and generalizes across cell lines. Method-prioritized top gRNA(s) are 4.6-fold more likely to exert effects, compared to other gRNAs in the same cis-regulatory region. Furthermore, launch-dCas9 identifies the most critical sequence-related features and functional annotations from >40 features considered. Our results establish launch-dCas9 as a promising approach to design gRNAs for CRISPR epigenomic experiments.

DOI: https://doi.org/10.1101/2024.04.18.590188