bims-novged 2023-10-29 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2023–10–29
eight papers selected by
the Merkel lab, Ludwig-Maximilians University

Inhaled mRNA nanoparticles dual-targeting cancer cells and macrophages in the lung for effective transfection.
Dendritic Cell-Mimicking Nanoparticles Promote mRNA Delivery to Lymphoid Organs.
Inverse Cubic and Hexagonal Mesophase Evolution within Ionizable Lipid Nanoparticles Correlates with mRNA Transfection in Macrophages.
Mechanistic pharmacokinetics and pharmacodynamics of GalNAc-siRNA: Translational model involving competitive receptor-mediated disposition and RISC-dependent gene silencing applied to givosiran.
Delivery of Plasmid DNA by Ionizable Lipid Nanoparticles to Induce CAR Expression in T Cells.
M2pep-Modified Cyclodextrin-siRNA Nanoparticles Modulate the Immunosuppressive Tumor Microenvironment for Prostate Cancer Therapy.
RNA-based translation activators for targeted gene upregulation.
Transient inhibition of lysosomal functions potentiates nucleic acid vaccines.

Proc Natl Acad Sci U S A. 2023 Oct 31. 120(44): e2304966120

Inhaled mRNA nanoparticles dual-targeting cancer cells and macrophages in the lung for effective transfection.

Zhongmin Tang, Xinru You, Yufen Xiao, Wei Chen, Yongjiang Li, Xiangang Huang, Haijun Liu, Fan Xiao, Chuang Liu, Seyoung Koo, Na Kong, Wei Tao.

  Messenger RNA (mRNA)-based therapeutics are transforming the landscapes of medicine, yet targeted delivery of mRNA to specific cell types while minimizing off-target accumulation remains challenging for mRNA-mediated therapy. In this study, we report an innovative design of a cationic lipid- and hyaluronic acid-based, dual-targeted mRNA nanoformulation that can display the desirable stability and efficiently transfect the targeted proteins into lung tissues. More importantly, the optimized dual-targeted mRNA nanoparticles (NPs) can not only accumulate primarily in lung tumor cells and inflammatory macrophages after inhalation delivery but also efficiently express any desirable proteins (e.g., p53 tumor suppressor for therapy, as well as luciferase and green fluorescence protein for imaging as examples in this study) and achieve efficacious lung tissue transfection in vivo. Overall, our findings provide proof-of-principle evidence for the design and use of dual-targeted mRNA NPs in homing to specific cell types to up-regulate target proteins in lung tissues, which may hold great potential for the future development of mRNA-based inhaled medicines or vaccines in treating various lung-related diseases.

Keywords:  dual-targeted mRNA nanoparticles; inhalation delivery; lung cancer; lung tissue transfection; pneumonia

DOI:  https://doi.org/10.1073/pnas.2304966120
Adv Sci (Weinh). 2023 Oct 22. e2302423

Dendritic Cell-Mimicking Nanoparticles Promote mRNA Delivery to Lymphoid Organs.

Yiming Cao, Jinrong Long, Huisheng Sun, Yiqi Miao, Ye Sang, Haitao Lu, Changxiao Yu, Zhen Zhang, Lin Wang, Jing Yang, Shengqi Wang.

  Spleen and lymphoid organs are important targets for messenger RNA (mRNA) delivery in various applications. Current nanoparticle delivery methods rely on drainage to lymph nodes from intramuscular or subcutaneous injections. In difficult-to-transfect antigen-presenting cells (APCs), such as dendritic cells (DCs), effective mRNA transfection remains a significant challenge. In this study, a lymphatic targeting carrier using DC membranes is developed, that efficiently migrated to lymphoid organs, such as the spleen and lymph nodes. The nanoparticles contained an ionizable lipid (YK009), which ensured a high encapsulation efficacy of mRNA and assisted mRNA with endosomal escape after cellular uptake. Dendritic cell-mimicking nanoparticles (DCMNPs) showed efficient protein expression in both the spleen and lymph nodes after intramuscular injections. Moreover, in immunized mice, DCMNP vaccination elicited Spike-specific IgG antibodies, neutralizing antibodies, and Th1-biased SARS-CoV-2-specific cellular immunity. This work presents a powerful vaccine formula using DCMNPs, which represents a promising vaccine candidate for further research and development.

Keywords:  delivery; dendritic cell membrane; lymphoid organs; mRNA vaccine

DOI:  https://doi.org/10.1002/advs.202302423
J Am Chem Soc. 2023 Oct 23.

Inverse Cubic and Hexagonal Mesophase Evolution within Ionizable Lipid Nanoparticles Correlates with mRNA Transfection in Macrophages.

Haitao Yu, Joshua Iscaro, Brendan Dyett, Yiran Zhang, Susanne Seibt, Natalia Martinez, Jacinta White, Calum J Drummond, Steven Bozinovski, Jiali Zhai.

mRNA lipid nanoparticle (LNP) technology presents enormous opportunities to prevent and treat various diseases. Here, we developed a novel series of LNPs containing ionizable amino-lipids showing a remarkable array of tunable and pH-sensitive lyotropic liquid crystalline mesophases including the inverse bicontinuous cubic and hexagonal phases characterized by high-throughput synchrotron radiation X-ray scattering. Furthermore, with an interest in developing mRNA therapeutics for lung macrophage targeting, we discovered that there is a strong correlation between the mesophase transition of the LNPs during acidification and the macrophage association/transfection efficiency of mRNAs. The slight molecular structural differences between the SM-102 and ALC-0315 ionizable lipids are linked to the LNP's ability to transform their internal structures from an amorphous state to the inverse micellar, hexagonal, and finally cubic structures during endosomal maturation. SM-102 LNPs showed exceptionally improved transfection efficiency due to their ability to form a cubic structure at a lower pH than the ALC-0315 analogues, which remained within the hexagonal structure, previously attributed to promoting endosomal escape of the ionizable LNPs. Overall, the new knowledge draws our attention to the important role of mesophase transition in endosomal escape, and the novel LNP libraries reported herein have broad prospects for advancing mRNA therapeutics.

DOI: https://doi.org/10.1021/jacs.3c08729
J Pharm Sci. 2023 Oct 21. pii: S0022-3549(23)00435-5. [Epub ahead of print]

Mechanistic pharmacokinetics and pharmacodynamics of GalNAc-siRNA: Translational model involving competitive receptor-mediated disposition and RISC-dependent gene silencing applied to givosiran.

Vivaswath S Ayyar, Dawei Song.

  Triantennary N-acetyl-D galactosamine (GalNAc)3-conjugated small interfering RNA (siRNA) have majorly advanced the development of RNA-based therapeutics. Chemically stabilized GalNAc-siRNAs exhibit extensive albeit capacity-limited (nonlinear) distribution into hepatocytes with additional complexities in intracellular liver disposition and pharmacology. A mechanism-based pharmacokinetic-pharmacodynamic (PK-PD) model of GalNAc-siRNA was developed to i) quantitate ASGPR-mediated disposition and downstream RNA-induced silencing complex (RISC)-dependent pharmacology following intravenous (IV) and subcutaneous (SC) dosing, ii) assess the kinetics of formed active metabolite, iii) leverage, as an example, published experimental data for givosiran, and iv) demonstrate PK translation across two preclinical species (rat and monkey) with subsequent prediction of human plasma PK. The structural model is based on competition between parent and formed active metabolite for occupancy and uptake via ASGPR into hepatocytes, intracellular sequestration and degradation, and downstream engagement of RNA-induced silencing complex (RISC) governing target mRNA degradation. The model jointly and accurately captured available concentration-time profiles of givosiran and/or AS(N-1)3' givosiran in rat and/or monkey plasma, liver, and/or kidney following givosiran administered both IV and SC. RISC-dependent gene silencing of ALAS1 mRNA was well-characterized. The model estimated an in vivo affinity (KD) value of 27.7 nM for GalNAc-ASGPR and weight-based allometric exponents of -0.27 and -0.24 for SC absorption and intracellular (endolysosomal) degradation rate constants. The model well-predicted reported givosiran plasma PK profiles in humans. PK simulations revealed net-shifts in liver-to-kidney distribution ratios with increasing IV and SC dose. Importantly, decreases in the relative liver uptake efficiency were demonstrated following IV and, to a lesser extent, following SC dosing explained by differential ASGPR occupancy profiles over time.

Keywords:  Clinical pharmacokinetics; Hepatocyte(s); Nonlinear pharmacokinetics; Pharmacokinetic/pharmacodynamic (PK/PD) modeling; RNA interference (RNAi); Small interfering RNA (siRNA); Targeted drug delivery; Translational pharmacokinetics

DOI:  https://doi.org/10.1016/j.xphs.2023.10.026
Int J Nanomedicine. 2023 ;18 5891-5904

Delivery of Plasmid DNA by Ionizable Lipid Nanoparticles to Induce CAR Expression in T Cells.

Pedro Henrique Dias Moura Prazeres, Heloísa Ferreira, Pedro Augusto Carvalho Costa, Walison da Silva, Marco Túllio Alves, Marshall Padilla, Ajay Thatte, Anderson Kenedy Santos, Anderson Oliveira Lobo, Adriano Sabino, Helen Lima Del Puerto, Michael J Mitchell, Pedro Pires Goulart Guimaraes.

   Introduction: Chimeric antigen receptor (CAR) cell therapy represents a hallmark in cancer immunotherapy, with significant clinical results in the treatment of hematological tumors. However, current approved methods to engineer T cells to express CAR use viral vectors, which are integrative and have been associated with severe adverse effects due to constitutive expression of CAR. In this context, non-viral vectors such as ionizable lipid nanoparticles (LNPs) arise as an alternative to engineer CAR T cells with transient expression of CAR.
Methods: Here, we formulated a mini-library of LNPs to deliver pDNA to T cells by varying the molar ratios of excipient lipids in each formulation. LNPs were characterized and screened in vitro using a T cell line (Jurkat). The optimized formulation was used ex vivo to engineer T cells derived from human peripheral blood mononuclear cells (PBMCs) for the expression of an anti-CD19 CAR (CAR-CD19BBz). The effectiveness of these CAR T cells was assessed in vitro against Raji (CD19+) cells.
Results: LNPs formulated with different molar ratios of excipient lipids efficiently delivered pDNA to Jurkat cells with low cytotoxicity compared to conventional transfection methods, such as electroporation and lipofectamine. We show that CAR-CD19BBz expression in T cells was transient after transfection with LNPs. Jurkat cells transfected with our top-performing LNPs underwent activation when exposed to CD19+ target cells. Using our top-performing LNP-9-CAR, we were able to engineer human primary T cells to express CAR-CD19BBz, which elicited significant specific killing of CD19+ target cells in vitro.
Conclusion: Collectively, our results show that LNP-mediated delivery of pDNA is a suitable method to engineer human T cells to express CAR, which holds promise for improving the production methods and broader application of this therapy in the future.

Keywords:  CAR T cells; cancer immunotherapy; cell engineering; lipid nanoparticles; pDNA delivery

DOI:  https://doi.org/10.2147/IJN.S424723
Mol Pharm. 2023 Oct 24.

M2pep-Modified Cyclodextrin-siRNA Nanoparticles Modulate the Immunosuppressive Tumor Microenvironment for Prostate Cancer Therapy.

Yao Sun, Michael F Cronin, Monique C P Mendonça, Jianfeng Guo, Caitriona M O'Driscoll.

  Prostate cancer (PCa) is the most prevalent cause of cancer deaths in men. Conventional strategies, such as surgery, radiation, or chemotherapy, face challenges including poor prognosis and resistance. Therefore, the development of new improved strategies is vital to enhance patient outcomes. Recently, immunotherapy has shown potential in the treatment of a range of cancers, including PCa. Tumor-associated macrophages (TAMs) play an important role in the tumor microenvironment (TME) and reprogramming of TAMs is associated with remodeling the TME. The colony-stimulating factor-1/colony stimulating factor-1 receptor (CSF-1/CSF-1R) signaling pathway is closely related to the polarization of TAMs. The downregulation of CSF-1R, using small interfering RNA (siRNA), has been shown to achieve the reprogramming of TAMs, from the immunosuppressive M2 phenotype to the immunostimulatory M1 one. To maximize specific cellular delivery an M2 macrophage-targeting peptide, M2pep, was formulated with an amphiphilic cationic β-Cyclodextrin (CD) incorporating CSF-1R siRNA. The resulting nanoparticles (NPs) increased M2 macrophage targeting both in vitro and in vivo, promoting the release of M1 factors and simultaneously downregulating the levels of M2 factors through TAM reprogramming. The subsequent remodeling of the TME resulted in a reduction in tumor growth in a subcutaneous PCa mouse model mainly mediated through the recruitment of cytotoxic T cells. In summary, this M2pep-targeted CD-based delivery system demonstrated significant antitumor efficacy, thus presenting an alternative immunotherapeutic strategy for PCa treatment.

Keywords:  CSF-1R; M2pep, M2 macrophages; functionalized cyclodextrin; immunotherapy; prostate cancer; targeted nanoparticles; tumor microenvironment

DOI:  https://doi.org/10.1021/acs.molpharmaceut.3c00769
Nat Commun. 2023 Oct 26. 14(1): 6827

RNA-based translation activators for targeted gene upregulation.

Yang Cao, Huachun Liu, Shannon S Lu, Krysten A Jones, Anitha P Govind, Okunola Jeyifous, Christine Q Simmons, Negar Tabatabaei, William N Green, Jimmy L Holder, Soroush Tahmasebi, Alfred L George, Bryan C Dickinson.

Technologies capable of programmable translation activation offer strategies to develop therapeutics for diseases caused by insufficient gene expression. Here, we present "translation-activating RNAs" (taRNAs), a bifunctional RNA-based molecular technology that binds to a specific mRNA of interest and directly upregulates its translation. taRNAs are constructed from a variety of viral or mammalian RNA internal ribosome entry sites (IRESs) and upregulate translation for a suite of target mRNAs. We minimize the taRNA scaffold to 94 nucleotides, identify two translation initiation factor proteins responsible for taRNA activity, and validate the technology by amplifying SYNGAP1 expression, a haploinsufficiency disease target, in patient-derived cells. Finally, taRNAs are suitable for delivery as RNA molecules by lipid nanoparticles (LNPs) to cell lines, primary neurons, and mouse liver in vivo. taRNAs provide a general and compact nucleic acid-based technology to upregulate protein production from endogenous mRNAs, and may open up possibilities for therapeutic RNA research.

DOI: https://doi.org/10.1038/s41467-023-42252-z
Proc Natl Acad Sci U S A. 2023 Oct 31. 120(44): e2306465120

Transient inhibition of lysosomal functions potentiates nucleic acid vaccines.

Chunxi Wang, Amelia Karlsson, Thomas H Oguin, Andrew N Macintyre, Gregory D Sempowski, Kevin R McCarthy, Yifei Wang, M Anthony Moody, Fan Yuan.

  Nucleic acid vaccines have shown promising results in the clinic against infectious diseases and cancers. To robustly improve the vaccine efficacy and safety, we developed an approach to increase the intracellular stability of nucleic acids by transiently inhibiting lysosomal function in targeted tissues using sucrose. To achieve efficient and localized delivery of sucrose in animals, we designed a biomimetic lipid nanoparticle (LNP) to target the delivery of sucrose into mouse muscle cells. Using this approach, viral antigen expression in mouse muscle after DNA vaccination was substantially increased and prolonged without inducing local or systemic inflammation or toxicity. The same change in antigen expression would be achieved if the vaccine dose could be increased by 3,000 folds, which is experimentally and clinically impractical due to material restrictions and severe toxicity that will be induced by such a high dose of nucleic acids. The increase in antigen expression augmented the infiltration and activation of antigen-presenting cells, significantly improved vaccine-elicited humoral and T cell responses, and fully protected mice against the viral challenge at a low dose of vaccine. Based on these observations, we conclude that transient inhibition of lysosome function in target tissue by sucrose LNPs is a safe and potent approach to substantially improve nucleic acid-based vaccines.

Keywords:  biomimetic nanoparticles; lysosomal function; nucleic acid vaccine delivery; nucleic acid vaccines

DOI:  https://doi.org/10.1073/pnas.2306465120