bims-novged 2023-06-11 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2023–06–11
ten papers selected by
the Merkel lab, Ludwig-Maximilians University

GL67 lipid-based liposomal formulation for efficient siRNA delivery into human lung cancer cells.
Determination of mRNA copy number in degradable lipid nanoparticles via density contrast analytical ultracentrifugation.
Polymeric nanoparticle gel for intracellular mRNA delivery and immunological reprogramming of tumors.
Recent advances in stimuli-responsive polymeric carriers for controllable CRISPR/Cas9 gene editing system delivery.
Correlating the Structure and Gene Silencing Activity of Oligonucleotide-Loaded Lipid Nanoparticles Using Small-Angle X-ray Scattering.
Unlocking Delivery Strategies for mRNA Therapeutics.
Current state of RNA delivery using lipid nanoparticles to extrahepatic tissues: A review towards clinical translation.
Biomaterial-based gene therapy.
Extended Nucleic Acid (exNA): A Novel, Biologically Compatible Backbone that Significantly Enhances Oligonucleotide Efficacy in vivo.
Oversized liposomes boost macrophage-targeted RNA delivery to regulate macrophage polarity.

Saudi Pharm J. 2023 Jul;31(7): 1139-1148

GL67 lipid-based liposomal formulation for efficient siRNA delivery into human lung cancer cells.

Somayah J Jarallah, Ahmad M Aldossary, Essam A Tawfik, Reem M Altamimi, Wijdan K Alsharif, Nouf M Alzahrani, Homood M As Sobeai, Wajhul Qamar, Ahmed J Alfahad, Manal A Alshabibi, Sarah H Alqahtani, Abdullah A Alshehri, Fahad A Almughem.

  The efficient delivery of small interfering RNA (siRNA) to the targeted cells significantly affects the regulation of the overexpressed proteins involved in the progression of several genetic diseases. SiRNA molecules in naked form suffer from low internalization across the cell membrane, high susceptibility to degradation by nuclease enzyme and low stability, which hinder their efficacy. Therefore, there is an urge to develop a delivery system that can protect siRNA from degradation and facilitate their uptake across the cell membrane. In this study, the cationic lipid (GL67) was exploited, in addition to DC-Chol and DOPE lipids, to design an efficient liposomal nanocarrier for siRNA delivery. The physiochemical characterizations demonstrated that the molar ratio of 3:1 has proper particle size measurements from 144 nm to 332 nm and zeta potential of -9 mV to 47 mV that depends on the ratio of the GL67 in the liposomal formulation. Gel retardation assay exhibited that increasing the percentage of GL67 in the formulations has a good impact on the encapsulation efficiency compared to DC-Chol. The optimal formulations of the 3:1 M ratio also showed high metabolic activity against A549 cells following a 24 h cell exposure. Flow cytometry findings showed that the highest GL67 lipid ratio (100 % GL67 and 0 % DC-Chol) had the highest percentage of cellular uptake. The lipoplex nanocarriers based on GL67 lipid could potentially influence treating genetic diseases owing to the high internalization efficiency and safety profile.

Keywords:  A549 cells; GL67 lipid; Gene therapy; Liposomes; siRNA delivery

DOI:  https://doi.org/10.1016/j.jsps.2023.05.017
Eur Biophys J. 2023 Jun 08.

Determination of mRNA copy number in degradable lipid nanoparticles via density contrast analytical ultracentrifugation.

Alexander Bepperling, Gesa Richter.

  Lipid nanoparticles as delivery system for mRNA have recently attracted attention to a broader audience as COVID-19 mRNA vaccines. Their low immunogenicity and capability to deliver a variety of nucleic acids renders them an interesting and complementary alternative to gene therapy vectors like AAVs. An important quality attribute of LNPs is the copy number of the encapsulated cargo molecule. This work describes how density and molecular weight distributions obtained by density contrast sedimentation velocity can be used to calculate the mRNA copy number of a degradable lipid nanoparticle formulation. The determined average copy number of 5 mRNA molecules per LNP is consistent with the previous studies using other biophysical techniques, such as single particle imaging microscopy and multi-laser cylindrical illumination confocal spectroscopy (CICS).

Keywords:  Analytical ultracentrifugation; Density matching; Gene therapy; Lipid nanoparticle; mRNA

DOI:  https://doi.org/10.1007/s00249-023-01663-y
Biomaterials. 2023 May 31. pii: S0142-9612(23)00193-X. [Epub ahead of print]300 122185

Polymeric nanoparticle gel for intracellular mRNA delivery and immunological reprogramming of tumors.

Sarah Y Neshat, Chun Hei Ryan Chan, Jawaun Harris, Osamah M Zmily, Savannah Est-Witte, Johan Karlsson, Sydney R Shannon, Manav Jain, Joshua C Doloff, Jordan J Green, Stephany Y Tzeng.

  Immuno-oncology therapies have been of great interest with the goal of inducing sustained tumor regression, but clinical results have demonstrated the need for improved and widely applicable methods. An antigen-free method of cancer immunotherapy can stimulate the immune system to recruit lymphocytes and produce immunostimulatory factors without prior knowledge of neoantigens, while local delivery reduces the risk of systemic toxicity. To improve the interactions between tumor cells and cytotoxic lymphocytes, a gene delivery nanoparticle platform was engineered to reprogram the tumor microenvironment (TME) in situ to be more immunostimulatory by inducing tumor-associated antigen-presenting cells (tAPCs) to activate cytotoxic lymphocytes against the tumor. Biodegradable, lipophilic poly (beta-amino ester) (PBAE) nanoparticles were synthesized and used to co-deliver mRNA constructs encoding a signal 2 co-stimulatory molecule (4-1BBL) and a signal 3 immuno-stimulatory cytokine (IL-12), along with a nucleic acid-based immunomodulatory adjuvant. Nanoparticles are combined with a thermoresponsive block copolymer for gelation at the injection site for local NP retention at the tumor. The reprogramming nanoparticle gel synergizes with immune checkpoint blockade (ICB) to induce tumor regression and clearance in addition to resistance to tumor rechallenge at a distant site. In vitro and in vivo studies reveal increases in immunostimulatory cytokine production and recruitment of immune cells as a result of the nanoparticles. Intratumoral injection of nanoparticles encapsulating mRNA encoding immunostimulatory agents and adjuvants via an injectable thermoresponsive gel has great translational potential as an immuno-oncology therapy that can be accessible to a wide range of patients.

Keywords:  Immuno-oncology; Immunoengineering; Immunotherapy; Nanoparticles; Nonviral gene delivery; mRNA

DOI:  https://doi.org/10.1016/j.biomaterials.2023.122185
Biomater Sci. 2023 Jun 07.

Recent advances in stimuli-responsive polymeric carriers for controllable CRISPR/Cas9 gene editing system delivery.

Panqin Ma, Qi Wang, Xi Luo, Liuzhou Mao, Zhanxiang Wang, Enyi Ye, Xian Jun Loh, Zibiao Li, Yun-Long Wu.

Non-viral polymeric vectors with good biocompatibility have been recently explored as delivery systems for clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nucleases. In this review, based on current limitations and critical barriers, we summarize the advantages of stimulus-responsive polymeric delivery vectors (i.e., pH, redox, or enzymes) towards controllable CRISPR/Cas9 genome editing system delivery as well as the advances in using stimulus-responsive CRISPR/Cas9 polymeric carriers towards cancer treatment. Last but not least, the key challenges and promising development strategies of stimulus-responsive polymeric vector designs for CRISPR/Cas9 systems will also be discussed.

DOI: https://doi.org/10.1039/d3bm00529a
ACS Nano. 2023 Jun 06.

Correlating the Structure and Gene Silencing Activity of Oligonucleotide-Loaded Lipid Nanoparticles Using Small-Angle X-ray Scattering.

Michal Hammel, Yuchen Fan, Apoorva Sarode, Amy E Byrnes, Nanzhi Zang, Ponien Kou, Karthik Nagapudi, Dennis Leung, Casper C Hoogenraad, Tao Chen, Chun-Wan Yen, Greg L Hura.

  With three FDA-approved products, lipid nanoparticles (LNPs) are under intensive development for delivering wide-ranging nucleic acid therapeutics. A significant challenge for LNP development is insufficient understanding of structure-activity relationship (SAR). Small changes in chemical composition and process parameters can affect LNP structure, significantly impacting performance in vitro and in vivo. The choice of polyethylene glycol lipid (PEG-lipid), one of the essential lipids for LNP, has been proven to govern particle size. Here we find that PEG-lipids can further modify the core organization of antisense oligonucleotide (ASO)-loaded LNPs to govern its gene silencing activity. Furthermore, we also have found that the extent of compartmentalization, measured by the ratio of disordered vs ordered inverted hexagonal phases within an ASO-lipid core, is predictive of in vitro gene silencing. In this work, we propose that a lower ratio of disordered/ordered core phases correlates with stronger gene knockdown efficacy. To establish these findings, we developed a seamless high-throughput screening approach that integrated an automated LNP formulation system with structural analysis by small-angle X-ray scattering (SAXS) and in vitro TMEM106b mRNA knockdown assessment. We applied this approach to screen 54 ASO-LNP formulations while varying the type and concentration of PEG-lipids. Representative formulations with diverse SAXS profiles were further visualized using cryogenic electron microscopy (cryo-EM) to help structural elucidation. The proposed SAR was built by combining this structural analysis with in vitro data. Our integrated methods, analysis, and resulting findings on PEG-lipid can be applied to rapidly optimize other LNP formulations in a complex design space.

Keywords:  PEG-lipid; cryogenic electron microscopy; high-throughput screening; lipid nanoparticle; small-angle X-ray scattering; structure−activity relationship

DOI:  https://doi.org/10.1021/acsnano.3c01186
Recent Pat Anticancer Drug Discov. 2023 Jun 07.

Unlocking Delivery Strategies for mRNA Therapeutics.

Zhiyuan Luo, Yangchao Chen.

  mRNA emerged as an attractive therapy modality with the development of mRNA structure engineering techniques and delivery platforms. mRNA therapeutics, applied for vaccine therapy, protein replacement therapy, and chimeric antigen receptor (CAR) T cell-based therapy, has shown huge potential in treating a wide range of diseases, such as cancer and rare genetic diseases, with successful and exciting preclinical and clinical progress. In mRNA therapeutics, a potent delivery system is key to the success of its application for disease treatment. Herein, different types of mRNA delivery strategies, including nanoparticles produced from lipid or polymer materials, virus-based platforms, and exosome-based platforms, are mainly focused.

Keywords:  delivery system; gene therapy; mRNA delivery; mRNA therapeutics; nanoparticle; nucleic acid delivery

DOI:  https://doi.org/10.2174/1574892818666230607093231
Int J Biol Macromol. 2023 Jun 03. pii: S0141-8130(23)02079-2. [Epub ahead of print]242(Pt 4): 125185

Current state of RNA delivery using lipid nanoparticles to extrahepatic tissues: A review towards clinical translation.

Linh B Truong, David Medina-Cruz, Ebrahim Mostafavi.

  Genetic medicine, including ribonucleic acid (RNA) therapy, has delivered numerous progresses to the treatment of diseases thanks to the development of lipid nanoparticles (LNPs) as a delivery vehicle. However, RNA therapeutics are still limited by the lack of safe, precise, and efficient delivery outside of the liver. Thus, to fully realize the potential of genetic medicine, strategies to arm LNPs with extrahepatic targeting capabilities are urgently needed. This review explores the current state of next-generation LNPs that can bring RNA biomolecules to their targeted organ. The main approaches commonly used are described, including the modulation of internal lipid chemistries, the use of conjugated targeting moieties, and the designs of clinical administration. This work will demonstrate the advances in each approach and the remaining challenges in the field, focusing on clinical translation.

Keywords:  Extrahepatic delivery; Extrahepatic tissues; Genetic medicine; Lipid nanoparticles; RNA delivery

DOI:  https://doi.org/10.1016/j.ijbiomac.2023.125185
MedComm (2020). 2023 Jun;4(3): e259

Biomaterial-based gene therapy.

Yi Yu, Yijun Gao, Liming He, Bairong Fang, Wenhui Ge, Pu Yang, Yikun Ju, Xiaoyan Xie, Lanjie Lei.

  Gene therapy, a medical approach that involves the correction or replacement of defective and abnormal genes, plays an essential role in the treatment of complex and refractory diseases, such as hereditary diseases, cancer, and rheumatic immune diseases. Nucleic acids alone do not easily enter the target cells due to their easy degradation in vivo and the structure of the target cell membranes. The introduction of genes into biological cells is often dependent on gene delivery vectors, such as adenoviral vectors, which are commonly used in gene therapy. However, traditional viral vectors have strong immunogenicity while also presenting a potential infection risk. Recently, biomaterials have attracted attention for use as efficient gene delivery vehicles, because they can avoid the drawbacks associated with viral vectors. Biomaterials can improve the biological stability of nucleic acids and the efficiency of intracellular gene delivery. This review is focused on biomaterial-based delivery systems in gene therapy and disease treatment. Herein, we review the recent developments and modalities of gene therapy. Additionally, we discuss nucleic acid delivery strategies, with a focus on biomaterial-based gene delivery systems. Furthermore, the current applications of biomaterial-based gene therapy are summarized.

Keywords:  biomaterial; gene therapy; nonviral vector; viral vector

DOI:  https://doi.org/10.1002/mco2.259
bioRxiv. 2023 May 26. pii: 2023.05.26.542506. [Epub ahead of print]

Extended Nucleic Acid (exNA): A Novel, Biologically Compatible Backbone that Significantly Enhances Oligonucleotide Efficacy in vivo.

Ken Yamada, Vignesh Narayan Hariharan, Jillian Caiazzi, Rachael Miller, Chantal Furguson, Ellen Sapp, Hassan Fakih, Qi Tan, Nozomi Yamada, Raymond C Furgal, Joseph Paquette, Brianna Bramato, Nicholas McHugh, Ashley Summers, Clemens Lochmann, Bruno M D C Godinho, Samuel Hildebrand, Dimas Echeverria, Matthew R Hassler, Julia F Alterman, Marian DiFiglia, Neil Aronin, Anastasia Khvorova.

Metabolic stabilization of therapeutic oligonucleotides requires both sugar and backbone modifications, where phosphorothioate (PS) is the only backbone chemistry used in the clinic. Here, we describe the discovery, synthesis, and characterization of a novel biologically compatible backbone, extended nucleic acid (exNA). Upon exNA precursor scale up, exNA incorporation is fully compatible with common nucleic acid synthetic protocols. The novel backbone is orthogonal to PS and shows profound stabilization against 3'- and 5'-exonucleases. Using small interfering RNAs (siRNAs) as an example, we show exNA is tolerated at most nucleotide positions and profoundly improves in vivo efficacy. A combined exNA-PS backbone enhances siRNA resistance to serum 3'-exonuclease by ∼32-fold over PS backbone and >1000-fold over the natural phosphodiester backbone, thereby enhancing tissue exposure (∼6-fold), tissues accumulation (4- to 20-fold), and potency both systemically and in brain. The improved potency and durability imparted by exNA opens more tissues and indications to oligonucleotide-driven therapeutic interventions.

DOI: https://doi.org/10.1101/2023.05.26.542506
Mol Ther Nucleic Acids. 2023 Jun 13. 32 877-878

Oversized liposomes boost macrophage-targeted RNA delivery to regulate macrophage polarity.

Dongqing Wang, Jiayu Zhang, Jun Chen.



Keywords:  M1/M2; inflammation; macrophages; microRNAs; size-controlled liposomes

DOI:  https://doi.org/10.1016/j.omtn.2023.05.010