bims-novged 2024-01-28 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2024–01–28
twelve papers selected by
the Merkel lab, Ludwig-Maximilians University

Enhanced gene transfection and induction of apoptosis in melanoma cells by branched poly(β-amino ester)s with uniformly distributed branching units.
Development of Ionizable Lipid Nanoparticles and a Lyophilized Formulation for Potent CRISPR-Cas9 Delivery and Genome Editing.
Predictive High-Throughput Platform for Dual Screening of mRNA Lipid Nanoparticle Blood-Brain Barrier Transfection and Crossing.
Airway epithelial-targeted nanoparticle reverses asthma in inhalation therapy.
Backbone cationized highly branched poly(β-amino ester)s as enhanced delivery vectors in non-viral gene therapy.
Modifying Peptide/Lipid-Associated Nucleic Acids (PLANAs) for CRISPR/Cas9 Ribonucleoprotein Delivery.
An intranasally delivered ultra-conserved siRNA prophylactically represses SARS-CoV-2 infection in the lung and nasal cavity.
Chitosan-Functionalized Poly(β-Amino Ester) Hybrid System for Gene Delivery in Vaginal Mucosal Epithelial Cells.
Tet1 peptide and zinc (II)-adenine multifunctional module functionalized polycations as efficient siRNA carriers for Parkinson's disease.
Reversible Stabilization of Nanofiber-Polyplexes through Introducing Cross-Linkages.
The Efficacy and Safety of Polyethylene Glycol Cholesterol- and Tocopherol Polyethylene Glycol 1000 Succinate-Modified Transforming Growth Factor β1 Small Interfering RNA Lipid Nanoparticles in the Treatment of Paclitaxel-Resistant Non-Small-Cell Lung Cancer.
Quality assessment of LNP-RNA therapeutics with orthogonal analytical techniques.

J Control Release. 2024 Jan 25. pii: S0168-3659(24)00038-5. [Epub ahead of print]367 197-208

Enhanced gene transfection and induction of apoptosis in melanoma cells by branched poly(β-amino ester)s with uniformly distributed branching units.

Zhaowei Chu, Zhili Li, Haiyang Yong, Delu Che, Bingjie Li, Cong Yan, Tong Zhou, Xi Wang, Yuqing Feng, Kun Guo, Songmei Geng.

  Melanoma, one of the most devastating forms of skin cancer, currently lacks effective clinical treatments. Delivery of functional genes to modulate specific protein expression to induce melanoma cell apoptosis could be a promising therapeutic approach. However, transfecting melanoma cells using non-viral methods, particularly with cationic polymers, presents significant challenges. In this study, we synthesized three branched poly(β-amino ester)s (HPAEs) with evenly distributed branching units but varying space lengths through a two-step "oligomer combination" strategy. The unique topological structure enables HPAEs to condense DNA to form nano-sized polyplexes with favorable physiochemical properties. Notably, HPAEs, especially HPAE-2 with intermediate branching unit space length, demonstrated significantly higher gene transfection efficiency than the leading commercial gene transfection reagent, jetPRIME, in human melanoma cells. Furthermore, HPAE-2 efficiently delivered the Bax-encoding plasmid into melanoma cells, leading to a pronounced pro-apoptotic effect without causing noticeable cytotoxicity. This study establishes a potent non-viral platform for gene transfection of melanoma cells by harnessing the distribution of branching units, paving the way for potential clinical applications of gene therapy in melanoma treatment.

Keywords:  Branched poly(β-amino ester)s; Gene therapy; Melanoma cells; Non-viral vector; Transfection efficiency

DOI:  https://doi.org/10.1016/j.jconrel.2024.01.026
Int J Pharm. 2024 Jan 22. pii: S0378-5173(24)00079-6. [Epub ahead of print] 123845

Development of Ionizable Lipid Nanoparticles and a Lyophilized Formulation for Potent CRISPR-Cas9 Delivery and Genome Editing.

Qian Sun, Hongqian Zhang, Feng Ding, Xue Gao, Zongwei Zhu, Chuanxu Yang.

  CRISPR-Cas genome editing technology holds great promise for wide-ranging biomedical applications. However, the development of efficient delivery system for CRISPR-Cas components remains challenging. Herein, we synthesized a series of ionizable lipids by conjugation of alkyl-acrylate to different amine molecules and further assembled ionizable lipid nanoparticles (iLNPs) for co-delivery of Cas9 mRNA and sgRNA. Among all the iLNP candidates, 1A14-iLNP with lipids containing spermine as amine head, demonstrated the highest cellular uptake, endosomal escape and mRNA expression in vitro. Co-delivery of Cas9 mRNA and sgRNA targeting EGFP by 1A14-iLNP achieved the highest EGFP knockout efficiency up to 70% in HeLa-EGFP cells. In addition, 1A14-iLNP displayed passive liver-targeting delivery of Cas9 mRNA in vivo with good biocompatibility. Moreover, we developed a simple method of lyophilization-mediated reverse transfection of CRISPR-Cas9 components for efficient genome editing. Therefore, the developed 1A14-iLNP and the lyophilization formulation, represent a potent solution for CRISPR-Cas9 delivery, which might broaden the future of biomedical applications of both mRNA and CRISPR-based therapies.

Keywords:  CRISPR-Cas9; genome editing; lipid nanoparticle; lyophilization; mRNA delivery

DOI:  https://doi.org/10.1016/j.ijpharm.2024.123845
Nano Lett. 2024 Jan 23.

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

Emily L Han, Marshall S Padilla, Rohan Palanki, Dongyoon Kim, Kaitlin Mrksich, Jacqueline J Li, Sophia Tang, Il-Chul Yoon, Michael J Mitchell.

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

Keywords:  blood−brain barrier; brain delivery; lipid nanoparticle; mRNA

DOI:  https://doi.org/10.1021/acs.nanolett.3c03509
J Control Release. 2024 Jan 23. pii: S0168-3659(24)00059-2. [Epub ahead of print]

Airway epithelial-targeted nanoparticle reverses asthma in inhalation therapy.

Yinglan Yu, Mingjie Ni, Yaxian Zheng, Yuan Huang.

  Despite extensive research on corticosteroids for treating asthma, their short residence time in the lungs has limited their therapeutic effects in vivo. Nanoparticles have been widely investigated for inhaled drug delivery due to their potential benefits in prolonging drugs' residence time in the lungs. However, the retention of nanoparticles may be limited by mucus and ciliated epithelium clearance mechanisms in the airway. Herein, we anchored a neonatal-Fc-receptor-targeted peptide (FcBP) onto "mucus-penetrating" polyethylene glycol (PEG) nanoparticles (PEG-NP). Interestingly, the mucus-permeability of PEG-NP was not impaired by FcBP-functionalization. Moreover, FcBP modification enhanced cellular internalization and exocytosis via specific receptor-mediated processes, which subsequently ameliorated transepithelial transport and prolonged pulmonary retention. Importantly, after loading dexamethasone, FcBP-functionalization could effectively help nanoparticles cross the airway epithelial layer and be endocytosed by inflammatory cells, resulting in a marked decrease in inflammatory cytokines. Finally, FcBP modification significantly enhanced the therapeutic effect of dexamethasone-loaded nanoparticles in asthma mice. This study demonstrates that FcBP-functionalized PEG-NP can overcome multiple obstacles in the airway to prolong the pulmonary retention of drugs, providing a promising strategy for inhalation therapy.

Keywords:  Asthma; Epithelial barrier; Inhalation; Mucus barrier; Nanoparticles

DOI:  https://doi.org/10.1016/j.jconrel.2024.01.044
J Control Release. 2024 Jan 23. pii: S0168-3659(24)00061-0. [Epub ahead of print]

Backbone cationized highly branched poly(β-amino ester)s as enhanced delivery vectors in non-viral gene therapy.

Yinghao Li, Bei Qiu, Zishan Li, Xianqing Wang, Zhonglei He, Darío Manzanares Sandoval, Rijian Song, A Sigen, Chunyu Zhao, Melissa Johnson, Jing Lyu, Irene Lara-Sáez, Wenxin Wang.

  Gene therapy holds great potential for treating Lung Cystic Fibrosis (CF) which is a fatal hereditary condition arising from mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in dysfunctional CFTR protein. However, the advancement and clinical application of CF gene therapy systems have been hindered due to the absence of a highly efficient delivery vector. In this work, we introduce a new generation of highly branched poly(β-amino ester) (HPAE) gene delivery vectors for CF treatment. Building upon the classical chemical composition of HPAE, a novel backbone cationization strategy was developed to incorporate additional functional amine groups into HPAE without altering their branching degree. By carefully adjusting the type, proportion, and backbone distribution of the added cationic groups, a series of highly effective HPAE gene delivery vectors were successfully constructed for CF disease gene therapy. In vitro assessment results showed that the backbone cationized HPAEs with randomly distributed 10% proportion of 1-(3-aminopropyl)-4-methylpiperazine (E7) amine groups exhibited superior transfection performance than their counterparts. Furthermore, the top-performed backbone cationized HPAEs, when loaded with therapeutic plasmids, successfully reinstated CFTR protein expression in the CFBE41o- disease model, achieving levels 20-23 times higher than that of normal human bronchial epithelial (HBE) cells. Their therapeutic effectiveness significantly surpassed that of the currently advanced commercial vectors, Xfect and Lipofectamine 3000.

Keywords:  Backbone cationization; Cystic fibrosis; Highly branched structure; Non-viral gene delivery; Poly(β-amino ester)

DOI:  https://doi.org/10.1016/j.jconrel.2024.01.046
Eur J Pharm Sci. 2024 Jan 21. pii: S0928-0987(24)00019-8. [Epub ahead of print] 106708

Modifying Peptide/Lipid-Associated Nucleic Acids (PLANAs) for CRISPR/Cas9 Ribonucleoprotein Delivery.

Abdulelah Alhazza, Parvin Mahdipoor, Ryley Hall, Arthur Manda, Sandeep Lohan, Keykavous Parang, Hamidreza Montazeri Aliabadi.

  With the first reports on the possibility of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas)9 surfacing in 2005, the enthusiasm for protein silencing via nucleic acid delivery experienced a resurgence following a period of diminished enthusiasm due to challenges in delivering small interfering RNAs (siRNA), especially in vivo. However, delivering the components necessary for this approach into the nucleus is challenging, maybe even more than the cytoplasmic delivery of siRNA. We previously reported the birth of peptide/lipid-associated nucleic acids (PLANAs) for siRNA delivery. This project was designed to investigate the efficiency of these nanoparticles for in vitro delivery of CRISPR/Cas9 ribonucleoproteins. Our initial experiments indicated higher toxicity for PLANAs with the more efficient reverse transfection method. Therefore, polyethylene glycol (PEG) was added to the composition for PEGylation of the nanoparticles by partially replacing two of the lipid components with the PEG-conjugated counterparts. The results indicated a more significant reduction in the toxicity of the nanoparticle, less compromise in encapsulation efficiency and more PEGylation of the surface of the nanoparticles using DOPE-PEG2000 at 50% replacement of the naïve lipid. The cell internalization and transfection efficiency showed a comparable efficiency for the PEGylated and non-PEGylated PLANAs and the commercially available LipofectamineTM CRISPRMAXTM. Next Generation Sequencing of the cloned cells showed a variety of indels in the transfected cell population. Overall, our results indicate the efficiency and safety of PEGylated PLANAs for in vitro transfection with CRISPR/Cas9 ribonucleoproteins. PEGylation has been studied extensively for in vivo delivery, and PEGylated PLANAs will be candidates for future in vivo studies.

Keywords:  CRISPR/Cas9; Lipids; PLANA; Polyethylene glycol; Transfection; in vitro

DOI:  https://doi.org/10.1016/j.ejps.2024.106708
Antiviral Res. 2024 Jan 19. pii: S0166-3542(24)00023-8. [Epub ahead of print]222 105815

An intranasally delivered ultra-conserved siRNA prophylactically represses SARS-CoV-2 infection in the lung and nasal cavity.

Adi Idris, Aroon Supramaniam, Yaman Tayyar, Gabrielle Kelly, Nigel A J McMillan, Kevin V Morris.

  There remains a striking overall mortality burden of COVID-19 worldwide. Given the waning effectiveness of current SARS-CoV-2 antivirals due to the rapid emergence of new variants of concern (VOC), we employed a direct-acting molecular therapy approach using gene silencing RNA interference (RNAi) technology. In this study, we developed and screened several ultra-conserved small-interfering RNAs (siRNAs) before selecting one potent siRNA candidate for pre-clinical in vivo testing. This non-immunostimulatory, anti-SARS-CoV-2 siRNA candidate maintains its antiviral activity against all tested SARS-CoV-2 VOC and works effectively as a single agent. For the first time, significant antiviral effects in both the lungs and nasal cavities of SARS-CoV-2 infected mice were observed when this siRNA candidate was delivered intranasally (IN) as a prophylactic agent with the aid of lipid nanoparticles (LNPs). Importantly, a pre-exposure prophylactic IN-delivered anti-SARS-CoV-2 siRNA antiviral that can ameliorate viral replication in the nasal cavity could potentially prevent aerosol spread of respiratory viruses. An IN delivery approach would allow for the development of a direct-acting nasal spray approach that could be self-administered prophylactically.

Keywords:  Intranasal; Lipid nanoparticles; RNAi; SARS-CoV-2; siRNA

DOI:  https://doi.org/10.1016/j.antiviral.2024.105815
Pharmaceutics. 2024 Jan 22. pii: 154. [Epub ahead of print]16(1):

Chitosan-Functionalized Poly(β-Amino Ester) Hybrid System for Gene Delivery in Vaginal Mucosal Epithelial Cells.

Xueqin Gao, Dirong Dong, Chong Zhang, Yuxing Deng, Jiahui Ding, Shiqi Niu, Songwei Tan, Lili Sun.

  Gene therapy displays great promise in the treatment of cervical cancer. The occurrence of cervical cancer is highly related to persistent human papilloma virus (HPV) infection. The HPV oncogene can be cleaved via gene editing technology to eliminate carcinogenic elements. However, the successful application of the gene therapy method depends on effective gene delivery into the vagina. To improve mucosal penetration and adhesion ability, quaternized chitosan was introduced into the poly(β-amino ester) (PBAE) gene-delivery system in the form of quaternized chitosan-g-PBAE (QCP). At a mass ratio of PBAE:QCP of 2:1, the polymers exhibited the highest green fluorescent protein (GFP) transfection efficiency in HEK293T and ME180 cells, which was 1.1 and 5.4 times higher than that of PEI 25 kD. At this mass ratio, PBAE-QCP effectively compressed the GFP into spherical polyplex nanoparticles (PQ-GFP NPs) with a diameter of 255.5 nm. In vivo results indicated that owing to the mucopenetration and adhesion capability of quaternized CS, the GFP transfection efficiency of the PBAE-QCP hybrid system was considerably higher than those of PBAE and PEI 25 kD in the vaginal epithelial cells of Sprague-Dawley rats. Furthermore, the new system demonstrated low toxicity and good safety, laying an effective foundation for its further application in gene therapy.

Keywords:  chitosan; gene delivery; mucoadhesion; poly(β-amino ester)

DOI:  https://doi.org/10.3390/pharmaceutics16010154
J Control Release. 2024 Jan 20. pii: S0168-3659(24)00057-9. [Epub ahead of print]

Tet1 peptide and zinc (II)-adenine multifunctional module functionalized polycations as efficient siRNA carriers for Parkinson's disease.

Weijie Liu, Chunchao Ma, Junpeng Cao, Hao Zhou, Tianying Guo.

  A bioreducible Zn (II)-adenine multifunctional module (BS) and Tet1 peptide were used to modify low molecular weight PEI3.5k into a siRNA vector Zn-PB-T with high transfection efficiency in neurons. A GHS-responsive breakable disulfide spacer was introduced into BS to realize the controlled release of siRNA from the polyplexes in cytoplasm. Zn-PB showed >90% transfection rates in multiple cell lines (3 T3, HK-2, HepG2, 293 T, HeLa, PANC-1)，and 1.8-folds higher EGFP knockdown rates than commercial Lipo2k in normal cell line 293 T and cancer cell line HepG2. And Zn-PB-T1 showed 4.7-4.9- and 8.0-8.1-folds high transfection efficiency comparing to commercial Lipo2k and PEI25k in PC12 cells respectively, 2.1-fold EGFP gene silencing efficiency (96.6% EGFP knockdown rates) superior to commercial Lipo2k in neurons. In Parkinson's model, Zn-PB-T1/SNCA-siRNA can effectively protect neurons against MPP+-induced cell death and apoptosis, increasing the cell survival rate to 84.6% and reducing the cell apoptosis rate to 10.8%. This work demonstrated the promising application prospects of the resulting efficient siRNA carriers in siRNA-mediated gene therapy of Parkinson's disease.

Keywords:  Gene therapy; Parkinson's disease; Polycation modification; siRNA delivery vector

DOI:  https://doi.org/10.1016/j.jconrel.2024.01.042
J Funct Biomater. 2023 Dec 30. pii: 14. [Epub ahead of print]15(1):

Reversible Stabilization of Nanofiber-Polyplexes through Introducing Cross-Linkages.

Ryuta Aono, Kenta Nomura, Eiji Yuba, Atsushi Harada.

  Non-viral gene delivery systems are typically designed vector systems with contradictory properties, namely sufficient stability before cellular uptake and instability to ensure the release of nucleic acid cargoes in the transcription process after being taken up into cells. We reported previously that poly-(L-lysine) terminally bearing a multi-arm PEG (maPEG-PLL) formed nanofiber-polyplexes that suppressed excessive DNA condensation via steric repulsion among maPEGs and exhibited effective transcriptional capability in PCR amplification experiments and a cell-free gene expression system. In this study, the reversible stabilization of a nanofiber-polyplex without impairing the effective transcriptional capability was investigated by introducing cross-links between the PLL side chains within the polyplex using a cross-linking reagent with disulfide (SS) bonds that can be disrupted under reducing conditions. In the presence of dextran sulfate and/or dithiothreitol, the stability of the polyplex and the reactivity of the pDNA were evaluated using agarose gel electrophoresis and real-time PCR. We succeeded in reversibly stabilizing nanofiber-polyplexes using dithiobis (succinimidyl propionate) (DSP) as the cross-linking reagent. The effect of the reversible stabilization was confirmed in experiments using cultured cells, and the DSP-crosslinked polyplexes exhibited gene expression superior to that of polyethyleneimine polyplexes, which are typical polyplexes.

Keywords:  cross-linking; disulfide bond; gene delivery; polyplex; reversible stabilization

DOI:  https://doi.org/10.3390/jfb15010014
Pharmaceutics. 2024 Jan 04. pii: 75. [Epub ahead of print]16(1):

The Efficacy and Safety of Polyethylene Glycol Cholesterol- and Tocopherol Polyethylene Glycol 1000 Succinate-Modified Transforming Growth Factor β1 Small Interfering RNA Lipid Nanoparticles in the Treatment of Paclitaxel-Resistant Non-Small-Cell Lung Cancer.

Zhaowu Zeng, Xianglong Zeng, Xinyi Li, Yuxin Feng, Yue Kan, Xingyan Liu, Yiying Zeng.

  The aim of this study was to explore the efficacy and safety of TGFβ1 siRNA lipid nanoparticles (LNPs) modified with different PEG derivatives (PEG5000 cholesterol, abbreviated as CE; tocopherol polyethylene glycol 1000 succinate, abbreviated as TPGS) in the treatment of paclitaxel-resistant non-small-cell lung cancer. Three kinds of TGFβ1 siRNA LNPs were prepared via microfluidics technology, using different PEG derivatives and dosages (CE1.5, CE2.5, TPGS2.5) as variables. Their particle size, zeta potential, contents, and encapsulation efficiencies were determined. The inhibition of TGFβ1 mRNA and protein expression and the effects of the three kinds of LNPs on the proliferation of paclitaxel-resistant non-small-cell lung cancer cells (A549/T cell) were characterized. The distributions of the three siRNA LNPs in nude mice bearing A549/T tumors, especially at the tumor site, were observed using in vivo mouse imaging technology, and their corresponding efficacies were evaluated. The average particle size of the three kinds of TGFβ1 siRNA LNPs was about 70-80 nm, and they were capable of charge flipping. All three siRNA LNPs could effectively inhibit the expression of TGFβ1 mRNA and protein in A549/T cells and inhibit the proliferation of A549/T cells in vitro. The results of in vivo mice imaging showed that the three kinds of siRNA LNPs, when labeled with cypate, retain strong fluorescence in the tumor at 24 h. The pharmacodynamic results, such as for relative tumor volumes and tumor inhibition rates, reveal that TGFβ1 siRNA LNPs modified with CE1.5, CE2.5, or TPGS2.5 can be used to effectively treat paclitaxel-resistant lung adenocarcinoma. The histopathological results showed that the three kinds of LNPs have a certain toxicity but are relatively safe compared to common forms of chemotherapy such as cabazitaxel. TGFβ1 siRNA LNPs modified with CE1.5, CE2.5, and TPGS2.5 can inhibit TGFβ1 mRNA and protein expression in A549/T cells in vitro and can accumulate and play a role in the tumor tissue of nude mice, features that can be exploited for treating paclitaxel-resistant lung adenocarcinoma.

Keywords:  PEG cholesterol; TGFβ1; lipid nanoparticles; paclitaxel-resistant non-small-cell lung cancer; siRNA; tocopherol polyethylene glycol succinate (TPGS)

DOI:  https://doi.org/10.3390/pharmaceutics16010075
J Control Release. 2024 Jan 20. pii: S0168-3659(24)00050-6. [Epub ahead of print]

Quality assessment of LNP-RNA therapeutics with orthogonal analytical techniques.

Jeremie Parot, Dora Mehn, Hanna Jankevics, Natalia Markova, Michele Carboni, Camilla Olaisen, Andrea D Hoel, Margrét S Sigfúsdóttir, Florian Meier, Roland Drexel, Gabriele Vella, Birgitte McDonagh, Terkel Hansen, Huong Bui, Geir Klinkenberg, Torkild Visnes, Sabrina Gioria, Patricia Urban-Lopez, Adriele Prina-Mello, Sven Even Borgos, Fanny Caputo, Luigi Calzolai.

  The availability of analytical methods for the characterization of lipid nanoparticles (LNPs) for in-vivo intracellular delivery of nucleic acids is critical for the fast development of innovative RNA therapies. In this study, analytical protocols to measure (i) chemical composition, (ii) drug loading, (iii) particle size, concentration, and stability as well as (iv) structure and morphology were evaluated and compared based on a comprehensive characterization strategy linking key physical and chemical properties to in-vitro efficacy and toxicity. Furthermore, the measurement protocols were assessed either by testing the reproducibility and robustness of the same technique in different laboratories, or by a correlative approach, comparing measurement results of the same attribute with orthogonal techniques. The characterization strategy and the analytical measurements described here will have an important role during formulation development and in determining robust quality attributes ultimately supporting the quality assessment of these innovative RNA therapeutics.

Keywords:  Covid-19; Lipid based nanoparticles; Particle structure; Physico-chemical properties; Quality control; Vaccines; mRNA-LNPs

DOI:  https://doi.org/10.1016/j.jconrel.2024.01.037