bims-novged 2022-04-24 papers

on Non-viral vectors for gene delivery

Issue of 2022–04–24
ten papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Targeting nucleic acid-based therapeutics to tumors: Challenges and strategies for polyplexes.
Oleyl Conjugated Histidine-Arginine Cell-Penetrating Peptides as Promising Agents for siRNA Delivery.
Brush-Structured Chitosan/PolyHEMA with Thymine and Its Synergistic Effect on the Specific Interaction with ssDNA and Cellular Uptake.
Targeted Transposition of Minicircle DNA Using Single-Chain Antibody Conjugated Cyclodextrin-Modified Poly (Propylene Imine) Nanocarriers.
Dendron-functionalised hyperbranched bis-MPA polyesters as efficient non-viral vectors for gene therapy in different cell lines.
Development of a Spray-Dried Formulation of Peptide-Dna Nanoparticles into a Dry Powder for Pulmonary Delivery Using Factorial Design.
Poly-L-Lysine/Hyaluronan Nanocarriers As a Novel Nanosystem for Gene Delivery.
Arginine-Rich Polymers with Pore-forming Capability Enable Efficient Intracellular Delivery via Direct Translocation Across Cell Membrane.
Polycatechol-Derived Mesoporous Polydopamine Nanoparticles for Combined ROS Scavenging and Gene Interference Therapy in Inflammatory Bowel Disease.
Blood-brain barrier-penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy.

J Control Release. 2022 Apr 15. pii: S0168-3659(22)00207-3. [Epub ahead of print]

Targeting nucleic acid-based therapeutics to tumors: Challenges and strategies for polyplexes.

Victoria C Vetter, Ernst Wagner.

  The current medical reality of cancer gene therapy is reflected by more than ten approved products on the global market, including oncolytic and other viral vectors and CAR T-cells as ex vivo gene-modified cell therapeutics. The development of synthetic antitumoral nucleic acid therapeutics has been proceeding at a lower but steady pace, fueled by a plethora of alternative nucleic acid platforms (from various antisense oligonucleotides, siRNA, microRNA, lncRNA, sgRNA, to larger mRNA and DNA) and several classes of physical and chemical delivery technologies. This review summarizes the challenges and strategies for tumor-targeted nucleic acid delivery. Focusing primarily on polyplexes (polycation complexes) as nanocarriers, delivery options across multiple barriers into tumor cells are illustrated.

Keywords:  Cancer; Nanoparticle; Polyplex; Targeting; pDNA; siRNA

DOI:  https://doi.org/10.1016/j.jconrel.2022.04.013
Pharmaceutics. 2022 Apr 18. pii: 881. [Epub ahead of print]14(4):

Oleyl Conjugated Histidine-Arginine Cell-Penetrating Peptides as Promising Agents for siRNA Delivery.

Muhammad Imran Sajid, Dindyal Mandal, Naglaa Salem El-Sayed, Sandeep Lohan, Jonathan Moreno, Rakesh Kumar Tiwari.

  Recent approvals of siRNA-based products motivated the scientific community to explore siRNA as a treatment option for several intractable ailments, especially cancer. The success of approved siRNA therapy requires a suitable and safer drug delivery agent. Herein, we report a series of oleyl conjugated histidine-arginine peptides as a promising nonviral siRNA delivery tool. The conjugated peptides were found to bind with the siRNA at N/P ratio ≥ 2 and demonstrated complete protection for the siRNA from early enzymatic degradation at N/P ratio ≥ 20. Oleyl-conjugated peptide -siRNA complexes were found to be noncytotoxic in breast cancer cells (MCF-7 and MDA-MB-231) and normal breast epithelial cells (MCF 10A) at N/P ratio of ~40. The oleyl-R3-(HR)4 and oleyl-R4-(HR)4 showed ~80-fold increased cellular uptake in MDA-MB-231 cells at N/P 40. Moreover, the conjugated peptides-siRNA complexes form nanocomplexes (~115 nm in size) and have an appropriate surface charge to interact with the cell membrane and cause cellular internalization. Furthermore, this study provides a proof-of-concept that oleyl-R5-(HR)4 can efficiently silence STAT-3 gene (~80% inhibition) in MDA-MB-231 cells with similar effectiveness to Lipofectamine. Further exploration of this approach holds a great promise in discovering a successful in vivo siRNA delivery agent with a favorable pharmacokinetic profile.

Keywords:  RNA interference; STAT-3; Western blotting; cell-penetrating peptides; siRNA; siRNA delivery

DOI:  https://doi.org/10.3390/pharmaceutics14040881
Langmuir. 2022 Apr 19.

Brush-Structured Chitosan/PolyHEMA with Thymine and Its Synergistic Effect on the Specific Interaction with ssDNA and Cellular Uptake.

Sarunya Phunpee, Suwabun Chirachanchai, Uracha R Ruktanonchai.

Cationic polymers are known to attach on an anionic cell surface and favor gene transportation/transfection into the cells. However, when the positive charges accumulate, they tend to cause cell damage and delivery failure. Chitosan (CS) is a potential cationic bio-derived polymer whose chemical structures can be modified to fine-tune the charges as well as the add-on functions. The present work demonstrates (i) the decoration of a nucleic acid sequence-like brush structure on CS to allow the specific interaction with DNA and (ii) delivery into the cell. By simply applying mercaptoacetic acid as the chain transfer agent, the grafting of poly(hydroxyethyl methacrylate) (PHEMA) containing Thy (P(HEMA-Thy)) on CS is possible. The brush-like P(HEMA-Thy) leads Thy moieties to be in sequences. The Thy sequences perform as poly[T] for the specific interaction with ssDNA. The synergistic effect of CS and Thy sequences, i.e., electrostatic and base pairing interactions, results in an effective and efficient binding with ssDNA as well as significant delivery, especially in cellular uptake and cell viability. The use of CS in combination with Thy sequences in brush-like structures on CS is a model for other polysaccharides to be conjugated with the as-designed nucleic acid sequences for potential gene delivery.

DOI: https://doi.org/10.1021/acs.langmuir.2c00559
Cancers (Basel). 2022 Apr 11. pii: 1925. [Epub ahead of print]14(8):

Targeted Transposition of Minicircle DNA Using Single-Chain Antibody Conjugated Cyclodextrin-Modified Poly (Propylene Imine) Nanocarriers.

Willi Jugel, Stefanie Tietze, Jennifer Daeg, Dietmar Appelhans, Felix Broghammer, Achim Aigner, Michael Karimov, Gabriele Schackert, Achim Temme.

  Among non-viral vectors, cationic polymers, such as poly(propylene imine) (PPI), play a prominent role in nucleic acid delivery. However, limitations of polycationic polymer-based DNA delivery systems are (i) insufficient target specificity, (ii) unsatisfactory transgene expression, and (iii) undesired transfer of therapeutic DNA into non-target cells. We developed single-chain antibody fragment (scFv)-directed hybrid polyplexes for targeted gene therapy of prostate stem cell antigen (PSCA)-positive tumors. Besides mono-biotinylated PSCA-specific single-chain antibodies (scFv(AM1-P-BAP)) conjugated to neutravidin, the hybrid polyplexes comprise β-cyclodextrin-modified PPI as well as biotin/maltose-modified PPI as carriers for minicircle DNAs encoding for Sleeping Beauty transposase and a transposon encoding the gene of interest. The PSCA-specific hybrid polyplexes efficiently delivered a GFP gene in PSCA-positive tumor cells, whereas control hybrid polyplexes showed low gene transfer efficiency. In an experimental gene therapy approach, targeted transposition of a codon-optimized p53 into p53-deficient HCT116p53-/-/PSCA cells demonstrated decreased clonogenic survival when compared to mock controls. Noteworthily, p53 transposition in PTEN-deficient H4PSCA glioma cells caused nearly complete loss of clonogenic survival. These results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition, which, due to the modular design, can be extended to other target genes and tumor entities.

Keywords:  DNA delivery; p53; poly(propylene imine); β-cyclodextrin

DOI:  https://doi.org/10.3390/cancers14081925
Biomater Sci. 2022 Apr 20.

Dendron-functionalised hyperbranched bis-MPA polyesters as efficient non-viral vectors for gene therapy in different cell lines.

María San Anselmo, Alejandro Postigo, Alexandre Lancelot, José Luis Serrano, Teresa Sierra, Silvia Hernández-Ainsa.

Gene therapy has become a relevant tool in the biomedical field to treat or even prevent some diseases. The effective delivery of genetic material into the cell remains a crucial step to succeed in this purpose. In the search for efficient non-viral vectors, a series of amino-terminated dendronized hyperbranched polymers (DHPs) of different generations based either on bis-MPA or bis-GMPA have been designed. All of them have demonstrated an accurate ability to complex two types of genetic materials, a plasmid DNA and a siGFP, yielding dendriplexes. Moreover, some of them have proved to be able to deliver the genetic material inside the cells, resulting in the effective accomplishment of the desired genetic modification and improving the activity of some commercial transfection reagents. Different cell lines, including cancer and mesenchymal stem cells, have been studied here to evaluate the ability of DHPs as vectors for transfection. Treatments based on mesenchymal stem cells are gaining importance due to their pluripotency. Thus, it is of special relevance to introduce a genetic modification into a mesenchymal cell line as it allows it to act over a wide spectrum of tissues after inducing cellular differentiation.

DOI: https://doi.org/10.1039/d2bm00365a
Pharm Res. 2022 Apr 19.

Development of a Spray-Dried Formulation of Peptide-Dna Nanoparticles into a Dry Powder for Pulmonary Delivery Using Factorial Design.

Miftakul Munir, Vicky L Kett, Nicholas J Dunne, Helen O McCarthy.

   BACKGROUND: Gene therapy via pulmonary delivery holds the potential to treat various lung pathologies. To date, spray drying has been the most promising method to produce inhalable powders. The present study determined the parameters required to spray dry nanoparticles (NPs) that contain the delivery peptide, termed RALA (N-WEARLARALARALARHLARALARALRACEA-C), complexed with plasmid DNA into a dry powder form designed for inhalation.
METHODS: The spray drying process was optimised using full factorial design with 19 randomly ordered experiments based on the combination of four parameters and three centre points per block. Specifically, mannitol concentration, inlet temperature, spray rate, and spray frequency were varied to observe their effects on process yield, moisture content, a median of particle size distribution, Z-average, zeta potential, encapsulation efficiency of DNA NPs, and DNA recovery. The impact of mannitol concentration was also examined on the spray-dried NPs and evaluated via biological functionality in vitro.
RESULTS: The results demonstrated that mannitol concentration was the strongest variable impacting all responses apart from encapsulation efficiency. All measured responses demonstrated a strong dependency on the experimental variables. Furthermore, spray drying with the optimal variables in combination with a low mannitol concentration (1% and 3%, w/v) produced functional RALA/pDNA NPs.
CONCLUSION: The optimal parameters have been determined to spray dry RALA/pDNA NPs into an dry powder with excellent biological functionality, which have the potential to be used for gene therapy applications via pulmonary delivery.

Keywords:  cell-penetrating peptide; dry powder formulation; gene delivery; pulmonary delivery; spray drying

DOI:  https://doi.org/10.1007/s11095-022-03256-4
J Microsc. 2022 Apr 20.

Poly-L-Lysine/Hyaluronan Nanocarriers As a Novel Nanosystem for Gene Delivery.

Masoumeh Souri, Mohammad Aref Bagherzadeh, Mirza Ali Mofazzal Jahromi, Hossein Mohammad-Beigi, Amir Abdoli, Hamed Mir, Abazar Roustazadeh, Majid Pirestani, Parham Sahandi Zangabad, Jafar Kiani, Amirmahmoud Bakhshayesh, Mehdi Jahani, Mohammad Taghi Joghataei, Mahdi Karimi.

  The present research comes up with a novel DNA-loaded poly-l-lysine (PLL) / hyaluronan (HA) nanocarrier (DNA-loaded PLL/HA NCs) for gene delivery applications, as a promising candidate for gene delivery into diverse cells. A straightforward approach was employed to prepare such a nanosystem through masking DNA-loaded PLL molecules by HA. Fourier-transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), field emission-scanning electron microscopy (FE-SEM), and transmission electron microscopy (TEM) were used to analyze the interaction of the molecules as well as the physicochemical properties of the NCs. The NCs showed a negative charge of -24 ± 3 mV, with an average size of 138 ± 6 nm, in a ellipsoid-shape with smooth surfaces. The DNA loading efficiency (LE) measured by DNA absorbance was around 95 %. The MTT assay showed that the developed NCs are non-toxic to the cells. Furthermore,the uptake of the DNA-loaded PLL/HA NCs by the human embryonic kidney (HEK)-293T cells was evaluated by a flow cytometry method, and demonstrated high potential cellular uptake over 90% for transferring the gene to HEK-293T cells at the optimized conditions. Therefore, the DNA-loaded PLL/HA NCs are the potent strategy for developing nanosystems for gene delivery applications. This article is protected by copyright. All rights reserved.

Keywords:  gene delivery; hyaluronan; nanocarriers; poly-l-lysine

DOI:  https://doi.org/10.1111/jmi.13107
Adv Healthc Mater. 2022 Apr 23. e2200371

Arginine-Rich Polymers with Pore-forming Capability Enable Efficient Intracellular Delivery via Direct Translocation Across Cell Membrane.

Ziyao Kang, Qi Liu, Zhanzhan Zhang, Yadan Zheng, Chun Wang, Zheng Pan, Qiushi Li, Yang Liu, Linqi Shi.

  Efficient delivery of biomacromolecules or drugs across cell membrane via endocytosis usually encounter inevitable entrapment in endosomes and subsequent degradation in lyso-endosome. To address this issue, we designed and synthesized a series of arginine-rich cell penetrating polymers, which internalize into cells by inducing the formation of pores on cell membrane, thereby crossing cell membrane via direct translocation that fundamentally avoids endo/lysosomal entrapment. The structure-activity relationship studies have shown that PTn-R2-C6, which is a type of polymers that have two arginine residues and a flexible hexanoic acid linker in each side chain, exhibited excellent pore-formation ability on cell membrane. Further investigations indicated that PTn-R2-C6 rapidly transported plasmid DNAs into cytosol through a similar endocytosis-independent pathway, thereby achieving significantly higher transfection efficiency and lower cytotoxicity than the gold-standard transfection reagent PEI 25K. These results suggest the great potential of PTn-R2-C6 as a safe and efficient gene transfection reagent for wide applications including disease treatments, vaccine development, and biomedical research purposes. This article is protected by copyright. All rights reserved.

Keywords:  arginine-rich polymers; cell penetration; direct transloction; endocytosis-independent pathway; gene delivery

DOI:  https://doi.org/10.1002/adhm.202200371
ACS Appl Mater Interfaces. 2022 Apr 20.

Polycatechol-Derived Mesoporous Polydopamine Nanoparticles for Combined ROS Scavenging and Gene Interference Therapy in Inflammatory Bowel Disease.

Liucan Wang, Zhenqiang Wang, Yiyang Pan, Shuaishuai Chen, Xin Fan, Xiaolong Li, Guoqing Chen, Yuanhang Ma, Yujiao Cai, Jixi Zhang, Hua Yang, Weidong Xiao, Min Yu.

  Benefiting from the evolution of nanotechnology, the combination therapy by gene interference and reactive oxygen species (ROS) scavenging are expected, which holds great potential in inflammatory bowel disease (IBD) therapy. However, the functional integration of different therapeutic modules through interface modification of gene vectors for safe and efficient treatment is urgently needed. Herein, we present a catechol chemistry-mediated core-shell nanoplatform for ROS scavenging-mediated oxidative stress alleviation and siRNA-mediated gene interference in a dextran sulfate sodium (DSS)-induced colitis model. The nanoplatform is constructed by employing mesoporous polydopamine nanoparticles (MPDA NPs) with surface modification of amines as the porous core for TNF-α-siRNA loading (31 wt %) and exerts an antioxidant function, while PDA-induced biomineralization of the calcium phosphate (CaP) coating is used as the pH-sensitive protective shell to prevent siRNA from premature release. The CaP layer degraded under weakly acidic subcellular conditions (lysosomes); thus, the synergistic integration of catechol and cation moieties on the exposed surface of MPDA resulted in an efficient lysosomal escape. Subsequently, effective ROS scavenging caused by the electron-donating ability of MPDA and efficient knocking down (40.5%) of tumor necrosis factor-α (TNF-α) via sufficient cytosolic gene delivery resulted in a synergistic anti-inflammation therapeutic effect both in vitro and in vivo. This work establishes the first paradigm of synergistic therapy in IBD by ROS scavenging and gene interference.

Keywords:  ROS scavenging; combination therapy; gene interference; inflammatory bowel disease; mesoporous polydopamine nanoparticles

DOI:  https://doi.org/10.1021/acsami.1c25180
Sci Adv. 2022 Apr 22. 8(16): eabm8011

Blood-brain barrier-penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy.

Yan Zou, Xinhong Sun, Qingshan Yang, Meng Zheng, Olga Shimoni, Weimin Ruan, Yibin Wang, Dongya Zhang, Jinlong Yin, Xiangang Huang, Wei Tao, Jong Bae Park, Xing-Jie Liang, Kam W Leong, Bingyang Shi.

We designed a unique nanocapsule for efficient single CRISPR-Cas9 capsuling, noninvasive brain delivery and tumor cell targeting, demonstrating an effective and safe strategy for glioblastoma gene therapy. Our CRISPR-Cas9 nanocapsules can be simply fabricated by encapsulating the single Cas9/sgRNA complex within a glutathione-sensitive polymer shell incorporating a dual-action ligand that facilitates BBB penetration, tumor cell targeting, and Cas9/sgRNA selective release. Our encapsulating nanocapsules evidenced promising glioblastoma tissue targeting that led to high PLK1 gene editing efficiency in a brain tumor (up to 38.1%) with negligible (less than 0.5%) off-target gene editing in high-risk tissues. Treatment with nanocapsules extended median survival time (68 days versus 24 days in nonfunctional sgRNA-treated mice). Our new CRISPR-Cas9 delivery system thus addresses various delivery challenges to demonstrate safe and tumor-specific delivery of gene editing Cas9 ribonucleoprotein for improved glioblastoma treatment that may potentially be therapeutically useful in other brain diseases.

DOI: https://doi.org/10.1126/sciadv.abm8011