Gene therapy promises accurate, targeted administration and overcoming drug resistance in diverse cancer cells

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Gene Therapy ◽  
Drug Resistance ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Clinical Research ◽  
Cancer Cells ◽  
Cationic Lipids ◽  
Target Cells ◽  
Genetic Components ◽  
Wide Range

Nucleic acid-based therapeutics such as siRNA and miRNA employ the silencing capabilities of the RNAi mechanism to affect the expression of one gene or several genes in target cells. Nucleic acid-based therapies enable accurate, targeted administration and overcoming drug resistance in diverse cancer cells. Several studies have shown that they can be utilized alongside pharmacological therapy to increase the efficacy of existing therapies. In addition, nucleic acid-based therapies have the potential to widen the spectrum of druggable targets for a range of diseases and emerge as a novel therapeutic technique for treating a number of diseases that are today untreatable. Nucleic acids are dependent on their effective distribution to target cells, which need correct complexation and encapsulation in a delivery mechanism. Although nucleic acids exist in a variety of forms and sizes, their physical and chemical commonality allow them to be loaded into a wide range of delivery vehicles. The primary biomaterials used to encapsulate genetic components were cationic lipids and polymers. Furthermore, the experiments focused particularly on effective transfection in target cells.Recent breakthroughs in NP-based RNA therapeutics have spurred a flood of clinical research, facing many challenges. In vivo, pharmacokinetics of different RNA-based medications must be researched to establish the viability and therapeutic potential of nucleic acid-based therapeutics. The U.S. Food and Drug Administration recently authorized many NP-based gene therapy. In 2019, Novartis authorized Zolgensma (onasemnogene abeparvovec-xioi) to treat spinal muscle atrophy. The first clinical research employing siRNA began in 2004 and is considered a milestone in nucleic acid-based drug development. Thirty clinical investigations have subsequently been completed. In 2018, the US FDA cleared Onpattro (Patisiran, Alnylam Pharmaceuticals) for the treatment of polyneuropathy caused by transthyretin amyloidosis.Several new generations of nucleic acid compositions employing polymer nanoparticles or liposomes are presently undergoing clinical testing. If allowed, the debut of nucleic acid-based treatments would represent a watershed event in immunotherapy. Advances in the design and development of biocompatible nanomaterials would allow us to overcome the above-mentioned problems and so show the potential to deliver nucleic acids in the treatment of a number of illnesses.

Kaempferol Improves TRAIL-Mediated Apoptosis in Leukemia MOLT-4 Cells by the Inhibition of Anti-apoptotic Proteins and Promotion of Death Receptors Expression

2019 ◽  
Vol 19 (15) ◽  
pp. 1835-1845
Author(s):  
Ali Hassanzadeh ◽  
Adel Naimi ◽  
Majid F. Hagh ◽  
Raedeh Saraei ◽  
Faroogh Marofi ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Cancer Cells ◽  
Tumor Necrosis ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Death Receptors ◽  
Target Cells ◽  
Wide Range ◽  
Apoptotic Proteins ◽  
Necrosis Factor

Introduction: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a member of the tumor necrosis factor (TNF) superfamily, which stimulates apoptosis in a wide range of cancer cells via binding to death receptors 4 and 5 (DR4/5). Nevertheless, TRAIL has noticeable anti-cancer abilities; some cancer cells acquire resistance to TRAIL, and consequently its potential for inducing apoptosis in target cells is strongly diminished. Acute lymphoblastic leukemia MOLT-4 cell line is one of the most resistant cells to TRAIL that developed resistance to TRAIL via different pathways. We used TRAIL plus kaempferol to eliminate resistance of the MOLT-4 cells to TRAIL. Material and Methods: First, IC50 for kaempferol (95 µM) was determined by using the MTT assay. Second, the viability of the MOLT-4 cells was assayed by FACS after Annexin V/PI staining, following treatment with TRAIL (50 and 100 nM) and kaempferol (95 µM) alone and together. Finally, the expression levels of the candidate genes involved in resistance to TRAIL were assayed by real-time PCR technique. Results: Kaempferol plus TRAIL induced apoptosis robustly in MOLT-4 cells at 12, 24 and 48 hours after treatment. Additionally, we found that kaempferol could inhibit expression of the c-FLIP, X-IAP, cIAP1/2, FGF-8 and VEGF-beta, and conversely augment expression of the DR4/5 in MOLT-4 cells. Conclusion: We suggest that co-treatment of MOLT-4 cells with TRAIL plus kaempferol is a practical and attractive approach to eliminate cancers’ resistance to TRAIL via inhibition of the intracellular anti-apoptotic proteins, upregulation of DR4/5 and also by suppression of the VEGF-beta (VEGFB) and FGF-8 expressions.

scholarly journals Сationic liposomes as delivery systems for nucleic acids

2020 ◽  
Vol 15 (1) ◽  
pp. 7-27
Author(s):  
A. A. Mikheev ◽  
E. V. Shmendel ◽  
E. S. Zhestovskaya ◽  
G. V. Nazarov ◽  
M. A. Maslov
Keyword(s):  
Gene Therapy ◽  
Nucleic Acids ◽  
Serum Proteins ◽  
Transfection Efficiency ◽  
Biological Fluids ◽  
Genetic Material ◽  
Cationic Liposomes ◽  
Delivery Systems ◽  
Cationic Lipids

Objectives. Gene therapy is based on the introduction of genetic material into cells, tissues, or organs for the treatment of hereditary or acquired diseases. A key factor in the success of gene therapy is the development of delivery systems that can efficiently transfer genetic material to the place of their therapeutic action without causing any associated side effects. Over the past 10 years, significant effort has been directed toward creating more efficient and biocompatible vectors capable of transferring nucleic acids (NAs) into cells without inducing an immune response. Cationic liposomes are among the most versatile tools for delivering NAs into cells; however, the use of liposomes for gene therapy is limited by their low specificity. This is due to the presence of various biological barriers to the complex of liposomes with NA, including instability in biological fluids, interaction with serum proteins, plasma and nuclear membranes, and endosomal degradation. This review summarizes the results of research in recent years on the development of cationic liposomes that are effective in vitro and in vivo. Particular attention is paid to the individual structural elements of cationic liposomes that determine the transfection efficiency and cytotoxicity. The purpose of this review was to provide a theoretical justification of the most promising choice of cationic liposomes for the delivery of NAs into eukaryotic cells and study the effect of the composition of cationic lipids (CLs) on the transfection efficiency in vitro.Results. As a result of the analysis of the related literature, it can be argued that one of the most promising delivery systems of NAs is CL based on cholesterol and spermine with the addition of a helper lipid DOPE. In addition, it was found that varying the composition of cationic liposomes, the ratio of CL to NA, or the size and zeta potential of liposomes has a significant effect on the transfection efficiency.Conclusions. Further studies in this direction should include optimization of the conditions for obtaining cationic liposomes, taking into account the physicochemical properties and established laws. It is necessary to identify mechanisms that increase the efficiency of NA delivery in vitro by searching for optimal structures of cationic liposomes, determining the ratio of lipoplex components, and studying the delivery efficiency and properties of multicomponent liposomes.

scholarly journals An optimized live bacterial delivery platform for the production and delivery of therapeutic nucleic acids and proteins

2021 ◽  
Author(s):  
Darcy S.O. Mora ◽  
Madeline Cox ◽  
Forgivemore Magunda ◽  
Ashley B. Williams ◽  
Lyndsey Linke
Keyword(s):  
Epithelial Cells ◽  
Mouse Model ◽  
Nucleic Acids ◽  
Endosomal Escape ◽  
Therapeutic Window ◽  
Target Cells ◽  
Full Potential ◽  
Wide Range ◽  
Delivery Platform

There is an unmet need for delivery platforms that realize the full potential of next-generation therapeutic and vaccine technologies, especially those that require intracellular delivery of nucleic acids. The in vivo usefulness of the current state-of-the-art delivery systems is limited by numerous intrinsic weaknesses, including lack of targeting specificity, inefficient entry and endosomal escape into target cells, undesirable immune activation, off-target effects, a small therapeutic window, limited genetic encoding and cargo capacity, and manufacturing challenges. Here we present our characterization of a delivery platform based on the use of engineered live, tissue-targeting, non-pathogenic bacteria (Escherichia coli strain SVC1) for intracellular cargo delivery. The SVC1 bacteria are engineered to specifically bind to epithelial cells via a surface-expressed targeting ligand, to escape the endosome upon intracellularization, and to have minimal immunogenicity. Here we report findings on key features of this system. First, we demonstrated that bacterial delivery of a short hairpin RNA (shRNA) can target and silence a gene in an in vitro mammalian respiratory cell model. Next, we used an in vivo mouse model to demonstrate that SVC1 bacteria are invasive to epithelial cells of various tissues and organs (eye, nose, mouth, stomach, vagina, skeletal muscle, and lungs) via local administration. We also showed that repeat dosing of SVC1 bacteria to the lungs is minimally immunogenic and that it does not have adverse effects on tissue homeostasis. Finally, to validate the potential of SVC1 bacteria in therapeutic applications, we demonstrated that bacterial delivery of influenza- targeting shRNAs to the respiratory tissues can mitigate viral replication in a mouse model of influenza infection. Our ongoing work is focused on further refining this platform for efficient delivery of nucleic acids, gene editing machinery, and therapeutic proteins, and we expect that this platform technology will enable a wide range of advanced therapeutic approaches.

scholarly journals Role of Lipid-Based and Polymer-Based Non-Viral Vectors in Nucleic Acid Delivery for Next-Generation Gene Therapy

Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2866 ◽  
Author(s):  
Aniket Wahane ◽  
Akaash Waghmode ◽  
Alexander Kapphahn ◽  
Karishma Dhuri ◽  
Anisha Gupta ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Nucleic Acid ◽  
Messenger Rna ◽  
Viral Vectors ◽  
Cationic Lipids ◽  
Nucleic Acid Delivery ◽  
Nucleic Acid Analogs ◽  
Organ Specific ◽  
Interfering Rna

The field of gene therapy has experienced an insurgence of attention for its widespread ability to regulate gene expression by targeting genomic DNA, messenger RNA, microRNA, and short-interfering RNA for treating malignant and non-malignant disorders. Numerous nucleic acid analogs have been developed to target coding or non-coding sequences of the human genome for gene regulation. However, broader clinical applications of nucleic acid analogs have been limited due to their poor cell or organ-specific delivery. To resolve these issues, non-viral vectors based on nanoparticles, liposomes, and polyplexes have been developed to date. This review is centered on non-viral vectors mainly comprising of cationic lipids and polymers for nucleic acid-based delivery for numerous gene therapy-based applications.

Binding Of Immune Serine Proteases To Nucleic Acids Enhances Their Nuclear Localization and Promotes Their Cleavage Of Nucleic Acid-Binding Protein Substrates

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3471-3471
Author(s):  
Jennifer Whangbo ◽  
Marshall Thomas ◽  
Geoffrey McCrossan ◽  
Aaron Deutsch ◽  
Kimberly Martinod ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nuclear Localization ◽  
Serine Proteases ◽  
Rna Binding ◽  
Nuclear Accumulation ◽  
Target Cells ◽  
Nucleic Acid Binding ◽  
High Affinity ◽  
Nucleic Acid Binding Proteins

Abstract When released from cytotoxic T lymphocytes and natural killer cells, Granzyme (Gzm) serine proteases induce programmed cell death of pathogen-infected cells and tumor cells. The Gzms rapidly accumulate in the target cell nucleus by an unknown mechanism. Many of the known substrates of GzmA and GzmB, the most abundant killer cell proteases, bind to DNA or RNA. Gzm substrates predicted by unbiased proteomics studies are also highly enriched for nucleic acid binding proteins. Here we show by fluorescence polarization assays that Gzms bind DNA and RNA with nanomolar affinity. We hypothesized that Gzm binding to nucleic acids enhances nuclear accumulation in target cells and facilitates their cleavage of nucleic acid-binding substrates. In fact, RNase treatment of cell lysates reduced cleavage of RNA binding protein (RBP) targets by GzmA and GzmB. Moreover, adding RNA to recombinant RBP substrates greatly enhanced in vitro cleavage by GzmB, but adding RNA to non-nucleic acid binding proteins did not. For example, exogenous RNA enhanced GzmB cleavage of recombinant hnRNP C1 (an RBP) but not LMNB1 (a non-RBP). In addition, GzmB cleaved the RNA-binding HuR protein efficiently only when it was bound to an HuR-binding RNA oligonucleotide, but not in the presence of an equal amount of non-binding RNA. Thus, nucleic acids facilitate Gzm cleavage of nucleic acid binding substrates. To evaluate whether nucleic acid binding influences Gzm trafficking in target cells, we incubated fixed target cells with RNase and then added Gzms. RNA degradation in target cells reduced Gzm cytosolic localization and increased nuclear accumulation. Similarly, pre-incubating Gzms with exogenous competitor DNA reduced Gzm nuclear localization. The Gzms form a monophyletic clade with other immune serine proteases including neutrophil elastase (NE) and cathepsin G (CATG). Upon neutrophil activation, NE translocates to the nucleus to drive the formation of neutrophil extracellular traps (NETs). NE and CATG, but not non-immune serine proteases such as trypsin and pancreatic elastase, also bind DNA with high affinity and localize to the nucleus of permeabilized cells. Consistent with this finding, competitor DNA also blocks the nuclear localization of NE. Moreover NE and CATG localization to NETs depends on DNA binding. Thus the antimicrobial activity of NETs may depend in part upon the affinity of these proteases for DNA. Our findings indicate that high affinity nucleic acid binding is a conserved and functionally important property of serine proteases involved in cell-mediated immunity. Disclosures: Lieberman: Alnylam Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.

Superfamily I helicases as modular components of DNA-processing machines

2011 ◽  
Vol 39 (2) ◽  
pp. 413-423 ◽  
Author(s):  
Mark S. Dillingham
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Molecular Mechanisms ◽  
Acid Metabolism ◽  
Nucleic Acid Metabolism ◽  
Cellular Functions ◽  
Dna Helicases ◽  
Wide Range ◽  
Look Back ◽  
Partner Proteins

Helicases are a ubiquitous and abundant group of motor proteins that couple NTP binding and hydrolysis to processive unwinding of nucleic acids. By targeting this activity to a wide range of specific substrates, and by coupling it with other catalytic functionality, helicases fulfil diverse roles in virtually all aspects of nucleic acid metabolism. The present review takes a look back at our efforts to elucidate the molecular mechanisms of UvrD-like DNA helicases. Using these well-studied enzymes as examples, we also discuss how helicases are programmed by interactions with partner proteins to participate in specific cellular functions.

scholarly journals Oligonucleotide-based systems: DNA, microRNAs, DNA/RNA aptamers

2016 ◽  
Vol 60 (1) ◽  
pp. 27-35 ◽  
Author(s):  
Pawan Jolly ◽  
Pedro Estrela ◽  
Michael Ladomery
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Rna Aptamers ◽  
Locked Nucleic Acid ◽  
Synthetic Analogue ◽  
Dna And Rna ◽  
Naturally Occurring ◽  
Wide Range ◽  
Synthetic Oligonucleotides ◽  
Shed Light

There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a synthetic analogue of naturally occurring nucleic acids. This review will shed light on various types of nucleic acids such as DNA and RNA (particularly microRNAs), their role and their application in biosensing. It will also cover DNA/RNA aptamers, which can be used as bioreceptors for a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the laboratory-based status and the reality of biomedical applications.

scholarly journals Tuning the Immunostimulation Properties of Cationic Lipid Nanocarriers for Nucleic Acid Delivery

2021 ◽  
Vol 12 ◽  
Author(s):  
Arindam K. Dey ◽  
Adrien Nougarède ◽  
Flora Clément ◽  
Carole Fournier ◽  
Evelyne Jouvin-Marche ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Messenger Rna ◽  
Cationic Lipid ◽  
Lipid Nanoparticles ◽  
Cationic Lipids ◽  
Fine Tuning ◽  
Target Cells ◽  
Substantial Impact ◽  
Lipid Nanocarriers ◽  
The Impact

Nonviral systems, such as lipid nanoparticles, have emerged as reliable methods to enable nucleic acid intracellular delivery. The use of cationic lipids in various formulations of lipid nanoparticles enables the formation of complexes with nucleic acid cargo and facilitates their uptake by target cells. However, due to their small size and highly charged nature, these nanocarrier systems can interact in vivo with antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. As this might prove to be a safety concern for developing therapies based on lipid nanocarriers, we sought to understand how they could affect the physiology of APCs. In the present study, we investigate the cellular and metabolic response of primary macrophages or DCs exposed to the neutral or cationic variant of the same lipid nanoparticle formulation. We demonstrate that macrophages are the cells affected most significantly and that the cationic nanocarrier has a substantial impact on their physiology, depending on the positive surface charge. Our study provides a first model explaining the impact of charged lipid materials on immune cells and demonstrates that the primary adverse effects observed can be prevented by fine-tuning the load of nucleic acid cargo. Finally, we bring rationale to calibrate the nucleic acid load of cationic lipid nanocarriers depending on whether immunostimulation is desirable with the intended therapeutic application, for instance, gene delivery or messenger RNA vaccines.

scholarly journals Non-Viral Delivery of RNA Gene Therapy to the Central Nervous System

Pharmaceutics ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 165
Author(s):  
Ellen S. Hauck ◽  
James G. Hecker
Keyword(s):  
Gene Therapy ◽  
Central Nervous System ◽  
Nervous System ◽  
Nucleic Acid ◽  
Gene Delivery ◽  
Dna Delivery ◽  
Clinical Applications ◽  
Cationic Lipids ◽  
Viral Dna ◽  
The Central Nervous System

Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Lipid-mediated nucleic acid delivery is an alternative to viral vector-mediated gene delivery and has the following advantages. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, since transit across the nuclear membrane is not necessary, and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Delivery of RNA to target organ(s) has previously been challenging due to RNA’s rapid degradation in biological systems, but cationic lipids complexed with RNA, as well as lipid nanoparticles (LNPs), have allowed for delivery and expression of the complexed RNA both in vitro and in vivo. This review will focus on the non-viral lipid-mediated delivery of RNAs, including mRNA, siRNA, shRNA, and microRNA, to the central nervous system (CNS), an organ with at least two unique challenges. The CNS contains a large number of slowly dividing or non-dividing cell types and is protected by the blood brain barrier (BBB). In non-dividing cells, RNA-lipid complexes demonstrated increased transfection efficiency relative to DNA transfection. The efficiency, timing of the onset, and duration of expression after transfection may determine which nucleic acid is best for which proposed therapy. Expression can be seen as soon as 1 h after RNA delivery, but duration of expression has been limited to 5–7 h. In contrast, transfection with a DNA lipoplex demonstrates protein expression within 5 h and lasts as long as several weeks after transfection.

scholarly journals Tuning the immunostimulation properties of cationic lipid nanocarriers for nucleic acid delivery

2021 ◽  
Author(s):  
Arindam K Dey ◽  
Adrien Nougarede ◽  
Flora Clement ◽  
Carole Fournier ◽  
Evelyne Jouvin-Marche ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Messenger Rna ◽  
Cationic Lipid ◽  
Lipid Nanoparticles ◽  
Cationic Lipids ◽  
Fine Tuning ◽  
Target Cells ◽  
Substantial Impact ◽  
Lipid Nanocarriers ◽  
The Impact

Nonviral systems, such as lipid nanoparticles, have emerged as reliable methods to enable nucleic acid intracellular delivery. The use of cationic lipids in various formulations of lipid nanoparticles enables the formation of complexes with nucleic acid cargo and facilitates their uptake by target cells. However, due to their small size and highly charged nature, these nanocarrier systems can interact in vivo with antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. As this might prove to be a safety concern for developing therapies based on lipid nanocarriers, we sought to understand how they could affect the physiology of APCs. In the present study, we investigate the cellular and metabolic response of primary macrophages or DCs exposed to the neutral or cationic variant of the same lipid nanoparticle formulation. We demonstrate that macrophages are the cells affected most significantly and that the cationic nanocarrier has a substantial impact on their physiology, depending on the positive surface charge. Our study provides a first model explaining the impact of charged lipid materials on immune cells and demonstrates that the primary adverse effects observed can be prevented by fine-tuning the load of nucleic acid cargo. Finally, we bring rationale to calibrate the nucleic acid load of cationic lipid nanocarriers depending on whether immunostimulation is desirable with the intended therapeutic application, for instance, gene delivery or messenger RNA vaccines.

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