Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 31
Filter
1.
Sci Rep ; 12(1): 5424, 2022 Mar 31.
Article in English | MEDLINE | ID: covidwho-1768856

ABSTRACT

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene "landing pad" composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies > 40% with up to ~ 43 kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~ 4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds.


Subject(s)
Bacteriophages , COVID-19 , Animals , Bacteriophages/genetics , DNA , Gene Transfer Techniques , Genetic Background , Integrases/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Inbred NOD , RNA, Viral , SARS-CoV-2
2.
Int J Mol Sci ; 23(6)2022 Mar 11.
Article in English | MEDLINE | ID: covidwho-1742489

ABSTRACT

The pandemic emergency determined by the spreading worldwide of the SARS-CoV-2 virus has focused the scientific and economic efforts of the pharmaceutical industry and governments on the possibility to fight the virus by genetic immunization. The genetic material must be delivered inside the cells by means of vectors. Due to the risk of adverse or immunogenic reaction or replication connected with the more efficient viral vectors, non-viral vectors are in many cases considered as a preferred strategy for gene delivery into eukaryotic cells. This paper is devoted to the evaluation of the gene delivery ability of new synthesized gemini bis-pyridinium surfactants with six methylene spacers, both hydrogenated and fluorinated, in comparison with compounds with spacers of different lengths, previously studied. Results from MTT proliferation assay, electrophoresis mobility shift assay (EMSA), transient transfection assay tests and atomic force microscopy (AFM) imaging confirm that pyridinium gemini surfactants could be a valuable tool for gene delivery purposes, but their performance is highly dependent on the spacer length and strictly related to their structure in solution. All the fluorinated compounds are unable to transfect RD-4 cells, if used alone, but they are all able to deliver a plasmid carrying an enhanced green fluorescent protein (EGFP) expression cassette, when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:2 ratio. The fluorinated compounds with spacers formed by six (FGP6) and eight carbon atoms (FGP8) give rise to a very interesting gene delivery activity, greater to that of the commercial reagent, when formulated with DOPE. The hydrogenated compound GP16_6 is unable to sufficiently compact the DNA, as shown by AFM images.


Subject(s)
DNA/genetics , Gene Transfer Techniques , Methane/chemistry , Pyridinium Compounds/chemistry , Surface-Active Agents/chemistry , Transfection/methods , A549 Cells , Cell Survival , DNA/chemistry , DNA/metabolism , Genetic Therapy/methods , Halogenation , Humans , Hydrogenation , Methane/metabolism , Microscopy, Atomic Force , Molecular Structure , Plasmids/chemistry , Plasmids/genetics , Plasmids/metabolism , Pyridinium Compounds/metabolism , Reproducibility of Results , Surface-Active Agents/metabolism
3.
Biomed Pharmacother ; 145: 112385, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1565522

ABSTRACT

Chemically modified mRNA represents a unique, efficient, and straightforward approach to produce a class of biopharmaceutical agents. It has been already approved as a vaccination-based method for targeting SARS-CoV-2 virus. The COVID-19 pandemic has highlighted the prospect of synthetic modified mRNA to efficiently and safely combat various diseases. Recently, various optimization advances have been adopted to overcome the limitations associated with conventional gene therapeutics leading to wide-ranging applications in different disease conditions. This review sheds light on emerging directions of chemically modified mRNAs to prevent and treat widespread chronic diseases, including metabolic disorders, cancer vaccination and immunotherapy, musculoskeletal disorders, respiratory conditions, cardiovascular diseases, and liver diseases.


Subject(s)
COVID-19/prevention & control , Chronic Disease/prevention & control , Chronic Disease/therapy , Genetic Therapy/methods , Immunotherapy/methods , Pandemics/prevention & control , RNA, Messenger/chemistry , SARS-CoV-2/immunology , Vaccines, Synthetic , Biological Availability , Drug Carriers , Forecasting , Gene Transfer Techniques , Genetic Vectors/administration & dosage , Genetic Vectors/therapeutic use , Humans , Immunotherapy, Active , RNA Stability , RNA, Messenger/administration & dosage , RNA, Messenger/immunology , RNA, Messenger/therapeutic use , SARS-CoV-2/genetics , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology , /immunology
5.
Bioelectrochemistry ; 144: 107994, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1499650

ABSTRACT

Gene therapies are revolutionizing medicine by providing a way to cure hitherto incurable diseases. The scientific and technological advances have enabled the first gene therapies to become clinically approved. In addition, with the ongoing COVID-19 pandemic, we are witnessing record speeds in the development and distribution of gene-based vaccines. For gene therapy to take effect, the therapeutic nucleic acids (RNA or DNA) need to overcome several barriers before they can execute their function of producing a protein or silencing a defective or overexpressing gene. This includes the barriers of the interstitium, the cell membrane, the cytoplasmic barriers and (in case of DNA) the nuclear envelope. Gene electrotransfer (GET), i.e., transfection by means of pulsed electric fields, is a non-viral technique that can overcome these barriers in a safe and effective manner. GET has reached the clinical stage of investigations where it is currently being evaluated for its therapeutic benefits across a wide variety of indications. In this review, we formalize our current understanding of GET from a biophysical perspective and critically discuss the mechanisms by which electric field can aid in overcoming the barriers. We also identify the gaps in knowledge that are hindering optimization of GET in vivo.


Subject(s)
Electroporation , Gene Transfer Techniques , Genetic Therapy , Animals , COVID-19/prevention & control , Electroporation/instrumentation , Electroporation/methods , Equipment Design , Gene Transfer Techniques/instrumentation , Genetic Therapy/methods , Humans , Vaccines, DNA/administration & dosage , Vaccines, DNA/genetics , Vaccines, DNA/therapeutic use , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/therapeutic use , /genetics , /therapeutic use
6.
Adv Drug Deliv Rev ; 179: 114007, 2021 12.
Article in English | MEDLINE | ID: covidwho-1482395

ABSTRACT

In recent years, nucleic acid-based therapeutics have gained increasing importance as novel treatment options for disease prevention and treatment. Synthetic messenger RNAs (mRNAs) are promising nucleic acid-based drugs to transiently express desired proteins that are missing or defective. Recently, synthetic mRNA-based vaccines encoding viral proteins have been approved for emergency use against COVID-19. Various types of vehicles, such as lipid nanoparticles (LNPs) and liposomes, are being investigated to enable the efficient uptake of mRNA molecules into desired cells. In addition, the introduction of novel chemical modifications into mRNAs increased the stability, enabled the modulation of nucleic acid-based drugs, and increased the efficiency of mRNA-based therapeutic approaches. In this review, novel and innovative strategies for the delivery of synthetic mRNA-based therapeutics for tissue regeneration are discussed. Moreover, with this review, we aim to highlight the versatility of synthetic mRNA molecules for various applications in the field of regenerative medicine and also discuss translational challenges and required improvements for mRNA-based drugs.


Subject(s)
Drug Delivery Systems , RNA, Messenger/administration & dosage , Regeneration , Regenerative Medicine/trends , Animals , COVID-19 Vaccines/administration & dosage , Gene Transfer Techniques , Genetic Therapy , Humans , RNA, Messenger/immunology
7.
Acc Chem Res ; 54(21): 4001-4011, 2021 11 02.
Article in English | MEDLINE | ID: covidwho-1475239

ABSTRACT

Since the U.S. Food and Drug Administration (FDA) granted emergency use authorization for two mRNA vaccines against SARS-CoV-2, mRNA-based technology has attracted broad attention from the scientific community to investors. When delivered intracellularly, mRNA has the ability to produce various therapeutic proteins, enabling the treatment of a variety of illnesses, including but not limited to infectious diseases, cancers, and genetic diseases. Accordingly, mRNA holds significant therapeutic potential and provides a promising means to target historically hard-to-treat diseases. Current clinical efforts harnessing mRNA-based technology are focused on vaccination, cancer immunotherapy, protein replacement therapy, and genome editing. The clinical translation of mRNA-based technology has been made possible by leveraging nanoparticle delivery methods. However, the application of mRNA for therapeutic purposes is still challenged by the need for specific, efficient, and safe delivery systems.This Account highlights key advances in designing and developing combinatorial synthetic lipid nanoparticles (LNPs) with distinct chemical structures and properties for in vitro and in vivo intracellular mRNA delivery. LNPs represent the most advanced nonviral nanoparticle delivery systems that have been extensively investigated for nucleic acid delivery. The aforementioned COVID-19 mRNA vaccines and one LNP-based small interfering RNA (siRNA) drug (ONPATTRO) have received clinical approval from the FDA, highlighting the success of synthetic ionizable lipids for in vivo nucleic acid delivery. In this Account, we first summarize the research efforts from our group on the development of bioreducible and biodegradable LNPs by leveraging the combinatorial chemistry strategy, such as the Michael addition reaction, which allows us to easily generate a large set of lipidoids with diverse chemical structures. Next, we discuss the utilization of a library screening strategy to identify optimal LNPs for targeted mRNA delivery and showcase the applications of the optimized LNPs in cell engineering and genome editing. Finally, we outline key challenges to the clinical translation of mRNA-based therapies and propose an outlook for future directions of the chemical design and optimization of LNPs to improve the safety and specificity of mRNA drugs. We hope this Account provides insight into the rational design of LNPs for facilitating the development of mRNA therapeutics, a transformative technology that promises to revolutionize future medicine.


Subject(s)
COVID-19 Vaccines/pharmacology , Gene Editing , Gene Transfer Techniques , Lipids/chemistry , Nanoparticles/chemistry , RNA, Messenger/pharmacology , COVID-19/drug therapy , COVID-19 Vaccines/chemistry , Genetic Therapy , Humans , RNA, Messenger/chemistry , SARS-CoV-2/drug effects
8.
Nucleic Acid Ther ; 31(5): 321-323, 2021 10.
Article in English | MEDLINE | ID: covidwho-1467290

ABSTRACT

The utilization of the mRNA-based Pfizer-BioNTech and Moderna coronavirus disease 2019 (COVID-19) vaccines represents the culmination of many years of nonviral nucleic acid delivery, but more importantly, they signify a massive clinical scientific success. Scientists working in the area of nucleic acid delivery using lipid nanoparticles will undoubtedly be energized by the success of these vaccines and begin to collect much needed data in the realm of nonviral-based RNA and DNA delivery, specifically, the use of lipid nanoparticles, the immune response, safety, and efficacy. It is easily conceivable that in the future we can utilize these data to help streamline our approach for the delivery of DNA for gene therapy and regulatory RNAs for therapeutic and regenerative medicine (ie, wound repair) applications.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , DNA/pharmacokinetics , Gene Transfer Techniques , RNA, Messenger/pharmacokinetics , Biotechnology/trends , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/biosynthesis , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/genetics , DNA/chemistry , Data Mining , Dependovirus/genetics , Dependovirus/immunology , Humans , Liposomes/chemistry , Liposomes/pharmacokinetics , Nanoparticles/administration & dosage , Nanoparticles/chemistry , RNA, Messenger/chemistry , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity
9.
Adv Drug Deliv Rev ; 179: 113998, 2021 12.
Article in English | MEDLINE | ID: covidwho-1465980

ABSTRACT

Gene therapy has been widely investigated for the treatment of genetic, acquired, and infectious diseases. Pioneering work utilized viral vectors; however, these are suspected of causing serious adverse events, resulting in the termination of several clinical trials. Non-viral vectors, such as lipid nanoparticles, have attracted significant interest, mainly due to their successful use in vaccines in the current COVID-19 pandemic. Although they allow safe delivery, they come with the disadvantage of off-target delivery. The application of ultrasound to ultrasound-sensitive particles allows for a direct, site-specific transfer of genetic materials into the organ/site of interest. This process, termed ultrasound-targeted gene delivery (UTGD), also increases cell membrane permeability and enhances gene uptake. This review focuses on the advances in ultrasound and the development of ultrasonic particles for UTGD across a range of diseases. Furthermore, we discuss the limitations and future perspectives of UTGD.


Subject(s)
Gene Transfer Techniques , Genetic Therapy/methods , Molecular Targeted Therapy/methods , Ultrasonics , Animals , COVID-19 , Humans , Liposomes , Nanoparticles
10.
Adv Drug Deliv Rev ; 177: 113930, 2021 10.
Article in English | MEDLINE | ID: covidwho-1355525

ABSTRACT

Messenger RNAs (mRNAs) present a great potential as therapeutics for the treatment and prevention of a wide range of human pathologies, allowing for protein replacement, vaccination, cancer immunotherapy, and genomic engineering. Despite advances in the design of mRNA-based therapeutics, a key aspect for their widespread translation to clinic is the development of safe and effective delivery strategies. To this end, non-viral delivery systems including peptide-based complexes, lipidic or polymeric nanoparticles, and hybrid formulations are attracting growing interest. Despite displaying somewhat reduced efficacy compared to viral-based systems, non-viral carriers offer important advantages in terms of biosafety and versatility. In this review, we provide an overview of current mRNA therapeutic applications and discuss key biological barriers to delivery and recent advances in the development of non-viral systems. Challenges and future applications of this novel therapeutic modality are also discussed.


Subject(s)
RNA, Messenger/administration & dosage , Animals , Drug Delivery Systems , Gene Transfer Techniques , Humans
11.
J Biol Chem ; 297(5): 101266, 2021 11.
Article in English | MEDLINE | ID: covidwho-1446794

ABSTRACT

Functional delivery of mRNA has high clinical potential. Previous studies established that mRNAs can be delivered to cells in vitro and in vivo via RNA-loaded lipid nanoparticles (LNPs). Here we describe an alternative approach using exosomes, the only biologically normal nanovesicle. In contrast to LNPs, which elicited pronounced cellular toxicity, exosomes had no adverse effects in vitro or in vivo at any dose tested. Moreover, mRNA-loaded exosomes were characterized by efficient mRNA encapsulation (∼90%), high mRNA content, consistent size, and a polydispersity index under 0.2. Using an mRNA encoding the red light-emitting luciferase Antares2, we observed that mRNA-loaded exosomes were superior to mRNA-loaded LNPs at delivering functional mRNA into human cells in vitro. Injection of Antares2 mRNA-loaded exosomes also led to strong light emission following injection into the vitreous fluid of the eye or into the tissue of skeletal muscle in mice. Furthermore, we show that repeated injection of Antares2 mRNA-loaded exosomes drove sustained luciferase expression across six injections spanning at least 10 weeks, without evidence of signal attenuation or adverse injection site responses. Consistent with these findings, we observed that exosomes loaded with mRNAs encoding immunogenic forms of the SARS-CoV-2 Spike and Nucleocapsid proteins induced long-lasting cellular and humoral responses to both. Taken together, these results demonstrate that exosomes can be used to deliver functional mRNA to and into cells in vivo.


Subject(s)
Exosomes/immunology , RNA, Messenger/genetics , SARS-CoV-2/immunology , Cells, Cultured , Gene Transfer Techniques , HEK293 Cells , Humans , Lipids/chemistry , Nanoparticles/chemistry , RNA, Messenger/immunology , SARS-CoV-2/genetics
12.
N Biotechnol ; 66: 25-35, 2022 Jan 25.
Article in English | MEDLINE | ID: covidwho-1428279

ABSTRACT

The aim of this survey is to identify and characterize new products in plant biotechnology since 2015, especially in relation to the advent of New Breeding Techniques (NBTs) such as gene editing based on the CRISPR-Cas system. Transgenic (gene transfer or gene silencing) and gene edited traits which are approved or marketed in at least one country, or which have a non-regulated status in the USA, are collected, as well as related patents worldwide. In addition, to shed light on potential innovation for Africa, field trials on the continent are examined. The compiled data are classified in application categories, including agronomic improvements, industrial use and medical use, namely production of recombinant therapeutic molecules or vaccines (including against Covid-19). The data indicate that gene editing appears to be an effective complement to 'classical' transgenesis, the use of which is not declining, rather than a replacement, a trend also observed in the patenting landscape. Nevertheless, increased use of gene editing is apparent. Compared to transgenesis, gene editing has increased the proportion of some crop species and decreased others amongst approved, non-regulated or marketed products. A similar differential trend is observed for breeding traits. Gene editing has also favored the emergence of new private companies. China, and prevalently its public sector, overwhelmingly dominates the patenting landscape, but not the approved/marketed one, which is dominated by the USA. The data point in the direction that regulatory environments will favor or discourage innovation.


Subject(s)
Gene Editing , Plant Breeding , Plants, Genetically Modified , Biotechnology , CRISPR-Cas Systems , Gene Transfer Techniques , Genome, Plant , Plants, Genetically Modified/genetics , Recombinant Proteins/biosynthesis , Vaccines/biosynthesis
13.
Int J Nanomedicine ; 16: 5981-6002, 2021.
Article in English | MEDLINE | ID: covidwho-1399046

ABSTRACT

Gene therapy is a promising approach for the treatment of several diseases, such as chronic or viral infections, inherited disorders, and cancer. The cellular internalization of exogenous nucleic acids (NA) requires efficient delivery vehicles to overcome their inherent pharmacokinetic drawbacks, e.g. electrostatic repulsions, enzymatic degradation, limited cellular uptake, fast clearance, etc. Nanotechnological advancements have enabled the use of polymer-based nanostructured biomaterials as safe and effective gene delivery systems, in addition to viral vector delivery methods. Among the plethora of polymeric nanoparticles (NPs), this review will provide a comprehensive and in-depth summary of the polyester-based nanovehicles, including poly(lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA) NPs, used to deliver a variety of foreign NA, e.g. short interfering RNA (siRNA), messenger RNA (mRNA), and plasmid DNA (pDNA). The article will review the versatility of polyester-based nanocarriers including their recent application in the delivery of the clustered, regularly-interspaced, short palindromic repeats/Cas (CRISPR/Cas) genome editing system for treating gene-related diseases. The remaining challenges and future trend of the targeted delivery of this revolutionary genome-editing system will be discussed. Special attention will be given to the pivotal role of nanotechnology in tackling emerging infections such as coronavirus disease 2019 (COVID-19): ground-breaking mRNA vaccines delivered by NPs are currently used worldwide to fight the pandemic, pushing the boundaries of gene therapy.


Subject(s)
COVID-19 , Nanoparticles , CRISPR-Cas Systems , Gene Transfer Techniques , Genetic Therapy , Humans , Polyesters , SARS-CoV-2
14.
Stem Cell Res Ther ; 11(1): 448, 2020 10 23.
Article in English | MEDLINE | ID: covidwho-1388825

ABSTRACT

Gene therapy is being investigated for a range of serious lung diseases, such as cystic fibrosis and emphysema. Recombinant adeno-associated virus (rAAV) is a well-established, safe, viral vector for gene delivery with multiple naturally occurring and artificial serotypes available displaying alternate cell, tissue, and species-specific tropisms. Efficient AAV serotypes for the transduction of the conducting airways have been identified for several species; however, efficient serotypes for human lung parenchyma have not yet been identified. Here, we screened the ability of multiple AAV serotypes to transduce lung bud organoids (LBOs)-a model of human lung parenchyma generated from human embryonic stem cells. Microinjection of LBOs allowed us to model transduction from the luminal surface, similar to dosing via vector inhalation. We identified the naturally occurring rAAV2 and rAAV6 serotypes, along with synthetic rAAV6 variants, as having tropism for the human lung parenchyma. Positive staining of LBOs for surfactant proteins B and C confirmed distal lung identity and suggested the suitability of these vectors for the transduction of alveolar type II cells. Our findings establish LBOs as a new model for pulmonary gene therapy and stress the relevance of LBOs as a viral infection model of the lung parenchyma as relevant in SARS-CoV-2 research.


Subject(s)
Dependovirus/genetics , Genetic Therapy/methods , Human Embryonic Stem Cells/cytology , Lung Diseases/therapy , Organoids/cytology , Cell Line , Dependovirus/immunology , Gene Transfer Techniques , Genetic Vectors/genetics , Humans , Lung/metabolism , Models, Biological , Parenchymal Tissue/cytology
15.
Molecules ; 26(2)2021 Jan 15.
Article in English | MEDLINE | ID: covidwho-1389464

ABSTRACT

Peptides are fragments of proteins that carry out biological functions. They act as signaling entities via all domains of life and interfere with protein-protein interactions, which are indispensable in bio-processes. Short peptides include fundamental molecular information for a prelude to the symphony of life. They have aroused considerable interest due to their unique features and great promise in innovative bio-therapies. This work focusing on the current state-of-the-art short peptide-based therapeutical developments is the first global review written by researchers from all continents, as a celebration of 100 years of peptide therapeutics since the commencement of insulin therapy in the 1920s. Peptide "drugs" initially played only the role of hormone analogs to balance disorders. Nowadays, they achieve numerous biomedical tasks, can cross membranes, or reach intracellular targets. The role of peptides in bio-processes can hardly be mimicked by other chemical substances. The article is divided into independent sections, which are related to either the progress in short peptide-based theranostics or the problems posing challenge to bio-medicine. In particular, the SWOT analysis of short peptides, their relevance in therapies of diverse diseases, improvements in (bio)synthesis platforms, advanced nano-supramolecular technologies, aptamers, altered peptide ligands and in silico methodologies to overcome peptide limitations, modern smart bio-functional materials, vaccines, and drug/gene-targeted delivery systems are discussed.


Subject(s)
Anti-Infective Agents/pharmacology , Antiviral Agents/pharmacology , Peptides/chemistry , Peptides/pharmacology , Peptides/therapeutic use , Amino Acids/chemistry , Anti-Infective Agents/chemistry , Antiviral Agents/chemistry , COVID-19/drug therapy , Computer Simulation , Cosmeceuticals/chemistry , Cosmeceuticals/therapeutic use , Dietary Supplements , Gene Transfer Techniques , Humans , Lactoferrin/chemistry , Lipid Bilayers , Nanostructures/administration & dosage , Nanostructures/chemistry , Peptides/administration & dosage , Stem Cells , Vaccines, Subunit/chemistry , Vaccines, Subunit/pharmacology
16.
Int J Mol Sci ; 22(14)2021 Jul 14.
Article in English | MEDLINE | ID: covidwho-1323263

ABSTRACT

Efficient delivery of genetic material into cells is a critical process to translate gene therapy into clinical practice. In this sense, the increased knowledge acquired during past years in the molecular biology and nanotechnology fields has contributed to the development of different kinds of non-viral vector systems as a promising alternative to virus-based gene delivery counterparts. Consequently, the development of non-viral vectors has gained attention, and nowadays, gene delivery mediated by these systems is considered as the cornerstone of modern gene therapy due to relevant advantages such as low toxicity, poor immunogenicity and high packing capacity. However, despite these relevant advantages, non-viral vectors have been poorly translated into clinical success. This review addresses some critical issues that need to be considered for clinical practice application of non-viral vectors in mainstream medicine, such as efficiency, biocompatibility, long-lasting effect, route of administration, design of experimental condition or commercialization process. In addition, potential strategies for overcoming main hurdles are also addressed. Overall, this review aims to raise awareness among the scientific community and help researchers gain knowledge in the design of safe and efficient non-viral gene delivery systems for clinical applications to progress in the gene therapy field.


Subject(s)
Gene Transfer Techniques , Genetic Diseases, Inborn/therapy , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Nanoparticles/administration & dosage , Animals , Genetic Diseases, Inborn/genetics , Genetic Vectors/genetics , Humans
17.
Adv Mater ; 33(34): e2101707, 2021 Aug.
Article in English | MEDLINE | ID: covidwho-1316189

ABSTRACT

The transfer of foreign synthetic messenger RNA (mRNA) into cells is essential for mRNA-based protein-replacement therapies. Prophylactic mRNA COVID-19 vaccines commonly utilize nanotechnology to deliver mRNA encoding SARS-CoV-2 vaccine antigens, thereby triggering the body's immune response and preventing infections. In this study, a new combinatorial library of symmetric lipid-like compounds is constructed, and among which a lead compound is selected to prepare lipid-like nanoassemblies (LLNs) for intracellular delivery of mRNA. After multiround optimization, the mRNA formulated into core-shell-structured LLNs exhibits more than three orders of magnitude higher resistance to serum than the unprotected mRNA, and leads to sustained and high-level protein expression in mammalian cells. A single intravenous injection of LLNs into mice achieves over 95% mRNA translation in the spleen, without causing significant hematological and histological changes. Delivery of in-vitro-transcribed mRNA that encodes high-affinity truncated ACE2 variants (tACE2v mRNA) through LLNs induces elevated expression and secretion of tACE2v decoys, which is able to effectively block the binding of the receptor-binding domain of the SARS-CoV-2 to the human ACE2 receptor. The robust neutralization activity in vitro suggests that intracellular delivery of mRNA encoding ACE2 receptor mimics via LLNs may represent a potential intervention strategy for COVID-19.


Subject(s)
COVID-19 Vaccines/genetics , Galactosidases/chemistry , Nanoparticles/chemistry , Phosphatidylethanolamines/chemistry , RNA, Messenger/chemistry , SARS-CoV-2/genetics , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/prevention & control , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/metabolism , Cell Membrane Permeability , Cell Survival/drug effects , Female , Galactosidases/metabolism , Gene Expression Regulation , Gene Transfer Techniques , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Phosphatidylethanolamines/metabolism , Protein Binding , RNA, Messenger/genetics
18.
Int J Mol Sci ; 21(19)2020 Oct 05.
Article in English | MEDLINE | ID: covidwho-1299431

ABSTRACT

CRISPR/Cas technologies have advanced dramatically in recent years. Many different systems with new properties have been characterized and a plethora of hybrid CRISPR/Cas systems able to modify the epigenome, regulate transcription, and correct mutations in DNA and RNA have been devised. However, practical application of CRISPR/Cas systems is severely limited by the lack of effective delivery tools. In this review, recent advances in developing vehicles for the delivery of CRISPR/Cas in the form of ribonucleoprotein complexes are outlined. Most importantly, we emphasize the use of extracellular vesicles (EVs) for CRISPR/Cas delivery and describe their unique properties: biocompatibility, safety, capacity for rational design, and ability to cross biological barriers. Available molecular tools that enable loading of desired protein and/or RNA cargo into the vesicles in a controllable manner and shape the surface of EVs for targeted delivery into specific tissues (e.g., using targeting ligands, peptides, or nanobodies) are discussed. Opportunities for both endogenous (intracellular production of CRISPR/Cas) and exogenous (post-production) loading of EVs are presented.


Subject(s)
Extracellular Vesicles/genetics , Gene Editing/trends , Gene Transfer Techniques , RNA/genetics , CRISPR-Cas Systems , Humans , Mutation/genetics
19.
Exp Biol Med (Maywood) ; 246(14): 1643-1649, 2021 07.
Article in English | MEDLINE | ID: covidwho-1201841

ABSTRACT

The year 2020 witnessed an unpredictable pandemic situation due to novel coronavirus (COVID-19) outbreaks. This condition can be more severe if the patient has comorbidities. Failure of viable treatment for such viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is due to lack of identification. Thus, modern and productive biotechnology-based tools are being used to manipulate target genes by introducing the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated) system. Moreover, it has now been used as a tool to inhibit viral replication. Hence, it can be hypothesized that the CRISPR/Cas system can be a viable tool to target both the SARS-CoV-2 genome with specific target RNA sequence and host factors to destroy the SARS-CoV-2 community via inhibition of viral replication and infection. Moreover, comorbidities and COVID-19 escalate the rate of mortality globally, and as a result, we have faced this pandemic. CRISPR/Cas-mediated genetic manipulation to knockdown viral sequences may be a preventive strategy against such pandemic caused by SARS-CoV-2. Furthermore, prophylactic antiviral CRISPR in human cells (PAC-MAN) along with CRISPR/Cas13d efficiently degrades the specific RNA sequence to inhibit viral replication. Therefore, we suggest that CRISPR/Cas system with PAC-MAN could be a useful tool to fight against such a global pandemic caused by SARS-CoV-2. This is an alternative preventive approach of management against the pandemic to destroy the target sequence of RNA in SARS-CoV-2 by viral inhibition.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/virology , Gene Transfer Techniques , Host-Pathogen Interactions/physiology , SARS-CoV-2/genetics , Antiviral Agents/administration & dosage , COVID-19/epidemiology , COVID-19/genetics , CRISPR-Cas Systems , Gene Editing/methods , Host-Pathogen Interactions/genetics , Humans , RNA, Guide/administration & dosage , RNA, Guide/pharmacology , SARS-CoV-2/pathogenicity
SELECTION OF CITATIONS
SEARCH DETAIL