Development Of Non-viral Vectors Research Articles

Nanoplatform-Based In Vivo Gene Delivery Systems for Cancer Therapy.

Gene therapy uses modern molecular biology methods to repair disease-causing genes. As a burgeoning therapeutic, it has been widely applied for cancer therapy. Since 1989, there have been numerous clinical gene therapy cases worldwide. However, a few are successful. The main challenge of clinical gene therapy is the lack of efficient and safe vectors. Although viral vectors show high transfection efficiency, their application is still limited by immune rejection and packaging capacity. Therefore, the development of non-viral vectors is overwhelming. Nanoplatform-based non-viral vectors become a hotspot in gene therapy. The reasons are mainly as follows. 1) Non-viral vectors can be engineered to be uptaken by specific types of cells or tissues, providing effective targeting capability. 2) Non-viral vectors can protect goods that need to be delivered from degradation. 3) Nanoparticles can transport large-sized cargo such as CRISPR/Cas9 plasmids and nucleoprotein complexes. 4) Nanoparticles are highly biosafe, and they are not mutagenic in themselves compared to viral vectors. 5) Nanoparticles are easy to scale preparation, which is conducive to clinical conversion and application. Here, an overview of the categories of nanoplatform-based non-viral gene vectors, the limitations on their development, and their applications in cancer therapy.

Designed Peptide Assemblies for Efficient Gene Delivery.

The safe and efficient delivery of nucleic acids including DNA, mRNA, siRNA, and miRNA into targeted cells is critical for gene therapy. Currently, viral gene vectors are very popular, but they have potential toxicity and insecurity. Therefore, the development of nonviral vectors has attracted considerable research attention. Peptide assemblies are superior candidates for being used as gene vectors by having good biocompatibility, versatile molecular design, excellent assembly capacity, ease of modification, and stimuli responsivity. The de novo designed peptides not only can induce efficient condensation of nucleic acids into compacted nanoparticles and protect them from enzymatic digestion but also can effectively overcome biological barriers and improve gene delivery efficiency through targeted delivery, enhanced cellular uptake, improved endolysosomal escape, and nuclear importation. By having these merits, peptidic gene vectors are developing fast, showing outstanding advantages compared to liposome and polymer vectors. This Perspective focuses on peptidic gene delivery systems by emphasizing the molecular design strategies for meeting the criteria of gene condensation, protection from nuclease degradation, cellular uptake, endolysosomal escape, and so on. The new arising research area of peptide-based artificial viruses for gene and ribonucleoprotein delivery has also been reviewed. The challenges and future perspectives are put forward, aiming to provide a conclusive guide for the development of peptidic delivery systems to achieve efficient gene therapy.

A Quality by Design Approach in Pharmaceutical Development of Non-Viral Vectors with a Focus on miRNA.

Cancer is the leading cause of death worldwide. Tumors consist of heterogeneous cell populations that have different biological properties. While conventional cancer therapy such as chemotherapy, radiotherapy, and surgery does not target cancer cells specifically, gene therapy is attracting increasing attention as an alternative capable of overcoming these limitations. With the advent of gene therapy, there is increasing interest in developing non-viral vectors for genetic material delivery in cancer therapy. Nanosystems, both organic and inorganic, are the most common non-viral vectors used in gene therapy. The most used organic vectors are polymeric and lipid-based delivery systems. These nanostructures are designed to bind and protect the genetic material, leading to high efficiency, prolonged gene expression, and low toxicity. Quality by Design (QbD) is a step-by-step approach that investigates all the factors that may affect the quality of the final product, leading to efficient pharmaceutical development. This paper aims to provide a new perspective regarding the use of the QbD approach for improving the quality of non-viral vectors for genetic material delivery and their application in cancer therapy.

Development of non-viral vectors for neuronal-targeted delivery of CRISPR-Cas9 RNA-proteins as a therapeutic strategy for neurological disorders.

The aging population contributes to an increase in the prevalence of neurodegenerative diseases, such as Parkinson's disease (PD). Due to the progressive nature of these diseases and an incomplete understanding of their pathophysiology, current drugs are inefficient, with a limited efficacy and major side effects. In this study, CRISPR-Cas9 RNA-proteins (RNP) composed of a Cas9 nuclease and single-guide RNA were delivered with a non-viral targeted delivery system to rescue the PD-associated phenotype in neuronal cells. Here, we fused the cell-penetrating amphipathic peptide, PepFect14 (PF14), with a short fragment of the rabies virus glycoprotein (C2) previously shown to have an affinity towards nicotinic acetylcholine receptors expressed on neuronal cells and on the blood-brain barrier. The resultant peptide, C2-PF14, was used to complex with and deliver RNPs to neuronal cells. We observed that RNP/C2-PF14 complexes formed nanosized, monodispersed, and nontoxic nanoparticles that led to a specific delivery into neuronal cells. α-Synuclein (α-syn) plays a major role in the pathology of PD and is considered to be a target for therapy. We demonstrated that CRISPR/Cas9 RNP delivered by C2-PF14 achieved α-syn gene (SNCA) editing in neuronal cells as determined by T7EI assay and western blotting. Furthermore, RNP/C2-PF14 relieved PD-associated toxicity in neuronal cells in vitro. This is a proof-of-concept towards simple and safe targeted genome-editing for treating PD and other neurological disorders.

Dendrimers as Non-Viral Vectors in Gene-Directed Enzyme Prodrug Therapy.

Gene-directed enzyme prodrug therapy (GDEPT) has been intensively studied as a promising new strategy of prodrug delivery, with its main advantages being represented by an enhanced efficacy and a reduced off-target toxicity of the active drug. In recent years, numerous therapeutic systems based on GDEPT strategy have entered clinical trials. In order to deliver the desired gene at a specific site of action, this therapeutic approach uses vectors divided in two major categories, viral vectors and non-viral vectors, with the latter being represented by chemical delivery agents. There is considerable interest in the development of non-viral vectors due to their decreased immunogenicity, higher specificity, ease of synthesis and greater flexibility for subsequent modulations. Dendrimers used as delivery vehicles offer many advantages, such as: nanoscale size, precise molecular weight, increased solubility, high load capacity, high bioavailability and low immunogenicity. The aim of the present work was to provide a comprehensive overview of the recent advances regarding the use of dendrimers as non-viral carriers in the GDEPT therapy.

Application of Non-Viral Vectors in Drug Delivery and Gene Therapy.

Vectors and carriers play an indispensable role in gene therapy and drug delivery. Non-viral vectors are widely developed and applied in clinical practice due to their low immunogenicity, good biocompatibility, easy synthesis and modification, and low cost of production. This review summarized a variety of non-viral vectors and carriers including polymers, liposomes, gold nanoparticles, mesoporous silica nanoparticles and carbon nanotubes from the aspects of physicochemical characteristics, synthesis methods, functional modifications, and research applications. Notably, non-viral vectors can enhance the absorption of cargos, prolong the circulation time, improve therapeutic effects, and provide targeted delivery. Additional studies focused on recent innovation of novel synthesis techniques for vector materials. We also elaborated on the problems and future research directions in the development of non-viral vectors, which provided a theoretical basis for their broad applications.

How Far Are Non-Viral Vectors to Come of Age and Reach Clinical Translation in Gene Therapy?

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.

In silico identification and experimental validation of cellular uptake by a new cell penetrating peptide P1 derived from MARCKS

Viral vectors for vaccine delivery are challenged by recently reported safety issues like immunogenicity and risk for cancer development, and thus there is a growing need for the development of non-viral vectors. Cell penetrating peptides (CPPs) are non-viral vectors that can enter plasma membranes efficiently and deliver a broad range of cargoes. Our bioinformatic prediction and wet-lab validation data suggested that peptide P1 derived from MARCKS protein phosphorylation site domain is a new potential CPP candidate. We found that peptide P1 can efficiently internalize into various cell lines in a concentration-dependent manner. Receptor-mediated endocytosis pathway is the major mechanism of P1 penetration, although P1 also directly penetrates the plasma membrane. We also found that peptide P1 has low cytotoxicity in cultured cell lines as well as mouse red blood cells. Furthermore, peptide P1 not only can enter into cultured cells itself, but it also can interact with plasmid DNA and mediate the functional delivery of plasmid DNA into cultured cells, even in hard-to-transfect cells. Combined, these findings indicate that P1 may be a promising vector for efficient intracellular delivery of bioactive cargos.

Oncogenic Epidermal Growth Factor Receptor Silencing in Cervical Carcinoma Mediated by Dynamic Sugar-Benzoxaborole Polyplexes.

Although, various types of pharmaceuticals have been developed for cervical carcinomas, treatment with these drugs often results in a number of undesirable side effects, toxicity and multidrug resistance. Here, we aimed at modifying the genetic profiling of cancer cells by silencing the expression of the epidermal growth factor receptor (EGFR) gene. We have synthesized two kinds of RAFT-made, biocompatible, and cationic polymers for the encapsulation of silencing RNA (siRNA). This vector has a dual capability: it contains a cationic segment to complex with the siRNA and an omega-end modified with an oxaborole group via thiol-ene click chemistry that responds to the acidic tumor microenvironment. This structural innovation enables this macromolecule to interact with multiple polyplexes and release the siRNA in a mild acidic environment. A strategy that has shown enhanced gene silencing without elevating the cytotoxicity of the system, as determined by Western blot analysis. The success of this approach has afforded further interest in utilizing boron-carbohydrate interaction in the development of nonviral vectors for gene therapy.

Design of non-viral vector with improved regulatory features towards therapeutic application.

Viral vectors based gene therapy is often compromised by adverse immunological reactions raising safety concerns. Hence, improved design and development of non-viral vectors with strong regulatory regions is desired. We describe the design of a non-viral mammalian expression vector in which the primary transgene (a truncated dystrophin gene linked with Duchenne muscular dystrophy (DMD)) named microdystrophin delR4-R23/delCT (MD1) is under the transcriptional control of elements of desmin locus control region (DES-LCR). The designed vector, named as DES-LCR/MD1-EGFP, was constructed by cloning two fragments into the pBluescript backbone. Fragment 1 contains DES-LCR enhancer and DES-LCR promoter region while fragment 2 contains MD1 transgene and reporter EGFP (enhanced green fluorescent protein) gene separated by linker P2A (2A peptide). This vector design provides a framework for strong regulation with non-viral features. This design forms the foundation for application in conditions linked to multisystem diseases.

Research progress on non-viral vectors of CRISPR/Cas9 system

In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system has become one of the most powerful technologies in biomedical research and shown great potentials in the establishment of disease models as well as the development of new treatments. However, efficient delivery of CRISPR/Cas9 systems to target organs and cells remains challenging. With the rapid development of non-viral vectors, nanocarriers based on liposomes, polymers, and inorganic nanoparticles have shown great potentials for the delivery of CRISPR/Cas9 systems. In this review, we first summarize the potential of the CRISPR/Cas9 system in disease modeling and disease treatment, as well as the limitations. The advantages and disadvantages of delivering the CRISPR/Cas9 system in the form of plasmid DNA, mRNA or protein are then analyzed and the non-viral vectors that have emerged are reviewed. Finally, we summarize the key barriers currently faced by non-viral vectors in the delivery of CRISPR/Cas9 systems and propose strategies that are expected to overcome them.

Synthesis and Characterization of Quaternized Poly(β-amino ester) for Highly Efficient Delivery of Small Interfering RNA

The development of non-viral vectors for gene delivery has gained attention over the past decades. Specifically, poly(β-amino ester) (PBAE) has shown great potential for improving the delivery of gene therapeutics. It has been observed that low-molecular-weight PBAE displayed low transfection activities, while quaternization could enhance the transgene expression efficacy of PBAE. Herein, PBAE quaternary ammonium salt (PBAEQAS) was synthesized to increase the positive charge of the polymers, which resulted in an increase in siRNA binding efficiency based on self-assembly electrostatic interaction. Specifically, the nanoparticle surface was positively charged, which increased the uptake ability of siRNA. Compared with acrylate-PBAEQAS/siNC nanoparticles and amine-PBAEQAS/siNC nanoparticles, acrylate-PBAEQAS/siSurvivin nanoparticles and amine-PBAEQAS/siSurvivin nanoparticles induced more-efficient cell apoptosis and gene silencing. All these results suggest that PBAEQAS would be a promising gene delivery vector for cancer treatment.

Recent Advances in Nucleic Acid-Based Delivery: From Bench to Clinical Trials in Genetic Diseases.

Delivery of nucleic acids is the most promising therapy for many diseases that remain untreatable. Therefore, many research efforts have been put on finding a safe and efficient delivery system able to provide a sustained response. Viral vectors have proved to be the most efficient for delivery of nucleic acids and, thus, stand as the foremost vector used in current clinical trials. However, safety issues arise as a main concern and mitigate their use, impelling the improvement of non-viral alternatives. This review focuses on the recent advances in pre-clinical development of non-viral polyplexes and lipoplexes for nucleic acid-based delivery, in contrast with vectors being used in present clinical trials. Nucleic acid vectors for neurodegenerative ataxias, Parkinson's disease, retinitis pigmentosa, cystic fibrosis, hemophilia, pancreatic and lung cancer, and rheumatoid arthritis are discussed to illustrate current state of pre-clinical and clinical studies. Thereby, denoting the prospects for treatment of genetic diseases and elucidating the trend in non-viral vector development and improvement which is expected to significantly increase disease rescue exceeding the modest clinical successes observed so far.

Graphene Nanoribbon-Based Platform for Highly Efficacious Nuclear Gene Delivery.

Current efforts in the design and development of nonviral vectors for gene delivery and transfection have focused on the development of versatile agents that can load short or large sized genetic material, and are efficacious without eliciting toxicity in dividing and nondividing cells. Herein, we have investigated oxidized graphene nanoribbons (O-GNRs) as nonviral vectors for gene therapy and report in vitro studies that detail their cytotoxicity, intracellular and nuclear uptake, and gene delivery and transfection efficiencies. Our results indicate that, without additional functionalization with positively charged groups or other nonviral vectors, O-GNRs could load large amounts of small-sized single-stranded or large-sized double stranded genetic materials. O-GNRs at potential therapeutic doses (20-60 μg/mL) elicited lower cytotoxicity compared to widely used commercial nonviral gene delivery vectors (Polyethylenimine and Fugene 6). The O-GNR-plasmid DNA complexes showed uptake into vesicular structures of dividing Henrietta Lacks (HeLa) and nondividing Human umbilical vein endothelial cells (HUVEC), release into the cell's cytoplasm and entry into the nucleus. In these cells, O-GNRs loaded with enhanced green fluorescence protein (EGFP) plasmid or siRNA against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed a concentration- and time- dependent increase in gene delivery and gene transfection efficiencies up to 96-98%. The results suggest that O-GNRs are promising candidates as versatile and efficient nonviral vectors of small- or large-sized genetic material in primary and secondary cell types for gene therapy.

RETRACTED: Development of Nonviral Vectors Targeting the Brain as a Therapeutic Approach For Parkinson's Disease and Other Brain Disorders

Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by tremor, rigidity, bradykinesia, and postural instability, for which there is no effective treatment available till date. Here, we report the development of nonviral vectors specific for neuronal cells that can deliver short interfering RNA (siRNA) against the α-synuclein gene (SNCA), and prevent PD-like symptoms both in vitro and in vivo. These vectors not only help siRNA duplexes cross the blood-brain barrier in mice, but also stabilize these siRNAs leading to a sustainable 60-90% knockdown of α-synuclein protein. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine rapidly develop PD-like symptoms which were significantly alleviated when SNCA was knocked down using our vectors. Together, our data not only confirm the central role of α-synuclein in the onset of PD, but also provide a proof of principle that these nonviral vectors can be used as novel tools to design effective strategies to combat central nervous system diseases.

RETRACTED: Corrigendum to “Development of Nonviral Vectors Targeting the Brain as a Therapeutic Approach For Parkinson's Disease and Other Brain Disorders”

RETRACTED: Corrigendum to “Development of Nonviral Vectors Targeting the Brain as a Therapeutic Approach For Parkinson's Disease and Other Brain Disorders”

Cytoplasmic dynein associated peptide-linked nanoparticles for non-viral vector applications

Event Abstract Back to Event Cytoplasmic dynein associated peptide-linked nanoparticles for non-viral vector applications Ercia Carbone1, Komal Rajpura1, Ho Man Kan1, Tao Jiang1 and Wai Hong Lo1 1 University of Connecticut Health Center, Institute for Regenerative Engineering, United States Statement of Purpose: Poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NP) hold great promise for gene delivery applications. Non-viral polymeric nanoscale vectors offer several advantages over viral vectors[1]. However, most non-viral vectors suffer from significantly lower transfection efficiencies than viral vectors. This is because during their way to the nuclei of cells, non-viral vectors must overcome a series of diffusional and enzymatic barriers [1]. Interestingly, it has been shown that a number of viruses exploit microtubules by interacting with cytoplasmic dynein motor protein (a motor protein responsible for retrograde movement along microtubules) in order to enter the nucleus of the host cell. Cytoplasmic dynein is a large, multisubunit molecular motor that translocates various cargoes toward the nucleus. The consensus peptide sequence “K/R-X-T-Q-T” has been shown to be important for interacting with one of the dynein subunits [2]. Here, we develop dynein associated peptide-linked polymeric nanoparticles for non-viral vector applications. It is hypothesized that the novel dynein peptide-linked nanoparticles will hijack the endogenous dynein motor system intracellularly, and consequently move along microtubules toward the nucleus (-ve end of microtubule) (Fig.1). Methods: Dynein-associated peptide “LysGluThrGlnThrCys (KETQTC)” and the control peptide “LysGluThrAlaThrCys (KETATC)” were synthesized and labeled with FITC commercially at the N-terminus of the peptides (LifeTein, Inc). Note that the additional Cys residue is to facilitate the conjugation of the Asp to PLGA NPs; Maleimide-PEG-PLGA and Methoxy-PEG-PLGA were purchased from Polyscitech. This NP was fabricated using a water-in-oil-in-water (W/O/W) double emulsion technique using a mixture of 1:9 (Maleimide-PEG-PLGA : Methoxy-PEG-PLGA) [3]. The particle size distribution and zeta potential measurements of the NPs were determined by dynamic laser scattering. Conjugation of the peptides to the NP was performed as previously described [3]. Equal amounts of peptide-linked NPs or control peptide-linked NPs were mixed with purified dynein LC8 protein (1 mg) (ProSpec) for 1 hour at 4°C. The complex, i.e. peptide-linked NPs bound to LC8, was precipitated by centrifugation. The precipitated NPs were washed 3x with PBS and subsequently boiled with 2x sample buffer (Bio-rad). The proteins were resolved by SDS-PAGE (Bio-rad) and subsequently analyzed by immunoblot analysis with anti-LC8 antibody (Millipore). Human MG63 osteoblasts (ATCC) were cultured on coverslips with dynein peptide-linked NPs or control peptide-linked NPs for various time points. After removing the NPs and washing the coverslips 4x with PBS, the cells were fixed with 4% PFA (Santa Cruz). Nuclei were stained with popdium iodide (PI) nucleic acid stain. Cells were then mounted by a mounting solution, and fluorescent signals on the cells were observed via confocal microscopy (Zeiss LSM ConfoCor 3). Results: The average diameter of the NPs is 143.8 nm and the zeta potential is -26.43 mV. Biochemical binding analysis showed that purified recombinant dynein LC8 protein efficiently binds to KETQT-NPs. In contrast, LC8 protein does not bind well to the control peptide (KETAT)-NPs (Fig.2A). We further demonstrated that the KETQT-NPs can interact with endogenous dynein LC8 protein in MG63 cell lysates (Fig. 2B). To investigate the localization of the peptide-NPs in cells, we incubated the NPs with cells for various time points. Figure 2C shows that the FITC signals from KETQT-NPs (green) are colocalized with cells’ nuclei (red), whereas the FITC signals from the control NPs do not. Conclusions: Taken together, our data demonstrated that the KETQT-NPs can bind to dynein subunit (LC8) in vitro and in vivo, and these NPs can be enriched in nuclei region when they were incubated with cell culture. Future research including live-cell imaging will be used to trace the movement of the NPs in cells. Our dynein associated NPs may have a significant impact in non-viral vector development for gene therapy applications. This work was supported by funding from the State of Connecticut Stem Cell Research Foundation.

The influence of the polar head-group of synthetic cationic lipids on the transfection efficiency mediated by niosomes in rat retina and brain

The development of novel non-viral delivery vehicles is essential in the search of more efficient strategies for retina and brain diseases. Herein, optimized niosome formulations prepared by oil-in water (o/w) and film-hydration techniques were characterized in terms of size, PDI, zeta potential, morphology and stability. Three ionizable glycerol-based cationic lipids containing a primary amine group (lipid 1), a triglycine group (lipid 2) and a dimethylamino ethyl pendent group (lipid 3) as polar head-groups were part of such niosomes. Upon the addition of pCMS-EGFP plasmid, nioplexes were obtained at different cationic lipid/DNA ratios (w/w). The resultant nioplexes were further physicochemically characterized and evaluated to condense, release and protect the DNA against enzymatic digestion. In vitro experiments were performed to evaluate transfection efficiency and cell viability in HEK-293, ARPE-19 and PECC cells. Interestingly, niosome formulations based on lipid 3 showed better transfection efficiencies in ARPE-19 and PECC cells than the rest of cationic lipids showed in this study. In vivo experiments in rat retina after intravitreal and subretinal injections together with in rat brain after cerebral cortex administration showed promising transfection efficiencies when niosome formulations based on lipid 3 were used. These results provide new insights for the development of non-viral vectors based on cationic lipids and their applications for efficient delivery of genetic material to the retina and brain.

Challenges in CRISPR/CAS9 Delivery: Potential Roles of Nonviral Vectors.

CRISPR/Cas9 genome editing platforms are widely applied as powerful tools in basic research and potential therapeutics for genome regulation. The appropriate alternative of delivery system is critical if genome editing systems are to be effectively performed in the targeted cells or organisms. To date, the in vivo delivery of the Cas9 system remains challenging. Both physical methods and viral vectors are adopted in the delivery of the Cas9-based gene editing platform. However, physical methods are more applicable for in vitro delivery, while viral vectors are generally concerned with safety issues, limited packing capacities, and so on. With the robust development of nonviral drug delivery systems, lipid- or polymer-based nanocarriers might be potent vectors for the delivery of CRISPR/Cas9 systems. In this review, we look back at the delivery approaches that have been used for the delivery of the Cas9 system and outline the recent development of nonviral vectors that might be potential carriers for the genome editing platform in the future. The efforts in optimizing cationic nanocarriers with structural modification are described and promising nonviral vectors under clinical investigations are highlighted.

Anatomy of plasmid DNAs with anti-silencing elements

The transience of transgene expression is a major obstacle in the development of nonviral vectors. The CpG-free and pLIVE plasmids reportedly achieve long-term transgene expression in mouse liver. In this work, the anti-silencing elements within these plasmids were studied. The effects of plasmid that was being silenced on transgene expression from the CpG-free plasmid and those of transgene expression at early time points on silencing were also examined. The results suggested that the backbone sequence of the CpG-free plasmid and the 3' untranslated region of the albumin gene of the pLIVE plasmid contribute to durable expression. In addition, no influence of the silencing of another plasmid on the duration of CpG-free plasmid expression or of transgene expression at early time points on silencing was detected.