Nucleic Acid Vectors Research Articles

BioSpotlight

BioTechniquesVol. 50, No. 3 BioSpotlightOpen AccessBioSpotlightPatrick C.H. Lo & Kristie NyboPatrick C.H. LoSearch for more papers by this author & Kristie NyboSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000113620AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Diving BAC into the Pool with MicroarraysSecond-generation sequencing with its short read lengths is not practicable when determining whole-genome sequences from species possessing large genomes with a high content of repetitive DNA. Realistically, reference sequences for these species can only be obtained using physical map–based sequencing of BAC clones. A key step to this genome sequencing methodology is the ordering and orientation of BAC clones through their anchoring to a genetic map. This approach typically requires screening BAC libraries for clones with specific gene sequences using DNA hybridization or PCR, rendering it low-throughput and labor-intensive. In this issue, P. Hedley and colleagues at the Scottish Crop Research Institute (Dundee, Scotland) describe a new strategy for the high-throughput addressing of thousands of genes to thousands of BACs in parallel using a novel application of DNA microarray technology developed for the unsequenced 5.3-Gb genome of barley. Whole genome–amplified DNA derived from multidimensional pools of barley BAC clones was screened on a custom microarray of 42,000 expressed barley genes. The BACs were pooled using a previously developed “matrix” pooling strategy whereby the location of a clone along a particular dimension (plate, row, or column of 384-well plates) of a Super Pool (SP) of seven 384-well plates could be determined by the presence of positive signals in two separate matrix pools in that dimension. As a proof of principle, the authors tested 2 SPs out of the 55 SPs from a barley BAC library with ~148,000 clones. All matrix pools from the first SP were labeled with Cy3 while those of the second SP were labeled with Cy5. Pairs of labeled matrix pools, one from each SP, were then cohybridized to a microarray. Signals extracted from the scanned images of the hybridized microarrays were analyzed using customized scripts to first identify those probes with a high signal in only two matrix pools in a particular dimension, and then to combine the information from all three dimensions for deconvolution and identification of specific BAC clones containing the microarray probe sequence. This cost-effective, high-throughput, and accurate method for anchoring BACs should be applicable to any species where a whole-genome sequence is lacking.(See “Highly parallel gene-to-BAC addressing using microarrays”)Magnetic AttractionDiscovering the molecular mechanisms governing the differentiation of neural stem cells (NSCs) is essential before these cells can be used to develop therapies for neurodegenerative diseases. For those researchers wishing study specific genes that could induce or suppress differentiation of NSCs, an obstacle has been the difficulty in transfecting NSCs using conventional methods. Nucleofection and lipofection are commonly used for delivering genes to cells, but when applied to NSCs, they exhibit extensive toxicity soon after transfection and may interfere with studies of differentiation. While nucleofection does result in a reasonable percentage of transfected cells, it is costly and most cells die during electroporation. Writing in this issue of BioTechniques, Sapet et al. from OZ Biosciences (Marseille, France) along with colleagues from the National Center for Scientific Research in Paris demonstrate that magnetofection is a viable alternative method for efficiently delivering DNA to NSCs that minimizes undesired toxic effects and does not appear to affect differentiation. Magnetofection is accomplished by associating nucleic acid vectors carrying the desired sequences with super-paramagnetic nanoparticles, which are added to a cell culture. When a magnetic field is then applied to the culture particles rapidly accumulate around the cells, leading to uptake of the DNA. The authors present an optimized protocol for transfecting NSCs using magnetofection, and show that the magnetofection method can be used to identify genes involved in the neuronal differentiation of NSCs. Because of the simplicity, low cost, reduced toxicity, and reliability of this method, the approach has the potential to be valuable for both small scale and high throughput projects seeking to identify new regulators of neuronal differentiation facilitating understanding of the differentiation of these cells and adding further insight into their potential for therapeutic development.Neural stem cell differentiation.Images of neural stem cells acquired 4 days after NeuroMag transfection of a mix between a GFP vector together with either an empty vector (A) or a NeuroD1 expressing vector (B).(See “High transfection efficiency of neural stem cells with magnetofection”)FiguresReferencesRelatedDetails Vol. 50, No. 3 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print March 2011 Information© 2011 Author(s)PDF download

Magnétofection™ in vitro et in vivo: une voie vers la thérapie génique

Gene therapy offers exciting opportunities for the treatment of innate or acquired genetic diseases. However, there is still a need for a safe and efficient strategy to deliver nucleic acids into cells while overcoming the current limitations faced with standard viral vectors. Intensive researches have been carried out over the past decade, focusing both on viral and non-viral (i.e. physical or chemical) strategies. Of these numerous attempts, magnetofection, defined as the combination of nucleic acid vectors with magnetic nanoparticles, holds the promise to achieve high transfection efficiency with reduced toxicity by magnetically focusing the genetic material to be delivered on its cellular target. In vitro as well as in vivo results already demonstrated that this strategy may become a valuable tool towards practical gene therapy.

New Degradable Cationic Lipid-DNA Complexes for Gene Delivery

Cationic lipids (CLs) continue to attract attention as synthetic nucleic acid (NA) vectors, and are broadly used for gene transfection and silencing including applications in clinical trials. However, these complexes exhibit suboptimal gene expression due to inefficiencies in overcoming the cellular barriers to transfection (Ahmad et al., J. Gene Med. 2005, 7, 739). After entry of the CL-DNA complex into the cell, two major barriers are efficient dissociation of DNA from the complex and the cytotoxicity of CLs. To address both these issues, we have synthesized a novel series of multi¬valent lipids (CMVLs) with degradable disulfide bonds linking the positively charged headgroups of the CMVLs to their hydrophobic tails. The linker is designed to be cleaved in the reducing milieu of the cytoplasm thus facilitating CL-NA complex degradation, reducing cytotoxicity and improving NA release. X-ray scattering demonstrates that CMVLs form lamellar complexes with DNA, which degrade in reducing environments mimicking the cytoplasmic milieu. For lipids with highly charged headgroups such as CMVL5 (5+), X-ray diffraction under reducing conditions shows DNA condensed by the cleaved headgroup. No such condensation is observed for smaller headgroup charge, as in CMVL2 (2+). Most significantly, we observed an unexpectedly large reduction in cytotoxicity of degradable CMVL-vectors compared to vectors prepared from corresponding lipids without degradable bonds (MVLs). This is of particular importance for applications in gene silencing, where the delivery of short interfering RNAs (siRNAs) requires large CL/NA charge ratios. Correspondingly, the transfection efficiency of CMVL-DNA complexes remains high for high CL/DNA charge ratios. In summary, the much reduced cytotoxicity of these new degradable multivalent lipids and their propensity for DNA release in the cytoplasm open the way for the development of efficient non toxic CL-vectors of NAs. Funding provided by NIH GM-59288.

A Peptide-Based Dendrimer That Enhances the Splice-Redirecting Activity of PNA Conjugates in Cells

The full therapeutic potential of oligonucleotide (ON)-based agents has been hampered by cellular delivery challenges. Cell-penetrating peptides (CPP) represent promising delivery vectors for nucleic acids, and their potential has recently been evaluated using a functional splicing redirection assay, which capitalizes on the nuclear delivery of splice-correcting steric-block ON analogues such as peptide nucleic acids (PNA). Despite encouraging in vitro and in vivo data with arginine-rich CPP-steric block conjugates, mechanistic studies have shown that entrapment within the endosome/lysosome compartment after endocytosis remains a limiting factor. Previous work from our group has shown that CPP oligomerization greatly improves cellular delivery and increases transfection of plasmid DNA. We now report the chemical synthesis and the evaluation of multivalent CPP-PNA constructs incorporating monomeric (p53(mono)) and dendrimer-like tetrameric (p53(tet)) forms of the p53 tetramerization domain containing peptide, a 10 arginine CPP domain (R10), and a splice redirecting PNA (PNA705). These CPP-PNA conjugates were termed R10p53(tet)-PNA705 and R10p53(mono)-PNA705, referring to their oligomerization state. The present study demonstrates that the splicing redirection efficiency of PNA705 is much greater in the context of the tetrameric R10p53(tet)-PNA705 construct than for the monomeric and occurs at nanomolar concentrations, demonstrating that multivalency is an important factor in delivering PNA into cells.

Magnetofection: The Use of Magnetic Nanoparticles for Nucleic Acid Delivery: Figure 1.

INTRODUCTIONMagnetofection is defined as nucleic acid delivery guided and mediated by magnetic force acting on associations of magnetic particles and nucleic acids or nucleic acid vectors. Vectors are bound to magnetic, usually iron oxide, nanoparticles, in most cases by noncovalent bonds. Magnetic force accumulates and/or holds magnetic vectors in a target tissue against hydrodynamic forces. In cell culture, magnetic vectors are magnetically sedimented on the target cells within minutes. Thus, the diffusion barrier to nucleic acid delivery is overcome, the full vector dose comes in contact with the target cells, and introduction of genetic material is synchronized. Nucleic acid delivery is greatly accelerated and its efficiency with many, if not most, vector types is improved. Magnetofection is applicable to small and large nucleic acids. Other advantages include low-dose requirements, the possibility of confining nucleic acid introduction to a localized area (magnetic targeting), and the amenability to high-throughput automation. Due to the favorable dose-response profile and the rapid kinetics, vector-related toxicity can be kept low. This protocol describes the use of magnetic nanoparticles for delivery of nucleic acid to target cells, using either nonviral or viral vectors.

Efficient nucleic acid transduction with lipoplexes containing novel piperazine- and polyamine-conjugated cholesterol derivatives

To advance the use of cationic lipids for non-viral nucleic acid vector formulation, a panel of novel nitrogen heterocycle cholesteryl derivatives containing a biodegradable carbamate linker was synthesised. Optimally acting piperazine and cyclen compounds had nucleic acid-binding and lipoplex nanoparticle formation properties that were suitable for their use as non-viral vectors. It was found that the lipoplexes formed were capable of efficient non-toxic nucleic acid delivery to cells in culture. The chemical structure of individual cationic lipids, which is likely to influence lipoplex formation, affected efficiency of DNA or RNA transfection. The results indicated that the cyclen containing compound possessing two cholesteryl moieties resulted in efficient siRNA-mediated target gene silencing but was a poor reagent for DNA transfection.

A Fibrin Glue Composition as Carrier for Nucleic Acid Vectors

Gene delivery from biomaterials has become an important tool in tissue engineering. The purpose of this study was to generate a gene vector-doted fibrin glue as a versatile injectable implant to be used in gene therapy supported tissue regeneration. Copolymer-protected polyethylenimine(PEI)-DNA vectors (COPROGs), naked DNA and PEI-DNA were formulated with the fibrinogen component of the fibrin glue TISSUCOL and lyophilized. Clotting parameters upon rehydration and thrombin addition were measured, vector release from fibrin clots was determined. Structural characterizations were carried out by electron microscopy. Reporter and growth factor gene delivery to primary keratinocytes and chondrocytes in vitro was examined. Finally,chondrocyte colonized clots were tested for their potency in cartilage regeneration in a osteochondral defect model. The optimized glue is based on the fibrinogen component of TISSUCOL, a fibrin glue widely used in the clinics, co-lyophilized with copolymer-protected polyethylenimine(PEI)- DNA vectors (COPROGs). This material, when rehydrated, forms vector-containing clots in situ upon thrombin addition and is suitable to mediate growth factor gene delivery to primary keratinocytes and primary chondrocytes admixed before clotting. Unprotected PEI-DNA in the same setup was comparatively unsuitable for clot formation while naked DNA was ineffective in transfection. Naked DNA was released rapidly from fibrin clots (>70% within the first seven days) in contrast to COPROGs which remained tightly immobilized over extended periods of time (0.29% release per day). Electron microscopy of chondrocytecolonized COPROG-clots revealed avid endocytotic vector uptake. In situ BMP-2 gene transfection and subsequent expression in chondrocytes grown in COPROG clots resulted in the upregulation of alkaline phosphatase expression and increased extracellular matrix formation in vitro. COPROG-fibrinogen preparations with admixed autologous chondrocytes when clotted in situ in osteochondral defects in the patellar grooves of rabbit femura gave rise to luciferase reporter gene expression detectable for two weeks (n=3 animals per group). However, no significant improvement in cartilage formation in osteochondral defects filled with autologous chondrocytes in BMP-2-COPROG clots was achieved in comparison to controls (n=8 animals per group). COPROGs co-lyophilized with fibrinogen are a simple basis for an injectable fibrin gluebased gene-activated matrix. The preparation can be used is complete analogy to fibrin glue preparations that are used in the clinics. However, further improvements in transgene expression levels and persistence are required to yield cartilage regeneration in the osteochondral defect model chosen in this study.

Functionalized Carbon Nanotubes in Drug Design and Discovery

Carbon nanotubes (CNTs) have been proposed and actively explored as multipurpose innovative carriers for drug delivery and diagnostic applications. Their versatile physicochemical features enable the covalent and noncovalent introduction of several pharmaceutically relevant entities and allow for rational design of novel candidate nanoscale constructs for drug development. CNTs can be functionalized with different functional groups to carry simultaneously several moieties for targeting, imaging, and therapy. Among the most interesting examples of such multimodal CNT constructs described in this Account is one carrying a fluorescein probe together with the antifungal drug amphotericin B or fluorescein and the antitumor agent methotrexate. The biological action of the drug in these cases is retained or, as in the case of amphotericin B constructs, enhanced, while CNTs are able to reduce the unwanted toxicity of the drug administered alone. Ammonium-functionalized CNTs can also be considered very promising vectors for gene-encoding nucleic acids. Indeed, we have formed stable complexes between cationic CNTs and plasmid DNA and demonstrated the enhancement of the gene therapeutic capacity in comparison to DNA alone. On the other hand, CNTs conjugated with antigenic peptides can be developed as a new and effective system for synthetic vaccine applications. What makes CNTs quite unique is their ability, first shown by our groups in 2004, to passively cross membranes of many different types of cells following a translocation mechanism that has been termed the nanoneedle mechanism. In that way, CNTs open innumerable possibilities for future drug discovery based on intracellular targets that have been hard to reach until today. Moreover, adequately functionalized CNTs as those shown in this Account can be rapidly eliminated from the body following systemic administration offering further encouragment for their development. CNT excretion rates and accumulation in organs and any reactivity with the immune system will determine the CNT safety profile and, consequently, any further pharmaceutical development. Caution is advised about the need for systematic data on the long-term fate of these very interesting and versatile nano-objects in correlation with the type of CNT material used. CNTs are gradually plyaing a bigger and more important role in the emerging field of nanomedicine; however, we need to guarantee that the great opportunities they offer will be translated into feasible and safe constructs to be included in drug discovery and development pipelines.

Metallic Colloid Nanotechnology, Applications in Diagnosis and Therapeutics

In recent years the fields of medicine and biology assist to an ever-growing innovation related to the development of nanotechnologies. In the pharmaceutical domain, for example, liposomes, polymer based micro and nanoparticles have been subjects of intense research and development during the last three decades. In this scenario metallic particles, which use was already suggested in the first half of the '80, are now experiencing a real renaissance. In the field of diagnosis, magnetic resonance imaging is one of the first and up to now the most developed application of metallic particles. But beside this application, a very new generation of biosensors based on the optical properties of colloidal gold and fluorescent nanocrystals, called quantum dots seems to be ready to be implemented in diagnosis and medical imaging. Concerning therapeutic applications, the potentialities of metal nanoparticles to help fulfilling the need of time and space controlled release of drugs has been intuited for a long time. Nowadays, magnetically guided carriers or thermal responsive matrices, in which drug release is triggered by the heating of metal nanoparticles, are effective examples of their application in drug delivery, while more recently efforts to develop metallic nanoobjects to be used as vectors of nucleic acids for vaccination and transfection have been multiplied. In the future, one of the most interesting challenges is certainly the use of metallic nanoparticles for an innovating, effective and selective physical treatment of solid tumors via targeted intracellular hyperthermia.

DNA vaccination against tumors

DNA vaccines have been used to generate protective immunity against tumors in a variety of experimental models. The favorite target antigens have been those that are frequently expressed by human tumors, such as carcinoembryonic antigen (CEA), ErbB2/neu, and melanoma-associated antigens. DNA vaccines have the advantage of being simple to construct, produce and deliver. They can activate all arms of the immune system, and allow substantial flexibility in modifying the type of immune response generated through codelivery of cytokine genes. DNA vaccines can be applied by intramuscular, dermal/epidermal, oral, respiratory and other routes, and pose relatively few safety concerns. Compared to other nucleic acid vectors, they are usually devoid of viral or bacterial antigens and can be designed to deliver only the target tumor antigen(s). This is likely to be important when priming a response against weak tumor antigens. DNA vaccines have been more effective in rodents than in larger mammals or humans. However, a large number of methods that might be applied clinically have been shown to ameliorate these vaccines. This includes in vivo electroporation, and/or inclusion of various immunostimulatory molecules, xenoantigens (or their epitopes), antigen-cytokine fusion genes, agents that improve antigen uptake or presentation, and molecules that activate innate immunity mechanisms. In addition, CpG motifs carried by plasmids can overcome the negative effects of regulatory T cells. There have been few studies in humans, but recent clinical trials suggest that plasmid/virus, or plasmid/antigen-adjuvant, prime-boost strategies generate strong immune responses, and confirm the usefulness of plasmid-based vaccination.

Enhancing and targeting nucleic acid delivery by magnetic force

Insufficient contact of inherently highly active nucleic acid delivery systems with target cells is a primary reason for their often observed limited efficacy. Physical methods of targeting can overcome this limitation and reduce the risk of undesired side effects due to non-target site delivery. The authors and others have developed a novel means of physical targeting, exploiting magnetic force acting on nucleic acid vectors associated with magnetic particles in order to mediate the rapid contact of vectors with target cells. Here, the principles of magnetic drug and nucleic acid delivery are reviewed, and the facts and potentials of the technique for research and therapeutic applications are discussed. Magnetically enhanced nucleic acid delivery – magnetofection – is universally applicable to viral and non-viral vectors, is extraordinarily rapid, simple and yields saturation level transfection at low dose in vitro. The method is useful for site-specific vector targeting in vivo. Exploiting the full potential of the technique requires an interdisciplinary research effort in magnetic field physics, magnetic particle chemistry, pharmaceutical formulation and medical application.

Histidine containing peptides and polypeptides as nucleic acid vectors.

Nucleic acid transfer in mammalian cels is drastically improved with devices which increase their delivery in the cytosol upon endocytosis. In this chapter, we describe the effect on plasmid DNA (pDNA) and oligonucleotide (ODN) transfer, of an histidine-rich peptide (H5WYG), histidylated oligolysine (HoK), and histidylated polylysine (HpK) designed on the basis of the membrane destabilization capacity of poly-L-histidine at a pH dose to that of the endosomes. We report that H5WYG, which permeabilizes the cell membrane at pH 6.4, favors the transfection mediated by lactosylated polylysine/pDNA complexes and, by lowering the pH of extracellular medium, allows the loading of the cytosol and the cell nucleus with ODN. We show that HoK forms small cationic spherical particles of 35 nm with ODN and HpK rod or toroid cationic particles of 100 nm with pDNA. PEGylation stabilizes these particles at physiological salt concentration. We also show that (i) HoK/ODN complexes yield a more than 20-fold increase of the biological activity of antisense ODN towards the inhibition of transient as well as constitutive gene expression and (ii) HpK/pDNA complexes yield a transfection efficiency of 3-4.5 order of magnitude higher than do polylysine/pDNA complexes. We also provide evidence that the effect of these polyhistidylated molecules is mediated by imidazole protonation in endosomes. Overall our data show that polyhistidylated molecules constitute interesting devices for an efficient cytosolic delivery of nucleic acids, and that ionic complexes between histidylated polylysine and a pDNA are attractive for developing a nonviral gene delivery system.

Alphavirus vectors: from protein production to gene therapy

Alphaviruses are enveloped viruses containing a single positive strand RNA molecule as genome. Several vectors derived from alphaviruses have been developed, which include Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEE). Alphavirus self-replicating RNA containing heterologous genes can be synthesized in vitro from plasmids having the recombinant alphavirus replicon sequences under the control of a prokaryotic promoter, such as SP6 or T7. High level expression of the heterologous proteins/RNA is obtained in cells transfected with this RNA. Although the system can be used for gene delivery directly as naked RNA, several packaging systems have been developed which allow the encapsidation of the alphaviral recombinant RNA into suicidal viral particles, increasing the efficiency of delivery of the recombinant genome into cells. A more recent variant of the system is based on a DNA/RNA layered alphaviral vector in which the recombinant replicon is transcribed from an RNA polymerase-II promoter, allowing direct delivery of DNA into cells. Alphavirus vectors have been used to express a great number of proteins with many different purposes, including protein production and characterization, functional studies, vaccination, and gene therapy. Both the recombinant alphavirus particles and the alphavirus nucleic acid vectors have shown to be able to induce protective immune responses in animal models. The possible application of these vectors in gene therapy faces, however, two limitations, which are the lack of specific targeting and the transient nature of the vector, due to the induction of apoptosis by the alphavirus replicon. Several strategies have been recently described to improve the targeting of alphavirus vectors and to develop noncytopathic vectors with potential use in gene therapy.

The promise of nucleic acid vaccines.

Establishing the effective use of 'naked' nucleic acids as vaccines would undoubtedly be one of the most important advances in the history of vaccinology. While nucleic acids show much promise for use as vaccine vectors in experimental animals, not a single naked nucleic acid vector has been approved for use in humans. Indeed, data from human clinical trials is scant: nucleic acid vaccines have not been clearly demonstrated to have any convincing efficacy in the prevention or treatment of infectious disease or cancer. Here we illustrate possible mechanisms underlying effective nucleic acid vaccination. We focus on progress that has been made in the improvement of their function. Additionally, we identify promising new strategies and try to forecast future developments that could lead to the real success of nucleic acid vaccines in the prevention and treatment of human disease.

Herpesvirus Vaccines

The development of vaccines against the herpesviruses has major public health importance because of the wide spectrum of associated clinical disease with this virus in both immunocompetent and immunocompromised populations. Because these viruses establish latent infections capable of subsequent reactivation, both immunotherapeutic and prophylactic vaccine strategies are needed. A range of vaccine formulations has been devised, largely as a result of the rapid growth in knowledge in molecular microbiology and genetic engineering, including live and inactivated whole virus vaccines, and subunit vaccines consisting of recombinant viral glycoproteins in various adjuvants. The live attenuated virus Oka strain vaccine is now licensed for prophylaxis against varicella (VZV) in some countries. Recent studies with herpes simplex viruses (HSV) have demonstrated immunogenicity with glycoprotein vaccines; however, these studies have also highlighted their failure to reduce seroconversion to HSV-2 in high-risk populations. Nevertheless, his work has helped develop comprehensive methodologies for the clinical amd immunological assessment of newer vaccine formulations which have proved successful in animal models. The live attenuated Towne strain vaccine has been shown to prevent severe human cytomegalovirus (CMV) infection in transplant recipients. More recently, subunit antigens and virus vector vaccines against CMV and Epstein Barr virus (EBV) have been devised. Novel attempts to present viral antigens now include the development of plasmid expression vectors for viral nucleic acids and genes as well as engineered live vectors for viral antigens. These new technologies may allow future vaccines to be devised, not only against the 5 well characterised herpes viruses, but also against the recently recognised herpes viruses 6, 7 and 8 whose full clinical spectrum is still unknown.