Nucleic acid transfer in mammalian cells 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 close 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.
Gene transfer into skeletal muscle cells by direct injection of naked plasmid DNA results in sustained gene expression. Intramuscular injection of plasmid DNA might thus be used to correct myopathies, to secrete locally or systematic therapeutic proteins and to elicit an immune response against specific antigens. However, the potential utility of this technique for gene application in humans is limited by the poor transduction efficiency and the low and highly variable level of gene expression. Different methods are thus being developed to increase the efficiency of gene transfer in muscles. It has been recently reported that a dramatic improvement of DNA transfer is achieved by applying an electric field to the muscle fibers subsequent to local DNA injection. Electro-gene-transfer increases gene expression by several orders of magnitude and strongly reduces interindividual variability. Electroinjection of genes encoding for secreted proteins resulted in sustained expression and disease correction in animal models of gene therapy. Moreover, the immunogenicity of DNA vaccines is dramatically increased when antigen-encoding plasmids are delivered by this technique. This technique may thus have broad and important applications in human gene therapy. This review provides a brief overview of the theory of electro-gene-transfer and describes parameters governing its efficiency in muscle. We also summarize the results obtained with electro-gene-transfer in animal models to date and the technical issues that must be solved before its use for human therapy can be considered.
Synthetic gene delivery vehicles are a highly promising approach to gene delivery; however, several problems must still be overcome before they can begin to enjoy clinical success. A number of these problems can be addressed by engineering and optimizing the properties of the vector surface, the component of the particle that interacts and “communicates” with tissues and cells during the delivery process. Surfaces must be engineered to satisfy two ostensibly conflicting constraints: the ability to interact specifically with a target cell while avoiding nonspecific protein interactions, particularly with components of the immune system. We summarize progress that has been made in both these areas and discuss several approaches where the intersection of biological and chemical solutions promises to significantly advance the engineering of synthetic vehicles.
Gene delivery into cultured cells or in vivo is a promising approach to the treatment of diseases. Several gene delivery systems have been developed to promote gene expression either in vivo or in vivo. Concerns about viral-induced immune responses, the risk associated with replication-competent viruses, and production issues have stimulated efforts toward the development of alternative gene delivery systems such as cationic lipids and polymers. These positively charged molecules interact through electrostatic forces with DNA. This results in the formation of highly organized supramolecular structures where DNA molecules are condensed and protected against DNAses degradation. Association of DNA with cationic lipids under a micellar or liposomal form leads to lamellar organization with DNA molecules sandwiched between lipid bilayers. Although the lamellar phase is the common described structure, as evidenced by small-angle X-ray scattering and electron microscopy, monovalent cationic lipid combined with a hexagonal forming lipid resulted with DNA in an inverted hexagonal structure. Despite a lot of effort, the mechanism of gene transfer with cationic carrier is still ill-defined. Therefore, correlations need to be established between physicochemical properties of synthetic DNA delivery systems and in vivo and in vivo transfection efficiency.
Systemic gene delivery systems are needed for therapeutic applications; in some situations, target cells might be spread throughout the organism, as in the case of cancer metastases, which can be reached only via the systemic route. Within the class of nonviral vectors, polymer-based transfection particles named DNA polyplexes and lipid-based systems named DNA lipoplexes are being developed for this purpose. For systemic circulation, masking the surface charge of DNA complexes has to be accomplished to avoid interactions with plasma components, erythrocytes, and the reticuloendothelial system. Among other vector formulations, polyplexes based on polyethylenimine (PEI), shielded with polyethylene glycol (PEG), and linked to the receptor binding ligands transferrin (Tf) or epidermal growth factor (EGF) have been developed. Complexes were found to mediate efficient gene transfer into tumor cell lines in a receptor-dependent and cell-cycle-dependent manner. Systemic administration of surface-shielded Tf-PEI polyplexes into the tail vein of mice resulted in preferential gene delivery into distantly growing subcutaneous tumors. In contrast, application of positively charged PEI polyplexes directed gene transfer primarily to the lung.
All discussions on the intracellular delivery of DNA are based on a seemingly evident assumption that the key task is to bring the intact DNA into the cell cytoplasmic compartment, and then the DNA will find its way to a right place. The nuclear genome is usually considered to be this “right place.” However, until recently, in numerous experiments on the intracellular DNA delivery, it has been almost completely neglected that cells contain another genome, the mitochondrial one. And, in many cases, this genome should become a therapeutic target. Being delivered inside the cell, DNA actually has two ways to go—to nuclei and to mitchondria, and the proper choice between these two ways may be decisive for the success of gene therapy. Certainly, nuclear DNA delivery is far more advanced than mitochondrial delivery one. In addition, free DNA from the cytoplasm has a strong tendency to spontaneously associate with the nuclear genome. Mitochondria as a target for DNA have much less accessibility, still remaining an important site to reach. Whereas the nuclear delivery of DNA is under active investigation and just awaits better protocols to be elaborated, practically applicable mitochondrial DNA delivery is at its early stage and must be developed almost from scratch. In our studies on intracellular DNA delivery, we have attempted to develop new protocols for targeting DNA to nuclei and to mitochondria. In this chapter we provide a brief description of our recent experiments in both of these important areas.
Development of nonviral gene transfer methods would be a valuable addition to the gene-therapy armamentarium, particularly for localized targeting of specific tissue volumes. Ultrasound can produce a variety of nonthermal bioeffects via acoustic cavitation including DNA delivery. Cavitation bubbles may induce cell death or transient membrane permeabilization (sonoporation) on a single cell level, as well as microvascular hemorrhage and disruption of tissue structure. Application of sonoporation for gene delivery to cells requires control of cavitation activity. Many studies have been performed using in vitro exposure systems, for which cavitation is virtually ubiquitous. In vivo, cavitation initiation and control is more difficult, but can be enhanced by cavitation nucleation agents, such as an ultrasound contrast agent. Sonoporation and ultrasonically enhanced gene delivery has been reported for a wide range of conditions including low frequency sonication (kilohertz frequencies), lithotripter shockwaves, HIFU, and even diagnostic ultrasound (megahertz frequencies). In vitro, a variety of cell lines has been successfully transfected, with concomitant cell killing. In vivo, initial applications have been to cancer gene therapy, for which cell killing can be a useful simultaneous treatment, and to cardiovascular disease. The use of ultrasound for nonviral gene delivery has been demonstrated for a robust array of in vitro and mammalian systems, which provides a fundamental basis and strong promise for development of new gene therapy methods for clinical medicine.
Dendritic cells are professional antigen presenting cells and are unique in their ability to prime naíve T cells. Gene modification of dendritic cells is of particular interest for immunotherapy of diseases where the immune system has failed or is aberrantly regulated, such as in cancer or autoimmune disease, respectively. Dendritic cells abundantly express mannose receptor and mannose receptor-related receptors, and receptor-mediated gene transfer via mannose receptor offers a versatile tool for targeted gene delivery into these cells. Accordingly, mannose polyethylenimine DNA transfer complexes were generated and used for gene delivery into dendritic cells. Mannose receptor belongs to the group of scavenger receptors that allow dendritic cells to take up pathogenic material, which is directed for degradation and MHC class II presentation. Therefore, a limiting step of transgene expression by mannose receptor-mediated gene delivery is endosomal degradation of DNA. Several strategies have been explored to overcome this limitation including the addition of endosomolytic components to DNA transfer complexes like adenovirus particles and influenza peptides. Here, we review the current understanding of mannose receptor-mediated gene delivery into dendritic cells and discuss strategies to identify appropriate endosomolytic agents to improve DNA transfer efficacy.
Most synthetic gene delivery vectors are taken up in the cell by endocytosis, and inefficient escape of the transgene from endocytic vesicles often is a major barrier for gene transfer by such vectors. To improve endosomal release we have developed a new technology, named photochemical internalization (PCI). PCI is based on photochemical reactions initiated by photosensitizing compounds localized in endocytic vesicles, inducing rupture of these vesicles upon light exposure. PCI constitutes an efficient light-inducible gene transfer method in vivo , which potentially can be developed into a site-specific method for gene delivery in in vivo gene therapy. In this paper the principle behind the PCI technology and the effect of PCI on transfection with different synthetic gene delivery vectors are reviewed. PCI treatment by the photosensitizer aluminum phthalocyanine (AlPcS 2 a ) strongly improves transfection mediated by cationic polymers (e.g., poly-L-lysine and polyethylenimine), while the effect on transfection with cationic lipids is more variable. The timing of the light treatment relative to the transfection period was also important, indicating that release of the DNA from early endosomes is important for the outcome of PCI-induced transfection. The possibilities of using PCI as a technology for efficient, site-specific gene delivery in in vivo gene therapy is discussed.
Recombinant vectors derived from murine leukemia virus (MLV) have been widely used to introduce genes in human gene therapy clinical trials and have shown the potential for medical applications and the promise of significantly improving medical therapies. Yet, the demonstrated limitations of these vectors support the need for continued development of improved vectors. The intrinsic properties associated with the MLV genome and its life cycle do not favor the successful application of this vector system in certain human gene transfer applications. Since MLV integrates randomly into the host genome, transgene expression is frequently affected by the flanking host chromatin.1 MLV insertions can often result in silencing or position effect variation of gene expression either immediately after insertion or following cell expansion in culture or in vivo.2–6 Migration of the MLV pre-integration complex from the cytoplasm into the nucleus of infected cells requires mitosis for nuclear membrane breakdown.7 Since a majority of human cells exist in a quiescent state in vivo, it is unlikely that direct in vivo gene delivery into target tissues can be achieved with the MLV vector system. Finally, insertion of tissue-specific cis-regulatory sequences to direct transgene expression frequently results in either the rearrangement of the vector sequence or disruption of the cis-regulatory sequence functions.8–12 The long terminal repeat (LTR) of MLV, which contains a ubiquitously active enhancer/promoter element, may partially account for this problem. Together, these problems pose a major obstacle for the use of MLV vectors in the treatment of human diseases. This Chapter discusses some of the potential targets to which HIV vectors might be applied in clinical settings and some of the issues surrounding use of HIV vectors in gene transfer clinical trials.
The inadvertent production of replication competent retrovirus (RCR) constitutes the principal safety concern for the use of lentiviral vectors in human clinical protocols. Because of limitations in animal models to evaluate lentiviral vectors for their potential to recombine and induce disease, the vector design itself should ensure against the emergence of RCR in vivo. Issues related to RCR generation and one approach to dealing with this problem are discussed in this chapter. To assess the risk of generating RCR, a highly sensitive biological assay was developed to specifically detect vector recombination in transduced cells. Analysis of lentiviral vector stocks has shown that recombination occurs during reverse transcription in primary target cells. Rejoining of viral protein-coding sequences of the packaging construct and cis-acting sequences of the vector was demonstrated to generate env-minus recombinants (LTR-gag-pol-LTR). Mobilization of recombinant lentiviral genomes was also demonstrated but was dependent on pseudotyping of the vector core with an exogenous envelope protein. 5′ sequence analysis has demonstrated that recombinants consist of U3, R, U5, and the ψ packaging signal joined with an open gag coding region. Analysis of the 3′ end has mapped the point of vector recombination to the poly(A) tract of the packaging construct's mRNA. The state-of-the-art third generation packaging construct and SIN vector also have been shown to generate env-minus proviral recombinants capable of mobilizing retroviral DNA when pseudotyped with an exogenous envelope protein. A new class of HIV-based vector (trans-vector) was recently developed that splits the gag-pol component of the packaging construct into two parts: one that expresses Gag/Gag-Pro and another that expresses Pol (RT and IN) fused with Vpr. Unlike other lentiviral vectors, the trans-vector has not been shown to form recombinants capable of DNA mobilization. These results indicate the trans-vector design prevents the generation of env-minus recombinant lentivirus containing a functional gag-pol structure (LTR-gag-pol-LTR), which is absolutely required for retroviral DNA mobilization and the emergence of RCR. Quality assurance based on monitoring for RCR may have limitations as a predictor of safety in vivo, especially in the long term. The demonstration of lentivirus infection via alternative entry mechanisms supports this notion. Therefore, the approach of monitoring trans-vector stocks for env-minus recombinant virus in vitro as a surrogate marker for the possible emergence of RCR in vivo should represent a significant advancement in vector safety quality assurance.
This chapter will outline the various concerns which have been raised in scientific, bioethics, and lay communities about the use of lentiviral vectors for purposes of gene therapy. Many of these concerns are ranged around gene therapy itself; others are concerns particular to using this sort of vector for genetic modification of human cells. These concerns are outlined within the chapter, and arguments are given in favor and against various approaches to these concerns. Lastly, it is noted throughout that at this stage of research into gene therapy, the most practical approach to these dilemmas is to maintain awareness of the ethical problems and provide information to those concerned with all aspects of the development of this set of technologies.
Lentiviruses share the common characteristic of infecting non-dividing target cells, distinguishing them from the oncogenic retroviruses which only productively infect dividing cells. The search for determinants for infection of non-dividing cells has produced a number of candidates. From HIV-1, the viral proteins matrix, integrase and Vpr have all been implicated. A structural determinant, the central DNA flap, has also been implicated. The supporting evidence for each of these proposed determinants will be examined and compared to how other viruses, non-retroviruses, transport their genomes to the nucleus. With currently available data, integrase and the central DNA flap appear to be the key players, and yet the mechanism for infection of non-dividing cells remains undefined.
Why is feline immunodeficiency virus (FIV) such an appealing candidate for gene therapy vector development? Phylogenetic analysis suggests FIV is only distantly related to the primate lentiviruses, and despite repeated exposure, neither seroconversion nor other detectable evidence of human infection occurs. FIV naturally infects diverse Felidae worldwide, including the domestic cat. Here, the disease progression parallels the immunodeficiency caused by HIV, and for that reason, FIV and the cat provide an excellent model for anti-virals and AIDS vaccine research. Simple genome organization also facilitates vector development and analysis: FIV has only three accessory/regulatory proteins. To overcome FIV's cat-specific tropism, feline vectors are equipped with hybrid LTRs, since the FIV LTR shows low activity in human cells. Recombinant FIV vectors generate titers comparable to other lentiviral systems, are capable of incorporating heterologous envelopes and efficiently transduce dividing and nondividing cells in the presence and absence of the accessory proteins in vitro. Compared to HIV vectors, FIV vector development is still in its infancy, but initial in vivo data in various species and tissues indicate long-term gene expression at therapeutic levels, and thus FIV vectors hold great promise. Future efficacy studies in animal models and primates will determine the FIV vectors' suitability for gene therapy. The design of recombinant FIV vectors incorporates safety features described for primate lentiviral vectors with the benefit that biosafety testing of FIV vectors can occur in the natural host. Currently, FIV vectors are generated in a transient fashion, but the availability of a stable producer system amenable to better characterization and scale-up will considerably increase the potential for use of FIV vectors in the clinic.
As various viral vector systems for gene transfer are developed, interest in using such systems in applied settings continues to grow. This Chapter is designed to provide background information for readers interested in learning about lentiviral vector systems for gene transfer applications but who lack a background in retrovirology. To assist those readers who are unfamiliar with retroviral vector systems, basic outlines of the retroviral replication cycle and of characteristics of retroviral vector systems are introduced here in order to present and define concepts and terms that are discussed in subsequent Chapters.
In general terms, the replication cycle of lentiviruses, including HIV-1, closely resembles that of other retroviruses.1 There are, however, a number of unique aspects of HIV replication; for example, the HIVs and SIVs target receptors and coreceptors distinct from those used by other retroviruses. Lentiviruses encode a number of regulatory and accessory proteins not encoded by the genomes of the prototypical “simple” retroviruses. Of particular interest from the gene therapy perspective, lentiviruses possess the ability to productively infect some types of non-dividing cells. This chapter, while reiterating certain points discussed in Chapter 1, will attempt to focus on issues unique to HIV-1 replication.
Lentiviral vectors have received much attention in recent years due to their ability to efficiently transduce non-dividing cells. Of the lentiviruses HIV-2 and SIV offer several unique benefits as the basis for lentiviral vector design. HIV-1, HIV-2 and SIV remain the only known primate lentiviruses, and consequently are among the most extensively studied viruses known. Substantial effort has been devoted towards identifying the pathogenic determinants of the primate lentiviruses and towards understanding their replication within primates. Of the primate lentiviruses, the pathogenicity and rates of transmission of HIV-2 and SIV fall far below that of HIV-1, potentially providing vectors based upon HIV-2/SIV with a greater degree of biosafety. Last, and perhaps most importantly, HIV-2 and SIV are viruses which may be studied within non-human primate models susceptible to AIDS-like disease, making vectors based upon these viruses accessible to substantial preclinical evaluation. We approach this Chapter presenting information regarding the basic biology of HIV-2 and SIV and conclude by pointing to how unique features of HIV-2 and SIV are well suited to vector design, hoping to leave the reader with a greater appreciation of the potential these viruses offer within the field of gene transfer applications.
Lentiviruses that infect non-primates make up a diverse collection of viruses. Although these viruses have some features in common with HIV and other primate viruses, differences in genome organization and viral gene function have made the successful derivation of vectors from non-primate lentiviruses unpredictable. This Chapter discusses the construction and application of gene transfer systems derived from four non-primate lentiviruses including equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), visna virus, and Jembrana disease virus (JDV).
Human immunodeficiency virus type 1 (HIV-1) based gene transfer systems are gaining in popularity due to their ability to transduce terminally differentiated and non-dividing cells. Oncoretroviral vectors based on Moloney murine leukemia virus (MoMLV), on the other hand, can only transduce dividing cells. The reasons for increased ability of lentivirus vectors to transduce such cells has been attributed to several of the viral proteins (integrase, matrix and Vpr) that are purported to be involved in the nuclear import of the pre-integration complex (PIC). Nuclear import is also augmented by a unique triple stranded DNA region created during reverse transcription of the incoming viral RNA in the target cell (discussed in chapter 3). This chapter deals with the rationale behind the design of human immunodeficiency virus type 1 (HIV-1) based packaging systems with an emphasis on some recent advances in the field for the creation of safe and efficient HIV-1 based vectors. The review covers trans-acting proteins and cis-sequences required for the deployment of HIV-1 vectors for gene transfer. This is a rapidly advancing field that with further refinements may soon allow the utilization of HIV-1 based and/or other lentivirus vectors in a clinical setting.
We use an Epstein-Barr virus (EBV) plasmid model chromosome system to study how different donor plasmid constructs affect recombination stimulated by an I-SceI-induced double-strand break in the target sequence in human cells. The entire 3.5 kb lacZ gene was efficiently recombined into a target EBV vector lacking lacZ sequences, but having limited homology to the donor plasmid. A donor plasmid with lacZ flanked by sequence homologous to the target consistently generated gene conversion events and was more effective than a donor carrying lacZ outside the same sequence homology. Reducing the length of homology between the target and donor from 5.5 kb to 1 kb caused only a 3-fold drop in recombination frequency, contrasting with the exponential dependence on homology length seen when no DSB is present in the target. These results document a DSB-induced 175-fold increase in recombination of a heterologous gene into a target, requiring only limited flanking homology.
