Potential Gene Delivery Research Articles

Genetic engineering within the adult brain: Implications for molecular approaches to behavioral neuroscience

Currently, the most popular technology used to modify the molecular makeup of the nervous system is through germline modifications of early embryos. This allows to construct gene ‘knock-ins’ (gene overexpression) or ‘knock-outs’ (gene deletions). This technology leads to gene additions or deletions from the earliest developmental stages. This can potentially lead to compensatory genetic changes. The technology to achieve inducible and cell-type-specific changes in gene expression in transgenic animals has been established. However, it is not yet possible, to reliably turn a particular gene ‘on’ or ‘off’ exclusively in adult animals. Alternatively, the use of gene transfer technology in fully mature animals could overcome many of these shortcomings. Gene therapy is the use of nucleic acids as drugs, and uses gene transfer technology to genetically engineer adult animals. Viral and nonviral vectors have been modified to serve as vectors for nucleic acid sequences of interest. Thus, over the last two decades, methods have been developed to deliver particular nucleic acids directly to target tissues. Further technological advances allow delivery of transgenes or antisense mRNAs directly to predetermined cell types, as well as their delivery under the control of inducible promoter elements. Combined transgenic (i.e., germline modifications) and viral vector technology will also be very powerful in allowing the genetic modification of selected neuronal populations in adult animals. In this review, we discuss the potential of gene delivery to the brain to analyze the effect of genetic engineering of particular neuronal groups on behavior, as well as recent developments and applications of newly engineered vector systems to allow transgenesis within nervous structures of adult animals.

An in vivo and in vitro assessment of integrity of plasmid DNA and cells after passage through the NOGA needle catheter: A potential gene delivery system Michael A. Kuliszewski, Michael J. B. Kutryk, David Fitchett, Duncan J. Stewart. Terrence Donnelly Heart Center, St. Michael's Hospital, Toronto, ON, Canada

An in vivo and in vitro assessment of integrity of plasmid DNA and cells after passage through the NOGA needle catheter: A potential gene delivery system Michael A. Kuliszewski, Michael J. B. Kutryk, David Fitchett, Duncan J. Stewart. Terrence Donnelly Heart Center, St. Michael's Hospital, Toronto, ON, Canada

A Potent New Class of Reductively Activated Peptide Gene Delivery Agents

A new class of peptide gene delivery agents were developed by inserting multiple cysteine residues into short (dp 20) synthetic peptides. Substitution of one to four cysteine residues for lysine residues in Cys-Trp-Lys(18) resulted in low molecular weight DNA condensing peptides that spontaneously oxidize after binding to plasmid DNA to form interpeptide disulfide bonds. The stability of cross-linked peptide DNA condensates increased in proportion to the number of cysteines incorporated into the peptide. Disulfide bond formation led to a decrease in particle size relative to control peptide DNA condensates and prevented dissociation of peptide DNA condensates in concentrated sodium chloride. Cross-linked peptide DNA condensates were 5-60-fold more potent at mediating gene expression in HepG2 and COS 7 cells relative to uncross-linked peptide DNA condensates. The enhanced gene expression was dependent on the number of cysteine residues incorporated, with a peptide containing two cysteines mediating maximal gene expression. Cross-linking peptides caused elevated gene expression without increasing DNA uptake by cells, suggesting a mechanism involving intracellular release of DNA triggered by disulfide bond reduction. The results establish cross-linking peptides as a novel class of potent gene delivery agents that enhance gene expression through a new mechanism of action.

DQAsomes: a novel potential drug and gene delivery system made from Dequalinium.

Dequalinium, a drug known for over 30 years, is a dicationic amphiphile compound resembling bolaform electrolytes. The purpose of our work was to determine the state of aggregation of dequalinium in aqueous medium and to investigate both, its ability to bind DNA and its potential to serve as a novel non-viral transfection vector. The form of aggregation was determined employing electron microscopic techniques. The DNA binding capacity of dequalinium was assayed using SYBR Green I stain. For in vitro cell transfection experiments plasmid DNA encoding for firefly luciferase was used. Dequalinium forms in aqueous medium liposome-like aggregates, which we term DQAsomes. These dequalinium vesicles bind DNA and they are able to transfect cells in vitro with an efficiency comparable to Lipofectin. Based on the intrinsic properties of dequalinium such as the in vivo selectivity for carcinoma cells and selective accumulation in mitochondria we propose DQAsomes as a novel and unique drug and gene delivery system.

A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.

Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se--i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its gene-delivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.

Gene replacement therapy in the CNS: A view from the retina

AbstractGene replacement therapy holds great promise in the treatment of many genetic CNS disorders. This commentary discusses the feasibility of gene replacement therapy in the unique context of the retina, with regard to: (1) the genetics of retinal neoplasia and degeneration, (2) available gene transfer technology, and (3) potential gene delivery vehicles.