Gene Delivery Strategies

Gene delivery is the process of introducing foreign DNA into host cells. Gene delivery is, for example, one of the steps necessary for gene therapy and the genetic modification of crops. There are many different methods of gene delivery developed for a various types of cells and tissues, from bacterial to mammalian. Generally, the methods can be divided into two categories, viral and non-viral. Virus mediated gene delivery utilizes ability of a virus to inject its DNA inside a host cell. A gene that is intended for delivery is packaged into a viral particle. Non-viral methods include physical methods such as microinjection, gene gun, impalefection, hydrostatic pressure, electroporation, continuous infusion, and sonication and chemical, such as lipofection. It can also include the use of polymeric gene carriers. Microinjection refers to the process of using a very fine needle to insert substances at a microscopic or borderline macroscopic level into a single living cell. It is a simple mechanical process in which a needle roughly 0.5 to 5 micrometers in diameter penetrates the cell membrane and/or the nuclear envelope. The desired contents are then injected into the desired sub-cellular compartment and the needle is removed. Microinjection is normally performed under a specialized optical microscope setup called a micromanipulator. The process is frequently used as a vector in genetic engineering and transgenetics to insert genetic material into a single cell. Microinjection can also be used in the cloning of organisms, and in the study of cell biology and viruses. Examples •

Scientists can create simple transgenic organisms by injecting genes into the testicle of a nematode at a point where the cells that will become its sperm are undergoing meiosis. Since the developing gametes share a common cytoplasm, all of the nematode's gametes will carry a foreign gene as the result of a single injection.

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Microinjection is used as a vector in transgenic plant production.

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Microinjection of genes into fertilized eggs is a common vector used in the production of higher forms of transgenic animals.

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Microinjection of a gene knockdown reagent such as a Morpholino oligo into eggs or early zygotes is commonly used to probe the function of a gene during development of embryos.

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The gene gun or the Biolistic Particle Delivery System, originally designed for plant transformation, is a device for injecting cells with genetic information . The payload is an elemental particle of a heavy metal coated with plasmid DNA. This technique is often simply referred to as biolistics. Another instrument that uses biolistics technology is the PSD-1000/He particle delivery system (pictured).

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This device is able to transform almost any type of cell, including plants, and is not limited to genetic material of the nucleus: it can also transform organelles, including plastids.

Design The gene gun was originally a Crosman air pistol modified to fire dense tungsten particles. The design was first used on onions to deliver particles coated with a marker gene. Genetic transformation can then be proven when the onion tissue expresses the gene. The earliest custom manufactured geneguns (Fabricated by Nelson Allen) used a 22 caliber nailgun cartridge to propel an extruded polyethylene cylinder (bullet) down a 22 cal. Douglas barrel. A droplet of the tungsten powder and genetic material was placed on the bullet and shot down the barrel at a lexan "stopping" disk with a petri dish below. The bullet welded to the disk and

the genetic information blasted into the sample in the dish with a doughnut effect (devastation in the middle, a ring of good transformation and little around the edge). The gun was connected to a vacuum pump and was under vacuum while firing. The early design was put into limited production by a Rumsey-Loomis (a local machine shop then at Mecklenburg Rd in Ithaca, NY, USA). Later the design was refined by removing the "surge tank" and changing to nonexplosive propellants. DuPont added a plastic extrusion to the exterior to visually improve the machine for mass production to the scientific community. Biorad contracted with Dupont to manufacture and distribute the device. Improvements include the use of helium propellant and a multi-disk-collision delivery mechanism. Other heavy metals such as gold and silver are also used. Gold may be favored because it has better uniformity than tungsten and tungsten can be toxic to cells, but its use may be limited due to availability and cost. The primary inventor of the gene gun is horticultural scientist John C. Sanford together with Edward Wolf, who was the Director of Cornell's Submicron Facility at the time but now at Nanofabrication facility, and Nelson Allen. As an electrical engineer, Wolf is familiar with making and using small structures. He bought the Crosman air pistol and performed the first genegun experiments with it in his basement. Sanford would come to his house with the genetic material and then take the transformed cells back to his lab. Horticultural scientist Theodore Klein at Cornell University worked closely with John Sanford on experiments using and proving the genegun. They had support from co-inventor Nelson Allen of the Cornell Nanofabrication Facilities Machine shop who had an instrumental role in changing the genegun from the air pistol prototype to a working scientific device. The rights to commercial use of the gene gun were sold by Wolf, Sanford and Cornell University to DuPont in 1990. Application Gene guns are so far mostly applied for plants cells. However, there is much potential use in animals and humans as well. Plants The target of a gene gun is often a callus of undifferentiated plant cells growing on gel medium in a petri dish. After the gold particles have impacted the dish, the gel and callus are largely disrupted. However, some cells were not obliterated in the impact, and have successfully enveloped a DNA coated tungsten particle, who’s DNA eventually migrates to and integrates into a plant chromosome. Cells from the entire petri dish can be re-collected and selected for successful integration and expression of new DNA using modern biochemical techniques, such as a using a tandem selectable gene and northern blots. Selected single cells from the callus can be treated with a series of plant hormones, such as auxins and gibberellins, and each may divide and differentiate into the organized, specialized, tissue cells of an entire plant. This capability of total re-generation is called totipotency. The new plant that originated from a successfully shot cell may have new genetic (heritable) traits. The use of the gene gun may be contrasted with the use of Agrobacterium tumefaciens and its Ti plasmid to insert genetic information into plant cells. See transformation for different methods of transformation in different species. Humans and animals Gene guns have also been used to deliver DNA vaccines to experimental animals. Theoretically, it may be used in humans as well.

The delivery of plasmids into rat neurons through the use of a gene gun, specifically DRG neurons, is also used as a pharmacological precursor in studying the effects of neurodegenerative diseases such as Alzheimer's Disease. The Gene gun technique is also popularly used in Edible vaccine production technique, where the nano gold particles coated with plant gene under the high vacuum pressurized chamber is transformed into suitable plant tissues. Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA.[1] Electroporation is a dynamic phenomenon that depends on the local transmembrane voltage at each cell membrane point. It is generally accepted that for a given pulse duration and shape, a specific transmembrane voltage threshold exists for the manifestation of the electroporation phenomenon (from 0.5V to 1V). This leads to the definition of an electric field magnitude threshold for electroporation (Eth). That is, only the cells within areas where E≧Eth are electroporated. If a second threshold (Eir) is reached or surpassed, electroporation will compromise the viability of the cells, i.e., irreversible electroporation.[2] In molecular biology, the process of electroporation is often used for the transformation of bacteria, yeast, and plant protoplasts. In addition to the lipid membranes, bacteria also have cell walls which are different from the lipid membranes and are made of peptidoglycan and its derivatives. However, the walls are naturally porous and only act as stiff shells that protect bacteria from severe environmental impacts. If bacteria and plasmids are mixed together, the plasmids can be transferred into the cell after electroporation. Several hundred volts across a distance of several millimeters are typically used in this process. Afterwards, the cells have to be handled carefully until they have had a chance to divide producing new cells that contain reproduced plasmids. This process is approximately ten times as effective as chemical transformation.[1][3] This procedure is also highly efficient for the introduction of foreign genes in tissue culture cells, especially mammalian cells. For example, it is used in the process of producing knockout mice, as well as in tumor treatment, gene therapy, and cell-based therapy. The process of introducing foreign DNAs into eukaryotic cells is known as transfection. Laboratory Practice Cuvettes for electroporation. These are plastic with aluminium electrodes and a blue lid. They hold a maximum of 400 μl. Electroporation is done with electroporators, appliances which create an electro-magnetic field in the cell solution. The cell suspension is pipetted into a glass or plastic cuvette which has two aluminum electrodes on its sides. For bacterial electroporation, typically a suspension of around 50 microliters is used. Prior to electroporation it is mixed with the plasmid to be transformed. The mixture is pipetted into the cuvette, the voltage and capacitance is set and the cuvette inserted into the electroporator. Immediately after electroporation, one milliliter of liquid medium is added to the bacteria (in the cuvette or in an eppendorf tube), and the tube is incubated at the bacteria's optimal temperature for an hour or more to allow recovery of the cells and expression of antibiotic resistance, followed by spreading on agar plates.

The success of the elecroporation depends greatly on the purity of the plasmid solution, especially on its salt content. Solutions with high salt concentrations might cause an electrical discharge (known as arcing), which often reduces the viability of the bacteria. For a further detailed investigation of the process more attention should be paid to the output impedance of the porator device and the input impedance of the cells suspension (e.g. salt content). As the process needs direct electrical contact between the electrodes and the suspension, and is inoperable with isolated electrodes, obviously the process involves certain electrolythic effects, due to small currents and not only fields. The Electroporators Electroporators come in two flavors - hand-held and bench-tops. Benchtop electroporators are generally used as common lab equipments, residing atop a central bench or hood. They offer the advantage of electroporating multiple samples at the same time. They can also be set to different operating parameters depending on whether the cell has a cell-wall or not. Unlike them, the handheld electroporators are cordless, rechargeable and use disposable pipectrodes, which combine elements of both cuvettes and pipettes. It's operating parameters are pre-set to the optimal parameters for transforming either bacteria or mammalian cells. Both types of electoporators have been used on a wide range of cells - including E. coli (for transformation) and the mammalian cells such as neurons, astrocytes, neuroglia, lymphocytes, monocytes, fibroblasts, epithelial and endothelial cells from humans, mice, rats and monkeys (for transfection). Medical Applications A higher voltage of electroporation was found in pigs to irreversibly destroy target cells within a narrow range while leaving neighboring cells unnaffected, and thus represents a promising new treatment for cancer, heart disease and other disease states that require removal of tissue Physical Mechanism Further information: Lipid bilayer mechanics Electroporation allows cellular introduction of large highly charged molecules such as DNA which would never passively diffuse across the hydrophobic bilayer core.[1] This phenomenon indicates that the mechanism is the creation of nm-scale water-filled holes in the membrane. Although electroporation and dielectric breakdown both result from application of an electric field, the mechanisms involved are fundamentally different. In dielectric breakdown the barrier material is ionized, creating a conductive pathway. The material alteration is thus chemical in nature. In contrast, during electroporation the lipid molecules are not chemically altered but simply shift position, opening up a pore which acts as the conductive pathway through the bilayer as it is filled with water. Electroporation is a multi-step process with several distinct phases.[5] First, a short electrical pulse must be applied. Typical parameters would be 300-400mV for <1ms across the membrane (note- the voltages used in cell experiments are typically much larger because they are being applied across large distances to the bulk solution so the resulting field across the actual membrane is only a small fraction of the applied bias). Upon application of this potential the membrane charges like a capacitor through the migration of ions from the surrounding solution. Once the critical field is achieved there is a rapid localized rearrangement in lipid morphology. The resulting structure is believed to be a “pre-pore” since it is not electrically conductive but leads rapidly to the creation of a conductive pore. Evidence for the existence of such pre-pores comes mostly from the “flickering” of pores, which suggests a transition between conductive and insulating states.[6] It has been suggested

that these pre-pores are small (~3Å) hydrophobic defects. If this theory is correct, then the transition to a conductive state could be explained by a rearrangement at the pore edge, in which the lipid heads fold over to create a hydrophilic interface. Finally, these conductive pores can either heal, resealing the bilayer or expand, eventually rupturing it. The resultant fate depends on whether the critical defect size was exceeded[7] which in turn depends on the applied field, local mechanical stress and bilayer edge energy. Lipofection (or liposome transfection) is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer. Lipofection is a lipid-based transfection technology which belongs to biochemical methods including also polymers, DEAE dextran and calcium phosphate. The main advantages of lipofection are its high efficiency, its ability to transfect all types of nucleic acids in a wide range of cell types, its ease of use, reproducibility and low toxicity. In addition this method is suitable for all transfection applications (transient, stable, co-transfection, reverse, sequential or multiple transfections…), high throughput screening assay and has also shown good efficiency in some in vivo models. Sonication is the act of applying sound (usually ultrasound) energy to agitate particles in a sample, for various purposes. Sonication can be used to speed dissolution, by breaking intermolecular interactions. In biological applications, sonication may be sufficient to disrupt or deactivate a biological material. For example, sonication is often used to disrupt cell membranes and release cellular contents. This process is called sonoporation. Sonication can also refer to buzz pollination - the process that bees use to shake pollen from flowers by vibrating their wing muscles.