Non Viral Gene Delivery Approaches In Fish

Priyanka C. Nandanpawar, Mohan R. Badhe

Central Institute of Fisheries Education, Mumbai

Gene delivery using nonviral approaches has been extensively studied as a basic tool for intracellular gene transfer and gene therapy. Since its development, gene therapy has held great promise in the field of medicine. The ability to introduce functional genes into mammalian cells containing otherwise defective copies offers many new possibilities for the treatment of commonly acquired and inherited fish diseases such as autoimmune disorders and monogenic diseases. The development of efficient gene therapy depends on an efficient transfer of therapeutic genes into a cell to replace or silence defective ones associated with fish disease. Viral vectors like adenoviruses and retroviruses are commonly used in gene therapy due to their high efficiency of gene delivery. However, there are several recurring issues that have led to a reconsideration of the use of viral vectors in clinical trials, such as immunological problems, insertional mutagenesis and limitations in the size of the carried therapeutic genes. The ultimate goal of gene therapy is to allow for continuous expression of transgenes in specific target tissues with minimal toxicity. Gene therapy has many benefits over other therapeutic methods; most importantly, it allows for selective treatment of the genetic defects which cause a disease in the specific cells or tissues that are effected. Recently, non-viral particles have been receiving increasing attention in gene therapy, since they can overcome major viral delivery toxicity issues, it remains a great challenge to find a carrier that will

(1) load genetic materials,

(2) pass the material through cellular barriers without causing a foreign body immune response,

(3) release it into the cell nucleus,

(4) Allow the visualization of this entire process without degrading the genetic materials.

Gene delivery refers to the transmission of nucleic acid (DNA and RNA) encoding a therapeutic gene of interest into the targeted cells or organs with consequent expression of the transgene. Due to electronegativity of naked DNA, the negatively charged cell membrane inhibits the entry of DNA, also the unprotected DNA will be rapidly degraded by nucleases present in plasma.so there is a need to develop a safe and efficient gene transfection therapy system. Recently many techniques have been developed for the introduction of nucleic acids includes both viral and non-viral vectors, but viral vectors may induce a range of fish infections, so non viral gene delivery systems are getting more attention from the viewpoint of increased transfection efficiency and reduced immunogenic risks. Methods of nonviral gene delivery have also been explored using physical (carrier-free gene delivery) and chemical approaches (synthetic vector-based gene delivery). Physical approaches, including needle injection, electroporation, gene gun, ultrasound, and hydrodynamic delivery employ a physical force that permeates the cell membrane and facilitates intracellular gene transfer. The chemical approaches use synthetic or naturally occurring compounds as carriers to deliver the transgene into cells. The earliest chemical methods for DNA delivery were introduced in the late 1950s. These techniques used high salt concentration and polycationic proteins to enhance nucleic acid entry into the cell. Over a 10-year period starting in 1965, a variety of other chemicals were introduced, including DEAE dextran and calcium phosphate, which interact with DNA to form DEAE-dextran—DNA and calcium phosphate—DNA complexes, respectively. After the complexes are deposited onto cells, they are internalized by endocytosis

MICROINJECTION:

Microinjection simply involves using remote-control levers to operate a micropipette tipped with a very fine glass needle. Individual fertilised eggs are held steady by suction against a blunt ended tube and the micropipette needle is inserted into the egg . A small volume of buffer (usually 1—2 nl) containing a high copy number (106—107 copies) of the transgene construct is then injected into the egg, the needle is removed and the egg incubated in the normal way. Fish eggs are surrounded by a chorion that rapidly hardens after fertilisation, making it difficult to penetrate. Partly owing to the hardening of the chorion, it is also extremely difficult to identify the position of the chromosomes in fertilised eggs. the transgene should be injected at the single cell stage to ensure integration in all the cells of the GMO. However, even when this is done, integration (if it happens at all) often only takes place after some cleavage divisions have occurred. This produces mosaic embryos where the transgene is present in some cells but not others. One drawback of the approach, however, is that microinjection can be achieved only one cell at a time, which limits its use to applications in which individual cell manipulation is desired and possible, such as producing transgenic organisms. Though relatively efficient, the method is also rather slow and laborious and therefore neither appropriate for research with large numbers of cells nor practical for DNA delivery in vivo. Injection can cause damage that affects embryonic survival and can result in quite high mortalities. Often fewer than 50% of eggs which have been microinjected express the transgenic DNA in early embryos and expression can decrease significantly after a few days. Better results can be obtained experimentally with smaller fish species such as zebrafish (Danio rerio) and medaka (Oryzias latipes).

ELECTROPORATION:

Electroporation is a method which was first developed for work with cells in tissue culture and it involves subjecting the cells to a short burst of electrical impulse. When cells are treated like this, their cell walls become temporarily much more porous and larger molecules than usual can pass through. Cells to be treated are suspended at high concentration in a solution of high copy number DNA construct and held in a 1—2 ml container with flat electrodes on each side. The transient current is passed and DNA construct molecules pass through the cell membrane. The electrical pulse can have a range of voltages and different rates of decay after the pulse and these are characteristics that are varied in order to produce optimum electroporation results. A major advantage of electroporation over microinjection is that there is no need to handle and manipulate eggs individually. Treatment of cells with colcemid before they are electroporated increases the frequency of transfection. This is most likely due to the arrest of cells at metaphase and the associated absence of nuclear envelope or to an unusual permeability of the plasma membranes. Linearized DNA is far more efficient in transfection than circular supercoiled DNA.

BIOLISTICS:

Particle bombardment, which is also called biolistic particle delivery, can introduce DNA into many cells simultaneously. In this procedure, DNA-coated microparticles (composed of metals such as gold or tungsten) are accelerated to high velocity to penetrate cell membranes or cell walls. Bombardment is widely employed in DNA vaccination, where limited local expression of delivered DNA (in cells of the epidermis or muscle) is adequate to achieve immune responses. Because of the difficulty in controlling the DNA entry pathway, this procedure is applied mainly adherent cell cultures and has yet to be widely used systemically. In plants, where there is a tough cell wall, experimenters have resorted to brute force to get foreign DNA into the cells. Biolistic involves coating microscopic particles, usually of gold, with DNA construct and explosively firing these particles directly into the cell through the cell membrane. This method has been tried with fish, sea urchins and oysters and results suggest that viable transgenic embryos can be produced. However, there is currently no evidence that biolistics offers a more effective or more efficient method of producing transgenic fish than microinjection.

Ultrasound-Facilitated Gene Transfer:

The discovery that ultrasound can facilitate gene transfer at cellular and tissue levels expands the methodology of gene transfer by physical methods. A 10- to 20-fold enhancement of reporter gene expression over that of naked DNA has been achieved. The transfection efficiency of this system is determined by several factors, including the frequency, the output strength of the ultrasound applied, the duration of ultrasound treatment, and the amount of plasmid DNA used. The efficiency can be enhanced by the use of contrast agents or conditions that make membranes more fluidic. The contrast agents are air-filled microbubbles that rapidly expand and shrink under ultrasound irritation, generating local shock waves that transiently permeate the nearby cell membranes. Unlike electroporation, which moves DNA along the electric field, ultrasound creates membrane pores and facilitates intracellular gene transfer through passive diffusion of DNA across the membrane pores. Consequently, the size and local concentration of plasmid DNA play an important role in determining the transfection efficiency. Efforts to reduce DNA size for gene transfer by methods of standard molecular biology or through proper formulation could result in further improvement. Interestingly, significant enhancement has been reported in cell culture and in vivo when complexes of DNA and cationic lipids have been used. Since ultrasound can penetrate soft tissue and be applied to a specific area, it could become an ideal method for noninvasive gene transfer into cells of the internal organs. Evidence supporting this possibility has been presented: in one study, plasmid DNA was co-administered with a contrast agent to blood circulation, and this was followed by ultrasound treatment of a selected tissue. So far, the major problem for ultrasound-facilitated gene delivery is low gene delivery efficiency.

Calcium Phosphate Precipitation:

In this approach, the DNA preparation to be used for transfection is first dissolved in a phosphate buffer. Calcium chloride solution is then added to the DNA solution; this leads to the formation of insoluble calcium phosphate which co-precipitates with the DNA. The calcium phosphate-DNA precipitate is added to the cells to be transfected. The precipitate particles are taken in by the cells by phagocytosis . Initially, 1-2% of the cells were transfected by this approach. But the procedure has now been modified to obtain transfection of upto 20% of the cells. In a small proportion of the transfected cells, the DNA becomes integrated into the cell genome producing stable or permanent transfection.

Deae-dextran Mediated Transfection:

DEAE-dextran (dimethylaminoethyl-dextran) is water soluble and polycationic, i.e., has a multiple positive charge. It is added to the transfection solution containing the DNA. In some unknown way, DEAE-dextran brings about DNA uptake by the cells through endocytosis. Possibly its interaction with the negatively charged DNA molecules and with the components of cell surface plays an important role.

Lipids, liposomes and polymers:

A number of cationic substances, such as liposomes, lipids and polymers, have been investigated for their capacity to improve efficiency of gene delivery. Cationic lipids and liposomes are the most widely used and several have been shown to raise the efficiency of in vitro gene delivery in many cell types. The mechanisms by which lipids and liposomes improve gene transfer is not clearly understood, but several factors, such as the type of cell and their proliferative status, appear to be involved. The positively charged lipids and liposomes are thought to improve transgene delivery mainly through binding to and condensing negatively charged DNA, forming a complex called lipoplex in which the DNA is protected against extracellular degradation. Moreover, the positively charged lipoplex binds to the negatively charged cell surface molecules facilitating endocytosis. Once in the endosome, some lipids may destabilize the endosomal membrane and encourage the release of DNA into the cytosol, thus avoiding the lysosomal degradation pathway. Such lipoplexes can effectively deliver transgenes to myoblasts in culture and many different types of cells in vivo, including epithelial cells and hepatocytes. non-ionic polymers such as poly N-vinyl pyrrolidone (PVP) and some co-polymers have been shown in vivo to enhance transduction of muscle fibres significantly. Based on the use of PVP, Rolland and colleagues established the concept of the 'protective, interactive, non-condensing' (PINC) delivery system, and eported up to 10-fold enhancement of transgene expression over naked plasmid alone.

POLYMERIC GENE CARRIERS

The poor transfer efficiency and transient gene expression of polymer-DNA complexes continue to stimulate the development of more effective and less-toxic polymeric gene carriers. A number of polycations have been tested to transfer genes in vitro or in vivo, including poly-L-lysine (PLL) ,PEI, polyamidoamine (PAMAM) dendrimers, poly(a-(4-aminobutyl)-L-glycolic acid) (PAGA), poly((2-dimethylami-no)ethyl methacrylate) (PDMAEMA) ,chitosan etc.

Poly-L-Lysine

Poly-L-lysine has been widely used in the early days as a gene delivery carrier because of its excellent DNA condensation ability and efficient protection of DNA from nuclease digestion. However, its cytotoxicity and low transfection problems remain to be solved. Moreover, PLL-DNA complexes tend to aggregate under physiological conditions. Efforts have focused on improving the solubility of PLL by the introduction of hydrophilic groups, such as dextran and PEG. Both PEG-g-PLL and PEG-b-PLL have shown lower cytotoxicity and enhanced transfection efficiency than PLL.

Polyethylenimine

Polyethylenimine-DNA complex is another potent synthetic gene transfer vector which has the ability to transfect a wide variety of cells. The high transfection efficiency of PEI relates to its buffering capacity, which leads to the proton accumulation due to endosomal ATPase and an influx of chloride anions, triggering endosome swelling and disruption, followed by the release of DNA into cytoplasm. There are considerable differences in the cytotoxicity and transfection efficiency of branched and linear PEI. Toxicity remains one of the main concerns for PEI to be used in gene delivery. Cytotoxicity of PEI is thought to be derived from its ability to permeabilize cell membranes. Moreover, PEI is not degradable and high molecular weight PEI may accumulate in the body. Several approaches have been explored to reduce the cytotoxicity, such as PEGylation and conjugation of low molecular weight PEI with cleavable cross-links such as disulfide bonds, which can be cleaved in the reducing environment of the cytoplasm.

Chitosan:
Chitosan, a biodegradable polysaccharide composed of D-glucosamine repeating units, has been explored by several research groups as a nonviral gene carrier. Chitosan can efficiently bind DNA and protect DNA from nuclease degradation. Moreover, chitosan has good biocompatibility and toxicity profile, rendering it a safe biomedical material for clinical applications. Chitosan-DNA nano-particles can transfect several different cell types. However, the transfection efficiency is relatively poor. Chitosan can be readily modified. For example, trimethy-lated chitosan can be prepared with different quarter-nization degree to increase the solubility of chitosan at neutral pH,or chitosan can be conjugated with deoxycholic acid to become a colloidal gene carrier.Both quaternarized chitosan and deoxylic acid-modified chitosan can efficiently transfect COS-1 cells. Chitosans with different molecular weights have different DNA binding affinities and exhibit different transfection efficiencies, indicating that particle stability may be one of the rate-limiting steps in the overall transfection process.The effect of degree of deacetylation on the stability of chitosan-DNA nanoparticles is being investigated.

Polyphosphoester:

Polyphosphoesters (PPE), particularly the ones with a backbone analogous to nucleic acids and teichoic acids show promise in different biomedical applications because of their biocompatibility, biodegradability, and pendent chain functionality. Several polyphosphoesters with positive charges either in the backbone or in the side chain were synthesized and evaluated as nonviral gene car-riers. These polyphosphoesters can efficiently bind DNA and protect DNA from nuclease degradation. Efficient transfection was found in a number of cell lines, with some of them comparable to Lipofectamine. The transfection is cell-type dependant and can be improved with the incorporation of chloroquine. All the polypho-sphoesters exhibit a significantly lower cytotoxicity than PLL or PEI in vitro and in vivo. With the structural versatility, polyphosphoester can be designed to have different hydophilicity-hydrophobicity properties. One interesting feature is that the PPE can provide extra cellular sustained release of the DNA, leading to prolonged and enhanced transgene expression in the muscle compared to naked DNA administration.

DEAE-dextran and calcium phosphate methods are simple, effective, and still widely used in the laboratory for in vitro transfection. Even so, both methods are hampered by cytotoxicity and the difficulty of applying them to in vivo studies. In addition, DEAE dextran can be used neither with serum in culture medium nor for stable transfection. The calcium phosphate method also suffers from variations in calcium phosphate—DNA sizes, which causes variation among experiments. Since the early 1970s, many other polymers have been demonstrated to increase DNA uptake by cells. The most noticeable improvement is the development of artificial lipid-based DNA delivery systems. Felgner and colleagues developed the cationic lipid Lipofectin in 1987. Lipofectin—DNA complexes can be handled easily and, therefore, became one of the first chemical systems that could be used in animals and fishes.

Advantages of non viral gene delivery systems:

• No viral components

• Low or no immunogenicity

• No limit to DNA insert size

• Cell specificity possible with targeted ligands

• Relatively simple preparation procedures

• Standardized homogenous, stable reagents

• Scale up possible

Disadvantages of non viral gene delivery systems:

• Low transfection efficiency

• Transient gene expression-episomal expression

• Intracellular barrier- may require additional agents

• Cellular toxicity with some vectors (PEI,liposomes)

• Inflammation due to unmethylated CpG DNA sequences

Conclusion

The efficiency of transgene delivery and expression by non-viral delivery systems in muscle has been improved significantly. This is mainly due to the construction of new vectors, the use of gene delivery enhancing reagents and the development of novel delivery systems. Nearly all of the measures taken so far focus on tackling obstacles which prevent the efficient entry of plasmid into cytosols and nuclei, or which cause DNA degradation. However, although it is known that plasmid DNA can persist in myonuclei for long periods, little is known about how the expression of transgenes in episomal DNA is controlled by cellular transcriptional and translational machineries. Nor do we know how the protein products of transgenes are transported from muscle fibre cytoplasm into the circulation. The ultimate efficiency of transgene expression and delivery of the gene products could therefore be interfered with or improved at each of a number of stages. For example, recent evidence suggests that active RNA polymerases are concentrated in discrete 'factories' where they work together on many different templates. The evidence that such factories specialize in the transcription of particular groups of genes prompts the speculation that these factories could be targeted for efficient transcription.

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