Non Viral Vectors

One of the greatest concerns that researchers are faced with in gene therapy is the safety of viral vectors as gene delivery vehicles. Consequently, considerable effort has been devoted to evaluating and designing alternative strategies for gene delivery. Nonviral approaches that are currently in devel opment take into consideration the size of the therapeutic gene to be delivered, targeting specificity, immunogenicity, and toxicity.

a. Naked DNA. Perhaps the simplest nonviral gene delivery system in use today is the transfer of naked DNA directly into cells. The overall efficiency of this method, however, is very poor when compared to viral gene transfer. Without mechanical or chemical help, naked DNA will not enter cells rapidly, and once inside, the nucleic acid is exposed and susceptible to enzymatic degradation. In addition, plasmid DNA carrying therapeutic gene does not usually integrate into the host genome, and gene expression is transient in those cells that are successfully transfected. In spite of these limitations, surprisingly high levels of gene expression have been obtained in a few accessible tissues, such as skin and muscle, using plasmid DNA. In such cases, treatment is carried out by directly injecting the plasmid DNA into the tissue because the DNA is vulnerable to degradation in body fluids. So far, the method is safe and nontoxic, but it lacks the ability to transduce a large number of cells and requires surgical procedures to access internal tissues.

An improved strategy for delivery nucleic acid directly into cells is by high velocity bombardment of the cells with DNA attached to gold particles using a "gene gun'' approach. Microparticle bombardment has shown some impressive results in focal delivery of naked DNA to corneal cells with little damage or irritation to the tissue (144). It is a method that is being developed for more widespread use and may be a solution to some of the problems encountered with viral vectors. Most gene transfer studies in the eye are carried out using viral vector, but plasmid delivery of a few therapeutic genes, such as tissue plasminogen activator and IFN-a, has been tested and shows potential benefits in treating corneal-related pathologies (145-157). A few studies have also reported successful gene expression in the retina using plasmid DNA. In one case it was demonstrated that condensed plasmids containing the human fibroblast growth factor genes were able to transduce a small population of choroidal and RPE cells after subretinal injections into RCS rat eyes. FGF gene expression in those tissues consequently resulted in a delay in photoreceptor degeneration (148). While the current methods of delivering naked DNA are still very inefficient, the eye is in a prime location to benefit from improvements in mechanical delivery strategies that increase the therapeutic index of this approach.

b. Liposomes. Liposomes are probably the most widely known non-viral vectors used to transfer DNA into cells. The strategy involves encapsulation of plasmid DNA in lipid complexes that are capable of fusing with the cell membrane and delivering the therapeutic genes intracellu-larly. Initially, this approach has encountered difficulties because classical liposomes are negatively charged lipids that do not interact spontaneously with DNA. Charge limitations and the need to separate DNA-liposome complexes after delivery have led to the development of positively charged cationic lipids. These interact with DNA more readily and have proven to be valuable tools that can compact and deliver DNA across the cell membrane with greater efficacy (149-170). Cationic liposomes are typically formulated using a positively charged lipid and a co-lipid that will stabilize the DNA complex. A commonly used formulation is a mixture of a cyto-fectin with a neutral lipid component such as DOPE. This combination can be formulated into unilammellar vesicles by several methods, including reverse phase evaporation and microfluidization. Stable complexes are formed when DNA is combined with the vesicles. The DNA is subsequently condensed in the vesicles, forming nanometric particles that are referred to as lipoplexes. These complexes protect the DNA, interact with cell surface proteoglycans, and enter the cell by endocytosis (170) (Fig. 7).

Figure 7 Liposome-mediated transfer of nucleic acid. The nucleic acid is condensed in the liposome to form lipoplexes. These enter the cell by endocytosis. The majority of lipoplexes are trapped in late endosome. A small percentage can either be released into the cytoplasm (mRNA), where they are functional, or traffic non-specifically to the nucleus, where they may form episomes.

Figure 7 Liposome-mediated transfer of nucleic acid. The nucleic acid is condensed in the liposome to form lipoplexes. These enter the cell by endocytosis. The majority of lipoplexes are trapped in late endosome. A small percentage can either be released into the cytoplasm (mRNA), where they are functional, or traffic non-specifically to the nucleus, where they may form episomes.

Currently, no more than 30 genes transfer-competent cationic liposomes have been developed and are commercially available. Perhaps the most widely used formulations are DOTAP and DOTMA, the latter of which is sold as Lipofectin, an in vitro transfecting agent.

A disadvantage of current methods of liposome-mediated gene transfer is due to the large percentage of the DNA-bound complexes trapped in late endosomes, where they undergo enzymatic degradation and are no longer therapeutically useful. Only a small percentage of the bound nucleic acid escapes systemic inactivation and endosome entrapment. Those that manage to escape face yet another hurdle of getting to the nucleus and maintaining their functional integrity. As with viral delivery, in vitro effectiveness of liposome-mediated gene transfer is often misleading and is a poor guide for clinical efficacy. Conventional liposome formulations lack cell specificity and can take hours for uptake into the cell. They are highly susceptible to inactivation by a number of serum proteins that bind and cause membrane destabilization, a major obstacle for systemic administration of liposomes. A current research focus in the pharmaceutical industry is the development of sterically stable liposome formulations that are resistant to serum disruption and that will not aggregate prior to delivery. One modification currently in development uses conventional liposome lipid membranes to covalently attach polymers such as polyethylene glycol (PEG-lipid) to create stealth liposomes (152,155,156,162,169). Properly formulated polymer-grafted liposomes are shown to be sterically stabilized compounds that have long residence times in circulation, increased biodistribution, and reduction in uptake by cells of the reticuloendothelial system. Other clinically advantageous features of pegylated liposome pharmacokinetics include dose independence and increased efficacy as a slow release system for ther-apeutically active drugs. This is a fascinating technology that has the potential of being a tailor-made delivery system that will improve the therapeutic index of a number of drugs.

Another modification strategy under active investigation is the manufacture of ligand-targeted liposomal drugs using combinatorial approaches. Such molecular conjugates could potentially be more versatile than the conventional systems (159-164). In a recent study, transfection was observed to be increased in hepatoma cells after the administration of modified lipoplexes containing triantennary galactosyl residues that specifically target hepatoma cells (172). Targeted delivery of doxorubicin to human umbilical vein endothelial cells and subsequent decrease in the survival of the cells were also achieved with immunoliposomes that were conjugated to a monoclonal antibody against E-selectin, a surface marker of HUVECs (173). While targeting will increase transfection efficiency to specific tissues, it does not address problems of DNA release encountered in the endosomes.

Some researchers have shown that the association of amphiphilic peptides, such as GALA, a pH-sensitive peptide, with cationic liposomes can induce fusion and permeabilization at acidic pH values and improve release of the DNA from endosomes. The peptides induce osmotic swelling and subsequent rupture of the endosomal membranes so that the DNA can escape easily (174). These new modifications, however, are not without problems. Competition between ligand-mediated processes and nonspecific interactions with the cell membrane can hinder the efficacy of gene delivery and must be resolved before ligand-modified liposomes are of clinical relevance.

In addition to engineering modifications that result in specific cell targeting and more efficient DNA release, mechanisms that will increase nucleic acid condensation and promote nuclear targeting are promising areas of research that will improve liposome-mediated gene delivery. Modifications using DOTAP liposome-protamine sulfate-DNA (LPD) formulations are shown to produce denser particles when bound to DNA and result in consistently higher gene expression levels (175,176). Complexes formed with polycations are also observed to be much smaller than those formed with liposomes alone and have increased resistance to nuclease degradation. Smaller-size complexes may allow for higher levels of gene expression because of increased cellular internalization. An interesting variation to this hybrid concept is the use of UV-irradiated Japanese Sendai virus (HVJ)-cationic liposome to facilitate nuclear targeting. The binding of high mobility group 1 protein (HMG-1) increase the potency of the complex and enhances nuclear targeting and stability of the DNA after delivery into the nuclear envelope. The success of HVJ-liposome complexes in cancer applications is thought to result from the ability of the complex to bypass the endocytosis process, thereby minimizing the difficulties encountered when the DNA is released from the endosomes (171). The development of "synthetic chemical viruses'' that are capable of (a) extended blood circulation, (b) increased DNA microparticle condensation, (c) improved cellular uptake, (d) flexible tropism, (e) escaping enzymatic degradation, and (f) nuclear targeting is an attractive challenge in the area of biopharmaceutics. If realized, such compounds have enormous potential in gene therapy protocols and may surpass the clinical usefulness of viral vectors.

Liposome-based techniques have been optimized to successfully transfer functional genes into human primary RPE cells. In one study, differences in the efficiency of transfection were observed between the types of liposomes used in the assay. Nontoxic transfer was achieved after each liposome treatment, but the Tfx-50 formulation showed the most significant results when compared to transfection of the RPE cells with other liposome variations, including lipofectin, lipofectamine, Cellfectin, and DMRIE-C (177). A fascinating variation of gene transfer by liposomes was achieved by a group of researchers who used liposome eye drops to transfer rat retinal ganglion cells. Transfection was reported to be efficient and nontoxic to ocular tissues. This approach represents an interesting development in nonsurgical gene delivery for retinal diseases (178). The use of another liposome method, hemagglutinating virus of Japan liposomes, was tested for efficacy in delivering tissue inhibitor of metalloproteinase-3 gene into rat RPE cells. Not only was the transfection successful, but expression of the introduced gene inhibited the development of experimental choroidal neovascularization induced by laser photocoagulation after transfection of the tissue (179). These are only a few examples showing the feasibility of using, nonviral, nontoxic synthetic DNA-complexing derivatives to transfer therapeutic genes to the retina.

The development of innovative nonviral delivery system is still in its infancy, but many advantages are associated with their use in gene transfer applications: (a) they can package and deliver a transgene of any size; (b) packaging cell lines are not required to generate high titers; (c) they are nonpathogenic and cannot replicate; (d) immunogenicity, toxicity, and inflammation are minimized with their use; and (e) they can become completely synthetic. While these are safe gene delivery systems, the disadvantages currently lie in their overall inefficiency of transfection and their inability to achieve cell-specific and nuclear targeting. Modifications that improve these features will allow synthetic polymer-based gene vectors to be the candidates of choice for pharmacological intervention in many diseases.

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