Integrated gene delivery vectors—Evolution and prospects

Aditya Pattani, Manan Desai and Deepti Panicker

Treating human genetic diseases poses new challenges for modern medicine. Genetic diseases are mainly caused by mutation or deletion of genes, being inherited or transferred from the parent, usually leading to the impairment of the otherwise normal metabolic pathway.

Gene delivery system

Genetic disorders have existed since long; however, no adequate tools were available for their treatment till date. Gene therapy, although still in its infancy, has provided the correct tool. Gene therapy is considered to cure common diseases, as well as cystic fibrosis, SCID, haemophilia, muscular dystrophies, and so on. While new generation nucleic acid based therapies are emerging rapidly, a complete transfer of gene (DNA) into a cell, which is imperative for therapy, is proving to be a difficult hurdle to overcome. The journey of the gene from the needle into the nucleus of the cell is fraught with barriers. The human system naturally contains nucleases, which degrade the gene. That apart, the lack of hydrophobicity and a large size prevents them from reaching the cell and further into the nucleus, imposing a formidable challenge to gene delivery technologists. Efforts are now being directed, across the globe towards efficient and safe gene delivery.

One of the first methods for gene delivery was the use of viral vector. As described by Wolff and Lederberg, viral genomes have been used for the development of the first relatively efficient methods of in vitro gene transfer into mammalian cells. In the late 1970s, transfection techniques were combined with selection systems for cultured cells and recombinant DNA technology. With the development of retroviral vectors in the early 1980s, the possibility of efficient gene transfer into mammalian cells became accepted widely.

Viruses are well known to be able to penetrate the mammalian cell membrane and deliver their nucleic acid to the mammalian cells in an efficient manner. Thus, it was obvious to try gene delivery using such viruses. Various viruses have been used for this purpose. These include a wide range of viruses-non-pathogenic adenovirus, adenoassociated virus, and retrovirus. Although adenoviruses appear to be the most efficient among the viruses, they are reported to cause immune reactions. Next in the efficiency hierarchy come the retroviruses, which have a good efficiency, but suffer from a major limitation of requiring cell division for effective transfection.

Adenoassociated virus comes next in the in the degree of efficiency, while reportedly safer than the adenoviruses and retroviruses, these are difficult to produce. Thus, despite high efficacy, safety and handling difficulties impede their use in practical gene therapy. This is exemplified in one of the earliest clinical trials, which included the transfection of haematopoietic cells with retroviruses carrying a sequence of lambda c-chain cytokine subunit for interleukins, conducted on patients with severe immunodeficiency lacking functional T-cells and natural killers. Although promising results were obtained, a rare form of leukaemia developed in some cases. Such an outcome highlighted not only the risks associated with viral gene delivery but also jeopardised further virus based gene delivery trials.

Non-viral vectors as gene delivery vehicles

Such safety issues combined with the lack of scalability of viral vectors as vehicles for gene delivery prompted the development of non-viral vectors as gene delivery vehicles. The simplest non-vectors include cationic-polymer-DNA complexes, also known as polyplexes; can be used to deliver DNA into cells. Polyplexes are positively charged complexes of cationic polymers with anionic DNA. Use of cationic materials help condense the negatively charged DNA and reduce its susceptibility to nucleases. In addition, positive charges aid to bind the complex to the negatively charged cell surface and improve the chances of internalisation. Similarly, cationic lipids may be used and the complexes so formed are called lipoplexes. With the advent of nanoscience, the DNA may be complexed with nanoparticles made up of cationic polymers like chitosan or polyethylene imine or cationic lipids like lipofectin. Due to their small size, nanoparticles are deemed to be more efficacious than the simple complexes. Cationic liposomes, owing to their typical bi-layer structure, have also improved the gene delivery efficacy. Liposomes remain one of the most worked upon vehicles from DNA delivery. Many cationic materials are known to cause inflammatory responses and efficacy remains moderate. In spite of these drawbacks, the major achievement of this generation of gene delivery vehicles was of increasing the possible size of the gene they could transfer. While, the highest possible gene size that can be delivered by a virus is eight kda, that with a non-viral vector is at least 50 kda.

With exponential increase in the understanding of molecular biology, it was shown that viruses possessed certain proteins, which helped them to get across the cell membranes efficiently. Scientists have further modified the above carriers with these proteins (Their derivatives are called cell penetrating peptides-CPP) to efficaciously transfer the genes intracellularly. The most common cell penetrating peptides are HIV TAT derived peptide, pentratin and transportan. Contemporary research involves coupling these peptides with the nanoparticles or liposomes to improve their intracellular delivery of the genes. Apart from these CPP's other agents such as transferrin, which are internalised via a receptor mediated endocytosis may also be used.

In addition to ensuring the gene crosses into the cell it is essential to ensure their rapid escape from endosomal degradation, once the gene enters the cell. Inclusion of fusogenic peptides such as Hemagglutinin HA2 and those derived from adenoviruses help in endosomal escape. Hemagglutinin HA2 undergoes conformational transition which leads to the destruction of the endosome, Instead of using fusogenic peptides, fusogenic lipids such as 1, 2-dioleoyl-sn-glycero-3-phosphoethano-lamine (DOPE) may also be used in liposomes as a supporting lipid. It disrupts the endosomal membrane upon endosomal acidification by the formation of lipid hexagonal phases. Agents such as monensin and chloroquine, which raise the endosomal pH, block acidification, and thus inhibit lysozyme activity, have also been used to facilitate endosomal release of DNA. Cationic liposomes have also been widely been conjugated to fusogenic peptides to facilitate endosomal escape.

The nuclear envelope is the final obstacle to gene delivery into the nucleus. Passage of molecules into the nucleus is controlled by the nuclear pore complexes that only allow free passage of molecules smaller than approximately 40 kda. Larger molecules and particles up to approximately 40 nm can be actively transported through the nuclear pore complexes only if they comprise a nuclear localisation signal; which is a short stretch of amino acids that mediates the active transport of nuclear proteins into the nucleus.

Thus, gene delivery is an elaborate task and requires the surpassing of multiple barriers. The challenge begins from protection from nucleases and enhancing cellular uptake, to ensuring endosomal escape and subsequent active transport of the DNA through the cytosol and into the nucleus. Clearly, no single material or a simple drug delivery system is capable of effectively achieving such a feat. Non-viral gene delivery systems have been unable to match the efficiency of the viral vectors. Consequently, today gene delivery scientists are working on integrated vectors-vectors, which have the efficacy and complexity of the viral systems while being safe and economically feasible like the non-viral systems.

One such vector that has been experimented is composed of polylysine (a cationic polymer) to condense the DNA; transferrin to obtain cell attachment and subsequent endocytosis; and synthetic peptides resembling the N-terminal fusion domain of the influenza virus hemagglutinin to induce endosomal escape. This integrated system or artificial virus has been shown to be efficiently internalised by cells expressing the transferrin receptor, while the presence of the membrane disrupting peptides enhances the transfection efficiency of these complexes via endosomal escape. Such systems are safe in terms of being non-infectious, and simpler to produce than viruses. They also have a substantially higher capacity of carrying large amounts of DNA. Although much more improvisation needs to be done on such systems, we have come a long way towards a time where gene delivery will see the light of the clinics.

(Aditya Pattani is from UICT , Mumbai , Manan Desai was formerly with Institute of Science , Mumbai and DrDeepti Panicker, B H M S,Mumbai)