Generally speaking, transfection is understood as involving the introduction of a foreign material (such as nucleic acid or proteins) into a cell. Transfection as discussed herein involves introduction of nucleic acid into a cell.
Delivery of nucleic acid to cells is a powerful tool for study and treatment of various medical conditions, as well as a basic research tool. General methods for introducing nucleic acids into mammalian cells include chemically-mediated transfection (such as calcium phosphate), lipid-mediated transfection (such as Lipofectamine™), transfection via cationic polymers (such as poly(ethyleneimine) (PEI) and poly-L-lysine (PLL)), instrument-mediated transfection (for example, using such known devices as Electroporator, Nucleofector®, and Gene Gun), magnetofection, and viral transduction. Of these methods, transfection reagents employing chemicals, lipids, polymers, or combinations thereof that can efficiently deliver nucleic acids to cells offer the greatest convenience while avoiding the risks, toxicity, and regulations associated with the use of viruses, as well as the physical cellular trauma associated with the use of ballistic or electroporation techniques.
Cationic polymers such as PEI and PLL have become commercially available for transfection of mammalian cells. These positively charged polymers are efficient at forming polyplex nanoparticle complexes with negatively charged nucleic acids, including plasmid DNA and siRNA. More recent PEI formulations have in many instances been found to be less toxic and more efficient than lipid-based transfection reagents. Amine-rich polymers such as PEI (mixture of primary, secondary, and tertiary amines) facilitate endosomal escape through the so-called proton-sponge effect, whereby amine buffering of protons pumped into endosomal compartments during acidification causes their swelling and rupture, releasing the carrier and nucleic acid(s) into the cytoplasm within about four hours. This mechanism may be primarily responsible for more efficient transfection of mammalian cells by PEI-based vectors when compared to cationic lipid-based vectors.
Delivery of nucleic acid to neuronal cells is critical for the study and understanding of neuronal cell function in both healthy and disease states. However, transfection of developmentally mature cultured neurons (greater than approximately 9 days in vitro), as well in vivo neuronal transfection, present special challenges. Non-viral transfection reagents for the delivery of nucleic acids to neurons or neural circuits in vivo, or to mature neurons in vitro, are notably inefficient. Thus, reagent-based methods for the genetic manipulation of neurons are typically limited in use to developmentally immature cultured neurons.
Difficulties in transfecting developmentally mature neurons can be illustrated by work done with some common transfection reagents. For in vitro use, commercially available transfection reagents such as calcium phosphate and cationic lipids (such as Lipofectamine 2000™) have been the state of the art for many years, achieving transfection rates of greater than 50% in various non-neuronal mammalian cells, for example HEK-293 and fibroblasts. These transfection reagents are also used with fair success in dissociated early-postnatal or embryonic primary neuronal cultures when used during the first few days in culture and prior to the development of a mature network of functional synapses (up to approximately 20-25% transfection efficiency). However, these reagents typically produce transfection rates of much less than 5% for developmentally mature neurons in dissociated cultures (greater than 7-14 DIV). This low efficiency transfection of developmentally mature neurons, coupled with inherent serum instability, makes these reagents poorly suited for many, if not most, in vivo applications. This shortcoming has in turn curtailed the application of powerful molecular genetic tools in neuroscience research beyond their application to developmentally immature dissociated cultures.
Moreover, a common observation in the field is that lipid-transfected neurons in vitro present challenges in electrophysiological procedures, due to increased membrane fragility and leak currents, and a trend towards depolarized resting potentials when compared to untransfected cells. This observation suggests that these reagents posses an inherent toxicity to neurons.
In further studies, it has been demonstrated that the efficiency of PEI- and PLL-mediated transfection in mature cultured neurons is stubbornly low. Accordingly, these techniques have found limited utility as a neuronal transfection reagent either in vitro or in the more challenging environment of the central nervous system (CNS). Indeed, one effort found that laser-induced stress waves were required for efficient transfection with a PEI gene carrier in mouse CNS. Other studies have shown that differentiation of neurotypic cells results in markedly decreased uptake of transfection reagents. It appears likely that mature neurons are fundamentally resistant to transfection with cationic reagents, because these reagents do not cross the neuronal plasma membrane efficiently on their own.
In general, there are a number of barriers for transfection-based delivery of nucleic acids to mammalian cells in vivo, including, for example: nucleic acid particle stability in the presence of serum proteins, protection from nucleases and acid hydrolysis, nucleic acid particle interaction with plasma membrane, nucleic acid particle internalization, escape from endocytic vesicles, efficient nucleic acid dissociation, and acute or chronic toxicity.
In addition to the above-mentioned barriers encountered in delivery of nucleic acid to mammalian cells, neurons present further challenges to transfection techniques. Developmentally mature neurons appear to have unique requirements for the internalization of nanoparticles, with cationic substances generally performing poorly. The unique characteristics and composition of mature neuronal plasma membrane lipids, associated membrane proteins, and variations in the structure and sulfonation of extracellular glycoseaminoglycans such as heparin sulfate (which is known to be cell type dependent) may decrease the binding affinity/avidity or uptake of conventional non-viral gene vectors by mature neurons. These barriers to non-viral gene transfer, especially in mature neurons, are a major bottleneck as the efficiency of the gene expression in vivo often lies below the threshold efficiency for functional or therapeutic changes. Further, for delivery of nucleic acids to neuronal soma, the ability for the vector to undergo retrograde transport could be a significant advantage, given the unique, highly elongated and branched morphology typical of mature neurons, and the relative scarcity of neuronal soma versus neurophil in brain parenchyma. However, this intracellular transport may present further challenges, as it involves further targeting of the nucleic acid even after it has been taken up into a neuronal cell.
On a separate subject, neuroanatomical tract-tracing involves methods to label and follow the course of neural pathways by axonal transport of injected neuronal tract-tracers. Neuronal tract-tracing materials generally comprise markers that can be stained or fluoresce, to enable their visualization. Neural tract-tracing can be retrograde or anterograde. Negatively charged tract-tracers are not, on their own, considered suitable nucleic acid carriers because they have not been found to efficiently condense nucleic acids.
Agents capable of condensing and maintaining nucleic acids in a form suitable for transfection under physiological conditions are generally cationic. Such agents commonly used for the condensation of nucleic acids in vitro include multivalent cations, basic proteins or peptides, cationic polymers and copolymers (polycations), cationic liposomes, or combinations thereof. Cationic carriers may also include suspensions of suitable sized nanoparticles bearing a high density of cationic charges on their surface and which are capable of forming a complex with nucleic acids that is stable under physiological conditions.