Curso: Functional Genomic of Central Cardiovascular Control.
Coordenador: Ruy R Campos Jr.1
Convidados: Sergey Kasparov, Anja G.Teschemacher.2
1. Departamento de Fisiologia – UNIFESP – Sao Paulo – SP - Brasil
2. Department of Physiology and Pharmacology, University of Bristol, Bristol UK.
Viral vectors as tools for neuroscience:
Viral vectors (VV) have a great potential for neuroscience research and future gene therapy of brain diseases. VV are derived from pathogenic viruses which have evolved as intracellular parasites and are therefore remarkably efficient in delivering their genomes into host cells. Almost all VV used in research are to make them non-pathogenic, typically by removing critical parts of the wild type viral genome. Therefore, even if accidentally inhaled or injected into the body, VV cannot cause infection because they do not replicate and are promptly destroyed by the immune system. They retain, however, their ability to introduce DNA or RNA into the host cells. We make use of this ability by loading them with foreign genes (transgenes) in order to be delivered to and expressed in mammalian cells.
VV have some very important advantages over drugs for research into brain functions. First, they can modify a cellular function for many weeks, thus allowing study effects in a chronic situation. Second, they can be designed to modulate cell function in a manner which no available drug can, for example to artificially hyperpolarize neurons and reduce their electrical activity. Third, they can be made selective and only allow gene expression in one particular subtype of cells. Numerous examples of VV applications in vivo and in vitro will be discussed.
We will mainly focus on two types of VV which have proven particularly useful for imaging and functional studies of brain cells. These are adenoviral vectors (AVV) derived from the human adenovirus serotype 5, and lentiviral vectors (LVV) developed from the human immunodeficiency virus. AVV and LVV allow introduction of various genes into a range of cells in the body. In the brain, they can transduce (e.g. introduce the foreign genes) neuronal, glial, and vasculature-related cells. Once in the cell, the expression of these virally-delivered genes is controlled by the expression machinery of the host cell. This opens up a way for specific expression of genes in selected cell types within the brain, for example, production of specific proteins only in glia or only in neurons, or even restricted to a particular subtype of neurons, such as those releasing noradrenaline or GABA.
A number of practical considerations apply when choosing the right VV for a particular experimental approach. VVneed to be delivered directly into the brain tissue. For in vivo approaches, this can be achieved via microinjection into specific brain areas of interest several days to weeks prior to experimentation. Expression of transgenes then occurs without further invasive procedures. Hence experiments can be carried out in freely moving conscious experimental animals. Alternatively, in vitro brain preparations, for example organotypic slice cultures can be easily transduced by VV for studies of cell function.
While AVV and LVV with their different structures and modes of operation are both useful for transduction of a range of brain cells, they have different properties important for their use and production. For example, AVV initially take longer to generate, but they then can be produced easily in comparatively large amounts. In contrast, a small amount of LVV sufficient for a few brain microinjections can be produced within 1 to 2 weeks but the preparation has to be repeated for each batch. Sterile stocks, in particular AVV, are stable when in small aliquots at -80C. Suspensions are diluted immediately prior to use in saline or culture media. Activity of the viral suspension depends on density of the active viral particles (titre). Titre is determined using special protocols and has a direct effect on the physiological outcome of the experiments. According to the experience of our laboratories, optimal titres for AVV and LVV in vivo are in the range of 1 – 5 x10^9 TU/ml. It is important to note that higher titres of AVV may cause immune response and thus give worse results. Immune reaction to AVV limits their use even more for peripheral tissues, although they are still used for that purpose. LVV, in contrast, are non-immunogenic.
As such, both VV types are harmless. However, it has to be remembered that they carry and introduce foreign genes and this is where safety considerations must be a priority. Different protocols for AVV and LVV production and titration, as well as safety issues will be introduced.
VV are being more and more intensively used for studies of various functions of the central nervous system, including central cardiovascular regulation. They are used in a wide range of in vivo and in vitro applications, either on their own or in combination with drugs.