Recombinant viral vectors are commonly used for the delivery of genetic material to the nervous system. Viruses have evolved over eons to bypass a host’s immune system and deliver its genetic material in to a host cell. Recombinant viral genomes are engineered to take advantage of the virus ’ ability to infect a host cell while removing the ability of the virus to replicate itself, or to produce damage to the host cell [1]. This book covers the three most commonly used viral vectors: adeno-associated virus (AAV ), lentiviruses (LV)/retroviruses, and adenovirus (Ad). Each of these vectors has its distinct advantages and disadvantages that need to be considered when planning an experiment.

In contrast to the use of viral vectors, non-viral vectors do not rely upon the evolved capabilities of viruses to insert genetic material in to the target cell. Instead, non-viral gene-therapy relies upon the use of a physical force, or DNA conjugated to branched chemicals, in order to force the cell to take up the exogenous DNA, typically in the form of linear DNA, plasmid DNA, or siRNA . A variety of techniques have been devised to achieve this, including the gene gun, electroporation , or the use of branched molecules such as polyethylenimine [15]. Although this approach is beneficial in that it does not require the complexities involved in viral production, the efficacy and longevity of non-viral gene therapy are often inferior to viral-mediated gene therapy.

Once a gene delivery vehicle has been selected, the second decision is whether to produce the vector in-house, or to utilize a core service (either academic or industry) to produce the vector. Moreover, if the choice to outsource vector production is made, what type of service is needed: Will you design, clone, grow, and send the viral genome to a core for packaging or will you utilize a “boutique” service where the complete process (genomic design, cloning , and packaging of the vector) is outsourced? This may depend on how frequently viral vectors will be utilized, as setting up production in-house can be both costly and time-consuming. However, once a laboratory has a working production protocol, outsourcing production is often more expensive and time consuming.

If one chooses to clone and grow the viral genome, there is a certain degree of esoteric knowledge that must be understood, as the cloning and handling of each type of viral vector genome carries with it certain nuances that are unique to that virus . For instance, the AAV and lentiviral genomes are contained within standard plasmids, and during the production of the recombinant virus these plasmids are inserted in to the production cell (e.g., HEK 293 cells) together with plasmids encoding helper functions and essential viral components (see Chapters 7 and 8). The AAV ITRs are genetic structural elements that are relatively unstable and readily recombine. However, this poses a problem as the ITRs are absolutely required for packaging of viable particles, and the functional titer is directly related to the ratio of plasmids with intact versus recombined ITRs. In order to grow these AAV genome plasmids, one must therefore utilize recombination deficient bacterial cells (e.g., SURE cells from Agilent). Moreover, reducing the growing temperature (from 37 °C to 30 °C) and the time of plasmid growth (from roughly 16 to 12–14 h) further improves the integrity of the population of ITRs. Finally, it is imperative that the integrity of the ITRs is checked prior to packaging using a restriction enzyme that gives a unique patterning following recombination (e.g., sma1 for AAV2 ITRs). The ITRs also create a structural barrier for certain manipulations of the viral genome. For instance, any type of mutagenesis that involves a PCR amplification of the entire bacterial plasmid is unlikely to succeed as the complimentary terminal repeats may cause PCR “skipping.” Thus, sub-cloning of the area of interest into a holding plasmid should be done prior to such manipulations. Similarly, the lentiviral LTRs (contained in the transfer plasmid) are also prone to recombination, thus, care should be taken while growing this plasmid in bacteria as well. In addition to the genomic component, when producing lentivirus, one must pay attention to what components must be provided in trans during packaging as the cis acting elements change with each generation of lentiviruses. For instance, the third generation of lentivirus requires the concomitant transfection of four plasmids during production: one transfer plasmid, two packaging plasmids, and one envelope plasmid.

Many plasmids containing viral genomes, helper and transfer plasmids, envelope plasmids, and various expression cassettes can be found at Addgene (http://​www.​addgene.​org) or the National Gene Vector Repository (https://​www.​ngvbcc.​org).

One of the greatest sources of discrepancies between seemingly identical gene therapy studies lies in how the virus is handled once it is produced. Again, this involves esoteric knowledge that is rarely published, if ever. For instance, both AAV and LV are sensitive to freeze-thawing, where the titer drops with each subsequent freeze cycle. In fact, in our hands we never freeze AAV, as this virus is stable at 4 °C for years. Similarly, following production, LV is immediately aliquoted and stored at −80 °C, but each aliquot is never re-frozen.

Outside a host cell, a viral vector can be defined as a complex protein/molecule. The protein structures that envelope these viruses are incredibly “sticky.” Accordingly, viral particles will aggregate in solutions with low ionic strength, or via the adherence of capsids to any contact surfaces. In order to prevent the loss of viral particles (and to avoid re-titering of the vector preparation) we have implemented certain precautions outlined in Chapter 14. Briefly, every surface that comes into contact with the virus is siliconized (e.g., by treating the surface with Sigmacote^® (Sigma-Aldrich)). This reduces the surface charges which otherwise would facilitate virus binding. Alternatively, one can use an identical virus to “coat” surfaces that will come in contact with the vector. Regardless, despite taking all these measures, it is safe to assume that if a virus is transferred (e.g., aliquoted), that some virus will be lost to the procedure itself.

In designing your gene therapy experiment another important consideration is what population of cells you are aiming to transduce: Do I need to transduce only neurons, or astrocytes, or both? Is off-target transduction acceptable? How much volume of the brain do I need to cover? These questions highlight several variables that must be considered when designing your experiment in order to successfully implement a rational approach that best fits your particular situation: (1) the type and pseudotype of viral vector injected, (2) the expression cassette utilized to express your genetic payload, and (3) delivery method.