Viral Vectors
Gene therapy covers a broad spectrum of applications, from gene replacement and knockdown for genetic or acquired diseases to vaccination, each with different requirements for gene delivery. Viral vectors have emerged as the vehicles of choice for many applications today. One way viral vectors are categorized is as integrating or non-integrating, based on whether the recombinant vectors integrate their viral genome into the cell genome of the host. Integrating vectors, such as gamma-retroviral vectors, are generally used to transduce actively dividing cells. Lentiviruses, another integrating vector, transduce non-dividing and dividing cells. Non-integrating vectors, such as adeno-associated virus (AAV) vectors and adenovirus (Ad) vectors, transduce slowly dividing cells, as they can be quickly lost from rapidly dividing cells.
Figure 1. Converting a virus into a vector.
Vectors Based on Gammaretroviruses
Retroviral vectors are mostly derived from murine leukemia virus. In first generation vectors, transgenes were inserted between long terminal repeats (LTRs) in place of gag, pol, and env genes. Deletion of gag, pol, and env and other nonrelevant sequences enables retroviral vectors to support an expression cassette of up to 9 kb. In second-generation retroviral vector packaging systems, env was separated from gag and pol to avoid recombination and creation of replication-competent particles. Thus recombinant retrovirus particles are generated by triple transfection with plasmids encoding the transgene, gal/pol, or env. More recent vector constructs use self-inactivating (SIN) vectors. SIN vectors, in which the enhancer or promoter of the LTR is deleted, decrease the risk of activating a nearby gene.
Vectors Based on Lentiviruses
Lentiviral vectors are commonly derived from human immunodeficiency virus (HIV), feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV). Initially, lentiviral vectors were packaged using three plasmids: a vector plasmid with the transgene between the HIV LTRs, a packaging plasmid containing gag, pol, and the accessory genes, and a separate envelope plasmid with an env gene. To improve safety, the latest generation of lentiviral packaging systems uses four plasmids, one for rev, one for gag and pol, one for the transgene, and one for encoding the envelope. Furthermore, tat is eliminated through the addition of a chimeric 5' LTR fused to a heterologous promoter on the vector plasmid.
Vectors Based on Adenoviruses
First-generation Ad vectors had E1 and E3 genes deleted, allowing for an insert of up to 7.5 kb. Generally, the expression of Ad vectors in vivo is transient due to the expression of non-deleted viral genes and ensuing immune responses to the expressed viral proteins. More useful Ad vectors for sustained expression in vivo are known as helper-dependent adenoviral vectors, in which all viral genes are excised from the vector backbone except for the inverted terminal repeats (ITRs) and the packaging signals of the wild-type adenovirus. In addition to first-generation Ad or fully gutted Ad, replication competent (or oncolytic) Ads are also used.
Vectors Based on the Adeno-Associated Virus (AAV)
AAVs are generated by inserting a transgene expression cassette between the ITRs in place of the rep and cap genes. The rep and cap genes are provided along with helper viral genes in trans during vector production. There are 12 serotypes and 100 variants of AAVs, which mediate a broad range of tissue or cellular tropisms. For example, AAV8 has been shown to effectively transduce the liver, whereas AAV1 and AAV5 effectively transduce cells in the central nervous system. In addition, the rational design approach utilizes knowledge of AAV to make targeted changes to the capsid to alter transduction efficiency or specificity. Approaches to broaden the range of tissues that can be targeted by AAV include directed evolution and insertion of peptides into capsid surface loops.
Vectors Based on the Herpes Simplex Virus (HSV)
Two types of HSV-1 based vectors have been developed: replication defective vectors and amplicon vectors. As with retroviral and adenoviral vectors, it is relatively easy to establish replication defective HSV vectors. Part or all of the essential IE genes of the viral genome are deleted and the gene of interest is inserted, followed by transduction of a helper cell line that complements the deleted essential genes. The result is a stock of replication defective vectors. Amplicon vectors, on the other hand, are derived from plasmids that contain the HSV-1 origin of replication, the HSV-1 packaging signal and the gene of interest. The plasmid and an HSV-1 helper virus are then co-delivered into a cell line that supports the growth of the helper virus.
Vectors Based on the Vaccinia Virus
Vaccinia virus (VACV) is one of the most widely studied viruses of the Poxviridae family with a linear, double-stranded DNA genome of approximately 190 kb in length, encoding for around 250 genes. It has become a suitable viral vector in vaccine design or cancer gene therapy because of its large packing capacity (up to 25 kb of foreign DNA), stable integration and expression, completely independent of the replication and transcription machinery of the host cell.
Vectors Based on the Baculovirus
Baculovirus is enveloped viruses with a circular double-stranded DNA genome and is widely used to express heterologous genes in cultured insect cells and insect larvae. Baculovirus particles can accommodate large amounts of foreign DNA, because the size of these nucleocapsids is flexible. Moreover, this type of vector provides appropriate post-translational modifications. Insect cells can be adapted to serum-free media, and recombinant protein production can be scaled up to pilot plant or larger bioreactors.
References
- Walther, W.; et al. (2000). Viral vectors for gene transfer. Drugs. 60(2): 249-271.
- Zhang, X.; et al. (2006). Viral vectors for gene delivery in tissue engineering. Advanced drug delivery reviews. 58(4): 515-534.
