The exploitation of living bacteria for gene transfer is central to the genetic manipulation of plants. The majority of Chapter 12 in the 6th edition of Principles of Gene Manipulation is devoted to the subject of plant genetic engineering by two species of bacteria: Agrobacterium tumefaciens and, to a lesser extent, Agrobacterium rhizogenes. Recently, it has been shown that A. tumefaciens can transfer DNA to cultured human cells (Kunik et al. 2001). As discussed below, however, this is not an isolated observation. Indeed there is growing evidence that highly efficient gene transfer to animals can be achieved using a variety of bacterial species.

The transfer of recombinant DNA from bacterial to animal cells was first achieved in 1980 using a technique called protoplast fusion (Schaffner 1980). In this method, bacterial cells containing recombinant plasmids were treated with lysozyme and chloramphenicol. The lysozyme digested away the bacterial cell wall, leaving behind a membrane-bound protoplast, while the chloramphenicol amplified the plasmid copy number. The resulting plasmid-filled sacs were centrifuged onto a monolayer of mammalian cells and induced to fuse with them using polyethylene glycol (PEG).

The protoplast fusion technique can be regarded as highly artificial due to the amount of human intervention required to achieve gene transfer. In contrast, Agrobacterium species can transfer DNA to plants without any human intervention. The first reports of similarly natural gene transfer between bacterial and animal cells were published in the mid 1990s (e.g. Sizemore et al. 1995, reviewed by Higgins & Portnoy 1998). Typically, the bacteria invade the host animal cells and undergo lysis within them, releasing plasmid DNA. In the case of Salmonella species, lysis occurs in the phagocytic vesicle, while for other species (e.g. Listeria monocytogenes and Shigella flexneri) lysis occurs after the bacterium has escaped from the vesicle. The plasmid DNA then finds its way to the nucleus, where it is incorporated into the host cell's genome and expressed. In contrast, A. tumefaciens has been shown to transfer DNA to mammalian (and plant) cells without invading them. In this case, transfer occurs by attachment to the outside of the cell followed by conjugation (the transfer of DNA through a conduit called a pilus, which is assembled by the bacterial cell).

An important principle in the use of live bacteria as invasive gene transfer vehicles is that they must be attenuated. This is because the gene transfer system exploits the natural ability of the bacteria to infect and subvert the activity of eukaryotic cells. Without attenuation, the bacteria would multiply and destroy the host cells. Attenuation is achieved in two ways. The first is to use auxotrophic mutants, i.e. bacterial strains that are unable to manufacture essential molecules such as amino acids, nucleotides or components of the cell wall. For example, aroA mutants are unable to synthesize aromatic amino acids, and Salmonella typhimurium and Shigella flexneri strains carrying this mutation have been used for gene transfer (reviewed by Weiss & Chakraborty 2001). Alternatively, the bacteria can be engineered so that they undergo inducible autolysis. There are no auxotrophic strains of Listeria monocytogenes available, so attenuation has been achieved by induced suicide, i.e. introducing an autolysin-encoding gene that is activated once the bacterium is inside the host cell (Dietrich et al. 1998). In vitro, lysis can also be induced by treating cells with antibiotics.

Thus far, bacteria-mediated gene transfer has been used not only as a general transfection method for the introduction of DNA into cultured cells, but also as a high-efficiency method for gene transfer in vivo. There have been many reports of bacterial gene transfer as a method for the delivery of recombinant DNA vaccines (e.g. see Xiang et al. 2000, Woo et al. 2001) and comparative studies indicate that bacterial transfer is more efficient than equivalent naked DNA vaccines, which are discussed in Chapter 12 of Principles of Gene Manipulation (Zoller & Christ 2001). The potential of bacterial gene transfer in gene therapy has also been explored (Paglia et al. 2000).

References:

Dietrich G, Bubert A, Gentschev I, Sokolovic Z et al. (1998) Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by attenuated suicide Listeria monocytogenes. Nature Biotechnol 16, 181-5.

Higgins DE, Portnoy DA (1998) Bacterial delivery of DNA evolves. Nature Biotechnol 16, 138-9.

Kunik T, Tzfira T, Kapulnik Y, Gafni Y et al. (2001) Genetic transformation of HeLa cells by Agrobacterium. Proc Natl Acad Sci USA 98, 1871-6.

Paglia P, Terrazzini N, Schulze K, Guzman CA, Colombo MP (2000) In vivo correction of genetic defects of monocyte/macrophages using attenuated Salmonella as oral vectors for targeted gene delivery. Gene Therapy 7, 1725-30.

Schffner W (1980) Direct transfer of cloned genes from bacteria to mammalian cells. Proc Natl Acad Sci USA 77, 2163-7.

Sizemore DR, Branstrom AA, Sadoff JC (1995) Attenuated Shigella as a DNA delivery vehicle for DNA-mediated immunization. Science 270, 299-302.

Weiss S, Chakraborty T (2001) Transfer of eukaryotic expression plasmids to mammalian host cells by bacterial carriers. Curr Opin Biotechnol 12, 467-72.

Woo PCY, Wong LP, Zheng BJ, Yuen KY (2001) Unique immunogenicity of hepatitis B virus DNA vaccine presented by live attenuated Salmonella typhimurium. Vaccine 19, 2945-54.

Xiang R, Lods HN, Chao TH, Ruehlmann JM et al. (2000) An autologous oral DNA vaccine protects against murine melanoma. Proc Natl Acad Sci USA 97, 5492-7.

Zoller M, Christ O (2001) Prophylactic tumor vaccination: comparison of effector mechanisms initiated by protein versus DNA vaccination. J Immunol 166, 3440-50.

Baculoviruses for gene transfer to mammalian cells

Chapter 10 of the 6th edition of Principles of Gene Manipulation discusses viral and non-viral vectors for gene transfer to animal cells. The section on baculoviruses (pp 190-193) deals extensively with the use of baculovirus vectors for gene transfer to insect cells. Baculovirus vectors replicate in insect cells and can produce large amounts of recombinant protein under the control of endogenous promoters, such as the polyhedrin promoter. Briefly, we mention that such vectors can also infect, but not replicate within, mammalian cells. Recently, there have been a number of reports in which baculovirus vectors containing genes under the control of mammalian promoters have been transferred to mammalian cells and expressed at high levels. Importantly, gene transfer has been achieved not only to cultured cells but also to cells in vivo. There have also been recent developments in the use of hybrid viral vectors based on baculoviruses, some of which are discussed below. For recent reviews on baculovirus vectors, see Kost & Condreay (1999, 2002).

The ability of baculoviruses to infect mammalian cells was first demonstrated more than 35 years ago (Himeno et al. 1967). However, it was not until the mid 1990s that recombinant baculovirus vectors containing genes under the control of mammalian promoters were first used to transduce and express foreign proteins in mammalian cells. In the first reports, the human cytomegalovirus (CMV) promoter was used to drive luciferase gene expression (Hofmann et al. 1995) and the Rous sarcoma virus long terminal repeat promoter (RSV-LTR) was used to drive lacZ gene expression (Boyce & Bucher 1996). A variety of cell lines was tested and it appeared that efficient gene expression was possible only in cells of hepatic origin. Since then, however, many different mammalian promoters have been used and the range of amenable cell lines has been vastly extended (see for example Shoji et al. 1997, Condreay et al. 1999, Sarkis et al. 2000). A two-component system has also been developed in which one vector expresses RNA polymerase from bacteriophage T7, and another contains a gene driven by the T7 promoter (Yap et al. 1997).

While representing an efficient in vitro transduction system, the inability of baculoviruses to replicate in mammalian cells makes them especially valuable as a safe alternative for in vivo applications such as DNA vaccination and gene therapy. Unfortunately, early studies of in vivo gene transfer using baculoviruses were hampered by the sensitivity of the virus to complement-mediated inactivation (Sandig et al. 1996). The introduction of baculovirus vectors into mice in which the complement system had been inactivated resulted in efficient gene transfer and expression (Hoffman & Strauss 1998, Hoffman et al. 1999). Therefore, a baculovirus vector has been developed expressing its own regulator of complement activity, decay accelerating factor, and has been shown to be highly efficient for in vivo gene delivery and expression (Huser et al. 2001). Efficient gene transfer has also been achieved by pseudotyping the baculovirus with a vesicular stomatitis virus G glycoprotein (Barsoum et al. 1997). Transfer by unmodified baculovirus vectors is efficient if the virus is not exposed to high levels of complement, as occurs for example in the brain.

Baculoviruses are useful not only for the delivery of foreign genes into mammalian cells, but also for the delivery of other viruses. For example, hepatitis C virus does not infect cultured cells, but a hybrid baculovirus containing the entire HCV genome can initiate an HCV infection (Fipaldini et al. 1999). Baculoviruses can also be used to improve the production of recombinant viral vectors. As discussed in Chapter 10 of Principles of Gene Manipulation (pp 188-189), vectors based on adenoviruses are generally replication-defective because they lack one or more essential viral gene products. Adenovirus vectors lacking the E1a and E1b genes (E1 replacement vectors) can be propagated on a helper cell line (a packaging line) which supplies these functions, but the capacity of such vectors is limited to about 7 kb. Higher capacity vectors, known as gutted vectors, amplicons or fully deleted adenoviruses (FD-AdV), contain no viral genes at all, only those cis-acting elements required for replication and packaging. Cell lines are not available for the packaging of such vectors so helper viruses are normally required, which leads to contamination of recombinant stocks. Recently, however, a recombinant baculovirus vector has been developed which carries a packaging-deficient copy of the entire adenovirus genome (Cheshenko et al. 2001). This hybrid is maintained stably in insect cells and supplies all the required adenoviral functions when introduced into 293 cells, allowing the FD-AdVs to be efficiently packaged. Currently, some replication-competent adenoviruses are produced by recombination in this system, but further refinements are likely to eliminate this problem in the future.

References:

Barsoum J, Brown R, McKee M, Boyce FM (1997) Efficient transduction of mammalian cells by a recombinant baculovirus having the vesicular stomatitis virus G glycoprotein. Hum Gene Therapy 8, 2011-8.

Boyce FM, Bucher N (1996) Baculovirus mediated gene transfer into mammalian cells. Proc Natl Acad Sci USA 93, 2348-52.

Cheshenko N, Krougliak N, Eisensmith RC, Krougliak VA (2001) A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus. Gene Therapy 8, 846-54.

Condreay JP, Witherspoon SM, Clay WC, Kost TA (1999) Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. Proc Natl Acad Sci USA 96, 127-32.

Fipaldini C, Bellei B, La Monica N (1999) Expression of hepatitis C virus cDNA in human hepatoma cell line mediated by a hybrid baculovirus-HCV vector. Virology 255, 302-11.

Himeno M, Sakai F, Onodera K, Nakai H, Fukada T (1967) Formation of nuclear polyhedral bodies and nuclear polyhedrosis virus of silkworm in mammalian cells infected with viral DNA. Virology 33, 507-12.

Hofmann C, Huser A, Lehnert W, Strauss M (1999) Protection of baculovirus-vectors against complement-mediated inactivation by recombinant soluble complement receptor type 1. Biol Chem 380, 393-5.

Hofmann C, Sandig V, Jennings G, Rudolph M et al. (1995) Efficient gene transfer into human hepatocytes by baculovirus vectors. Proc Natl Acad Sci USA 92, 10099-103.

Hofmann C, Strauss M (1998) Baculovirus-mediated gene transfer in the presence of human serum or blood facilitated by inhibition of the complement system. Gene Therapy 5, 531-6.

Huser A, Rudolph M, Hofmann C (2001) Incorporation of decay-accelerating factor into the baculovirus envelope generates complement-resistant gene transfer vectors. Nature Biotechnol 19, 451-5.

Kost TA, Condreay JP (1999) Recombinant baculoviruses as expression vectors for insect and mammalian cells. Curr Opin Biotechnol 10, 428-33.

Kost TA, Condreay JP (2002) Recombinant baculoviruses as mammalian cell gene-delivery vectors. Trends Biotechnol 20, 173-80.

Sandig V, Hofmann C, Steinert S, Jennings G et al. (1996) Gene transfer into hepatocytes and human liver tissue by baculovirus vectors. Hum Gene Therapy 7, 1937-45.

Sarkis C, Serguera C, Petres S, Buchet D et al. (2000) Efficient transduction of neural cells in vitro and in vivo by a baculovirus-derived vector. Proc Natl Acad Sci USA 97, 14638-43.

Shoji I, Aizaki H, Tani H, Ishii K et al. (1997) Efficient gene transfer into various mammalian cells, including non-hepatic cells, by baculovirus vectors. J Gen Virol 78, 2657-64.

Yap CC, Ishii K, Aoki Y, Aizaki H et al. (1997) A hybrid baculovirus-T7 RNA polymerase system for recovery of an infectious virus from cDNA. Virology 231, 192-200.