General Position Statement of the ZKBS on Frequently Carried Out

Ref. no. 6790-10-41 Update November 2011 General position statement of the ZKBS on frequently carried out genetic engineering operations based on the criteria of comparability: Gene transfers using retroviral vectors 1. Description of the retroviral system 1.1. General introduction Retroviruses (family: Retroviridae ) are enveloped RNA viruses which the International Commit- tee on Taxonomy of Viruses (ICTV) divided, on account of their genetic proximity to each other, in two subfamilies, i.e. Orthoretrovirinae and Spumaretrovirinae, and in seven genera. The genome of replication-competent retroviruses consists of two identical, single-stranded RNA molecules possessing a length of 7 - 15 Kb [1]. They replicate via a double-strand DNA intermediate (provirus), which stably integrates into the genome of the infected cell. The three genes gag , pol and env are fundamental components of every retroviral genome (Fig. 1). a) ψ gag pol env LTR LTR PBS vpu tat vpr rev vif b) gag pol env nef ψ LTR LTR tar PBS RRE Fig. 1: a) Genetic map of a simple retrovirus, showing the Moloney mouse leukemia virus (MoMLV) provirus as an example. PBS (primer binding site) is the binding site for the tRNA primer; Ψ denotes the packaging signal; gag (group-specific antigens), pol (reverse transcriptase and integrase) and env (envelope proteins) are coding regions, LTR (long terminal repeats) contain repetitive sequences which appear in both terminal ends of the DNA genome. The figure was modified after [1]. b) Genetic map of a complex retrovirus, showing the HIV provirus as an example. Apart from regulatory elements such as LTR, packaging signal ψ, the primer binding site (PBS), the trans-activation response element ( tar ) and the rev-responsive element (RRE) as well as the genes gag , pol and env , HIV possesses several additional reading frames that code for regulatory proteins. Figure derived from and modified after http://hiv-web.lanl.gov/MAP/landmark.html BVL_FO_05_4100_402_V1.1 At both terminal ends of the DNA genome (provirus), there are so-called LTR (long terminal repeats) with promoter and enhancer properties. They contain repetitive sequences U3RU5, 1 whereby the repetition R also occurs at both terminal ends of the RNA genome. U5 (unique sequence 5) designates a nucleotide sequence which only occurs at the 5' end in the RNA genome, whereas the sequence designated U3 (unique sequence 3) is encountered at the 3’ end only. They are of significance for the reverse transcription of the genome. Apart from these sequences, complex retroviruses possess several other reading frames whose gene products take over regulatory functions (cf. Fig. 1). 1.2. Gene transfers using retroviral vectors Recombinant retroviruses to be applied in gene transfers were originally developed based on murine retroviruses, in particular the Moloney mouse leukemia virus (MoMLV). They exclusively transduce cells undergoing cell division. They can be taken up by non-dividing cells, however, their nucleocapsid is not able to cross the nuclear membrane. It is able to approach the nucleus only after the nuclear membrane dissolves [2]. Retroviral vectors derived from lentiviruses are used in order to stably transduce non-dividing cells or terminally differentiated cells. Whereas the early lentiviral vectors were derived from HIV-1 [3] and SIV ( Simian immunodeficiency virus ), vector systems have also been by now developed on the basis of the FIV ( Feline immunodeficiency virus ) [4], EIAV ( Equine infectious anemia virus ) [5], CAEV ( caprine arthritis encephalitis virus ) [6], BIV ( Bovine immunodeficiency virus ) [7] and MVV ( Visna/maedi virus ) [8]. Murine retroviral vectors The creation of recombinant retroviruses derived from murine retroviruses includes two components, i.e. the retroviral vector and the packaging cell line (Fig. 2). The retroviral vector is a plasmid which is usually derived from pBR328. It does not encode retroviral proteins, instead, it only disposes of the packaging signal ψ, the primer binding site PBS, and the retroviral 5’ and 3’ LTR, which flank an insertion site for the gene to be transferred as well as a selection marker, if applicable. The expression of more than one transgene is achieved by means of a bicistronic expressions cassette, which contains the IRES (internal ribosome entry site) sequence from picornaviruses. A cap-independent translation of the proteins is enabled by ribosomes binding to the IRES. The packaging cell line provides the retroviral proteins required for vector RNA packaging and thus for the formation of retroviral particles. The packaging cell line is created by introducing nucleic acid (helper genome) which codes for the retroviral structural proteins into a cell line. The packaging signal Ψ in this helper genome is deleted, so that the viral RNA which is produced from the helper genome in the packaging cell line will not be packaged into virions. While all structural genes were formerly introduced together into the first packaging cell lines constructed, nowadays gag-pol and env are transfected into the cell separately in order to min- imize recombination events during cell proliferation [9]. Depending on the type of retroviral envelope proteins which the helper genome expresses, we now distinguish ecotropic and amphotropic packaging cell lines. The host range of ecotropic murine retroviruses is confined to cells of mice and rats. Amphotropic murine retroviruses have a broader host range which includes both murine and non-murine, e.g. human, cells. In the development of recombinant retroviruses, the envelope proteins of murine retroviruses have been increasingly modified or exchanged in order to obtain a broader host range, a host tropism adjusted to a certain cell type, or a higher stability of the virion (pseudotyping) [10]. A number of heterologously expressed viral envelope proteins have already been successfully applied for pseudotyping. Among the donors are, for example, arenaviruses [11], alphaviruses [12] and representatives of the families Hepadnaviridae [13], Flaviviridae [14] and Rhabdoviridae . In particular, the G glycoprotein (VSV-G) of the Vesicular stomatitis virus (VSV) is used in a variety of ways in order to fit out murine retroviruses with a broader host range which also includes human cells. In this process, the glycoproteins of the enveloped virus particles bind to specific cell- BVL_FO_05_4100_402_V1.1 surface receptors and initiate membrane fusion. The receptors of the retroviral envelope proteins mostly used are functionally expressed only basolaterally in polarized epithelial cells [15]. For example, an in vivo transduction of epithelial lung-tissue cells by VSV-G-pseudotyped ret- 2 roviral vectors could only be demonstrated from the apical side if access to the basolateral surface was enabled by agents which opened the tight junctions [16, 17]. In addition, VSV-G- pseudotype retroviruses possess a higher particle stability which permits a concentration of the infectious virus particles by ultracentrifugation. A specific cell tropism can be obtained by inserting a ligand for a cell-surface protein, e.g. the recognition domain of an antibody, into the envelope protein of murine retroviruses [18]. Since recombinant retroviruses with a specific cell tropism are primarily supposed to be applied in somatic gene therapy, ligands for human-cell epitopes are used to this end. packaging construct vector construct with transfer gene gag pol An env virions Fig. 2: Production of recombinant replication-defective retroviruses. The retroviral vector with the transgene to be transferred is transfected into the packaging cell line con- stitutively expressing the retroviral structural proteins. Transcripts of the retroviral vector are then produced which are both translated and packaged with the help of viral structural proteins as genomic RNA into the new virions. The latter are then released from the packaging cell line. The figure was modified after [19]. These recombinant retroviruses, which are usually replication-defective, can be used to infect target cells into which the provirus, which had been reverse transcribed from the transferred retroviral RNA, is then stably integrated. The transferred gene as well as selection markers, if any, are expressed in the target cells. The so-called "ping-pong amplification" mechanism was developed in order to obtain higher virus titers. This is a method which achieves an amplification of the recombinant retrovirus by means of an alternating transfer to an amphotropic and ecotropic packaging cell line. To this end, the two packaging cell lines are co-cultivated [20, 21, 22]. Lentiviral vectors Lentiviruses possess a complex genome (Fig. 1). The possibility of transducing non-dividing cells relies on the ability of the lentiviral pre-integration complex to cross the intact nuclear membrane [3, 23]. This pre-integration complex is composed of the enzyme integrase encoded by the pol gene, the vpr gene product, the matrix protein encoded by the gag gene, the reverse BVL_FO_05_4100_402_V1.1 transcriptase also encoded by the pol gene, and the viral RNAs. In addition to the regulatory elements usually known to occur in retroviruses, such as LTR, the packaging signal ψ and the structural genes gag , pol and env , lentiviruses dispose of further elements and reading frames. 3 The lentiviral transcriptional transactivator (Tat) activates viral transcription by the Tat protein binding to the trans-activation response element (tar), a cis-regulatory sequence

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