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 which is located at the 5‘ end of the viral RNA and DNA. Rev (regulator of expression of the virion) mediates the transport of single-spliced and unspliced RNA from the nucleus by interacting with the rev responsive element, RRE, and enables its translation in the cytoplasm. The so-called ac- cessory genes vif , vpr , vpu and nef are not essential for viral replication in cell cultures. Vif (viral infectivity factor) increases the infectivity of the virions in primary T cells. Vpr (virion-associated protein) is involved in the nuclear import of the pre-integration complex. Nef and Vpu have an influence on the infectivity of the virions. In this context, Nef has been brought in connection with a reduced expression of CD4 receptors and MHC-I antigens located on the cell surface of infected cells, and Vpu degrades CD4 receptors in the ER and thus prevents complexation with the envelope protein. The development of lentiviral vectors is gaining importance especially for gene therapy. How- ever, because of the pathogenicity of lentiviruses, there are concerns about the safety of these vectors. Here, especially the potential formation of replication-competent lentiviruses during vector production presents a problem. By applying a three or four plasmid system, which comprises one vector plasmid, one packaging plasmid, and one or two further plasmids (envelope protein gene and Rev protein gene), the possibility of recombination to replication-competent lentiviruses could be minimized. The minimum vector plasmid contains the cis -active sequences which are essential for packaging, reverse transcription, and integration. They are limited to the LTR, the packaging signal Ψ, the primer binding site (PBS), and the polypurine tract (PPT), and the corresponding transgene perhaps with a heterologous promoter (cf. Fig. 3a). Current constructs are also based on systems inducible either by tetracycline or ecdysone [24, 25], for example, and/or tissue-specific promoters [26]. If the rev-RRE system of HIV is used to export the mRNA molecules into the cytoplasm the vector will have to additionally contain the RRE sequence. The proteins needed for target cell infection are provided in trans . However, in order to increase the efficiency of gene transfer regulatory sequences may also be introduced into the vector. The central poly-purine-tract (cPPT), for example, supports the translocation of the pre- integration complex into the nucleus [27]. Another example is the posttranscriptional regulatory element of the woodchuck hepatitis virus (WPRE). It increases RNA stability and thus mediates a higher expression rate of the transgene [28, 29]. As lentiviruses display a limited cell tropism, the vectors derived from them are usually pseudotyped with envelope proteins which extend cellular tropism. The env gene of the amphotropic MLV is often used to this end, however, VSV-G is used even more often [30]. Besides, a number of other heterologously expressed viral envelope proteins have already been successfully applied in pseudotyping (e.g. lyssaviruses [31], baculoviruses [32], filoviruses [33] and alphaviruses [34]). The establishment of packaging cell lines for lentiviral vector systems is only possible in inducible or attenuated systems because of the cytotoxic properties of the HIV Gag/ Pol proteins [35]. Usually, three or four plasmids carrying the genes and regulatory sequences needed for vector production are co-transfected into a cell line (cf. Fig 3). This is where replication-defective lentiviral vectors are packaged and released from the cell line.

BVL_FO_05_4100_402_V1.1

4 CMV/ 5‘ LTR ψ transgene 3‘ LTR a) PBS RRE PPT

promoter b) gag/ pol polyA

RRE

promoter rev polyA c)

d) promoter envelope gene polyA

Fig. 3: Components needed for the production of lentiviral vectors based on HIV. Four plasmids are used for production: a) The minimum HIV vector consists of the CMV/5’ LTR hybrid promoter, which enables tat-independent transcription, 3’ LTR, the packaging signal Ψ, the Rev-binding element RRE for the cytoplasmic export of the RNA, the PBS and PPT sequences necessary for reverse transcription and the transgenic expression cassette, which might also contain internal promoters as well as several genes to be transferred. All genes of the enzymatic or structural HIV proteins have been removed. b) The HIV packaging plasmid contains the genes of HIV, which provide the proteins necessary for packaging the retroviral vector RNA in trans : 5’ and 3’ LTR are replaced by a heterologous promoter or a heterologous polyadenylation signal. c) The plasmid carries the rev gene. The gene is under control of a heterologous promoter, the polyadenylation site is also heterologous. d) The plasmid carries an envelope protein. The gene is controlled by a heterologous promoter, the polyadenylation site is also heterologous. The figure was modified after [36].

Another safety concern which, however, not only involves lentiviral vectors but also MLV-derived vectors is the possibility of activating cellular oncogenes by means of an accidental integration of the vector provirus into the host genome. Activation might proceed on three levels: (1) The vector exerts an influence on neighboring enhancers or promotion elements of the cell and thus on gene expression, (2) promoter and enhancer of the integrated provirus activate neighboring cellular oncogenes, (3) the presence of the vector DNA might produce alterations in the chro- matin structure of regulatory domains and thus affect gene expression. In case of the so-called self-inactivating (SIN) vectors, the U3 region of the 3‘ LTR has been deleted, resulting in a loss of the promoter and enhancer properties it normally contains [3, 27, 37]. This limits a potential activation of neighboring cellular genes, however, the deletion would reach to the 5' LTR during a viral replication cycle which would also turn off the expression of the vector and additionally prevent the mobilization of the integrated vector provirus (cf. Fig. 4). BVL_FO_05_4100_402_V1.1

5 ∆U3

R U3 R U5 ψ transgen e U5

PBS LTR RRE LTR

Fig. 4: Self-inactivating (SIN) vector

The PBS of the vectors might be mutated for reasons of additional safety, so that no cellular tRNA will be able to attach. The creation of such vectors requires an adequately mutated tRNA which is provided in trans either as synthetized tRNA or as Pol-III gene on a co-transfected plasmid [38]. The introduction of chromosomal insulators into the vector construct and the application of cell and tissue specific promoters are further strategies to prevent an activation of cellular oncogenes by means of unspecific integration. Currently, the development of vectors pursues the deliberate integration of the vectors into innocuous regions of the human genome. The experi- ments are directed at a modification of the integrase, for example, by insertion of a specific DNA- binding domain [39, 40, 41].

Adenovirus/retrovirus hybrid vectors In order to accomplish an efficient gene transfer and sustainable gene expression, chimeric vectors are being developed on the basis of adenoviral and retroviral vectors [42, 43]. After co- infection, three replication-defective adenoviral vectors respectively transfer the retroviral vector, the retroviral packaging functions ( gag/pol region), and one env gene to a cell thus becoming a retroviral producer cell. The released retroviral vectors can then stably transduce further cells. The risk assessment of adenoviral vector production proceeds according to the general position statement of the ZKBS on frequently carried out genetic engineering operations based on the criteria of comparability: gene transfer by applying adenovirus type 5, Ref. no. 6790-10-28, update from November 2011. Risk assessment of cells after co-infection of all three adenoviral vectors is based on the release of retroviral vectors — as is the case with a packaging cell line transfected with a retroviral plasmid.

2. Summary of criteria relevant to the safety classification of genetic engineering operations involving gene transfers using retroviral vectors

The hazard potential of handling recombinant retroviruses derived from murine retroviruses is assessed to be low, even though they are not human pathogens. In the course of an infection the proviral genome integrates nondirectionally into the genome of the host cell and in individual cases might induce, by way of insertional mutagenesis, the activation of cellular oncogenes or a change in the transcriptional activity of other regulatory genes. The risk of this event depends on the replication capacity in the host, the potential target cells, and natural defense mecha- nisms of the organism infected. Naturally occurring infections of humans with murine retrovi-

BVL_FO_05_4100_402_V1.1 ruses are not known. In addition, these retroviruses possess a low physical stability. An airborne transmission has not been described. The host range acquired by the packaged recombinant retroviruses is essential to the safety classification of genetic engineering operations with recombinant murine retroviruses in the individual case.

6 As far as ecotropic murine retroviruses are concerned, a hazard potential for humans or animals must not be assumed (cf. ZKBS, Position statement on risk assessment of ecotropic murine C- type retroviruses). A minor hazard potential for humans cannot be ruled out in case of amphotropic murine retroviruses, as primate cells can be infected by amphotropic retroviruses both in vitro and under certain in vivo conditions [1, 44]. However, humans and Old World monkeys possess some protection against an infection with murine retroviruses. The glycosylation system in murine cells is responsible for the formation of the so-called Gal epitope in both cellular and retroviral glycoproteins which are constituents of the viral envelope. The interaction with anti-α- Gal antibodies circulating in human blood activates the complement system, resulting in the lysis of the retroviral particles [45]. A replication capacity of the recombinant retroviruses is given when replication-competent retroviruses are formed as a result of recombination events between homologous sequences of the helper genome of the packaging cell line and the vector genome. Such an event depends on the vector/packaging cell-line system used. Safety can be guaranteed by eliminating sequence homologies between helper and vector genome and by introducingstop codons, muta- tions and deletions, which in case of recombination produce a replication defect. The amphotropic packaging cell line PA317 may serve as an example. Although the formation of replication- competent retroviruses has been described after transfection of the cell line with the vector N2 [46], LN-derived vectors are constructed in such a fashion that only replication-defective retroviruses are built in case recombination happens [47]. Furthermore, a recombination event will be less likely if packaging cell lines are used carrying the genes gag/pol and the envelope protein gene env separately from each other. This applies especially whenever recombinant retroviruses with modified envelope proteins are created. Certainty regarding the occurrence of replication-competent amphotropic retroviruses is provided, for example, by applying the S +L--as- say [48]. In it, the cell-culture supernatant to be tested is placed on top of PG-4 cells. PG-4 cells contain a defective Moloney mouse sarcoma virus (M-MSV). If replication-competent retroviruses are present in the supernatant the defects of M-MSV can be complemented and foci formed. There will be no hazard potential for humans and animals if ecotropic packaging cell lines able to produce replication-competent retroviruses are used. Retroviruses with modified envelope proteins usually infect human cells and other cell types. The hazard potential resulting from them equals that of amphotropic murine retroviruses. How- ever, if the envelope protein of the feline virus RD114 or monkey SSAV is used the corresponding pseudotypes will not be inactivated efficiently [49, 50]. An efficient inactivation by the complement system was shown to occur in case of pseudotypes with the VSV-G protein [51]. When working experimentally with pseudotypes, it must be considered that the vector is capable of using other transmission routes than those characteristic of the wildtype virus [10]. When lentiviral vectors are produced, the replication incompetence of these vectors are safety- relevant. The possibility of a homologous recombination event between overlapping retroviral DNA sequences in the vector and the helper constructs could give rise to the formation of replication-competent lentiviruses which are pathogenic and assigned to risk group 3** as wildtype viruses. The application of codon-optimized packaging plasmids reduces the degree of homol- ogy and the length of homologous regions between packaging plasmid and vector and can therefore contribute to reducing the risk of creating replication-competent lentiviruses [52]. Only the hazard potential of replication-defective lentiviral vectors will be equivalent to the hazard potential of amphotropic murine retroviral vectors.

BVL_FO_05_4100_402_V1.1

7 3. Criteria for the comparability of genetic engineering operations designed to carry out gene transfers using retroviral vectors

General criteria for the comparability of genetic engineering operations designed to carry out gene transfers using retroviral vectors will be summarized below.

Note: a. The assessed retroviral vectors are murine retroviral or lentiviral vectors which are derived from pBR328 and the 5’ and 3’ LTR of retroviruses, and contain the packaging signal ψ, which might be extended by additional nucleotide sequences of the gag region, the cloning site for the nucleic acid segment to be transferred, and, if applicable, selection markers. The selection marker or the nucleic acid segment to be transferred might be under the control of a further promoter active in the mammalian cell. An envelope protein gene does not exist here. In case of lentiviral vectors a RRE regulatory sequence might be additionally present. b. Packaging cell lines are established cell lines belonging to risk group 1 containing the gag/pol genes of murine leukemia viruses (MLV) (without packaging signal ψ) and, if applicable, genes for unmodified envelope proteins from ecotropic MLV, amphotropic MLV, other viruses such as GaLV or VSV or genes for recombinant envelope proteins with ligands for cellular surface proteins. The genes for the recombinant envelope proteins occur either as a single envelope protein gene or together with the unmodified envelope protein genes of the MLV. c. The genes ( gag/pol and env ) necessary for packaging the lentiviral vectors and further regulatory reading frames exist separately from each other on two or three additional pBR328 plasmid derivatives which are transfected together with the lentiviral vector into the cell. The plasmids do not contain any packaging signal, for which reason the transcripts are not packaged into particles. A recombination between the constructs to yield replication-competent lentiviruses must not be assumed.

Criteria:

Introduction of retroviral, including lentiviral vectors into E. coli : 3.1. If subgenomic viral or cellular nucleic acid segments are introduced into an E. coli K12 derivative using the abovementioned murine retroviral or lentiviral vectors, the genetically modified organisms are to be assigned to risk group 1 . Genetic engineering operations with genetically modified organisms fulfilling the stated criteria are comparable and assigned to containment level 1.

Production of ecotropic retroviruses in a cell line: 3.2. If subgenomic viral or cellular nucleic acid segments are introduced into an ecotropic packaging cell line belonging to risk group 1 using the abovementioned murine retroviral vectors, the genetically modified organisms are assigned to risk group 1 . Genetic engineering operations with genetically modified organisms fulfilling the stated criteria are comparable and assigned to containment level 1. 3.3. Recombinant replication-defective ecotropic murine retroviruses, which have been re- BVL_FO_05_4100_402_V1.1 leased by the packaging cell lines described under 3.2., are assigned to risk group 1, even if a contamination with replication-competent ecotropic retroviruses has to be anticipated. Genetic engineering operations with genetically modified organisms fulfilling the stated

8 criteria, inclusive of the infection of other cells belonging to risk group 1 and the inoculation of animals, are comparable and assigned to containment level 1. 3.4. Cells belonging to risk group 1, which have been infected with recombinant replication- defective ecotropic retroviruses described under 3.3 are assigned to risk group 1 under the provision that the cells infected do not release any retroviruses possessing an extended host range. Genetic engineering operations with genetically modified organisms fulfilling the stated criteria are comparable and assigned to containment level 1.

Production of amphotropic retroviruses in a cell line: 3.5. If amphotropic packaging cell lines belonging to risk group 1 are infected with the ecotropic retroviruses described under 3.3. the genetically modified organisms are assigned to risk group 2 . Genetic engineering operations with genetically modified organisms fulfilling the stated criteria are comparable and assigned to containment level 2. 3.6. If subgenomic viral or cellular nucleic acid segments are introduced into amphotropic packaging cell lines belonging to risk group 1 using the abovementioned retroviral vectors, the genetically modified organisms are assigned to risk group 2 . Genetic engineering operations with genetically modified organisms fulfilling the stated criteria are comparable and assigned to containment level 2. 3.7. If subgenomic viral or cellular nucleic acid segments are introduced into a co-culture of ecotropic and amphotropic packaging cell lines belonging to risk group 1 using the abovementioned retroviral vectors, the genetically modified organisms are assigned to risk group 2 . Genetic engineering operations with genetically modified organisms fulfilling the stated criteria are comparable and assigned to containment level 2. 3.8. Recombinant replication-defective murine retroviruses, which have been released from the packaging cell lines described under 3.5., 3.6. or 3.7. are assigned to risk group 2, even if a contamination with replication-competent amphotropic retroviruses must be anticipated. Genetic engineering operations with genetically modified organisms fulfilling the stated criteria, including the infection of other cells belonging to risk group 1 and the inoculation of animals, are comparable and assigned to containment level 2.

Production of lentiviruses in a cell line 3.9. If the lentiviral vectors mentioned under 3.1 are introduced into cell lines belonging to risk group 1 together with further pBR328-derived vectors containing subgenomic nucleic acid segments which serve the purpose of lentiviral packaging, the genetically modified organisms are assigned to risk group 2 . Genetic engineering operations with genetically modified organisms fulfilling the stated criteria are comparable and assigned to containment level 2. 3.10. Recombinant replication-defective lentiviruses, which have been released by the cell lines described under 3.9., are assigned to risk group 2 . Genetic engineering operations with genetically modified organisms fulfilling the stated criteria, including the infection of other cells belonging to risk group 1 and the inoculation of animals, are comparable and assigned to containment level 2.

Infection of cells with recombinant retroviruses including lentiviruses 3.11. Cells belonging to risk group 1 which have been infected with recombinant replication- defective retroviruses described under 3.8., 3.10., 3.17 or 3.19., in which a contamination BVL_FO_05_4100_402_V1.1 with replication-competent retroviruses does not have to be expected, are assigned to risk

9 group 1 , under the condition that the cells do not complement the replication defect. Ge- netic engineering operations with genetically modified organisms fulfilling the stated criteria are comparable and assigned to containment level 1. 3.12. Cells belonging to risk group 1, which have been infected with the recombinant replication- defective amphotropic retroviruses described under 3.8., are assigned to risk group 2, if a contamination with replication-competent retroviruses must be assumed or if the cells complement the replication defect. Genetic engineering operations with genetically modified organisms fulfilling the stated criteria are comparable and assigned to containment level 2. 3.13. Primary human cells belonging to risk group 2, which have been infected with recombinant retroviruses described under 3.3, 3.8., 3.10, 3.17. or 3.19. are assigned to risk group 2 . Genetic engineering operations with genetically modified organisms fulfilling the stated criteria are comparable and should be assigned to containment level 2.

Retroviruses including lentiviruses with modified envelopes: 3.14. If subgenomic viral or cellular nucleic acid segments are introduced into cell lines belonging to risk group 1 using the abovementioned lentiviral vectors in cell lines belonging to risk group 1, and if replication-defective lentiviral particles are formed with an envelope protein of ecotropic MLV, then the genetically modified organisms are assigned to risk group 1 . Genetic engineering operations with genetically modified organisms fulfilling the stated criteria are comparable and assigned to containment level 1. 3.15. Recombinant replication-defective lentiviruses, which have been released by the cell lines described under 3.14., are assigned to risk group 1 . Genetic engineering operations with genetically modified organisms fulfilling the stated criteria, inclusive of the infection of other cells belonging to risk group 1 and the inoculation of animals, are comparable and assigned to containment level 1. 3.16. If subgenomic viral or cellular nucleic acid segments are introduced into packaging cell lines belonging to risk group 1 by using the abovementioned murine retroviral vectors which express gag/pol from the MLV and the envelope proteins from other viruses, e.g. GaLV, VSV or recombinant MLV envelope proteins, replication-defective, phenotyped retroviral particles will be produced. The genetically modified organisms are assigned to risk group 2 . Genetic engineering operations with genetically modified organisms fulfilling the stated criteria are comparable and to be assigned to containment level 2. 3.17. Recombinant replication-defective retroviruses, which have been released by the packaging cell lines described under 3.16., are assigned to risk group 2 . Genetic engineering operations with genetically modified organisms, fulfilling the stated criteria inclusive of the infection of other cells belonging to risk group 1 and the inoculation of animals, are comparable and assigned to containment level 2. 3.18. If subgenomic viral or cellular nucleic acid segments are introduced into cell lines belonging to risk group 1 with the aid of the abovementioned lentiviral vectors, and if replication- defective lentiviral particles are formed with envelope proteins of other viruses, such as GaLV, VSV, RD114, or with recombinant MLV envelope proteins which contain the ligand for a cellular surface protein, then the genetically modified organisms are assigned to risk group 2 . Genetic engineering operations with genetically modified organisms fulfilling the stated criteria are comparable and assigned to containment level 2. 3.19. Recombinant replication-defective lentiviruses, which are released by the cell lines described under 3.18., are assigned to risk group 2 . Genetic engineering operations with genetically modified organisms, fulfilling the stated criteria, including the infection of other BVL_FO_05_4100_402_V1.1 cells belonging to risk group 1 and the inoculation of animals, are comparable and assigned to containment level 2.

10 Note: If nucleic acid segments of oncogenic potential are transferred, the same precautionary personal safety measures must be observed for working with these GMOs as those which the ZKBS has listed in its "Position statement of the ZKBS on safety measures for handling nucleic acids with potential for neoplastic transformation " (updated in December 2016). If, in genetic engineering operations designed to transfer nucleic acid segments of oncogenic potential, retroviral vectors are used which, due to phenotyping, obtain a reinforced particle stability or a host range including human epithelial cells, or which the human complement system will fail to recognize because of an altered glycosylation pattern, it will be recommended to wear a surgical mask in addition to level-2 safety measures in order to prevent smear infections. The criteria applicable to nucleic acids of oncogenic potential have been defined in the general position statement of the ZKBS on adenoviral and AAV-derived replication-defective viral particles which transfer a nucleic acid segment with potential for neoplastic transformation (updated in December 2016).

Genetic engineering operations with retroviruses including lentiviruses in animals: 1. Animals infected with such recombinant retroviruses including lentiviruses, in which a contamination with replication-competent retroviruses must be assumed, are carriers of these GMOs, without becoming new, genetically modified organisms themselves. The safety measures applicable to the animal housing units depend on the risk group assign- able to the recombinant retroviruses. 2. No transgenic animals will be created when replication-defective recombinant viruses are transferred to animals. These animals are also not able to produce or transmit GMOs. Recombinant replication-defective retroviruses including lentiviruses transmit a heterologous nucleic acid segment and subgenomic retroviral nucleic acid segments. The heterologous nucleic acid segment does not complement the replication defect. If the animal does not complement the replication defect either, an abortive infection will be the result. The viral nucleic acid will not be mobilized and thus transferred to other cells. New virions will not develop. The nucleic acid is transferred to the animal's somatic cells. There will only be a transient retention of the transgene. It must not be assumed that a transfer and integration of the nucleic acid into germ line cells will take place. 3. Animals to which cells described under 3.11. have been transferred are not GMOs and also incapable of producing GMOs. The cells do not complement the viral replication defect. The viral nucleic acid will not be mobilized and thus transferred to other cells. New retroviral particles will not be created. 4. Animals to which cells described under 3.12. and 3.13. have been transferred are not GMOs. The evaluation proceeds on the basis of the hazard potential of the replication- competent viruses imported by way of the transduced cells. These viruses may either be amphotropic replication-competent retroviruses resulting from a contamination during the production of the viral particles (GMOs of risk group 2) or replication-competent viruses which had infected the primary cells used before transduction. Safety measures applicable to the animal housing units depend on the hazard potential of the replication- competent viruses (S2 or L2).

4. References

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