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Ref. No.: H2016-003 IN CONFIDENCE Risk Assessment for Genetic Modification Project to be considered by the Biological Safety Committee of the Hutchison-MRC Research Centre, Cambridge. 1. Name of applicant: Prof. Rebecca Fitzgerald Division or Unit: MRC Cancer Unit Group Leader responsible (if not applicant): Prof. Rebecca Fitzgerald ____________________ ______________________________________ 2. Functional Analysis of Oesophageal Adenocarcinoma Genomic variants ______________________________________ Title of project: ____________________ 3. Overview, including details of insert(s): This project aims to better understand the functional effects of genomic aberrations in oesophageal adenocarcinoma by permanently or transiently altering the genomic and transcriptomic state of in vitro models including oesophageal cell lines and organoids derived from primary tumour tissue. This requires the use of lentiviral RNAi and cDNA over-expression vectors as well as expression of targeted nucleases that will permanently alter cancer cell genomes. Inserts will encode reporters (e.g. EGFP, DsRed, luciferase); transcriptional regulators (e.g. Tet-On); normal and modified transcription factors or epigenetic regulators and other cancer-associated transcripts, including known oncogenes; shRNAi constructs, or targeted nucleases and their associated RNA molecules (CRISPR/Cas9), including constructs that functionally ablate known tumour suppressor genes or activate known oncogenes. Genes will be expressed under the control of viral and mammalian promoters, both constitutive (e.g. CMV or E2F) and/or inducible (e.g. regulated by Tet-On transactivator protein). Ref. No.: H2016-003 IN CONFIDENCE ____________________ ______________________________________ 4. Plasmid production Host/ vector system(s): K12-derived E. coli strains (e.g. DH5alpha) of bacteria are used for transformations and amplification of plasmids. No constructs will contain mammalian extra-cellular protein encoding genes driven by bacterial promoters as these could potentially enhance E.coli pathogenicity. lentiviral production Stable gene transduction will be achieved by HIV1-derived 2nd generation lentiviral vectors (e.g. psPAX2, psMD2.G). Later generation systems with further safety mechanisms and higher efficacy may be adopted as they become established. 293T human embryonic kidney cells and derivatives will be used as packaging/producer cells. Viral particles will be filtered to remove any possible contamination by 293T cells, which could allow continued viral production. Targeted nuclease-mediated genome engineering The CRISPR/Cas9 system will be used for targeted genome editing in somatic cells both by transient transfection and stable integration. For transient transfection, plasmids such as but not limited to pX330, pX260, and pX335 will be used. Cas9 nuclease and guide RNAs will also be stably integrated in the target cells using lentiviral gene delivery (e.g. but not limited to using pCW57.1 and pLX304-based vectors). Genetically manipulated cell lines will be propagated as regular cancer cell lines in containment level 1 once confirmed lentiviral negative using reverse transcriptase activity assays. Genetically manipulated organoid cultures will be maintained in containment level 2. Target cells 1. Established human tumour cell lines and 2. Oesophageal organoid cultures derived directly from patient samples. These can be passaged >30 times in many cases, without Ref. No.: H2016-003 IN CONFIDENCE transformation. The patient samples are not in general screened for blood-borne viruses, and we are not permitted to screen them, for ethical/consent reasons. There is a separate risk assessment for these cultures. ____________________ ______________________________________ 5. E. coli Hazard identification in respect of human health and environmental safety. (Consider host, vector, insert and final GMM.) Estimation of the severity or consequence of the harmful effect were it to occur. Laboratory strains of E. coli K12 are recognised as non-colonising and may be considered to be ACDP hazard group 1. Viral vectors We will use 2nd generation lentiviral vectors containing several safety mechanisms: • Reduced number of genes from HIV-1 in lenti-viral vectors (i.e. gag, pol, tat and rev are absent). • Separation of genes encoding the structural and other components required for packaging the virus to substantially reduce the risk of undesirable recombination events that could lead to the generation of a replicationcompetent virus (Dull et al., J Virology 72 8463-8471 1998). • None of the HIV-1 structural genes are present in the packaged viral genome and are therefore never expressed in the transduced target cell. • The lentiviral particles produced are replicationincompetent and only carry the gene of interest. No other viral species are produced. • In some cases expression of the gene insert of interest will be dependent on a tet-responsive promoter, and regulated by a co-transduced lentivirus doxycyclinresponsive Tet regulator, giving an added level of safety for insert expression. These vectors contain the WPRE (Woodchuck hepatitis B virus post-transcriptional regulatory element), which may have oncogenic properties, so, whether or not the inserts are oncogenic, all these lentiviruses are potentially oncogenic. Ref. No.: H2016-003 IN CONFIDENCE Viral vectors in unscreened human oesophageal organoid cultures In principle, there is a small risk that organoid cultures might carry HIV, which, by acting as a helper virus, would make the lentivirus inserts replication-competent. However, to our knowledge HIV does not replicate in cells such as oesophageal epithelium, so, provided the organoids have been passaged by disaggregation so that they do not also carry macrophages or lymphocytes, the risk of HIV is low. ____________________ After infected organoids have been passaged further, the risk of active virus will be very low. ______________________________________ 6. Provisional Class/containment level (in particular taking account of the biological agents hazard group and other classification scheme for pathogens). E. coli This step will often involve considering the containment level necessary to control the risk of the host and making a judgement about whether the modification will result in a GMM which is more hazardous, less hazardous or about the same. Sometimes it might help to compare the GMM with the relative hazard presented by other organisms. Viral vectors and infection The E. coli and tissue culture cells are disabled and belong to Hazard Group 1. The risk of Lentiviral infection of a worker during the packaging/infection step is small, but still not negligible. As the vector contains the complete WPRE element as well as tumorigenic inserts this part of the work is Class 2. However any theoretical hazard is only during initial contact since such viruses could not propagate. Given these non-negligible hazards, particular attention will be paid to staff awareness when working with genes with a potential growth-promoting function (virus production, labelling, storage). No sharps will be used when working with lentiviruses. Also, appropriate personal protective equipment will be used (gloves, lab coats). DNA grown up from clones should also be handled with care as it is potentially oncogenic because of the WRPE element and oncogenic inserts; i.e. gloves should be worn, sharps avoided and all wastes be rendered harmless before disposal. Virus storage For some experiments, virus will be stored outside the Class 2 lab. For this, it will be in a designated section of a clearly IN CONFIDENCE Ref. No.: H2016-003 designated freezer, in double containers, according to the Hutchison-MRC Research Centre Class 2 Code of Practice. Moving Infected Cell Lines to CL1 After infection, selection and passaging at least once, for cell line cultures only, NOT organoids, cells that are virus negative may be transferred to CL1. To show that cells are negative for replicating lentivirus, we will use well characterised methods such as the Molecular Probes' EnzChek® Reverse Transcriptase Assay. All organoids (prior to and after infection) will remain in CL2.Cell storage To cryopreserve cells in CL2, storage will be at -80 outside the CL2 lab as specified above for virus storage. When it is really necessary for long-term preservation to store cells in a vapour phase liquid nitrogen refrigerator this will be in a clearly designated location, in a double container, according to an agreed Hutchison-MRC Research Centre Class 2 Code of Practice and with explicit permission from the Hutchison Lab Manager responsible. ____________________ ______________________________________ 7. The prokaryotic cells are highly disabled and the plasmid vectors are non-mobilisable, therefore neither has any possibility of further spread. Environment and activity considerations. This includes an estimation of the likelihood that hazards will be realised. Given that the provisional Class (and hence containment level) has already been decided it helps to bear this in mind when deciding how likely a harmful event is. Use these considerations of likelihood to revise the provisional containment so that all risks are controlled to low or effectively zero. Double check that all hazards are properly controlled by the proposed containment. The principle hazard in the human cell work is the low risk of blood-borne virus and particularly HIV. Neither are expected to survive passage of cultures and reverse transcriptase assay should confirm the absence of HIV in cultures maintained after infection. We are not going to produce aerosols, which contain viral particles. Also, the amounts of virus produced are modest. The lentiviruses have unstable infectivity and infection is only obtained by co-cultivation of the packaging cell supernatant with the recipient cells. There will be no animals present in the tissue culture facilities. The risk to the environment is therefore effectively zero. Ref. No.: H2016-003 IN CONFIDENCE ____________________ ______________________________________ 8. Class 1, with no additional precautions required, for the work with E. coli. Assign final activity Class. This is done by comparing the containment and control measures identified as necessary to control the risk with the table of containment in the Regulations. Class 2, with no additional precautions required for the packaging/infection steps with the retro and lentivirus. Potentially growth-promoting DNA will be handled appropriately (see above). Class 1, with no additional precautions required, for the work with stably infected or genetically engineered cells. _____________________________ _____________________________ Final Class/containment level: _____________________________ 2 and 1 _____________________________ 9. ALL persons involved in GM work (give experience or name of trainer if not already experienced): Xiaodun Li Post-Doctoral Researcher. More than 8 years of experience in molecular biology. Gianmarco Contino Post-Doctoral Researcher. More than 8 years of experience in molecular biology. Alex Frankell of those above) PhD student (under the direction Nuria Galeano-Dalmau Research Assistant (under the direction of those above) ____________________ ______________________________________ 10. Notes (including any abbreviations No animals are involved in this work. used): If work involves transgenic animals and is not exempt, give Licence number: __________________________________________________________ IN CONFIDENCE 11. Ref. No.: H2016-003 Signed: Date: (Group Leader) __________________________________________________________ 12. Comments of Biological Safety Committee: This is principally a project to use lentivirus to manipulate cancer-relevant genes in human cells. The non-standard element of the proposal is that organoid cultures of human epithelium from unscreened patients are among the target cells to be used. Screening is not permissible for ethical reasons. Since such cultures might rarely be contaminated with HIV, the general principle that modern lentivirus constructs cannot propagate could break down, because the HIV could package the lentivirus vector. Self-inactivating constructs would be preferable in this case, since they are less likely to transcribe packageable RNAs. An SOP for cryopreserving CL2 cells needs to be developed. Clearance given to start work/Notified to HSE 13. Signed: Date: (Chairman of BSC) __________________________________________________________ 14. Agreement of person responsible for supervision and safety: Signed: Date: (Competent person under MHSW Regs 1999/Biological Safety Officer)