Team:Freiburg/HumanPracticeAndSafety/Safety/viralvector

Viral Vector Safety

1: Are we creating amphotropic MuLV derived retroviruses during our research?

No, we are creating ecotropic replication-deficient MuLV derived retroviruses. In contrast to amphotropic viruses these are not able to infect human cells under normal conditions.

Explanation:

In order to produce our viral vector we are inserting one plasmids into a human packaging cell line (Phoenix eco). This cell line is carrying the gag, pol and env genes, which are needed for the synthesis of the virion. When transfected with a so called transfer plasmid these cells produce replication-deficient viral particles that can be harvested from the cell culture supernatant. The env gene is responsible for the ecotropic nature of our viral vector capsid. The ecotropic MuLV is highly specific for the mCAT1 receptor that is only found on rodent cells (mouse and rat). Cells that were transduced are not able to generate viral particles because they are lacking the required genes for the production of the virion, even after stable integration. The work with ecotropic MuLV can therefore be done under BSL-1 requirements.

We tested the specificity with different human cell lines to validate that normally human cells cannot be infected by our viral vector.

2. Is it possible to infect human cells with these viral vectors under experimental conditions?

Yes, we infected HEK293T (Human Embryonic Kidney) cells with our ecotropic MuLV.

Explanation:

In order to infect non-rodent cells we decided not to pseudotype the virus itself, but the target cells. This allows us to create a safe and controlled environment. Manipulating the capsid itself would have led to an increased need of safety. Because we wanted to distribute our viral vector throughout the iGEM community, design it as safe as possible was our major goal. To achieve this, we used the murine ecotropic retrovirus receptor (mCAT-1) to transfect HEK293T cells allowing targeted precision of viral transduction.  

3. How did we measure the safety of our viral vector in detail?

During our research we intensively concerned with existing biosafety regulations and created a catalog of characteristics describing the safety of our viral vector. As a basis of valuation we studied the law of prevention and fighting of infection disease (IfSG) as well as the genetechnology safety edict (GenTSV).

Following the description of the law the criteria for human pathogenic genetically engineered organisms are: Transferability, infective dose, host range, possibility of survival outside of human host, the presence of vectors and means of dissemination, biological stability, allergenicity and toxicity.

3.1. Transferabiity, infective dose and host range:

Rodents are the target of our MuLV, there are no cases described in which non-rodent cells were infected by an ecotropic MuLV. MuLVs are transferred by fluids. Non-rodent cells carry different glycosylation patterns at their CAT-1 receptor and can therefore not be targeted by the ecotropic MuLV. The immune system of non-rodent mammalians provides different other barriers preventing an infection. The complement system of these mammalians is recognizing the MuLV and is inactivating it. Furthermore a number of restriction factors are prohibiting an infection. Possible is a receptor blockage, avoidance of replication after penetration (Trim5alpha) or impede activation of retroviral elements in the genome (deaminases, Zink-Finger-proteins, micro-RNA, siRNA).

Our retroviral vectors are not air transmittable and are unstable under different conditions. For transduction a direct contact between virus and cell is mandatory. Integration of the virus into the host genome can only take place when the target cell is in the mitotic phase. Human skin cells which are in the mitotic phase are located in the basal lamina. Above those is a layer of non-mitotic cells protecting the skin from infections by viruses. Only with a damaged upper layer a hypothetical infection would be possible. Besides the fact that cells without the mCAT receptor cannot be transduced at all shows how unlikely a accidental infection with a retroviral vector in general would be.

We tested the half life of our viral vector at 37°C.

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