Generic environmental risk assessment of gene therapies that use GM cells containing free lentiviral particles
A generic environmental risk assessment has been prepared for gene therapy trials in which use is made of human cells that have been genetically modified ex vivo and then returned to the patient. The risk assessment, which is for use in the European Union (EU), was designed to simplify licence applications for these trials.
The risk assessment contains a number of requirements that must be met. One of these is the absence of ‘free’ viral vector particles, which may remain as ‘residues’ after the cells have been genetically modified. The absence of vector particles can be demonstrated experimentally, but also estimated theoretically. To make this estimation, the EU risk assessment refers to a formula developed earlier by COGEM. This formula is used to estimate the reduction in the number of free vector particles during the preparation of the genetically modified (GM) cells in relation to the number of vector particles added during transduction. Factors that contribute to this reduction are the ‘natural’ inactivation of the particles (based on the halftime), the inactivation of particles under the influence of trypsin or human serum, and the loss of particles during the washing or passaging of the cell cultures. These factors are included in the formula in the form of parameters.
COGEM has had the values of these parameters experimentally validated for the most commonly used viral vectors, the lentiviral vectors. Lentiviral vectors were pseudotyped with the envelope protein G of the Vesicular stomatitis virus (VSV-G), Feline endogenous virus RD114, Gibbon ape leukaemia virus, Moloney murine leukaemia virus (both ecotrope and amphotrope), the hemagglutinin (H) and fusion (F) glycoproteins of Measles morbillivirus (Measles virus) and the glycoprotein of Rabies lyssavirus (Rabies virus).
The study showed that the values used in the formula need to be adjusted and that in the past the number of free vector particles has been underestimated. This is because the stability (halftime) of the vector particles is strongly influenced by the pseudotyping and by the culture conditions under which the transduction took place and the halftime of VSV-G pseudotyped particles in the chosen experimental design is longer than reported in the literature. Additionally, in practice trypsin is less effective at inactivating the vector particles than was assumed and the degree of inactivation is also heavily dependent on the pseudotyping. Also, the complement-mediated inactivation differs considerably between serum donors and titres determined for each vector batch depend on the assay that is used. This means that some titre calculations may lead to an underestimation of the initial inoculum.
A consequence of the research results is that when assessing future licence applications, it will probably not always be possible to ascertain with certainty that medicinal products administered to a patient may still contain vector particles, in which case the application would not meet the requirements of the EU environmental risk assessment.
Based on a theoretical worst case scenario, COGEM calculated the probability of third parties being exposed to free vector particles during such trials. The scenario makes various assumptions. Important assumptions are that inactivation occurs in the blood stream in the same way as in serum in vitro, and that the reduction of free vector particles during preparation of the medicinal product – resulting from biological decay and washing steps, among other factors – is not taken into account. The theoretical scenario therefore heavily overestimates the actual number of free vector particles present.
It can be concluded from the calculation that 16 hours after administering the medicinal product, the viral particles appeared to be cleared from the patient. The first 16 hours, transmission of viral particles can be prevented by taking into account standard hospital hygiene measures plus a few additional measures. In view of this, COGEM advises that patients should remain in the hospital for at least 16 hours after administration of the lentiviral transduced cells.
Taking the conditions described above into account, COGEM is of the opinion that the risks to third parties in trials with GM cells in which free lentiviral particles are still present are negligible, irrespective of which transgene sequences are inserted.
The Dutch advisory report issued at the same time as the publication of the research report, is available here. The English version of the advisory report is available. The research report itself is available here.