Summary of the standard environmental risk assessments (ERAs) belonging to applications for an environmental release permit for other purposes of medical applications of naked DNA.

The medical practice uses so-called naked DNA to insert certain genes into human cells. The objective of the treatment with naked DNA molecules is to insert one or more genes into the body cells of a test subject that subsequently are expressed, after which the created gene products have a direct therapeutic effect. Naked DNA can also be applied as a vaccine, against tumours or certain infectious diseases.

DNA that is injected into the human body will be immediately absorbed by the body cells. This DNA is administered in ‘naked’ form: it is not surrounded by a capsid, as in the case of medical and veterinarian applications of genetically modified viruses. 
This naked DNA is not a genetically modified organism (GMO Genetically Modified Organism (Genetically Modified Organism)). However, as soon as the naked DNA is absorbed by the genetic material of a body cell, this cell is considered a GMO. For medical applications of naked DNA, therefore, GMOs are considered to be formed within the body of the test subject. Such applications are subject to a permit under the GMO Environmental Management Act and the corresponding regulation. The outcome of a so-called environmental risk assessment is one of the foundations for such a permit. Closer inspection of the environmental risk assessment for naked DNA shows that, in many cases, the assessment can be carried out without taking the exact composition of the DNA into account.

Standard environmental risk assessment for naked DNA

In the environmental risk assessment (ERA environmental risk assessment (environmental risk assessment)) for medical applications of naked DNA, just as for viral therapies and vaccines, the aim is to determine and assess the risks with regard to humans other than the test subject, and the environment. The effects of the treatment experienced by the test subject are the responsibility of the attending physician, who is bound to other rules and regulations within the medical field. Risks to other people and the environment may emerge when GMOs are released from the test subject and spread into the test subject’s surrounding environment.

For naked DNA, the most obvious transmission route would be the following: the administered DNA or the cells that carry this DNA are released from the test subject and are subsequently absorbed into the body of another human or an animal, where they may form a GMO and in whom the cells could continue to exist as GMOs. However, in practice this transmission route is non-existent. The human cells that are released from the test subject cannot survive in the environment – and even if they were absorbed as living cells by another human or animal, such as via the mouth, their chance of survival is negligible. If the administered naked DNA were released into the environment, it could not end up inside the body of another human or animal in such a way that a GMO could be created. This is only possible if the DNA is inserted into the body intentionally, such as via injection.

The conclusion here is that naked DNA that has been inserted into a test subject does not pose a risk to the environment if this DNA, or cells containing it, were to be released from the test subject. These conclusions remain valid, irrespective of the composition of the naked DNA. After all, the reasoning is based on the general properties of DNA and how DNA and human or animal cells behave in the environment.

The above argument does not consider certain special cases. These are discussed below and lead to a refinement of the ERA.

Refinement of the environmental risk assessment for naked DNA

The standard environmental risk assessment applies to the absorption of naked DNA into body cells. If the DNA were absorbed into germ cells, then genetically modified germ cells (sperm or egg cells) could be created, which after fertilisation could grow into genetically modified offspring. The standard environmental risk assessment does not apply to this unintended and unchecked route of GMO transmission. This is why the standard ERA specifies that measures must be taken to prevent transmission, by excluding the administration of DNA into the reproductive glands of test subjects.

Furthermore, the possibility must be taken into account of the naked DNA interacting with other organisms (or their DNA) found inside or on the test subject.

Interaction with bacteria

The DNA can come into contact with the bacteria on the skin of the test subject, particularly during administration. Certain bacteria are naturally able to absorb naked DNA from their environment. Transmission of the naked DNA along this route does not necessarily pose an environmental risk, but the risk depends on the nature of the genetic information present on the naked DNA. This standard ERA is not suitable to address these risks. Therefore, this route of transmission is excluded from this environmental risk assessment. To ensure this exclusion, the bacteria on the administering location of the skin are to be killed by way of disinfection before administration.

Antibiotic resistance

A special case that must be taken into account in the environmental risk assessment is the situation in which the naked DNA carries a gene that codes for antibiotic resistance. The chances of naked DNA being transmitted to bacteria in the environment are extremely small. However, in the unlikely event of transmission of antibiotic-resistant genes, this may lead to unwanted effects. The selection pressure for antibiotic-resistant bacteria in the environment is high due to the widespread use of antibiotics. The standard environmental risk assessment is therefore limited to cases in which the naked DNA does not carry antibiotic-resistance genes. Only genes that code for resistance to the antibiotics kanamycin and neomycin are permitted, and not genes conferring resistance to any other antibiotics. The consideration here is that the importance of kanamycin and neomycin has been strongly reduced in medical and veterinarian practice.

Interaction with viruses

Another possible route of transmission of naked DNA to the environment is that of interaction with the genome of a virus inside the cell into which the DNA is inserted. If there is compatibility between parts of the sequence of the naked DNA and that of the viral genome, recombination may occur between these two. And if the recombined DNA molecule can become surrounded by the capsid of the virus, an infectious virus particle may even be created. In contrast to the naked DNA, there is a very small chance of such a virus particle being released from the cell and transmitted to another human or animal. The newly formed virus particle is in fact a GMO, and may pose a risk to the environment, depending on the properties encoded by the naked DNA. This specific risk is not addressed in the standard environmental risk assessment, but must be assessed in a separate ERA.  

The naked DNA, however, cannot recombine with a viral genome if it does not contain any viral sequences. The standard environmental risk assessment therefore states that it is only applicable in cases where the naked DNA does not contain any viral sequences.

The potential consequences of a recombination between naked DNA and a viral genome must be assessed in an environmental risk assessment that has been drafted particularly for such cases. This may lead to the conclusion that the presence of a certain sequence of viral DNA does not result in any transmission risks; for example, because there are grounds to assume that the supposed recombination event will not occur.

Environmental risk assessment for specific viral sequences

A number of such risk assessments have been drafted for certain viral DNA sequences that are often applied in naked DNA. This applies to the following viral sequences:

  • a CMV promoter, applied in humans with the exception of immunocompromised test subjects and newborns, or in animals with the exception of non-human primates; 
  • an RSV promoter, applied in humans or in animals with the exception of chickens; 
  • an SV40 polyadenylation signal, applied in humans or in animals with the exception of non-human primates;
  • an SV40 nuclear targeting sequence, applied in humans or in animals with the exception of non-human primates.  

These DNA sequences and the related environmental risk assessments have been included in the standard environmental risk assessment (in Dutch)

In principle, a general, standard environmental risk assessment may be drawn up for an Environmental Release (ER) permit for the medical application of naked DNA. Because, to date, experience has only been gained in environmental risk assessments in relation to naked DNA insertion by tattooing or by direct injection into the skin or striated muscle tissue, the standard environmental risk assessment is limited to these methods of administration. Should other administration methods present themselves in the future, then it will be assessed whether the standard environmental risk assessment could also be applied in those cases. 

Background documents

This environmental risk assessment is partly based on advice (in Dutch) provided by the Netherlands Commission on Genetic Modification (COGEM Netherlands Commission on Genetic Modification (Netherlands Commission on Genetic Modification)):

  • CGM/041223-02 of 23 December 2004, ‘Integration and transmission of naked DNA’
  • CGM/101026-06 of 26 October 2010, ‘Simplified procedure for some of clinical gene therapy research using naked DNA’ and the related report CGM2010-06, ‘Gene therapy with naked DNA: potential steps towards deregulation’
  • CGM/120919-01 of 19 September 2012, ‘Application of viral sequences in clinical research using naked DNA – for simplified permit issuing’
  • CGM/120927-01 of 27 September 2012, ‘Simplified procedure for issuing permits for clinical research using naked DNA’.