Work with Recombinant & Synthetic Nucleic Acids

Introduction

The purpose of the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (known as the NIH Guidelines) is to specify the practices for constructing and handling: (i) recombinant nucleic acid molecules, (ii) synthetic nucleic acid molecules, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules, and (iii) cells, organisms, and viruses containing such molecules.  

In this section, we will summarize some key concepts related to the safe use of recombinant and synthetic nucleic acid molecules, as well as viral vectors, in research.  

Experiments Exempt from the NIH Guidelines

Safety Risks of Viral Vectors

 

Adeno-Associated Viral Vectors

Adenoviral Vectors

Lentiviral Vectors

 

Definition of Recombinant and Synthetic Nucleic Acid Molecules

The NIH Guidelines defines recombinant and synthetic nucleic acids as:

  1. Molecules that are constructed by joining nucleic acid molecules and that can replicate in a living cell (i.e., recombinant nucleic acids);
  2. Nucleic acid molecules that are chemically or by other means synthesized or amplified, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules (i.e., synthetic nucleic acids), or
  3. Molecules that result from the replication of recombinant or synthetic nucleic acids.
 
Some examples of recombinant nucleic acids frequently used in research include plasmids, viral vectors, and shRNAs. Chemically synthesized molecules such as primers, modified analogs of nucleotides (e.g., morpholinos), and siRNA are examples of synthetic nucleic acids.

 

 

Aplicabiliy of the NIH Guidelines and Compliance at UVM

The NIH Guidelines apply to all recombinant or synthetic nucleic acid research conducted at, or sponsored by, an institution that receives support from the NIH for such purposes. Since The University of Vermont receives funding from the NIH for research with recombinant or synthetic nucleic acids, all research with recombinant or synthetic nucleic acids at UVM must comply with the NIH Guidelines regardless of the source of funding.

There are six categories of experiments involving recombinant or synthetic nucleic acid molecules:

  1. Experiments that require Institutional Biosafety Committee (IBC) approval, Recombinant DNA Advisory Committee (RAC) review, and NIH Director approval before initiation;
  2. Experiments that require NIH/OBA (Office of Biotechnology Activities) and IBC approval before initiation;
  3. Experiments that require IBC and IRB (Institutional Review Board) approval and RAC review before research participation enrollment;
  4. Experiments that require IBC approval before initiation;
  5. Experiments that require IBC notification simultaneous with initiation, and
  6. Experiments that are exempt from the NIH Guidelines.

 

Most of the research with recombinant or synthetic nucleic acid molecules at UVM requires only IBC approval or is exempt. In addition, gene transfer trials in humans require, at a minimum, IBC and IRB approval.

 

Non-compliance with the NIH Guidelines may result in: 

 

  1. Suspension, limitation, or termination of financial assistance from the NIH for recombinant or synthetic nucleic acid molecule research at UVM, or
  2. A requirement for prior NIH approval or any or all recombinant or synthetic nucleic acid molecule projects at UVM.

 

 

Experiments Exempt from the NIH Guidelines

The following recombinant or synthetic nucleic acid molecules are exempt from the NIH Guidelines and do not require IBC registration:

 

  1. Synthetic nucleic acids that: (a) can neither replicate nor generate nucleic acids than can replicate in any living cell (e.g., oligonucleotides or other synthetic nucleic acids that do not contain an origin of replication or elements known to interact with either DNA or RNA polymerase), and (b) are not designed to integrate into DNA, and (c) do not produce a toxin that is lethal for vertebrates at an LD50 < 100 ng/Kg body weight. NOTE: Deliberate transfer of synthetic nucleic acids into one or more human research participants may not be exempt.
  2. Nucleic acid molecules that are not in organisms, cells, or viruses and have not been modified or manipulated (e.g., encapsulated into synthetic or natural vehicles) to render them capable of penetrating cellular membranes.
  3. Those that consist solely of the exact nucleic acid sequence from a single source that exists contemporaneously in nature.
  4. Those that consist entirely of nucleic acids from a prokaryotic host, including its indigenous plasmids or viruses when propagated in that host - or a closely related strain of the same species - or when transferred to another host by well-established physiological means.
  5. Those that consist entirely of nucleic acids from a eukaryotic host, including its chloroplasts, mitochondria, or plasmid (excluding viruses) when propagated only in that host - or a closely related strain of the same species -.
  6. Those that consist entirely of DNA segments from different species that exchange DNA by known physiological processes - one or more of the segments may be a synthetic equivalent -.
  7. Genomic DNA molecules that have acquired a transposable element, provided the transposable element does not contain any recombinant and/or synthetic DNA.
  8. Those molecules that do not present a significant risk to the health or the environment, as determined by the NIH director, with advice of the RAC, and following appropriate notice and opportunity for public comment.

 

Experiments that are exempt from the NIH Guidelines may still be required to follow other federal and state biosafety standards from the latest edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) manual.

 

 

Safety Risks of Viral Vectors

Viral vectors are increasingly being used in genetic engineering since they deliver genes into many types of cells with high efficiency. This has open the door to a new search for medical treatments based on gene therapy. However, despite their success as molecular biology tools, viral vectors are not exempt of risks. Thus, researchers who work with viral vectors must understand how those viral vectors function and their potential safety risks and limitations. The sections below highlight some important concepts you should be familiar with before using viral vectors in order to conduct a risk assessment of your work. 

 

Risk Group of Viral Vectors

 

Viral vectors are derived from wild-type parent viruses that have been engineered to make room for a transgene and be safer. Enhanced safety is usually accomplished by removing key genes. However, it is still possible that a viral vector regains deleted genes thus reverting to its original form. Though the probability of reversion is very low, reversion can lead to serious consequences and should not be disregarded. Hence, the risk group of the parent virus should always be taken into consideration when conducting a risk assessment of work involving viral vectors. The 2013 NIH Guidelines classifies biological agents into four Risk Groups (RGs) according to their relative pathogenicity for healthy adult humans as shown in the table below: 

 

RISK GROUP 

BIOLOGICAL AGENT RISK GROUP DEFINITION

EXAMPLES

RG1

Agents that are not associated with disease in healthy adult humans

Adeno-associated virus, E. coli K12

RG2

Agents that are associated with human disease that is rarely serious and for which preventive or therapeutic interventions are often available

Adenovirus, Salmonella sp.

RG3

Agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available

HIV (lentivirus), Mycobacterium tuberculosis

RG4

Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available

Ebola virus

 

This classification is based on the potential effect of a biological agent on a healthy human adult and does not account for situations in which a person may have increased susceptibility to such agents due to, for instance, preexisting disease, use of certain medications, compromised immune status, pregnancy or breast feeding (which may increase exposure of infants to some agents).

 

Remember that the Risk Group (RG) and Biosafety Level (BSL) are two different things. The RG applies to the biological agent, while the BSL pertains to the containment level needed to work with that biological agent after the completion of a thorough risk assessment. In practice, the RG and BSL often match though that is not always the case. For instance, a RG2 agent may need to be handled at BSL-3 containment.

 

Route of Transmission of Viral Vectors

 

The route of transmission of the wild-type virus must be taken into consideration when assessing the risk of using viral vectors derived from it. For example, some viruses are spread through aerosols, while others need to come in direct contact with a person's blood in order to cause an infection. 

 

Cell Tropism of Viral Vectors

 

Viruses and derived viral vectors preferentially target certain host species or cell types within those species in a process known as tropism. While some viral vectors are attracted to - and efficiently infect - a limited range of cells, others have broader specificity. Tropism is determined by a precise fit between the surface proteins of the virus or viral vector and receptors expressed on the outside of target cells. Frequently, researchers replace the viral vector's surface proteins with those of other viruses to widen or limit the range of cells susceptible to infection. This procedure is called pseudotyping and results in the generation of a pseudotyped virus. Depending on their tropism, viral vectors can be classified as:

 

  • Ecotropic vectors - only infect murine cells (mice and rats)
  • Amphotropic vectors - infect mammalian cells, including human cells
  • Pantropic vectors - infect any type of cells of any species (e.g., vectors pseudotyped with VSV glycoprotein G)

 

Special precautions should be taken when working with pantropic and amphotropic vectors, which are capable of infecting human cells.

 

Integration and Mutagenesis Potential of Viral Vectors

 

The DNA of some viruses, such as lentivirus and gammaretrovirus, is able to integrate in the genome of the host cells as a provirus. Integration of the viral genome may disrupt endogenous genes resulting in mutations. This can lead to a wide range of disorders depending on the site of integration. Of special concern are the activation of proto-oncogenes and inactivation of tumor-suppressor genes, which can lead to the development of cancer.

 

Environmental Stability of Viral Vectors

 

Viruses may be more or less sensitive to external factors depending on the presence or absence of viral envelopes. Naked or non-enveloped viruses - e.g., adenovirus - exit the host cell by lysis. Enveloped viruses - e.g., retroviruses - exit the host cell by budding through the cell membrane. The envelope is composed of fragments from the host cell membranes - phospholipids and proteins - and viral glycoproteins. The lipid bilayer envelope is relatively sensitive to heat, desiccation, and detergents, making them easier to inactivate than naked viruses. Enveloped viruses have limited survival outside of the host and usually transfer from host to host in order to survive.

 

Nature of the Transgene/Oligonucleotide Inserted into Viral Vectors

 

The genes involved in replication have been deleted in most viral vectors thus rendering them replication-incompetent or deficient. However, such vectors are still able to infect host cells and express the transgene or oligonucleotide they are carrying. This may result in gene activation or gene suppression - e.g., shRNA or microRNA -. Genes that alter the cell cycle when overexpressed - e.g., growth factors, transcription factors, kinases, antiapoptotic molecules, oncogenes... - or genes whose inhibition may promote carcinogenesis - e.g., tumor suppressors - are of special concern.   

 

Replication-Competent Virus Breakthroughs

 

The genes necessary for virus replication are deleted in replication-incompetent viral vectors. However, such genes need to be supplied by other means - through plasmids, helper viruses, or packages cell lines - in order to produce the viral vector particles and infect cells. The risk resides in that replication-deficient viral vectors can regain the genes required for replication through recombination in a process known as replication-competent virus (RCV) breakthroughs. That is an important concern for researchers using lentiviral vectors. The following safety measures can be implemented to avoid the generation of RCV breakthroughs:

 
  • Removing viral regulatory regions to decrease the risk of homologous recombination.
  • Splitting the viral replication genes into several plasmids - the larger the number of plasmids, the more recombination events needed to generate RCV breakthroughs.
  • Producing virus as a transient single batch (simultaneous transfection of plasmids) instead of as a continuous culture (using a packaging cell line with replication genes integrated into the cell line's genome).

 

 

Adeno-Associated Viral Vectors

Adeno-Associated Viruses (AAV) are helper-dependent members of the Parvoviridae family and Dependovirus genus. Productive AAV infection requires helper functions supplied by coinfection with other viruses - such as Adenovirus (Ad) and Herpes Simplex Virus (HSV) - or the use of DNA damaging agents. Although ubiquitous in humans, AAV have not been associated with any human disease, thus being classified as Risk Group 1 agents. AAV are small viruses with limited coding capacity that rely heavily on the cellular environment and machinery. In the absence of helper virus, AAV can remain latent in many cell types. They can be rescued from latency by subsequent infection with helper virus. AAV2, for instance, remains latent by integrating at a specific locus on human chromosome 19. There are multiple serotypes of AAV, with AAV2 being the most frequently used as a viral vector. To learn more about adeno-associated viruses and derived vectors, having AAV2 as a model, please click on the Adeno-Associated Virus and AAV Vectors Fact Sheet

 

 

Adenoviral Vectors

Human adenoviruses (Ad) used in the construction of adenoviral vectors - generally Ad 2 and Ad 5 - are members of the Adenoviridae family and the human Mastadenovirus C genus. They usually target the respiratory tract and/or conjunctiva of the eye, resulting in acute respiratory disease and/or pharyngoconjunctival fever. They are classified as Risk Group 2 agents and third-generation (gutless) adenoviral vectors have become very attractive for the development of new gene therapies. Gutless vectors elicit a much weaker immune response than first- and secon-generation vectors, while achieving high transduction efficiency and tropism. They can be delivered to different organs - such as the liver, muscle, and the central nervous system - resulting in high-level and long-term transgene expression in rodent and primates. However, they require the presence of a helper virus. To learn more about adenoviruses and derived vectors, please click on the Adenovirus and Adenoviral Vectors Fact Sheet

 

 

Lentiviral Vectors

Lentiviruses, such as Human Immunodeficiency Virus (HIV), Feline Immunodeficiency Virus (FIV), and Equine Infectious Anemia Virus (EIAV) are enveloped viruses of the Retroviridae family with a genome composed of plus-stranded RNA. Lentiviruses have become very popular for the development of viral vectors because they can infect both dividing and non-dividing cells. Lentiviral vectors derived from the HIV virus are frequently used in biomedical research. As is the case with adenoviral vectors, different generations of lentiviral vectors have been developed to improve their safety profile. To learn more about lentiviral vectors, please click on the Lentivirus and Lentiviral Vectors Fact Sheet

 

Last updated: November 2014