In laboratory for  forensic genetics  scientific projects, supported by the respective ministries and foreign institutions, are implemented or are under realization. The focus of scientific research is directed towards  genetic analysis  of  archaeological   skeletal  samples, forensic genetic parameters testing of Bosnian population, as well as towards a target oriented expansion of previously initiated population genetic research.


This laboratory represents the organizational segment of the Institute which is dealing with genetic characterisation of  DNA  of human origin for the purpose of basic and applied research. We use molecular-genetics approach mainly  PCR  based in investigating the genetic structure.


Main activities of the laboratory comprise research directions:


Other important aspect is participation in higher education programs at  University of Sarajevo  as well as public engagement in developing  molecular-genetics  methods for support of  medical diagnostics . [ 13 ]


The laboratory scope includes wide array of activities mainly focused on the issues of food  biosafety  and plant  biotechnology . It provides qualitative and quantitative analysis of specific  DNA sequences  in various food matrices, provides advice and correct interpretation of  GMO  related data to consumers and food safety authority and promotes science based approach to biosafety. In that respect the Laboratory has established communication with JRC-EURL-GMFF and follows the published guidelines. Also, the Laboratory develops new analytical methods, where appropriate, to bridge the gaps in the available methodology.


Research aspect of the Laboratory is mainly focused on  endemic  and endangered plant  species  with bioactive potential. Simultaneously with bioactive potential of a species, which is explored in in vitro and in vivo models,  molecular markers  are employed to evaluate its  genetic diversity  for the purpose of conservation.


Research activities of the Laboratory for Cytogenetics and Genotoxicology are based on:


Expert activity of the Laboratory for Cytogenetics and Genotoxicology mainly includes  chromosome  analysis and  karyotyping  of human samples. The most frequently used tests in research projects of this lab are based on cell culture and include:  chromosome aberrations  analysis, cytokinesis-block  micronucleus  cytome assay and sister  chromatids  exchange assay. Evaluation of cytotoxic and cytostatic potential of various chemical agents includes application of colorimetric method in different cell lines. Research capacities are significantly used for academic education and the realization of final thesis of Sarajevo University students.


Expertises :


Projects :


The  International Centre for Genetic Engineering and Biotechnology  ( ICGEB ) was established as a project of the  United Nations Industrial Development Organization  (UNIDO) in 1983. The Organisation has three Component laboratories with over 45 ongoing research projects in Infectious and Non-communicable diseases, Medical, Industrial and Plant Biology Biotechnology in:  Trieste, Italy ,  New Delhi, India  and  Cape Town, South Africa .


On February 3, 1994, under the direction of  Arturo Falaschi  the ICGEB became an autonomous International Organisation and now has over 65 Member States across world regions.


Its main pillars of action comprise: Research, Advanced Education through PhD and Postdoctoral Fellowships, International Scientific Meetings and Courses, competitive Grants for scientists in Member States and Technology Transfer to industry.
 [ 1 ]


The  International Society for Transgenic Technologies (ISTT)  is an organization dedicated to advancing research, communication, and technology exchange regarding  transgenic technologies . [ 1 ] [ 2 ]


Every one and a half years the ISTT organizes an international scientific conference, the  Transgenic Technology Meeting , also known as the  TT Meeting . [ 3 ]  To promote communication and technology exchange, the website of the society publishes information and protocols related to transgenic technologies as well as the locations of transgenic service facilities, recognized as a valuable resource in the  scientific literature . [ 9 ] [ 10 ] [ 11 ] [ 12 ] [ 13 ]  A collection of ISTT subject-related protocols has been published in the book  Advanced Protocols for Animal Transgenesis – an ISTT Manual . [ 2 ]  The society is also associated with the peer-reviewed scientific journal  Transgenic Research , which publishes scientific findings on transgenic and genome-edited higher model organisms. [ 14 ] [ 3 ]  As a platform for the rapid exchange of scientific information, the ISTT hosts two  mailing lists , the public  transgenic-list  (often referred to as  tg-l ) and the  ISTT-list  reserved for ISTT members with around 1500 and 660 participants (April 2024). [ 15 ] [ 16 ] [ 17 ]


The ISTT Prize recognizes individuals for their outstanding contributions to the field of transgenic technologies and is presented at the Transgenic Technology Meeting. Prominent winners included  Ralph Brinster  (2011),  Janet Rossant  (2014),  Mario Capecchi  (2017) and  Rudolf Jaenisch (2025) . [ 3 ] [ 25 ] [ 27 ] [ 33 ]


The ISTT Young Investigator Award recognizes outstanding achievements by young scientists whose work is advancing the field of transgenic technologies with new ideas and who have recently received an academic degree. The ISTT Young Investigator Award is presented at the  Transgenic Technology Meetings . Prominent winners included  Feng Zhang  (2014) and Alexis Komor (2017) for their work on  genome editing  in model organisms. [ 3 ] [ 25 ]


The 3Rs Award recognizes outstanding achievements by a researcher or research team that advances the field of transgenic technologies with new methods and improvements in strict accordance with the 3Rs principles for reduction, refinement, and replacement of animals used in research. The prize is awarded during the  Transgenic Technology Meetings . [ 34 ] [ 3 ]


A  knockout moss  is a kind of genetically modified  moss . One or more of the moss's specific  genes  are deleted or inactivated (" knocked out "), for example by  gene targeting  or other methods. After the deletion of a gene, the knockout moss has lost the trait encoded by this gene. Thus, the function of this gene can be inferred. This scientific approach is called  reverse genetics  because the scientist wants to understand the function of a specific gene. In  classical genetics , the scientist starts with a  phenotype  of interest and searches for the gene that causes this phenotype. Knockout mosses are relevant for  basic research  in  biology  as well as in  biotechnology .


The targeted deletion or alteration of genes relies on the integration of a  DNA  strand at a specific and predictable position into the  genome  of the host cell. This DNA strand must be engineered in such a way that both ends are identical to this specific  gene locus . This is a prerequisite for being efficiently integrated via  homologous recombination  (HR). This is similar to the process used for creating  knockout mice .
So far, this method of gene targeting in  land plants  has been carried out in the mosses  Physcomitrella patens  and  Ceratodon purpureus , [ 2 ]  since in these  non-seed  plant  species  the efficiency of HR is several orders of magnitude higher than in  seed plants . [ 3 ]


Knockout mosses are stored at and distributed by a specialized  biobank , the  International Moss Stock Center .


For altering moss genes in a targeted way, the DNA-construct needs to be incubated together with moss  protoplasts  and with  polyethylene glycol  (PEG). Because mosses are  haploid  organisms, the regenerating moss filaments ( protonemata ) can be directly assayed for gene targeting within six weeks when utilizing  PCR  methods. [ 4 ]


The first scientific publication in which knockout moss was used to identify the function of a hitherto-unknown gene appeared in 1998, and was authored by  Ralf Reski  and coworkers. They deleted the  ftsZ -gene and thus functionally identified the first gene pivotal for the division of an  organelle  in any  eukaryote . [ 5 ]


Physcomitrella  plants were engineered with multiple knockouts to prevent the plant-specific  glycosylation  of proteins, an important  post-translational modification . These knockout mosses are used to produce complex  biopharmaceuticals  in the field of  molecular farming . [ 6 ]


In cooperation with the chemical company  BASF , Ralf Reski and coworkers established a collection of knockout mosses to use for gene identification. [ 1 ] [ 7 ]


LEAPER  ( L everaging  e ndogenous  A DAR for  p rogrammable  e diting of  R NA) is a  genetic engineering  technique in  molecular biology  by which  RNA  can be edited. The technique relies on engineered strands of RNA to recruit native ADAR enzymes to swap out different compounds in RNA. Developed by researchers at  Peking University  in 2019, the technique, some have claimed, is more efficient than the  CRISPR gene editing technique . [ 1 ]  Initial studies have claimed that editing efficiencies of up to 80%.


As opposed to DNA gene editing techniques (e.g., using CRISPR-Cas proteins to make modifications directly to a defective gene), LEAPER targets editing messenger RNA (mRNA) for the same gene which is transcribed into a protein. [ 3 ]  Post-transcriptional RNA modification typically involves the strategy of converting adenosine-to-inosine (A-to-I) since inosine (I) demonstrably mimics guanosine (G) during translation into a protein. A-to-I editing is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes, whose substrates are double-stranded RNAs. [ 4 ]  Three human ADAR genes have been identified with ADAR1 (official symbol ADAR) and ADAR2 (ADARB1) proteins developed activity profiles. LEAPER achieves this targeted RNA editing through the use of short engineered ADAR-recruiting RNAs (arRNAs). arRNAs consist of endogenous ADAR1 proteins with several RNA binding domains (RBDs) fused with a peptide, CRISPR-Cas13b protein, and a guide RNA (gRNA) between 100 and 150 nt in length for high editing efficiency designed to recruit the chimeric ADAR protein to a target site. [ 2 ]


This results in a change in which protein is synthesized during  translation .


The technique was discovered by a team of researchers at Peking University in  Beijing , China. The discovery was announced in the journal  Nature Biotechnology  in July 2019. [ 5 ]


Chinese researchers have utilized LEAPER to restore functional enzyme activity in cells from patients with  Hurler syndrome . They have claimed that LEAPER could have the potential to treat almost half of all known hereditary disorders. [ 5 ]


Highly specific editing efficiencies of up to 80% can be achieved when LEAPER editing using arRNA151 is delivered via a plasmid or viral vector or as a synthetic  oligonucleotide , though this efficiency varied significantly across cell types. [ 4 ]   Based on these preliminary results, LEAPER may have the most therapeutic promise with no production of functional protein but if a partial restoration of protein expression would provide therapeutic benefit. For example, in human cells with defective α-L-iduronidase (IDUA) expression in cells from patients with IDUA-defective Hurler syndrome, LEAPER resulted in a W53X truncation mutant of p53 being edited using arRNA151 to achieve a "normal" p53 translation and functional p53-mediated transcriptional responses. [ 4 ]


LEAPER is analogous to  CRISPR Cas-13  in that it targets RNA before proteins are synthesized. However, LEAPER is simpler and more efficient as it only requires arRNA, rather than Cas and a guide RNA. [ 5 ]  According to the developers of LEAPER, it has the potential to be easier and more precise than any CRISPR technique. [ 6 ]


LEAPER also eliminates health concerns and technical barriers arising from the introduction of exogenous proteins. [ 7 ]


It has also been called more  ethical  as it does not change DNA and thus does not result in heritable changes, unlike methods using CRISPR Cas-9. [ 8 ]


In  molecular biology , a  library  is a collection of  genetic material  fragments that are stored and propagated in a population of microbes through the process of  molecular cloning . There are different types of DNA libraries, including  cDNA libraries  (formed from  reverse-transcribed RNA ),  genomic libraries  (formed from genomic DNA) and randomized mutant libraries (formed by de novo gene synthesis where alternative nucleotides or codons are incorporated). DNA library technology is a mainstay of current  molecular biology ,  genetic engineering , and  protein engineering , and the applications of these libraries depend on the source of the original DNA fragments. There are differences in the  cloning vectors  and techniques used in library preparation, but in general each DNA fragment is uniquely inserted into a cloning vector and the pool of recombinant DNA molecules is then transferred into a population of  bacteria  (a  Bacterial Artificial Chromosome  or BAC library) or yeast such that each organism contains on average one construct (vector + insert). As the population of organisms is grown in culture, the DNA molecules contained within them are copied and propagated (thus, "cloned").


The term "library" can refer to a population of organisms, each of which carries a DNA molecule inserted into a cloning vector, or alternatively to the collection of all of the cloned vector molecules.


A  cDNA library  represents a sample of the  mRNA  purified from a particular source (either a collection of cells, a particular tissue, or an entire organism), which has been converted back to a DNA template by the use of the enzyme  reverse transcriptase . It thus represents the genes that were being actively transcribed in that particular source under the physiological, developmental, or environmental conditions that existed when the mRNA was purified. cDNA libraries can be generated using techniques that promote "full-length" clones or under conditions that generate shorter fragments used for the identification of " expressed sequence tags ".


cDNA libraries are useful in reverse genetics, but they only represent a very small (less than 1%) portion of the overall genome in a given organism.


Applications of cDNA libraries include:


A  genomic library  is a set of clones that together represents the entire genome of a given organism. The number of clones that constitute a genomic library depends on (1) the size of the genome in question and (2) the insert size tolerated by the particular  cloning vector  system. For most practical purposes, the tissue source of the genomic DNA is unimportant because each cell of the body contains virtually identical DNA (with some exceptions).


Applications of genomic libraries include:


In contrast to the library types described above, a variety of artificial methods exist for making libraries of variant genes. [ 1 ]  Variation throughout the gene can be introduced randomly by either  error-prone PCR , [ 2 ]   DNA shuffling  to recombine parts of similar genes together, [ 3 ]  or transposon-based methods to introduce  indels . [ 4 ]
Alternatively, mutations can be targeted to specific codons during  de novo  synthesis  or  saturation mutagenesis  to construct one or more  point mutants  of a gene in a controlled way. [ 5 ]  This results in a mixture of double stranded DNA molecules which represent variants of the original gene.


The  expressed  proteins from these libraries can then be screened for variants which exhibit favorable properties (e.g. stability, binding affinity or enzyme activity). This can be repeated in cycles of creating gene variants and screening the expression products in a  directed evolution  process. [ 1 ]


If creating an mRNA library (i.e. with cDNA clones), there are several possible protocols for isolating full length mRNA. To extract DNA for genomic DNA (also known as gDNA) libraries, a DNA mini-prep may be useful.


cDNA libraries require care to ensure that full length clones of mRNA are captured as cDNA (which will later be inserted into vectors). Several protocols have been designed to optimise the synthesis of the 1st cDNA strand and the 2nd cDNA strand for this reason, and also to make directional cloning into the vector more likely.


gDNA fragments are generated from the extracted gDNA by using non-specific frequent cutter restriction enzymes.


The nucleotide sequences of interest are preserved as inserts to a  plasmid  or the genome of a  bacteriophage  that has been used to infect bacterial cells.


Vectors are propagated most commonly in bacterial cells, but if using a YAC (Yeast Artificial Chromosome) then yeast cells may be used. Vectors could also be propagated in viruses, but this can be time-consuming and tedious. However, the high transfection efficiency achieved by using viruses (often phages) makes them useful for packaging the vector (with the ligated insert) and then introducing them into the bacterial (or yeast) cell.


Additionally, for cDNA libraries, a system using the Lambda Zap II phage, ExAssist, and 2 E. coli species has been developed. A Cre-Lox system using loxP sites and the in vivo expression of the recombinase enzyme can also be used instead. These are examples of in vivo excision systems. In vitro excision involves subcloning often using traditional restriction enzymes and cloning strategies. In vitro excision can be more time-consuming and may require more "hands-on" work than in vivo excision systems. In either case, the systems allow the movement of the vector from the phage into a live cell, where the vector can replicate and propagate until the library is to be used.


This involves "screening" for the sequences of interest. There are multiple possible methods to achieve this.


The following is a  list of  hybrid entities  from the  folklore  record grouped morphologically. Hybrids not found in  classical mythology  but developed in the context of modern  popular culture  are listed in  § Modern fiction .


The following hybrid creatures appear in modern fiction:


The  Jurassic Park  franchise had these hybrids in the films, toylines, and video games.


The word  metagenics  uses the prefix  meta  and the suffix  gen . Literally, it means "the creation of something which creates". In the context of biotechnology, metagenics is the practice of engineering organisms to create a specific enzyme, protein, or other biochemicals from simpler starting materials. The genetic engineering of  E. coli  with the specific task of producing human  insulin  from starting amino acids is an example.  E. coli  has also been engineered to digest plant biomass and use it to produce hydrocarbons in order to synthesize biofuels. The applications of metagenics on  E. coli  also include higher alcohols, fatty-acid based chemicals and terpenes. [ 1 ]


The depletion of petroleum sources and increase in greenhouse gas emissions in the twenty and twenty-first centuries has been the driving factor behind the development of biofuels from microorganisms.  E. coli  is currently regarded as the best option for biofuel production because of the amount of knowledge available about its genome. The process converts biomass into fuels, and has proven successful on an industrial scale, with the United States having produced 6.4 billion gallons of bioethanol in 2007. Bioethenol is currently the front-runner for alternative fuel production and uses  S.cerevisiae  and  Zymomonas mobilis  to create ethanol through fermentation. However, maximum productivity is limited due to the fact that these organisms cannot use pentose sugars, leading to consideration of  E.coli  and  Clostridia. E.coli  is capable of producing ethanol under anaerobic conditions through metabolizing glucose into two moles of formate, two moles of acetate, and one mole of ethanol. While bioethanol has proved to be a successful alternative fuel source on an industrial scale, it also has its shortcomings, namely, its low energy density, high vapor pressure, and  hygroscopicity . Current alternatives to bioethanol include biobutanol, biodiesel, propanol, and synthetic hydrocarbons. [ 2 ]  The most common form of biodiesels is fatty acid methyl esters and current synthesis strategies involve transesterification of triacylglycerols from plant oils. However, plant oils have a major limitation in availability of oil-seed supplies at competitive prices, leading to an interest in direct synthesis of fatty acid methyl esters in bacteria. This process bypasses transesterification, leading to higher energy yields and lower production cost. [ 3 ]  One of the principal obstacles in production of viable biofuels is that the maximum blend ratio of biofuel to petroleum is between 10% and 20%, Current biofuels are not compatible with high-performance, low-emission engines and costly changes in infrastructure and engine remodeling would be required. A University of Exeter study sought to overcome this obstacle through production of biofuels that can replace current fossil fuels through sustainable means, namely, the production of  n -alkanes,  iso -alkanes, and  n -alkenes, as these are the hydrocarbons that compose current retail transport fuels. The study found suitable substrates for production of the aforementioned hydrocarbons by means of the  P. luminescens  fatty acid reductase (FAR) complex. [ 4 ]  A study published in Biotechnology for Biofuels used  S. cerevisiae  to produce short- and branched-chain alkyl esters biodiesel through metabolic engineering. Negative regulators for the  INO1  gene, Rpd3 and Opi1 were deleted to boost  S. cerevisiae's  ability to produce fatty acid esters. To increase the production of alcohol precursors, five isobutanol pathway enzymes were overexpressed. [ 5 ]


Increase in the demand for recombinant insulin can be explained by an increase in the number of diabetic patients globally, as well as alternative delivery methods such as inhalation and oral routes, which require higher doses. [ 6 ]  Through the use of recombinant DNA technology,  E. coli  can be used for the production of human insulin. The biosynthesis of insulin within the human body confers a significant advantage over bovine or porcine synthesis, which are often immunogenic in diabetic patients. [ 7 ]  To accomplish this, synthetic genes for human insulin are fused with the β-galactosidase gene of  E.coli , where they undergo transcription and ultimately translation into proteins. [ 8 ]  The limiting factor for the use of  microorganisms  like  E. coli  in biosynthesis of gene products like insulin is time, yet due to advancements in the synthesis of  oligonucleotides  and liquid chromatography, the production time needed for DNA fragments has greatly decreased. [ 9 ]  Recombinant human insulin was first approved for clinical trials in 1980. At this time the A and B chains of insulin were produced separately and then chemically joined. [ 10 ]  Joining of the two chains was often carried out through air oxidation with low efficiency. A 1978 study by Goedell et al. successfully accomplished correct joining of the A and B chains through S-sulfonated derivatives and an excess of the A chain, resulting in 50-80% correct joining. [ 8 ]  Recent advances have allowed the chains to be synthesized together by inserting the human proinsulin gene into  E. coli  cells, which produce proinsulin through fermentation. [ 10 ]


The  NK-92  cell line is an  immortalised cell line  that has the characteristics of a type of  immune cell  found in human blood called ’ natural killer ’ (NK) cells. Blood NK cells and NK-92 cells recognize and attack  cancer cells  as well as cells that have been infected with a  virus ,  bacteria , or  fungus . [ 1 ]  NK-92 cells were first isolated in 1992 in the laboratory of Hans Klingemann at the  British Columbia Cancer Agency  in Vancouver, Canada, from a patient who had a rare NK cell  non-Hodgkin-lymphoma . [ 2 ]  These cells were subsequently developed into a continuously growing cell line. NK-92 cells are distinguished by their suitability for expansion to large numbers, ability to consistently kill cancer cells and testing in clinical trials. When NK-92 cells recognize a cancerous or infected cell, they secrete  perforin  that opens holes into the diseased cells and releases  granzymes  that kill the target cells. NK-92 cells are also capable of producing cytokines such as  tumor necrosis factor alpha  (TNF-a) and  interferon gamma  (IFN-y), [ 3 ]  which stimulates proliferation and activation of other immune cells.


Several phase 1 clinical trials have been performed by experts in the field of  adoptive immunotherapy  of cancer. Hans Klingemann and Sally Arai completed a US trial at  Rush University Medical Center  ( Chicago ) in  renal cell cancer  and melanoma patients in 2008, [ 4 ]  and Torsten Tonn, MD and Oliver Ottmann, MD completed the European trial at the  University of Frankfurt  in patients with various solid and hematological malignancies in 2013. [ 5 ]  Armand Keating at  Princess Margaret Hospital  in  Toronto  conducted a trial in which NK-92 cells were given to patients who had relapsed after  autologous  bone marrow transplants for  leukemia  or lymphoma. In all clinical trials so far, NK-92 cells were administered as a simple intravenous infusion, dosed two or three times per treatment course, and given in the outpatient setting.


Of the 39 patients enrolled across the three studies, 2 serious (grade 3–4) side-effects occurred during or after the infusion of NK-92 cells, the side effects disappeared afterward. The doses given to patients ranged from 1x10 8  cells/m 2  to 1x10 10  cells/m 2  per infusion. Patients received between two and three infusions over a period of less than a week. About one-third of the treated patients had clinically meaningful responses with some of them fully recovering.


In a 2017 study by Congcong Zhang and Winfried S. Wels, NK-92 cells were genetically engineered to recognize and kill specific human cancers by expressing  chimeric antigen receptors  (CARs). [ 6 ]  CAR-engineered  T-lymphocytes  (CAR-T) have garnered attention in immuno-oncology, as the infusion of CAR-T cells has been shown to induce remissions in some patients with acute and chronic  leukemia  and  lymphoma . However, CAR-T cells can cause  cytokine release syndrome  (CRS). CAR-engineered NK cells from either peripheral or cord blood have not proved to be as feasible for use to treat diseases as they are difficult to expand to get sufficient numbers, and the yields can be variable and/or too low. Also,  genetic transduction  to introduce the CAR into blood NK cells requires  lentiviral  or  retroviral  vectors, which are only moderately efficient.


NK-92 cells, in contrast to NK-92 CAR-T cells, have predictable expansion kinetics and can be grown in bioreactors that produce billions of cells within a couple of weeks. [ 7 ]  Further, NK-92 cells can easily be transduced by physical methods, and  mRNA  can be shuttled into NK-92 cells with high efficiency. CAR-expressing NK-92 have been generated to target a number of cancer surface receptors [ 8 ]  such as programmed death domain ligand 1 (PD-L1),  CD19  (a type of B cell receptor), [ 9 ] [ 10 ]   human epidermal growth factor receptor 2  (HER2/ErbB2) and  epidermal growth factor receptor  (EGFR, aka HER1); and many of these engineered NK-92 cells are currently in clinical trials for the treatment of cancer. [ 11 ]


NK-92 cells, which require interleukin-2 (IL-2) for growth, have also been genetically altered with an IL-2 gene to allow them to grow in culture without the addition of IL-2. [ 12 ]  They have also been engineered to express a high-affinity  Fc-receptor  which is the main receptor for  monoclonal antibodies  to bind to NK-92 and use their cytotoxic load to kill cancer cells. [ 13 ] [ 14 ]  The cells have been further engineered to express Chimeric Antigen Receptors (CARs) such as programmed death domain ligand 1 (PD-L1). [ 15 ]  During the course of development, NK-92 cells were renamed  activated  NK cells (aNK) and the different variants have been designated as follows:


NK-92 = parental cells, later designated  aNK


NK-92ci = NK-92 cells transfected with an episomal vector for expression of IL-2


NK-92 mi = NK-92 cells transfected with an MFG vector for expression of IL-2


haNK = NK-92 (aNK) transfected with a plasmid expressing high affinity CD16 FcR and erIL-2


taNK =  NK-92 (aNK) transfected with either a plasmid or lentiviral vector expressing a CAR


t-haNK = NK-92 (aNK) transfected with a plasmid expressing a CAR and CD16 FcR erIL-2


qt-haNK = NK-92 (aNK) transfected with a plasmid expressing a 4th gene in addition to a CAR, the CD16 FcR, and erIL-2 :  examples: homing receptor of the CXCR family or immune-active cytokines


The high affinity Fc-receptor-expressing NK (haNK) cells were administered to patients with advanced  Merkel cell carcinoma  (MCC) and there were some notable responses. Currently, a HER2-targeted aNK (taNK) line and various t-haNK (CAR and Fc-receptor expressing) cell lines are in clinical trials in patients with various cancers, as described in the review “The NK-92 cell line 30 years later: its impact on natural killer cell research and treatment of cancer." [ 16 ]


Global rights to the NK-92 cell line were assigned to  ImmunityBio  Inc. (formerly NantKwest, Inc.). ImmunityBio's only authorized NK-92 distributor is Brink Biologics, Inc. (San Diego), which makes NK-92 cells and certain genetically modified CD16+ variants available to third parties for non-clinical research under a limited use license agreement.


Nucleofection  is an  electroporation -based  transfection  method which enables transfer of  nucleic acids  such as  DNA  and  RNA  into  cells  by applying a specific voltage and reagents. Nucleofection, also referred to as  nucleofector technology , was invented by the biotechnology company Amaxa. "Nucleofector" and "nucleofection" are  trademarks  owned by Lonza Cologne AG, part of the  Lonza Group .


Nucleofection is a method to transfer substrates into  mammalian cells  so far considered difficult or even impossible to transfect. Examples for such substrates are nucleic acids, like the DNA of an isolated  gene   cloned  into a  plasmid , or  small interfering RNA  (siRNA) for knocking down expression of a specific  endogenous  gene.


Primary cells, for example  stem cells , especially fall into this category, although many other  cell lines  are also difficult to transfect. Primary cells are freshly isolated from  body tissue  and thus cells are unchanged, closely resembling the  in-vivo  situation, and are therefore of particular relevance for  medical research  purposes. In contrast, cell lines have often been cultured for decades and may significantly differ from their origin.


Based on the physical method of  electroporation , nucleofection uses a combination of electrical parameters, generated by a device called Nucleofector, with cell-type specific reagents. The substrate is transferred directly into the  cell nucleus  and the  cytoplasm . In contrast, other commonly used non-viral transfection methods rely on  cell division  for the transfer of DNA into the nucleus. Thus, nucleofection provides the ability to transfect even non-dividing cells, such as  neuron  and resting  blood cells . Before the introduction of the Nucleofector Technology, efficient  gene transfer  into primary cells had been restricted to the use of  viral vectors , which typically involve disadvantages such as safety risks, lack of reliability, and high cost. The non-viral gene transfer methods available were not suitable for the efficient transfection of primary cells. Non-viral delivery methods may require cell division for completion of transfection, since the DNA enters the nucleus during breakdown of the  nuclear envelope  upon  cell division  or by a specific localization sequence.
Optimal nucleofection conditions depend upon the individual  cell type , not on the substrate being transfected. This means that identical conditions are used for the nucleofection of DNA, RNA, siRNAs,  shRNAs ,  mRNAs  and  pre-mRNAs ,  BACs ,  peptides ,  morpholinos ,  PNA , or other biologically active molecules.


The  overlap extension polymerase chain reaction  (or  OE-PCR ) is a variant of  PCR . It is also referred to as  Splicing by overlap extension  /  Splicing by overhang extension (SOE) PCR . It is used to assemble multiple smaller double stranded DNA fragments into a larger DNA sequence.  OE-PCR  is widely used to insert  mutations  at specific points in a sequence or to assemble custom DNA sequence from smaller DNA fragments into a larger  polynucleotide . [ 1 ]


As in most PCR reactions, two primers—one for each end—are used per sequence. To splice two DNA molecules, special primers are used at the ends that are to be joined. For each molecule, the primer at the end to be joined is constructed such that it has a 5' overhang complementary to the end of the other molecule. Following annealing when replication occurs, the DNA is extended by a new sequence that is complementary to the molecule it is to be joined to. Once both DNA molecules are extended in such a manner, they are mixed and a PCR is carried out with only the primers for the far ends. The overlapping complementary sequences introduced will serve as primers and the two sequences will be fused. This method has an advantage over other gene splicing techniques in not requiring restriction sites.


To get higher yields, some primers are used in excess as in  asymmetric PCR .


To insert a mutation into a DNA sequence, a specific  primer  is designed. The primer may contain a single substitution or contain a new sequence at its 5' end. If a  deletion  is required, a sequence that is 5' of the deletion is added, because the 3' end of the primer must have complementarity to the template strand so that the primer can sufficiently  anneal  to the template DNA.


Following annealing of the primer to the template,  DNA replication  proceeds to the end of the template. The duplex is denatured and the second primer anneals to the newly formed DNA strand, containing sequence from the first primer. Replication proceeds to produce a strand of the required sequence, containing the mutation.


The duplex is denatured again and the first primer can now bind to the latest DNA strand. The replication reaction continues to produce a fully  dimerised  DNA fragment. After further PCR cycles, to amplify the DNA, the sample can be separated by  agarose gel electrophoresis , followed by electroelution for collection.


Efficiently generating  oligonucleotides  beyond ~110 nucleotides in length is very difficult, so to insert a mutation further into a sequence than a 110 nt primer will allow, it is necessary to employ overlap extension PCR. In OE-PCR the sequence being modified is used to make two modified strands with the mutation at opposite ends, using the technique described above. After mixing and denaturation, the strands are allowed to anneal to produce three different combinations as detailed in the diagram. Only the duplex without overlap at the 5' end will allow extension by DNA polymerase in 3' to 5' direction.


Following the extension of the OE-PCR reaction, the PCR mix or the eluted fragments of appropriate size are subject to normal PCR, using the outermost primers used in the initial, mutagenic PCR reactions.


In addition, the combination of OE-PCR and  asymmetric PCR  could be used to improved the efficiency of  site-directed mutagenesis . [ 2 ]


Besides the introduction of mutations, Overlap Extension PCR is widely used to assemble complex DNA sequences without the introduction of undesired nucleotides at any position. This is possible since OE-PCR relies on the utilization of complementary overhangs to guide the scarless splicing of custom DNA fragments in a desired order. This is the main advantage of OE-PCR and other long-homology based cloning methods such as  Gibson assembly , which overcome the limitations of traditional  restriction enzyme digestion  and  ligation  cloning methods. [ 3 ]


Assembly of custom DNA sequences with OE-PCR consists on three main steps. First, individual DNA sequences are amplified by PCR from different templates and flanked with the required complementary overhangs. Second, the formerly obtained PCR products are combined together into the overlap extension PCR reaction, where the complementary overhangs bind pair-wise allowing the polymerase to extend the DNA strand. Eventually, outer primers targeting the external overhangs are used and the desired DNA product is amplified in the final PCR reaction.


The overall success of OE-PCR based DNA assemblies relies on several factors, being the most relevant ones the instrinsic features of the DNA sequence to assemble, the sequence and length of the overlapping overhangs, the design of outer primers for the final amplification and the conditions of the PCR reaction. Normally, from 2 to 6 fragments can be spliced simultaneously into a single OE-PCR reaction. [ 4 ]  Overhangs should be at least 40 nucleotides long to ensure adequate interaction between fragments. Final amplification primers are commonly designed following general guidelines for PCR, however they are used in 2 to 5 times lower concentration than in standard PCR reactions, as it this has been shown to reduce undesired amplifications. [ 5 ]  Additionally the utilization of  proofreading  DNA polymerases is highly recommended.


Plant expressed vaccine  or  project GreenVax [ 1 ]  In 2005  DARPA ’s Accelerated Manufacture of Pharmaceuticals (AMP) program was created In response to emerging and novel biologic threats. [ 2 ]  In 2009  DARPA  offered a government contract for a  Non-GMO  plant-based systems expressing recombinant proteins, due to The 2009 H1N1  swine flu  pandemic that highlighted the national need for rapid and agile vaccine manufacturing capabilities. [ 3 ]  The  Texas A&M University  and a Texas company (GreenVax LLC, later renamed to Caliber Biotherapeutics LLC and ultimately acquired by iBio, Inc.) have been awarded a $40 million U.S.  Department of Defense  grant to develop a plant expressed vaccine made from tobacco. [ 4 ]  While egg-based vaccines typically take more than six months to develop after a virus is isolated, the new process will take only four to six weeks. [ 4 ]  The vice chancellor for research at A&M System declared that if the project works it will be one of the largest and most capable vaccine facilities in the world. [ 4 ]  However the major problem is the public acceptance of this technology, many of the companies are looking for the FDA approval  [ 5 ]


The plant-based vaccine production method works by isolating a specific antigen protein, one that triggers a human immune response from the targeted virus. A gene from the protein is transferred to bacteria, which are then used to “infect” plant cells. The plants then start producing the exact protein that will be used for vaccinations. [ 6 ]  Other uses of plant-expressed vaccines including the successful creation of edible bananas that protect against the  Norwalk virus . [ 7 ]


Passive immunization  is a medical strategy long employed to provide temporary protection against pathogens. Early implementations involved recovering ostensibly cell-free plasma from the blood of human survivors or from non-human animals deliberately exposed to a specific pathogen or toxin. These approaches resulted in crude purifications of plasma-soluble proteins including  antibodies .


Antibodies (also known as an immunoglobulins) are complex proteins produced by vertebrates [ 1 ]  that recognize  antigens  (or molecular patterns) on pathogens and some dangerous compounds in order to alert the  adaptive immune system  that there are pathogens within the body. [ 2 ]


A  plantibody  is an  antibody  that is produced by plants that have been  genetically engineered  with animal DNA encoding a specific human antibody known to  neutralize  a particular pathogen or toxin. The transgenic plants produce antibodies that are similar to their human counterparts, and following purification, plantibodies can be administered therapeutically to acutely ill patients or prophylactically to at-risk individuals (such as healthcare workers). The term plantibody and the concept are trademarked by the company  Biolex .


A plantibody is produced by insertion of genes encoding antibodies into a  transgenic  plant. The plantibodies are then modified by intrinsic plant mechanisms (N-glycosylation). [ 3 ]  Plantibodies are purified from plant tissues by mechanical disruption and denaturation/removal of intrinsic plant proteins by treatment at high temperature and low pH, as antibodies tend to be stable under these conditions. Antibodies can further be purified away from other acid- and temperature- stable proteins by capture on commercially produced  protein A  resins. Production of antibodies in whole transgenic plants, such as species in the genus Nicotiana, is cheaper and safer than in cultured animal cells. [ 4 ]


Transgenic plants offer an attractive method for large-scale production of antibodies for  immunotherapy . [ 5 ]  Antibodies produced in plants have many advantages that are beneficial to humans, plants, and the economy as well. They can be purified cheaply and in large numbers. The many seeds of plants allow for ample storage, and they have no risk of transmitting diseases to humans because the antibodies are produced without the need of the  antigen  or infectious microorganisms. Plants could be engineered to produce antibodies which fight off their own plant diseases and pests, for example, nematodes, and eliminate the need for toxic pesticides.


Antibodies generated by plants are cheaper, easier to manage, and safer to use than those obtained from animals. [ 6 ]  The applications are increasing because  recombinant DNA  (rDNA) is very useful in creating proteins that are identical when exposed into a plant's. A recombinant DNA is an artificial DNA that is created by combining two or more sequences that would not normally come together. In this way, DNA injected into a plant is turned into recombinant DNA and manipulated. The favorable properties of plants are likely to make the plant systems a useful alternative for small, medium and large scale production throughout the development of new antibody-based pharmaceuticals. [ 7 ]


The main reason plants are being used to produce antibodies is for treatment of illnesses such as immune disorders, cancer, and inflammatory diseases, given the fact that the plantibodies also have no risk of spreading diseases to humans. [ 5 ]
In the past 2 decades, research has shown that plant-derived antibodies have become easier to produce. [ 8 ]


Plantibodies are close to passing clinical trials and becoming approved commercially because of key points. [ citation needed ]  Plants are more economical than most forms of creating antibodies and the technology for harvesting and maintaining them is already present. Plants also reduce the chance of coming in contact with  pathogens , making their antibodies safer to use. Plantibodies can be made at an affordable cost and easier manufacturing due to the availability and relatively easy manipulation of genetic information in crops such as potatoes, soybean, alfalfa, rice, wheat and tobacco.


Commercial use is not yet legalized [ where? ] , but clinical trials are underway to implement the use of plantibodies for humans as injections. So far, companies have started conducting human tests of pharmaceutical products, creating plantibodies that include:


By being able to genetically alter plants to create specific antibodies, it is easier to produce antibodies that will fight diseases not only for plants but for human as well. For that reason, plantibody applications will move more towards the medicinal field.


Prime editing  is a 'search-and-replace'  genome editing  technology in  molecular biology  by which the genome of living organisms may be modified. The technology directly writes new genetic information into a targeted DNA site. It uses a  fusion protein , consisting of a catalytically impaired  Cas9   endonuclease  fused to an engineered  reverse transcriptase  enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. It mediates targeted  insertions ,  deletions , and base-to-base conversions without the need for  double strand breaks  (DSBs) or donor DNA templates. [ 1 ]


The technology has received mainstream press attention due to its potential uses in medical genetics. It utilizes methodologies similar to precursor genome editing technologies, including  CRISPR/Cas9  and  base editors . Prime editing has been used on some animal models of genetic disease [ 2 ] [ 3 ] [ 4 ]  and plants. [ 5 ]


Prime editing involves three major components: [ 1 ]


Genomic editing takes place by transfecting cells with the pegRNA and the fusion protein.  Transfection  is often accomplished by introducing vectors into a cell. Once internalized, the fusion protein nicks the target DNA sequence, exposing a 3’-hydroxyl group that can be used to initiate (prime) the  reverse transcription  of the RT template portion of the pegRNA. This results in a branched intermediate that contains two DNA flaps: a 3’ flap that contains the newly synthesized (edited) sequence, and a 5’ flap that contains the dispensable, unedited DNA sequence. The 5’ flap is then cleaved by structure-specific  endonucleases  or 5’  exonucleases . This process allows 3’ flap ligation, and creates a heteroduplex DNA composed of one edited strand and one unedited strand. The reannealed double stranded DNA contains nucleotide mismatches at the location where editing took place. In order to correct the mismatches, the cells exploit the intrinsic  mismatch repair   (MMR)  mechanism, with two possible outcomes: (i) the information in the edited strand is copied into the complementary strand, permanently installing the edit; (ii) the original nucleotides are re-incorporated into the edited strand, excluding the edit. [ 1 ]


During the development of this technology, several modifications were done to the components, in order to increase its effectiveness. [ 1 ]


In the first system, a wild-type Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase was fused to the Cas9 H840A nickase C-terminus. Detectable editing efficiencies were observed. [ 1 ]


In order to enhance DNA-RNA affinity, enzyme processivity, and thermostability, five amino acid substitutions were incorporated into the M-MLV reverse transcriptase. The mutant M-MLV RT was then incorporated into PE1 to give rise to (Cas9 (H840A)-M-MLV RT(D200N/L603W/T330P/T306K/W313F)). Efficiency improvement was observed over PE1. [ 1 ]


Despite its increased efficacy, the edit inserted by PE2 might still be removed due to DNA mismatch repair of the edited strand. To avoid this problem during DNA heteroduplex resolution, an additional single guide RNA (sgRNA) is introduced. This sgRNA is designed to match the edited sequence introduced by the pegRNA, but not the original allele. It directs the Cas9 nickase portion of the fusion protein to nick the unedited strand at a nearby site, opposite to the original nick. Nicking the non-edited strand causes the cell's natural repair system to copy the information in the edited strand to the complementary strand, permanently installing the edit. [ 1 ]  However, there are drawbacks to this system as nicking the unaltered strand can lead to additional undesired  indels .  [ 9 ]


Prime editor 4 utilizes the same machinery as PE2, but also includes a plasmid that encodes for dominant negative MMR protein  MLH1 . Dominant negative MLH1 is able to essentially knock out endogenous MLH1 by inhibition, thereby reducing cellular MMR response and increasing prime editing efficiency. [ 9 ]


Prime editor 5 utilizes the same machinery as PE3, but also includes a plasmid that encodes for dominant negative MLH1. Like PE4, this allows for a knockdown of endogenous MMR response, increasing the efficiency of prime editing. [ 9 ]


Nuclease Prime Editor uses Cas9 nuclease instead of Cas9(H840A) nickase. Unlike prime editor 3 (PE3) that requires dual-nick at both DNA strands to induce efficient prime editing, Nuclease Prime Editor requires only a single pegRNA since the single-gRNA already creates double-strand break instead of single-strand nick. [ 10 ]


The "twin prime editing" (twinPE) mechanism reported in 2021 allows editing large sequences of DNA – sequences as large as genes – which addresses the method's key drawback. It uses a prime editor protein and two prime editing guide RNAs. [ 11 ] [ 12 ] [ more detail needed ]


Prime editing was developed in the lab of  David R. Liu  at the  Broad Institute  and disclosed in Anzalone et al. (2019). [ 13 ]  Since then prime editing and the research that produced it have received widespread scientific acclaim, [ 14 ] [ 6 ] [ 15 ]  being called "revolutionary" [ 7 ]  and an important part of the future of editing. [ 13 ]


Prime editing efficiency can be increased with the use of engineered pegRNAs (epegRNAs). One common issue with traditional pegRNAs is degradation of the 3' end, leading to decreased PE efficiency. epegRNAs have a structured RNA motif added to their 3' end to prevent degradation. [ 16 ]


Although additional research is required to improve the efficiency of prime editing, the technology offers promising scientific improvements over other gene editing tools. The prime editing technology has the potential to correct the vast majority of pathogenic alleles that cause genetic diseases, as it can repair insertions, deletions, and nucleotide substitutions. [ 1 ]


The prime editing tool offers advantages over traditional gene editing technologies.  CRISPR/Cas9  edits rely on  non-homologous end joining (NHEJ)  or  homology-directed repair (HDR)  to fix DNA breaks, while the prime editing system employs  DNA mismatch repair . This is an important feature of this technology given that DNA repair mechanisms such as NHEJ and HDR, generate unwanted, random  insertions  or  deletions  (INDELs). These are byproducts that complicate the retrieval of cells carrying the correct edit. [ 1 ] [ 17 ]


The prime system introduces single-stranded DNA breaks instead of the double-stranded DNA breaks observed in other editing tools, such as base editors. Collectively, base editing and prime editing offer complementary strengths and weaknesses for making targeted transition mutations. Base editors offer higher editing efficiency and fewer INDEL byproducts if the desired edit is a transition point mutation and a  PAM sequence  exists roughly 15 bases from the target site. However, because the prime editing technology does not require a precisely positioned PAM sequence to target a nucleotide sequence, it offers more flexibility and editing precision. Remarkably, prime editors allow all types of substitutions,  transitions  and  transversions  to be inserted into the target sequence. [ 1 ] [ 17 ]   Cytosine base editing  and  adenine BE  can already perform precise  base transitions  but for  base transversions  there have been no good options. Prime editing performs transversions with good usability. PE can insert up to 44bp, delete up to 80, or combinations thereof. [ 7 ]


Because the prime system involves three separate DNA binding events (between (i) the guide sequence and the target DNA, (ii) the primer binding site and the target DNA, and (iii) the 3’ end of the nicked DNA strand and the pegRNA), it has been suggested to have fewer undesirable off-target effects than  CRISPR/Cas9 . [ 1 ] [ 17 ]


There is considerable interest in applying gene-editing methods to the treatment of diseases with a genetic component. However, there are multiple challenges associated with this approach. An effective treatment would require editing of a large number of target cells, which in turn would require an effective method of delivery and a great level of tissue specificity. [ 1 ] [ 18 ]


As of 2019, prime editing looks promising for relatively small genetic alterations, but more research needs to be conducted to evaluate whether the technology is efficient in making larger alterations, such as targeted insertions and deletions. Larger genetic alterations would require a longer RT template, which could hinder the efficient delivery of pegRNA to target cells. Furthermore, a pegRNA containing a long RT template could become vulnerable to damage caused by cellular enzymes. [ 1 ] [ 18 ]  Prime editing in plants suffers from low efficiency ranging from zero to a few percent and needs significant improvement. [ 19 ]


Some of these limitations have been mitigated by recent improvements to the prime editors, [ 2 ] [ 20 ]  including motifs that protect pegRNAs from degradation. [ 21 ]  Further research is needed before prime editing could be used to correct pathogenic alleles in humans. [ 1 ] [ 18 ]  Research has also shown that inhibition of certain  MMR  proteins, including MLH1 can improve prime editing efficiency.  [ 9 ]


Base editors used for prime editing require delivery of both a protein and RNA molecule into living cells. Introducing exogenous gene editing technologies into living organisms is a significant challenge. One potential way to introduce a base editor into animals and plants is to package the base editor into a viral capsid. The target organism can then be transduced by the virus to synthesize the base editor  in vivo . Common laboratory vectors of transduction such as  lentivirus  cause immune responses in humans, so proposed human therapies often centered around  adeno-associated virus  (AAV) because AAV infections are largely asymptomatic. Unfortunately, the effective packaging capacity of AAV vectors is small, approximately 4.4kb not including inverted terminal repeats. [ 22 ]  As a comparison, an SpCas9-reverse transcriptase fusion protein is 6.3kb, [ 1 ] [ 23 ]  which does not even account for the lengthened guide RNA necessary for targeting and priming the site of interest. However, successful delivery in mice has been achieved by splitting the editor into two AAV vectors [ 2 ] [ 3 ] [ 4 ] [ 24 ]  or by using an adenovirus, [ 3 ]  which has a larger packaging capacity.


Prime editors may be used in  gene drives . A prime editor may be incorporated into the  Cleaver  half of a  Cleave and Rescue / ClvR  system. In this case it is not meant to perform a precise alteration but instead to merely disrupt. [ 25 ]


PE is among recently introduced technologies which allow the transfer of  single-nucleotide polymorphisms  (SNPs) from one individual  crop  plant to another. PE is precise enough to be used to recreate an arbitrary SNP in an arbitrary target, [ 14 ]  including deletions, insertions, and all 12 point mutations without also needing to perform a  double-stranded break  or carry a donating template. [ 6 ]


Provirus  silencing, or proviral silencing, is the repression of expression of proviral genes in cells.
A provirus is a viral DNA that has been incorporated into the chromosome of a host cell, often by retroviruses such as HIV.  [ 1 ]
Endogenous  retroviruses  are always in the provirus state in the host cell and replicate through  reverse transcription . By integrating their genome into the host cell genome, they make use of the host cell's transcription and translation mechanisms to achieve their own propagation. This often leads to harmful impact on the host. However, in recent gene therapy techniques, retroviruses are often used to deliver desired genes instead of their own viral genome into the host genome. As such, researchers are interested in the host cell's mechanisms to silence such gene expressions to find out firstly, how the host cell manages provirus transcription to eliminate the deleterious effects of retroviruses; and secondly, how can researchers ensure stable and long-term expression of  retrovirus-mediated gene transfer .


It has been found that the level transcription of integrated retroviruses depends on both genetic and  chromatin remodeling  at the site of integration. Mechanisms such as  DNA methylation  and  histone modification  seem to play important roles in the suppression provirus transcription, such that proviral activity can be silenced.  [ 2 ]   The location of integration also plays a crucial role with the level of silencing that is observed. For example, integration into the  H3K4me3 regions , areas of the genome that are twisted around histone H3 proteins that are tri-methylated at the 4 th  lysine residue. It has been reported that the manipulation or insertion of  CpG dinucleotide  islands can lead to the disruption of proviral silencing. [ 3 ] [ 4 ] [ 5 ]  Silencing frequently begins with the binding of a zinc finger DNA-binding protein to the primer sequence, targeting more the expression of the provirus itself rather than attempting to curtail the sequence. [ 6 ]  The protein then proceeds to recruit other enzymes that complete the silencing through DNA or histone methylation.
However, studies within the field do note that the patterns are species-specific with regards to the virus in question, thus caution should be taken when attempting to generalize to all cases. Additionally, many studies focus on proviral silencing within murine embryonic cells as opposed to human cells. Some researchers also posit that proviral silencing may be more complex than a simple question of whether the virus is repressed or not. [ 6 ]  They suggest that proviruses played more of a role with transcriptional regulation as they integrated and evolved with the host sequence over time, occasionally serving as promoters or enhancers.


It has been shown that the orientation of proviruses can have dramatic effects on the expression of proviruses. [ 7 ]  With regard to HIV-1, the viral genome is frequently inserted into the introns of active genes. Perhaps unsurprisingly, when the viral genome is oriented in the same direction as the host gene, expression is increased. The converse is also true, with genes that are oriented in the opposite direction of the gene showing reduced expression. This produces challenges for effective therapeutics to aid in treatment for the disease because it can lead to large variations in detectability. This can lead to struggles for physicians who are attempting to maintain  HIV latency . HIV reservoirs, or cells that are infected with HIV but not actively producing viral particles, additionally contribute to this problem. CD4+ T cells are considered to be the main reservoir and are reported to have a half-life of over three years. [ 8 ]  While the cells are effectively temporarily silencing the expression of HIV, this results in the condition being essentially impossible to eradicate.


Additionally, DNA methylation has been linked to aging and geriatric disease. Increases in DNA methylation have been linked to diseases including various types of cancer, Alzheimer’s disease, Type 2 Diabetes, and cardiovascular disease. [ 9 ]  From a proviral silencing standpoint, this does make logical sense as individuals would naturally accumulate more proviruses over their lifetimes. This does pose a slight concern because as groups have researched the utility of DNA methylation clocks to predict age, there is the risk that treatments which treat DNA methylation with the goal of reducing biological age inadvertently result in the increase of proviral expression within their patients. Additionally, it must be emphasized that most of the work in this field is correlational rather than causational.


The expression of  transgenes  is often hindered by mechanisms associated with proviral silencing. This naturally proves to be an issue when attempting to create longer-lasting  gene therapies  or transgenic cell lines. Most methods center around choosing a specific locus of integration.


Recently, researchers have demonstrated that targeted integration of a lentiviral payload using homology-directed repair can result in stable integration and expression. In this approach,  CRISPR-associated ribonucleoprotein complexes  (CRISPR RNP complexes) are used to create double-stranded breaks upstream of an endogenously promoted essential gene. [ 10 ]  The payload is designed to where it contains the transgene flanked by two regions of DNA that are homologous/identical to the regions upstream of the gene, enabling it to integrate in the same reading frame as the gene. This approach is similar to other strategies that seek to integrate in areas that are less susceptible to silencing through more mechanistic methods.


Public Law 114-216  is a federal law of the  United States  that regulates  GMO food  labeling. It was enacted on July 29, 2016 when  President Obama  signed then  Senate Bill  764 (S.764) and is codified at  7 U.S.C.   ch. 38, subch. V  and  VI . While the law is officially termed  A bill to reauthorize and amend the  National Sea Grant College Program  Act, and for other purposes , it evolved over time into "the legislative vehicle for a measure concerning bioengineered food disclosure", [ 1 ]  which opponents have called the "DARK Act", an acronym for "Deny Americans the Right to Know Act". [ 2 ] [ 3 ] [ 4 ]


The bill was crafted by Senator  Pat Roberts  (R-KS) and  Debbie Stabenow  (D-MI). [ 5 ]  The  "GMO labeling bill" [ 6 ]  was introduced on 17 March 2015 by its sponsor, Sen.  Roger F. Wicker  (R-MS), [ 1 ]  cosponsored by Sen.  Dan Sullivan  (R-AK), and passed Senate and House in June 2016. The law overturned relevant state laws such as Vermont's GMO labeling law that had called for strict and transparent GMO food labeling in Vermont after July 1, 2016. [ 7 ]


Labeling of GMO food is mandated in at least 64 countries, including most European countries, China, Russia, Japan, Brazil, South Africa, and Australia. [ 2 ]


Senate resolution S.744 as originally introduced on 17 March 2015 contained no language to regulate bioengineered foods. [ 8 ]  A bill entitled " Safe and Accurate Food Labeling Act of 2015 " was received in the Senate on 24 July 2015 and referred to the  Committee on Agriculture, Nutrition, and Forestry  (ANF), which resulted in the amendments to Public Law 114-216 seen now; this was done on 7 July 2016 [ 9 ]  to "establish a national bioengineered food disclosure standard", whereby bioengineered food (commonly referred to as genetically modified organism or GMO food) is defined as "food that has been genetically engineered in a way that could not be obtained through conventional breeding or found in nature". [ 1 ]


Mitch McConnell  introduced on 29 June 2016 the "Roberts GMO bill" named after  Pat Roberts  who was then Chair of the Committee on ANF, [ 10 ]  and Public Law 114-216 thereafter suffered through no less than 40 amendments over the course of one week. [ 11 ]


Public Law 114-216 charges the  U. S. Department of Agriculture  (USDA) to establish a national mandatory bioengineered food disclosure standard within two years with certain provisions: [ 1 ]


While the  FDA  is responsible for protecting and promoting  public health  through the control and supervision of  food safety , the agency holds the position that "the use of  genetic engineering  in the production of food does not present any safety concerns for such foods as a class", and , as there is "an absence of reliable data indicating safety concerns" with GMO foods as a class, voiced no opposition for USDA having the responsibility of regulating GMO food labeling. [ 12 ]   The agency commented that the bill has language that may allow GMO material to escape labeling: the bill requires labeling if the food contains "genetic material", but that may exempt secondary products like oil, starches, sweeteners, or proteins derived from GMO substrates. [ 12 ]  The agency  questioned the specificity of  the definition of bioengineered food when it would not apply to GMOs that could also be achieved by  "conventional breeding". [ 12 ]  The FDA has also voiced some concerns about food information being presented in electronic codes. [ 12 ]


As the details of the law need to be worked out, USDA established a working group by September 2016 "to develop a timeline for rulemaking and to ensure an open and transparent process for effectively establishing this new program, which will increase consumer confidence and understanding of the foods they buy, and avoid uncertainty for food companies and farmers". [ 13 ]


Public Law 114-216 was passed after previous attempts to introduce a national GMO labeling bill had failed. It was fast-tracked without debate or committee review. [ 5 ] [ 14 ]  The original bill S. 764  - "A bill to reauthorize and amend the National Sea Grant College Program Act, and for other purposes" -  had nothing to do with food and stalled after having passed the Senate. Hollowed out of its content it was replaced with a bill to defund  Planned Parenthood . [ 15 ]  This bill was then replaced with a bill creating to outlaw state-level GMO labeling and setting a voluntary GMO labeling bill. [ 15 ]  When this bill failed, the S. 764 husk was used to rush through the present bill, just in time before the Vermont GMO food labeling requirement would have been activated on July 1. [ 14 ]


Previous attempts to enact a national GMO labeling law included H. R. 1599 in 2017 –  the  Safe and Accurate Food Labeling Act of 2017 . It was a proposed legislative amendment to the United States  Federal Food, Drug and Cosmetic Act . [ 16 ]  The act passed the House of  Representatives on July 23, 2017 but failed in the Senate. An earlier version of the bill had been originally introduced as H. R. 4432 in 2004. [ 17 ]  and attempted to regulate food labeling specifically in view of the introduction of  GMO food  in the United States.


Katie Hill, White House spokesperson, lauded the bill, "(t)his measure will provide new opportunities for consumers to have access to information about their food". [ 6 ]  Proponents have argued for a comprehensive labeling law that applies nationwide instead of a "patchwork" approach state-by-state. [ 18 ]  They also feel that a  proposed bill will enhance  agricultural biotechnology. [ 18 ]


Proponents argue that approved GMO food has undergone extensive testing, is "safe" and that basically labeling is unnecessary. Labeling may discourage consumers to use GMO products when such a choice may be irrational. A lot of consumers express fears that have not been substantiated by science. [ 19 ]


The bill is backed by  Grocery Manufacturers Association ,  Monsanto , and other large food and beverage corporations. [ 3 ]


While GMOs are present in 75-80% of food Americans consume and have been termed "substantially equivalent" to the corresponding non-GMO foods by the FDA, consumers believe that they have a right to know what is in their food. [ 6 ]   Thus, a 2013 poll by the  New York Times  indicated that ninety-three percent of American consumers would like to know if their food has been genetically modified. [ 19 ]


The primary objection to the bill is that manufacturers have the option to use electronic codes in lieu of clear readable labels placed directly on the food package, which they argue hides the information. [ 13 ]  The bill allows the use codes such as the  QR code  as a form of labeling, and opponents see this as impractical as well as discriminatory. They argue that, for instance, low-income families may not be able to access the information. [ 6 ]  Critics also oppose the fact that no fines or penalties are included when companies do not follow the law. [ 5 ]  Additionally, they are concerned that the bill's stipulation that labeling will be required when foods contain genetic material from genetic modification may exempt many of the highly processed foods and ingredients that are usually derived from genetically-modified crops (such as many seed oils,  high-fructose corn syrup , and some refined starches and sweeteners), because such foods are often sufficiently refined that no genetic material remains in them. [ 20 ]  Senator Stabenow  dismissed this interpretation when it was advanced by the FDA. [ 21 ]


Because clear and accessible labeling is not mandated, some opponents have called this bill and its predecessors the "DARK act" as in "Deny Americans the Right to Know" or "Keep Americans in the DARK". [ 2 ] [ 3 ] [ 4 ]


The  Pusztai affair  is a controversy that began in 1998. The  protein  scientist  Árpád Pusztai  went public with the initial results of unpublished research he was conducting at the  Rowett Institute  in Aberdeen, Scotland, investigating the possible effects of  genetically modified potatoes  upon rats. Pusztai claimed that the genetically modified potatoes had stunted growth and repressed the rats' immune systems while thickening their gut mucosa. Initially supported by the Rowett Institute, his  comments on a British television programme caused a storm of controversy, and the Rowett Institute withdrew its support. Pusztai was suspended and misconduct procedures were used to seize his data and ban him from speaking publicly. The institute did not renew his annual contract and Pusztai was criticized by the  Royal Society  and some other scientists for making an announcement before his experiment was complete or peer-reviewed and for the experiment's design, methodology and analysis. Some of the data from the study was eventually published in  The Lancet  in 1999 after five out of six peer reviewers approved of the study [ 1 ]  – triggering further controversy.


Before 1995, no peer-reviewed studies had been published investigating the safety of  genetically modified food  using human or animal feeding trials. [ 2 ]  In 1995 the Scottish  Agriculture Environment and Fisheries Department  commissioned a £1.6 million three-year research study to assess the safety of  genetically engineered   Desiree Red potatoes . [ 3 ]  The potatoes had been developed by biochemist [ 4 ]  John Gatehouse at Cambridge Agricultural Genetics (later renamed Axis Genetics) and had recently completed two years of field trials at  Rothamsted Experimental Station . [ 3 ]  The GNA gene from the  Galanthus  (snowdrop) plant was inserted into the potato, allowing the GNA  lectin protein  to be  synthesised . [ 5 ]  This lectin has been shown to be toxic to some insects. [ 6 ]  (Further research along the same lines -  inserting  further antifeedant-producing genes - was performed over the next few years. This was the work of a Durham/Axis team: At  University of Durham , Gatehouse, his wife  Angharad  and others; and Axis; and a few others.) [ 7 ]


Twenty-eight studies were proposed, of which eight were  selected for peer review by the  Biotechnology and Biological Sciences Research Council . From these eight the  Rowett Research Institute 's proposal was chosen and a combined team of academics from the  Scottish Crop Research Institute , the  Durham University Department of Biology  and the Rowett Institute was assembled, and coordinated by Pusztai. [ 3 ]


Although the tested potatoes were not a commercial variety and not intended for human consumption [ 8 ]  a contract was signed with Cambridge Agricultural Genetics, which included a profit-sharing agreement, if potatoes developed using this technology were approved and released commercially. [ 3 ]  In earlier ten-day feeding trials on GNA-fed rats, Pusztai concluded that they did not significantly affect growth, despite some  hypertrophy  of the small intestine and a slight decrease of gut  enzyme  activity. [ 9 ]


The experimental potatoes had been  transformed  with the  Galanthus nivalis   agglutinin  (GNA)  gene  from the  Galanthus  (snowdrop) plant, allowing the GNA  lectin protein  to be  synthesised . This lectin is toxic to some insects. Rats were fed raw and cooked genetically modified potatoes, using unmodified  Desiree Red potatoes  as  controls . One control group ate an unmodified Desiree Red potato spiked with the GNA snowdrop lectin. [ 5 ]  Twelve feeding experiments were conducted, ten short-term (10 days) and two long-term (110 days). [ 10 ]  Before the experiment Pusztai and his team said they expected no differences between the rats fed modified potatoes and rats fed the non-modified ones. [ 11 ] [ 12 ]


The potatoes were chosen because they were deemed  substantially equivalent  to non-genetically modified Desiree Red potatoes. [ 13 ]  The study used two  transgenic  lines of potato, both with the GNA gene inserted. They were grown in the same conditions as the non-genetically modified parent plant. [ 10 ]  According to Pusztai, the potatoes were not substantially equivalent, as one of the transgenic lines contained 20 percent less  protein  than the other, and the  starch  and sugar contents varied by up to 20 percent among the three lines. [ 14 ]  Pusztai claimed that these differences were reason enough to discontinue further experimentation. [ 14 ]


Their experiment showed a  statistically significant  difference in the thickness of the stomach  mucosa . The mucosa of rats fed raw or cooked potato modified with the GNA gene was thicker than that of rats fed the unmodified potato. [ 5 ]  The  crypt  length in the  jejunum  was greater on rats fed the raw modified potato, although there was no statistical difference observed in the rats fed the cooked potato. [ 5 ]  As these effects were not observed in rats fed the control potatoes spiked with GNA, Pusztai concluded that the differences were a result of the transformation procedure, rather than the presence of GNA. [ 5 ] [ 12 ]  Pusztai's collaborator Stanley Ewen said that the  cauliflower mosaic virus  used as a  promoter  could likely be the cause of the observed changes. [ 15 ]


On June 22, 1998 Pusztai revealed his research findings during an interview on  Granada Television 's current affairs programme  World in Action [ 16 ]  titled "Eat up your genes". [ 17 ]  He was given permission to do the interview by Rowett Institute Director Philip James. Rowett's press officer was present at the start of filming. During the interview Pusztai said he had "concerns that some of the testing techniques are not up to what we thought it was necessary to do, and therefore we should have more testing." [ 18 ]  When asked why he felt concerned, he said "it was because we had done some experiments which made us feel concerned" and discussed his results in general terms. [ 18 ]


Pusztai later said that at the time of the interview he was not sure if he should reveal results from experiments that had not been completed and did not think the programme would be hostile toward genetically modified food. He estimated that the experiments were 99 percent complete when the interview was conducted. [ 18 ]  He said that the rats in his experiments suffered stunted growth and had suppressed immune systems and that more safety research was required. [ 19 ]  He also said, "If you gave me the choice now, I wouldn't eat it" [ 19 ]  and it was "very, very unfair to use our fellow citizens as guinea pigs". [ 20 ]


World in Action  issued a press release the day before the broadcast, [ 21 ]  stimulating numerous phone calls to Pusztai and the Institute from government, industrial, non-governmental and media organisations. James says he was dismayed that unpublished data had been released and withdrew Pusztai from any further media commitments that morning. [ 18 ]  He eventually suspended Pusztai, used misconduct procedures to seize his data, banned him from speaking publicly and did not renew his annual contract. [ 8 ]


Confusion reigned over just what experiments had been conducted. Pusztai had mentioned two lines of genetically modified potatoes, meaning the two GNA lines, and this was reported by the media. The Rowett institute mistakenly assumed the media was talking about a second line transformed with  concanavalin A  (ConA), a  Jack Bean  lectin that is toxic to mammals. Transgenic ConA Potatoes had been developed, but had never been tested. [ 18 ]  Two press releases issued by the Rowett Institute on the 10th and 11th praised Pusztai's research [ 22 ]  and supported increased safety tests on  genetically modified food . [ 18 ]  The press releases also said that the potatoes were modified with ConA, adding to the confusion. Pusztai claimed that he had not seen the press releases before they went out and had no opportunity to correct the mistake. James says that he drafted it and Pusztai rewrote one section, but did not see the final copy. [ 18 ]  The mistaken belief that the ConA gene was inserted into the potato led scientist  Sir Robert May  and Agricultural Minister  Jack Cunningham  to release statements to the media saying that the findings were not surprising, as a known poison had been added to the potato. [ 21 ]  Some scientists still dismiss Pusztai's work over this error. [ 8 ]


The Rowett Institute audited Pusztai's work on  22 October 1998. It concluded that his data did not support his conclusions. [ 23 ] [ 24 ]  In February 1999, 22 scientists from 13 countries, organised by  Friends of the Earth , [ 25 ]  published a memo responding to the audit. [ 26 ]  It stated that their independent examination supported Pusztai's conclusions and that he should have been concerned by his findings.


On 19 February the  Royal Society  publicly announced that a committee would review his work.  World in Action  reporters Laurie Flynn  and  Michael Sean Gillard claimed that this was an unusual step, as the Royal Society did not normally conduct peer reviews. [ 27 ]  The data were sent to six anonymous reviewers [ 8 ]  and the resulting review was published in June 1999. [ 28 ]  It stated that Pusztai's experiments were poorly designed, contained uncertainties in the composition of diets, tested too few rats, used incorrect statistical methods and lacked consistency within experiments. Pusztai responded by saying the reviewers had reviewed only internal Rowett reports, which did not include the design or methodology of the experiments. [ 3 ]


The editors of  The Lancet  published an editorial in May 1999 in which they denounced all parties involved, criticizing Pusztai for "unwisely" announcing his results on television and stating that scientists should publish "results in the scientific press, not through the popular media"; the editorial also denounced the Royal Society's review as "breathtaking impertinence". [ 29 ]


The data were published as a letter in  The Lancet  in October 1999, co-authored by Ewen. [ 5 ]  It reported significant differences in the thickness of the gut  epithelium  of rats fed genetically modified potatoes (compared to those fed the control diet), but no differences in growth or immune system function were suggested.


The letter was reviewed by six reviewers – three times the Lancet's usual number. Four reviewers found it acceptable after revisions. A fifth thought it was flawed, but wanted it published "to avoid suspicions of a conspiracy against Pusztai and to give colleagues a chance to see the data for themselves". The sixth, John Pickett of the  Institute of Arable Crops Research , also said it was flawed. [ 30 ]  After consulting with the Royal Society, Pickett publicly criticised  The Lancet  for agreeing to publish the study. The study, which used data held by Ewen, [ 31 ]  who was not subject to the veto of Pusztai's work, [ 8 ]  reported  significant  differences in thickness of the gut  epithelium  between control and test subjects, but did not mention growth or immunity problems. [ 5 ]


The published work was criticised on the grounds that the unmodified potatoes were not a fair control diet and that any rats fed only potatoes would suffer from protein deficiency. [ 32 ]  Pusztai responded to these criticisms by saying that all the experimental diets had the same protein and energy content, and that the food intake of all rats was the same. [ 33 ]  In an interview, Pickett later said that  Lancet  editor  Richard Horton  must have had a political motive for publishing the paper because the referees had rejected it. According to Pusztai this claim was repeated by academic critics who assumed that Pickett's use of the plural suggested that the study had failed peer review. [ 3 ]


Horton claimed that he had received a "very aggressive" phone call calling him "immoral" and threatening that if he published the paper it would "have implications for his personal position" as editor. [ 27 ]   Peter Lachmann , the former vice-president and biological secretary of the Royal Society and president of the Academy of Medical Sciences, acknowledged making the call but denies that he threatened Horton and says the call was to "discuss his error of judgment" in publishing the letter and to discuss the "moral difficulties about publishing bad science". [ 27 ]


Ewen retired following publication, claiming that his career options had been "blocked at a very high level". [ 21 ]


A survey by the  European Food Safety Authority  GMO Panel Working Group on Animal Feeding Trials concluded: "Results obtained from testing GM food and feed in rodents indicate that large (at least 100-fold) 'safety' margins exist between animal exposure levels without observed adverse effects and estimated human daily intake. The studies did not show any biologically relevant differences in the parameters tested between control and test animals." [ 34 ]


In 2005 Pusztai was given a  whistleblower  award from the  Federation of German Scientists . [ 8 ]


The regulation of  genetic engineering  varies widely by country. Countries such as the United States, Canada, Lebanon and Egypt use  substantial equivalence  as the starting point when assessing safety, while many countries such as those in the European Union, Brazil and China authorize GMO cultivation on a case-by-case basis.  Many countries allow the import of GM food with authorization, but either do not allow its cultivation (Russia, Norway, Israel) or have provisions for cultivation, but no GM products are yet produced (Japan, South Korea).  Most countries that do not allow for GMO cultivation do permit research. [ 2 ]  Most (85%) of the world's GMO crops are grown in the Americas (North and South). [ 1 ]  One of the key issues concerning regulators is whether GM products should be labeled. Labeling of GMO products in the marketplace is required in 64 countries. [ 3 ]   Labeling can be mandatory up to a threshold GM content level (which varies between countries) or voluntary. A study investigating voluntary labeling in South Africa found that 31% of products labeled as GMO-free had a GM content above 1.0%. [ 4 ]  In Canada and the US labeling of GM food is voluntary, [ 5 ]  while in Europe all food (including  processed food ) or  feed  which contains greater than 0.9% of approved GMOs must be labelled. [ 6 ] [ 7 ]


There is no evidence to support the idea that the consumption of approved GM food has a detrimental effect on human health. [ 28 ] [ 29 ] [ 30 ]  Some scientists and advocacy groups, such as  Greenpeace  and  World Wildlife Fund , have however called for additional and more rigorous testing for GM food. [ 29 ]


The development of a regulatory framework concerning genetic engineering began in 1975, at  Asilomar , California. The first use of  Recombinant DNA  (rDNA) technology had just been successfully accomplished by  Stanley Cohen  and  Herbert Boyer  two years previously and the scientific community recognized that as well as benefits this technology could also pose some risks. [ 31 ]  The  Asilomar meeting  recommended a set of guidelines regarding the cautious use of recombinant technology and any products resulting from that technology. [ 32 ]  The Asilomar recommendations were voluntary, but in 1976 the  US National Institute of Health  (NIH) formed a rDNA advisory committee. [ 33 ]  This was followed by other regulatory offices (the  United States Department of Agriculture  (USDA),  Environmental Protection Agency  (EPA) and  Food and Drug Administration  (FDA)), effectively making all rDNA research tightly regulated in the US. [ 34 ]


In 1982 the  Organisation for Economic Co-operation and Development  (OECD) released a report into the potential hazards of releasing  genetically modified organisms  (GMOs) into the environment as the first  transgenic plants  were being developed. [ 35 ]  As the technology improved and genetically organisms moved from  model organisms  to potential commercial products the US established a committee at the  Office of Science and Technology  (OSTP) to develop mechanisms to regulate the developing technology. [ 34 ]  In 1986 the OSTP assigned regulatory approval of genetically modified plants in the US to the USDA, FDA and EPA. [ 36 ]


The basic concepts for the safety assessment of foods derived from GMOs have been developed in close collaboration under the auspices of the OECD, the  World Health Organization  (WHO) and  Food and Agriculture Organization  (FAO). A first joint FAO/WHO consultation in 1990 resulted in the publication of the report ‘Strategies for Assessing the Safety of Foods Produced by Biotechnology’ in 1991. [ 37 ]   Building on that, an international consensus was reached by the OECD’s Group of National Experts on Safety in Biotechnology, for assessing biotechnology in general, including field testing GM crops. [ 38 ]   That Group met again in Bergen, Norway in 1992 and reached consensus on principles for evaluating the safety of GM food; its report, ‘The safety evaluation of foods derived by modern technology – concepts and principles’ was published in 1993. [ 39 ]  That report recommends conducting the safety assessment of a GM food on a case-by-case basis through comparison to an existing food with a long history of safe use. This basic concept has been refined in subsequent workshops and consultations organized by the OECD, WHO, and FAO, and the OECD in particular has taken the lead in acquiring data and developing standards for conventional foods to be used in assessing  substantial equivalence . [ 40 ] [ 41 ]


The  Cartagena Protocol on Biosafety  was adopted on 29 January 2000 and entered into force on 11 September 2003. [ 42 ]  It is an international treaty that governs the transfer, handling, and use of genetically modified (GM) organisms. It is focused on movement of GMOs between countries and has been called a de facto trade agreement. [ 43 ]  One hundred and seventy-two countries [ 44 ]  are members of the Protocol and many use it as a reference point for their own regulations. [ 45 ]  Also in 2003 the Codex Alimentarius Commission of the FAO/WHO adopted a set of "Principles and Guidelines on foods derived from biotechnology" to help countries coordinate and standardize regulation of GM food to help ensure public safety and facilitate international trade. [ 46 ]  and updated its guidelines for import and export of food in 2004, [ 47 ]


The  European Union  first introduced laws requiring GMOs to be labelled in 1997. [ 48 ]   In 2013, Connecticut became the first state to enact a labeling law in the US, although it would not take effect until other states followed suit. [ 49 ]


Institutions that conduct certain types of scientific research must obtain permission from government authorities and ethical committees before they conduct any experiments. Universities and research institutes generally have a special committee that is responsible for approving any experiments that involve  genetic engineering . Many experiments also need permission from a national regulatory group or legislation. All staff must be trained in the use of GMOs and in some laboratories a biological control safety officer is appointed. All laboratories must gain approval from their regulatory agency to work with GMOs and all experiments must be documented. [ 50 ]   As of 2008 there have been no major accidents with GMOs in the lab. [ 51 ]


The legislation covering GMOS was initially covered by adapting existing regulations in place for chemicals or other purposes, with many countries later developing specific policies aimed at genetic engineering. [ 52 ]  These are often derived from regulations and guidelines in place for the non-GMO version of the organism, although they are more severe. In many countries now the regulations are diverging, even though many of the risks and procedures are similar. Sometimes even different agencies are responsible, notably in the Netherlands where the  Ministry of the Environment  covers GMOs and the  Ministry of Social Affairs  covers the human pathogens they are derived from. [ 51 ]


There is a near universal system for assessing the relative risks associated with GMOs and other agents to laboratory staff and the community. They are then assigned to one of four risk categories based on their virulence, the severity of disease, the mode of transmission, and the availability of preventive measures or treatments. There are some differences in how these categories are defined, such as the  World Health Organisation  (WHO) including dangers to animals and the environment in their assessments. When there are varying levels of virulence the regulators base their classification on the highest. Accordingly there are four  biosafety levels  that a laboratory can fall into, ranging from level 1 (which is suitable for working with agents not associated with disease) to level 4 (working with life threatening agents). Different countries use different nomenclature to describe the levels and can have different requirements for what can be done at each level. [ 51 ]


In Europe the use of living GMOs are regulated by the  European Directive on the contained use of genetically modified microorganisms (GMMs). [ 50 ]  The regulations require risk assessments before use of any contained GMOs is started and assurances that the correct controls are in place. It provides the minimal standards for using GMMs, with individual countries allowed to enforce stronger controls. [ 53 ]  In the UK the  Genetically Modified Organisms (Contained Use) Regulations 2014  provides the framework researchers must follow when using GMOs. Other legislation may be applicable depending on what research is carried out. For workplace safety these include the  Health and Safety at Work Act 1974 , the  Management of Health and Safety at Work Regulations 1999 , the  Carriage of Dangerous Goods legislation  and the  Control of Substances Hazardous to Health Regulations 2002 . Environmental risks are covered by Section 108(1) of the  Environmental Protection Act 1990  and The Genetically Modified Organisms (Risk assessment) (Records and Exemptions) Regulations 1996. [ 54 ]


In the US the  National Institute of Health  (NIH) classifies GMOs into four risk groups. Risk group one is not associated with any diseases, risk group 2 is associated with diseases that are not serious, risk group 3 is associated with serious diseases where treatments are available and risk group 4 is for serious diseases with no known treatments. [ 50 ]  In 1992 the  Occupational Safety and Health Administration  determined that its current legislation already adequately covers the safety of laboratory workers using GMOs. [ 52 ]


Australia has an exempt dealing for genetically modified organisms that only pose a low risk. These include systems using standard laboratory strains as the hosts,  recombinant DNA  that does not code for a  vertebrate  toxin or is not derived from a  micro-organism  that can cause disease in humans. Exempt dealings usually do not require approval from the national regulator. GMOs that pose a low risk if certain management practices are complied with are classified as notifiable low risk dealings. The final classification is for any uses of GMOs that do not meet the previous criteria. These are known as licensed dealings and include  cloning  any genes that code for vertebrate toxins or using hosts that are capable of causing disease in humans. Licensed dealings require the approval of the national regulator. [ 55 ]


Work with exempt GMOs do not need to be carried out in certified laboratories. All others must be contained in a Physical Containment level 1 (PC1) or Physical Containment level 2 (PC2) laboratories. Laboratory work with GMOs classified as low risk, which include  knockout mice , are carried out in PC1 lab. This is the case for modifications that do not confer an  advantage  to the animal or doesn't secrete any infectious agents. If a laboratory strain that is used isn't covered by exempt dealings or the inserted DNA could code for a pathogenic gene, it must be carried out in a PC2 laboratory. [ 55 ]


The approaches taken by governments to assess and manage the risks associated with the use of  genetic engineering  technology and the development and release of GMOs vary from country to country, with some of the most marked differences occurring between the United States and Europe. [ 56 ]  The United States takes on a less hands-on approach to the regulation of GMOs than in Europe, with the FDA and USDA only looking over pesticide and plant health facets of GMOs. [ 57 ]  Despite the overall global increase in the production in GMOs, the European Union has still stalled GMOs fully integrating into its food supply. [ 58 ]  This has definitely affected various countries, including the United States, when trading with the EU. [ 58 ] [ 59 ]


The  European Union  enacted regulatory laws in 2003 that provided possibly the most stringent GMO regulations in the world. [ 6 ]  All GMOs, along with  irradiated food , are considered "new food" and subject to extensive, case-by-case, science-based food evaluation by the  European Food Safety Authority  (EFSA).  The criteria for authorization fall in four broad categories: "safety", "freedom of choice", "labelling", and "traceability". [ 60 ]


The European Parliament's Committee on the Environmental, Public Health, and Consumer Protection pushed forward and adopted a "safety first" principle regarding the case of GMOs, calling for any negative health consequences from GMOs to be held liable.


The history of the development of GM crop and GM food regulations in the EU has been challenged to develop a policy environment that is (a) efficient, (b) predicable, (c) accountable, (d) durable or (e) inter- jurisdictionally aligned. [ 61 ]  However, although the European Union has had relatively strict regulations regarding genetically modified food,  Europe is now allowing newer versions of modified maize and other agricultural produce. Also, the level of GMO acceptance in the European Union varies across its countries with Spain and Portugal being more permissive of GMOs than France and the Nordic population. [ 62 ]  One notable exception however is Sweden. In this country, the government has declared that the GMO definition (according to Directive 2001/18/EC [ 63 ] ) stipulates that foreign DNA needs to be present in an organism for it to qualify as a genetically modified organisms. Organisms that have the foreign DNA removed (for example via selective breeding [ 64 ] ) therefore do not qualify as GMOs, even if  gene editing  has been used to make the organism. [ 65 ]


In June 2014 the  European Parliament  approved that individual member states are allowed to restrict or ban the growth of GM crops within their territory. Austria, France, Greece, Hungary, Germany, and Luxembourg had prohibited the growth or sale of bioengineered foods in their territory in 2015. [ 66 ]  Scotland also announced its rejection. By 2015, sixteen countries declared they want to opt out of EU-approved GM crops, including GMOs from major companies like  Monsanto ,  Dow ,  Syngenta  and Pioneer. [ 67 ]


The U.S. regulatory policy is governed by the  Coordinated Framework for Regulation of Biotechnology [ 68 ]  The policy has three tenets:  "(1) U.S. policy would focus on the product of genetic modification (GM) techniques, not the process itself, (2) only regulation grounded in verifiable scientific risks would be tolerated, and (3) GM products are on a continuum with existing products and, therefore, existing statutes are sufficient to review the products." [ 69 ]


For a genetically modified organism to be approved for release in the U.S., it must be assessed under the  Plant Protection Act  by the  Animal and Plant Health Inspection Service  (APHIS) agency within the USDA [ 70 ]  and may also be assessed by the FDA and the EPA, depending on the intended use of the organism. The USDA evaluate the plants potential to become weeds, [ 70 ]  the FDA reviews plants that could enter or alter the food supply, [ 71 ]  and the EPA regulates genetically modified plants with pesticide properties, as well as agrochemical residues. [ 72 ]


In 2017 a proposed rule was withdrawn by APHIS after public comment. Agricultural stakeholders especially felt it would have excessively restricted genetic engineering and even new methods of conventional  plant breeding . [ 73 ] [ 70 ]


The level of regulation in other countries lies in between Europe and the United States.


Common Market for Eastern and Southern Africa  (COMASA) is responsible for assessing the safety of GMOs in most of Africa, although the final decision lies with each individual country. [ 74 ]


India and China are the two largest producers of genetically modified products in Asia. [ 75 ]  The Office of Agricultural Genetic Engineering Biosafety Administration (OAGEBA) is responsible for regulation in China, [ 76 ]  while in India it is the Institutional Biosafety Committee (IBSC), Review Committee on Genetic Manipulation (RCGM) and Genetic Engineering Approval Committee (GEAC). [ 77 ]


Brazil and Argentina are the 2nd and 3rd largest producers of GM food. [ 78 ]  In Argentine assessment of GM products for release is provided by the National Agricultural Biotechnology Advisory Committee (environmental impact), the National Service of Health and Agrifood Quality (food safety) and the National  Agribusiness  Direction (effect on trade), with the final decision made by the Secretariat of Agriculture, Livestock, Fishery and Food. [ 79 ]  In Brazil the National Biosafety Technical Commission is responsible for assessing environmental and food safety and prepares guidelines for transport, importation and field experiments involving GM products, while the Council of Ministers evaluates the commercial and economical issues with release. [ 79 ]


Health Canada  and the  Canadian Food Inspection Agency [ 80 ]  are responsible for evaluating the safety and nutritional value of genetically modified foods released in Canada. [ 81 ]