Although the law's wording is unambiguous, it was used by local authorities as justification for measures enforcing  forced sterilization  and abortions upon people with certain  genetic disorders , as well as  leprosy , as well as an excuse for legalized discrimination against people with physical and mental handicaps. [ 186 ]


In Russia, one supporter of preventive eugenics is the president of the  Independent Psychiatric Association of Russia   Yuri Savenko , who justifies  forced sterilization  of women, which is practiced in Moscow psychoneurological nursing homes. He states that “one needs a more strictly adjusted and open control for the practice of preventive eugenics, which, in itself, is, in its turn, justifiable.” [ 187 ]  In 1993, the  health minister  of the  Russian Federation  issued the order that determined the procedure of  forced abortion  and sterilization of disabled women and the need for court decision to perform them. [ 188 ]  The order was repealed by the head of  Ministry of Health and Social Development of the Russian Federation   Tatyana Golikova  in 2009. [ 188 ]  Therefore, now women can be subjected to compulsory sterilization without court decision, according to the  Perm Krai   ombudswoman  Tatyana Margolina. [ 188 ]  In 2008, Tatyana Margolina reported that 14 women with disabilities were subjected to compulsory medical sterilization in Ozyorskiy psychoneurological nursing home whose director was Grigori Bannikov. [ 188 ]  The sterilizations were performed not on the basis mandatory court decision appropriate for them, but only on the basis of the application by the guardian Bannikov. [ 189 ]  On 2 December 2010, the court has not found  corpus delicti  in the compulsory medical sterilizations performed by his consent. [ 188 ]


Dor Yeshorim, a program which seeks to reduce the incidence of  Tay–Sachs disease ,  cystic fibrosis ,  Canavan disease ,  Fanconi anemia ,  familial dysautonomia ,  glycogen storage disease ,  Bloom Syndrome ,  Gaucher disease ,  Niemann-Pick disease , and  mucolipidosis IV  among certain  Jewish communities , is another screening program which has drawn comparisons with  liberal eugenics . [ 190 ]  In  Israel , at the expense of the state, the general public is advised to carry out genetic tests to diagnose these diseases early in the pregnancy. If a fetus is diagnosed with one of these diseases, among which Tay–Sachs is the most commonly known, the pregnancy may be terminated, subject to consent.


Most other  Ashkenazi  Jewish communities also run screening programs because of the higher incidence of genetic diseases. In some Jewish communities, the ancient custom of matchmaking ( shidduch ) is still practiced, and some matchmakers require blood tests so that they can avoid making matches between individuals who share the same recessive disease traits. In order to attempt to prevent the tragedy of infant death which always results from being  homozygous  for Tay–Sachs, associations such as the strongly observant Dor Yeshorim (which was founded by Rabbi Joseph Ekstein, who lost four children to the disease) with the purpose of preventing others from suffering the same tragedy test young couples to check whether they carry a risk of passing on fatal conditions.


If both the young man and woman are Tay–Sachs carriers, it is common for the match to be broken off. Judaism, [ dubious  –  discuss ]  like numerous other religions, discourages abortion unless there is a risk to the woman, in which case her needs take precedence. The effort is not aimed at eradicating the hereditary traits, but rather at the occurrence of homozygosity. The actual impact of this program on  allele  frequencies is unknown, but little impact would be expected because the program does not impose genetic selection. Instead, it encourages  disassortative mating .


Which ideas should be described as "eugenic" are still controversial. Bio-ethicists Stephen Wilkinson and Eve Garrard note that due to its history, "there's no overwhelming argument for completely abandoning the term 'eugenics', but concerns remain about ambiguity, confusion and manipulation, and the consequent failure to respect people's autonomy." The pair argue that anyone using the term "must at least be clear about what they mean by it and be prepared to offer a clear definition. Otherwise unnecessary confusion and disagreement may ensue." [ 191 ]


Modern inquiries into the potential use of genetic engineering have led to an increased invocation of the history of eugenics in discussions of  bioethics , most often as a cautionary tale. Some suggest that even non-coercive eugenics programs are inherently unethical. [ 192 ]


James D. Watson , the first director of the  Human Genome Project , initiated the  Ethical, Legal and Social Implications Program  (ELSI) which has funded a number of studies into the implications of human genetic engineering (along with a prominent website on the history of eugenics), because:


In putting ethics so soon into the genome agenda, I was responding to my own personal fear that all too soon critics of the Genome Project would point out that I was a representative of the  Cold Spring Harbor Laboratory  that once housed the controversial  Eugenics Record Office . My not forming a genome ethics program quickly might be falsely used as evidence that I was a closet eugenicist, having as my real long-term purpose the unambiguous identification of genes that lead to social and occupational stratification as well as genes justifying racial discrimination. [ 193 ]


Philosopher  Philip Kitcher , writing in 1997, has described the use of  genetic screening  by parents as making possible a form of "voluntary" eugenics. [ 194 ]  In 2006,  Richard Dawkins  stated that breeding humans for traits is possible and society should not be afraid to debate the ethical differences between breeding a child for an ability versus forcing a child to gain an ability through training. [ 195 ]


Historian  Nathaniel C. Comfort  wrote in 2012, "The eugenic impulse drives us to eliminate disease, live longer and healthier, with greater intelligence, and a better adjustment to the conditions of society." [ 196 ]  Comfort claims, "the health benefits, the intellectual thrill and the profits of genetic biomedicine are too great for us to do otherwise." [ 196 ]


Bill McKibben  suggests that emerging reprogenetic technologies would be disproportionately available to those with greater financial resources, thereby exacerbating the gap between rich and poor and creating a "genetic divide". [ 197 ]   Lee M. Silver , a biologist and science writer who coined the term "reprogenetics" and supports its applications, has expressed concern that these methods could create a two-tiered society of genetically-engineered "haves" and "have nots" if  social democratic reforms  lag behind implementation of reprogenetic technologies. [ 198 ]


Lentiviral vectors in gene therapy  is a method by which genes can be inserted, modified, or deleted in organisms using  lentiviruses .


Lentiviruses are a family of  viruses  that are responsible for diseases like  AIDS , which infect by inserting  DNA  into their host cells'  genome . [ 1 ]  Many such viruses have been the basis of research using viruses in  gene  therapy, but the lentivirus is unique in its ability to infect non-dividing cells, and therefore has a wider range of potential applications. [ 2 ]   Lentiviruses can become  endogenous  (ERV), integrating their genome into the host germline genome, so that the virus is henceforth inherited by the host's descendants. Scientists use the lentivirus' mechanisms of infection to achieve a desired outcome to gene therapy. Lentiviral vectors in gene therapy have been pioneered by  Luigi Naldini . [ 3 ] [ 4 ]


The lentivirus is a  retrovirus , meaning it has a single stranded  RNA   genome  with a reverse transcriptase enzyme. Lentiviruses also have a  viral envelope  with protruding  glycoproteins  that aid in attachment to the host cell's outer membrane. The virus contains a  reverse transcriptase  molecule found to perform  transcription  of the viral genetic material upon entering the cell. Within the viral genome are RNA sequences that code for specific proteins that facilitate the incorporation of the viral sequences into the host cell genome. The  "gag" gene  codes for the structural components of the viral nucleocapsid proteins: the matrix (MA/p17), the capsid (CA/p24) and the nucleocapsid (NC/p7) proteins. The "pol" domain codes for the reverse transcriptase and integrase enzymes. Lastly, the "env" domain of the viral genome encodes for the glycoproteins and envelope on the surface of the virus. [1] [ full citation needed ]


There are multiple steps involved in the infection and replication of a lentivirus in a host cell. In the first step the virus uses its surface glycoproteins for attachment to the outer surface of a cell. More specifically, lentiviruses attach to the  CD4  glycoproteins on the surface of a host's target cell. The viral material is then injected into the host cell's cytoplasm. Within the cytoplasm, the viral reverse transcriptase enzyme performs reverse transcription of the viral RNA genome to create a viral DNA genome. The viral DNA is then sent into the nucleus of the host cell where it is incorporated into the host cell's genome with the help of the viral enzyme  integrase . From now on, the host cell starts to transcribe the entire viral RNA and express the structural viral proteins, in particular those that form the viral capsid and the envelope. The lentiviral RNA and the viral proteins then assemble and the newly formed  virions   leave the host cell  when enough are made. [ citation needed ]


Two methods of gene therapy using lentiviruses have been proposed. In the ex vivo methodology, cells are extracted from a patient and then cultured. A lentiviral vector carrying therapeutic transgenes are then introduced to the culture to infect them. The now modified cells continue to be cultured until they can be infused into the patient. In vivo gene therapy is the sample injection of viral vectors containing transgenes into the patient. [ 5 ]


Lentiviruses are modified to act as a  vector  to insert beneficial genes into cells.  Unlike other retroviruses, which cannot penetrate the  nuclear envelope  and can therefore only act on cells while they are undergoing  mitosis , lentiviruses can infect cells whether or not they are dividing (shown to be largely due to the capsid protein). [ 6 ]   Many cell types, like  neurons , do not divide in adult organisms, so lentiviral gene therapy is a good candidate for treating conditions that affect those cell types. [ 7 ]


Some experimental applications of lentiviral vectors [ 8 ]  have been done in gene therapy in order to cure diseases like  Diabetes mellitus , Murine haemophilia A,  prostate cancer ,  chronic granulomatous disease , and  vascular diseases . [ citation needed ]


HIV -derived lentiviral vectors have been widely developed for their ability to target specific genes through the  coactivator   PSIP1 . [ 9 ]  This target specificity allows for the development of lentiviral gene vectors that do not carry the risk of randomly inserting themselves into normally functioning genes. As HIV is pathogenic, it must be genetically modified to remove its disease-causing properties and its ability to replicate itself. This can be achieved by deleting viral genes that are unnecessary for transduction of therapeutic transgenes. It has been proposed that by targeting the "gag" and "env" domains, enough of the HIV-1 genome can be deleted without losing its effectiveness in gene therapy while minimizing viral genes integrated into the patient. [ 10 ]  Genes may also be replaced rather than disrupted as another method to reduce the risks associated with the use of HIV-1. [ 7 ]


Other lentiviruses such as  Feline immunodeficiency virus [ 11 ]  and  Equine infectious anemia  virus [ 12 ]  have been developed for use in gene therapy and are of interest due to the inability to cause serious disease in human hosts. Equine infectious anemia virus in particular has been shown to perform somewhat better than HIV-1 in  hematopoietic stem cells [ 13 ]


Historically, lentiviral vectors included strong viral promoters which had a side effect of  insertional mutagenesis , nuclear DNA mutations that affect the function of a gene. [ 14 ]  These strong viral promotors were shown to be the main cause of  cancer formation . [ 14 ]  As a result, viral promotors have been replaced by cellular promotors and regulatory sequences. [ 14 ]


As mentioned, lentiviruses have the unique ability to infect non-dividing cells. Beyond that, there are several other properties that distinguish lentiviral vectors from other viral vectors. Such properties are important to consider when determining whether lentiviruses are appropriate for a given treatment.


Gammaretroviruses are retroviruses like lentiviruses.  Murine leukemia viruses  (MLVs) were among the first to be investigated for their use in gene therapy. However, recent research has favored lentiviruses for their ability to integrate into non-dividing cells. More practically, gammaretroviruses have an affinity for integrating themselves near  oncogene  promoters, bringing forward an adverse risk of tumors. [ 15 ]  MLVs may be replication competent, meaning they can replicate in the host cell. These replication-competent viruses offer stable gene transfer and tumor and tissue specific targeting. [ 16 ]


In gene therapy,  adenoviruses  differ from lentiviruses in many ways, some of which provide advantages over lentiviruses. Transduction efficiency is higher in adenoviruses compared to lentiviruses. [ 5 ]  Secondly, most human cells have receptors for adenoviruses [ 17 ]  likely as a result of the wide variety of  adenovirus diseases  in humans. As adenoviruses frequently infect humans, this could build an immune response in the body. Such a response can reduce the efficiency of adenoviral vector therapies and can result in adverse reactions such as inflammation of tissues. [ 18 ]  Research has been conducted to exploit this immune response to target cancerous cells and to develop vaccines. [ 19 ]  Hybrid adenovirus-retroviruses (specifically MLVs) have also been developed to exploit the benefits of MLVs and adenoviruses. [ 20 ]


The ADA deficient variant of  severe combined immunodeficiency  (SCID) was treated highly successfully in a multi-year study reported in 2021. Over 95% of treated patients continued to be event free after 36 months, and 100% of patients survived this normally lethal disease. A self-inactivating lentiviral vector, EFS-ADA LV, was used to insert a functional ADA gene in autologous CD34+ hematopoietic stem and progenitor cells (HSPCs). [ 21 ]


In a study designed to enhance the outcomes of vascular transplant through vascular endothelial cell gene therapy, the third generation of Lentivirus showed to be effective in the delivery of genes to moderate venous grafts and transplants in procedures like coronary artery bypass. Because the virus has been adapted to lose most of its genome, the virus becomes safer and more effective in transplanting the required genes into the host cell. A drawback to this therapy is explained in the study that long-term gene expression may require the use of promoters and can aid in a greater trans-gene expression. The researchers accomplished this by the addition of self-inactivating plasmids and creating a more universal tropism by pseudotyping a vesicular stomatitis virus glycoprotein. [ 22 ]


In  chronic granulomatous disease  (CGD), immune functioning is deficient as a result of the mutations in components of the nicotinamide adenine dinucleotide phosphate oxidase ( NADPH oxidase ) enzyme in  phagocyte  cells, which catalyzes the production of  superoxide  free radicals. If this enzyme becomes deficient, the phagocytes can't kill effectively the engulfed bacteria, so  granulomas  can be formed. Study performed in mice emphasizes the use of lineage-specific lentiviral vectors to express a normal version of one of the mutant CGD proteins, allowing white blood cells to now make a functional version of the NADPH oxidase. Scientists developed this strain of lentivirus by transinfecting 293T cells with pseudotyped virus with the vesicular stomatitis G protein. The viral vector's responsibility was to increase the production of a functional NADPH oxidase gene in these phagocytic cells. They did this to create an affinity for myeloid cells. [ 23 ]


With prostate cancer, the lentivirus is transformed by being bound to  trastuzumab  to attach to androgen-sensitive LNCaP and castration-resistant C4-2 human prostate cancer cell lines. These two cells are primarily responsible for secretion of excess human epidermal growth factor receptor 2 ( HER-2 ), which is a hormone linked to prostate cancer. By attaching to these cells and changing their genomes, the lentivirus can slow down, and even kill, the cancer-causing cells. Researchers caused the specificity of the vector by manipulating the Fab region of the viral genome and pseudotyped it with the Sindbis virus. [ 24 ]


Haemophilia A has also been studied in gene therapy with a lentiviral vector in mice. The vector targets the haematopoietic cells in order to increase the amount of factor VIII, which is affected in haemophilia A. But this continues to be a subject of study as the lentivirus vector was not completely successful in achieving this goal. They did this by trans-infecting the virus in a 293T cell, creating a virus known as 2bF8 expressing generation of viral vectors. [ 25 ]


Studies have also found that injection of a lentiviral vector with  IL-10  expressing genes in utero in mice can suppress, and prevent, rheumatoid arthritis and create new cells with constant gene expression. This contributes to the data on stem cells and in utero inoculation of viral vectors for gene therapy. The target for the viral vector in this study, were the synovial cells. Normally functioning synovial cells produce  TNFα  and  IL-1 . [ 26 ]


Like many of the in utero studies, the lentiviral vector gene therapy for diabetes mellitus is more effective in utero as the stem cells that become affected by the gene therapy create new cells with the new gene created by the actual viral intervention. The vector targets the cells within the pancreas to add insulin secreting genes to help control diabetes mellitus. Vectors were cloned using a  cytomegalovirus  promoter and then co-transinfected in the 293T cell. [ 27 ]


As mature neurons do not divide, lentiviruses are ideal for division independent gene therapy. Studies of lentiviral gene therapy have been conducted on patients with advanced  Parkinson's disease [ 28 ]  and aging-related atrophy of neurons in primates. [ 29 ]


A  liposome  extruder  is a device that prepares cell membranes,  exosomes  and also generates nanoscale liposome formulations. [ 1 ] [ 2 ]  The liposome extruder employs the track-etched  membrane  to filter huge particles and achieve sterile filtration. [ 3 ] [ 4 ]


A  liposome  is made up of  phospholipid bilayers , with the liposome being a spherical vesicle. Phospholipid bilayers have both  hydrophobic  and  hydrophilic  properties, which are important characteristics of  cell membranes . [ 5 ]  The hydrophobic ends of phospholipid molecules are constrained [ clarification needed ] , often to each other, creating spherical liposomes that are smaller when the hydrophobic ends are exposed to a solution that is aqueous in nature. The preparation of liposomes results in the formation of the liposome extruder [ clarification needed ] . A liposome extruder is characterized by the uniform, narrow size distribution of its output, and has a particle-size control mechanism that is highly precise. Complex, toxic, injectable products such as the  antifungal  liposomal  Amphotericin B , or the liposomal  cytotoxic   anticancer agents   doxorubicin ,  paclitaxel ,  irinotecan ,  Adriamycin , and  cytarabine  contain liposomes which are prepared using the liposome extruder. [ 6 ]


The technology for extruding liposomes relies on the performance and structural characteristics of the  lipid bilayers  in the liposomal  phospholipids . [ how? ] [ clarification needed ] [ 7 ]  An external extrusion force pushes the vesicles of liposomes that are large through the polycarbonate membranes with pore sizes that are specific when the  transition temperature  of the phospholipids rises slightly due to the change in operating temperature [ further explanation needed ] . Re- polymerization  of the multiple compartments pr liposomes that are large in particle size occurs, [ 8 ] [ 9 ]  and smaller liposomes are created due to the rupturing of the membrane [ which? ]  pores. Extrusion of liposomes occurs at a uniform size, based on the pore size in the  polycarbonate   membrane . [ 10 ]  This happens when the big vesicles are passed through the  cell membrane  with a nanopore size specified in size several times [ 10 ]  due to the extrusion of polycarbonate membranes having uniform and vertical nanopore distribution on the surface of the membrane. [ 11 ] [ 12 ]


Liposome extruders are applied in the formulation of liposomes of homogeneous size distributions. [ 3 ] [ 13 ] [ 14 ] [ 15 ]


This type of liposome extruder is primarily used in research laboratories, as it can process mini-sample volumes between 0.25 ml and 2.5 ml. The hand-driven liposome extruders are further categorized into liposome extruders with a thermal-jacketed option and liposome extruders under ambient temperature. They are operated by manually by  pushing a plunger. Liposome extruders under ambient temperature can be fitted with a cooling jacket to regulate temperatures during liposome extrusion. [ 16 ] [ 17 ]


Jacketed liposome extruders are applied in laboratories and in pilot-scale research phases. They process volumes between 2 mL and 3 L. The jacketed extruders are fitted with barrels [ clarification needed ]  to regulate the temperatures of the samples. To drive this extruder, a compressed nitrogen cylinder is used. [ 18 ]


Online liposome extruders process volumes of between 2 ml and 20L. They are driven by a high-pressure electric pump, making them appropriate for use in pilot-scale liposome production. [ 2 ]


A multiple liposome extruder system is fitted with pressure and temperature sensors and a control panel to regulate liposome production. it processes capacities of between 1L and 200L. [ 19 ]


This article contains a list of commercially available  gene therapies .


Living DNA  is a UK-based company that specialises in  DNA testing  and analysis whose head office is in the UK with facilities in the USA and Denmark. The service is to provide deep ancestry details from all around the world, using a unique process of analysis and using linked DNA. It is one of the major DNA testing services in the world. [ 1 ] [ 2 ]   The company conducts three types of DNA analyses: autosomal, Y-chromosome and mitochrondrial. [ 3 ]  However, while the DNA test results provide information about the origins of a person, genealogy, i.e. finding relatives in historic time, is not yet part of the company's portfolio. [ citation needed ]


In 2016, Living DNA was co-founded by Tricia Nicholson and husband-and-wife team, David Nicholson and Hannah Morden-Nicholson, [ 4 ]  in  Frome ,  Somerset , England. [ 5 ] [ 6 ] [ 7 ] [ 8 ]  The company began after extensive research and work along with a team of around 100 genealogists around the world. [ 9 ]  In 1999 Nicholson founded another company, DNA Worldwide, which he has been running since. [ 5 ]


In July 2018, Living DNA announced and signed a partnership agreement with  Findmypast , also a British genealogy company. [ 10 ]  By working together, their mission was to provide an extensive and detailed family roots and history. [ 11 ]  Unfortunately, this partnership ended in 2023.


In 2019, Living DNA was reported to provide, for each DNA sample tested, recent (less than 80,00 years) ethnic breakdown for 80 regions in the world with the UK broken down in to 21 regions. [ 12 ]   They also provided insight into female and paternal (for males) heritage going back about 200,000 years showing migration patterns out of Africa. [ 12 ]


Research published in the scientific journal  eLife  by geneticist Michael Edge from the University of California uncovered security concerns with customers DNA data held online by the smaller genealogy companies, including Living DNA. It was found that hackers using creative means could easily exploit these upload-based services. Biostatistician  Sharon Browning  from the University of Washington said that if consumers "care about their DNA's privacy, then they shouldn't upload [their DNA] to these databases." [ 13 ]


Living DNA has gotten a positive review from PCWorld. [ 3 ]  Tech Radar commented that "..the vagueness of some of its results combined with its relatively high price mean it doesn’t stand out from the crowd." [ 14 ]


After getting DNA tests results from three different companies to know if his "dad's family came from Russia",  David Gewirtz  says, "the results I got back from  Ancestry  and  23andMe  were shocking and upsetting would be an understatement." However, "the results from Living DNA were substantially different and led to some fascinating insights that were actually really cool, rather than painful." [ 15 ]


Director Hannah Morden-Nicholson, stepped down from the Frome Chamber of Commerce committee in early 2019 after being associated with a locally established "cult"  Universal Medicine . This followed on from a  BBC  investigation into the "socially harmful" group. [ 16 ]  Co-director David Nicholson is also dedicated to the sect and its leader’s teachings, [ 17 ]  and ex-director and co-founder Tricia Nicholson declares a 'lifelong family friendship' with the sect's leader. [ 18 ] [ 4 ]


Lord Morton’s mare  was an  equid hybrid  and once an often-noticed example in the history of  evolutionary theory .


In 1820,  George Douglas, 16th Earl of Morton , F.R.S., reported to the President of the  Royal Society  that, being desirous of domesticating the  quagga  (a now extinct subspecies of the  plains zebra ), he had bred an Arabian chestnut mare with a quagga  stallion  and that, subsequently, the same  mare  was bred with a black stallion and Lord Morton found that the  offspring  had strange stripes in the legs like the quagga. The Royal Society published Lord Morton's letter in its  Philosophical Transactions , 1821. [ 1 ]  In the same issue "Particulars of a Fact, nearly similar to that related by Lord Morton, communicated to the President, in a letter from Daniel Giles, Esq." reported that in a litter of a black and white sow, by a "boar of the wild breed, the chestnut colour of the boar strongly prevailed" in some of the piglets, even to the two subsequent litters of that sow. [ 2 ]


These circumstantial reports seemed to confirm the ancient idea of  telegony  in heritability:  Charles Darwin  cited the example in  On the Origin of Species  (1859) and  The Variation of Animals and Plants Under Domestication  (1868). The concept of telegony, that the seed of a male could continue to affect the offspring of a female, whether animal or human, had been inherited from  Aristotle  and remained a legitimate theory until experiments in the 1890s confirmed  Mendelian inheritance . Biologists now explain the phenomenon of Lord Morton's mare as the result of dominant and recessive  alleles . The mare and black stallion each carried genes for the striped markings on the foal, but the markings were hidden in the parent animals by dominant genes for normal color. Striped " primitive markings " are in fact commonly seen in domesticated horses, particularly those with a  dun  coat color.


Multiplexed Accurate Genome Editing with Short, Trackable, Integrated Cellular barcodes  ( MAGESTIC ) is a platform that builds on the  CRISPR /Cas technique. It further improves CRISPR/Cas by making the gene-editing process more precise. It also increases cell survival during the editing process up to sevenfold. [ 1 ] [ 2 ]


This technology was invented at the Stanford Genome Technology Center in collaboration with the Joint Initiative for Metrology in Biology (JIMB) [ 3 ]  which is a coalition of  Stanford University  and the  National Institute of Standards and Technology .


Gene editing  is used for a variety of tasks including the modifying of crops, the modifying of bacteria, and the modifying of  disease-causing genetic mutations in patients . When only a single edited cell line is required, CRISPR/Cas combined with the endogenous DNA repair efficiency is sufficient to obtain an edited cell line.  However, when trying to introduce many edits in multiplex, a higher efficiency of  Homology directed repair  is required.  The MAGESTIC technology has multiple components.  One component, the LexA-Fkh1 protein is involved in the process of Donor Recruitment that increases the efficiency of homology directed repair.  The second component is a library of CRISPR  Guide RNAs  paired with donor DNA which encodes for specified edited to be integrated through homology directed repair.  This in turn is linked to a   DNA barcodes  that allows for specific variants to be tracked in pools, similar to how  Genome-wide CRISPR-Cas9 knockout screens  work, only MAGESTIC is more versatile as it allows for not only loss of function edits, but also  DNA Codon  changes,  Single-nucleotide polymorphism ,  Indels , and other types of genetic changes to be introduced and tracked. By improving DNA repair efficiency, using array-synthesized guide–donor oligos for the plasmid-based high-throughput editing, and integrating a genomic barcode to prevent plasmid barcode loss, MAGESTIC leads to more uniform pools with genome integrated stable single copy barcodes and enables robust phenotyping.


Because editing multiple sites in pools can be impacted by a number of factors including ineffective CRISPR  Guide RNA ,  DNA synthesis  errors, competition with  Non-homologous end joining  and other challenges that occur when building multiplex libraries, MAGESTIC screens required improved DNA repair. This is where the donor recruitment aspect of MAGESTIC comes in. MAGESTIC achieves greater editing efficiency by localizing donor DNA to the site of DNA breaks introduced by a CRISPR cut.


A CRISPR machinery cuts at desired locations in the genome, and then MAGESTIC direct the donor DNA to the site of this cut to direct cells to introduce designed edits at the DNA cut sites.  This technology is called donor recruitment and relies on a fusion protein that contains one domain recruited to DNA breaks and another domain that binds to the donor DNA.  This allows for the production of high quality precision edit pools in yeast, where each cells contains a single edit and a DNA barcode. The donor recruitment aspect of the technology also holds the potential to improve editing efficiency in additional cell types, such as mammalian cells.  This may one day prove beneficial to  gene therapies  or other therapeutic editing.


Male egg  can refer to either:


This article focuses on the first definition.


Male eggs are the result of a process in which the eggs of a female would be emptied of their genetic contents (a technique similar to that used in the  cloning  process), and those contents would be replaced with male DNA. Such eggs could then be fertilized by sperm.  The procedure was conceived by  Calum MacKellar , a Scottish bioethicist. With this technique, two males could be the biological parents of a child. However, such a procedure would additionally require an  artificial womb  or a female  gestational carrier . [ 4 ] [ 5 ] [ 1 ]


In 2023, male eggs from male mice cells were developed and used to create bi-paternal mice that grew into adulthood; [ 6 ] [ 7 ]  bi-paternal mice had been obtained in 2008, but they only survived for a few days. [ 8 ] [ 9 ] [ 10 ]


Minicircles  are small (~4 kb ) circular  replicons . They occur naturally in some  eukaryotic   organelle   genomes . In the  mitochondria -derived kinetoplast of  trypanosomes , minicircles encode  guide RNAs  for  RNA editing . [ 1 ]  In  Amphidinium , the  chloroplast  genome is made of minicircles that encode chloroplast proteins. [ 2 ] [ 3 ]


Minicircles are small (~4kb) circular  plasmid  derivatives that have been freed from all  prokaryotic   vector  parts. They have been applied as  transgene  carriers for the  genetic modification  of mammalian cells, with the advantage that, since they contain no bacterial  DNA  sequences, they are less likely to be perceived as foreign and destroyed. (Typical transgene delivery methods involve plasmids, which contain foreign DNA.) The smaller size of minicircles also extends their  cloning  capacity and facilitates their delivery into cells.


Their preparation usually follows a two-step procedure: [ 4 ] [ 5 ]


The purified minicircle can be transferred into the recipient cell by  transfection  or  lipofection  and into a differentiated tissue by, for instance,  jet injection .


Conventional minicircles lack an  origin of replication , so they do not replicate within the target cells and the encoded genes will disappear as the cell divides (which can be either an advantage or disadvantage depending on whether the application demands persistent or transient expression). A novel addition to the field are nonviral  self-replicating  minicircles, which owe this property to the presence of a  S/MAR-Element . Self-replicating minicircles hold great promise for the systematic modification of stem cells and will significantly extend the potential of their plasmidal precursor forms ("parental plasmids"), the more as the principal feasibility of such an approach has amply been demonstrated for their plasmidal precursor forms. [ 6 ] [ 7 ] [ 8 ] [ 9 ]


A  miniemulsion  (also known as  nanoemulsion ) is a particular type of  emulsion . A miniemulsion is obtained by ultrasonicating a mixture comprising two  immiscible  liquid phases (for example, oil and water), one or more  surfactants  and, possibly, one or more co-surfactants (typical examples are hexadecane or cetyl alcohol). They usually have nanodroplets with uniform size distribution (20–500 nm) and are also known as sub-micron, mini-, and ultra-fine grain emulsions. [ 1 ]


Mini-emulsion : emulsion in which the particles of the  dispersed phase  have diameters in the range from approximately 50 nm to 1 μm.


Note 1 : Mini-emulsions are usually stabilized against diffusion degradation (Ostwald ripening (ref. [ 6 ]  )) by a compound insoluble in the  continuous phase .


Note 2 : The dispersed phase contains mixed stabilizers, e.g., an ionic surfactant, such as sodium dodecyl sulfate ( n -dodecyl sulfate sodium) and a short aliphatic chain alcohol ("co-surfactant") for colloidal stability, or a water-insoluble compound, such as a hydrocarbon ("co-stabilizer" frequently and improperly called a "co-surfactant") limiting diffusion degradation. Mini-emulsions are usually stable for at least several days. [ 7 ]


Mini-emulsion polymerization : Polymerization of a mini-emulsion of monomer in which all of the polymerization occurs within preexisting monomer particles without the formation of new particles. [ 8 ]


There are two general types of methods for preparing miniemulsions:


Miniemulsions are kinetically stable but thermodynamically unstable. [ 13 ]  Oil and water are incompatible in nature, and the interface between them is not favored. Therefore, given a sufficient amount of time, the oil and water in miniemulsions separate again. Various mechanisms such as gravitational  separation,  flocculation ,  coalescence , and  Ostwald ripening  result in instability. [ 14 ]  In an ideal miniemulsion system,  coalescence  and  Ostwald ripening  are suppressed thanks to the presence of the surfactant and co-surfactant. [ 9 ]  With the addition of  surfactants , stable  droplets  are then obtained, which have typically a size between 50 and 500 nm. [ 15 ] [ 16 ]


A  sterile filter  is a device used to remove microorganisms and other contaminants from a liquid or gas, making it sterile. [ 17 ] [ 18 ]  Sterile filters are commonly used in the medical, pharmaceutical, and biotech industries to ensure that the products produced are free of bacteria and other harmful organisms.


There are different types of filters which include:


A nanogenizer, also known as a high-pressure  homogenizer  or a microfluidizer, is a device used to create small droplets or particles by applying high pressure to a liquid mixture. [ 24 ]  These devices can be used to produce nanoemulsions, as well as other types of  emulsions  and suspensions. [ 25 ]  They work by passing the mixture through a small orifice under high pressure, which causes the liquid to be sheared and broken into small droplets or particles. The size of the droplets or particles can be controlled by adjusting the pressure and the design of the orifice. [ 26 ]


A  nanoparticle  sizer, also known as a nanoparticle analyzer, is a device used to measure the size, size distribution, and concentration of  nanoparticles  in a sample. [ 27 ] [ 28 ]  The size of nanoparticles is typically in the range of 1 to 100 nanometers (nm), and they are much smaller than the particles that can be measured with conventional particle size  analyzers . [ 29 ]


Miniemulsions have wide application in the synthesis of nanomaterials and in the pharmaceutical and food industries. [ 30 ] [ 31 ]  For example, miniemulsion-based processes are, therefore, particularly adapted for the generation of  nanomaterials . There is a fundamental difference between traditional emulsion polymerisation and a miniemulsion polymerisation. Particle formation in the former is a mixture of micellar and  homogeneous nucleation , particles formed via miniemulsion however are mainly formed by droplet nucleation. In the pharmaceutical industry, oil droplets act as tiny containers that carry water-insoluble drugs, and the water provides a mild environment that is compatible with the human body. [ 32 ] [ 33 ]  Moreover, nanoemulsions that carry drugs allow the drugs to crystallize in a controlled size with a good dissolution rate. [ 34 ] [ 35 ]  Finally, in the food industry, miniemulsions can not only be loaded with water-insoluble nutrients, such as  beta-carotene  and  curcumin , but also improve the nutrients' digestibility. [ 12 ]  Miniemulsions are also used in the creation of cannabinoid infused beverages and foods. Emulsifying cannabiniods has shown to increase bioavailability and digestion time. [ 36 ]


New eugenics , also known as  liberal eugenics  (a term coined by bioethicist  Nicholas Agar ), [ 1 ]  advocates  enhancing human characteristics and capacities  through the use of  reproductive technology  and  human genetic engineering . Those who advocate new eugenics generally think selecting or altering embryos should be left to the preferences of  parents , rather than forbidden (or left to the preferences of the  state ). "New" eugenics purports to distinguish itself from the forms of  eugenics  practiced and advocated in the 20th century, which fell into disrepute after  World War II . [ 2 ]


Eugenics is sometimes broken into the categories of positive eugenics (encouraging reproduction among the designated "fit") and negative eugenics (discouraging or prohibiting reproduction among those designated "unfit"). Both positive and negative eugenic programs were advocated and pursued during the early 20th century. Negative programs were responsible for the  compulsory sterilization  of hundreds of thousands of persons in many countries, and were included in much of the rhetoric of  Nazi eugenic  policies of  racial hygiene  and  genocide . [ 3 ]  According to its proponents, new eugenics belongs in the category of positive eugenics. [ citation needed ]


New eugenics generally supports  genetic modification  or genetic selection of individuals for traits that are supposed to improve human welfare. The underlying idea is to improve the genetic basis of future generations and reduce incidence of genetic diseases and other undesirable traits. Some of the practices included in new eugenics are: pre-implantation diagnosis and embryo selection, [ 4 ]  selective breeding, [ 5 ]  and human embryo engineering and gene therapy. [ 6 ] [ 7 ]


New eugenics was founded under the liberal ethical values of  pluralism , which advocates for the respect of  personal autonomy , and  egalitarianism , which represents the idea of equality for all people. Arguments used in favor of new eugenics include that it is in the best interest of society that life succeeds rather than fail, and that it is acceptable to ensure that progeny has a chance of achieving this success. [ 7 ]  Ethical arguments against new eugenics include the claim that creating designer babies is not in the best interest of society as it might create a breach between genetically modified individuals and natural individuals. [ 8 ]  Additionally, some of these technologies might be economically restrictive further increasing the socio-economical gap. [ 5 ]


Dov Fox, a law professor at the  University of San Diego , argues that liberal eugenics cannot be justified on the basis of the underlying  liberal theory  which inspires its name. Instead he favors traditional, coersive eugenics, arguing that reprogenetic technologies like embryo selection, cellular surgery, and human genetic engineering, which aim to enhance general purpose traits in offspring, are not  practices a liberal government leaves to the discretion of parents, but practices the  state  makes compulsory. [ 9 ]  Fox argues that if the liberal commitment to  autonomy  is important enough for the state to mandate childrearing practices such as health care and basic education, that very same interest is important enough for the state to mandate safe, effective, and functionally integrated genetic practices that act on analogous all-purpose traits such as resistance to disease and general cognitive functioning. He concludes that the liberal case for compulsory eugenics is a  reductio ad absurdum  against liberal theory. [ 9 ]


The United Nations  International Bioethics Committee  wrote that new eugenics should not be confused with the ethical problems of the 20th century eugenics movements. They have also stated the notion is nevertheless problematic as it challenges the idea of human equality and opens up new ways of discrimination and stigmatization against those who do not want or cannot afford the enhancements. [ 10 ]


Oncogenomics  is a sub-field of  genomics  that characterizes  cancer -associated  genes . It focuses on genomic, epigenomic and transcript alterations in cancer.


Cancer  is a genetic disease caused by accumulation of  DNA mutations  and  epigenetic  alterations leading to unrestrained cell proliferation and  neoplasm  formation. The goal of oncogenomics is to identify new  oncogenes  or  tumor suppressor genes  that may provide new insights into cancer diagnosis, predicting clinical outcome of cancers and new targets for cancer therapies. The success of targeted cancer therapies such as  Gleevec ,  Herceptin  and  Avastin  raised the hope for oncogenomics to elucidate new targets for cancer treatment. [ 1 ]


Besides understanding the underlying genetic mechanisms that initiate or drive cancer progression, oncogenomics targets personalized cancer treatment. Cancer develops due to  DNA mutations  and epigenetic alterations that accumulate randomly. Identifying and targeting the mutations in an individual patient may lead to increased treatment efficacy.


The completion of the  Human Genome Project  facilitated the field of oncogenomics and increased the abilities of researchers to find oncogenes. Sequencing technologies and global methylation profiling techniques have been applied to the study of oncogenomics.


The genomics era began in the 1990s, with the generation of DNA sequences of many organisms. In the 21st century, the completion of the  Human Genome Project  enabled the study of functional genomics and examining tumor genomes. Cancer is a main focus.


The epigenomics era largely began more recently, about 2000. [ 2 ] [ 3 ]  One major source of epigenetic change is altered methylation of  CpG islands  at the  promoter  region of genes (see  DNA methylation in cancer ). A number of recently devised methods can assess the DNA methylation status in cancers versus normal tissues. [ 4 ]  Some methods assess methylation of CpGs located in different classes of loci, including CpG islands, shores, and shelves as well as promoters, gene bodies, and intergenic regions. [ 5 ]  Cancer is also a major focus of epigenetic studies.


Access to whole  cancer genome sequencing  is important to cancer (or cancer genome) research because:


Access to methylation profiling is important to cancer research because:


The first cancer genome was sequenced in 2008. [ 6 ]  This study sequenced a typical  acute myeloid leukaemia  (AML) genome and its normal counterpart genome obtained from the same patient. The comparison revealed ten mutated genes. Two were already thought to contribute to tumor progression: an internal tandem duplication of the  FLT3  receptor  tyrosine kinase  gene, which activates kinase signaling and is associated with a poor prognosis and a four base insertion in exon 12 of the  NPM1  gene (NPMc). These mutations are found in 25–30% of AML tumors and are thought to contribute to disease progression rather than to cause it directly.


The remaining 8 were new mutations and all were single base changes: Four were in families that are strongly associated with cancer pathogenesis ( PTPRT , CDH24,  PCLKC  and  SLC15A1 ). The other four had no previous association with cancer pathogenesis. They did have potential functions in  metabolic pathways  that suggested mechanisms by which they could act to promote cancer (KNDC1,  GPR124 , EB12, GRINC1B)


These genes are involved in pathways known to contribute to cancer pathogenesis, but before this study most would not have been candidates for targeted gene therapy. This analysis validated the approach of whole  cancer genome sequencing  in identifying somatic mutations and the importance of parallel sequencing of normal and tumor cell genomes. [ 9 ]


In 2011, the genome of an exceptional bladder cancer patient whose tumor had been eliminated by the drug  everolimus  was sequenced, revealing mutations in two genes,  TSC1  and  NF2 . The mutations disregulated  mTOR , the protein inhibited by everolimus, allowing it to reproduce without limit. As a result, in 2015, the Exceptional Responders Initiative was created at the National Cancer Institute. The initiative allows such exceptional patients (who have responded positively for at least six months to a cancer drug that usually fails) to have their genomes sequenced to identify the relevant mutations. Once identified, other patients could be screened for those mutations and then be given the drug. In 2016 To that end, a nationwide cancer drug trial began in 2015, involving up to twenty-four hundred centers. Patients with appropriate mutations are matched with one of more than forty drugs. [ 10 ]


In 2014 the Center for Molecular Oncology rolled out the MSK-IMPACT test, a screening tool that looks for mutations in 341 cancer-associated genes. By 2015 more than five thousand patients had been screened. Patients with appropriate mutations are eligible to enroll in clinical trials that provide targeted therapy. [ 10 ]


Genomics technologies include:


Bioinformatics  technologies allow the statistical analysis of genomic data. The functional characteristics of oncogenes has yet to be established. Potential functions include their transformational capabilities relating to tumour formation and specific roles at each stage of cancer development.


After the detection of  somatic  cancer mutations across a cohort of cancer samples, bioinformatic computational analyses can be carried out to identify likely functional and likely driver mutations. There are three main approaches routinely used for this identification: mapping mutations, assessing the effect of mutation of the function of a protein or a  regulatory element  and finding signs of positive selection across a cohort of tumors. The approaches are not necessarily sequential however, there are important relationships of precedence between elements from the different approaches. Different tools are used at each step. [ 22 ]


Operomics aims to integrate genomics, transcriptomics and  proteomics  to understand the molecular mechanisms that underlie the cancer development. [ 23 ]


Comparative oncogenomics uses cross-species comparisons to identify oncogenes. This research involves studying cancer genomes, transcriptomes and proteomes in model organisms such as mice, identifying potential oncogenes and referring back to human cancer samples to see whether homologues of these oncogenes are important in causing human cancers. [ 24 ]  Genetic alterations in mouse models are similar to those found in human cancers. These models are generated by methods including  retroviral  insertion  mutagenesis  or graft transplantation of cancerous cells.


Mutations provide the raw material for natural selection in evolution and can be caused by errors of DNA replication, the action of exogenous mutagens or endogenous DNA damage. The machinery of replication and genome maintenance can be damaged by mutations, or altered by physiological conditions and differential levels of expression in cancer (see references in [ 25 ] ).


As pointed out by Gao et al., [ 26 ]  the stability and integrity of the human genome are maintained by the  DNA-damage response  (DDR) system. Un-repaired DNA damage is a major cause of mutations that drive carcinogenesis. [ 27 ] [ 28 ]  If DNA repair is deficient, DNA damage tends to accumulate. Such excess DNA damage can increase  mutational  errors during  DNA replication  due to error-prone  translesion synthesis . Excess DNA damage can also increase  epigenetic  alterations due to errors during DNA repair. [ 29 ] [ 30 ]  Such mutations and epigenetic alterations can give rise to  cancer . DDR genes are often repressed in human cancer by epigenetic mechanisms. Such repression may involve DNA methylation of promoter regions or repression of DDR genes by a microRNA. Epigenetic repression of DDR genes occurs more frequently than gene mutation in many types of cancer (see  Cancer epigenetics ). Thus, epigenetic repression often plays a more important role than mutation in reducing expression of DDR genes. This reduced expression of DDR genes is likely an important driver of carcinogenesis.


Nucleotide sequence context influences mutation probability [ 31 ] [ 32 ] [ 33 ]  and analysis of mutational (mutable) DNA motifs can be essential for understanding the mechanisms of mutagenesis in cancer. Such motifs represent the fingerprints of interactions between DNA and mutagens, between DNA and repair/replication/modification enzymes. Examples of motifs are the AID motif WRCY/RGYW (W = A or T, R = purine and Y = pyrimidine) with C to T/G/A mutations, [ 33 ]  and error-prone DNA pol η attributed AID-related mutations (A to G/C/G) in WA/TW motifs. [ 34 ]


Another (agnostic) way to analyze the observed mutational spectra and DNA sequence context of mutations in tumors involves pooling all mutations of different types and contexts from cancer samples into a discrete distribution. If multiple cancer samples are available, their context-dependent mutations can be represented in the form of a nonnegative matrix. This matrix can be further decomposed into components (mutational signatures) which ideally should describe individual mutagenic factors. [ 35 ]  Several computational methods have been proposed for solving this decomposition problem. The first implementation of Non-negative Matrix Factorization (NMF) method is available in Sanger Institute Mutational Signature Framework in the form of a MATLAB package. [ 36 ]  On the other hand, if mutations from a single tumor sample are only available, the DeconstructSigs R package [ 37 ]  and  MutaGene server [ 38 ]  may provide the identification of contributions of different mutational signatures for a single tumor sample. In addition, MutaGene server provides mutagen or cancer-specific mutational background models and signatures that can be applied to calculate expected DNA and protein site mutability to decouple relative contributions of mutagenesis and selection in carcinogenesis.


Synthetic lethality arises when a combination of deficiencies in the expression of two or more genes leads to cell death, whereas a deficiency in only one of these genes does not. The deficiencies can arise through mutations, epigenetic alterations or inhibitors of one of the genes.


The therapeutic potential of synthetic lethality as an efficacious anti-cancer strategy is continually improving. Recently, the applicability of synthetic lethality to targeted cancer therapy has heightened due to the recent work of scientists including  Ronald A. DePinho  and colleagues, in what is termed 'collateral lethality'. Muller et al. found that passenger genes, with chromosomal proximity to tumor suppressor genes, are collaterally deleted in some cancers. [ 39 ]  Thus, the identification of collaterally deleted redundant genes carrying out an essential cellular function may be the untapped reservoir for then pursuing a  synthetic lethality  approach. Collateral lethality therefore holds great potential in identification of novel and selective therapeutic targets in oncology. [ 40 ]  In 2012, Muller et al. identified that homozygous deletion of redundant-essential glycolytic  ENO1  gene in human  glioblastoma  (GBM) is the consequence of proximity to 1p36 tumor suppressor locus deletions and may hold potential for a synthetic lethality approach to GBM inhibition. [ 39 ]  ENO1 is one of three homologous genes ( ENO2 ,  ENO3 ) that encodes the mammalian  alpha-enolase  enzyme. [ 41 ]  ENO2, which encodes  enolase 2 , is mostly expressed in neural tissues, leading to the postulation that in  ENO1 -deleted GBM, ENO2 may be the ideal target as the redundant homologue of ENO1. [ 42 ]  Muller found that both genetic and pharmacological ENO2 inhibition in GBM cells with homozygous ENO1 deletion elicits a synthetic lethality outcome by selective killing of GBM cells. [ 39 ]  In 2016, Muller and colleagues discovered antibiotic SF2312 as a highly potent nanomolar-range  enolase  inhibitor which preferentially inhibits glioma cell proliferation and glycolytic flux in ENO1-deleted cells. [ 43 ]  SF2312 was shown to be more efficacious than pan-enolase inhibitor PhAH and have more specificity for ENO2 inhibition over ENO1. [ 43 ]  Subsequent work by the same team showed that the same approach could be applied to  pancreatic cancer , whereby homozygously deleted  SMAD4  results in the collateral deletion of mitochondrial malic enzyme 2 ( ME2 ), an oxidative decarboxylase essential for  redox  homeostasis. [ 44 ]  Dey et al. show that ME2 genomic deletion in pancreatic ductal adenocarcinoma cells results in high endogenous reactive oxygen species, consistent with KRAS-driven  pancreatic cancer , and essentially primes ME2-null cells for synthetic lethality by depletion of redundant NAD(P)+-dependent isoform ME3. The effects of ME3 depletion were found to be mediated by inhibition of de novo nucleotide synthesis resulting from AMPK activation and mitochondrial ROS-mediated apoptosis. [ 45 ] [ 44 ]  Meanwhile, Oike et al. demonstrated the generalizability of the concept by targeting redundant essential-genes in process other than metabolism, namely the  SMARCA4  and  SMARCA2  subunits in the chromatin-remodeling  SWI/SNF  complex. [ 46 ]


Some oncogenes are essential for survival of all cells (not only cancer cells). Thus, drugs that knock out these oncogenes (and thereby kill cancer cells) may also damage normal cells, inducing significant illness. However, other genes may be essential to cancer cells but not to healthy cells.


Treatments based on the principle of synthetic lethality have prolonged the survival of cancer patients, and show promise for future advances in reversal of carcinogenesis. A major type of synthetic lethality operates on the DNA repair defect that often initiates a cancer, and is still present in the tumor cells. Some examples are given here.


BRCA1  or  BRCA2  expression is deficient in a majority of high-grade breast and ovarian cancers, usually due to epigenetic methylation of its promoter or epigenetic repression by an over-expressed microRNA (see articles  BRCA1  and  BRCA2 ). BRCA1 and BRCA2 are important components of the major pathway for  homologous recombinational  repair of double-strand breaks. If one or the other is deficient, it increases the risk of cancer, especially breast or ovarian cancer. A back-up DNA repair pathway, for some of the damages usually repaired by BRCA1 and BRCA2, depends on  PARP1 . Thus, many ovarian cancers respond to an FDA-approved treatment with a PARP inhibitor, causing synthetic lethality to cancer cells deficient in BRCA1 or BRCA2. This treatment is also being evaluated for breast cancer and numerous other cancers in Phase III clinical trials in 2016. [ 47 ]


There are two pathways for  homologous recombinational  repair of double-strand breaks. The major pathway depends on BRCA1,  PALB2  and BRCA2 while an alternative pathway depends on RAD52. [ 48 ]  Pre-clinical studies, involving epigenetically reduced or mutated BRCA-deficient cells (in culture or injected into mice), show that inhibition of RAD52 is synthetically lethal with BRCA-deficiency. [ 49 ]


Mutations in genes employed in DNA mismatch repair (MMR) cause a high mutation rate. [ 50 ]  In tumors, such frequent subsequent mutations often generate "non-self" immunogenic antigens. A human Phase II clinical trial, with 41 patients, evaluated one synthetic lethal approach for tumors with or without MMR defects. [ 51 ]  The product of gene  PD-1  ordinarily represses cytotoxic immune responses. Inhibition of this gene allows a greater immune response. When cancer patients with a defect in MMR in their tumors were exposed to an inhibitor of PD-1, 67–78% of patients experienced immune-related progression-free survival. In contrast, for patients without defective MMR, addition of PD-1 inhibitor generated only 11% of patients with immune-related progression-free survival. Thus inhibition of PD-1 is primarily synthetically lethal with MMR defects.


ARID1A , a chromatin modifier, is required for  non-homologous end joining , a major pathway that repairs double-strand breaks in DNA, [ 52 ]  and also has transcription regulatory roles. [ 53 ]  ARID1A mutations are one of the 12 most common carcinogenic mutations. [ 54 ]  Mutation or epigenetically decreased expression [ 55 ]  of ARID1A has been found in 17 types of cancer. [ 56 ]  Pre-clinical studies in cells and in mice show that synthetic lethality for ARID1A deficiency occurs by either inhibition of the methyltransferase activity of EZH2, [ 57 ] [ 58 ]  or with addition of the kinase inhibitor dasatinib. [ 59 ]


Another approach is to individually knock out each gene in a genome and observe the effect on normal and cancerous cells. [ 60 ] [ 61 ]  If the knockout of an otherwise nonessential gene has little or no effect on healthy cells, but is lethal to cancerous cells containing a mutated oncogene, then the system-wide suppression of the suppressed gene can destroy cancerous cells while leaving healthy ones relatively undamaged. The technique was used to identify  PARP-1  inhibitors to treat BRCA1/BRCA2-associated cancers. [ 62 ] [ 63 ]  In this case, the combined presence of PARP-1 inhibition and of the cancer-associated mutations in BRCA genes is lethal only to the cancerous cells.


The  Cancer Genome Project  is an initiative to map out all somatic mutations in cancer. The project systematically sequences the exons and flanking splice junctions of the genomes of primary tumors and cancerous cell lines. COSMIC software displays the data generated from these experiments. As of February 2008, the CGP had identified 4,746 genes and 2,985 mutations in 1,848 tumours.


The  Cancer Genome Anatomy Project  includes information of research on cancer genomes, transcriptomes and proteomes.


Progenetix is an oncogenomic reference database, presenting cytogenetic and molecular-cytogenetic tumor data.


Oncomine has compiled data from cancer transcriptome profiles.


The integrative oncogenomics database IntOGen and the Gitools datasets integrate multidimensional human oncogenomic data classified by tumor type. The first version of IntOGen focused on the role of deregulated  gene expression  and  CNV  in cancer. [ 64 ]  A later version emphasized mutational cancer driver genes across 28 tumor types,. [ 65 ] [ 66 ]  All releases of IntOGen data are made available at the IntOGen database.


The  International Cancer Genome Consortium  is the biggest project to collect human cancer genome data. The data is accessible through the ICGC website. The BioExpress® Oncology Suite contains gene expression data from primary, metastatic and benign tumor samples and normal samples, including matched adjacent controls. The suite includes hematological malignancy samples for many well-known cancers.


Specific databases for model animals include the Retrovirus Tagged Cancer Gene Database (RTCGD) that compiled research on retroviral and transposon insertional mutagenesis in mouse tumors.


Mutational analysis of entire gene families revealed that genes of the same family have similar functions, as predicted by similar coding sequences and  protein domains . Two such classes are the  kinase  family, involved in adding phosphate groups to proteins and the  phosphatase  family, involved with removing phosphate groups from proteins. [ 67 ]  These families were first examined because of their apparent role in transducing cellular signals of cell growth or death. In particular, more than 50% of colorectal cancers carry a mutation in a kinase or phosphatase gene.  Phosphatidylinositold 3-kinases  ( PIK3CA ) gene encodes for lipid kinases that commonly contain mutations in colorectal, breast, gastric, lung and various other cancers. [ 68 ] [ 69 ]  Drug therapies can inhibit PIK3CA. Another example is the  BRAF  gene, one of the first to be implicated in melanomas. [ 70 ]  BRAF encodes a  serine / threonine  kinase that is involved in the RAS-RAF- MAPK  growth signaling pathway. Mutations in BRAF cause constitutive phosphorylation and activity in 59% of melanomas. Before BRAF, the genetic mechanism of melanoma development was unknown and therefore prognosis for patients was poor. [ 71 ]


Mitochondrial DNA  (mtDNA) mutations are linked the formation of tumors. Four types of mtDNA mutations have been identified: [ 72 ]


Point mutations have been observed in the  coding  and  non-coding  region of the mtDNA contained in cancer cells. In individuals with bladder, head/neck and lung cancers, the point mutations within the coding region show signs of resembling each other. This suggests that when a healthy cell transforms into a tumor cell (a neoplastic transformation) the  mitochondria  seem to become homogenous. Abundant point mutations located within the non-coding region,  D-loop , of the cancerous mitochondria suggest that mutations within this region might be an important characteristic in some cancers. [ 72 ]


This type of mutation is sporadically detected due to its small size ( < 1 kb). The appearance of certain specific mtDNA mutations (264-bp deletion and 66-bp deletion in the complex 1 subunit gene ND1) in multiple types of cancer provide some evidence that small mtDNA deletions might appear at the beginning of  tumorigenesis . It also suggests that the amount of mitochondria containing these deletions increases as the tumor progresses. An exception is a relatively large deletion that appears in many cancers (known as the "common deletion"), but more mtDNA large scale deletions have been found in normal cells compared to tumor cells. This may be due to a seemingly adaptive process of tumor cells to eliminate any mitochondria that contain these large scale deletions (the "common deletion" is > 4 kb). [ 72 ]


Two small mtDNA insertions of ~260 and ~520 bp can be present in breast cancer, gastric cancer, hepatocellular carcinoma (HCC) and colon cancer and in normal cells. No correlation between these insertions and cancer are established. [ 73 ]


The characterization of mtDNA via real-time  polymerase chain reaction  assays shows the presence of quantitative alteration of mtDNA copy number in many cancers. Increase in copy number is expected to occur because of oxidative stress. On the other hand, decrease is thought to be caused by somatic point mutations in the replication origin site of the  H-strand  and/or the D310 homopolymeric c-stretch in the D-loop region, mutations in the  p53  (tumor suppressor gene) mediated pathway and/or inefficient enzyme activity due to  POLG  mutations. Any increase/decrease in copy number then remains constant within tumor cells. The fact that the amount of mtDNA is constant in tumor cells suggests that the amount of mtDNA is controlled by a much more complicated system in tumor cells, rather than simply altered as a consequence of abnormal cell proliferation. The role of mtDNA content in human cancers apparently varies for particular tumor types or sites. [ 72 ]


57.7% (500/867) contained somatic point putations and of the 1172 mutations surveyed 37.8% (443/1127) were located in the D-loop control region, 13.1% (154/1172) were located in the tRNA or rRNA genes and 49.1% (575/1127) were found in the mRNA genes needed for producing complexes required for mitochondrial respiration.


Some anticancer drugs target mtDNA and have shown positive results in killing tumor cells. Research has used mitochondrial mutations as biomarkers for cancer cell therapy. It is easier to target mutation within mitochondrial DNA versus nuclear DNA because the mitochondrial genome is much smaller and easier to screen for specific mutations. MtDNA content alterations found in blood samples might be able to serve as a screening marker for predicting future cancer susceptibility as well as tracking malignant tumor progression. Along with these potential helpful characteristics of mtDNA, it is not under the control of the cell cycle and is important for maintaining  ATP  generation and mitochondrial homeostasis. These characteristics make targeting mtDNA a practical therapeutic strategy. [ 72 ]


Several  biomarkers  can be useful in cancer staging, prognosis and treatment. They can range from  single-nucleotide polymorphisms  (SNPs),  chromosomal aberrations , changes in DNA copy number, microsatellite instability, promoter region  methylation , or even high or low protein levels. [ 90 ]  Between 2013 and 2019 only 6.8% of people with cancer in 2 US states underwent genetic testing, suggesting broad under-utilization of information that could improve treatment decisions and patient outcomes. [ 91 ]


Paratransgenesis  is a technique that attempts to eliminate a  pathogen  from  vector  populations through  transgenesis  of a symbiont of the vector. The goal of this technique is to control vector-borne  diseases . The first step is to identify  proteins  that prevent the vector species from transmitting the pathogen. The  genes  coding for these proteins are then introduced into the  symbiont , so that they can be expressed in the vector. The final step in the strategy is to introduce these transgenic symbionts into vector populations in the wild. One use of this technique is to prevent mortality for humans from insect-borne diseases. Preventive methods and current controls against vector-borne diseases depend on insecticides, [ 1 ]  even though some mosquito breeds may be resistant to them. There are other ways to fully eliminate them. [ 2 ]  “Paratransgenesis focuses on utilizing genetically modified insect symbionts to express molecules within the vector that are deleterious to pathogens they transmit.” [ 1 ]  The acidic bacteria Asaia symbionts are beneficial in the normal development of mosquito larvae; however, it is unknown what Asais symbionts do to adult mosquitoes. [ 1 ]


The first example of this technique used  Rhodnius prolixus  which is associated with the symbiont  Rhodococcus rhodnii .  R. prolixus  is an important insect vector of  Chagas disease  that is caused by  Trypanosoma cruzi . The strategy was to engineer  R. rhodnii  to express proteins such as Cecropin A that are toxic to  T. cruzi  or that block the transmission of  T. cruzi . [ 3 ]


Attempts are also made in  Tse-tse flies  using bacteria [ 4 ] [ 5 ]  and in  malaria mosquitoes  using fungi, [ 6 ]  viruses, [ 7 ]  or bacteria. [ 8 ]


Although the use of paratransgenesis can serve many different purposes, one of the main purposes is “breaking the disease cycle”. This study focuses on the experiments with tsetse flies and trypanosomes, which cause sleeping sickness in Subsaharan Africa. The tsetse fly’s transmission biology was studied to learn how it transmits the disease. This was done inn order to find the best way to use paratransgenesis, which could help solve transmission. In this case, paratransgenesis was used to create trypanocides which stop the transmission of trypanosomes in the tsetse fly vector. [ 4 ]


Another disease caused by the transmission of mosquitoes to humans is malaria. This has been an ongoing health issue as there is not an effective vaccine and malaria is deadly. “The development of innovative control measures is an imperative to reduce malaria transmission.” [ 9 ]  In this study, it was found that when using paratransgenesis of Asaia (gfp) in these mosquitoes, there was a lower chance of the disease. [ 9 ]  They are using anti-pathogen effector molecules. [ 9 ]


Another example is in honey bees. A study done in 2012 found that using lactic acid bacteria could improve or help with honey bee’s health and digestion. [ 10 ]  This is a different use of paratransgenesis and was suggested as the Lactobacillus was an easy target for paratransgenesis. The scientists wanted to see if maintaining the microbiome in the insect model’s guts would work to keep the bees and the entire colony healthy. [ 10 ]  There has been a major decrease in honey bee populations and colonies in recent years. By using paratransgenesis, scientists and beekeepers hope to increase the population of honey bees.


Experiments have shown that the spread through mosquito populations is resistant to parasites engineered through symbiotic bacterium Serratia AS1. Major concerns of regulators for the release of such engineered bacteria into the field shows there were zero options for “recall”. “Serratia AS1 loses plasmids as it replicates in mosquitoes and in culture, reverting to wild type and that horizontal transfer of the plasmid from Serratia AS1 to other bacteria is difficult to detect.” [ 11 ]  This means the initial field trials can be used in the reversible system besides the released recombinant bacteria expressing antiplasmodial compounds from a plasmid revert to wild type at a certain rate. [ 11 ]


“Paratransgenesis is the genetically modified symbiotic organisms that block pathogen development or transmission by vectors using expressing molecules”. Figure 2 shows An. gambiae [ 7 ]  and Ae. aegypti [ 12 ]  symbiotic viruses using bacteria symbionts blood-sucking, [ 3 ]  tsetse flies [ 13 ]  and mosquitoes. [ 14 ]  Symbionts expressing molecules targeting pathogen development can have transmission in endemic regions. [ 13 ]  As with transgenesis, The spread of transformed symbionts benefits from the availability of a gene drive system to replace non-transformed symbionts present in natural vector populations is also seen in transgenesis. [ 13 ]  Paratransgenesis reduces African trypanosomes transmission by tsetse flies. It has transformed  Sodalis , a symbiont of tsetse flies found in the midgut and hemolymph of  Glossina m. morsitans ,  Glossina p. palpalis ,  Glossina austeni , and  Glossina brevipalpis , and the salivary glands of  Gl. p. palpalis , which all have transmitted vertically via the female milk glands. [ 13 ]  Vertical transmission has GFP-transformed (recSodalis) that was detected in 9 out of 12 F1 offspring and eight out of 12 F2 descendents, which has transformed symbiont to be spread across tsetse populations. [ 13 ]  This resulted in  Sodalis  being isolated from  Gl. m. morsitans  and  Gl. fuscipes  transforming with GFP, the recSodalis obtained colonized septic non-native tsetse host species at a density similar to native colonization. [ 14 ] [ 15 ]


A future direction on vector paratransgenesis is within the natural insect populations and it has not been determined if transformed symbionts can replace non-transformed symbionts. There are no effects on insect hosts and are capable of being transmitted vertically (via trans-ovarian transmission) or laterally (due to feeding habits) when it comes to symbionts.  Wolbachia  endosymbionts is a gene driven system and can also affect paratransgenesis. [ 4 ]


Wolbachia  are intracellular transitional bacteria that control the reproduction of insects via cytoplasmic incompatibility (CI). [ 16 ]  “ Wolbachia -uninfected females will not breed with infected males, which reduces the frequency of uninfected individuals and increases the frequency of  Wolbachia -infected insects in a population.” [ 16 ]  This effect will cause other transitional controlled transformed symbionts to spread within an insect population which expands the frequency. [ 4 ]  These insects include: Ae. aegypti, Aedes albopictus, and Culex quinquefasciatus. [ 16 ]  Densovirus is an example of how the spread is transformed through symbionts, occurring in the natural populations of mosquitoes.


During the process of conducting as a gene driven mechanism, the  Wolbachia  strain reduces the mosquito lifespan for pathogen development inside the mosquito (known as the extrinsic incubation period or EIP). [ 17 ]  Elimination of the disease vectors is hard to treat due to reduced vector lifespan for its own growth from a shorter growth time. [ 18 ]  This means it targets older mosquitoes over younger ones and this also implies evolution-proof of mosquitocidal biocontrols agents. [ 19 ]  Time already exists for a selective pressure on pathogen development in Plasmodium-infected mosquitoes for Anopheles (marsh mosquitos) from 20% to 40% per gonotrophic cycle [ 20 ] [ 19 ]  resulting in a shortening of the parasite life cycle within the vector.


“One approach is to reduce vector competence (linear parameter), and vector survivorship (exponential parameter). Both effects together should reduce vectorial capacity and disease burden in endemic areas and prevent transmission.” [ 14 ]


Vector-borne diseases are common; therefore working to understand how these diseases are transmitted can lead to better prevention of or treatment for these illnesses. Vector borne diseases such as malaria are passed from mosquitoes to humans. [ 21 ]  Trypanosoma Cruz causes Chagas disease, and there are efforts to use paratransgenesis to prevent the spread of this disease. The strategy is to alter the microbe then reinsert it into the insect that has been genetically modified to alter pathogens. The article “Paratransgenic Control of Vector Borne Diseases'' discusses the approach to understanding these diseases. [ 22 ]  Human African Trypanosomiasis (sleeping sickness) is an illness that affects many individuals in sub-Saharan Africa. In the last decade the numbers have come close to an elimination. This illness is passed by flies and the past few efforts on controlling this disease is less than 10,000 cases per year. [ 23 ]


There are many diseases in which paratransgenesis can occur, with the most common being malaria. The paper “Evaluating the usefulness of paratransgenesis for malaria control,” describes the global problem of malaria, a cause of significant health issues. [ 2 ]  It is carried by mosquitoes and although the most useful way to eliminate them is to use insecticides, some mosquito species are resistant to insecticide. In order to combat insecticide-resistant mosquitos, there are genetically engineered plasmodium that has been created to help destroy the mosquito gut. [ 2 ]  Another study “Using infection to fight infection: paratransgenesis fungi can block malaria transmission in mosquitoes” demonstrates anti-malaria effector genes that were injected into entomopathogenic fungus, Metarhizium anisopliae. [ 24 ]  Next, the fungus was injected into non-infected mosquitoes and expressed in the hemolymph. The interesting thing about this is when other molecules were coexpressed, the salivary glands expressed Malaria levels up to 98%. [ 24 ]


In order to perform paratransgenesis, there are several requirements:


Pharming , a  portmanteau  of  farming  and  pharmaceutical , refers to the use of  genetic engineering  to insert  genes  that code for useful pharmaceuticals into host animals or plants that would otherwise not express those genes, thus creating a  genetically modified organism  (GMO). [ 1 ] [ 2 ]  Pharming is also known as  molecular farming ,  molecular pharming , [ 3 ]  or  biopharming . [ 4 ]


The products of pharming are recombinant proteins or their metabolic products.  Recombinant proteins are most commonly produced using  bacteria  or  yeast  in a  bioreactor , but pharming offers the advantage to the producer that it does not require expensive infrastructure, and production capacity can be quickly scaled to meet demand, at greatly reduced cost. [ 5 ]


The first  recombinant  plant-derived protein (PDP) was human  serum albumin , initially produced in 1990 in transgenic  tobacco  and  potato  plants. [ 6 ]   Open field growing trials of these crops began in the United States in 1992 and have taken place every year since.  While the  United States Department of Agriculture  has approved planting of pharma crops in every state, most testing has taken place in Hawaii, Nebraska, Iowa, and Wisconsin. [ 7 ]


In the early 2000s, the pharming industry was robust.  Proof of concept has been established for the production of many  therapeutic proteins , including  antibodies ,  blood products ,  cytokines ,  growth factors ,  hormones ,  recombinant enzymes  and human and  veterinary   vaccines . [ 8 ]   By 2003 several PDP products for the treatment of  human diseases  were under development by nearly 200 biotech companies, including  recombinant   gastric lipase  for the treatment of  cystic fibrosis , and  antibodies  for the prevention of  dental caries  and the treatment of  non-Hodgkin's lymphoma . [ 9 ]


However, in late 2002, just as ProdiGene was ramping up production of trypsin for commercial launch [ 10 ]  it was discovered that  volunteer plants  (left over from the prior harvest) of one of their GM corn products were harvested with the conventional soybean crop later planted in that field. [ 11 ] [ unreliable source? ]   ProdiGene was fined $250,000 and ordered by the USDA to pay over $3 million in cleanup costs.  This raised a furor and set the pharming field back, dramatically. [ 5 ]  Many companies went bankrupt as companies faced difficulties getting permits for field trials and investors fled. [ 5 ]   In reaction, APHIS introduced more strict regulations for pharming field trials in the US in 2003. [ 12 ]   In 2005,  Anheuser-Busch  threatened to boycott rice grown in Missouri because of plans by  Ventria Bioscience  to grow pharm rice in the state.  A compromise was reached, but Ventria withdrew its permit to plant in Missouri due to unrelated circumstances.


The industry has slowly recovered, by focusing on pharming in simple plants grown in  bioreactors  and on growing GM crops in greenhouses. [ 13 ]  Some companies and academic groups have continued with open-field trials of GM crops that produce drugs.  In 2006  Dow AgroSciences  received USDA approval to market a vaccine for poultry against  Newcastle disease , produced in plant cell culture – the first plant-produced vaccine approved in the U.S. [ 14 ] [ 15 ]


Milk is presently the most mature system to produce recombinant proteins from transgenic organisms. Blood, egg white,  seminal plasma , and urine are other theoretically possible systems, but all have drawbacks. Blood, for instance, as of 2012 cannot store high levels of stable recombinant proteins, and biologically active proteins in blood may alter the health of the animals. [ 16 ]   Expression in the  milk of a mammal, such as a cow, sheep, or goat, is a common application, as milk production is plentiful and purification from milk is relatively easy.  Hamsters and rabbits have also been used in preliminary studies because of their faster breeding.


One  approach to this technology is the creation of a transgenic mammal that can produce the biopharmaceutical in its milk (or blood or urine). Once an animal is produced, typically using the pronuclear microinjection method, it becomes efficacious to use cloning technology to create additional offspring that carry the favorable modified genome. [ 17 ]   In February 2009 the US FDA granted marketing approval for the first drug to be produced in genetically modified livestock. [ 18 ]  The drug is called  ATryn , which is antithrombin protein purified from the milk of genetically modified  goats .  Marketing permission was granted by the  European Medicines Agency  in August 2006. [ 19 ]


As indicated above, some mammals typically used for food production (such as goats, sheep, pigs, and cows) have been modified to produce non-food products, a practice sometimes called pharming. Use of genetically modified goats has been approved by the FDA and EMA to produce  ATryn , i.e. recombinant  antithrombin , an  anticoagulant   protein drug . [ 20 ]  These products "produced by turning animals into drug-manufacturing 'machines' by genetically modifying them" are sometimes termed  biopharmaceuticals .


The patentability of such biopharmaceuticals and their process of manufacture is uncertain. Probably, the biopharmaceuticals themselves so made are unpatentable, assuming that they are chemically identical to the preexisting drugs that they imitate. Several 19th century  United States Supreme Court  decisions hold that a previously known natural product manufactured by artificial means cannot be patented. [ 21 ]  An argument can be made for the patentability of the process for manufacturing a biopharmaceutical, however, because genetically modifying animals so that they will produce the drug is dissimilar to previous methods of manufacture; moreover, one Supreme Court decision seems to hold open that possibility. [ 22 ]


On the other hand, it has been suggested that the recent Supreme Court decision in  Mayo v. Prometheus [ 23 ]  may create a problem in that, in accordance with the ruling in that case, "it may be said that such and such genes manufacture this protein in the same way they always did in a mammal, they produce the same product, and the genetic modification technology used is conventional, so that the steps of the process 'add nothing to the laws of nature that is not already present. [ 24 ]  If the argument prevailed in court, the process would also be ineligible for patent protection. This issue has not yet been decided in the courts.


Plant-made pharmaceuticals (PMPs), also referred to as pharming, is a sub-sector of the  biotechnology  industry that involves the process of genetically engineering plants so that they can produce certain types of therapeutically important  proteins  and associated molecules such as peptides and secondary metabolites.  The proteins and molecules can then be harvested and used to produce pharmaceuticals. [ 25 ]


Arabidopsis  is often used as a  model organism  to study gene expression in plants, while actual production may be carried out in  maize ,  rice ,  potatoes ,  tobacco ,  flax  or  safflower . [ 26 ]   Tobacco has been a highly popular choice of organism for the expression of transgenes, as it is easily transformed, produces abundant tissues, and survives well  in vitro  and in greenhouses. [ 27 ]  The advantage of rice and flax is that they are self-pollinating, and thus  gene flow  issues (see below) are avoided.  However, human error could still result in modified crops entering the food supply.  Using a minor crop such as safflower or tobacco avoids the greater political pressures and risk to the food supply involved with using staple crops such as beans or rice. Expression of proteins in plant cell or hairy root cultures also minimizes risk of gene transfer, but at a higher cost of production. Sterile hybrids may also be used for the bioconfinement of transgenic plants, although stable lines cannot be established. [ 28 ]  Grain crops are sometimes chosen for pharming because protein products targeted to the endosperm of cereals have been shown to have high heat stability. This characteristic makes them an appealing target for the production of  edible vaccines , as viral coat proteins stored in grains do not require cold storage the way many vaccines currently do. Maintaining a temperature controlled supply chain of vaccines is often difficult when delivering vaccines to developing countries. [ 29 ]


Most commonly, plant transformation is carried out using  Agrobacterium tumefaciens . The protein of interest is often expressed under the control of the  cauliflower mosaic virus  35S promoter ( CaMV35S ), a powerful constitutive promoter for driving expression in plants. [ 30 ]  Localization signals may be attached to the protein of interest to cause accumulation to occur in a specific sub-cellular location, such as chloroplasts or vacuoles. This is done in order to improve yields, simplify purification, or so that the protein folds properly. [ 31 ] [ 32 ]  Recently, the inclusion of antisense genes in expression cassettes has been shown to have potential for improving the plant pharming process. Researchers in Japan transformed rice with an antisense SPK gene, which disrupts starch accumulation in rice seeds, so that products would accumulate in a watery sap that is easier to purify. [ 33 ]


Recently, several non-crop plants such as the  duckweed   Lemna minor  or the  moss   Physcomitrella patens  have shown to be useful for the production of biopharmaceuticals. These frugal organisms can be cultivated in  bioreactors  (as opposed to being grown in fields), secrete the transformed proteins into the growth medium and, thus, substantially reduce the burden of  protein purification  in preparing recombinant proteins for medical use. [ 34 ] [ 35 ] [ 36 ]  In addition, both species can be engineered to cause secretion of proteins with human patterns of  glycosylation , an improvement over conventional plant gene-expression systems. [ 37 ] [ 38 ]   Biolex  Therapeutics developed a duckweed-based expression platform; it sold the business to  Synthon  and declared bankruptcy in 2012. [ citation needed ]


Additionally, an Israeli company, Protalix, has developed a method to produce therapeutics in cultured transgenic carrot or tobacco cells. [ 39 ]   Protalix and its partner, Pfizer, received FDA approval to market its drug,  taliglucerase alfa  (Elelyso), as a treatment for  Gaucher's disease , in 2012. [ 40 ]


The regulation of genetic engineering concerns the approaches taken by governments to assess and manage the risks associated with the development and release of  genetically modified crops . There are differences in the regulation of GM crops – including those used for pharming – between countries, with some of the most marked differences occurring between the USA and Europe. Regulation varies in a given country depending on the intended use of the products of the genetic engineering. For example, a crop not intended for food use is generally not reviewed by authorities responsible for food safety.


There are controversies around GMOs generally on several levels, including whether making them is ethical, issues concerning intellectual property and market dynamics; environmental effects of GM crops; and GM crops' role in industrial agricultural more generally.  There are also specific controversies around pharming.


Plants  do not carry  pathogens  that might be dangerous to human  health . Additionally, on the level of  pharmacologically  active  proteins , there are no proteins in plants that are similar to human proteins. On the other hand, plants are still sufficiently closely related to animals and humans that they are able to correctly process and configure both animal and human proteins. Their seeds and fruits also provide sterile packaging containers for the valuable therapeutics and guarantee a certain storage life. [ 41 ]


Global demand  for  pharmaceuticals  is at unprecedented levels.  Expanding the existing  microbial  systems, although feasible for some therapeutic products, is not a satisfactory option on several grounds. [ 8 ]   Many proteins of interest are too complex to be made by microbial systems or by  protein synthesis . [ 6 ] [ 41 ]  These proteins are currently being produced in animal  cell cultures , but the resulting product is often prohibitively expensive for many patients.  For these reasons, science has been exploring other options for producing proteins of therapeutic value. [ 2 ] [ 8 ] [ 15 ]


These pharmaceutical crops could become extremely beneficial in developing countries.  The  World Health Organization  estimates that nearly 3 million people die each year from vaccine preventable disease, mostly in Africa.  Diseases such as measles and hepatitis lead to deaths in countries where the people cannot afford the high costs of vaccines, but pharm crops could help solve this problem. [ 42 ]


While molecular farming is one application of  genetic engineering , there are concerns that are unique to it. In the case of  genetically modified  (GM) foods, concerns focus on the safety of the food for  human consumption . In response, it has been argued that the genes that enhance a  crop  in some way, such as  drought resistance  or  pesticide resistance , are not believed to affect the food itself. Other GM foods in development, such as fruits designed to ripen faster or grow larger, are believed not to affect humans any differently from non-GM varieties. [ 2 ] [ 15 ] [ 41 ] [ 43 ]


In contrast, molecular farming is not intended for crops destined for the  food chain . It produces plants that contain  physiologically  active compounds that accumulate in the plant’s tissues. Considerable attention is focused, therefore, on the restraint and caution necessary to protect both consumer health and environmental  biodiversity . [ 2 ]


The fact that the plants are used to produce drugs alarms  activists . They worry that once production begins, the altered plants might find their way into the food supply or  cross-pollinate  with conventional, non-GM crops. [ 43 ]   These concerns have historical validation from the ProdiGene incident, and from the  StarLink  incident, in which  GMO  corn accidentally ended up in commercial food products.  Activists also are concerned about the power of business. According to the  Canadian Food Inspection Agency , in a recent report, says that U.S. demand alone for biotech pharmaceuticals is expanding at 13 percent annually and to reach a market value of $28.6 billion in 2004. [ 43 ]  Pharming is expected to be worth $100 billion globally by 2020. [ 44 ]


Please note that this list is by no means exhaustive.


Projects known to be abandoned


Prenetics  (NASDAQ:PRE) is a health sciences company headquartered in Hong Kong with a significant presence in the United States. It is best known for its innovations in consumer and clinical health, focusing on early detection, prevention, and treatment of diseases, particularly cancer.


Prenetics' consumer initiatives are spearheaded by IM8, a health and wellness brand. Another key offering is CircleDNA, which utilizes next-generation sequencing (NGS) technology to provide comprehensive DNA testing for consumers, offering insights into health, wellness, and genetic predispositions.


The company's clinical arm includes Insighta, a joint venture focused on developing multi-cancer early detection technologies. Additionally, ACT Genomics develops and provides a variety of genomic profiling panels, including ACTOnco, ACT HRD, ACTFusion, and ACTDrug tests, tailored to meet specific clinical needs. ACTLiquid Pro, a sequencing-based liquid biopsy assay, and ACTMonitor, which tracks drug resistance, treatment response, and cancer recurrence in real-time, are among its notable products. ACT Risk offers management solutions for assessing and mitigating cancer risks.


One of its major investors was  Alibaba  founder Jack Ma. [ 1 ]


Since listing, it has not been able to turn a profit. [ 2 ] [ 3 ]  In October 2023 it announced a 1 for 15 reverse stock split so it could regain the stock exchange's required US$1 per share stock price. [ 4 ]


Danny Yeung serves as CEO of Prenetics. [ 5 ]


Prenetics has become the first Hong Kong–based unicorn to list on Nasdaq following a merger with a  Special-purpose acquisition company  Artisan Acquisition Corp. [ 6 ] [ 7 ]


Its subsidiary CircleDNA focuses on disease prevention rather than genealogy. For example, in 2016 it launched a rapid DNA-based colon cancer screening product. [ 8 ]
In 2019 it launched a line of at-home tests to be sold through retailers in Asia. [ 9 ]


In 2023 it announced $200 million joint venture with the  Chinese University of Hong Kong  and molecular biologist  Dennis Lo   to launch DNA testing for several major diseases and cancers. [ 10 ] [ 11 ]  Lo and Prenetics are working together to create a new company, Insighta, that will focus on their cancer detection work. [ 12 ]  The early detection cancer test, Presight, will begin clinical trials in 2024. [ 12 ]  The company claims that by 2027 patients will be able to test for more than 10 types of cancer with a single blood test. [ 13 ]  Danny Yeung will serve as CEO of Insighta and Lo will serve as chairman. [ 12 ]


Prenetics was aggressive in marketing Covid testing capacity.  It was one of three companies, and the only one from Hong Kong, selected to provide COVID-19 testing support to the Hong Kong government. [ 1 ] [ 14 ]   It also had plans to participate in a Covid passporting scheme. [ 15 ]


Its UK arm provided COVID-19 testing to the Premier League to help them restart football amid the pandemic. [ 16 ] [ 17 ]  As part of so-called "project restart" the company hired former FIFA player  Rio Ferdinand  to reassure players about the safety of the plan. [ 18 ]  Around the same time, the company also launched pop-up testing sites for a flexible workspace provider. [ 19 ]


<reflist>


Prevention of autosomal recessive disorders is focused on making it less likely that two carriers for the same  hereditary disease  will have children together.


Some  genetic disorders  are caused by having two "bad" copies of a recessive allele. When the gene is located on an  autosome  (as opposed to a sex chromosome), it is possible for both men and women to be  carriers . A child of two carriers has a 1/4 chance of being affected by the disorder.


Due to carriers being unaffected (or barely affected), the bad recessive alleles can persist in the gene pool for quite a while, even if the disorder is 100% lethal. [ medical citation needed ]


Most modern societies have  laws regarding incest , [ 1 ]  with avoiding the  genetic disorders  caused by  inbreeding  as one of the major motivations. [ 2 ]


Both social acceptance and legality of first- cousin marriage  is mixed. Some jurisdictions  narrowly tailor  their laws to preventing inbreeding: in Maine, [ 3 ]  first cousins can marry with proof of  genetic counseling , while in Arizona [ 4 ]  and several other states, first cousins can marry if they are old or infertile. [ 5 ]


Carrier testing  can help guide the decisions of couples who are at high risk, e.g.:


Couples who learn that they are both carriers may decide to part ways, adopt, or use  preimplantation genetic diagnosis  to select unaffected embryos. [ citation needed ]


These practices are not designed to change  allele frequencies  and therefore have little impact on  future generations  beyond the first. As a result, these practices are generally not considered to be a form of  eugenics , despite overlapping goals. [ 9 ]


A  pseudodeficiency allele  or  pseudodeficiency mutation  is a mutation that alters the protein product or changes the gene's expression, but without causing disease. For example, in the  lysosomal storage diseases , patients with a pseudodeficiency allele show greatly reduced enzyme activity, yet they remain clinically healthy.


In medical genetics, a  false positive  result occurs in an  enzyme assay  test when test results are positive, but disease or morbidity is not present. One possible cause of false positive results is a pseudodeficiency allele. Disease may also be present, but at a subclinical level.


A pseudodeficiency allele may indicate a deficiency of the enzyme assay method, or it may reflect incomplete understanding of the enzyme's activity.


Because of pseudodeficiency alleles, the results of enzyme assay testing in one population cannot be generalized to other populations. For example, while Tay–Sachs screening was able to nearly eliminate Tay–Sachs disease among  Ashkenazi Jews , similar screening in the general population has proven less effective. [ 1 ]


For some genetic diseases, especially those that have low  penetrance  or are easily treatable,  carrier screening  may be of questionable value when pseudodeficiency alleles are present at high frequency. [ 4 ]


Repoxygen  was the tradename for a type of  gene therapy  to produce  erythropoietin  (EPO). It was under preclinical development by Oxford Biomedica as a possible treatment for  anaemia  but was abandoned in 2003. [ 1 ]


The project became infamous when it was mentioned during the criminal trial of Thomas Springstein, a former track coach for some German athletes, who was found guilty of giving athletes  performance enhancing drugs  without their knowledge.  An email in which Springstein attempted to obtain Repoxygen was read by a prosecutor, which led to a flurry of media coverage. [ 2 ]


The  World Anti-Doping Agency  banned "gene doping" in 2003 [ 2 ]  and as of 2009 was researching detection methods for substances such as repoxygen. [ 3 ]


The Resilience Project  is a project, undertaken by the  Icahn School of Medicine at Mount Sinai  in collaboration with  Sage Bionetworks . [ 1 ]