Molecular BIO- Unit 12

Genetically Modified Humans: 4. HIV Gene Therapy Continued. (pt2)

- 2 people have been "cured" of HIV through bone marrow ablation and allogenic bone marrow transplant from patients with CCR5 mutations. •The patients were HIV positive but unfortunately were also diagnosed with non-Hodgkin lymphoma. •To treat the cancer they underwent high dose chemotherapy which dramatically, almost fully, destroyed their immune system. •They needed allogenic bone marrow transplantation. •The donor for the transplant was a CCR5 mutant individual who was resistant to HIV infection. •When the donors bone marrow repopulated the patients hematopoietic system, the new cells were resistant to HIV, and the few remaining HIV infected recipient cells died, but the virus couldn't infect the donors cells.

Genetically Modified Mammals 1. Enviropig

- A pig was genetically modified so that it more efficiently absorbs phosphorus from it diet. •This dramatically reduces the amount of phosphorus in their manure which in turn doesn't contaminate water sources with high phosphorus run off. •This helps prevent algal blooms, since P is a limiting nutrient for algae. •Algae consume a ton of oxygen and cause death of fish in water consumed by algae. •They added an E. coli phytase gene driven by a parotid salivary gland promoter by microinjection of a fertilized oocyte. •The funding for the research ended in 2012 and they couldn't find a company to partner with so the pigs were slaughtered after genetic material was stored away.

Genetically Modified Plants 7. Pharming

- Creating genetically modified organisms that produce therapeutic agents for use in biomedicine. •Examples: 1.Antibodies for immunotherapy 2.Cytokines 3.Hormones 4.Enzymes 5.Vaccines

Genetically Modified Fish 2. Double Muscling

- Genetically modified fish in which the follistatin gene is mutated. •This prevents myostatin inhibition which allows a double layer of muscle to form in the fish increasing meat yield per fish.

Genetically Modified Fish 1. AquaAdvantage Salmon

- AquaBounty developed a genetically modified salmon that produces GH year round. •This allows them to grow at an expedited rate and grow larger in size than wildtype atlantic salmon. •They added a GH regulating gene from a Chinook salmon fused to an Ocean Pout promoter. •To prevent breeding with wildtype salmon the AquaAdvantage salmon are all female and are triploid making them 99% sterile. •Similar research is underway for trout, and tilapia.

Genetically Modified Plants 3. Arctic Apple

- Similar to the "browning resistant" mushrooms, artic apple company has genetically engineered to silence the polyphenol oxidase gene. •This provides resistance to the oxidase-mediated browning of apples. •They have since introduced this method into granny smith, fuji, and golden delicious apple species.

Reasons to do genetic engineering include

1. Drug manufacturing - vaccines, insulin, HGH 2. Loss of function research - Gene knockout studies 3. Gain of function research - Gene knock in studies 4. Lineage tracing research - in developmental biology 5. Localization research - identifying localization of proteins within cells. 6. Increasing agriculture/aquaculture yields - larger crops, resistant crops, increased milk production, etc. 7. Altering nutrients in food - Golden rice

Genetically Modified Plants

•Manipulation is more difficult than single celled organisms, •Their ability to facilitate post-translational modification allows for more complex proteins to be synthesized. •Many plants can be genetically engineered using agrobacterium fairly easily. •Many species of plants are able to grow from a single plant cell from tissue culture. •The highest number of genetically modified plants are crops for agriculture. •Genetic modification of crop plants increase nutritional load, resistance to pests, and tolerating environmental stresses allowing for broader cultivation regions.

Genetically Modified Humans: 6. Many other examples are being investigated but have yet to get FDA approval.

•There is promise in this field, it just needs to mature. •Unlike in lower organisms, we care if our genetic manipulation kills or harms the subject.

Genetically Modified Humans

•Until 2018 when Professor He genetically modified human embryos to make them more resistant to HIV infection, human genetic engineering occurred only in cultured human cells for research or in post natal humans in attempts to treat genetic disorders.

Genetically Modified Fish

•Zebrafish (Danio rerio) are a commonly used organisms in research labs. •Genetic manipulation is not too difficult and they develop very quickly. •The chorionic membrane is clear so their development can be observed fairly easily. •They are routinely used in developmental biology investigations. •In 2015 the USDA approved the cultivation, sale and consumption of AquaAdvantage fish. •Glofish were developed for studying pollution but have made their way into pet stores.

Genetically Modified Humans: Tangent: GUIDE-Seq

•dsOligoDeoxynucleotide (ODN) is incorporated into sites of DSB. - ODN is known sequence •DNA is fragmented via sonication. •Adaptors are ligated onto ends of fragment to create "library" •Library is amplified up using ODN specific primer to yield a final library. •Final library is run on a NextGen sequencer to identify sites where the ODN incorporated (IE off target sites of the genome editing vector).

Genetically Modified Plants 6. Flavr Savr Tomatoes

- Genetically modified tomatoes were the first GMO to be USDA approved for human consumption. •Polygalacturonase antisense gene was introduced into tomatoes to improve shelf life. •This was accomplished using agrobacterium. •Originally developed by the Company Calgene, they were poor marketers and went out of business and the patent for Flavr Savr tomatoes was sold to Monsanto.

Genetically Modified Fish 3. Glofish

- Genetically modified zebra fish engineered to express GFP, RFP, YFP, etc. •They were trying to engineer fish that would produce fluorescence when subject to pollution in water. •They signed a distribution deal with Yorktown Technologies which has since sold the fish as pets to fish enthusiasts. •The researchers haven't finished their work in creating a pollution reporter line, but have made a bunch of money selling them. •Other species of Glofish have since been created.

Genetically Modified Humans: 3. Immunotherapy

- Hematology researchers have long sought a way to have our own immune systems clear cancer from our body. •Problem is the cancer is recognized as "self" by the immune system therefore the immune system doesn't attack it. •Genetically modified T and B-cells have been used in clinical trials to treat various forms of cancers including: •B-cell Acute Lymphoblastic Leukemia •Non-Hodgkin Lymphoma •Multiple Myeloma •Chronic Lymphocytic Leukemia •Melanoma •Head and Neck Squamous cell carcinoma

Genetically Modified Mammals 4. The lysozyme goat

- In 1999 Artemis the goat that was genetically modified to express human lysozyme. •They introduced the human lysozyme gene into a fertilized goat oocyte via microinjection. •They thought that by making goats milk that contained lysozyme would help bolster our immune system and prevent digestive diseases caused by bacteria. •They haven't started producing large quantities of their milk but the goat has been bred and given rise to many offspring as of 2016. •A similar attempt in cows was made in 2014. •In this case they were trying to make the cows mastitis resistant, but they haven't reported their findings on whether or not they were indeed mastitis resistant. •Another group did the same thing to make "human breast milk from cows", which we'll discuss in a second.

Genetically Modified Mammals 7. The Parkinson's Marmoset

- In 2009 scientists introduced a gene (alpha synyclein) thought to contribute to Parkinson's disease in an attempt to create a non-human primate model of the disease for research purposes. •They haven't published any findings yet but at a conference in 2016 they claim their animals get Parkinson's like symptoms at about 3 years old. •Current models use MPTP, a chemical agent that destroys the dopamine neurons of the substantia niagra of the brain. •This would be a better genetic model more similar to what is observed in humans.

Genetically Modified Mammals 3. The silk goat

- In 2010 a goat was genetically modified to express spider silk in milk. •They introduced spider silk genes into a fertilized goat oocyte via microinjection. •The hope was they could extract enough spider silk fibers from the goats milk to make artificial ligaments, tendons, bulletproof vests, and airbags. •There aren't enough silk spiders to meet demands and this offered a possible alternative. •The status of this is unknown, but the researchers thought that alfalfa would be better alternative... so why start with goat???

Genetically Modified Mammals 5. The hornless bull

- In 2013 a red Holstein cow named Randy was genetically modified so that the a gene from red angus cows that inhibit horn development was introduced. •Their idea was to generate hornless dairy cattle to make the animals safer for the handlers. •Cattle injuring dairy farmers with their horns is apparently a MAJOR concern J. •Levity aside, cattle without horns will pose less of a threat to the dairy farmers. •Currently the horns are removed via a painful dehorning process, so this method is more humane. •This research is still ongoing.

Genetically Modified Mammals 8. The Antitrypsin Goat

- In the first drug derived from a GMO was FDA approved for use in humans. •Patients suffering from hereditary antithrombin deficiency require alpha-1 antitrypsin. •Goats were genetically modified to introduce this gene driven by a promoter which allows the protein to be expressed in the milk. •The drug is extracted from the milk. •Expressing this gene in goats allow it to be post-translationally modified in a way more similar to humans. •An example of Pharming.

Genetically Modified Plants 4. Drought-resistant maize

- Monsanto developed a genetically modified maize plant that is resistant to drought. •They manipulated the genes that play a role in Crassulacean acid metabolism which allows a plant to survive in low water conditions. •This was developed in 2011, has USDA approval, but large scale cultivation has yet to occur. Monsanto, makers of drought-resistant maize AND glyphosate resistant crops is distrusted by the public given the recent classification of glyphosate as a probable carcinogen.

Genetically Modified Humans: 4. HIV Gene Therapy (pt1)

- Professor He genetically modified human embryos to give them a non-functional CCR5 gene. •CCR5 is a receptor that HIV needs to infect T-cells. •CCR5 mutant individuals are resistant to HIV infection. •CCR5 mutations are also associated with increased cognitive ability. •The father of twin girls had HIV so he wanted to make the children resistant to HIV (he recruited patients in which the father had HIV but the mother did not... why???) •Babies blood and mothers blood are more likely to mix during birth. •Speculation is that he wanted to create a super-intelligent human by mutating CCR5... this is SPECULTATION, we cannot be sure of his true intent, only God knows.

Genetically Modified Plants 1. Golden Rice

- Rice was genetically engineered such that the edible parts express the genes necessary for the synthesis of beta-carotene. •Phytoene synthase (from daffodil) •Phyotene desaturase (from soil bacteria) •This was meant to curtail lethality and blindness in undeveloped nations in which there was high levels of vitamin A deficiency. • The rice haven't been grown commercially yet as there are issues with yield, and the beta-carotene levels are not high enough yet to warrant large scale production.

Genetically Modified Humans: 1. SCID Gene Therapy

- Severe Combined Immunodeficiency (Aka bubble boy syndrome) results in patients having essentially no immune system (B and T cell development doesn't occur). •This disease can be caused by a couple of mutations but one of the most common is in adenosine deaminase gene (ADA). •Researchers used retroviruses to introduce ADA back into white blood cells of patients during clinical trials. •It did cure the disease in a number of the patients but in a few others it caused leukemia due to transgene integration near an oncogene. •The trials were promising but because of the leukemia they were stopped. •If a more specific approach can be used (crispr/cas9 with HDR), this approach could see its way into the clinic.

Genetically Modified Humans: 2. Sickle Cell Cure

- Sickle cell anemia is caused by a mutation in the beta-globin gene and causes clumping of the protein causing abnormal erythrocyte shape. •This disease was "cured" in 2017 by a French group. •This was announced via press release and not in a peer reviewed journal. •They "introduced wildtype copies of beta globin into hematopoietic stem cells and transplanted them back into the patient." •Not sure how they introduced the beta globin into the patient, whether it replaced the mutant versions, and whether or not bone marrow ablation was done prior to transplantation. •This is the problem with non-peer reviewed press releases.

Genetically Modified Plants 5. Biofuel

- Syngenta has USDA approval to market maize which has been genetically modified to convert its starch into simple sugars for ease in converting it to ethanol for fuel. -This has not been adopted on a large scale yet

Genetically Modified Mammals 2. Hermann the bull

- The first bovine genetically engineered to express a human protein, lactotransferrin. •The hope was that this protein would be produced and could be extracted from female offspring of Hermann. •The protein was expressed at such low levels it was never useful. •Hermann received a stay of execution from the Dutch government and lived until he was 14 years old when he got severe osteoarthritis. •He was taxidermied and he is on display in Naturalis Biodiversity Center

Genetically Modified Humans: 4. HIV Gene Therapy Continued. (pt3)

- This is a possible case where the cure is worse than the disease according to the researchers. •They only got the high dose chemotherapy to treat an unrelated disease (non-Hodgkin lymphoma). •They wouldn't recommend this to other HIV patients. Although theoretically scientists could... •Take out an HIV patient's bone marrow •Use genome editing to disrupt the HIV virus' genome •Use genome editing to mutate CCR5 in the cells •Put them back into the patient without bone marrow ablation (high dose chemotherapy). •Eventually the HIV infected cells will all die, the "edited" cells will survive to repopulate the patients hematopoietic system

Genetically Modified Mammals 6. The human breast milk cow

- This is the same as the human lysozyme cow and goat already discussed but done by 2 different groups. •They claim the cow milk is "more chemically similar to human milk", but don't go so far as to say it is human milk. •They haven't published any results yet so the information is from media reports only. •They hope in "10 years" their milk will be on supermarket shelves ready for human consumption... I for one don't love the idea of drinking human breast milk... I'll stick with cow milk. •Also, one of the most important things from breast milk is the maternal antibodies which promote natural immunity. •They did nothing to introduce human immunoglobulin genes into cow B-cells to promote "human" antibody production.

Genetically Modified Plants 2. Glyphosate-resistant crops

-Glyphosate is broad range herbicide that will kill most crops and weeds. Crop plants have been genetically engineered to be resistant to glyphosate so fields can be treated and the weeds will die. •Over 90% of US grown soybeans and maize is glyphosate-resistant. •Glyphosate was recently labeled as a probable carcinogen by the world health organization. •Farmers are pumping this chemical all over fields and suffering from increased rates of cancer. •This will most likely lead to alternative genetically modified crops that are resistant to other non-carcinogenic broad range herbicides.

Examples of Genetically Modified Fungi=

1. As pesticides - yeast can kill insects on contact, but not as efficiently as commercial pesticides. Yeast have been genetically modified to increase their virulence. 2. In Wine Fermentation - strains of yeast have been created which dramatically reduce the amount of dangerous ethyl carbamate compounds. 3. In disease prevention - plasmodium in mosquitos causes dengue fever, malaria and yellow fever. Yeast are being genetically engineered which will block these diseases from being transmitted. 4. Mushrooms devoid of polyphenol oxidase don't turn brown and have a longer shelf life.

Examples of Genetically Modified Bacteria=

1. Insulin - the first bacterial expressed protein for use in medicine. 2. HGH - used to treat patients with pituitary dwarfism (and for athletes J) 3. Erythropoietin - used to treat anemia (and for blood doping J) 4. In Food - alpha-amylase from bacteria used to convert starch into simple sugars 5. Ice-free bacteria - pseudomonas bacteria have been genetically modified which prevent ice crystal formation. The have been applied to crops to allow them to grow in colder climates.

Hurdles to Overcome When Genetically Modifying Humans

1. Integration of genetic material is necessary to achieve long term expression of transgenes. - Choose an integrating virus 2. Silencing of transgenes - Target to euchromatin or endogenous locus 3. Immune responses to viral delivery (has caused deaths) - Suppress the immune system during viral delivery 4. Many diseases are polygenic, delivery of one transgene will not cure the disease. - If there is a driver mutation, fix that mutation. 5. Random integration of transgene can disrupt oncogenes and tumor suppressors which can lead to cancer. - Target specific genomic loci (safe havens or endogenous locus) 6. Gene therapies can possibly cross the blood testis barrier creating genetically modified offspring (ethical concern). - Not sure how to get around, but with Dr. He's work this may not be a concern in the future.

Examples of genetically modified viruses

1. Oncolytic viruses - attack tumor cells. 2. Gene Therapy Viruses - cure genetic disorders (cystic fibrosis, hemophilia, glioblastoma, etc.) 3. Immunocontraception viruses - developed to make animals sterile (control pest populations) 4. Biomaterial producing viruses - (viruses that aggregate to form biomaterials)

Concerns include but are not limited to

1. Scientists "playing God" 2. Horizontal gene transfer between GMOs and wild organisms - creation of "superweeds" 3. Escape of GMOs from captivity - knockout mice getting out in the wild and trashing the wild mouse population, fast developing large salmon making their way into native populations 4. Patenting of life 5. Product labeling

Genetically Modified Fungi=

Can be manipulated in much the same way as bacteria. Yeast have been used as pesticides as they can kill insects on contact. GMO yeast has also been developed for use in wine fermentation.

Genetically Modified Bacteria=

The first genetically modified organism. Bacteria are easily genetically modified because of their single chromosome - Hfr strains

____________ of transgenes via non-integrative approaches is generally NOT considered genetic engineering.

Transient expression

Genetically Modified Viruses=

Used mainly for the introduction of genetic material into host organisms (used to genetic engineer other species).

What makes viruses "more safe" to use?

Viruses must be modified to remove their ability to replicate

Genes are expressed in _________ to synthesize various proteins.

bacteria

genetic engineering

direct manipulation of an organisms genome using biotechnology.

In a way, ____________ is a more rapid way to alter phenotypes than breeding.

genetic engineering

The techniques by which genomes are edited depend on the _________ being genetically engineered.

organism

Additional Hurdles to Overcome When Genetically Modifying Humans

•Crispr/Cas9 and TALEN technology can be used to specifically target transgenes to specific genomic locations when combined with HDR. •These have limitations though as well... they have off target effects that could cause mutations in tumor suppressors and oncogenes and cause cancer. •To check for off target effects Keith Joung's lab developed a technique called GUIDE-Seq, (Genome-wide Unbiased Identification of Double Stranded Breaks Enabled by Sequencing). •Before genome-editing vectors are introduced into humans, they should be screened for off targets using GUIDE-Seq. •Even this has limitations in that the off target sites may be different in different cell types (heterochromatin vs euchromatin in different cells)

Genetically Modified Mammals

•House mouse (Mus musculus) is the most commonly genetically modified mammal. •Genetic manipulation in mice is done predominantly for research purposes and is complicated (remember our last discussion J). •Other mammals have also been genetically modified for various reasons including research but also for agricultural purposes.

Genetically Modified Humans: Tragic Story of Jessie Gelsinger

•Jessie suffered from a rare metabolic disorder called OTC deficiency (ornithine transcarbamylase deficiency). •The disease is caused by a mutation in ornithine transcarbamylase gene. •He was treated with an adenovirus encoding the OTC gene. •He suffered a severe immune reaction to treatment with virus which caused multiple organ failure resulting in death 4 days after treatment. •The researchers and university were investigated and found to have had: 1.Financial stake in the success of the trial. (compromised their morals???) 2.Failure to disclose the death of monkeys who received the treatment in preclinical trials. 3.Failure of the university to disclose 2 other patients had experienced serious side effects from the treatment. 4.Gelsinger should have been excluded from the study because he didn't meet the approved conditions. Moral of the story = trust the preclinical trial data and only move forward when animals aren't dying of the treatment.