1. I want to use a human artificial chromosome to improve the ability of cytoreductive gene therapy to “alert” the immune system. The reason I choose this is because we would be able to signal the immune system to attack the cancer cell anywhere in the body. That is achieved by introducing HAC that contain cytokine genes into a harvested host’s cancer cell. The introduced HAC will be active most of the time in the cell. The reason is that HAC does not contain any protein portion such as histones that may silence the gene. With the cytokine being expressed by the cancer cell, the immune system will be alerted, and they will create antibody against the cancer cell. Since the immune system (finally) recognized the cancerous cell, the hope is that the immune system will clear up any other cancer cell that linger somewhere else in the body.
I would improve the ability of cytoreductive gene therapy by incorporating several elements into the HAC. Firstly, I would include genes that code for RNAi that might be used for cancer for “tumor escape mechanisms” as described by Igney and Krammer (2002). That is to make sure that cancer that is re-injected into the body do not escape from the immune system and successfully attacked by the host immune system. Also, I would also like to incorporate RNAi code for VEGF gene; this is to serve as a double layer protection. Just in case if cancer somehow not detected and destroyed by the immune system, the injected cancer do not cause extra trouble in the body.
2. Let’s assume that the defective gene result in a non-functional protein only. The non-functional protein does not cause any pathological/phenotype problem. By inserting some of the correct blastomeres into the defective one, although why not use the correct blastomere (I don’t understand), it will provide a cell line that contain the working gene. Hopefully, if all procedure is working just fine, the blastomeres will grow into an embryo that then become a person. As a result, mosaicism of the functional gene in his/her body might overcome the original defect. Hopefully, the functional gene will be kept on being expressed in the body for the entire patient’s life. Therefore, in this scenario, the treatment might work.
However, there are several things that may result into a problem. The procedure of transplanting the new blastomere. Would it be possible to introduce the new blastomere and let them proceed cell replication process?
Then, the reaction of the entire cell/individual/embryo to the entirely new different genetic makeup of the “correct” blastomere. The problem is that the “correct” blastomere will have a very different genetic makeup than the “defective” blastomere. This is because of the very unlikely, or impossible, event of the fertilization of 2 exactly identical germ cell.
Lastly, it depends on the location whereby the protein needs to be expressed; in other words, the fate of the gene depends on cellular differentiation and epigenetic factors. For example, if the “correct” blastomere cell were differentiated into an ectoderm, while the protein needed to be expressed in endoderm, it would be pointless.
Since there are so many possible hurdles, I would say that the treatment would not work.
3. I would use a blastomere engineering technique to fix the promotor. Working with a blastomere would similar to that working with a somatic cell. I would fix it by using a vector that would infect replicating cells, such as lentivirus. The vector would have to be injected into the fertilization envelope (the cavity between the blastomeres). If virus delivery could not be done, I would continue trying using microinjection, injecting the DNA engineering system directly into the blastomere. Those (both viral delivery and microinjection) things are to ensure the delivery of vector able to reach all blastomeres. Also, I would use a CRISPR/Cas9 for the targeted genomic editing. The gRNA will target the defective promotor gene and replace it with the correct promotor sequence. As a result, hopefully, the whole mice (the cells) will have a working VEGF promotor gene.
4. We now know that any material that is made into a nano-sized particle behaves differently than their normal-sized counterparts. The use of carbon nanotubes offers a tremendous promise for the delivery of therapeutic agents. The “pro” article claims that the carbon nanotubes do not accumulate in vital organs. The carbon nanotubes will be excreted out of the body in feces and urine after they “unpack” its payload (Bergeron, 2009). Also, a similar form of carbon nanotubes, nanosphere, also has shown promises (Namm, 2015). Meanwhile the “cons” article emprise that no matter what kind of nanoparticle/structure might have some effect in living tissue especially in vivo (Berger, 2008). My solution to both of this contradictory situation is that although it has been shown that carbon nanotubes or nanosphere (nanostructure) were excreted from the body, the study has only been done in rodent subject. Therefore, the concern from the “cons” article are still legitimate. There will be a need to test the efficacy, efficiency, safety and toxicity in a higher level of the animal model (Friedman, Furberg, & DeMets, 2010). When all gone well, then we may go on a higher stage of the clinical trial.
5. For the hardware, I would use SWNT, mobile nanobots, and nanomotors. The goal with those three nano-hardware is that I will try to form a nanorobot. The goal is that the nanorobot would be able to swim move through the entire body (using nanomotors and mobile nanobots). Lastly, they will be able to inject (moved using nanomotors) a loaded SWNT containing an artificial gene into tumor’s nucleus.
For the biological supplies, I will be using a promotor and p53 gene. I will construct a linear DNA construct followed by a promotor followed by the p53 gene.
What I am hoping is that with the modified mobile nanobots would be able to detect which cell are cancerous and inject them with the DNA construct directly into the cancer nucleus. p53 gene will be activated; thus, activating the cellular apoptosis. Since mobile nanobots deliver the gene, remote cancers could also be dealt with; resulting in complete resolution to the cancer crisis.
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EXTRA CREDIT: WORTH ZERO OR ONE POINT EXTRA CREDIT
There is something wrong with the absorption and retention of the nanoparticle. How would the coating know and able to distinguish between the normal cell vs the cancerous cell. Let’s say, when tumor are destroyed, then the iron oxide nanoparticle are released. When most (not all) tumor cell are destroyed, some of the nanoparticle would have entered into the normal cell and killing it as well-that is if the particle don’t know the difference between the cancerous cell and the normal. Also, how come if the nanoparticle able to be discharged from the body, that means, the nanoparticle should be able to pass through the blood-brain barrier. But here is the catch, the particle is only injected once. I don’t get how would the particle would be able to be maintained in the brain while being discharged by the body through normal process in the same time.
References
Berger, M. (2008). Nanotechnology and toxicity: the growing need for in vivo study. from http://www.nanowerk.com/spotlight/spotid=4132.php
Bergeron, L. (2009). Researchers’ nanotube findings give boost to potential biomedical applications. Stanford Report. Retrieved July 22, 2015, from http://news.stanford.edu/news/2008/january30/tube-013008.html
Friedman, L. M., Furberg, C. D., & DeMets, D. (2010). Fundamentals of Clinical Trials: Springer New York.
Igney, F. H., & Krammer, P. H. (2002). Immune escape of tumors: apoptosis resistance and tumor counterattack. Journal of Leukocyte Biology, 71(6), 907-920.
Namm, T. (2015). Gene Therapy – Protocols and Prognoses. Retrieved July 22, 2015, from https://uml.umassonline.net/webapps/blackboard/execute/content/file?cmd=view&content_id=_5444605_1&course_id=_66158_1&framesetWrapped=true
Week 11 assignment
Christianto Putra (01526759)
1. Firstly, I would gather all the mice into a compound group. Every mouse will be tagged and identified by numerical sequence. Then, the genetic sample would be collected from each mouse. All the genetic material then would be run through a genetic analysis (OLA – oligonucleotide ligation assay). The OLA will be tested on the known SNP that causes the mutated protein.
OLA procedure work by amplifying the genetic sample from the mice and allowed to hybridize with a designed oligomers. The designed oligomers, also called as Allele-specific oligonucleotide (ASO), will have a unique sequence to the target SNP. In addition, the 5′ end of the ASO will also be attached with differing length of oligo (typically mutant oligo will be longer than wild-type oligo) and a molecule of biotin (act as chemical hook) (Namm, 2015).
Common oligomer that is attached to a fluorescent label would also be present in the OLA reaction. The common oligomer would hybridize right next to the targeted SNP. Then, all the DNA hybrids will then be ligated using ligase to combine the designed oligomers and the common oligomer (MacDonald, 2007). Next, the sample washed under buffer and analyzed using capillary electrophoresis. The wild-type allele would have shorter oligos compared to the mutant-type allele.
I will be expecting there will be three different results based on the genetic hereditary of the mice. There would be normal genotype, heterozygous genotype, and homozygous mutant genotype. Then, I would confirm each mice’s genotype using PCR. From here, I would assign each mouse to its assigned group based on its genotype.
From the genotype groups, I would administer the drug and record the result. If it is true that the SNP result to ineffective “mouse-alive”, then the whole group of the homozygous mutant genotype group would have the same adverse reaction. Whereas, the variable result should be observed in the heterozygous genotype and the effective result observed in the homozygous wild-type group.
2. Firstly, I would group the family member into two groups. Those with desired result (group 1), lowered blood pressure, and those not affected or with an adverse effect (group 2). Those people who have no effect or developed an adverse reaction to the beta-blocker would have either issue with the target of the drug or cellular binding site of the beta-blocker. From here, I would split the group 2 into another group, group 2a and 2b. Group 2a would consist of people that is not affected by the beta-blocker and without any adverse effect. Group 2b would consist of people who had an adverse effect from the beta-blocker. Then, I would conduct analysis using “Toxchip”, PCR and OLA to detect the gene that might affect the activity of the drug.
If the problem is caused by the target of the drug, the “Toxchip” will show indifferent mRNA pattern to those untreated individuals. Hence, group 2a should show the indifferent pattern of mRNA because although with the presence of beta-blocker, the cells are somehow not affected by it.
If the problem is caused by the cellular binding site for the beta-blocker, the raw form of the drug will cause a problem in the body; resulting in the adverse reaction (Namm, 2015). For this case, group 2b would be analyzed by “Toxchip” and the mRNA pattern should show an abnormal mRNA production. The abnormal mRNA production would indicate the toxic reaction of the beta-blocker in the cell.
Lastly, I would conduct PCR and OLA analysis to confirm any gene that is accountable for the modification of the drug activity. Any SNP or mutation that is associated with a similar response would indicate that that gene is the source of the problem. It is noteworthy that since the participants are related to each other, we could also confirm the genetic inheritance pattern of the gene that is responsible for the modification of the drug activity.
3. Optogenetics would work better for spinal cord injury compared to cerebral cortex neuron malfunction. Spinal cord injuries involve in injury, due to physical or disease, of the nerve connection in the spine that leads to loss of connectivity from the CNS. Optogenetics could work by diagnosing spinal cord injuries; to check which nerve cell got damaged. I can’t see other application for spinal cord injury using optogenetics because spinal cord injury only results in loss of action from the nerve impulse. Even if optogenetics able to solve spinal cord injury caused either by physical or disease, some controllable light stimuli are needed to activate that particular neuron cell. For example, if the spinal cord injury results in the loss of function in the left leg. A light switch need to be turned on every time the person need to move his/her left leg. Personally, I think it is impractical. But still, it is doable.
However, unlike spinal cord injuries that only result in loss of nerve impulse, cerebral cortex neuron malfunction is a disorder that could be a result from over-reaction or under-reaction of activating or deactivating types of neuron cells. Although, optogenetics would work much better in diagnosing and studying cerebral cortex neuron malfunction due to the nature of the disease, I could not see how optogenetics would solve brain malfunction. We know that optogenetics could turn on or off whichever cell are desired in the brain, which allow the scientist to pinpoint the root of the problem. However, scientist still needs to figure out how to match the complex brain activity. For example, if the sensory part of cerebral cortex got affected, how would the scientist know when to turn on or off the light when the sensory stimuli received? Which of the nerve cell suppose to give sour taste? Which nerve cell is required to be active to feel a sensation on the tip of the toe?
Therefore, although optogenetics offers much more diagnostic and exploratory function in the brain, dealing with a simpler problem such as spinal cord injuries would, in my opinion, would be more approachable.
4.Not only that CRISPR/Cas9 technology able to silencing a gene like RNA interference, CRISPR/Cas9 able to introduce nucleotide resulting in gene editing.
CRISPR/Cas9 utilize a gRNA that enable a very precise gene silencing or gene editing.
CRISPR/Cas9 result in long-lasting changes, unlike RNA interference. The RNA produced by RNA interference might still be degraded by RNAse. Whereas CRISPR/Cas9 edit the DNA itself.
CRISPR/Cas9 has 3 three different kinds of endonuclease that allow flexible utilization of the technology whereas RNAi only serve to silence the gene.
Due to the nature of RNAi, it is possible that the RNAi might silence a non-target gene (Semizarov, et al., 2003) Whereas CRISPR/Cas9 system is a guided gene specific silencing/editing, the risk of non-target silencing are be greatly minimized.
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References
Macdonald, S. J. (2007). Genotyping by Oligonucleotide Ligation Assay (OLA).Cold Spring Harbor Protocols, 2007(9), pdb-prot4843.
Namm, Theodore. (2015). Week 11 – Today’s hottest topics. https://uml.umassonline.net/webapps/blackboard/content/listContent.jsp?course_id=_66158_1&content_id=_5444525_1&mode=reset
Semizarov, D., Frost, L., Sarthy, A., Kroeger, P., Halbert, D. N., & Fesik, S. W. (2003). Specificity of short interfering RNA determined through gene expression signatures. Proceedings of the National Academy of Sciences,100(11), 6347-6352.
Karen Roa Assignment 10 (Exam 2)
2.
(1). If I were in charge of creating and maintaining a line of stem cells to intergrade tumor gene p53 when mutates the best cells to use would be to use human embryonic stem cells specifically pluripotent skin stem cells. Pluripotent stem cells can have high yields for differentiating. Pluripotent connotes the ability of a cell to give rise to multiple cell types, including all three embryonic lineages forming the body’s organs, nervous system, skin, muscle and skeleton. This is a good choice for the reasons you provide.
(2). A way to engineer embryonic stem cells so that p53 gene is highly up regulated by inserting the correct p53 gene to the skin cell. Adding the p53 gene essentially erases the skin cell programming and will reinstall the embryonic stem programming (that is the hope, but doing this for a single gene might be next to impossible). Once the cell recognizes the p53 gene it will read the instructions to make any specific proteins, in this case the tp53. But in order to hyperproduce the p53 would be by hyperphosphorylating p53 protein at the posttranslational level by protein-to-protein interaction. (Interesting theory – OK, this might work, but I am not completely sure. However, that doesn’t matter – it is very well thought out.
(3). Stem cell self-renewal is controlled by intrinsic factors and signals within a niche. In order for stem cells in the niche to self renew are anchored in the niche by adhersions. Once the cells have changed to its cancerous conditions in its niche with the mutant p53 gene and its apoptotic activity of uncontrolled proliferation. For the niche to be able to control these cancer cells forcing a decrease in cadherins from their niche. If cadherins can be induced to decrease this will cause a delay in the correction of localized adhersion junctions of alpha and beta catenin. Good1
25 points out of 25
3.
(a). Muscle stem cells can be ideal for cloning, because they have the ability to divide and produce more cells to repair damage (Some are, and some aren’t. Certainly, the muscle cells themselves would not be a good choice, but their precursor cells do have stem cell properties. Satellite cells or cardiac stem cells would be good; smooth muscle precursors would not be a good choice) A benefit of using a muscle cell is that allows for technology to create or modify cells genetically to a patient need depending of a disease or disorder like muscular dystrophy. In cases it has been seen that patients reject foreign tissues or cells because the body thinks they are invader and thus creating muscle stem cells using the patients DNA to overcome this barrier, of rejection (1).
(b). Mammary gland cell are not ideal for cloning. In Dolly’s case (sheep that cloned) the mammary cell can only replicate themselves for a certain period of time since they have shorter telomeres perhaps causing them to be susceptible to DNA damage. Good!
20 points out of 25
4.
(1). I believe leukemia would respond well Aponte’s white blood cell protocol since it has worked previously with patients (I guess that is a good reason!). It seems that in this protocol white blood cells are taken from the patient to use a retrovirus to change them into target and destroy cancer cells. It seems that this protocol works with certain patients and in these patients the cancer seems to be going away in a matter or days or weeks. Ok – this one is fine,
(2). I believe that anti-VEGF can be used to help treat numerous cancers like lung cancer. VEGF normal function is to supply blood flow to tissues but in having an intact tumor and cutting off the blood supply essentially the nutrients and oxygen (life’s supply) it cannot sustain itself therefore reducing the size of the tumor. When a tissue does not receive a certain amount of blood flow and oxygen necrosis occurs which you would imagine would also occur in a tumor. This is the most straightforward response, and also provides the common denominator for most cancers.
25 points out of 25
6.
(a). Johannes P. Muller was born on July 14, 1801 and died on April 28, 1858. He was born in Germany where he became a doctor and studied physiology.
(b). His work was done between 1823 through 1852 (3). He was a physiologist that increased the understanding of the voice, speech, hearing, and the chemical and physical properties of the lymphatic and hematology. He established basic anatomical facts on human and animal physiology. His work of these general principals published as the Elements of Physiology in 1837 showed physiologist how to apply physics and chemistry, which marked a new era in the study of physiology.
He was one of the true pioneers of both physical and life sciences.
(C)
Question 1: In using the bar coding method to treat cancer how does it affect the cellular mechanism in the body? A fair question from a scientist of that era.
Question 2: Have scientist or researcher been able to find a consistent pattern of any gene sequence coding? That might be beyond his area of expertise to even ask. However, if you explained all those details, then it might elicit that kind of question.
(D)
Answer to Question 1: It will affect the cellular mechanism of the body by ribonucleic acid (RNA) interference. It is a biological process in which the RNA molecules will inhibit gene expressions by destroying specific messenger RNA molecules. It can be treated for cancer in a similar way it is used to fight viruses by RNA interference identifying short segments of suspicious looking RNA and later then destroying any identical copies of that RNA (4). Therefore that type of suspicious looking protein will not encoded later on and can be a promising way to treat cancer by silencing different genes that are up-regulated in tumor cells or cell division. Too complex for J.M., even though it is accurate.
Answer to Question 2: It seems like there is no specific gene sequence in RNA interference. Scientist have paired the first 3,000 genes that are vital in cell signaling and growth factor but the results are random if they are part of the RNA interference that may cause an up-regulated cell division. Again, if your initial explanation supported this, then J.M. might be able to understand this.
25 points out of 25
Work Cited
1. Stem Cells used to treat muscular dystrophy in mice. http://hsci.harvard.edu/stem-cells-used-treat-muscular-dystrophy-mice
2. Everyday Mysteries. Stem Cells. http://www.loc.gov/rr/scitech/mysteries/stemcells.html
3. Johannes P. Muller. http://www.britannica.com/biography/Johannes-Peter-Muller
4. New Genetic Barcoding Technique Identifies Dozens of Targets for Cancer Drugs. 2008. http://www.hhmi.org/news/new-genetic-barcoding-technique-identifies-dozens-targets-cancer-drugs
CTC technology locates cancer cells in patients. The CTC chip allows for the detection of cancer cells without sifting through blood vial samples collected by patients. I believe that CTC technology specifically the digital tomosynthesis will work best with breast cancer because it allows a clearer image to allow radiologist not to miss tumors. Radiologist may miss tumors due to the overlapping of dense tissue (which may contain a tumor) to regular breast tissue. The patient too will have a reduce exposure to radiation.
It seems that CTC technology will work best in detecting p53 tumor suppressor protein since it is mutated in about 85 % of all cancers (1). According to the table it seems that the there are p53 mutations in all BRCA1 and BRCA2 mutations. Its amazing that in the future the CTC technology will be able to detect cancer cells in vials of blood collected from patients at annual doctor visits. When catching the cancer early it will allow for cancer treatments to work better and more effectively.
CTC technology for early detection of cancer can be used as a marker for overall survival and assessment of a therapeutic response. The benefits of CTC detection in early breast cancer and other solid tumours are reduction of tumors. In dealing with proto-oncogenes CTC technology would work best with EpCAM as a binding receptor in genes n-ras, h-ras, and k-ras. EpcAM is a transmembrane glycoprotein involved in cell signaling, migration, proliferation, and differentiation. EpCAM is expressed in the epithelial cells specifically at the cell membrane. Therefore if any mutation were to occur they would most likely occur in the colon or small intestine. OK – this makes sense in terms of the common denominator, as well as the EPCAM factor.
10 points out of 10
Cited Work
Dr. Namm. 2015. Week 10. What so devastating about a mutated p53 gene? https://uml.umassonline.net/webapps/blackboard/execute/content/file?cmd=view&content_id=_5776659_1&course_id=_69823_1&framesetWrapped=true
Week 10 (exam 2) score: 95 + 10 = 105
Wednesday, March 11, 2009
Part IV
The President Executive Order 13505—Removing Barriers to Responsible Scientific Research Involving Human Stem Cells Memorandum of March 9, 2009— Presidential Signing Statements Memorandum of March 9, 2009— Scientific Integrity
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Presidential Documents
10667
Federal Register Vol. 74, No. 46
Wednesday, March 11, 2009
Title 3—
The President
Executive Order 13505 of March 9, 2009
Removing Barriers to Responsible Scientific Research Involv- ing Human Stem Cells
By the authority vested in me as President by the Constitution and the laws of the United States of America, it is hereby ordered as follows: Section 1. Policy. Research involving human embryonic stem cells and human non-embryonic stem cells has the potential to lead to better understanding and treatment of many disabling diseases and conditions. Advances over the past decade in this promising scientific field have been encouraging, leading to broad agreement in the scientific community that the research should be supported by Federal funds. For the past 8 years, the authority of the Department of Health and Human Services, including the National Institutes of Health (NIH), to fund and conduct human embryonic stem cell research has been limited by Presidential actions. The purpose of this order is to remove these limitations on scientific inquiry, to expand NIH support for the exploration of human stem cell research, and in so doing to enhance the contribution of America’s scientists to important new discoveries and new therapies for the benefit of humankind. Sec. 2. Research. The Secretary of Health and Human Services (Secretary), through the Director of NIH, may support and conduct responsible, scientif- ically worthy human stem cell research, including human embryonic stem cell research, to the extent permitted by law. Sec. 3. Guidance. Within 120 days from the date of this order, the Secretary, through the Director of NIH, shall review existing NIH guidance and other widely recognized guidelines on human stem cell research, including provi- sions establishing appropriate safeguards, and issue new NIH guidance on such research that is consistent with this order. The Secretary, through NIH, shall review and update such guidance periodically, as appropriate. Sec. 4. General Provisions. (a) This order shall be implemented consistent with applicable law and subject to the availability of appropriations. (b) Nothing in this order shall be construed to impair or otherwise affect: (i) authority granted by law to an executive department, agency, or the head thereof; or (ii) functions of the Director of the Office of Management and Budget relating to budgetary, administrative, or legislative proposals. (c) This order is not intended to, and does not, create any right or benefit, substantive or procedural, enforceable at law or in equity, by any party against the United States, its departments, agencies, or entities, its officers, employees, or agents, or any other person.
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10668 Federal Register/Vol. 74, No. 46/Wednesday, March 11, 2009/Presidential Documents
Sec. 5. Revocations. (a) The Presidential statement of August 9, 2001, limiting Federal funding for research involving human embryonic stem cells, shall have no further effect as a statement of governmental policy. (b) Executive Order 13435 of June 20, 2007, which supplements the August 9, 2001, statement on human embryonic stem cell research, is revoked.
THE WHITE HOUSE, March 9, 2009.
[FR Doc. E9–5441 Filed 3–10–09; 11:15 am] Billing code 3195–W9–P
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