Genetic Mapping Proposal: Fighting Tuberculosis with CRISPR and Nanotech
Maui Arcuri, Angel Herrera, Shazad Outar, Jasper Saffa, Loadys Vargas, XiaoYing Li
The City College of New York
Summary:
Genetic mapping is a way to map genes in order to determine things such as the risk of developing disease. Genetic mapping can even be used for gene editing where genes can be cut out or replaced to stop or perform new functions. A good method of gene editing is with the tool CRISPR, which uses an enzyme that searches for specific genes to cut out or replace. Using nano technology that can help CRISPR to perform gene editing in humans, disease can be stopped from spreading and also permanently erase the disease among future generations.
The planned starting point would be to target Tuberculosis, a deadly and contagious disease that affects the lungs. It is caused by a bacteria and is characterized by the production of an enzyme called MMP-1. Ideally, research and clinical trials would be performed to pinpoint the gene that causes the increased production of MMP-1 in humans. The genes would be edited using CRISPR and nanotech. The estimated costs of the proposal is to be upwards of $300 million as lengthy research and trials will be needed which could take a few years. In addition, nanobots would need to be developed to help deliver CRISPR in humans. The goal would be to start first tests in an area with high cases of Tuberculosis such as Bangladesh and bring it to other areas upon successful trials.
Background – Genetic Mapping:
Even with the huge advances in health practices and techniques over the past decades, life-threatening illness and disease continue to affect millions around the world. It is estimated that a little over 600 thousand people will die of cancer in 2019 in the US alone (Simon, 2019). The good thing is there is lots of research being poured into treating and curing many of today’s problematic diseases and health issues. One breakthrough method of treating disease is treating them at the gene level of human biology. This method involves the process of genetic mapping.
Genetic mapping is the process by which one can determine the position of certain genes in a chromosome that carries specific traits or diseases. Chromosomes are structures found in the nucleus of most living cells. They contain most of the DNA of living organisms. Researchers have tried genetic mapping on plants, animals, and humans. In humans, researches collect blood samples of many people from the same family. Then, they isolate the DNA from the samples and look for markers (Genetic Mapping, 2015). Markers are the unique patterns or sequences that are found on the family members that share the same disease or traits. Markers give researches an approximation of the location of the gene that causes the specific disease or trait in a chromosome. These genes can be identified through positional cloning which roughly approximates the chromosomal location of the gene on the candidate region. The candidate region is found through linkage analysis which identifies when two genetic markers are close to each other because these are more likely to be transmitted to the next generation (Genetic Mapping, 2015).
The benefit of genetic mapping today is that it can tell people how likely they are to develop certain diseases. This is very important nowadays when the health care system cares more about treating diseases rather than preventing them in the first place (Drell, 2013). But thanks to genetic mapping, which is now more affordable, people can have access to their genetic information and try to prevent getting the high-risk diseases shown on their reports. However, genetic mapping is still in progress and there are more important achievements that are yet to come.
Our group will focus on taking genetic mapping to a new level where people would not only see their genetic information but would be able to modify it to prevent themselves and their next generations from developing unwanted diseases. We would in particular like to focus on treating Tuberculosis (TB) with gene modification as a starting point or first stage of our proposal. Although tuberculosis is treatable and curable, millions are still affected by it to this day with 10 million affected in 2017 (10 facts on TB, 2018). TB causes a deadly breakdown of lung tissues with symptoms such as bad coughing, a painful chest, coughing blood among others (TB Disease: Symptoms and Risk Factors, 2019). If we can successfully stop TB with gene modification, this could pave the way for many other diseases to be treated by gene modification.
Nanotechnology and CRISPR:
Our proposed method of gene modification is through the use of CRISPR methods and nanotechnology. Before we explain what CRISPR is, we’d like to clear up misconceptions on nanotechnology. When someone says that, what may come to mind are tiny, microscopic robots that can do all sorts of things. However, the reality is this isn’t possible due to physics limitations and may not be possible for many more decades (Bhat, 46). Nanotechnology refers to things in nanoscale, around 1 to 100 nanometers. To put this in scale, a sheet of paper is about 100,000 nanometers thick. Much smaller is a strand of DNA at 2.5 nanometers thick. Our cells are kind of like nanobots themselves which perform specific biological tasks. Nanobots that exists today are usually made of organic compounds such as proteins and bacteria which perform simple tasks such as delivering medicine or detecting certain particles (Diamandis, 2016).
CRISPR stands for Clustered regular short palindromic repeats. It can be thought of as the library of the bacteria in the immune system. When a person gets sick and successfully fights off the infection, the DNA fragment affected is stored in this “library” for the immune system to refer back to if faced with the same or similar infection. If invaded again with a similar infection, the bacteria will produce an enzyme called Cas9 and together with CRISPR can identify and effectively cut the invading DNA. With the use of this system, scientists can effectively locate, cut and even replace parts of our DNA with ease. This modified DNA can be replicated and passed down depending on the location of DNA modified (Ryan, 2019).
This opens up an array of possibilities, providing the ability to cure diseases that are caused by a single-cell such as sickle cell anemia, and Duchenne muscular dystrophy. This use of CRISPR is commonly known as gene therapy. Often, diseases are characterized by a signature pattern of DNA. By cutting away the “infectious” parts, one can prevent the disease from ever forming. However, all of these applications haven’t been thoroughly tested on humans and one small change to our DNA can have a ripple effect on the rest of the body. Meaning that we simply don’t know all the implications that come with cutting large strands of DNA. The biggest barriers with CRISPR are the “off-target defects” these are instances in which Cas9 cuts another gene similar to the targeted one. An unintended cut or a gap in the DNA can lead to mutations in other genes, and in some cases even cancer (Ryan, 2019).
There is promising research though that can improve the accuracy and reduce unintended side effects from CRISPR. On Feb. 4 of this year, researchers at UC Berkeley, including CRISPR pioneer, Jennifer Douda identified another enzyme that works better than Cas9 called CasX. CasX is an enzyme that is not usually present in humans, however, it is small enough to be unnoticed by our immune system. This means that it can make more accurate and precise adjustments to the human DNA (Ryan, 2019).
Tuberculosis:
Tuberculosis makes a good candidate to target because of the way it spreads and initiates. When one is exposed to the disease, the deposition of the bacteria, Mycobacterium tuberculosis (M. tuberculosis) is what causes the disease. As M. tuberculosis spreads, lung tissue damage can get worse depending on the state of the host (Smith, 1). Contained in victims of TB are increased levels of an enzyme, MMP-1 which degrade collagens in the lungs. When cells are infected with TB, researchers found that the cells increased production of MMP-1 (How TB deystroys lungs, 2011).
Scope and Costs:
With the necessary background explained, our plan of action is to use CRISPR along with nanotechnology to target the genes that cause the increased production of MMP-1 in cells to stop TB. Nanobots would be used as a way to effectively deliver Cas9 or CasX enzymes to the target genes more quickly. First, we would have to have research done on the genes that cause the increased production of MMP-1. Then Cas enzymes would have to be edited to target that particular gene with a possible suitable replacement for the gene. Nanobots would then be developed in a way that they can carry the Cas to be deployed in a human. The treatment would be administered through multiple injections in individuals with TB.
While CRISPR kit costs are relatively cheap, about $100, research and trial experiments for developing new drugs are very costly (Ryan, 2019). They have a median cost of $19 million with some research going into the hundreds of millions (Cost of Clinical Trials, 2018). Because CRISPR and nanotechnology are relatively new and breakthrough, we predict that the total cost of research and trials would be an upwards of $300 million. We would need multiple teams of doctors, scientists, engineers, and more to develop the methods and nanotechnology for our proposal. After successful research and trials, we would like to implement the technology in a country with high TB rates such as Bangladesh (Countries with TB). To those administered with the technology, the disease would be stopped and ideally, new generations would be immune to TB. Some precautions of our proposal is that cutting out genes that cause MMP in TB or cutting our genes that cause other disease may yield unpredictable results. It could be a long, windy, and costly spanned over multiple years. However, the benefits gained if our plan is successful would be immeasurable as it would lead the way to treating many other diseases.
Technical Description:
There are several physical components to our proposal. This includes CRISPR / Cas enzymes, and nanobots. This section will detail exactly how these components work.
CRISPR / CAS9:
CRISPR stands for Clustered regular short palindromic repeats. This is essentially, bits of recurring DNA. As mentioned before, when our body is hit with a virus, we have bacteria that acts as a defense system to fight back. They use proteins to cut out the invader’s DNA. If the bacteria survives the viral attack, they store a snapshot of the virus DNA, so that next time they encounter the virus, it is quickly identified as an enemy. To organize the DNA library, the virus-DNA snapshot is stored in between the palindromic repeats as to not confuse this DNA with any other important DNA.
Above in Figure 1A, a Cas9 protein enzyme is shown. The RNA is the guide or snapshot it follows. When the protein recognizes instances on the DNA, it cuts out the DNA. On the opposite end of the guiding RNA, two pincer like appendages do the actual cutting of the DNA once the protein checks for a match along a DNA strand (Molteni, 2017). At this point, the Cas9 may only render out the matching DNA or it could optionally replace it with new DNA. We would replace the guiding RNA with the MMP-1 gene sequence that causes TB. When the Cas protein recognizes instances of the TB causing gene, it would be cut out.
Nanobot Assist:
Nanobots would play the crucial role of carrying the Cas proteins inside a human. There are several different designs and types of nanobots that could be used for this purpose. One type of nanobot could be a nanobot built out of organic components such as proteins or even out of DNA strands. A nanobot exists made out of DNA enzymes that has three arms and four legs. It moves around using its DNA legs which are attracted to longer DNA strings using it as a track (Diaz, 10).
Another option is a nanobot that is highly flexible and adaptable. They aren’t quite as small as DNA nanobots, but they are quite mobile and adaptable to changes in pressure and environment size. These nanobots are mass producible at reasonable costs being made out of hydrogel nanocomposites. They look like similar to strings of DNA and employ an origami-like folding mechanism to change size. The nanobots can be programmed to move with electromagnetic fields or to move on its own utilizing natural blood flow (Smart microrobots, 2019). This type of microrobot is the more viable option because it is faster and provides more function over the DNA nanobot.
Innovation Process:
This section details the process of the proposal with a general timeline and plan of action after funding.
Phase 1:
6 months – Establish contracts and hires with scientists and researchers knowledgeable with
CRISPR. Work with scientists researching hydrogel nanobots.
1 year – Create first Cas model that targets MMP-1 of TB, controlled testing
2 years – Develop improved nanobot with the ability to transport Cas proteins
3 years – Testing CRISPR with nanobots in controlled environments. Connect with health
organizations in Bangladesh and plan first trial testings.
4 years – Testing in mice with TB. Start manufacturing products in greater quantities.
Phase 2:
5 years – Carry out first human tests in volunteers. Implement operation in Bangladesh upon
successful trials.
6 years and beyond – Continue treatments and operations in Bangladesh and move to other
countries. Possibly expand operation to treat other diseases.
Phase 1 will be the more costly part of the operation. This part includes research, testing, building and manufacturing. Phase 1 is estimated to cost from 50 million to 150 million dollars. Phase 2 involves human testing and actual mass implementation of the treatment in Bangladesh. The costs here (30 million – 80 million) include manufacturing costs and administering of the treatments. We will use volunteers and / or local organizations to do the actual work of finding individuals fit to go through the treatment. It will be first used to treat people of a young adult age range and the demographic will expand through more trials. At this point, we will have started research to treat other diseases as well bringing a whole other dimension of possibilities.
References:
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