Mapping into Genomics Potential: A promising breakthrough or another technology landmine

Affordable and rapid DNA sequencing has caused a revolution in the medicine and health-care sectors globally, while genomics can be seen as a paradigm shift in health practices and outcomes, as it ushers in a new era of precision healthcare in which preventive methods, treatments, and health advice reach the right person at the right time. The cost of reading DNA sequencing that is, genomics has shot up to about $100 million in the last 15 years. Its implications can be found in genetic diagnosis and the innovative treatment of cancer and other rare diseases.

Genomics is the leading technology unfolding a revolution in healthcare because its study and applications consistently reflect changes in clinical trials and consumer health. The very first study of genomics published in 1977 introduced the concept of DNA sequencing to develop the first full genome of a virus (phiX174) (1). But little was known about of what has now become a breakthrough strategy to prevent disease and late treatments: advanced genetic research.

Genomics is of interest to everyone, as not only does it forecast change in the health-care sector, but one can also discern it happening now, as stated by Dr. Barlow. The FDA has approved over 250 drugs now prescribed based on patient genetics, and it is no surprise that the number has tripled since 2014 (2).

Everything you should know about Genes:-

Genes, a fundamental unit of hereditary in humans, resides on the chromosome, it is a long filamentous structure within the cells that comprise tens of thousands of genes linked together in chains (3). Humans own 46 chromosomes in total, in two pairs of 23 from each parent. So, an entire set of genetic instructions carried by an organism is genome (bibliography of all genes with footnotes, references, annotations etc). With advanced technology one can alter some of these genes, thereby modifying human states and physiologies deliberately.

This transformation from evolution to manipulation makes genetics an unparalleled aspect beyond the realm of science. Our reckoning of genes has now reached such a level of dexterity that power is not restricted to altering genes in a test tube but also in their native context in human cells. The potential to predict the fate of an individual from his/her genome has become advanced (although its true potential remains unknown).

Before you start: An insight into key terms:

Gene- A fundamental unit of hereditary.

Genome- It encompasses total genetic material of an organism which cache biological information. It may be either DNA or RNA.

Prokaryotic genome- Possess DNA genome

Eukaryotic genome- DNA genome that consists of two distinct parts: nuclear genome and organelle genome (mitochondrial and chloroplast).

Genomics*- It is the study of genes, their functions and related techniques. It encompasses all genes and their interrelationships in order to discern their combined effect on the growth and development of the organism.

Genetics**- It is the study of heredity. It probes into the functioning and composition of a single gene.

Genome Sequencing- A study of the composition of a person’s DNA in to look for a wide diversity of mutations and variants, using a microarray, a DNA chip or whole sequencing of the entire genome.

 A Milestone in the evolution of Genomics Research-

Many early discoveries led to the study of genomes as it is applied today. Charles Darwin work, entitled “On the Origin of species by means of natural selection” in 1859. Studies on the origin of genetics started with Gregory Mendel, when in 1865, he introduced fundamental laws of inheritance, this was crucial for understanding that characteristics of an organism are inherited through genes. Mendel’s rule was further experimented by Thomas Hunt Morgan in 1910, which first showed that genes are located on chromosomes. Here’s a closer look at where it all started, from the earliest documented studies on genomics and other ‘firsts’ through where we are today (4).

1888- The term ‘chromosome’ described by Waldeyer.

1890- Hugo de Vries first introduced the concept of the gene (a fundamental unit of hereditary).

1902- Boveri proposed a relationship between chromosomes and cancer.

1940- Human eugenics used to explain experiments, sterilization etc.

1953- TheDouble helical structure of DNA was described by James Watson and Francis Crick.

1956- Levan and Tijo reported human chromosome number that is total 46.

1970- Two technologies evolved in genetics namely, gene cloning (writing) and gene sequencing (reading) of genes.

1977- Sanger proposed and developed the method of sequencing.

1980- Use of gene cloning to map genes linked diseases such as Cystic fibrosis, Down’s syndrome, Tay Sach’s disease or screening of BRCA1 or BRCA2, if carrying deleterious mutation can be aborted that involves genetic diagnosis, management, and optimization. This picture is the presence of genetics advancement, not a distant future.

1982- Fluorescence in situ hybridization (FISH) method was developed.

1990- Human Genome Project (HGP) was launched, it isa joint venture of international, government and private sponsored effort to map and order the entire human genome. It was developed by the US Department of National Institutes of Health (NIH) & Energy to make approximately 35,000 human genes accessible for further biological study.

1992- Comparative genomic hybridization (CGH) was developed.

2000- Massive parallel sequencing (MPS) developed by Lynx Therapeutics.

2001- The first draft of The Human Genome Project was reported.

2003– The ‘Human Genome Project’ was officially completed.

2004- 2nd and 3rd generation sequencing was launched by Applied Biosystems, Roche company, Illumina, Oxford technologies Nanopore, Pacific Biosciences, Helicos Biosciences, and Solex.

CRISPR- A revolutionary technology

In 1990’s there was a concise foray into genetic engineering in humans but gene editing was expensive, complicated and time-consuming but now it has transformed to a revolutionary technology named CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) which is now on center stage (5). With the advent of CRISPR, cost of gene editing plummeted to around 99% less as previously compared cost. Now the process takes few weeks rather years and can be performed in the lab by anyone. It has changed human potential to develop a new era of designer babies to ending diseases (though in near future).

Genome engineering-Decoding Science of CRISPR

CRISPR is a unique blend of being extremely potent yet easy to access genome-editing technique in a scientist’s arsenal for treatment. The technique works on two basic methods: a CRISPR associated (Cas9) nuclease incise DNA and a guide RNA that inform nuclease accurately where to cut the genome. To enable real-world application of CRISPR in a lab, a researcher must be equipped with the basics of CRISPR.

Everything you need to know about CRISPR- Cas9

In microbial immunity, CRISPR plays an important role, for instance, when a virus infects a bacterium, the surviving bacterium activates their antivirus system by saving part of DNA in their old genetic code until needed. The bacterium employs DNA archive known as CRISPR-Cas9 to cut off viral DNA and direct nuclease to make RNA copy through guide RNA (gRNA) that is similar to the target part of the viral genome. Cas9 as GPS scan signs of virus invader by comparing every DNA, when matched, gets activated and cuts off a fragment of a viral genome.

The snipped DNA portion is thus stored in the palindromic CRISPR sequences to store genetic material as cache memory to disable future attack from the same virus.

CRISPR acts as a precise DNA surgeon which is reprogrammable, that is a copy of DNA can be put in a living cell to be modified and as can edit a live cell, target and study every type of DNA sequences not only in humans but also in micro-organisms plants and animals.

Biohacking CRISPR Potential to End Genetic Diseases

The rituals of medical care via learning the genetic code of an individual may be a powerful prevention tool of genetic disease, according to some studies. Taking it seriously, even if it is not explainable, may be worth the effort. Every day, everywhere, our connection to nature (gene) and nurture (triggers) are infinite.

In 2015, a study published the application of CRISPR technology in removing HIV virus out of the living cell in lab condition. Later in 2016, the study was performed on a larger scale, in which CRISPR was inserted in tails of a rat having HIV virus, resulted in the removal of about 50% of the virus from the cells (6).

The first trial of CRISPR technology in cancer patients was approved by US National Institutes of Health (NIH) in 2016 (7), whereas first time in the world treatment of patients with lung cancer with CRISPR was announced in China, a team led by Lu You, an oncologist (8).

In practice, CRISPR is at a nascent stage as a therapy, scientists are doing clinical trials with the help of CRISPR to end genetic diseases more in animal models than humans but there is more to unravel. The following chronic and genetic diseases are explored by researchers to apply the promising effects of CRISPR to treat the disease.

1. Cancer

Doctors at The University of Pennsylvania have planned to use CRISPR to alter human immune cells to kill tumor cells. This is first of a kind of human test in the US that will take place later in the year 2018 that already in process in China. For this, around 18 patients battling with three different kinds of cancer- sarcoma, melanoma and multiple myeloma will be enrolled. Application of CRISPR will involve removing a gene that encodes for a receptor known as PD-1, tumor cells bind to it and employ immune system not to attack. Thus engineering T cells of cancer patients in ex-vivo and then re-infusing those in the patients will wipe out unhealthy tumor cells (9). In China, the CRISPR engineered T-cells have been applied in 86 patients whose results will be out soon.

Study 1-: Biomarkers of a rare mutation in cancer cells:

A new computational method called Lancet has been devised by researchers at the New York Genome Centre (NYGC) recognizes rare gene mutations in cancer cells with a precision and responsiveness greater than those of the current available procedures. It identifies mutations in tumor cells, using a process known as a somatic variant calling. Lancet is an important toolkit that allows scientists to detect accurate somatic variations, forming the foundation for personalized medicine which improves the conditions of cancer patients (10).

How it works-: Unlike the sequencing of tumor and normal cell genomes through present computational methods, Lancet uses a micro-assembly procedure to reconstruct a complete sequence of small areas of the genome without the use of a reference to determine variants. With the application of a colored deBruijn graph, Lancet analyzes tumor and normal DNA resulting in the differentiation of tumor variants from those present in an individual’s healthy tissue.

2. Blood Disorders- β-Thalassemia, a genetic disease would be the first clinical trial in Europe to be treated using CRISPR Cas9 gene editing tool in 2018. The therapy is a joint venture of CRISPR Therapeutics and Vertex Pharmaceuticals in 2015, and their second trial would be for sickle cell anemia (11). The process involves removal of hematopoietic stem cells from patient and editing outside the patient’s body in the lab to develop red blood cells with high levels of fetal hemoglobin, a naturally produced protein in newborns, which bind to oxygen more than adult form of hemoglobin. Edited cells are then transfused back in the patient to treat a genetic disorder that impairs oxygen transport in the blood. Using CTX001, over 80% of human hematopoietic stem cells engineered and their infusion in mice model showed increased production of fetal hemoglobin. Now, the trial will be done in humans in Europe, but these clinical trials are already in process for over a year in China.

Study 2: Precise gene therapy to cut unwanted genetic mutations in blood disorders

A study published in Genome Research, researchers reports a modified version of CRISPR Cas9, a gene editing tool to correct defective gene with fewer errors (12). They demonstrate the precise role of Single Nicking in the target gene and donor (SNGD) that suppresses unintended genetic mutation over the conventional method. In one of their experiment the conventional method made potentially harmful errors about 90% of the time, whereas SNGD reported harmful errors less than 5% of the time as it snips one strand of the DNA strand, not both of the strands to achieve desired genetic edits.

3. AIDS- The gene editing technique CRISPR works in two possible ways to treat the HIV virus. Firstly, it cuts the HIV virus out of DNA of the immune cells. Secondly, to make one resistant to HIV infection, individuals are born with natural resistance to HIV because of mutation in gene CCR5, which bind to a receptor on the surface of immune cells that HIV penetrates inside the cells. The mutated gene changes structure so virus no longer binds to the receptor. There have been clinical trials in animal models but not in humans yet.

Study: CRISPR Cas9 a promising gene editing tool to deactivate HIV-1 gene

Worldwide around 35 million people are affected by HIV 1 (Human immunodeficiency virus) which is a chronic disease that proliferates in a patient’s body and gets inserted to the chromosome in the infected cell. Antiretroviral therapy (ART) can control the infection to a certain level but it is not the complete cure (13).

The two genes known as tat and rev regulates the proliferation HIV-1 was targeted and subsequently, six types of guide RNA were designed based on genetic information from major six HIV-1 subtypes. The researchers created a lentiviral vector in which Cas9 and g- RNA gets expressed. There was a significant decline in the expression and function of both tat and rev when the vector was introduced in cultured cells that expressed regulatory gene product of tat and rev. No off-targets were reported along with any harmful effect on the survival rate of the cultured cells, these findings were published in Scientific Reports on May 2107.

4. Hungtington’s Disorder- A neurodegenerative genetic condition that results from an abnormal repetition of specific DNA sequences within which the Huntington gene. Higher the number of copies early will be the onset of the disease. Treating this disease should be precise as any off-target effects and immune reactions cause more dangerous complication in the brain. Researchers have developed KamiCas9, another version of Cas9 that have a ‘self- destruct button’ for a Cas9 enzyme, wherein CRISPR is encoded with instructions to cut the sequence of its own cas9 enzyme (14).

Study 2: New improved gene editing technique for inactivating defective gene in Huntington Disease

A study published in Frontiers in Neuroscience in 2018 reported a new variant of CRISPR Cas9 which is more specific and safer for treating Huntington’s disease. Till date, there is no proper treatment for this disease. To make excision of repeat gene of the disease requires much more improvement to cut specific DNA sequence and not other correct DNA sequences that can trigger other unwanted changes before it can be applied to humans directly.

How it works: The process involves snipping patient’s defective gene which is the prime treatment that requires accurate and effective methods (15). Scientists have tested Cas9 nickase pair that removes toxic protein synthesis in cellular models of the Huntington’s patients along with inactivation of Huntington gene.

5. Cardiovascular Disease- The American Heart Association in their study reports the potential of genomics to understand cardiovascular disease at the molecular level (16). A person’s unique genes can make personalized predictions about risk factors and cardiovascular traits that are likely to develop heart disease in future and subsequent treatment. DNA carries genetic information that is ‘translated’ via RNA into proteins and molecules, which is responsible to carry out specific roles in the body. Genes made up of DNA carry hereditary traits of our ancestors that are stable during one’s lifespan but can undergo certain environmental triggers such as smoking, unhealthy diet and lack of physical activity.

Genomics establishes an association between patterns of molecular variations including DNA, RNA, and microorganisms in the gut to maintain optimal health. At present, non-invasive blood test an example of genomic medicine is available for heart transplant patients. Now, researchers are keen to find the outcome of induced pluripotent stem cells (iPSCs), which is grown from skin or blood cells of the body and can be converted to any type of cell, a noninvasive method, these are developed to determine the best treatment course to be given to a patient. Although it is in the early stage of development is a promising tool for cardiovascular health.

Contraindications:-

Application of genetic engineering through CRISPR is limited to the individual and dies with the patient excluding in reproductive cells and embryos, where they can be reprogrammed. CRISPR technology can trigger wrong edits or even unwanted changes that may remain unnoticed while editing of the mutated gene to disable disease, this corresponds to the complex interplay of our genes.

The Takeaway-:

As the human lifespan has ascended due to advanced medical interventions, scientists have become much more interested in extending the scope of genomics. Scientists now have the genomic tool that will unveil future risk factors for disease and be touted as a personalized cure.

Bottom line about Genomics:-

Both clinical and animal trials indicate that in terms of prevention or remedy there is a lot to be achieved in the future because many facets of genomics are much more beneficially modifiable than people are familiar with. One major bottleneck that is needed to be widened is the establishment of a universal set of biomarkers or guidelines to hack the potential of genomics in medical and human health, though it remains undefined.

References:

1. Heather J M, Chain B (2016). The Sequence of Sequencers: The history of sequencing DNA. Genomics. 107(1):1-8. DOI: 10.1016/j.ygeno.2015.11.003.

2. http://www.sciencedaily.com/releases/2018/05/180504103811.htm.

3.https://www.encyclopedia.com/science-and-technology/biology-and-genetics/genetics-and-genetic-engineering/chromosomes.

4. Durmaz AA et al (2015). Evolution of Genetic Techniques: Past, Present and Beyond. Biomed Res Int. DOI: 10.1155/2015/461524.

5. https://futurism.com/crispr-tested-on-a-person.

6. Kaminski R, Chen Y, Fischer T et al (2016) Removal of HIV-1 Genomes from Human T lymphoid cells by CRISPR/Cas9 gene editing. Sci Rep.2016; 6:22555. doi: 0.1038/srep22555. 

7.https://www.nature.com/news/first-crispr-clinical-trial-gets-green-light-from-us-panel-1.20137.

8.https://www.nature.com/news/chinese-scientists-to-pioneer-first-human-crispr-trial-.20302.

9. Ratan ZA et al (2018) CRISPR-Cas9: a promising genetic engineering approach in cancer research. Ther Adv Med Oncol. DOI: 10.1758834018755089.

10. Giuseppe Narzisi, André Corvelo, Kanika Arora, Ewa A. Bergmann, Minita Shah, Rajeeva Musunuri, Anne-Katrin Emde, Nicolas Robine, Vladimir Vacic, Michael C. Zody. Genome-wide somatic variant calling using localized colored de Bruijn graphs. Communications Biology, 2018; 1 (1) DOI: 10.1038/s42003-018-0023-9.

11. https://clinicaltrials.gov/ct2/show/NCT03655678.

12. Nakajima K, Yue Zhou Y, Tomita A, Hirade Y, Gurumurthy CB, Nakada S (2017) Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair. Genome Research. DOI: 10.1101/gr.226027.117.

13. Youdiil Ophinni, Mari Inoue, Tomohiro Kotaki, Masanori Kameoka. CRISPR/Cas9 system targeting regulatory genes of HIV-1 inhibits viral replication in infected T-cell cultures. Scientific Reports, 2018; 8 (1) DOI: 10.1038/s41598-018-26190-1.

14. Merienne N et al (2017) The self-inactivating KamiCas 9 System for the Editing of CNS Disease Genes.  Cell Rep. 20(12):2980-2991. Doi. 10.1016/j.celrep.2017.08.075.

15. Magdalena Dabrowska, Wojciech Juzwa, Wlodzimierz J. Krzyzosiak and Marta Olejniczak. Precise Excision of the CAG Tract from the Huntingtin Gene by Cas9 Nickases. Frontiers in Neuroscience, 2018 DOI: 10.3389/fnins.2018.00075.

16. Kiran Musunuru K (2018) Interdisciplinary Models for Research and Clinical Endeavors in Genomic Medicine: A Scientific Statement from the American Heart Association. Circulation: Genomic and Precision Medicine. DOI: 10.1161/HCG.0000000000000046.

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