DNA editing is the science of the future wherein it will impact on disease treatment thereby promoting the well being of people. Thus, the discovery of the CRISPR-Cas9 system as a potential tool for DNA editing has revolutionized the prospects of biotechnology industry. Dr. Doudna highlighted the crucial role of Cas9 protein enzyme in DNA editing in her lecture while also highlighting laboratory studies that she and her colleagues have done in the past decades that resulted to promising information of the enzyme. It has the ability to cleave double stranded DNAs, target locations within a DNA to change, and its structural rearrangement as it binds the DNA are Cas9’s characterization that provides a clearer picture on its ability to be an important technology in DNA editing.
Dr. Jennifer A. Doudna, a specialist in ribonucleic acid (RNA) study, presented a lecture entitled “CRISPR-Cas genome surveillance: From basic biology to transformative technology” (nhivcast, 2015) during the annual Margaret Pittman Lecture as part of the Wednesday Afternoon Lectures of the National Institute of Health last March 2015. It highlights the role of CRISPR-Cas9 on DNA editing technology. CRISPR stands for clustered regularly interspaced short palindromic repeats which are found in bacteria that manifest acquired immunity from a virus in order to protect its host (Hsu, Lander & Zhang, 2014). Cas stands for CRISPR associated protein enzyme. The CRISPR-Cas system has been revolutionizing medical research and is expected to advance the search for genome editing (Sontheimer & Barrangou, 2015). Target editing to the DNA in terms of contexts and output in a simplified and efficient manner particularly in relation to human cells will truly revolutionalize medicine and biotechnology (Hsu et al., 2014).
Dr. Doudna interestingly introduced the topic by sharing a brief history that led to the discovery of the processes of CRISPR-Cas9. The manner in which she explained such processes made the audience appreciate the tons of work that have been put up in order to discover such break through biological process in relation to DNA editing technology.
It started with the discovery in 2005 of bacterial genomes that have the CRISPR loci which was hypothesized at that time to have developed some sort of an immune system to protect the genome through acquisition of tiny RNA sequences from foreign invaders. Experimental data supported this hypothesis years later when studies revealed that the CRISPR loci do have absolute capability to obtain immunity to plasma. Such immunity enables bacteria to identify foreign DNA that enters into cells while at the same time obtain small sequences from the foreign DNA into the CRISPR locus. The activities of the CRISPR-Cas directs the molecular destruction of this foreign DNA. Thus, in the year 2011, Cas9 is identified as genetically vital for the CRISPR system to operate. (nhivcast, 2015)
Sontheimer and Barrangou (2015) corroborate the historical information provided by Dr. Doudna that 2005 was the key year in determining the function of the CRISPR-Cas locus (p. 413) with the discovery of its immunity acquired function by independent reports from three groups which presumably included that of Jillian Banfield that Dr. Doudna shared in her lecture. They noted, though, that medical science first encountered it in 1987 from a K-12 obtained strain of E. coli (p. 413). Importantly, immunity that CRISPR system mediated is driven by three molecular processes namely, “acquisition, expression and interference” (p.414).
Function and Processes of Cas9 Protein Enzyme
Dr. Doudna then proceeded to discuss the processes involve on how Cas9 became a genetically vital inclusion in the operation of the CRISPR system by presenting the results of biochemical and cell based experiments that test its activities in an effort to figure out its function. She did this in a very systematic manner and related the experiment methods and results thoroughly to let the audience appreciate the functions and processes of Cas9 protein enzyme.
- Cleaving double stranded DNAs
These experiments showed that Cas9 cleaved double stranded DNAs at locations that are guided by the sequence of RNA molecules it binds. These locations must be near the location of a short motif known as the PAM. As it turned out, the Cas9 protein enzyme needed two RNAs where the second RNA molecule which is known as the tracer is vital in processing the CRISPR RNA. It then formed a structure that is important in engaging Cas9 protein enzyme that will eventually bind RNA molecules. This process made Cas9 a programmable protein enzyme that cleaved DNAs on locations programmable by such RNA sequences. In a related discovery, Moineau et al (cited in Hsu et al, 2014) revealed that Cas9 is the sole enzyme that provides mediation to the targeted DNA cleavage
Cas9 can function with only a single RNA sequences. Further investigations led to the discovery of a simplified system to program Cas9. It happened when a linker was created between CRISPR RNA and tracer RNA that consequently created a single guided RNA that gave information on targeting and protein binding in a single molecule. This was revealed in an experiment wherein five versions of the single guide RNA was designed and tested to a purified plasma DNA molecule. The result showed small piece of the DNA released from the plasma that match the expected size of the site that Cas9 was programmed to identify it. Thus, Cas9 became a completely programmable protein through this system.
Dr. Doudna then related that this can be a prospective system for an RNA programmable editing of a genome that begins with dsDNA cleavage. It was at this point that she mentioned other studies from previous decades on how to engineer DNA such as the study on a double stranded repair model for DNA where double stranded breaks received by DNAs and cells create a route to repair these breaks by cutting on the location. Different experiments were done on ways to launch double stranded breaks on locations that can create changes in a genome through the use of proteins. These proteins were programmed to identify a specific DNA sequence and paired with the nuclei’s cleavage domain to become programmable endonuclease which actually worked. However, there is the problem of generation of new protein for every location in a genome that needs change. The system of the Cas9 protein enzyme is the answer to such problem since it provides a single protein that is the same for every experiment and makes targeted changes in different DNA sequences. Various studies around the world have supported this thesis.
Such presentation of facts with highlights from few studies mentioned by Dr. Doudna made the information about Cas9 protein enzyme’s role in genome editing legitimate and valid given the correlating results of various studies that confirmed the role of protein and later on specifically identified Cas9 to create changes in genomes of cells and organisms. The presentation of this information jumpstart from the discovery of certain information such as the earlier discovery of protein without naming specific type of protein that can help create changes in the genome which led to the inquiry of whether it is a possible to reengineer bounded short RNA molecules to identify different sequences of DNA. This eventually led to testing of Cas9 for targeted changes in the genome.
Just like Dr. Doudna, Hsu et al (2014) expressed the medical science community’s attraction to the idea that Cas9 can be a potential tool for an RNA- guided system for editing of the DNA. Many studies were conducted to harness or test this idea which included Dr. Doudna and her colleagues work. Thus, various characterization of Cas9 were revealed in these studies such as the revelation that it is transferrable (Siksnys et al 2011 cited in Hsu et al, 2014) and can be guided by crRNA to cut targeted DNA in cultured dish (Carpenter et al 2012; cited in Hsu et al 2014).
- Finding target locations
Dr. Doudna shared current works about Cas9 in the laboratory to understand how it locates targets within the DNA. They experimented on one molecule curtains of DNA. It showed that Cas9 interacts with locations that are not target sequence or are not a match to a guide RNA known as PAM sequence, a complex protein RNA. It revealed that the non-specific binding of RNA is actually binding of PAM. Also, it was observed that there was an increase in the competition as the number of PAMs increased. Thus, Cas9 do really interact with DNA but it is done only after its interaction with the PAM. Afterwards, it examines the nearby sequence for base paired maps to the guided RNA.
Thus, it was concluded that the process of DNA targeting occurs only in the following conditions:
- When there is a highly similar product binding to the DNA which is the protein that binds the DNA and cleaved it while remaining tightly associated with those cleaved ends;
- Binding first occurred at PAM motifs;
- The binding of PAM triggers Cas9 activities due to the structural rearrangement happening in the protein.
Hsu et al (2014), reiterates the importance of the ability of Cas9 to be able to target locations correctly since the editing of the DNA will result to its permanent modification. This is particularly crucial in its application in a clinical environment and to gene therapy; hence it is important to be able to achieve this. They pointed out, however, that the target identification of Cas9 can be tracked experimentally and evaluated systematically in terms of a mismatch (Hsu et al, 2014).
- Structural rearrangement binding to DNA
Another work highlighted by Dr. Doudna was a low resolution work they conducted wherein they used a negative stain electron microscopy to investigate the different forms of Cas9. They looked at the protein alone, and then looked at the protein bounded to a guide RNA and eventually they looked at protein RNA complex that was bounded to a substrate. The result of this observation was that the activity of RNA induced conversion of Cas9 into a dynamic conformation for the surveillance of DNA.
Cas9 started as closely conformed to a protein’s structural lobes containing catalytic centers which is mostly responsible for RNA binding. It was observed that as this was happening, there was a huge structural rearrangement happening that led to the creation of a pathway towards the center of these two lobes. DNA binding resulted to the creation of an RNA-DNA hybrid in this particular center. The model for this experiment was used in a high resolution work and resulted to the same structural rearrangement of Cas9 protein as it binds to the DNA.
- Activation of Cas9 nuclease domain as a result of PAM binding
Dr. Doudna’s group also programmed Cas9 by adding the PAM-mer which actually triggers Cas9 activities in terms of binding and cutting. A single strand of RNA can actually be cleaved when the PAM-mer is present. This is a programmable activity; hence Cas9 can be programmed with different RNAs.
New Aspects of CRISPR System
Dr. Doudna ended her lecture on the new biological aspects of CRISPR systems that currently emerged contributing to its high technological potential. They probed the manner of acquisition of a viral space sequences. It was found out that there were actually two genes found in all CRISPR-Cas system namely Cas1 and Cas 2. These have complex function. A biochemical experiment using purified Cas1 and Cas 2 proteins was conducted to determine how this complex insert spacers into the CRISPR locus. It resulted to substrates of reactions that were inserted into the CRISPR locus resulting to observed changes in the plasma. The formation of Cas1 and Cas2 is necessary for the invitro integration of DNA. Majority of the integration occur in the CRISPR locus where CRISPR repeats are necessary for integration particularly its sequence and structure.
Such updated aspects of the CRISPR system is promising enough to merit another round of studies until it is known without a doubt that it is a perfect tool for DNA editing. Dr. Doudna is giving her audience something to look forward to in the current and future studies of the function and characterization of the CRISPR system.
Application of Cas9
Dr. Doudna’s lecture implied that Cas9 has been and can be used intensively in research, biotechnology and medicine. Hsu et al (2014) illustrates the various applications of Cas9 in these fields as shown below.
- Facilitate various applications that target the engineering of the genome.
- Large scale DNA experiments to investigate the function of genes and clarify underlying variants in genetics.
- The ability of Cas9 to send multiple signals at the same time offers the potential to use it in the study of human diseases that are normally polygenic such as heart disease, diabetes, autism and schizophrenia.
Using Cas9 in the study of human diseases is the ultimate advantage it offers to the medical community particularly for diseases that covered much of the population today. Imagine the impact of this possibility to the future well being of the earth’s population.
Sontheimer and Barrangou (2015) acknowledged the DNA editing capabilities of CRISPR-Cas9 system which has been successfully established in both academic and industrial laboratories. The most crucial development that the medical science community awaits is its use in its development for clinical treatments for diseases. However, a lot of work has to be done yet to be able to reach that level given that the sgRNA and Cas9 technology is just three years old (p. 421). The works of Dr. Doudna and her colleagues as well as of other laboratories around the world has provided a stack of knowledge that further cleared the functional capability of the CRISPR-Cas9 system. In less than 30 years a lot has been discovered about the CRISPR system. Thus, it is a wonder what discoveries can still be made in the years to come.
Finally using the CRISPR-Cas system in clinical environment will have a huge impact in medical science and the health of the world population. Just imagine the possibilities of the use of this system in treating diseases particularly the leading diseases of our time such as cancer and other autoimmune diseases. But then again, the ultimate concern is the cost of the application of this system in the health care system of the world.
It can revolutionize the manner in which diseases are treated or prevented in the future if it is finally used in clinical environment. However, the concern of making it mass accessible in order to create greater impact to the larger population is another matter. It would be a waste that this technology would become so expensive that it can only be accessed by higher income individuals. Thus, the prospects of DNA editing to be possible using the CRISPR-Cas system is just another bigger concern in the future since the much bigger concern is whether it will be available to all at a cheaper price. If not, then it is just something that will not be practically appreciated by most of the populace.
The commercial prospect of DNA editing is very huge such that it can truly revolutionize biotechnology and medicine. However, there is also the danger of using this technology in manners that are not originally intended such as harming certain population in order to take advantage of them. A measure in proper implementation and legal guidelines is highly recommended in its future implementation in order to deter any negative implementation of this tool.
Doudna, J.A. [nihvcast]. (2015 March 16). CRISPR-Cas Genome Surveillance: From Basic Biology to Transformative Technology [Video File]. Retrieved from https://www.youtube.com/watch?v=WnP4384WWTM
Hsu, D., Lander, E.S., & Zhang, F. (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6), 1262-1278
Sontheimer, E.J. & Barrangou, R. (2015). Bacterial origins of the CRISPR genome-editing revolution. Human Gene Therapy, 26(7), 413-424.
Novartis’ Drug Development for Neurodevelopmental Disorders in Children
Novartis’s return to research and development in the field of neuroscience is a welcome turnaround from the pharmaceutical industry. The community is hopeful that this development will again lead the industry in making a dent in the field of neuroscience. The current time is faced with several neurological disorders that will define a large chunk of its population in the future such as depression, alzheimer’s disease, timothy syndrome, phelan mcdermid syndrome and other neurological development disorders, among others. These disorders affect a large spectrum of the society which makes it an imperative initiative to find ways on how to treat these diseases. Treating these diseases, however, require a broad base of understanding on its occurrence and mechanism in order to create an effective treatment intervention.
Dr. Ricardo E. Dolmetsch is the Global Head of Neuroscience at Novartis Institute for Biomedical Research. He gave a lecture entitled “A New Day for Drug Development in Neuroscience” (nihvcast 2015) which discusses the current progress made by Novartis in the development of drugs to combat neurological disorders. He specifically studies the neurodevelopmental disorders in children and is trying to come up with a drug for it. He introduced his lecture by presenting the goal of Novartis supported by statistics on neurological disorders.
Novartis recognized the human suffering caused by neurological disorders with the large number of people suffering from it. He cited, for example, Europe’s’ 50,000 deaths from suicide and the 15 million people suffering from some form of addiction. He shared that it is predicted that by 2050 Alzheimer’s disease (AD) will claim 130 million people by the year 2050. He also cited autism as a neurodevelopmental disorder in children wherein one among one hundred children will need lifetime care. Dr. Dolmetsch admitted that finding treatments for these diseases has not been successful which actually not the case years ago were. He said that pharmaceutical companies are moving out of the field of neuroscience in terms of research to find a drug to treat various neurological disorders. Novartis’ reentry into this field is good news in the pharmaceutical industry. In fact, Haberman (2013) echoed the thoughts of many if Novartis reengagement with research and development in neuroscience will also lead back the entire pharmaceutical industry to such endeavor.
However, the breakthroughs in neuroscience are difficult if not scarce. Thus, pharmaceutical companies negatively relate their success with neuroscience. (nihvcast, 2015). This is confirmed by the Forum on Neuroscience and Nervous System Disorders (2011) by the Institute of Medicine by the National Academe when it pointed out the lack of consistency between academic research and product research and development by private sector which is represented by the pharmaceutical industry. The latter have become risk averse due to higher costs and higher rate of failures.
Why is it difficult to find drugs for neuroscience?
Dr. Dolmetsch outlined the reasons why is it difficult for pharmaceutical companies to discover drugs for neuroscience (nihvcast 2015). Presenting this almost at the start of his lecture provided interesting rapport with the audience which kept their interest towards his main presentation on the programs of Novartis which presumably had taken in mind his points for this particular presentation in order to effectively develop drugs for neurodevelopmental disorders.
- Disease and target selection
Pharmaceutical companies choose diseases that they do not understand thoroughly. To some extent, they are fooled by previous discoveries on these diseases. Depression, for example, was being treated as a single disease when in fact it is not. Thus, it is the aim of Novartis to match each of its programs for drug development to a patient and is population. Also, there is a need to take advantage of current drugs that actually alleviate symptoms of neurological disorders even though it was not developed specifically for such diseases. Thus, it is necessary to further develop such drugs to specifically coincide with neurological disorder treatment. Further, pharmaceutical companies must seize the opportunity to study human genetics in relation to neuroscience through observation of behaviors. This is particularly timely because of the large reduction on the cost of genome sequencing making it least costly to launch neuroscience experiments.
- Preclinical modeling and assay development
Previous preclinical models were not actually predictive of future of the disease due to the lack of assay development. Pharmaceutical companies must find ways on how to convert genes effect on behavior into an assay.
- Screening and optimization
It takes a lot of time for drugs for neurological disorders to get into the brain and at the same time be really safe.
- Clinical development
It is very difficult to attain clinical development due to the diverse population of patients suffering from neurological disorders; hence final stages of clinical development are very hard to measure.
Novartis Programs for Neurodevelopment Diseases
Dr. Dolmetsch (nihvcast 2015) outlined the various programs being undertaken by Novartis in relation to trying to find treatments for neurodevelopment diseases in children.
- Timothy syndrome (TS)
Cutaneous syndactyly, hyperglycemia, autism and cardiac arrhythmia characterized TS as a neurodevelopmental disease. Mark Keating of Novartis discovered an increasing mutation in the Cav1.2 gene, a voltage expressed in both the heart and the brain resulting to changes in its channel forces wherein it continue to open instead of opening and closing.
Novartis studied the finite type of cells from children suffering from TS through gathering of neurons that form part of the development of a series of cellular assays. For example, the assay on dendtridec carbonization identified L type channels that form dendrites in the developing brain of these children with TS. A light activated ion channels were introduced into the cells to stimulate it to discharge any potential for action which was observed for 12 hours. A software was prepared to extract participants from any extension and retraction activities. Dendrites normally expand over time in TS mutation as seen in the cells of seven control individuals. That is why retractions are received immediately after the cells are stimulated. Calcium independence, ectopic recruitment of related proteins is the indentified mechanism for this activity. Dr. Dolmetsch used this information to develop a phenotopic acid sensing ion channel (ASIC).
Dr. Dolmetsch’s team developed a chemical screen targeted at agents that reverse TS dendritic phenotype wherein light stimulation were done to untamed cells resulting to a few hits. For example, a block of CDK5 known as roscovitine derivatives can be reversed independent of CDK5 by binding the mutant L type channel that reverses the molecular defect deactivating the mutating L type channel.
- Phelan McDermid syndrome (PMDS)
PMDS is also known as 22Q13 deletion syndrome which is caused by deletion of a large amount of genes in chromosome 22. It is characterized by neonatal hypotonia and lack of or severely delayed speech.
Dolmetsch’s team was interested in identifying the gene significant for its underlying phenotype. Protein Shank 3 was used for this observation. It is a synaptic scuffle protein where a wide range of proteins are bounded at a densely synaptic. An assay was developed by looking at the synaptic transmission. It was then immediately discovered that the synapsis in cells are very variable. The density, age and health of the cells are important factors. An assay was eventually created that contains control cells and PMDS cells in the same experimental plate at the same time to allow for simultaneous observation and reporting. Harsh phenotypes of young developing neurons were immediately observed. Attractive responses from ampha receptors and NMDA receptors were observed in controlled cells while shorted synaptic responses were observed in PMDS cells; hence it is observed that there are defects in the latter cells.
Interventions were then given to the cells to determine if the defects in the cells are rescued through the expression of Shank 3 and IGF1. Indeed, synaptic transmission defects in PMDS neurons were rescued by Shank3 and IGF1expressions. However, the former did not rescue all response specifically unable to rescue plasma cells. The team then tried ways to rescue these plasma cells through the expression of IGF1, a growth factor which is a heterozygote mutation of Shank 3. However, as it turned out IGF1 reduced the amount of Shank 3 thereby creating a set of synapse leading to the conclusion that human neurons have an entire synaptic complements compared to rat neurons. Patients of PMDS have severe phenotypes probably because they lack a whole set of synapses.
Such discovery led to the creation of a speculative model that a PMDS patient have a half amount of Shank 3 proteins with few immature synapsis. IGF1 is able to acquire these immature synapsis to become mature synapsis which is the reason why the defect is rescued. However, small clinical trial testing on the effect of IGF1 on the social withdrawals of children with PMDS showed in the negative leading to the conclusion that IGF1 in itself is not an effective drug to treat PMDS because it does not immerse in the brain with a lot of side effects. This leads Dr. Dolmetsch’s team to work on another program similar to IGF1 but is safer and more effective.
- 22Q11 deletion syndrome
22Q11 deletion syndrome is a congenital disease of the heart with abnormalities in the palate and auto immunity. Novartis became interest with this disease since it was found out that 25% of children afflicted with this condition have autism while another 25% have schizophrenia. It is the most common cause of psychosis based on genes.
Dr. Dolmetsch’s team were making IPS cells from individuals suffering from this disease which partially explains what is amiss in children suffering from 22Q11 deletion syndrome. It was observed that a large number of their genes are wrongly regulated. However, there was an overlapped of these genes with the genes connected to wider schizophrenia. It was further found out that there are physiological defects in the neurons of patients with 22Q11 syndrome due to smaller calcium level. This low level of calcium, however, is not due to the impairment of calcium expressions. It is actually secondary to the decreased potassium channel. Some of the cells’ phenotypes are reversed when the antipsychotic drug dopamine D2 was used for treatment.
- Dravet syndrome (DS)
DS is an intractable early onset of epilepsy. Novartis is interested in finding a drug for this disease since it is not just characterized by epilepsy but also connected to severe abnormalities in cognition such autism and developmental delay. Such abnormalities are due to heterozygous loss of functional mutation in the sodium channel Nav1.1. A member of Novartis, Yishan Sun, created protocols for inhibitory and excited turnarounds as well as markers to identify mature inhibitory neurons. At the same time he developed a sodium current through the use of automated electrophysiology. Testing this protocols resulted to reduced Nav1.1. currents on inhibitory neurons of DS patients as well as reduced defects in excitability neurons. It also revealed that DS patients have excess levels of sodium currents in their inhibitory neurons while having a normal sodium currents level in their excitability neurons. This discovery supported the thesis that epilepsy in these children is due to a discriminating effect in the inhibitory interneurons.
Clinical trials for this model showed that a derivative of cannabis acts as anti-epileptic. However, therapeutic doses of this derivative of cannabis did not rescue all the phenotypes concluding that it is not the perfect treatment for DS. Currently, the group of Dr. Dolmetsch is developing their own compounds to hopefully all the electronic activity in DS cells.
Dr. Dolmetsch concluded his lecture by providing tips on how to improve clinical trials for treatments of neurodevelopmental diseases in children and for other diseases. An improved system to measure outcomes is necessary through patient stratification based on genetics and pathophysiology and not based on behavior which is important if a drug is being tested for mutation. Also, developing a final outcome that continuously measures behavior in real life and not measuring it on every episode of trials. Further, there is a need to implement the placebo effect and evaluate individual improvement of patients involved in the study.
Dr. Dolmetsch lecture was very enlightening in terms of the effort that a pharmaceutical company takes in researching and developing a drug to treat neurological disorders. He is right that there must be an improved system to measure outcomes of clinical trials to ensure a high rate of success thereby limiting losses on the part of the pharmaceutical companies. Also, an improved outcome measurement will also pave the way for a potential increase in the rates of success from disease exploration and understanding to treatment.
Neuroscience is very important in today’s clinical society particularly with the increase in the cases of neurological disorders plaguing the world’s population. It is imperative that research and development should focus on this area. The academe and the private sector must be aided by the government in finding ways to work with each other. Also, such government intervention should find ways to lessen the burden of cost on the part of the private sector in the form of government sponsorship, subsidy or donations to ensure that research and development on the treatment of neurological disorders are expanded and focused. Lastly, the government can partner with the private sector to co-share cost or co-used facilities and human resources to intensify neuroscience research and development.
Neuroscience can become the future brand of pharmaceutical companies wherein research and development provide opportunities to fully understand the human brain and its underlying mechanisms. This way creation of treatment is direct and successful.
Dolmetsch, R. [nihvcast]. (2015 June 24). A new day in drug development in Neuroscience (Neuroscience Reborn) [Video File]. Retrieved from https://www.youtube.com/watch?v=3ShXtXKjpc8
Institute of Medicine (2011). Forum on Neuroscience and Nervous System Disorders: Future Opportunities to Leverage the Alzheimer’s Disease Neuroimaging Initiative. National Academic Press.
Haberman, A. (2013). Will Novartis lead a pharma return industry return to neuroscience R&D?, Biopharma Consortium Blog. Retrieved from http://biopharmconsortium.com/2013/12/10/will-novartis-lead-a-pharma-industry-return-to-neuroscience-rd/