How will the worlds most powerful democracies deal with the ethical and legal dilemmas posed by the development of so-called killer robots, or lethal autonomous weapons systems (LAWS)? On the one hand, LAWS promise unparalleled operational advantages, like acting as a force multiplier, expanding the battlefield, and removing humans from dull, dirty, or dangerous missions. Authoritarian powers like China and Russia appear to be dedicating tremendous resources into pursuing these capabilities. On the other hand, giving autonomous weapons the authority to determine who lives or dies is an ethical, practical, and legal nightmare.A couple of states have well-documented policies, most notably the United States and the United Kingdom.

This article focuses on France, which has begun the difficult work of thinking through the ethical problems associated with lethal autonomous weapons systems. Im a member of Frances Defense Ethics Committee, which reports directly to the countrys defense minister. Last year, the committee submitted an opinion on the enhanced soldier, which drew a red line between acceptable, non-invasive practices, and unacceptable ones such as genetic engineering.

In April, the French defense ministry published another Defense Ethics Committee opinion on the integration of autonomy into lethal weapon systems. We argue that LAWS should be understood as fully autonomous weapons, which are ethically unacceptable for a number of reasons, but that partially autonomous lethal weapon systems (PALWS), which present both potential benefits and risks, could be ethically acceptable under certain conditions.

Why does it matter? Not only because, to the knowledge of this author, no other major military power has such an ethics committee playing such a role within their Ministry of Defense, and that itself tells something about the French ethos. The Defense Ethics Committees opinion on autonomous weapons is likely to be scrutinized for a number of other reasons: it was France that, in 2013, initiated the multilateral debate on autonomous weapons; it has been an active participant in the debate since then (see for example the 2017 French-German proposal); and also because France will chair the next Convention on Certain Conventional Weapons Review Conference in December 2021.

LAWS Versus PALWS

The committees first and arguably most important initial task was to define key terms. It decided to define LAWS as:

A lethal weapon system programmed to be capable of changing its own rules of operation particularly as regards target engagement, beyond a determined framework of use, and capable of computing decisions to perform actions without any assessment of the situation by human military command.

The most important aspect of this definition is its narrowness: in line with Frances position at U.N. meetings, LAWS are considered as fully autonomous systems. Defining LAWS has always been a challenge, because if understood as fully autonomous we are talking about weapons that do not yet exist. As a result, there is no shared experience or understanding with these weapons. In the history of arms control, that makes them quite unique. In multilateral fora (e.g., the United Nations), some states have used the challenge of defining LAWS as an excuse to obstruct or redirect debate. By limiting LAWS to fully autonomous weapons, France defends a restrictive approach avoiding those weapons being confused with remotely operated or supervised weapons systems, which always involve a human operator.

The committee then introduced the category of partially autonomous lethal weapon systems (PALWS). It decided to define it as:

[I]ntegrating automation and software: [1] to which, after assessing the situation and under their responsibility, the military command can assign the computation and execution of tasks related to critical functions such as identification, classification, interception and engagement within time and space limits and under conditions; [2] which include technical safeguards or intrinsic characteristics to prevent failures misuse and relinquishment by the command of two vital duties, namely situation assessment and reporting.

PALWS are an in-between category, distinct from two others. On the one hand, PALWS are not LAWS because they cannot change their own rules of operation, they cannot take lethal initiatives. On the other, PALWS are not automated weapon systems either. The difference between autonomy and automation is foundational. Once deployed, both autonomous and automated weapons can function without human involvement. However, while automation refers to the performance of a limited number of repetitive and pre-determined tasks (the system always reacts the same way to the same stimulus), autonomy involves an ability to learn and adjust in a changing environment. For example, mines and some air defense systems are automated in that they act in a reactive and repetitive way, by detonating or firing, when their sensors detect an object. They do not learn or adapt, and they do not need to because they do not have to face unexpected situations. Their environment does not change. PALWS are not LAWS in that they are not fully autonomous. However, because they are still (partially) autonomous, they are not automated weapons either. Several existing weapons could be categorized as PALWS, among which loitering munitions such as the Israeli IAI Harop, the Turkish STM Kargu-2, and a non-identified Chinese model used in swarms; the American Collaborative Small Diameter Bombs (CSDB), or the drone warship Sea Hunter.

Now, two objections could be raised at this point. First, that such a distinction between LAWS and PALWS is certainly not new in the international debate, nor in national doctrines. Indeed, as early as in 2012, when the United States was the first country to establish guidelines for the development and use of autonomy in weapon systems, they had already distinguished between an autonomous weapon system and a semi-autonomous one. If the PALWS category is a terminological innovation in French especially in a ministerial document partially and semi autonomous weapon systems, while being defined differently, do refer to the same challenge of describing what lies under the threshold of full autonomy.

Second, by adopting a narrow definition of LAWS limited to fully autonomous weapons, isnt France defining them as something nobody ever wanted? Under the appearance of rejecting LAWS, a category of systems that havent really been under consideration, isnt France actually legitimizing the more realistic category of PALWS? This is a legitimate concern. However, as a member of the committee that drafted the opinion, the intention was not to legitimize whatever category of autonomous weapons it may be. Instead, the goal was to add some needed intellectual rigor. The problem with the LAWS terminology is that autonomous is presumed to be dichotomic: a system is, or is not, autonomous. And if it is autonomous, it is presumed to be fully autonomous which, for good reasons, no one really wants. Therefore, it is more useful to adopt an alternative terminology based on the idea that the integration of autonomy in weapon systems can and will be gradual. Rejecting LAWS and focusing on PALWS for that reason does not mean that PALWS cannot be ethically problematic. It is not legitimizing them. Rather, their legitimacy depends on a number of criteria. The distinction offered by the committee simply reorients the discussion to center on the relevant category.

LAWS Are Not Acceptable

France has publicly renounced the use of fully autonomous lethal weapons, for both ethical and operational reasons, since 2013. In 2018, President Emmanuel Macron said he was categorically opposed to LAWS, to the extent they would abolish all accountability. He added, the decision to give the green light has to be made by a human being because you need someone to take responsibility for it. In May 2021, French Defense Minister Florence Parlyconfirmed that France says and will always say no to killer robots. France refuses to entrust the decision of life or death to a machine that would act in a fully autonomous way and would escape any human control.

Frances position on LAWS is in line with its closest allies. The U.S. Department of Defenses 2012 directive explicitly stated that their weapon systems should allow commanders and operators to exercise appropriate levels of human judgment in the use of force. This is, of course, another way to say they should not be fully autonomous. Similarly, the United Kingdom repeatedly expressed that it is not developing lethal autonomous weapons systems, and the operation of weapons systems by the UK armed forces will always be under human oversight and control. Many other states made similar remarks. This is indeed one of the few points of consensus in the U.N. debate on LAWS: in one way or another, everyone insists on retaining human control. No one wants a fully autonomous weapon, as full autonomy, literally the ability to set ones own rules, would mean unpredictability, which would make such systems militarily useless.

But this only begs a more difficult question: Should countries preventatively ban LAWS? This is where the disagreement lies.

The French Defense Ethics Committee also rejected incorporating LAWS into the countrys military for a number of reasons. LAWS would:

[B]reak the chain of command; run counter to the constitutional principle of having liberty of action to dispose of the armed forces; not provide any assurance as to compliance with the principles of international humanitarian law; be contrary to our military ethics and the fundamental commitments made by French soldiers, i.e. honour, dignity, controlled use of force and humanity.

The committee considered it legitimate and vital to continue research in the area of autonomy in lethal weapons, a research focused on ways and means of enabling French forces to counter the use of LAWS by states or other enemies, but without using LAWS ourselves.

PALWS Are Interesting and Risky

PALWS offer a number of advantages in terms of performance, precision, pertinence, protection, and permanence (the 5 Ps). In terms of performance, they will provide means to gain speed, in particular by shortening the observe-orient-decide-act loop. Also, one of the greatest challenges of the future of warfare will be defense against incoming conventional or nuclear strikes at hypersonic speed (of at least Mach 5, with some of them reportedly reaching Mach 20), leaving very little time to react and therefore requiring a greater autonomization. The same is true regarding defense against a saturation attack, or swarming. PALWS will also be useful to monitor very vast areas in all environments (land, air, sea, cyber, space) that cannot be covered without a certain degree of autonomy.

PALWS will also help to deal with the increasing mass of information (data deluge) that confront command centers and individual soldiers. Autonomous systems can help decision-making on an increasingly interconnected battlefield. They will also help penetrate highly defended areas physically and virtually; improve the precision of strikes; and protect soldiers, especially against improvised explosive devices or in contaminated environments. Finally, PALWS will last longer than human teams at sea, in the air, or on the ground, especially in dangerous or dirty environments, and they will therefore provide a greater permanence in a given area.

At the same time, PALWSs present a number of risks. Deploying autonomous weapons even if they are only partially autonomous tests the moral and social acceptability of using force without human intervention. Domestic opposition to the use of PALWS, including among the soldiers themselves, could undermine confidence in the states actions and legitimacy. Machine Learning may also lead to unexpected and unwanted behavior, as it raises issues on the long-term reliability of the systems.

There is also the issue of accountability: In the event of an incident (e.g., friendly fire or civilian casualties), who should be considered responsible? This is indeed one of the main criticisms directed at autonomous weapons and invoked by opponents as a reason to ask for a preventative ban. The integration of autonomy in weapon systems will inevitably make it more difficult to establish responsibility as there are many layers of control (state, manufacturer, programmer, system integrator, contractor, and military commander). Establishing responsibility will be difficult but not impossible, because an autonomous decision-making capacity does not break the causal chain allowing attribution and responsibility, as professor Marco Sassli explained in 2014. Moreover, such a dilution of responsibility is not unheard of, as it is already what happens when a plane on automatic pilot crashes, or when a self-driving car has an accident.

Among other risks of incorporating PALWS, the Defense Ethics Committee identified hacking (thereby hijacking those systems); the psychological impact on humans, especially those excluded from the decision-making process or no longer able to understand what the system is doing, potentially causing a lack of involvement or a loss of humanity in combat; and other psychological risks such as blindly trusting the machine, losing confidence in the human ability to deal with a complex situation, and developing all kinds of cognitive biases. There is also a risk of lowering the threshold of the use of force, and a risk of global proliferation, including acquisition by non-state actors.

How PALWS Could Be Ethically Acceptable

It is essential to delineate conditions under which it would be ethically acceptable to design, develop, and deploy PALWS. This is what the Committee called the 5Cs: command, risk control, compliance, competence, and confidence.

For each mission, PALWS should have rules set up by the human command (in terms of its target, spatial and temporal limits, rules of engagement, and other constraints); they should not be able to change those rules themselves (only human command can); they should not be able to assign a mission departing from what was initially programmed to another PALWS, or only after validation by the human command; and what they acquire through machine learning during a mission should not be used to program new tasks without human involvement.

Additionally, military personnel deploying PALWS (not only operators but also tactical leaders, theatre commanders and strategic leaders) should be prepared and trained accordingly. Similarly, any personnel involved in the design, development and promotion of those weapons (e.g., engineers, researchers, diplomats, politicians) should be made aware of the various risks and issues their use involve. Public authorities should be informed as well. Furthermore, mechanisms such as emergency deactivation or self-destruction should be implemented in the systems, in the event of a communication loss, as well as a device for aborting a mission in progress.

The French Defense Ethics Committee also recommended conducting a complete legal review whenever decision-making autonomy is developed in a lethal weapon system, especially as far as identification, classification and opening fire functions are concerned. Last but not least, it also advocated for international transparency.

Looking Ahead

There is nothing radically new in this French Defense Ethics Committee opinion for those closely following the decade-long, international debate on more or less autonomous weapons. Most, if not all, of these recommendations have been made by scholars and non-governmental organizations. What is interesting in this ethical opinion is that it also involves legal, scientific, and operational arguments, and that this comes from a committee set up by the French Ministry of Defense. However, what is at stake here is not just one state. The more individual states develop a clear and detailed public policy, the easier it will be to agree on a normative framework at the global level.

Jean-Baptiste Jeangene Vilmer, Ph.D., a member of the French Defense Ethics Committee, is the director of the Institute for Strategic Research (IRSEM) at the French Ministry of the Armed Forces, and a nonresident senior fellow at the Atlantic Council, Washington, D.C. He is also an adjunct professor at the Paris School of International Affairs (PSIA), Sciences Po, and an Honorary Ancien of the NATO Defense College. The views and opinions expressed in this article are the authors alone and do not necessarily reflect the official position of the French Defense Ethics Committee or the French Ministry of the Armed Forces.

Image: U.S. Navy

Read more here:
A French Opinion on the Ethics of Autonomous Weapons - War on the Rocks

Markeshaw Tiruneh G/Medhin,1 Endeshaw Chekol Abebe,2 Tekeba Sisay,3 Nega Berhane,3 Tesfahun Bekele Snr,1 Tadesse Asmamaw Dejenie1

1Department of Biochemistry, School of Medicine, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia; 2Department of Biochemistry, College of Health Sciences, Debre Tabor University, Debre Tabor, Ethiopia; 3Institute of Biotechnology, College of Natural Science, University of Gondar, Gondar, Ethiopia

Correspondence: Markeshaw Tiruneh G/Medhin Tel +251922712112Email [emailprotected]

Abstract: Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins are referred to as CRISPR-Cas9. Bacteria and archaea have an adaptive (acquired) immune system. As a result, developing the best single regulated RNA and Cas9 endonuclease proteins and implementing the method in clinical practice would aid in the treatment of diseases of various origins, including lung cancers. This seminar aims to provide an overview of CRISPR-Cas9 technology, as well as current and potential applications and perspectives for the method, as well as its mechanism of action in lung cancer therapy. This technology can be used to treat lung cancer in two different ways. The first approach involves creating single directed RNA and Cas9 proteins and then distributing them to cancer cells using suitable methods. Single directed RNA looks directly at the lungs mutated epidermal growth factor receptor and makes a complementary match, which is then cleaved with Cas9 protein, slowing cancer progression. The second method is to manipulate the expression of ligand-receptors on immune lymphocytic cells. For example, if the CRISPR-Cas9 system disables the expression of cancer receptors on lymphocytes, it decreases the contact between the tumor cell and its ligand-receptor, thus slowing cancer progression.

Keywords: CRISPR, Cas9, CRISPR-Cas9 technology, cancer, lung cancer, cancer treatment

The word CRISPR-Cas9 refers to Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins.15 CRISPR-Cas9 system is a kind of acquired immunity possessed by most bacteria and archaea (prokaryotes) to act against their enemies (bacteriophages).4,6 It is a ribonucleic acid (RNA) guided, convenient, and versatile endonuclease platform for site-specific genome editing,1,7,8 which can play a tremendous role in the application of cancer therapy.1 The application of this technology can be used to resolve mutations and to introduce site-specific therapeutic genes in human cells so that, correcting disease-causing mutations, and alleviate disease-related symptoms. This system is also a useful tool for delineating molecular mechanisms involving hematological malignancies.4 Sequence-specific gene editing using CRISPR-Cas9 shows promise as a novel therapeutic approach for the treatment of a variety of diseases that currently lack effective treatments like cancers.3,9 To accomplish its task, it requires Cas9 DNA endonuclease protein and single guided RNA (sgRNA) that can produce precise gene matching for editing and correction techniques.2 So the system has enabled easy manipulation of genes for the scientific community by making the hybrid to the target sequence and cleaving the double-strand DNA.10

Additionally, the CRISPR-Cas9 technology is increasingly feasible to overcome drug resistance in breast cancer therapy and will become an essential tool for personalized medicine.4 It is a technological breakthrough that facilitates the ability to change nucleic acids,11 and with continued improvement in the function, the system can help to develop best treatment options to a variety of genetic disease which affects several tissues in our body.12 Gene manipulation using CRISPR-Cas9 system has revolutionized and made it easy to study the work of genes and importantly opens the new era of treatment mechanisms for different disease conditions including cancer.13 Technologies like this are a simple and efficient method of targeting the required DNA regions.14 Thus, scientists have designed two main components of the system for easy detection and alteration of gene function one component is a protein Cas9 that enzymatically cleave the desired gene and the sgRNA which scans and determines where the gene of interest will be cleaved by Cas9 protein.3,12,15 The system has been scientifically optimized and developed to regulate expression of the gene, modify and edit the desired locus and this makes the technology of choice seen by the scientific community to treat or edit disease-causing mutations more efficiently than ever before. Furthermore, its application is encouraging for more vigorous gene therapy in clinical setups.16 Based on the discovery, there are three main types of CRISPR-Cas9 system (I to III) and with three additional types (IV to VI) being identified more recently.17 They are different during the processes of immunity, adaptation, expression, and interference, each type acts in distinct mechanisms to ensure genetic manipulation. Type I employs a large complex of Cas9 proteins with distinct helicase and DNase activities, while type III employs repeat-associated mysterious proteins, which form a large Cas9 superfamily. Another classification is based on subunit effectors, with multi-subunit effector complexes being the most common. Types I, III, and IV are grouped in class 1. Those systems, on the other hand, that have a single subunit effector are categorized as class 2 comprising types II, V, and VI.17,18 Type II uses only a Single protein (Cas9) for its nuclease activity and has got more attention and adopted for genome engineering.5,17,19 Thus, the objective of this seminar is to introduce CRISPR-Cas9 technology and describe current applications and future perspectives of the system with its mechanism of action on lung cancer therapy.

The tracrRNA gene will be transcribed to tracrRNA, the crRNA gene will be transcribed to pre crRNA, and the Cas9 gene will be transcribed to Cas9 messenger RNA and converted to Cas9 protein, all of which will be post-transcribed and chopped off to form the mature CRISPR-Cas9 complex.20 Cas1 and Cas2 integrase, which are present in all CRISPR forms, catalyze spacer integration on the CRISPR array especially on the leader end of the repeat there will be a nucleophilic attack of the 3 OH of the protospacers followed by the same practice on the spacer end of the repeat.21

The CRISPR-Cas9 method has a variety of formulation methods for genome editing. The use of a plasmid-based CRISPR-Cas9 system encoding both the Cas9 protein and sgRNA from the same vector, which is necessary to avoid multiple transfections of different components of the technology, is the leading and possibly the easiest technique. The Cas9 protein and sgRNA will be expressed in the vector, which will form the sgRNA-Cas9 complex within cells to edit the target genomic sequences.3,12,15,18 The second approach involves combining Cas9 mRNA and sgRNA. The sgRNA-Cas9 complex will be formed when Cas9 mRNA is converted into Cas9 protein in cells. The third strategy is to deliver the in vitro assembled sgRNA-Cas9 complex directly to the cell.18

It is difficult to transmit nucleic acid in general, and CRISPR-Cas9 in particular, to the target tissue or cell. Physical and vector (viral or non-viral) approaches are two of the most widely used distribution strategies.11,22,23 Electroporation and microinjections are used in physical methods, while viral delivery strategies such as adeno associated virus (AAV) are widely used in vector-based methods since they are not disease-causing agents and can infect both dividing and non-dividing cells25 and lentivirus with inactivated integrase enzymes are under investigation.24

Another technique is lipofection (lipid-mediated nanoparticle transfection), which is possibly the most efficient CRISPR-Cas9 in vivo delivery method.22 This technique was further developed by26 and is currently being tested in clinical trials.13,24

The CRISPR-Cas9 system, as discussed, a little earlier, is made up of two main components that work together to accomplish its goal.19 The sgRNA contains crRNA, which scans and identifies the target DNA sequences that must be cleaved and corrected, and transactivated crRNA (tracrRNA), which recruits component two, the Cas9 protein DNA endonuclease, which can sense, identify, and establish site-specific double-strand DNA breaks (DSB).15 Because of its simplicity and convenience, the bacterial type II CRISPR-Cas9 system has been used for RNA-guided engineering nucleases.4,18 However, the proto-spacer adjacent motifs (PAM) sequences are required by the method. After recognition, two Cas9 domains cleave double-stranded DNA: the endonuclease domain named for characteristic histidine and asparagine residues (HNH) domain, which cleaves the complementary strand, and the endonuclease domain named for an E. coli protein involved in DNA repair (RuvC-like) domain, which cleaves the non-complementary strand.17 As a result, the host DNA repair machinery introduces numerous mutations such as substitutions, deletions, and insertions in the target genome, including non-homologous end joining (NHEJ) or homologous-dependent repair (HDR).1518 Another paper, CRISPR-Cas9 for Cancer Therapy: Hopes and Challenges, supports this theory by demonstrating that the sgRNA-Cas9 complex scans and anneals to the genomic target sequence with base-pairing complementarity and precisely cleaves double-stranded DNA of the target cell after identification of the protospacer adjacent motif (PAM) sequence adjacent to the target sequence. NHEJ or HDR pathways are activated as a result of double-strand breaks. NHEJ is an error-prone repair mechanism that results in indels (insertions or deletions) of random base pairs disrupting the target sequence in the absence of a homologous repair prototype with more specific repair mechanisms.23,27

Lung cancer is the major cause of death in the United States and a significant public health concern worldwide.5 In both developed and developing countries, it is a common cause of morbidity and mortality.28 According to a study conducted by the American Lung Cancer Society in 2015, lung cancer claims the lives of almost 150,000 people each year. However, surgery and radiation were used as treatment options. The treatment was later changed to selective Tyrosine kinase inhibitors (TKIs) like gefitinib and erlotinib to inhibit the tyrosine kinase activity of epidermal growth factor receptor, which has technical difficulties and nonspecific cytotoxicity (EGFR).29,30 Extracellular ligand binding, transmembrane, and intracellular tyrosine kinase domains are found in this membrane glycoprotein. When the ligand activator binds to the extracellular ligand domain, it transduces and initiates intracellular kinase activities, which cause cellular proliferation, neovascularization, invasion, and metastasis, as well as reduced apoptosis and glycolysis activation. These medications, however, have encountered drug resistance.28,29

The CRISPR-Cas9 device is the start of a new biotechnological era and a groundbreaking technology that is being used to treat lung cancer.6,29 The system works in two ways. The first is by designing sgRNA that looks for the mutated EGFR sequence, which is then accompanied by Cas9 protein. To do so, scientists created a CRISPR device that has complementary sequences with the mutated EGFR and introduced it into the patient, as mentioned earlier which has complementary sequences with the mutated EGFR and introducing this into the patient. As this complementary sequence binds to the mutated EGFR, the Cas9 protein (endonuclease) creates a double-stranded or single-stranded DNA break, depending on the type of enzyme used, followed by DNA repair mechanisms such as homologous or non-homologous DNA repair.29 If the receptor mutation is limited, there will be no contact between the ligand activator, resulting in no cell proliferation, neovascularization, or cancer metastasis, and the problem will be solved. The inhibition of EGFR by CRISPR-Cas9 increases the expression of major histocompatibility complex class I, which improves cytotoxic lymphocyte recognition and lysis of tumor cells.30,31 Off-target effects, which can induce genome instability, gene functional disturbances, and epigenetic alterations, are a challenge. Off-target effects of CRISPR-Cas9 systems, particularly when used for therapeutic purposes, should be minimized and precisely profiled. Off-target effects are separated into two categories: off-target binding and off-target cleavage. Cas9 can bind to target sequences that are partially complementary to sgRNA and inhibit target gene transcription without cleaving them.8 Off-target binding effects may thus be removed in traditional off-target identification approaches, such as using in vitro assembled sgRNA with a long-lasting association with cas9, which also has a high proportion of on-target and high efficiency for genome editing. Another technique is to use a Cas9 variant or modified Cas9 that can generate a single nick at one strand.23 So that the off-target effect is reduced.

The second, and equally significant, strategy for using this biotechnological method to treat lung cancer is to search for immune cells like lymphocytes. T cells are immune cells that are extracted from the blood of patients engaged in a clinical trial for lung cancer treatment in China, and then CRISPR-Cas9 is used to knock out a gene in the cells that encodes a protein called PD-1. The edited gene cells would then be propagated in the lab before being injected back into the patients bloodstream.6,25 Scientists took blood from the patient and extracted lymphocytes, which were then treated with a CRISPR-Cas9 gene-editing system containing a sgRNA sequence with a pattern identical to lymphocytes programmable death 1 protein (PD 1). When the system detects its target sequence, cas9 would sever the DNA, which is then repaired by cell repair mechanisms. When the expression of the PD 1 gene is blocked or disabled, cancerous cells lack the receptor on immune lymphocytes.6,25 As a result, if lymphocytes do not express the PD 1 receptor well, there will be less contact between the cancerous ligand and receptor, causing the T cell receptor to identify the problematic cell and perform its function. Naturally, these manipulated lymphocytes were screened for viability and lympho-proliferation to rule out new mutations, and only those cells that passed the test were returned to the patient.6,25 Furthermore, knocking out the PD-1 protein on immune cells is necessary for caspase activation, which is needed for programmed cell death and enhanced apoptosis in cancerous cells.31 It also concludes that PD-1-deficient cells have potent antitumor activity of cytotoxic lymphocytes. The hyperactivity of the manipulated T cells is one of the technologys drawbacks for use in this way6 and obtaining a safe and efficient delivery method, as well as some side effects Patients with advanced NSCLC with positive PD-1 expression were assigned to a Phase I clinical trial to assess the safety of CRISPR-Cas9-mediated knockout of PD-1 gene therapy in patients with metastatic non-small cell lung cancer. Nine patients were enrolled, and eight patients received PD-1 deficient T cell therapy, and the patients were manifested with PD-1 deficient T cell therapy.25 Patients undergoing PD-1 deficient T cell therapy, on the other hand, appeared to be healthy, and researchers recommended that broader studies be conducted to determine the most appropriate dosage and immune response.

In cancer biology, the CRISPR-Cas9 device has a bright future ahead of it,9, because it is a technology that is adaptable, simple, convenient and efficient.32,33 The method introduces a novel approach to cancer treatment by allowing for modifications to the genome of target cells, which was previously difficult to achieve.3436 the technologys versatility, effectiveness, and flexibility would make it the best form of cancer care in the future.4,37,38 It will affect cancer biology as a whole in the future,34 and if researchers have devised well-organized strategies and instruments for delivering the technology to the target cell or tissue, as well as effective methods and instructions for controlling and eliminating the technologys off-target effects.

The CRISPR-Cas9 device is a recent biotechnological breakthrough and scientific achievement. This technology has created a new treatment option for diseases of various origins, such as cancer and infectious disease. To solve the problem, the best sgRNA must be designed using a CRISPR tool (http://crispr.mit.edu) and its associated endonuclease cas9 protein against the target sequence. However, ethical concerns, the need for the best delivery strategies, and the risk of off-target effects are only a few of the problems that must be addressed. Since the technology is still in its infancy, researchers must devise simple methods and mechanisms to track and test its protection and efficacy. For a simple comparison, the benefits of this technology are simple, fast, relatively effective, relatively precise, and versatile, while the drawbacks are distribution is difficult, ethical problems are highly conservative, some off-target effects, and some adverse effects.

ATP, Adenosine triphosphate; CRISPR, Clustered regularly interspaced short palindromic repeat; CRISPR-Cas, Clustered regularly interspaced short palindromic repeat-associated; CrRNA, Clustered regularly interspaced short palindromic repeat ribonucleic acid; DNA, Deoxyribonucleic acid; DSB, Double-stranded break; EGFR, epidermal growth factor receptor; HDR, Homologous directed repair; mRNA, Messenger ribonucleic acid; NHEJ, Non-homologous end-joining; PD 1, Programmable death protein 1; RNA, Ribonucleic acid; SgRNA, Single guided ribonucleic acid; TracrRNA, Trans activating clustered regularly short palindromic repeat ribonucleic acid; TKIs, Tyrosine kinase inhibitors.

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.

There is no funding to report.

The authors declare that they have no conflicts of interest for this work.

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8 of 11 Patients in Dose Escalation Cohorts 2 and 3 Achieved Objective Response

6 of 11 Patients Achieved Complete Response, including 2 Patients Previously Treated with Autologous CD19 CAR T-cell Therapy

Favorable FT516 Safety Profile Was Observed; No FT516-related Serious Adverse Events or FT516-related Grade 3 or Greater Adverse Events

Outpatient Treatment Regimen Was Well-tolerated; No Events of Any Grade of Cytokine Release Syndrome, Immune Effector Cell-Associated Neurotoxicity Syndrome, or Graft-vs-Host Disease

SAN DIEGO, June 04, 2021 (GLOBE NEWSWIRE) -- Fate Therapeutics, Inc. (NASDAQ: FATE), a clinical-stage biopharmaceutical company dedicated to the development of programmed cellular immunotherapies for cancer, today highlighted positive interim Phase 1 data from the Companys FT516 program for patients with relapsed / refractory B-cell lymphoma at the 2021 American Society of Clinical Oncology (ASCO) Annual Meeting being held virtually June 4-8, 2021. FT516 is the Companys universal, off-the-shelf natural killer (NK) cell product candidate derived from a clonal master induced pluripotent stem cell (iPSC) line engineered with a novel high-affinity, non-cleavable CD16 (hnCD16) Fc receptor, which is designed to maximize antibody-dependent cellular cytotoxicity (ADCC), a potent anti-tumor mechanism by which NK cells recognize, bind and kill antibody-coated cancer cells. The ongoing Phase 1 dose-escalation study of FT516 is currently enrolling patients in the fourth dose cohort of 900 million cells per dose.

As of the data cutoff date of March 11, 2021, four patients in the second dose cohort of 90 million cells per dose and seven patients in the third dose cohort of 300 million cells per dose were evaluable for assessment of safety and efficacy. Eight of eleven patients achieved an objective response, including six patients who achieved a complete response, as assessed by PET-CT scan per Lugano 2014 criteria (see Table 1). Patients had received a median of three prior lines of therapy and a median of two prior lines containing CD20-targeted therapy. Of the eleven patients, eight patients had aggressive B-cell lymphoma, five patients were refractory to their most recent prior therapy, and four patients were previously treated with autologous CD19 CAR-T cell therapy.

These additional data from our Phase 1 study of FT516 administered off-the-shelf in the outpatient setting continue to reinforce its differentiated safety profile and underscore its potential clinical benefit, said Wayne Chu, M.D., Senior Vice President of Clinical Development of Fate Therapeutics. Based on the favorable therapeutic profile of FT516 that continues to emerge and the potential to treat patients on-demand without delay, we plan to initiate multiple indication-specific, dose-expansion cohorts for patients with B-cell lymphomas to broadly assess FT516 in combination with CD20-targeted monoclonal antibody regimens, including those used as standard-of-care in earlier-line settings.

The ongoing Phase 1 clinical trial in relapsed / refractory B-cell lymphoma is assessing FT516 in an off-the-shelf treatment regimen of up to two cycles, with each cycle consisting of three days of conditioning chemotherapy (500 mg/m2 of cyclophosphamide and 30 mg/m2 of fludarabine), a single-dose of rituximab (375 mg/m2), and three weekly doses of FT516 each with IL-2 cytokine support. The FT516 treatment regimen is designed to be administered in the outpatient setting.

Safety DataNo dose-limiting toxicities, and no FT516-related serious adverse events or FT516-related Grade 3 or greater adverse events, were observed. The FT516 treatment regimen was well tolerated, and no treatment-emergent adverse events (TEAEs) of any grade of cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, or graft-versus-host disease were reported by investigators (see Table 2). All Grade 3 or greater TEAEs were consistent with lympho-conditioning chemotherapy and underlying disease. Of note, a Grade 3 or greater TEAE of infection was reported in one patient only. There were no discontinuations due to adverse events, and no patients withdrew from the study except in the setting of disease progression. In addition, no evidence of anti-product T- or B-cell mediated host-versus-product alloreactivity was detected, supporting the potential to safely administer up to six doses of FT516 in the outpatient setting without the need for patient matching.

Activity DataAs of the data cutoff date of March 11, 2021, eleven relapsed / refractory patients in the second and third dose cohorts were evaluable for assessment of safety and efficacy. Of the eleven patients, nine patients completed both FT516 treatment cycles and eight patients achieved an objective response, including six patients who achieved a complete response, as assessed by PET-CT scan per Lugano 2014 criteria. Notably, two of four patients previously treated with autologous CD19 CAR-T cell therapy achieved a complete response. Two patients showed progressive disease following the first FT516 treatment cycle and discontinued treatment. The Company previously reported that two patients treated in the first dose cohort (30 million cells per dose) showed progressive disease.

Patient Case StudyThe ASCO presentation featured a case study of a 36-year old male with triple-hit, high-grade B-cell lymphoma with rearrangements of MYC, BCL2, and BCL6 genes. The patient was refractory to all prior lines of therapy with the exception of autologous CD19 CAR T-cell therapy, for which a complete response of two months duration was achieved. The patient was most recently refractory to an investigational CD20-targeted T-cell engager and presented with bulky lymphadenopathy with the largest lesion measuring approximately 10 centimeters. The first FT516 treatment cycle resulted in a complete response with resolution of all metabolically active disease and 85% reduction in the size of target lesions. Subsequent to the data cutoff date of March 11, 2021, the patient completed a second FT516 treatment cycle after which the response assessment continued to show complete response.

As of March 11, 2021 database entry. Data subject to source document verification.CR = Complete Response; PR = Partial Response; PD = Progressive DiseaseCAR = Chimeric antigen receptor; DH/DE = Double-hit / double expressor; DLBCL = Diffuse large B-cell lymphoma; FL = Follicular lymphoma; Gr = Grade; HGBCL = High-grade B-cell lymphoma; iNHL = Indolent non-Hodgkin lymphoma; TH = Triple-hit; Transformed iNHL = Aggressive B-cell lymphoma transformed from indolent non-Hodgkin lymphoma1 Cycle 2 Day 29 protocol-defined response assessment per Lugano 2014 criteria2 Subject did not proceed to Cycle 23 Confirmed DLBCL (transformation from Gr3A FL) subsequent to the data cutoff date of March 11, 20214 Cycle 2 Day 29 protocol-defined response assessment reported subsequent to the data cutoff date of March 11, 2021

CRS = Cytokine Release Syndrome; DL = Dose Level; GvHD = Graft vs. Host Disease; ICANS = Immune Cell-Associated Neurotoxicity Syndrome;M = Million; SAE = Serious Adverse Event; TEAE = Treatment-Emergent Adverse Event1 Includes two subjects in the first dose cohort of 30 million cells per dose

About Fate Therapeutics iPSC Product PlatformThe Companys proprietary induced pluripotent stem cell (iPSC) product platform enables mass production of off-the-shelf, engineered, homogeneous cell products that are designed to be administered with multiple doses to deliver more effective pharmacologic activity, including in combination with other cancer treatments. Human iPSCs possess the unique dual properties of unlimited self-renewal and differentiation potential into all cell types of the body. The Companys first-of-kind approach involves engineering human iPSCs in a one-time genetic modification event and selecting a single engineered iPSC for maintenance as a clonal master iPSC line. Analogous to master cell lines used to manufacture biopharmaceutical drug products such as monoclonal antibodies, clonal master iPSC lines are a renewable source for manufacturing cell therapy products which are well-defined and uniform in composition, can be mass produced at significant scale in a cost-effective manner, and can be delivered off-the-shelf for patient treatment. As a result, the Companys platform is uniquely designed to overcome numerous limitations associated with the production of cell therapies using patient- or donor-sourced cells, which is logistically complex and expensive and is subject to batch-to-batch and cell-to-cell variability that can affect clinical safety and efficacy. Fate Therapeutics iPSC product platform is supported by an intellectual property portfolio of over 350 issued patents and 150 pending patent applications.

About FT516FT516 is an investigational, universal, off-the-shelf natural killer (NK) cell cancer immunotherapy derived from a clonal master induced pluripotent stem cell (iPSC) line engineered to express a novel high-affinity 158V, non-cleavable CD16 (hnCD16) Fc receptor, which has been modified to prevent its down-regulation and to enhance its binding to tumor-targeting antibodies. CD16 mediates antibody-dependent cellular cytotoxicity (ADCC), a potent anti-tumor mechanism by which NK cells recognize, bind and kill antibody-coated cancer cells. ADCC is dependent on NK cells maintaining stable and effective expression of CD16, which has been shown to undergo considerable down-regulation in cancer patients. In addition, CD16 occurs in two variants, 158V or 158F, that elicit high or low binding affinity, respectively, to the Fc domain of IgG1 antibodies. Scientists from the Company have shown in a peer-reviewed publication (Blood. 2020;135(6):399-410) that hnCD16 iPSC-derived NK cells, compared to peripheral blood NK cells, elicit a more durable anti-tumor response and extend survival in combination with anti-CD20 monoclonal antibodies in an in vivo xenograft mouse model of human lymphoma. Numerous clinical studies with FDA-approved tumor-targeting antibodies, including rituximab, trastuzumab and cetuximab, have demonstrated that patients homozygous for the 158V variant, which is present in only about 15% of patients, have improved clinical outcomes. FT516 is being investigated in a multi-dose Phase 1 clinical trial as a monotherapy for the treatment of acute myeloid leukemia and in combination with CD20-targeted monoclonal antibodies for the treatment of advanced B-cell lymphoma (NCT04023071). Additionally, FT516 is being investigated in a multi-dose Phase 1 clinical trial in combination with avelumab for the treatment of advanced solid tumor resistant to anti-PDL1 checkpoint inhibitor therapy (NCT04551885).

About Fate Therapeutics, Inc.Fate Therapeutics is a clinical-stage biopharmaceutical company dedicated to the development of first-in-class cellular immunotherapies for patients with cancer. The Company has established a leadership position in the clinical development and manufacture of universal, off-the-shelf cell products using its proprietary induced pluripotent stem cell (iPSC) product platform. The Companys immuno-oncology pipeline includes off-the-shelf, iPSC-derived natural killer (NK) cell and T-cell product candidates, which are designed to synergize with well-established cancer therapies, including immune checkpoint inhibitors and monoclonal antibodies, and to target tumor-associated antigens using chimeric antigen receptors (CARs). Fate Therapeutics is headquartered in San Diego, CA. For more information, please visit http://www.fatetherapeutics.com.

Forward-Looking StatementsThis release contains "forward-looking statements" within the meaning of the Private Securities Litigation Reform Act of 1995 including statements regarding the safety and therapeutic potential of the Companys iPSC-derived NK cell product candidates, including FT516, its ongoing and planned clinical studies, and the expected clinical development plans for FT516. These and any other forward-looking statements in this release are based on management's current expectations of future events and are subject to a number of risks and uncertainties that could cause actual results to differ materially and adversely from those set forth in or implied by such forward-looking statements. These risks and uncertainties include, but are not limited to, the risk that results observed in studies of its product candidates, including preclinical studies and clinical trials of any of its product candidates, will not be observed in ongoing or future studies involving these product candidates, the risk that the Company may cease or delay clinical development of any of its product candidates for a variety of reasons (including requirements that may be imposed by regulatory authorities on the initiation or conduct of clinical trials, the amount and type of data to be generated, or otherwise to support regulatory approval, difficulties or delays in subject enrollment and continuation in current and planned clinical trials, difficulties in manufacturing or supplying the Companys product candidates for clinical testing, and any adverse events or other negative results that may be observed during preclinical or clinical development), and the risk that its product candidates may not produce therapeutic benefits or may cause other unanticipated adverse effects. For a discussion of other risks and uncertainties, and other important factors, any of which could cause the Companys actual results to differ from those contained in the forward-looking statements, see the risks and uncertainties detailed in the Companys periodic filings with the Securities and Exchange Commission, including but not limited to the Companys most recently filed periodic report, and from time to time in the Companys press releases and other investor communications.Fate Therapeutics is providing the information in this release as of this date and does not undertake any obligation to update any forward-looking statements contained in this release as a result of new information, future events or otherwise.

Contact:Christina TartagliaStern Investor Relations, Inc.212.362.1200christina@sternir.com

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Fate Therapeutics Highlights Positive Interim Data from its Phase 1 Study of FT516 in Combination with - GlobeNewswire