The Forefront of a Revolution: The Applications of CRISPR-Cas9 Technology to Treat Multiple Sclerosis

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Abstract

The advent of CRISPR-Cas9 gene-editing technology has revolutionized the biomedical field, reshaping and elevating the possibilities in which medicine approaches diagnosis, treatment, prevention, and potentially curing certain diseases. The prospect of CRISPR-Cas9 gene-editing has transformed the seemingly abstract hypothetical into practical solutions that have the scope to possibly cure an extensive range of currently “incurable” genetic diseases. This list includes Multiple Sclerosis (MS), a debilitating and chronic autoimmune disorder that causes rapid neurodegeneration in the central nervous system. Although the etiological cause of MS remains unknown, CRISPR-Cas9 has the potential to effectively ameliorate its clinical symptoms by acting on several molecular factors that result in the progression of the disease. When attempting to achieve successful genome editing, several factors need to be taken into consideration. This paper highlights the importance of different stages involved in the gene- editing process, including components of CRISPR – Cas9 technology, effective expression and delivery methods, novel approaches to treat MS, as well as the ethical aspects surrounding the therapeutic use of gene-editing to cure and treat an extensive range of diseases including MS.

Introduction

The ability to manipulate the very basis of human life — our genetics — carries tremendous potential for applications in various biological systems for therapeutic and biomedical purposes. The CRISPR-Cas9 system has allowed for a breakthrough method of precise, cost-effective, and targeted genome editing that has the potential to revolutionize and change the face of modern medicine. In the past few decades, scientists have drawn upon the idea that genome editing can be achieved by generating double-stranded breaks in the DNA to produce precise edits to targeted mutations, resulting in permanent changes in the genome1.

In nature, the CRISPR-Cas9 system is derived from bacterial/archaeal immunity and was exploited for genome editing in eukaryotes. Microbiologist Francisco Mojica first discovered the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system and Cas (CRISPR-associated) proteins as mechanisms that prokaryotes used to defend themselves and eliminate foreign genetic material from cellular invaders such as bacteriophages, a type of virus that infects bacteria1. In the case of prokaryotes, the cell employs different mechanisms to protect themselves against invaders. The primary stages include adaptation, CRISPR RNA (crRNA) biogenesis, and interference, as shown in Figure 12.

In the first stage of adaptation, upon cellular attack, the DNA ejected from the invading virus or plasmid is broken into smaller sequences that are integrated into the prokaryote’s CRISPR archive, called a CRISPR array. The CRISPR locus contains the code for the Cas proteins, palindromic repeats, and spacer DNA (i.e., history of previous infections). Following this, under the stage of crRNA biogenesis, the CRISPR system transcribes crRNA along with an RNA-guided endonuclease, the Cas9 protein, to break the viral sequences apart and copy the DNA into the CRISPR system1,2.

During the interference stage, Cas9 carries out the degradation process by searching the DNA of the cellular invader for sequences that are complementary to the crRNA complexed with the endonuclease. Cas9 recognizes and binds to the Proto-spacer Adjacent Motif (PAM) within the invader DNA. The Cas9 protein that was derived from S. pyogenes, SpCas9, recognizes a PAM sequence of 5’-NGG-3’, which is most commonly used for gene editing3,4 PAMs are an essential element in this process; even when Cas9 is able to find complete complementary sequences, the protein is unable to make edits without the presence of a PAM sequence following the 3’ of the crRNA4.

Upon binding to the PAM sequence, the Cas9 endonuclease cleaves the viral DNA and generates double-stranded breaks through the use of the HNH domain and the RuvC-like domain. The HNH domain cleaves the sequence that is complementary to the crRNA strand, whereas the RuvC domain generates a break in the non-complementary strand. This disruption in the invader DNA effectively results in its degradation and inability to infect the cell2,4. Prokaryotes have employed this mechanism for centuries to protect themselves from cellular invaders.

Figure 1 | The stages of the CRISPR defense pathway in prokaryotes: adaptation, crRNA biogenesis, and interference.

Gene-editing in eukaryotes

Upon discovering this phenomenon, researchers Jennifer Doudna and Emmanuelle Charpentier found that the CRISPR-Cas9 system could be adapted for effective gene editing at specific and targeted sites in multicellular organisms4 It was already known that the double-stranded breaks in DNA that are generated by the Cas9 endonuclease could induce two primary repair pathway responses from targeted cells. This includes the use of either homology-directed repair (HDR) or non-homologous end joining (NHEJ) by the cell to repair the damage to the breaks in the DNA (Figure 2)5.

Figure 2 | CRISPR-Cas9 gene-editing in eukaryotes. The Cas9 endonuclease cleaves the target sequence and employs one of two repair pathways: homology directed repair (HDR) or non-homologous end joining (NHEJ). To induce HDR, a donor template is introduced to replace the double stranded break in DNA for precise replacement. For NHEJ to occur, cells inaccurately re-ligate the broken ends of DNA together, often resulting in indel mutations.

NHEJ is a highly error-prone pathway for cells to respond to breakage in the DNA wherein the cell re-ligates the bases and inaccurately joins the broken ends of the DNA back together. This often results in both insertion or deletion (indel) mutations that can interrupt the coding sequence of a gene, rendering it broken6. However, a comparatively safer and high-fidelity alternative repair pathway for cells to employ is HDR. HDR can be conducted through two primary mechanisms. Firstly, sequences that share homologous DNA and are in close proximity to the region of the double-stranded break can be used as HDR templates to repair the double-stranded break. Secondly, the preferred method for gene editing is when an artificial donor template is introduced to repair the damage. This system can be exploited for gene editing by generating double-stranded breaks (via the Cas9 endonuclease) and using the homology-directed repair mechanism to introduce a sequence of interest in the damaged region1,6.

To effectively carry out the gene-editing process in eukaryotes, multiple factors and guidelines need to be taken into consideration.

A notable factor is the difference between genetic engineering in eukaryotes versus the adaptive immunity in prokaryotes. Perhaps one of the most prominent differences is that, unlike bacterial/archaeal immunity, gene editing in multicellular organisms involves the role of the crRNA:tracrRNA complex. It was then discovered that the number of components involved in the gene-editing process could be reduced by condensing the crRNA:tracrRNA complex into one guide RNA for effective delivery (gRNA)1,4.

In addition, there are three different stages incorporated in the process of gene-editing within eukaryotic cells. This includes 1) target and guide RNA selection, 2) generation and delivery of components; and 3) the identification of the desired mutation1,7.

The selection of gRNA plays a major role in generating mutations in areas of interest to conduct the gene-editing process. The design of the gRNA must be specific and adhere to multiple guidelines. For mutations to be successfully generated, there must be the presence of a 5’-NGG-3’ PAM site directly proximal to the 20-nucleotide targeted region, when using SpCas94.

The specificity of the target sequence plays an integral role in both the efficacy of the edit as well as safety concerns as a result of using this technology. When target sequences are not highly specific there is an increase in the chance of off-target edits. These edits occur when the Cas9 endonuclease binds to non-target sequences and generates double-stranded breaks, causing mutations in different areas of the genome8. Several tools and techniques have been developed that aim to scan and analyze the target specificity of gRNA to reduce the chance of off-target edits9.

The second stage of inducing a specific mutation in a region of interest is the generation and delivery of the components. After the first stage, the CRISPR- Cas9 components such as the gRNA specific to the target site and Cas9 protein have been developed. These macromolecules can be introduced to the body through either DNA, RNA, or RNA/protein complexes as expression methods. Once the expression method has been selected, the optimum approach for therapeutic delivery must be considered, taking into account the balance between cost and efficiency1,4.

Delivery of gene-editing technology

A primary barrier to the eradication of multiple diseases is the effectiveness of delivery at the level of the organelle, cell, organ, tissue, and the organism itself, especially for genome-editing therapeutics.10 Although gene-editing technology presents a revolutionary breakthrough method of curing diseases, delivery still remains to be an essential factor in determining the scope of its effectiveness. In theory, the mechanisms behind gene-editing are straightforward; however, the effective delivery of these macromolecules in complex organisms poses an additional and highly difficult challenge for research scientists to overcome.11 Recent studies have highlighted the discovery that earlier viral methods of delivery may pose unintended and adverse effects on the body, wherein the risk of the therapy is greater than the severity of the condition itself. The extremely promising potential that gene editing proposes can only be fulfilled by its ability to safely treat non-life-threatening diseases without initiating additional risks10, and effective delivery plays a fundamental role in this process.

The challenge of delivery entails a variety of different factors to take into consideration. Firstly, the optimal use of CRISPR-Cas9 as a gene-editing tool requires the delivery of large macromolecules (gRNA and Cas9) into targeted cells, either in vivo or ex vivo. Secondly, the macromolecules need to be delivered to cells simultaneously to ensure the consistency of the editing within the set of targets.10,11.

Despite the challenges, there is a light at the end of the tunnel as clinical successes of gene-editing technologies have ushered the development of an expanded range of delivery strategies, which can be categorized into two primary approaches: viral and non-viral methods of delivery.

Viral methods of delivery in-vivo

Viral vectors are the most preferred means of delivery, because viruses have specifically evolved to target and efficiently infect human beings at the genetic level. The effectiveness of viral methods of delivery lies in the fact that researchers are leveraging the pre-existing evolutionary traits of viruses that enable their ability to enter human cells and introduce their genetic material effectively.10 The realization that the properties of viruses can be harnessed and engineered to be used as a platform for delivery was a key breakthrough that helped researchers overcome multiple barriers concerning the challenge of delivery.

In the 1990s, Perricaudet et al. found that viruses allowed for recombinant engineering and that they could harness this property by replacing the viral genome with that of the therapeutic genetic sequences for safe and programmable delivery.12 Delivery through viral vectors also allows for both transient and stable expression. Foreign nucleases are either expressed for a temporary period in case of transient expression, or in the case of stable expression, the nucleases are integrated into the target cell and expressed indefinitely. Currently, the most commonly used and advantageous methods of delivery include the engineering of Adenoviruses, Lentiviruses, and Adeno-associated viruses to cater to an extensive variety of therapeutic needs and clinical applications (Table 1).10


Figure 3 | Summary of key features, advantages, and challenges for in vivo viral gene-editing therapeutic approaches.

Adeno-associated virus

Adeno-associated viruses (AAV) are currently at the forefront of the preferred methods of delivery primarily due to their lack of pathogenicity, non-integrating nature, ability to target quiescent cells, and high target specificity. AAVs encode single-stranded DNA (ssDNA), a feature that can be highly leveraged for HDR to create knock-in mutations.10 Perhaps the most fundamental advantage of engineering an AAV for therapeutics is their lack of pathogenicity and low immunogenicity, making them a safer delivery vehicle than the majority of other viral vectors.10,13

Another advantageous characteristic that AAVs possess is the ability to persist outside of the genome of the host cell in an extrachromosomal state, or integrate into a neutral area of the host genome preferentially. Additionally, a primary advantage of using AAV vectors is that they possess highly specific tropism towards different tissue cells, increasing their effectiveness as a method of delivery. Different variations, or serotypes, of the virus are either inherently tailored or can be engineered towards tissue-specific delivery.10

However, AAVs also present two major drawbacks. Firstly, they have an extremely limited carrying capacity of around 4.4 to 4.8 kilobases. This poses a significant challenge because the sequence for Cas9 protein is around 4.1 kilobases in itself, excluding the small- guide RNA (sgRNA) that needs to be delivered along with it10,11  This means that larger genes and macromolecules cannot be fitted into a single AAV vector due to its reduced coding capacity.

Although AAVs propose significant drawbacks and challenges in terms of limited size and coding capacity they are still currently reigning as the prime candidate for the delivery of gene-editing technologies due to the extensive variety of advantages they propose.

Lentivirus

Another popular method for the delivery of gene-editing technologies is the use of lentiviruses. The lentivirus used for therapeutic purposes is an engineered version of the human immunodeficiency virus (HIV-1), which has been made comparatively safe for use by removing the genes of the parent HIV and replacing them with the genome of the biologic to produce a replication-incompetent vector.10 Modified HIV lentiviruses propose numerous advantages that can be harnessed for clinical usage. This includes its ability to transduce both dividing and quiescent cells, having an extremely high carrying capacity of around 10 kb of exogenous DNA, and having cost-effectiveness for high-scale clinical usage.14

The key advantage that makes lentiviruses another top candidate for the delivery of gene-editing technology is that they contain two copies of the RNA genome with a capacity of 10 kb, which allows for the co-delivering of the CRISPR-Cas9 system as well as the sgRNA donor template within a single infection. In addition, another advantage of lentiviruses is their low inherent immunogenicity, but this is dependent on the body’s immune response to the artificial transgene being introduced via the delivery of the lentivirus.10,13

However, one of the most significant drawbacks of the use of lentiviral systems is the semi-random integration of the lentiviral genome into the host genome, resulting in stable expression. Stable expression increases the risk of off- target edits and genotoxicity in cells. The permanent expression of these genes could stimulate the irregular expression of proto-oncogenes which in turn may lead to abnormal cell transformation, resulting in different forms of Cancer.10 Although lentiviral vectors propose key safety concerns, an increasing amount of research has fuelled the advancement of  the design and production of the virus, in order to find prospective ways to improve the safety and efficacy of this method of delivery.13

Overall, lentiviruses show promising prospects for the future of the delivery of gene-editing tools, and still have an enormous role to play in the developments in the field of delivery that are currently being made.

Adenovirus

Adenoviruses were the first viral vectors to be harnessed as a delivery strategy for gene therapy, as they were licensed for clinical trials in 1990. Adenoviruses function as linear and double-stranded DNA vectors with a non-integrating nature by remaining as an episome within the targeted cell.10 Furthermore, adenoviruses also have the advantage of a much higher carrying capacity than that of other viral vectors, as they are able to encode around 35 kilobases of DNA.15 However, this poses the fundamental challenge of inducing immunogenic responses from the body that causes inflammatory responses in animals.10,14

Electroporation

Several non-viral methods of delivery have also been developed over recent years, which could provide the significant advantage of transient nuclease expression for certain therapeutic applications. The primary and most advantageous non-viral method for delivery is the use of the physical transfection process of electroporation. Essentially, the induction of high-voltage electrical pulses through the targeted cells creates temporary and transient pores in their outer membrane.10 This allows for foreign DNA or proteins to enter the cells through the pores, wherein different lengths of DNA or sizes of proteins can be introduced without size limitations.16

Electroporation offers an extensive range of advantages including its versatility in its application on any cell type, high efficiency, and target specificity, unlimited size restrictions for DNA, and its transient expression ability.10,16. However, it does present the primary drawback of inducing substantial cell death due to the use of high voltage pulses on targeted cells. In addition, electroporation is a physical process that can only be implemented in cells ex vivo. This poses a major disadvantage as the process of drawing cells out of the body and injecting them into areas that are harder to access, such as the brain, presents a primary challenge.10

In summary, delivery poses an extensive set of barriers for researchers to overcome to fulfil the potential that this CRISPR- Cas9 technology has to offer. Both viral and non-viral methods of delivery in vivo and ex vivo pose their own set of unique challenges, but provide the opportunity for rapid scientific developments to take place.

Multiple Sclerosis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease characterized by the demyelination of neurons and axonal damage in the central nervous system. Although the etiology of MS remains unknown, the disease progression is mediated by the attack of innate, as well as acquired, immune responses directed towards the antigens in the myelin sheath. This is caused due to the failure of the immune system to discriminate between self and non-self antigens, resulting in the lack of self-tolerance.17 In MS, effector CD8+ and CD4+ T-cells permeate through the blood-brain barrier and attack the antigens of the lipid-rich coating around nerve fibers — the myelin sheath. The myelin sheath plays a vital role in the body as it is responsible for the insulation of neurons.18

In Multiple Sclerosis, however, the damage to the myelin sheath results in the breakdown of signalling as a consequence of the disruption of the usual pathway for nerves to send impulses to each other causing an extensive range of progressive symptoms. This includes cognitive degeneration, abnormal sensations, muscle spasticity, fatigue, loss of balance, and decreased mobility among others. These symptoms primarily manifest as a result of the weakening of the blood-brain barrier, and demyelination leading to the damaging of nerve cells.17,18

Although the underlying genetic cause of MS still remains unknown and the rate of progression is difficult to determine, several molecular factors have been identified that activate the neuroinflammatory symptoms of the disease, and show promise for the potential use of genetic engineering to alleviate symptoms and disrupt the progression of the disorder.

Recent studies have identified the expression of the Glutathione S-transferase 4? (GSTA4) gene in controlling oligodendrocyte apoptosis during differentiation as a primary factor that can be attributed towards causing the debilitating effects of MS.19

Oligodendrocytes and oligodendrocyte precursor cells are found in abundance in the central nervous system and are responsible for producing the myelin sheath that insulates neuronal axons, providing steady transmission of signals along neuronal networks. However, in MS, the ability of oligodendrocytes to remyelinate axons is inhibited. Recent studies have shown that the depletion of oligodendrocyte cells in the neocortex during developmental phases hinders their response to remyelinate damaged lesions.19 The depletion of oligodendrocytes occurs as a result of the restriction in the amount of support received for the cells to survive leading to programmed cell death.

Although the underlying mechanisms that result in the apoptosis of oligodendrocytes during this phase are still unestablished, studies have identified the correlation between the expression of the GSTA4  gene and oligodendrocyte differentiation. A study conducted on a mouse model of MS, experimental autoimmune encephalomyelitis (EAE) showed that when overexpressed, GSTA4 promotes remyelination by increasing the number of precursor cells that develop into myelinating oligodendrocytes, and it also regulates the rate of apoptosis of pre myelinating oligodendrocytes.19 Due to the more effective remyelination of rats in which GSTA4 was overexpressed, the clinical symptoms of EAE were alleviated and the rats exhibited a milder and shorter disease timeline.19 The results further validate the direct correlation between the overexpression of GSTA4 and remyelination in Multiple Sclerosis.

On the other hand, studies have also highlighted the role of active suppression by regulatory T cells (Tregs) in the maintenance of immune tolerance by controlling the autoreactive T cells that mediate the autoimmune response in MS. Recent investigations have shown that selectively inducing autoantigen-specific Tregs is an effective method of preventing the damage that is caused by the T cells that have escaped the body’s immune regulatory mechanisms.20 Studies conducted have also suggested that the introduction of CD4+ and CD25+ Tregs have the ability to reduce the neurodegenerative symptoms of EAE in mice by secreting anti-inflammatory cytokines that inhibit autoimmunity.20 Myelin Oligodendrocyte Proteins (MOG) are antigens present within the myelin sheath that produce a highly potent T cell responses, leading to demyelination.

This investigation showed that MOG immunotherapy using Tregs has the ability to not only restrict the progression of the EAE model of MS in mice, but it can also reverse the symptoms of EAE in early stages of the disease.20

Both of these findings through clinical investigations have shown promise for the use of gene-editing technology to ameliorate symptoms of MS.

Current therapies for Multiple Sclerosis treatment

Although the cause of Multiple Sclerosis remains unknown, multiple therapies and treatments are currently available that aim to alleviate symptoms and improve the quality of life of patients. The prevention of demyelination, promotion of the regeneration of the myelin sheath, and immune regulation/immunosuppression are three primary target areas for the treatment of the chronic disease. A variety of different therapies are directed towards commercial usage that address the neurodegenerative symptoms, as well as rapid progression of Multiple Sclerosis. These current treatment options include antibody therapy, symptomatic therapy, plasma exchange, as well as pharmacotherapy.21

Antibody-mediated therapy

Antibody-mediated therapy primarily functions as an immunosuppressive method of treating Multiple Sclerosis by preventing damage to neurons in the central nervous system. Antibody-mediated therapy uses monoclonal antibodies to target and bind to T cells mono-specifically.22 Recent studies have elucidated the idea that using antibody-mediated therapy in the earlier stages of Multiple Sclerosis can inhibit or even delay the disease’s progression. Currently, the only licensed monoclonal antibody with the purpose of treating Multiple Sclerosis is natalizumab.21

Although antibody-mediated therapy has proven to be an effective method and shows hopeful prospects for being able to treat Multiple Sclerosis, it does pose various drawbacks and side effects to take into consideration. Firstly, due to the exertion of immunosuppressive effects, antibody therapy could decrease the body’s resistance towards foreign antigens because the immune system has been weakened, thereby increasing a patient’s risk of viral infection. In addition to being more susceptible to infection due to the body’s weakened defences patients may also experience side effects due to the intravenous means of administration. Some of the side effects include infusion reactions in the blood, toxicity in the liver, or adverse immune reactions that could inadvertently cause secondary immune responses, because of the introduction of foreign antibodies that are immunogenic.21,22

Symptomatic Therapy

Although there have been numerous developments and breakthroughs in the field of disease-modifying therapies, the majority of patients with MS suffer from an extensive range of symptoms that hinder their quality of life. Hence, symptom management also plays an essential role in the treatment of MS along with disease-modifying therapies.21

Plasma Exchange

Plasma Exchange (PE) is another second-line treatment option that aims to alleviate symptoms to improve the quality of life for patients. Most patients opt for PE to manage severe or sudden attacks and relapses; it is a blood purification and filtration technique that enables the removal of soluble mediators and heavy molecular particles from plasma.23

Although PE is effective in disrupting the recurrence of MS and ameliorating clinical symptoms, it still poses a set of drawbacks and limitations to consider.

An important limitation of the intervention is the frequency of plasma exchange. Patients that receive PE may require treatment sessions at least five times per week, wherein each procedure can last for around 1 to 3 hours.23 In addition, there is ambiguity surrounding the effectiveness of PE regarding the severity of MS within the patient, as patients who are in the more progressive stages of the disease seem to benefit more from plasma exchange.21,23

Pharmacotherapy

Pharmacotherapy through the use of immunoregulatory and immunosuppressive drugs has proven to be substantially beneficial in ameliorating the clinical symptoms of MS. Studies have shown that various immunomodulatory drugs are effective in significantly reducing demyelination, and also propose the advantage of being able to inhibit the frequency of relapses in MS.21

However, the use of immunosuppressive therapeutics has a high chance of increasing the body’s risk of viral infection, as highlighted above. Furthermore, studies have illustrated that pharmacological methods of treatments may aggravate the pre-existing conditions that patients may have which significantly decreases the safety of its use on a diverse range of patients.21,24

Although several developments in the area of clinical treatments of MS have effectively aided patients in minimizing symptoms and improving neuronal functions this is just the tip of the iceberg. Although therapeutics through pharmacotherapy, plasma exchange, and antibody-mediated therapy, etc. help improve symptom management, MS patients are still susceptible to the harsh progression of the disorder.21 This entails an increased risk for behavioural, cognitive, and neurological disorders that can lead to permanent disability. These risk factors elucidate the vital need for bolder and more robust technologies that can approach the disease head-on while addressing its underlying pathogenetic causes.

Proposal

A prime area for implementing and developing innovative ways to address the molecular and genetic basis of MS is with CRISPR- Cas9 gene- editing. It is essential that we harness our current understanding of the underlying mechanisms that cause MS to be able to create innovative solutions. Because of the limitations on our current understanding of MS, having multiple approaches to combat the disease is crucial because it is challenging to guarantee that only one option will work effectively. I propose two primary novel applications of gene-editing technologies that aim to address the underlying mechanisms that mediate the autoimmune responses in MS.

Adeno-associated virus in combination with dead Cas9

The first approach of treating MS with CRISPR-cas9 technology is to use a neurotropic AAV9 containing a dead Cas9 (dCas9), fused to an activation domain. This AAV will encode a gRNA targeting GSTA4, and the dCas9 fused to an activation domain to increase the expression of GSTA4. As previously highlighted, studies have shown that the overexpression of Gtsa4 prevents the apoptosis of oligodendrocytes during differentiation, regenerating optimal nerve signalling in the body, and therefore ameliorating the symptoms of MS.19

The proposal leverages the underlying relationship between GSTA4 and oligodendrocyte differentiation by heavily increasing the expression of the gene.

Research in recent years has shown that when fused to different proteins, dCas9 can be used for novel applications that entail specific molecular activities.1,24 Cas9 ordinarily has two endonuclease domains: HNH and RuvC, that can cleave DNA to produce double-stranded breaks for gene-editing4 The introduction of mutations in these domains produces dCas9, wherein the endonuclease activity of the protein is disabled and it can instead be used to deliver functional domains to target regions within the genome.24  In this case, we can tether a transcriptional activator (i.e., activation domain that functions by enhancing RNA polymerase binding) with the dead cas9 (Figure 3). This system can then be accompanied by the specific guide RNA for GSTA4, and if successfully delivered, can activate transcription, and overexpress the gene. Increased oligodendrocyte differentiation, as a result of the overexpression of GSTA4, could promote remyelination, which could reverse the damage observed in MS.


Figure 3 | Novel approach of combining dCas9 with a transcriptional activator to treat Multiple Sclerosis. This system includes a transcriptional activator bound to dCas9, accompanied with a specific gRNA for GSTA4. The dCas9 encoding the gRNA will be delivered to the CNS using an AAV vector, resulting in overexpression of GSTA4.

However, specific tissues present within the central nervous system have certain physiologies that require unique methods of delivery. Previous literature has showcased that the brain is perhaps the most difficult organ for delivery10,11,13 This is due to the presence of the blood-brain barrier, which is highly selectively permeable against the foreign molecules that try to infiltrate the membrane. This essentially makes the delivery of both small molecules and gene-editing tools in the form of biologics into the brain extremely challenging. Although some developments have been made in being able to predict the permeability of the blood-brain barrier to unique types of small molecules, the majority of the drugs that are able to infiltrate the membrane are unable to accumulate in the brain.10

Because of this, the proposal aims to leverage the neurotropic property of AAV9 vectors for delivery into the central nervous system. The AAV9 serotype has the ability to distribute around the brain and spinal cord, and is also capable of infecting neurons and oligodendrocytes.25 Therefore, the AAV9 serotype surpasses the distribution capability of any other neurotropic AAV serotype. The tropism of AAV9 was earlier observed in an investigation on mice, and recent studies have shown that the same principle can be translated to primates.26 Due to its preferential neurotropism, AAV9 vectors are currently considered to be “the gold standard”26 for the prospect of gene therapy in the Central Nervous System.

While this approach may fulfill the prospect of effectively treating MS through CRISPR-Cas9 technology, it is also important to consider diverse approaches that target different regions in the body to increase the probability that one option will work most effectively.

T regulatory cell gene-editing

Another prospective approach to combat the autoimmune pathogenesis of MS is through engineering Treg receptors to recognize MOG via the use of HDR. As reviewed previously, the autoimmune pathogenesis of MS is thought to be characterized by effector T cells incorrectly attacking the MOG antigen present in the myelin sheath.17

Tregs on the other hand, are the counterbalance to the damage caused, as they play a pivotal role in maintaining immune tolerance by regulating the autoreactive CD4+ T cells that are responsible for the autoimmune responses that cause MS. Investigations have highlighted that MOG immunotherapy on Tregs also has the ability to reverse clinical symptoms in mice, as a result of the suppression of inflammation.20

Figure 5 | Novel approach to treat Multiple Sclerosis using regulatory T cell gene-editing. Tregs will be removed and isolated from the patient, then HDR with a template encoding MOG will be applied to the receptor of the cells via electroporation ex vivo. The reprogrammed Tregs that can recognize MOG will be reinfused into the patient, resulting in the protection of the myelin sheath from effector T cells.

The proposal aims to conduct homology-directed repair on the receptors of Tregs to identify MOG and protect them from effector T cells that recognize the proteins as non-self-antigens. This can be done by introducing a replacement sequence/HDR template that recognizes the receptor for MOG, using CRISPR-Cas9 gene-editing technology. Once cells are extracted from patients, Tregs can be isolated using the separation technique of flow cytometry. The Cas9 and gRNA encoding MOG will then be delivered into the isolated solution of Tregs through the delivery method of electroporation and, when introduced back into the body, reprogrammed Tregs will recognize MOG and be able to protect the antigen from the effector T cells (Figure 4).

Discussion

Despite being a relatively recent discovery, the use of CRISPR-Cas9 has shown unprecedented potential for various different applications and has transformed the ways through which modern medicine uses innovative techniques for therapeutic needs. Although the advent of CRISPR-Cas9 gene-editing technology has been extremely beneficial in advancing the field of biomedicine, it has also raised several bioethical questions surrounding the use of gene-editing technology on human beings. The extremely high efficacy of CRISPR- Cas9 has raised a range of legal, ethical, social, and political issues that require extensive consideration and oversight.27

A study conducted in 2015 highlighted that genome editing was used to manipulate the genetic information of early-stage human embryos.28 While this discovery reduced some of the ambiguity around the extent to which the technology can be applied on humans, it also ushered in the cascade of ethical questions and moral considerations surrounding the use of CRISPR- Cas9 on human embryos and germline cells.

However, it is important to take into consideration that while CRISPR-Cas9 technology can be used on embryos its practice in the early stages will primarily focus on non-heritable gene editing. This idea highlights the important difference between somatic and germline genetic engineering. Essentially, somatic gene-engineering entails edits in the genomes of sole individuals, without affecting gamete formation, and therefore, the changes introduced are not heritable. On the other hand, germline genetic engineering refers to the manipulation of the genes in either gametes or early-stage embryos, wherein the changes are heritable and likely to be passed on from the modified individual to subsequent generations.29 The ethical dilemma around germline genetic engineering has elicited greater controversy and skepticism, as the edits to the genome are not limited to the individual, but could instead compromise the entire human gene pool.27

Another ethical consideration to take into account is the gaps in scientific knowledge in the current model of genome editing through CRISPR-Cas9.29 Although this technology works extremely well through a versatile range of different types of cells, research has suggested that the probability of off-target edits and unwanted mutations are highly likely, producing heterogeneous cells. This presents a key safety concerns regarding the accuracy of the edits.

While most off-target edits do not have prominent side effects in research application purposes, the guidelines for clinical trials are extremely strict regarding the safety concerns that unwanted and off-target edits propose.29 Although developments have been made in trying to predict and eliminate the possibility of off-target edits9, there is still ambiguity around the long term effects of the these  unwanted mutations on the patient and, in the case of germline genome editing, the entire human gene pool.

Our relatively limited understanding of the nature of these unwanted edits not only complicates moral decision making by making it difficult to weigh the risks versus the benefits, but also reinforces the need for safeguarding and oversight regarding premature attempts of using this technology.

In addition, there are also gaps in scientific knowledge concerning the complex interplay of genes that cause a particular disease.27,29 Our understanding of the relationship between genetic expression and its result on the observable phenotype still remains convoluted and difficult to gauge. Therefore, it is extremely challenging to be able to induce changes on the whole human body based on our current understanding of the interplay between genes. It is highly uncommon that a complex disease is caused due to the expression and/or inexpression of a singular gene, especially in the case of MS wherein the genetic cause still remains unknown. It is likely that a variety of genes, as well as continuous factors such as the environment plays a role in the progression of the disease.29  Although this serves as a technical challenge for the prospect of gene therapy to treat MS it also sheds light on the primary need for genetic engineering because the cause is unknown.

From an ethical perspective one could argue that genetic screens of embryos have the potential to identify faulty genes in earlier stages of development, and therefore diseases can be eliminated from the human gene pool without the intervention of genome editing. However, this is not the case for MS as no singular gene has been determined to cause the disease which is why no faulty genes can be identified in genetic screenings of embryos either. Thus, this challenge further elucidates the requirement of gene editing through CRISPR- Cas9 to treat MS due to its complex and undetermined etiology.

In conclusion, CRISPR-Cas9 gene editing has the potential to revolutionize the face of modern medicine and presents limitless benefits for the future, perhaps even being able to eliminate genetic diseases from the entire human gene pool altogether. The application of gene-editing to treat Multiple Sclerosis is incredibly promising and shows hopeful prospects for the future as an innovative and efficient technique to combat the debilitating neurodegenerative disease. In the future, it is extremely important that we address the prospect of this technology by investing in extensive research. While CRISPR-Cas9 technology plays a pivotal role in translational biology for therapeutic needs it can also be extremely beneficial for basic biological research. Although this is beyond the scope of this paper, the future application of CRISPR technology can also include its potential to help discover the specific interplay of genes which results in MS, and therefore help researchers demystify the genetic etiology and gain a better understanding of the autoimmune disease.

Additionally, although the governing systems of some countries and international laws discourage research into this field, it is important to consider the motivations behind it to ensure that the restrictions are not bound by fear, or controversy without rational justification.27,29 While CRISPR-Cas9 has unprecedented potential for its therapeutic and biomedical applications in the future its truest scope can only be fulfilled when guided with careful deliberation, transparency, and oversight.

Acknowledgments

Thank you for the guidance of Mr. Connor Tsuchida mentor from UC Berkeley and Ms. Natalie Murphy from Johns Hopkins University in the development of this research paper.

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