Challenges and Advances with the Use of CRISPR/Cas9 Technology for Mitochondrial Disease Treatment

Abstract

Mitochondrial diseases are becoming an ever-growing problem within the medical community. The variation of disorders classified under this title occurs from mutations within a patient's mitochondrial DNA. Unfortunately, this can cause symptoms that make it difficult for a person to continue their life normally, as symptoms can progress to debilitating states. Current treatment only focuses on reducing a patient’s pain after it has already emerged, making it too late for prevention. This highlights the importance of research for new treatment options that can tackle the root of this disease. CRISPR/Cas9 has become an up-and-coming technology that many researchers are saying could be a potential treatment for these mitochondrial mutations. This new possibility of cutting-edge treatment has become very popular as new studies and experiments on its use have been released, especially in relation to mitochondrial diseases. This literature review aims to condense and explain the current state of research on CRISPR/Cas9 use for treating mitochondrial ailments.

Keywords: mitochondrial diseases, CRISPR/Cas9, gene therapy, mitochondrial DNA editing

Introduction

Mitochondrial diseases (MD) are a group of disorders caused by genetic mutations. This leads to a dysfunction in the mitochondria's ability to produce energy. Mitochondria are organelles found in all eukaryotic cells of a body, each plays the important role of generating the energy required for a person and their cells to function and survive. The depletion of energy caused by mitochondrial disorders oftentimes can have a detrimental effect on the functions of organs within the body. This deficit can present itself most commonly as Leber hereditary optic neuropathy, Leigh syndrome, and Kearns-Sayre syndrome just to name a few. These varying types of mitochondrial diseases can give rise to a wide range of symptoms, depending on the patient, that may start mild but have the potential to mature into debilitating conditions. Current research has not shown promising signs of finding a cure for these mitochondrial ailments; however, despite that, several treatments have been developed to reduce and manage symptoms from escalating to life-threatening conditions. Mitochondrial treatments still remain limited, further highlighting the critical need for progressive therapeutic interventions and technology. 

CRISPR/Cas9 technology has become a new revolutionary part of genetic research. Its innovative techniques of gene editing technology have become a vital tool for the selective correction and modification of an organism's genome/DNA in a detailed and efficient manner. This groundbreaking potential has been slowly integrated into clinical and laboratory applications as possibilities for the correction of genetic disorders. The concept of targeted modifications to DNA has been a profound idea that scientists are beginning to merge with its possibility of treating immune deficiencies, specifically its prospect of addressing mitochondrial ailments and offering corrections for its conditions.

Addressing mitochondrial diseases with new technology such as CRISPR/Cas9 is crucial not only for reducing the detrimental impact on quality of life caused by these diseases' symptoms but also for advancing effective treatments that target the underlying roots of mitochondrial and other genetic-related disorders. Additionally, having a greater insight into the genetic mutations and details behind technology for mitochondrial disease treatment can pave the way for a broader application into treatment for other conditions as well.

Within this literature review, an aim will be to explore the current state of research on the use of CRISPR/Cas9 technology to address and treat mitochondrial diseases. Furthermore, it will provide an overview of the applications and main challenges faced within the context of this research, as well as possible future directions.

Methodology

This literature review focused on collecting several comprehensive review papers, relevant case studies, and other articles applicable to the topic of mitochondrial diseases and their connection to CRISPR/Cas9 technology. In order to gather these related studies, two main databases were utilized: PubMed and Google Scholar. Within each database, several keywords were implemented into the search including ‘CRISPR/Cas9’, ‘mitochondrial diseases’, ‘mitochondrial DNA editing’, ‘gene therapy’, ‘CRISPR treatment’, ‘mitochondria gRNA’, as well as additional variations and combinations of these search terms. A few criteria were considered to determine whether an article would suit this literature review's use. One factor accounted for was the article's publication date, meaning each article was checked to have at least been published within the last 10-15 years. Each source’s keywords were also checked to have relevance and additive information that would be helpful for this review.

Assessment of each article started with an initial screening of the title, keywords, and abstract. From there, suitable studies that met the criteria were then fully reviewed through its text. When analyzing each article, the goal was to detect common findings, results, and challenges being faced within this topic.

Results

Mitochondrial mutations that lead to the formation of mitochondrial ailments have the ability to induce several symptoms that can be incapacitating to a patient's life. Clinical signs that arise due to these disorders can be neurological, muscular, sensory, or even present themselves in a patient's organs causing things such as diabetes, infertility, kidney dysfunction, and more. From these symptoms alone, living with any mitochondrial disease can be incredibly tough to battle physically but also mentally, which is why many doctors and researchers are urgent in finding options for treatment that can minimize this pain.

Current standard treatment options often come in the form of working to reduce the symptoms after they have already risen, which does not help in finding a curative for the root of the problem. These prescribed treatments can be medications, vitamins, supplements, a change in nutrition or lifestyle, physical therapy, and even assistive devices. However, these recommended treatments often have been reported by patients to be poorly effective and unresponsive. In response to these difficulties, a more recent dive into the use of gene therapy has been explored as an alternative to selectively modify mitochondrial mutations. Specifically with CRISPR/Cas9, this gene editing technology requires a guiding RNA that matches the target gene sequence and a Cas9 protein to create DNA double-stranded breaks, allowing for the genome to be modified, therefore addressing the mutation. These important factors on both sides of CRISPR/Cas9 and mitochondrial ailments are heavily considered when determining a new treatment.

Advances in the technology of CRISPR/Cas9 have been rapidly improving, which has boosted expectations for its use in clinical settings. However, deeper research on its use must be examined as several studies on this system in mitochondrial diseases have produced differing and inconsistent results. In a study conducted by the Samsung Biomedical Research Institute (SBRI) in 2015, the group supposedly demonstrated that using CRISPR/Cas9 for mitochondrial DNA editing with specific localization was possible. The ability to manipulate and cleave the mitochondrial genome using CRISPR/Cas9 systems also revealed alterations in proteins associated with mitochondria. This entailed unique characteristics of the mitochondria in response to its DNA damage, which could further be explored as helpful knowledge for the regulation of mitochondrial functions. However, apart from this extra knowledge learned, this successful demonstration suggests its prospect of being a therapeutic treatment to correct mitochondrial DNA mutations.

Despite these promising results, reports of persistent technical challenges with this CRISPR/Cas9 approach have been revealed and directly contradict successful experiments like the ones previously mentioned. One significant obstacle has been the inability to import the component of RNA into the mitochondrial DNA. A critical part of the CRISPR system deals with the use of guide RNA (gRNA) sequences, which lead to target DNA sequences, where the enzyme, Cas9, that is released then controls the gene’s expression and modifies its function. While CRISPR/Cas9 has been successful in gRNA import within nucleus DNA, its transfer into the mitochondria’s separate mitochondrial DNA has raised concern. Some studies have not been able to consistently produce any results that foresee the ability of gRNA to reach or edit mammalian mitochondrial DNA and guide the Cas9 enzyme.

Discussion and Conclusion

This great contradiction to previous supposed successful attempts has still led many researchers to explore new ways of delivery in order to overcome this obstacle. Advancements in methods for gRNA delivery have expanded, for example, researchers have been testing a possible solution in using mitochondrial localization signals (MLS) to help direct gRNA into the mitochondria. Which is essentially a stem-loop motif that guides molecules or proteins, and may help improve gRNA delivery for the purpose of CRISPR editing. Additionally, other approaches being looked at to improve CRISPR as a whole include the use of cytosine base editors also known as Cas9-BE3. The idea of Cas9-BE3 is to help make more precise genetic edits to mitochondrial DNA mutations without cutting the DNA, like in typical CRISPR methods, and instead only making base changes to the DNA strands.

While new advancements for this technology are constantly being studied and oftentimes do show great potential, they still reveal their limitations. Being able to efficiently and successfully deliver gRNA to mitochondrial DNA would enable a significant number of possibilities for precisely treating mitochondrial diseases. This idea contributes to and explains why more current research is being done to find strategies that overcome this challenge for the future.

In summary, there is significant promise in the use of CRISPR/Cas9 technology and other gene editing techniques for treating mitochondrial diseases. Although there are several challenges that prevent consistent results in CRISPR gene editing within the mitochondria, its gradual improvement over time in correcting mitochondrial mutations shows current research going in the right direction. Moreover, a focus on the known limitations that must be tackled signifies the development of more successful CRISPR/Cas9 technology as a suitable treatment for mitochondrial ailments.

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