Genetics Research And Textbook 3

By Aliasghar Tabatabaei Mohammadi, Masoumeh Mahdilou, Majid Sadeghpour and 6 more

Research And Textbook about Genetics for medical students and researchers.
 

• Language: English
• Publisher: Nobel Sciences
• Release date: Jul 7, 2023
• ISBN: 9791222424255

Book preview

Genetics

Research And Textbook

3

Chapter1: CRISPR and treating cancer1

Chapter2: CRISPR and treating cancer2

Chapter3: miRNA Pathway Gene Therapy 1

Chapter4: miRNA Pathway Gene Therapy 2

Chapter5: Nanosystems for gene therapy

Chapter6: Nucleic acid drug vectors

Author in Chief: Aliasghar Tabatabaei Mohammadi

Gmail: [e-mail]

Melorin Biotech, London, UK

https://orcid.org/ 0000-0002-3285-8701

Authors

Majid Sadeghpour

Affiliation: school of medicine of isfahan University of medical science

Gmail: [e-mail]

Chapter: 6

Zahra Mehraji

Affiliation: Department of cellular and molecular biology, kish International Campous, University of Tehran

Gmail: [e-mail]

ORCID: 0000-0003-1538-269X

Chapter: 1,2

Sina Etemadifar

Affiliation: Tehran medical sciences islamic azad university

Gmail: [e-mail]

ORCID: 0009-0005-4417-5317

Chapter: 3

Paria Arab Amiri

Affiliation: Department of biology, Central Tehran Branch, Islamic Azad University, Tehran, Iran

Gmail: [e-mail]

ORCID: 0009_0003_1123_0821

Chapter: 4

Masoumeh Nejatollahi

Gmail: [e-mail]

ORCID: 0009-0000-3806-8607

Chapter: 3

Ali Mehrani

Affiliation: Islamic Azad University Gorgan

Gmail: [e-mail]

ORCID: 0009-0009-1036-4311

Chapter: 2

Masoumeh Mahdilou

Chapter: 5

Erfan Ghanbarzadeh

Affiliation: Melorin Biotech, London, UK

Gmail: [e-mail]

Chapter: 5

Chapter1: CRISPR and treating cancer1

Cancer treatment has seen a revolution with the advent of immuno-oncology (IO) and targeted therapies such as small molecule inhibitors. However, while these treatments have shown remarkable success in improving the prognosis of cancer patients, achieving durable responses remains an uphill task due to several factors such as tumor heterogeneity, development of drug resistance, and adverse effects that limit dosing and prolonged drug use.

To address these challenges and further enhance the medicinal armamentarium, there is an urgent need for innovative approaches to understand, reverse, and treat carcinogenesis. One such approach is the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) 9 technology for genome editing, which has shown tremendous promise in developing new therapeutic strategies.

While CRISPR/Cas9 has been successfully used in pre-clinical cancer research, its application in the clinical setting is still in its early stages of development. However, the potential of this technology cannot be overstated, given its high efficiency and precision in gene editing.

The review aims to provide a comprehensive overview of the CRISPR technology, its current applications, and future potential as cancer therapies. With CRISPR/Cas9 technology, it is now possible to selectively target and modify specific genes that are involved in tumorigenesis, thereby opening up new possibilities for personalized cancer treatment.

As the field of cancer research continues to evolve, the use of CRISPR/Cas9 technology offers immense hope for developing novel cancer therapeutics that are not only more effective but also more tolerable for patients.

Cancer remains a pressing public health issue globally, with millions of new cases diagnosed each year. Despite the progress made in cancer treatment, it continues to be the second leading cause of death, following closely behind cardiovascular disease.

In recent years, there has been a significant decrease in cancer-related deaths, with over 2 million men and 1 million women avoiding such fatalities between 1975 and 2018. This achievement is largely attributed to the efficacy of novel treatments, including breakthroughs in immunotherapy, targeted therapy, and radiation therapy.

Despite these remarkable advances, there is still much work to be done. In the United States alone, approximately 2 million new cancer cases occurred in 2021, highlighting the urgent need for continued research efforts to develop effective treatments and prevention strategies.

Women aged 40-60, as well as all individuals aged 60-80, continue to face a particularly high risk of succumbing to cancer, with the disease being the leading cause of death in these groups.

Therefore, there is an urgent need for continued investment in cancer research, particularly towards developing new approaches that can improve detection, prevention, and treatment outcomes. Advances in genomics, proteomics, and immunology offer immense promise for personalized cancer therapies that are tailored to individual patients' needs.

By prioritizing funding towards research and development, we can hope to make further progress in the fight against cancer and ultimately save more lives.

Immunotherapy has emerged as one of the most promising front line agents in clinical practice for cancer treatment in recent years. Despite the effectiveness of immunotherapeutic regimens, a significant challenge that remains is predicting the response to these treatments accurately. One major contributing factor to this difficulty is that the response to immunotherapy is not linear or directly proportional to the dose of the treatment administered. Additionally, there is currently a lack of reliable biomarkers for predicting response, further complicating the task of assessing the efficacy of immunotherapy.

To address this challenge, there is an increasing need to establish new criteria and tools that can help select appropriate patients for immunotherapy treatment and quantify its beneficial effects accurately. By doing so, we can optimize treatment outcomes and minimize the risk of toxicities associated with these therapies.

Several factors positively affect the efficacy of immunotherapy, including PD-L1 status, tumor mutation burden, and gene alterations such as microsatellite instability. Studies have shown that tumors that express high levels of PD-L1 are more likely to respond to immune checkpoint inhibitors. Similarly, high tumor mutation burden has been associated with improved response rates and survival in patients treated with immunotherapy.

Moreover, gene alterations such as microsatellite instability have also been found to predict response to immunotherapy. This is because microsatellite instability results in an increased number of neoantigens, which can stimulate the immune system to attack cancer cells more effectively.

In conclusion, while immunotherapy has revolutionized cancer treatment, accurately assessing its clinical outcomes remains a significant challenge. Establishing new criteria and reliable biomarkers to select appropriate patients and quantify the beneficial effects of these therapies is crucial to optimizing treatment outcomes and reducing the risks associated with immunotherapy.

The assessment of programmed death ligand 1 (PD-L1) positivity is an essential aspect of determining the eligibility of cancer patients for immunotherapy treatment. However, the multiplicity of antibody assays and heterogeneous PD-L1 expression levels within tumors have made it challenging to establish a standardized methodology for assessing PD-L1 positivity accurately.

Despite the lack of a standardized methodology, tumor biopsies are currently considered the most reliable method for assessing PD-L1 status in cancer patients. This is because both tumor-infiltrating lymphocytes (TILs) and the immune profile of the tumor are key determinants of clinical outcome, making it essential to obtain tissue samples from the tumor site.

While there have been efforts to identify and analyze circulating tumor cells (CTCs), these methods have not yet become standard practice in assessing PD-L1 positivity. CTC analysis is still being explored as a potential non-invasive alternative to tumor biopsies, especially for cases where it may be difficult or risky to obtain a biopsy sample.

Moreover, recent advances in imaging technology, such as positron emission tomography (PET) scans, have shown promise in predicting response to immunotherapy by evaluating PD-L1 expression levels in real-time. However, these methods require further validation before they can be adopted as standard practice.

In conclusion, while there is no standardized methodology for assessing PD-L1 positivity, tumor biopsies remain the gold standard for determining patient eligibility for immunotherapy treatment. The heterogeneity of PD-L1 expression levels within tumors and the influence of TILs and immune profiles on clinical outcomes make it essential to obtain tissue samples from the tumor site. Nonetheless, emerging technologies such as CTC analysis and PET scans offer hope for potential non-invasive alternatives in the future.

Identifying biomarkers of durable response is crucial in the development and optimization of chemotherapeutic agents and targeted therapies. However, there are several challenges that hinder the identification of such biomarkers among these treatments. For instance, inhibitors of vascular epidermal growth factor (VEGF), mammalian target of rapamycin (mTOR), and cyclin-dependent kinase 4/6 (CDK4/6) have shown significant efficacy in treating various cancers. Still, identifying biomarkers to predict which patients will benefit from these treatments and the duration of response remains a significant challenge.

Moreover, dose-limiting toxicity is a common issue with cancer treatments, limiting their clinical benefits and leading to early treatment discontinuation. Therefore, finding ways to overcome dose-limiting toxicity while maximizing therapeutic efficacy is a critical area of focus in cancer research.

Furthermore, predicting toxicity in the future is also essential in optimizing cancer treatments. The use of predictive biomarkers can aid in identifying patients who are at higher risk of developing adverse events and enable clinicians to modify treatment protocols accordingly.

To address these challenges, there is a need for continued investment in research aimed at identifying reliable biomarkers of durable response and toxicity prediction. Advances in molecular profiling techniques such as next-generation sequencing (NGS) and liquid biopsy offer immense promise in this area, enabling the identification of actionable biomarkers and facilitating the development of personalized cancer therapies.

In conclusion, identifying biomarkers of durable response, overcoming dose-limiting toxicity, and predicting toxicity in the future are critical areas of focus in cancer research. Developing innovative approaches to address these challenges will be beneficial in enhancing clinical benefits, preventing early treatment discontinuation, and improving overall patient outcomes.

The field of cancer therapeutics faces numerous obstacles, including the heterogeneity of tumors, the lack of reliable biomarkers, and the need for personalized treatments that improve survival rates while reducing treatment tolerability. To overcome these hurdles, there is an urgent need to explore novel avenues for developing effective cancer therapies.

One promising platform that has emerged in recent years is the CRISPR/Cas system, which offers a versatile tool for gene editing. This innovative technology works by selectively modifying DNA sequences at specific sites in the genome with higher accuracy than conventional genome modification techniques.

The CRISPR/Cas system holds immense promise for advancing the field of cancer therapeutics, as it could potentially be used to develop highly targeted approaches to treat various types of cancer. Current efforts are focused on identifying clinical applications of this readily available pre-clinical tool, with several studies yielding promising results.

One potential application of the CRISPR/Cas system is in the development of personalized cancer therapies. By selectively targeting specific genes that contribute to tumorigenesis, it may be possible to develop more effective and tolerable treatments tailored to individual patient needs.

Moreover, the high precision and efficiency of the CRISPR/Cas system also offer new opportunities for improving the accuracy of cancer diagnosis, which, in turn, could lead to earlier interventions and better clinical outcomes.

In conclusion, identifying novel approaches to advancing the field of cancer therapeutics is essential to address the current challenges faced in treating this disease. The CRISPR/Cas system is one such platform that shows immense promise for developing highly targeted and personalized cancer therapies. Continued research efforts aimed at uncovering the full potential of this technology will undoubtedly yield exciting new therapeutic options for cancer patients in the future.

CRISPR

CRISPR (clustered regularly interspaced short palindromic repeats) technology rose to prominence as a gene-editing tool in 2012, primarily due to its low cost and relatively easy-to-use nature compared to other genome editing technologies available at that time. The key to CRISPR's relative simplicity lies in its unique DNA-targeting mechanism.

CRISPR uses a short strand of RNA that is complementary to the target DNA sequence as a homing beacon for the rest of the CRISPR protein complex. This RNA strand acts as a guide molecule that leads the endonuclease enzyme Cas9 to the specific site on the DNA where it performs the gene-editing function. In contrast, other DNA editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), use proteins to target DNA sites of interest.

The use of RNA as the target proved to be revolutionary, as designing custom CRISPR guide RNAs is significantly simpler than synthesizing custom ZFN/TALEN proteins. This ease of customization makes CRISPR an attractive option for gene editing in a wide range of applications, particularly in the field of cancer research.

Moreover, CRISPR's precision and high efficiency in targeting specific genes make it an essential tool for identifying the genetic drivers of cancer and developing more effective targeted therapies. Additionally, CRISPR can be used to create animal models with specific genetic mutations, which can help researchers understand the mechanisms of cancer progression and test new therapies.

In conclusion, CRISPR technology has revolutionized gene editing due to its low cost, ease of use, and ability to custom-design guide RNAs for specific targets. The use of RNA as a target instead of proteins has reduced the complexity of the gene editing process and made it more accessible for researchers. With its high precision and efficiency, CRISPR holds immense promise for developing new cancer therapies and unlocking the mysteries of cancer progression.

The origins of CRISPR lie in biology, where it was originally discovered as a bacterial defense mechanism against invading viruses, also known as bacteriophages. Since then, diverse CRISPR systems have been characterized in archaea and phages, highlighting the broader potential of this technology beyond its original discovery.

Despite its origin as a bacterial defense mechanism, the potential of CRISPR as a genome editing tool was quickly recognized and adapted. At its core, CRISPR consists of two fundamental components: a guide RNA (gRNA) that targets the gene of interest, and a protein complex (called Cas9) that contains a nuclease. Together, these two components act as molecular scissors to achieve double-stranded DNA cleavage at the target site.

The precise nature of CRISPR-mediated genome editing has made it an invaluable tool in the field of biomedicine, with numerous applications ranging from basic research to therapeutic interventions. In particular, CRISPR has shown immense promise in developing new cancer therapies, where precision editing of cancer-causing genes can potentially eliminate tumors while minimizing off-target effects.

Additionally, CRISPR has been used to generate animal models for studying various diseases, including cancer, which has allowed researchers to better understand disease mechanisms and test potential treatments.

As the field of CRISPR research continues to evolve, new applications are being explored, such as gene therapy and epigenetic modifications. The versatility of CRISPR technology makes it a powerful tool for exploring the complexities of genetic and epigenetic regulation, ultimately leading to more effective treatments for a wide range of diseases.

In conclusion, while CRISPR has its origins in biology as a bacterial defense mechanism against invading viruses, its transformative potential as a genome editing tool has revolutionized the biomedical field. With its precise and efficient editing capabilities, CRISPR holds immense promise for developing new cancer therapies, generating animal models for disease research, and exploring the complexities of genetic and epigenetic regulation.

The guide RNA serves as the link between the target DNA sequence and the Cas9 endonuclease in the CRISPR system. The gRNA contains a DNA complementarity sequence and a conserved tracrRNA sequence that binds with Cas9 to direct it to the DNA sequence of interest, resulting in cleavage.

However, gene editing is not as simple as just inducing CRISPR-mediated DNA cleavage. Our cells have two primary defense mechanisms against double-stranded DNA damage: non-homologous end joining (NHEJ) and homology-directed repair (HDR). Following CRISPR DNA cleavage, human cells typically undergo NHEJ, which is an error-prone process that restores the double-strand break with indels resulting in a non-functional gene.

On the other hand, HDR offers higher fidelity and can even introduce new functional genes in place of the cleaved gene when provided with an appropriate donor sequence. Consequently, following CRISPR gene cleavage, the action of NHEJ or HDR-mediated DNA repair ultimately achieves the desired gene-editing effect of either gene-knockdown or complete gene replacement (knock-in).

The decision of whether the cell undergoes NHEJ or HDR can be influenced by several factors, including the cell state, and is an area of active research. Gene knock-in via HDR is highly coveted in clinical applications because of its precision and high fidelity.

Given its versatility, CRISPR naturally lends itself to many imaginative applications in cancer diagnostics and treatment. For example, CRISPR-based technologies can detect and quantify cancer-specific mutations and gene fusions in bodily fluids such as blood plasma or urine. Additionally, CRISPR can be used to engineer T-cells for immunotherapy, allowing for more precise targeting of cancer cells while sparing healthy cells.

In conclusion, CRISPR technology has revolutionized gene editing due to its precision, efficiency, and versatility. The interplay between the gRNA and Cas9 in the CRISPR system is essential for targeted DNA cleavage, which subsequently induces DNA repair mechanisms such as NHEJ or HDR. Optimizing this process will be crucial in achieving the desired gene-editing effect, especially for clinical applications. With its vast potential, CRISPR technology offers a wide range of imaginative applications in cancer diagnostics and treatment, providing renewed hope for many patients who are fighting this disease.

Preclinical Use of CRISPR

CRISPR technology has shown immense potential in various preclinical applications, including mutation repair, gene editing, oncogene knockdown, and engineered T cell immunotherapy. One early use case of CRISPR was in the identification of a causative mutation in retinitis pigmentosa (RP), a model of inherited blindness.

In this study, Wu et al. used CRISPR-mediated repair to demonstrate that a point mutation was the causative variant of disease in the rodless mouse model of RP. This model had two homozygous mutations that were suspected to be the cause of retinal degeneration: a nonsense point mutation and an intronic insertion of a leukemia virus.

Using CRISPR, the researchers repaired the point mutation in a stepwise fashion. The first generation of animals showed mosaic correction of the allele, while the second generation of homozygous CRISPR-repaired mice exhibited rescue and disease amelioration. This achievement demonstrates the precision and potential of CRISPR for repairing mutations implicated in genetic diseases.

CRISPR has also been used to develop novel cancer therapies, such as oncogene knockdown through targeted disruption of cancer-driving genes. Additionally, engineered T cell immunotherapy using CRISPR-edited T cells has shown promise in treating various types of cancer.

Furthermore, CRISPR has been used to engineer animal models with specific gene mutations that recapitulate human diseases, allowing researchers to better understand disease mechanisms and test potential treatments. For instance, the CRISPR-Cas9 system has been used to create mouse models of human cancers, which have facilitated the development of more effective cancer therapies.

In conclusion, the versatility and precision of CRISPR technology have revolutionized preclinical research, opening up new avenues for studying genetic diseases and developing novel therapies. From identifying causative mutations in genetic disorders to engineering animal models of human diseases, CRISPR technology has shown immense promise in preclinical research. As the field continues to evolve, CRISPR will undoubtedly play a crucial role in advancing our understanding