TALENs - Molecular Medicine PDF

Molecular Medicine Genome Editing Technology TALENS Dr Temba Mudariki  Overview Introduction Structure and mechanism of genome editing tools Genome editing for disease modeling and gene therapy Future application prospects Application for gene...

Molecular Medicine Genome Editing Technology TALENS Dr Temba Mudariki  Overview Introduction Structure and mechanism of genome editing tools Genome editing for disease modeling and gene therapy Future application prospects Application for gene editing in clinical trials Changes in therapeutic targeting Future prospects  Introduction Historical Context 1970s: Advent of Genetic Engineering Established the foundation for genome editing. Evolution of Genome Editing Rapid developments over the past decade From basic research to biotech and biomedical applications. Mechanism of Action In Vitro & In Vivo editing capabilities Precise gene addition, ablation, and correction.  Introduction Nuclease-Induced DNA Breaks Generation of double-stranded breaks (DSBs) Triggers cellular DNA recombination in mammalian cells. DNA Repair Pathways Homology-Directed Repair (HDR) Non-Homologous End-Joining (NHEJ) Resulting in targeted gene integration or disruption.  Gene Editing Platforms and Mechanism Genome Editing Nucleases ZFNs: Zinc-Finger Nucleases TALENs: Transcription Activator-Like Effector Nucleases CRISPR/Cas9: Clustered Regularly Interspaced Short Palindromic Repeats / Cas9 Nuclease Inducing Double-Stranded Breaks (DSBs) Targeted induction of DSBs at specific genomic sites. DNA Repair Pathways Non-Homologous End-Joining (NHEJ): Gene disruption via indel formation at the DSB site.  Gene Editing Platforms and Mechanism Homology-Directed Repair (HDR): Precise gene correction or addition with donor template. Therapeutic Applications NHEJ Gene Correction: Deletion of pathogenic sequences by targeting DSBs on both sides. HDR Gene Correction: Introduction of a corrected gene sequence using a donor template. Gene Editing Platforms and Mechanism  The Role of DSBs in Genome Editing Efficacy Comparing HR and NHEJ in Genetic Alteration Homologous Recombination (HR) Traditional Method: Uses undamaged homologous DNA as a template. Limitation: Inherently inefficient in mammalian cells. Double-Stranded Breaks (DSBs) Enhancement Discovery- DSBs significantly increase HDR incidence. Advancement- Targeted nucleases boost HDR efficiency.  The Role of DSBs in Genome Editing Efficacy HDR vs. NHEJ Mechanisms HDR-Mediated Alterations Precise gene modification with an exogenous DNA template. Utilized for mutation correction or sequence insertion. NHEJ-Mediated Alterations: Often results in indels, leading to gene inactivation. Indels in coding sequences may cause frameshift mutations. Simpler than HR: no repair matrix needed; less cell-type dependent. Applications and Implications NHEJ- Ideal for creating loss-of-function mutations. Permanent gene inactivation similar to RNAi effects. Applicable in immortalized cell lines for single or multiple gene inactivation.  Evolution of Genome Editing Technologies Navigating the Milestones from ZFNs to CRISPR/Cas9 Early Genome Editing Zinc-Finger Nucleases (ZFNs): Customizable DNA-binding proteins. Meganucleases: Highly specific but challenging to engineer. Advancement Through TALENs TALENs- Derived from Transcription Activator-Like Effectors (TALEs). Customization- Complex molecular cloning for new targets. Challenge- Low efficiency in genome screening.  Evolution of Genome Editing Technologies Advancement Through TALENs TALENs- Derived from Transcription Activator-Like Effectors (TALEs). Customization- Complex molecular cloning for new targets. Challenge- Low efficiency in genome screening.  Evolution of Genome Editing Technologies CRISPR/Cas9 Revolution Discovery- Bacterial adaptive immune mechanism. Versatility- RNA-guided DNA cleavage. Simplicity- Easier programming compared to ZFNs and TALENs. Broad Applications- From sequence correction to transcription modulation. Comparison of Genome Editing Technologies  The Clinical Horizon of Genome Editing Technologies From Benchtop to Bedside- A New Era of Genetic Medicine The Rise of Programmable Nucleases Impact: Revolutionized gene editing — from theory to therapy. Tool: Versatile manipulation of genes across species and cell types. Preclinical Research Focus Areas: Viral infections Cardiovascular diseases (CVDs) Metabolic disorders Immune system defects Haemophilia Muscular dystrophy T cell-based anticancer immunotherapies  The Clinical Horizon of Genome Editing Technologies Progression to Clinical Trials Current Status: Phase I/II clinical trials for various diseases. Advancements: ZFN, TALENs, and CRISPR/Cas9 platforms improving rapidly.  TALENs- A Protein-Based DNA targeting system The Essence of TALENs Design: Fusion of non-specific DNA-cleaving nuclease with customizable DNA-binding domain. Flexibility: Engineered to target virtually any DNA sequence. The Promise of Genome Engineering Speed and Efficiency: Rapid alteration of gene sequences. Impact: Significant advancements in biological research. Therapeutic Potential Genetic Diseases: New strategies for treatment via gene correction. Research Applications: Understanding gene function, creating disease models. Advantages of TALENs Specificity: High target sequence specificity. Customizability: Tailored design for individual gene targets. TALENS  A Breakthrough in Genome Editing TALENs - The Code Unlocked TALENs Technology: Modular assembly allows for quick design. Protein-DNA Code: Direct correlation between TALE repeat domains and DNA bases. TALENs in Action Broad Host Range: Demonstrated success in a variety of organisms: Yeast Fruit Fly Roundworm Crickets Zebrafish Frog Rat Pig Cow Thale Cress Rice Silkworm Human Cells Pluripotent Stem Cells  A Breakthrough in Genome Editing The Success Story High Success Rate: Large-scale tests confirm TALENs' effectiveness in human cells. Direct Comparisons: TALENs match or exceed ZFNs in efficiency. The Advantages User-Friendly: Accessible to researchers without specialized expertise. Targeting Range: Capable of targeting nearly any DNA sequence.  The Blueprint of TALENs: Derived from Nature's Toolkit Natural Roots of TALENs Source: TALEs from Xanthomonas proteobacteria. Function in Nature: Alter host plant cell transcription to aid bacterial colonization. Mechanism of DNA Binding TALE Repeats: 33–35 amino acids, highly conserved. Binding Precision: Each repeat corresponds to a single DNA base pair. Engineering of TALENs Building Blocks: Repeats are modular, enabling custom design of DNA-binding domains. Flanking Domains: Additional TALE-derived sequences provide stability and specificity.  Pioneers in Precision Gene Editing Disruption of Human Genes  Indels: Non-homologous end joining (NHEJ) introduces small insertions/deletions (indels).^(15, 18, 19, 68-71)  Loss-of-Function: Mimicking disease conditions in somatic cells. Precise Genome Engineering  Homology-Directed Repair (HDR): Accurate insertion using TALENs and donor templates.^(19, 20)  Applications: Endogenous gene fusions (e.g., fluorescent proteins, epitope tags) Visualization of protein dynamics and interactions in live cells. Advanced Modelling with HDR  Creation of Isogenic Cell Lines: Integration of specific single nucleotide polymorphisms (SNPs). Investigation of genetic variants from GWAS, ENCODE, etc. Potential Outcomes  Functional Studies: Determining the impact of SNPs on gene function. Understanding disease mechanisms at a cellular level. Challenges and Solutions in TALEN Assembly Crafting the DNA Sequence for Custom TALENs Assembly Challenge High Similarity: Engineering TALE repeat arrays is complex due to nearly identical sequences. Assembly Platforms Strategies Developed: Various methods for constructing plasmids encoding TALE repeat arrays. Challenges and Solutions in TALEN Assembly Broad Categories of Assembly Methods 1. Standard Cloning: Traditional cut-and-paste approach using restriction enzymes and ligase. 2. Golden Gate Cloning: Modular, uses Type IIS restriction enzymes allowing for the directionally oriented assembly of multiple fragments simultaneously. 3. Solid-Phase Assembly: Synthesizing TALENs on a solid support, often automated and high- throughput.  Platforms for Engineering TALENS Nuclease Domain Innovations Homodimeric to Heterodimeric FokI Domains Initial use of wild-type homodimeric FokI nuclease domains in TALEN design Shift to obligate heterodimeric FokI domains, improving specificity and reducing off-target effects Comparison with similar developments in ZFN technology  Platforms for Engineering TALENS Choosing the Right TALEN Framework Selecting a TALEN Assembly Method Not all TALEN architectures perform equally—consider activity levels and specificity. Importance of reviewing literature to choose the most appropriate and validated framework. Mention of the most extensively tested and validated TALEN framework by Rebar et al. as a benchmark  Platforms for Engineering TALENS Final Thoughts on TALEN Selection Making an Informed Choice in TALEN Design Summary of factors influencing TALEN performance. Encouragement to consider both historical and latest developments in TALEN architecture. Reminder of the ongoing evolution of TALEN technology and the importance of staying informed about the latest research. Therapeutic Potential of Targeted Nucleases Revolutionizing Genetic Disease Treatment Targeted Nucleases Contrast with traditional therapies: Addressing the root cause vs treating symptoms. Introduction to ZFNs and TALENs as tools for precise genetic modifications. Correcting Genetic Mutations with HDR Targeted Gene Correction via Homology-Directed Repair (HDR) Explanation of HDR and its role in precise genetic correction. Examples of ZFN-induced HDR in treating: Sickle cell anaemia Alpha1-antitrypsin deficiency Parkinson's disease by targeting alpha-synuclein gene Therapeutic Potential of Targeted Nucleases TALEN-Induced HDR in Human Cells Advancements with TALENs in Gene Correction Achievements of TALEN-induced HDR in human pluripotent stem and somatic cells Potential of TALENs to expand the scope of gene-correction therapies Gene Disruption via NHEJ for Therapy Nuclease-Induced Gene Disruption Therapy Basics of NHEJ-mediated repair and its use in gene disruption. Case study: ZFN-mediated disruption of CCR5 gene for AIDS therapy Advantages over traditional therapies in terms of specificity and immune compatibility.  Therapeutics What TALENs Can Do: TALENs are like precise molecular scissors; they can be designed to target almost any gene we want to edit. Gene Correction with TALENs: They can fix mutations in genes by accurately cutting the DNA and utilizing the cell's natural repair mechanisms to correct the faulty part, like fixing a typo in a crucial email. Gene Disruption with TALENs: TALENs can also disable a problematic gene by creating cuts that the cell repairs in a way that stops the gene from working, like switching off a broken alarm clock. Hope for Genetic Diseases: This technology opens up new possibilities for treating many genetic diseases, potentially offering personalized medicine solutions that were not possible before. From Lab to Clinic: Researchers are working hard to take TALENs from the lab bench to the patient's bedside, ensuring that these therapies are safe and effective. Looking Ahead: As we learn more, we'll see TALENs being used to tackle an increasing number of conditions, transforming how we think about and treat genetic diseases.  SUMMARY Revolutionizing Treatment: ZFNs and TALENs have the power to change the landscape of medical therapy Beyond Symptom Management: Instead of just managing symptoms, these tools aim to correct or disrupt the genes causing those symptoms Ethical Considerations: we must consider the ethical implications of gene editing, such as consent, accessibility, and potential long-term effects on individuals and the human gene pool. Practical Challenges: There are practical hurdles as well, including ensuring safety, avoiding off-target effects, and effectively delivering these therapies to the right cells in the body. Future of Medicine: Personalized medicine Call to Action: Continued research and development are crucial.