🦠Regenerative Medicine Engineering Unit 10 – Gene Therapy & Genetic Engineering

Fundamentals of Genetics

• Genetics is the study of genes, genetic variation, and heredity in living organisms
• DNA (deoxyribonucleic acid) is the hereditary material in humans and almost all other organisms
  • Consists of four nucleotide bases: adenine (A), guanine (G), cytosine (C), and thymine (T)
  • Bases pair up with each other (A with T and C with G) to form units called base pairs
• Genes are segments of DNA that encode specific proteins and determine an organism's traits
• Chromosomes are structures found in the nucleus of cells that contain an organism's genetic material
  • Humans have 23 pairs of chromosomes (46 total), with one set inherited from each parent
• Mutations are changes in the DNA sequence that can lead to altered gene function
  • Can be caused by errors during DNA replication, exposure to mutagens (UV radiation, chemicals), or viral infections
• Genetic disorders result from mutations in one or more genes (sickle cell anemia, cystic fibrosis)

Introduction to Gene Therapy

• Gene therapy involves the introduction of genetic material into cells to treat or prevent diseases
• Aims to correct defective genes or introduce new genes to compensate for missing or malfunctioning proteins
• Can be used to treat monogenic disorders caused by a single gene mutation (Huntington's disease, hemophilia)
• Also has potential applications in complex diseases involving multiple genes (cancer, cardiovascular diseases)
• Two main approaches: in vivo gene therapy (direct delivery to target cells) and ex vivo gene therapy (modification of cells outside the body)
• Requires efficient delivery methods to ensure the therapeutic gene reaches the target cells
• Offers the potential for long-term or even permanent treatment by addressing the underlying genetic cause of a disease

Genetic Engineering Techniques

• Genetic engineering involves the manipulation of an organism's genetic material using biotechnology
• Recombinant DNA technology combines DNA molecules from different sources to create new genetic sequences
  • Involves the use of restriction enzymes to cut DNA at specific sites and DNA ligase to join DNA fragments
• Polymerase Chain Reaction (PCR) is a technique used to amplify specific DNA sequences
  • Allows for the rapid production of millions of copies of a target DNA sequence
• CRISPR-Cas9 is a powerful gene-editing tool derived from the bacterial immune system
  • Uses a guide RNA to direct the Cas9 enzyme to a specific DNA sequence, where it creates a double-strand break
  • Enables precise gene editing, including gene knockout, gene insertion, and gene correction
• Viral vectors are commonly used to deliver genetic material into target cells (retroviruses, adenoviruses, adeno-associated viruses)
• Non-viral vectors, such as liposomes and nanoparticles, offer an alternative delivery method with lower immunogenicity

Delivery Methods in Gene Therapy

• Efficient delivery of therapeutic genes to target cells is crucial for successful gene therapy
• Viral vectors are the most commonly used delivery method due to their natural ability to infect cells
  • Retroviruses integrate the therapeutic gene into the host cell's genome, allowing for long-term expression
  • Adenoviruses do not integrate into the genome but can infect a wide range of cell types
  • Adeno-associated viruses (AAVs) are non-pathogenic and can provide long-term gene expression
• Non-viral vectors offer advantages such as lower immunogenicity and easier large-scale production
  • Liposomes are spherical vesicles composed of lipid bilayers that can encapsulate and deliver genetic material
  • Nanoparticles, such as gold nanoparticles and polymeric nanoparticles, can be engineered to target specific cell types
• Physical methods, such as electroporation and ultrasound, can temporarily permeabilize cell membranes to facilitate gene delivery
• Tissue-specific promoters can be used to restrict gene expression to the desired target cells

Applications in Regenerative Medicine

• Gene therapy has significant potential in regenerative medicine, which aims to replace, regenerate, or repair damaged tissues and organs
• Can be used to deliver growth factors, transcription factors, or other signaling molecules to stimulate tissue regeneration
  • Delivery of VEGF gene to promote angiogenesis and improve blood supply to damaged tissues
  • Introduction of BMP genes to enhance bone regeneration in fractures or skeletal defects
• Offers a promising approach for the treatment of genetic disorders affecting regenerative processes (Duchenne muscular dystrophy, epidermolysis bullosa)
• Can be combined with stem cell therapies to enhance their regenerative potential
  • Genetic modification of mesenchymal stem cells to overexpress therapeutic factors (neurotrophic factors for neural regeneration)
• Potential applications in the regeneration of various tissues, including skin, cartilage, and nervous tissue
• May enable the development of personalized regenerative therapies tailored to individual patient needs

Ethical Considerations

• Gene therapy raises several ethical concerns that must be carefully addressed
• Informed consent is crucial, as patients must fully understand the risks and benefits of the treatment
  • Challenges arise when treating children or individuals with impaired decision-making capacity
• Germline gene therapy, which involves modifying the genes in reproductive cells, is controversial due to its potential impact on future generations
  • Raises concerns about the long-term safety and unintended consequences of altering the human germline
• Equitable access to gene therapies is a significant concern, as high costs may limit availability to certain populations
• Potential for misuse or abuse of genetic engineering technologies (creation of "designer babies" with enhanced traits)
• Need for robust regulatory frameworks to ensure the safety, efficacy, and ethical use of gene therapies

Current Challenges and Limitations

• Efficient delivery of therapeutic genes to target cells remains a major challenge
  • Viral vectors can trigger immune responses and have limited cargo capacity
  • Non-viral vectors often have lower transfection efficiency compared to viral vectors
• Long-term safety and efficacy of gene therapies need to be thoroughly evaluated
  • Potential for insertional mutagenesis, where the therapeutic gene integrates into the host genome and disrupts normal gene function
• Immune responses against the delivered genetic material or the vectors can limit the effectiveness of gene therapy
• Scaling up the production of gene therapy vectors for clinical use can be challenging and expensive
• Regulatory hurdles and the need for extensive preclinical and clinical testing can slow down the development and approval of gene therapies

Future Directions and Emerging Technologies

• Advancement of CRISPR-based gene editing technologies for more precise and efficient gene modification
  • Development of novel CRISPR systems with improved specificity and reduced off-target effects
• Exploration of alternative gene editing tools, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)
• Development of targeted delivery systems to improve the specificity and efficiency of gene delivery
  • Use of aptamers, peptides, or antibodies to direct vectors to specific cell types or tissues
• Combination of gene therapy with other regenerative medicine approaches, such as tissue engineering and biomaterials
• Expansion of gene therapy applications to a wider range of diseases, including complex disorders and age-related conditions
• Integration of gene therapy with advanced technologies, such as artificial intelligence and big data analytics, to optimize treatment strategies
• Continued research to address the current challenges and limitations of gene therapy, ensuring its safe and effective translation into clinical practice