Genetic Therapy and Stem Cells Review
Genetic Therapy Overview
This set of notes encapsulates the essential aspects of genetic therapy and related cellular models as presented in the transcript.
Page 1: Introduction to Genetic Therapy
Page 2: Key Themes from Previous Discussions
Biological Treatment: The treatment of monogenic diseases, particularly metabolic disorders, can involve strategies like:
Protein replacement
Substrate removal
Small Molecules: Increasingly used for monogenic diseases therapy.
Gene and Cell Therapies: Heavily rely on the delivery of either genetic materials or live cells into an organism.
Vectors: Delivery can utilize both viral and non-viral vectors.
Page 3: Learning Outcomes
In Vitro vs. In Vivo Models: Understand their significance in research and therapy.
Pluripotent Stem Cells: Recognize their role in creating in vitro models for drug screening and development.
Recommended Readings:
Strachan & Read, Human Molecular Genetics, 5th ed., 2019.
Scherman, (editor), Gene Transfer, Gene Therapy and Genetic Pharmacology, 2nd ed., 2020.
Page 4: Perspectives on Genetic Therapies
Gene Therapy Approaches:
Ex vivo Gene Therapy: Utilizing haematopoietic stem cells.
In vivo Gene Therapy: Utilizing vectors such as adenoviruses and adeno-associated viruses.
Emerging Strategies:
Therapeutic use of embryonic stem cells
Therapeutic use of induced pluripotent stem cells (iPSCs)
Genome Editing
Germ-line Gene Therapy
Page 5: Stem Cells
An introduction to the role STEM CELLS play in genetic therapy.
Page 6: Disease Models
In vitro Models: Based on knowledge of tissues and affected cell types; may require advanced systems like co-culture and 3D organoid cultures.
Limitations: Animal experiments are still essential to assess systemic effects prior to clinical applications.
Page 7: Stem Cell Generation
Development Stages: Understanding gastrulation and germ layers which lead to stem cellular variants.
Page 8: Stem Cell Potential
Pluripotent Cells: Can differentiate into three germ layers – ectoderm, mesoderm, endoderm.
Methylation and Inducibility: Ability to generate pluripotent stem cells from adult cells by inducing pluripotency.
Page 9: Isolation of Pluripotent Cells
Describes methods to isolate and generate induced pluripotent stem cells (iPSCs) from:
Healthy Individuals
Mutation Carriers
Technology: Nuclear transfer and somatic cell reprogramming.
Page 10: Inducing Pluripotency
iPS Cells Creation: Reprogramming skin fibroblasts with pluripotency factors for applications like:
Autologous cell therapy
Ex vivo gene therapy
Drug screening
Page 11: Pluripotency Factors
Key factors involved in pluripotency:
OCT3/4 (POU5F1): Maintains pluripotency.
SOX2: Regulates embryonic development.
KLF4: Function in regulation remains unclear.
MYC: Involved in cell proliferation and differentiation.
Reprogramming Method: Popular cocktail known as “OSKM” used in lentiviral transduction.
Page 12: Cellular Models
The need and structure of cellular disease models.
Page 13: Importance of Disease Models
Rationale for using cellular models to understand diseases.
Page 14: Need for Cellular Models
‘3R’ Principles: Refine, Reduce, Replace animal experiments.
Objectives include studying disease molecular basis, drug screening, and toxicity testing.
Cell Culture: Essential for systematic approaches in experimentation.
Page 15: Comparison of Models
Cellular vs. Animal Models:
Advantages: Faster analysis of pathology, cost-effective, and ethical acceptance for cellular models.
Disadvantages: Limited analysis depth and cellular variety for cellular models, and ethical concerns with animal models.
Page 16: Therapeutic Cloning
Process: Nuclear transplantation into an enucleated egg; resulting cells genetically identical to donor.
Ethical Considerations: Cloning for reproductive purposes is considered unacceptable.
Page 17: Directed Differentiation
Engineering differentiation of pluripotent cells using defined conditions.
Page 18: Applications of iPS Cells
Uses in generating models and genetic therapies, emphasizing drug testing and personalized treatments.
Page 19: Differentiation of Pluripotent Stem Cells
Successful differentiation into various therapeutically relevant cells such as:
Dopaminergic neurons (Parkinson’s disease)
Striatal neurons (Huntington’s disease)
Oligodendrocytes (multiple sclerosis)
Page 20: Further Differentiation
Continued successful differentiation into other important cell types such as:
Cardiomyocytes (heart conditions)
Pancreatic precursors (type I diabetes).
Page 21: Organoids
Development of organoids through self-organization of mixed cell types.
Staining Techniques: H&E staining used to visualize structures.
Page 22: Culturing Organoids
PLURIPOTENT STEM CELLS can be directed into organoid cultures using specific growth factors and conditions.
Page 23: Need for Animal Models
Even with advances in cell models, animal models still play a critical role in studying complex diseases, physiological responses, and treatments.
Page 24: Today's Learning Summary
Mimetization of human diseases through in vitro and in vivo models.
Induction of pluripotency in adult cells.
Differentiation potential of iPSCs.
Ethical dilemmas associated with genome cloning and targeting.