Gene therapy

1 Effect of medicine: → improvement of the quality of individual life → genetic of deterioration of modern human population

(2) Preimplantation diagnostics (3) Intrauterine diagnostics Solution: (1) Gene therapy (2) Preimplantation diagnostics (3) Intrauterine diagnostics amniocentesis chordocentesis

3 Gene therapy Germline gene therapy Somatic gene therapy

Germline gene therapy 4 – retrovirus vector Baby with genetic disease Parents with genetic disease Zygote with mutant gene Therapeutic gene Figure: FIGURE 13.13 Title: Human cloning technology might allow permanent correction of genetic defects Caption: Researchers might derive human embryos from eggs fertilized in culture dishes, using sperm and eggs from the natural parents, one or both of whom have a genetic disorder. When an embryo containing a defective gene grows into a small cluster of cells, a single cell could be removed from the embryo and the defective gene replaced by means of an appropriate vector. Then the repaired nucleus could be implanted into another egg (taken from the mother) whose nucleus had been removed. The repaired, diploid egg cell could then be implanted in the mother’s uterus for normal development. Virus vector Treated culture Cell culture Embryo with genetic disease Enucleated egg cell Genetically improved embryo Genetically modified Cell from the culture Genetically improved zygote Healthy baby 5

5 Somatic gene therapy in vivo gene therapy Virus vector Liposome Gene gun Ex-vivo gene therapy is performed by transfecting or infecting patient-derived cells in culture with vector DNA and then reimplanting the transfected cells into the patient. Two types of ex-vivo gene therapies under development are those directed at fibroblasts and hematopoietic stem cells.

6 Somatic gene therapy ex vivo gene therapy virus vector liposome

7 Somatic gene therapy in vivo gene delivery ex vivo gene delivery Genetically modified cells - Viral and other types of gene transfer Neurodegenerative diseases represent an heterogeneous group of disorders that usually strike in mid-life, causing progressive loss of motor and cognitive function. Although clinical manifestations vary, the outcome is the same: patients become incapacitated over a period of years and finally die. Tackling neurodegenerative diseases thus represents a formidable challenge for our ageing society. A first axis of our research is focused on the development of rodent and primate animal models to improve our understanding of the molecular basis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). On the other hand, we are developing gene therapy strategies aimed at correcting gene defects or treating its consequences, especially for gene deletion-based genetic disorders. To that purpose, we are using two distinct gene therapy approaches to deliver candidate genes in animal models of the diseases: (i) the encapsulation of genetically engineered cell lines releasing the therapeutic molecules (Ex vivo gene therapy); and (ii) direct in vivo lentiviral vector delivery (In vivo gene therapy). Stem cell therapy: Genetically non-modified stem cells Genetically modified stem cells (gene therapy)

8 Somatic gene therapy Direct (in vivo) gene delivery Cell-based (ex vivo) Gene delivery Therapeutic gene Therapeutic gene ES cells Retrovirus Retrovirus In vitro differentiated cells Adult stem cells Neurodegenerative diseases represent an heterogeneous group of disorders that usually strike in mid-life, causing progressive loss of motor and cognitive function. Although clinical manifestations vary, the outcome is the same: patients become incapacitated over a period of years and finally die. Tackling neurodegenerative diseases thus represents a formidable challenge for our ageing society. A first axis of our research is focused on the development of rodent and primate animal models to improve our understanding of the molecular basis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). On the other hand, we are developing gene therapy strategies aimed at correcting gene defects or treating its consequences, especially for gene deletion-based genetic disorders. To that purpose, we are using two distinct gene therapy approaches to deliver candidate genes in animal models of the diseases: (i) the encapsulation of genetically engineered cell lines releasing the therapeutic molecules (Ex vivo gene therapy); and (ii) direct in vivo lentiviral vector delivery (In vivo gene therapy). Genetically modified cells 9

2 important steps of gene therapy 5 2 important steps of gene therapy 1. Delivery of foreign genes to the cells a. Cultured cells: electroporation, lipofectamine, Ca-PO4, viruses b. Cells of the body: viruses: retroviruses, adenoviruses, AAV 2. Integration of foreign DNA into the host genome: a. Cultured cells: no need specific technique for the enhanced efficiency b. Cells of the body: CRISP/R technique

Successes and failures of gene therapy Neurodegenerative diseases represent an heterogeneous group of disorders that usually strike in mid-life, causing progressive loss of motor and cognitive function. Although clinical manifestations vary, the outcome is the same: patients become incapacitated over a period of years and finally die. Tackling neurodegenerative diseases thus represents a formidable challenge for our ageing society. A first axis of our research is focused on the development of rodent and primate animal models to improve our understanding of the molecular basis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). On the other hand, we are developing gene therapy strategies aimed at correcting gene defects or treating its consequences, especially for gene deletion-based genetic disorders. To that purpose, we are using two distinct gene therapy approaches to deliver candidate genes in animal models of the diseases: (i) the encapsulation of genetically engineered cell lines releasing the therapeutic molecules (Ex vivo gene therapy); and (ii) direct in vivo lentiviral vector delivery (In vivo gene therapy). 11

9 Gene therapy of ADA David the bubble boy SCID (severe combined immunodeficiency disease): non-functioning B and T lymphocytes One of its type is caused by the mutation of ADA (adenosin deaminase) gene Neurodegenerative diseases represent an heterogeneous group of disorders that usually strike in mid-life, causing progressive loss of motor and cognitive function. Although clinical manifestations vary, the outcome is the same: patients become incapacitated over a period of years and finally die. Tackling neurodegenerative diseases thus represents a formidable challenge for our ageing society. A first axis of our research is focused on the development of rodent and primate animal models to improve our understanding of the molecular basis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). On the other hand, we are developing gene therapy strategies aimed at correcting gene defects or treating its consequences, especially for gene deletion-based genetic disorders. To that purpose, we are using two distinct gene therapy approaches to deliver candidate genes in animal models of the diseases: (i) the encapsulation of genetically engineered cell lines releasing the therapeutic molecules (Ex vivo gene therapy); and (ii) direct in vivo lentiviral vector delivery (In vivo gene therapy). Earlier: bone marrow transplantation (bone marrow of relatives) Gene therapy: ADA gene delivery to lymphocyte by retrovirus vector 12

Cystic fibrosis 10 CFTR: ABC transporter chloride channel adenovirus CFTR gene 7th chromosome nucleus mucosals cell CFTR gene lung CFTR: cystic fibrosis transmembrane conductance regulator

Trichromatic vision for squirrel monkey 11 Trichromatic vision for squirrel monkey L-opsin gene delivery to retinal cones by adeno-associated virus (AAV) vector

12 Gene therapy of Parkison’ s disease Michael Kaplitt AAV-2 – GAD gene → subthalamic nucleus AAV inoculation AAV vector for foreign gene receptor protein Nucleus subthalamicus . foreign gene rekombinant AAV Secreted protein AAV-2 : adeno-associated virus (type 2) GAD: glutamate dehydrogenase (GABA synthesis)

The first victim of gene therapy 13 The first victim of gene therapy Jesse Gelsinger Ornithine transcarbamylase deficiency (X chr.-linked disease; playing a role in ammonia metabolism) Death was caused by the side effects of adenovirus vector

The debate NO 9 Do not interfere with the Creator’s business gene therapy