HC70A & SAS70A Winter 2011 Genetic Engineering in Medicine, Agriculture, and Law Professors John Harada & Bob Goldberg Lecture 8 Human Genetic Engineering and Gene TherapyTHEMES 1. Review Genetic Engineering Applications (Bacteria to Animals & Plants) 2. Human Gene Therapy-Genetic Engineering Humans a. What is Gene Therapy? i. Germ Line ii. Somatic Cell b. Types of Somatic Cell Gene Therapy i. Ex Vivo Gene Therapy ii. In Vivo Gene Therapy c. Example of Ex Vivo Gene Therapy i. Severe Combined Immunodeficiency (SCID) ii. Using Retroviruses For Gene Therapy iii. β-Thalasemmia d. Examples of In Vivo Gene Therapy i. Leber Congenital Amaurosis ii. Brain Tumors iii. Cystic Fibrosis iv. Gene Therapy Trials & Recent Advances e. Problems and Issues With Human Gene Therapy 3. Using Gene Therapy to Deliver “Molecular Drugs” a. Anti-Sense and RNAi b. RibozymesSome Uses of Genetic Engineering Review Of Genetic Engineering Applications Parts One & Two 1. Bacteria 2. Fungi a. Drugs b. Fermentation 3. Animals a. Mouse Model-Knock-Outs-Human Gene Functons b. Farm Animals-Drugs 4. Plants a. Spectrum of Genes Engineered b. Specific Examples of Genetically Engineered Crops c. The GMO Crop Landscape d. Reasons For Opposition to GMO Crops e. GMO “Logic” Based on Science & What We Know About Genes & Gene FunctionHuman Genetic Engineering and Gene TherapyIssues Regulation? NIH Guidelines? Human Experimentation? Ethics? Eugenics?Which type(s) of gene therapy should be allowed? a. Germline cell gene therapy b. Somatic cell gene therapy c. Both d. NeitherQuestions to Consider Before Initiating Gene Therapy 1. Does the condition result from a mutation of one or more genes? 2. What is known about the biology of the disorder? 3. Has the gene been cloned? 4. Will adding a normal copy of the gene fix the problem in the affected tissue? 5. Can you deliver the gene to cells of the affected tissue? http://learn.genetics.utah.eduEx Vivo vs In Vivo Somatic Cell Gene Therapy In vivo Ex vivoEx Vivo vs In Vivo Somatic Cell Gene TherapyEx Vivo Gene Therapy ExampleAdenosine Deaminase Gene (ADA) Deficiency and Severe Combined Immunodeficiency (SCID) Disease 32,213 kb Gene Chromosome 20 12 Exons 1,092 kb mRNA 323 aa protein David Vetter-Died at Age 12 Degradation of Purine • ADA deficiency results in elevated adenosine and deoxyadenosine levels • Abnormal levels impair lymphocyte development and function • The immune system is severely compromised or completely defectiveHumans Have Been Genetically Engineered To Cure a Lethal Genetic Disease (SCID) Several Teenagers Are Alive Because They Have Been Engineered With an ADA Gene That They Were Not Born With!!! The Age of Human Genetic Engineering Began Almost Twenty Years Ago Treating SCID With Normal ADA Genes!!! Adenosine Deaminase Gene (ADA)Ex Vivo Gene Therapy for Severe Combined Immunodeficiency (SCID)Animal Viruses are Used as Vectors to Deliver Genes for Gene TherapyComparison of Virus and Cell Sizes Note: 1 nm = 10-9 mHuman Retroviruses Are Used As Gene Therapy VectorsHIV is a Retrovirus T-CellRetrovirusesFigure 5-72 Molecular Biology of the Cell (© Garland Science 2008) Reverse Transcriptase is Encoded by a Retrovirus Genome and Converts the RNA Genome into a Double-Stranded DNA Genome That is Integrated Into a Host Cell Reverse TranscriptaseRetrovirus Life Cycle Retroviruses Replicate Using Reverse Transcriptase David Baltimore & Howard Temin-Nobel Prize 1975 Modified the Central Dogma of Molecular Biology Use For Genetic Engineering?Using a Retrovirus as a Vector For Human Ex Vivo Gene Therapy • Gag = Capid Protein • Pol = Reverse Transcriptase • Env = Envelope Protein • Ψ (Psi) = Packaging Sequence Env Gag Pol EndocytosisUsing Retroviruses for Ex Vivo Gene Therapy 1. Cloning in Bacteria 2. DNA Transformation into Packaging Cell 1. Packaging Cells Makes Viral Proteins 2. Cannot Package (Ψ-Minus) 3. Packages Therapeutic Transcript (Ψ-Plus) 1. Infect Target Cells 2. Check For Presence of Gene 3. Transfer To Patient A. B. C. Packaging Cell Line (Made Previously)It Works! Ashanthi DeSilva • Hematopoietic stem cells (HSCs) were obtained from the patient • HSCs were transduced with the ADA-containing retrovirus. • Transgenic HSCs were infused back into the patientEx-vivo Gene Therapy for β-Thalassemia • Recessive mutation in β-globin gene causes reduced rates of synthesis and formation of abnormal hemoglobin • Disease is treated with regular blood transfusions • Gene therapy – transduced hematopoietic stem cells (HSC) with lentivirus (HIV) engineered with β-globin gene • Transplanted therapeutic HSCs into patient following chemotherapy to destroy diseased HSCsIn Vivo Gene Therapy ExamplesLeber Congenital Amaurosis (LCA) • Degenerative diseases of the retina • The most common cause of congenital blindness in children Type 2 LCA is caused by recessive mutations in the RPE65 isomerase gene Normal retina LCA retina How We See Moiseyev G et al. PNAS 2005;102:12413-12418 Cideciyan et al. PNAS 2008;105:15112LCA Gene Therapy Using RPE65 & AAV ALESSANDRO CANNATA Adeno-associated viruses (AAV) • Does not generally provoke antibody formation • Infects nondividing cells of many different tissues • Integrates its DNA into a single site in the genome of animal cells • Has a small genome and can carry only short segments of DNA SUCCESS! – sort of Cideciyan et al. PNAS 2008;105:15112In Vivo Suicide Gene Therapy for Brain Cancer Retrovirus Vector for Hs-tk Gene1. The retrovirus carrying the therapeutic gene is incorporated into the genome of the tumor cells and expresses a protein encoded by the new gene [herpes simplex virus thymidine kinase gene-(HS-tk)] 2. The protein (the herpes simplex virus enzyme thymidine kinase, HS-tk) encoded by the HS-tk gene sensitizes the tumor cells to an antiviral drug (ganciclovir, GCV) which is a substrate for HS-tk. Human tk is not affected by GCV (i.e., normal cells surrounding a tumor remain healthy). 3. The enzymatic process induced by GCV leads to death of the cell expressing the herpes TK activity, i.e., death of the tumor cells. 4. Because the human HS-tk enzyme has very low affinity for GCV, systemic toxicity related to this mechanism is not