“Technology always goes forward. There are radical new technologies that surprise us all the time. And we’ve got a long time in the future to go. This is my conclusion: Human evolution will be self-driven.” Lee Silver, PhD, 3/98Objectives: Gene Therapy See lecture objectives on web Read pages 311-327 (chapter 13) in textSlide 3Early Human Gene Therapy ExperimentsSlide 5ADA DeficiencyGene Therapy in the NewsSlide 8Concerns about Genetic EngineeringSlide 10Slide 11Gene Transfer MethodsProblemsSlide 14Retroviral VectorsAdenovirus VectorsSlide 17Gene Therapy VectorsSlide 19Slide 20Somatic Gene Therapy Treatment of human diseases by gene transferSlide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43“It is the prospect of genetic engineering that helps us appreciate what it means to be human: It means to be mortal, to be imperfect, and to be flawed. It also means to wish to be better”Slide 45Slide 46Slide 47Slide 48Slide 49Alternatives to Gene TherapyPharmaceuticals Produced by Recombinant DNA TechnologySlide 52Slide 53Slide 54Slide 55Slide 56Slide 57Slide 58Slide 59How much do we value science and technology advances? How do we view quality of human life in past, present, and future generations? How can we comfortably merge our desire for scientific advances with our respect for human life and diversity within our own value system and ethical frameworks?Slide 61Review: Gene TherapySlide 63“Technology always goes forward. There are radical new technologies that surprise us all the time. And we’ve got a long time in the future to go. This is my conclusion: Human evolution will be self-driven.”Lee Silver, PhD, 3/98Objectives: Gene TherapySee lecture objectives on webRead pages 311-327 (chapter 13) in text•Germline vs. somatic gene therapy •Gene therapy vectors (advantages and disadvantages):–Retrovirus –Adenovirus –Adeno-associated virus (AAV)–Non-viral vectors •in vivo vs ex vivo gene therapy•Current status of human gene therapy experimentation •Stem cell therapy•Pharmaceuticals produced by recombinant DNA technologyEarly Human Gene Therapy Experiments•Marty Cline human experiments-- 1980•NeoR/TIL marking studies-- 1989•ADA/peripheral blood T cells-- 1990•LDL receptor/ex vivo hepatocytes-- 1992•HLA-B7 Melanoma-- 1992•ADA/bone marrow, cystic fibrosis, multiple cancer protocols, HIVADA Deficiency•Rare Immunodeficiency (fatal in childhood)•Advantages as model for gene therapy:–Regulated expression not necessary–Low level expression sufficient–Site of synthesis not critical–Potential for in vivo selection–Bone marrow suitable target•Problems:–Difficulty achieving high level, stable expression–Other effective therapy:•PEG-ADA therapy, allogeneic/haploidentical BMT•First human experiments performed 1991 (2 patients)–?successful; simultaneous PEG-ADA therapyGene Therapy in the News•October 1999-- 1st reported death due to gene therapy•November 1999-- Failure of scientists to report gene therapy trial deaths to FDA/RAC•April 2000-- 1st definite success of human gene therapy (SCID-X1) Cavazzana-Calvo, et al. Science 288:669.•Factor IX gene therapy (hemophilia B) ? promising–In vivo AAV: Kay et al. Nat.Gen. 24:257, 2000.–Ex vivo fibroblast: Roth et al. NEJM 344:1735, 2001.Heard at the Genetics Clinic:“Can you take out the bad gene?”“Can you fix that gene?”“Can you remove the extra chromosome?”“By the time my daughter gets the disease, will be there be gene therapy to treat it, or at least to her babies”“Are doctors working on gene therapy for this?”Concerns about Genetic EngineeringThe Council for Responsible Genetics“in utero gene therapy efforts will result in eugenic practices”Mothers for Natural Law“fundamental weaknesses of genetic concepts and health hazards”Washington Biotechnology Action Council“Genetic engineering is a big business...major decisions are made in the boardrooms of corporations and by a handful of scientists and gene-splicing entrepreneurs…critical information is hidden from the public”Physicians and Scientists for Responsible Application of Science and Technology“We demand a global moratorium on the release of genetically engineered organisms and on the use of genetically engineered foods…..there are reasons to expect potentially serious hazards...”Gene Transfer Methods•Retroviral vectors–Lentiviruses•Adenovirus •Adeno-associated virus (AAV)•Other viral vectors–Vaccinia – Hepatitis virus–Herpes virus – Polio virus–Papilloma virus – Sindbis and other RNA viruses•Non viral methods–Ligand-DNA conjugates – Adenovirus- ligand-DNA –Lipofection – Direct DNA injection–CaPO4 precipitation – Ribozymes–chimeric oligo/gene correctionProblems•Delivery of DNA•Achieving high level expression•Maintaining stable expression•Tissue-specific expression•in vivo regulationRetroviral Vectors•Replace viral genes with therapeutic gene –Limited size (<8 kb)•Limited cell targets–Require dividing cells –Specific cellular receptors•High efficiency (1 virus/cell)•Stable integration into genome–Potential for insertional mutagenesisAdenovirus Vectors•Respiratory diseases in man–type 2 and 5•~36 kb, linear, double stranded DNA–Early genes (E1-E4)–Late genes (L1-L5)•Replication deficient viruses- –Delete E1a and part of E1b–grow on Ad transformed cell line (293), which contains E1 region and complements in trans–Infect target cell, but no replication•Infects broad range of cells–liver – CNS–lung – endothelial cells–muscle – othersIn addition to being safe and cost-effective, the most important properties of an efficacious gene transfer system will be; 1) target cell selective. 2) transcriptionally competent for the desired length of time.3) available in a highly concentrated active form.4) immunologically neutral.Gene Therapy VectorsVector Advantages DisadvantagesRetrovirus High efficiency transduction of appropriate target cells. Long-term expression- integration into chromosomal DNA).Potential for insertional mutagenesis.Requires dividing cells.Limited size of DNA insert.Adenovirus High transduction efficiency.Broad range of target cells.Does not require cell division.Low risk of insertional mutagenesis.Transient