Biology 311 Human Genetics Fall 2004 Lecture Gene Therapy Readings Mountain A 2000 Gene therapy the first decade TIBTECH 18 119 128 Wu N and Ataai M M 2000 Production of viral vectors for gene therapy applications Current opinion in biotechnology 11 205 208 Chapter 21 pp 616 629 Presentation Presentation on gene therapy by Nicholas Simon Spring 2004 Biology of Cancer Outline 1 Gene therapy strategies 2 Disease targets 3 Gene therapy vectors a viruses b non viral 4 Future prospects Lecture 1 Gene therapy strategies Gene therapy The treatment or prevention of disease by gene transfer Two major approaches a Gene addition supplementation Introduction of a new copy of a gene to supplement an existing copy Gene therapy protocols in clinical trials all use this strategy b Gene replacement Correction or replacement of a defective gene by a functioning gene Technically much more difficult Other strategies are shown in Fig 21 4 2 Disease targets Existing therapies are directed at somatic cells not germ line gamete cells Cancer cells are a major target most gene therapies for cancer introduce versions of the tumor suppressor gene p53 in order to restore normal cell control Some diseases caused by single gene defects monogenic diseases are targets for gene therapy For example cystic fibrosis Some infectious diseases are being treated with gene therapy approaches Some genetic diseases are not readily approachable with gene therapies yet due to the large size of the gene muscular dystrophy or inability of the vectors to direct the gene to the correct location Drug and therapeutic testing Clinical trials Phase I Small trials of 10 15 individuals to determine toxicity of a treatment Phase II Small trials of 10 50 individuals to determine toxicity to test possible dosages or delivery methods and to obtain preliminary results on effectiveness Phase III Large trials of several hundred to a thousand individuals to determine whether a treatment strategy is safe and effective A successful trial provides results that lead to approval of drugs or more widespread applications of new therapies 3 Gene therapy vectors a viruses Adenovirus Human virus causes respiratory tract illness Double stranded DNA virus of 36 kb Can be grown to high titer Can infect a variety of cell types Infection can trigger an adverse immune response Size limit of introduced gene about 7 5 kb Most successful vector so far for human gene therapies Most useful versions are gutted removing viral genes that trigger the immune response but require growth in presence of helper virus Short duration of expression Adeno associated virus AAV Human virus not toxic to infected cells doesn t trigger an immune response A single stranded DNA virus with genome 4680 bases long Replication requires functions of a helper virus such as adenovirus or herpes simplex Can infect both dividing and non dividing cells Can only carry about a 4 5 kb gene Low production yield New generation vectors engineer helper functions on other plasmids or viruses Retroviral vectors Most are based on a mouse leukemia virus MMLV Moloney murine leukemia virus Only infect actively dividing cells especially useful for tumor therapy Single stranded RNA virus DNA copy integrates into host genome Size limit 8 kb Vector replication and packaging functions are provided by engineered cell line Problems Recovery of replication competent retrovirus and low titer Lentivirus HIV vectors HIV causes lethal disease safety concerns a disadvantage Single stranded RNA virus DNA copy integrates into host genome Infect proliferating and non proliferating cells Can infect blood forming hematopoietic stem cells Size limit of 8 kb Herpes simplex virus HSV causes many different diseases in humans cytotoxic Double stranded DNA virus 150 kb viral genome with 80 viral genes Capacity as a vector is about 30 kb Can infect a wide range of cells including neurons Does not insert its DNA into host genome b non viral Low immunogenicity safe Easy to manufacture and store Inefficient transfection and short duration of expression Naked DNA Works well for muscle heart skin DNA vaccinations can induce immune response against encoded antigens Not suitable for targeted therapies New methods allow injection into tissues without needles gene gun or Intraject Cationic lipids or polymers Positively charged molecules that bind to negatively charged DNA Protect DNA from degradation Can be used to transfect cells in lung in cystic fibrosis treatments Poor targeting Inactivated in the blood RNA based therapies Antisense oligonucleotides Ribozyme RNA enzymes RNAi 4 Future prospects Greatest limitations of current technologies Small capacity of available vectors Targeting of vectors to particular cells or tissues Safety especially cytotoxicity or immunogenicity Death of Jesse Gelsinger in 1999 in gene therapy trial with adenoviral vector for ornithine transcarbamylase deficiency Need repeated doses for many of the types of gene therapy in early trial one dose of adenovirus gene therapy for cystic fibrosis only lasted 2 4 weeks Most strategies not widely applicable to many genes Promising gene therapy concepts Direct killing of tumor cells with genes delivered by Ad vectors Ad vectors may be most applicable for short term expression Delivery of naked DNA for preventative vaccination against infectious diseases Naked DNA delivery of genes for cardiovascular disorders AAV delivery for chronic single gene disorders such as hemophilia Human hereditary diseases SCID severe combined immunodeficiency ADA 1990 Retroviral vector used to deliver adenosine deaminase gene to Tlymphocytes of affected child Used with an enzyme therapy how much of improvement due to gene therapy X linked SCID 2000 Retroviral vector Deliver portion of the cytokine receptor gene IL2R 9 11 patients were cured Complications 2 11 patients developed leukemia most likely due to insertional activation of oncogene Cystic Fibrosis Only 5 10 of normal CFTR levels needed to see improvement 18 different clinical trials of therapies initial therapies used adenovirus concern about immune response recent trials used liposomes or AAV problem in getting around mucus barrier in lungs and into correct subset of glandular epithelial cells