1 crispr 472016 Genetic manipulations Gene therapy CRISPR

1 crispr 4/7/2016 Genetic manipulations, Gene therapy CRISPR revolution OLIVER RÁCZ, 2016

Gene manipulations – the beginnings? 2 crispr 4/7/2016 Agriculture more than 12 000 years ago – Mesopotamia, Europe Breeding, crossing of plants, animals The current maize, corn, rice is very different from the original natural types. High yield, some negative characteristics The same in domestic animals (dogs, cats, horses, pigs, cows, poultry) SLOW GENETIC MANIPULATION

Recombinant DNA technology – restriction enzymes from bacteria Cutting out genes from different species Insertion of genes into the genome of viruses, bacteria, yeast Infection of the target with the virus – gene transfer – small probability of success and temporary effect OR: Production of proteins from inserted genes in vitro 3 crispr 4/7/2016 Direct gene manipulation from 1970

th 20 Century 4 crispr 4/7/2016 Drug production in yeast and bacteria Human Some insulin, growth hormone, erythropoetin products from milk or urine of domestic animals Gene therapy – only “somatic”, not very successful (cystic fibrosis, immunodeficiencies) Gene Modified Organisms (GMO) = agricultural products and food Anxiety and fear of lay (not educated) people Business and politics

Most widespread in USA 5 crispr 4/7/2016 Soya 89 % Maize 61 % Corn – increasing And also in Argentina, Brazilia, Canada, China, India – together 114 mil. ha. Estimated increase to 2025 cca tenfold but in 2016 a small decrease The solution of starvation in developing countries? Where is Africa?

The problems – 1 the first years no “improved” products but resistance against chemical pesticides! Sowing of seeds, strong spraying the fields against weed and pests. The soya is growing… but! The second step – seeds resistant against pests but only one generation (suicidial genes) The possibilities of horizontal transfer? Arise of resistent weeds (they are here anyway) crispr 4/7/2016 In 6

The problems – 2 7 crispr 4/7/2016 Monopoly Only of some companies for big producers, not for small farms Monocultures – biodiversity violation (already present but GMOs increase the danger) Pest spread in association with monocultures BUT NO DANGER OF CONSUMPTION OF FOODS FROM MODIFIED FRUITS OR VEGETABLES

The public is against USA 55 %, CAN 61 % D 81 %, F 89 % EU embargo 1998 – protection of EU market? ? ? SK two thirds of population have no idea about it MAIZE MON 810 IS PERMITTED – RESISTAND AGAINS AN IMPORTANT PEST. ONLY FOR ANIMAL FEED SK 1 900 hectares from 120 000, BETTER YIELDS, BUT A LOT OF PAPERWORK New trends – Biofoods, fair trade, etc… Slogan or truth? Only for rich people? crispr 4/7/2016 8

Gene therapy 3 scientific journals, 30 companies, 300 protocols and more than 3000 probands In vivo and ex vivo methods Broad scale of vectors and their delivery to the target cells but they were far from perfect Later disillusionment A new start not long ago – better results in some rare diseases, no breakthrough in everyday medicine crispr 4/7/2016 Gene therapy = treatment or prevention of diseases through gene transfer into somatic cells – not only for hereditary diseases! n First years – big expectations n 9

Gene therapy crispr 4/7/2016 n 10 Clinical protocols Malignant tumoursy 216 (suicidial genes) Monogenic diseases Infectious diseases 24 Cardiovascular 8 49 (SCID ADA def. ) Vectors: mostly retro- or adenoviruses n Switching on and off the genes (expression regulation) is a problem n Problems – 700 side effects, some fatalities. Lack of informed consent. Falsified results! n

CRISPR-Cas 9 revolution 11 crispr 4/7/2016 What is it? Clustered regularly interspaced short palindromic repeats A defense mechanism of bacteria against viruses (recognition of viral genes through complementarity) Cas 9 = enzyme cutting both chains of DNA How can we use it in human biology and medicine? Exactly directed cuts of DNA, simple manipulation The possibility of gene editing (mutation repair) SOMATIC: OK – GAMETIC ? ? ? OLD METHODS – SCISSORS; CRISPR: INTELLIGENT SCISSORS!

“All hands on the desk” ADDGENE – FROM 2013 60 000 ORDERS OF ASSAY KITS GENE EDITING AS A CURE OF THALASSEMIA (EXPERIMENTAL) OTHER POSSIBILITIES: BROKEN SCISSORS – CAS 9 DOES NOT CUT BLOCKS THE GENE EXPRESSION. STUDY OF ALZHEIMER AND OTHER DISEASES CRISPR AND GENE ACTIVATION CRISPR AND BLOCK OR ACTIVATION OF EPIGENETIC FACTORS CRISPR AND OPTOGENETICS – GENE SWITCH WITH LIGHT IMPULSES STUDY OF NONCODING REGIONS NEGOTIATIONS ON ETHICAL ISSUES (GAMETIC GENE REPAIR) crispr 4/7/2016 12

CRISPR ZOO 13 crispr 4/7/2016 EGG ALERGY – BLOCK OF EGG PROTEIN EXPRESSION IN CHICKEN DIFFERENT „IMPROVEMENTS“ OF DOMESTIC ANIMALS (MORE MEAT)? ? ? BULLS WITHOUT HORNS? ? ? DIEASE RESISTANT BEES MALARIA OR ZIKA RESISTANT MOSQITOES DE-EXTINCTION OF MAMMOOTHS? MUSHROOMS WITH SLOW DECAY – ALREADY APPROVED ANIMAL MODELS OF HUMAN DISEASES