Principles and perspectives of gene therapy

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2 Principles and perspectives of gene therapy 05th October 2015 Prof. Józef Dulak, PhD, DSc Department of Medical Biotechnology Faculty of Biochemistry, Biophysics and Biotechnology Room 3.025/3.07 Phone

3 Conditions for positive outcome Attending, learning and understanding the information delivered during the lectures 2. Asking the questions during and after the lecture! 3. Supplementary materials: a) articles distributed during the course b) Gene transfer to animal cells several copies are in the library For Polish students: b) Terapia genowa red. Stanisław Szala (PWN, 2003) c) Biotechnologia No 3/2007 (several copies are available in the library) 4. Passing the final exam (end of January/begining of February 2016) Test multiple choice questions will concern the information presented at the slides and those which will be explained in more details during the lectures (also based on additional materials) hence attending all lectures is strongly adivised (and is reasonable)

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6 Special issue of the journal GENE More - later.

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8 At the Department of Medical Biotechnology website there will be slides from each lecture, indicating the major aspects discussed during the course. The slides will be published at the website at the end of the course

9 Parallel course Gene transfer techniques in vitro seminars and practical course (Viral vectors in medical biotechnology) Prof. Alicja Józkowicz Dr Magdalena Kozakowska

10 What s more? During the lecture: 1. Drinking coffee not to fall asleep is allowed 2. Eating is not allowed 3. Using phones not allowed 4. If you want to chat to your colleague please leave the room and do not waste your time

11 What is gene therapy? Application of nucleic acids for treatment of diseases M. Kay, Nature Reviews Genetics, 2011

12 Gene therapy Protein Therapeutic gene (transgene) Vector Patient Expression of therapeutic gene

13 Gene genetic therapy Not only protein-coding sequences can be used in genetic therapies

14 Therapeutic nucleic acids DNA RNA Genes (DNA vectors) Non-coding sequences Genes (RNA vectors) Non-coding sequences Antisense oligonucleotides DNA decoys ribozymes sirna microrna

15 Which diseases could be cured with gene therapy? Is gene therapy necessary?

16 Gene therapy overview (1)

17 Potentially, every disease can be treated with gene therapy Every disease is characterized by the disturbances in the expression of genes: - correct version of gene is lacking (inherited diseases or acquired mutations); - expression of genes is changed due to to the disturbances in homeostasis; - even the injuries and their consequences can be (potentially) cured by gene therapy; - infection as well (genetic therapy can target both the pathogen and organisms response to the infection)

18 Gene therapy overview (2) Number of gene therapy clinical trials

19 Clinical trials The strongest evidence for therapeutic interventions is provided by systematic review of randomized, double-blind, placebo-controlled trials involving a homogeneous patient population and medical condition. Randomized: Each study subject is randomly assigned to receive either the study treatment or a placebo. Double-blind: The subjects involved in the study and the researchers do not know which study treatment is being given. Placebo-controlled: The use of a placebo (fake treatment) allows the researchers to isolate the effect of the study treatment. Some Phase II and most Phase III drug trials 19

20 Phases of clinical trials disease Some Phase II and most Phase III drug trials are randomized, double-blind and placebo-controlled 20

21 VERY IMPORTANT INFORMATION 1. The effectiveness of any treatment (drugs, gene therapy) must be confirmed in controlled clinical trials. 2. In case of diseases, which development and progression is multifactorial, and the course of disease can vary (significantly) between individual patients, and the number of patients is large (eg. cardiovascular diseases, cancer) it is necessary to peform clinical trials at all steps/stages, before any definitive conclusions on the efficacy can be presented! 3. The composition of the study group is very important! Eg. it cannot be conclusively stated that treatment is effective in a given type of cancer, if the classification of the stage and patient recrutiment was not proper 4. It is always necessary to record and control the other type of treatment when the given therapy is tested: the patients may benefit from the other drugs used at the same time 5. In case of rare diseases, in which the other treatment is not effective, the improvement after well-performed gene therapy trial can be a strong indication for the effectiveness of therapy even when observed in single patients. 21

22 Gene therapy overview (3)

23 Gene therapy overview (4)

24 When and how it has started?

25 Gene therapy was born in HPRT -/- cells HAT medium Prof.Wacław Szybalski McArdle Laboratory for Cancer Research, Wisconsin, Madison, USA HPRT +/+ cells

26 Professor Waclaw Szybalski Prof.Wacław Szybalski McArdle Laboratory for Cancer Research, Wisconsin, Madison, USA Szybalska, E. H., Szybalski, W Genetics of human cell lines. IV. DNA mediated heritable transformation of a biochemical trait. Proc. Nad. Acad. Sci. 48,

27 De novo and salvage pathways for nucleotide synthesis De novo pathways Salvage pathways

28 HAT medium A selection medium for hybrid cell lines; contains hypoxanthine; aminopterin; thymidine. Only cell lines expressing both hypoxanthine phosphoribosyl transferase (HPRT+) and thymidine kinase (TK+) can survive in this medium. Aminopterin inhibits de novo synthesis of nucleosides, while HPRT and TK supply them from hypoxanthine and thymidine. HAT medium is used to select hybridoma cells producing monoclonal antibodies

29 Inborn error of metabolism deficiency of HPRT HPRT allopurinol

30 Lesch-Nyhan Syndrome Michael Lesch HPRT allopurinol William L. Nyhan 1926-

31 Lesch-Nyhan Syndrome What is Lesch-Nyhan Syndrome? Lesch-Nyhan syndrome (LNS) is a rare, inherited disorder caused by a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). LNS is an X-linked recessive disease-- the gene is carried by the mother and passed on to her son. The lack of HPRT causes a build-up of uric acid in all body fluids, and leads to symptoms such as severe gout, poor muscle control, and moderate retardation, which appear in the first year of life. A striking feature of LNS is self-mutilating behaviors characterized by lip and finger biting that begin in the second year of life. Abnormally high uric acid levels can cause sodium urate crystals to form in the joints, kidneys, central nervous system, and other tissues of the body, leading to gout-like swelling in the joints and severe kidney problems. Neurological symptoms include facial grimacing, involuntary writhing, and repetitive movements of the arms and legs similar to those seen in Huntington s disease. Because a lack of HPRT causes the body to poorly utilize vitamin B12, some boys may develop a rare disorder called megaloblastic anemia. Is there any treatment? Treatment for LNS is symptomatic. Gout can be treated with allopurinol to control excessive amounts of uric acid. Kidney stones may be treated by breaking up kidney stones using shock waves or laser beams. There is no standard treatment for the neurological symptoms of LNS. Some may be relieved with the drugs carbidopa/levodopa, diazepam, phenobarbital, or haloperidol. What is the prognosis? The prognosis for individuals with LNS is poor. Death is usually due to renal failure in the first or second decade of life.

32 Development of gene therapy - Mechanisms of diseases: genes are known - Tools: vectors

33 vector nucleic acid, which is used to deliver the therapeutic gene/therapeutic nucleic acid Vehicle A chemical substance, which improves delivery of nucleic acid to the cells

34 Vectors Carriers of the therapeutic nucleic acids

35 Cellular barriers for successful gene therapy Kay M, Nature Rev Genetics, 2011

36 Bariers decreasing transfection efficiency in vivo I. Extracellular barriers II. Cellular/intracellular barriers 1. Opsonins 1. Cell membrane 2. Phagocytic cells 2. Endosomes/lizosomes 3. Extracellular matrix/mucus 3. Nuclear membrane 4. Digestion enzymes

37 Strategies of gene therapy in vivo and ex vivo Kaufmann et al., EMBO Mol Med. Nov 2013

38 Types of gene therapy -Somatic - germ line Germ line gene therapy is not allowed

39 Genetic therapy Substituting delivery of proper version of mutated gene Gene addition Enhancing delivery of gene which expression is impaired Suppresive delivery of gene killing the cells Gene inhibition Silencing of gene expression with non-coding nucleic acids (antisense DNA decoys sirna/shrna microrna) Gene repair Correction of the mutated gene Inherited diseases Acquired diseases Acquired diseases

40 Main problems to solve in gene therapy 1. Efficient delivery of therapeutic gene 2. Safe delivery All is about vectors

41 Vectors Non-viral/plasmids Viral naked DNA Lipoplexes RNA Retroviruses (including lentiviruses) DNA Adenoviral AAV Herpes complexes with Viroplexes other chemicals (lipoplexes enriched in specific viral proteins)

42 Nucleic acids can be introduced to the cells using * physical methods (transfection) * chemical methods (transfection) * biological methods (transduction)

43 Other useful terms Transformation of bacteria Introduction of foreign genes into bacteria Transfection Introduction of foreign genes to eucaryotic cells by means of plasmid vectors (using physical or chemical methods) Transduction Introduction of genes by means of viral vectors (biological methods) Molecular cloning Production of numerous copies of a gene, by methods of genetic engineering, eg. transformation of bacteria with plasmid, PCR cloning

44 Genetic syringes - vectors AAV vectors (adeno-associated) Retroviral vectors Adenoviral vectors Plasmid DNA

45 Viral vectors follow the rule of their parent viruses Nature,

46 Physical and chemical methods of transfection will be discussed next week

47 A visit of Dr. Waclaw Szybalski and Dr. James D. Watson at Jagiellonian University Krakow, 28 June 2008

48 A visit of Dr. Waclaw Szybalski and Dr. James D. Watson at Jagiellonian University Krakow, 28 June 2008

49 Conferment of Doctorate honoris causa for Professor Waclaw Szybalski Jagiellonian University. 28th September 2012

50 Conferment of Doctorate honoris causa to Professor Waclaw Szybalski Jagiellonian University. 28th September 2012

51 Conferment of Doctorate honoris causa to Professor Waclaw Szybalski Jagiellonian University. 28th September 2012

52 Conferment of Doctorate honoris causa to Professor Waclaw Szybalski Jagiellonian University. 28th September 2012

53 Conferment of Doctorate honoris causa to Professor Waclaw Szybalski Jagiellonian University. 28th September 2012

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55 Special issue of the journal GENE Available through the Jagiellonian Library Printed issue at the institute library