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In vivo and ex vivo gene therapy

Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s P rogrammes at the University of Pécs and at the University of Debrecen Identification number : TÁMOP-4.1.2-08/1/A-2009-0011.

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In vivo and ex vivo gene therapy

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  1. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011

  2. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011 Zoltan Balajthy Molecular Therapies- Lecture 4 In vivo and ex vivo genetherapy

  3. TÁMOP-4.1.2-08/1/A-2009-0011 Learning objectives of chapter 4. We are going to learn several methods by which we can introduce genetic material into a cell to treat disease, and in this manner, the aim of gene therapy is to introduce therapeutic material into the target cells, for this to become active inside the patient and exert the intended therapeutic effect. Topics in chapter 4 4.1. Gene therapy Main criterias of the effective gene therapy In vivo gene therapy Ex vivo gene therapy 4.2. Types of gene transfer, vectors for gene therapy Liposomes, naked DNA Retrovirus vector Adenovirus vector Adeno-associated viral vector (AAV) 4.3. General gene therapy strategies Targeted killing of specific cells Targeted inhibition of gene expression Targeted mutation correction 4.4. 3.4. Human gene therapy Severe combined immunodeficiency (SCID) Genetic defects in the LDL receptor Cystic fibrosis (CF)

  4. TÁMOP-4.1.2-08/1/A-2009-0011 4.1. Gene Therapy • Gene therapy may be used for treating, or even curing, • genetic and acquired diseases by using normal genes • to supplement or replace defective genes or • to bolster a normal function. • Somatic : gene is introduced into specific somatic cells; • not heritable • Germline : gene is introduced into emryonic cells or fertilized • egg; heritable (ethical, legal and religious • questions in human use)

  5. TÁMOP-4.1.2-08/1/A-2009-0011 Main criterias of the effective gene therapy Well-designed and manufactured gene The gene introduction into the right cells Safe integration of the gene into the chromosome without disturbing the surrounding genes The control of the gene, so the protein is produced only when it is needed

  6. TÁMOP-4.1.2-08/1/A-2009-0011 Somatic gene therapy targets • Cancer: main target of the current clinical trials • Muscle: easily accessible, has a good blood supply and • abundant tissue • Endothelium: can directly secrete the therapeutic protein • into the bloodstream • Skin: skin grafts also can secrete the necessary proteins • Liver: has many functions and great regeneration • Lung: easily accessible with aerosol sprays • Nervous tissues: many illnesses and injuries can affect it, • not easy to modify the neurons

  7. TÁMOP-4.1.2-08/1/A-2009-0011 In vivo gene therapy Recombinant virus DNA liposome Plasmid DNA

  8. TÁMOP-4.1.2-08/1/A-2009-0011 Ex vivo gene therapy Therapeutic gene Therapeutic gene is inserted into a specially engineered virus Target cells are removed from the patient and grown in a large number in tissue culture plates Cultured cells are mixed with the virus The cells are returned to the patient to replace the function lost due to the inheritance of mutant gene

  9. TÁMOP-4.1.2-08/1/A-2009-0011 Properties of the ideal gene therapy vector • Safe (no side effects) • Immunologically inert • Can be targeted to a specific cell type or tissue • Can be used to deliver any gene whatever its size • Easy large scale production • Cost effective • The ideal vector does not exist (yet)!

  10. TÁMOP-4.1.2-08/1/A-2009-0011 4.2. Types of gene transfer • Non-viral gene transfer • Liposomes • Naked DNA • Viral gene transfer • Retroviruses • Adenoviruses • Other viruses (Herpes simplex virus etc.)

  11. TÁMOP-4.1.2-08/1/A-2009-0011 hidrophylic head Liposomes + H2O phospholipid bilayer phospholipid hidrophobic tail Liposome Advantages: non-pathogenic, no immunity problems, no gene size limit Disadvantages: low transfection efficiency, low rate of stable integration

  12. TÁMOP-4.1.2-08/1/A-2009-0011 Naked DNA Plasmids, PCR products • Advantages: simplest • Disadvantages: very low transfection effectivity

  13. TÁMOP-4.1.2-08/1/A-2009-0011 Gene particle bombardment (bioballistic delivery system) Advantages: same as liposome-mediated transfer, promising as a vaccination method Disadvantages: limited to dermal tissue, low rate of stable integration, difficult to QC

  14. TÁMOP-4.1.2-08/1/A-2009-0011 Main criteria of the viral vectors Well-designed and manufactured gene The gene introduction into the right cells Safe integration of the gene into the chromosome without disturbing the surrounding genes The control of the gene, so the protein is produced only when it is needed

  15. TÁMOP-4.1.2-08/1/A-2009-0011 Retroviruses surface glycoprotein transmembrane protein Env integrase reverse transcriptase protease Pol matrix capsid nucleocapsid Gag RNA genome

  16. TÁMOP-4.1.2-08/1/A-2009-0011 Life cycle of retroviruses 1. Attachment 2. Penetration and uncoating 3. Reverse Transcription 4. Transport of PIC to the nucleus 5. Integration 6. Transcription 7. Translation 8. Assembly 9. Budding 10. Maturation Gene therapy constructs maintained at this stage

  17. TÁMOP-4.1.2-08/1/A-2009-0011 Moloney Murine Leukemia Virus Based Retroviral Vector I. A retroviral genome ψ B THERAPEUTIC GENE LTR LTR C infection reverse transcription LTR gag pol env LTR integration ψ integrated provirus transcription infected cell translation virus assembly 3’ LTR LTR pol gag env ψ replication-competent retrovirus

  18. TÁMOP-4.1.2-08/1/A-2009-0011 Moloney Murine Leukemia Virus Based Retroviral Vector II. ψ LTR LTR THERAPEUTIC GENE proviral therapeutic plasmid transfection THERAPEUTIC GENE gag/pol proteins gagpol ψ packaging cell line LTR LTR env envelop proteins retroviral vector infectious, but replication incompetent

  19. TÁMOP-4.1.2-08/1/A-2009-0011 MoMLV-based retroviral vector (A) The retroviral genome contains the genes gag (structural proteins), pol (reverse polymerase), and env (envelope proteins). Ψ the packaging signal distinguishing cellular RNA from viral packaging proteins. The viral genome 5 flanked by long terminal repeats (LTR). (B) Gag, pol, and env in the vector genome have been replaced by a therapeutic gene. (C) Gag, pol, env are expressed by separate genes that are transfected into the packaging cell. lf the viral vector construct is cotransfected with the transgene into the packaging cell, the protein products of the vector genome recombine with gag/pol to form infectious viruses that cannot replicate.

  20. TÁMOP-4.1.2-08/1/A-2009-0011 Retroviral gene therapy

  21. TÁMOP-4.1.2-08/1/A-2009-0011 Properties of retroviral gene therapy vectors • Advantages: • stable and long term expression of transgene • Very effective gene delivery for dividing cells (e.g. tumors) • easy production • Disadvantages: • maximum gene size 7-8kb • difficult to purify so only used for ex-vivo methods • can not be used for differentiated and non-dividing cells * • complement cascade can inactivate it • random integration may led to oncogenic activation

  22. TÁMOP-4.1.2-08/1/A-2009-0011 Lentiviral vector RSV polyA rev A gag CMV RRE polyA pol wild type HIV B lentiviral vector gag vif env 5’ 3’ LTR LTR ψ pol vpr nef tat C rev 3’ 5’ THERAP. GENE WPRE LTR CMV/LTR RRE cPPT CMV ψ RSV polyA VSV-G packaging constructs packaging cell

  23. Lentiviral vector (A) Schematic representation of the wild type HIV provirus. The HIV genome codes not only for gag, pol, and env, but also for proteins such as tat, rev, nef, vif, vpu and vpr. None of those, apart from rev, and tat, is needed for the in vitro propagation of the virus. (B) The latest generation of SIN lentiviral contains a central polypurine tract (cPPT) to support the translocation of the vector into the nucleus. An additional WPRE sequence enhances the expression of the transgene. Nearly all viral elements have been deleted, apart from the LTRs (with a SIN deletion in the 3’-LTR, see arrow), RRE (essential for the nuclear export of viral RNA), and W which is needed for packaging. (C) Gag, pol, tat (transactivates the HIV-LTR-promoter), while rev, enhances the export of unspliced genomic RNA from the nucleus after binding to RRE, and the envelope protein VSV-C are expressed by separate genes that are cotransfected into the packaging cell.

  24. TÁMOP-4.1.2-08/1/A-2009-0011 Adenoviruses

  25. TÁMOP-4.1.2-08/1/A-2009-0011 Evolution of adenoviral vectors VA E3 E1A,B L1 L2 L3 L4 L5 ITR ITR ψ E2 E4 IVa2 Ad5 genome VA L1 L2 L3 L4 L5 T. gén ITR ITR ψ E2 E4 IVa2 First generation vector; removed E1/E3 VA L1 L2 L3 L4 ITR ITR CMV T. gén ψ ψ ITR E2 E4 IVa2 Second generation vector; removed E1/E3/L5 CMV T. gén E4 ITR Third generation vector; most of the genes are removed, helper-dependent

  26. TÁMOP-4.1.2-08/1/A-2009-0011 Adenoviral vectors (A) Schematic representation of a serotype 5 adenovirus (Ad5) on which most of the adenoviral vectors described here are based. The vector genome is flanked by inverted terminal repeats (ITRs). Ψ is the packaging signal. The adenoviral genes are highlighted in boxes. (B) First-generation adenoviral vector in which the genes E1 and E3 have been deleted. The E1A plays a decisive part in viral replication as the initiator of the transcription of other viral transcription units. However, the gene is not needed for adenoviral replication within 293 cells, which makes those cells ideal for virus production. The E3 gene product is not essential for viral reproduction, although its role in immune modulation and suppression is important. The therapeutic gene is simultaneously transfected into the packaging cell, using a shuttle vector. It is than inserted into the adenoviral vector in exchange for the E1 gene. (C) Helper-dependent adenoviral vectors in which parts of the adenoviral genome (flanked by loxP recognition sites, triangles) have been excised in order to avoid immune reactions in the host. The expression of the therapeutic gene is driven by a promoter such as CMV. (D) In order to avoid potentially violent immune reactions of the host to adenoviral proteins, mini or gutless adenoviral vectors have been produced in which most of the adenoviral genes have been deleted.

  27. TÁMOP-4.1.2-08/1/A-2009-0011 Adenoviral gene therapy

  28. TÁMOP-4.1.2-08/1/A-2009-0011 Properties of adenoviral gene therapy vectors • Advanteges: • no risk of oncogenic activation (no DNA integration) • can carry large genes (30 kb) • infect dividing and non-dividing cells • high level gene expression • easy production • Dissadvantages: • short term gene expression • induce inflammatory and immune response • Cell-specific targeting difficult to achieve

  29. TÁMOP-4.1.2-08/1/A-2009-0011 Adeno-associated viruses A B C vector genomes viral vector construct ITR ITR Rep/Cap construct Packaging and replication proteins rep Theraupeticgene cap ITR ITR Recombinant vector genome Adeno-associated viral genome Packaging cell Adeno-associated viruses

  30. TÁMOP-4.1.2-08/1/A-2009-0011 Adeno-associated viral vector (A) The AAV genome contains sequences which are essential for the transduction process, such as inverted terminal repetitions (ITRs) and the genes rep and cap. (B) In the vector genome, rep and cap have been replaced by a therapeutic gene. lf the therapeutic gene is larger than 4.5 kb, it is distributed over two concatemeric vector constructs. (C) The REP and CAP proteins are expressed by the packaging cells and are needed for the production of single-stranded DNA genomes in a capsule consisting of proteins. A non-enveloped AAV virus collects in the nucleus. Helper proteins from adenoviruses, which are needed for replication, are also expressed in the packaging cell (not shown here). The AAV are released from the packaging cell through the lytic adenoviral replication process.

  31. TÁMOP-4.1.2-08/1/A-2009-0011 Properties of adeno-associated viral vectors • Advantages: • not associated to human diseases • effectively infects dividing and non-dividing cells • able to integrate into the specific site (chromosome 19) • small genom and easy to manipulate • can obtain high titered virus stock (109-1010/ml) • Dissadvantages: • limited gene size ~ 4.5kb

  32. TÁMOP-4.1.2-08/1/A-2009-0011 3.3 Gene therapy strategies I. Gene augmentation prodrug gene X gene toxin gene normal phenotype disease cells disease cells disease cells Direct cell killing cells killed by toxin cells killed by drug drug

  33. TÁMOP-4.1.2-08/1/A-2009-0011 Gene therapy strategies II. Indirect cell killing by immunostimulation foreign antigene gene disease cells cytokine gene disease cells killing of disease cells because of enhanced immune response immun cells

  34. TÁMOP-4.1.2-08/1/A-2009-0011 Gene therapy strategies III. Targeted inhibition of gene expression x gene antisense gene AAAA or disease cells with mutant gene x inhibition disease cells with mutant or harmful gene block expression of pathogenic gene N C antisense TFO, ODN m m m m Targeted gene mutation correction X X normal phenotype corrected gene

  35. TÁMOP-4.1.2-08/1/A-2009-0011 4.4. Gene therapy targets infectious diseases monogenic diseases other cardiovascular diseases cancer diseases

  36. TÁMOP-4.1.2-08/1/A-2009-0011 Targeting of different organs by viral vectors Adenoviruses (tumors, hematopoietic cells) Hematopoietic cells AAV (liver, muscle, retina) Lentiviruses (CNS, liver, muscle) Alphaviruses (tumors) Retroviruses (tumors, stem cells, hematopoetic cells) stem cells Herpes simplex virus (CNS, hematopoietic cells, muscle, stem cells)

  37. TÁMOP-4.1.2-08/1/A-2009-0011 Severe combined immunodeficiency (SCID): Lack of adenosine deaminase (ADA) deoxyadenosine SYMPTOMS deoxy-ATP ADA deficiency STOP Accumulation of dATP blocks the development of T and B-cells which leads severe immunodeficiency „bubble boy” disease deoxyinosine hipoxanthine xanthine uric acid

  38. TÁMOP-4.1.2-08/1/A-2009-0011 Possible therapies for ADA-SCID • Life long germ-free tent (David Vetter) • Regular injections of PEG-ADA • ADA is isolated from cow and conjugated with PEG • Bone marrow transplantation • No rejection because of the defective immune system • Transplanted T-cells can attack the graft recipient • Donor cells may be infected (David Vetter) • T-cell gene therapy • Retroviral vectors, repeated injections because T-cells live • 6-12 months • Stem cell gene therapy • Blood stem cells of the patients are transformed with ADA gene, • while some of the bone marrow cells are destroyed. Then • transduced cells are injected back to build up new normal bone • marrow cells

  39. TÁMOP-4.1.2-08/1/A-2009-0011 Gene therapy for severe immunodeficiency syndrom I. Isolation of DNA from normal cells isolation of normal T-lymphocytes Restriction cleavage and isolation of ADA gene lymphocyte growing isolation of viral DNA and same restriction cleavage Ligation of DNA fragments

  40. TÁMOP-4.1.2-08/1/A-2009-0011 Gene therapy for severe immunodeficiency syndrom II. T lymphocyte isolation from patient injection of engineered lymphocytes into the patient viral vector production growing and testing lymphocytes (ADA) T lymphocyte infection with virus

  41. TÁMOP-4.1.2-08/1/A-2009-0011 Ornithine transcarbamoilase (OTC)deficiency liver symptoms amino acids from food ammonia mental retardation ATP carbamoil-phosphate urea STOP OTC ornithine arginine urea cycle citrulline • most common disorder of urea cycle • X-linked recessive disorder • Low-protein diet and administration • of medications scavenging nitrogen arginino- succinate

  42. TÁMOP-4.1.2-08/1/A-2009-0011 Setbacks in gene therapy Jesse Gelsinger (1999) • had a mild OTC deficiency, which was controlled by diet and regular therapy • volunteered for the OTC gene therapy where normal OTC gene was in vivo transferred • into his liver using by adenovirus • felt in coma after few hours of treatment then died 3 days afterwards • he had extreme high virus level which caused strong immune response led to his death • French X-SCID (2002) • one of the eleven „bubble boys” did not respond to the treatment • eight children cured • leukemia was developed in two children

  43. TÁMOP-4.1.2-08/1/A-2009-0011 Limitating factors of gene therapy • short-lived nature of therapy • - multiple periodic treatments required • immune response • - hard for repeated treatments • problems with viral vectors • - could regain virulence • - could cause toxicity, immun and imflammation response • - could control and activate genes • multi-gene disorders • - challenge for gene therapy (high blood pressure, Alzheimer)

  44. TÁMOP-4.1.2-08/1/A-2009-0011 • Genetic disorder • - mostly resulted by the mutation of the LDL receptor gene • - homozygous frequency 1: 500 • - heterozygous frequency 1: 1 000 000 • High cholesterol and LDL levels in blood • homozygous have 6-7 X higher • heterozygous have 2.5 X higher compared to normal values • Early cardiovascular diseases (heart attack / stroke) • for homozygous in childhood at the age of 5 and 10 • for heterozygous at the age of 35 and 40 Familial hypercholesterolemia

  45. TÁMOP-4.1.2-08/1/A-2009-0011 Transport of lipids with plasma lipoproteins

  46. TÁMOP-4.1.2-08/1/A-2009-0011 Regulation of the mevalonate pathway

  47. TÁMOP-4.1.2-08/1/A-2009-0011 Main facts of the cholesterol question • most of the cells permanently synthetize cholesterol • daily cholesterol intake is significant even from a normal diet • cholesterol does not degrade, removed with biles • inhibition of the cholesterol synthesis could block the formation • of other important compounds which may lead severe side effects

  48. TÁMOP-4.1.2-08/1/A-2009-0011 Levels of the genetic deficiencies of LDL receptor

  49. TÁMOP-4.1.2-08/1/A-2009-0011 Correlation between the LDL cholesterol level of blood and the number of LDL receptors in liver

  50. TÁMOP-4.1.2-08/1/A-2009-0011 General treatments of the high plasma cholesterole level • high HDL level • change of the dietary mode • block of the entherohepatic circulation of bile acids • inhibition of HMG-CoA reductase by statins bile acid depletion + reductase inhibitor no drugs bile acid depletion HMG CoA LDL LDL LDL LDL LDL LDL plasma HMG CoA HMG CoA cholesterol bile acids liver cholesterol cholesterol bile acids bile acids intestine

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