Gene therapy involves transferring therapeutic genes into diseased tissues to treat genetic disorders. The document discusses using gene therapy to treat hemophilia in dogs by infusing genes encoding coagulation factor IX via a one-hour procedure, resulting in gene expression for 15 months and blood clotting within 20 minutes. It also lists several diseases that may be treated with gene therapy and discusses general concerns with the approach, such as its short-lived nature and immune responses.

1. GENE THERAPY

2. Gene therapy: Transfer of therapeutic gene into the diseased tissue Hemophiliac dogs with coagulation factor IX deficiency Normal dog: blood clots in about 8 to 10 minutes Diseased dog: blood clots in about 50 to 60 minutes Dr. Kenneth Brinkhous (North Carolina Univ) 1-hour procedure of GT infusion, 15 month of expression, 20 min for clotting

3. Diseases for applying gene therapy Disease Defect Target cell Severe combined Adenosine deaminase 4 Bone marrow cells or immunodeficiency T-lymphocytes Hemophilia Factor VIII, Factor IX deficiency Liver, muscle, fibrob. Cystic fibrosis Loss of CFTR gene Airspaces in the lung Hemoglobulinpathies  or  globulin gene Bone-marrow cells  1-antitrypsin deficiency  1-antitrypsin Lung or liver cells Cancer Many causes Many cell types Neurological diseases Parkinson’s, Alzheimers Direct injection into the brain Cardiovascular Restenosis, arteriosclerosis Vascular endothelium Infectious diseases AIDS, hepatitis B T cells, macrophages, Liver cirrhosis Fibrogenesis Hepatocyte growth factor Autoimmune disease Lupus, diabetes MHC,  2-microglobulin

4. In humans Cancer 69%

8. Gene therapy In vivo Ex vivo

9. in vivo and ex vivo schemes http://laxmi.nuc.ucla.edu:8237/M288/SChow_4_10/sld005.htm IN VIVO EX VIVO

10. 1. The genetic material is transferred directly into the body of the patient 2. More or less random process ; small ability to control; less manipulations 3. Only available option for tissues that can not be grown in vitro; or if grown cells can not be transferred back In vivo gene therapy

12. Select normal cells that are GT resistant to chemotherapy Select hematopoietic cells resistant to paclitaxel (taxol) after introducing an MDR1 pump Resistant to alkylating agents after introducing an O6-alkylguanine-DNA-alkyltransferase Resistant to methothrexate after introducing a mutant DHFR enzyme Blood vessel

13. Transgenes Integrated Not integrated - stable expression; may provide a cure - expression is transient; repeated treatments nesessary - random insertions in heterochomatin can be inactivated; In euchromatin -- Can disrupt important host genes; Long-term consequences are unknown for episomes (plasmids) random mutagenesis not an issue

14. How episomes and integrated trasgenes behave in dividing cells Integral transgene Episome Loss of plasmid

15. Influences on choice of vector high efficiency viral vectors for gene replacement therapy of monogenic diseases (cystic fibrosis; SCID; hemophilia…) short term gene expression To prime an immune response To sensitise cells to radiotherapy … Liposomal Delivery…

16. Desirable characteristics of gene delivery vector 1. High titer or concentrations (>10 8 particles/ml) 3. Precise and stable introduction of transgene 2. Easy and reproducible method of production 4. Vector should not elicit immune response in the host 6. Vector should be able to target specific cell types 5. Transgene should be responsible for its regulatory elements (on/off system)

17. Methods of gene delivery (therapeutic constructs) -- Injection of naked DNA into tumor by simple needle and syringe -- DNA transfer by liposomes (delivered by the intravascular, intratracheal, intraperitoneal or intracolonic routes) -- DNA coated on the surface of gold pellets which are air-propelled into the epidermis (gene-gun), mainly non applicable to cancer -- Biological vehicles (vectors) such as viruses and bacteria. Viruses are genetically engineered so as not to replicate once inside the host. They are currently the most efficient means of gene transfer .

19. Injections of naked DNA

21. DNA vaccines Cancer immunotherapy Antiviral and antibacterial (traditional vaccines are better when available) Passive to increase the pre-existing immune response to the cancer Active initiates an immune response against an unrecognised or poorly antigenic tumor

22. Current attempts with naked DNA vaccination in infectious diseases HIV hepatitis B and C Influenza Papilloma Cytomegalovirus Tuberculosis, Lyme disease Helicobacter pylori Malaria T cells recognise liver cell with malarial parasite inside Produce IFN-gamma IFN-gamma stimulate antigen presentation Important: too much IFN-gamma is also too bad. (it is pro-inflammatory) www.malaria-vaccines.org.uk

23. DNA vaccines (and other vaccines too) prime immune system with properly presented antigen immunologically important components of the malaria pathogen whole protein called thrombospondin related adhesion protein (TRAP). DNA vaccine encoding an immuno recognisable insert several peptide epitopes that we know are recognised by T-cells (a so-called multi-epitope string)

24. Ballistic DNA Injection , particle bombardment, microprojectile gene transfer (gene guns) Invented for DNA transfer to plant cells Fully applicable to mamalian cells Light micrograph of DNA-coated gold beads in the skin after gene-gun vaccination plasmid DNA is precipitated onto 1-3 micron sized gold or tungsten particles. Discharge: helium pressure, or high-voltage electronic

25. Duchenne muscular dystrophy (DMD) 1. Generalized weakness and muscle wasting affecting limb and trunk muscles first. Calves often enlarged. Wheels at 12 y.o. X-linked recessive disorder; 1/3500 boys worldwide About 30% of cases represent new mutations. Life threatening dysrhythmia or heart failure develops in about 10 %. Absence of dystrophin, a cell membrane protein (approximately 0.01 % of skeletal muscle protein). All muscles involved Death ay 10 th -20 th after pulmonary problems (breathing)

26. Why muscles are enlarged in DM patients? Increased fibrous connective tissue revealed by this trichrome stain. There are larger overly contracted muscle fibers with scattered small degenerating or regenerating fibers Degenerated muscles contain lots of fibrous and adipose tissue

27. Normal muscles and DM muscle muscles stained for dystrophin with monoclonal antibodies myofibers are circumscribed by the darkly-staining dystrophin dystrophin is not evident wider variation in myofiber diameters increased connective tissue

28. Dystrophin Provide links between the intracellular cytoskeleton and the actin filaments with the extracellular matrix Duchenne and Becker MDs Sarcoglicans: Limb Girdle MDs (4 types) Laminin2 α 2: congenital MD chr 6 Whole complex stabilizes the membrane.

29. DM is good model disease as ballistic GT is available for muscles Problem: Native gene is 2,4 Mb in size (quite unusual) mRNA is 14 kb in size (also too big for any vector) IDEA: Dystrophin can retain significant function even when missing large portions of its sequence (Becker’s phenotype) Becker’s phenotype is anyway better than complete Duchenne ! Patient: exon 17–48 removed (48% of the coding region ), ambulatory before age 61

30. Deletion variants of dystrophin for GT ABD= actin-binding domains Scott Harper et al., 2002 most, but not all, of the spectrin-like repeats are dispensable for the function of dystrophin.

31. GT with dystrophin minigene in mice with DM phenotype GT treated Non-treated MDX mice with premature stop codon in exon 23; no dystrophin

32. Jun. 05, 2003 French Muscular Dystrophy Association (AFM) and Transgene announced that the results of their Phase I trial on gene transfer for Duchenne/Becker's Muscular Dystrophy Nine patients in three groups: a single injection of 200 mkg of plasmid with dystrophin a single injection of 600 mkg of plasmid with dystrophin Two injections of 600 mkg each of plasmid with dystrophin Muscle segment were taken out for examination Expression of dystrophin is found in 1 to 10 percent of muscle fibers for group 1 and 2 ; for all 3 patient in group 3 No immune reactions; no side effects !!!! Phase 1 trial for safety

33. Liposomes Next level idea – why naked DNA? Lets’ wrap it in something safe to increase transfection rate Therapeutic drugs Lipids – is an obvious idea !

34. Liposomes are formed by the self-assembly of phospholipid molecules in an aqueous environment. www.emc.maricopa.edu/faculty/ farabee/BIOBK/ Anionic liposome

36. Lab procedure for liposome preparation

37. Liposome disadvantages Liposomes are rapidly cleared from the circulation and largely taken up by the liver macrophages liposome surface ligands decrease degradation (monosialoganglioside or polyoxyethyle) How to overcome it?

38. Modified liposomes (stealth liposomes) hydrophilic polyoxyethylene lipids incorporated into liposome Increased half-life is be due to a reduced coating (opsonisation) of these liposomes by plasma proteins So liver cells not able to uptake them cholesterol, polyvinyl-pyrrolidone polyacrylamide lipids, glucoronic acid lipids are working the same….

39. Complex multilayer liposomes (Piedmont) Able to transport medication through the epidermal and dermal layers of the skin via the lipid-rich intercellular channels. The medication can be directed specifically to the targeted area

40. Cochleates – multilayer lipid rolls 1. Storageable without any problems – could be lyophilized (at least one year as a lyophilized powder at room temperature)! 2. Durable – survive multiple membrane fusion event (fuse-release drug-disengage-fuse-release..) 3. Can survive in GI tract Cochleates have been shown to be an effective oral delivery system.

41. Immunoliposomes for active targeting Antibodies to intracellular myosin target liposomes to infarcted areas of heart Antibody against tumor specific molecules will target them to tumors

42. Liposomes could serve as tumor specific vehicles (even without special targeting) www.pharmj.com/Editorial/19990828/ education/ parenteral.html Liposomes better penetrate into tissues with disrupted endothelial lining

43. DNA delivery of genes by liposomes Cheaper than viruses No immune response Especially good for in-lung delivery (cystic fibrosis) 100-1000 times more plasmid DNA needed for the same transfer efficiency as for viral vector

44. Cystic fibrosis most common lethal genetic disorder in Caucasian populations (1 in 2000 live births.) . Among African and Asian is really rare a defect in the CFTR gene (cystic fibrosis transmembrane conductance regulator ) irregular chloride and sodium ion conductance in epithelial cells of many organs (increased uptake of sodium ions). Sweat glands (too much salty secretion) Pancreas is damaged (leads to diabetes) Lungs create thick mucus secretion (prone to infections, constant cough, leading cause of death) Digestive tract (constipation)

45. Lungs in cystic fibrosis Normal lung CF lungs dilated crypts filled with mucus and bacteria. Normal alveolar appearance CF lungs filled with mucus lung did not collapse when it was removed postmortem

46. Cystic fibrosis lungs are prone to infections The battle between neutrophils and bacteria leads ultimately to lung fibrosis and damage Pseudomonas aeruginosa easily colonise mucus in the dilated lungs Neutrofiles are activated, then overactivated "Hyperinflammation" as recruited neutrophils unable to eradicate bugs, instead damage lung tissue. Mucus protects bugs and promotes hypermutation

47. Pancreas in cystic fibrosis Normal pancreas Distended CF cripts filled with mucus Impaired glucose tolerance and diabetes Survivors to 25 years old: 1/3 with impaired glucose tolerance and with 1/3 diabetes Pancreatic enzymes not able to leave the gland; they damage the gland Less insuline prodiced

48. Treatment of CF Staphylococcus aureus , Haemophilius influenzae , Aspergillus fumigatus - the same picture… 1. Many different antibiotics are required to clear infections.

49. CFTR gene (chromosome 7) 27 exons, 1480 aa ATP binding domain

50. CFTR product Delta F508 mutation 50% of all patients are homozygotes with this mutation 30% are heterozygotes, delta F508/X an incompletely folded, protease-sensitive form; Rapidly degrades before entering Goldgi complex

51. Cystic fibrosis Gene Therapy January 1995. Results of intranasal CFTR-liposome spaying in CF patients. 12 patients, Temporary relief in 20% of patients. Maximum on day 3, faded away on day 7. No immune reasctions. Monthly applications also work, 2000

53. MOST COMMON VIRAL VECTORS Retroviruses Adenoviruses Adeno-associated viruses Herpes simplex viruses can create double-stranded DNA copies of their RNA genomes . Can integrate into genome. HIV, MoMuLV, v-src, Rous sarcoma virus dsDNA viruses that cause respiratory, intestinal, and eye infections in humans. Virus for common cold ssDNA viruses that can insert their genetic material at a specific site on chromosome 19 dsDNA viruses that infect a neurons. Cold sores virus

54. Retroviral vectors are able to infect dividing cells only Good for cancer gene therapy Nevertheless, retroviruses are most often used vectors for common disease gene therapy Every therapeutic construct should include safety features In dividing cells nuclear membranes are broken down, so viral genome can enter and integrate into the chromosome Preintegration complex of retroviruses non able to penetrate nuclear membrane . Infection of dividing cells only

55. Amphotropic retroviruses capable of infecting both mouse cells and human cells Moloney murine leukaemia virus (Mo-MLV), 2. All regions of homology with the packaging virus should be removed to prevent recombination resulting in replication competent retroviruses Treatment could be tested in mouse Safety features: 1. Propagation only in packaging cells … .Anyway, some replication competent retroviruses do occur at a low frequency…. After removing of all non-essential parts carrying capacity for retroviral vectors is approximately 7.5 kb (not enough for some approaches)

56. Tissue tropism still a major issue even for amphotropic retroviruses In humans, retroviruses use sodium-dependent phosphate transporters Pit-1 and Pit-2 for entry Unfortunately, in humans this receptor expressed too widely. (With ironical exception of hematopoietic stem cells) Many approaches invented to improve and target the viral delivery

57. Modify env gene by c reation a pseudotyped vector Vesicular stomatitis virus (VSV): phospholipid component of membrane as a receptor (Rhabdoviridae) www.urmc.rochester.edu/smd/ hybrid virion with “mixed” envelope VSV G protein

58. What we gain : 1) Host range now determined by both envelopes 2. VSV envelope is very durable () pseudotyped virus has the ability to withstand the shearing forces encountered during ultracentrifugation Retro-VSV hybryds are able to infect even Fish, Xenopus, Mosquito, Butterflyes…. high-titer retroviral vector stocks could be generated

61. Equipping retroviral particles with cell-specific ligands 1 . Addition of part of the ligand to create env interaction with cell specific receptor 50 aa from EPO added to env makes it interacting with EPO-receptor on EPO receptor bearing cells Specific binding is easy to achieve ; but virus uptake become less efficient . Other additions: heregulin. Binds to HER-2 and HER-4 receptors overexpressed on breast cancer cells

62. Complete substitution of env surface subunit (SU) by cell specific ligand Problem: conformational changes in Env are strong, so resulting chimera not able to effeclively trigger internalisation Single-chain antibodies as a ligands are especially perspective Linker cleavable by protease Binary systems (ligand for binding; env for internalisation) After cleavage, local titer of virus is high Drawback: systemic applications of protease is no good. 1. Made this sequence cleavable for internal human protease 2. Made the linker flexible (Proline – rich) , to move away without cleavage

63. Real treatments performed with retroviral system Severe Combined Immunodoficiency (SCID): ADA-SCID and X-linked SCID

64. What is Severe Combined Immunodoficiency (SCID)? > 8 new ear infections per year > 2 serious sinus infections per year > 2 month on antibiotics with little effect > 2 pneumonias per year -- failure to gain weight and grow -- recurrent deep skin and organ abscesses lymphopenia (absolute lymphocyte count less than 200)

66. SCID treatments An excess of adenosine deaminase leads to hemolytic anemia Life in germ-free envinronment Histocompatible bone-marrow transplantations (with potential graft vs host disease) Enzyme replacement therapy with weekly injections of the PEG-ADA (ADAGEN) VERY expensive; not a cure; temporary effect GENE THERAPY

67. ADA gene therapy story Three separate laboratories published the gene sequence in 1983 ADA protein has been characterized in the late 1970s W. French Anderson (NIH); in the late summer of 1990, the FDA was sufficiently convinced by the preliminary laboratory data to approve the first human gene therapy trials using the MoMLV-based delivery vector September 14, 1990. Mature T-cells GT Ashanti DeSilva ; advanced stage of SCID; 4 yr old; Cynthia Cutshall January 31, 1991

68. What precisely has been done to Ashanti DeSilva? Her T cells were: -- placed in tissue culture -- stimulated to proliferate (by treating them with the IL-2) -- infected with the retroviral vector MoMLV-ADA -- returned to her in a series of treatments the injections had to be repeated because T cells live for only 6-12 months in the blood Both girls continued to receive ADA-PEG so the actual benefit of the gene therapy was unclear

69. Radical approach: make more room for transgenic T-cells by suppressing host bone marrow Aiuti A et al., 2002 (Science) By non-myeloablative conditioning “ you don't really wipe out the bone marrow, you just give one of the drugs used in for a transplant, at a much lower dose, to make 'space' for engineered marrow to seize, expand and grow better," Two children in this study never got PEG-ADA Results: improved immune functions (including antigen-specific responses), lower toxic metabolites . Both patients are currently at home and clinically well, with normal growth and development.

70. Umbilical cord blood (gene therapy of stem cells) Donald Kohn , a pediatrician, diagnosed 3 children with ADA-SCID in utero. early 1993 As the PEG-ADA has been reduced, the overall proportion of T cells  1-10% - a 100-1,000-fold increase! Shortly after infusion of the altered cord blood cells about .01 to .10 percent of the T cells in these infants were expressing the transgene. Umbilical cord blood samples were collected

71. X-linked SCID (bubble disease) Photo: Courtesy of Duke Medical Center News Office Gene therapy trial for X-linked SCID successed in 2000; 8 of 10 patients significantly improved and live normal life. More severe than ADA-SCID, as X-SCIDs have no B-, T-, NK cells David got a bone marrow transplant from his sister; she was EBV positive. David dies. Il-7 needed for T-cell proliferation; T cell helps B-cell "bubble boy" disease, named after David Vetter, a Texan who lived out his 12 years in a plastic, germ-free bubble.

73. Leukemia in X-SCID treated patients One of them underwent gene therapy at the age of six months, and contracted chicken pox at two-and-a-half. Probable reason of stimulation…. In 2 of 15 cases therapeutic gene insert itself near the LMO2 proto-oncogene The US Food and Drug Administration (FDA) halted 27 gene therapy LMO2 = LIM domain transcription regulator playing role in angiogenesis Rearranged in T-ALL. Transgenic mice with enforced expression of LMO2 in their thymocytes develop T cell leukemias…

74. Lentiviral vectors Lentiviruses are retroviruses that can infect both dividing and nondividing cells Preintegration complex of lentiviruses can get through the intact membrane of the nucleus of the target cell. Able to infect nondividing or terminally differentiated cells such as neurons, macrophages, hematopoietic stem cells, retinal photoreceptors, and muscle and liver cells Example of lentiviruses: HIV-1 (infects T-helper cells) – AIDS. Good feature – no immune response!

76. ADENOVIRUSES non-enveloped viruses containing a linear double stranded DNA genome 40 serotypes known; most producing respiratory infections in humans subgroup C serotypes 2 or 5 are predominantly used as vectors can infect both dividing and nondividing cells 12 antenna-like fiber projections for virus attachment www.nobel.se

77. Problems with adenoviral vectors 1. Cannot integrate with the host cell genome expression from adenoviral vectors is transient (5-10 days) due to immunoclearance of the virus in vivo hepatic gene delivery to hemophilia B dogs. Days posttreatment

78. Transient nature of expression in adenoviral vector any therapy based on adenoviral gene transfer would require long term application of the vector increased risk of recombination , especially if wild type infection occur simultaneously severe inflammatory cellular and serological immune responses possible

79. Adenovirus is very promiscous MHC class I molecule coxsackievirus-adenovirus receptor (CAR) Adenoviral receptors Very common everywhere Less common in the airway epithelium and cancer cells Topically administered Adenovirus anyway will move to other tissues, that produces distant toxic effects, especially in the liver (where virus is cleared) Needs escalating doses More toxicity CAR important for cell-cell adhesions

80. Safety features for adenovirural GT 1. Should not able to propagate itself (E1A deletion) 2. Should be as non-immunogenic as possible (get rid of most of the viral proteins) 3. Should be as non-recombinable as possible (get rid of most of the viral proteins that could be homologous to wt) at high titres (>10 11 /ml) viruses are produced in special cell lines with a helper virus (episomal or intergated in genome). E1A integration in 293 cells

81. How to suppress immunoclearance of adenoviral vector 1. transient immunosuppressive therapies to patient 2. induce oral tolerance by feeding the host UV inactivated vector Better to manipulate with vector, not with the host "gutless" vectors which contain no viral coding sequences The helper virus supplies the structural proteins required for gutless vector replication and packaging (293 kidney) In preparates helper virus represents less than 0,5% of particles, but they are immunogenic anyway Helps to overcome a liver toxicity

82. Attachment via CAR, internalization via integrins Adenoviral particles are disrupted in endosome

83. Cells that have less than normal CAR expression Mature skeletal muscle and smooth muscle (DM gene therapy) !!! Endothelial cells (all cardio diseases) Airway epithelium (cystic fibrosis) !!! Lymphocytes Fibroblasts Hematopoietic cells !!! Dendritic cells Most cancer cells !!! In the same time other, non-target cells actively sequester the virus !!!! Unwanted side effects again

85. Comparison of targeting startegies Population of chemical conjugates is always heterogenous, so clinical certification is difficult Exogenous recombinant genes (anti-knob scFv + anti-receptor scFv) are homogenous (CAR as anti-knob part could be used) Changes in the adenoviral knob itself

86. Clinical gene therapy with adenoviruses ornithine transcarbamylase (OTC) gene for OTC deficiency ( X-linked disorder ) OTC is a key urea cycle enzyme (break down and removal of nitrogen from the body ) hyperammonemia in the blood OTC deficiency vomiting, refusal to eat meat, progressive lethargy, and coma Ammonia is neurotoxic www.med.monash.edu.au/biochem/

87. OTC deficiency: treatment options 1. restriction of dietary protein 2. L-citrullin (to provide substrate for arginine synthesis) 3. During viral infections (when body produce more ammonia) protein intake should be stopped, and glucose is given either by mouth or intravenously. incidence of 1:30,000 in the U.S OTC frequency: Severe form of disease in boys; mild in girls, often indetected. Reye syndrom in children and young adults (encephalitis + liver failure after aspirin + viral infection ) Mortality 15 - 85% is caused by white matter edema and demyelination Even with this treatment, mortality rates in these children are about 50%.

88. Jesse Gelsinger , an 18 year old from Arizona died after fast developing fever and organ failures OTC-deficient sparse fur mouse as a model available . Mice treatment with Ad-OTC vector was very successful. Human trial for OTC deficiency 6 escalation doses i.h. ; up to 10 13 at the dose level 6 E-1, E-4-deleted third generation Ad-OTC vector NIH's National Gene Vector Laboratories' facility in UPenn 1) Grade 3 toxicities in two patients at the 4th dose level (level 6 should never be administered) 2) High level of ammonia in J.G. Probable source of problem 3) Probable undetected parvoviral infection in J.G. 4) Recombination of adenovirus to wild-type

90. Other adenoviral trials on their way Atherosclerosis : regional angiogenesis Goal: improve perfusion of ischemic limbs or heart by the induction of collateral vessel formation AdV with VEGF-121 in patients with intermittent claudication of limb arterias Claudication A single dose of Ad-VEGF will be administered as 20 intramuscular injections throughout the area of the lower limb Walking impairment will be compared in low-dose(10 9 ), high-dose (10 10 )and placebo groups University of Michigan Health System

91. Adeno-associated virus (AAV) -- does not stimulate inflammation in the host -- does not elicit antibodies against itself -- can enter non-dividing cells -- integrates successfully into one spot in the genome of its host (on chromosome 19 in humans). Can be ideal as: How to make expression tissue specific?

92. Binary system of AAV-based vectors 1 January 1999 issue of Science, James M. Wilson http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/ Carry genes for the components of the transcription factors needed to turn the target gene on. Chimeric gene that encode p65 (transcativator, not able to bind DNA) + FRB that binds the drug rapamycin . Chimeric gene that encode ZFHD1 (binds specifically EPO promoter ) but that by itself cannot activate transcription of the gene; + FKBP12 that also binds to rapamycin .

93. Vector 2 Target gene encodes EPO – erythropoietin – that stimulate production of red blood cells -- Treat severe anemia, e.g. after chemotherapy; -- Doping in sport (cyclists) -- Instead of blood transfusions for Jehovah's Witnesses -- Protect neurons during the stroke To enchance EPO expression

94. Experimental animals injected with both vectors into skeletal muscles Than injected with rapamycin (clinical immunosuppressant) If both vectors are in the same cell  EPO transcription is activated

95. RESULTS of experiment: Two vectors without rapamycin – harmless and no influence on Hb level In mice: Hematocrits (number of red blood cells) increase from 42% to 60% After injection: Fast production of EPO (200 times induction) Stable effect – still working 5 month after vector injection So, with this system we can deliver therapeutic construct once – but have a prolonged effect

96. Curing Insulin-Dependent Diabetes Mellitus (IDDM) in mice and rats Mice with inherited diabetes Rats after chemical destruction of their insulin-secreting beta cells Animals gained control over their blood sugar level and kept this control for over 8 months. Constructs injected into hepatic portal vein Intronless insulin gene Glucose -sensitive promoter AAV Signal sequence for secretion enhancer

99. Hemophilia treatments: 3) Transgenic animals. female sheep transgenic for the human factor 9 gene. The human gene is coupled to the promoter for the ovine milk protein beta-lactoglobulin. 4) Liver transplants 5) Gene therapy Avigen, Inc

101. CANCER GENE THERAPY And other experimetal cancer therapies (113 trials currently open in US in immunotherapy of cancer) 54% of immunotherapy trials dedicated to melanoma Delivery of the tumour-suppressor gene TP53 accounts for the next largest group Suicide GT

102. Genetic prodrug activation therapy (GPAT) tumor-specific promotor + drug activating gene http://www.sghms.ac.uk/depts/ogem Normal breast cells do not possess factors that lead to overexpression of ERBB2. Cytosine Deaminase gene under ERBB2 promotor. Active only in tumor cells. It allows activation of the harmless 5-FC prodrug to the cytotoxic 5-FU and consequent cell death. Major flaw of the current chemotherapy: lack of selectivity. If drug-activating genes could be inserted and expressed only in cancer cells , then treatment with an appropriate prodrug could be highly selective. Tumor-specific Suicide

104. Ganciclovir conversion by HSV-TK

105. Examples of suicide schemes Linamarase = beta-glucosidase, to convert the cyanogenic glucoside substrate, linamarin, into glucose and cyanide . From cassava Production of the cyanide ion that diffuses freely across membranes. In culture 10% lis-positive glioma cells are sufficient to eliminate the entire glioma cell culture in 96 h. Examples of suicide gene/prodrug combinations and the active cytotoxic drug selectively produced in the target cell. nitrobenzamidine CB 1954 nitroreductase cyanide Amygdalin Linamarase 5-fluorouracil 5-fluorocytosine Cytosine deaminase Ganciclovir triphosphate Ganciclovir Viral thymidine kinase Active drug Prodrug Suicide gene

106. Two targeting strategies of suicide gene Transcriptional targeting regulatory sequences of genes overexpressed in cancer cells (promotor) + suicide gene e.g. ERBB2 promoter in breast cancer or tyrosinase promoter in melanoma. Transduction targeting relies on preferential delivery of vectors constitutively expressing a suicide gene into actively dividing cells only. e.g. glioma cells vs normal neighbouring central nervous system cells. Like chemotherapy but may be topical (theoretically) 1. 2.