Chapter 14                Gene Cloning in                      Medicine        14.1 Production of recombinant             ...
14.1.1 Recombinant Insulin   Insulin controls level of glucose in blood      by β-cells of islets of Langerhans in pancr...
Synthesis & expression of    artificial insulin genes (1978)   (a) Artificial gene synthesis      Amino acid sequences ...
Synthesis & expression of     artificial insulin genes A subsequent improvement  Artificial gene synthesis contain B-C-A...
Fig 14.3 Production of     recombinant somatostatin Insertion of artificial  gene into a lacZ’  vector (pBR322 type) Syn...
Fig 14.4 Production of   recombinant somatotropinmRNA  cDNASmaller segment replaced by artificial DNA14.1.3 Recombinant...
Fig 14.5 Factor VIII gene & its     translation product                  Impossible to synthesize an active               ...
Fig 14.6 Expression signals used inproduction of recombinant factor VIII A & C separated: downstream of Ag promoter &  up...
14.1.4 Synthesis of other recombinant human proteins Human proteins synthesized by recombinant  technology continues to g...
14.1.5 Recombinant Vaccines Vaccine  An antigenic preparation  injection into bloodstream    stimulate immune system t...
Fig 14.7 Use ofrecombinant proteins                                  a preparation of                                   is...
Live recombinant virus        牛痘 vaccines       天花Use live vaccinia virus as a vaccine for smallpox   1796, Edward Jenne...
Table 14.2 Some foreign genes that have been expressed in recombinant vaccinia viruses               狂犬病                  ...
14.2 Identification of Genes  Responsible for Human Disease A genetic or inherited disease      Cause by a defect in a s...
Why identifying the gene responsible for a genetic disease is important?• Provide an indication of biochemical basis  ena...
Locating approximate position of    gene in human genomeIf: no information about the desired gene  How can it be located...
Locating approximate position of    gene in human genomeHuman: impossible to carry out directed breeding programsMapping...
Fig 14.10 Mapping breastcancer gene  Initial: gene was mapped to                       a 20 Mb segment of                ...
Approaches can be used to   identify the disease gene Expression profiles of candidate genes   By hybridization analysis...
14.3 Gene Therapy Original name    Aim to cure an inherited     disease by providing     patient with a correct     copy...
14.3.1 Gene therapy for       inherited diseases Somatic cell therapy  Cells remove cell from organism, transfected, the...
Somatic cell therapyPotential in treatment of cystic fibrosis  DNA cloned in adenovirus vectors or   contained in liposo...
Fig 14.12 Antisense RNA can beused to silence a cellular mRNA                                           DS                ...
14.3.3 The ethical issuesSomatic cell therapy     raised by gene therapy Should gene therapy be used to cure human  disea...
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Transgenic animals

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Transgenic animals

  1. 1. Chapter 14 Gene Cloning in Medicine 14.1 Production of recombinant pharmaceuticals 14.2 Identification of genes responsible for human disease 14.3 Gene therapy 14.1 Production ofrecombinant pharmaceuticalsTreatment of disorders absence or malfunction protein Supply by human protein Need large amount Supply by animal protein Side effects: an allergenic responseHow are these techniques being applied to the production of proteins for use as pharmaceuticals 1
  2. 2. 14.1.1 Recombinant Insulin  Insulin controls level of glucose in blood  by β-cells of islets of Langerhans in pancreas  Disease of insulin deficiency: diabetes mellitus  Treatment of DM: pig or cow insulin  Slight differences between animal & human proteins  Potentially dangerous contaminants  Two features facilitate production of insulin by recombinant DNA techniques 1. Human protein is not modified after translation  Synthesized by a bacterium should therefore be active 2. Relative small protein (A: 21, B: 30 amino acids) Fig 14.1 Structure of insulin molecule & a summary of itssynthesis by processing from preproinsulin One of the first projects  Synthesis of artificial gene for A & B chains  Production of fusion proteins in E. coli 2
  3. 3. Synthesis & expression of artificial insulin genes (1978)  (a) Artificial gene synthesis Amino acid sequences  trinucleotides codons Two recombinant plasmids: each carry artificial gene for A & B pBR322-type vector 提供E.coli 轉錄及轉譯所需訊息 1. lac P For RNA polymerase 2. lacZ’ For ribosome binding p.233Fig 14.2 Synthesis of recombinant insulin from artificial A & B  The final step is actually rather inefficient chain genes 3
  4. 4. Synthesis & expression of artificial insulin genes A subsequent improvement Artificial gene synthesis contain B-C-A proinsulin chain A more daunting proposition in DNA synthesis The prohormone: folding spontaneously into correct disulphide-bonded structure C-chain: excised relatively easily by proteolytic cleavage在此章中要注意的重點是,用何種載體、何種宿主以及如何表現?一開始如何進行? 到何種困難?如何改進? 14.1.2 Synthesis of human growth hormones in E. coli Somatostatin & Somatotropin Control growth processes in human body Malfunction: painful & disabling disorders such as acromegaly (uncontrolled bone growth) & dwarfism Somatostatin or Growth hormone-inhibiting hormone (GHIH) :   acromegaly First human protein synthesized by E. coli A very short protein: only 14 amino acids Ideally suited for artificial gene synthesis Use the same strategy for recombinant insulin 4
  5. 5. Fig 14.3 Production of recombinant somatostatin Insertion of artificial gene into a lacZ’ vector (pBR322 type) Synthesis of a fusion protein Cleavage with cyanogen bromide 14.1.2 Synthesis of Human Growth Hormones in E. coli Somatotropin or Growth Hormone   dwarfism; 191 amino acids, almost 600 bp Out of DNA synthesis capabilities (late 1970s) Artificial gene synthesis + cDNA cloning Obtain mRNA from pituitary  RT-PCR  cDNA Cut by restriction endonuclease (Hae III) Longer segment: condons 24-191, retain Smaller segment replaced by artificial DNA  provided correct signals for translation in E. coli Insert into an expression vector carrying lac promoter 5
  6. 6. Fig 14.4 Production of recombinant somatotropinmRNA  cDNASmaller segment replaced by artificial DNA14.1.3 Recombinant factor VIII Human factor VIII: role in blood clotting An inability to synthesize factor VIII  commonest form of hemophilia Treat: injection of purified factor VIII protein (donors) ? Hepatitis & AIDS Factor VIII gene: very large (>186 kb) 26 exons & 25 introns  2351 AA A complex series of post-translational processing events Dimeric protein 17 disulphide bonds & a number of glycosylated sites 6
  7. 7. Fig 14.5 Factor VIII gene & its translation product Impossible to synthesize an active version in E. coli  Mammalian cells14.1.3 Recombinant factor VIII Initial attempts Entire cDNA was cloned in hamster cell But yields of protein were disappointingly low Probably because post-translational events  Did not convert all initial product into an active form An alternative Two separated segments from cDNA were used Each fragment: ligated into an expression vector  Downstream of Ag promoter & upstream of SV40 polyadenylation signal Introduced into a hamster cell line & recombinant protein obtained > 10X yields; function as native form 7
  8. 8. Fig 14.6 Expression signals used inproduction of recombinant factor VIII A & C separated: downstream of Ag promoter & upstream of SV40 polyadenylation signal Ag promoter: an artificial hybrid of chicken β-actin & rabbit β-globin sequences Polyadenylation signal (needed for correct processing of mRNA before translation into protein) is obtained from SV40 virus14.1.3 Recombinant factor VIII Most recent technology: pharming Complete human cDNA: promoter for whey (乳漿) acidic protein gene of pig Leading to synthesis of Fig 13.20 human factor VIII in pig  Exactly same as native mammary tissue protein & fully functional in Subsequent secretion blood clotting assays of protein in the milk  2002 台灣首例第九凝血因子 及乳鐵蛋白霜轉殖基因猪 「酷比」: #1~4 8
  9. 9. 14.1.4 Synthesis of other recombinant human proteins Human proteins synthesized by recombinant technology continues to grow (Table 14.1) Proteins used to treat  Proteins are very disorders limited amount in body Replacement or Interferons & supplementation of interleukins dysfunction  Proteins need very Potential uses in cancer large quantities therapy Serum albumin Some growth factors (interferons & interleukins) Table 14.1 Some human proteins that have been synthesized from genes cloned in bacteria &/or eukaryotic cells or by pharming 9
  10. 10. 14.1.5 Recombinant Vaccines Vaccine An antigenic preparation  injection into bloodstream  stimulate immune system to synthesize antibodies  protect body against infection Two problems have hindered preparation of attenuated viral vaccines (減毒疫苗) Inactivation must be 100% efficient whole virus  Just one live virus particle could result in infection  口服沙賓疫苗, cattle disease foot-&-mouth particle Need large amounts of virus particles  Some virus do not grow in tissue culture (HBV) Producing vaccines as recombinant proteins Isolate viral components also can induce virus- specific antibody (Fig 14.7) Advantages: free of intact virus particle & could be obtain in large quantities Greatest success: hepatitis B virus (42 nm) Hepatitis B surface antigen (HBsAg, 22 nm ) Synthesized in both Saccharomyces cerevisiae (vector based on 2 µm plasmid) & Chinese hamster ovary (CHO) cells Both have been approved for use in humans WHO is promoting their use 10
  11. 11. Fig 14.7 Use ofrecombinant proteins a preparation of isolated virus coat proteins as a vaccine • Gene cloning its gene • Expression & purification Recombinant vaccines in transgenic plants Tobacco, tomato, & rice plants pharmingOral administration  Immunity could be acquired simply by eating part or all of the transgenic plant: simpler, cheaper  HBsAg & coat proteins of measles virus & Respiratory syncytial virus 11
  12. 12. Live recombinant virus 牛痘 vaccines 天花Use live vaccinia virus as a vaccine for smallpox 1796, Edward Jenner  1980, eradication smallpoxUse recombinant vaccinia virus as live vaccine against other diseases (Fig 14.8) A gene coding for HBsAg is ligated into vaccinia genome under control of a vaccinia promoter  the gene will be expressed Immunity against both smallpox & hepatitis B Fig 14.8 Potential use of a recombinant vaccina virus Immunity against both smallpox & hepatitis B 12
  13. 13. Table 14.2 Some foreign genes that have been expressed in recombinant vaccinia viruses 狂犬病 皰疹 口腔炎 Live recombinant virus vaccinesPossibility of broad spectrum vaccines is raised A single recombinant vaccinia virus expresses influenza virus HA, HBsAg, HSV glycoprotein Immunity against each diseases in monkeys 避免接觸此活疫苗的Vaccinia viruses expressing rabies 動物得到牛痘狂犬病 glycoprotein Deletion of vaccinia gene for thymidine kinase Prevent virus from replicating Now being used in Europe & north America 13
  14. 14. 14.2 Identification of Genes Responsible for Human Disease A genetic or inherited disease  Cause by a defect in a specific gene  Table 14.3 Individuals carry the defective gene  Predisposed toward developing the disease  X-linked: hemophilia, male  Autosomal recessive: most  Autosomal dominant: a few disease, Huntignton’s chorea Table 14.3 Some commonest genetic diseases in the UKDisease Symptoms FrequencyInherited breast cancer Cancer 1 in 300 femalesCystic fibrosis Lung disease 1 in 2000Huntington’s chorea Neurodegeneration 1 in 2000Duchenne muscular Progressive muscle weakness 1 in 3000 malesdystrophyHaemophilia A Blood disorder 1 in 4000 malesSickle cell anaemia Blood disorder 1 in 10 000Phenylketonuria Mental retardation 1 in 12 000β-thalassaemia Blood disorder 1 in 20 000Retinoblastoma Cancer of the eye 1 in 20 000Haemophilia B Blood disorder 1 in 25 000 malesTay-Sachs disease Blindness, loss of motor control 1 in 200 000 14
  15. 15. Why identifying the gene responsible for a genetic disease is important?• Provide an indication of biochemical basis enable therapies to be designed• Be used to devise a screening program Mutant gene can be identified in individuals Carrier or who have not yet developed the disease Counseling for carrier Early identification in individuals: precautions• A prerequisite for gene therapy 14.2.1 How to identify a gene for a genetic diseaseNo single strategyBest approach Depend on the available information about the diseaseBreast cancer Most common & most difficult scenario 方案 To understand its principle 15
  16. 16. Locating approximate position of gene in human genomeIf: no information about the desired gene How can it be located in human genome?Genetic mapping: by linkage Analysis Target gene; genetic loci whose map positions are already known Comparing their inheritance pattern (Fig 14.9)Key to understand its chromosome position Demonstration of linkage with one or more mapping genetic lociFig 14.9 Inheritance patterns for linked & unlinked genes (a) Two closely linked genes are almost always inherited together. (b) Two genes on different chromosomes display random segregation. (c) Two genes that are far apart on a single chromosome are often inherited together, but recombination may unlink them. 16
  17. 17. Locating approximate position of gene in human genomeHuman: impossible to carry out directed breeding programsMapping of disease gene: data from pedigree analysis Inheritance of gene is examined in families with a high incidence of disease being studies Obtain DNA samples from at least 3 generations of each families More family members: better How linkage analysis is used? Linkage to DNA markers is more usually tested One of the susceptible genes to human breast cancer was mapped 1990, U. C. Berkeley: first breakthrough A result of restriction fragment length polymorphism (RFLP) Linkage analysis Families w/ a high incidence  same version of an RFLP (Fig 14.10) D17S74, long arm of chromosome 17 It was far from the end of the story >1000 genes in this 20 Mb stretch Carry out more linkage studies: short tandem repeated (STRs)  just 600 kb This approach to locate a gene: positional cloning 17
  18. 18. Fig 14.10 Mapping breastcancer gene  Initial: gene was mapped to a 20 Mb segment of chromosome 17  Additional mapping experiments narrowed this down to a 600 kb region flanked by two previously mapped loci, D17S1321 & D17S1325  After examination of expressed sequences, a strong candidate for BRCA1 was eventually identified Identification of candidates for disease geneIn breast cancer project Fortunate: just 600 kb  >60 genes Any one of which could have been BRCA1Other projects Often 10 Mb or more of DNA sequences has be examined 18
  19. 19. Approaches can be used to identify the disease gene Expression profiles of candidate genes By hybridization analysis or RT-PCR of RNA from different tissues BRCA1: hybridize to RNA from breast & ovary tissues Southern hybridization analysis for different species DNA (zoo blots) human gene has homologs in other mammals Compare sequences between cancer & non- cancer women: mutations Confirm identify of a candidate gene Prepare a knockout mouse  display disease symptoms Identification of a candidate BRCA1 geneBRCA1: a ~100 kb gene, 22 exons, coding for a 1863 amino acids protein 回應上頁的四點 Its transcripts were detectable in breast & ovary tissues Homologs were present in mice, rats, rabbits, sheep & pigs, but not chickens Most important: the genes from five susceptible families contained mutations Evidence was sufficiently overwhelming Subsequent research: BRCA2, transcription regulation & DNA repair 19
  20. 20. 14.3 Gene Therapy Original name Aim to cure an inherited disease by providing patient with a correct copy of defective gene Now extend to Attempts to cure any disease by introduction of cloned gene into patient 14.3.1 Gene therapy for inherited diseases Germline therapy A fertilized egg is provided with a copy of correct version of relevant gene & reimplanted into mother Fig 13.19 Resulting individual: the gene is present & Usual: microinjection of a expressed in all cells somatic cell  nuclear transfer into an oocyte (桃利羊) Could be used to treat any inherited disease 在道德倫理宗教上,尚具爭議性;理論上可行,但是科學家們約定不做 20
  21. 21. 14.3.1 Gene therapy for inherited diseases Somatic cell therapy Cells remove cell from organism, transfected, then place back in body Cells transfected in situ without removal 目前容許之治療方法 Somatic cell therapy  Most promise for inherited blood disease hemophilia & thalassemia Genes are introduced into stem cells from BM High transfection血液方面的疾病比較可行 Which give rise to all frequency specialized cell types in blood Retrovirus-based vector Fig 14.11 Differentiation of a transfected stem cell leads to new gene being present in all mature blood cells 21
  22. 22. Somatic cell therapyPotential in treatment of cystic fibrosis DNA cloned in adenovirus vectors or contained in liposomes Introduction into respiratory tract via inhaler Take up by epithelial cells in lungs Gene expression occurs for only a few weeks Has not been developed into an effective means14.3.2 Gene therapy & cancer Most intensive area of current research Inactivation of a tumor suppress gene Introduction of a correct version gene Activation of an oncogene Prevent expression of oncogene  Not to replace it with a non-defective copy One possible way: introduce an antisense version of its mRNA (Fig 14.12) An alternative: suicide gene therapy Another approach: improve natural killing of cancer cells by patients immune system 22
  23. 23. Fig 14.12 Antisense RNA can beused to silence a cellular mRNA DS  Prevent protein synthesis  The target is therefore inactivated Suicide gene therapy An effective general approach Introduce a gene: selectively kills cancer cells or promotes their destruction by drugs Many genes code for toxic proteins Enzymes convert non-toxic prodrugs into toxic Under control of human telomerase promoter Active only in cancerous tissues 23
  24. 24. 14.3.3 The ethical issuesSomatic cell therapy raised by gene therapy Should gene therapy be used to cure human disease?  There is no simple answer No justifiable objection Routine application via a respiratory inhaler of correct versions of CF gene If bone marrow transplants are acceptable It is difficult to argue: gene therapies aimed at correction of blood disorders via stem cell transfection 癌症是如此恐怖的疾病 若是以道德的立場反對基因治療這種有效的治療,那 它本身是否可以被批評為不道德 14.3.3 The ethical issues raised by gene therapy Germline therapy is a more difficult issue Aims being to ‘improve’ farm animals Make genetic changes result in lower fat content Germline manipulation of inherited characteristics Development of this technique with animals has not prompted by any desire to cure genetic disease Exactly same techniques Genetic constitution of an organism is changed in a directed, heritable fashion This type of manipulation is clearly unacceptable with humans 24

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