Chapter 14 Gene cloning in medicine

Transcription

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 of recombinant pharmaceuticals Treatment of absence or malfunction protein Supply by human protein Supply by animal protein Production of recombinant protein as pharmaceuticals 1

2 14.1 Production of recombinant pharmaceuticals Recombinant insulin Synthesis of human growth hormones in E. coli Recombinant Factor VIII Synthesis of other recombinant human protein Recombinant vaccines Recombinant Insulin Disease of insulin deficiency: Treatment of DM: pig or cow insulin Slight differences between animal and human protein Potentially dangerous contaminants Advantage for recombination Human protein is not modified after translation Relative small protein (A: 21, B: 30 amino acid) 2

3 Insulin and Its Synthesis Disease of insulin deficiency: DM (diabetes mellitus) Treatment of DM Synthesis & expression of artificial insulin genes (a) Artificial gene synthesis Two recombinant plasmids each contain one of artificial gene of A and B 3

4 Synthesis and expression of artificial insulin genes (b) Purified A and B chain then attach each other by disulphide bond formation Synthesis and expression of artificial insulin genes Improvement Artificial gene synthesis A recombinant plasmid contain B-C-A proinsulin chain Folding spontaneously into correct structure Remove C-chain easily by proteolytic cleavage 4

5 Synthesis of Human Growth Hormones in E. coli Malfunction of growth hormones Acromegaly & dwarfism Somatostatin (SS): acromegaly Growth hormone-inhibiting hormone (GHIH) First human protein synthesized by E. coli 14 amino acids Synthesis like insulin Production of recombinant somatostatin 5

6 Production of recombinant somatostatin pbr322 first 7 triplets of bgalactosidase structure gene Sequence of chemically synthesized somatostatin Exogenous somatostatin was degraded rapidly in E.coli extract Such proteolytic degradation might be prevented by attachment of somatostatin to a large protein b-galactosidase 6

7 Formation of psom II Formation of psom III 7

8 Cleavage of chimaeric protein by cyanogen bromide to yield active somatostatin Synthesis of Human Growth Hormones in E. coli Somatotropin or Growth Hormone: dwarfism 191 amino acids; almost 600 bp Out of DNA synthesis capabilities Prepare cdna Obtain mrna from pituitary: RT-PCR! cdna Cut by restriction endonuclease (Hae III) Smaller segment replaced by artificial DNA Insert into an expression vector carrying lac promoter 8

9 (a) Prepare the somatotropin cdna fragment Obtain mrna cdna Cut by restriction endonuclease (Hae III) Smaller segment replaced by artificial DNA Recombinant somatotropin Reproduced start of somatotrophin gene & provided correct signals for translation in E. coli 9

10 Recombinant Factor VIII Instability of Factor VIII: hemophilia Injection of purified factor VIII protein (AIDS) Very large protein 186 kb DNA, mrna: 26 exons & 25 introns Post-translational processing events Primary: 2351 amino acid Dimeric protein 17 disulphide bonds & glycosylated sites Factor VIII gene and expression 10

11 Recombinant Factor VIII Entire cdna were cloned in hamster cell But yields were low Two separated segments were used Downstream of Ag promoter Upstream of SV40 polyadenylation signal > 10X yields Function as native form Expression signals of recombinant Factor VIII Ag promoter is an artificial hybrid Chicken b-actin & rabbit b-globin hybrid Introduced into a hamster cell line & recombinant protein obtained 11

12 Recombinant Factor VIII Most recent technology: pharming Complete human cdna has been attached to promoter for whey acidic protein gene of pig Leading to synthesis of human factor VIII in pig mammary tissue Subsequent secretion of protein in the milk Exactly same as native protein Synthesis of Other Recombinant Human Protein Proteins are very limited amount in body Interferons and interleukins Proteins need very large quantities Serum albumin Table

13 Recombinant Human Proteins Recombinant Vaccines Two problems have hindered prepararion of attenuated viral vaccines 1. Inactivation must be 100% efficient 2. Need large amounts of virus particles Some virus do not grow in tissue culture (HBV) a) Producing vaccines as recombinant proteins b) Live recombinant vaccines 13

14 A) Producing vaccines as recombinant proteins Isolate viral component induced virusspecific antibody Has not been entirely successful Lack of full antigenic properties of intact virus Success examples: Hepatitis B major surface antigen coat protein could synthesis by Saccharomyces cerevisiae used as vaccine On 2 mm plasmid Isolated virus coat proteins as a vaccine 14

15 B) Live recombinant vaccines Use recombinant vaccinia virus as live vaccine Fig Immunity against both smallpox & hepatitis B Broad spectrum vaccines by single recombinant vaccinia virus Influenza virus HA, HBV sag, HSV glycoprotein Table 14.2 Unknown result for wide spreading of recombinant vaccinia virus Potential use of recombinant vaccina virus 15

16 Table 14.2 Foreign genes expressed by recombinant vaccinia viruses Plasmodium falciparum (malaria parasite) surface antigen Influenza virus coat proteins Rabies virus G protein Hepatitis B major surface antigen Herpes simplex glycoproteins HIV envelope proteins Vesicular stomatitis coat proteins Sindbis virus proteins 14.2 Identification of Genes Responsible for Human Disease A genetic or inherited disease Cause by a defect in a specific gene 16

17 Table 14.3 Some of the commonest genetic diseases in the UK Disease Inherited breast cancer Cystic fibrosis Huntington s chorea Duchenne muscular dystrophy Haemophilia A Sickle cell anaemia Phenylketonuria b-thalassaemia Retinoblastoma Haemophilia B Tay-Sachs disease Symptoms Cancer Lung disease Neurodegeneration Progressive muscle weakness Blood disorder Blood disorder Mental retardation Blood disorder Cancer of the eye Blood disorder Blindness, loss of motor control Frequency 1 in 300 females 1 in in in 3000 males 1 in 4000 males 1 in in in in in males 1 in Identification of Genes Responsible for Human Disease Why identifying gene responsible for a genetic disease is important? 1. Provide an indication of biochemical basis enable therapies to design 2. Be used to devise a screening program counseling for carrier Early identification in individuals 3. A prerequisite for gene therapy 17

18 How to identify a gene for a genetic disease Locating approximate position of gene in human genome Identification of candidates for disease gene Locating approximate position of gene in human genome Linkage Analysis Comparing inheritance pattern for target gene with inheritance patterns for genetic loci whose map positions are already known Pedigree analysis Inheritance of gene is examined in families with a high incidence of disease being studies 18

19 (a) Genes are linked Linkage Analysis (c) Genes are on the same chromosome, but far apart (b) Genes are on different chromosomes * Product of recombination Mapping breast cancer gene BRCA1: first breakthrough occurred in 1990 RFLP (restriction fragment length polymorphism) Linkage analysis U. C. Berkeley D17S74, long arm of chromosome Mb, >1000 genes STRs (short tandem repeatd) 19

20 Identification of candidates for disease gene Genetic mapping only gives an approximate indication of location of gene In breast cancer project Narrow search area down to just 600 kb Often 10 Mb or more of DNA sequences has be examined Contained over 60 genes Any one of which could have been BRCA1 Identification of a candidate gene 1. Expression profiles can be examined by hybridization analysis or RT-PCR of mrna from different tissue Breast & ovary tissue 2. Southern hybridization analysis for different species DNA (these are called zoo blots) Other mammals 3. Compare sequences between cancer & noncancer women 4. To confirm identify of a candidate gene, it might be possible to prepare a knockout mouse 20

21 Identification of a candidate BRCA1 gene ~100 kb, 22 exons, coding for a 1863 amino acids protein Transcripts of the gene were detectable in breast & ovary tissues, & homogues are present in mice, rats, rabbits, sheep and pigs, but not chickens The genes from five susceptible families contained mutations 14.3 Gene Therapy Gene therapy for inherited diseases Gene therapy & cancer The ethical issues raised by gene therapy 21

22 Gene therapy for inherited diseases Germline therapy A fertilized egg is provided with a copy of correct version of relevant gene & reimplanted into mother Microinjection of DNA into isolated egg cell Somatic cell therapy Remove cell from organism, transfected, then place back in the body Inherited blood disease: haemophilia and thalassaemia Retrovirus-based vector Figure Differentiation of a transfected stem cell leads to new gene being present in all mature blood cells 22

23 Gene therapy & cancer Most intensive area of current research into gene therapy concerns its potential use as a treatment for cancer Most cancers result from activation of an oncogene that leads to tumor formation, or inactivation of a gene that normally suppresses formation of a tumor Gene therapy and cancer In both cases a gene therapy could be envisaged formation of a tumor Introduction of a gene for an antisense RNA copy of an oncogene could reduce or prevent expression of the oncogene & reverse its tumorigenic activity To introduce a gene that selectively kills cancer cells To improve natural killing of cancer cells by the patient s immune system With a gene that causes tumor cells to synthesize strong antigens that are efficiently recognized by immune system 23

24 The ethical issues raised by gene therapy Should gene therapy be used to cure human disease? If bone marrow transplants are acceptable It is difficult to argue Gene therapies aimed at correction of blood disorders Via stem cell transfection The ethical issues raised by gene therapy Germline therapy is a more difficult issue Techniques used for germline correction of inherited diseases are exactly same techniques that could be used for germline manipulation of other inherited characteristics Development of this technique with animals has not prompted by any desire to cure genetic disease 24