Medical Biotechnology. Insulin - první gen biotech 1982. - презентация

1 Medical Biotechnology

2 Insulin - první gen biotech 1982

3 Recombinant proteins for human use ~2003 Approved in US or EU

4 Recombinant interferon: isolation of cDNA Strategies for isolating either the genes or cDNAs for human proteins 1) Isolate target protein and determine partial AAc sequence Synthesize oligo as probe to screen cDNA library 2) Generate Ab against purified proteins Screen gene library Interferon strategy above, pre-human genome sequence 6,000 clones

5 Hybrid products: INF Interferons assist the immune response by inhibiting viral replication within host cells, activating natural killer cells, increasing antigen presentation to lymphocytes, and inducing the resistance of host cells to viral infection IFN cDNA isolated early 80s Now, three groups of IFN genes identified:,, IFN family of 13 genes; IFN family of 2 genes; IFN of 1 genes IFN 1 and 2 have common RE sites Hybrid INFs demonstrate potential therapeutics by combining functional domains Some (2003)- successful clinical trials, approved for use as human therapeutic agents

6 Site-specific directed mutagenesis: hGH hGH: 191 AAc, 22,1 kDa One of first therapeutic proteins approved for human use Recombinant form produced in E. coli, identical to native pituitary-derived hGH Native binds to growth hormone receptor and prolactin receptor Side effects Prolactin receptor binding function of Zn ++ binding Domain: His-18, His-21, Glu , testing mutants

7 Optimizing gene expression Multistep process: Design a protein, construct a recombinant molecule, express and characterize Need to optimize expression First, either prokaryote or eukaryote host Comparative analysis of host and expression ex., interleukin-3 expression Best in Bacillus licheniformis Balance with glycosylation in eukaryotic hosts But, glycosylation is not essential for interleukin-3 activity

8 Cystic fibrosis Genetic disease affecting lungs and digestive system Average life span 37 years, extended and extending In US, ~1/3,900; 1/22 are carriers Most common in Europeans and Ashkenazi Jews Cystic fibrosis transmembrane conductance regulator (CFTR) Chloride ion channel, sweat, digestive juices and mucus thick, sticky mucus to build up in the lungs and digestive tract 7q31.2 -> 180,000 bp gene, 1,480 AAc Most common mutation DF508; 1,400 other mutations DF508: missense, not folded correctly Lungs susceptible to bacterial infection Antibiotics treatment results in resistance and combination with DNA from bacteria and leukocytes causes pulmonary problems (mucus) wikipedia

9 Treatment Genentech: hDNase I in CHO cells Not a cure, but alleviates symptoms Purified protein delivered via aerosol mist to lungs of CF - Approved by FDA in 1994

10 Optimizing treatment Another symptom, In response to bacteria in lungs, leukocytes cluster and lyse bacteria (and leukocytes) Lysed leukocytes release actin Monomeric actin binds DNase I very tightly and inhibits Limits effectiveness X-ray structure data suggested Ala-144 required for binding or Tyr-65 Changing either to Arg decreases actin binding by 10,000x Clinical efficacy of mutants to be determined (2003)

11 Clearing the lungs 2 with alginate lyase Alginate produced by seaweeds, soil and marine bacteria P. aeruginosa excretion in lungs contributes to viscosity of mucus In addition to DNase I treatment, alginate lysate can be used as therapeutic agent

12 Cloning alginate lyase Flavobacterium sp. Clone bank in E. coli Screen by plating onto medium plus alginate +/- Ca ++ Ca ++ + alginate = cross-linked opaque Hydrolyzed alginate does not cross-link Analysis and characterization of clones and alginate lyase

13 Alginate lyase[s] ORF 69,000 Da Precursor of three alginate lyases -> 3,000 Da + 63,000 Da 63,000 Da lyses both bacterial and seaweed alginates 63,000 Da -> 23,000 Da seaweed effective+ 40,000 Da bacterial effective Clone bacterial activity portion

14 Optimization of activity Increase expression of 40,000 Da protein PCR amplify and insertion behind strong promoter B. subtilis plasmid, fused to a B. subtilis a-amylase leader peptide, directs secretion and penicillinase gene promoter Expressed and assayed for halo phenotype Liquifies alginates produced by P. aeruginosa isolated from lungs of CF patients 2003, additional trials to determine if effective therapeutic agent

15 Phenylketonuria (PKU) Autosomal recessive genetic disorder in phenylalaniine hydroxylase Phe accumulation, decreases other large, neutral AAc in brain, needed for protein and neurotransmitter synthesis Brain development; progressive mental retardation and seizures Incidence ~1/15,000; varies: 1/4,500 Ireland and 1/100,000 Finland 12q22-q24.1 Macaque genome: PAH gene sequence identical to a human PKU mutation wikipedia

16 Phenylketonuria treatment[s] Traditional treatment: diagnosis at birth or prenatal Controlled semi-synthetic diet with low levels of Phe Possible treatment: metabolism of Phe PAH multienzyme complex, requiring cofactor Phe ammonia lyase (PAL) converts Phe as well Stable and does not require cofactor To test concept, yPAL cloned and overexpressed in E. coli Preclinical studies (2003) with mice deficient in PAL See lower plasma levels of Phe when PAL injected or administered as oral encapsulated enzyme

17 Monoclonal antibodies (mAb) as therapeutic agents Mouse mAb OKT3 first to be approved by FDA Immunosuppressive agent after organ transplant in humans

18 Antibody molecular structure CDRs variable portions of the protein, both H and L Fc elicits immunological responses after Ag-Ab Complement cascade

19 Polyclonal antibodies (Ab)

20 Monoclonal antibodies (mAb)

21 Monoclonal antibodies (theoretical) Monoclonal Antibodies: A Manual of Techniques. HZola

22 Monoclonal antibodies (mAb) protocol Monoclonal Antibodies: A Manual of Techniques. HZola

23 Herceptin ® Magic bullet Genentech. FDA 9/98; Aullrich/Genentech and DSlamon/UCLA Jonsson Cancer Ctr Trastuzumab (trade name Herceptin) Humanized monoclonal antibody Target is HER2/neu receptor (erbB2) HER2-positive metastatic breast cancer Anti-cancer therapy in breast cancer, over-expressing erbB2 receptor ErbB2 receptor amplification occurs in 25-30% of early-stage breast cancers Transmembrane Tyr kinase, activating PI3K/Akt pathway and MAP pathway Overexpression promotes invasion, survival and angiogenesis of cells Also confers therapeutic resistance to cancer therapies Herceptin binds to extracellular domain of erbB2 receptor, Arresting cell at G1 phase wikipedia

24 Magic bullet: delivery of drug to site Binding of mAb requires second step 1) delivery of drug 2) delivery of enzyme to convert pro-drug

25 Magic bullet: delivery of active agent to site Binding of mAb requires second step variations

26 Priklad ANTISENSE delivery

27 Human mAb problem Drawbacks to immunotherapeutic agents use Chemical couplings problem Yields low; coupling at random sites; chemical portion may inactive attached enzyme Nonhuman mAb If condition requires multiple treatments, nonhuman mAb causes immune response Human mAb Human chromosomes of fused human lymphocyte-mouse myeloma cells are unstable No human myeloma cell line can replace mouse myeloma cell line Ethics of injecting human subject to generate Ab-producing cells and doing partial splenectomy to collect Ab-producing cell

28 Hybrid human-mouse mAb: chimeric Genetic engineering to convert mouse mAb into a hybrid Exchange Fc portions Using oligonucleotides and in vitro DNA replication or cloned segments Construct in expression vector; transfect into cultured B lymphocytes Chimeric Abs are 70% human/30% mouse

29 Hybrid human-mouse mAb: chimeric Ex., chimera of mouse mAb against surface of human colon cancer cells Tested in patients with colorectal cancer Half-life in blood system 6x longer 1/10 patients developed mild response against chimera But, no anti-tumor activity observed (2003) Low dosage and/or advanced state of the cancer?

30 Jak je to delano

31 Hybrid human-mouse mAb: humanized Humanized Ab Substitute CDRs into human Ab 95 %human/5 % mouse Construction by isolating cDNAs for L and H chains Amplify variable regions using PCR protocol Primers are complementary to ends of variable regions, conserved CDRs are highly variable sequences

32 PCR amplification of CDR Primers are hybrids, with 12 bases at ends corresponding to human mAb L chain cDNAs Six pairs of primers: 3 for V L and 3 for V H PCR protocol to splice these segments into human Ab, replacing CDRs different mAbs have been humanized Technology is effective and widely applicable Time-consuming and expensive procedure

33 E. coli production of mAb: phage display Protocol for creating phage combinatorial libraries Hybridoma cells grow slowly, do not reach high cell densities, expensive to maintain Bioreactors: bacteria, plants and animals

34 DNA constructs of Fv combinatorial gene library Lambda phage Clones each L and H into two separate libraries Cut with common RE Directionally clone into third library: H -> L Combinations random RBS= ribosome binding site

35 Combinatorial library in M13 PCR amplify VH and VL separately Add linker Ligate into M13 genome Displays on surface

36 Phage Display Display peptide or protein on surface of bacterial virus (in principle can use other viruses but phage viruses easiest to prepare etc.) Some proteins on viral coats can accommodate peptides or proteins and will present them on the surface. The phage genome (or alternatively phagemid) contains the sequence for the protein or peptide so isolation of the phage with desired phenotype will also provide the genotype. Most popular is filamentous phage f1 or M13. pIII on the end or pVIII along the length of the rod-like virion for pVIII ~10% can be loaded with alternate peptide Advantage of phage display: easy to screen over 10 9 sequences Can either clone library directly into phage genome or use a phagemid (plasmid that contains f1 ori) with replication deficient helper phage

37 5 copies of pIII and pVI 2800 copies pVIII - all can accommodate peptides

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39 Combinatorial library in M13 Assay expressed mAb by Immunological screening ELISA-like system Multiwell plate coated with target Ag Bind, wash Score with chromogenic substrate cleaved by Ab-enzyme complex

40 Shuffling CDR sequences Very large libraries yield wider range of Abs B cells from several non-immunized individuals collected and pooled mRNA isolated; cDNA synthesized PCR amplify all six CDR regions separately Pool with oligos encoding the framework regions and linker Overlap extension PCR gives variable L and H domains At 2x10 9 different single-chain Ab

41 Using PCR to Detect for HIV RT-PCR (reverse transcriptase PCR). HIV has a ssRNA genome. Lyse plasma cells from the potentially infected person to release HIV RNA genome. The RNA is precipitated using isopropanol. Reverse transciptase is used to make a cDNA copy of the RNA of the virus. This cDNA is used as a template to make dsDNA. Diagnostics

42 RT-PCR Diagnosis of HIV

43 Using PCR to Detect for HIV Specific primers are used to amplify a 156 bp portion of the HIV gag gene. Using standards the amount of PCR product can be used to determine the viral load. PCR can also be used as a prognostic tool to determine viral load. This method can also be used to determine the effectiveness antiviral therapy.

44 Polymorphic refers to the existence of two or more forms of the same gene, or genetic marker Each form must be too common in a population to be merely attributable to a new mutation One type of polymorphism, Single Nucleotide Polymorphisms (SNPs), can be used as a diagnostic tool Gene polymorphism

45 SNPs are the 3.2 million single nucleotide changes that differ between genomes Most SNPs occur outside of genes, but some occur in gene promoters & a few occur in genes themselves For SNPs to be useful, a person's DNA must be examined for the presence of specific SNPs

46 SNPs

47 How do we identify SNPs? DNA microarrays (DNA chips) make it possible to examine person for the presence of specific SNPs quickly and affordably A single microarray can now be used to screen 100,000 SNPs found in a patient's genome in a matter of hours

48 How Are Microarrays Made? Short fragments of DNA (oligonucleotides) corresponding to each version of all known SNPs are spotted onto a glass slide in a known order A patients DNA is fragmented and each fragment is linked to a fluorescent dye This pool of fragments is allowed to hybridize to its corresponding oligonucleotide on the chip The pattern of fluorescence determines which SNPs are found in the patient

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50 Whole Human Genome Microarray by Agilent Technologies 1 x 3 glass slide with 44,000 genes dotted

51 What Can SNPs Be Used to Predict?

52 A persons susceptibility to disease is linked to which alleles they carry as well as how those alleles interact with the environment SNPs can be used to build a profile of a persons susceptibility to various diseases Example: Craig Venter (Celera genomics) has an increased risk of heart attack based on a SNP in the promoter of the MMP-3 gene

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55 Drug Dosing/Reaction

56 Average patient There is no simple way to determine how particular patient will respond to a medication Adverse Drug Reactions (ADRs) one of the important causes of hospitalization and death in the United States Medical drugs are developed using a average patient Pharmacogenomics examines the DNA variations that is correlated to drug response Can be used to predict if a patient will have a good response to a drug, a bad response to a drug, or no response at all 1

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59 Testing for Variation Cytochrome p450 (CYP450) involved in drug metabolism Four major types; CYP3A, CYP2C9, CYP2D6 & CYP2C19 Variations in at least 3 genes regulate drug metabolism By looking at the alleles a person has of these genes it is possible to predict how a patient will react to a drug Dosing can be regulated so that a patient gets the maximum benefit without possible toxic side effects

60 CYP3A CYP2D6 AntihistaminesCodeine StatinsBeta-Blockers Ca + Channel BlockersTricyclic Antidepressants BenzodiazepinesTamoxifen HIV protease inhibitors CYP2C9CYP2C19 (Missing in 30% of Asians) NSAIDs Proton pump inhibitors Anti-epileptics Valium Warfarin CYP Genes & Their Metabolites

61 Herceptin targets HER2 & is effective in stopping breast cancer growth Only 25 to 30% of breast cancers overexpress HER2 Erbitux effective in colorectal cancers by stopping signaling through EGFR Not all colorectal cancers overepress EGFR Diagnostic tests are used to detect which tumors will benefit from treatment allowing better use of treatment time & money

62 1733 Genes 84 breast tumor samples Red =gene induction Green = gene repression Tumors Are Not Identical So Why Should Every Patient be Treated the Same?

63 Recombinant DNA Drugs

64 Chinese Hamster Ovary Cells Most popular cells for producing proteins that are not able to be produced in E. coli These are proteins that are difficult to fold, glycosylated, or even toxic to the bacteria

65 Mammalian Cell Bioreactor

66 Protein Drugs Made in CHO Cells Avonex (Interferon Beta-1a) Multiple Sclerosis - Biogen Herceptin (Trastuzumab) Breast Cancer - Genentech Humira (Adalimumab) Rheumatoid Arthritis - Abbott Labs Remicade (Infliximab) Crohns Disease - Centocor Embrel (Etanercept) Rheumatoid Arthritis - Amgen

67 GENE THERAPY

68 Gene Therapy Cells are removed from a patient and modified either by having a working copy of a defective gene inserted or a therapeutic gene added Once the cells are expressing the new gene correctly, they are inserted back into the patient (ex vivo) The gene is usually delivered using a defective virus Sometimes the virus is delivered directly into the patient (in vivo)

69 Jak je to delano, priklady, doplnit

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71 Gene-Therapy and SCIDs Severe Combined Immune Defiency (SCID): no T cells Two types: ADA-SCID & SCID-X1 >20 SCID patients have been successfully treated The FDA has not approved any human gene therapy

72 Current State of Gene Therapy Little progress has been made since the first gene therapy clinical trials begun in 1990 In 1999, gene therapy suffered a major setback with the death of 18-year-old Jesse Gelsinger Part of a gene therapy trial for ornithine transcarboxylase deficiency (OTCD) Died from multiple organ failures 4 days post-treatment Death was caused by a severe immune response

73 In 2003, the FDA placed a temporary halt on all gene therapy trials using retroviral vectors in blood stem cells FDA took this action after it learned that two childern treated in a French gene therapy trial had developed a leukemia-like condition These children in August 2002 had been successfully treated by gene therapy (SCID-X1)

74 Stem Cells

75 Stem cells are unspecialized cells that renew themselves for long periods through cell division. Under certain physiologic or experimental conditions, they can be induced to become cells with special functions such as the beating cells of the heart muscle or the insulin-producing cells of the pancreas These cells could then be used to repair or replace damaged organs or tissues

76 Three Types of Stem Cells Embryonic Adult/Somatic Induced Pluripotent

77 In 1998, human embryonic stem cells (hES) were isolated and grown in the laboratory hES cells are derived from the ICM of human blastocysts These cells are pluripotent just like mouse ES cells The embryos used in these studies were created for infertility purposes through in vitro fertilization They were donated for research with the informed consent of the donor Human Embryonic Stem Cells

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79 Therapeutic Cloning Isolation of cloned cells/tissue for curing disease or injury The nucleus from an adult cell is placed in an enucleated egg Instead of implanting the egg and letting it grow into a fetus, it is cultured until the blastocyst stage where ES cells are removed and cultured These ES cells are coaxed down a specific developmental pathway such that they differentiate into a specific tissue This allows for the creation of cells identical to the donor thus preventing rejection

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81 An adult or somatic stem cell is an undifferentiated cell found among differentiated cells in a tissue or organ It can renew itself, and can differentiate to yield the major specialized cell types of the tissue or organ The primary roles of adult stem cells are to maintain and repair the tissue in which they are found These cells are more restricted as to what cell types they can become & are thus said to be multipotent Adult Stem Cells

82 1960s, two stem cell populations identified in bone marrow One population, called hematopoietic stem cells, forms all the types of blood cells in the body The second, called mesenchymal stem cells generate bone, cartilage, fat, & connective tissue Hematopoietic stem cells have also been isolated from umbilical cord blood Mesenchymal stem cells have now been isolated from amniotic fluid, umbilical cord blood, and adipose tissue

83 Bone Marrow Stem Cells Mesenchymal

84 Tissue Engineering

85 Tissue engineering or regenerative medicine is a multidisciplinary field combining biology, medicine, and engineering & involving the restoration, maintenance, or enhancement tissue & organ function Often involves the growth of new tissue or organs within a 3D matrix to mimic natural organ growth

86 Generation of Replacement Knee Cartilage

87 Valvular heart disease is a major cause of mortality Currently available substitutes for failing heart valves have serious limitations An alternative is to tissue engineer heart valves

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89 Cartilage scaffold of a human ear implanted under the skin of a mouse