Lession 4.4 Molecular Biology in Medical and Pharmacology PDF

SESSION 4: APPLICATION OF MOLECULAR BIOLOGY 1. Western blotting 2. mRNA vaccine 3. Hormone/Enzyme Production (Insulin) 4. Monoclonal Antibody 5. Genome editing and Gene therapy 6. Stem cells technology Department of Cell Biology...

SESSION 4: APPLICATION OF MOLECULAR BIOLOGY 1. Western blotting 2. mRNA vaccine 3. Hormone/Enzyme Production (Insulin) 4. Monoclonal Antibody 5. Genome editing and Gene therapy 6. Stem cells technology Department of Cell Biology Le Duy Duc COVID-19 VACCINES What do nucleic acids DNA is found in the cell nucleus Cell DNA (DNA and RNA) do? DNA is the code for making RNA DNA in the nucleus is stable HUMAN CELL Fatty coat (membrane) codes for Cell RNA Cell RNA RNA is found in the cell cytoplasm Cell DNA RNA is the code for making proteins Cell RNA is unstable and falls apart in hours nucleus proteins codes for cytoplasm Cell proteins Proteins are found in the cell cytoplasm Proteins build cell machinery  How do viruses work? RNA 1. Virus enters cell 2. Viral DNA enters nucleus 1. DNA virus Adenovirus 3. Viral DNA makes viral RNA (eg. adenovirus) proteins 4. Viral RNA makes proteins DNA 5. Proteins make new viruses Protein shell 6. Cell bursts releasing viruses Spike protein nucleus 7. Viruses infect new cells DNA code 8. Infection eliminated by immune response cytoplasm 2. RNA virus (eg. coronavirus) RNA 1. Virus enters cell 2. Viral RNA enters cytoplasm Fatty coat stolen Coronavirus 3. Viral RNA makes viral protein from infected cell RNA proteins 4. Proteins assemble into viruses Protein shell 5. Cell releases viruses Spike protein 6. Viruses infect new cells RNA code nucleus 7. Infection eliminated by immune response cytoplasm  What kind of coronavirus RNA vaccines have we made? 1. Oxford/AstraZeneca vaccine (adenovirus vaccine) coronavirus DNA spike Has adenovirus protein shell empty of natural virus DNA Synthetic DNA coding for coronavirus spike protein is inserted into otherwise empty adenovirus shell nucleus Virus shell carries coronavirus spike DNA into cell nucleus Coronavirus spike DNA makes spike RNA and spike protein in cytoplasm cytoplasm Spike protein is released to activate immune system Neither virus shell nor coronavirus spike DNA persist or cause disease RNA 2. BioNTech Pfizer/Moderna vaccines (RNA vaccines) RNA Has synthetic fatty coat similar to cell membrane coronavirus Contains synthetic RNA coding for coronavirus spike proteins spike Coronavirus spike RNA enters cell cytoplasm Coronavirus spike RNA makes coronavirus spike protein nucleus Spike protein is released to activate immune system Neither fatty coat nor spike RNA persist or cause disease cytoplasm  How do COVID19 vaccines stimulate an immune response? Vaccine makes coronavirus spike protein in cells, which is presented to immune system Immune cells detect spike protein and make antibodies specific for spike protein During infection, antibodies bind spike protein on real virus and protect against infection and/or disease Antibodies speed up elimination of the coronavirus from the body Vaccination Coronavirus Vaccine antibody Coronavirus Strong antibody Virus spike protein response to spike infection response eradicated YYY YY Y Y days weeks 1 week days YY RNA Y Y HOW LONG HAVE WE BEEN STUDYING RNA VACCINES?  Some conclusions and considerations Oxford vaccine Is a protein shell from a harmless adenovirus into which synthetic DNA coding for coronavirus spike protein is added Is highly purified virus particles without contaminants or additional chemicals (except possibly a harmless preservative) Synthetic DNA from the vaccine persists briefly in the cell nucleus then the cell dies and the DNA is digested by enzymes RNA vaccines Are made of synthetic RNA coding for coronavirus spike proteins surrounded by a synthetic fatty coat (like our cell membranes) Are highly purified fatty particles without contaminants, containing small amounts of polyethylene glycol, a harmless chemical Coronavirus RNA persists briefly in the cell cytoplasm and is then naturally degraded by enzymes within hours Other considerations Unlike previous generations of vaccines that are contaminated with other proteins (eg. egg proteins in influenza vaccines) or mixed with adjuvants such as aluminium salts, or preservatives containing traces of mercury, these COVID19 vaccines are clean. There is no known mechanism by which vaccine RNA can interfere with our own genetic code, and there is no evidence from animal studies or clinical trials (where they exist) that this can occur. There is no known mechanism by which DNA introduced into the cell by an adenovirus can interfere with our own genetic information, and natural adenovirus infections are commonplace and harmless.  Q&A Can you get COVID from these vaccines? No.  Q&A Why do these RNA vaccines require two shots? The second shot is like a “booster shot” for your immune system.  Q&A Why do these vaccines need to be kept so cold? Because RNA is fragile. RNAse can easily chop it up at warmer temperatures, and even water can start to break the backbone down if not kept cold.  INSULIN Insulin is a hormone central regulating carbohydrate and fat metabolism in the body. Insulin is secreted by the Islets of Langerhans of pancreas which catabolizes glucose in blood. Insulin causes liver cells, muscle cells and fat tissue to take up glucose from the blood and store it as glycogen in the liver and muscle. REGULATION OF BLOOD GLUCOSE LEVEL INSULIN PRODUCTION  Human Insulin Production by Bacteria The final steps are to collect the bacteria, break open the cells, and purify the insulin protein expressed from the recombinant human insulin gene. INSULIN PRODUCTION INSULIN PRODUCTION  STRUCTURE Insulin consists of two polypeptide chains, Chain A ( 21 amino acid long) and B ( 30 amino acid long). Its precursor is proinsulin which also contains two polypeptide chains, A and B, and is connected with a third peptide chain –C (35 amino acid long). PRODUCTION OF RECOMBINANT INSULIN  PRODUCTION OF INSULIN In the Islets of Langerhans, insulin accumulates in secretary vesicles as a single polypeptide chain called proinsulin. Before secretion into the bloodstream the third C chain of the proinsulin molecule is excised, leaving the A and B chains joined by disulphide bridges as the active insulin. E. coli is not capable of removing the C chain. There are several strategies for producing insulin from bacteria, but the most successful is to synthesize the A and B separately and then join them together.  PRODUCTION OF INSULIN The gene sequence of determining the A chain has been fused to the ß-galactosidase gene (lac Z) of E.coli. The whole lac-Z-A chain fusion is cloned into pBR322. Bacteria with this plasmid synthesize ß-galactosidase with the insulin A chain. The B chain is produced in an identical manner. After purification of the two chains they are mixed , oxidized and then reduced which allows the disulphide bridges to form and active insulin to be produced. INSULIN PRODUCTION OF GROWTH HORMONE FUNCTIONING OF THE IMMUNE SYSTEM HUMORAL (ANTIBODY MEDIATED) IMMUNE RESPONSE CELL MEDIATED IMMUNE RESPONSE ANTIGEN (1ST EXPOSURE) ENGULFED BY MACROPHAGE FREE ANTIGENS ANTIGENS BECOMES DISPLAYED DIRECTLY BY INFECTED ACTIVATE APC CELLS ACTIVATE STIMULATES HELPER CYTOTOXIC B CELLS T CELLS STIMULATES STIMULATES T CELL MEMORY GIVES RISE TO HELPER T GIVES RISE TO CELLS STIMULATES STIMULATES STIMULATES ANTIGEN (2nd EXPOSURE) PLASMA MEMORY ACTIVE STIMULATES MEMORY CELLS B CELLS CYTOTOXIC T T CELLS CELL SECRETE ANTIBODIES  WHAT IS AN ANTIBODY? An antibody is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target. Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. Polyclonal antibodies are antibodies that are derived from different cell lines. Isotypes According to differences in their heavy chain constant domains, immunoglobulins are grouped into five classes, or isotypes: IgG, IgA, IgM, IgD, and IgE. IgG: IgG1 (66%), IgG2 (23%), IgG3 (7%) and IgG4 (4%) , blood and tissue liquid. IgA:IgA1 (90%) and IgA2 (10%), stomach and intestines IgM: normally pentamer, ocassionally hexamer, multiple immunoglobins linked with disulfide bonds IgD:1% of proteins in the plasma membranes of B-lymphocytes, function unknown IgE: on the surface of plasma membrane of mast cells, play a role in immediate hypersensitive and denfensive for parasite  MONOCLONAL ANTIBODIES Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. Given (almost) any substance, it is possible to create monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine.  THE IGG CLASS OF ANTIBODIES All current therapeutic antibodies are of the IgG class. When the objective of antibody therapy is to directly kill the target cell, the isotype of choice is IgG1, since this isotype is optimal for complement fixation.  THE STRUCTURE OF ANTIBODIES http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html  NOMENCLATURE OF THERAPEUTIC ANTIBODIES Terminate the name in –ximab for chimeric antibodies and –umab for humanized antibodies. COMMON CHEMOTHERAPY IN TREATMENT OF CANCER Shortcomings: A. Nature of cytotoxin B. Lack of in vivo selectivity C. The mechanism of anti-proliferation on cells cycle, rather than specific toxicity directed towards particular cancer cell D. Host toxicity: treatment discontinued, most of them had bad side- effects, such as no appetites, omit, lose hair  MONOCLONAL ANTIBODIES FOR C ANCER TREATMENT Three mechanisms that could be responsible for the cancer treatment. A. mAbs act directly when binding to a cancer specific antigens and induce immunological response to cancer cells. Such as inducing cancer cell apoptosis, inhibiting growth, or interfering with a key function. B. mAbs was modified for delivery of a toxin, radioisotope, cytokine or other active conjugates. C. it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell  mAbs treatment for cancer cells ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment. INTRODUCTION TO MABS  WHAT ARE MONOCLONAL ANTIBODIES? I. Antibody II. MABS III. Advantages Proteins from Same as normal Can clone itself amino acids antibodies Selective Made in response Made in lab setting towards target to antigens and mass High quality produced amongst all Develop from batches single selected cell MAB’S ARE CURRENTLY PRODUCED USING ANIMALS' CELLS Common Host Cell Lines Potential Host Cell Lines Animal based cell host lines dominate current protein therapeutics Insect cells Mouse Low production cost CHO – Chinese Hamster Ovary Plant-cell-culture (ex: Agenus/iBio) NS0 – Murine Myeloma Cells Cheaper and faster Sp2/0 0 – Spleen of a mouse Fungus-based Human 2-10x productivity HEK293 – kidney PER.C6 – embryonic retinal cells KEY ACTIVITIES OF MABS Block Binds to hazardous cell to prevent it from interacting with target Flag Binds to receptors of hazardous cells to signal for destruction of those cells to immune system Deliver Binds to a cell and delivers toxic chemotherapy drug that kills cell Binding Binding between the antibody and antigen is result of Dipole-Dipole interactions, H-Bonding, VDW forces, and Nonpolar interactions Main function The amino acid sequence of the light chains are what determine selectivity for a particular antigen MABS ARE COMMONLY USED FOR DETECTION AND TREATMENT Detection Prevention/Treatment Targeted detection kits Asthma Pregnancy/Ovulation Cancer COVID Autoimmune diseases HIV Viral Disease Treatment Malaria (ex: AIDS) Chlamydia Organ Rejection Drugs MARKET OF MABS 19.3 million new cancer cases in 2020, expected increase by 47% by 2040 (IARC) to ~28.4 million new cases Surging growth of cancer cases indicates increase in demands of mAbs MAbs applicable to broad range of viruses Monoclonal antibodies market (by source: Humanized Mab, human MAB, murine MAB, chimeric MAB; by indication: Cancer, autoimmune diseases, inflammatory diseases, infectious diseases, others; by Distribution Channel: Hospital Pharmacy, online pharmacy, retail pharmacy; by production methods: In vivo, in vitro) - global industry analysis, size, share, growth, trends, Regional Outlook, and forecast 2022 – 2030. Graphical Research. (2022, April). North America monoclonal antibodies market 2022-2028: Growth outlook. Precedence Research. (2022, May). Retrieved February 6, 2023. Graphical Research. Retrieved February 6, 2023. MANUFACTURING PROCESS  OVERALL DEVELOPMENT PROCESS Production in Purification bioreactor Formulation Note that during (downstream) (Upstream) the development process pre- clinical and clinical studies Analytical and Product are being run Final Product simultaneously stability market Fill and Finish testing release Goldberg, D. (2022, Nov). Module 2. College Park, MD, United States UPSTREAM PROCESS DEVELOPMENT GOALS The goal of upstream process development is 1. Antigen Selectivity to generate cells which produce the protein of interest with the highest 2. Survivability possible productivity while maintaining the appropriate product quality attributes 3. Effectiveness  OVERVIEW OF THE UPSTREAM PROCESS Development of Establishment of Selection of host Clone selection master cell bank cell culture cell line (MCB) conditions Seed train Production of Upstream development cells Harvest Goldberg, D. (2022, Nov). Module 2 [Paper presentation]. College Park, MD, United States  DOWNSTREAM GOALS The goal of downstream process development is to purify the upstream product so that the level of impurities is at or below specifications Unchained Labs. (n.d.). Ultrafiltration [Review of Ultrafiltration]. Retrieved February 3, 2023, from https://www.unchainedlabs.com/ultrafiltration-diafiltration-uf-df/  DOWNSTREAM PROCESS OVERVIEW Step1: Harvest Centrifugation/filtration Removal of cells and cell debris Step 2: Protein A Chromatography Purify Product Step 3: Low pH hold for viral inactivation Inactivates viruses Step 4: Additional Polishing Chromatography Removal of impurities and viruses Step 5: Viral filtration Remove viruses Step 6: Ultrafiltration/diafiltration Achieve final protein concentration within (UFDF) desired drug substance buffer Liu, H. F.; Ma, J.; Winter, C.; Bayer, R. Recovery and Purification Process Development for Monoclonal Antibody Production. mAbs 2010, 2 (5), 480–499. https://doi.org/10.4161/mabs.2.5.12645. FORMULATION DRUG FORMULATION RESULTS IN A FINAL MEDICINAL PRODUCT Methodology: Goal: Mixing active pharmaceutical Find the best way to ingredient (API) with inactive deliver a molecule components (excipients) Administration route Safety and stability Packaging Taste, smell, appearance, etc. Dosage Formulation occurs throughout the entire manufacturing process!!  TYPICAL FORMULATION DEVELOPMENT FOR MABS Optimal Excipient Stability Administration Characteristics Addition Evaluation Specifications: Rheological Mechanical Container Closure pH Structure Transportation Integrity testing Concentration Viscosity Thermal Administration Physical Functional Chemical characteristics Permeability Acidic / basic Dosage Appearance Solubility environments Homogenous Antimicrobial UV AID concentration Shelf life Each individual stage always considers safety and efficacy What you need to know about drug formulation. https://www.contractpharma.com/issues/2016-05-01/view_columns/what-you-need-to-know-about-drug-formulation/ (accessed Feb 3, 2023).  FUTURE DIRECTION Bispecific Antibody-Drug Conjugates (ADC) Combine antigen binding from different antibodies Enables use normally intolerable cytotoxic agents, to enable dual targeting up to 1000x more potent (more killing potential) Challenges Challenges Purification strategies to isolate desired bispecific Extreme toxicity of product requires special handling from random combination and expertise  ‘NAKED’ MONOCLONAL ANTIBODIES ‘Naked’ means these antibodies are not fused to a toxin. Naked Monoclonal antibodies can kill cells via a variety of mechanisms, including: Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), and direct induction of apoptosis. However, the precise clinical mechanisms often remain uncertain  Antibody-dependent cellular cytotoxicity From Wikipedia, the free encyclopedia Antibody-Dependant Cellular Cytotoxicity ADCC is the least understood of the three mechanisms, it is mediated by either NK cells or CTL. The action of ADCC is dependant on the recognition of the objective cell by antibodies attached on the surface of the effector cell (terminally differentiated leukocyte). This process is part of the adaptive immune response due to the dependence on antibodies and therefore a former anti-body response is required for this mechanism to take effect and be effective against an invading pathogen.  RITUXIMAB (RITUXAN) Rituximab is a chimeric monoclonal antibody that targets the CD20 B-cell antigen. This antigen is expressed on 90% of B-cell neoplasms The precise biological functions of CD20 are uncertain, but the antibody is believed to function by flagging the B-cells for destruction by the body’s own immune system, including ADCC, CDC, and apoptosis. This antibody thus leads to the elimination of all B-cells from the body (including cancerous ones), allowing new, healthy B-cells to be produced from lymphoid stem cells.  TRASTUZUMAB (HERCEPTIN) Herceptin is an anti-cancer antibody that acts on HER2/neu (erbB2) receptor, which is overexpressed in breast cancer. Only cells overexpressing this receptor are susceptible. Such cells, when treated with Herceptin, undergo arrest in the G1 phase of the cell cycle and experience a reduction in proliferation. This can reduce the rate of relapse of breast cancer by 50% during the first year. The precise mechanism of action is still under investigation. MONOCLONAL ANTIBODIES WHICH DELIVER A TOXIN Monoclonal antibodies can be utilized to selectively deliver a toxin to a malignant cell.  GEMTUZUMAB OZOGAMICIN (MYLOTARG) This monoclonal antibody is conjugated to the cytotoxic agent calicheamycin It is used to treat acute myelogenous leukemia (AML), which is a cancer of the myeloid line of blood cells. This monoclonal antibody attacks the CD33 receptor, which is found in most leukemic blast cells, but not in normal hematopoietic stem cells  GEMTUZUMAB OZOGAMICIN (MYLOTARG) Once bound to CD33, the antibody-calicheamycin complex is transported inside of the AML cells by lysosomes. To facilitate selective release inside of the cancer cells, calicheamycin is connected to gemtuzumab by a chemical linker that is stable at physiologic pH but is hydrolyzed in the acidic pH of the lysosomes that transport the antibody-calicheamycin complex into the cell. GENOME EDITING GENOME EDITING TOOLS ARE SEQUENCE-SPECIFIC NUCLEASES Genome editing tools have two features: 1) Recognize specific DNA sequences (i.e. specific genes or non-coding elements) van der Oost. Science (2013) 339:768. GENOME EDITING TOOLS ARE SEQUENCE-SPECIFIC NUCLEASES Genome editing tools have two features: 1) Recognize specific DNA sequences (i.e. specific genes or non-coding elements) van der Oost. Science (2013) 339:768. GENOME EDITING TOOLS ARE SEQUENCE-SPECIFIC NUCLEASES Genome editing tools have two features: 1) Recognize specific DNA sequences (i.e. specific genes or non-coding elements) van der Oost. Science (2013) 339:768. GENOME EDITING TOOLS ARE SEQUENCE-SPECIFIC NUCLEASES Genome editing tools have two features: 1)Recognize specific DNA sequences (i.e. specific genes or non-coding elements) 2)Cut DNA (“nuclease”), then a scar is left behind van der Oost. Science (2013) 339:768. GENOME EDITING: CLEAVAGE REPAIR CAN EITHER DISRUPT ORIGINAL SEQUENCE OR REPLACE IT WITH A NEW COPY NEB.com  GENOME EDITING: CLEAVAGE REPAIR CAN EITHER DISRUPT ORIGINAL SEQUENCE OR REPLACE IT WITH A NEW COPY “delete” NEB.com GENOME EDITING: CLEAVAGE REPAIR CAN EITHER DISRUPT ORIGINAL SEQUENCE OR REPLACE IT WITH A NEW COPY “delete” “copy and paste”  TWO STRATEGIES FOR GENETIC THERAPY: GENE ADDITION AND GENOME EDITING Fischer. Nature (2014) 510:226. TWO STRATEGIES FOR GENETIC THERAPY: ADDITION AND EDITING Gene addition: Feasible with existing technology; clinical trials ongoing. Early trial results appear exciting. Challenges: 1. Will enough of the added gene be made in the cells with the integration? Will enough of the blood stem cells have the added gene? 2. Is the benefit durable? Will the added gene continue to function over days, weeks, months, years, decades? 3. Is the added gene safe? Will its semi-random integration into the genome change the function of other genes in the genome? TWO STRATEGIES FOR GENETIC THERAPY: ADDITION AND EDITING Gene editing: Promise of permanent repair of the underlying disease-causing mutation. Promise of specific beneficial change at the intended genomic site (e.g. β- globin gene) without impacting remainder of genome. Challenges: 1. Technology is in a relatively early stage and needs to be further developed. 2. Can enough cells be edited to have therapeutic impact? 3. Will the editing be exquisitely specific, or will other regions of the genome aside from the target be affected? KEY FEATURES OF STEM CELLS  OUTCOMES OF STEM CELL DIVISION Division of a stem cell can result in one of three outcomes. (a) Division produces two new stem cells – called symmetric self-renewal. (b) Division produces two differentiated (non-stem) cells. (c) Division produces one stem cell and one non-stem cell – called asymmetric self- renewal.  HIERARCHY OF POTENCY Type of cell Ability to make cell types Example in humans Totipotent cell All cell types in body and cell Zygote (only) types that are important for development of embryo, e.g. placenta Pluripotent cell All cell types in body Embryonic stem cell (cell from inner cell mass of blastocyst) Multipotent cell Members of a group of cell Haematopoietic cell (cell from types bone marrow cell able to make all blood cell types) Unipotent cell Only one cell type Germinal layer of the skin STEM CELLS THROUGHOUT LIFE  EMBRYONIC STEM CELLS (ESCS) Pluripotent Blastocyst stem cells ONE TYPE OF ADULT STEM CELL  BONE MARROW TRANSPLANTS Type of transplant Donor and Issues recipient Autologous Same person Tissue rejection unlikely Period of immunosuppression is relatively short Allogeneic Donor is different Tissue rejection more likely from recipient Tissue matching needed Longer period of immunosuppression ALLOGENEIC TRANSPLANT  UMBILICAL CORD STEM CELLS Blood from an umbilical cord is also a source of haematopoietic cells. Blood collected, at birth, from a vein in the umbilical cord, usually has the red blood cells removed before being stored cryogenically in a cord blood bank. Removal of red blood cells increases the chance of stem cell recovery. The stored haematopoietic cells could be used in regenerative medicine, later in the same person’s life. Being their own cells, there is a low risk of tissue rejection. It is estimated that, worldwide, over 4 million couples have chosen to have their child’s cord blood stored. Storage of cord stem cells is tightly regulated. In the UK, the NHS cord blood bank is a public service, but private cord blood bank services also exist. In March 2004, the European Union Group on Ethics concluded in a report that the ‘legitimacy of commercial cord blood banks for autologous use should be questioned as they sell a service, which has presently no real use regarding therapeutic options. Thus they promise more than they can deliver. The activities of such banks raise serious ethical criticisms.’  INDUCED PLURIPOTENT STEM CELLS In 2006, a team in a laboratory in Kyoto showed how adult cells could be reprogrammed to become induced pluripotent stem cells (iPS cells). The team hypothesised that genes that are important to the functioning of embryonic stem cells might be able to produce an embryonic state in adult cells. They used retroviruses to introduce 24 of these genes into mouse fibroblast cells. Of the 24, they found four genes that when used together would reprogramme a mouse fibroblast to become a pluripotent cell – an iPS cell. These genes encoded four transcription factors: KLF4, SOX2, c-Myc and Oct4. In 2007, the same Japanese team transformed human fibroblasts into pluripotent stem cells using the same four transcription factors. In the same year, a team at the University of Wisconsin-Madison achieved the same feat using a different combination of four transcription factors, OCT4, SOX2, NANOG and LIN28. PRODUCING IPS CELLS  USES OF IPS CELLS In theory, iPS cells could be used in regenerative medicine without the ethical considerations involved with the use of embryonic stem cells. Using a patient’s own fibroblasts would lower the risk of tissue rejection. There are issues to be overcome, however, before iPS cells can be used successfully. The conversion rate is very low (in the original mouse study, only 0.01% – 0.1% of the fibroblasts were reprogrammed into iPS cells). In humans, the reprogramming of adult cells could trigger the expression of oncogenes. Insertion of genes risks inserting mutations into the target cell’s genome. Reprogramming – especially of the epigenetic code – might be incomplete. Le Duy Duc Department of Cell Biology, Faculty of Biology, HUS, VNU Email: [email protected]