Antibodies / Gene Therapy Quiz – Flashcards
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Vaccinations have been effective for...
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protection
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Which types of vaccines are being explored?
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cancer
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T or F: The cancer vaccines being explored are therapeutic and not preventive.
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True
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How are cancer vaccines administered?
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patients injected with cancer cell antigens to stimulate patient's immune system to attack existing cancer cells.
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What is the relationship between vaccines and antibodies?
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vaccines stimulate antibody production by the immune system antibodies themselves may be used to treat existing conditions.
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How are antibodies specified?
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specified based on molecules or pathogens to which they are produced
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What are some characteristics and uses of antibodies?
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can find and bind to their targets with great affinity many techniques to diagnose and treat use antibodies, including Monoclonal Antibodies & ELISA.
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What do antibodies look like?
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See picture. (notice locations of variable and constant regions)
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What does MAbs stand for?
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Monoclonal Antibodies
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What are MAbs
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purified antibodies that are very specific for certain molecules
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What are they also considered? (nickname)
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"magic bullets" for disease treatment
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How are MAbs administered and what do they do?
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injected into patients where they seek out and target antigens
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What antigens do they target?
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Those of Cancerous tumors, Arthritis, Alzheimer's disease
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What are the initial steps in the procedure for making a MAb?
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Mouse/rat injected with purified antigen and makes antibodies to antigen (over several weeks)
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What are the next steps in the procedure for making a MAb?
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Animal's spleen removed (rich source of B lymphocytes) In culture dish, B cells mixed with cancerous cells that grow & divide indefinitely Certain B cells & myeloma cells will fuse to create hybridomas
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What are myeloma cells?
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Cancerous cells that grow & divide indefinitely
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What are hybridomas?
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hybrid cells that act as factories for making antibodies
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How are hybridomas grown?
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chemically selected and grown in liquid culture
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What do hybridomas do?
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secrete antibodies into liquid culture surrounding cells
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What is an advantage of hybridomas?
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can be transferred and frozen (so permanent stock always available)
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Where are antibodies isolated in/from?
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cultures in large batches using bioreactors
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What are some MAbs attached to?
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snake venom (to cause lysis of the cell)
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What further research is need regarding MAbs?
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ensure that they only attack intended cells
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How do MAbs have value in therapy?
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treating people addicted to harmful drugs (preventing damage)
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What MAbs applicational products are on the market now?
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strep tests and home pregnancy kits
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What does ELISA stand for?
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Enzyme Linked Immunosorbent Assay
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What is the ELISA technique?
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fundamental tool of clinical immunology
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For what is ELISA technique used?
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initial screen for HIV detection
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What principle is the ELISA technique based upon?
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antibody-antibody interaction
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What does ELISA allow for?
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easy visualization of results without use of radioactive materials
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In what is the ELISA technique performed?
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microplates
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What are the first steps in the procedure of the ELISA technique?
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Bottom of each well is coated with a protein (ex. antigen) (which will bind the antibody you want to measure) Whole blood is allowed to clot and the cells are centrifuged out to obtain the clear serum with antibodies (called primary antibodies) Serum is incubated in a well, and each well contains a different serum. (Positive and negative control serum samples tested)
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What are the next steps in the procedure of the ELISA technique?
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The serum is removed and weakly adherent antibodies are washed off with a series of buffer rinses To detect the bound antibodies, a secondary antibody is added to each well The secondary antibody would bind to all human antibodies and is typically produced in a rodent
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What are the final steps in the procedure of the ELISA technique?
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Attached to the secondary antibody is an enzyme such as peroxidase or alkaline phosphatase These enzymes can metabolize colorless substrates (sometimes called chromagens) into colored products
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What is indirect ELISA?
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After an incubation period, the secondary antibody solution is removed and loosely adherent ones are washed off as before The final step is the addition the enzyme substrate and the production of colored product in wells with secondary antibodies bound The amount of color produced is proportional to the amount of primary antibody bound to the proteins on the bottom of the wells
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What is gene therapy?
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Involves delivery of therapeutic genes into the human body to correct disease conditions created by faulty genes
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What are the two primary strategies of gene therapy?
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Ex vivo gene therapy and In vivo gene therapy
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Ex vivo gene therapy
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cells from diseased person are removed and are treated in a lab (using techniques similar to bacterial transformation) and reintroduced to the patient
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T or F: Ex vivo is more effective than in vivo.
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True
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What is transfection?
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introduction of DNA into animal or plant cells
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In vivo gene therapy
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introducing genes directly into tissues or organs without removing body cells
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What is the challenge of In vivo gene therapy?
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delivery to only intended tissues
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What do viruses act as for gene delivery?
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Vectors (HW chart) but some are injected directly into tissue
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What is payload?
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therapeutic genes
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What do payloads possibly require?
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May require long-term expression of corrective gene Others require rapid expression for short periods of time
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What do viral vectors do?
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use viral genome to carry therapeutic gene(s) and to infect human body cells
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What are the types of viral vectors?
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Adenovirus (common cold) Adeno-Associated Virus Retrovirus (HIV) Herpes simplex virus (cold sores)
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Why must viruses be engineered for viral vectors?
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so that they can neither produce disease nor spread (extremely effective at infecting human cells)
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Describe Adenoviruses.
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infect both dividing & non-dividing cells effectively
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Describe Adeno-Associated viruses.
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do not cause illness in humans, can infect a wide variety of cells, & integrate 95% of time in same location
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Describe Retroviruses.
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of interest because they insert DNA into the genome of host where it remains permanently (integration), but often, randomly
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Describe Herpes virus (HSV-1)
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strain primarily affects central nervous system (CNS) and may help develop treatments for Alzheimer's, Parkinson's, and other genetic neurodegenerative diseases
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What is one major setback to viral vectors?
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not easily transfected
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Describe Liposomes.
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small diameter, hollow particles made of lipid molecules and packaged with genes - injected into tissues (gene guns also used)
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Describe "Naked" DNA.
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DNA injected directly into body tissues (ex. effective in liver/muscle), but not enough cells express gene to have affect
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Describe Artificial chromosomes.
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Non-protein coding DNA with therapeutic gene with a similar construction to normal chromosomes (designed for permanent incorporation)
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About how many genetic disease conditions are caused by single genes?
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More than 3,000
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Candidates for treatment by gene therapy include:
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Cystic Fibrosis Huntington's disease Tay-Sachs Hemophilia Sickle cell disease Phenylketonuria (PKU)
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Who was Ashanti de Silva?
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(4 years old) with severe combined immunodeficiency (SCID) treated in 1990 at NIH in Maryland Lack functioning immune system because of defect in gene called adenosine deaminase (ADA), which is involved in metabolism of dATP (nucleotide precursor used for DNA synthesis) Accumulations of dATP are toxic to T cells Normal gene cloned into vector introduced into nonpathogenic retrovirus
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First human gene therapy success
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Ex vivo approach used T cells isolated from blood Multiple treatments required Within a few months, T cell numbers increased After 2 years, ADA enzyme activity was high
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Cystic Fibrosis
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occurs with 2 defective copies of gene encoding the protein called cystic fibrosis transmembrane conductance (CFTR) serves as a pump at the cell membrane to move chloride ions out of the cells if cells can't move chloride out, they absorb water trying to dilute the chloride in the cell. leads to the production of thick sticky mucus that clogs airways; ideal environment for infections (leading to pneumonia, etc...)
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Treatments for Cystic Fibrosis
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Back clapping Drugs that thin mucus Antibiotic treatment (to fight infections) One form of gene therapy has helped
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Cystic Fibrosis Gene Therapy
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Uses viruses and liposomes sprayed into nose & mouth Expensive treatment NOT a reliable cure yet Requires multiple applications (DNA doesn't integrate) May not produce adequate protein
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Risk of Gene Therapy
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Discussions of safety intensified when 18-year-old Jesse Gelsinger died during a clinical trial @ Upenn in 1999 Complications related to adenovirus vector that was used Ornithine transcarbamylase deficiency (affects ability to break down dietary amino acids) 1st person to die as a result of gene therapy
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Success of Gene Therapy
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Success in Rhys Evans, a child born with X-linked Severe Combined Immunodeficiency Syndrome (SCIDS - aka bubble boy), in 2002 The team took stem cells that give rise to immune cells from the boy's bone marrow They used a modified form of a retrovirus as a vector The engineered stem cells were then returned to the boy's body Now, he has normal levels of T cells
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Unresolved Questions
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Can gene expression be controlled in the patient? What happens if normal gene is overexpressed? How long will the therapy last? What is the best vector to use? What is the minimum number of cells needed to infect to achieve success?
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God Bless America.
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And God Bless Phusko Pharmaceuticals.
