Molecular Medicine II Flashcards
How could you test if someone still has a BRCA1- dependent cancer?
If new variant - analyze grandmother’s DNA and see if grandma has variant
It could be methylation of BRCA1 or BRCA2 promoter, which would shut down gene expression - find out with methylation sequencing since methylation BRCA1 cancer is sporadic and not familial
Or could analyze the transcriptome to see if BRCA1 and BRCA2 are expressed in tumor tissue
How do we analyze the transcriptome?
qRT-PCR and RNA sequencing
An analysis of RNA is necessary to learn about gene expressions and gene expression is tissue specific
How is qRT-PCR and RNA sequencing done?
RNA cannot be amplified by PCR or reliably sequenced - analysis requires reverse transcription, in vitro synthesis of cDNA and RNA template
What does Quantitative Reverse Transcriptase PCR (qRT-PCR) show?
Shows amount of a few select RNA molecules in a sample - expression of selected genes
What does RNA sequencing show?
Shows the amount of all RNA molecules in a sample - global gene expression
What does RNA seq capture? What can it inform?
Captures gene expression and mutations across the entire transcriptome
Gene expression data informa cancer diagnosis, prognosis, and treatment
What is BRCA1 negatively correlated with?
Survival in breast cancer
What is MSH2 expression positively correlated with?
Survival in colon cancer
What are the most important tools for protein analysis?
Antibodies
Antibodies bind to antigens with high specificity
Antibodies can be labeled, traced and precipitated
How is monoclonal antibody production done?
Challenge animal with antigen and isolate the B cells
Fuse B cells with immortal myeloma cells
Separate hybridoma cells and select the clone that produces the best antibody
What is ELISA and what is it used for?
Enzyme-Linked Immuno-Sorbent Assay (ELISA) is used to quantify proteins
What does Sandwich ELISA identify?
Identifies antigen in sample
Ex. Peptide hormones, HbA1c
What does Indirect ELISA identify?
Identifies antibodies in sample
Ex. HIV core proteins in AIDS diagnosis
What does ELISA not provide?
Gives no information about size or localization of proteins
Ex. purely quantitative
What are ELISA techniques commonly used for?
Diagnosis and management of diabetes
GAD autoantibody detection to distinguish between type I and type II diabetes
C-peptide and insulin detection to determine pancreatic activity
What is the procedure for blotting techniques?
Separate molecules in a gel
Transfer blot onto membrane
Detect molecule of interest
What do blotting techniques work with and what do they detect?
Work with DNA (southern blot), RNA (northern blot), and protein (western blot)
Detects molecule size and molecule quantity
What can Western blotting show?
If protein is properly glycosylated
If protein is properly cleaved
What does mass spectroscopy do and show?
Small molecules are ionized and separated by size
Detector shows extremely accurate molecular weight
Molecules in sample are identified by weight
What are two molecular therapies?
Targeting genes - replacing a defective gene to cure a genetic defect. inserting a new gene to reprogram T-cells
Targeting proteins - using antibodies to identify and destroy malignant cells
What is the aim of gene therapy and how does it work?
Aims at inserting a functional copy of a defective gene into the genome - integration of manufactured DNA into genome through viruses or increase target specificity with CRISPR/Cas9 guide RNA
Requires efficient gene delivery systems to reach as many cells as possible
Requires safe delivery - cannot trigger fatal immune response or transform cells into cancer cells
What is ADA deficiency?
Adenosine deaminase (ADA) deficiency causes an excess of cellular dATP, inhibits DNA synthesis
Results in severe combined immunodeficiency (SCID)
Affected patients must live germ free
Recessive disorder - ideal for gene therapy because disease will disappear in hetero state
What occured with the First Gene Therapy with ADA gene transfer?
T cells were isolated from patient and transfect with virus carrying normal ADA gene
Patient transfused with transfected cells
Patient survived and had some immune function restored
What are the T Cells (CART) and what can they do?
Genetically engineered T cells can detect and destroy cancer cells
T cells are isolated from the patient and then stimulate and transfect T cells with lentiviral vector
Vector integration creates a chimeric receptor for a tumor cell antigen (CD19) - CAR-T cells will now recognize lymphoblasts in acute lymphoblastic leukemia (ALL)
Patient infused with modified cells
Next generation - modify the CAR-T cells with CRISR- Cas9 gene editing - avoid immune destruction and exhaustion
What are the antibodies for cancer immunotherapy?
Naked antibodies
Conjugated antibodies
What are the naked antibodies in relation to cancer immunotherapy?
Can bind to tumor cells and attract immune cells to destroy the malignant cells
Can unblock the immune system (nonspecific immunotherapy)
Can block receptors on cancer cells and shut off growth signals
What are conjugated antibodies in relation to cancer immunotherapy?
Can be radio labeled or chemo labeled to deliver cytotoxic drug into center of tumor
What is an example of antibodies targeting tumor cells?
Breast cancer - some breast cancers are characterized by overexpression of growth factors receptor ErbB2/HER2
Poor prognosis
Herceptin (trastuzumab) is an antibody that binds to ErbB2/HER2 expressing cells
Immune system recognizes and destroys labeled cells
