Molecular Medicine II

Question 1

How could you test if someone still has a BRCA1- dependent cancer?

Answer 1

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

Question 2

How do we analyze the transcriptome?

Answer 2

qRT-PCR and RNA sequencing

An analysis of RNA is necessary to learn about gene expressions and gene expression is tissue specific

Question 3

How is qRT-PCR and RNA sequencing done?

Answer 3

RNA cannot be amplified by PCR or reliably sequenced - analysis requires reverse transcription, in vitro synthesis of cDNA and RNA template

Question 4

What does Quantitative Reverse Transcriptase PCR (qRT-PCR) show?

Answer 4

Shows amount of a few select RNA molecules in a sample - expression of selected genes

Question 5

What does RNA sequencing show?

Answer 5

Shows the amount of all RNA molecules in a sample - global gene expression

Question 6

What does RNA seq capture? What can it inform?

Answer 6

Captures gene expression and mutations across the entire transcriptome

Gene expression data informa cancer diagnosis, prognosis, and treatment

Question 7

What is BRCA1 negatively correlated with?

Answer 7

Survival in breast cancer

Question 8

What is MSH2 expression positively correlated with?

Answer 8

Survival in colon cancer

Question 9

What are the most important tools for protein analysis?

Answer 9

Antibodies

Antibodies bind to antigens with high specificity

Antibodies can be labeled, traced and precipitated

Question 10

How is monoclonal antibody production done?

Answer 10

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

Question 11

What is ELISA and what is it used for?

Answer 11

Enzyme-Linked Immuno-Sorbent Assay (ELISA) is used to quantify proteins

Question 12

What does Sandwich ELISA identify?

Answer 12

Identifies antigen in sample

Ex. Peptide hormones, HbA1c

Question 13

What does Indirect ELISA identify?

Answer 13

Identifies antibodies in sample

Ex. HIV core proteins in AIDS diagnosis

Question 14

What does ELISA not provide?

Answer 14

Gives no information about size or localization of proteins

Ex. purely quantitative

Question 15

What are ELISA techniques commonly used for?

Answer 15

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

Question 16

What is the procedure for blotting techniques?

Answer 16

Separate molecules in a gel

Transfer blot onto membrane

Detect molecule of interest

Question 17

What do blotting techniques work with and what do they detect?

Answer 17

Work with DNA (southern blot), RNA (northern blot), and protein (western blot)

Detects molecule size and molecule quantity

Question 18

What can Western blotting show?

Answer 18

If protein is properly glycosylated

If protein is properly cleaved

Question 19

What does mass spectroscopy do and show?

Answer 19

Small molecules are ionized and separated by size

Detector shows extremely accurate molecular weight

Molecules in sample are identified by weight

Question 20

What are two molecular therapies?

Answer 20

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

Question 21

What is the aim of gene therapy and how does it work?

Answer 21

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

Question 22

What is ADA deficiency?

Answer 22

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

Question 23

What occured with the First Gene Therapy with ADA gene transfer?

Answer 23

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

Question 24

What are the T Cells (CART) and what can they do?

Answer 24

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

Question 25

What are the antibodies for cancer immunotherapy?

Answer 25

Naked antibodies

Conjugated antibodies

Question 26

What are the naked antibodies in relation to cancer immunotherapy?

Answer 26

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

Question 27

What are conjugated antibodies in relation to cancer immunotherapy?

Answer 27

Can be radio labeled or chemo labeled to deliver cytotoxic drug into center of tumor

Question 28

What is an example of antibodies targeting tumor cells?

Answer 28

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