MAB Ch 11

myeloma

cancer cell + b cell

model organisms

organisms that help scientists study human diseases 

homologues

genes we have in common with other organisms based on DNA sequence

Ob gene

(in fat tissue)

• codes for leptin hormone (tells hypothalamus in brain that it’s full )

• if defunct → mouse becomes obese

pre-clinical trials

testing on model organisms

clinical trials

testing on humans

Biomarkers

indicators of disease

• Ex: PSA (prostate-specific antigen) present when the prostate is inflamed

• Ex: Circulating Tumor DNA (ctDNA)

  • when blood is drawn, you can get some cancerous DNA, if you see none, you know that the treatment is effective

• Ex: Nanotech Tattoo for Glucose

  • tattoo that can measure blood sugar levels

Prognosis

likelihood of developing disease

Diagnosis

identifying gene/disease

Amniocentesis

• from amniotic fluid surrounding the embryo

• 16 week fetus

  • amni. fluid removed w/ needle

  • Cells cultured/chromosomes stained

• takes longer than CVS

Chorionic Villus Sampling (CVS)

• from portion of the placenta

• 8-10 week fetus

• Suction tube removes outer layer of chorion

  • more invasive

Noninvasive Prenatal Genetic Diagnosis (NIPD)

also known as prenatal cell free (cfDNA)

mother’s blood can be used to detect DNA of the baby

The Cancer Genome Atlas Project (TCGA)

• identify how genetic changes are linked to cancer

• Cells of interest: brain / mammary glands / ovaries / pancreas / liver / lungs

Karyotype

→ all chromosomes

• Fluorescent in situ hybridization (FISH)

• fluorescent probes bind to the ssDNA (chromosome), when binding it fluoresces, stuck to a slide

• identifies translocations (crossing over, genes from certain chrms. swap with each other )

  • chromosome painting

ASO

• can detect single nucleotide changes in a gene, even if restriction site is the same

• can measure SNPs

• is labeled with fluorescent tag

• used for preimplantation genetic diagnosis/testing (PGD/PGT)

Allele Specific Oligonucleotide (ASO) analysis

If A=Has Dominant Trait

If B=Has Recessive Trait

then what is the genotype for 5?

Hh

DNA microarray

• Gene chip, screens for diseases

  • single stranded cDNA attached in each well (probe)

  • nothing binds → black

  • Healthy tissue binds → green

  • yellow (red + green) → both binds

  • diseased tissue → red

Protein microarray

• antibodies attached to chip

• run patient proteins over chip

  • binds = fluorescence

How would you describe a gene’s presence in cell if it binds to cDNA in a microarray and the well turns red?

the gene is mostly present in diseased cells

Whole Genome Sequencing (WGS)

Anorexia, Alzheimer’s, Autism, detecting foodborne pathogens

Whole Exome Sequencing (WES)

Identify cancers (melanoma, breast, colorectal, ovarian)

Undiagnosed Disease Network

NIH initiative- diagnose rare diseases

Genome Wide Association Studies (GWAS)

Genomes of several 100 (or 1000) individuals analyzed to compare to individuals without a particular disease

Oncogenes

cause cancer (Ex: BRCA1/2)

small molecule inhibitors

target cancer w/o hurting other cells

• Magic bullet” drugs (look for proteins only on cancer cells)

Herceptin

inhibits HER-2 (aggressive, metastatic prone breast cancer)

Gleevec

targets BCR-ABL protein, treats chronic myelogenous leukemia (CML)

CML (chronic myelogenous leukemia)

• Translocation between 9 and 22 chromosome

• Identified in 1960

• 90% survival rate after 10 years

Microspheres (1-100 nm)

• used as a mist to treat lung cancer and administer insulin

• Usually made of lipids

nanoparticles

• can be made of lipids, silica, gold, graphite

• potential future → removing cholesterol

Provenge

→ therapeutic cancer vaccine, treat prostate cancer

1. harvest immune cells

2. induce them into dendritic cells

   1. Expose to prostatic acid phosphatase (PAP) → causes immune cells to fight prostate cancer

Monoclonal Antibodies (mab) → for specific diseases

1. inject mouse w antigen

2. remove mouse spleen (rich in B cells)

3. Mix b cells w cancer cells (in culture)

4. fusion = hybridomas → secret antibodies in liquid culture

5. delivering radioactive materials to treat cancer

• treating substance addiction

• pregnancy test

• strep throat

• monoclonal ab treatment

Immunotherapy

using immune system to attack cells

Chimeric Antigen Receptor (car-t)

extracellular antigens (leukemias/lymphomas)

• Kymriah for ALL

Recombinant T cell receptors (TCRs)

can target intracellular antigens and solid tumors

Oncolytic Viruses

engineered to bind to and infect cancer cells,without causing illness

Vectors

• Adenovirus (common cold)

• Adeno Associated Virus (AAV; the flu)

• Retrovirus

• Integration → permanently becomes a part of the genome

• Viral DNA → provirus

• Lentivirus

• Liposomes → lipids packaged w/ genes

Antisense RNA

complementary strand to mRNA, blocks translation

RNAi

• dicer (enzyme) chops delivered dsRNA → siRNA

• siRNA joins w/RISC (RNA induced silencing complex) → siRNA is single stranded, looks for target mRNA

• Triggers miRNA (micro), inhibits gene expression

• Hematopoietic Stem Cell (HSC)

• in bone marrow, becomes blood cells

• Isolated from patient, modified (in vitro), given back to patient

Severe Combined immunodeficiency (SCID)

• Lacking immune system bc of faulty ADA (adenosine deaminase) production

• ADA breaks down dATP (deoxyadenosine triphosphate)

• Buildup of dATP is toxic to T cells

Leber’s congenital amaurosis (LCA)

• Degenerative retinal disease, misshapen rods & cones

• RPE65 gene breaks down retinol (allows rods/cones to detect light)

Hemophilia

• Faulty FIX gene → Factor IX → blood clotting

  • Modified AAD delivered into liver cells

Lipoprotein lipase deficiency (LPLD)

high levels of triglycerides in blood → pancreatic inflammation → pancreatitis

Glycera → modified AAV → delivers LPL gene

Regen. Medicine

growing cells/tissue to replace damaged tissue/organ

Fetal Tissue Transplantation

• used since 80’s

• from accident victims/abortions

• possibly for parkinson’s/alzheimer’s

• new cells can’t produce growth hormone

Organ Transplant

first → liver transplant, 1963

must take anti-rejection drugs (immunosuppresive)

autografting → transplant of tissue from one part of body from another

must be typed

Major Histocompatibility complex (MHC)

• tissue typing proteins from 70+ genes

• donor & recipient must be a match

Xenotransplantation

→ donation from another species

1984 → human gets a baboon heart, died 3 weeks later

possibility of using pigs?

Knockout Pig

→ get rid of GGTA1

doesn’t produce sugars on tissue cells

lower chance of rejection

Anti-Zoonotic

CRISPR pigs, released by eGenesis

Eliminated 62 copies of porcine (pig) endogenous retrovirus (PERV)

Biocapsules

used to deliver therapeutic proteins/nutrients

Tissue Engineering

• create a scaffolding

• put the cells on it → seeding

• bathe in nutrients

Embryonic → ESC

Cell lines- cultured stem cells that continually grow (hela)

Development

• 1 cell → zygote

• 16 cells → morula

• 32 cells → blastocyst

directed differentiation

• Transcription factors → turn off Nanog gene → stem cells is no longer pluripotent

Nuclear Reprogramming

→ using genes to turn somatic cells pluripotent

retroviruses used to deliver 4 transgenes, from fibroblasts to stem cell

OCT3/4, SOX2, c-MYC, KLF4

Cloning

• reproductive → create a fetus

  • Somatic cell nuclear transfer → enucleated egg + somatic cell nucleus, grow on plate, implant in surrogate

• therapeutic, enucleated egg + somatic cell nucleus, embryo used for stem cells