FINAL exam (3 lectures) Flashcards
Three complications in treating monogenic disorders
1.) gene may not have been identified
2.) Fetal damage
3.) most severe clinical phenotypes are less ammenable to intervention
Levels of intervention for treatment strategy
-mutant gene modification
-mutant mRNA modification
-mutant protein modification
-disease-specific compensation
-clinical phenotype modification (medical/surgical)
-genetic counseling
Long-term complications of gene therapy
1.) deficiencies have a late onset
2.) successful in one tissue but harmful to another
3.) side effects have a late onset
Dominant negative
effect of genetic heterogeneity on therapy, new wildtype replacements my be “poisoned” by a still-present mutant gene
The most successful gene therapy
treatment of metabolic deficiencies (strategies include: dietary modification, avoidance, diversion, inhibition. and depletion)
Biotinidase deficiency
prenatal biotin administration
Cobalamin-responsive methylmalonic adicuria
prenatal maternal cobalamin administration
Congenital adrenal hyperplasia
Dexamethasone, a cortisol analogue
Phosphoglycerate dehydrogenase (PGDH) deficiency, a disorder of L-serine synthesis
Prenatal L-serine administration
Protein mutation genetic therapies
1.) add a cofactor
2.) replace the defective protein
Problems:
1.) proteins with a short half-life require frequent treatments and replacement –> insufficent supplies and harmful immune responses
Ivacaftor
For CF patients, restores lung function by permitting CTFR protein to be integrates into the cell membrane (augmentation)
Gaucher Disease
Lysosomal storage disease, mutation in glucocerebrosidase causing accumulation of glucocerebrosides in reticuloendothelial system
treated successfully with enzyme replacement (imiglucerase, miglustat) therapy (expensive and weekly infusions)
Beta-globin deficiencies– sickle cell
inducing the expression of gamma-globin (noramally only found in fetus) can partially rescue beta-globin deficiency phenotype
Hematopoietic stem cell (HSC) transplantation
bone marrow transplant, is risky due to infection
placental cord blood is more desirable over bone marrow, rich in HSCs, graft vs host risk is greatly reduced
gene therapy
modification of cells to produce a therapeutic effect, based on recombinant DNA technology that permits the introduction of new genes and possibly the removal of damaged genes
reasons to do gene therapy
- compensate for a mutant cellular gene with a loss of function mutation
- replace or inactivate a dominant mutant gene
- Pharmacological effect (like cancer)
Ex vivo gene transfer
transfer outside the body or a stem cell, followed by introduction into the body
ADVANTAGES: does not require an efficient means to enter a cell because it can be engineered for
DISADVANTAGES: difficult and time consumig
In vivo gene transfer:
direct injection into the body using a vector
ADVANTAGES: quick and easy
DISADVANTAGES: targeting proper cells, immune responses, safety
Retroviral vectors
-Can enter virtually all target cells
-made simple and replication-defective
-Easy to engineer
-Introduce DNA into host genome
-accomodate large transgenes
Problems:
-require dividing cells to introduce DNA into host genome (lentiviruses, HIV can get around this)
- Safety due to mutagenesis
adenoviral vectors
Advantages:
- generated at high titer
- infect wide range of cell types
- accomodate large genes
Disadvantages:
- does not integrate into the genome
- expression is transient
-strong immune responses
- result in cell toxicity sometimes
Adeno-associated vectors (AAVs)
Advantages:
- can infect both dividing and non-dividing cells
- integrate host genome
- EX: CF, Factor IX, muscular dystrophy, CNS diseases
Disadvantages:
- Small transgenes (5KB)
—- Preferred viral vector for clinical trials
non-viral vectors
Advantages:
- lack biological risks associated with viral vectors
Disadvantages:
- not successful, DNA degraded in lysosomes
EX: naked DNA, liposomes, protein-DNA conjugates, artificial chromosomes, nanoparticles
Herpesviruses
central nervous system tropism, have large packing capacity, however they have strong inflammation and neurotoxic responses
CRISPR/CAS-9 example
in vivo-gene editing of transthyretin amyloidosis (ATTR), KO the mutant gene with 87% response positive
siRNA gene therapy
designed to target a range of tissues
ex studies: SARS, pathology of lungs in CF, KO of Huntington mRNA
Hemophilia B gene therapy
- Factor IX replacement
- Liver-specific
-AAV2, AAV9
and Hemophilia A (factor VIII)
Lysosomal storage disease gene therapy
- enzyme replacement therapy, Gaucher disease)
- hematopoietic cell transplantation
- AAVs and other vectors
Parkinson’s disease gene therapy
- Viral vectors targeting the neuronal population that is affected in PD
Leber congenital amaurosis gene therapy
early onset photoreceptor degeneration
- RPE65 gene (RA metabolism)
-AAV2 vectors
DMD gene therapy
-mini-dystrophin genes delivered by AAVs
- mutation suppression by STOP codon read through
- Exon skipping
SCID gene therapy treatment
OTC deficiency gene therapy, Jesse Gelsinger, X-SCID –> fatality and Leukemia (LMO2 gene)
Terminal differentiation
As cells differentiate, they usually stop dividing permanently.
Two features of stem cells that are consistently present
1.) stem cells retain the capacity to proliferate and self-renew
2.) stem cells produce daughter cells that can terminally differentiate into specific cell types
Hematopoiesis
the ground floor of stem cells; hierarchy of cell types
multipotent SC > multipotent progenitor > common myeloid/lymphoid progenitor > erythrocytes/leukocytes
transit amplifying cells/progenitors
transient between stem cell and only proliferative for a limited time
Mechanisms to promote stem cell DNA health
1.) use progenitor cells to divide to create tissues so that stem cells don’t have to divide as frequently (dividing leads to errors)
2.) exceptions to random assortment because they give ALL parental (non-copied) chromosomes to one daughter cell to reduce the chance of them being altered during synthesis– known as “the immortal strand hypothesis”
Embryonic stem cells
obtained from inner cell mass, are pluripotent. Primarily obtained from unneeded extra blastocytes created for IVF. Ethical considerations
Tissue-specific stem cells
Somatic/adult stem cells: multi or uni-potent and usually exist in small numbers. Can be difficult to isolate and grow in cultures
Mesenchymal stem cells
multipotent stromal cells from various locations including: bone marrow, connective tissue, and adipose tissue. Can be induced into bone, cartilage, and fat cells
Cancer stem cells
undifferentiated cells, contain a mix of pleomorphic and relatively undifferentiated cells
Induced pluripotent stem cells (iPS/iPSCs)
Differentiated cells engineered in the lab to resemble embryonic stem cells.
Regulation of Embryonic pluripotency
– Oct4, Sox2, NANOG, KLF4 forming a core Pluripotency Gene Regulatory Network
- miR-302 regulating the inhibition of Oct4 and NANOG
PGRN
transcription factors acting as hubs, core intact=pluripotency
PGRN
transcription factors acting as hubs, core intact=pluripotency
Three general levels:
1.) signaling pathways
2.) Transcriptional networks
3.) Epigenetic factors
Stem cell niche
stem cell regions that allow for controlled stem cell proliferation (uses Hh and Wnt). They produce paracrine factors that regulate stem cell proliferation and prevent differentiation
The 4 R’s of regenerative medicine
1.) Repair
2.) Replace
3.) Restore
4.) Regenerative
OKSM method
Certain transcription factors were strongly expressed in pluripotent embryonic stem cells, but suppressed in differentiating cells: Oct4/Sox2/Klf4/Myc or Oct4/Sox2/Klf4/NANOG because overexpression of Myc increases the risk of oncogenic transformation
Direct reprogramming examples
Fibroblasts –> Neurons
Pancreatic exocrine cells –> Beta-cells
Fibroblasts –> cardiac muscle cells
SCNT cloning
replacing the genome of an oocyte with that of an adult cell
Ex. Dolly ant the mammary cell
Antigen assay
Search for microbial or virus antigens, using fluorescent antibody, or EIA
Molecular assay
 Search for key genes of pathogen, nucleic acid hybridization, PCR
Blood sample
search for antibodies using agglutination, RIA, EIA, and so on 
Direct microscopic examination of clinical specimen
• gram stain, acid fast stain
• India ink, CSF for cryptococcus neoformans
• KOH treatment for fungal forms,
blue
• fluorescent antibodies
• Electron microscopy for viral infection
Most common direct microscopic exams
Bright field, and fluorescence
Culture media
Supplies the nutritional needs of micro organisms and can either be chemically defined or undefined (complex media) 
Selective media
Contains compounds that selectively inhibit growth of some microbes but not others
Differential media
Contains an indicator, usually a guy, that to text chemical reactions were occurring during growth
Anaerobic micro organisms
Isolation, growth, and identification must be kept in anoxic conditions
Aerobes
Require oxygen to live, whereas anaerobes do not and may even be killed by oxygen
Facultative organisms
Can live with or without oxygen
Aerotolerant anaerobes
Can tolerate oxygen and grow and its presence even though they cannot use it
Microaerophiles
Arabs that can use oxygen only when it is present levels reduced from that an air
Most common culture medias
Blood agar, enteric agar
Sheep’s blood agar
Nonselective nutrient base with 5% sheep‘s blood added. Used for the cultivation of non-fastidious micro organisms. Measures hemolytic capacity, and classifies into alpha beta or gamma
EMB agar
Enteric media,eosin- methylene blue, selective and differential media. Inhibits growth of gram-positive bacteria and distinguishes gram-negative bacteria based on their ability to ferment lactose (metallic sheen= can ferment, blue = cannot ferment) 
EMB appearance for Escherichia coli
Dark center with greenish metallic Sheen
EMB appearance of Enterobacter
Similar to E. coli, but colonies are larger
EMB appearance of klebsiella
Large, mucoid, brownish
ENB appearance of proteus
Translucent, colorless
EMB appearance of pseudomonas, salmonella, Shigella
Translucent, colorless to Gold
MacConkey agar
Selective and differential, enteric agar inhibits the growth of G positive, distinguishes G negative by lactose fermenters. Lactose fermenter will be deep purple/pink and non-fermenter will be colorless
Thayer-Martin media
Used for isolating Neisseria bacteria. Media inhibits the growth of most other organisms by using:
Vancomycin to kill G positive
Colistin to kill G negative
Nystatin to kill fungi
SXT to inhibit G negative, swarming Proteus
Nutrients: chocolate chips blood, beef infusion, casein hydrolysate, and starch
Example identification of N. gonorrhea
Look for cytochrome C oxidase, oxidase positive means gonorrhea positive
On Thayer-Martin media
API strip
Purified colony growths added to solutions, looking for biochemical pathways (changes the color of the liquid)
Serology
The study of antigen antibody reactions in vitro, looking for particular antigen using antibodies or looking for antibodies against a particular antigen
Agglutination
The visible clumping of a particular antigen when mixed with antibodies specific for the particulate antigens
Typically used to identify blood group antigens and many pathogens
Direct Agglutination
When soluble antibody causes clumping due to interaction with an antigen that is an integral part of the surface of a cell or other insoluble particle. Used for classification of antigens found on RBCs
Passive agglutination
When it is too small, colorless: chemically couple to cells or insoluble particles like latex beads or charcoal. Clumping gives you a positive test, while diffusion is a negative result
Direct ELISA test
Antibodies bound to the wells, allow for virus antigen binding, add antivirus antibody containing conjugated enzyme and look for fluorescent colors
Indirect ELISA test
Wells coated with antigen preparation, looking for antibodies from the patient (allowing them to bind antigen), add fluorescent conjugated enzyme
Search for pathogenic nucleic acid
Finding Jean on present, deep sequencing/next generation sequencing
