Gene Therapy (+antipathogenic)

1. Understand how Gene therapy works

2. Learn about specific gene therapy examples

3. learn about specific advantages/Disadvantages

4. Learn about gene editing

Goals of therapy

Transplantation of normal genes into cells in place of missing or defective ones in order to correct genetic disorders

Definition of Gene therapy

adding new genes to current genes; editing current genes without adding new ones

Gene therapy is ________ while Gene editing is _______

to give DNA that makes proteins more functional in individuals who make defective protein or low protein expression

An example of gene therapy would Be:

reengineered; DNA/RNA into a cell

Viruses can be _____ to deliver ________ , to which the virus binds and causes cell to make new proteins with the gene

Retinal Dystrophy - RPE65 gene being delivered correctly (by a virus) into the eye (delivers to the retinal pigment epithelium) - delivered by virus bc the protein is too big to deliver into eye alone

Causes the eye to produce more of the protein over time 

Called LUXTURNA - too expensive! 

A real-world example of gene therapy: 

1. Pt’s WBC extracted and programmed for reprogramming

2. Vector/Virus genetically encoded to recognize cancer cells/antigen expressing cells

3. Car-T cells undergo Expansion

4. Quality check the cells

5. Patient undergoes lymphodepletion (to allow CAR-T cells to be accepted)

6. CAR-T cells are given back to patient (infused)

Steps to CAR-T cell therapy for ALL (acute lymphoblastic Leukemia)

• takes 3 weeks for CAR-T cells to be produced

• Cytokine Release Syndrome (CRS) - inflammatory response

• ICANS (Neurotoxicity Syndrome) - 20-70% of CAR-T patients

• Increased infection susceptibility

• EXPENSIVE 

Major issues with CAR-T therapy

Gene EDITING

The replacing of Valine with Glutamic Acid in patients with sickle cell, is called _____ and is done by CRISPR - Called Casgevy

1. learn about new strategies to combat pathogens as needed

2. Learn about Liposomes as cell mimics to combat bacteria and virsues

3. Antibacterial proteins

4. Learn about the discovery and use of PHAGE therapy treat bacteria

Learning Goals for Antipathogenic therapy

dwindling

New antibiotic approvals are _____

pore-forming proteins/bacteria

Gram-Positive

bacteria that injects virulence proteins

Gram-negative

Aqueous cores that are enclosed by a lipid bilayer- used for drug delivery

Liposomes are

Liposomes that mimic host cells can act as bait for gram-positive bacteria, which causes them to use their pore-forming toxins in unproductive manner, allowing immune system to clear bacteria

Drug is inside liposome

Gram positive bacteria cause pores in cells

Gram + causes pore/opening in liposome cell, unknowingly releasing the drug that kills the bacteria

Liposome hypothesis:

Liposomes with sialic acid on surface of cell reduce flu infection - gives the flu something UNPRODUCTIVE to bind to (instead of human cells, which can be infected) - Liposomes mimic human cells, and the flu binds to it, and can’t bind to human cells

Example of Liposomes

Lactoferrin

perhaps the most important antibacterial protein

human milk, tears, saliva, and amniotic fluid - also 80kDa size

Lactoferrin is present in ______

unusually stable; gastric transit

Lactoferrin is ______ , and is not unstable or denatured in _______

• Iron binding protein

• Bacteria, require iron for survival, so lactoferrin starves bacteria from Iron

• Lactoferrin has antioxidant activity AND promotes bone growth

• Has antiviral activity - binds to surface cells occupying sites that viruses would normal attack

• Can also hydrolyze RNA (hydrolytic activity)

Important notes about lactoferrin’s biological activity

Human Milk Lysozyme

_____ is a antibiotic precursor, and is thought to be a key defense factor in protecting the GI tract of newborns against bacterial infection 

Has novel antimicrobial properties within N-terminal domain that target bacterial respiration

Platypus

____ milk contains a unique antibacterial protein

Streptomycin (bad bacteria) 

____ bacteria can wipe out good bacteria in the gut, and put humans/mice (in the stud) at risk for more infection

Bacteriophages

How does nature kill cells? This “biologic” are viruses that specialize in INFECTING BACTERIA

• Ernest Hanbury Hankin (1896) discovered waters from ganga and Yamuna river could kill cholera

• Frederick Twort (1915) also reported a small agent capable of lysing cells

• Felix d’Herelle (1917) was studying patients that had dysentery, and filtered things smaller than bacteria in their stool

Discovery of bacteriophages

temperate (Symbiotic); lysogenic (killing cell by lysing)

Bacteriophages can be _____ or ______

specificity; infecting human cells

Bacteriophages have high ____ and are incapable of _______

• Using phages in agriculture in place of antibiotics due to antibiotic resistance

• Starting to gain more interest recently 

potential benefits of bacteriophages

abundant genetic entities on the planet - a single mL of ocean water contains approximately 50,000,000 bacteriophages!!! 

We have MORE in our body than bacteria (over a quadrillion) 

Bacteriophages are the most ____

screened; cost and time

Bacteriophages can be ____ and selected to specifically lyse cells that we want them too (phage database)

However, the disadvantage can be due to _____ 

• can penetrate biofilms, offering solution where antibiotics fail

• Lower risk of disrupting host microbiome

• can still be effective against drug-resistant pathogens

• Limited need for repeated doses (phages multiply on their own)

• Capable of evolving along with bacteria

Overall bacteriophage advantages

• can cause immune response in some people - antibodies will bind to phages and prevent them from being effective 

• Complicated from a regulatory standpoint

• Time and cost

• limited awareness of phages among public

Overall bacteriophage disadvantages