2.2 Gene Therapy

gene therapy

The alteration of the genes of a person afflicted with a genetic disease

gene editing

Cut defective gene so that it repairs to a correct version
CRISPR

stem cells

unspecialized cells that are able to renew themselves for long periods of time by cell division
help develop cells into other cells

vectors

an agent that contains extra/ modified genetic material
viruses- main vector in gene therapy

Insert

functional gene is given to patient

disable

A dysfunctional gene is disabled, eliminating the impact of the protein

repair

dysfunctional gene is repaired so it can function properly

diseases that can be cured through gene therapy

cancer, hemophilia, blood disease

plasmid dna (vector)

any size; DNA; no specificity; unlikely to trigger response; will not integrate into host cells; temporary effects

liposome (vector)

any size; DNA; no specificity; unlikely to trigger response; will not integrate into host cells; temporary effects; less efficient; lipids

herpes virus (vector)

20k bp; dna; nervous system cells only; can cause immune response; will not integrate into host cells; remains active in cell for a long time

Adeno-associated virus (vector)

5k bp; dna; dividing and non-dividing cell types, varied cell types; unlikely to trigger immune response; will integrate into the host cell; needs helper virus; effects are long lasting

adenovirus (vector)

7500 bp; dna; dividing and non-dividing cell types; varied cell types; will cause immune response; will not integrate into host cell; effects are temporary

retro-virus (vector)

8k bp; rna; dividing cells only; will cause immune response; will integrate into host cell; can cause cancer; not efficient at entering cells

lentivirus (vector)

virosome

live liposome w/ viral surface proteins
carrying capacity and immune advantages of plasmids

Vectors characteristics

fit, target, correct, integrate, activate

ex vivo

taking place outside a living organism; Latin: from life
benefits: no risk to patient, faster
drawbacks: artificial environment, not as specific

in vivo

taking place inside a living organism; Latin: in living
benefits: view side-effects in all parts of the body, more specific, easier to control
drawbacks: long time, live test subjects

Step 1: remove pathogenic genes from virus

used to prevent virus from causing disease

Step 2: insert the CFTR gene into viral genome

cannot cause disease and contains new gene

Step 3: replicate the virus

use the helper virus to replicate the genes
replicate genetically modified ones

Step 4: collect the virus

can now be used to teat for the ability to infect

Step 5: testing

add genetically modified virus to a culture to lungs cells that have a mutation
integrates functional genes into cells genome

Step 6: make a plan to administer modified virus to patient

consider how to deliver meds

CRISPR - Cas9

tool used to edit DNA in cells and living organisms cheaply and quickly
type of immune system discovered in bacteria

CRISPR uses

treat genetic diseases, defeat viruses, produce better crops, and create designer babies

Cas-9 RNA

programs the CRISPR- Cas9 molecule to seek out target gene

PAM

Cas-9 recognizes and binds to three-nucleotide sequence

After Cas-9 and PAM binds

it unwinds,
if the guide RNA matches the DNA sequence next to PAM, the RNA will bind to the complimentary strand.
if not, the DNA will zip back together and Cas-9 will keep binding to other PAM sequences until it finds its matching target DNA

Homology-Directed Repair (HDR)

used to repair a gene by adding donor DNA to replace mutated DNA (Knock-in)
used to repair gene by adding random DNA to replace the mutated DNA (Knock-out)

CRISPR elements

Cas-9 : DNA cutting enzyme
- Helicase: unwind
- Nuclease: cuts DNA
Guide RNA: programmable RNA module used to precisely target any gene

CRISPR step 1

targeting
Cas-9 searches to find a match so it can bind to PAM

CRISPR step 2

Binding
Cas-9 binds to PAM and unwinds

CRISPR step 3

Cleaving
DNA-RNA pairing triggers Cas-9 to change its 3D structure and activate its nuclease activity

CRISPR step 4

DNA Repair
rush to fix the break and use random pieces, can be error prone

in-vitro fertilization (IVF)

egg and sperm cells are combined outside the body in a laboratory dish to facilitate fertilization

Preimplantation Genetic Diagnosis (PGD)

laboratory procedure used to detect genetic abnormalities prior to in vitro fertilization

Sperm Sorting

separate sperm by their genetic material in a tube

Andrologist

Diagnose and treat male reproductive and urinary system disorders, diseases, and injuries. They may conduct physical examinations, perform laboratory tests, or other medical interventions to diagnose these problems. They may treat these
problems using hormone therapy, medication, or surgery.

reproductive endocrinologist

diagnose and treat patients dealing with infertility. Evaluates women's reproductive organs

Embryologist

physician who specializes in the study and treatment of the growth and development of the human organism

reproductive cloning

Using a somatic cell from a multicellular organism to make one or more genetically identical individuals.

theraputic cloning

attempt to create new individual but also produce embryonic stem cells

Oligonucleotides

a short series of DNA bases that contains the desired sequence

dNTs

deoxynucleotides
free nucleotides

CRISPR advantages

target specifically
efficient (time)
relatively simple
edit more than one at a time

CRISPR disadvantages

still pretty new so there are lots of unknowns
efficiency (repair times)
unwanted changes to the gene
ethics of gene editing