module one review

What is gene therapy

a technique used to alter genetic material in a persons cells to treat or prevent disease

how is gene therapy done

insertion of genes

repair of damage genes

inactivate problematic genes

what is gene editing

it involved the deliberate changes in gees often in context of their normal chromosome location

what is needed to accomplish gene editing

an efficient and accurate delivery system

the therapeutic genes need to be delivered to the correct target cells

what makes viruses a good delivery mechanism

they bind and enter specific cells

they uncoat during entry

some even integrate host chromosome (better for long term expression)

what does replication defective for virus vectors mean

the essential genes have been removed from the virus genome and replaced with therapeutic genes. The viruses cannot replicate or produce new viruses after they’ve enter

how are replication defensive cels grown in lab

using complementation cell lines

how does complementation cells work

the host supplies essential proteins that the virus lacks which allows the virus to replicate inside those specific cells

example is HEK293 cells have an E1 gene integrated already which allows E1 replication defense to grow

how are viral vectors made for gene therapy purposes

delete viral genes

leave the origin of replication intact

transfect into packaging cell line

what four objectives are virus vectors developed for

protein expression in the lab

vaccination

defective gene replacement

anti cancer agents

(the last three can also be considered gene therapy

criteria for successful intervention of viral vectors in gene therapy

there has to be effective therapeutic gene, appropriate tissue for delivery, an animal model and a delivery mechanism

non viral gene delivery methods

DNA injection and lipid nanoparticles

viral vectors of gene delivery (most efficient)

ex vivo (delivered outside the body)

in vivo (delivered into cells in the body)

retroviruses

stable integration of DNA into host chromosome and efficient packaging cell lines = beneficial features for delivery

acting in trans

a factor can be expressed from a different genetic location and still function

packaging cell lines

cells that provide viral genes in trans

these allow replication defective viruses to grow

transfection

introducing nucleic acids into mammalian cells

equal of bacterial transformationt

transduction

used in gene therapy field to indicate delivery of therapeutic gene to target cells via viral vector

advantages to retroviruses

integrates into host genome for long term expression

viral proteins not expressed into host so not very innumogenic

retrovirus disadvantages

need dividing cells for some vectors

integration is random and maybe mutagenic

adenovirus vectors

infects a wide variety of cells, differentiated as well as replicating and grow go high titers

replication selective adenovirus vectors

use the killing capability of the virus in therapies directed against cancers

selective for only killing tumor cells

advantages of adenoviruses

high transduction efficiency and infects both the replicating and differentiated cells

disadvantages to adenoviruses

does not integrate, potentially immunogenic and can have toxic inflammatory effects at high doses

adeno associated virus

requires adenovirus as a helper to carry out productive infections

infects both dividing and non dividing cells

lower risk of mutagenesis

advantages of aden associated vectors

infects both dividing and non

nonpathogenic

disadvantages to aden associated vectors

mutagenesis is rare but possible

can only accept 5kb sequences

how to use viral vectors for preventing/treating disease

they can be engineered to express a gene from a pathogenic organism in order to induce immune response (vaccine)

Cas3

promotes DNA degradation of the target sequence instead of making a double stranded break as Cas9 does

useful to destroy the target cell wh

what does crispr cas 3 require

the cascade complex in addition to cas3

gene transfer/editing to treat genetic disorder

casgevy is used for sickle cell disease since it affects the function of hemoglobin Hb in red blood cells

there is a switch from fetal HbF to adult HbA hemoglobin

so by inactivating the HbF repressor, the HbF expression would be unregulated and alleviate symptoms

using gene transfer to treat cancer

T lymphocytes of the immune system normally have receptors that recognize cells that express foreign stuff and destroy them

cancer develops because this occurs but nothing is ever recognized and destroyed

CAR

chimeric Antigen Receptor genes are modified to change the T cell receptor to recognize a marker protein specific to the cancer cell being targeted

then the chimeric antigen receptor cels can be used to treat cancers

basic procedure for CAR T

get the patients cells, transduce with viral vector ex vivo, grow the CAR T cells then return to patient where the cells will be targeted and eliminated

how is gene transfer used in agriculture

introduces new DNA into an important crop or livestock animal to confer a desirable trait

how does Bt gene work in agriculture

it produces a protein toxin that kills pest larval caterpillars that are bad for crops

this isnt toxic to humans or plants

concerns with GMO in good

some allergic reactions

outcrossing of the engineered gene into other crops

gene editing in agriculture

does not rely on introduction of foreign gene, can be done to repair, silence or knock out certain genes that make for better desires

crispr

clustered regularly interspaced short palindromic repeats

cas

crispr associated

PAM

photospacer adjacent motif

crRNA

crispr RNA

spacer sequence

specific targeting sequence that codes for the crRNA

repeat sequence

sequences of repeats that divide the specific crispr spacers

leader sequence

promoter for transcription

MOI

multiplicity of infection

challenges associated with crispr to target and eliminate pathogens

not fully characterized and use endogeneous systems

large and may impair the phage

can be resisted by bacteria

negative control

shows no result

positive control

shows expected result and indicated that the experiment worked correctly

what is the main concern of gene therapy

the expense

some cost one million per dose

Glybera case

first gene therapy drug approved in europe, it was developed to supply a gene that degrades triglycerides, it helped but symptoms were not eliminated, it was then withdrawn because of high cost and lack of demand

ethics of germ line editing in humans

not allowed in United States, current resource focuses on editing of somatic cells and this has been done in plants and animals

Dr. He

used crispr to edit embryos

the goal was to edit receptor used by HIV to make children resistant to the infection

how is crispr used to create precise genome edits

the crispr recognizes and cuts the foreign DNA and inserts the foreign DNA into the crispr locus

this protects the bacteria from foreign DNA that it has previously been exposed to

non homologous end joining

double stranded break yields a knock out

is rapid and error prone

insertions and deletions (Indels)

homologous recombinations

double stranded break allows for a knock in

depends on the presence of homologous sequences that are around and available

accurate

what impact can NHEJ result in

gene disruption which is fine if the goal is to disrupt the gene in cells/animals

what repair is more accurate for gene replacement therapy

homologous recombination

can reduce mutations as well

what is a newer strategy to promote HR and refine the editing process

up regulate HR and inhibit NHEJ

RAD51 promotes HR while SCR7 inhibits NHEJ enzymes

what is a second new strategy to promote HR in editing processes (Nickase Cas9)

(cas9n) enzymes

they have mutants that nick DNA at the target site rather than creating a double stranded break

nicked DNA will repair better by HR

what is the third strategy to promote HR in the editing process (NICER)

(nicking inducing corrective editing for recombination)

creates nick near the mutation so that it will be corrected

what is the fourth strategy to promote HR in the editing process (coupling base editors to crispr)

cas9 nickase is coupled to base editors which removes an animo group from the base converting it to a different base

limitations of promoting HR for editing processes

there can be mutations in bases that are nearby target regions

the target sequence needs to be near a PAM sequence for the guide RNA to bind to

current and future challenges within crispr

possible increases cancer (safety)

delivery challenges (efficiency)

class one system

are multiple proteins as part of the effector complex

type of crispr system most commonly found in bacteria

class two system

a single cas protein is the effector protein

includes crispr cas 9 which is most commonly used for gene editing in eukaryotes and humans