Exam3Lec1GeneTherapyandGeneEng

Question 1

What are the genetic therapies that we have?

Answer 1

1. Production of recombinant therapeutic proteins: if we identify a protein that is impt and hard to come by, we can generate a recomb version and express it in milk of large animal
2. production of genetically engineered antibodies
3. production of genetically engineered vaccines
4. production of reagents for gene therapies
5. testing of treatments in genetically modified animals: human protein in a mouse then use a drug to target it and see the side effects (pre-clinical trial)
6. genetic modification of patient or donor cells in gene and cell therapy

Question 2

What is the effect of using siRNA inside a cell

Answer 2

is it used to silence a sequence (no transcription. There are two ways, the exogenous way and through the use of a vector

Question 3

Explain using siRNA exogenously

Answer 3

siRNA is directly added to the cell and is taken up by risc pathway, then binds to the sequence, and cleaves RNA out. This is NOT permanent

#3

temporary

Question 4

Explain using siRNA with a vector

Answer 4

1. Incorportate siRNA ds DNA vector and is now integrated into the genome, making it permanent.
2. We cleave vector and get shRNA, and this is turned into siRNA
3. Continues same process as exo, pathway. Binds to risc comples, binds to seq, cleaves RNA out

#2

note that come cells might not make shRNA but can use CRISPR/CAS 9 to integrate it
THIS IS PERMANENT

Question 5

__ is a valid targeting technique to induce KO of a dominant negative mutant to resolve the normal phenotype

Answer 5

shRNA

Question 6

The artificial (exogenous)__ would become a part of RISC complex just like natural siRNA from a ds RNA virus

Answer 6

siRNA

Question 7

What is this picture an example of?

Answer 7

Transcription and cellular processing of the shRNA primary transcript

shRNA integrate into DNA, and will always create siRNA, will always knock out . The artificial (exogenous) siRNA would become a part of RISC complex just like natural siRNA from a double stranded RNA virus

Question 8

In Dr. Blanck’s study what was it an example of?

Answer 8

example use of shRNAi vector to knock-down expression of the transcroption factor, YY1. So he silence a gene,that gene is a repressor, it incr gene expresssion

shRNAi equals shRNA

Question 9

Explain the results of George’s study in relation to YY1

Answer 9

• YY1 shRNAi expression vector resulted in no protein because shRNA targeted mRNA and silenced it. (shRNA makes siRNA, and siRNA cleaves it out, no gene expression)
• Empty expression vector as a control (no sequence incorporated), there is protein
• No transfection (no vector): there is protein

Question 10

Explain the real time PCR assay for HLA-DRA mRNA

Answer 10

When YY1 is eliminated, the mRNA for HLA-DRA gene is increased, which encodes for MHC class II protein. Since we had more mRNA for HLA-DRA genes we needed less cycles of PCR.

when we add shRNA, we remove YY1 (which is a repressor of HLA-DRA gen) so we increase HLA-DRA transcription, and we need less cycles of PCR.

Question 11

What is transfection?

Answer 11

adding nucleic acids (DNA or RNA) to cell but doesn’t tell you about the fate or effect

Question 12

What is transformation?

Answer 12

The DNA integrates into the genome, changing cell fate (PERMANENT)
shRNAi expression vector integrated into genome
There is NO transformation with siRNA treatment (this is NOT integrated into genome)

Question 13

What is exogenous DNA, gene, protein, etc

Answer 13

this is not natural/produces in cell=exogenous

Question 14

What does knock out (KO) mean?

Answer 14

you took out a specific gene

Question 15

Explain the example given with a KO mouse lacking EVC gene with associated dysplasia

Answer 15

There is the wild type mouse which is normal, and the EVC (-/-) mouse which is a gene associated with dysplasia. The neg/neg shows that we took out the gene and there is no more dysplasia. With this procedure, the added gene could integrate anywhere in the genome.

Question 16

True or false the First generation of mice is not going to give you a KO in every cell

Answer 16

True

Question 17

What is knock in?

Answer 17

put something in place of the endogenous gene

means taking exons and replacing exons

Question 18

Gene targeting vecotors can alter the natural gene in many different ways, What are two ways?

Answer 18

Some vectors will be used to “knock out” gene (remove sequence)
Other gene targeting vectors could replace exons that have as little as one base pair change, which is “knock in”

Question 19

In order for knock in to occur, what two things are needed?

Answer 19

Both neo and tk. Neo is incorporated, TK is not.

When you get correct Homologous Recombination (HR), Tk is not included but Neo is; the Tk gene is outside of the Homologous region since Tk is not flanked on both sides by the “A” and “B” sequences and is therefore not included during HR

Question 20

When knocking-in, we need a piece of DNA that will recombine at the proper position in the genome. This is really hard to do by itlsef, how can it wcbp at the right position?

Answer 20

Crisper Cas 9 ( randomly putting it in, not every singel cell is going to uptake it and wcbp at the right postion so we use crisper cas 9)

Question 21

Making a knock-out or knock-in mouse summary

Answer 21

1. The recombinant gene is first generated by transfecting DNA into embryonic stem cells (ES cells) in a laboratory dish.
2. Very few transformed ES cells have the proper recombination of vector with host DNA. The cells that do are selected with antibiotic treatments that kill off the cells that do NOT have the proper recombination.
3. The desired gene structure is verified by PCR and DNA sequencing of the ES cell DNA, ie, PCR of the section of the DNA that contains the desired gene structure.
4. The ES cells with proper gene structure are injected into an embryo.
5. A mouse is obtained with desired gene structure in the germ cells. That mouse is termed a “founder mouse” and is used for subsequent breeding to generate mice where every cell has the genetic alteration.

1. Recomb gene generated via transfecting (adding Nucleic acids) to embryonic stem cell whihc is pluripotent in a petri dish
2. Few have the PROPER recomb, destroy the rest
3. Verify and amplified by PCR and DNA seq
4. Inject ES into embryo and this developed mouse is the FOUNDER MOUSE
   Note that after a few generation of mice, we get mice with every cell and every gene that we want ki or ko

Question 22

Explain this picture

Answer 22

1. Take out stem cell (isolate blastocyst eg. eggs, sperm)
2. add DNA (recombinant gene)
3. Look to see which cells took it at the right place and incorporate into genome
4. Once we find it we select and expand
5. Put back into mouse
6. Founder mouse has ko gene in the germ cells
7. Founder used to make heterozygous mice mate to make homozygous muce

Generation of embryonic stem (ES) cells, usually for the purpose of mutating or “knocking out” a particular gene, and then using the ES cells lacking the gene to generate a mouse.

Not every cell will have the ko at first, long process

Question 23

Why do we knock in a gene?

Answer 23

We want to figure out where a gene is expressed. So we ko a gene, (in this case EVC) and ki a diff gene (in this case lacz) and note with flourescence. We see where it lights up and where it was expressed.

KI and replaced EVC gene with lac z to see where expressed

Gene targeting vectors can also carry a completely new coding sequence (in this case, one that, when expressed, turns cells blue) to the site of the original gene, which can allow for a very simple method of learning where that gene is expressed.

Question 24

Genetic engineering that is directly relevant to human medicine

Answer 24

• Genetically engineered cells, especially stem cells
• Pharmaceuticals produced from genetically engineered bacteria

Take stem cells and genetically engineer them
Take bacteria and genetically engineer them

Question 25

What are the two parts of CRISPR/cas9?

Answer 25

1. guide RNA guiding to right complimentary sequence
2. Cas9 for cleavage of seq (uses RNP targeting RNA)

Question 26

Cas 9 system allows for

Answer 26

Site specific DNA targeting

Question 27

Explain CRISPR/Cas9 approach to genome editing

Answer 27

1. Guide RNA guides the endonuclease cas 9 to the complimentary sequence creating a triple helix
2. Cas9 cleaves seq
3. If you have donor DNA you replace seq.
4. If you donot have donor DNA you have NHEJ which ends up being degrades

A

RNP with targeting RNA and Cas9 for DNA cleavage.

Question 28

How do you you use CRISPR/Cas9 over a long stretch or portion of nucleotide?

Answer 28

You do crispr/cas9 at two postions

this is genome engineering by double nicking with paired cas9 nickases

Question 29

Explain using CRISPR/cas9 to insert a fully formed (recombined) T-cell receptor gene into a T-cell.

Answer 29

You have a cancer with a specific proteins and we can sequence the proteins and edit T-cell receptor portion of the gene and take a segments and use crisper/cas9. Where the seq recognized the cancer, when it recombines to make t-cell, it will find the specifc cancer.

(Fixing to correctly target the tumor cell antigen: Take t-cells out of a pt, put it in and recombines T-cell receptor gene in T-cells, put T cells back into pt)

T-cell will have a T-cell receptor that binds a tumor antigen, namely NY-ESO-1. We NEED to have this in the same place in the genome, so it can be regulated effectively-how? Crisper /Cas9

Question 30

Explain Recombinant DNA cloning

similar to bac clones process

Answer 30

We can use bacteria to produce proteins for human purposes (proteins like insulin and growth hormone)

If we want a specific protein or DNA for protein, we can take a gene and insert it into receptor (replication origin). So when the bacteria replicates, so does the DNA that we want.

EX: get gene that makes insulin, put into bacteria, let bacteria divide, insulin is produces and we extract insulin

amplyfing protein

The cloning process ultimately leads to one bacterial clone that is producing the protein insulin. The bacterial clone can be amplified extensively, particularly in a liquid culture, and the insulin recovered from the liquid media.

Question 31

With Recombinant DNA cloning, do we want cDNA? Why or Why not?

Answer 31

YES because bacteria doesn’t splice so we dont want the introns. We want to attach our cDNA to the bacteria origin of replication so the cDNA will replicate in the bacteria.

Question 32

Which of the following pharmaceuticals is generated with recombinant DNA and Expression in Bacteria??

Answer 32

Growth hormone and Insulin

Question 33

Explain humanized, recombinant antibodies produced by DNA cloning

Answer 33

We can take the rodents antibody and humanize it to where our body wont attack it. How?

We want a specific abs, we use rodents abs and humanize it by taking the binding site from rodent antigen and integrating it with the sequences present in the rest of human abs. We inject abs into pt

Just taking the specific antigen binding regions that are mouse-specific and putting them in the humanized antibody

Question 34

We can use engineered antibodies to target ligands to treat diseases. What are some examples of drugs that do this and specify what ligand they target

Answer 34

Humira targets TNFalpha to treat crohns and rhem. arthritis
Avastin targets VEGF to treat diff cancer

take rodent abs, genetically modify it to attack a specific target and inject medication into pt.

Question 35

Stem cells have self-renewal, what does this mean?

Answer 35

As stem cells divide into 2 cells, 1 daugter cell stays a stem cell, and the other cell differentiates.

Stem Cells produce BOTH stem cells and differentiated cells

Question 36

What are the best stem cells and why are they the best?

Answer 36

Embryonic stem cells because they could become ANY cell (pluripotent). So we can add a geene to it, use crisper cas9 and every cell is going to have that gene (ki or ko)

Question 37

What is alloreaction?

Answer 37

Bad Reaction, t cell of one person reacts w/ HLA of another
person.

have a organ transplant and you reject (graft rejection)

Question 38

True or False: 20% of T-cells are alloreactive (FATAL)

Answer 38

True

Question 39

How do we prevent graft rejection?

Answer 39

there is a match with autologous cells

Question 40

What is the name of the drug used as an immunosuppressant?

Answer 40

Cyclosporine

Immunosuppresive medication to prevel alloreaction/graft rejection (even if you have a match).

Question 41

What are Autologous cells?

Answer 41

Involves using patients own stem cells
If we take stem cells from ourselves, it prevent immune reaction/rejection

we want this

Question 42

Subject of 2012 nobel prize, cloning of a sheep

Answer 42

Take the nuclei of sheep, put into oocyte (eeg)
Will make a whole cloned sheep with EXACT MHC match/ autologous cells

Question 43

Explain therapeutic cloning

Answer 43

1. remove nucelus from unfertilized egg
2. Insert nucleus from somatic cells into enucleated egg
3. get a blastocyst which has a lot of embryonic stem cells in inner mass
4. Take stem cells cells ko/ki and then we differentiate, these are pluripotent.

Question 44

Explain induced puripotency

this we do

Answer 44

1. Take somatic cells from pt ( ex: fibroblasts) which have specific transcription factors and induce them to become pluripotents and then differentiate. (IPS)

Question 45

What is transdifferentation?

Answer 45

We take a close cell ( in this case, cardiac fibroblasts), add some transcripton factors to it (TBX5), then it transdifferentiates into cardiomyocytes. This means that we are changing the phenotype.

transdifferentiate for cells that are close

Remember TBX5 is important for….. HEART MORPHOLOGY

We can transfect the coding sequences for those transcription factors into cell types that are readily available they will transform these cell types and we get other types of differentiated cells
Changing cell phenotype; causing expression of different genes to produce a different cell

Question 45

What is dedifferentiation?

Answer 45

We can dediffeerentiate cells that are far/completely diff from each other by taking the cell (in this case skin fibroblasts) and we completely de-differentiate and induce them to be pluripotents (iPS cells) and then you can get a neuron

dedifferentiation for cells that are far from each other (stripping away differentation)

We start with differentiated cell, use transcription factors to get a different cell type with same MHC (no rejection), so we use own embryonic stem cells for no allorxn

Question 46

What are two ways to get stem cells?

Answer 46

Dedifferentiating w/ transcription Factors
CD34+ cells if blood cells

Question 47

How can we get stem cells from CD34+ cells If blood cell)

Answer 47

Hematopoietic stem cells (in bone marrow) are relatively accessible from the body. We can look for CD34+ cells/ arbitrary markers and extract them and get stem cells/

Remove bone marrow, induce to what we want, inject back in

Question 48

Explain using vivo gene thearapy facilitated by a viral vector

Answer 48

1. Take bone marrow and find CD34+ cells
2. Put them in a retroviral vector carrying cytokine receptor gene
3. Inject back in w/ gnes that we want

Viruses are good at getting DNA into cells, instead of incorporating harmful viral proteins, it incorporates genes that we want

Question 49

What three things can we do with stem cells to rescue a cell defect?

Answer 49

1. Gene augmentation (add a gene)
2. Gene silencing
3. Repair of a mutant gene (w/ crispr/cas9)

Question 50

What is used for gene therapy?

Answer 50

a viral vector

Question 51

What sequence is needed to package a therapeutic gene into a virus?

Answer 51

psi sequence needed for packaging the RNA into the capsid. We take natual viral genome with psi, gag, pol, env and replace it therapeutic recombinant genome (psi + therapeutic gene) and insert it back in so virus can go in cell and incorportate itslef into our genome

Question 52

Which virus is best at integrating itslef into our genome?

Answer 52

Lentiviruses, more specifically HIV

specific virus attakes a specific sytem, and we can target specifc gene in that system using the virus.

Question 53

How can we use lipids to carry DNA through cell membrane?

Answer 53

We can coat the DNA with lipids so it can get through the lipid bi-layer and incorporate itself.

not as good as viruses

Question 54

What two types of deletions cause muscular dystrophy?

Answer 54

1. In-frame deletion of multple central exons: still in the right frame, just lost a few, this is MILD, and leads to BECKER muscular dys.
2. Out-of-frame deletion of central exon or frameshift: coding for a diff aa, diff protein, this is SEVERE, and leads to DUCHENNE muscular dys

know this photo

Question 55

How do we treat muscular dystrophy?

Answer 55

by using direct administration of an antisense oligonucloetide (AO) to block splicing and put it back in-frame

Question 56

Explain the study where researchers inserted HLA genes into mesenchymal stromal cells (stem cells) and looked at allogenic rxn and how to decrease the rxn

Answer 56

Mesenchymal stromal cells (MSCs) are stem cells and we use them for cellular therapy. The issue is that we have diff type of stem cells , so can we produce enough cells, is the timing and technique right. These problems(immune rejection) are caused by human leukocyte antigen (HLA) mismatches.

Method: So we are trying to avoid allogenic rxn (immune rxn) that is going to attack cells. So we are going to KO beta-2-microglobulin (B2M) gene and KI B2M-HLA-G fusion gene with CRISPR/Cas9 system and AAV.

Results: It retained the potential to differentiate into adipocytes, osteoblasts, and chondrocytes. We ko a gene and then replaced gene BY KO to try to have no immune rxn, which protected cells from Tcells and NK cells,

Conclusion: CRISPR/Cas9 system combined with AAV can be used to effectively disrupt/introduce any gene into UC-MSCs

Everyone has different HLA, recognized by t cells as none self. If put someone else’s cells into you, that’s allogenic (Reaction, they’ll attack the cell)

Question 57

Case questions about mesenchymal stromal cells.
1. What is alloreactivity?
2. Explain the relationship between the MSCs and chondrocytes, adiptocytes etc.
3. What was the purpose of the AAV vector?

Answer 57

1. Immune RXN to not self
2. MSC=stem cells that are undifferentiated (pluripotent)
   Adipocytes/chondrocytes: Differentiated (they dont naturally turn into any type of cell)
3. Need a vector to Knock in/out

Question 58

Two common ways of providing exogenous siRNA to cells for experimental purposes are to transform the cells with an __________ vector for the siRNA or to simply treat the cells with __________.

Answer 58

shRNA, siRNA (not perm)

Question 59

KO mice are generated by first generating _______ cells lacking the gene of interest and then adding the ES cells to a ___________.

Answer 59

embryonic stem, blastocyst

Question 60

_____ can be generated to knock out a gene, to “knock-in” a new gene.

Answer 60

KO ES cells

Question 61

Many human pharmaceuticals are produced in bacteria by using ________________ cloning techniques. Two good examples are insulin and avastin.

Answer 61

recombinant

Question 62

Human stem cells can be autologous and can be altered by addition of exogenous DNA to repair a gene defect. An autologous stem cell will not be ______________ by the immune system. Some autologous stem cells may be generated with somatic nuclei placed into ______________ for expansion into a blastocyst, from which stem cells may be recovered.

Answer 62

rejected, enucleated (no nucleus) egg cell
(could be on exam)

Question 63

DNA or RNA can be added to cells with a viral vector, by using a _____________for transiting the cell membrane, and by direct addition to tissue.

Answer 63

lipid coating

Question 64

____________ is the less severe form of muscular dystrophy because the dystrophin gene is deleted “in frame”. In-frame splicing can be therapeutically induced with an _________ in the case of the ___________ form of muscular dystrophy, the more severe form due to an out of frame deletion and a truncated dystrophin protein.

Answer 64

Beckers, antisense risc digonucelotide, mutant

Question 65

The _____ system facilitates human cell knock-outs and knock-in’s, i.e., sequence specific insertions into, or deletions of the genome, unlike a retroviral vector, which will insert anywhere..

Answer 65

CRISPR/Cas9