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Gene Therapy Flashcards

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1
Q

What is Gene therapy?

A

Gene therapy: The medical field that uses which focuses on genetic modification of cells to treat or prevent disease

ex: mutations play a role in causing minor problems (like color blindness) or serious problems (cancer risks)

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2
Q

Explain the challenges that may be seen in gene therapy?

A

Challenges of gene therapy
* How do we safely edit human DNA
- restoring good copies of genes with recessive alleles
-correcting bad copies of genes with dangerous dominant alleles
(Harder problem)
*How can we correct enough cells to heal the patient ?
*Do we try to heal a patient, or to help the patient produce a generally normal child?

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3
Q

What is the most common way of treating diseases?

A

(directly providing missing proteins) to affected individuals) is the most common and important way of treating disease

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4
Q

What is the role of growth hormones?

A

Growth hormones act through a network of hormones made by different parts of the body (anterior pituitary, hypothalamus)
They affect how hormones can stunt growth or cause other problems

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5
Q

What is Growth Hormone deficiency? What may affect growth hormone ?

A

Growth hormone deficiency occurs in people with genetic diseases that result in too little growth hormone
-GH1 mutations affect the growth of hormone itself
-GHRHR mutations affect the growth hormone releasing hormone receptor. The GHRHR will also lower the production of growth hormone
-Each disorder is autosomal recessive
-Tumors can also affect growth hormone production

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6
Q

Explain how human growth hormone can be made using bacteria?

A

Human growth hormone gene has been cloned in bacteria
process;
you can take human gene (like growth hormone) that we identify and insert gene into bacterial plasmid (small circular chromosome in bacteria)
-Then you transform the gene in bacteria (shock it) and the bacteria will now have human growth gene .
This gene can then be isolated fro bacteria and given to patients
(better method of obtaining GH than isolating it from animals; for ethical and expensive reasons)
bacteria can be grown on industrial scale

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7
Q

Explain what is used in the treatment of Growth disorders. How is treatment administered?

A

Treatment of Growth Disorders
-if patients do Not make enough human growth hormone, they can have extra provided by doctors
Growth hormone is produced in large amounts using vats of bacteria that contain the human GH1 gene (gene will be purified)
-Growth hormone is administered by injection
(this can be used for genes in growth hormone or complement–> clotting factor; or any hormone that circulates through the blood)

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8
Q

How can we manipulate genes in humans ?

A

We can deliver DNA using viral vectors
(have to be process about this method; so viruses used must be carefully altered)

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9
Q

Describe the characteristics of the Adeno-associated virus

A

Adeno-associated virus
-infects humans
-Not very dangerous
-DNA can often be maintained in an extra-chromosomal state
-can be modified to carry human genes

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10
Q

What are the features of the Wild type Adeno-associated virus (AAV) ? What are the genes in its genome?

A

Wild type AAV is a Simple virus
-its genome has inverted terminal repeats that mark DNA for:
-replication
-packaging into viruses
-integration
-p5 is a promoter
-the rep gene controls replication
-The cap gene makes the capsid

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11
Q

Describe how the Altered AAV (adeno-associated virus) can carry human genes

A

Altered AAV can carry human genes
-p5 is replaced with the appropriate human gene promoter
-the rep and cap genes are replaced with a human gene
- The ITRs (inverters terminal repeats) are removed, so this virus CANNOT replicate in cells. Thus it is not dangerous.
-How to make it ?
-you create second form of virus that they grow (cell culture lines) with only replication gene and capsid gene no ITRS, so they cannot be packaged into any viruses.

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12
Q

what happens if plasmid lacks the ITRs (inverted terminal repeats) ? how is DNA packing affected

A

A plasmid carrying the viral genes can direct the replication and packaging
-if the plasmid lacks the ITRs, then it CANNOT be packaged itself.
- But it can help package vector DNA in the same cell.
(makes lots of viruses that can carry human disease gene in to cells)
helper viruses help make vectors.

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13
Q

How are viral vectors produced?

A

viral vectors made by combing the AAV vector plasmid and Helper Plasmid, and growing them on the same cell.

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14
Q

How can manipulation of genes occur in humans ?

A

You can modify genes with CRISPR/Cas9
(Emmanuelle Charpentier and Jennifer Doudna won Nobel prize in 2020)

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15
Q

What is the CRISPR system? how do bacteria use this system>

A

CRISPR System- a precise way of making cuts in specific places in DNA sequences
bacteria use it to remember viral genomes that they do not want infecting them (ex; if they find new virus, they’ll use a copy from old virus to find new one, and chop it up and destroy that new virus)

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16
Q

Explain how CRISPR/Cas9 is used in human cells

A

CRISPR/Cas 9 allows precise editing in living cells
- we can use it to make cuts in any gene to edit and change and try and repair something.

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17
Q

What is CRISPR system useful for?

A

Using the CRISPR system:
*To make a double-strand break in the DNA, which use:
-Cas9 + tracer RNA
-Guide RNA that targets your gene
*To change the sequence of the target site
-Repair the template for the DNA with the change you want to make
This is easy to do in cells, but hard for whole people
(cannot do CRISPR on whole human cells)

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18
Q

How could we get good copies of genes into humans?

A

By putting the good copies into STEM CELLS
(germline cells give rise to many sperm in male, many eggs in female) (blood cells give rise to lots or red and blood cells)

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19
Q

What is ADA-SCID (ADA- severe combined immunodeficiency syndrome) . What type of mutations cause it? What happens to these patients?

A

ADA- SCID
-Adenosine Deaminase Deficiency- Severe Combined Immunodeficiency
-Caused by mutations in the ADA gene
-The ADA gene makes Adenosine Deaminase, which is needed to break down deoxy adenosine, a by product of DNA processing
-*ADA is critical in lymphocytes (white blood cells) *
-Patients with ADA fail to fight infections, which are usually Lethal.
-hence patients with ADA must be protected from infectious
(ex; in the past, kid name David Vitter lived in bubbles to prevent infections from others)

20
Q

How are lymphocytes produced?

A

Lymphocytes are produced by hematopoietic stem cells (blood stem cells)

(hence if you could correct blood stem cells you put them in patients)

21
Q

What is the purpose of hematopoietic stem transplants? Why is it important for their to be a match between donor and patients ?

A

heamtopoietic stem cells are found in bone marrow
-transplants can regenerate all types of blood cells.
-Donors should match patients, so their cells do NOT attack the patient’s body
(hematopoietic stem transplants generate all blood cell types)

22
Q

Explain how transplants can be dangerous

A

Transplants can be dangerous
-initially, doctors tried bone marrow transplants fro relatives with similar antigen types
-David received a bone marrow transplant from his sister to try and provide healthy immune cells.
He died form cancer caused by a dormant Epstein-Barr virus that had been in sister’s cells

23
Q

Explain the process of putting human genes in stem cells.

A

We can edit human genes in cells grown in the lab
-by editing hemapoietic stem cells, we ca use someone’s own cells as donor material.
-isolate cells
-Edit DNA to correct problem
-reintroduce cells to body

24
Q

How else can viruses be used human cells.

A

Viruses can be used to move genes into human cells

25
Q

Explain the gene therapy procedure for ADA (adenosine deaminase)

A

Gene therapy procedure for ADA
1. Remove bone marrow stem cells (that has defective ADA gene)
2. Use retroviruses to bring the normal gene into the bone marrow stem cells
3. Viral recombination DNA carries normal gene into genome
4. Return genetically engineered cells to patient

26
Q

Explain why Gene therapy is partially successful (describing positive and negative results)

A

Gene therapy is partially successful
-The first person treated with a successful gene therapy was Ashanti De Silva in 1990
-This technique has now been used to treat several patients
-A few patients have developed leukemia caused by the viral vector DNA

27
Q

What can epidermal stem cells be used for?

A

You can treat patients with epidermolysis bullosa with epidermal stem cells

(however most work on gene therapy involve bone marrow cells )

28
Q

Can we treat diseases that do not affect stem cells?

A

yes, we can but it is hard to do
(possible to provide good copies of genes in other types of cells—> spinal muscular atrophy type 1)

(easy and cheap to use bacteria to inject cells in blood, and can correct blood stem cells (expensive, but useful) for SCID patients)

29
Q

What is Spinal muscular atrophy type 1? What causes this disease? What happens to patients with this disease?

A

Spinal muscular atrophy type 1
- humans have two Survival of Motor Neuron genes: SMN1 and SMN2
-Mutations in SMN1 are recessive
-Absence of the SMN1 protein causes:
-many motor neurons to gradually die
-muscles that lack signals from these neurons then atrophy and die
-Eventually, patients die from an inability to breathe
-in milder cases, patients can live past 2 years

30
Q

What happens to infants who are given Adeno associated virus with good SMN1 gene ?

A

Studies show that if patients (who have spinal muscular atrophy type 1) were given Adeno-associated virus with a good SMN1 gene, you could see major improvement and outcome in patients.
Low dose of virus with good copy of gene (SMN1) : some children function good, some children do not function well
High dose of virus with good copy of gene: patients do better for many months.

(putting live virus into one’s body for treatment)

31
Q

Describe the early success seen with AAV administration of health genes

A

Early successes with AAV administration of healthy genes
-All patients in the spinal muscular atrophy study survived!!
-but they were not completely cured
-Other genes are being targeted too:
-Muscular dystrophy (which requires making a small working copy of the gene)

32
Q

Discuss the gene therapy that is currently being used for muscular dystrophy ?

A

Gene therapy for muscular dystrophy
-Samulski’s group is using AAV to deliver a working copy of the mini dystrophin gene to muscle cells throughout the body

(since muscular dystrophy is a very large gene, need to devise a way to have fragment of gene small enough to go in a virus, and big enough to be helpful for treatment)
-Initial results are promising

33
Q

How can harmful genes be knocked down?

A

Can known down harmful genes with ANTISENSE
(seen in Huntington’s Disease)

(inject anti-RNA which binds to regular RNA to make double helix, the helix cannot be used to make a protein)

34
Q

What is the role of Antisense molecules? What is an affect of this?

A

Antisense molecules bind a specific messenger RNA
-For a gene to make a harmful protein, it first needs to make an mRNA
-Custom antisense molecules bind specific mRNAs and disable them
-If theses can be administered, they can block harmful dominant genes

35
Q

What are the technical challenges that arise for antisense ? What are solutions to these challenges?

A

Technical challenges for antisense:
-Small RNAs are unstable
solution: Chemically modify the RNAs to stabilize them
-It can be difficult to target the molecules to the right cells
Solution:
-Chemically modify the RNAs to stabilize them
-The molecules can be linked to a chemical that targets them to the right cell
-The molecules can be injected in a specific part of the body.

36
Q

Explain how an antisense drug may help patient with Huntington’s disease

A

many antisense drugs have Not been very successful
-A new trial for IONIS-HTT (Rx) shows promising results
-it involves injections of the drug into the spinal cord

37
Q

How can gene therapy be used to make Healthy babies?

A

germ line therapy: take an egg and isolate it, alter its genetic makeup and put it into mother. The mother will then give rise to healthy child
(hence you can use germ line therapy and cloning to to help parents have a healthy child)

38
Q

What occurs in cloning frogs? What are the results ?

A

Cloning Frog cells
-people have cloned frogs for decades
can get normal healthy frogs (some not healthy)
Adults DNA
Take DNA from adult skin cell and put into frog egg and get a tadpole growing up (to eventually a frog)
also dolly the sheep has been done
,mice: take an egg remove its nucleus, put edited nucleus into egg cell and put back Into mouse to grow up. reguwies foster mother to nurse egg (since we need a placenta to nurture within mother for health egg.

39
Q

How do Adult cells know how to make Frogs?

A

Adult DNA can make frogs
process;
Take DNA from adult skin cell and put into frog egg and get a tadpole growing up (to eventually a frog)

(also, dolly the sheep has been created using cloying, and cloning done in mice too)

40
Q

Explain how cloning by nuclear transfer occurs in mammals. what does cloning mammals require?

A

Cloning by nuclear transfer in mammals
ex: in mice:
-you take an egg and remove its nucleus (do some editing, modifications) .
Then you put edited nucleus into egg cell and put back into mouse to grow up.
This cloning requires a FOSTER MOTHER to nurse egg (since mammals,(like us) need a placenta to nurture the child within mother for healthy growth.

41
Q

What does cloning mammals require ?

A

cloning mammals requires A FOSTER mother

42
Q

Explain what occurs in mitochondrial replacement therapy?

A

Mitochondria replacement therapy:
Some patients have defective mitochondria, and would pass it on the mitochondrial disease to their children
Therapy:
take patient with defective mitochondria, and have donor who provides egg with healthy mitochondria.
- you remove the nucleus from donor egg, and add nucleus from patient (with defective mito) into this egg
-now you have, patients DNA in egg cell full of healthy mitochondria. you put it back into patient and let it grow up Into a child

43
Q

Discuss success seen with mitochondrial replacements

A

Mitochondrial replacements are successful
-First baby was born in 2016.
-Children are healthy so far.
-However, small numbers of defective mitochondria are carried over during the technique
-will these eventually cause problems?

44
Q

how can you modify the genome throughout the body?

A

By CLONING

45
Q

Discuss the CRISPR editing of human genomes that have occurred? Which protein is involved? What is its role?

A

CRISPR editing of human genomes (can edit gene and put it back in egg)
-in 2018, He Janjui (China) announced the creation of twins with inactivated CCR5
(nuclear gene controlling CCR5 gene was altered in Petri dish and put back into mother)
-CRISPR/Cas9 was used to produce altered embryos but seems to have been only partially successful
This protein is expressed on T-cells and a few other cell types.
CCR5= C-C chemokine receptor type 5
-The HIV-1 virus can enter cells by binding this receptor

46
Q

What are the prospects for gene therapy?

A

Prospects of for gene therapy
-making human proteins to treat disease –> successful, but only works for some diseases
*Adding back a good copy of defective gene * –> successful but works best for Recessive diseases for which we can manipulate stem cells
other diseases are now being targeted with AAV
-Knowing down defective mRNA with antisense (for dominant diseases like Huntington’s– –> promising but difficult
replacing mitochondria through cloning–> seems to be successful
-Germline therapy —> Feasible but hard and full of ethical problems

Human Genetics (13 decks)

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