Treatment of Genetic Diseases Oct 9

Question 1

What are three complications in treating monogenic disorders?

Answer 1

1. we may not know the gene
2. we may know the gene but not understand the pathophysiology
3. Fetal damage may have occured prior to diagnosis and it may not be reversible

Question 2

As of now, do most therapies treat symptoms or th eunderlying genetic mutation?

Answer 2

symptoms

Question 3

What are some additional long term complications in attempting to treat genetic diseases?

Answer 3

1. the treatment may be successful, but sublte deficiencys may develop later
2. Treatment may be successful in one tissue, but problems may arise in other tissues later
3. the treatment doesn’t show harmful affects at first, but they develop later

Question 4

Why does genetic heterogeneity make treatment more difficult?

Answer 4

If multiple mutations can lead to a certian disease phenotype, you may try to treat the wrong mutation

Question 5

Agagin, what is allelic heterogeneity

Answer 5

different mutant alleles of the same gene give rise to different disease phenotypes

PKU

Question 6

Again, what is locus heterogeneity?

Answer 6

Different mutations can result in the same disease

like hyperphenylalaninemia:

defect in PAH or in biopterin

Question 7

Why has the treatment of metabolic deficiencies been most succesful historically?

Answer 7

You can do diet modification like avoidance

you can do diversion (think ura cycle)

You can inhibit certain enzymes - like using statins against HMG CoA Reductase in familial hypercholesteromeia

Question 8

What are two ways you can utilize “protein” treatments?

Answer 8

1. add a cofactor
2. Replace the defective protein (like alpha antitrypsin in emphysema)

Question 9

What are the main problems with protein treatments?

Answer 9

Proteins have a short half life, so patients need freuquent transfussion - costly and time

Immune responses can develop

risk for viral contamination

sometimes supplies are insufficient

Question 10

How has Gaucher disease been treated with protein therapy?

Answer 10

Gacher disease is a lysoomal storage disease that is cause by the lack of the enzyme glucocerebrosidase which diegrase glucocerebrosides in the lysosome

the glcocerebrosides build up - especially in macrophages and cause gross enlargment of spleen and liver

the acrophages slowly replace bone marrow, leading to anemia and immune suppression

Protein treatment involves glucocerebrosidase enzyme replacement therapy (expensive and rrequires weekly transfusiosn)

has been super successful - probably because the disease doesn’t affect the brain

Question 11

What are some ways you can modulate gene expression through treatment?

Answer 11

1. increase the mRNA of either the wildtype or (if it still makes a functional protein just not enough) ,the mutant gene
2. Induces expression of a different gene that could rescue the mutatnt phenotype (hydroxyurea for fetal Hb in sickle cell)
3. Repress expression of a dominal negative mutation using RNAi

Question 12

What can bone marrow transplantation be used for?

Answer 12

when the disease stems from a defect in the bone marrow stem cells - like hematopoitic cancers, immune disorders, lysoeomal storage diseases, and thalassemias

problem: you have to destroy the patient’s immune system for them to acept the new bone marrow, so they often die of infection

Question 13

Why is cord blood preferable to a bone marrow transplant in HSC transplantation?

Answer 13

Recipients tend to be more tolerant of histocompatible placental blood than other allogenic donor cells

there’s more cord blood available

risk of host-vs-grat disease greatly reduced

Question 14

Besides protein replacement therapy, wha tis another way we can treat Gaucher disease?

Answer 14

bone marrow transplants

Question 15

What’s the goal in gene therapy? why would you do it?

Answer 15

THe goal is to modify cells to produce a therapeutic effect based on recombinant DNA tecnology - it introduces new genes and hopefully removes the damaged genes

this can compensate for a loss of fucntion mutation

it may replace or inactivate a cominant mutant gene

Question 16

What are some guildelines for gene therapy? What do you need before you can start?

Answer 16

 defective gene & biochemistry has been described
 cDNA or functional version of the gene exists
 substantial disease burden & favorable risk-benefit
 confidence that the gene therapy will work based on knowledge of the defect
 reliable promoter to regulate gene expression
 good target cell
 data from model systems (eg., mouse) that gene, vector and delivery system all work
 protocol review by government

Question 17

What are the usual target cells for gene therapy?

Answer 17

You want them to be long-lived so it will last - preferably a stem cell

right now bone marrow is the only current example being used clinically

Question 18

What’s the difference between ex vivo and in vivo gene transfer strategies?

what are the advantages and disadvantages?

Answer 18

Ex vivo invovles the transfer of stem cells out of the body, modification to remove the mutation and put in the wildtype, amplify, introduce back into the body. This is good because it allows for direct targeting, but it’s difficult and takes time.

in vivo: direct injection into the body using a vector . This is good because it’s quick and easy, but it’s harder to target the right cells, you can get immune responses, and there are safety risks

Question 19

What is a vector?

Answer 19

It’s what we package the gene into in order to introduce it to the cell

You want them to be safe, easily introduced, and promote expression of the transferred gene for the life of the cell

can be viral and non-viral

Question 20

What are some examples of viral vectors?

nonviral vectors?

which class has been more successful?

Answer 20

 viral vectors include: retroviruses, adenoviruses, adeno-associated viruses, herpesviruses

 non-viral vectors: naked DNA, liposomes, protein-DNA conjugates, artificial chromosomes

THe viral vectors have been orme successful

Question 21

How do the retroviral vectors work?

What are the advantages and disadvantages?

Answer 21

Retroviruses are RNA viruses

Advantages: they will integrate the gene into the host genome, so that if it works it will essentially fix the mutation for the entire lifetime of that cell. THere is very low inflammatory potential. They are easy to make. Can accomodate large trangenes

Disadvantages: they target dividing cells only AND because they integrate into the genome randomly they can turn a cancer gene on (this is what happened with the SCID trial)

Question 22

Why are lentiviruses considered better than regular retrovirus vectors?

Answer 22

They can targe quiescent cells as well, so they have a broader tropism

However, they can still result in oncogenesis

Question 23

How do the adenovirus vectors work?

Why don’t we use them anymore?

Answer 23

The Adenovirus vectors were dsDNA

Advantages: they had very broad tropism, can be generated at high titer, can accomodate large genes.

Disadvantages: However, they could not integrate into the host genome, so the fix wasn’t perfect

Also, the biger issue was that they had very high inflammatory potential and people have died because of the immune response tot he vector itself

because of this, they don’t use these anymore

Question 24

How are adeno-associated vectors different and better than adenovirus vectors?

What’ stheir main fallback?

Answer 24

AAV are ssDNA instead of dsDNA

Advantage: They do not have the inflammatory potential like adenoviral vectors do. They can infect both dividing and nondividing cells.

Disadvantage: although some do, most do not incorporate into the host genome :(

they have VERY low carrying capacity :(

Question 25

What is the main issue with the Herpes Viral Vectors?

Answer 25

The HSV vectors ar enice because they have CNS tropism and a large packaging capacity, but they can cause severe immune repsonse and neurotoxicity

Question 26

In what diseases have gene therapy been having some success?

Answer 26

heophilia A

lysosomal storage diseases

Parkinson DIseases

ADA-SCID

Question 27

What gene therapy is used to treat hemophilia B?

Answer 27

They use gene therapy for Factor 9 replacement in the liver

they use AAV vectors

Question 28

Why are lysosomal storage diseases comon targets for gene therapies?

Answer 28

Because only low levels of wildtype protein are needed to significantly improve the clinical phenotype