From Iusmgenetics

[edit] Gene Therapy

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 6:48:32 PM First we realized that bacteria took up plasmids and thats how they became resistant.

• So we wondered if we can do this to our own cells.
• We see that with plasmids in human cells we get expression, for a couple days but eventually it goes away.
• Integration is a rare event, perhaps 1 in 10K on the best cell line; so not very efficient.
• How do we make this more efficient?

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 6:49:16 PM

• So we try to use viruses which are really good at integrating their DNA.
• We remove the gag, pol, and env genes which make it infectious and put in our own genes of interest.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 6:52:59 PM

• The trick now is to get our material into the virus
  • use packaging cell lines which are immortalized
  • gag is expressed; capsid protein for virus
  • pol is expressed; integrase gene
  • put in the vector material (often rna for rna viruses)
• So this cell is infected with a virus and is then a little factor that makes virus particles that are set up to infect our target cells.
• They carry capsid for update, RT for converting rna to DNA and integrase for putting the DNA in the target cell.
• These then infect the target cell.
• Then when the target cell divides, all the daughter cell will have the delivered DNA, too.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:03:22 PM

• So we tried this and even got it to trial.
• But we wanted to try other viruses.
• These are actually be used at the trial level.

• Retrovirus / Lentivirus:
  • Murine retrovirus was the first tried.
    • Turns out murine virus can cause leukemia because of the enhancer region on the virus.
  • HIV is a lentivirus
    • Doesn't have the enhancer region of murine retrovius so it will decrease the chance of leukemia.
  • Used in bone marrow and T cells
    • Used here because T cells and bone marrow have many, many offspring so the treatment must involve integration into the genome.

• Adenovirus:
  • Gives flu-like symptoms and that's it so you can give a high titer without much side effect.
  • Can integrate without regard to the cell cycle of the target cell
  • However, it doesn't integrate so it isn't so useful for tx with longevity
  • Applications are cancer and immunization because they are short-term objectives that don't need integration.

• AAV
  • Adeno-associated virus
  • Challenge is that it is a very small virus, so not much material fits in the virus
  • While AAV doesn't integrate, you can get it into slow-dividing cells like muscle , liver, and brain
  • We have many different serotypes that can be used to target specific tissues.

• Herpes
  • Huge virus so you can put tons of stuff in there
  • Non integrating
  • First application was for cancer pain syndrome: inject in skin, neruons will pick it up (because that's what herpes likes to infect) and then

express some anti-pain proteins

• • Now being used for CNS tumor destruction.

• The concept here is that there won't be one vector but many depending on the target cell and how long you want to express your material.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:03:57 PM

• HIV, adenovirus, Lentivirus, and herpes are increasing. *Changing with time

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:07:20 PM

• The first gene therapy that was approved was for the treatment of adenosine deaminase deficiency or bubble baby deficiency.
• When T cells leave BM to visit thymus for education, there is rapid turnover of the T cells as they are educated to antigens.
• Without ADA to breakdown the biproducts of cell apoptosis, the T cells die without making it out of the thymus.
• Pts have very low T cells and without T cells you can't educate B cells so they have combined immune deficiency.
• Usually die of infection in the first year.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:10:00 PM

• Why was ADA picked for gene therapy?
  • Can simply plug in the protein.
  • Ex vivo can be done by extracting the pt's bone marrow, exposing to vector, put back in, hope that the protein is expressed.
  • Selective advantage because T cells normally die in the thymus (especially in SCID pts) so there are not many cells to compete against.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:13:28 PM

• Wide range of expression is important because it allows us to be generous in the expression we end up providing without causing a different

disease.

• Selective advantage is important when you contrast with something like sickle cell diease:
  • There must be a selective advantage for the transfected cells because we can only reasonable transfect a small proportion.
  • In sickle cell, for example, the cells are only at a disadvantage once they get out into the peripheral blood--before that they are at the same

selectivity as transfected cells.

• • Once in the peripheral blood, it's too late--that is the disease state.

• Initially we took blood stem cells out, transfected, and re-infused the pts with the treated cells.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:14:50 PM

• This study shows **10 pts treated **9 pts have normal immune function **T cell numbers are in the normal range **Kids have normal immunization reaction **kids went from being in bubble to going to school and stuff. *This project used lentivirus.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:18:01 PM

• This study: **used AAV **Leber's congenital amaurosis **Causes blindness **At birth, the children are pretty normal in terms of site **Without this gene, by 8 or 9 they are blind **In this case, used AAV to express gene **Injected in retina **Showed improvement in sight **Showed little disruption in the retina **Legally blind to playing baseball. **Putting in a good copy.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:20:11 PM

• This study: **Used lentivirus **Treating adrenoleukodystrophy **Major affect over time is deterioration of the brain **Lack of enzyme that helps maintain myelin, thus bad demyelination **Treated not the brain but the bone marrow because that's where glial cells come from **Glial cells eventually ended up in the brain and helped. **Published in Science **Much slower progression, perhaps arrested.
• Lorenzo's oil was hogwash.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:20:31 PM

• We can also think about this technology as drug delivery, too.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:24:05 PM

• Hemophilia
  • Very expensive to treat
  • Disruptive to the pt's life to have to give themselves factor IX ir IV
  • Risk of HIV transmission for these people.
• Inject intravenously, let it get taken up by the liver, let it integrate, let the liver then make the clotting protein like it should.
• When we did this, we saw that factor IX expression went up after being taken up by the liver
• But then it went down after 4-6 weeks.
• This happened at the same time that the liver function tests went high and back to normal.
• So we wondered if this was an immune thing.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:29:33 PM

• Perhaps the immune response is due to the transgene.
• Two animal models are the evidence:
  • One model: knockout mouse, treated with gene therapy, showed immune response on the transgenetic product
  • Two model: non-fxnal factor IX mouse, treated with gene therapy, no immune response (because even though the factor wasn't functional it

was still present during immune cell training to present as an epitope and therefore generate central tolerance that leads to no immune reaction upon gene therapy correction)

• So this is what we thought was going on.
• Turns out it was that the vector (AAV) was leaving it's capsid protein around for weeks / months and upon detection the transfected cells were

killed off one by one.

• • Hence the reduction in factor IX expression
  • Hence the hepatitis-like elevation of liver fxn tests.

• Because of this reaction, they tried the eye because they knew it was an immune priviledged site.

• The brain is protected, too, so they are trying to work on Parkinsons.

• They are also thinking aobut tx profilactically with predisone or some immune supporessor.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:32:51 PM

• Gene therapy: Huntington disease
• This work is on a mouse model of HD.
• Here they put the mice on a rotorod and they look at how quickly the mice fall off.
  • Faster fall off times in those with poorer neurological function.
• Putting in the lentivirus causes delayed onset of the dieases.
• They put in glial-cell derived neurotrophic factor
  • A biologic factor that is endogenous
  • This is truley drug therapy
  • This is good because giving it systemically causes lots of bad systemic side effects.
• So we aren't replacing HD gene but we delay the onset by making the microenvironment more suitable to handle the disease.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:33:48 PM

• There are a bunch of clinical trials using gt for acquired diseases.
• Heart failure, parkinsons, macular degeneration, and rheumatoid arthritis.
• These are delivery of different compounds.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:39:49 PM

• Here's another twist on how gt can be used to treat.
• We can inactive harmful genetic defects.
• We can try to soak up the transcription factors that cause transcription of that gene.
  • Probably not clinically applicable
• Triplex: we can try to make stable oligonucleotides that are not degraded that sit between the two strands of DNA at the site of the defunct

gene such that when polymerase comes along it gets stuck and won't transcribe.

• • Not to the clinic yet.
• Antisense:
  • Short, complimentary sequence to the defunction mRNA sequence
  • This ends up making dsmRNA which is natveily rapidly degraded.
  • In clinical trials.
• Ribozymes
  • short squences of RNA that you can make to compliment your target mRNA
  • They pair and are actually enzymatic so they directly cut up the target mRNA.
  • Folks who discovered this got the nobel prize for showing that a non-protein entity could be enzymatic.
  • This is in clinical trials
• RNAi
  • can cause degredation at the RNA level or potentially even at the translational level
  • In clinical trials

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:42:32 PM

• How might we use these RNAis?
• Treating HIV
• Usually BM stem cells are not infected with HIV.
• So let's take out the bone marrow, get the stem cells, introduce a lentivirus with a RNA that will get turned into DNA that will get integrated,

that will get expressed as RNA(i) which can interfere with the HIV's RNA products.

• The lentivirus also has ribozymes in it to directly cut up the HIV RNA (before it has even integrated).

• And we're thinking about using this on HepC, too.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:44:02 PM

• miRNA
• We realize that lots of gene regulation is via miRNAs.
  • Probably lots of the "filler genome" is very important.
  • Propably processed through RISC and DICER.
• Basically, miRNA bind to RNA and cause cleavage or cause translation repression by preventing the ribosome from functioning.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:44:52 PM

• Do not memorize. *miRNA is really hot right now. *Applications in: **retinal disorders **cancers **kidney diseases **HIV **RSV **HepC **Hypercholesterolemia *These tirals are completed or ongoing

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:49:36 PM

• How might you want to engineer a cell?
• One idea is in chemotherapy.
  • Chemo has lots of side effects, especially in BM.
  • BM is usually the limiting factor as chemo will kill it to the pt of having bad infections and bleeding.
• So, we'd like to make the bm resistant to BCNU therapy.
  • BCNU is a methyl donor, it donates the methyl to guanine.
  • After 18 hours, BCNU and guanine are irreversibly bound across cytosines.
  • And that's how it kills tumor cells because upon division, DNA synthesis fails.
• We know of a protein that can demethylate (MGMT); a methyl acceptor.
  • It is a one-time use protein (and therefore not an enzyme).
  • It is one of our endogenous DNA repair mechanisms.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:53:08 PM

• So normally we give BCNU to the highest dose we can without losing the pt to bleeding or infection.
• Now if we take out the BM cells and introduce the MGMT gene before we treat with BCNU, then the bone marrow cells will be more resilient to

the chemotherapy and we will have better outcomes.

• This can also be used as a selective advantage marker for other gt tx.
  • For example, if we replace the HgA gene in thalassemia, we still need a way to give that 5% of BM stem cells a selective advantage.
  • If we give them MGMT, also, then we can give chemo on top and select for the BM stem cells with both MGMT and the fixed HbA gene.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 7:59:19 PM

• This idea is the convers: suicide genes.
• Rather than protecting the cell, we might want to kill the cells.
• We can target bad cells and give them suicide genes.
• One way is to use thymidine kinase
  • Herpes virus has thymidine kinase.
  • TK will help phosphorylate some compounds (like gancyclovir, etc.).
  • These products only get phosed in infected cells because only infected cells have thymidine kinase.
    • It kill by integrating into DNA synthesis and screwing stuff up.
• So we can put thymidine kinase in our vectors so that if the cells we've engineered start screwing up, we can kill them with ganciclovir, etc.

• TRial here at IU
  • In pts with BM transplant we often give T cells from their donor to help with graft-verus-tumor.
  • Problem is, at the doses at whcih one has to give T cells it can also cause GVHD which is often fatal.
• So we put into the T cells TK we can turn them off if they start GVHD.
• Used in the clinic.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 8:02:46 PM

• Melanoma
  • There are many T cells in the tumor but they aren't doing anything.
  • When he grew them and gave them back to the pt, they only sometimes did stuff.
  • He saw that the T cells were clonal (so they all have the same receptor).
  • He saw that the tumor has specific antigens.
  • So he cloned the appropriate T cell receptor for the melanoma antigen and ptu it in the vectyor.
  • Then he used retroviral to infect the T cells so they have the "right" receptor.
• 2 of 11 seem to have long-term affect.
• Now we are trying with lots of different antigens like CEA and lung stuff.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 8:04:45 PM

• This is similar to the melanoma slide.
• This is against leukemia.
• Reprogrammed T cells against a CD19 epitope.
• BM decreased in B cell congestion.
• Worked so well that there was tumor lysis syndrome.
• 3/3 had great response.
• Pts will probably need Ig administration for life because they probably won't have B cells anymore.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 8:05:09 PM

• TAke away:
  • Approved trials:
  • 2/3 in cancer
  • but lots of other stuff, too

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 8:07:32 PM

• Homologous recombination
• There are zinc finger nucleases that are used for DNA repair and sit down nicely on dNA.
• Can be designed to sit down on a specific sequence.
• Then it can remove a portion of DNA.
• Then flood with an engineered template, it will get integrated.
• Could be used for something like Sickle Cell where we know we only need to fix a single codon.

• This is attractive because viral integration can't be targeted to a certain integration site.

• Probably will be combined with stem cell developments (iPSCs).

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 8:09:10 PM

• All things we have done are somatic cell repair.
  • Must convince FDA when doing trials that there is little to no risk of altering germ line
  • People just aren't really to alter the gene pool.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 8:11:26 PM

• JG had modest problems, had severe immune reaction and died. *FDA investigated **Multiple protocol violations **Press had a hay day *Trial approvals went way down *Congressed pushed for more regulation *Also, a volunteer study fro an asthma drug resulted in several deaths. *Went from 2 IRBs to 5 IRBs

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 8:13:48 PM

• Raised ethical issues for many types of treatment.
• Most GT therapies are in phase 1 which is for toxicity
  • We don't think this will help, but we need to find out if it will help.
  • Increase dosage until you see harm.
• Phase II:
  • Safety is the number one criteria
• Phase III:

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 8:16:11 PM

• So we said, OK, we'll only try these things on people who have really no hope of getting better.
  • The problem is that ethically that doens't seem right; we should treat people who are likely to benefit.
  • That's how JG got on the study.
• But now we're much more cautious.
  • Hard to balance the risk and benefit.

• Therapeutic misconception
  • Tell the pt over and over that it won't help them.
  • Even still, 40% will believe that the study is helping them.
  • Ethicists say this happens because of poor informed conscent.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 8:18:42 PM

• What are the pros and cons of GT?
• Right to an open future:
  • You shouldn't do gt in kids because they should be able to decide for themselves if they want changed.
  • But parents will say that they are responsible for taking care of their children.

• Sperm, egg, embryo manipulation

• Unintended consequences
  • Recall that SC has an advantage in some places
  • What about other gene "defects"?
  • Hypertension

• Altered gene pool
  • we aren't perfect at insertion and we might get it wrong

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 8:20:31 PM

• The science fiction part!

• What about enhancement
  • Taller, smarter, etc.
  • Splitting of populations; rich get richer, etc.

• Resetting what is normal
  • Small people of america say they aren't broken

• Eugenics
  • We used to sterilize people who we though weren't smart enough or strong enough or whatever enough to bear children
  • If we weed them out we will become a better society.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 8:22:24 PM

• There is a balance, though.
• How do we define what is a disease and what is an enhancement **Study on Rat impotence: ***Used DNA to treat the impotent mice ***Some said this was enhancement and not necessary ***Some said that impotence is a real disease and it is right to treat it.

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Number: 1 Author: Peter Subject: Sticky Note Date: 11/29/2011 8:23:37 PM

• Bone marrow transplants **A review **Fist in 1950s **Took off after we knew more about the immune system **Now an accepted therapy. **Took 30-40 years.
• We are 20 years into GT
• Antibody therapy took is about 10 years ahead of GT.

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