Cystic fibrosis lecture notes

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Current revision as of 14:18, 17 March 2010

  • We'll go over the basics of the disease.
  • We'll talk about ethics.
  • There is a potential volunteer opportunity:
    • The state science fair is here at IUPUI on Saturday, March 28.
    • Judging is open to graduate students.
    • Email her or her husband if you're interest.

Contents

[edit] Cystic fibrosis

  • It is the most common genetic disease in the Caucasian population.
  • Many carriers.
  • Heterozygotes are not affected. This is the textbook form.
    • Many who are heterozygous have trouble with chronic pancreatitis.
  • It affects 1 in 2000 live, Cuacasian births.
  • It is an autosomal disease.
  • It is uniformly fatal for homozygotes.
  • 25-30 years ago the average lifespan was 5 years.
    • Now, treatments have expanded lifespan to late 30s, early 40s.
  • There are various treatments and we'll talk about each of the organs affected.
  • Regardless of the other organs, it is recurrent infections in the lungs that are the major cause of death.
    • Therefore, most treatments target the lungs and infection control.
    • One problem is antibiotic resistance which occurs when treating with large and long antibiotic treatment.

[edit] History

  • Don't worry about the dates, this is just a background.
  • By the 80s, we realized it had to do with ion transport.
    • Some thought problem was in sodium channel, some though it was in a chlorid channel.
    • At this point, the best diagnosis was to make them sweat in a bag and then test it.
      • As we got better, they could put a little skin chamber on them.
  • In 1984, there was a classic paper published
    • Two pages, one table, one author.
    • Author isolated sweat ducts and thus showed that the defect was in a chloride transporter.
  • In 1989 we isolated the gene for CF: CFTR.
    • It was a huge gene.
  • In 1992 we isolated the gene product.
    • Though we though we could predict the product, this one looked very different from any other chlorine channel that we knew of at the time.
    • So we called it cystic fibrosis transmembrane regulator.
    • Then we put it in a frog and did some patch channel experiments and showed it was a chloride channel.
  • In 1994 we started clinical trials.

[edit] Organ systems affected in CF

[edit] Sweat ducts

  • There is a coil where the sweat is formed.
  • Initially the sweat is a plasma filtrate (no cells).
  • This travels through the excretory duct.
  • As it travels we reabsorb both sodium and chloride.
  • The sweat is hypotonic on the skin.
    • This is good because it will evaporate well and not leave much salt behind.
  • So most of the Na and Cl has been reabsorbed.
  • We knew for a long time that patients with CF had sweat that was 3-5x as concentrated with NaCl than normal.
    • You could even see it on their skin!
  • They found that their sweat was isotonic (that is, the same as the plasma) indicating that no Na or Cl was being reabsorbed.
  • One early experiment:
    • Either side of the sweat gland is canulated to a pipette.
    • One pipette infuses fluid that travels through the sweat duct and the other sucks it out.
    • This whole system is immersed in the bath.
    • Researchers have control of what is going through and over the bath.
    • There is an electrode in the pipette so we can measure the volts over the lumen and bath.
    • We saw 10 mV in normal patients and 80 mV in patients with CF.
    • So they perfused lots of different stuff and changed the bath with and without Na and / or Cl.
    • They used samples from the lab members, including the diseases tissues which came from the PI (who is the longest living patient with CF).
    • And thus they could show which ion mattered: Cl-.

[edit] Pancreas

  • In the pancreas, there are two parts: exocrine and endocrine.
    • Endocrine = islets of langerhans, make hormones.
    • Exocrine = enzymes made in acini, secreted via ducts into GI tract to help with digestion.
  • A sodium bicarbonate rich fluid is also secreted along with the enzymes.
    • Note that CFTR is a Na / bicarbonate channel.
    • Bicarbonate serves as a buffer, too.
  • CFTR is used to generate this secretion.
  • When ions are secreted into the lumen, water follows.
  • So if you cannot secrete ions, you cannot get water to move.
  • So if water doesn't join, all the enzymes and such will just sit there and it will eat away at the pancreas.
    • This causes generation of diabetes as the pancreas' function decreases.

[edit] Colon

  • Not really a problem in CF.
  • However, an explanation of CF's prevalence in Caucasians might be explained via the colon.
  • 1:2000 births is high for a fatal disease.
  • So the theory is that CF yields a selective advantage for heterozygous people.
    • 1 in 20 might be protected against cholera which was killing boat-loads of people at the same time that CF arose.
    • CFTR is turned on by phosphorylation by a kinase that is activated by cAMP.
    • Cholera toxin constitutively activates the stimulatory g-protein of adenylase cyclase causing an increase of cAMP.
    • Then, CFTR gets turned on which secretes chloride which causes water secretion, which, when overdone causes diahrea and death.
  • So if you have CF or are a carrier of CF, the chloride channels may not work so you don't die of diahhrea.

[edit] Salivary glands

  • CF causes a lack of secretion.
  • This includes male reproductive tract secretions.
  • This causes an increase in viscocity and a decrease in organ function.

[edit] Lung

  • CF will cause mucus and submucus layers to be much thicker.
  • The mucus that sits on top of the epithelium of the airway helps to clear stuff and sits on the cilia.
  • In CF the mucus is thick and it bends the cilia and the mucus cannot be moved.
  • So we do percussion therapy to help clear the mucus from the lungs.
  • Mucus is a great place for bacteria to colonize.
    • Once you have biofilms building up in the lungs you get scarring and loss of air exchange function.
[edit] Apical membrane
  • Epithelial cells line the lungs.
    • They have tight junctions that define the luminal side from the basolateral membrane (blood side).
  • There are transporters on both membranes and they are different.
    • This allows polarized transport (the movement of ions in one direction or another).
  • A classic hallmark of an absorptive lumen epitheial membrane is sodium channel which it is pumping into the cell (down it's chemical gradient).
    • This channel doesn't require energy.
  • There is a Na/K atp-ase on the basolateral membrane moving Na out of the cell and into the blood.
  • When we say absorption and secretion, we are talking about from and to the blood.
  • Secretry epithelial cells
    • There is a tripple transporter: moving Cl, Na, and K all in.
    • We care most about the chloride which is moved into the lumen from the blood.
  • The sweat duct is impermiable to water (one of the few tissues of the body with this property).
  • CFTR can transprot Cl in either direction depending on the driving forces.
  • In a normal sweat gland:
    • Both Na and Cl are moving in an absorptive direction.
      • This is normal for Na but abnormal for Cl. Na is going with its concentration and electrical gradient.
      • Cl is going with its concentration gradient and against its electrical gradient.
  • In a CF sweat gland:
    • Now you have a huge 80 mV potential because Chloride is not being moved into the cell.
    • And then Na won't move much either because the electrical balance of the sweat must remain neutral and if chloride isn't moving then sodium can't move either.

[edit] Gene therapy clinical trials

  • The rational was that the lungs are relatively accessible and that's what kills most people.
  • So we thought that if we could introduce a non-mutated CFTR gene, we could save the patients.
  • Rodent experiments were relatively successful.
  • Then they went on to primates.
  • But how do you get the gene into the cell?
    • They had adenoviruses and retroviruses.

[edit] Choice of vectors for CF gene therapy

  • Retroviruses:
    • Good: They are well understood.
    • Bad: They insert into the genome in a random fashion.
    • So, this could cause oncogenesis because of interuption or changing of a genome product.
      • This was only theory at the time.
    • Also, once the lungs are formed, a barrier is put up and the cells stop dividing.
      • This is bad because retroviruses infect dividing cells, mainly.
    • Retrovirues have been used in gene therapy for SCIDs.
      • They took the blood of the kids out and put in the mutated gene and put the blood back in.
      • This cured them. Yay!
      • However, it caused cancer in 3 of the 20 patients.
      • So all those trials were stopped.
  • Adenoviruses:
    • They are fairly benign.
      • 75% of us have had an infection via one of these yielding only mild infection.
    • Adenoviruses like lung cells.
    • They can infect non-dividing cells.
    • Also, a very good thing (in theory), is that these vectors will not insert their DNA into the host DNA but will be expressed (episomal).
      • Insertion will follow the host DNA upon division.
      • If it is only in episomal form, you have to have multiple doses as infected cells turn over.
      • Lung cells turn over slowly, but the do turn over.
    • Adenovirus has been reproduced in large quantities which is good.
    • We've also engineered them to be replication defective so they don't run rampant.

[edit] Preliminary animal studies

  • We did primate studies to assess safety and efficacy.
  • Safety comes in two forms:
    • To the organism receiving the treatment,
      • We knew that there must be no toxicity to the host.
    • To the environment
      • We know we had to make sure that the virus couldn't recombine with a WT virus and start infecting other people.
  • They wanted to make sure that the treatment worked, too.
  • In checking for safety they look for:
    • inflammation response of the lungs
      • They administer it via direct application.
    • clinical evaluation of the lungs
      • via x-rays over time
      • via autopsy of animals over time
    • check for dissemination of the virus throughout the body
      • looking for escape of the mutated virus into the environment or throughout the animal
    • how much of the vector you get into the cells
    • they looked at stability of vector expression
    • how functional the vector was in the cells

[edit] Results of primate studies

  • In general, no adverse affect on health.
  • All blood work was normal.
  • Urinalysis was normal.
  • Blood gasses were some changes but were not statistically significance.
  • They found no virus in other tissue of the animal (so it wasn't moving around or escaping into the environment).
  • Chest x-ray results:
    • Showed (especially in the long term animals) that in the highest doses the infiltrates took 30 to 70 days to clear.
  • When they autopsied, they found severe inflammation that moved from the infiltrate and moved outward to other tissue of the lung.
  • They said "it was unable to determine if inflammed area would recover and be able to participate in gas exchange".
  • So we see that there are some problems in the lung.
  • But did it work?
  • The highest doses expressed the gene at 4 days but at 21 days they found no expression.
  • So, it isn't really working.
  • So it is safe for the environment, it is not safe for the animals, and it doesn't really work.
  • Their conclusion was that gene transfer was possible and we should start human trials.

[edit] Article: Administration of adenovirus (by Crystal)

  • This clinical trial got the furthest and was the reason all three others were stopped.
  • The patients were in pretty good health to begin, even though they had CF.
  • They administered stuff through the nose.
  • They found that:
    • the treatment (the vector) caused an immune response,
    • there was inflammation,
    • no matter how hard they looked they only found a very small amount of gene expression,
    • that they could not dose a second time because of immune response,
    • there was low rate of transfer, not enough to take an effect.
  • Conclusions:
    • "Correction of the CF phenotype of the airway epithelium might be achieved with this strategy".
    • We'll probably have to readminister the treatment.
  • Then the scientists became media stars!

[edit] Public perception of the human trials

  • This was a huge deal; scientists on the cover of newsweek.
  • Scientists were worried.
  • Overplaying this type of "breakthrough" is bad because when it doesn't deliver, scientists and researchers are looked down on.
  • NIH convened an investigation and concluded that commercial interest was pushing science too fast without heeding the results.
  • So NIH put money into developing better vectors instead of into the trials.
    • This went on for several years.

[edit] Jesse Gelsinger case

  • This was part of the attempt to build new vectors.
  • This version was supposed to reduce immune reaction so we could do multiple dosing.
  • Jesse Gelsinger was 18.
    • He had ornithine transcarbamylase deficiency.
    • His older sibling had died before he was born.
    • His diet was controlled so he could live (with medicine).
    • He was "normal" and an athlete.
  • He was recruited into the trial.
  • He knew this vector would do nothing for his disease.
  • The adenovirus they were using was targeting the liver.
  • Leading up to this human trial:
    • Mice, then monkeys, then baboons.
    • It had been shown that there was some toxicity with the vector.
    • They were turned down the first time they submitted for human trial permission.
    • So they resubmitted and said they would only do low doses because primates had die at high dose.
    • So they got permission to start at 5% of the highest dose.
    • If they got no bad results they were allowed to increase dosage up to 75% of highest dosage.
    • They had three patients in each group.
    • Most patients in lowest group showed fever and moderate immune response.
    • The 10th and 11th volunteers showed substantial increase in liver enzymes which shows change of function of liver, which was recovered.
    • 18th patient died of immune response to vector.
  • The vector was patented by the lead investigator and the university (University of Pennsylvania).
    • Now this cannot happen, which is one good thing about this whole fiasco.
  • Five years later, the university settled (1 million to the Gelsingers).
  • Lead investigator was barred from doing clinical trial for 5 years.
  • Under the aggreement, however, the researchers do not admit responsibility for Gelsinger's death.

[edit] Where do we go to here?

  • We're trying to make new vectors.
  • We've made some progress with a small molecule that helps to move the mutated form into the plasma membrane.
    • This affects the major CFTR mutant (F508) which breaks because the protein gets stuck in the ER.
  • Test on Monday.
  • No class on Wednesday (snow make-up day).
  • stopped here on 03/03/10.
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