Cystic fibrosis lecture notes

From Biol557

• continued here from Respiration lectures on 03/03/10.

• 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 1 Cystic fibrosis 1.1 History 1.2 Organ systems affected in CF 1.2.1 Sweat ducts 1.2.2 Pancreas 1.2.3 Colon 1.2.4 Salivary glands 1.2.5 Lung 1.2.5.1 Apical membrane 1.3 Gene therapy clinical trials 1.3.1 Choice of vectors for CF gene therapy 1.3.2 Preliminary animal studies 1.3.3 Results of primate studies 1.3.4 Article: Administration of adenovirus (by Crystal) 1.3.5 Public perception of the human trials 1.3.6 Jesse Gelsinger case 1.3.7 Where do we go to here?

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.

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.

Organ systems affected in CF

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-.

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.

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.

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.

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.

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.

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.

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.

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

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.

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!

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.

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.

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.