CF Genes & Genetics

 ian.pringle@trinity.ox.ac.uk

  All references used and a copy of this file are online

  http://users.ox.ac.uk/~trin0791/Lecture/References.htm

 What do I do for the Consortium?

  Job title?

  Job description?

  Try to advance or support our research wherever I think I can

 What do I do for the Consortium?

  Job title?

  Job description?

  Try to advance or support our research wherever I think I can   Gene therapy vector design

 What do I do for the Consortium?

  Job title?

  Job description?

  Try to advance or support our research wherever I think I can   Gene therapy vector design   Molecular assay development

 What do I do for the Consortium?

  Job title?

  Job description?

  Try to advance or support our research wherever I think I can   Gene therapy vector design   Molecular assay development   Laboratory automation

 What do I do for the Consortium?

  Job title?

  Job description?

  Try to advance or support our research wherever I think I can   Gene therapy vector design   Molecular assay development   Laboratory automation   Animal studies – mouse

 What do I do for the Consortium?

  Job title?

  Job description?

  Try to advance or support our research wherever I think I can   Gene therapy vector design   Molecular assay development   Laboratory automation   Animal studies – mouse   Software tools, databases, websites

 Working for the Consortium

 Thanks to the rest of the Consortium

 CF Genes & Genetics

  Background

  Genetics of CF

  How it affects us in the lab?

  The future

 James Watson & Francis Crick 1953

"We wish to suggest a structure for the salt of deoxyribose nucleic acid (DNA). This structure has novel features which are of considerable

biological interest."

 What is the structure of DNA?

  Four different nucleoside bases

  Adenine A

  Cytosine C

  Thymine T   Guanine G

  Very strong sugar-phosphate backbone   ACGTCTGAGTATGGGAGT

  Base-pairing between the two strands

 What is the structure of DNA?

  Four different nucleoside bases

  Adenine A - T

  Cytosine C - G

  Thymine T - A   Guanine G - C

  Very strong sugar-phosphate backbone   ACGTCTGAGTATGGGAGT

  Base-pairing between the two strands

 How does DNA code for proteins?

  Was not immediately obvious

 Amino acid triplet code

  Crick, Brenner et al., 1961

  DNA is copied (transcribed) into RNA

  RNA is read (translated) and a protein produced

  3 DNA bases (codon) = 1 Amino acid (the building blocks of proteins)

  4 bases maximum   3 in a codon   64 possible triplet codes

 Each triplet codes for only 1 amino acid

  Some amino acids are coded for by multiple codons

  This code is almost completely universal across all forms of life

 1 gene codes for 1* protein

  *There may be multiple versions of the protein made by the gene

  A gene may code for 100s or 1000s of amino acids

 Every human cell has exactly the same genes

  Prior to the human genome project, estimate was 100,000+ genes

Nerve cell Photoreceptors Macrophage

 Every human cell has exactly the same genes

  It turns out, we have just over 30,000

  Mutations in just 1 gene – CFTR causes CF

  DNA from blood – screen for CFTR mutations

Nerve cell Photoreceptors Macrophage

 Arranged on 23 chromosomes

  3000,000,000 base pairs of DNA

 Only 2% of it actually codes for proteins

 98% is ‘junk DNA’

 98% regulates the 2% that does the work

 Human genome headlines

  On average any two humans differ only once in 1500 bases

  Still means 1.4 million common mutations   Men have slightly more

  Everyone has the same genes

  In the same order

  No genetic basis for race

 Variations stand out

 Variations stand out

  When they occur, mutations can be easily identified

  Direct sequencing of DNA

 Variations stand out

  Design assays to specifically amplify the mutation/wt sequence

  There are systems that can screen for 100s of mutations at a time

 CFTR is located on chromosome 7

 Total sequence around 250,000 bp

  Only the red sections here code for the protein   4400 bp (1.8%)   1480 amino acid protein

 CFTR is completely unremarkable

  Known as a ‘housekeeping’ gene

  Typically expressed at very low levels in airway cells

  1-2 copies of mRNA per cell (my own estimate from nasal epithelia)

  There are over 1900 mutations that can cause CF

 Why are there so many mutations in CFTR?

  1/25 Caucasians are carriers – don’t get CF

  Must be some evolutionary advantage to being a carrier

 Carriers were more resistant to diarrhea

  Carrier mice have lower CFTR activity in the intestine

  Secrete less water in response to intestinal infections

  Medieval European carriers less likely to die from dehydration?

 Nice theory but - less evidence of in humans

  Carriers seem to have normal CFTR chloride activity in intestine

  Frequency of CF too high for late emergence of cholera epidemics

 Theory makes a lot of sense, but not clear

  Still most likely explanation but not 100%

  ‘Founder effects’ can be very subtle over 1000s of years

  Definitive experiment?   1000 carriers and 1000 non carriers   Give them cholera   Any volunteers?

 Why are mutations of this gene such an issue?

 CFTR is a real ‘keystone’ protein

  Very complex trans-membrane protein

  Folding crucial

 Regulates chloride transport across epithelia

 Is CFTR just a chloride channel?

  No, other ions as well – bicarbonate

  Evidence that its involved in Pseudomonas killing as well

 Interacts with many other proteins

  Loss of CFTR function will influence the function of these proteins too

 No-one fully understands the role(s) of CFTR

  If you think you do – you haven’t realised how complicated it is yet.

 Mutations fall into 6 different classes

Non-CF 1 2 3 4 5&6 Production Processing Regulation Activity Abundance

G542X ΔF508 G551D Q1412X

Stops translation early

Miss folding of the protein

Gets to surface No activity

Some functional protein

Mild/absent lung disease

Spectrum of severity

 G551D almost works!

1 2 3 G542X ΔF508 G551D

  Translated – Folds– Processed – Gets to membrane – No ATP binding

  Very specific issue for G551D CFTR – ‘just’ needs activation

 Kalydeco very specific for G551D CFTR

1 2 3 G542X ΔF508 G551D

  Random drug screening is expensive

  High chance of a specific activity on just G551D

 ΔF508 is much harder to fix

1 2 3 G542X ΔF508 G551D

  ΔF508 CFTR fails much earlier – exquisite removal of unfolded proteins

  You need a drug which can refold it and get it all the way to membrane

 ΔF508 – off target effects?

1 2 3 G542X ΔF508 G551D

  Far more likely to have non-specific effects

 How much random screening can you afford?

  Far more likely to have non-specific effects

 Genetics is usually very subtle

  CFTR is one of the few genes where it’s not

 This shouldn’t affect us?

  All gene therapists need to do is get a copy of the coding sequence

 You can buy virtually any gene online

 The CFTR sequence has been known since 1989

  Hasn’t it…?

 Spring 2006

  2-3 years work

  We had our clinical trial plasmid design

  Plasmid vector tested and ready – the CFTR sequence was functional

  Manufacturing slot booked

 Collaborators in Japan noticed something

  The CFTR sequence had subtly changed in March

  Two changes   Position 620 now Histidine not Asparagine (N620H)   Position 833 now Phenylalanine not Leucine (L833F)

 Sequencing genes is a numbers game

  N620H was simply an error in 1989 that took a long time to be noticed

  L833 was in the original 1989 sequence

  F833 is by far the more common variation (90%)

 The original sequence was functional

  Had normal CFTR electrics

  CFTR protein on a gel

  Used in every other CF gene therapy trial

 Lets not bother changing it!

  90% certain it would make no difference

  90% certain isn’t enough

  Risky – cheap – quick

  Guaranteed – expensive - slower

 Lets not bother changing it!

  90% certain it would make no difference

  90% certain isn’t enough

  Risky – cheap – quick

  Guaranteed – expensive – slower

BOTH!!!

 The result was our clinical trial plasmid pGM169

 We had to do more work – boo hoo!!

  Not all CFTR variations change its function

  The notion of a single fixed gene sequence is misleading

  Prone to human error

 Cost a lot of money

  Why is research so expensive sometimes   We noticed this issue really early but were on a tight timeline   For 6 weeks everything else got shelved   Had to spend money on a backup strategy

  What if the new ‘correct’ sequence was actually worse?   We had to test it in multiple models   A slight hint that it may be more active in cell culture models

 Was it worth it?

  25 previous gene therapy trial for CF

  None of them used the most common functional CFTR sequence

  Ours does

 The future?

  Mid 1990s – several hundred machines

  12 months - $ millions = The human genome project

 The 1000 Genome Project

  Sequence 1000 human genomes

  Map every common instanceof variation

  $1000-$10,000 each in 3-4 days

 Genome sequencing as a diagnostic tool

  Oxford Nanopore MinION

  Sequence a whole genome <$100

  Still a few years away

 Genome sequencing as a diagnostic tool

  Oxford Nanopore MinION

  Sequence a whole genome <$100

  Still a few years away

  Massive implications   Earlier diagnosis   Modifier genes   Better outcome predictions   ‘Smarter’ trials

 Are the NHS ready for this?

  Prof. Crook, NDM, Oxford

  Replace microbiological screening with genome sequencing

  Infrastructure in place, embedded within the hospital and GP surgeries   Tight geographical mapping of infections

 Are the NHS ready for this?

  Prof. Crook, NDM, Oxford

  Replace microbiological screening with genome sequencing

  Infrastructure in place, embedded within the hospital and GP surgeries   Tight geographical mapping of infections

 How will this affect CF?

  Routine genome based screening of sputum

  Better infection control

  Less infections

  Reduce the spread/development of antibiotic resistant strains

 Conclusions

  CF Mutations Database http://www.genet.sickkids.on.ca

  Scientists always say something revolutionary is around the corner

  In terms of the technology driving genetics it really is true

  Potentially huge benefits