The CFTR Protein

1
The CFTR Protein

• Secretes Cl- ions
• Downregulates Epithelial Na Channel
• Maintains airway surface liquid volume

2
What is CF?

• Absence of gene coding for CFTR protein
• ? Airway surface liquid
• Accumulation and desiccation of mucus
• Bacterial colonisation
• Chronic infection ? fibrosis in the lung
• ? Pulmonary resistance ? Cor pulmonale
• Heart and lung transplant is the only cure

3
Why gene therapy?

• CF is a single gene defect
• Accessibility of the lung
• Therapeutic window
• Low gene expression is curative
• What will the therapeutic product be like?
• It incorporates corrected gene into the nuclei of
  airway epithelial cells
• Non-immunogenic / non-inflammatory
• Safe for CF pts with existing damage
• Provokes CFTR expression curatively
• EITHER reaches stem cells one-hit
• OR can be safely readministered

4

• Adenoviral Vectors (Ad)
• Inefficient for 3 reasons
• Primary receptors are on the basolateral surface.
• Ad causes an immune response ? inflammation and
  cytotoxicity.
• Neutralizing antibodies present in humans for Ad.
• Adeno-associated Viral Vectors (AAV)
• Also have receptors on the basolateral surface.
• Do not have an inflammatory response like Ad.
• But neutralizing antibodies also exist in humans
  for AAV.
• Very small

5

• Administration by inhalation?...
• Modify vectors to allow uptake on apical surface.
• Break down tight junctions to allow access to
  basolateral membrane.
• Avoiding antibodies?
• Modify surfaces of vectors again with
  Polyethylene glycol (PEG).
• Eg. PEGylation of AAV vector by tresyl chloride
  PEG ? efficacy of AAV2 serotype after repeat
  administration.
• Suppress immune system during administration of
  vector.
• Future for Ad and AAVs?...
• Break down tight junctions of epithelia.
• Target Ad and AAV to airway epithelia.

6

• Lentiviruses
• Can integrate in non-dividing cells
• Can pseudotype
• Concerns with possible recombinant events in
  humans if readministered.
• Future for Lentiviruses?...
• Further trials into readministration and immune
  responses.
• Enter bloodstream ?infect stem cells ?Chronic
  infection One hit Cure!
• Could mean infecting patient with a dangerous
  virus.
• Viral vectors must overcome inflammation and have
  the ability to repeat doses but this could effect
  their efficacy.

7
Non-viral Vectors

• Naked DNA
• Easy to manufacture and store
• Safe and non-immunogenic
• Gets broken down by endonucleases
• Poor at penetrating the cell membrane

8
Lipoplexes

• Cationic lipids bound with DNA
• Particle size 100 500 nm
• Nuclear pore admits molecules lt25nm
• Endosomal escape
• Clinical trial in 1999 (Alton et al.)
• Partial correction of Cl- transport in lower lung
  and ? pseudomonas binding
• Inflammatory response at high dose

9
Polyplexes

• Poly-L-lysine (polyK) folds DNA
• Polyethylene glycol (PEG) allows
• Aerosolisation
• ? Stability
• Protection from immune surveillance
• Clinical Trial Konstan et al. 2004
• 12 CF pts, 6 ?dose and 6 ?dose
• Vector detected after 14 days in high dose group
• PD correction in 8/12 for six days

10
The Future!!

• Clinical trials are taking place right now
• Delivery enhanced by mechanical force e.g.
  magnetofection and ultrasound
• Gene repair
• Trans-splicing
• Foetal gene therapy
• Bone marrow stem cell therapy

11
References

• Ziady AG Davis PB Current prospects for gene
  therapy of cystic fibrosis Current opinion in
  Pharmacology (Oct 2006) 6 515-521
• Griesenbach U,Geddes DM, Alton EWFW Gene therapy
  progress and prospects Gene Therapy (Jul 2006)13
  1061-1067
• Ziady AG Davis PB Current prospects for gene
  therapy of cystic fibrosis Current opinion in
  Pharmacology (Oct 2006) 6 515-521
• Alton EWFW Use of non-viral vectors for cystic
  fibrosis gene therapy Proc Am Thorac Soc. 2004
  1(4) 296-301
• Davies JC New therapeutic approaches for cystic
  fibrosis lung disease Journal of the Royal
  Society of Medicine 2002 95(41) 58-67
• Lee TWR, Mattews DA Blair GE Novel molecular
  approaches to cystic fibrosis gene therapy
  Biochem. J. (2005) 387, 1-15
• Wilson JM Adeno-associated virus and lentivirus
  pseudotypes for lung-directed gene therapy Pro Am
  Thorac Soc. (2004) 1, 309-314
• Alton EW et al. Cationic lipid-mediated CFTR gene
  transfer to the lungs and nose of patients with
  cystic fibrosis a double blind placebo
  controlled trial. Lancet (1999) 353 947-954
• Konstan MW et al. Compacted DNA nanoparticles
  administered to the nasal mucosa of cystic
  fibrosis subjects are safe and demonstrate
  partial to complete CFTR reconstitution Hum Gene
  Ther (2004) 15 1255-1269

12

• Le HT et al. Utility of PEGylated recombinant
  adeno-associated viruses for gene transfer J.
  Control Release (2005) 108161-177
• Jooss K et al. Blunting of immune responses to
  adeno viral vectors in mouse liver and lung with
  CTLA4lg Gene Ther (1998) 5 309-319
• Crystal RG et al. Admisitration of an adeno virus
  containing human CFTR cDNA to the respiratory
  tract of individual with cystic fibrosis Nat.
  Genet. (1994) 8 42-51
• Hanrahan JW, Gentzsch M and Riordan JR ABC
  proteins from bacteria to man. Academic Press,
  New York pp589-607