• Research

Cystic Fibrosis

Cystic fibrosis is a devastating genetic diseases that affects 70,000 people worldwide and 34,000 people in the US. It is an autosomal recessive disease found in primarily people of North European heritage and occurs in 1:3500 live births, with an incidence of 1 in 1000 every year in the US. Over 100 mutations have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene which is responsible for the disease. CFTR is a chloride channel conductance regulator found on epithelial cell membranes that line the airways and gut. Mutations in CFTR disrupt mucous transport and antibacterial activity in the airway surface liquid. Abnormally thickened mucous, lowered pH and inflammation of the airways result in progressive respiratory failure and bronchiectasis. The average life expectancy of an adult with cystic fibrosis is 39 years.

A child with cystic fibrosis (https://www.medicalnewstoday.com/articles/147960.php):

 

Treatments for Cystic Fibrosis

Current treatments of cystic fibrosis target the symptoms of the disease rather than the cause. These including mucolytics, bronchodilators, antibiotics, osmotic agents, hypertonic saline, anti-inflammatory drugs and chest percussion therapy. In recent years several small molecule drugs have been approved by the FDA for treatment, including ivacaftor - Kaledyco (chloride channel potentiator) and lumacaftor (CFTR activity corrector) and the ivacaftor/lumacaftor combination drug - Orkambi. However, these drugs only treat a small proportion of patients with cystic fibrosis (those with the dF508 mutation).

Gene therapy has previously been tested in clinical trials to correct the CFTR defect. There are several advantages to using this method, including the ability to treat all people with cystic fibrosis regardless of specific mutation and the potential to establish a long term correction of CFTR expression. However, previous clinical trials using adenovirus, AAV and non-viral nanoparticles have failed due to a variety of reasons. The knowledge gained from previous trials will serve as a basis for our development to create better viral gene therapy products this time.

AAV

I am working on an Adeno-Associated Virus (AAV) that will deliver the CFTR gene into airway epithelial cells. This AAV gene therapy product has been patented by researchers at the University of Iowa and has several advantages over previous AAV constructs. These advantages include:

1. A highly functional CFTR minigene (CFTRΔR ) that fits into the limited packaging capacity of AAV vectors.

2. A synthetic promoter (SP183) that drives high level CFTR expression.

3. A chimeric vector with a hybrid capsid that is highly tropic for airway surface of human airway epithelial cells.

4. Use of proteasome inhibitors to enhance trafficking of recombinant AAVs to the nucleus to improve transduction efficiency by over 100 fold.

Lentivirus

Talee Bio is also developing a lentivirus vector with a large carrying capacity, high transduction efficiency, persistent expression and minimal immunogenicity and integration that will transduce progenitor cells to produce corrected CFTR in airway epithelia. The lentivirus is engineered with an envelope protein that will increase binding and entry to the surface of epithelial cells.

 

A little about viruses

Lentivirus - a genus of retroviridae family (80-100 nm diameter) derived from HIV-1 virus.  Lentiviruses are used to package a gene of interest into the lentiviral DNA construct.  To produce lentiviruses, HEK 293 cells are transfected with a packaging plasmid and the envelope vector (VSVG). Once viral particles are produced, the media from cells are purified by centrifuging through a sucrose gradient, desalted and titered.

Adeno Associated Virus (AAV) a genus of the parvoviridae family (20 nm diameter).  AAV is used by packaging a gene of interest into the ITR sites of the vector. AAV has limited packaging capacity compared with lentiviruses (<4000 bp). To produce AAV, HEK293 cells are transfected with a Helper vector and the Rep/Cap DNA integration vector. Once viral particles are produced, the cells are collected from the media, lysed and purified by centrifuging through multiple Cesium chloride gradients, desalted and titered.