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Cystic fibrosis : 

Cystic fibrosis Is the lethal inherited disease that effects -lungs -pancreas -sweat glands characterized by the production of abnormally viscous mucus by the affected glands Fibrous scar tissue develops in the pancreas

Epidemology : 

Epidemology Approximately 30,000 people in the U.S. Around 2,500 children with CF are born every year Over 10 million americans are unkown carriers

Molecular and genetic basis : 

Molecular and genetic basis Mutations in a single gene - the Cystic Fibrosis Transmembrane Regulator (CFTR) gene on chromosome 7 The CFTR gene: Locus 7q31.2 Is 250,000 bp long Contains 27 exons Protein has 1,480 aminoacids

The ∆F508 Mutation : 

The ∆F508 Mutation A 3 basepair deletion called ∆F508 is the most common mutation causing cystic fibrosis IN Normal CFTR: Nucleotide AAT ATC ATC TTT GGT GTT TCC Aminoacid Asn Ile Ile Phe Gly Val Ser In ∆F508 CFTR: Nucleotide AAT ATC ATC GGT GTT TCC Aminoacid Asn Ile Ile Gly Val Ser The mutation results in deletion of a single amino acid (Phe) at 508 position

Role of CFTR Protein : 

Role of CFTR Protein Protein moves chloride ions out of an epithelial cell to the covering mucus In CF No movement of cl This means water does not leave and it results in the mucus becoming thick

Gene therapy : 

Gene therapy Introduction of functional genetic material into the target cell to replace the defective genes Gene transfer techniques Exvivo: Cells removed, genetically modified and transplanted back in to the patient Invivo: Direct transfer of genetic material into patient

Gene transfer : 

Gene transfer Vectors -viral -non-viral Cells -germ cells -somatic cells

Viral vectors : 

Viral vectors

Non viral vectors : 

Non viral vectors

How it works : 

How it works A vector delivers the therapeutic gene into a patient’s target cell The target cells become infected with the viral vector The vector’s genetic material is inserted into the target cell Functional proteins are created from the therapeutic gene causing the cell to return to a normal state

Adenoviral vectors : 

Adenoviral vectors Double-stranded DNA viruses, usually cause benign respiratory disease Replication-deficient adenovirus vectors can be generated by replacing the E1 or E3 gene, The recombinant vectors are then replicated in cells that express the products of the E1 or E3 gene Cells infected with recombinant adenovirus can express the therapeutic gene, but because essential genes for replication are deleted, the vector can’t replicate.

Generation of non replicative adeno virus vector : 

Generation of non replicative adeno virus vector

Adenoviral vectors- Limitations : 

Adenoviral vectors- Limitations Expression lasts for only a short time Eliciting both the cellular and humoral response. Generation of antibodies to adenoviral proteins.

Adenoviral vectors : 

Adenoviral vectors Advantages: Higher titer Efficient transduction of nondividing cells in vitro and in vivo Disadvantages: Toxicity Immunological response Prior exposure

Adeno associated virus : 

Adeno associated virus Non-pathogenic, single stranded DNA virus dependent on the helper virus (usually adenovirus) to replicate . It has two genes (cap and rep), sandwiched between inverted terminal repeats The cap gene encodes viral capsid proteins and the rep is involved in viral replication and integration.   It can infect a variety of cell types

Adeno-associated viral vectors : 

Adeno-associated viral vectors To produce an AAV vector, the rep and cap genes are replaced with a transgene The total length of the insert cannot exceed 4.7 kb cotransfect two plasmids, one for the vector and another for rep and cap into cells infected with adenovirus. This method is cumbersome, low yielding and prone to contamination with adenovirus and wild type AAV. Interest in AAV vectors is due to their integration into the host genome allowing prolonged gene expression.

AAV Vectors : 

AAV Vectors

Adeno-associated virus vectors: : 

Adeno-associated virus vectors: Advantages: All viral genes removed Safe Transduction of nondividing cells Stable expression Disadvantages: Small genome limits size of foreign DNA Labor intensive production Status of genome not fully elucidated

Problems with gene therapy : 

Problems with gene therapy Short lived Immune response Viral vectors Multigene disorders

Major challenges : 

Major challenges Scientists need better ways to deliver genes to the body, more target specific with higher rate of DNA integration Ability to deliver genes consistently to a precise DNA location and ensure that the transplanted gene is controlled by the body’s physiologic signals

References : 

References Gibson RL, Burns JL, Ramsey BW: Pathophysiologyand management of pulmonaryinfections in cystic fibrosis. Am J Respir CritCare Med 2003 Smyth A, Walters S: Prophylactic antibioticsfor cystic fibrosis. Cochrane Database SystRev 2003;

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