Molecular Basis forRelationship between Genotype and Phenotype

DNA

RNA

protein

genotype

function

organismphenotype

DNA sequence

amino acidsequence

transcription

translation

Gene Therapy

Germ-line Therapy vs Somatic Therapy

Gene Therapy in Mammals

Human Gene Human Gene TherapyTherapy

Approaches Approaches

to to treatment treatment

of diseasesof diseases

DNA DNA transfer transfer

into into afflicted afflicted

individualindividual

1990 : First Clinical Trial

Severe Combined Immunodeficiency (SCID)

- defective adenosine deaminase (ADA) gene.

- afflicted patients express no functional T or B lymphocytes.

- “Bubble Boy Disease”

Delivery Approaches to Somatic TherapyDelivery Approaches to Somatic Therapy

• Direct Administration: Direct Administration:

Introduction of normal gene using a Introduction of normal gene using a virusvirus

retrovirusretrovirusadenovirusadenovirus

• Indirect Administration: Indirect Administration:

Reintroduction of cells that have Reintroduction of cells that have been made transgenicbeen made transgenic

Criteria for Gene Therapy:Criteria for Gene Therapy:

Candidacy of a Disease for ConsiderationCandidacy of a Disease for Consideration

1.1. Mendelian inheritanceMendelian inheritance

2.2. One gene product for the geneOne gene product for the gene

3.3. Knowledge of function of gene Knowledge of function of gene

productproduct

4.4. Availability of normal gene Availability of normal gene

sequencesequence

5.5. Localized symptoms for the Localized symptoms for the

diseasedisease

6.6. Availability of delivery systemAvailability of delivery system

~ 25,000 genes in humans are potential candidates for gene therapy.

Candidates for

Gene Therapy?

~ 25,000 genes in humans are potential candidates for gene therapy.

Cystic FibrosisCystic Fibrosis

Disease is usually Disease is usually fatalfatal..

Lungs, other organs are clogged with Lungs, other organs are clogged with mucus.mucus.

repeated bouts of repeated bouts of pneumoniapneumoniaheart stressedheart stressed by decreased lung by decreased lung

capacitycapacitydelivery delivery failurefailure of pancreatic enzymes of pancreatic enzymes

Life expectancy of 20-25 yearsLife expectancy of 20-25 years

~1 of 29 Caucasians are carriers~1 of 29 Caucasians are carriers~1 of 1600 Caucasians are affected~1 of 1600 Caucasians are affected~30,000 Americans are affected~30,000 Americans are affected

Cystic FibrosisCystic Fibrosis

• Caused by recessive mutation Caused by recessive mutation (chromosome 7)(chromosome 7)

• Failure to pump Cl- ions across cell Failure to pump Cl- ions across cell membranemembrane

• Missing Cl- ion channel protein (cystic Missing Cl- ion channel protein (cystic fibrosis fibrosis

transmembrane protein or CFTR)transmembrane protein or CFTR)

• Normal gene sequence available (cloned in Normal gene sequence available (cloned in 1989)1989)

• Normal gene 250 kb in sizeNormal gene 250 kb in size

• Transcript of 6 kb in sizeTranscript of 6 kb in size

• Gene product of 1480 amino acids in Gene product of 1480 amino acids in lengthlength

• Over 1500 mutations documentedOver 1500 mutations documented

Cystic Fibrosis as Candidate for Gene Cystic Fibrosis as Candidate for Gene TherapyTherapy

1.1. Recessive gene on chromosome 7.Recessive gene on chromosome 7.

2.2. CFTR is the gene product.CFTR is the gene product.

3.3. CFTR is an ion channel protein.CFTR is an ion channel protein.

4.4. Normal gene sequence has been Normal gene sequence has been cloned.cloned.

5.5. Symptoms are localized to specific Symptoms are localized to specific organs.organs.

6.6. Adenovirus (cold virus) can be used Adenovirus (cold virus) can be used to to

deliver normal sequence to lung deliver normal sequence to lung cells.cells.

Human DNA Information: Human DNA Information: Ethical ConcernsEthical Concerns

Who should have Who should have access to your DNA access to your DNA

information?information?