Gene Therapy

• Understand how “ normal ” (wildtype) genes can be introduced into the cells of a patient through the use of vectors or other delivery methods.• Explain what goes wrong in individuals who are deficient in a specific

protein (enzyme), and outline a possible gene therapy treatment for that specific disease or disorder.• Understand the characteristics of the different vectors used in gene

transfer, and appreciate the mechanisms used by the different vectors during the gene therapy process.• Describe how RNA interference (RNAi) technology is used as a gene

therapy application to treat disease.• Explain the medical risks and potential complications of gene therapy.

Key steps in a gene therapy treatment

Key steps in a gene therapy treatment. (1)Therapeutic (wildtype) gene (blue DNA helix). (2)Therapeutic gene (blue DNA) is inserted into a vector (yellow arrow). (3)The vector carrying the therapeutic gene is delivered into the host cell. (4)The vector DNA travels into the host cell nucleus. (5)The therapeutic gene is transcribed into mRNAs (not shown), and the mRNAs are translated to produce the therapeutic proteins.

Two main approaches used in gene therapyIn vivo gene therapy. Gene therapy performed on cells inside a patient’s body using therapeutic wildtype gene DNA.

Ex vivo gene therapy . Ex vivo gene therapy is performed on disease cells removed from the patient. Different gene therapy vectors and methods can be used to treat the cells depending on the genetic disease. After the cells are treated with the appropriate gene treatment, the cells are returned to the patient.

Two main approaches used in gene therapy

Delivery SystemAdenovirus

Therapeutic genes in viral DNA vectors are delivered into cells. (1)The wildtype (normal) gene is carried by the viral vector, which is packaged with virus capsid proteins into recombinant gene delivery system. (2)The recombinant virus carrying the wildtype gene is used to infect the diseased cells. (3)The viral vector and wildtype gene are inserted (integrated) into the chromosome DNA in the host cell (patient’s) genome. (4)The therapeutic wildtype gene in the cell is expressed as mRNAs and wildtype (normal) proteins

Delivery SystemRetrovirus

Building a retroviral vector. (1)The wildtype retrovirus genome contains viral genes (gag, pol, env) and has longterminal repeat (LTR) regulatory sequences located at the ends of the linear RNA genome. (2)To make the retroviral genome safe for use as a gene therapy vector, the genes that the virus uses to make more viruses during an infection were removed (deleted) from the retroviral genome (gag, pol, env). Without these three genes and their proteins, the retrovirus genome is not infectious. (3)The therapeutic gene needed for the gene therapy was inserted between the LTR sequences at each end of the retrovirus vector. Retrovirus gene therapy vectors retain the ability to integrate into the host cell chromosome, where the vectors can remain integrated in the genome for years without becoming infectious.

Delivery SystemGene Gun

Cells can be transformed using DNA fired by a gene gun. (A)DNA encoding a green fluorescent protein (GFP) gene was introduced into a nerve cell in the brain causing the body of the nerve cell and its extended axons to fluoresce with a green color. (B)Rice embryos were genetically modified by DNA from a gene gun. The transferred DNA carried with it a “ marker ” gene that was expressed in the cells, and caused the rice embryos to have a blue color

Delivery SystemLiposom

Liposomes deliver genes into the cell nucleus. 1.Liposome with DNA inside (DNA-lipid complex). 2.Cell takes up the liposome-DNA complex by endocytosis. 3.DNA-lipid complex enters cell nucleus. 4.Therapeutic gene is expressed in the nucleus.

Delivery System Human artificial chromosome

An artificial human chromosome (*) is shown among many native human chromosomes. The centromere regions of the chromosomes, including the artificial chromosome, show up as two bright dots and the artificial chromosome is a light green color

RNA interference mechanism (RNAi)1. dsRNA is cut into

short interfering RNAs (siRNAs) by Dicer enzyme,

2. RNAinduced Silencing Complex (RISC) binds to siRNA,

3. siRNA-RISC base pairs to target mRNA, and

4. the target mRNA is cut into nonfunctional fragments.

Jesse Gelsinger?

Severe Combined ImmunodeficiencyDisease

Flowchart of ex vivo gene therapy used to treat babies with SCID. The normal ADA gene was inserted into a retroviral vector and packaged into a viral capsid. T cells with the mutant ADA gene were taken from the babies with SCID and treated with the ADA retrovirus vector, which transferred the normal ADA gene into the T cells from the babies. The T cells expressed the wildtype ADA proteins and were transplanted back into the babies where they continued to produce functional ADA enzymes.

Restoring Sight to Blind

Restoring Sight to Blind

• The recovery of visual brain function persisted in one dog for at least two-and-a-half years after gene therapy.

• These amazing studies indicate that blindness in infancy does not permanently alter the structure and function of the blind brain. When the retina can again detect light, the brain can properly process the information and restore vision.

ENZYME REPLACEMENT THERAPY: ANALTERNATIVE TO GENE THERAPY?

Lysosome pathway in the cell. 1.The RER and Golgi are part of the membrane system used by cells to secrete newly made proteins out of the cell, or to incorporate the proteins into the plasma membrane. 2.Lysosome vacuoles vesicles) contain many different degradation enzymes (proteases, lipases, nucleases, and polysaccharidases)