Genetic Basis of Human Disease - Gene Therapy

______ ______ is a novel therapeutic branch of modern medicine for correcting a defective disease-causing gene

gene therapy

Types of Gene Therapy → Cell Types

1. ______ gene therapy: therapeutic gene —> somatic cells —> NOT inherited

2. ______ gene therapy: therapeutic gene —> germ cells —> inherited to later generations

1. somatic

2. germline

Types of Gene Therapy → # of Defective Copies

1. ______ ______ therapy: replacement of homozygous mutant gene

2. ______ ______ therapy: correction of heterozygous mutant gene

1. gene replacement

2. gene augmentation

______ ______ ______: a blocking gene is introduced to eliminate the activity of a gene that promotes the growth of disease-related cells (e.g., cancer causing genes, for example activation of an oncogene)

gene inhibition therapy

Gene therapy to kill specific cells can be achieved in one of two ways:

1. Introduction of “______” gene.

2. Introduction of “______” gene.

1. suicide

2. marker

______ gene therapy: introducing viral or bacterial genes to tumor cells —> allows conversion of a non-toxic compound (converts the pro-drug ______ to ______) to a lethal medication.

suicide, 5-fluorocytosine (5-FC), 5-fluorouracil (5-FU)

______ gene therapy: causes expression of a protein that marks the cells so that the diseased cells are attacked by the body’s natural immune system.

marker

Steps in Gene Therapy (5)

1. Defective gene identification

2. Cloning the identical healthy gene (therapeutic gene)

3. Loading the vector with the therapeutic gene

4. Gene delivery to the target cell

5. Functional activity (augmentation, suppression or repair)

Step 2: Cloning

• DNA cloning is the process of making multiple, ______ copies of a particular piece of DNA.

• ______ ______ cut open the plasmid and “paste” the therapeutic gene via ______ ______.

• the plasmid is introduced into bacteria (or other cell types) via a process called ______

• bacteria with the ______ (correct/incorrect) plasmid are used to make more plasmid DNA

• identical

• restriction enzymes, DNA ligase

• transformation

• correct

Step 3: Loading the Therapeutic Gene in Vectors for Gene Delivery

• The ______ acts as a vehicle to carry the therapeutic DNA into the cells of a patient with a genetic disease.

• Types of Vectors:

  1. ______ vectors —> (e.g., retrovirus, adeno virus, etc.)

  2. ______ vectors —> (e.g., naked DNA, liposome, etc.)

• vector

• viral

• non-viral

• ______ ______ gene therapy or direct therapy: direct delivery of a therapeutic gene into the cells of a particular organ in the body via viral or non-viral vector.

• ______ ______ gene therapy or cell-based delivery: transfer of therapeutic gene into cultured cells and then introduction of genetically modified cells into the body.

• in vivo

• ex vivo

Advantages of non-viral vectors > viral vectors (4)

• Have lower risk for immune reaction and cytotoxicity

• Have the potential to deliver larger genetic payloads

• Are cost effective and easier to synthesize

• Have no risk of insertional mutagenesis

Disadvantages of non-viral vectors > viral vectors (2)

• Have lower efficiency of transfecting host cells

• Have higher risk of degradation by nucleases

Targeted Gene Therapy to Treat Cancer: ______ ______

• a new receptor is added to a patient’s ______ ______

• CAR = ______ ______ ______

• once the T cell has the CAR —> ______ ______

• in 2017, FDA approved CAR T cell therapy for ______ ______ ______ and ______ ______

• CAR-T cell

• T cells

• chimeric antigen receptor

• CAR-T cell

• acute lymphoblastic leukemia, advanced lymphomas

Gene Therapy to Cure LCA

• about 2,000 individuals in the U.S. have ______ ______ ______

• retina degenerates due to mutation in ______ gene

• Spark Therapeutics clinical trial results demonstrated 93% Phase III subjects (27 out of 29 patients) responded ______ (positively/negatively) —> EXPENSIVE —> $425,000 PER EYE.

• Leber’s Congenital Amaurosis 

• RPE65

• positively

Gene Therapy to Treat AIDS

• HIV needs ______ to enter CD4 cells, so gene therapy blocks or removes it to stop the virus from infecting T cells.

• ZFN gene therapy —> ______ ______ —> cuts out the CCR5 gene from the surface of CD4 T cells, making them resistant to HIV, and then these modified T cells are returned to the patient.

• CCR5

• ZFN overexpression

Challenges of Gene Therapy (4)

• Delivering the therapeutic gene to the right place and switching it on are major challenges

• Therapeutic gene can induce an immune response in the patient

• There is a risk that the therapeutic gene may disrupt the activity of a normal gene

• Gene therapy is expensive

• ______ ______ ______ —> a procedure in which a patient receives healthy stem cells to replace damaged stem cells

• Stem cells have the ability to ______ into a large number of cell types

• Stem cells can be used for ______ ______

• stem cell transplation

• differentiate

• gene therapy

Types of Stem Cell Transplantation

• ______ → the donor is an identical twin of the patient

• ______ → the patient’s own marrow or peripheral blood is used

• ______ → an allogeneic donor is one other than the patient

• ______ → donor is a different species

• syngeneic

• autologous

• allogenic

• xenotransplant

stem cells have the capacity to:

1. ______ ______ → produce unaltered daughter cells

2. differentiate into specialized tissue cell types (potency)

   • ______ → can generate all cell types

   • ______ → can generate a limited number of cell types

   • ______ → can generate only one cell type

• self-renew

• pluripotent

• multipotent

• unipotent

Types of Stem Cells

• ______ stem cells → high capacity of self-renewal and pluripotent

• ______/______ stem cells → multipotent; hematopoietic or intestinal stem cells

• ______ ______ stem cells → pluripotent stem cell derived from adult
  somatic cells by overexpressing Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28

• embryonic

• adult/somatic

• induced pluripotent