Several factors must be considered for a disease to be a candidate for gene therapy:
-single gene causing a genetic disorder
-identified The normal and mutated genes must be well-studied
-disease caused by the mutation
must be well-understood
-gene delivery method must be available
-produces an immune response?
-delivered to the correct cells
-integrated into the host chromosome
For a disease to be a candidate for gene therapy...MAJOR ITEM
gene must be turned on (transcription) at the right time, and is also regulated properly
There are two major strategies for the insertion of genes into cells.
Ex vivo and In Vivo
Ex vivo gene therapy BIGEST ISSUE
The transplantation of the cells is the biggest technical problem
EX VIVO ADVANTAGE OVER IN VIVO
advantage over in vivo gene therapy is that rejection does not occur if the person’s own cells are used
EN VIVO....4 STEPS
Cells are removed from the body
The gene of interest is inserted into them
The cells are cultured for reproduction
The cells are returned to the body
IN VIVO...2 ISSUES
The transferred gene is unstable and the product produced temporarily
The methods are not as controlled as ex vivo gene therapy because cells are not removed from the body
Retrovirus:
RNA viruses that only infect ------ human cells
DNA of interest can only be up to ----- kb in size
dividing
8
NAME 6 WAYS TO TRANSFER DNA INTO CELLS
RETROVIRUS
ADENOVIRUS
ADENO-ASSOCIATED VIRUS
HERPIES SIMPLES VIRUS
LIPOSOMES
NAKED DNA
WHAT VIRUS CAN INVECT DIVIDING AND NONDIVIDING CELLS?
ADENOVIRUS AND ADENO-ASSOCIATED
Can engineer proteins on the virus surface to target specific cells
VIRUS TYPE?
ADENOVIRUS
ADENOVIRUS:
DNA of interest can only be up to ----- kb in size
7.4 KB AND PROTEIN WILL BE HIGHLY EXPRESSED
Does not integrate into the genome, so there is low risk of mutation...WHAT VIRUS?
ADENOVIRUS
95% of the time DNA integrates into a region in chromosome 19...WHAT VIRUS
ADENO-ASSOCIATED
DNA of interest can be up to 5 kb..WHAT VIRUS ?
ADENO-ASSOCIATED
Need a helper virus to infect host cells
WHAT VIRUS TYPE?
ADENO-ASSOCIATED
DEFINE LIPOSOMES
membrane-bound spheres that contain the DNA of interest...DELIEVERY METHOD
1ST GENE THERAPY LANDMARK
1990 4YR OLD GIRL WITH SCID.
treated with T-cells with adenosine deaminase gene in stem cells that permanently corrected the genetic disorder
The therapy had to be repeated because the enzyme would be produced for only a few
months and then stopped
More than 50% of the T cells in the girl contained the corrected gene
2003 her body still actively produces ADA
WHAT THE HELL IS SCID?
Defective adenosine deaminase (ADA) gene
= immune malfunction
Allows for build-up of toxic metabolic by-product
2ND GENE THERAPHY CASE
11-year-old girl
Immune system developed a reaction against the virus
Only 0.1% to 1.0% of her cells produced ADA
Therapies aimed at lowering LDL by transfecting liver cells with the gene for the LDL receptor using
---- & ------- vectors
retroviral and adenoviral.
IN SOME CASES....EX VIVO...BAD RECIEPTOR GENES.
Transfected ----------- have not been inserted into the liver with much success
hepatocytes (liver cells)
Only about 10% of the cells incorporating into the liver.
CF transmembrane transconductance regulator (CFTR) in the plasma membrane of a cell .....WHAT ORGANS?
airways,
intestines,
pancreas
NAME TWO TREATMENTS FOR CFTR
small amount of molecules need to be produced per cell
Spraying adenoviruses with the CFTR gene into the nose has had only temporary treatment
spraying a DNA-liposome aerosol into the nose, but it may not affect distant organs
CF transmembrane transconductance regulator (CFTR) in the plasma membrane of a cell..HOW DOES IT WORK?
NAME ORGANS USED IN GENE THERAPY
LIVER
LUNG
hematopoietic (BLOOD)
Circulated Gene Products (HORMONE IN CAPSULES)
DEFINE Hematopoietic diseases
Cells of circulatory system are produced by pluripotent, undifferentiated hematopoietic stem cells in bone marrow
Erythrocytes, leukocytes and platelets are made in response to erythropoietin and colony stimulating factor
Any stem cells that are transfected would give function for life of the individual
WHAT ARE Circulated Gene Products?
Encapsulated recombinant cells
-Uses include timed production of human growth hormone inside the capsule
Is Gene Therapy Safe?
NAME 2 CASES
Gelsinger An 18-year-old with ornithine transcarbamylase (OTC) deficiency. vectors used formed clotts--> death.
SCID/ Leukemia:
Retrovirus used inserted the DNA next to an oncogene (a gene that can lead to cancer) in a white blood cell-->uncontrollable growth
T OR F ?
No one has completely been cured by gene therapy
TRUE
The first commercially licensed gene therapy.
In October 2003 in China, under the name Gendicine,
after 5 years of clinical trials.
Treats head and neck squamous cell carcinoma.
ONLY SIDE EFFECT...LOW FEVER
The first commercially licensed gene therapy...WHAT VECTOR?
An adenovirus vector containing the
p53 tumor suppressor gene; Can be used along with chemotherapy and radiation therapy to increase effectiveness.
DEFINE dominant negative gene
Mutated gene can prevent a normal protein from functioning properly.
Cannot be corrected by simply inserting the correct gene
DEFINE SMaRT
Spliceosome Mediated RNA Trans-splicing.
Instead of correcting the gene, the region of mRNA that is affected is repaired
HOW DOES SMART WORK?
RNA strand pairs with intron (by base pairing) next to mutated region (exon) into cells
modified to contain the correct exon and a small region that binds to the neighboring intron.
strand binds to the intron, the duplex (section of double-stranded RNA) causes the spliceosome to cut and remove the intron and the defective exon from the mRNA.
exons are joined, with the corrected exon ligated into the mRNA, thereby generating a functional, mature mRNA and protein
single-stranded string of nucleotides, 15-21 bases long binds to the groove between the double strands of DNA where the mutated gene is used---TRIPLE HELIX FORMS..BLOCKS TRANSCRIPTION..NO mRNA.CORRECT GENE INTO CELL PRODUCE PROTEIN.
