Gene Therapy

Gene Therapy Uses genetic material as a therapeutic agent to combat genetic disorders Single Gene Diseases:
Cystic Fibrosis
Haemophilia
Muscular Dystrophy Most common approach : insert a normal copy of a gene into a diseased cell to compensate for the mutated one Targeted Cells Somatic Cell line

Germ Cell line GENE THERAPY Hala Al-Hafez
Nick DeGasperis
Katrina Farrell
Alyssa Pastic Candidate for Gene Therapy? 3) Do we know the affected tissues? 4) Do we know how the mutation affects protein function? 5) Will adding a normal copy of the gene solve the problem? 1) Is it a genetic condition? 2) Do we know the sequence of the gene? 6) Is the tissue accessible? If all conditions are met, gene therapy can be used! Vectors Viral Vectors Non-viral Vectors Retrovirus Adenovirus Adeno-associated virus Adenovirus

Double stranded DNA

Targets dividing and non-dividing cells

DNA does not integrate into host genome

Size is 7-8 kilobases Retrovirus

Single stranded RNA

Targets only dividing cells

Integrates RANDOMLY into the host genome

Genome size is 7-8 kilobases Naked DNA Naked DNA

Circular double stranded DNA

Non-specific targeting

Does not integrate into host genome Lipoplex Lipoplexes

Artificial vesicles composed of a lipid bilayer

Contains DNA of interest

Does not integrate into host genome Adeno-associated Virus

Single stranded DNA

Targets dividing and non-dividing cells

May insert into a specific region in chromosome 19

Dependent on helper virus

Genome size is 4-5 kilobases Pros of Viral Vectors Pros: More efficient in transfection

More easily targeted to specific tissues

Potential long term effect Cons: Potential to trigger an immune response

Limited to the size of the genome Methodology 1) Production of the viral vectors: Therapeutic gene is obtained

Pathogenic genes removed from virus

Therapeutic gene is ligated into viral genome

Recombinant viral genome inserted into "package cell lines"

"Package cell lines" mass produce non-pathogenic viruses that have the therapeutic gene

Cells are lysed and centrifuged to isolate viruses

Viruses are injected into diseased cells HEK293 cells 2) Deliver and target specific cells: Engineer surface proteins to target specific cells

Can inject in two ways :
a) in-vivo

b) ex-vivo

Cell infection is by endocytosis or membrane fusion Large Scale Paper Efficiency of eight different AAV serotypes in transducing rat myocardium in vivo Palomeque et al., 2007 Gene Therapy ,14: 989-997 Background

Gene therapy is a great treatment option for heart malfunctions.

AAV's can have different tissue targets determined by:

surface proteins
charges
hydrophobicity
specific affinity for extracellular matrices Purpose To look at the efficiency of 8 different adeno-associated viruses in transfecting heart cells. Method LacZ was inserted as the "therapeutic gene"

8 different serotypes of AAV were injected into rat hearts to see which ones were best suited to transfect heart cells 3 tests were used to determine transduction efficiency of each serotype:

B-galacosidase activity:
luminometer -> bioluminescent assay

X-gal:
blue/white test -> digested indole turns blue

qPCR of LacZ and AAV capsid genes


Each method was tested 1, 4, 12, and 24 weeks after transfection Results AAV5 was interesting because it was not previously known to target heart cells because the only other study of it involved GFP localization for analysis (LacZ insertion proved to be more accurate!).


No NAb's were found against any of the serotypes (which is good in rats, but maybe not in other organisms..?) Small Scale Paper Adenoviral delivery of human CDC5 promotes G2/M progression and cell division in neonatal ventricular cardiomyocytes

Williams et al., 2006

Gene Therapy, 13: 837-843 Background Cardiomyocytes are heart muscle cells

Cell division in cardiomyocytes ceases in neonatal period hence, no regeneration

Cardiomyocyte death causes an indefinite decrease in functional capacity

To determine whether recombinant adenoviruses can be used to overexpress hCDC5 in conjunction with E1A and E1B in cardiomyocytes in an attempt to induce cell division In past research:

Terminally differentiated cardiomyocytes have been induced to re-enter the cell cycle by overexpression of E1A and E1B viral proteins

BUT – these cells could not move past G1/S phase, DNA synthesis was achieved but entry into G2 and mitosis was blocked

New research: Cell division cycle -5 protein (hCDC5) is necessary for G2 progression and mitosis entry Purpose Methods E1A, E1B and hCDC5 genes were packaged into non-virulent adenoviral serotype 5 vectors

The vectors were injected into rat cardiomyocytes (ex vivo)

Successful transfection and expression of recombinant E1A, E1B and hCDC5 in cardiomyocytes verified by RT-PCR and immunoblot assays Results Overexpression of E1A/E1B and hCDC5 caused:

Nuclear localization of cyclin-dependant kinase 1 and cyclin B1
Increase in number of cardiomocyte nuclei
Increase in mitotic index
Increase in cardiomyocyte cell number Conclusions Overexpression of hCDC5 together with E1A and E1B can successfully drive cardiomyocytes through cell division

Potential treatment for heart disease patients in the future?

HOWEVER – cell numbers suggest that only one round of cell division occurred, so adenoviral vectors would have to be administered after every cell division in order for this treatment to be effective Limitations of Gene therapy Everything about the gene must be known

Potential for vector insertion into non-target cells

Regulation of gene expression is extremely complex

Risk of re-incorporation or reversion of pathogenic genes

Retroviruses insert at random

Ethics and Cost Advantages of Gene Therapy Treat problem from the root

Potentially no side effects

Potential to treat diseases that are not curable by drugs Thank you for listening!
Hope you enjoyed it. Limitations: Advantages: Cardiovascular disease is often caused by a decrease in contractile ability.


Contractile dysfunction is caused by mutations in genes that code for calcium handling proteins and other ion pumps in heart cells. AAV8 was most successful, while AAV3 and 4 were least efficient AAV8 was the only one that did not plateau (transfection efficiency) before the end of the experiment (proves that humoral immunity [genetic reversion] was not an issue for AAV8)

AAV 2 and 5 showed a slight decrease after the plateau (indicating possible humoral immunity). Serotypes 7 and 8 were shown to have different distributions within cryosections compared to the others, indicating specific tropism within the heart Cons of viral vectors