Any procedure intended to treat or alleviate disease by genetically modifying the cells of a patient

(Human Molecular Genetics)

Gene therapy involves delivery of genes that:

 Replace the defective genes causing the disease  Drives the cells with the defective genetic

structure to death

Genes, gene segments or oligonucleotides

Genetic disorders Cancer (oncogenes, tumor-suppressor genes, apoptosis genes) Infectious diseases (viral or bacterial) Immune system disorders (allergies, inflammations and also autoimmune diseases) Cardiovascular diseases Neurological diseases

In vivo gene therapy Direct delivery of genes

http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture25/Lecture25.html

In vitro gene therapy (Ex vivo gene therapy)

Removal of patient s cells, transfection of cells with the specific gene, re-introduction of the cells to the body

http://library.thinkquest.org/28000/load_image.php3?id=50

Gene augmentation therapy (GAT)

 Loss-of-function diseases autosomal recessive  Additional copies of wildtype gene  Enhanced gene product  Even slight expression make a considerable

difference no requirement for high expression

Example;
 Hemophilia

http://www.nwabr.org/studentbiotech/winners/studentwork/2007/WB_BA_TRONGTHAM/9_developing cures.htm

Direct killing of the disease cells

 Transgene expression cause cell death

Suicide genes: encode lethal toxins Prodrug genes: confer sensitivity to a subsequent drug treatment

or Indirect killing by provoking immune response against target cells

Targeted killing of diseased cells is popular with cancer treatments

Targeted Mutation Correction

 Gain-of-function diseases where GAT has no

help
 At gene level: via HR  At mRNA level: therapeutic ribozymes & RNA Editing

Targeted inhibition of gene expression

 Novel or inappropriate gene expression in

diseased cells  Block expression at either mRNA or protein level

ODN, oligodeoxynucleotide; TFO, triplex-forming oligonucleotide

Classical GT aims the introduction of genes that;

 expresses a product that the patient lacks  kill diseased cells directly by expressing lethal toxins  activate cells of the immune system to attack

diseased cells

Non-classical GT aims
 to inhibit the expression of genes related to

pathogenesis  to restore normal gene expression via correction of the mutation

High expression is usually desired Thus, cDNA with flanking regulatory sites are introduced Upon transfer, the inserted genes may
 integrate into the chromosomes of the cell  remain as extrachromosomal genetic elements --

called episomes

Long-term stable expression

In each cell divison, the inserted gene will be replicated too !

Also has drawbacks! Integration is almost alway random

 No expression of the inserted gene if integration

occurs on heterochromatin  Insertional inactivation of an essential gene resulting in cell death  Activation of oncogenes cancer development  Inactivation of tumor-suppressors or apoptosis genes cancer development

What can you do then?

 Ex vivo delivery screen the cells before after the

genome integration

For treatment of actively dividing cells, longterm expression and stable transmission to progeny cells may be problematic
 Repeated application

Thus, episomal expression is usually employed for targeted killing of the disease cells once they re dead, you don t need the therapeutic anymore

Gene Therapy

Reverse-transcriptase activity & genome integration up to 8 kb of exogenous DNA

Nucleus excludes preintegration complex Thus, only actively dividing cells are targets
 Certain blood cells  Cells lining the

gastrointestinal tract

Then, what s the use of retroviruses if target cells are limited?

 Ideal for cancerous cells of otherwise non-dividing

cells targeted killing approach

 Replication incompetent Transduction (infection + integration) capable

http://www.genetherapyreview.com/gene-therapy-education/gene-transfer-vectors/1-viral-vectors/8retrovirus.html

DNA viruses of the upper respiratory tract infections

 Receptor-mediated endocytosis  High transduction efficiency  Can infect non-dividing cells  Large viruses

accept large inserts, Up to ~35 kb

Episomal expression transient, may require repeated application Infects both dividing and non-dividing cells not suitable for targeted killing strategy Virtually non-specific May trigger serious inflammatory responses!

ssDNA viruses Requires co-infection by a helper virus to replicate

 In the absence of such a helper virus, AAVs integrate into

the host genome  Any subsequent infection by a helper virus can activate its reproduction

Safe - 96% of all AAV genes can be removed Long-term expression Up to ~4.5 kb

Complex retroviruses inc Capable of transducing non-dividing cells

 For HIV, preintegration complex contains nuclear

localization signal  Thus, HIV is actively transported through nuclear pores during interphase

Neurons, macrophages, lymphocytes, hematopoietic stem cells, retinal photoreceptors, muscle and liver cells

They do not trigger immune responses

Gene Therapy

Enclosed circle of synthetic lipid bilayers Endocytosis by the plasma membrane No size limit for transgenes, easy to prepare Pitfalls
 Episomal expression

transient  Low delivery efficiency

Injection by a syringe into a specific tissue

 Early mouse model: intramuscular injection of a

dystrophin minigene for DMD

Biolistics Naked DNA-coated metal particles Relatively safe Low efficiency Low level of integration

Reversible attachment of DNA to a targeting molecule Interaction with specific receptor for the targeting molecule Lysosomic degradation is a threat!

Akhtar S,2006, Non-viral cancer gene therapy: Beyond delivery

http://www.nature.com/gt/journal/v13/n9/full/3302692a.html

http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture25/Lecture25.html

Which route to take?

http://journals.cambridge.org/fulltext_content/ERM/ERM1_11/S1462399499000691sup007.pdf

GENE THERAPY APPLICATIONS

http://www.nature.com/nrg/journal/v4/n5/fig_tab/nrg1066_F2.html

Serious Combined Immunodeficiency

 25% of cases are autosomal
associated with chromosome 20, ADA mutation

 X-linked SCID
mutation in a receptor gene

 Polygenic

Treatment
   

Isolation from all pathogens Bone-marrow transplant Enzyme replacement GT

1990, 1st trial ADA adenosine deaminase

 ADA mutants: Adenosine compounds are

accumulated induce cell death in immune system cells  ADA gene is small and T-cells are easy to culture  Ex vivo GT

1990, Ashanthi DeSilva

 MoMLV (Retrovirus)

Simultaneously, enzyme replacement was continued.

Thus, there is a little controversy concerning the actual action of GT

2003

Bubble Boy David Vetter (1971-1984)

 Lived in a sterile plastic bubble for 12 years!  No B-cells, T-cells, NKs

Sadly, David died following bone marrow transplant from his sister

- His sister was later revealed to be + for EBV

SCID development

http://www.nature.com/ni/journal/v11/n6/pdf/ni0610-457.pdf

Alain Fischer, 2000

 10 affected babies 1-9 months IL-2 receptor- K gene is defected Kc cytokine receptor subunit is impaired  Retroviral application for 3 days Kc cDNA insert

2 out of 10 babies developed leukemia! 1 died

Ornithine transcarbamylase
 important enzyme in urea metabolism
OTC deficiency leads to hyperammonia in blood stream Affected people should avoid high protein diets

1999, Jesse Gelsinger

 Mosaic individual for OTC deficiency  Relatively mild symptoms

Volunteered for a clinical trial

 Single shot on hepatic artery using Adenoviral vectors  Fewer & sudden decrease in platelet count in 12 h  Death in 3 days as a result of Systemic Inflammatory

Response Syndrome

For more information: Human Molecular Genetics 2

http://www.ncbi.nlm.nih.gov/books/NBK7569/#A2865