Gene therapyWith Adeno-Associated Virus

Noushin rajaeie

What is Gene TherapyA technique for correcting defective genes that are

responsible for disease development.There are four approaches:

1. A normal gene inserted to compensate for a nonfunctional gene.

2. An abnormal gene traded for a normal gene3. An abnormal gene repaired through selective

reverse mutation4. Change the regulation of gene pairs

Gene therapy requires three things: the identification of thedefect at the molecular level a correcting genea way tointroduce the gene into appropriate host cells

(i.e., a vector).Choices of Vectors For Gene therapy:

• Retrovirus• adenovirus• Adeno-associated virus• Herpes Simplex Virus

Viral vectors:

• Liposome• DNA–polymer conjugates• Naked DNA

Non-viral vectors:

The ideal vector system for gene therapy:(1) an adequate carrying capacity(2) to be undetectable by the immune system(3) to be non-inflammatory(4) to be safe to the patients with pre-existing lung inflammation.(5) to have an efficiency sufficient to correct the cystic fibrosis phenotype.(6) to have long duration of expression and/or the ability to be safely re-administered.

The history of gene thropy: In the 1980:Scientists began to look into gene

therapy. In the 1990:The first gene therapy was performed for

treatment of SCID. In the1993 : Trials using liposome-mediated CFTR

gene transfer In the1995:The first cystic fibrosis gene therapy

clinical trials used an adenovirus vector In the 1998:Trials using adeno-associated virus to

deliver gene.

How Gene Therapy With Virus Works:1) A vector delivers the therapeutic gene into a

patient’s target cell.2) The target cells become infected with the viral

vector.3) The vector’s genetic material is inserted into

the target cell. 4) The therapeutic gene causing the cell to return

to a normal state.

A virus insert its genes into the host cell's genome. This virus has three genes - A, B and C:

1)Gene A encodes a protein which allows this virus to insert itself into the host's genome.

2)Genes B and C actually cause the disease this virus is associated with. 3)Replace B and C with a beneficial gene. Thus, the modified virus could introduce your 'good gene' into the host cell's genome without causing any disease.

AB C a beneficial geneA

virus modified virus

Adeno-associated virus (AAV)A small virus which infects humans and some

other primate species. AAV dose not cause disease AAV causes a very mild immune response.AAV is a candidate for gene therapy. AAV belongs to the genus Dependovirus,turn

belongs to the family Parvoviridae. The virus is a small (20 nm). AAV is a replication-defective, non envelopevirus.

AAV life cycle:AAV undergoes productive infection in the presence of adenovirus co infection. characterized by: genome replicationviral gene expressionvirion production

In the absence of helper virus infection: AAV binds to its

receptor, traffics to the nucleus, and integrates into the distal end of chromosome 19.

In the presence of an Ad coinfection:the AAV genome is replicated as replicative form monomers and dimers (RFM, RFD) yielding progeny virus.

AAV’s life regulated by:

1)complex interactions between the AAV genome and AAV 2) adenoviral 3)host protein

(AAV) vectors:

Adeno-associated viral (AAV) vectors are based on a non-pathogenic, replication deficient member of the parvovirus family with a 4.7 kb single-stranded DNA genome.

AAV-mediated gene transfer is under investigation for treatment of a large number of diseases.

Producing recombinant AAV vectors

The advantages of (AAV)vectors:AAV is non-pathogenic and safe for use as a

vector. All viral genes can be deleted from recombinant

AAV (rAAV) vectors.AAV can efficiently infect both dividing and

nondividing cells in muscle liver, brain, retina , heart , and pancreas.

Vector administration does not elicit a strong cellular immune reaction.

The disadvantages with AAV vectors: limited tissue tropism for serotypes targetedgene delivery to specific cell populations. preexisting immunity due to prior exposure of the

majority of the human population with multiple AAV serotypes and a limited transgene carrying capacity. 

AAV CLINICAL TRIALS:

AAV vectors have been used in over 38 clinical trials worldwide Including: retinal diseasesHemophilia congestive heart failure lipoprotein lipase deficiency Parkinson's diseaseCystic fibrosis

Gene Therapy for Cystic Fibrosis:CF, a lethal, autosomal recessive disease in CF the CF transmembrane regulator

(CFTR) is inactivated by mutationThis mutation leads to the accumulation of

thick secretions in the lungCF is an autosomal recessive disease and is

the most common lethal genetic disease among whites.

Cystic fibrosis as an ideal candidate for gene therapy:

1. it is a single gene defect 2. it is a recessive condition, with heterozygotes

being phenotypically normal3. the main pathology is in the lung, which is

accessible for treatment4. it is a progressive disease with a virtually

normal phenotype at birth

HemophiliaTwo of the required enzymes are factors VIII and

IX; a lack of the former results in hemophilia A, and a lack of the latter results in hemophilia B.

A factor IX AAV vector could be used to “cure” mice with hemophilia B.

Parkinson’s disease• It is a chronic neurodegenerative disease• In Parkinson’s disease, a loss of dopaminergic

neurons leads to the loss of inhibitory gamma aminobutyric acid-sensitive input to the subthalamic nucleus.

• a study in which 12 patients with advanced Parkinson’s disease had an AAV vector carrying a transgene encoding glutamic acid decarboxylase injected into the subthalamic nucleus on one side

References:1. Berns,K.Daya,sh.(2008) Gene Therapy Using Adeno-Associated Virus

Vectors, Clinical Microbiology Reviews, Vol. 21, No. 4:583–5932. Collaco,R.F.Tremp,J.P(2000) A Method for Helper Virus-Free Production

of Adeno-Associated Virus Vectors.3. Kenneth,H. Roland,W.Warrington,J.r (2006)Treatment of human disease

by adeno-associated viral gene transfer, Hum Genet ,119: 571–603.4. Kwon,I.Schaffer,D(2008) Designer Gene Delivery Vectors: Molecular

Engineering and Evolutionof Adeno-Associated Viral Vectors for Enhanced Gene Transfer, Pharmaceutical Research, Vol. 25, No. 3