Transcript Gene therapy With Adeno

Noushin rajaeie
What is Gene Therapy
 A 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 gene
3. An abnormal gene repaired through selective reverse
mutation
4. Change the regulation of gene pairs
 Gene therapy requires three things:
 the identification of thedefect at the molecular level
 a correcting gene
 a way tointroduce the gene into appropriate host cells (i.e., a
vector).
Choices of Vectors For Gene therapy:
Viral vectors:
Non-viral
vectors:
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Retrovirus
adenovirus
Adeno-associated virus
Herpes Simplex Virus
• Liposome
• DNA–polymer conjugates
• Naked DNA
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:
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.
1)
B C
A a beneficial gene
A
virus
modified virus
 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.
Adeno-associated virus (AAV)
 A small virus which infects humans and some other
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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 replication
 viral gene expression
 virion 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 diseases
 Hemophilia
 congestive heart failure
 lipoprotein lipase deficiency
 Parkinson's disease
 Cystic fibrosis
 Gene Therapy for Cystic Fibrosis:
 CF, a lethal, autosomal recessive disease
 in CF the CF transmembrane regulator (CFTR) is
inactivated by mutation
 This mutation leads to the accumulation of thick
secretions in the lung
 CF 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 normal
3. the main pathology is in the lung, which is accessible
for treatment
4. it is a progressive disease with a virtually normal
phenotype at birth
Hemophilia
 Two 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:
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Berns,K.Daya,sh.(2008) Gene Therapy Using Adeno-Associated Virus
Vectors, Clinical Microbiology Reviews, Vol. 21, No. 4:583–593
Collaco,R.F.Tremp,J.P(2000) A Method for Helper Virus-Free Production
of Adeno-Associated Virus Vectors.
Kenneth,H. Roland,W.Warrington,J.r (2006)Treatment of human disease
by adeno-associated viral gene transfer, Hum Genet ,119: 571–603.
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