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Gene Therapy and Viral Vector
Lecture 4
Adeno-Associated Viral Vectors
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Adeno-associated viruses belong to the parvovirus
family.
Small viruses with a genome of single stranded DNA.
These viruses can insert genetic material at a specific
site on chromosome 19 with near 100% certainty.
There are a few disadvantages to using AAV, including
the small amount of DNA it can carry (low capacity)
and the difficulty in producing it.
This type of virus is being used, however, because it is
non-pathogenic (most people carry this harmless
virus).
In contrast to adenoviruses, most people treated with
AAV will not build an immune response to remove
the virus and the cells that have been successfully
treated with it.
Comparison with Adeno Viruses
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Adeno-associated vectors (AAV) are like adenoviral
vectors in their features but because of having some
deficiency in their replication and pathogenicity, are
safer than adenoviral vectors.
In human, AAVs are not associated with any disease.
Another special character of AAV is their ability to
integrate into a specific site on chromosome 19 with no
noticeable effects cause long-term expression in vivo.
AAVs have been used in the treatment of some
diseases, such as CF, hemophilia B, Leber
congenital amaurosis, and AAT (Alpha-1
antitrypsine) deficiency.
Structure of the Genome
The AAV genome is built of single-stranded deoxyribonucleic acid
(ssDNA), either positive- or negative-sensed, which is about 4.7 kilobase
long. The genome comprises inverted terminal repeats (ITRs) at both ends
of the DNA strand, and two open reading frames (ORFs): rep and cap (see
figure 1). The former is composed of four overlapping genes encoding Rep
proteins required for the AAV life cycle, and the latter contains overlapping
nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact
together to form a capsid of an icosahedral symmetry.
 The Inverted Terminal Repeat (ITR) sequences comprise 145 bases each.
They were named so because of their symmetry, which was shown to be
required for efficient multiplication of the AAV genome. Another property
of these sequences is their ability to form a hairpin, which contributes to
so-called self-priming that allows primase-independent synthesis of the
second DNA strand. The ITRs were also shown to be required for both
integration of the AAV DNA into the host cell genome and rescue from it,
as well as for efficient encapsidation of the AAV DNA combined with
generation of a fully-assembled, deoxyribonuclease-resistant AAV particles.
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Recombinant Vector Genome
The use of antisense oligodeoxynucleotides targeted to the reninangiotensin system [hormone system that regulates blood pressure and
water (fluid) system] and adeno-associated virus vector delivery of
antisense DNA offers a new approach to prolonged hypertension
therapy with a single administration.
Drawbacks
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The major disadvantages of these vectors
are complicated process of vector
production and the limited transgene
capacity of the particles (up to 4.8 kb).
Comparison
Cystic Fibrosis
Average life span:25-30 years
Conventional Treatment
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The use of physiotherapy, antibiotics and pancreatic
supplements.
Many patients require treatment four times daily, including a
considerable time spent on physiotherapy.
This combination of treatment has helped increase life
expectancy considerably to the current median of
approximately 30 years of age.
Despite small further improvements, more recently it has
become apparent that there is a need for a more effective
and convenient therapy.
The identification of the gene responsible for CF (the cystic
fibrosis transmembrane conductance regulator or CFTR
protein) in 1989, was responsible for the advent of potential
new treatments for CF.
Gene therapy, the transfer of a normal copy of the CFTR
gene into the lungs of CF patients, was proposed as an
attractive new option.
CFTR Pathway
Cystic Fibrosis, a case study
http://learn.genetics.utah.edu/content/gen
etherapy/casestudy/
 For further reading:
 http://www.intechopen.com/books/genetherapy-tools-and-potentialapplications/targeting-the-lung-challengesin-gene-therapy-for-cystic-fibrosis
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News of Gene therapy in CF
http://www.bbc.com/news/scienceenvironment-32932922
 http://www.nhs.uk/news/2015/07July/Page
s/Gene-therapy-breakthrough-for-cysticfibrosis.aspx
 3 July 2015
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Lectures prepared from
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http://www.genetherapynet.com/viralvector/adeno-associated-viruses.html
Further reading on AAV
Efficient AAV Vector Production System:
Towards Gene Therapy For Duchenne
Muscular Dystrophy
 http://www.intechopen.com/books/genetherapy-tools-and-potentialapplications/efficient-aav-vectorproduction-system-towards-gene-therapyfor-duchenne-muscular-dystrophy
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