This document discusses the use of adeno-associated virus (AAV) as a vector for gene therapy. AAV is a promising delivery method due to its low immunogenicity, ability to target specific cell types, and lack of pathogenicity. The document focuses on how AAV may be used as a therapy for cystic fibrosis, cancer, and heart disease. It summarizes challenges with AAV therapy but concludes that AAV vectors appear to be among the safest methodologies for further developing therapies for many currently incurable diseases.

1. 2012
Molecular Biotechnology,
University of Central
Florida
Regina Gates
[THE USE OF ADENO-ASSOCIATED VIRUS
IN CYSTIC FIBROSIS AND OTHER HUMAN
DISEASES]
Viruses have a long use in the field of biotechnology, from bacteriophages, to plant viruses, to human
viruses. Human viruses have been experimentally retooled to treat human diseases.

2. Introduction
Viruses are widely used in the field of biotechnology, allowing transduction of various
species among a great variety of species. Applications exist in the laboratory and in clinical
practice, for the purposes of genome illumination, proteomics, diagnostics, including
nanodiagnostics, and therapy. In humans, deadly and nonlethal viruses have been retooled to
deliver new genes to replace defective ones. Human gene therapy stands to be the holy grail of
biotechnology, but its long history is spotted with inefficiency and even death. For instance,
Jesse Gelsinger had managed his Ornithine Transcarbamylase Deficiency (OTCD) during a
clinical trial in 1999, died at the age of 18 after being administered gene therapy to replace the
defective gene (Obasogie).
In spite of this, or perhaps, because of it, researchers continue to develop approaches and
increase our understanding of gene therapy, in the hopes that it will be the panacea the world is
looking for. Yet, while gene therapy is still a major focus of biotechnology, there are other
applications for viral therapy, such as delivery of RNAi and oncolytic genes. The focus of this
paper is the Adeno-Associated Virus (AAV) as a delivery vector for various therapies, why it is a
leader among viral vectors, and why this therapy may or may not be superior to other delivery
methods, with greater focus on Cystic Fibrosis, cancer, and heart disease.
Adeno-associated virus is a dependovirus of the Parvoviridae. Dependovirus is a genus
of Parvoviridae that are unable to replicate without the presence of a helper virus, the role of
which can be fulfilled by adenoviruses (where AAV gets its name), herpesviruses, or vaccinia.
Parvoviridae is a family of viruses which possess a single-stranded DNA genome. Parvoviruses,
one of the two genera of Parvoviridae, are responsible for childhood ailments and can cause
major disease in adults. The Adeno-associated virus itself, however, is not associated with any
disease, even in the presence of a helper virus (Siegel).
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3. Discussion and Conclusions
Adeno-associated virus is a promising delivery method for various therapies. It has low
immunogenic effects, and tends to be well-tolerated. While it does not have a large payload
capacity, methods are being devised to be as efficient as possible, including taking advantage of
AAV’s self-priming attribute of its 5’ and 3’ ITRs.
AAV’s surface proteins allow it to be targeted to various cell types, naturally.
Researchers can take advantage of this, engineering AAV vectors that will go to the specific cell-
type for integration. This makes AAV a versatile candidate for a variety of diseases, from Cystic
Fibrosis to cancers to heart disease, and many others, from the muscular system to the central
nervous system.
That it is not associated with any known disease means that AAV is inherently safe for
at-risk patients, helping to improve quality of life for even patients who require a very aggressive
therapeutic approach, where the therapy stands to do collateral damage, and not just targeting the
disease.
Given that AAV has two phases in its lifecycle, clinicians have options in their approach
to disease eradication. They can allow the vector to reside in the cell extrachromasomally, in the
lytic cycle. In this way, it is always ready to be transcribed and translated. In the lysogenic cycle,
it integrates into the host genome for long-term effectiveness. Recovering the vector seems to be
an issue, however. Recovery genes would need to be included in the construct, diminishing
payload capacity that much more.
Immunogenicity does not appear to be an issue for the patient, in terms of immune
system reaction. While there is an immune response, it does not appear to affect the patient’s
overall health adversely. Rather, the main concern is inactivation of the viral vector, rendering
the therapy ineffective. Methods have been employed to side-step this possibility, however,
which include growing up libraries of variants and amplifying them in the presence of mouse
monoclonal antibodies against the wild type exhibiting the epitope.
Many challenges have arisen in the pursuit of using AAV as a vector for therapeutic
delivery. In the case of Cystic Fibrosis, suitable models were difficult to find. There seems to
remain the problem, also, that the mucus build-up in the lungs interferes with transduction of the
lung cells.
Control of the vector may be an issue in cases where the vector is allowed to grow up in
the presence of disease, such as in the case of cancer. When the control is external to the host,
titration may be of concern to both the clinician and the patient. However, the specificity of the
vector, along with the fact that its tumor-dependent growth may be self-terminating, may
circumvent this issue.

4. Biotechnology Perspectives
Gene therapy and gene-control therapy seem to be a hotbed of research, especially where
Adeno-associated virus is concerned.
There are many issues regarding such therapies, however. A black mark was placed on
the field when gene therapy resulted in the deaths of research patients. However, researchers
seem determined to work out the issues that plagued them 13 years ago.
Choosing known delivery systems, such as AAV, which has shown no pathogenesis may
help ease the minds of those who have concerns of the safety of these therapies. Reducing the
immunologic response in patients can only help to ensure the safety of the therapy in the minds
of the public, and also boost the efficacy of the delivery system.
A natural product as a therapy can also help researchers, clinicians, patients and other
concerned individuals rest knowing that this is something that already occurs in our bodies. As
the environment is usually a concern in the case of biotechnology, AAV vectors have an
advantage over similar approaches using nanoparticles as delivery vehicles. What happens when
nanoparticles get into the environment? What if they are broken down in the body, instead of
outside the body? With AAV, we know the answer, already. We know, too, that even if it
integrates into the host genome by accident, it localizes in a specific position. At least, the wild-
type does, and the recombinant vectors are being engineered to do so, as well.
The sheer number of diseases being studied with this construct is encouraging. Among
methodologies, AAV vectors seem to be among the safest. Even compared to naked DNA
administration, AAV vectors seem to have many advantages, including specificity, ease of
developments, greater efficacy, and comparative safety levels.
In time, a large number of these diseases may be eradicated, but there is yet more work to
be done on the existing research. More optimization, more efficacy, more targeted delivery, and
once these are established, more diseases can be researched with such methods.

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