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Dissecting HIV-2: From a Study of Genetic Recombination to the Development of a Regulatory System for Gene Therapy

by
Hui-Ling Rose Lee
B.A. 2003
Rutgers University

Thesis Advisor: Joseph P. Dougherty, Ph.D.
Graduate Program in Molecular Genetics, Microbiology &
Immunology

Molecular Genetics, Microbiology & Immunology Department
Conference Room 747
7th floor, RWJMS, Piscataway

Wednesday, July 21, 2010
11:00 am


Abstract

More than 33.4 million people live with HIV/AIDS worldwide, of which one
million are infected with HIV-2. AIDS can be caused by two types of lentiviruses, HIV-1
and HIV-2, both are members of the retrovirus family. One important mechanism that
enables the virus to maintain viral fitness and evade eradication is to generate a wide
genetic diversity through retroviral recombination. HIV-1 has been well documented to
undergo an extremely high rate of crossover. Taking into consideration the similarities and
differences between the two lentiviruses, we carried out experiments to determine the
crossover rate for HIV-2. The results showed that HIV-2 exhibited a crossover rate similar
to that of HIV-1 and MLV, indicating that the extremely high rate of crossover is a
common retroviral feature. The results also suggest that the current model of reverse
transcription should be modified to incorporate multiple crossover events during minusstrand
DNA synthesis.
Currently, the lentiviral vectors used in clinical gene therapy studies are all HIV-1
based. A primary concern for HIV-positive patients is the risk of recombination between
the gene delivery vector and the provirus harbored in the patientís genome. To circumvent
this problem, we decided to develop HIV-2 based self-inactivating (SIN) vectors. This
should ensure that vector mobilization or proto-oncogene activation downstream of any
proviral integration site does not occur. Furthermore, the utilization of HIV-2 based
vectors appears to be safer than HIV-1 based vectors in preventing the occurrence of
recombination especially with the significant sequence divergence between the two types.
A regulatory system where the level of therapeutic gene expression could be
temporally regulated in accordance with the needs of the patient is of crucial importance in
the development of gene therapy, especially since some of the genes cannot be tolerated
constitutively or at certain stages of development due to toxicity. Current systems are
limited in that it requires a co-expression of the relevant transcriptional transactivator along
with the gene to be regulated. This constraint means that the transactivator will be
constitutively expressed within the patient or target of interest, which may provide potential
harm in the long-term. In order to circumvent this limitation we developed a posttranscriptionally
regulated inducible system via the exploitation of the premature
termination read-through mechanism. Pharmacological molecules targeting posttranscriptional
controls were then used as the ON/OFF switch regulators to control the
inducible system in vitro and in vivo in xenogeneic transplantation models.
In general, the basic mechanism of HIV-2 recombination was studied and
subsequently the HIV-2 backbone was exploited as the vehicle for the delivery of a posttranscriptionally
regulated transgene in vitro and in vivo.


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