Project Details


DESCRIPTION: (Adapted from Investigators' Abstract) The ultimate goal of HIV-1
directed gene therapy is to inhibit virus replication and the development of
AIDS. Clinical studies have focused on human cell and gene transfer for this
purpose, and studies have revealed both a low frequency of marked cells and a
very low level of gene expression. Thus, several essential issues will need to
be addressed before gene therapy will become a treatment reality, particularly
for HIV-1 infected children. As an alternative to ex vivo transduction of
hematopoietic stem cells (HSC), these investigators propose to investigate the
in vivo transduction of HSC in their native environment, the milieu of the bone
marrow. Studies in uninfected rhesus neonates will initially be performed to
determine the requirements for efficient gene transfer to the HSC. The
investigators will explore the most effective vector system (Moloney murine
leukemia virus {MLV} versus HIV-1-derived lentivirus), the effects of cytokines
on the in vivo transduction efficiency of HSC, and the number of intramarrow
infusions necessary for efficient marking of HSC and their progeny (Specific
Aim 1). Newborns will be transferred intramarrow with either MLV/VSV-G or
HIV/VSV-G each marked with the enhanced green fluorescent protein (EGFP) as a
reporter gene. Blood and marrow will be collected 2 weeks then monthly
post-transfer for 12 months. Samples will be assessed for transduction and
expression (flow cytometry, PCR, hematopoietic progenitor assays). While it is
clear that juvenile/adult animals generate a potent immune response to
retrovirus vectors and their transgenes, the immune consequences of the
proposed studies are uncertain due to the age of the recipients, and will be
investigated (Western blots, antibody titers, lymphocyte proliferation assays.
The investigators will then utilize the best in vivo transduction approach
developed in Specific Aim 1 to deliver an antisense Tat/Rev gene cassette to
the HSC of SIV-infected rhesus neonates (Specific Aim 2). Siv-infected newborns
on antiretroviral chemotherapy (PMPA) will receive intramarrow transfers, and
blood and marrow collected 2 weeks than monthly post-transfer for 12 months.
The presence of the vector transduced cells in samples will be detected by
Southern blot of DNA-PCR, and expression measured by Northern blot, RT-PCR, or
FACS analysis. Plasma- and PBMC-associated veremia, and levels of SIV in plasma
will be quantitated (bDNA assay). Studies will focus on the generation of
anti-SIV gene-marked HSC and mature lymphohematopoietic cells, and the
expression of the antiviral elements(s) in these cells. Thus, the goal of the
investigators is to explore a safe and efficacious approach for pediatric gene
therapy of HIV-1 infection targeting HSC using in vivo transduction for the
delivery of antiviral genes. These studies are directly relevant to infants and
adults infected with HIV-1.
Effective start/end date1/07/0031/05/01