Blood, 15 January 2002, Vol. 99, No. 2, pp. 397-397
Lentiviruses get specific
Lineage-specific and high-level expression of transgenes
introduced into hematopoietic stem cells have been the holy grail of
gene therapy for over a decade. Initial excitement and competitiveness to be first quickly faded into frustration and disappointment as 2 major hurdles were identified using standard retroviral vectors. First,
early human clinical trials demonstrated the deficiencies of murine and
in vitro models for predicting gene transfer efficiency into true
repopulating stem cells, and levels of gene transfer were far below
those required for therapeutic efficacy. Second, the inclusion of
elements required for high-level and tissue-specific expression in
retroviral vectors, studied most extensively in relation to hemoglobin
transgenes, resulted in unstable vector genomes and resulting low
vector production.
Progress toward clearing the first hurdle began several years ago, when
relatively simple modifications in culture conditions used during
transduction of primitive nonhuman primate and human hematopoietic
cells increased vector uptake and integration. These included the use
of flt3 ligand in combination with other early-acting cytokines
to stimulate the required passage through the cell cycle while
retaining in vivo engraftment ability, and use of a supportive surface composed of a carboxy-terminal fragment of fibronectin, important both for colocalizing vector particles and primitive cells,
and maintaining stem cell properties of cultured cells. These advances
resulted in encouraging results in nonhuman primates and, finally, an
unequivocally successful human clinical trial, with correction of the
immune defect in children with X-linked severe combined
immunodeficiency by Fischer and coworkers in France.
But overall levels of stem- and progenitor-cell retroviral transduction
are still insufficient for many applications, and recent interest has
focused on the development of safety-modified lentiviral vectors, based
on their ability to transduce cells without requirement for nuclear
membrane breakdown and passage through the M phase of the cell cycle.
Results in murine-human xenograft models are encouraging, and extensive
modifications of the HIV sequences in the helper and vector genomes at
this point reassure investigators that recombination and productive HIV
infection using these vector systems is extraordinarily unlikely. But
results in nonhuman primates to date have not indicated a significant
improvement over optimized standard retroviral vectors, and thus the
advantages of lentiviral vectors simply for increasing stem-cell
transduction efficiency are not yet clear.
The paper by Cui and colleagues (page 399), however, highlights
progress toward overcoming the second hurdle, utilizing a potentially
more uniquely useful property of lentiviral vectors for gene therapy
applications. They have focused on achieving lineage-specific
expression of a transgene in dendritic cells derived from transduced
hematopoietic stem and progenitor cells. Using modern safety-modified
self-inactivating (SIN) lentiviral vectors, the authors have been
able to accommodate the HLA-DR class II MHC promoter to drive
lineage-enhanced expression of a green fluorescent protein specifically
in dendritic cells. None of the problems encountered with the inclusion
of strong nonviral LTR promoter/enhancer elements in standard
retroviral vectors were encountered, such as a drop in titer or
recombination of the vector in the producer cells. The ratio of
expression of GFP in dendritic cells to other lineages generated from
transduced human CD34+ cells in vitro, or after engraftment
in NOD/SCID mice, was much higher using the MHC promoter than with a
constitutive housekeeping EF-1 alpha promoter. This is the second
demonstration that high-level, lineage-specific expression can be
achieved using lentiviral vectors, the first being the recent report
from Sadelain's group that lentiviral vectors can overcome the myriad
problems encountered using retroviral vectors to drive
erythroid-specific high-level hemoglobin gene expression.
Expression of transgenes specifically in dendritic cells may be
important for tumor vaccine strategies, but the results are more
important globally as a demonstration that lentiviral vectors may be
able to accommodate large, strong, and complex genetic regulatory
elements conferring high-level expression and lineage-specificity, critical to the success of many experimental and therapeutic
gene-transfer strategies.
Cynthia Dunbar
Hematology Branch, NHLBI,
NIH
