Current Gene Therapy, Volume 5, No. 4, 2005
Contents
Retrovirus Silencing and Vector Design:
Relevance to Normal and Cancer Stem Cells? Pp.367-373
James
Ellis and Shuyuan Yao
Genetic Manipulation of Human Embryonic Stem
Cells: A System to Study Early Human Development and Potential Therapeutic
Applications Pp.375-385
Pablo
Menendez, Lisheng Wang and Mickie Bhatia
Altering the Tropism of Lentiviral Vectors
through Pseudotyping
Pp.387-398
James
Cronin, Xian-Yang Zhang and Jakob Reiser
Delivering RNA Interference to the Mammalian
Brain Pp.399-410
Timothy
M. Fountaine, Matthew J.A. Wood and Richard Wade-Martins
Transcriptionally Targeted Adenovirus Vectors Pp.411-427
Hamid
Sadeghi and Mary M. Hitt
The Use of Oncolytic Vaccinia Viruses in the
Treatment of Cancer: A New Role for an Old Ally? Pp.429-443
Stephen
H. Thorne, David L. Bartlett and David H. Kirn
Abstracts
[Back to top] Retrovirus Silencing and Vector Design: Relevance to
Normal and Cancer Stem Cells?
James
Ellis and Shuyuan Yao
An obstacle
confronting gene therapy in stem cells is transcriptional silencing of the
vector. Here, we discuss recent data indicating that oncoretrovirus and
lentivirus vectors are silenced by multiple epigenetic pathways that result in
DNA methylation and histone modifications. Both vector types can be variegated
in stem cells and expression is often extinguished during differentiation. We
propose a novel model of retrovirus silencing in which epigenetic pathways
compete to recruit histone deacetylases, de novo methyltransferases, histone H1
and MeCP2 to the provirus. These chromatin modifications may act in concert
with heterochromatin at or near the integration site to establish silencing or
variegation respectively. Retrovirus vector designs for stem cells should
delete virus silencer elements, incorporate strong positive regulatory elements
and insulators, and avoid non-mammalian reporter genes. In addition, cancer
stem cells that continually repopulate a growing tumour may share silencing
pathways with normal stem cells. Ultimately, optimized vector designs may prove
to be valuable tools for gene therapy of both normal and cancer stem cells.
[Back to top] Genetic Manipulation of Human Embryonic Stem Cells: A
System to Study Early Human Development and Potential Therapeutic Applications
The successful
derivation of human embryonic stem cell (hESC) lines by Thomson and colleagues [Thomson et al., 1998] provided a new area of investigation in both
regenerative medicine and early human development. Fundamental study of the
molecular and cellular mechanisms responsible for normal lineage development
will rely on reproducible protocols to direct the differentiation of hESCs into
specific lineages of interest and genetically manipulate both hESCs and their
derivatives. Identifying standards for maintenance of hESCs, methods for
controlled differentiation and genetic manipulation of hESCs and their
derivatives will provide a foundation to explore their potential therapeutic
use in cell and gene therapy. In the present review, our goal is to outline the
latest advances in the field with particular focus on how hESCs and their
derivatives can be genetically altered, how this may be useful in better
understanding the cellular and molecular events of lineage differentiation, and
how deregulation of these cellular processes may lead to abnormal development
and disease.
[Back to top] Altering the Tropism of Lentiviral Vectors
through Pseudotyping
James
Cronin, Xian-Yang Zhang and Jakob Reiser
The host range of
retroviral vectors including lentiviral vectors can be expanded or altered by a
process known as pseudotyping. Pseudotyped lentiviral vectors consist of vector
particles bearing glycoproteins (GPs) derived from other enveloped viruses.
Such particles possess the tropism of the virus from which the GP was derived.
For example, to exploit the natural neural tropism of rabies virus, vectors
designed to target the central nervous system have been pseudotyped using
rabies virus-derived GPs. Among the first and still most widely used GPs for
pseudotyping lentiviral vectors is the vesicular stomatitis virus GP (VSV-G),
due to the very broad tropism and stability of the resulting pseudotypes.
Pseudotypes involving VSV-G have become effectively the standard for evaluating
the efficiency of other pseudotypes. This review samples a few of the more
prominent examples from the ever-expanding list of published lentiviral
pseudotypes, noting comparisons made with pseudotypes involving VSV-G in terms
of titer, viral particle stability, toxicity, and host-cell specificity.
Particular attention is paid to publications of successfully targeting a
specific organ or cell types.
[Back to top] Delivering RNA Interference to the Mammalian
Brain
Timothy
M. Fountaine, Matthew J.A. Wood and Richard Wade-Martins
RNA interference
(RNAi) is a new modality in gene therapy which can elicit down-regulation of
gene expression and has enormous potential in the treatment of neurological diseases.
RNAi is a conserved system through which double stranded RNA (dsRNA) guides
sequence specific mRNA degradation. The RNAi apparatus may be artificially
triggered by delivery of naked siRNA molecules or by plasmid-based expression
of dsRNA. Before these techniques can be used as effective treatments in the
brain, efficient methods of in vivo delivery must be devised. This
review first describes the mechanism of RNAi, and then critically examines both
viral and non-viral methods for delivery of RNAi to the mammalian brain. There
have been a number of important recent publications in this field and the
progress towards effective in vivo delivery of RNAi to the central
nervous system is discussed. Finally, potential problems that must be
considered before applying this technology to the human brain are outlined.
[Back to
top] Transcriptionally Targeted Adenovirus Vectors
Hamid
Sadeghi and Mary M. Hitt
Adenovirus vectors
are the most highly efficient vehicles currently available for gene transfer to
mammalian cells. Their ability to transduce both proliferating and non-dividing
cells allows in vivo gene delivery, but the wide spectrum of cell types
infected by adenovirus necessitates a requirement for targeting, particularly
if the transduced gene is detrimental when expressed in inappropriate tissues.
Over the past decade, numerous investigators have examined tissue- or
tumor-specific enhancer-promoters as a means to transcriptionally target genes
delivered by adenovirus vectors. We review here recent developments in
adenovirus vectors including improvements in the vector backbone to maintain
promoter specificity. In addition, we discuss the regulatory elements directing
cell-specific expression of genes encoding telomerase, prostate-specific
antigen, probasin, osteocalcin, tyrosinase, alpha-fetoprotein, surfactant B,
and mammaglobin. Recent results using these regulatory sequences to target Ad
vectors to cancer cells are highlighted.
[Back to top] The Use of Oncolytic Vaccinia Viruses in the
Treatment of Cancer: A New Role for an Old Ally?
Stephen
H. Thorne, David L. Bartlett and David H. Kirn
The use of
genetically engineered, tumor-targeting viruses as oncolytic agents has
recently emerged as a promising new area for the development of novel cancer
therapies. The first viruses to enter the clinic, such as ONYX-015 (an
oncolytic adenovirus), provided evidence both for the safety and for the
anti-tumor potential of this approach. The results of these early trials have
also allowed investigators to examine the limitations of these viruses and to
develop potentially far more effective approaches. In this review the
development of such next generation viruses, in particular the potential use of
strains of vaccinia virus, will be discussed. Vaccinia has an enormous history
of use in humans and possesses many of the features felt to be beneficial for
the creation of a successful virotherapy agent. It causes no known disease in
humans, yet is capable of infecting almost all cell types with a subsequent
rapid and lytic infection, which subsequently induces a vigorous local CTL
immune response at the site of infection. Vaccinia also displays natural tumor
tropism, and several approaches have been used to further limit viral
replication to tumor cells and to optimize the immune response induced at the
site of the tumor. Finally, the large cloning capacity of vaccinia allows for
the addition of multiple foreign genes into the viral genome. This has been exploited
to increase the bystander effect of the virus by immune modulation or by
expression of pro-drug converting enzymes as well as to incorporate safety
controls and reporters for in vivo molecular imaging. Initial clinical
trials with these viruses further highlights their potential as the next
generation of oncolytic agents and as highly effective future cancer therapies.