|
RESEARCH
SUMMARY 2001-2004
Molecular
Medicine
******
1. Effective gene transfer to human melanomas
via integrin-targeted adenoviral vectors.
The utility of recombinant adenoviral vectors (Adv) for gene therapy is limited
by their low transduction efficiency and lack of specificity for target cells.
The low transduction efficiency is often recognized as due to deficiency of the
primary adenoviral receptor, the coxsackievirus-adenovirus receptor (CAR). In
this paper, studies of CAR levels on human melanoma cell lines confirmed that
low transduction efficiency was closely related to deficiency of the adenoviral
receptor. To achieve CAR-independent gene transfer via Adv, we modified viral
tropism via genetic alteration of the adenovirus type 5 (Ad5) fiber protein.
Insertion of an Arg-Gly-Asp (RGD)-containing peptide in the HI loop of the
fiber knob domain allowed the virus to use an alternative receptor, the
integrin receptor, during the cell entry process. With this modified vector
(Adv-F/RGD) transduction was increased 5- to 96-fold relative to a vector
containing wild-type fiber (Adv-F/wt) in five human melanoma cells expressing
integrins of the alpha(v)beta(3), alpha(v)beta(5) class, which are recognized
by the RGD peptide motif. In contrast, no significant difference in
transduction efficiency between Adv-F/RGD and Adv-F/wt was observed in 293
cells, which show high-level expression of CAR. In this study, we attempted to
apply Adv-F/RGD for gene therapy for malignant melanoma. At the same
multiplicity of infection, melanoma cells infected with Adv-F/RGD carrying
human interleukin 2 (AxCAhIL2-F/RGD) produced a higher level of cytokine than
cells infected with AxCAhIL2-F/wt. Treatment by intratumoral injection of
AxCAhIL2-F/RGD was more effective than intratumoral injection of AxCAhIL2-F/wt
in regressing tumors in a melanoma xenograft model. These data suggest that
integrin-targeted adenoviral vectors may be a powerful tool in gene therapy for
CAR-deficient melanomas.
2. Reduction of natural adenovirus tropism to
the liver by both ablation of fiber-coxsackievirus and adenovirus receptor
interaction and use of replaceable short fiber.
The initial recognition and binding of adenovirus vector to the host cell
surface is mediated by interaction between the adenovirus fiber knob protein
and its receptor, the coxsackievirus and adenovirus receptor (CAR). This
natural tropism of adenovirus vector needs to be ablated in order to achieve
targeted gene transfer. To this end, we noted that adenovirus serotype 40 (Ad40)
contains two distinct long and short fibers; the short fiber is unable to
recognize CAR, while the long fiber binds CAR. We generated adenovirus serotype
5-based mutants with chimeric Ad40-derived fibers, which were composed of
either long or short shafts together with CAR binding or nonbinding knobs. The
capacity of these adenovirus mutants for in vitro and in vivo gene transfer to
liver cells was examined. In the case of primary human hepatocytes displaying a
high expression level of CAR and alphav integrin, both CAR binding ability and
fiber shaft length played important roles in efficient transduction. Most
significantly, the high transduction efficiency observed in the liver and
spleen following intravenous administration of adenovirus vector was dramatically
reduced by both ablation of fiber-CAR interaction and the use of replaceable
short fiber. In other tissues displaying a low level of transduction, no
significant differences in transduction efficiency were observed among
adenovirus vector mutants. Furthermore, incorporation of a 7-lysine-residue
motif at the C-terminal end of CAR-nonbinding short fiber efficiently achieved
transduction of target cells via the heparan-containing receptor. Our results
demonstrated that the natural tropism of adenovirus in vivo is influenced not
only by fiber-CAR interaction but also by fiber shaft length. Furthermore, our
strategy may be useful for retargeting adenovirus to particular tumors and
tissue types with specific receptors.
3. Pre-administration of angiopoietin-1
followed by VEGF induces functional and mature vascular formation in a rabbit
ischemic model.
BACKGROUND: Angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF)
play important roles in vascular formation and maturation, suggesting that the
combination of these two would be a promising therapy for ischemia. However, it
remains unclear what the best schedule of administration of these cytokines
might be. METHODS: Six experimental groups were used to prepare the rabbit
ischemic hindlimb model following naked plasmid intramuscular administration as
follows: empty vector (C), single gene (Ang1, A; VEGF, V), Ang-1 followed by
VEGF (A - V), co-administration of Ang1 and VEGF (A + V), and VEGF followed by
Ang1 (V - A). RESULTS: Thirty days after gene administration, A - V showed a
significantly increased blood pressure and blood-flow recovery in the ischemic
limb compared with the control group. Histological findings by alpha-smooth
muscle-actin (alpha-SMA) staining revealed that the two combination groups had
more mature vessels as compared with the control group. Significantly, A - V
revealed the highest density of alpha-SMA-positive vessels compared with VEGF
alone or Ang1 alone. Angiographic assessment revealed that A - V had a greater
increased arterial diameter compared with VEGF alone. Edema, one of the major
adverse effects induced by VEGF, was not found in A - V throughout the
experiments, while VEGF alone and V - A showed severe edema induced by VEGF.
CONCLUSIONS: The pre-administration of Ang1 followed by VEGF resulted in an
improvement of hemodynamic status, an increased number of vessels covered with
alpha-actin-positive mural cells, and prevention of VEGF-mediated edema. Thus,
priming by Ang1 gene administration would be beneficial for therapeutic
angiogenesis in VEGF gene therapy. Copyright 2003 John Wiley & Sons, Ltd.
4. Adenoviral-delivered angiopoietin-1 reduces the
infarction and attenuates the progression of cardiac dysfunction in the rat
model of acute myocardial infarction.
In acute myocardial infarction (AMI), prognosis and mortality rate are closely
related to the infarct size and the progression of postinfarction cardiac
failure. Angiogenic gene therapy has presented a new approach for the treatment
of AMI. Angiopoietin-1 (Ang1) is a critical angiogenic factor for vascular
maturation and enhances vascular endothelial growth factor (VEGF)-induced
angiogenesis in a complementary manner. We hypothesized that gene therapy using
Ang1 for AMI might promote angiogenesis cooperatively with intrinsic VEGF,
since high concentrations of circulating VEGF have been reported in AMI. To
evaluate our hypothesis, we employed a rat AMI model and adenoviral Ang1
(HGMW-approved gene symbol ANGPT1) gene transfer to the heart. A significant
increase in capillary density and reduction in infarct sizes were noted in the
infarcted hearts with adenoviral Ang1 gene treatment compared with control
infarcted hearts treated with saline or adenoviral vector containing the
beta-galactosidase gene. Furthermore, the Ang1 group showed significantly
higher cardiac performance in echocardiography (55.0% of ejection fraction, P
< 0.05 vs control) than the saline or adenoviral controls (36.0 or 40.5%,
respectively) 4 weeks after myocardial infarction. The adenoviral delivery of
Ang1 during the acute phase of myocardial infarction would be feasible to
attenuate the progression of cardiac dysfunction in the rat model.
5. Efficient BMP2 gene transfer and bone formation of
mesenchymal stem cells by a fiber-mutant adenoviral vector.
Strategies using mesenchymal stem cell (MSC)-mediated gene therapy have been
developed to improve bone healing. However, transduction efficiency into MSCs
by each vector is not always high. To overcome this problem, we used a modified
adenoviral vector (Adv-F/RGD) with an RGD-containing peptide in the HI loop of
the fiber knob domain of adenovirus type 5 (Ad5). Transduction efficiency into
bone marrow-derived MSCs with Adv-F/RGD increased 12-fold compared with a
vector containing the wild-type fiber (Adv-F/wt) by beta-galactosidase
chemiluminescent assay. As a next step, we constructed AxCAhBMP2-F/RGD and
AxCAhBMP2-F/wt carrying human bone morphogenetic protein 2 (BMP2). At the same
multiplicity of infection, MSCs infected with AxCAhBMP2-F/RGD produced higher
amounts of BMP2 than cells infected with AxCAhBMP2-F/wt, and also
differentiated towards the osteogenic lineage more efficiently in vitro.
Furthermore, using ex vivo gene transduction, we evaluated the potential for
ectopic bone formation by the transduced MSCs in vivo. Transduction with
AxCAhBMP2-F/RGD exhibited greatly enhanced new bone formation. These data
suggest that Adv-F/RGD is useful for introducing foreign genes into MSCs and
that it will be a powerful gene therapy tool for bone regeneration and other
tissue engineering.
6. Ex vivo expansion of human umbilical cord
hematopoietic progenitor cells using a coculture system with human telomerase
catalytic subunit (hTERT)-transfected human stromal cells.
We developed a new human stromal cell line that could expand human
hematopoietic progenitor/stem cells. Primary human bone marrow stromal cells
were infected with retrovirus containing the human telomerase catalytic subunit
(hTERT) gene, resulting in increased population doubling and the acquisition of
cell immortalization. Characteristics of the hTERT-transduced stromal
(hTERT-stromal) cells were identical with those of the primary stromal cells in
terms of morphologic appearance and expression of surface antigens. Human cord
blood (CB) CD34(+) cells were expanded by coculture with primary stromal or
hTERT-stromal cells in the presence of stem cell factor, thrombopoietin, and
Flk-2/Flt-3 ligand under serum-free condition. The degree of expansion of
CD34(+) cells and total number of colony-forming units in culture (CFU-Cs)
after 2 weeks' coculture with the hTERT-stromal cells were nearly the same as
those after 2 weeks' coculture with primary stromal cells (CD34(+) cells,
118-fold +/- 8-fold versus 117-fold +/- 13-fold; CFU-Cs, 71-fold +/- 5-fold
versus 67-fold +/- 5-fold of initial cell number). CB expansion on
hTERT-stromal cells occurred at a similar rate through 7 weeks. In contrast,
the rate of CB expansion on primary stromal cells had drastically declined at 7
weeks. In nonobese diabetic/severe combined immunodeficiency (SCID) mice, the
degree of engraftment of SCID-repopulating cells that had been cocultured with
hTERT-stromal cells for 4 weeks was significantly higher than that of
precocultured CB cells. These results indicate that this hTERT-stromal cell
line could be useful for ex vivo expansion of hematopoietic progenitor/stem
cells and for analyzing the microenvironment of human bone marrow.
7. Indian hedgehog gene transfer augments hematopoietic
support of human stromal cells including NOD/SCID-{beta}2m-/- repopulating
cells.
Hematopoietic stem cells (HSCs) are a subset of bone marrow cells that are
capable of self-renewal and of giving rise to all types of blood cells.
However, the mechanisms involved in controlling the number and abilities of
HSCs remain largely unknown. The Indian hedgehog (Ihh) signal has an essential
role in inducing hematopoietic tissue during embryogenesis. We investigated the
roles of the Ihh in coculture with CD34(+) cells and human stromal cells. Ihh
mRNA was expressed in primary and telomerized human (hTERT) stromal cells, and
its receptor molecules were detected in CD34(+) cells. Ihh gene transfer into
hTERT stromal cells enhanced their hematopoietic supporting potential, which
was elevated compared with control stromal cells, as indicated by the
colony-forming units in culture (CFU-Cs) (26-fold +/- 2-fold versus 59-fold +/-
3-fold of the initial cell number; mixed colony-forming units [CFU-Mix's],
63-fold +/- 37-fold versus 349-fold +/- 116-fold). Engraftments of nonobese
diabetic/severe combined immunodeficiency-ss(2)m(-/-) (NOD/SCID-ss(2)m(-/-))
repopulating cells (RCs) expanded on Ihh stromal cells were significantly
higher compared with control coculture results, and engraftment was neutralized
by addition of an antihedgehog antibody. Limiting dilution analysis indicated
that NOD/SCID-ss(2)m(-/-) RCs proliferated efficiently on Ihh stromal cells,
compared with control stromal cells. These results indicate that Ihh gene
transfer could enhance the primitive hematopoietic support ability of human
stromal cells.
8. BDNF gene-modified mesenchymal stem cells promote
functional recovery and reduce infarct size in the rat middle cerebral artery
occlusion model.
Examination of the clinical therapeutic efficacy of using bone marrow stromal
cells, including mesenchymal stem cells (MSC), has recently been the focus of
much investigation. MSC were reported to ameliorate functional deficits after
stroke in rats, with some of this improvement possibly resulting from the
action of cytokines secreted by these cells. To enhance such cytokine effects,
we transfected telomerized human MSC with the BDNF gene using a fiber-mutant
F/RGD adenovirus vector and investigated whether these cells contributed to
improved functional recovery in a rat transient middle cerebral artery
occlusion (MCAO) model. BDNF production by MSC-BDNF cells was 23-fold greater
than that seen in uninfected MSC. Rats that received MSC-BDNF showed
significantly more functional recovery than did control rats following MCAO.
Specifically, MRI analysis revealed that the rats in the MSC-BDNF group
exhibited more significant recovery from ischemia after 7 and 14 days. The
number of TUNEL-positive cells in the ischemic boundary zone was significantly
smaller in animals treated with MSC-BDNF compared to animals in the control
group. These data suggest that MSC transfected with the BDNF gene may be useful
in the treatment of cerebral ischemia and may represent a new strategy for the
treatment of stroke.
9. Antitumor effect of genetically engineered
mesenchymal stem cells in a rat glioma model.
The prognosis of patients with malignant glioma is extremely poor, despite the
extensive surgical treatment that they receive and recent improvements in
adjuvant radio- and chemotherapy. In the present study, we propose the use of
gene-modified mesenchymal stem cells (MSCs) as a new tool for gene therapy of
malignant brain neoplasms. Primary MSCs isolated from Fischer 344 rats
possessed excellent migratory ability and exerted inhibitory effects on the
proliferation of 9L glioma cell in vitro. We also confirmed the migratory
capacity of MSCs in vivo and showed that when they were inoculated into the
contralateral hemisphere, they migrated towards 9L glioma cells through the corpus
callosum. MSCs implanted directly into the tumor localized mainly at the border
between the 9L tumor cells and normal brain parenchyma, and also infiltrated
into the tumor bed. Intratumoral injection of MSCs caused significant
inhibition of 9L tumor growth and increased the survival of 9L glioma-bearing
rats. Gene-modification of MSCs by infection with an adenoviral vector encoding
human interleukin-2 (IL-2) clearly augmented the antitumor effect and further
prolonged the survival of tumor-bearing rats. Thus, gene therapy employing MSCs
as a targeting vehicle would be promising as a new therapeutic approach for
refractory brain tumor.
10. Adenovirus-mediated transfer of siRNA against
survivin induced apoptosis and attenuated tumor cell growth in vitro and in
vivo.
Gene targeting using short interfering RNA (siRNA) has become a common strategy
to explore gene function because of its prominent efficacy and specificity. For
the application of siRNA technology to gene therapy, however, still more
efficient transduction of siRNA into target cells is needed. In this study, we
developed an adenoviral vector harboring a tandem-type siRNA expression unit,
in which sense and antisense strands composing the siRNA duplex were separately
transcribed by two human U6 promoters. Targeting survivin, an antiapoptotic
molecule widely overexpressed in malignancies but not detected in terminally
differentiated adult tissues, this type of adenoviral vector (Adv-siSurv)
successfully exerted a gene knockdown effect and induced apoptosis in HeLa,
U251, and MCF-7 cells. These cancer cells, once infected with Adv-siSurv,
displayed remarkably attenuated growth potential, both in vitro and in vivo.
Moreover, intratumoral injection of Adv-siSurv significantly suppressed tumor
growth in a xenograft model using U251 glioma cells. This novel modality may be
a promising tool for cancer therapy.
******
Technology development for gene
therapy:
Nakamura
T, Sato K. and Hamada H. Effective
Gene Transfer to Human Melanomas via Integrin-Targeted Adenoviral Vectors. Hum. Gene Ther.,13(5): 613-626, 2002.
Nakamura
T, Sato K and Hamada H.
Reduction of natural adenovirus tropism to the liver by both ablation of
fiber-Coxsackievirus and adenovirus receptor interaction and use of replaceable
short fiber. J. Virol., 77(4): 2512-2521, 2003.
Regenerative medicine and gene
therapy for cardiovascular diseases:
Yamauchi,
A, Ito, Y, Morikawa, M, Kobune, M, Huang, J, Sasaki, K, Takahashi, K, Nakamura,
K, Dehari, H, Niitsu, Y, Abe, T and Hamada, H. Pre-administration of
angiopoietin-1 followed by VEGF induces functional and mature vascular
formation in a rabbit ischemic model. J.
Gene Med., 5(11):994-1004, 2003.
Takahashi
K, Ito Y, Morikawa M, Kobune M, Huang J, Tsukamoto M, Sasaki K, Nakamura K, Dehari
H, Ikeda K, Uchida H, Hirai S, Abe T and Hamada H Adenoviral delivered angiopoietin-1 reduces
the infarction and attenuates the progression of cardiac dysfunction in the rat
model of acute myocardial infarction. Mol. Ther. 8(4):584-592, 2003.
Regenerative medicine using bone
marrow stem cells:
Tsuda H., Wada T., Ito Y., Uchida H., Dehari H., Nakamura K., Sasaki K.,
Kobune M., Yamashita T. and Hamada H. Efficient
BMP2 gene transfer and bone formation of mesenchymal stem cells by a
fiber-mutant adenoviral vector. Mol. Ther., 7(3): 354-365, 2003.
Kawano
Y, Kobune M, Yamaguchi M, Nakamura K, Ito Y, Sasaki K, Takahashi S, Nakamura T,
Chiba H, Sato T, Matsunaga T, Azuma H, Ikebuchi K, Ikeda H, Kato J, Niitsu Y
and Hamada H. Ex vivo expansion
of human umbilical cord hematopoietic progenitor cells using a coculture system
with human telomerase catalytic subnit (hTERT)-transfected human stromal cells.
Blood, 101(2): 532-540, 2003.
Kobune M, Ito Y, Kawano Y, Sasaki K, Uchida
H, Nakamura K, Dehari H, Chiba H, Takimoto R, Matsunaga T, Terui T, Kato J,
Niitsu Y, Hamada H. Indian
hedgehog gene transfer augments hematopoietic support of human stromal cells
including NOD/SCID-{beta}2m-/- repopulating cells. Blood. 2004 Apr 22 [Epub ahead of print] Blood in press 2004.
Kurozumi,
K., Nakamura, K., Tamiya, T., Kawano, Y., Kobune, M., Hirai, S., Uchida, H.,
Sasaki, K., Ito, Y., Kato, K., Honmou, O., Houkin, H., Date, I., and Hamada,
H. BDNF gene-modified mesenchymal stem cells promote functional
recovery and reduce infarct size in the rat middle cerebral artery occlusion
model. Mol. Ther. 9(2):189-97, 2004.
Cancer Gene Therapy and Immunothrapy
Nakamura
K, Ito Y, Kawano Y, Kurozumi K, Kobune M, Tsuda H, Bizen A, Honmou O, Niitsu Y,
and Hamada, H. Anti-tumor effect of genetically engineered
mesenchymal stem cells in a rat glioma model.
Gene Ther. In press, 2004. 2004 May 13 [Gene Ther. , Epub
ahead of print]
Uchida H, Tanaka T, Sasaki K, Kato K,
Dehari H, Ito Y, Kobune M, Miyagishi M, Taira K, Tahara H, Hamada H. Adenovirus-Mediated Transfer of siRNA against Survivin Induced
Apoptosis and Attenuated Tumor Cell Growth in Vitro and in Vivo. Mol Ther.
10(1):162-71, 2004.
|
