An Emerging Strategy of Gene Therapy for Cardiac Disease


Yoshinori Yoshida,
Shinya Yamanaka

From the Center for iPS Cells Research and Application, Kyoto
University, Kyoto, Japan.

Heart disease is the major cause of morbidity and mortality
worldwide.1,2 The current therapeutic approaches for heart failure are limited because postnatal cardiomyocytes have little regenerative
capacity. Therefore, a new strategy needs to be established to improve the cardiac function.

Gene therapy is one of the most attractive new therapeutic strategies. Several kinds of cardiac gene therapy have so far been reported. Jeffrey M. Isner’s group reported that the administration of plasmid vectors encoding cDNA of the 165-amino acid isoform of vascular endothelial growth factor induced angiogenesis in patients with ischemic heart disease and with ischemic limbs.3,4 The effect of
vascular endothelial growth factor gene therapy is mediated by the
paracrine of cytokines.

Another type of gene therapy targets cardiomyocytes. The expression
and activity of sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) have
been observed to decrease in cardiomyocytes in a failing heart, and
the overexpression of SERCA2a could restore the cardiac function in
heart failure by improving calcium handling in the cardiomyocytes. Clinical trials for SERCA2A gene therapy were conducted
for patients with heart failure, and positive results have been

A new strategy for gene therapy is to restore the number of target
cells by direct conversion from other types of cells.
Twenty-five years ago, Weintraub et al7 demonstrated that a single
master transcription factor, MyoD, was capable of converting mouse
fibroblasts into skeletal muscle cells. This first demonstration of
cell fate conversion in mammalian cells was then applied in vivo.8,9
However, attempts to identify master factors for other cell lineages,
including cardiac myocytes, were painfully unsuccessful.10

This situation suddenly changed after the demonstration of induced
pluripotent stem cells; pluripotent state can be induced not by a
single factor but by a combination of 4 transcription factors.11–14 It did not take long for other researchers to identify other combinations of transcription factors that can induce direct conversion to pancreatic β cells,15 neurons,16,17 hepatocyte-like cells,18,19 as well as cardiac myocytes.20–22

The first report of direct in vitro reprogramming into cardiomyocytes
was published by Ieda et al.20 They demonstrated that the combination
of GATA4, MEF2C, and TBX5 (GMT) was able to reprogram cardiac
fibroblasts directly into cardiomyocytes in vitro. This cardiac direct reprogramming technology was reproduced with different factor
combinations by other groups.22,23

Earlier this year, 2 groups reported in vivo conversion of fibroblasts into cardiac myocytes. Song et al22 reported that the injection of retroviruses encoding GMT and Hand2 converted
β-galactosidase–expressing cardiac fibroblasts in Fsp1-Cre/Rosa26-LacZ mice into cardiomyocytes. Qian et al21 also reported that retroviral delivery of GMT in mice induced direct reprogramming of cardiac fibroblasts into cardiomyocytes. They used periostin-Cre/Rosa26-LacZ mice and Fsp1-Cre/Rosa26-LacZ mice, in which only descendants of the nonmyocyte population were β-galactosidase-positive and confirmed that the noncardiomyocytes were reprogrammed into cardiomyocytes. These reprogrammed cells revealed ventricular cardiomyocyte-like action potentials and a response to electrical stimulation, and electrical coupling with neighboring cells.

In this issue of Circulation Research, Inagawa et al24 reported
another evidence of direct reprogramming into cardiomyocytes. In
addition to the conventional retroviral vectors, they used a
polycistronic retrovirus expressing GMT by a 2A system, which was
previously reported to be useful for the efficient generation of
induced pluripotent stem cells.25,26 Introduction of a polycistronic
retrovirus to transduce 3 factors into fibroblasts generated more
matured cardiomyocytes compared with introducing 3 separate vectors.
The injection of polycistronic GMT (3F2A) converted around 1% of the
infected cells. Although the conversion efficiency was almost the same as that of the conventional retroviruses, 30% of the converted
cardiomyocytes showed cross striations in 3F2A-infected hearts,
whereas 15% of the converted cells were striated in the conventional
method. These findings suggested that efficient introduction of 3
genes by 3F2A resulted in generation of more matured cardiomyocytes.

The conversion efficiency from cardiac fibroblasts into cardiomyocytes was found to be 1%, and a large population of infected cells failed in the full conversion into cardiomyocytes and were thought to undergo only partial reprogramming. Do these partially reprogrammed cells finally revert to fibroblasts or remain in a partially reprogrammed cell state? Whether these partially reprogrammed cells play a role in the improvement or deterioration of the cardiac function is not known.
A genetic tracing analysis of the reprogrammed fibroblasts might be
informative to clarify the fate of reprogrammed cells. It is necessary to learn more about the molecular mechanisms and behavior of reprogrammed cells in cardiac direct reprogramming.

It is noteworthy that Inagawa et al24 observed no tumor formation in
the treated mouse hearts. Inagawa et al24 reported that most
retrovirus-infected cells were removed by an immune response within 4
weeks in immunocompetent mice. In such cases. many supportive effects, including mechanical support by induced cardiomyocytes, will be lost after reprogrammed cells disappear. To obtain long-lasting effects, other types of gene delivery methods may be desirable for such clinical application.

Conversion efficiency is very important to achieve the clinical
application of in vivo direct reprogramming technology. Another group
reported that the in vitro conversion from adult fibroblasts into
cardiomyocytes was very low.27 The combination of GMT with other
transcription factors, microRNAs, small molecules, or other devices
such as the 2A system, may increase the efficiency.

In this article, Inagawa et al reported that the polycistronic 2A
system can be used for in vivo direct reprogramming and succeed in
generating more matured cardiomyocytes in vivo. This polycistronic
system might facilitate the application of other types of gene
delivery methods which are less immunogenic or make no genomic
integration. Finally, the advance of direct cardiac reprogramming
technology could provide a new strategy for conducting gene therapy in patients with cardiac failure.

Sources of Funding

The authors were supported by the Lading Project of the Ministry of
Education, Culture, Sports, Science and Technology, and the Funding
Program for World-Leading Innovative R&D on Science and Technology
(FIRST Program) of the Japanese Society for the Promotion of Science.


S.Y. is a member without salary of the scientific advisory boards of
iPierian, iPS Academia Japan, Megakaryon Corporation, and Retina
Institute Japan. The other author has no conflicts to report.


The opinions expressed in this editorial are not necessarily those
of the editors or of the American Heart Association.

© 2012 American Heart Association, Inc.


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Induction of Cardiomyocyte-Like Cells in Infarct Hearts by Gene
Transfer of Gata4, Mef2c, and Tbx5
Kohei Inagawa,
Kazutaka Miyamoto,
Hiroyuki Yamakawa,
Naoto Muraoka,
Taketaro Sadahiro,
Tomohiko Umei,
Rie Wada,
Yoshinori Katsumata,
Ruri Kaneda,
Koji Nakade,
Chitose Kurihara,
Yuichi Obata,
Koichi Miyake,
Keiichi Fukuda,
and Masaki Ieda
Circulation Research. 2012;111:1147-1156, published online before
print August 28 2012, doi:10.1161/CIRCRESAHA.112.271148