Page 1 of 26
Human Gene Therapy Recent Advancements in Cardiovascular Gene Therapy and Vascular Biology (doi: 10.1089/hum.2015.095) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
1 Invited Review for Human Gene Therapy Nordic Issue
Recent Advancements in Cardiovascular Gene Therapy and Vascular Biology Johanna P. Laakkonen1, Seppo Ylä-Herttuala1-3 From the
1
Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for
Molecular Sciences, University of Eastern Finland, Kuopio, Finland, 2Department of Medicine, University of Eastern Finland, Kuopio, Finland, and
3
Gene Therapy Unit, Kuopio University
Hospital, Kuopio, Finland
Key words: cardiovascular diseases, cardiovascular gene transfer, gene therapy vectors, animal models for cardiovascular research, vascular biology, high resolution imaging of microvasculature
Address correspondence: PhD. Johanna Laakkonen, Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute
for
Molecular
[email protected]
Sciences,
University
of
Eastern
Finland,
Kuopio,
Finland,
Page 2 of 26
Human Gene Therapy Recent Advancements in Cardiovascular Gene Therapy and Vascular Biology (doi: 10.1089/hum.2015.095) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
2 ABSTRACT. Cardiovascular gene therapy aims to treat coronary and peripheral artery disease, heart failure and arrhythmia. Chosen transgene, delivery method, gene therapy vector type, high quality vector production and dose are all determining factors of the therapeutic outcome. High resolution vascular imaging and increased knowledge of vascular biology in physiological and pathological conditions enables finding of novel molecular targets for cardiovascular gene therapy. Transgenic and knockout mouse models have provided researchers several powerful experimental tools for studying the effects of single genes on cardiovascular diseases. For preclinical efficacy, safety and toxicology studies, large animal models are needed before entering to clinical testing. This review focuses on commonly used animal models in cardiovascular gene therapy and describes recent advancements in the field of vascular biology. Emphasis is also given for high resolution imaging of microvasculature and its impact on our knowledge of vascular function.
Page 3 of 26
Human Gene Therapy Recent Advancements in Cardiovascular Gene Therapy and Vascular Biology (doi: 10.1089/hum.2015.095) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
3 Introduction. Virus vectors are efficient tools for gene transfer. To date adenovirus (Ad), adeno-associated
virus
(AAV) and
lentivirus
(LV) vectors have been used for
cardiovascular gene transfer due to their ability to transduce non-dividing cells, as well as to obtain high gene transfer efficiency. Chosen transgene, delivery method, gene therapy vector type and dose, as well as high quality vector production all determine the therapeutic outcome. Most gene therapy applications currently rely on direct applications into the treated tissue with catheters, needle injections or equivalent techniques. For coronary and peripheral artery disease transgenes such as vascular endothelial growth factor (VEGF), fibroblast growth factor, hepatocyte growth factor (HGF) and hypoxiainducible factor (HIF-1α) have been used in clinical trials, whereas for heart failure, transgenes such as sarcoendoplasmic reticulum calcium ATPase 2a (Serca2a), adenylyl cyclase 6 and stromal-derived-factor 1 have been used 1. To date human Ad serotype 5 vector (Ad5) has been the most widely used vector for cardiovascular gene transfer. Localized intramuscular and intramyocardial delivery of Ad vectors has shown great promise in, e.g., developing treatment for peripheral and myocardial ischemia by using VEGFs.
During the last decade four phase I/II clinical trials with Ad vector expressing
VEGF-A have been conducted in Finland. Currently, a new phase I clinical trial is on-going with Ad vectors expressing VEGF-D 2. Emerging data on epigenetic regulation of vasculature, exosomes, non-coding RNAs, mechanotransduction and cell signaling in vivo have transformed our views on vascular biology. Growing knowledge of vascular function at the cell and molecular level enables also finding of new therapeutic targets for cardiovascular diseases (CVDs). Availability of several transgenic and knockout mouse models greatly enhances possibilities to obtain physiologically significant results using gene transfer applications. Besides ApoE and LDL receptor knockout mice for hyperlipidemia and atherosclerosis research 3, several mouse
Page 4 of 26
Human Gene Therapy Recent Advancements in Cardiovascular Gene Therapy and Vascular Biology (doi: 10.1089/hum.2015.095) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
4 models with deletion of specific apolipoproteins, cytokines, growth factors, or extracellular matrix components have been generated. New mouse models, e.g., for modeling vulnerable plaques have also been recently introduced 4. Final preclinical efficacy, safety, biodistribution and toxicology studies of gene therapy vectors are usually performed in larger animals, such as rabbits or pigs, since they produce a more realistic outcome about the usefulness of CVD gene transfer applications regarding human clinical applications. Particularly pigs have been increasingly used for myocardial infarction, cardiac ischemiareperfusion and in-stent restenosis studies. Pig heart is very similar to human heart and allows the use of similar gene vector dose, catheter interventions, and imaging techniques as for man. Detection of the therapeutic outcome in pre-clinical animal models by imaging of microvasculature
in three-dimension enables
better understanding of vascular
morphology and function, as well as biodistribution of the virus vectors after gene delivery. Fast development of new imaging modalities with higher spatial resolution is going to improve vascular imaging and analysis even further 5. The challenge is now how to interpret the most useful data, e.g., from bioinformatics and vascular imaging for development of new therapeutic vectors for the treatment of CVDs.
Virus Vectors for Cardiovascular Gene Transfer. Ad vectors have been the most commonly used vector type in clinical trials aiming to treat CVDs due to their high gene transfer efficiency and ease of production in good manufacturing practice (GMP) quality. Although systemic delivery of Ad vectors is limited due to induced immune response and virus interactions with circulating blood and cell components, localized intramuscular and intramyocardial delivery of Ad vectors have shown great promise in developing treatment for CVDs. Vascular gene transfer of endothelial cells, cardiomyocytes or medial smooth muscle cells have shown to be obtained after Ad vector administration by intramyocardial
Page 5 of 26
Human Gene Therapy Recent Advancements in Cardiovascular Gene Therapy and Vascular Biology (doi: 10.1089/hum.2015.095) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
5 injection, coronary perfusion or intravascular delivery to transiently isolated vessel segments
6-11
. To date Ad vectors have been used in clinical trials for coronary and
peripheral artery disease by expressing transgenes such as HGF, HIF-1α and VEGF
2,12-14
.
A clinical trial for heart failure by Ad vector expressing adenylyl cyclase type VI is ongoing. Besides Ad vectors, AAV and LV vectors have been used in cardiovascular gene transfer applications. With AAV vectors, high gene transfer efficiency has particularly been detected in cardiomyocytes after intramyocardial delivery. Persistent gene expression by AAV vectors have been shown in pre-clinical trials in cardiomyocytes and skeletal muscle 7,15
. AAV9 can efficiently target and transduce cardiomyocytes after systemic delivery
16
,
whereas AAV2 has been shown to have the most promising efficiency after intracoronary delivery to heart transplants ex vivo
17
. AAV1 instead has shown to be the most efficient
gene transfer vector after intramyocardial injection
18
and have been used in phase 2
clinical trials for heart failure by expressing Serca2a as a transgene
19
. Also, LV vectors
have been developed for cardiovascular gene transfer due to their ability to induce longterm gene expression in cardiomyocytes in pre-clinical trials
20
. Instead, in rabbit skeletal
muscle LV mediated transduction was shown to be short-term (
Human Gene Therapy Recent Advancements in Cardiovascular Gene Therapy and Vascular Biology (doi: 10.1089/hum.2015.095) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
1 Invited Review for Human Gene Therapy Nordic Issue
Recent Advancements in Cardiovascular Gene Therapy and Vascular Biology Johanna P. Laakkonen1, Seppo Ylä-Herttuala1-3 From the
1
Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for
Molecular Sciences, University of Eastern Finland, Kuopio, Finland, 2Department of Medicine, University of Eastern Finland, Kuopio, Finland, and
3
Gene Therapy Unit, Kuopio University
Hospital, Kuopio, Finland
Key words: cardiovascular diseases, cardiovascular gene transfer, gene therapy vectors, animal models for cardiovascular research, vascular biology, high resolution imaging of microvasculature
Address correspondence: PhD. Johanna Laakkonen, Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute
for
Molecular
[email protected]
Sciences,
University
of
Eastern
Finland,
Kuopio,
Finland,
Page 2 of 26
Human Gene Therapy Recent Advancements in Cardiovascular Gene Therapy and Vascular Biology (doi: 10.1089/hum.2015.095) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
2 ABSTRACT. Cardiovascular gene therapy aims to treat coronary and peripheral artery disease, heart failure and arrhythmia. Chosen transgene, delivery method, gene therapy vector type, high quality vector production and dose are all determining factors of the therapeutic outcome. High resolution vascular imaging and increased knowledge of vascular biology in physiological and pathological conditions enables finding of novel molecular targets for cardiovascular gene therapy. Transgenic and knockout mouse models have provided researchers several powerful experimental tools for studying the effects of single genes on cardiovascular diseases. For preclinical efficacy, safety and toxicology studies, large animal models are needed before entering to clinical testing. This review focuses on commonly used animal models in cardiovascular gene therapy and describes recent advancements in the field of vascular biology. Emphasis is also given for high resolution imaging of microvasculature and its impact on our knowledge of vascular function.
Page 3 of 26
Human Gene Therapy Recent Advancements in Cardiovascular Gene Therapy and Vascular Biology (doi: 10.1089/hum.2015.095) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
3 Introduction. Virus vectors are efficient tools for gene transfer. To date adenovirus (Ad), adeno-associated
virus
(AAV) and
lentivirus
(LV) vectors have been used for
cardiovascular gene transfer due to their ability to transduce non-dividing cells, as well as to obtain high gene transfer efficiency. Chosen transgene, delivery method, gene therapy vector type and dose, as well as high quality vector production all determine the therapeutic outcome. Most gene therapy applications currently rely on direct applications into the treated tissue with catheters, needle injections or equivalent techniques. For coronary and peripheral artery disease transgenes such as vascular endothelial growth factor (VEGF), fibroblast growth factor, hepatocyte growth factor (HGF) and hypoxiainducible factor (HIF-1α) have been used in clinical trials, whereas for heart failure, transgenes such as sarcoendoplasmic reticulum calcium ATPase 2a (Serca2a), adenylyl cyclase 6 and stromal-derived-factor 1 have been used 1. To date human Ad serotype 5 vector (Ad5) has been the most widely used vector for cardiovascular gene transfer. Localized intramuscular and intramyocardial delivery of Ad vectors has shown great promise in, e.g., developing treatment for peripheral and myocardial ischemia by using VEGFs.
During the last decade four phase I/II clinical trials with Ad vector expressing
VEGF-A have been conducted in Finland. Currently, a new phase I clinical trial is on-going with Ad vectors expressing VEGF-D 2. Emerging data on epigenetic regulation of vasculature, exosomes, non-coding RNAs, mechanotransduction and cell signaling in vivo have transformed our views on vascular biology. Growing knowledge of vascular function at the cell and molecular level enables also finding of new therapeutic targets for cardiovascular diseases (CVDs). Availability of several transgenic and knockout mouse models greatly enhances possibilities to obtain physiologically significant results using gene transfer applications. Besides ApoE and LDL receptor knockout mice for hyperlipidemia and atherosclerosis research 3, several mouse
Page 4 of 26
Human Gene Therapy Recent Advancements in Cardiovascular Gene Therapy and Vascular Biology (doi: 10.1089/hum.2015.095) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
4 models with deletion of specific apolipoproteins, cytokines, growth factors, or extracellular matrix components have been generated. New mouse models, e.g., for modeling vulnerable plaques have also been recently introduced 4. Final preclinical efficacy, safety, biodistribution and toxicology studies of gene therapy vectors are usually performed in larger animals, such as rabbits or pigs, since they produce a more realistic outcome about the usefulness of CVD gene transfer applications regarding human clinical applications. Particularly pigs have been increasingly used for myocardial infarction, cardiac ischemiareperfusion and in-stent restenosis studies. Pig heart is very similar to human heart and allows the use of similar gene vector dose, catheter interventions, and imaging techniques as for man. Detection of the therapeutic outcome in pre-clinical animal models by imaging of microvasculature
in three-dimension enables
better understanding of vascular
morphology and function, as well as biodistribution of the virus vectors after gene delivery. Fast development of new imaging modalities with higher spatial resolution is going to improve vascular imaging and analysis even further 5. The challenge is now how to interpret the most useful data, e.g., from bioinformatics and vascular imaging for development of new therapeutic vectors for the treatment of CVDs.
Virus Vectors for Cardiovascular Gene Transfer. Ad vectors have been the most commonly used vector type in clinical trials aiming to treat CVDs due to their high gene transfer efficiency and ease of production in good manufacturing practice (GMP) quality. Although systemic delivery of Ad vectors is limited due to induced immune response and virus interactions with circulating blood and cell components, localized intramuscular and intramyocardial delivery of Ad vectors have shown great promise in developing treatment for CVDs. Vascular gene transfer of endothelial cells, cardiomyocytes or medial smooth muscle cells have shown to be obtained after Ad vector administration by intramyocardial
Page 5 of 26
Human Gene Therapy Recent Advancements in Cardiovascular Gene Therapy and Vascular Biology (doi: 10.1089/hum.2015.095) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
5 injection, coronary perfusion or intravascular delivery to transiently isolated vessel segments
6-11
. To date Ad vectors have been used in clinical trials for coronary and
peripheral artery disease by expressing transgenes such as HGF, HIF-1α and VEGF
2,12-14
.
A clinical trial for heart failure by Ad vector expressing adenylyl cyclase type VI is ongoing. Besides Ad vectors, AAV and LV vectors have been used in cardiovascular gene transfer applications. With AAV vectors, high gene transfer efficiency has particularly been detected in cardiomyocytes after intramyocardial delivery. Persistent gene expression by AAV vectors have been shown in pre-clinical trials in cardiomyocytes and skeletal muscle 7,15
. AAV9 can efficiently target and transduce cardiomyocytes after systemic delivery
16
,
whereas AAV2 has been shown to have the most promising efficiency after intracoronary delivery to heart transplants ex vivo
17
. AAV1 instead has shown to be the most efficient
gene transfer vector after intramyocardial injection
18
and have been used in phase 2
clinical trials for heart failure by expressing Serca2a as a transgene
19
. Also, LV vectors
have been developed for cardiovascular gene transfer due to their ability to induce longterm gene expression in cardiomyocytes in pre-clinical trials
20
. Instead, in rabbit skeletal
muscle LV mediated transduction was shown to be short-term (
