Summary of common viral vectors used in gene therapy.
Gene therapy is an advanced treatment approach that alters the genetic composition of cells to confer therapeutic protein or RNA expression to the target organ. It has been successfully introduced into clinical practice for the treatment of various diseases. Cardiac transplantation stands to benefit from applications of gene therapy to prevent the onset of post-transplantation complications, such as primary graft dysfunction, cardiac allograft vasculopathy, and rejection. Additionally, gene therapy can be used to minimize or potentially eliminate the need for immunosuppression post-transplantation. Several animal models and delivery strategies have been developed over the years with the goal of achieving robust gene expression in the heart. However, a method for doing this has yet to be successfully translated into clinical practice. The recent advances in ex vivo perfusion for organ preservation provide potential ways to overcome several barriers to achieving gene therapy for cardiac transplantation into clinical practice. Optimizing the selection of the gene-carrying vector for gene delivery and selection of the therapeutic gene to be conferred is also crucial for being able to implement gene therapy in cardiac transplantation. Here, we discuss the history and current state of research on gene therapy for cardiac transplantation.
- gene therapy
- cardiac transplantation
- gene delivery
- viral vectors
- non-viral vectors
Cardiac transplantation is the gold standard therapy for end-stage heart failure. The perfection of surgical interventions, development of modern immunosuppressive therapies, and implementation of rigorous transplant care protocols have contributed to better outcomes over the last several years [1, 2]. However, cardiac transplantation is limited by the number of available donor hearts, primary graft dysfunction (PGD), rejection of the heart, as well as by the side effects caused by immunosuppression therapy . Gene therapy is an advanced treatment intervention that can potentially bridge the gap to overcome these common post-transplantation complications. The success of commercially available gene therapy interventions, such as Zolgensma for spinal muscular atrophy and Luxturna for Leber congenital amaurosis, demonstrates that gene therapy provides a viable treatment option for people who would otherwise suffer from diseases that have traditionally been thought of as impossible to treat.
Gene therapy works by altering the genetic composition of cells to confer therapeutic protein or RNA expression to the target organ. To date, it has been commercially used to treat spinal muscular atrophy, Duchenne muscular dystrophy, and for various types of ocular disorders . There are currently many gene therapy clinical trials underway and growing in number (clinicaltrials.gov). Gene therapy based interventions have been studied for various cardiovascular diseases, such as coronary artery disease (CAD), heart failure (HF), and myocardial ischemia (MI) . However, no intervention has yet been able to attain robust or long-term transgene expression in the heart in clinical practice. One promising intervention for HF was the AAV1-SERCA2a therapeutic which was evaluated in human clinical trials (CUPID, AGENT-HF, SERCA-LVAD). The trials, unfortunately, failed to demonstrate that the intervention led to a statistically significant difference in the primary endpoint of time to recurrent HF and secondary endpoint of time to first terminal events [6, 7, 8].
The heart is a complex target for gene therapy interventions due to its location in the body, the mechanical force of blood flow, endothelial barriers, cellular barriers, and the body’s immune response . A cardiac graft being treated prior to transplantation is uniquely amenable to gene therapy as most of these traditional barriers of gene delivery to the heart can be overcome. Through gene therapy, a cardiac allograft can be engineered to express selected therapeutic genes that could prevent the onset of post-transplantation complications and potentially minimize or eliminate the need for traditional systemic immunosuppression medications [10, 11]. Gene therapy for heart transplantation, though attractive, has not been translated clinically.
There are major challenges that need to be overcome for gene therapy to be able to be applied for cardiac transplantation. One of them is that, despite major advances in the understanding of transplant immunology, there remains an incomplete understanding of the mechanisms of both rejection and tolerance. This includes the understanding of the details of regulatory cytokine networks, MHC-antigen interactions during the rejection process, and a complete understanding of co-stimulatory factors and their functions . Another challenge is that most current gene delivery mechanisms confer a transient, low level of gene expression . With the current understanding of gene therapy in the context, it also is unclear what is the optimal dose of the therapeutic transgene needed to confer an appreciable clinical effect. However, recent investigations describe methods of robust and global gene delivery to cardiac grafts that offer promise to overcome this challenge. Similarly, viral vector delivery systems pose risks to the host and allograft via eliciting undesired immune reactions, off-target gene delivery, and genome integration. With the recent success and clinical adoption of
To achieve a successful gene therapy intervention for cardiac transplantation, several components need to be addressed: disease or indication and therapeutic target, use of an appropriate animal to test the therapeutic, selection of the vector for gene delivery, and method for vector delivery. Here we review select post-transplantation complications and potential targets where gene therapy can be implemented to prevent them. We will also review translational animal models that have been developed for investigating gene therapeutic targets. Finally, we will discuss the different viral and non-viral vectors that can be used for gene delivery, the selection of promoters, and the different modalities that have been investigated for the delivery of vectors to cardiac grafts.
2. Disease and therapeutic targets for cardiac transplantation
There are various insults that a cardiac graft experiences prior to, during, and after transplantation. Early damage to the cardiac graft can happen during the brain or cardiac death of the donor, organ procurement, organ preservation time, the implantation procedure, or as a result of reperfusion injury. These points of insult to the cardiac graft can trigger both innate and adaptive immune responses that result in injury. Common complications that occur following transplantation include primary graft dysfunction (PGD), coronary allograft vasculopathy (CAV), and rejection.
2.1 Primary graft dysfunction
PGD is a leading cause of early mortality post-transplantation . The diagnosis of PGD occurs in the first 24 hours following heart transplantation. It presents as severe ventricular dysfunction of the cardiac graft in the immediate post-transplant period, resulting in low cardiac output and hypotension despite the presence of adequate filling pressures . Either the left, right, or both ventricles can be involved, and the severity of the dysfunction can range from mild to moderate to severe depending on the extent of circulatory support that is needed to maintain hemodynamic stability .
Numerous causative factors, starting from donor death to weaning the heart from cardiopulmonary bypass in the recipient, have been identified that contribute to the cause of PGD . These factors relate to ischemic and ischemic-reperfusion injury of the cardiac graft. Additionally, the onset of systemic inflammatory response syndrome and the development of vasoplegic syndrome in the recipient have also been identified as significant causes . Finally, the use of extended criteria donors, such as donation after cardiac death (DCD), has also been identified as a significant risk factor for PGD .
The treatment of PGD is primarily through supportive care. It is typically initially managed with the use of inotropic support using catecholamines and phosphodiesterase inhibitors. The next escalation in care is typically the use of an intra-aortic balloon pump, followed by the initiation of advanced mechanical support using extracorporeal membranous oxygenation.
2.2 Cardiac allograft Vasculopathy
Cardiac allograft vasculopathy (CAV) is a major cause of late heart graft failure . It is characterized by diffuse and concentric narrowing of large epicardial and small intramyocardial arteries due to intimal fibromuscular hyperplasia, atherosclerosis, and vasculitis. As a result, the transplant recipient develops pathological changes within the donor blood vessels leading to a spectrum of diseases ranging from MI to HF. CAV is often unable to be diagnosed by coronary angiography and requires intravascular ultrasound for diagnosis.
The main driver of CAV is believed to be the immune system of the host. The intimal thickening seen in CAV results from an accumulation of smooth muscle cells (SMC) accompanied by the infiltration of T cells and macrophages which further contribute to intimal expansion [21, 22]. Yet CAV lesions characteristically stop at the suture line between the donor and the recipient. The endothelial lining of the vessels remains intact in CAV lesions suggesting that SMC injury may result from sterile inflammation as is seen during cold and warm ischemia effects and ischemia–reperfusion injuries .
Current treatments are based on vascular risk factor management and the use of statins and mTOR inhibitors (sirolimus and everolimus) to reduce the development of the disease. Percutaneous revascularization is used to treat focal obstructive coronary stenosis but repeat revascularization rates are high due to restenosis and disease progression . However, patients who go on to develop allograft dysfunction require re-transplantation [25, 26].
2.3 Rejection and immunosuppression
Cardiac allograft rejection is among the most common causes of death in heart transplant recipients . Acute rejection is categorized into hyperacute rejection acute cellular rejection (ACR), and antibody mediated rejection (AMR). Currently, recipients undergo routine screening for rejection with endomyocardial biopsies obtained by a bioptome. Hyperacute rejection is due to the presence of preformed host antibodies against the graft and portends an inevitable immediate immune rejection resulting in death . ACR and AMR take longer to manifest and are thus amenable to potential gene therapy intervention and we will focus our discussion on these forms of rejection. To prevent rejection of the cardiac allograft, patients are treated with systemic multidrug immunotherapies. Multidrug immunosuppressive regimens currently used in human transplant recipients are associated with an increased risk of malignancy and opportunistic infections, a metabolic syndrome characterized by insulin resistance and dyslipidemia, and drug-specific toxicity .
2.4 Potential targets for gene therapy
An understanding of the different insults that the cardiac graft experiences during the different steps of transplantation helps to identify potential targets for gene therapy for cardiac transplantation. The cardiac graft endothelium is vulnerable to ischemic reperfusion injury. In this setting, leukocytes adhere to the activated endothelium. The complement system becomes activated, neutrophils migrate into the cardiac graft, subsequently followed by natural killer cells and macrophage infiltration. These early non-specific inflammatory reactions are then followed by alloimmune reactions that result in massive graft infiltration by dendritic cells, T-cells, B-cells, and macrophages. Donor-derived dendritic cells leave the cardiac graft and migrate to recipient lymph nodes and the spleen. There they present donor antigen to recipient T cells directly and trigger acute rejection.
2.4.1 Inflammatory targets
Many candidate genes that interfere with one of these inflammatory mechanisms have been investigated in the context of cardiac transplantation. One such gene is endothelial nitric oxide synthase (eNOS). eNOS produces nitric oxide which is vasoprotective. Delivery of eNOS into the donor heart attenuated ischemic reperfusion injury, leukocyte infiltration, and cardiac graft rejection in a rabbit model . Similarly, superoxide dismutase (SOD) gene delivery into a donor heart attenuated ischemic reperfusion injury after organ preservation and transplantation in a rabbit model . SOD functions as a free radical scavenger that neutralizes reactive oxygen species generated during ischemic reperfusion injuries. Another target, nuclear factor-kappa B (NFkB), is a transcription factor involved in the up-regulation of pro-inflammatory gene products. One possible therapeutic intervention is to block NFkB in endothelial cells to attenuate ischemia–reperfusion injury in the myocardium. Sakaguchi et al. blocked NFkB by using double-stranded oligodeoxynucleotides with a specific affinity for NFkB (NFkB decoy group) to transduce rat hearts utilizing HVJ envelope . The hearts were then preserved for 16 hours in hypothermic preservation solution before being heterotopically transplanted into a recipient rat. What they found is that the intervention attenuated ischemic reperfusion injury after prolonged heart preservation in hypothermic solution. Another protein that is up-regulated during inflammation and serves as a potential target for gene therapy is heat shock protein-70 (HSP-70). It has an essential role in protein folding and translocation and as chaperones for intracellular proteins. HSP-70 has particularly been shown to be associated with protection against ischemia–reperfusion injury. Jayakumar et al. infused rat hearts using 1 mL of the gene vector solution then incubated the hearts on ice for 10 minutes before heterotopically transplanting them into a recipient rat . 4 days after the intervention, the hearts were perfused on a Langendorff apparatus for 45 minutes followed by reperfusion for 1 hour. They found that post-ischemic recovery of mechanical function was greater in the treatment arm versus control, recovery of coronary flow was greater as well. The conclusion was that HSP-70 gene transduction protects both the mechanical and endothelial function of the cardiac graft.
2.4.2 Rejection targets
The most direct and immediate barrier to the success of cardiac transplantation is the recipient immune response. Currently, the most effective clinical therapy is lifetime immunosuppressive therapy. Knowledge about the immune response in transplantation has grown tremendously in recent years such that gene therapy can be used to intervene on different targets of the immune response. Both cell and antibody mediated effector mechanisms are responsible for acute rejection . A strategy to protect the cardiac graft from recipient immune responses is through the delivery of genes that confer proteins to the graft that modulate host immune responses. These would include cytokines or soluble ligands. Qin et al. utilized a retrovirus and a plasmid delivery system to transfer genes that encode transforming growth factor beta-1 (TGF-β and interleukin 10 (IL-10) to a mouse myoblast and non-vascularized cardiac graft . Grafts transduced with either of these genes had significantly prolonged survival when compared with the vector alone (39 days with IL-10 vs. 26 days with TGF-β vs. 12 days with vector alone). The therapeutic effect of transduced IL-10 and TGF-β1 has been demonstrated in follow-up investigations using different types of vectors [34, 35, 36].
Another point of gene intervention would be at the point of T-cell costimulatory activation. Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a protein that modulates T-cell costimulatory activation. It becomes upregulated on T-cells upon T-cell activation. Gene delivery of a soluble CTLA-4 immunoglobulin fusion protein (CTLA4Ig) into the donor heart was associated with detectable CTLA4Ig serum levels 120 days after transplantation as well as long-term cardiac graft survival, >100 days in a rat model . However, the expression of CTLA4Ig did enter systemic circulation causing some systemic immunosuppression in the rats. Another similar target is the programmed death-1 (PD-1) gene. It is expressed on activated T-cells, B-cells, and myeloid cells. When PD-1 binds one of its ligands, PD-L1 or PD-L2, it leads to the inhibition of activated T-cells. PD-L1 and PD-1 play an important role in both acute and chronic rejection of transplanted hearts in animal studies [38, 39, 40]. In rejecting human transplanted hearts, PD-L1 expression is decreased relative to PD-1 expression . Gene delivery of soluble PDL1Ig fusion protein into the donor heart moderately prolonged cardiac graft survival in rats .
2.4.3 Cardiac ischemic disease targets
An additional example of gene therapy applied to treat cardiac disease involves targeting angiogenic gene therapy that facilitates neovascularization to augment blood flow in ischemic myocardium. These include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). In particular, these targets have been assessed for treating ischemic disease caused by MI or congestive heart failure. Rosengart et al. delivered 4 x 108 to 4 x 1010 particle units of an adenoviral vector encoding the VEGF gene to individuals undergoing bypass graft surgery and as the sole therapy to the experimental group via mini-thoracotomy. The intervention demonstrated no adverse events and there was symptomatic improvement in both groups .
The Angiogenic Gene Therapy (AGENT) clinical trials were the first randomized control trial studies investigating the benefits of stimulating coronary angiogenesis with gene therapy using FGF-4 . FGF-4 was delivered using adenovirus administered by infusion into the coronary arteries of patients with chronic stable angina. AGENT evaluated incremental doses of 3 x 108 to 1 x 1011 particle units. The overall improvement in exercise treadmill time was similar for those in the treatment and the control arms. However, post-hoc analysis showed that when baseline neutralizing antibody titer was controlled for, patients with titers less than 1:100, 44% had increased their exercise treadmill time by more than 30%. In patients with titers greater than 1:100, only 7% had increased their exercise treadmill time by more than 30%. AGENT 2 investigated whether FGF-4 improved myocardial perfusion compared with placebo. A significant decrease in ischemic defect size was observed in the treatment arm (21% relative decrease) that was not observed in the placebo group. AGENT 3 and 4 were planned to determine the efficacy and safety of FGF-4 in the larger population, however, an interim review of the data demonstrated no differences in exercise treadmill time and therefore recruitment was stopped.
HGF as a therapeutic target has been evaluated in numerous studies. In the context of therapy for MI, Jin et al. investigated the long-term effects of HGF in a rat MI model . Utilizing an adenoviral vector for delivery of HGR, the vector was injected directly into the infarct border zone immediately after permanent coronary ligation. 10 weeks post-intervention, there was no significant difference in the left ventricular ejection fraction, but capillary density was significantly higher in the treatment groups, whereas arteriole density was unchanged. Masahiro et al. describe the use of recombinant HGF delivered by HVJ envelope for prolonged cardiac graft preservation in rats during hypothermic storage . The rationale for this choice is that HGF functions as an antiapoptotic factor in the heart. They concluded that the administration of HGF prevented myocardial apoptosis and improved cardiac function after prolonged myocardial preservation in hypothermic solution.
3. Animal models
Selection of an appropriate animal model for heart transplantation is critical to be able to translate a potential gene therapy intervention from the laboratory bench to the patient bedside. Numerous small animal models using rodents have been described where the heart is transplanted either heterotopically or orthotopically in the recipient animal. Similarly, there have been numerous large animal models described using pigs, sheep, and non-human primates (NHP). We will discuss examples of different types of small and large animal models in heart transplantation and in what instances an investigator may choose one over the other.
3.1 Heterotopic heart transplantation
Heterotopic heart transplantation (HHT) is when the transplanted heart is placed in an ectopic position inside of the recipient without the removal of the recipient’s native heart. Intra-abdominal HHT is primarily used to investigate transplantation biology and is also suitable for studying unloading induced changes in the heart . The heart of a donor animal is explanted and subsequently transplanted into the abdomen of a recipient animal. To accomplish this the donor ascending aorta is anastomosed to the recipient infrarenal aorta and the donor pulmonary aorta is anastomosed to the recipient inferior vena cava. The result of this configuration is that the graft beats with reduced left ventricular filling while coronary perfusion is preserved. It offers several advantages over orthotopic transplantation in research applications such as technical simplicity, better accessibility for biopsies, and survival of the recipient even in cases of graft rejection .
The first heterotopic abdominal heart transplantation was published using rats by Abbott et al.  in 1964 and subsequently modified by Ono et al. . The technique of the latter has been widely adopted as the standard HHT rodent model. Heterotopic heart transplantation in mice is more challenging than in rats, however, testing mechanistic hypotheses is more practical in mice given the greater diversity of genetic modifications available in mice. The advantage of using a small animal model is that they are less costly when compared to the cost of a large animal. A larger number of small animals can be used to assess and describe the effects of a therapeutic transgene. It also allows for several transgenes to be tested in parallel to study the differences in efficacy between them. The main challenge in using small animals is that the micro-surgical implantation technique is very challenging given their smaller size. Another aspect that makes this surgery more challenging to do in smaller animals is that they have a lower tolerance for blood loss. Because of this, it is especially important that there be minimal blood loss during the procedure and that the anastomoses be hemostatic at the time of procedure completion.
The advantage of large animals is that the results of the gene therapy intervention are able to be translated more quickly into clinical practice than are the results obtained from small animal studies. However, large animals are very costly to acquire and maintain in comparison to small models. In the setting of small primates, Minanov et al. positioned NHP hearts into the iliac fossa of primate recipients . More recently this transplant configuration has been used to investigate interventions in xenotransplantation using a porcine heart transplanted into a baboon [52, 53, 54]. Porcine to porcine heart transplantation is also used in the research setting to investigate the immune system effects of cardiac transplantation as well as gene therapy interventions (Figure 1) [55, 56, 57]. This surgical research model is also amenable for modeling post-cardiac transplantation complications, such as CAV and rejection, without subjecting the animal to a high risk of morbidity or mortality [55, 58]. The recent success of a porcine to human xenotransplantation using genetically modified pigs to minimize rejection by the human immune system of the xenograft stresses the importance of the selection of the appropriate animal model. After procuring the heart, the xenograft was preserved utilizing an
3.2 Orthotopic heart transplantation
Orthotopic heart transplantation is when the transplanted heart is placed in the position of the recipient’s native heart. As such, the cardiac graft takes over providing the cardiovascular support of the recipient. This transplant configuration in research is most useful to investigate the cardiac graft’s overall ability to support the recipient following an administration of a new intervention. The pros of this design are that it most closely reflects clinical practice so one can investigate beyond the immunopathologic changes the heart undergoes after transplantation. This approach allows the investigator to determine whether an intervention permits the transplanted heart to perform its intended function to support the recipient’s cardiovascular system. This has been successfully described in porcine to porcine models, as well as in pig to baboon xenotransplantation models [60, 61, 62].
4. Vectors for gene delivery
Vectors for gene delivery comprise viral and non-viral vectors. Viral vectors are the more efficient of the two but are also associated with more side effects than non-viral vectors. Each type of viral vector confers different gene expression characteristics, such as the length of time for transgene expression and the intensity of transgene expression (Table 1). Additionally, when constructing the optimal vector for cardiac gene delivery consideration must be given to the selection of promoter. Constitutively active promoters, such as CMV or RSV promoters, confer broad tissue tropism and strong expression. However, cardiac-specific promoters, such as myosin heavy chain promoter, myosin light chain promoter, and troponin T promoter have been used to restrict transgene expression in the heart . While the cardiac-specific promoters focus gene delivery to cardiac tissue, they confer weaker expression when compared with constitutively active promoters (Table 2).
|Viral vector||Genetic material||Capacity||Transduction ability||Peak gene expression||Main advantages||Characteristics|
|Adenovirus||dsDNA||4.5–36 kb||Transduces both dividing and non-dividing cells.||1–7 days||Efficiently delivers genes to most tissues.||Short-term but highly efficient gene delivery. Can elicit a strong inflammatory response.|
|Adeno-associated virus||ssDNA||4.7 kb||Transduces both dividing and non-dividing cells.||2–4 weeks||Low immunogenicity. Broad but specific tropism.||Long-term gene expression. Low immunogenicity.|
|Lentivirus||RNA||8 kb||Transduces both dividing and non-dividing cells.||4–6 days||Can carry multiple transgenes. Persistent gene transfer in dividing cells.||Persistent gene expression in dividing cells. Low but potential risk of mutagenesis.|
|Reference||Transduced gene||Therapeutic mechanism||Transduction method||Key findings/conclusions|
|Iwata et al. ||eNOS||Attenuation of ischemia–reperfusion injury||Lipid/DNA complex via intra-op coronary infusion||Allogeneic rabbit heart transplant model demonstrated that intramyocardial neutrophil and T-cell populations were halved in eNOS transduced hearts. NF-kB activation in microvascular endothelial cells and cardiomyocytes was significantly reduced.|
|Abunasra et al. ||SOD||Attenuation of ischemia–reperfusion injury||Ad via ||Heterotopic heart transplant model in rats demonstrated positive immunoreactivity for SOD and 86.8% +/− recovery of pre-ischemic left ventricular pressure.|
|Jayakumar et al. ||HSP-70||Attenuation of ischemia–reperfusion injury||HVJ envelope via ||Heterotopic heart transplant model in rats demonstrated greater post-ischemic recovery of mechanical function and greater recovery of coronary flow in HSP-70 treated mice.|
|Sakaguchi et al. ||NF-kB decoy||Attenuation of ischemia–reperfusion injury||HVJ envelope via ||Heterotopic heart transplant model in rats demonstrated introduction of NF-kB decoy into the nuclei of endothelial cells and cardiomyocytes. After 1 hour of reperfusion the NF-kB decoy group showed significantly higher degrees of recovery of left ventricular function.|
|Guillot et al. ||CTLA4Ig||Attenuation of T-cell costimulatory pathway||Ad via intramyocardial injection||Heterotopic heart transplant model in rats demonstrated indefinite graft survival (>100 days) and could be detected in the graft at least 1 year after gene transfer. Evident suppression of antibody production against donor alloantigens up to at least 120 days after gene transfer.|
|Dudler et al. ||PD-L1Ig||Attenuation of T-cell costimulatory pathway||Ad via ||Heterotopic heart transplant model in rats demonstrated a prolonged median survival time (17 days vs. 11 days). Also demonstrated a decreased number of CD4 cells and monocytes/macrophages infiltrating the graft.|
|Grines et al. ||FGF||Angiogenic therapy||Ad via intracoronary infusion||Randomized controlled trial that enrolled patients with chronic stable angina demonstrated improved exercise time on a treadmill for those treated with intervention and had a baseline time < or equal to 10 minutes. Intervention decreased the ischemic defect size. Larger efficacy studies failed to demonstrate significant differences in exercise time on a treadmill so the trial was stopped.|
|Rosengart et al. ||VEGF||Angiogenic therapy||Ad via intramyocardial injection||Phase I clinical study that enrolled patients with clinically significant coronary artery disease. There were no systemic or cardiac related adverse events related to vector administration. Coronary angiography and stress sestamibi scan showed improvement in the treated area. All patients reported improvement in angina class after therapy.|
|Jin et al. ||HGF||Angiogenic therapy||Ad via intramyocardial injection||Myocardial infarction model in rats demonstrated no significant difference in the left ventricular ejection fraction. It did observe increased capillary density in the treatment group.|
|Ryugo et al. ||HGF||Angiogenic therapy/Antiapoptosis||HVJ via cold static storage||Cardiac grafts procured from rats demonstrated that HGF treated hearts had a significantly higher recovery rate of left ventricular developed pressure. c-MET/HGF receptor expression was stronger in the treatment group.|
4.1 Adenoviral vectors
Adenovirus (Ad) vectors have high transduction efficiency. They are able to transduce both quiescent and dividing cells and maintain epichromosomal persistence in the host cell . Ad vectors also have a broad tropism profile and large packaging capacity (4.5-36 kb). They offer efficient transduction of cardiomyocytes. However, gene expression is transient, peaking 1–7 days after delivery and then diminishing until it ceases at about 2–3 weeks after transduction . They carry double-stranded DNA. Their main disadvantage is the widely pre-existing viral immunity among the general population. Since Ad is strongly immunogenic it causes undesired immune responses in treated subjects . In order to overcome this and improve their capacity, Ad vectors have undergone several generations of engineering.
The first generation of Ad vectors was designed by removing the E1A gene which makes it so the recombinant Ad is unable to replicate within the host cell . With the deletion of this gene, complementary cell lines, such as HEK293, had to be designed to express E1A and E1B in order to produce the viral vector. The main disadvantages of the first generation of Ad were that de novo expression of Ad proteins could activate the host immune response and there was still the possibility of spontaneous homologous recombination between the vector and engineered E1 region from HEK293 that could generate replication-competent adenovirus .
In the second generation of Ad vectors, further early gene regions (E2a, E2b, or E4) of the vector were deleted to permit additional space for the transgenes. As in the first generation, the deleted genes needed to be complemented by engineered production cell lines. However, the deletion of these genes led to inefficient complementation of E2/4 with engineered cell lines thus negatively affecting viral vector amplification, resulting in lower titers. Another disadvantage was that the native Ad late genes that were still retained within the viral genome could trigger host immunogenicity and cellular toxicity .
Finally, the third generation of Ad vectors have all Ad viral sequences deleted except for the inverted terminal repeat sequences and packaging signal. As such, these are referred to as “gutless” or “high capacity” Ad vectors (HCAd). The production of HCAds in cell culture requires an adenoviral helper virus similar to the first-generation Ad vectors. Compared with the previous Ad vector generations, HCAds have reduced immunogenicity, prolonged transduction in the host cell, and a significantly larger transgene capacity . Their large transgene capacity makes it so that multiple transgenes could be delivered. The main disadvantage of HCAds is the challenge of ensuring that the helper virus is eliminated from the final vector preparation.
4.2 Adeno-associated viral vectors
Adeno-associated viral (AAV) vectors were discovered as a contaminant of Ad preparations in 1965 . They lack essential genes needed for replication and expression of their own genome. They are not known to cause any human diseases. AAV vector was first used in humans in 1995 to deliver the cystic fibrosis transmembrane regulator (CFTR) gene into a patient with cystic fibrosis using the AAV2 capsid . Today, recombinant AAVs are the leading vectors for the delivery of gene therapies. The first recombinant AAV gene therapy product, Glybera, was approved by the European Medicines Agency to treat lipoprotein lipase deficiency in 2012. Five years later, Luxturna was approved as the first recombinant AAV gene therapy product in the United States [72, 73].
AAVs carry single-stranded DNA (ssDNA). However, the efficiency of AAVs are limited by ssDNA in that it needs to be converted to double-stranded DNA (dsDNA) prior to expression. This step is circumvented through the use of self-complementary vectors which package an inverted repeat genome that can fold into dsDNA without the requirement for DNA synthesis or base-pairing between multiple vector genomes . Transgene expression peaks at around 2–4 weeks after delivery. AAVs can carry transgenes up to 4.7 kb in size.
There are 13 natural AAV serotypes. These have been isolated from laboratory Ad stocks and mostly from human or non-human primate origin . Engineering or recombinant AAV capsids confer the vector the capability to transduce multiple tissue types. Recombinant AAVs are composed of the same capsid sequence and structure as found in wild-type AAVs. Recombinant AAVs encapsidate genomes that are devoid of all AAV protein-coding sequences and that have therapeutic genes designed in their place. The complete removal of viral coding sequences maximizes the packaging capacity of these AAVs and contributes to their low immunogenicity and cytotoxicity .
Capsid development approaches are based on rational design and directed evolution. The rational design was among the first approaches to improve vector capsids. This entailed adding peptide sequences onto the surface of the capsid to direct the tropism of the vector and deter immunological recognition . While rational design allowed for the early development of specialized AAVs, a major limitation of that approach is that there oftentimes is insufficient knowledge regarding AAV cell surface binding, internalization, trafficking, uncoating, and gene expression. The basis of directed evolution is in the simulation of natural evolution. Capsid libraries are placed under selective pressure to yield genetic variants with specific biological properties and advantageous characteristics. This way directed evolution of the capsid does not require a prior understanding of the molecular mechanisms involved in the selection criteria .
Cell-type specific transgene expression, however, is conferred at the level of gene transcription by the promoters used in AAV vectors. The serotype AAV9 has been shown to have the highest cardiac gene transduction efficacy in mice and rats with either systemic or direct cardiac injection [77, 78]. Meanwhile, the serotype AAV6 has proven to be a more effective vector when injected into the myocardium of pigs and non-human primates [79, 80]. Piacentino et al. described a recombinant AAV serotype engineered via rational design, termed SASTG, which has extremely high-level cardiac transduction and tropism . A challenge for AAV-mediated gene therapy is overcoming the negative effect that innate immunity has on transgene expression. Yet adaptive immunity to the capsid and the foreign transgene is the main factor for decreased efficacy. Notwithstanding, recombinant AAVs are accepted as the least immunogenic when compared to other viral vectors. Patients that have been exposed to AAV serotypes that gene therapy is based on will have a high chance of forming antibodies against the vector capsid . One plausible way of removing these anti-AAV antibodies from the bloodstream is by using plasmapheresis . Another described pre-treatment is the use of IgG-cleaving endopeptidases which reduce IgG antibodies from the serum . Besides removing the neutralizing antibodies, investigators have also utilized rational design and directed evolution to develop AAV capsids that evade neutralizing antibodies [85, 86, 87, 88].
Lentiviral vectors constitute a genus of the retrovirus family. They permit long-term transgene expression by integrating the delivered genes into the host genome and can carry transgenes up to 8 kb in size . They can deliver single-stranded RNA to both dividing and non-dividing cells and display robust transduction efficiency . A unique advantage of lentiviral vectors is the ability to express multiple genes from a single vector [91, 92]. Transgene expression peaks after 4–6 days. The immune response to lentiviral vectors is low but concerns remain about potential insertional mutagenesis and off-target gene expression . They have a preference for targeting the coding regions of genes, carrying the risk of insertional oncogenesis . Additionally, the vector lacks tropism for the heart, making it unideal for heart-specific delivery through
4.4 Non-viral vectors
Naked nucleic acids allow for the delivery of large genes in high quantities. These include DNAs, mRNAs, micro RNAs, and siRNAs. However, the lack of protection from endonuclease degradation makes them unreliable with low cellular internalization of the transgene . Additionally, naked nucleic acids have an uncondensed shape and polyanionic charge that does not allow for their efficient uptake into cells. The half-life of plasmid DNA is about 10 minutes following systemic injection into mice .
Nanoparticles have been developed to interact with nucleic acids to protect them from degradation and condense them into nano-sized complexes that can be internalized by cells. Two main types of nanoparticles being used in investigations are lipid-based and cationic polymer-based. Another modification that is being used to improve the uptake of naked nucleic acids by cells is through chemical modification to mRNA to reduce the activation of the immune system and improve the stability of the RNA. These modified mRNAs are attractive agents for short-term gene delivery to the myocardium .
4.5 Hemagglutinating virus of Japan envelope vector
Wild-type hemagglutinating virus of Japan (HVJ) was discovered in 1953 and is a member of the paramyxovirus family. The envelope of HVJ is composed of a lipid bilayer and two integral membrane glycoproteins, F and HN, that project from the viral surface [100, 101]. HVJ envelope vector is constructed by incorporation of plasmid DNA into inactivated HVJ-containing liposomes . During the preparation of the envelope vector, HN and F are retained but all the genome inside of HVJ is removed. It has high efficacy to induce a molecule into a target cell by the strong action of fusing cells on its membrane. Additionally, the removal of all the virus genomes confers low immunogenicity to the vector and eliminates replication and viral gene expression in cells. It is in essence a “viral, non-viral hybrid vector” . HVJ can be used to deliver DNA, RNA, and oligonucleotides efficiently both
5. Methods for delivery of vectors for cardiac gene therapy
Gene delivery to any organ is a challenging feat. Gene delivery to the whole cardiac allograft is an especially challenging task given numerous obstacles.
5.1 Intramyocardial injection
Direct intramyocardial injection of the vector into the myocardium is one such technique for vector delivery. It is easy to perform the injections and could theoretically be performed during graft procurement or after cardiac transplantation. Guzman et al. described the use of this technique for the delivery of adenovirus injected through a 25-gauge needle into the cardiac apex . The intramyocardial injection has also been described in a clinical trial where subjects underwent a thoracotomy with the injection of vascular endothelial growth factor-2 naked deoxyribonucleic acid. They found that the procedure is well tolerated and reported few major adverse cardiac events at 1 year . The major limitation of this technique for cardiac transplantation is that it only allows for limited focal delivery and the inability to target deeper muscular structures of the heart, such as the septum. Additionally, it is challenging to keep all of the injected material inside of the myocardium, leading to leakage from the needle holes and causing injury to the heart .
5.2 Intracoronary infusion
Intracoronary infusion of the vector is another described technique. By this method, the vectors are infused directly into the coronary arteries and reach the target cells for transduction by transit through the coronary arterial tree. Intracoronary infusion can be achieved by several methods: coronary catheterization prior to procurement,
Catheterization of the coronary arteries for delivery and infusion through the cardioplegia catheter at the time of the graft procurement allows for a more dispersed and homogenous distribution of transgene delivery than is achieved through intramyocardial injections. Generally, transgene expression is able to be observed along with the distribution of the coronary arteries . Several disadvantages exist with these delivery techniques. One is the negative effect pre-existing coronary artery disease has on the ability of vectors to reach their cellular target. Another is that since the infusion of the vector is based on a single bolus delivery when using a catheter-based approach, there is a large amount of vector that is lost to the systemic circulation resulting in poor transduction efficacy of the heart and a significant amount of off-target transduction. Finally, transduction efficacy is hampered by the presence of circulating neutralizing antibodies in the recipient against viral vectors. Vector particles containing proteins that are similar to antigens that humans are exposed to following natural infection may be neutralized by antibodies upon injection in some humans because of pre-existing immunity .
5.3 Complete heart isolation by cardiopulmonary bypass
Administration of the vectors during cardiopulmonary bypass featuring complete heart isolation and continuous cardiac perfusion addresses the issues associated with the catheter-based intracoronary infusion. The technique for achieving this was described by Katz et al. using separate pumps for the systemic and cardiac circuits permitting continuous isolated arrested heart perfusion . This allows for the vectors to be recirculated through the coronary circulation of the heart, allowing for additional opportunities for the vectors to attach to cells and achieve entry. However, cardioplegia arrest requires for the heart and circulation to be maintained at a cold temperature (4°C) which is not favorable for vector attachment and entry into the target cells .
The procedure for cardiac transplantation offers a unique opportunity for gene delivery that does not exist for other indications for therapeutic intervention for heart disease. The cardiac graft is removed from the recipient and preserved for a period of time
Gene delivery to a whole cardiac graft has been described in both small and large animal models utilizing
There are several advantages that make ex vivo normothermic, sanguinous perfusion the ideal platform for translating gene therapy into clinical practice. The ability to recirculate the perfusate through the coronary arteries multiple times over a prolonged period of time optimizes the chances the delivery vectors attach to the target cells and enter. Normothermic perfusion provides a favorable environment for viral vectors to be able to efficiently transduce cells, enabling receptor-mediated vector entry and optimizing the downstream processes of transductions . The main obstacle to overcome with this vector delivery modality is the use of whole blood from the donor to make the circulating perfusate. The presence of preformed antibodies to different viral vectors could effectively neutralize the ability of the viral vectors to achieve cellular attachment. One successful intervention to overcome this is the addition of a blood washing step prior to adding the donor blood to the perfusion device and this way remove any neutralizing blood components .
Gene therapy for cardiac transplantation promises to transform clinical practice in the near future with cardiac grafts that are more robust and lasting than ever. However, in order to achieve its widespread adoption, there are various factors that need to be taken into consideration for how to achieve successful vector delivery and transgene expression to the cardiac graft. Here, we discussed several considerations such as choice of vector, choice of the therapeutic gene, and choice of vector delivery mechanism. Just as important is the selection of the appropriate animal model for determining the efficacy and therapeutic effect of a gene therapy construct. The successful translation of gene therapy interventions for cardiac transplantation can potentially minimize or eliminate the incidence of post-transplantation complications and the need for systemic immunosuppression therapy.
Stehlik J, Kobashigawa J, Hunt SA, Reichenspurner H, Kirklin JK. Honoring 50 Years of Clinical Heart Transplantation in Circulation: In-Depth State-of-the-Art Review. Circulation. 2018; 137(1):71-87
Li Y, Guo S, Liu G, Yuan Y, Wang W, Zheng Z, et al. Three preservation solutions for cold storage of heart allografts: A systematic review and meta-analysis. Artificial Organs. 2016; 40(5):489-496
Tonsho M, Michel S, Ahmed Z, Alessandrini A, Madsen JC. Heart transplantation: challenges facing the field. Cold Spring Harbor Perspectives in Medicine. 2014; 4(5):a015636
Mendell JR, Al-Zaidy SA, Rodino-Klapac LR, Goodspeed K, Gray SJ, Kay CN, et al. Current clinical applications of in vivo gene therapy with AAVs. Molecular Therapy. 2021; 29(2):464-488
Gaffney MM, Hynes SO, Barry F, O'Brien T. Cardiovascular gene therapy: Current status and therapeutic potential. British Journal of Pharmacology. 2007; 152(2):175-188
Hulot JS, Ishikawa K, Hajjar RJ. Gene therapy for the treatment of heart failure: Promise postponed. European Heart Journal. 2016; 37(21):1651-1658
Hulot JS, Salem JE, Redheuil A, Collet JP, Varnous S, Jourdain P, et al. Effect of intracoronary administration of AAV1/SERCA2a on ventricular remodelling in patients with advanced systolic heart failure: Results from the AGENT-HF randomized phase 2 trial. European Journal of Heart Failure. 2017; 19(11):1534-1541
Katz MG, Fargnoli AS, Yarnall C, Perez A, Isidro A, Hajjar RJ, et al. Technique of complete heart isolation with continuous cardiac perfusion during cardiopulmonary bypass: new opportunities for gene therapy. The Journal of Extra-Corporeal Technology. 2018; 50(3):193-198
Sahoo S, Kariya T, Ishikawa K. Targeted delivery of therapeutic agents to the heart. Nature Reviews. Cardiology. 2021; 18(6):389-399
Giannoukakis N, Thomson A, Robbins P. Gene therapy in transplantation. Gene Therapy. 1999; 6(9):1499-1511
Vassalli G, Roehrich M-E, Vogt P, Pedrazzini GB, Siclari F, Moccetti T, et al. Modalities and future prospects of gene therapy in heart transplantation. European Journal of Cardio-Thoracic Surgery. 2009; 35(6):1036-1044
Moore DJ, Markmann JF, Deng S. Avenues for immunomodulation and graft protection by gene therapy in transplantation. Transplant International. 2006; 19(6):435-445
Katz MG, Fargnoli AS, Kendle AP, Hajjar RJ, Bridges CR. Gene therapy in cardiac surgery: clinical trials, challenges, and perspectives. The Annals of Thoracic Surgery. 2016; 101(6):2407-2416
Singh SSA, Dalzell JR, Berry C, Al-Attar N. Primary graft dysfunction after heart transplantation: A thorn amongst the roses. Heart Failure Reviews. 2019; 24(5):805-820
Iyer A, Kumarasinghe G, Hicks M, Watson A, Gao L, Doyle A, et al. Primary graft failure after heart transplantation. J Transplant. 2011; 2011:175768
Kobashigawa J, Zuckermann A, Macdonald P, Leprince P, Esmailian F, Luu M, et al. Report from a consensus conference on primary graft dysfunction after cardiac transplantation. The Journal of Heart and Lung Transplantation. 2014; 33(4):327-340
Chew HC, Kumarasinghe G, Iyer A, Hicks M, Gao L, Doyle A, et al. Primary graft dysfunction after heart transplantation. Current Transplantation Reports. 2014; 1(4):257-265
Patarroyo M, Simbaqueba C, Shrestha K, Starling RC, Smedira N, Tang WH, et al. Pre-operative risk factors and clinical outcomes associated with vasoplegia in recipients of orthotopic heart transplantation in the contemporary era. The Journal of Heart and Lung Transplantation. 2012; 31(3):282-287
White CW, Messer SJ, Large SR, Conway J, Kim DH, Kutsogiannis DJ, et al. transplantation of hearts donated after circulatory death. Front Cardiovasc Med. 2018; 5:8
Pober JS, Jane-Wit D, Qin L, Tellides G. Interacting mechanisms in the pathogenesis of cardiac allograft vasculopathy. Arteriosclerosis, Thrombosis, and Vascular Biology. 2014; 34(8):1609-1614
Salomon RN, Hughes CC, Schoen FJ, Payne DD, Pober JS, Libby P. Human coronary transplantation-associated arteriosclerosis. Evidence for a chronic immune reaction to activated graft endothelial cells. The American Journal of Pathology. 1991; 138(4):791-798
Van Loosdregt J, Van Oosterhout MFM, Bruggink AH, Van Wichen DF, Van Kuik J, De Koning E, et al. The chemokine and chemokine receptor profile of infiltrating cells in the wall of arteries with cardiac allograft vasculopathy is indicative of a memory T–Helper 1 Response. Circulation. 2006; 114(15):1599-1607
Frye CC, Bery AI, Kreisel D, Kulkarni HS. Sterile inflammation in thoracic transplantation. Cellular and Molecular Life Sciences. 2021; 78(2):581-601
Lee MS, Lluri G, Finch W, Park KW. Role of percutaneous coronary intervention in the treatment of cardiac allograft vasculopathy. The American Journal of Cardiology. 2018; 121(9):1051-1055
Tremblay-Gravel M, Racine N, de Denus S, Ducharme A, Pelletier GB, Giraldeau G, et al. Changes in outcomes of cardiac allograft vasculopathy over 30 years following heart transplantation. JACC Heart Fail. 2017; 5(12):891-901
Khush KK, Cherikh WS, Chambers DC, Goldfarb S, Hayes D Jr, Kucheryavaya AY, et al. The International Thoracic Organ Transplant Registry of the International Society for Heart and Lung Transplantation: Thirty-fifth Adult Heart Transplantation Report-2018; Focus Theme: Multiorgan Transplantation. The Journal of Heart and Lung Transplantation. 2018; 37(10):1155-1168
Suarez-Pierre A, Kilic A. Surgical considerations for cardiac allograft rejection. Cardiovascular Pathology. 2019; 42:59-63
Iwata A, Sai S, Nitta Y, Chen M, de Fries-Hallstrand R, Dalesandro J, et al. Liposome-mediated gene transfection of endothelial nitric oxide synthase reduces endothelial activation and leukocyte infiltration in transplanted hearts. Circulation. 2001; 103(22):2753-2759
Abunasra HJ, Smolenski RT, Yap J, Sheppard M, O'Brien T, Yacoub MH. Multigene adenoviral therapy for the attenuation of ischemia-reperfusion injury after preservation for cardiac transplantation. The Journal of Thoracic and Cardiovascular Surgery. 2003; 125(5):998-1006
Sakaguchi T, Sawa Y, Fukushima N, Nishimura M, Ichikawa H, Kaneda Y, et al. A novel strategy of decoy transfection against nuclear factor-κB in myocardial preservation. The Annals of Thoracic Surgery. 2001; 71(2):624-629
Jayakumar J, Suzuki K, Khan M, Smolenski RT, Farrell A, Latif N, et al. Gene therapy for myocardial protection. Circulation. 2000; 102(suppl_3):Iii-302-IiIii-6
Hayry P. Molecular pathology of acute and chronic rejection. Transplantation Proceedings. 1994; 26(6):3280-3284
Qin L, Chavin KD, Ding Y, Favaro JP, Woodward JE, Lin J, et al. Multiple vectors effectively achieve gene transfer in a murine cardiac transplantation model. Immunosuppression with TGF-beta 1 or vIL-10. Transplantation. 1995; 59(6):809-816
Vassalli G, Fleury S, Li J, Goy J-J, Kappenberger L, Von Segesser LK. Gene transfer of cytoprotective and immunomodulatory molecules for prevention of cardiac allograft rejection. European Journal of Cardio-Thoracic Surgery. 2003; 24(5):794-806
Fujisawa K, Saito S, Okada Y, Fujiwara T, Yagi T, Iwagaki H, et al. Suppression of Allogeneic Response by Viral IL-10 Gene Transfer. Cell Transplantation. 2003; 12(4):379-387
Cheng J, Xia SS, Xie S, Tang LG, Shi XF. Transforming Growth Factor β1–Modified Donor Cell Transfusion Induced Allo-Heart Tolerance. Transplantation Proceedings. 2005; 37(5):2360-2364
Guillot C, Mathieu P, Coathalem H, Le Mauff B, Castro MG, Tesson L, et al. Tolerance to cardiac allografts via local and systemic mechanisms after adenovirus-mediated CTLA4Ig expression. Journal of Immunology. 2000; 164(10):5258-5268
Wang W, Carper K, Malone F, Latchman Y, Perkins J, Fu Y, et al. PD-L1/PD-1 Signal Deficiency Promotes Allogeneic Immune Responses and Accelerates Heart Allograft Rejection. Transplantation. 2008; 86(6):836-844
Özkaynak E, Wang L, Goodearl A, McDonald K, Qin S, O’Keefe T, et al. Programmed Death-1 Targeting Can Promote Allograft Survival. The Journal of Immunology. 2002; 169(11):6546-6553
Koga N, Suzuki J-I, Kosuge H, Haraguchi G, Onai Y, Futamatsu H, et al. Blockade of the interaction between PD-1 and PD-L1 accelerates graft arterial disease in cardiac allografts. Arteriosclerosis, Thrombosis, and Vascular Biology. 2004; 24(11):2057-2062
Bishawi M, Bowles D, Pla MM, Oakes F, Chiang Y, Schroder J, et al. PD-1 and PD-L1 expression in cardiac transplantation. Cardiovascular Pathology. 2021; 54:107331
Dudler J, Li J, Pagnotta M, Pascual M, von Segesser LK, Vassalli G. Gene transfer of programmed death ligand-1.Ig prolongs cardiac allograft survival. Transplantation. 2006; 82(12):1733-1737
Rosengart TK, Lee LY, Patel SR, Sanborn TA, Parikh M, Bergman GW, et al. Angiogenesis Gene Therapy. Circulation. 1999; 100(5):468-474
Grines CL. The AGENT clinical trials programme. European Heart Journal Supplements. 2004; 6(suppl_E):E18-E23
Jin YN, Inubushi M, Masamoto K, Odaka K, Aoki I, Tsuji AB, et al. Long-term effects of hepatocyte growth factor gene therapy in rat myocardial infarct model. Gene Therapy. 2012; 19(8):836-843
Ryugo M, Sawa Y, Ono M, Fukushima N, Aleshin AN, Mizuno S, et al. Myocardial Protective Effect of Human Recombinant Hepatocyte Growth Factor for Prolonged Heart Graft Preservation in Rats. Transplantation. 2004; 78(8):1153-1158
Westhofen S, Jelinek M, Dreher L, Biermann D, Martin J, Vitzhum H, et al. The heterotopic heart transplantation in mice as a small animal model to study mechanical unloading – Establishment of the procedure, perioperative management and postoperative scoring. PLoS One. 2019; 14(4):e0214513
Kadner A, Chen RH, Adams DH. Heterotopic heart transplantation: experimental development and clinical experience. European Journal of Cardio-Thoracic Surgery. 2000; 17(4):474-481
Abbott CP, Lindsey ES, Creech O Jr, Dewitt CW. A technique for heart transplantation in the rat. Archives of Surgery. 1964; 89:645-652
Ono K, Lindsey ES. Improved technique of heart transplantation in rats. The Journal of Thoracic and Cardiovascular Surgery. 1969; 57(2):225-229
Minanov OP, Kwiatkowski P, Popilskis S, Michler RE. Modified technique for heterotopic heart transplantation in small primates. The Annals of Thoracic Surgery. 1997; 63(1):258-260
Adams DH, Chen RH, Kadner A, Naficy S. Technique for heterotopic pig heart xenotransplantation in primates. The Annals of Thoracic Surgery. 1999; 68(1):265-268
Goerlich CE, DiChiacchio L, Zhang T, Singh AK, Lewis B, Tatarov I, et al. Heterotopic Porcine Cardiac Xenotransplantation in the Intra-Abdominal Position in a Non-Human Primate Model. Scientific Reports. 2020; 10(1):10709
Iwase H, Ekser B, Satyananda V, Bhama J, Hara H, Ezzelarab M, et al. Pig-to-baboon heterotopic heart transplantation--exploratory preliminary experience with pigs transgenic for human thrombomodulin and comparison of three costimulation blockade-based regimens. Xenotransplantation. 2015; 22(3):211-220
Madsen JC, Sachs DH, Fallon JT, Weissman NJ. Cardiac allograft vasculopathy in partially inbred miniature swine. I. Time course, pathology, and dependence on immune mechanisms. The Journal of Thoracic and Cardiovascular Surgery. 1996; 111(6):1230-1239
Bishawi M, Roan JN, Milano CA, Daneshmand MA, Schroder JN, Chiang Y, et al. A normothermic ex vivo organ perfusion delivery method for cardiac transplantation gene therapy. Scientific Reports. 2019; 9(1):8029
Au-Mendiola Pla M, Au-Evans A, Au-Lee FH, Au-Chiang Y, Au-Bishawi M, Au-Vekstein A, et al. A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft. JoVE. 2022; 180:e63114
Amano J, Akashima T, Terasaki T, Wada Y, Ito-Amano M, Suzuki J, et al. Characteristics of cardiac allograft vasculopathy induced by immunomodulation in the miniature Swine. Annals of Thoracic and Cardiovascular Surgery. 2015; 21(1):45-52
Singh AK, Chan JL, Dichiacchio L, Hardy NL, Corcoran PC, Lewis BGT, et al. Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs. Xenotransplantation. 2019; 26(2):e12465
Ali AA, White P, Xiang B, Lin HY, Tsui SS, Ashley E, et al. Hearts from DCD donors display acceptable biventricular function after heart transplantation in pigs. American Journal of Transplantation. 2011; 11(8):1621-1632
Langin M, Reichart B, Steen S, Sjoberg T, Paskevicius A, Liao Q , et al. Cold non-ischemic heart preservation with continuous perfusion prevents early graft failure in orthotopic pig-to-baboon xenotransplantation. Xenotransplantation. 2021; 28(1):e12636
Fukushima N, Bouchart F, Gundry SR, Nehlsen-Cannarella S, Gusewitch G, Chang L, et al. The role of anti-pig antibody in pig-to-baboon cardiac xenotransplant rejection. Transplantation. 1994; 57(6):923-928
Pacak CA, Sakai Y, Thattaliyath BD, Mah CS, Byrne BJ. Tissue specific promoters improve specificity of AAV9 mediated transgene expression following intra-vascular gene delivery in neonatal mice. Genet Vaccines Ther. 2008; 6:13
Bulcha JT, Wang Y, Ma H, Tai PWL, Gao G. Viral vector platforms within the gene therapy landscape. Signal Transduction and Targeted Therapy. 2021; 6(1):53
Chu D, Sullivan CC, Weitzman MD, Du L, Wolf PL, Jamieson SW, et al. Direct comparison of efficiency and stability of gene transfer into the mammalian heart using adeno-associated virus versus adenovirus vectors. The Journal of Thoracic and Cardiovascular Surgery. 2003; 126(3):671-679
Crystal RG. Adenovirus: the first effective in vivo gene delivery vector. Human Gene Therapy. 2014; 25(1):3-11
McGrory WJ, Bautista DS, Graham FL. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology. 1988; 163(2):614-617
Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proceedings of the National Academy of Sciences of the United States of America. 1994; 91(10):4407-4411
Wang Q , Finer MH. Second-generation adenovirus vectors. Nature Medicine. 1996; 2(6):714-716
Atchison RW, Casto BC, Hammon WM. Adenovirus-Associated Defective Virus Particles. Science. 1965; 149(3685):754-756
Flotte T, Carter B, Conrad C, Guggino W, Reynolds T, Rosenstein B, et al. A phase I study of an adeno-associated virus-CFTR gene vector in adult CF patients with mild lung disease. Human Gene Therapy. 1996; 7(9):1145-1159
Ylä-Herttuala S. Endgame: Glybera Finally Recommended for Approval as the First Gene Therapy Drug in the European Union. Molecular Therapy. 2012; 20(10):1831-1832
Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nature Reviews Drug Discovery. 2019; 18(5):358-378
McCarty DM, Self-complementary AAV. Vectors. Advances and Applications. Molecular Therapy. 2008; 16(10):1648-1656
Wu Z, Asokan A, Samulski RJ. Adeno-associated Virus Serotypes: Vector Toolkit for Human Gene Therapy. Molecular Therapy. 2006; 14(3):316-327
Chen YH, Chang M, Davidson BL. Molecular signatures of disease brain endothelia provide new sites for CNS-directed enzyme therapy. Nature Medicine. 2009; 15(10):1215-1218
Bish LT, Morine K, Sleeper MM, Sanmiguel J, Wu D, Gao G, et al. Adeno-associated virus (AAV) serotype 9 provides global cardiac gene transfer superior to AAV1, AAV6, AAV7, and AAV8 in the mouse and rat. Human Gene Therapy. 2008; 19(12):1359-1368
Zincarelli C, Soltys S, Rengo G, Rabinowitz JE. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Molecular Therapy. 2008; 16(6):1073-1080
Gabisonia K, Prosdocimo G, Aquaro GD, Carlucci L, Zentilin L, Secco I, et al. MicroRNA therapy stimulates uncontrolled cardiac repair after myocardial infarction in pigs. Nature. 2019; 569(7756):418-422
Gao G, Bish LT, Sleeper MM, Mu X, Sun L, Lou Y, et al. Transendocardial delivery of AAV6 results in highly efficient and global cardiac gene transfer in rhesus macaques. Human Gene Therapy. 2011; 22(8):979-984
Piacentino V, Milano CA, Bolanos M, Schroder J, Messina E, Cockrell AS, et al. X-linked inhibitor of apoptosis protein-mediated attenuation of apoptosis, using a novel cardiac-enhanced adeno-associated viral vector. Human gene therapy. 2012; 23(6):635-646
Mingozzi F, High KA. Immune responses to AAV vectors: Overcoming barriers to successful gene therapy. Blood. 2013; 122(1):23-36
Bertin B, Veron P, Leborgne C, Deschamps JY, Moullec S, Fromes Y, et al. Capsid-specific removal of circulating antibodies to adeno-associated virus vectors. Scientific Reports. 2020; 10(1):864
Leborgne C, Barbon E, Alexander JM, Hanby H, Delignat S, Cohen DM, et al. IgG-cleaving endopeptidase enables in vivo gene therapy in the presence of anti-AAV neutralizing antibodies. Nature Medicine. 2020; 26(7):1096-1101
Maheshri N, Koerber JT, Kaspar BK, Schaffer DV. Directed evolution of adeno-associated virus yields enhanced gene delivery vectors. Nature Biotechnology. 2006; 24(2):198-204
Bartel M, Schaffer D, Büning H. Enhancing the Clinical Potential of AAV Vectors by Capsid Engineering to Evade Pre-Existing Immunity. Frontiers in Microbiology. 2011; 2:204
Grimm D, Lee JS, Wang L, Desai T, Akache B, Storm TA, et al. In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses. Journal of Virology. 2008; 82(12):5887-5911
Biswas M, Marsic D, Li N, Zou C, Gonzalez-Aseguinolaza G, Zolotukhin I, et al. Engineering and In Vitro Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity. Molecular Therapy - Methods & Clinical Development. 2020; 19:347-361
Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996; 272(5259):263-267
Kieserman JM, Myers VD, Dubey P, Cheung JY, Feldman AM. Current Landscape of Heart Failure Gene Therapy. Journal of the American Heart Association. 2019; 8(10):e012239
Yu X, Zhan X, D'Costa J, Tanavde VM, Ye Z, Peng T, et al. Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells. Molecular Therapy. 2003; 7(6):827-838
Tian J, Andreadis ST. Independent and high-level dual-gene expression in adult stem-progenitor cells from a single lentiviral vector. Gene Therapy. 2009; 16(7):874-884
Di Pasquale E, Latronico MVG, Jotti GS, Condorelli G. Lentiviral vectors and cardiovascular diseases: a genetic tool for manipulating cardiomyocyte differentiation and function. Gene Therapy. 2012; 19(6):642-648
Papayannakos C, Daniel R. Understanding lentiviral vector chromatin targeting: working to reduce insertional mutagenic potential for gene therapy. Gene Therapy. 2013; 20(6):581-588
Kaiser RA, Mao SA, Glorioso J, Amiot B, Nicolas CT, Allen KL, et al. Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine. Journal of Visualized Experiments. 2018; 141:58399
Kohn DB, Booth C, Shaw KL, Xu-Bayford J, Garabedian E, Trevisan V, et al. Autologous Ex Vivo Lentiviral Gene Therapy for Adenosine Deaminase Deficiency. The New England Journal of Medicine. 2021; 384(21):2002-2013
Ramamoorth M, Narvekar A. Non viral vectors in gene therapy- an overview. Journal of Clinical and Diagnostic Research. 2015; 9(1):GE01-GGE6
Kawabata K, Takakura Y, Hashida M. The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake. Pharmaceutical Research. 1995; 12(6):825-830
Zangi L, Hajjar RJ. Synthetic MicroRNAs Stimulate Cardiac Repair. Circulation Research. 2017; 120(8):1222-1223
Zhang Q , Li Y, Shi Y, Zhang Y. HVJ envelope vector, a versatile delivery system: Its development, application, and perspectives. Biochemical and Biophysical Research Communications. 2008; 373(3):345-349
Yonemitsu Y, Kaneda Y. Hemagglutinating virus of Japan liposome-mediated gene delivery to vascular cells. Methods Mol Med. 1999; 30:295-306. DOI: 10.1385/1-59259-247-3:295. PM ID: 21341034
Kaneda Y, Nakajima T, Nishikawa T, Yamamoto S, Ikegami H, Suzuki N, et al. Hemagglutinating Virus of Japan (HVJ) Envelope Vector as a Versatile Gene Delivery System. Molecular Therapy. 2002; 6(2):219-226
Guzman RJ, Lemarchand P, Crystal RG, Epstein SE, Finkel T. Efficient gene transfer into myocardium by direct injection of adenovirus vectors. Circulation Research. 1993; 73(6):1202-1207
Fortuin FD, Vale P, Losordo DW, Symes J, DeLaria GA, Tyner JJ, et al. One-year follow-up of direct myocardial gene transfer of vascular endothelial growth factor-2 using naked plasmid deoxyribonucleic acid by way of thoracotomy in no-option patients**Drs. Isner and Schatz are stockholders of Vascular Genetics, Inc., the sponsor of this trial. The American Journal of Cardiology. 2003; 92(4):436-439
Grossman PM, Han Z, Palasis M, Barry JJ, Lederman RJ. Incomplete retention after direct myocardial injection. Catheterization and Cardiovascular Interventions. 2002; 55(3):392-397
Shirley JL, De Jong YP, Terhorst C, Herzog RW. Immune Responses to Viral Gene Therapy Vectors. Molecular Therapy. 2020; 28(3):709-722
Pellegrini C. Influence of temperature on adenovirus-mediated gene transfer. European Journal of Cardio-Thoracic Surgery. 1998; 13(5):599-603
Van Raemdonck D, Rega F, Rex S, Neyrinck A. Machine perfusion of thoracic organs. Journal of Thoracic Disease. 2018; 10(Suppl 8):S910-SS23
Ragalie WS, Ardehali A. Current status of normothermic ex-vivo perfusion of cardiac allografts. Current Opinion in Organ Transplantation. 2020; 25(3):237-240
Kypson AP, Peppel K, Akhter SA, Lilly RE, Glower DD, Lefkowitz RJ, et al. Ex vivo adenovirus-mediated gene transfer to the adult rat heart. The Journal of Thoracic and Cardiovascular Surgery. 1998; 115(3):623-630