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French, BA, Mazur, W, Geske, RS and Bolli, R. Direct in vivo gene transfer into porcine myocardium using replication-deficient adenoviral vectors. Circulation 90: 2414-2424
Abstract
Efficient methods of introducing genes into myocardial cells must be developed before local somatic cell gene therapy can be implemented against myocardial disease. Although adenoviral (Ad5) vectors have been used to target rodent hearts and plasmid DNA has been directly injected into the myocardium of rats and dogs, the amounts of recombinant protein produced by these procedures have not been reported, and adenoviral vectors have not been used in large mammalian hearts.
Replication-deficient recombinant adenoviral vectors carrying either the luciferase or lacZ reporter genes were injected directly into the ventricular myocardium of adult domestic swine for evaluation of reporter gene expression. This procedure did not affect regional myocardial function as assessed by systolic wall thickening using ultrasonic crystals. Luciferase activity was detected 3 days after injection, increased markedly at 7 days, and then declined progressively at 14 and 21 days. Luciferase production was comparable in the right and left ventricular walls and increased with increasing amounts of virus, reaching 61 +/- 21 ng at the highest dose examined (3.6 x 10(9) plaque-forming units). The injection of 200 micrograms of plasmid DNA (pRSVL) produced levels of luciferase comparable to 1.8 x 10(8) plaque-forming units of recombinant Ad5; however, when normalized to the number of genes injected, the adenovirus was 140,000 times more efficient than plasmid DNA. Histochemical analysis of beta-galactosidase activity produced by a second Ad5 vector demonstrated that nearly all (> 95%) of the stained cells were cardiomyocytes and that the percentage of cardiomyocytes infected by the virus could be quite high in microscopic regions adjacent to the needle track (up to 75% in fields of 60 to 70 cells); however, Ad5-infected cells were rarely observed farther than 5 mm from the injection site. Furthermore, the Ad5 vector induced pronounced leukocytic infiltration that was far in excess of that seen after injection of vehicle alone.
This study demonstrates for the first time that direct intramyocardial injection of replication-deficient adenovirus can program recombinant gene expression in the cardiomyocytes of a large animal species with relevance to human physiology. The efficiency of adenovirus-mediated gene transfer is far superior to that of plasmid DNA injection, and this method appears to be capable of producing more recombinant protein. However, the cell-mediated immune response to the Ad5 vector and the limited distribution of reporter gene expression suggest that less immunogenic recombinant vectors and more homogeneous administration methods will be required before Ad5 vectors can be successfully used for phenotypic modulation.
... Gene therapy applications use viral genomes which are highly edited, so that they become non-infectious and replication defective. Therefore, posing minimal risk to the host and surrounding environment [52][53][54]. ...
... Direct epicardial injections have also been used for vector delivery [52,80]. This process involves performing a thoracotomy to expose the epicardial surface of the heart. ...
... This increases the risk of major complications during the procedure, and in the postoperative period. There is also the probability of heterogeneous gene expression, injection-related tissue damage triggering an acute inflammatory response [52,83] as well as potential clinical issues such as ventricular fibrillation if the electroporation shocks are not synchronised with ventricular electrical activation [84]. ...
Bradycardia arising from pacemaker dysfunction can be debilitating and life threatening. Electronic pacemakers serve as effective treatment options for pacemaker dysfunction. They however present their own limitations and complications. This has motivated research into discovering more effective and innovative ways to treat pacemaker dysfunction. Gene therapy is being explored for its potential to treat various cardiac conditions including cardiac arrhythmias. Gene transfer vectors with increasing transduction efficiency and biosafety have been developed and trialed for cardiovascular disease treatment. With an improved understanding of the molecular mechanisms driving pacemaker development, several gene therapy targets have been identified to generate the phenotypic changes required to correct pacemaker dysfunction. This review will discuss the gene therapy vectors in use today along with methods for their delivery. Furthermore, it will evaluate several gene therapy strategies attempting to restore biological pacing, having the potential to emerge as viable therapies for pacemaker dysfunction.
... Another study featuring direct intramyocardial injection of replicationdeficient adenovirus demonstrated gene expression in a large animal model. However, the authors noted a robust T-cell-mediated immune response against the vector and limited distribution of the reporter gene (French et al. 1994). ...
... Hearts transfected with an adenovirus vector containing the β-galactosidase gene showed significantly increased β-galactosidase enzymatic activity compared with hearts injected with β-galactosidase plasmid. Unfortunately, the gene expression persisted for only 1 week after injection and it included acute inflammatory response, which the authors considered to be related to the injury produced by French et al. first demonstrated in a porcine model these important points relevant to needle injection: (1) Direct intramyocardial injection of replicationdeficient adenovirus is 140,000 times more efficient than injection of an equal number of genome copies of recombinant plasmid DNA, (2) The impact of this procedure on cardiac function appears to be negligible provided the number of injections is reasonable and across myocardial surface area, (3) The amount of recombinant protein produced increases with the amount of virus, but plateaus, (4) The expression of recombinant genes following intramyocardial injection is similar in the left and right ventricles; and (5) The percentage of cardiomyocytes expressing β-galactosidase in the needle track adjacent to the injection, but rarely are lacZ positive cells detected farther than 5 mm from any given injection site (French et al. 1994). ...
Heart failure is a significant burden to the global healthcare system and represents an underserved market for new pharmacologic strategies, especially therapies which can address root cause myocyte dysfunction. Modern drugs, surgeries, and state-of-the-art interventions are costly and do not improve survival outcome measures. Gene therapy is an attractive strategy, whereby selected gene targets and their associated regulatory mechanisms can be permanently managed therapeutically in a single treatment. This in theory could be sustainable for the patient's life. Despite the promise, however, gene therapy has numerous challenges that must be addressed together as a treatment plan comprising these key elements: myocyte physiologic target validation, gene target manipulation strategy, vector selection for the correct level of manipulation, and carefully utilizing an efficient delivery route that can be implemented in the clinic to efficiently transfer the therapy within safety limits. This chapter summarizes the key developments in cardiac gene therapy from the perspective of understanding each of these components of the treatment plan. The latest pharmacologic gene targets, gene therapy vectors, delivery routes, and strategies are reviewed.
... All previous studies have employed recombinant adenoviral (Adv) vectors for myocardial TBX18 gene transfer 14,15 . The known immune and inflammatory responses elicited by Adv vectors [17][18][19][20] , together with its systemic longevity and/or potential inefficacy due to antiviral neutralizing antibodies 21,22 , give pause to the use of viral vectors particularly for the aforementioned indications. ...
The adenovirus-mediated somatic transfer of the embryonic T-box transcription factor 18 (TBX18) gene can convert chamber cardiomyocytes into induced pacemaker cells. However, the translation of therapeutic TBX18-induced cardiac pacing faces safety challenges. Here we show that the myocardial expression of synthetic TBX18 mRNA in animals generates de novo pacing and limits innate and inflammatory immune responses. In rats, intramyocardially injected mRNA remained localized, whereas direct myocardial injection of an adenovirus carrying a reporter gene resulted in diffuse expression and in substantial spillover to the liver, spleen and lungs. Transient expression of TBX18 mRNA in rats led to de novo automaticity and pacemaker properties and, compared with the injection of adenovirus, to substantial reductions in the expression of inflammatory genes and in activated macrophage populations. In rodent and clinically relevant porcine models of complete heart block, intramyocardially injected TBX18 mRNA provided rate-adaptive cardiac pacing for one month that strongly correlated with the animal’s sinus rhythm and physical activity. TBX18 mRNA may aid the development of biological pacemakers.
... Ad vectors have many properties that make them ideal for the delivery of VEGF genes for therapeutic angiogenesis, namely, the robust but short-term transduction of cardiovascular tissues without integration into the human genome. Direct myocardial injection is the most certain method of myocardial gene transfer accomplished, for example, by thoracoscopy, left thoracotomy, or sternotomy, ideally involving minimally invasive approaches [7][8][9][10][11][12][13]. The advantages of a direct injection include (1) higher levels of localized transgene expression, (2) delivery of vectors with a high degree of precision, (3) suitability for multiple targeted injections, and (4) minimal systemic spread of the vector. ...
Adenovirus-mediated gene therapy holds promise for the treatment of cardiovascular diseases such as refractory angina. However, potential concerns around immunogenicity and vector dissemination from the target injected tissue require evaluation. This study was undertaken to evaluate the safety and biodistribution of XC001, a replication-deficient adenovirus serotype 5 vector expressing multiple isoforms of human vascular endothelial growth factor (VEGF), following direct administration into normal rat myocardium. Animals received the buffer formulation or increasing doses of XC001 (1 × 10⁷, 2.5 × 10⁸ or 2.5 × 10⁹ viral particles). Based on in-life parameters (general health, body weights, clinical pathology, serum cardiac troponin I, plasma VEGF, and gross necropsy), there were no findings of clinical concern. On Day 8, intramyocardial administration of XC001 was associated with dose-related, left ventricular myocardial inflammation at injection sites, resolving by Day 30. XC001 DNA was not detected in blood at any time but was present at Day 8 around the site of injection and to a much lesser extent in the spleen, liver, and lungs, persisting at low levels in the heart and spleen until at least Day 91. These findings demonstrate that intramyocardial injection of XC001 is supported for use in human studies.
... Others have reported achieving more extensive transgene expression through the use of multiple injection sites [36,37], potentially due to saturation of the available sites for AAV uptake [38][39][40]. To determine whether administering AAV6.2FF-MR78 at multiple intramuscular sites might boost serum MR78 expression levels without having to increase dose, AAV6.2FF-MR78 was administered IM at a dose of 1 × 10 11 vg to a group of female mice (n = 4) in one singular IM injection and another group of female mice (n = 4) spread across four IM injections. ...
Vectored monoclonal antibody (mAb) expression mediated by adeno-associated virus (AAV) gene delivery leads to sustained therapeutic mAb expression and protection against a wide range of infectious diseases in both small and large animal models, including nonhuman primates. Using our rationally engineered AAV6 triple mutant capsid, termed AAV6.2FF, we demonstrate rapid and robust expression of two potent human antibodies against Marburg virus, MR78 and MR191, following intramuscular (IM) administration. IM injection of mice with 1 × 1011 vector genomes (vg) of AAV6.2FF-MR78 and AAV6.2FF-MR191 resulted in serum concentrations of approximately 141 μg/mL and 195 μg/mL of human IgG, respectively, within the first four weeks. Mice receiving 1 × 1011 vg (high) and 1 × 1010 vg (medium) doses of AAV6.2FF-MR191 were completely protected against lethal Marburg virus challenge. No sex-based differences in serum human IgG concentrations were observed; however, administering the AAV-mAb over multiple injection sites significantly increased serum human IgG concentrations. IM administration of three two-week-old lambs with 5 × 1012 vg/kg of AAV6.2FF-MR191 resulted in serum human IgG expression that was sustained for more than 460 days, concomitant with low levels of anti-capsid and anti-drug antibodies. AAV-mAb expression is a viable method for prolonging the therapeutic effect of recombinant mAbs and represents a potential alternative “vaccine” strategy for those with compromised immune systems or in possible outbreak response scenarios.
... We had anticipated these findings, as previous studies using microscopy show that direct vector injections produce a focal gene expression pattern with high gene expression in regions adjacent to the needle track that decrease exponentially with distance from the needle track. 25 In addition, the onset and peak of 99m TcO 4 − uptake are in line with the previously reported in vivo kinetics of AAV9-mediated gene expression after injection. 24 AAV9 more efficiently transduces ischemic than nonischemic skeletal muscle. ...
Background:
We propose micro single-photon emission computed tomography/computed tomography imaging of the hNIS (human sodium/iodide symporter) to noninvasively quantify adeno-associated virus 9 (AAV9)-mediated gene expression in a murine model of peripheral artery disease.
Methods:
AAV9-hNIS (2×1011 viral genome particles) was injected into nonischemic or ischemic gastrocnemius muscles of C57Bl/6J mice following unilateral hindlimb ischemia ± the α-sialidase NA (neuraminidase). Control nonischemic limbs were injected with phosphate buffered saline or remained noninjected. Twelve mice underwent micro single-photon emission computed tomography/computed tomography imaging after serial injection of pertechnetate (99mTcO4-), a NIS substrate, up to 28 days after AAV9-hNIS injection. Twenty four animals were euthanized at selected times over 1 month for ex vivo validation. Forty-two animals were imaged with 99mTcO4- ± the selective NIS inhibitor perchlorate on day 10, to ascertain specificity of radiotracer uptake. Tissue was harvested for ex vivo validation. A modified version of the U-Net deep learning algorithm was used for image quantification.
Results:
As quantitated by standardized uptake value, there was a gradual temporal increase in 99mTcO4- uptake in muscles treated with AAV9-hNIS. Hindlimb ischemia, NA, and hindlimb ischemia plus NA increased the magnitude of 99mTcO4- uptake by 4- to 5-fold compared with nonischemic muscle treated with only AAV9-hNIS. Perchlorate treatment significantly reduced 99mTcO4- uptake in AAV9-hNIS-treated muscles, demonstrating uptake specificity. The imaging results correlated well with ex vivo well counting (r2=0.9375; P<0.0001) and immunoblot analysis of NIS protein (r2=0.65; P<0.0001).
Conclusions:
Micro single-photon emission computed tomography/computed tomography imaging of hNIS-mediated 99mTcO4- uptake allows for accurate in vivo quantification of AAV9-driven gene expression, which increases under ischemic conditions or neuraminidase desialylation in skeletal muscle.
... However, transfer of naked nucleic acids could offer positive benefit/risk balance. In this respect, different strategies for naked DNA delivery have been evaluated in liver [11,12] and heart, the most commonly used being: a) direct intramyocardial injection, which facilitates gene arriving in the target cell by avoiding the physical tissue barriers at the expense of being an invasive procedure that could alter the normal function of myocardium [13,14]; b) pericardial perfusion, which can be performed with a minimally invasive procedure in large animals but only achieves gene expression in superficial layers of myocardium [15]; c) coronary artery perfusion, which is a minimally invasive and safe procedure, but inefficient [16] due to the rapid arterial blood flow; d) coronary sinus retrograde injection, which has demonstrated [17,18] to be the most promising strategy when considering safety, clinical applicability and effectiveness. In our previous works, coronary sinus retrograde injection of green fluorescent protein (eGFP) gene mediated eGFP protein expression in heart tissue throughout the whole organ, as evidenced by immunohistochemistry and fluorescence [19] microscopy. ...
... The exceptional transduction efficiencies of adenoviruses and AAVs have resulted in these vectors being the most widely used for cardiovascular applications. Adenoviruses transfer genes efficiently into the myocardium in large animals, 24 but expression is transient, and these viruses trigger a strong immune response. 25 AAVs have low immunogenicity and are a widely used alternative for gene delivery to the heart. ...
Myocardial infarction (MI) and heart failure (HF) are the lead-ing causes of death in the United States and in most other industrialized nations. MI leads to a massive loss of cardiomyo-cytes (CMs), which are replaced with non-CM cells, leading to scarring and, in most cases, HF. The adult mammalian heart has a low intrinsic regenerative capacity, mainly because of cell-cycle arrest in CMs. No effective treatment promoting heart regeneration is currently available. Recent efforts to useDNA-based or viral gene therapy approaches to induce cardiac regeneration post-MI or in HF conditions have encountered major challenges, mostly because of the poor and uncontrolled delivery of the introduced genes. Modified mRNA (modRNA)is a safe, non-immunogenic, efficient, transient, local, and controlled nucleic acid delivery system that can overcome the obstacles to DNA-based or viral approaches for cardiac gene delivery. We here review the use of modRNA in cardia ctherapy, to induce cardioprotection and vascular or cardiac regeneration after MI. We discuss the current challenges in modRNA-based cardiac treatment, which will need to be over-come for the application of such treatment to ischemic heart disease
(PDF) mRNA-Based Protein Replacement Therapy for the Heart. Available from: https://www.researchgate.net/publication/329448176_mRNA-Based_Protein_Replacement_Therapy_for_the_Heart [accessed Jan 02 2019].
... A large number of gene transfer techniques have been studied in cardiovascular gene therapy. These findings indicate that the physical path of gene delivery is even more important than the vector system, and the target of gene delivery should be minimum or zero expression of the accessory organs [Lin 1990;Guzman 1993;Barr 1994;French 1994;Katz 2014b]. In recent years, Bridges and colleagues have established and done research on a molecular cardiac surgery delivery system for gene therapy. ...
Chronic heart failure (CHF) is still the leading cause of morbidity and mortality worldwide, and carries with it large economic and social burdens. Although steady and substantial progress has been made in reducing mortality from heart failure using conventional treatments, novel pharmacologic and surgical interventions have not been effective in extending five year survival rates. Therefore, it is necessary to explore new therapies. Gene therapy was introduced in 1970s with the development of recombinant DNA technology. Due to recent progress in the understanding of myocardial metabolism and application of vector based gene transfer strategies in animal models and initial clinical trials, gene therapy possibly affords an ideal treatment alternative for CHF. In last 2 decades, much research has been done on gene therapy, using various genes, signal transduction passages and delivery methods to treat advanced heart failure. Current research in ischemic heart disease (IHD) mainly focuses on stimulating angiogenesis, modifying the coronary vascular environment, and improving the vascular endothelial function with localized gene coated catheters and stents. Compared with standard ischemic heart disease treatment, the main goal of gene therapy for CHF is to inhibit apoptosis, reduce the undesirable remodeling and increase contractility through the most efficient cardiomyocyte transfection [Katz 2012a]. In this paper, we review various gene transfer technologies in ischemic heart disease and heart failure models, and discuss the advantages and disadvantages of these strategies in vector-mediated cardiac gene delivery, with the main focus on the high efficiency approach of a molecular cardiac surgery delivery system.
... For high-efficiency transduction, adenovirus vectors were selected as ideal gene therapy vehicles in the present study. In previous studies, adenovirus vectors have been used for a large amount of transgene therapies, due to their ease of production, high titers, stable inheritance, sufficient transduction efficiency and early peak expression compared with other vectors (32)(33)(34)(35). However, adenovirus-mediated gene expression may not last a long period of time; peaking at ~1 week, declining at 3-5 weeks and vanishing at ~10 weeks (36). ...
Bone mesenchymal stem cells (BMSCs) are currently considered the optimal stem cells for biological pacemaker cell transformation. The cardiac‑specific transcription factor T‑Box protein 18 (TBX18) is essential for sinoatrial node (SAN) formation, particularly formation of the head region that generates the electrical impulses that induce heart contraction. The present study aimed to confirm the effects of TBX18 on biological pacemaker differentiation of rat BMSCs. Flow cytometry was used to identify the surface markers of BMSCs, in order to acquire pure mesenchymal stem cells. Subsequently, BMSCs were transduced with TBX18 or green fluorescent protein adenovirus vectors. The effects of TBX18 were evaluated using SAN‑specific makers including TBX18, α‑actin, cardiac troponin I, hyperpolarization‑activated cyclic nucleotide‑gated channel 4 and connexin 43 by reverse transcription‑quantitative polymerase chain reaction, western blotting and immunofluorescence. The findings demonstrated that direct conversion of BMSCs to biological pacemaker cells via TBX18 is a feasible method in the field of cardiology.
... This adenoviral vector induces temporary transgene expression over a 2-to-3-week interval, which mirrors the time frame of greatest POAF risk. [12][13][14] KCNH2-G628S (G628S) is a dominant negative mutation that eliminates the function of a potassium channel that plays a central role in cardiac myocyte repolarization. 15 In a previous study, we demonstrated the efficacy of atrial painting of AdG628S with 200 mg/mL Pluronic P407 and 5 mg/mL trypsin. ...
Background:
Postoperative atrial fibrillation (POAF) is the most common complication occurring after cardiac surgery. Multiple studies have shown significantly increased risks of stroke, myocardial infarction, and death associated with POAF. Current prophylaxis strategies are inadequate to eliminate this problem. We examined the preclinical efficacy and safety of KCNH2-G628S gene transfer to prevent POAF.
Methods:
Domestic pigs received AdKCNH2-G628S by epicardial atrial gene painting and atrial pacemaker implantation for continuous-burst pacing to induce atrial fibrillation. In an initial dose-ranging evaluation, 3 pigs received 5 × 10(10) to 5 × 10(11) virus particles. In the formal study, 16 pigs were randomized to 3 groups: 5 × 10(11) virus particles of AdKCNH2-G628S with 20% Pluronic P407 in saline, 20% Pluronic P407 in saline with no virus, and saline alone. Animals were followed with daily efficacy and safety evaluations through the period of peak adenovirus-mediated transgene expression. After 14 days, pacing was discontinued, and the animals were followed in sinus rhythm for an additional 14 days to assess any longer-term toxicity.
Results:
In the primary efficacy analysis, the G628S animals exhibited a significant increase in the average time in sinus rhythm compared with the Pluronic control group (59 ± 7% vs 14 ± 6%; P = .009). There was no significant difference between the Pluronic and saline controls (14 ± 6% vs 32 ± 12%; P = .16). Safety assessment showed improved left ventricular function in the G628S animals; otherwise there were no significant differences among the groups in any safety measure.
Conclusions:
These data indicate that KCNH2-G628S gene therapy can successfully and safely reduce the risk of AF.
... 37,38 Intramyocardial injection leads to focused, high-density gene expression, but gene delivery is limited to the tissue volume within a few millimetres of the needle track. 39,40 Thus, multiple injection sites would be required to achieve sufficient gene delivery in the large mammalian heart, which increases both the risk of adverse events during the procedure and the probability of heterogeneous gene expression. Injection-related tissue damage also has a risk of triggering an acute inflammatory response. ...
Atrial fibrillation is the most common clinically significant cardiac arrhythmia, increasing the risk of stroke, heart failure and morbidity and mortality. Current therapies, including rate control and rhythm control by antiarrhythmic drugs or ablation therapy, are moderately effective but far from optimal. Gene therapy has the potential to become an attractive alternative to currently available therapies for atrial fibrillation. Various gene transfer vectors have been developed for cardiovascular disease with viral vectors being most widely used due to their high efficiency. Several gene delivery methods have been employed on different therapeutic targets. With increasing understanding of arrhythmia mechanisms, novel therapeutic targets have been discovered. This review will evaluate state-of-art gene therapy strategies and approaches including sinus rhythm restoration and ventricular rate control that could eventually prevent or eliminate atrial fibrillation in patients.
... The animal was warmed at 38°C (Bio Research Center Co., Ltd.). The ADV solution diluted by PBS () (viral titer, 10 11 -10 12 particles per ml) was injected into the epicardial surface of the central region of the LV (5 µl/mm 2 in 10 spots) by using a 1-ml syringe pump (MCIP-BOi; Minato Concept, Inc.) with a 32-gauge needle (Dentronics) under a stereoscopic microscope (SZ61; Olympus) (French et al., 1994;Fromes et al., 1999). One bolus injection of the ADV solution into the epicardial surface of the LV resulted stochastically in the expression of AcGFP in the Z-disks in approximately five cardiomyocytes per injected area. 2 d after chest closure, the mouse was anesthetized again with 2% isoflurane and ventilated, and the anterior thoracic wall was removed by cutting the ribs, muscles, and intercostal arteries with the electric scalpel for in vivo cardiac sarcomere imaging. ...
Sarcomeric contraction in cardiomyocytes serves as the basis for the heart’s pump functions in mammals. Although it plays a critical role in the circulatory system, myocardial sarcomere length (SL) change has not been directly measured in vivo under physiological conditions because of technical difficulties. In this study, we developed a high speed (100–frames per second), high resolution (20-nm) imaging system for myocardial sarcomeres in living mice. Using this system, we conducted three-dimensional analysis of sarcomere dynamics in left ventricular myocytes during the cardiac cycle, simultaneously with electrocardiogram and left ventricular pressure measurements. We found that (a) the working range of SL was on the shorter end of the resting distribution, and (b) the left ventricular–developed pressure was positively correlated with the SL change between diastole and systole. The present findings provide the first direct evidence for the tight coupling of sarcomere dynamics and ventricular pump functions in the physiology of the heart.
Introduction:
To date, the treatment option for tachyarrhythmia is classified into drug therapy, catheter ablation, and implantable device therapy. However, the efficacy of the antiarrhythmic drugs is limited. Although the indication of catheter ablation is expanding, several fatal tachyarrhythmias are still refractory to ablation. Implantable cardioverter-defibrillator increases survival, but it is not a curable treatment. Therefore, a novel therapy for tachyarrhythmias refractory to present treatments is desired. Gene therapy is being developed as a promising candidate for this purpose, and basic research and translational research have been accumulated in recent years.
Areas covered:
This paper reviews the current state of gene therapy for arrhythmias, including susceptible arrhythmias, the route of administration to the heart, and the type of vector to use. We also discuss the latest progress in the technology of gene delivery and genome editing.
Expert opinion:
Gene therapy is one of the most promising technologies for arrhythmia treatment. However, additional technological innovation to achieve safe, localized, homogeneous, and long-lasting gene transfer is required for its clinical application.
In Vivo Myocardial Gene Transfer: Optimization and Evaluation of Gene Transfer Models and Vectors Background: Acute myocardial infarction and its sequelae are recognised as the most common cause of mortality and morbidity in industrialised nations, and are predicted to continue to rise well into the 21st century. One of the most promising treatments that may be applied in the future is gene therapy. In order for this to become a reality a number of steps have to be mastered, with the initial problem being efficient and safe delivery of genetic material to the adult myocardium. Aim: This thesis examines in vivo myocardial gene transfer using a number of different gene transfer vectors and different models of vector delivery to the adult myocardium. Methods: In vivo gene transfer was attempted by two basic models of vector delivery, either direct intramyocardial injection or percutaneous transluminal intracoronary delivery. There are a number of different gene transfer vectors for use in either the heart or other organ systems, with differing properties. In order to perform rational experiments in the future a number of different vectors, including recombinant adenovirus, recombinant herpes simplex virus, recombinant adeno-associated virus, cationic liposomes, integrin targeting peptides and naked DNA, were tested in the direct intramyocardial injection model of gene transfer. Intracoronary infusion of the best vector from the direct comparison studies was used to improve this model of vector delivery. Gene transfer efficiency was determined by expression of an encoded reporter gene, with histological assessment of toxicity of the different vectors. Results: Direct intramyocardial injection of recombinant viral vectors resulted in gene transfer, with recombinant adenovirus being the most efficient, recombinant herpes simplex virus was both less efficient and more toxic to the myocardium, with subsequent shorter expression of the transgene. Transgene expression following direct intramyocardial injection of recombinant adeno- associated virus was delayed, until 21-28 days post gene transfer, in accordance with the lifecycle of the virus, however no toxicity was observed. Intracoronary infusion of lipoplexes and, contrary to previous work, recombinant adenovirus was inefficient. However, development of a closed chest model of myocardial infarction demonstrated that a false positive appearance of gene transfer is seen following infarction. Conclusions: Gene transfer using either recombinant adenoviral or recombinant adeno-associated virus offer 2 contrasting vectors for gene delivery, with the toxicity profile of the latter vector allowing direct intramyocardial injection of the vector, whereas the more efficient, but also more toxic recombinant adenovirus is best employed using high-pressure intracoronary delivery.
Congestive heart failure is the common end point for advanced coronary artery disease and the leading cause of mortality from heart disease. Stents and surgical bypass can address focal obstruction in larger coronary arteries, but diffuse small vessel disease is not amenable to these interventions. Intrinsic recovery is also limited, as adult cardiac muscle does not effectively regenerate after cardiomyocyte death. Cardiac gene therapy uses growth factors, genes or small molecules to alter gene expression for myocardial regeneration. Genes may be used to induce angiogenesis, reduce pathologic fibrosis, induce replication of endogenous cardiomyocytes, or expand existing cardiac progenitor cells into various cardiac subtypes. Delivery options include plasmids, integrative or non-integrative viruses, micro RNA or small molecules. Administration may be achieved systemically or by intracoronary or local injection, although local administration appears to provide key pharmacokinetic advantages. Initial attempts focused on creating new branches from existing blood vessels, often using vascular endothelial growth factor (VEGF). These demonstrated equivocal clinical results due, in part, to inconsistent study design, controls and clinically relevant endpoints as well as incomplete pharmacokinetics data on required gene “dose” or the ideal methods of gene delivery. Early lessons informed the development of cardiac cellular reprogramming, which transforms cardiac fibroblasts into induced cardiomyocytes using defined reprogramming factor cocktails. This approach has delivered improved post-infarct ejection fraction and reduced fibrosis in preclinical models. Gene therapy in cardiac disease is not yet ready for clinical application, but holds great promise for filling an important therapeutic gap in a growing patient population.
Cardiovascular diseases are the leading cause of death globally and are associated with increasing financial expenditure. With the availability of next-generation sequencing technologies since the early 2000s, non-coding RNAs such as microRNAs, long non-coding RNAs and circular RNAs have been assessed as potential therapeutic targets for numerous diseases, including cardiovascular diseases. In this Review, we summarize current approaches employed to screen for novel coding and non-coding RNA candidates with diagnostic and therapeutic potential in cardiovascular disease, including next-generation sequencing, functional high-throughput RNA screening and single-cell sequencing technologies. Furthermore, we highlight viral-based delivery tools that have been widely used to evaluate the therapeutic utility of both coding and non-coding RNAs in the context of cardiovascular disease. Finally, we discuss the potential of using oligonucleotide-based molecular products such as modified RNA, small interfering RNA and RNA mimics/inhibitors for the treatment of cardiovascular diseases. Given that many non-coding RNAs have not yet been functionally annotated, the number of potential RNA diagnostic and therapeutic targets for cardiovascular diseases will continue to expand for years to come.
Malfunction of nodal pacemaker (Pm) cardiomyocytes (CMs) due to diseases or aging leads to rhythm generation disorders, necessitating electronic Pm implantation. We functionally reprogrammed human pluripotent stem cell (hPSC) derived-ventricular (V) CMs into -PmCMs via recombinant adeno-associated virus serotype 9 (rAAV9)-mediated overexpression of engineered HCN1 channel (HCN1ΔΔΔ) whose S3-S4 linker has been strategically deleted by design to promote cardiac pacemaking. rAAV9-HCN1ΔΔΔ-reprogrammed hPSC-PmCMs converted from -VCMs showed automaticity and action potential parameters typical of native nodal PmCMs. Implantation of rAAV9-HCN1ΔΔΔ-based BPm in a preclinical porcine model of complete heart block significantly reduced the dependence on device-supported pacing and generated spontaneous heart rhythms from the BPm. Collectively, these results have further laid the groundwork on BPm for future translation.
Resumen
A pesar de los cuidados en unidades específicas, de los nuevos fármacos y de los dispositivos desarrollados en los últimos años, los pacientes con insuficiencia cardiaca presentan, no solo una esperanza de vida reducida, sino una baja calidad de vida, con constantes ingresos hospitalarios. El desarrollo de nuevas terapias continúa siendo fundamental, para tratar esta patología en constante crecimiento. A este respecto, el profundo conocimiento de los mecanismos moleculares implicados en el desarrollo de la insuficiencia cardiaca ha permitido desarrollar modelos de terapia génica mediante diferentes vectores dirigidos a estas dianas moleculares. Sin embargo, tras los prometedores resultados de estudios en animales, el salto traslacional mediante ensayos clínicos aleatorizados ha sido poco exitoso. Esta revisión repasa brevemente los principios que fundamentan la terapia de transferencia génica y su aplicación en el campo de la insuficiencia cardiaca, así como los recientes estudios llevados a cabo en pacientes con insuficiencia cardiaca.
Bio-motile systems have liquid-crystalline structures. This review first describes the contractile system of striated muscle having a smectic liquid crystalline structure. We here report the muscle's auto-oscillatory property named spontaneous oscillatory contraction (SPOC) [1] Ishiwata, S., Shimamoto, Y., & Fukuda, N. (2011). Prog. Biophys. Mol. Biol., 105, 187.[CrossRef], [PubMed], [Web of Science ®] [Google Scholar], and a mathematical model to explain its mechanism [2, 3] Sato, K., Ohtaki, M., Shimamoto, Y., & Ishiwata, S. (2011). Prog. Biophys. Mol. Biol., 105, 199.
Sato, K., Kuramoto, Y., Ohtaki, M., Shimamoto, Y., & Ishiwata, S. (2013). Phys. Rev. Lett., 111, 108104. . Also, sarcomere dynamics observed during heartbeat are described. The second topic is the micromechanics of the meiotic spindle, a bipolar assembly of microtubules with chromosomes [4] Takagi, J., Itabashi, T., Suzuki, K., Kapoor, T. M., Shimamoto, Y., & Ishiwata, S. (2013). Cell Rep., 5, 44.[CrossRef], [PubMed], [Web of Science ®] [Google Scholar]. The third topic is the demonstration of a contractile actin ring spontaneously formed inside a water-in-oil droplet, which can be considered as an artificial cell model [5] Miyazaki, M., Chiba, M., Eguchi, H., Ohki, T., & Ishiwata, S. (2015). Nat. Cell Biol., 17, 480.[CrossRef], [PubMed], [Web of Science ®] [Google Scholar].
There are a large number of cardiovascular diseases that could be treated by myocardial gene transfer (1,2). These include congestive heart failure, ischemic heart disease, and cardiomyopathy. In addition to its potential for treatment of disease, myocardial gene transfer is useful for the analysis of gene expression and promoter function and for generating animal models of human disease such as pulmonary hypertension. The ideal vector for myocardial gene therapy should give efficient and stable transduction of cardiomyocytes in vivo. Recombinant adenovirus vectors have been used to transduce cardiomyocytes in rodents, rabbits, pigs, and humans by both intramyocardial injection and intracoronary infusion (3–5). Although efficient transduction can be obtained with adenovirus vectors, immune responses and elimination of transduced cells results in only transient expression in immunocompetent hosts. Vectors based on recombinant adeno-associated virus (rAAV) offer a number of attractive features and are emerging as promising gene transfer vehicles for many in vivo applications.
Introduction: Local myocardial delivery (LMD) of therapeutic agents is a promising strategy that aims to treat various myocardial pathologies. It is designed to deliver agents directly to the myocardium and minimize their extracardiac concentrations and side effects. LMD aims to enhance outcomes of existing therapies by broadening their therapeutic window and to utilize new agents that could not be otherwise be implemented systemically.
Areas covered: This article provides a historical overview of six decades LMD evolution in terms of the approaches, including intrapericardial, epicardial, and intramyocardial delivery, and the wide array of classes of agents used to treat myocardial pathologies. We examines delivery of pharmaceutical compounds, targeted gene transfection and cell implantation techniques to produce therapeutic effects locally. We outline therapeutic indications, successes and failures as well as technical approaches for LMD.
Expert opinion: While LMD is more complicated than conventional oral or intravenous administration, given recent advances in interventional cardiology, it is safe and may provide better therapeutic outcomes. LMD is complex as many factors impact pharmacokinetics and biologic result. The choice between routes of LMD is largely driven not only by the myocardial pathology but also by the nature and physicochemical properties of the therapeutic agents.
In the myopathic heart, a number of abnormalities have been delineated (Fig. 1) at the cellular level [25-33, 35, 38-43]. These include changes at the level of the sarcolemma, sarcoplasmic reticulum, myofilaments, and mitochondria, all of which contribute to depressed contractile function and reserve [25-33, 35, 38-43]. Identifying the mechanisms by which these changes contribute to the observed pathology is frequently confounded by simultaneous alterations in multiple signaling pathways in the complex milieu of the failing heart. Targeting genes to the heart through somatic gene transfer allows us to identify and characterize the molecular changes of diseases as well as to manipulate the targeted pathways [34, 36, 37].
During the last decade, there has been a significant progress toward clinical translation in the field of cardiac gene therapy based on extensive preclinical data. However, despite encouraging positive results in early phase clinical trials, more recent larger trials reported only neutral results. Nevertheless, the field has gained important knowledge from these trials and is leading to the development of more cardiotropic vectors and improved delivery systems. It has become more evident that humans are more resistant to therapeutic transgene expression compared to experimental animals and thus refinement in gene delivery tools and methods are essential for future success. We provide an overview of the current status of cardiac gene therapy focusing on gene delivery tools and methods. Newer technologies, devices, and approaches will undoubtedly lead to more promising clinical results in the near future.
Electronic pacemakers have been used in patients with heart rhythm disorders for device-supported pacing. While effective, there are such shortcomings as limited battery life, permanent implantation of catheters, the lack of autonomic neurohumoral responses, and risks of lead dislodging. Here we describe protocols for establishing porcine models of sick sinus syndrome and complete heart block, and the generation of bioartificial pacemaker by delivering a strategically engineered form of hyperpolarization-activated cyclic nucleotide-gated pacemaker channel protein via somatic gene transfer to convert atrial or ventricular muscle cardiomyocytes into nodal-like cells that rhythmically fire action potentials.
Despite progress in clinical treatment, cardiovascular diseases are still the leading cause of morbidity and mortality worldwide. Therefore, novel therapeutic approaches are needed, targeting the underlying molecular mechanisms of disease with improved outcomes for patients. Gene therapy is one of the most promising fields for the development of new treatments for the advanced stages of cardiovascular diseases. The establishment of clinically relevant methods of gene transfer remains one of the principal limitations on the effectiveness of gene therapy. Recently, there have been significant advances in direct and transvascular gene delivery methods. The ideal gene transfer method should be explored in clinically relevant large animal models of heart disease to evaluate the roles of specific molecular pathways in disease pathogenesis. Characteristics of the optimal technique for gene delivery include low morbidity, an increased myocardial transcapillary gradient, esxtended vector residence time in the myocytes, and the exclusion of residual vector from the systemic circulation after delivery to minimize collateral expression and immune response. Here we describe myocardial gene transfer techniques with molecular cardiac surgery with recirculating delivery in a large animal model of post ischemic heart failure.
Heart disease remains the leading cause of mortality and morbidity worldwide, with 22 million new patients diagnosed annually. Essentially, all present therapies have significant cost burden to the healthcare system, yet fail to increase survival rates. One key employed strategy is the genetic reprogramming of cells to increase contractility via gene therapy, which has advanced to Phase IIb Clinical Trials for advanced heart failure patients. It has been argued that the most significant barrier preventing FDA approval are resolving problems with safe, efficient myocardial delivery, whereby direct injection in the infarct and remote tissue areas is not clinically feasible. Here, we aim to: (1) Improve direct cardiac gene delivery through the development of a novel liquid jet device approach (2) Compare the new method against traditional IM injection with two different vector constructions and evaluate outcome (3) Evaluate the host response resulting from both modes of direct cardiac injection, then advance a drug/gene combination with controlled release nanoparticle formulations.
The enhancement of myocardial tolerance mediated by heat-shock protein 70 (HSP70) can be utilized for further advancement in myocardial protection in clinical settings. Recently, we have developed a novel in vivo gene transfection method for the entire heart. We investigated the possibility of enhancing myocardial tolerance to ischemia-reperfusion injury by introducing the HSP70 gene into the whole heart by means of in vivo gene transfection with HVJ (Hemagglutinating virus of Japan)-liposome procedure. HVJ-liposome, either with the human HSP70 gene (H group; n = 5) or without the gene (C group; n = 5), was infused into rat hearts via the coronary arteries. The hearts obtained from nontreated rats (N group; n = 5) were also examined. Western blotting analysis clearly showed overexpression of HSP70 in the H group. Recovery rate of left ventricular developed pressure, rate-pressure product, and coronary flow after ischemia-reperfusion injury (37°C, 30 minutes) were significantly higher for the H group than for either the C or N group (p < 0.05). CK leakage for the first five minutes of reperfusion was lower in the H group than in the C and N groups (p < 0.05). HSP70 was overexpressed in rat hearts as a result of in vivo gene transfection with HVJ-liposome. Higher myocardial tolerance to ischemia-reperfusion injury was observed in the HSP70-overexpressing heart as compared to the control and even the nontreated hearts. Our results demonstrate the protective effect of gene-transfection-induced HSP70 against ischemia-reperfusion injury in the myocardium, suggesting the possibility of clinical application of gene therapy with HSP70 to ischemia-reperfusion injury.
The study of the effect of autocrine-paracrine vasoactive modulators (e.g., renin-angiotensin) on VSMC biology is very difficult in vivo because in vivo studies are limited. Recent progress in in vivo gene transfer technologies have provided us with the opportunity to study cellular responses to the manipulation of the individual components (i.e., by overexpression or inhibition). Currently, many researchers have developed many in vivo gene transfer techniques for cardiovascular application, including viral gene transfer and liposomal gene transfer. By using in vivo gene transfer approaches, the roles of the tissue renin-angiotensin system have been identified. Such an approach may increase our understanding of the biology and pathobiology of autocrine-paracrine system. This review has discussed the potential utility of in vivo gene transfer methods.
Gene therapy for cardiovascular diseases presents a novel opportunity to treat atherosclerosis, restenosis, acute coronary syndromes and the cardiomyopathies. However, gene therapy also presents significant challenges. Gene transfer technologies continue to evolve and have reached a level of efficiency that makes clinical applications potentially feasible although achieving therapeutic efficacy and specificity will require increased knowledge of the underlying molecular basis of disease. The rationale for gene therapy relies on manipulating the fonctions of specific genes to alter pathophysiology, by either augmenting normal functions, correcting deficiencies or inhibiting deleterious activities. The challenges to this field can be subdivided into three broad categories (Fig. 1). The first relates to the development of specific gene transfer vector and delivery systems suitable for genetic modification of the cardiovascular system, combining efficiency of delivery, ease of preparation, and an acceptable safety profile. The second relates to increasing the knowledge of the molecular mechanisms of cardiovascular disease. This requires the identification of the genes and their fonctions responsible for pathophysiology and the definition of specific gene activities as appropriate targets for manipulation. The third relates to developing suitable animal models of human disease. As suggested by the diagram, at the logical intersection of gene delivery vectors and molecular pathophysiology are vectors targeting specific genes. Similarly, reliable animal models are required in order to develop principles and protocols for in vivo gene transfer.
Despite significant advances in its prevention and treatment, coronary artery disease (CAD) remains the leading cause of death in the Western world today. The economic burden of CAD on society is significant. It has been estimated that the annual cost of treating the approximately 6.3 million Americans afflicted with this disease is $56 billion, with CAD accounting for a significant proportion of the total number of work days lost to illness in the United States [1,2]. Conventional treatment for CAD includes medical therapies designed to reduce hypercholesterolemia, prevent disease progression and reduce myocardial oxygen demand; and interventional therapies that restore blood flow to the epicardial coronary vessels, either by angioplasty or bypass surgery.
Cellular redox stress has been increasingly recognized as a critical component of cardiovascular diseases such as atherosclerosis, cardiac infarct, and cardiac hypertrophy. Oxidative injury can be caused by a number of reactive oxygen species (ROS), including superoxides, hydrogen peroxide, hydroxyl radicals, and nitric oxide, that mediate direct cellular injury and / or act as second messengers to stimulate signal transduction pathways affecting gene expression. These intracellular highways of communication are critical in determining cell fates and whole organ responses following environmental injury. Fundamental to the ability of cells to regulate their redox environment are enzymes capable of detoxifying ROS. These ROS scavengers such as superoxide dismutases, catalase, and glutathione peroxidases control the redox environment of the cell under normal conditions. However, in the setting of injury, many of these pathways can become overwhelmed, leading to alternations in cellular redox metabolism. This review will summarize the theoretical and practical applications of gene therapy for the treatment of cardiovascular ischemic disease by modulating redox activated cellular responses. Several approaches can be utilized to achieve this goal. These include the application of gene targeting to directly modulate the cellular redox environment by expressing recombinant genes capable of degrading reactive oxygen species (ROS) at pathophysiologic important subcellular sites. However, some ROS have been suggested to have beneficial effects on cellular responses following environmental injury. Hence, more sophisticated approaches have attempted to identify specific components of ROS signalling that contribute to damage in the setting of organ injury. These approaches attempt to intervene at the level of specific signal transduction pathways by expressing targeted dominant negative proteins to attenuate apoptosis and / or the expression of genes that are deleterious to organ repair. Development of these targeted approaches to modulate detrimental ROS regulated pathways, while leaving beneficial pathways intact, will only be realized through a comprehensive understanding the relevant pathophysiologic processes.
The incidence of cardiovascular disease (CVD) is increasing throughout the world and is associated with elevated morbidity and mortality. Gene therapy to treat cardiac dysfunction is gaining importance because of the limited therapeutic benefit offered by pharmacotherapies. The growing knowledge of the complex signaling pathways and the development of sophisticated vectors and delivery systems, are facilitating identification and targeting of specific molecular candidates involved in initiation and progression of CVDs. Several pre-clinical and clinical studies have shown the therapeutic efficiency of gene therapy in different disease models and patients. Hence, gene therapy might plausibly become an unconventional treatment modality for CVD patients. In this review, we summarize the gene delivery carriers, modes of delivery, recent preclinical/clinical studies and potential therapeutic targets. We also briefly discuss the existing limitations of gene therapy, technical challenges surrounding gene carriers and delivery systems, and some approaches to overcome these limitations for bringing CVD gene therapy one step closer to reality.Gene Therapy accepted article preview online, 29 April 2016. doi:10.1038/gt.2016.43.
Recently, gene therapy has been the center of interest in the treatment of cardiovascular diseases. One of the major targets for gene therapy is myocardial infarction. In this chapter, we discuss two potential gene therapy strategies:
(1)
replication of dead myocytes by genetically modified cardiac myocytes, and
(2)
prevention of myocardial infarction by NFкB decoy ODN. The use of genetically modified cells for the delivery of recombinant molecules has emerged as a powerful tool for ex vivo gene therapy. For the application of ex vivo gene therapy to cardiac diseases, we examined the potential of cell grafting into the intact heart. Of importance, survival of myocyte grafts was observed in the noninfarcted and border-zone areas but not in the infarcted area. Targeted expression of recombinant molecules in intracardiac grafts could induce a beneficial response in the myocardium. Another potential target is the essential transcription factor, NFкB, as NFкB plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes. In vivo transfection of NFкB decoy digodeoxynudeotides (ODN) into the coronary artery before as well as after reperfusion resulted in a marked decrease in the infarcted area. We have reported the first successful gene therapy for myocardial infarction using NFкB decoy ODN. In this chapter, we also discussed the future direction of gene therapy for myocardial infarction.
The ability to access the normal pericardium in the absence of any effusion induced increasing interest in the pericardial space as a location of and novel approach for various cardiac and intrapericardial applications. Intrapericardial drug delivery may be superior to other routes in special indications. Medication applied intrapericardially will maintain the effective concentration for a longer time. Drugs given in the pericardial space may access the vessel wall and the myocardial tissue directly for a much longer time, but also more predictably and consistently than by an intracoronary or intravenous injection [1]. The intrapericardial route might have additional important advantages for the application of pharmacological agents like an easier access to perivascular tissue.
The study of altered protein expression patterns in diseased myocardium has greatly contributed to our understanding of cardiac dysfunction. Adenovirus-mediated changes in this protein expression pattern in order to correct deregulated protein synthesis is currently undergoing widespread interest and is emerging as a possible future therapy. In an in vitro setting, most experiments to date have used primary adult myocyte cultures to study the impact of adenovirus-mediated gene transfer on the function of the myocardium. Several studies have already indicated that the isotonic shortening behavior of isolated myocytes can be successfully changed through adenoviralmediated gene transfer. However, under cell culture conditions active loaded contractions do not occur as they do in situ,thereby hampering interpretation to this in vivo situation. Although multicellular cardiac preparations may not represent every single aspect of the beating heart, loading conditions can be influenced and these preparations have a multicellular architecture contracting under loaded conditions as occur in heart. The latter implies that impact of secretory factors and interaction between different cell types is possible. Moreover, cell-to-cell connections allow for passing-on of intracellular signals and physical loading conditions, between myocytes specifically, in these multicellular preparations.
We have used a Langendorff-based adenoviral gene-transfer protocol to the rabbit heart and cultured multicellular preparations from these hearts for several days. Basic contractility parameters did not change over time when such preparations were cultured, and physiological and pharmacological responses were well preserved. Although after 48 hours efficient and high levels of the transfected reporter-gene LacZ were observed in the individual myocytes of the preparation, contractile parameters and physiological/pharmacological responses were unaltered.
Kaum ein Thema der Medizin hat in den letzten Jahren so viel Aufmerksamkeit in den Medien genossen wie das der Gentherapie. Das prinzipielle Ziel der Gentherapie besteht darin, durch Einbringen genetischer Information in den Empfängerorganismus Einbringen genetischer Information in den Empfängerorganismus Krankheitsprozesse gezielt zu unterbrechen. Am einfachsten läßt sich dies am Beispiel monogenetischer Erkrankungen nachvollziehen, wobei die Krankheit durch den Defekt eines bestimmten Gens und dem damit verbundenen Fehlen oder einer Fehlfunktion des Genprodukts verursacht ist. Das Einbringen einer funktionstüchtigen Variante des defekten Gens stellt die normale Organfunktion wieder her und verkörpert die klas-sische Form der Gentherapie. Monogenetische Erkrankungen sind allerdings relativ selten, und die aufwendige Entwicklung gentherapeutischer Verfahren ließe sich mit der Behandlung ausschließlich von monogenetischen Erkrankungen nicht rechtfertigen. Vielmehr ist ein großer Nutzen der Gentherapie langfristig für die Behandlung polygenetischer und erworbener Erkrankungen zu erwarten. Darunter befinden sich unzählige Krankheiten, deren herkömmliche pharmakologische Therapie unbefriedigend oder gar wirkungslos ist. Eine gezielte und wirksame Behandlung durch spezifische gentherapeutische Ansätze wäre deshalb sinnvoll. Doch liegt es in der Natur gerade der polygenetischen und erworbenen Erkrankungen, daß die zugrundeliegenden Mechanismen schwer zu erforschen und deshalb oft unbekanntsind. Wie wichtig die genauen Kenntnisse der Pathophysiologie als Grundlage gentherapeutischer Behandlungsansätze sind, läßt sich am gegenwärtigen internationalen Stand der Gentherapie ersehen. Die Anfang dieses Jahrzehnts durch die Möglichkeiten der Gentherapie ausgelöste Phase der Euphorie ist mittlerweile einer Phase der Ernüchterung gewichen. In der Tat ist bis heute nicht ein einziger Fall beschrieben, in dem eine Krankheit ausschließlich mit gentherapeutischen Verfahren geheilt werden konnte. Dies veranlaßte die FDA (Food and Drug Administration) — das zentrale Organ für die Genehmigung gentherapeutischer Behandlungen von Patienten in den USA -, die Mehrheit der über 100 inaugurierten Gentherapiestudien zu stoppen. Es seien noch mehr Kenntnisse aus der Grundlagenforschung erforderlich, so die Argumentation der FDA. Dennoch, die 1. Phase der Entwicklung der Gentherapie, die nun hinter uns liegt, hat uns die Erkenntnis gebracht, daß Gentherapie möglich und in vielen Fällen sicherlich sinnvoll ist. Außerdem wurde ersichtlich, daß das Einschleusen von rekombinanten Genen in Zellkulturen, Tiermodelle und sogar Patienten bisher ungeahnte Möglichkeiten der Erforschung biologischer Mechanismen ermöglicht. Sowohl Mediziner als auch Grundlagenforscher sind sich deshalb einig, daß Gentherapie in naher Zukunft an Patienten routinemäßig durchgeführt wird.
RNAi-based approaches show promising effects in preclinical models of cardiac diseases. However, a potential clinical use may be limited by the low efficiency of cardiac transfer. Efficient and sustained cardiac delivery in large animal models and finally clinical trials requires transfer of vectorized shRNA with suitable application systems. Packaging shRNA constructs in targeted adeno-associated viral (AAV) vectors may be versatile for systemic delivery into rodent hearts. Expressing microRNA under control of cardiac-specific promoters may further increase efficiency and specificity of delivery. For large animals and finally clinical studies, careful selection of the vector and application system will be necessary to obtain valid results.
Heart diseases are a major cause of morbidity and mortality in contemporary society. Advances in the understanding of the molecular basis of myocardial dysfunction have placed many acquired and congenital cardiovascular diseases within the reach of gene-based therapy. Four prerequisites are required for a successful clinical application of gene therapy: (1) an effective strategy for genetic manipulation, (2) availability of vectors with enhanced myocardial tropism, (3) a clinically translatable delivery technique that will result in global or regional expression, and (4) creation of therapeutic transgenes for selected molecular targets depending on the underlying pathological state of the heart. Despite significant promise, however, several obstacles exist with gene-based therapies. These obstacles are described in detail in this chapter, along with proposed solutions. We anticipate that advances in the field will improve cardiac gene therapy in future clinical approaches. © 2013 Springer Science+Business Media New York. All rights reserved.
Gene therapy is a novel method of treating some of the hitherto untreatable diseases. It involves the introduction of a functional gene to replace the activity of a resident defective gene so that biologically active proteins can be synthesized within the cells whose function is to be altered. Introduced as a concept about 2 decades ago it has become a reality today. A variety of DNA delivery systems have been developed involving biological, physical and chemical agents. Gene therapy was initially thought to be a treatment modality for inherited single gene defects however it has also found applications in acquired diseases. It's use is being studied in the treatment of cancer, immunodeficiency diseases, cardiovascular, metabolic and neurological disorders; hormones and blood factors deficiencies. It is also being developed as a 'gene' vaccine against influenza and malaria. Recently attempts have been made for its use in treatment of HIV infection. Gene therapy, although still in the infant stages of development offers the possibility for major advances in prevention and treatment of these diseases. Presently the clinical application of gene therapy is limited by the availability of suitable gene transfer methodology. This review describes some of these aspects of this new therapeutic modality.
Cardiovascular gene therapy applications began about 25 years ago. Since then, an in-depth understanding has accumulated on the underlying mechanisms of molecular structure as well as the development and function of the cardiovascular system in normal and disease states. In accordance with this, gene-based approaches have undergone substantial changes. Cardiovascular gene therapy should ideally deliver the genetic material to a specific target and reach a level of expression sufficient for therapeutic action. To achieve this, one needs to select a strategy with gene overexpression or gene silencing, suitable vectors and promoters, specific molecular targets known to be involved in a certain cardiovascular disease, and organ-targeted delivery techniques. Pharmacologic intervention has substantially increased survival and decreased morbidity in acquired and congenital cardiovascular diseases but still has multiple limitations including the targeting of symptoms rather than the pathological mechanism, difficulty in achieving efficacy, large variation between dose and concentration-dependent pharmacokinetics, and side effects. The progress in molecular biology and pharmacogenomics technology could allow for the development of gene containing drugs, which have the potential in the near future to momentously improve the management of a variety of clinical cardiovascular problems.
Our understanding of the molecular mechanisms that govern gene expression has been facilitated by the ability to introduce recombinant DNA molecules into heterologous cellular systems both in vitro and in vivo. One approach to defining DNA sequences important in the regulation of gene expression is to place controlling elements (e.g., promoter/enhancer sequences) upstream of a DNA coding sequence, introduce these constructs into transgenic animals or cells in culture, and analyze the levels of gene product produced by the introduced construct. Ideally, such a reporter gene should encode a product that is stable, innocuous to the cell or organism in which it is being expressed, and should be readily detectable, even when present in small quantities.
Adenoviruses have been isolated from a large number of different species (mammalian and fowl) and over 100 different serotypes have been reported, some 43 of them human. The human adenoviruses, particularly types 2, 5, and 12, have been the most extensively characterized, and these viruses have served as valuable tools in the study of the molecular biology of DNA replication, transcription, RNA processing, and protein synthesis in mammalian cells. Seeref. 1 for a general review.
RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and beta-galactosidase
were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no
special delivery system was required for these effects. The extent of expression from both the RNA and DNA constructs was
comparable to that obtained from fibroblasts transfected in vitro under optimal conditions. In situ cytochemical staining
for beta-galactosidase activity was localized to muscle cells following injection of the beta-galactosidase DNA vector. After
injection of the DNA luciferase expression vector, luciferase activity was present in the muscle for at least 2 months.
We demonstrate gene transfer into rat heart in vivo by the direct injection of plasmid DNA. Injection of gene constructs driven by retroviral and cellular promoters resulted in detectable levels of reporter gene activities. The cellular promoter and 5' flanking sequence (positions -613 to +32) were derived from the rat alpha-myosin heavy chain gene whose expression in vivo is restricted to cardiac muscle and is positively regulated by thyroid hormone. After DNA injection, activity of the firefly luciferase gene coupled to the myosin heavy chain promoter and regulatory sequence was detected in heart but not in skeletal muscle and was significantly increased in response to thyroid hormone treatment. Consequently, expression of injected genes can be targeted to specific cell types in vivo and can be modulated by the hormonal status of the animal. This approach provides a means of mapping the elements of genes that regulate their responses to complex stimuli that cannot be modeled in vitro.
Expression of the cardiac myosin isozymes is regulated during development, by hormonal stimuli and hemodynamic load. In this study, the levels of expression of the two isoforms (alpha and beta) of myosin heavy chain (MHC) during cardiac hypertrophy were investigated at the messenger RNA (mRNA) and protein levels. In normal control and sham-operated rats, the alpha-MHC mRNA predominated in the ventricular myocardium. In response to aortic coarctation, there was a rapid induction of the beta-MHC mRNA followed by the appearance of comparable levels of the beta-MHC protein in parallel to an increase in the left ventricular weight. Administration of thyroxine to coarctated animals caused a rapid deinduction of beta-MHC and induction of alpha-MHC, both at the mRNA and protein levels, despite progression of left ventricular hypertrophy. These results suggest that the MHC isozyme transition during hemodynamic overload is mainly regulated by pretranslational mechanisms, and that a complex interplay exists between hemodynamic and hormonal stimuli in MHC gene expression.
The cardiac ventricular myosin phenotype is developmentally and hormonally regulated. The genes coding for the two myosin heavy chains ( MHCs ), alpha and beta, have been recently isolated and characterized. In this study, we establish the precise temporal expression of these MHC genes in correlation with the myosin phenotype both during cardiac development and in response to different thyroid hormone levels and also document their expression in other muscle tissues. The close correlation observed between the relative abundance of the alpha- and beta-MHC mRNAs and corresponding isozymes demonstrates that the MHC phenotype is produced by the expression of the alpha- and beta-MHC genes and is regulated by changes in the level of their respective mRNAs. The opposite effect of thyroid hormone on the expression of the alpha- and beta-MHC genes in the ventricular myocardium indicates that these genes are regulated in an antithetic fashion. Finally, the MHC mRNAs encoded by the alpha- and beta-MHC genes are also present in the atrial myocardium and in the soleus, respectively.
100% of primary human hepatocytes infected with an adenoviral vector carrying beta-galactosidase expressed the exogenous gene. Expression was also achieved in > 40% of adult mouse hepatocytes in vivo. Normal levels of activity were achieved in mouse ornithine transcarbamylase (OTC)-deficient primary hepatocytes using another adenoviral vector carrying human OTC cDNA. Study of OTC-deficient primary human hepatocytes from a single patient confirmed the utility of adenoviral delivery of OTC. We describe adenoviral-mediated exogenous gene expression in human and mouse hepatocytes in vitro and in mouse liver in vivo. Data suggest that adenoviral vectors may be useful for correcting OTC deficiency.
We have explored the use of adenovirus-mediated gene transfer to transiently elicit production of low density lipoprotein (LDL) receptors in mice. A recombinant adenovirus carrying the human LDL receptor cDNA restored LDL receptor function in receptor-deficient cultured cells. Intravenous injection of recombinant virus acutely lowered plasma cholesterol levels and increased the rate of 125I-labeled LDL clearance from the circulation in normal mice. At 4 days after virus injection, the t1/2 of plasma LDL was reduced up to 10-fold. An estimated 90% of the parenchymal cells in liver expressed the adenovirus-transferred genes as judged by immunofluorescence of LDL receptors or by beta-galactosidase staining. These results demonstrate that adenovirus-mediated transfer of the LDL receptor gene provides an efficient way of augmenting LDL receptor gene function in the liver over the short term.
This chapter discusses the use of histochemical stains for macrophages in cell smears and tissue sections. Macrophages with strong microbicidal activity probably have high levels of mitochondrial enzymes because these enzymes are involved with oxygen transport. In tubercle bacilli (BCG) granulomas, the levels of specific digestive enzymes may vary with the substance ingested by the macrophage. In other words, the levels of acid phosphatase, but not β-galactosidase, may be high in certain macrophages, and vice versa. The indolyl method is used for the histochemical demonstration of β-galactosidase. This method has proved to be ideal for studying the digestive-type of activation of rabbit macrophages. The blue-green color resulting from β-galactosidase activity varies from zero in unactivated macrophages to almost black in strongly activated macrophages. This color is readily distinguishable from the pure blue color of the hematoxylin counterstain. In histochemistry, acid phosphatase is the classic lysosomal enzyme. The lead substitution method has localized acid phosphatase in the phagosomes of macrophages and can be used for electron microscopy.
Human gene therapy is a procedure that is being used in an attempt to treat genetic and other diseases. Eleven clinical protocols are under way at the present time, each with scientific and clinical objectives. Human genetic engineering raises unique safety, social, and ethical concerns.
In mice, rabbits, and pigs, two basic types of cardiac myosin isoenzymes were found by electrophoresis of native molecules: a fast-migrating form with high Ca(2+)-dependent ATPase activity and a slow-migrating form with low activity. According to the nomenclature of J. F. Y. Hoh, P. A. McGrath, and P. T. Hale (1978, J. Mol. Cell. Cardiol. 10, 1053-1076) these forms are called, respectively, V1 and V3. In all species, myosin was essentially V3 during fetal life, while V1 appeared around the time of birth. There were species differences in adults: mice remained V1, while rabbits and pigs returned to V3 after 3 weeks of age. Adult dog, beef, and human myosins were also composed of the V3 form only.
Since the first observation by Spann et al., it has become clear that in cardiac hypertrophy induced by a mechanical overloading, the velocity of shortening of the cardiac muscle (Vmax) is reduced (see ref. 2 for review). Most authors agree that this mechanical alteration is accompanied by a decrease in the Ca2+-dependent ATPase activity of myosin (see ref. 3 for review). The molecular basis of such changes was unknown because the structural modifications of the myosin molecule were ill-defined. Nevertheless, it has recently been shown that, like skeletal muscle myosin, cardiac myosin is composed of several polymorphic forms, comparable to isoenzymes. In the skeletal muscle, new functional requirements can induce changes in both contractile activity and type of myosin isoenzyme synthesised. We now report that an increase in cardiac work produced by mechanical overloading in rats induces the preferential synthesis of a cardiac myosin isoenzyme characterised by specific immunological and electrophoretic properties and exhibiting a lower ATPase activity. This adaptive change could account for the reduced shortening speed of this hypertrophied cardiac muscle.
Gene transfer can be achieved in the adult rat heart in vivo by direct injection of plasmid DNA. In this report we define the spatial and temporal limits of reporter gene expression after a single intracardiac injection. pRSVCAT (100 micrograms), in which the Rous sarcoma virus long terminal repeat is fused to the chloramphenicol acetyltransferase reporter gene, and p alpha MHCluc (100 micrograms), in which the alpha-cardiac myosin heavy chain promoter is fused to the firefly luciferase gene, were injected into hearts, and reporter gene activities were assayed at various times. Both chloramphenicol acetyltransferase and luciferase were detectable in 100% of the rats from 1 to 7 days, in 60% of the rats from 17 to 23 days, and in 30% of the rats from 38 to 60 days after injection. Reporter gene activity was largely limited to a 1-2-mm region of the ventricle surrounding the injection site. Closed circular DNA was far more effective than linear DNA in transfecting cells in vivo. The relative strengths of three different promoters, Rous sarcoma virus long terminal repeat, alpha-myosin heavy chain, and alpha 1-antitrypsin, all fused to the luciferase reporter gene were determined. The constitutive viral promoter was approximately 20-fold more active than the cardiac-specific cellular promoter, and the liver-specific cellular promoter was not active at all in the cardiac environment. Thus, direct injection of genes into the heart offers a simple and powerful tool with which to assess the behavior of genes in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
Genetic manipulation of the vasculature may offer insights into the pathogenesis of coronary artery disease and may lead to gene therapy for disorders such as restenosis after percutaneous coronary angioplasty. The goal of this study was to develop a percutaneous method for gene transfer into coronary arteries of intact animals. Liposomes were used to facilitate transfection in coronary arteries with a plasmid containing the cDNA encoding luciferase. This reporter was chosen since it is not expressed in mammalian cells, and it can be quantified using a sensitive assay (light production). Mongrel dogs were catheterized, and DNA was delivered to coronary arteries via a porous perfusion balloon system. Luciferase expression was measured 3-5 days after the procedure, when the dogs were killed. Luciferase activity in control arteries (n = 12) was no higher than average background activity. Eight of 12 transfected arteries exhibited gene expression, averaging 4.3 +/- 2.1 pg luciferase (p less than 0.01, transfected versus control arteries). In addition, the ability to transfect DNA into femoral arteries without a transfection vehicle was tested. Five dogs were subjected to surgical transfection attempts in their femoral arteries with either DNA alone or DNA plus liposomes. Luciferase was expressed in all 10 femoral arteries; those treated with DNA alone expressed 35.6 +/- 8 pg luciferase, and those treated with DNA plus liposomes expressed 42.3 +/- 14 pg luciferase (p = 0.70). These results demonstrate the use of a percutaneous catheter to achieve gene transfer and expression in coronary arteries of intact dogs and suggest that the efficiency of intra-arterial gene transfer may be similar whether or not a transfection vehicle is used.
Advances in the understanding of molecular biology of human disease and the development of efficient gene transfer techniques have resulted in practical approaches to human gene therapy, with new techniques being developed at an increasing rate. The first trials have now begun in humans and initial results are positive.
Successful treatment of muscular disorders awaits an adapted gene delivery protocol. The clinically applicable technique used for hematopoietic cells which is centered around implantation of retrovirally modified cells may not prove sufficient for a reversal of phenotype when muscle diseases are concerned. We report here efficient, long-term in vivo gene transfer throughout mouse skeletal and cardiac muscles after intravenous administration of a recombinant adenovirus. This simple, direct procedure raises the possibility that muscular degenerative diseases might one day be treatable by gene therapy.
We found previously that genes injected into skeletal muscle can be taken up by myofibers and expressed. In the present study we found that myocardial cells can also express a variety of reporter genes injected into myocardium as efficiently as skeletal myofibers, while the cells of several other tissues cannot. The inability of tissues other than striated muscle to express injected DNA is not due to technical difficulties of injection because injected DNA was detected in these other tissues by PCR analysis. These results suggest that skeletal and cardiac muscle cells have unique features such as T tubules that may play a critical role in DNA uptake. Expression in cardiac muscle was stable for only two weeks, possibly because of an immune response against the transfected cells. The ability to directly transfer genes into myocardial cells raises the possibility of gene therapy for both acquired and genetic heart diseases.
The ability to program recombinant gene expression in cardiac myocytes in vivo holds promise for the treatment of many inherited and acquired cardiovascular diseases. In this report, we demonstrate that a recombinant beta-galactosidase gene under the control of the Rous sarcoma virus promoter can be introduced into and expressed in adult rat cardiac myocytes in vivo by the injection of purified plasmid DNA directly into the left ventricular wall. Cardiac myocytes expressing recombinant beta-galactosidase were detected histochemically in rat hearts for at least 4 weeks after injection of the beta-galactosidase gene. These results demonstrate the potential of this method of somatic gene therapy for the treatment of cardiovascular disease.
In addition to its many other functions, the plasma membrane of eukaryotic cells serves as a barrier against invading parasites and viruses. It is not permeable to ions and to low molecular weight solutes, let alone to proteins and polynucleotides. Yet it is clear that viruses are capable of transferring their genome and accessory proteins into the cytosol or into the nucleus, and thus infect the cell. While the detailed mechanisms remain unclear for most animal viruses, a general theme is apparent like other stages in the replication cycle; their entry depends on the activities of the host cell. In order to take up nutrients, to communicate with other cells, to control the intracellular ion balance, and to secrete substances, cells have a variety of mechanisms for bypassing and modifying the barrier properties imposed by their plasma membrane. It is these mechanisms, and the molecules involved in them, that viruses exploit.
Recent studies suggest that oxygen free radicals may mediate postischemic myocardial dysfunction ("stunning"), but all the evidence for this hypothesis is indirect. Thus, we used electron paramagnetic resonance (EPR) spectroscopy and the spin trap, alpha-phenyl N-tert-butyl nitrone (PBN), to directly investigate whether free radicals are produced after a 15-min coronary artery occlusion and subsequent reperfusion in 30 open-chest dogs. After intracoronary infusion of PBN, EPR signals characteristic of oxygen- and carbon-centered radical adducts were detected in the venous blood draining from the ischemic/reperfused vascular bed. The myocardial release of PBN adducts began during coronary occlusion but increased dramatically in the first few minutes after reperfusion. After this initial burst, the production of radicals abated but did not cease, persisting up to 3 h after reflow. The EPR spectra (aH beta = 2.67-2.79 G, aN = 14.75-15.00 G) were consistent with the trapping by PBN of secondary oxygen- and carbon-centered radicals, such as alkoxy and alkyl radicals, which could be formed by reactions of primary oxygen radicals with membrane lipids. There was a linear, direct relationship between the magnitude of PBN adduct production and the degree of ischemic flow reduction. Recovery of contractile function (measured as systolic wall thickening) after reperfusion was greater (P less than 0.05) in dogs given PBN than in controls. This study demonstrates that reversible regional myocardial ischemia in the intact animal is associated with prolonged free radical generation, and that the intensity of such generation is related to the severity of ischemia. The results provide direct evidence to support the hypothesis that reactive oxygen metabolites contribute to the persistent contractile dysfunction (myocardial stunning) observed after brief ischemia in vivo.
Early region 1 (E1) of the human adenoviruses has many intriguing properties which have prompted numerous mutational studies to help delineate and characterize the domains responsible for these functions. In mutational analyses being done currently, the E1 region is usually cloned into a bacterial plasmid where it is mutated and then the altered E1 sequences are "rescued" back into infectious virus. The most frequently used rescue procedures are somewhat tedious, requiring the purification and fractionation of linear viral DNA or DNA fragments, and often involve the screening of numerous plaque isolates. Several observations we have made recently on the properties of adenovirus DNA in infected cells and on infectious plasmids in transfected cells led us to design a new approach for rescuing E1 mutations into infectious viral genomes. We constructed a plasmid, pJM17, containing the entire Ad5 DNA molecule, with an insert in the E1 region that exceeds the packaging constraints of the adenovirus capsid. Following transfection of pJM17 into 293 cells the plasmid DNA is able to replicate but cannot be packaged into infectious virions. In contrast cotransfection of 293 cells with pJM17 plus an E1-containing plasmid carrying mutated sequences produces recombinant virions at high efficiencies. Neither plasmid needs to be linearized prior to contransfection. The technique eliminates the need to purify and manipulate infectious virion DNA and since no unique restriction sites are needed, both E1A and E1B mutants' as well as foreign gene inserts in the E1 region can be easily rescued into virus.
Conventional measurement of wall thickening with two transit-time crystals requires considerable skill, is associated with myocardial trauma, and does not discern the function of different layers of the left ventricular (LV) wall. To overcome these limitations, we have developed a 10 MHz pulsed Doppler technique that measures thickening at any depth of the LV wall from a single crystal sutured to the epicardium. To verify its accuracy, we compared measurements of thickening fraction (TF) by pulsed Doppler and transit-time methods in 25 open-chest dogs. The epicardial Doppler crystal was placed over an intramyocardial crystal positioned either in the subendocardium or midwall. The epicardial crystal acted as both the Doppler transducer and the transit-time transmitter, so that TF was measured by each technique at the same site. A wide range of regional function (transmural TF:-28-42%, epicardial TF:-20-28%) was produced by coronary occlusion followed by reperfusion, and by isoproterenol and phenylephrine infusions. There was a good correlation between the two methods, both for transmural TF (r = 0.98, 107 paired measurements) and epicardial TF (r = 0.99, 70 paired measurements). Despite marked changes in function, the two techniques yielded similar measurements under all of the conditions tested (base line, ischemia, 5, 15, 30, 60, 120, and 180 min of reperfusion, isoproterenol and phenylephrine). One-millimeter errors in selecting the depth of the Doppler sample volume did not significantly affect the accuracy of TF measurements. Thus the single pulsed Doppler crystal provides a simple, atraumatic, and accurate means for measuring myocardial function, both transmurally and in selected layers of the LV wall.
Although several enzymes known to reside in peroxisomes have been studied extensively, no cis-acting amino acid sequences involved in the transport of these proteins to peroxisomes have been described. As a first step towards the determination of a putative peroxisomal targeting sequence, we have expressed the cDNA encoding the firefly luciferase [Photinus-luciferin:oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolyzing), EC 1.13.12.7] in monkey kidney cells and found that the product of the gene is transported to peroxisomes. Luciferase is derived from the firefly (Photinus pyralis) and is synthesized and stored in the cells of the firefly's lantern organ, where it is also found in peroxisomes. The fact that this protein is similarly targeted in cells from such different organisms suggests that the process of protein transport to peroxisomes has been highly conserved through evolution.
Some statistical techniques for analyzing the kinds of studies typically reported in Circulation Research are described. Particular emphasis is given to the comparison of means from more than two populations, the joint effect of several experimentally controlled variables, and the analysis of studies with repeated measurements on the same experimental units.
The ability to express recombinant genes in the coronary vasculature and the myocardium holds promise for the treatment of a number of acquired and inherited cardiovascular diseases. Previous in vivo gene transfer approaches in the heart have been limited by relatively low efficiencies of gene transduction. In this report, we demonstrate that catheter-mediated infusion of replication-defective adenovirus into the coronary arterial circulation in vivo represents a novel and efficient method for the induction of recombinant gene expression in both the coronary arteries and the myocardium. A single intracoronary infusion of 2 x 10(9) - 1 x 10(10) p.f.u. of adenovirus resulted in high level recombinant gene expression in both the coronary arteries and surrounding myocardium of adult rabbits for at least 2 weeks. No inflammatory response or myocardial necrosis was observed following the adenovirus infusions. The polymerase chain reaction (PCR) was used to assess the tissue distribution of infection following intracoronary infusion of adenovirus. Adenovirus DNA was detected by PCR in the livers, kidneys, lungs, brains and testes of animals 5 days after virus infusion. Percutaneous transluminal gene transfer (PTGT) into the heart by intracoronary infusion of replication-defective adenovirus represents a relatively non-invasive and efficient method of inducing recombinant gene expression both in the coronary arterial wall and in the surrounding myocardium.
We describe the use of a human bronchial xenograft model for studying the efficiency and biology of in vivo gene transfer into human bronchial epithelia with recombinant E1 deleted adenoviruses. All cell types in the surface epithelium except basal cells efficiently expressed the adenoviral transduced recombinant genes, lacZ and CFTR, for 3-5 weeks. Stable transgene expression was associated with high level expression of the early adenoviral gene, E2a, in a subset of transgene expressing cells and virtually undetectable expression of the late adenoviral genes encoding the structural proteins, hexon and fiber. These studies begin to address important issues that relate to safety and in vivo efficacy of recombinant adenoviruses for gene delivery into the human airway.
To evaluate the potential of direct transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA for the treatment of cystic fibrosis (CF), we administered an E1-deficient adenovirus, encoding CFTR, to a defined area of nasal airway epithelium of three individuals with CF. This treatment corrected the Cl- transport defect that is characteristic of CF-affected epithelia. After treatment, there was a decrease in the elevated basal transepithelial voltage, and the normal response to a cAMP agonist was restored. We found no evidence of viral replication or virus-associated adverse effects, even at the highest dose tested (25 MOI). These data represent a small step in achieving long-term improvement of CF lung function by gene therapy.
A new adenovirus-based vector (Ad2/CFTR-1) has been constructed in which the cDNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis (CF) gene product, replaces the early region 1 coding sequences, E1a and E1b. The virus retains the E3 region. Ad2/CFTR-1 and a related construct encoding beta-galactosidase replicate in human 293 cells which provide E1 gene functions in trans. Replication of these recombinant viruses was not detected in a variety of other cells, although very limited viral DNA synthesis and transcription from the E4 and L5 regions could be measured. These E1-deletion vectors were also deficient in cellular transformation, shut-off of host cell protein synthesis, and production of cytopathic effects, even at high multiplicities of infection. Ad2/CFTR-1 produced CFTR protein in a variety of cells including airway epithelia from CF patients. Expression of functional CFTR protein in a CF airway epithelial monolayer was detected by correction of the Cl- transport defect characteristic of CF. Surprisingly low multiplicities of infection (0.1 moi) were sufficient to generate CFTR Cl- current across a CF epithelial monolayer in vitro. These data, together with the lack of obvious toxicity, suggest that Ad2/CFTR-1 should be suitable for CF gene therapy.
First-generation recombinant adenoviruses that lack E1 sequences have shown tremendous promise in animal and human models of gene therapy. Important limitations of these vectors are that recombinant gene expression is transient and inflammation occurs at the site of gene transfer. Our hypothesis for generating vectors with increased persistence is that present recombinant adenoviruses express viral proteins that stimulate cellular immune responses leading to destruction of the infected cells and repopulation of the organ with non-transgene-containing cells. This model predicts that further crippling of the virus will improve persistence and diminish pathology. We describe in this report second-generation recombinant adenoviruses harboring a beta-galactosidase-expressing transgene in which a temperature-sensitive mutation has been introduced into the E2A gene of an E1-deleted recombinant. At nonpermissive temperature, this virus fails to express late gene products, even when E1 is expressed in trans. The biology of this recombinant was studied in vivo in the context of mouse liver, a setting that is permissive for adenovirus type 5 replication. Animals that received the second-generation virus expressed the transgene for at least 70 days, whereas expression of the first-generation virus was no longer than 14 days. In addition, the inflammatory response, as measured by infiltration of CD8+ T cells, was blunted and delayed in livers infected with second-generation virus. These studies illustrate that modifications that disrupt structural protein expression in recombinant adenoviruses may be useful in enhancing their utility for gene therapy.
Previous studies have established that gene transfer into myocardial cells in vivo is detectable after direct injection of plasmid DNA. Recently, adenovirus vectors have been shown to provide an efficient method for gene transfer into a wide range of tissues. Therefore, this study sought to assess the efficiency and stability of adenovirus-mediated gene transfer into myocardium and to compare this method with that using plasmid-based gene transfer techniques. Adult rats underwent myocardial injection via a subdiaphragmatic approach. Gene transfer efficiency was compared using direct injection of an adenovirus vector encoding for the marker gene beta-galactosidase (beta-gal), a control adenovirus vector encoding for the cystic fibrosis transmembrane conductance regulator gene, a plasmid encoding for beta-gal, or a control plasmid. Hearts infected with an adenovirus vector containing the beta-gal gene showed significantly increased beta-gal enzymatic activity compared with hearts injected with beta-gal plasmid. Histological examination revealed that cardiac myocytes were the target of adenovirus-mediated gene transfer. A time course of gene expression showed that beta-gal enzymatic activity peaked during the first week following injection. Adenovirus vectors provide an efficient but transient method for in vivo gene expression in myocardium.
The efficient introduction of genetic material into quiescent nerve cells is important in the study of brain function and for gene therapy of neurological disorders. A replication-deficient adenoviral vector that contained a reporter gene encoding beta-galactosidase infected rat nerve cells in vitro and in vivo. beta-Galactosidase was expressed in almost all sympathetic neurons and astrocytes in culture. After stereotactic inoculations into the rat hippocampus and the substantia nigra, beta-galactosidase activity was detected for 2 months. Infected cells were identified as microglial cells, astrocytes, or neurons with anatomical, morphological, and immunohistochemical criteria. No obvious cytopathic effect was observed.
We have constructed a helper-independent adenovirus type 5-luciferase recombinant (Ad5-Luc 3) containing the firefly luciferase gene flanked by simian virus 40 (SV40) regulatory sequences inserted in the early region 3 (E3) of the Ad5 genome. Expression of luciferase in cells infected with Ad5-Luc3 was relatively efficient. In HeLa cells approximately 20 micrograms luciferase per 10(6) cells was made by 36 h post-infection and a 62 kilo-Dalton (kDa) luciferase band was clearly visible in a [35S]methionine-labeled Ad5-Luc 3-infected cell extract analyzed directly by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of experiments in which cultured cells were infected with Ad5-Luc 3 in the presence or absence of 1-beta-D-arabinofuranosyl cytosine (AraC) showed that the majority of luciferase expression was dependent on viral DNA replication. This suggested that the enzyme was probably translated primarily from mRNA derived from transcripts expressed from the major late promoter of Ad5. An anti-luciferase antibody was raised in a rabbit and used to further characterize the luciferase expressed in HeLa cells infected with Ad5-Luc 3 by immunoprecipitations and Western blot analyses. The half-life of luciferase expressed in HeLa cells infected with Ad5-Luc 3 was calculated to be approximately 6-8 h by pulse chase analysis. Luciferase is likely to be a useful marker for monitoring virus dissemination and gene expression in experimental animals because assays for enzymatic activity are extremely sensitive and backgrounds are low in all tissues. In mice inoculated intraperitoneally (i.p.) with Ad5-Luc 3, luciferase activity was detected in the liver, spleen, kidney, and lung. A single i.p. inoculation of mice with Ad5-Luc 3 was sufficient to raise anti-luciferase antibody and Ad5 neutralizing antibody which persisted for at least 8 weeks. Even in the presence of circulating anti-luciferase and Ad5 neutralizing antibodies, luciferase activity could be detected in the livers, spleens, and kidneys of mice inoculated i.p. a second time with Ad5-Luc 3.
We studied the ability of adenoviral vectors to achieve gene transfer into injured arteries. A recombinant adenoviral vector expressing a nuclear-targeted beta-galactosidase gene was constructed and infused into balloon-injured rat carotid arteries. Three days after gene transfer, recombinant gene expression was assessed quantitatively by (1) measuring beta-galactosidase antigen and activity in tissue extracts and (2) histochemical staining and counting of cells expressing beta-galactosidase. Exposure of injured carotid arteries to increasing concentrations of the vector (10(8) to 10(10) plaque-forming units per milliliter) resulted in a dose-responsive increase in beta-galactosidase expression, with peak expression of approximately 43 mU or 25 ng beta-galactosidase per vessel. Microscopic examination of histochemically stained arteries demonstrated gene transfer limited to the vascular media; transduced cells were identified immunohistochemically as smooth muscle cells. Counting of both histochemically stained and total nuclei in the media revealed that approximately 30% of the cells in the media of the injured vessels were transduced. Calculations based on both counting cells and on the level of beta-galactosidase expression in tissue extracts suggested the presence of 5000 to 10,000 transduced cells per 10 mm of vessel. Arteries infused with either vehicle only, a control adenoviral vector, or liposomes combined with the vector plasmid contained little or no evidence of beta-galactosidase expression. High levels of in vivo beta-galactosidase expression persisted for at least 7 days after gene transfer but declined significantly by day 14. We conclude that adenoviral vector-mediated gene transfer into the injured rat carotid artery results in efficient gene transfer into the vascular media, with levels of recombinant protein production significantly higher than any previously reported in arterial gene transfer studies. Adenoviral vectors appear to be particularly useful agents for in vivo arterial gene transfer.
We have investigated the regulated expression of genes injected into the heart of large mammals in situ. Reporter constructs using the chloramphenicol acetyltransferase gene under the control of muscle-specific beta-myosin heavy chain (beta-MHC) or promiscuous (mouse sarcoma virus) promoters were injected into the canine myocardium. There was a linear dose-response relation between the level of gene expression and the quantity of plasmid DNA injected between 10 and 200 micrograms per injection site. The level of reporter gene expression did not correlate with the amount of injury imposed on the cardiac tissue. There was no regional variation in expression of injected reporter genes throughout the left ventricular wall. By use of both the mouse sarcoma virus and a muscle-specific beta-MHC promoter, reporter gene expression was one to two orders of magnitude greater in the heart than in skeletal muscle. Expression in the left ventricle was threefold higher than in the right ventricle. Chloramphenicol acetyltransferase activity was detected at 3, 7, 14, and 21 days after injection, with maximal expression at 7 days after injection. Statistical analysis of coinjection experiments revealed that coinjection of a second gene construct (Rous sarcoma virus-luciferase) is useful in the control of transfection efficiency in vivo. Furthermore, using reporter constructs containing serial deletions of the 5' flanking region of the beta-MHC gene, we performed a series of experiments that demonstrate the utility of this model in mapping promoter regions and identifying important regulatory gene sequences in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
Replication-deficient recombinant adenovirus vectors do not require target cell replication for transfer and expression of exogenous genes and thus may be useful for in vivo gene therapy in the endothelium. To evaluate the feasibility of adenovirus-mediated gene transfer in vivo in normal intact blood vessels, adenovirus vectors containing the Escherichia coli lacZ gene or a human alpha 1-antitrypsin (alpha 1AT) cDNA were injected in vivo into the lumen of an occluded vessel segment of sheep jugular vein and/or carotid artery. After 15 minutes of incubation, circulation was restored; the vessels were harvested 1-28 days later and evaluated for gene transfer and expression. Three days after in vivo exposure to the lacZ adenovirus vector, the endothelium of jugular veins and carotid arteries expressed beta-galactosidase. Exposure of jugular veins and carotid arteries in vivo to the alpha 1AT adenovirus vector resulted in the expression of alpha 1AT mRNA transcripts detected by Northern analysis and in the synthesis and secretion of alpha 1AT detected by ex vivo [35S]methionine labeling. Expression with the adenovirus vectors was efficient and easily detectable 1-14 days after injection, with maximum expression at 7 days. Expression was no longer evident at 28 days. Thus, adenovirus vectors are capable of transferring exogenous genes to the endothelium of normal arteries and veins with expression for at least 2 weeks, suggesting that these vectors have the potential for a variety of cardiovascular experimental and clinical applications.
Manipulation of adenovirus vectors Gene Transfer and Expression Protocols
- Fl Graham
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Validation of a single crystal for measurement of transmural and epicardial thickening Some statistical methods useful in circulation research Demonstration of free radical generation in 'stunned' myocardium of intact dogs with the use of the spin trap alpha-phenyl N-tert-butyl nitrone
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Second generation adenovirus vectors for cystic fibrosis gene therapy
- Armentano D
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The origin of beetle Iuciferase
- K V Wood