In cardiovascular diseases treatment, gene therapy is used to insert copies of a heathy gene into the chromosomes of a failed heart of an individual to replace faulty ones. The assessment gene delivery efficiency in large animals was performed. The stranded adeno-associated virus (ssAAV) and the self-complementary adeno-associated virus (scAAV) solution combined with a growth factor in phosphate-buffered saline were investigated. The molecular cardiac surgery with recirculating delivery (MCARD) and intramuscular injection (IM) injection were employed. The results exhibited that gene expression and transduction efficiency of scAAV6 was greater than ssAAV9 and ssAAV6 using MCARD system.
1. INTRODUCTION
The advancement in genetic engineering and gene therapy was firstly evolved in 1960’s after the discovery which enabled the use of enzymes to manipulate the DNA in the laboratories. In this development, scientists managed to process the DNA sequences. It was then realised that engineered human genes which transferred into the cell made some changes in instructing the cell to make protein. The idea of treating genetic diseases using a healthy DNA to replace the defective DNA originated by the scientist called Stanfield Rogers in 1970s.
Gene therapy is a genetic engineering technique which manually manipulates genes to modify the functions and activities of DNA for medical purposes (Kelemen, et al, 2015). This procedure for the first time attempted to treat a patient with congenital disease in 1990. In this trial, the blood with faulty gene was taken from the patient and then the blood with corrected DNA was returned back. The results showed some success to the patient and inspired many researchers to increase their trials in other related inherited diseases.
Structures of DNA, chromosome and a gene are explained in (Fig. 4). The DNA, deoxyribonucleic acid is a hereditary biomolecule that holds a blueprint for life of a living organism (Kelemen, et al, 2015). A gene is simple a section of DNA and an inherited trait transmitted from parents to offspring by reproduction (Heartl and Jones, 2006). A specific gene codes will guide instructions to postulate the order in which the amino acids should form a specific protein. The ability of DNA to replicate the same copy is fundamental for proper functions and health of the organism (Kelemen, et al, 2015).
Likewise, a chromosome is made of a very long single molecule of DNA rapped by a protein molecules. During the cell division chromosomes ensures that DNA codes correctly to avoid mistakes. When DNA is unable to code amino acids correctly, then incorrect and harmful protein molecules will be formed. In this case a particular gene is said to have undergone mutation and the hereditary disorder will occur. The preclinical studies in animals models has identified that this therapy will provide a hope in treating the inherited and acquired cardiovascular diseases (Rincon et al, 2015).
This comparative study aimed at assessing the relative effectiveness of gene delivery of the molecular cardiac surgery with recirculating delivery (MCARD)-AAV and intramuscular injection (IM)-AAV injection approaches in delivering foreign genes to the host cells. It will determine gene expression and transduction efficiency of stranded adeno-associated virus (ssAAV9), (ssAAV6) and self-complementary adeno-associated virus (scAAV6).
2. STUDY REVIEW AND DISCUSSION
1. GENE THERAPY FOR HEART FAILURE
It has been agreed that heart failure occurs when the heart fails to pump inadequate blood required for the body metabolism. This disorder eventually prompts calcium cycle dysregulation to the myocytes resulting in disruptions of other body functions. The cardiovascular related diseases such as hypertension, coronary artery disease, diabetes and infection are thought to influence this ailment (Hayward et al, 2014).
The heart failure gene therapy involves insertion of copies of a heathy gene into the chromosomes of an individual who carries a faulty allele to replace faulty genes to the heart ( Gwathmey et al,.2011; Pleger et al 2013).
2. FUNDAMENTALS OF GENE THERAPY
1. Gene selection and gene delivery
The selection of right gene vector and correct delivery strategy ensures the high transduction of the cardiomyocytes and minimizing the spillage of virus. The study associated the transduction to the correlation of the amount of injected virus to the rate of the expression within the cardiac tissue (Scimia et al, 2014). Along with another observation (Hayward et al, 2014) narrated the importance of identifying a gene which match with its modification of a target pathophysiology to produce expected results as well as avoiding an adverse cardiac adjustment (Hayward et al, 2014).
In this biological treatment, various methods were mentioned in gene transfer. Simple microinjection, bioballistics, electro and chemical poration are among these techniques. Specific virus and bacterial species are used as plasmid and vectors for preparing a recombinant DNA because of their ability to carry foreign genes into the host cells and eventually releasing the chosen gene to replace the faulty gene (Hayward et al, 2014).
1. The electro and chemical poration
The electro and chemical poration method is designed to create pores in the membrane of the cell so as genes can be transferred more easily. The pores are made by exposing the surface of the cells to a chemical or diminutive electric current for the relaxed entry of gene into the cell (Hayward et al, 2014).
2. Bioballistics method
In this method, small silver particles are combined with foreign genes and then inserted into the recipient cells. A small amount of mixture is then transferred is by a short gun in one projectile into the host cells (Hayward et al, 2014).
3. Microinjection
The alternative gene delivery method to replace the use of vectors and plasmid for transferring peripheral genes into the host cells is microinjection. However, when this procedure involves large cell of plants and animals, a fine glass needle is applied to deliver the genes into the cell nucleus (Hayward et al, 2014).
3. GENE DELIVERY
According to (Naim et al, 2013; Mason D et al, 2015), the most common virus vectors used to repair and regenerate heart tissue in cardiovascular diseases in preclinical models and in human trials of gene therapy are retrovirus, lentivirus, adenovirus and adeno-associated virus. The recombinant viruses were reported to be the most successful virus vector in gene transfer. (Paul and Paul, 2011).
1. Non-viral vectors and gene transfer without a vector (naked DNA)
Naked DNA has been uncommon gene transfer method due to its negatively charged membrane. The non-virus vectors have impressed many researchers for the reason that they are cost more effective. Even if non-virus vectors are insufficient in transduction efficiency, they have no size limitation in transgene delivery systems (Hayward et al, 2014).
2. Viral vectors
The natural ability of viruses to deliver therapeutic genes to cells with greater transduction efficiency and cardiotropism has given them advantages over non-viral vectors. It has been noted that adenoviruses are widely used in therapy clinical trials due to their wide target cell tropism and high transduction efficiency to cardiac cells (Hayward et al, 2014).
In various studies, adeno-associated virus (AAV) transduction has been linked to respiratory epithelial cells, bone marrow and lymphocyte-derived cells. In the lungs, direct gene delivery to the airway in rodents and non-human primates showed success without any detectable toxicity. Based on these findings, the use of AAV in human trial has been approved for gene therapy (Flotte and Carter, 1995).
1. Adeno-associated virus (AAV)
The AAV vector remained the best option for providing long-term expression, safety and efficacy (Fargnoli et al, 2013). The AAV vector exhibited a robust cardiac transduction and diverse tissue tropisms in different hosts (Asokan and Samulski, 2013).
In accordance with recent study which specified that AAV6 is more suitable vector for cardiac gene transfer through intramyocardial, intrapericardial, and intracoronary routes, while AAV9 achieved a significant cardiac transduction with intravenous method. Direct myocardial injections of AAV6 in nonhuman primate and intravenous administration of AAV9 in human tissue related to a stout cardiac transduction (Gao et al,. 2011)..
In other observations, studies (Gao et al, 2011) discovered that AAV9 vector has little impact on mediating cardiac gene transfer if compared with AAV6. The single tyrosine mutations in AAV9 capsids were described to incapable of improving the cardiac transduction efficiency in strains of mice (Qiao et al, 2012).
According to (Fargnoli et al, 2013) the acute inflammatory responses caused by the needle injury has been described as weakness of intramuscular injection. The undetected immunogenicity of the AAV capsid has been described to obstruct translation results of preclinical results to human patients (Mueller and Flotte, 2008). Considering the induction of a T-cell response against the efficient transgene, it has been emphasized to avoid neutralizing antibodies in the circulation and in transduction of antigen presenting cells (Oliver et al, 2007).
3. Plasmid vectors
Plasmid is known to be the simplest form of vector in gene transfer. Among its essential attributes include its circular DNA molecule, stability at room temperatures, and also its ability to produce proteins with antibiotic resistance. Although poor gene transfer efficiency in protein production has been perceived, plasmid has been described to have the capacity of encoding multiple proteins regardless of their sizes (Paul and Paul, 2011).
4. CLINICAL EXPERIMENT
1. Aim and Objectives
The clinical trials aimed at determining gene expression and transduction efficiency of stranded adeno-associated virus (ssAAV6), (ssAAV9) and self-complementary adeno-associated virus (scAAV6) vectors in gene transfer. The studies attempted to assess the relative effectiveness of the molecular cardiac surgery with recirculating delivery (MCARD)-AAV, intramuscular injection (IM)-AAV injection
2. Molecular cardiac surgery with recirculating delivery (MCARD)
Consistent with the clinical trials (White, J. 2011) which referred to ten sheep of the same age were given induced anaesthesia before being taken for median sternotomy incision. The heart was isolated from each animal and the flow was started to balance with coronary sinus at constant pressure (White, J. 2011).
The recombinant virus solution of 1014GC of scAAV6.CMV.EGFP, ssAAV6.CMV.EGFP and ssAAV9.CMV.EGFP combined with sixty micrograms of growth factor in phosphate-buffered saline was injected through coronary sinus and allowed to stay for ten minutes to ensure that intravascular space was saturated with the vector solution (White, J. 2011).
Through the isolated heart, the vector solution was recirculated through cardiac circulation for thirty minutes. The vectors were then allowed to remain in the heart for ten minutes before recirculating through coronary sinus for twenty minutes. The circulation was allowed to start for twenty minutes at the same pressure before the coronary circuit being flushed out and cleaned from the virus solution (White, J. 2011).
3. Intramuscular injection (IM)
In same experiment, IM injection was applied to a controlled group to deliver 1013GC of scAAV6.CMV.EGFP and Necropsy for four weeks. On the wall of LV the injection spacing measured about 1cm. The spacemen from all parts of the heart were taken for examination through direct fluorescence and GFP (White, J. 2011).
4. Analysis
The Microsoft Excel using unpaired Student’s t-tests was applied to analyse data for recombinant vectors scAAV and ssAAV.
3. RESULTA AND CONCLUSION
In this review study, the direct fluorescence photography and high-power photomicrographs of the cross-section of the LV showed a resilient green fluorescent protein expression (GFP) in the MCARD/scAAV6 while a weak and inadequate gene expression in the MCARD/ssAAV9 and MCARD/ssAAV6. This indicated high transduction efficiency when nearly all cardiomyocytes were transduced in MCARD/scAAV6 (White et al, 2011).
Corresponding to the clinical trials (Zincarelli et al; 2010), the results indicated that the scAAV6 are more effective in cardiac myocyte transduction for large animals and predicted to give remarkable results with human trials than its counterpart ssAAV6 and ssAAV9 (Gao et al,. 2011). In other observations (Fig.1, Graph 1,2 and 3) of the AAV serotype 1-9, the AAV6 revealed the higher transduction efficiency in the myocardium of the mouse (Zacchigna et al, 2014).
Although it is difficult to find a reliable and clinically effective cardiac gene delivery, results illustrated in (Fig.1 and 2) has shown the MCARD delivery system to be suitable if it is used with scAAV6 (Zacchigna et al, 2014; White, J et al. 2011).
Higher transduction efficiency of adeno-associated virus vectors in post mitotic tissues of the heart was reported to attract gene therapy applications in ischemic cardiomyopathy and heart failure. Protein-coding complementary DNAs, short hairpin RNAs and microRNA genes were some constraints mentioned to limit its range of applicability (Zacchigna et al, 2014). This approach has been recommended for use to patients with end stage heart failure who will undergo cardiac surgery for ventricular assist device placement and valve repair (White et al, 2011).
The IM method presented despondent gene expression results and inconsistent gene expression and transduction efficient (White et al, 2011). Consequently, this method was illustrated as ineffective and instead to find an alternative cardiac gene transfer models with an exceptional clinically translatable quality (White et al, 2011).
This study does not suggest the use of ssAA6 and ssAAV9 in human clinical trials, due to inadequate gene expression on ssAA6 and ssAAV9 recombinants as indicated in Fig. 1. The use of AVV vectors are limited due to its low transduction efficiency caused by the accumulation of recombinant AAV vectors in the perinuclear region of cells which creates a cellular barrier that obstructs a subcellular trafficking into the nucleus (Nicolson and Samulski, 2014). While the transfer across the endothelial barrier has been known to encounter viral vector-mediated gene delivery (Fargnoli et al, 2013).
Finally, the MCARD mediated vector transfer has been recommended for use to patients with end stage heart failure and those who needed valve repair, replacement or ventricular assist device placement (White et al, 2011).
The advancement in genetic engineering and gene therapy was firstly evolved in 1960’s after the discovery which enabled the use of enzymes to manipulate the DNA in the laboratories. In this development, scientists managed to process the DNA sequences. It was then realised that engineered human genes which transferred into the cell made some changes in instructing the cell to make protein. The idea of treating genetic diseases using a healthy DNA to replace the defective DNA originated by the scientist called Stanfield Rogers in 1970s.
Gene therapy is a genetic engineering technique which manually manipulates genes to modify the functions and activities of DNA for medical purposes (Kelemen, et al, 2015). This procedure for the first time attempted to treat a patient with congenital disease in 1990. In this trial, the blood with faulty gene was taken from the patient and then the blood with corrected DNA was returned back. The results showed some success to the patient and inspired many researchers to increase their trials in other related inherited diseases.
Structures of DNA, chromosome and a gene are explained in (Fig. 4). The DNA, deoxyribonucleic acid is a hereditary biomolecule that holds a blueprint for life of a living organism (Kelemen, et al, 2015). A gene is simple a section of DNA and an inherited trait transmitted from parents to offspring by reproduction (Heartl and Jones, 2006). A specific gene codes will guide instructions to postulate the order in which the amino acids should form a specific protein. The ability of DNA to replicate the same copy is fundamental for proper functions and health of the organism (Kelemen, et al, 2015).
Likewise, a chromosome is made of a very long single molecule of DNA rapped by a protein molecules. During the cell division chromosomes ensures that DNA codes correctly to avoid mistakes. When DNA is unable to code amino acids correctly, then incorrect and harmful protein molecules will be formed. In this case a particular gene is said to have undergone mutation and the hereditary disorder will occur. The preclinical studies in animals models has identified that this therapy will provide a hope in treating the inherited and acquired cardiovascular diseases (Rincon et al, 2015).
This comparative study aimed at assessing the relative effectiveness of gene delivery of the molecular cardiac surgery with recirculating delivery (MCARD)-AAV and intramuscular injection (IM)-AAV injection approaches in delivering foreign genes to the host cells. It will determine gene expression and transduction efficiency of stranded adeno-associated virus (ssAAV9), (ssAAV6) and self-complementary adeno-associated virus (scAAV6).
2. STUDY REVIEW AND DISCUSSION
1. GENE THERAPY FOR HEART FAILURE
It has been agreed that heart failure occurs when the heart fails to pump inadequate blood required for the body metabolism. This disorder eventually prompts calcium cycle dysregulation to the myocytes resulting in disruptions of other body functions. The cardiovascular related diseases such as hypertension, coronary artery disease, diabetes and infection are thought to influence this ailment (Hayward et al, 2014).
The heart failure gene therapy involves insertion of copies of a heathy gene into the chromosomes of an individual who carries a faulty allele to replace faulty genes to the heart ( Gwathmey et al,.2011; Pleger et al 2013).
2. FUNDAMENTALS OF GENE THERAPY
1. Gene selection and gene delivery
The selection of right gene vector and correct delivery strategy ensures the high transduction of the cardiomyocytes and minimizing the spillage of virus. The study associated the transduction to the correlation of the amount of injected virus to the rate of the expression within the cardiac tissue (Scimia et al, 2014). Along with another observation (Hayward et al, 2014) narrated the importance of identifying a gene which match with its modification of a target pathophysiology to produce expected results as well as avoiding an adverse cardiac adjustment (Hayward et al, 2014).
In this biological treatment, various methods were mentioned in gene transfer. Simple microinjection, bioballistics, electro and chemical poration are among these techniques. Specific virus and bacterial species are used as plasmid and vectors for preparing a recombinant DNA because of their ability to carry foreign genes into the host cells and eventually releasing the chosen gene to replace the faulty gene (Hayward et al, 2014).
1. The electro and chemical poration
The electro and chemical poration method is designed to create pores in the membrane of the cell so as genes can be transferred more easily. The pores are made by exposing the surface of the cells to a chemical or diminutive electric current for the relaxed entry of gene into the cell (Hayward et al, 2014).
2. Bioballistics method
In this method, small silver particles are combined with foreign genes and then inserted into the recipient cells. A small amount of mixture is then transferred is by a short gun in one projectile into the host cells (Hayward et al, 2014).
3. Microinjection
The alternative gene delivery method to replace the use of vectors and plasmid for transferring peripheral genes into the host cells is microinjection. However, when this procedure involves large cell of plants and animals, a fine glass needle is applied to deliver the genes into the cell nucleus (Hayward et al, 2014).
3. GENE DELIVERY
According to (Naim et al, 2013; Mason D et al, 2015), the most common virus vectors used to repair and regenerate heart tissue in cardiovascular diseases in preclinical models and in human trials of gene therapy are retrovirus, lentivirus, adenovirus and adeno-associated virus. The recombinant viruses were reported to be the most successful virus vector in gene transfer. (Paul and Paul, 2011).
1. Non-viral vectors and gene transfer without a vector (naked DNA)
Naked DNA has been uncommon gene transfer method due to its negatively charged membrane. The non-virus vectors have impressed many researchers for the reason that they are cost more effective. Even if non-virus vectors are insufficient in transduction efficiency, they have no size limitation in transgene delivery systems (Hayward et al, 2014).
2. Viral vectors
The natural ability of viruses to deliver therapeutic genes to cells with greater transduction efficiency and cardiotropism has given them advantages over non-viral vectors. It has been noted that adenoviruses are widely used in therapy clinical trials due to their wide target cell tropism and high transduction efficiency to cardiac cells (Hayward et al, 2014).
In various studies, adeno-associated virus (AAV) transduction has been linked to respiratory epithelial cells, bone marrow and lymphocyte-derived cells. In the lungs, direct gene delivery to the airway in rodents and non-human primates showed success without any detectable toxicity. Based on these findings, the use of AAV in human trial has been approved for gene therapy (Flotte and Carter, 1995).
1. Adeno-associated virus (AAV)
The AAV vector remained the best option for providing long-term expression, safety and efficacy (Fargnoli et al, 2013). The AAV vector exhibited a robust cardiac transduction and diverse tissue tropisms in different hosts (Asokan and Samulski, 2013).
In accordance with recent study which specified that AAV6 is more suitable vector for cardiac gene transfer through intramyocardial, intrapericardial, and intracoronary routes, while AAV9 achieved a significant cardiac transduction with intravenous method. Direct myocardial injections of AAV6 in nonhuman primate and intravenous administration of AAV9 in human tissue related to a stout cardiac transduction (Gao et al,. 2011)..
In other observations, studies (Gao et al, 2011) discovered that AAV9 vector has little impact on mediating cardiac gene transfer if compared with AAV6. The single tyrosine mutations in AAV9 capsids were described to incapable of improving the cardiac transduction efficiency in strains of mice (Qiao et al, 2012).
According to (Fargnoli et al, 2013) the acute inflammatory responses caused by the needle injury has been described as weakness of intramuscular injection. The undetected immunogenicity of the AAV capsid has been described to obstruct translation results of preclinical results to human patients (Mueller and Flotte, 2008). Considering the induction of a T-cell response against the efficient transgene, it has been emphasized to avoid neutralizing antibodies in the circulation and in transduction of antigen presenting cells (Oliver et al, 2007).
3. Plasmid vectors
Plasmid is known to be the simplest form of vector in gene transfer. Among its essential attributes include its circular DNA molecule, stability at room temperatures, and also its ability to produce proteins with antibiotic resistance. Although poor gene transfer efficiency in protein production has been perceived, plasmid has been described to have the capacity of encoding multiple proteins regardless of their sizes (Paul and Paul, 2011).
4. CLINICAL EXPERIMENT
1. Aim and Objectives
The clinical trials aimed at determining gene expression and transduction efficiency of stranded adeno-associated virus (ssAAV6), (ssAAV9) and self-complementary adeno-associated virus (scAAV6) vectors in gene transfer. The studies attempted to assess the relative effectiveness of the molecular cardiac surgery with recirculating delivery (MCARD)-AAV, intramuscular injection (IM)-AAV injection
2. Molecular cardiac surgery with recirculating delivery (MCARD)
Consistent with the clinical trials (White, J. 2011) which referred to ten sheep of the same age were given induced anaesthesia before being taken for median sternotomy incision. The heart was isolated from each animal and the flow was started to balance with coronary sinus at constant pressure (White, J. 2011).
The recombinant virus solution of 1014GC of scAAV6.CMV.EGFP, ssAAV6.CMV.EGFP and ssAAV9.CMV.EGFP combined with sixty micrograms of growth factor in phosphate-buffered saline was injected through coronary sinus and allowed to stay for ten minutes to ensure that intravascular space was saturated with the vector solution (White, J. 2011).
Through the isolated heart, the vector solution was recirculated through cardiac circulation for thirty minutes. The vectors were then allowed to remain in the heart for ten minutes before recirculating through coronary sinus for twenty minutes. The circulation was allowed to start for twenty minutes at the same pressure before the coronary circuit being flushed out and cleaned from the virus solution (White, J. 2011).
3. Intramuscular injection (IM)
In same experiment, IM injection was applied to a controlled group to deliver 1013GC of scAAV6.CMV.EGFP and Necropsy for four weeks. On the wall of LV the injection spacing measured about 1cm. The spacemen from all parts of the heart were taken for examination through direct fluorescence and GFP (White, J. 2011).
4. Analysis
The Microsoft Excel using unpaired Student’s t-tests was applied to analyse data for recombinant vectors scAAV and ssAAV.
3. RESULTA AND CONCLUSION
In this review study, the direct fluorescence photography and high-power photomicrographs of the cross-section of the LV showed a resilient green fluorescent protein expression (GFP) in the MCARD/scAAV6 while a weak and inadequate gene expression in the MCARD/ssAAV9 and MCARD/ssAAV6. This indicated high transduction efficiency when nearly all cardiomyocytes were transduced in MCARD/scAAV6 (White et al, 2011).
Corresponding to the clinical trials (Zincarelli et al; 2010), the results indicated that the scAAV6 are more effective in cardiac myocyte transduction for large animals and predicted to give remarkable results with human trials than its counterpart ssAAV6 and ssAAV9 (Gao et al,. 2011). In other observations (Fig.1, Graph 1,2 and 3) of the AAV serotype 1-9, the AAV6 revealed the higher transduction efficiency in the myocardium of the mouse (Zacchigna et al, 2014).
Although it is difficult to find a reliable and clinically effective cardiac gene delivery, results illustrated in (Fig.1 and 2) has shown the MCARD delivery system to be suitable if it is used with scAAV6 (Zacchigna et al, 2014; White, J et al. 2011).
Higher transduction efficiency of adeno-associated virus vectors in post mitotic tissues of the heart was reported to attract gene therapy applications in ischemic cardiomyopathy and heart failure. Protein-coding complementary DNAs, short hairpin RNAs and microRNA genes were some constraints mentioned to limit its range of applicability (Zacchigna et al, 2014). This approach has been recommended for use to patients with end stage heart failure who will undergo cardiac surgery for ventricular assist device placement and valve repair (White et al, 2011).
The IM method presented despondent gene expression results and inconsistent gene expression and transduction efficient (White et al, 2011). Consequently, this method was illustrated as ineffective and instead to find an alternative cardiac gene transfer models with an exceptional clinically translatable quality (White et al, 2011).
This study does not suggest the use of ssAA6 and ssAAV9 in human clinical trials, due to inadequate gene expression on ssAA6 and ssAAV9 recombinants as indicated in Fig. 1. The use of AVV vectors are limited due to its low transduction efficiency caused by the accumulation of recombinant AAV vectors in the perinuclear region of cells which creates a cellular barrier that obstructs a subcellular trafficking into the nucleus (Nicolson and Samulski, 2014). While the transfer across the endothelial barrier has been known to encounter viral vector-mediated gene delivery (Fargnoli et al, 2013).
Finally, the MCARD mediated vector transfer has been recommended for use to patients with end stage heart failure and those who needed valve repair, replacement or ventricular assist device placement (White et al, 2011).