Adeno associated virus (AAV) vectors

In cardiac clinical trials and animal models experiments, functional foreign genes were transferred into cardiomyocytes of patients and in animal hearts respectively using adeno associated virus (AAV) vectors. The delivery routes included were intracoronary injection, myocardia injection, sarcoplasmic reticulum calcium ATPase (SERCA2a) pump and molecular cardiac surgery with recirculating delivery (MCARD). Adeno associated virus (AAV) serotypes 1-9 were applied in experiments studies. The AAV6 and AAV7 serotypes shown significant progress in animal models while AAV1 denoted improvement in cardiac conditions in clinical trials.  Low scores for other serotypes were due to lack of suitable receptors in gene transfer process.
 
1 INTRODUCTION

Progress in genetic engineering and gene therapy technique were firstly evolved in 1960’s after the breakthrough in biology which enabled scientists to use enzymes to manipulate DNA sequences. Unlike conventional breeding where the desired traits of organisms were selected within or close species, genetic engineering employs biotechnology to change genetic information of organisms regardless of their species (Brooker et al, 2009).

The artificial manipulation of genomes is made for specific reasons and it rely on a simple principle that DNA is a universal language of life which can be communicated among organisms. In practical, genes can be crossed between any organisms, for example jelly fish and cat, cabbage and scorpion as well as goat and spider.

In modern world, genetic engineering found many of its applications in agriculture and in medications. In agriculture, genetic engineering is interchangeably referred to genetic modification (GM) or genetic improvement (GI). According to (FAO, 2004) biotechnology is an essential tool of genetic engineering which uses biological procedures, living things and their byproducts to alter systems and ultimately makes new product for specific use.

It helps farmers to improve quality of their animals and crops to increase volumes of livestock and crops respectively. Moreover (GE) increases the productivity by strengthening crop resistance to cope with damage associated with weeds and diseases (FAO, 2004).

In medical care, genetic engineering reflects to therapeutic techniques such as stem cells, gene therapy and cloning which strive for treating genetic disorders instigated from mistakes in DNA sequences. In this scenario, (GE) is anticipated to replace mutant gene for a functional gene to cure inherited disorders by coding specific functional proteins needed for synthesis of hormones and enzymes. Moreover, some (GE) techniques are intended to grow certain cells to replace the defective ones.

In that view, basic understanding of deoxyribonucleic acid, DNA, chromosomes and gene as illustrated in Fig. 4 is essential. The DNA and a gene are interchangeably used to express a hereditary strand that controls a blueprint of our life (Kelemen, et al. 2015; Heartl and Jones, 2006).

2 STUDY REVIEW AND DISCUSSION

2.1 Fundamentals of gene therapy

The rationale of gene therapy is to transfer genetic material to cure genetic disorders. When a mutant gene is identified, collective procedures in gene therapy are followed where copies of functional gene are introduced into non-specific areas in DNA to substitute malfunctioned gene (Fig. 5), sometime it is intended to repair abnormal gene using reverse mutation technique. Furthermore, gene therapy involves swapping of foreign gene in a homologous recombination (Mammen, B. et al. 2007).

2.2 Gene selection and Gene delivery

The selection of appropriate vector and correct delivery system has been emphasized to ensure high transduction of the cardiomyocytes and robust gene expression. In another observation it has been underlined the significance of identifying a gene to match with its modification of target pathology to generate expected results while avoiding an adverse cardiac adjustment (Gwathmey et al, 2011; Pleger et al, 2013).

2.3 Vectors

2.3.1 Non-Viral Vectors

Non- virus vectors are known to impressed many researchers because of their broader size in transgenic delivery system besides their cost effectiveness. But the use of naked DNA has been uncommon gene transfer method due to its interference of negatively charged membrane (Hayward et al, 2014).

2.3.2 Viral Vectors

Consistent with various gene therapy studies, the most common virus vectors used to repair and regenerate heart tissues in preclinical models and human trials are adeno viruses, adeno-associated viruses, retroviruses and lentiviruses. The recombinant viruses were reported to be the most successful virus vectors in gene transfer (Flotte and Carter, 1995).

The natural ability of viruses in delivering therapeutic genes with greater transduction and cardiotropism has given them advantages over non-viral vectors. It has been distinguished that wider target cell tropism and high transduction efficiency to cardiac cells, made adenoviruses a distinctive instrument in clinical trials (Hayward et al, 2014).

2.3.3 Virus binding and gene transfer mechanisms

The virus transport mechanism in gene therapy depends on virus interaction with receptors on the surface of the cell which bring intracellular signal to trigger a specific reaction in the cell.

When virus transfers gene to host cells, it changes metabolic patterns of the cells. It has been distinguished that for adeno associated virus, a virus activated signal improves the process of gene transfer, even though there are doubts about stimulation of innate and adaptive immunity in the host cells (Gerber, U. 2002).

Various virus receptors have been mentioned in gene expression despite the fact that their proper functions in virus binding are unknown. Other studies proposed that sugar moieties can prompt cell signaling (Gerber, U. 2002; Mark, A. et al. 2001). Some findings admitted that virus changes its structure when it binds to primary and secondary receptor sites and then exploit its primary receptors before advancing activated core receptors on the surface of the cell (Gerber, U. 2002).

2.3.4 Transfer efficiency of Adeno associated Virus

The recent studies concluded that adeno associated virus is the only viral vector currently used for cardiac gene transfer experiments due to its sustained long term gene expression, nonpathogenic, high cardiotropism and higher transduction (Kenneth, F. 2015).

The previous studies advocated that application of AAV gene transfer for cardiac disorders has demonstrated auspicious results in large animals (Hammoundi and hajjar, 2015). While the evaluation of therapeutic effects of AAV mediated gene therapy in animal models, indicated that repaired myocardium produced an effective expression in the sarcolemma of the mice heart (Yue, Y. et al. 2003). According to (Gao, et al. 2011), direct myocardial injection of AAV6 and AAV9 in human primate have exhibited a robust cardiac transduction (Gao, et al, 2011).

2.3.5 Transfer Barriers and Immune responses

In some cases, accumulation of AAV vectors in perinuclear region of the cell creates a cellular barrier which obstructs a subcellular trafficking into the nucleus. Also, acute inflammatory responses caused by the needle injury have been depicted as limitations of intramuscular injection (Nicolson and Samulski, 2014)

In contrast, circumventing the neutralization of antibodies recounted to stop induction of a T-cell response against the efficient transfer process in the circulation and in the antigen presenting cells (Nicolson and Samulski, 2014).

Mark, A. et al. (2001) concluded that the absence of viral coding sequence in AAV, ensures that the virus neither has been linked to toxicity nor inflammatory responses; hovwever some studies are skeptical about neutralizing antibodies generated from virus which can limit its dispensation.

3 ANIMAL MODELS EXPERIMENTS

This assay aimed at assessing the efficiency of adeno associated virus (AAV) in gene therapy. It attempted to evaluate the effectiveness of AAV serotypes in gene delivery to cardiac cells in animal models and clinical trials. The following are few experiments reviewed:

3.1 Clinical trials 1

In investigating the efficiency of AAV vector to reinstate functions of contractile in cardiomyocytes, samples from failing heart patients and healthy volunteers were taken.

Viable cells were isolated from endocardial tissues using digestive enzymes. The adeno associated virus with sarcoplasmic reticulum Ca+ ATPase (AAV1-SERCA2a) was applied to the samples (Federica, M et al. (1999).

3.2 Clinical trials 2

Two groups of heart failing patients were given intracoronary infusion of high and low dose of AAV1-SERCA2a and then compared with placebo group to assess the repeated heart conditions such as myocardial infarction and death. The experimental groups were monitored for almost three years (Zsebo, K et al. 2013).

3.3 Animal models 1

The experimental guidance was followed on preparation of animals and hearts were isolated from sheep. Gene expression and transduction efficiency of single stranded adeno associated virus (ssAAV) and self-complementary adeno associated virus (scAAV) were examined for their relative effectiveness in gene delivery. Cytomegalovirus (CMV) was used as promoter. Virus solution of 1014GC was prepared and then recirculated through cardiac circulation.

3.4 Animal models 2

In a comparative study of the effectiveness of adeno associated virus in heart transduction. The AAV vectors were prepared and the CMV promoter was combined in AAV serotypes 1-9 and then delivered through indirect intracoronary injection into the heart of adult mice trials (Zincarelli et al; 2010).

4 RESULT

i. In human cardiomyocytes, the activities of sarcoplasmic reticulum Ca2+ pump showed significant contraction induced by overexpression of SERCA2a gene received by AAV1 serotype (Fig. 4). Failing heart had reduced curbing and long relaxation nearly similar to non-failing cardiomyocytes (Federica, M. et al. 1999).

ii. After monitoring of the repeating cardiac cases such as myocardial infarction, heart failure and death, it was revealed that higher number of cases was reported in placebo than in controlled group in which the number was lowered by at least 80% compared to placebo

iii. The results illustrated that direct fluorescence photography showed a strong green fluorescent protein expression (GFP) in the Molecular cardiac surgery with recirculating delivery (MCARD)/ssAAV6.

iv. The higher scores were indicated in AAV6 and AAV7, with AAV6 transduced to the maximum highest mark (Fig.1).

5 CONCLUSION

Observed results revealed that gene transfer of AAV1-SERCA2a improved the ventricular arrhythmias and heart failure disorders (Federica, M et al. 1999; Yue, Y et al. 2003). Patients were described as safe and in stable conditions with higher survival rate resembling to that of placebo group as shown in (Fig. 4.). In clinical trials, effectiveness of AAV1 serotype corresponded to that of AAV6 and AAV7 in animal models as indicated in (Fig. 1).

In other annotations, results highlighted that application of high dose of AAV-SERCA2a to heart patients indicated a significant progress of cardiac conditions to patients because it reduced frequent clinical episodes (mariell, J. 2011; Zsebo, K et al. 2014). The results have proven a progress of gene therapy in all experiments reviewed without any safety issues identified, besides the advantages of adeno-associated virus AAV1 in delivering SERCA2a gene to improve cardiac diseases (Mariell J et al, 2011).

The evidence gathered from the heart cross-section indicated high transduction efficiency ssAAV6 as shown in graph 4. (White et al, 2011). These results were agreed with the clinical trials (Zincarelli et al; 2010) results indicating that AAV6 were more effective in cardiac myocyte transduction for large animals. This predicts remarkable results in human trials (Gao et al, 2011). In other remarks elucidated in (Fig.1, Graph 1, 2, 3and 4) of the AAV serotype 1-9, the AAV6 and AAV7 revealed higher transduction efficiency and over expression of gene in the myocardium of the mice (Zacchigna et al, 2014).

This observation is matched with (Kenneth, F. 2015; Hammoundi and Hajjar, 2015) comments on AAV cardiac gene expression in animal models and (Gao et al, 2011) for higher cardiac transduction of AAV6 in human primate. But the results in animal models contradicted with findings (Gao, et al, 2011) which suggested robust cardiac transduction of AAV9. Low efficiencies for other serotype could be due to cellular barriers, also the accumulation of vectors in the perinuclear of the cell hinders the entry of virus into the nucleus (Nicolson and Samulski, 2014) and inappropriate receptors for the AAV vectors in gene transfer (Geber, U. 2002).

The first principle found in the literature, gave general approval for the use of AAV in human trial of gene therapy. Therefore the assay concluded that compatible pairs of vector-route of serotype AAV1, AAV6 and AAV7 to be used in cardiovascular conditions. This is because in many occasions in this review have shown positive indications of improving cardiovascular disease.

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