Gene therapy for heart failure

Gene therapy is a genetic engineering technique which manually manipulates genes to modify the functions and activities of DNA for medical purposes. The DNA, deoxyribonucleic acid is a hereditary biomolecule that holds a blueprint for life of a living organism. It has been depicted that growth and development of living organisms requires the harmonious expression of genes which is controlled by sequence-specific DNA binding proteins or transcription factors (Kelemen, Z et al, 2015).

A gene is a length of a DNA in which instructions for protein synthesis are processed. A specific gene codes this information to postulate the order in which the amino acid should form a specific protein molecule. The ability of DNA to replicate exactly the same copy is fundamental for proper functions and health of the organism (Kelemen, Z et al, 2015).

Likewise, chromosome consists of protein and a single molecule of DNA. During the cell division chromosomes ensures that DNA codes correctly to avoid mistakes. Nevertheless during replication process occasionally mistakes happen. When a gene is unable to code amino acids correctly, the incorrect and harmful protein molecules will be formed. In this case a gene is said to have undergone mutation and the hereditary disorder will occur.

It is believed that gene therapy can resolve these shortcomings by delivering DNA remedies to faulty human cells to correct these genetic defects (Carl H et al, 2014). Regarding to the recent preclinical studies in animals models which identified molecular pathways in the pathophysiology of the heart failure, this therapy will provide hope in treating inherited and acquired cardiovascular diseases (Rincon et al, 2015).

This comparative study aims at assessing relative efficacy of the MCARD-AAV and IM-AAV injection approaches in delivering foreign genes to the host cells. It will determine the effectiveness of stranded adeno-associated virus (ssAAV) and self-complementary adeno-associated virus (scAAV) for gene transfer.



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 (Carl H et al, 2014).

The Heart failure gene therapy involves insertion of copies of a heathy peripheral gene into the chromosomes of an individual who carries a faulty allele to replace faulty genes to the heart. This practice is renowned to correct the major pathologies which are out of reach for conventional drugs and the application of gene therapy has shown some success in tackling heart failure ( Gwathmey K et al,.2011; Pleger et al 2013).


Gene selection

It is crucial to identify a gene that will match its modification with a target pathophysiology to produce expected results to avoid undesirable changes in heart failure trials that would result in an adverse cardiac adjustment (Carl H et al, 2014).

Vector selection and gene delivery

In recombinant DNA technology, various genetic engineering techniques are involved in the formulation of recombinant DNA. The restriction enzymes are used to cut a DNA molecule into small pieces. Simple microinjection, bioballistics, electro and chemical poration are among techniques used in gene delivery. It is important to understand that gene therapy is not a separate procedure; instead it is a genetic engineering method which uses these genetic tools to correct the DNA for medical purposes.

Specific virus and bacterial species are used as plasmid and vectors for preparing a recombinant DNA because of their ability to carry peripheral genes into the host cells and eventually releasing the chosen gene to replace the faulty gene.

In the preparation of a recombinant DNA, a gene is isolated from foreign DNA molecule by using restriction enzymes and then the gene is inserted into a vector to make multiple copies of the desired gene before a cloned genes being incorporated into the plasmid. An incorporated gene or DNA with the plasmid will be a recombinant DNA.

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. To achieve this, cells are exposed to chemical or to a very small electric current to make pores on the surface of the cells for the easy entry of genes.

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.


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 to merge with the host genetic material to start replication

Gene delivery

The common virus vectors used to repair and regenerate heart tissue in cardiovascular diseases in preclinical models of gene therapy and in human clinical trials are retrovirus, lentivirus, adenovirus, and adeno-associated virus (Naim C. et al, 2013; Mason D et al, 2015).

The recombinant viruses were reported to be the most successful virus vector in gene transfer. Some inflammatory responses have been known to contribute a cause of death in a clinical trial due to innate immune response during gene delivery. For that reason many preclinical studies use non- viral vectors therapeutic gene transfer to the heart. The plasmid DNA has been studied to find way for plasmid-mediated gene therapy vectors (Paul W and Paul K, 2011).

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 effective. Even if non-virus vectors are insufficient in transduction efficiency, they have no size limitation in transgene delivery systems (Carl H et al, 2014).

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. Adenoviruses are selected species and widely used in cardiovascular gene therapy clinical trials due to their wide target cell tropism and high transduction efficiency to cardiac cells (Carl H et al, 2014).

The studies portrayed the features of retrovirus that it contains single-stranded positive-sense RNA which generates double stranded DNA which uses to insert genetic sequences directly into the host genome. In contrast, it requires active mitosis for viral integration into the genome when it is used for cardiovascular gene therapy. Also it has been conveyed that the virus is incapable of transducing properly on non-dividing cell of cardiomyocytes and smooth muscle cells (Naim, C. et al,. 2013).

In vitro clinical trials, adeno-associated virus (AAV) have been used to transduce a variety of cells to respiratory epithelial cells, bone marrow and lymphocyte-derived cells. While in in vivo transduction and expression studies in the lungs, AAV has been observed in rodents and non-human primates after direct delivery to the airway surface 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).

Plasmid vectors

The simplest form of vector used to transfer the DNA into the cell reported is the plasmid. It’s essential attributes include a circular DNA molecule and stability at room temperatures, and also its ability to produce proteins with antibiotic resistance. Moreover, the plasmid consists of only one transgene but its multiple polycitrionic expression cassettes have the capacity of encoding multiple proteins regardless of their sizes, although poor gene transfer efficiency in protein production has been perceived (Paul W and Paul K, 2011).


Aim and Objectives

1. The clinical trials aimed at determining the efficiency of stranded adeno-associated virus (ssAAV) and self-complementary adeno-associated virus (scAAV) vectors for gene transfer. The studies attempted to:

i. the relative effectiveness of the molecular cardiac surgery with recirculating delivery (MCARD)-AAV, intramuscular injection (IM)-AAV injection


Molecular cardiac surgery with recirculating delivery (MCARD)

1. In this comparative study of the relative effectiveness of single stranded adeno-associated virus ( ssAAV) and self-complementary adeno-associated virus (scAAV) vectors, a molecular cardiac surgery with recirculating delivery (MCARD) were employed.

2. Ten sheep of the same age weighed between 30 to 50kg 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 of 80mmHg.

3. 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.

4. Through the isolated heart from the systematic circulation, the vector solution was recirculated through cardiac circulation for thirty minutes. It was reported that about forty eight green fluorescent protein genome copies per cell were delivered into the sheep left ventricular (LV) myocardium using MCARD technique.

5. The vectors were then allowed to remain in the heart for ten minutes before recirculating through coronary sinus for twenty minutes at the flow of 80cc per minutes with a controlled volume of 300cc and a constant delivery pressure of 80mmHg.

6. 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.

Intramuscular injection (IM)

In this same experiemnt,

1. IM injection was applied to a controlled group to deliver 1013GC of scAAV6.CMV.EGFP and

2. Necropsy for four weeks.

3. On the wall of LV the injection spacing measured about 1cm

4. The spacemen from all parts of the heart were taken for examination through direct fluorescence and GFP


The Microsoft Excel using unpaired Student’s t-tests was applied to analyse data for recombinant vectors scAAV and ssAAV.


Comparatively, with the MCARD technique, the effeteness of scAAV6 is greater than ssAAV9 greater than ssAAV6. The direct fluorescence photography and high-power photomicrographs of the cross-section of the LV showed a strong green fluorescent protein expression (GFP) in the MCARD/scAAV6 group while a weak gene expression in the MCARD/ssAAV9 and MCARD/ssAAV6. This indicated that that nearly all cardiomyocytes in the LV anterior wall were transduced in MCARD/scAAV6 (White, J. 2011).

Significant transduction efficiency was indicated on MCARD/scAAV6 (White et al, 2011). The IM/scAAV6 presented an inconsistent gene expression with substantial values of transduction efficient. Also, the results specified that considerable hepatic gene expression of IM/scAAV6 with a 10-fold lower vector dose of 1013GC was generated (White et al, 2011).


The clinical trials conducted from large animal models using AAV- mediated gene indicated that the scAAV6 is more effective in cardiac myocyte transduction for large animals and expected to give remarkable results with human trials than its counterpart ssAAV6 and ssAAV9, The MCARD delivery system is more suitable if it is used with scAAV6 (White et al, 2011).

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. The intravenous injection delivery methods intramuscular (IM) injection for cardiac organ-specific approach, illustrated as ineffective and the need for an alternative cardiac gene transfer models with excellent clinically translatable quality was anticipated (White et al, 2011).

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