1. INTRODUCTION:

Before understanding the non viral gene therapy we should first be clear of the terms genes and gene therapy.

What are actually genes?

· As we all know, genes, which are carried on chromosomes, are the basic physical and functional units of heredity.

· Genes are the specific sequences of bases of DNA that encode instructions on how to make proteins and these proteins perform most of the life functions.

· Therefore genes are playing a mojor role in every function of human body.

· Whenever any defect or mutations occur in a gene, encoded proteins are unable to carry out their normal functions, and genetic disorders result.

Definition of Gene therapy:

Gene therapy is a technique for correcting defective gene responsible for disease development. Gene therapy attempts to treat the disease in an individual by administration of DNA rather than a drug.

Purpose of gene therapy:

· Standard drug therapy is effective in treating the symptoms of, the disorder, but a patient is required to take the drugs for an extended period of time and also there may be serious side effects.

· However, the patient may be cured with few negative symptoms if the treatment can be targeted directly, at the specific cause of the disease.

· Gene therapy has given an opportunity to fight the cause the disease, rather than its symptoms.

Because only the somatic cells and not the germ cells are targets to these efforts , gene transfer, affects only individual under treatment and not their offspring.

Gene Medicines used in gene therapy consists of a therapeutic gene + delivery system (vector). Ideal gene delivery system/vector should be bio degradable, non toxic, non immunogenic, stable during storage and after administration and able to access to target cells and suitable for efficient gene expression. [1]

Mechanism of operation of gene therapy:

· Target cells such as patient’s liver/lung cells are infected with the vector. The vector then unloads its genetic material containing therapeutic gene into target cell.

· The generation of a functional protein product restores the target cell to normal state. [2]

The delivery System/Vector can be Either Viral/Non-Viral

When the delivery vector used for Gene therapy is non viral we can call this as “non viral gene therapy”

2. Viral/Non-Viral Vector:

A number of methods have been developed for transecting eukaryotic cells[3]. These can be viral or non viral vectors.

These vectors primarily differ in their assembling process:

A VIRAL vector is assembled in the cell, whereas NON-VIRAL is constructed in a test tube.

Viral vectors currently in use are adenovirus, retrovirus, adeno associated virus and others [4]. Viral vectors are highly efficient in transducing cells. However, due to following disadvantages of viral mediated gene therapy non viral delivery system has become an attractive alternative.

These are:

· First of all, because of the safety concerns, there is always a fear that viral vector once inside the patient, may recover its ability to cause the disease.

· Retroviral vectors function as single hit gene transfer system.

· Retroviral vectors can only infect dividing cells.

· Difficulty in targeting to certain cells.

· They possess a low DNA transfer.

· Size limitation of DNA constructs.

· Production of high titer viral vectors is difficult.

· Requirement of the packaging cell line.

· Possibility of insertional mutation.

Since viral vectors are posing many disadvantages we are going to study the depth of non viral gene therapy i.e Gene Therapy using non viral vectors

3. Non Viral vector Mediated Gene therapy:

a. Naked plasmid DNA:

The simplest approach to non viral delivery system is direct gene transfer with naked plasmid DNA.

The level of gene expression although relatively low, was sufficient for vaccination. It is seen that level of gene expression can be significantly increased by improving the method of injection. The use of myotoxic agents also improved the gene expression. Furthermore , it has been observed that polymers that interact with DNA such as PVP can improve the efficiency of gene therapy by direct intra-muscular injection. These polymers protect DNA from enzymatic degradation They may also serve as depot system allowing DNA to be released over a prolonged period of time.

Currently, intra muscular injection of plasmid DNA is also being used to express pharmacologically active molecules such as erythropoietin and leptin with encouraging results.

Intra vascular injection of plasmid DNA results in limited gene expression in all major organs. However, intravascular injection of naked DNA under high hydrostatic pressure leads to high level of foreign expression.

b. Electroporation:

In this process , DNA is transferred into cells in suspension by applying short electrical impulses of high field strength that induces transmembrane potential.

Induced potential of sufficient magnitude will trigger a dielectric breakdown of the membrane and creates pores in the cell membrane thereby facilitating entry of DNA into the cells. It was observed that molecules did not pass through the membrane gained intra cellular access after the cells were subjected to electric fields.

Cell electropermeabilization can be achieved locally in vivo by means of simple electrodes [5]. The pulse required for an efficient transfer of the DNA is generated by discharging a capacitor across the electrodes from a specially generated Electroporation chamber. The generated pulse may be either a high voltage (1.5 kV) rectangular wave pulse for a short duration or a low voltage (350 V) pulse for a longer duration.

DNA transfer is carried out by suspending the protoplast containing vector DNA in an ionic solution between the electrodes. Using Electroporation method, successful transfer of genes was achieved with the protoplasts of tobacco, maize, rice, etc.

Relevant clinical application , electro chemotherapy , investigated by using cell electropermeabilization, to facilitate the cellular entry of hydrophilic anticancer agents such as bleomycin.

c. Gene gun:

Gene delivery using this technology is achieved by a physical force.

A compressed shock wave of helium gas is created, accelerating DNA- coated gold particles of varying sizes to high speed. This force is often of sufficient momentum to penetrate substantial physical barriers such as plant cell walls, cell membranes and stratum corneum of mammalian epidermis.

This method has been successfully used to deliver DNA in vivo into liver, skin, pancreas, muscle, spleen and tumours.

Major advantage of this method is that it does not require use of complex delivery system.

Major application of gene gun is genetic vaccination . Gene gun vaccination has shown to be effective in eliciting immune responses.

Gene gun method may be employed in combination with intramuscular injection of DNA to achieve an optimal vaccination effect.

d. Metallic nanoparticles:

The advantages of using metallic nanoparticles are that they are not susceptible to microbial attack , there is no swelling with change in ph , they can be prepared at low temperature, some these can be used as biomaterials and are non toxic and highly biocompatible, they exhibit excellent storage stability , and preparation of these nanoparticles is cost effective.

Divalent metal ions like Calcium, Magnesium, Manganese, Barium, Strontium, have been tried as carriers for gene delivery

One of the conventional methods which have been attempted for the gene transfer is the calcium phosphate precipitation method, which was first described by Graham and colleagues. In this procedure, water insoluble calcium phosphate precipitate which can bind to DNA is formed by reacting calcium chloride with sodium phosphate. When added to a cell monolayer, this insoluble precipitate is taken up by cells in a calcium ion dependent manner. This complex is broken down, wherein the DNA is released, which can be incorpated in host cell genome under suitable circumstances. The limitation of this method of gene delivery was the transfection efficiency, which was only 10% compared to other methods of gene delivery such as viral delivery systems.

Recently, Kam W. Leong and collegues developed multifunction bimetallic nanorods for gene delivery. These Au/Ni bimetallic nanorods could simultaneously bind compacted DNA plasmids and targeting ligands in a spatially defined manner. This approach allowed precise control of composition, size and malfunctionality of the gene delivery system.

Transfection experiments which were performed in vitro and in vivo showed romising results that suggested potential in genetic vaccination applications .[6,7]

e. Oligonucleotides:

Introduction of a potent gene inhibitor is equally important to that of introducing a missing gene or to correct a mutant gene. These are especially required in cases like neoplastic infections.

Oligonucleotides are short strands of nucleic acid which bind to DNA, mRNA, or extracellular proteins in a complimentary fashion and arrest protein synthesis by inhibiting transcription or translation

Three basic approaches have been explored in this area.

1. Antisense oligonuleotides bind to the mRNA and block translation. This approach was used to inhibit translation of HIV.

2. Triple helix forming oligonucleotides which binds in a sequence specific masses in the major groove of the duplex DNA.

3. Oligonucleotides which bind to extracellur proteins and inhibit their enzymatic activity.

The rapid degradation in vivo by 3’ exonuclease digestion limits the use of natural oligodeoxynucleotides. Therefore, nuclease resistant oligonucleotides were synthesized modifying the phosphodiester backbone. Among them , the widely investigated are phosphorothioates (anionic) and methylphosphonate (non ionic)

The major problem of oligonuleotide delivery is their limited access to the intracellular (and intranulear) space . Chemical means to overcome cell exclusion eg: the design of more lipophilic compounds, are limited as they may compromise the selectivity and binding affinity for the intended target DNA or RNA.

Both cellular as well as intranuclear delivery of an antisnse oligonucleotide hybridized to ICAM-1 was found to be increased in the presence of a cationic liposome carrier. [8]

f. Electronic pulse delivery (EPD):

The EPD technology can be used as effective, efficient and safe means of delivery system. The target cells suitable for EPD delivery include haematopoietic stem cells, hepatocytes, fibroblasts and myoblasts.

EPD is a sophisticated method which subjects the targetted cells to a precisely controlled pulses of electric field in a computer controlled molecular transfer system.

EPD system consists of a controller and a reaction chamber. The controller, driven by an EPD computer accurately controlls the output of electronic pulses which in turn are controlled by different parameters. The reaction chamber where the DNA transfer takes place holds the target cells and therapeutic gene as a mixture.The target cells and the transfer material are placed in a reaction chamber with a movable conductive probe isolated from the cewll/DNA solution.

EPD has been sometimes confused with electroporation procedure [9]. In electroporation the target cells are damaged after which the molecular transfer takes place. Furthermore, electroporation often applies a significant electric current through the cells. In contrast, EPD technology limits electric current and needs no contact by electrode to the mixture of the genes and the target cells.

Advantages of EPD technology

1. Using this technology, large molecules can be transferred into the target cells (eg: DNA constructs~ 15kb).

2. EPD inserts only the therapeutic genes into the target cells without the involvement of any other molecule.

3. EPD technology has a remarkable efficiency (ranging 80-90%).

4. It has no toxic effects on the target cells and is completely free from potential hazards.

5. It can be operated in a batch or continous process.

6. The time taken by this method for transfer is very short.

g. Aquasomes:

A self assembled molecular carrier “Aquasomes” formed with preformed carbon ceramic nanoparticles and self assembled carbon phosphate dehydrate particles with a glassy carbohydrate coating can be studied for the delivery of the genes.

In vitro studies have been carried out by immobilizing DNAase, a therapeutic enzyme used in the treatment of cystic fibrosis, on to a ceramic carbon nanocrystalline particulate core coated with pyridoxal-5-pyrophosphate. A marked retention of biological activity was observed with surface immobilized DNAase on the solid phase of a colloidal calcium phosphate nanoparticle coated with a polyhydroxyl oligomeric film.

Kossovsky (1996) has envisioned a model for how the synthetic product of self assembling chemistry using non covalent forces comprising a ceramic nanocrystalline core with a polyhydroxy oloigomeric film coating will appear. [10]

h. Polymeric delivery system:

Polymers display striking advantages as they can specifically be tailored for desired application. They also demonstrate adequate safety levels.

Cationic polymers are commonly used in gene delivery because they easily complex with anionic DNA molecules. Polyplexes which are polymer - DNA complexes, can be used to deliver DNA into cells. Due to the electrostatic interaction, of cationic polymers with anionic DNA a positively charged complex is generated. This cationic polyplex then interacts with negatively charged cell surface to improve DNA uptake. On the other hand, presence of positive charges on polymer can also promote non-specific interactions with serum proteins. To prevent this, hydrophilic groups like polyethylene glycol have been conjugated to the transfection polyplexes. However, pegylation reduces DNA binding capacity of the polymer and it sterically hinders the interaction of copolymer/DNA complexes with the target cells.

In vitro transfection efficiencies of polyethylenimine (PEI) with PEG were found to considerably lower than corresponding non-modified polymers.

Some of the advantages of polymeric delivery systems are that they have versatile physicochemical properties which can be easily manipulated, polymeric matrices with various designs can be made, feasibly with low cost industrial manufacturing and agents such as transferring, folates, antibodies, or sugars like mannose and galactose could be incorporated for tissue targeting.

Polymeric delivery systems have their advantages but are not completely ideal. Their limitations include low transfection capabilities, control of molecular weight distributions, dispersities of polyplexes, issues with quality control and inherent potent pharmacological problems of some polymers which make them unfavourable for human use.

Commonly used polymers are PEI, PLL, PLGA and dendrimers. [11]

i. Lipidic gene delivery system:

Many different lipidic systems have been developed as vectors for gene transfer in vitro and in vivo such as liposomes, micelles, emulsions, and other organized structures of lipids.

Early studies used neutral and anionic liposomes. Plasmid DNA is encapsulated inside the vesicles and the structures of liposome’s remain unchanged.

Cationic liposomes interact with DNA through charge during complex formation [12]. The resulting complexes may bear little resemblance to the starting liposomes. Thus cationic liposome/DNA complex is somewhat of a misnomer. A term lipoplex has been recommended.

Recently several new types of lipidic vectors have been developed to improve the efficiency of in vivo gene delivery. One of these vectors has a unique structure with lipid bilayers. This type of vector is called lipopolyplex

4. Application of non viral:

Gene Therapy:

Human beings suffer from more than 5000 different diseases caused by the single gene mutation. Also many disorders seen to have the genetic components. Here, the “non viral gene therapy” finds its application in the treatment of these disorders.

Diseases focused by the non viral gene therapy include:

1. Cystic fibrosis.

2. Thalassaemia.

3. Malignant melonoma.

4. Pediatric AML.

5. Neuroblastoma.

6. Adenosine deaminase deficiency SCID.

7. Hemophilia B.

8. Chronic myleogenous leukemia.

9. Hepatitis B.

10. Hypercholestolemia.

11. Cardiovascular diseases.

12. Phenyl ketonuria.

13. Insulin dependent diabetes mellitus.

14. Cancer.

15. AIDS.

The ultimate goal is to replace a defective gene with a normal one via targeted insertion into the genome by homologous recombination.

Non viral gene therapy will have a major impact on the health care of our population only when the non viral vectors are developed that can safely and efficiently be injected directly into the patients as drugs. Non viral vectors need to be engineered that will target specific cell types, insert their genetic information’s to a safe site in the genome, and be regulated by normal physiological signals.

Non viral gene delivery strategies have been fairly successful in cell culture systems and animal models.

Two types of the clinical trials are being conducted in humans using these non viral vectors- gene marking and therapeutic studies [13, 14]. Gene marking involves areas in which potential therapeutic application are involved such as hematopoietic stem cells, T cells, etc, which are removed from the patient, cultured and transfected with non viral vectors encoding gene markers which are again reintroduced into the patient. The presences of gene markers are examined time to time by recovering the cells and are observed for the presence of marker gene. Cell marking studies are used to detect residual cancer cells in the marrow infused patients during autologous bone marrow transplantation. Therapeutic trials, are aimed at transfer of therapeutic genes into the patients for the treatment of specific genetic disorders and cancer.

This field of non viral gene therapy is still at its infancy and relevant but has witnessed a rapid growth towards the end of this century and hopefully the progress would continue.

CONCLUSION:

In conclusion, it can be stated that the opportunities for gene delivery using non-viral delivery systems seem to be abundant and the ultimate goal of providing tailor-made individualized medicines seems to be a very bright prospect indeed.

Nevertheless, it is a fact that no current synthetic gene delivery has all the ideal properties for efficient gene delivery.

Non-viral DNA-based therapeutics is still in a nascent stage. Lessons from viruses have immensely helped in improving synthetic delivery systems.

By learning from nature and understanding and incorpating the extremely efficient mechanisms of infection , the future gene delivery systems will be virus like in function by non-viral in nature.

REFERENCES:

1. Textbook of pharmaceutical biotechnology Vyas and Dixit chapter Drug Delivery System in Gene therapy.

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6. Metallic nano particles Dragomir Minela (pdf) , University of Nova Gonica , doctoral study, programme physics).

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14. Morishital, et al. Phase I/IIa clinical trial of therapeutic angiogenesis using hepatocyte growth factor gene transfer to treat critical limb ischemia. Arterioscler Thromb Vasc Biol. 2011;31:713-20.

Received on 21.10.2020 Modified on 09.11.2020

Research J. Science and Tech. 2021; 13(1):13-22.

DOI: 10.5958/2349-2988.2021.00003.6