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Concepts of Gene Therapy

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Genes - the units of heredity

“There can be little doubt that the idea of ‘the gene’ has been the central organizing theme of twentieth century biology” philosopher and biochemist Lenny Moss claimed in 2003. A gene is the basic unit of heredity in a living organism. Genes hold the information to build and maintain their cells and pass genetic traits to offspring. In general terms, a gene is a segment of nucleic acid that, taken as a whole, specifies a trait. The biological entity responsible for defining traits was termed a gene, but the biological basis for inheritance remained unknown until DNA (Deoxyribonucleic acid) was identified as the genetic material in the 1940s. In cells, a gene is a portion of DNA that contains both "coding" sequences that determine what the gene does, and "non-coding" sequences that determine when the gene is active (expressed). When a gene is active, the coding and non-coding sequences are copied in a process called transcription, producing an RNA (Ribonucleic acid) copy of the gene's information. RNA can then direct synthesis of proteins via the genetic code. In other cases, the RNA is used directly, for example, as part of the ribosome. The molecules resulting from gene expression, whether RNA or protein, are known as gene products, and are responsible for the development and functioning of all living things. Every cell requires a host of genes that act as blueprints of all the proteins essential for its proper functioning.

DNA is a linear polymer of deoxynucleotide monomers. Chemically speaking, it has a double helical structure of two polynucleotide chains held together by hydrogen bonds between the complementary base pairs of the nucleotide strands. Each nucleotide in DNA is comprised of three components, a heterocyclic base, a sugar (2-deoxyribose) and a phosphate group. The nucleotides in a polynucleotide chain are connected through phosphodiester bonds. The nitrogenous bases are of two types, namely, purine based adenine (A) & guanine (G), and pyrimidine based cytosine (C) & thymine (T). In DNA, base pairs form only between A & T and G & C and thus the base sequence of each single strand can be deduced from that of its complementary strand.

Gene Therapy: Molecular Bandage?

Gene therapy is believed by many to be the therapy of the twenty first century because it aims to eradicate cause rather than symptoms of diseases by delivering a normal functioning copy of the mutated gene and its associated regulatory elements into the cell nucleus (1-3). It is a technique whereby an absent or a faulty gene is replaced by a working gene, so that the body can make the correct enzyme or protein and consequently eliminate the root cause of the disease. A potential approach for treating genetic disorders is gene therapy. The most likely candidates for future gene therapy trials will be single gene disorders like, cystic fibrosis, hemophilia, familial hypercholesterolemia, ADA deficiency, Gaucher disease, alpha-1-antitrypsin deficiency etc. Apart from these monogenic disorders, gene therapy also holds the potential of treating acquired diseases such as cancer, by inhibiting oncogene expression or by restoring tumor suppressor genes or through immunomodulation (i.e. by increasing immune response to tumor antigens). Cardiovascular diseases too remain as one of the most “promising” targets for gene therapy because of the ready accessibility of the vascular system for gene transfer (4).

Types of Gene Therapy:

Depending on the type of cells into which genes are transferred a process popularly known as “transfection”. Gene therapy can be broadly classified into two types: Somatic cell & Germline gene therapy.

Somatic Cell gene therapy: This type of therapy involves the transfection of somatic (non-reproductive) cells especially of those tissues in which expression of the concerned gene is critical for health. Expression of the introduced gene relieves/eliminates symptoms of the disorder, but this effect is not heritable.

Germline gene therapy: This type of therapy involves gene transfer into reproductive cells (egg or sperm cells). Here germ cells are modified by the introduction of functional genes, which are ordinarily integrated into their genomes. It would change the genetic pool of the entire human species, and future generations would have to live with that change.

Depending upon the method of correcting the faulty gene, gene therapy can be classified into the following categories:

Gene augmentation therapy: This type of therapy is the most appropriate one for the treatment of inherited diseases caused by the loss of a functional gene. It involves supplementing the body cells with a functional copy of the lost gene so that the missing protein is expressed at sufficient levels in the body. It is only suitable if the pathogenic effects of the disease are reversible.

Gene inhibition therapy: Its aim is to introduce a gene whose product inhibits the expression of the pathogenic gene or interferes with the activity of its product (5).

Suicide gene therapy: This method is best suited for a disease like cancer where the aim is to eliminate a certain population of cells. It involves the transfection of such cells with a suicide gene, whose product is toxic. The suicide genes should be appropriately targeted to avoid widespread cell death (6).

Depending upon the mode of delivering genes into a patient’s body, gene therapy can be classified into the following two types:

Ex vivo gene therapy: In ex vivo gene therapy, gene transfer occurs outside the patient’s body. This is again sub-divided into two types viz, Autologous & Non-autologous. Autologous gene therapy involves the transfection of cells derived from the patient followed by the re-introduction of these cells into the patient’s body. Non-autologous gene therapy involves the transfection of cells not derived from the patient’s body.

In vivo gene therapy: In this method the therapeutic gene is directly introduced into the body by injection or by inhalation with the help of a suitably designed vector.

Gene Delivery Vectors: Key to Success in Gene therapy

Gene therapy, as a novel therapeutic modality, holds enormous promise for the treatment of a multitude of human diseases. However, till date it has failed regrettably in spite of

more than 1500 clinical trials completed or currently underway around the world. The primary reason for the failure of the clinical success of gene therapy is the lack of efficient gene delivery agents, commonly referred to as transfection vectors. However, since the biological cell surfaces are negatively charged (due to the presence of glycoproteins and glycolipids containing negatively charged sialic acid residues on cell surface), spontaneous entry of polyanionic naked genes (DNA) into body cells is an inefficient process. Hence “transfer vehicle” or a “vector” in needed to condense the macromolecular DNA and to help it in crossing the plasma membrane barrier. Again delivery of therapeutic DNA to the desired body tissue is important to overcome adverse affects. In other words, the problems of developing clinically viable gene therapy methods and designing safe & efficient gene delivery reagents are inseparable: shortcomings in one is going to adversely affect the success of the other. Hence, realization of the full potential of gene therapy will depend, in a major way, on the future development of safe and efficient gene delivery vectors.

The Ideal Vector!!! A “perfect” or an “ideal” vector would resemble a traditional pharmaceutical and should have the following characteristics: (a) should be capable of efficiently delivering to its target an expression cassette carrying one or more genes of the size suitable for clinical application, (b) must not elicit an immune response, (c) should not induce inflammation and thus be safe for the recipient, (d) can be produced in bulk at an acceptable cost with reproducibility, (e) should be stable on storage, and finally, it should express the gene (or genes) it carries for as long as required in a strictly regulated manner. No single vector currently available has all these desired properties and each vector presently in use has its own pros and cons. However, it is important to realize that there cannot be a “universal” vector, optimally useful for all gene therapy applications. This is due to the fact that each disease will have a unique set of technical requirements, and the “perfect” vector for a specific disease should be optimized in accordance with these requirements. For example, some diseases will require local delivery of the transgene (e.g., ischemia, retinitis pigmentosa, parkinson’s disease, etc.) while others likecancer and atherosclerosis necessitate systemic delivery. In some cases, only a transient, short-lived gene expression will be needed (e.g., therapeutic angiogenesis, cancer) while in monogenic disorders, such as familial hypercholesterolemia, hemophilia and SCID a long term (sometimes life long) gene expression is mandatory (1). The future clinical success of gene therapy will certainly depend on the uphill task of designing “tailor-made” vector systems for the treatment of specific diseases.

The efforts to design a “perfect vehicle” for the membrane-impermeable DNA have so far led to the development of many methods based on the principles of biology (viral vectors), physics (microinjection, electroporation, particle bombardment, hydrostatic pressure, and ultrasound) and chemistry (synthetic vectors like cationic lipids & polymers). Each of these methods has its intrinsic advantages and disadvantages.

Viral Vectors: Nature’s Own Infecting Vehicles

Viruses have evolved specific mechanisms through the course of evolution to deliver their genetic material into host cells and then hijack the cell’s biosynthetic machinery to produce new viral particles (7). Thus, owing to their natural ability to infect cells, they can be used as vectors in gene therapy by replacing the genes that are essential for replication phase of their life cycle with the therapeutic genes of interest. Majority of the clinical trials currently underway around the world are based on the use of mainly five categories of viruses, namely, retrovirus, adenovirus, adeno-associated virus, lentivirus and herpes simplex virus.

Retroviruses: These are a class of enveloped viruses containing a single stranded RNA molecule (approximately 10 kb). Inside the host cell, the RNA is reverse transcribed into double stranded DNA, which in turn integrates into the host genome and is expressed as viral proteins (8). These are the most promising and widely used viral vectors used for gene therapy applications to date.

Advantages: Rapidly dividing cancer cells can be targeted by using these viruses. Enters into cells efficiently and gives long lasting gene expression due to stable integration.

Disadvantages: Only infects dividing cells, capable of producing tumorigenic mutagenesis due to random integration, unable to deliver larger genomic sequences. Again, it can insert the genetic material of the virus in any arbitrary position in the genome of the host- it randomly shoves the genetic material into a chromosome.

Adenoviruses: These are the second most commonly used viruses for gene delivery. They carry a double stranded linear DNA chromosome of approximately 36 kb. Unlike retroviruses, adenoviruses deliver their genetic payload outside the chromosome and are thus less likely to disrupt the cell’s genome (9). But it is immunogenic and may cause inflammation and tissue damage.

Adeno-associated viruses (AAV): They contain a single stranded DNA of approximately 4.7 kb surrounded by a protein coat (10) and can integrate at a specific site in human chromosome 19. AAV does not contain any viral genes and contains only the therapeutic gene and it does not integrate into the genome. It requires co-infection with a “helper” adenovirus for propagation. The advantage of AAV is that it is a non-pathogenic virus but the size for the exogenous DNA it can deliver is limited due to its smaller genome. The difficulty in large scale production is an additional disadvantage.

Envelope protein pseudo typing of viral vectors: The envelope proteins on each of these viruses bind to cell-surface molecules make facile attachment to and entry into a susceptible cell. The potential for off-target cell modification would be limited, and many concerns from the medical community would be alleviated.

Although viruses are the most efficient gene transfer vehicles available to date, their widespread clinical success has been impeded by the following major drawbacks:

(a) Viruses are notorious for eliciting an immune response which, apart from posing a serious threat to the host, also makes a second dose of the same viral vector ineffective due to the production of high level of antibodies against the viral structural components following its initial administration. In 1999, the death of 18-year old Jesse Gelsinger, undergoing gene therapy for ornithine transcarboxylase deficiency, was believed to be triggered by a severe immune response to the adenoviral vector used.

(b) Size restriction on the genetic material that can be encapsulated within the viral particles.

(c) Possibility of random integration into host genome leading to the risk of inducing tumorigenic mutations

(d) Purification of recombinant vector, verifying the sequence, transfecting the packaging cells, isolating and titering the transgenic virus and finally transducing the target cells are time consuming and labor intensive steps.

Collectively, all of these complications associated with the use of viral vectors have prompted researchers around the world to develop artificial non-viral transfection vectors.

Non-Viral Methods:

Although the gene transfer efficacies of the viral vectors are unmatched till date, the above mentioned serious immunogenic concerns associated with their use have led to the development of non-viral methods for gene therapy. The non-viral vectors offer many advantages over their viral counterparts including significantly lower toxicity and immunogenicity, size independent transfer of nucleic acids, very low frequency of integration, relative ease of large-scale production, simpler quality control and substantially easier pharmaceutical and regulatory requirements. The non-viral transfection methods could be broadly classified into two types: Physical methods and Chemical methods.

Physical Methods for Gene Delivery: Physical methods involve the direct introduction of genes into the target cells or tissues thereby avoiding the introduction of any foreign substance like a virus or a synthetic vector. Hence, no serious immunogenic concerns are associated with their application. The required genes are inserted via microinjection, electroporation or particle bombardment (gene gun).

Microinjection: In this method, the DNA is directly injected into the nuclei of target cells using a fine glass needle under microscope. Although this method is seductively simple, it is difficult to apply clinically. While this method of gene transfer is nearly 100% efficient, it is laborious and time-consuming, typically allowing only a few hundred cells (< 500) to be transfected per experiment (11).

Electroporation: This technique involves the perturbation of the cell membrane by an electric pulse for a few microseconds resulting in the formation of transient pores thereby allowing the exogenous DNA to enter the cell cytoplasm. Although there is no limit on the size of DNA that could be delivered via electroporation, the gene transfer efficiency is low and there is high incidence of cell death (12).

Gene Gun: In this method, plasmid DNA is coated onto micron size tungsten or gold micro particles and then propelled into cells using either electrostatic force or gas (Helium) pressure. The high velocity results in some DNA being trapped by a few cells and then it may be expressed at sufficient levels. This technique is fast, simple and safe and has been successfully employed to deliver nucleic acids to cultured cells as well as to cells in vivo especially gene transfer to skin (13) and superficial wounds.

Chemical Methods of Gene Delivery:

DEAE-Dextran: Diethylaminoethyl-dextran (DEAE-dextran) is a polycationic derivative of the carbohydrate polymer, dextran and was one of the first chemical reagents used for transfer of nucleic acids into mammalian cells (14). Owing to its positive charge, DEAE-dextran forms an electrostatic complex with the polyanionic DNA. This technique of delivering genes into cells is simple, reproducible and cost effective. However, it could prove toxic to the target cells especially when DMSO or glycerol is used as a supplementary chemical shock to increase gene transfer efficiency. Secondly, this method is not generally useful for stable transfection studies that require integration of the transferred DNA into the chromosome. A major disadvantage of this method is its ability to transfect a limited variety of cells, e.g. phagocytic cells.

Calcium Phosphate: Calcium phosphate co-precipitation method for DNA delivery was first introduced by Graham and Van Der Eb in 1972 (15). This technique involves mixing of DNA with calcium chloride and then carefully adding this mixture to a phosphate buffered saline solution followed by incubation at room temperature. The finely divided DNA containing precipitate thus formed is taken up by the cells via endocytosis or phagocytosis. The main advantages of the calcium phosphate method are its simplicity, low cost, and its applicability to a wide variety of cell types. Moreover, it could be used for transient as well as stable transfection studies. The main drawbacks of the technique involve its sensitivity to slight changes in buffer salt concentrations, temperature, and pH, as well as its relatively poor transfection efficiency compared to newer transfection methods.

Cationic Polymers: A wide range of organic polymers has been used for gene transfection, the most popular being polylysine & polyethylenimine (PEI) (16). These have a high cationic charge density that condenses DNA into positively charged particles capable of interacting with anionic cell surfaces and entering cells via endocytosis. PEI also exhibits extensive buffering capacity across a wide range of pH which protects DNA inside the endosome from degradation via endosomal swelling and rupture. Dendrimers represent another class of polymers used for gene delivery. They consist of three-dimensional, bifurcated, branched structures called dendrons. The polyamidoamine (PAMAM) family of dendrimers has been shown to be very useful for transfection (17).

Cationic Liposomes: “The Artificial Fat Bubbles”

Liposomes, in general, have long been viewed as bio-compatible drug/gene delivery reagents owing to their structural similarity to cell membranes. They are spherical bilayers composed of individual lipids enclosing a watery interior. Each lipid possesses a hydrophilic head group attached via a linker to a large hydrophobic domain. When exposed to an aqueous environment, these amphiphiles spontaneously form large spherical structures known as liposomes above a certain critical vesicular concentration (CVC). Within the sphere, lipids are arranged back-to-back in bilayers with the polar hydrophilic group facing outwards shielding the hydrophobic domain from the aqueous solution. Liposomes may be unilamellar (composed of a single bilayer) or multilamellar (composed of many concentric bilayers). The multilamellar liposome (MLV) upon sonication followed by repeated extrusion through polycarbonate membranes of defined pore size assume the size of small unilamellar vesicle (SUV, 30-100 nm) or large unilamellar vesicle (LUV, 150-250 nm) (Figure 1).


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