GENE THERAPY TARGETING AND DELIVERY

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The efficient delivery of therapeutic proteins or DNA into specific cells or tissues of an organism to correct a mutant gene is paramount to the success of any gene therapy procedure. The inserted therapeutic DNA or desired gene must be continuously expressed in vivo at appropriate physiological level in order to correct the mutated gene and thus restore the normal genetic composition of the affected individual. Gene therapy generally involves the delivery of one or more desired genes and/or proteins into the body so that the inserted gene will spur the synthesis of missing protein or enzymes (whose lack caused the genetic disorder) in the affected individual. But this process of gene delivery is often the most difficult aspect of gene therapy because some of the available methods of delivering desired genes into the body have some deficiencies. For example, some of the vectors used for gene delivery are viral particles. These viruses can become pathogenic to the host receiving the gene insert as well as cause several other adverse immunological and inflammatory reactions in the body. Direct delivery (i.e. in vivo gene therapy) and cell-based delivery (i.e. ex vivo gene therapy) are usually the two main approaches used for delivering therapeutic gene or DNA meant to repair a faulty gene in the body (Figure 1). In direct delivery or in vivo gene therapy, desired genes are delivered into the body by means of viral vectors. But non-viral vectors as shall be seen later in this section are required for the delivery of desired genes or DNA in cell-based delivery or ex vivo gene therapy techniques. 

IN VIVO DELIVERY

In vivo delivery is the gene therapy technique that delivers DNA, RNA or therapeutic protein directly into the cell or tissue of an organism. Desired or therapeutic genes are delivered in this technique through the genetic transfer method known as transduction. Transduction is the genetic process of transferring genes of interest from one cell to another using viruses or bacteriophages. It is usually achieved using viral vectors. The delivery of desired gene into the cell of the recipient host using this technique is specific. In vivo gene therapy is the most feasible strategy for delivering desired genes or DNA into the cell or tissues. The cells of the body targeted by in vivo gene therapy include those of the brain, lungs, blood vessels, liver and muscle cells. In vivo gene delivery to a particular organ of the body is usually achieved through the catheterization of that organ by surgical approaches that employ invasive medical devices and other advanced medical techniques that ensure that the desired gene is delivered properly. Some in vivo gene therapy techniques may also be guided by computerized techniques that ensure the delivery of the desired genes or therapeutic DNA to target host cells where they are expected to efficiently repair a mutant DNA or gene.

Figure 1. Illustration of approaches or techniques used to achieve gene therapy. Gene therapy techniques is usually achieved via in vivo techniques (in which the therapeutic gene or DNA is directly injected into the body) and ex vivo techniques (in which cells are genetically altered in vitro and then injected back into the body). When cells are extracted or removed from the body of genetic disorder patients and genetically modified in vitro and then transferred back into the same body, the process is known as ex vivo gene therapy. But in the in vivo gene therapy techniques, therapeutic DNA or genes are inserted directly into the body of genetic disorder patients using viral vectors. Photo courtesy: Proceedings of the Royal Society.

Though finding an efficient delivery system for gene therapy may still not be possible; viruses are the best vectors for in vivo gene therapy because viral particles or viruses are efficient in transducing (i.e. transforming) host cells. This is because viruses have a history of penetrating host cells and transforming same by inserting their own genome into target host cells, a phenomenon that gives them an edge over other means of delivering therapeutic genes.

Most of the viruses used for in vivo gene therapy are “harmless viruses” that have been attenuated and made to lose their virulence so that they do not become pathogenic when used as vectors to deliver therapeutic genes to host cells.Examples of viruses considered for in vivo gene therapy include adenoviruses (adenoviral vectors), retroviruses (retroviral vectors) and adeno-associated viral vectors. The virulent genes of these viruses have been altered and made apathogenic. They are used to deliver therapeutic DNA or genes into a target host cell. The viral vector can be given intravenously or injected directly into a specific tissue in the host body, where it is then taken up by individual target cells that will be transformed and made to start secreting the correct type of proteins or enzymes they encode. However, the use of viruses for in vivo gene therapy has some safety concerns that limit their usage for gene delivery. The viral particles used for in vivo gene therapy (though apathogenic or non-virulent) may become pathogenic on entering the host’s body. Also, the virus may cause other adverse reactions in the individual taking the therapy.

EX VIVO DELIVERY

 Ex vivo delivery is the gene therapy technique in which cells extracted from a patient are genetically engineered in vitro and then re-introduced into the host’s body. Such extracted cells must be capable of survival outside the host’s body and re-implantation into the body (after they must have been transformed) before they can qualify to be used for ex vivo gene therapy. Desired genes or proteins known as transgenes (i.e. genetically modified genes) are inserted into the body where they are expected to bring out the desired response after being transformed in vitro. The cells of the body usually targeted by the ex vivo gene therapy technique include cells of the bone marrow, muscles, liver and fibroblasts. Ex vivo gene therapy employ non-viral vectors or techniques to deliver therapeutic genes or DNA into the cells of a host. They are normally easy to use. Unlike the viral vectors that is normally associated with adverse host cell response; non-viral vectors used for ex vivo gene techniques are not capable of stimulating the host immune response. Ex vivo gene therapy delivers desired genes that have been extracted and modified outside the body. They are usually not as specific as the in vivo delivery method that uses viruses as their vector.

Ex vivo gene therapy is generally achieved through the process of transfection which is the in vitro introduction of proteins or DNA into cells. Non-viral techniques used for ex vivo gene therapy include lipofection, microinjection, electroporation, the use of naked DNA or plasmids and the use of calcium phosphate precipitate and liposome’s. Though much safer than the use of viruses, non-viral vectors are usually inefficient at transforming genes and some of the techniques or approaches used are not specific in action. Non-viral vectors or delivery systems as exemplified in this section are fast becoming the alternatives to the use of viral vectors in delivering therapeutic genes or DNA into hosts cells due to the safety and health risks or concerns of the later (i.e. the viral vectors). Understanding the in vivo cellular barriers and other cellular restrictions in the host cell that impede the efficient delivery of therapeutic genes or DNA will help in developing efficient gene delivery systems that will ensure proficient expression of the delivered gene within the limits of the targeted hosts cells. After their successful delivery, it is critical that the inserted therapeutic DNA or genes continue to express their encoded gene products (e.g. enzymes or proteins) within the cells or tissues of the host because this will help to guarantee a thriving repair of the mutated gene responsible for the genetic disorder.        

Further reading

Cooper G.M and Hausman R.E (2004). The cell: A Molecular Approach. Third edition. ASM Press.

Das H.K (2010). Textbook of Biotechnology. Fourth edition. Wiley edition. Wiley India Pvt, Ltd, New Delhi, India.

Davis J.M (2002). Basic Cell Culture, A Practical Approach. Oxford University Press, Oxford, UK. 

Mather J and Barnes D (1998). Animal cell culture methods, Methods in cell biology. 2rd eds, Academic press, San Diego.

Noguchi P (2003).  Risks and benefits of gene therapy.  N  Engl J Med, 348:193-194.

Sambrook, J., Russell, D.W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York.

Tamarin Robert H (2002). Principles of Genetics. Seventh edition. Tata McGraw-Hill Publishing Co Ltd, Delhi.     

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