An Overview of Gene Therapy

Ashanthi, a four year old girl, was suffering from an immune deficiency disorder called SCID (Severe Combined Immune Deficiency). Due to the lack of a healthy immune system, she was susceptible to infections even from germs which otherwise would not affect healthy persons. She was confined to her room, met no one outside her family, and had to take heavy doses of antibiotics to fight the microbes on behalf of her dilapidated immune system. A team of doctors from the National Institutes of Health, in the United States, drew blood from the patient’s body, and separated the WBCs (white blood cells; cells which fight infections). They then cultured the WBCs, inserted the missing gene and then infused the blood back into Ashanthi’s bloodstream. The girl survived, she no longer lived a recluse life and antibiotics were no longer a ritual. That was the first approved gene therapy (ex vivo, as the engineering was done outside the body) procedure carried out in a human.

Gene therapy is the procedure of replacement of faulty genes (nucleic acid sequences) by healthy ones. Frequently, a normal gene is added to an existing faulty allele, rather than a replacement of the gene at fault. Genes consist of stretches of deoxy-ribonucleic acids (DNA). The nucleic acid sequences in the DNA dictate the formation of proteins via the mediation of ribonucleic acids (RNA). Information contained in the DNA is passed on to the RNA by a process called ‘transcription’, which occur in the nucleus of the cell. RNA then goes to the cytoplasm of the cell where it forms a protein, in a process called ‘translation’; the functional product of that gene, its spokesman! The DNA sequence determines the sequence of amino acids in the protein, which is important in that any mistake in having the right amino acid in the right place may yield a non-functional protein with an abnormal configuration. Thus, an abnormal DNA sequence might (not always) produce a non functioning enzyme (a protein), causing diseases of immunity, metabolism and cancer.

We can ‘insert’ a normal functional gene into the genome containing an abnormal one; exchange an abnormal gene for its normal counterpart by homologous recombination; we could even ‘regulate’ the ‘expression’ of a particular gene. Inherited genetic diseases like thalassaemia, sickle cell anemia and cystic fibrosis could best be tackled by manipulating the ‘germ cells’ (sperms and ova) and this not only would ensure that the progeny was healthy but would also be passed (this new gene) onto the next progeny. Such heritable ‘germ line therapy’ despite sounding promising, is prohibited due to ethical concerns and the lack of expert technical knowhow. ‘Somatic cell gene therapy’, the gene therapy practiced these days, however, is not heritable.

Now that we know the basics, we should find a suitable carrier (vector) to deliver the goods inside the cell. Viral vectors are the most commonly used. Retroviruses, for example, take with them 2 identical copies of single stranded RNA (ssRNA); an enzyme called ‘reverse transcriptase’ and ‘integrase’, another enzyme, when it enters a cell. Reverse transcriptase or RNA dependent DNA polymerase converts the RNA sequences into DNA. The double stranded DNA then integrates with the host genome by the mediation of ‘integrase’. A therapeutic gene could now express itself in the form of a usable protein, via the integrated viral genome. Since viruses may cause disease, researchers must ensure that the disease causing genes of the virus are deleted. For example, AIDS is caused by a retrovirus (HIV). Another cause for concern is that retroviruses integrate randomly in the human genome. If they sat close to a proto-oncogene, or in the middle of a tumor suppressor gene (this might disable the suppressor gene), it might cause cancer.

Adenovirus is another option. A double stranded DNA (dsDNA) virus, adenovirus, does NOT integrate with the host cell, hangs free in the nucleus and just carries out transcription. Frequent administration is necessary, as the gene does not replicate with the host cell. Adeno-associated virus (AAV), an ssDNA virus, may also be used as a vector. The recombinant type (rAAV) carries NO viral gene & does NOT integrate. But they can infect quiescent (non-dividing) cells, hence may prove useful in neural/neurodegenerative diseases.

Non viral vectors include:
Naked DNA: Transfection (using phosphate-DNA mixture), Electroporation (use of electrical pulse for better membrane permeability), Sonoporation (using ultrasound for facilitation of DNA delivery), gene gun (DNA coated gold nanoparticles ejecting out along with high velocity gas) are some techniques for delivering DNA fragments.
Oligonucleotides: Antisense nucleotide sequences for the target gene. Being antisense, the nucleotides will latch onto the sense strand, just like the opposite poles of a magnet, thus preventing its translation. Fomivirsen is one such drug which is used in cytomegalo virus (CMV) retinitis. 
Short interfering RNA (siRNA); Small nucleotide sequences which tell the cell to cleave faulty mRNA.
DNA-lipid complexes (lipoplexes): here the DNA molecule is covered with an arrangement of lipids in the form of a micelle. Using a nonionic surfactant such as Tween 80 in addition, gave a better yield.

The challenges are still great. Our immune system and the genome do not take these pieces of DNA easily. For example, the gene transfer frequency (in hematopoietic stem cells of dogs and monkeys) for adenosine deaminase, the deficiency of which causes SCID, was only 3%. Still scientists hoped that the healthy cells would outgrow diseased cells as they had distinct survival advantages. But the efficacy of delivery didn't improve.
As of today, most major trials on gene therapy are on pluripotent hematopoietic stem cells (PHSC) and cancer cells. It is only natural to assume that genetic manipulations on blood stem cells (PHSC) would cure a variety of diseases affecting the blood cell lineages. And the quest goes on.

1. Mark A. Kay*,, 2. Dexi Liu, and, 3. Peter M. Hoogerbrugge (1997). Gene therapy PNAS , 94
Last modified: never
References: Gene therapy
Gene therapy PNAS November 25, 1997 vol. 94 no. 24 12744-12746