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I’m writing this article on request after a friend asked me to, and what follows is a very simple introduction to what gene therapy entails, some notable examples and a very brief evaluation of the prospects of the field. I will also be looking at the challenges that must be met before gene therapy becomes an integral part of medicine.
At the outset, I would like to lay out a picture of disease pathology from a molecular biological perspective. In both genetic disorders, to which gene therapy is particularly relevant, and communicable disorders there is a certain degree of involvement of genes and genomes, and of transcription and translation. While extant chemotherapy usually ends up targeting the protein products of genes to prevent them from leading to disease, gene therapy focuses on correcting things well before translation even occurs.
With gene therapy, three approaches are possible, one is to introduce a properly functioning gene when there is none (and this is perhaps the most well-established principle) , one is to turn off genes that lead to an abnormal phenotype and the last one is to alter gene expression so that disorders can be circumvented. Where possible I will provide references to illuminating examples for all these approaches.
Before moving on, though, gene therapy can be classified broadly into ex-vivo or in-vivo gene therapy (ex-vivo gene therapy is administered to cells that are taken outside the body and in-vivo gene therapy takes place inside the body) and alternatively as somatic gene therapy and germline gene therapy (the former is not heritable, the latter is)
Now I think it is time to move on to the approaches themselves, with the apposite examples included…
[I] Introducing properly functional genes when there are none.
There are lots of genetic disorders wherein a non-functional gene can cause real problems by affecting body function, the examples I shall use to illustrate the principles of gene therapy in this case involve two such examples, one is Cystic Fibrosis and the other is X-linked Severe Combined Immunodeficiency Syndrome.
Cystic fibrosis is an autosomal recessive disorder (that is, both copies of the relevant gene need to be dysfunctional for symptoms to occur) that is caused by mutations that produce a non-functional CFTR (Cystric Fibrosis Transmembrane Conductance Regulator) gene. When both copies of this gene are abnormal it results in water balance in mucus not being properly regulated, which translates to extremely thick mucus that can block airways, impair breathing, affect digestion and endocrine function. The disease is also associated with a markedly higher propensity towards acquiring bacterial infections.
While there is no cure there are several management methods around, but usually lung transplants become necessary at some stage in life. Treating this disorder is a challenge that gene therapy tried to meet.
The technology is nascent and the processes are inefficient, but are nonetheless promising. The gene therapy trials that have been carried out to treat CF have involved the use of a specific kind of virus, called an adenovirus, to deliver functional copies of the CFTR gene to cells or alternatively the use of bubbles of fatty acid that contain the gene construct inside (we call these liposomes).
Results are apparently promising from Phase I Clinical Trials. How things go in the future remains to be seen, but one thing is certain, we do know that targeting genes is a viable strategy at least in this case, we know that there are methods that can be used to an extent where clinical impact is possible, and we have a procedure and a set of methods that can be improved.
This is a sex-linked disorder and involves dysfunctional versions of the Interleukin-2 receptor, which plays a very important role in how the body responds to infections. People who lack functional IL-2 receptors end up dying of infections due to compromised immunity in the early years of life. This is because IL-2 signalling is responsible for the maturation and differentiation of progenitors into T-Lymphocytes, functional B-lymphocytes and NK Cells. All of these are extremely vital components of the immune system and not having them opens the body to all kinds of pathogens.
SCID has been treated using ex-vivo gene therapy, where a lentiviral vector containing a copy of a fully functional IL-2 receptors was used to induce the gene expression required to rescue immune function.
The problem with using retroviral vectors is that they integrate randomly into host genomes, and this means it may affect normal gene expression and trigger cancers, for instance. In fact, this did happen and at least one baby in the original trial died, the rest though received chemotherapy and survived. See reference here
A similar approach has also been tried for versions of SCID which are due to a deficiency of functional Adenosine Deaminase, which leads to similarly dysfunctional immune systems.
You can find a lovely review of twenty years of SCID and gene therapy here
So there are two examples where people have attempted to fix genetic disorders by inserting functional copies of genes when there were none. What about the other two cases?
[II] Knocking out dysfunctional genes.
This approach to gene therapy relies on the use of agents to target genes directly and to knock them out. There are several methods that can be used to do this. RNAi (RNA interference) is one of these, the other is the use of antisense oligonucleotides and yet one more is the expression of antisense transcripts.
Ideal cases for the use of gene silencing (which is a form of gene therapy insofar I am concerned) would include viral infections and cancer. Examples of current use would include the use of RNAi in a clinical trial to treat a viral infection by RSV
However, the whole thing is very nascent and much of the work here has been in mouse models/cell lines. You may find references that are useful here (It is a Google Scholar Search List)
[III] Modifying gene expression.
The example I would like to give here is of Duchenne Muscular Dystrophy, which is a disorder in which muscles progressively die out and become weaker, eventually culminating in death. The modification of gene expression in this case is carried out using exon skipping.
So what happens is that Dystrophin, the protein whose mutant varieties are implicated in DMD, is synthesized from mRNA that is first cut from longer pre mRNA and then joined. Some people lack one of the exons and as a result the exons that follow cannot be joined, thus producing a short mRNA and a short, useless protein. However, not all exons have this problem, and some exons can be joined to later exons even if there is something missing. The idea is to block exons that truncate the mRNA using antisense oligomers so that exons that can bind to later exons are at the end. This way, the mutant, dysfunctional regions of dystrophin can be skipped over by blocking that particular region using an antisense oligomer. This prevents frameshifts from resulting in a dysfunctional protein and enables a functional protein to be expressed.
So that is a very brief (lol) introduction to what gene therapy is, what it entails and some of the things that demonstrate its potential. While there is great potential, we need to keep figuring out ways to improve gene expression using vectors, we must ensure that these vectors get safer, cheaper and can be produced more easily and most importantly of all, try to ensure that gene therapy does actually work when called upon, and when that point is reached I suspect the face of medicine may change forever, since we will be able to address diseases at their very roots.
That is all from me this time round, happy learning.
-Ankur “Exploreable” Chakravarthy