Molecular Virology In Course Assessment

"In light of the recent positive results (N. Cartier et al., Science 326, 818 (2009)), comment on the suitability and the problems related to the use of lentiviral and retroviral vectors for gene therapy"


As genetics become progressive field, scientists start to explore the possibilities of applying genes. Gene therapy is a technique of future that able to cure diseases incurable before. Insertions of a functional gene or a gene that bring advantageous properties are not fantasy nowadays. Indeed, human health can be much improved and there is a possibility even to "design" babies. As well as many new technologies, gene therapy also creates concern about ethics of its utilisation. But the ethics can be put apart, when we are speaking about fatal diseases, such as cancers or Severe combined immunodeficiency (SCID). Fortunately, nowadays medicine can help the sufferers of genetic diseases. According to Gene Therapy Clinical Trials Worldwide in 2009, great number of the therapy trials devoted to cancers (64.5%). Cardiovascular diseases are on the second place followed by rare disorders, 8.7% and 7.9%, respectively. (Gene Therapy Clinical Trials Worldwide, 2009 ) Recent positive results were reported during treatment of X-linked adrenoleukodystrophy (ALD), when the group of scientists managed to prevent brain demyelination. (Cartier et al., 2009) However, gene therapy was not always a success story. There are technical difficulties and safety issues, because the most efficient vectors are viral origin, with adenoviruses and retroviruses on periphery. When retrovirus designed as a vector, a limitation of it became apparent since they cannot infect non-dividing cells, leaving tissues such as brain, eye and lungs untreatable. But, the solution was, HIV, a lentivirus from Retroviridae family. The main problem of the viruses of this family is their high integration rate causing mutations. Nevertheless, gene therapy brings new prospect to genetically correct diseases and save people's life. Although, this is one side of the scales, whereas, the safety implications, such as insertional mutagenesis is on the other side. Thus, the usage of retrovirus-derived vectors can be reasonable, when a vast majority of patients are healed. Perhaps, we can overcome the problems by more analysing them.

How gene therapy works?

As was mentioned above many kinds of tumours and cancers can be cured by gene therapy. It seems that there is no hope to live a healthy life, when patients diagnosed with cancer, but yet gene therapy can provide with life-long treatment, when the basis of the life is under control. In those particular fields of oncogenetics, it is found to be a valuable technique, which is designed to sort out malignant cells from normal cells in elegant way. A gene can be subtitled with normal gene, or other possibility to activate toxin with pro-ddrug delivery. Also genes can be manipulated in such way that they activate the host immune response against tumour cells and induce apoptosis. The initial steps were not so successful in 1990s due to the interference of genes with patients' stem cells. (Cavazzana-Calvo et al., 2004) The therapy was done in patients with genetic diseases, such as ALD and SCID, where immune system is failed. (Somia and Verma, 2000) Recently many trials were approved in the UK to cure Cystic fibrosis, when patients can inhale vectors, but they use RNA (Gene Therapy Clinical Trials Worldwide, 2009) In some cases of HIV infections, gene therapy helps to elevate the degree of efficacy during drug treatment.

Gene therapy begins with an investigation of the genetics of a disease. The principal of how gene works is analysed. Depending on the case, a normal gene will be introduced as in Adenosine deaminase in Adenosine deaminase deficiency or trigger the host immune system to attack malicious tumour cell. For example, the strategy to cure tumours and cancers is to introduce a gene that tags the cell for destruction. (Kay et al., 2001) Therefore, a choice of an inserted gene lies within the function it provides.

Then the chosen material should be incorporated into a vector. The vector can be virus derived or non-viral; the chosen option will affect delivery into the host. Majority of doctors use viral-derived vectors. (Gene Therapy Clinical Trials Worldwide, 2009)

When the vectors are sorted, they are inserted ex vivo into fibroblasts or blood CD34+ cells. Those cells are taken from a patient. It is vital that the cells from patient are well growing and can be multiplied in laboratory. For that purposes bone marrow cells are usually used. (Cartier et al., 2009)

At this stage, different kinds of biochemical laboratory work can be done here. Most of the vectors are designed in such way, so they express the required protein in the right time and place. For example, switching on and off the expression by a promoter. Other tool is an injection of a pro-drug that activates toxins inside the cells.

The efficiency of the host's cells to take up and express proteins is checked with replication-competent lentivirus assay. Then the cells are incubated, so they can multiply 5-8 folds. Eventually, proliferated cells that can express the target protein injected in the patient. The amount of the protein should be then taken into account to avoid toxicity, because virus-derived vectors tend to trigger immune response.

After the injection of cells back into the patients, it is vital to control the expression and monitor the fluctuations in the titre. Further analysis show how long the protein is circulating within the body and if the next phase of treatment is needed.

Technical problems

There are technical problems associated with gene therapy, when viral vectors are used. Despite the recent progress, there is always need to increase efficiency of delivery. Retroviruses can provide around 70% successful insertion. (Somia and Verma, 2000) Even those vectors can be controlled by promoter, the duration of expression vary among viruses. Retroviruses insert themselves into the host genome, so persist a lot longer, which is vital for CFTR in cystic fibrosis patients. Some vectors, however, give high level transient expression. That is why they used to destroy cancer cells.

Apart from the technical difficulties, viral vectors cause significant safety issues. Generally, all viruses lead to the host immune response. That is especially concern of adenoviral vector usage, whereas retroviruses do not usually cause such problems. The tragic case of Jesse Gelsinger in 1999 showed undoubtedly that the use of viral-derived vector can lead to the death. (Somia and Verma, 2000) Those were drawbacks of all viral vectors, but further discussion will dwell on retroviruses and lentiviruses only.

Retroviruses are widely used to design vectors from the very beginning due to their ability to transfect cells in very efficient way. Even so, it was known that retroviruses do not transfect non-dividing cells, leaving a huge pool of unreachable cells. However, lentivirus proposed the solution to the problem. That is why scientists construct a vector based on HIV-1. But from that point, the safety problem raised sharply. HIV-1 genome was cut down maximum, so to reduce any risk of pathogenic effect. So, when it comes to delivery, viral vectors are good, however technical and safety problems are still there.

Insertional mutagenesis of Retroviruses and lentiviruses

The ability of retroviruses to integrate into the host genome is both beneficial and dreadful. This type of replication within cells make it paramount tool for laboratory. On the other hand, this behaviour causes an enormous problem of mutagenesis.

Consequence of the insertional mutagenesis is an activation of protogenes leading to leukaemia. The insertional mutagenesis resulted in leukaemia in two boys, which previously were treated with murine-derived leukaemia virus vectors. The doctors noticed abnormal expression of LMO-2 transcript in T-cells, which caused acute lymphoblastic leukaemia in patients. (Hacein-Bey-Abina et al., 2003a) Further investigations revealed the viral LTR integrations on the distal LMO2 promoter region. They suggested that abnormal T-cell proliferation were because of the activation of LMO2 enhancer or disruption of silencer with viral LTR. (Hacein-Bey-Abina et al., 2003b) Later, the same group reported another 2 abnormalities among their patients. So overall, in 2008, 4 patients were identified having leukaemia. Three of them were successfully cured with chemotherapy. Thus, safety issues are under study yet. (Hacein-Bey-Abina et al., 2008)

It was assumed that insertions of retroviruses occurred at random sites. However, using new technological advances, such as PCR and computational sequencing, the integration sites were determined. Figure 1 depicts, where the insertions occur commonly. Although that experiment was in vivo, this helps to identify clinical signs. (Fischer and Cavazzana-Calvo, 2005)

During insertional mutagenesis, LTR of lentiviruses affect the transcriptional sites, which determines the genotoxicity of those vectors. Cartier (2009) utilised self-inactivating vectors which reduced the rate of the insertions and thus risk of leukaemia. Insertional mutagenesis is still a problem, thus, more studies need to be done in order to improve viral-vector therapy.

Positive results

Eventually, recent paper published that they cured patients with X-linked adrenoleukodystrophy (ALD). Moreover, their analysis after 30 months showed an elevation of ALD protein in the blood samples. (Cartier et al., 2009) The advantages brought by gene therapy were striking. The techniques and protocols of gene therapy have changed dramatically, since the safety issues of retroviruses were revealed. The most beneficial characteristic of gene therapy that alternation of a gene can now be achieved in more or less efficient way, so humankind can now handle genetic disorders. Moreover, being relatively new scope, gene therapy already shows some insights in of otherwise fatal diseases, such as cancer or ALD. Without such treatment, the patients suffer and often have very little time surviving. Therefore, the results of 2000s gene therapy progression demonstrated that 18 patients in Europe had benefitted from the life-saving therapy. (Cavazzana-Calvo et al., 2004)

Indeed, careful analysis and design lead to proficient results. For example, it was suggested that in SCID trials the patients, who had leukaemia, were introduced very high dosage of vectors. So, in Cartier (2009) they carefully addressed previous problems with the lentiviral vector and no clonal skewing were detected in the patients. However, the function of a transgene and a pathophysiology of a disease should be taken into account too. (Hacein-Bey-Abina et al., 2008) Most importantly that gene therapy benefit for life-long period.


To conclude, gene therapy has suited in our clinical medicine, despite some problems associated with delivery and toxicity of retroviral vectors. Also, there is no ideal viral vector, they all comprise concerns. Gene therapy widely recruits vectors based on viruses from Retroviridae family. An insertion of a target gene into patients results in the healing from disorders. When using retroviruses and lentiviruses, technical problems and toxicity go in line with efficient treatment. Therefore, the future plan is to match the efficacy to safetyof such vectors. Nevertheless, there are prospects of more safe and effective vectors, as some experiments showed recently in ex-ALD patients.

Reference List

1. Cartier,N., Hacein-Bey-Abina,S., Bartholomae,C.C., Veres,G., Schmidt,M., Kutschera,I., Vidaud,M., Abel,U., Dal-Cortivo,L., Caccavelli,L., Mahlaoui,N., Kiermer,V., Mittelstaedt,D., Bellesme,C., Lahlou,N., Lefrere,F., Blanche,S., Audit,M., Payen,E., Leboulch,P., l'Homme,B., Bougneres,P., Von,K.C., Fischer,A., Cavazzana-Calvo,M., and Aubourg,P. (2009). Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 326, 818-823.

2. Cavazzana-Calvo,M., Thrasher,A., and Mavilio,F. (2004). The future of gene therapy. Nature 427, 779-781.

3. Fischer,A. and Cavazzana-Calvo,M. (2005). Integration of retroviruses: a fine balance between efficiency and danger. PLoS. Med. 2, e10.

4. Gene Therapy Clinical Trials Worldwide, 2009 online accessed 10/01/01

5. Hacein-Bey-Abina,S., Garrigue,A., Wang,G.P., Soulier,J., Lim,A., Morillon,E., Clappier,E., Caccavelli,L., Delabesse,E., Beldjord,K., Asnafi,V., MacIntyre,E., Dal,C.L., Radford,I., Brousse,N., Sigaux,F., Moshous,D., Hauer,J., Borkhardt,A., Belohradsky,B.H., Wintergerst,U., Velez,M.C., Leiva,L., Sorensen,R., Wulffraat,N., Blanche,S., Bushman,F.D., Fischer,A., and Cavazzana-Calvo,M. (2008). Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J. Clin. Invest 118, 3132-3142.

6. Hacein-Bey-Abina,S., Von,K.C., Schmidt,M., Le,D.F., Wulffraat,N., McIntyre,E., Radford,I., Villeval,J.L., Fraser,C.C., Cavazzana-Calvo,M., and Fischer,A. (2003a). A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N. Engl. J. Med. 348, 255-256.

7. Hacein-Bey-Abina,S., Von,K.C., Schmidt,M., McCormack,M.P., Wulffraat,N., Leboulch,P., Lim,A., Osborne,C.S., Pawliuk,R., Morillon,E., Sorensen,R., Forster,A., Fraser,P., Cohen,J.I., de Saint,B.G., Alexander,I., Wintergerst,U., Frebourg,T., Aurias,A., Stoppa-Lyonnet,D., Romana,S., Radford-Weiss,I., Gross,F., Valensi,F., Delabesse,E., MacIntyre,E., Sigaux,F., Soulier,J., Leiva,L.E., Wissler,M., Prinz,C., Rabbitts,T.H., Le,D.F., Fischer,A., and Cavazzana-Calvo,M. (2003b). LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415-419.

8. Kay,M.A., Glorioso,J.C., and Naldini,L. (2001). Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nat. Med. 7, 33-40.

9. Somia,N. and Verma,I.M. (2000). Gene therapy: trials and tribulations. Nat. Rev. Genet. 1, 91-99.

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