Recent developments and current status of gene therapy using viral vectors in the United Kingdom

Viral vectors remain the vehicles of choice for gene transfer in clinical trials of gene therapy in the United Kingdom and worldwide. In the United Kingdom alone, 88 clinical trials are registered with the Gene Therapy Advisory Committee (GTAC, www.advisorybodies.doh.gov.uk/genetics/gtac).

Although notable successes have been achieved in some inherited diseases, viral vectors have not been without their problems. We review the adverse events encountered to date and describe lessons that can be learnt from them to improve vector biology and pharmacology in order to make gene therapy with viral vectors a viable treatment for the future.

Sources and selection criteria

We searched Medline and Google, using the terms “gene therapy” and “viral vectors.” We also studied the websites www.wiley.co.uk/genmed/clinical and www.advisorybodies.doh.gov.uk/genetics/gtac for information on ongoing trials using viral vectors in the United Kingdom.

Viral gene therapy: successes and failures

In April 2002 the BBC online news service in the United Kingdom carried an emotive headline: “Bubble boy saved by gene therapy.”¹ The 18 month old infant featured had a rare immune disorder, X linked severe combined immunodeficiency (X-SCID), caused by a defective gene on the X chromosome. This gene encodes the common γC chain, an essential component of five cytokine receptors, all of which are necessary for the development of T cells and natural killer cells. Without the γC chain, mature T cells and natural killer cells are completely absent, whereas B cells are usually present in normal or increased numbers. X-SCID is fatal during the first year of life because of severe recurrent infections. As a result, much of the child's short life is spent in a sterile plastic bubble, a barrier to potentially life threatening infectious organisms. A collaboration of scientists from London, Paris, and Milan used a retroviral vector to introduce a functional copy of the defective gene into bone marrow stem cells taken from the patient in the hope that after re-infusion they would engraft and reconstitute a normal immune system.²,³ Integration and expression of the γC transgene and development of the lymphocyte subgroups and their functions were analysed over a period of up to 2.5 years after gene transfer. No immediate adverse effects resulted from the procedure. The numbers and repertoire of T cells and their in vitro proliferative response to several antigens were nearly normal after two years. The therapy was hailed as a success and an important breakthrough for gene therapy as it pioneered an ex vivo procedure that avoided direct in vivo transfer of the vector.

Summary points

In the United Kingdom, 74% of all gene therapy trials entail the use of viral vectors; most of these are for the treatment of cancer

Given recent setbacks, more efforts are required to improve the safety of viral vectors

These include reducing the risk of insertional mutagenesis and directing the expression of therapeutic genes to specific tissues using both transcriptional and transductional targeting

No serious adverse events have so far been reported in clinical trials of gene therapy in the United Kingdom

With efficient monitoring of trials and continuous improvement of viral vectors, gene therapy may still represent a real alternative to conventional therapies for a range of diseases

However, within a year, two of the 10 children treated in France for X-SCID had developed a leukaemia-like disease.⁴ Genetic analysis of the malignant cells showed that the retroviral vector, used to carry a functional copy of the defective γC chain gene into the bone marrow stem cells, had inserted into the patients' DNA and activated an oncogene, LIM-only2 (LMO2), which is associated with childhood leukaemia. Although this insertional activation was not the only cause of the malignancy, it was considered to be one of the events that triggered it. However, it is important to note that these leukaemias associated with the gene therapy are so far clinically manageable. Similar concerns have recently been raised by Mark Kay at Stanford University in the United States. In March 2000 encouraging results were reported, describing a phase I clinical trial using an adeno-associated virus (AAV) expressing the gene for blood coagulation factor IX (FIX): recombinant AAV vectors are considered to be one of the safest for use in gene therapy as the virus does not cause disease in humans naturally and rarely integrates randomly into the genome. One patient on the trial had a 50% reduction in the need to administer factor IX and a second an 80% reduction.⁵ However, in June 2003 Kay et al published a study conducted on mice, which found that the vector used in these trials integrates itself into regions of DNA containing genes more often than into non-coding regions.⁶ The integration sites were distributed on the mouse genome, with no particular bias to one particular chromosome. However, it was clear that integration was occurring in active genes. This showed that the AAV vector could possibly cause similar cellular defects to those that led to cancer in the SCID patients.

In addition to retroviral and AAV vectors, problems have also been encountered with adenoviral vectors. The death of a young man in the United States, Jesse Gelsinger, was attributed to the toxic effects of an adenoviral vector used to treat ornithine transcarbamylase (OTC) deficiency.⁷

Current status of viral gene therapy in the United Kingdom

Despite these recent reports, the field of gene therapy has come a very long way over the past decade. Gene transfer efficiencies have improved, and many new vectors are in preclinical studies. Viral vectors remain the gene transfer vehicles of choice for clinical trials in the United Kingdom and worldwide. Table 1 shows clinical trials in the United Kingdom that are open and ongoing. Of all 88 UK trials registered with GTAC, 75% (66) entail the use of viral vectors; most of these are for the treatment of cancer.

Table 1.

Ongoing clinical trials in the United Kingdom, March 2004 (sources: website of Journal of Gene Medicine, www.wiley.co.uk/genmed/clinical; and of Gene Therapy Advisory Committee www.advisorybodies.doh.gov.uk/genetics/gta)

Investigator	Centre	Virus	Disease	Gene	Phase	Year approved
James	Queen Elizabeth Hospital, Birmingham	Adenovirus	Head and neck cancer	Nitroreductase	1	1999
James	Queen Elizabeth Hospital, Birmingham	Adenovirus	Liver cancer	Nitroreductase	1	1999
Riddell	St Georges Hospital, London	Adenovirus	Peripheral arterial occlusive disease	Fibroblast growth factor (FGF)	1	2000
Link	Beatson Oncology Centre, Glasgow	Adenovirus	Head and neck cancer	E1b deleted	II	1997
Morris	University Hospital NHS Trust, Birmingham	Adenovirus	Ovarian cancer	Nitroreductase	I	1998
Chanon	John Radcliffe Hospital, Oxford	Adenovirus	Incomplete revascularisation after coronary bypass surgery	Hypoxia inducible factor (HIF)-1alpha	I	2000
James	Queen Elizabeth Hospital, Birmingham	Adenovirus	Prostate cancer	Nitroreductase	I	2001
Habib	Hammersmith Hospital	Adenovirus	Hepatocellular carcinoma	E1b-deleted	I	ns
Rampling	Beatson Oncology Centre, Glasgow	Herpes simplex virus	High grade glioma	ICP34.5 deleted	I	2000
Harris	John Radcliffe Hospital, Oxford	Herpes simplex virus	Malignant melanoma	Granulocyte macrophage colony stimulating factor (GM-CSF)	I	2000
Brown	University of Glasgow	Herpes simplex virus	Head and neck cancer	ICP34.5 deleted	II	2001
Coombes, Pandha, Harrington	St George's Hospital Medical School, London	Herpes simplex virus	Melanoma, breast cancer, head and neck cancer, non-Hodgkin's lymphoma	ICP34.5 deleted ICP47 deleted GM-CSF	I	2001
Evans	University of Glasgow, Beatson Oncology Centre, Glasgow	Herpes simplex virus	Mesothelioma	ICP34.5 deleted	I	2002
Harris	John Radcliffe Hospital, Oxford	Retrovirus	Metastatic melanoma	HSV-tk	I	-
	Hammersmith Hospital, London	Retrovirus	Chronic myeloid leukaemia	HSV-tk	I	2000
Thrasher	Institute of Child Health, London	Retrovirus	X-SCID	Gamma c common chain receptor	I	2001
Thrasher	Institute of Child Health, London	Retrovirus	X linked chronic granulomatous disease (X-CGD)	gp91phox	I	2000
Steven	Queen Elizabeth Hospital, Birmingham; Churchill Hospital, Oxford	Retrovirus	Breast cancer, melanoma	Cytochrome p450	I	2001
—	Churchill Hospital, Oxford	Retrovirus	Prostate cancer	Cytochrome p450	I	2001
Lotze	Queen Elizabeth Hospital, Birmingham	Vaccinia virus	Colorectal cancer	Carcinoembryonic antigen (CEA)	I	1998
Freeman	University of Cardiff, Cardiff	Vaccinia virus	Cervical intraepithelial neoplasia III	Human papilloma virus (HPV) E6 and E7	I	1996
Goh	GlaxoSmithKline	Vaccinia virus	Human immunodeficiency virus (HIV) infection	HIV1-Env	III	2000
—	—	Vaccinia virus	Advanced colorectal cancer	Oncofetal antigen 5T4	II	2004
Harris	Churchill Hospital, Oxford	Vaccinia virus	Melanoma	Mel3	I	2000
—	—	Vaccinia virus	Cervical cancer	Human papilloma virus (HPV) E6 and E7	I	2001
Steven	Institute of Cancer Studies, Birmingham	Vaccinia virus	Nasopharyngeal carcinoma	—	I	2002

Adenoviral vectors

Adenoviruses remain the most popular viral vectors in clinical trials because of their ability to transduce a broad range of dividing and non-dividing cells. In addition they replicate episomally and do not insert their genome into that of the host cell, which ensures less disruption of vital cellular genes. However, adenoviral vectors remain the most immunogenic of all the viral vectors currently in use. In the Gelsinger case, death was directly attributable to a massive inflammatory response to the first generation adenoviral vector. Scientists have therefore done their utmost to solve these problems and remove all non-essential viral genes to create a third generation of “gutless” vectors. However, studies in the rat brain have shown that an immune response can still be elicited by the adenoviral capsid proteins. The extent to which the vector elicits an immune response is linked to the dose of vector administered: the urgent challenge is to establish the dose at which the vector should be administered. However, assessing the amount of vector to deliver is made difficult by the variability of the immune response in humans. This was shown in the Gelsinger case. Jesse Gelsinger received the highest dose of vector in the trial (3.8×10¹³ viral particles), whereas another patient received a similar dose (3.6×10¹³ viral particles) but showed no side effects. As a result of the events in the United States, the UK Gene Therapy Advisory Committee assembled an adenoviral working committee, which made several recommendations including that the immune status and cytokine profile of the patients enrolled on the trial should be monitored before and after administration of the vector, to try to assess the patients' reaction to the treatment. Another recommendation was that the research community work towards a standardised method of assessing viral titre to ensure consistency through all clinical trials.⁸

Retroviral vectors

Retroviral vectors are being used in a range of clinical trials across the United Kingdom. One of the advantages of using retroviruses is their ability to integrate into the genome and maintain expression for long periods of time. However, given recent reports, the risk of oncogenesis induced by retroviral integration is clearly higher than thought previously. These risks must therefore be reduced by strategies such as targeting integration to inactive regions of the host genome.⁸ One approach could be to use the site specific integration machinery of bacteriophage C31, which has recently been used in non-viral delivery approaches to achieve targeted integration in mouse and human cells.⁹

Replication competent viral vectors

The use of non-replicating viral vectors can severely limit the number of cells that are targeted. As a result, replication competent viral vectors have been developed that allow the delivery of genes to a small number of cells and their subsequent transfer to surrounding cells as the infection spreads. These are proving particularly effective for treating cancer in preclinical models. The adenoviral vector, ONYX-015, is deleted in the E1B region that binds to and inactivates p53. Under normal viral replication an adenovirus binds to and inactivates p53 to ensure efficient replication. Therefore, in theory, ONYX-015 is unable to replicate in normal cells, but in cancer cells, in which p53 is deleted, the virus is able to replicate and cause cell lysis.¹⁰ However, this issue is not as clear as originally thought since the virus has been shown to replicate in p53 wild type cells. This approach is currently being tested in the United Kingdom to treat cancers of the head and neck, and liver. However, studies have indicated its limitations and areas where improvement is needed. For example, problems include a low transduction of tumour cells because of their low expression of the Coxsackie adenovirus receptor (CAR) required for adenoviral entry. New versions of ONYX-015 are being designed that remove the need to infect all tumour cells to gain complete remission. They have been engineered to deliver prodrug converting enzymes to tumour cells. The replication competent oncolytic herpes simplex virus (HSV) is also a popular vector currently in use in several clinical trials of cancer gene therapy in the United Kingdom. HSV1 is a large, double stranded DNA virus encoding at least 89 proteins. It has a wide cell tropism and remains extra-chromosomal, therefore minimising the risk of insertional mutagenesis. Many of the replication competent viruses under investigation have had the ICP34.5 gene deleted and are predisposed to replicate in cancer cells but not in adjacent normal tissues. Viral replication leads to cell lysis, which releases a second wave of the virus that can infect adjacent tumour cells. These vectors show promise, and no adverse side effects have been reported to date.

Future prospects for viral gene therapy

Despite the X-SCID and Gelsinger cases no serious adverse events have so far been reported in gene therapy trials in the United Kingdom. This is at least in part due to the small numbers of patients treated. It is important that lessons are learnt from these cases and that they are used to improve our understanding of vector biology and pharmacology. Although GTAC has a central role in controlling gene therapy studies in the United Kingdom, calls have materialised for a harmonisation of legislation among European states and between Europe and the United States.¹¹ Clearly, more efforts are needed to improve vectors to prevent the risk of insertional mutagenesis and also to direct the expression of therapeutic genes to specific tissues using both transcriptional and transductional targeting. This, in turn, will reduce vector doses and potentially immunogenicity.

Additional educational resources

Review articles

Cavazzana-Calvo M, Thrasher A, Mavilio F. The future of gene therapy. Nature 2004;427: 779-81.

Thomas CE, Ehrhardt A, Kay MA. Progress and problems with the use of viral vectors for gene therapy. Nature Rev Genet 2003;4: 346-58.

Lundstrom K. Latest development in viral vectors for gene therapy. Trends Biotechnol 2003;213: 117-22.

Useful sources of information and their websites

Journal of Gene Medicine (www.wiley.co.uk/genmed/clinical)—Contains information on all completed and ongoing clinical trials worldwide

Gene Therapy Advisory Committee (www.advisorybodies.doh.gov.uk/genetics/gtac)—Contains information on all completed and ongoing clinical trials in the United Kingdom

Information for patients

www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml—Contains basic information on gene therapy and links to other websites

Cancer BacUp (www.cancerbacup.org.uk—Contains information on all types of cancers as well as information on gene therapy trials for cancer

Despite the setbacks successes have been achieved. Of the 18 SCID patients treated for their life threatening condition, 17 are still alive and have had a healthy, functioning immune system for up to five years and a good quality of life. In addition to monogenic conditions, gene therapy also represents a viable treatment for multigenic disorders, such as cancer, either as a standalone treatment or in combination with chemotherapy or radiotherapy. A huge amount of work is yet to be done, but with efficient monitoring of trials and continuous improvements of viral vectors, gene therapy may still represent an important addition to the treatment armamentarium for a range of diseases.