Initial information provided by the French Medicines Agency AFSSAPS, oral presentation by the investigator during the 12th Annual Meeting of the American Society of Gene Therapy on 27th May 2009
A biological observation termed clonal cell dominance has been made in a gene therapy patient in France treated with cells modified by what was presumed to be a safer lentiviral vector. Lentiviral vectors are a sub-group of retroviral vectors. The vector used here contains two genetic safety elements known as SIN-LTR and insulator (see question 11), which has been non-clinically tested to reduce the likelihood of the occurrence of clonal cell dominance. As clonal cell dominance has preceded the very few cases of lymphoproliferative disease in similar trials in the last years, monitoring of the patients in the trial has been intensified. Klaus Cichutek, Vice President of the Paul-Ehrlich-Institut, stated: "No lentiviral vectors and no SIN vectors are currently being used in clinical trials in Germany. The investigators are aware of the issue and the patients have been informed accordingly. No non-anticipated risk is foreseen for on-going gene therapy trials in Germany. The molecular analysis of the observation of clonal cell dominance in the French trial will be evaluated in detail to understand its implications."
The French Medicines Agency stated on 28th May 2009 that a clonal cell dominance had been observed for the past five months in the blood of a patient treated in a gene therapy clinical trial two years earlier. Clonal cell dominance is a biological anomaly that preceded malignant disease in two earlier gene therapy clinical trials (see below). The patient is currently fine and, in terms of his disease, has shown improvement. The investigator has stopped enrolling patients. Monitoring of the patient for any signs of benign tumour growth or malignancy will continue, in order to initiate timely treatment.
Clonal cell dominance preceded the development of a lymphoproliferative disease in gene therapy patients treated for type X1 severe combined immunodeficiency disease (SCID-X1) in clinical trials in France and the UK (see press release). In contrast to these trials, the retroviral vector used in the new trial in France (termed lentiviral SIN vector) has an improved design which, based on scientific experiments with comparable vectors in tissue culture and in mice, is believed to be safer. The observation of clonal cell dominance in this new trial is therefore unexpected and needs further investigation.
The clinical trial is being conducted in France to evaluate the safety, tolerance and efficacy of gene therapy in patients with severe forms of Sickle Cell Anemia and β-Thalassemia Major, which are inherited disorders affecting red blood cells. The gene therapy medicinal product administered to patients contains genetically modified haematopoietic stem cells. The cells were taken from the patient, a therapeutic gene was transferred into the cells by using gene transfer vector particles, and the resulting genetically modified cells were re-administered to the patient for treatment. The gene transfer vector particles were designed to be replication-incompetent, they were derived from the lentivirus HIV-1 and contained only the therapeutic gene as the transfer vector, but none of the HIV-1 genes. The lentiviral vector is one of two types of replication-incompetent retroviral vectors used in a variety of clinical gene therapy trials in the EU and in the US. Retroviral vectors are derived either from gamma-retroviruses (gamma-retroviral vectors) or from lentiviruses such as HIV-1 (lentiviral vectors). The lentiviral vector used in this trial, what is known as a SIN vector containing insulators, has a design which is considered safer.
In France, the sponsor of the clinical trial has already committed to not enrolling further patients into this trial. In Germany a review of the risk-benefit of the ongoing clinical trials using gamma-retroviral vectors (explained in question 9) had previously been initiated. The sponsors and principal investigators in preparation of or leading this and other trials with gamma-retroviral vectors are aware of the problem, treated patients are being screened for the occurrence of dominant cell clones on a regular basis. This issue has already been addressed by regulatory guidance documents based on earlier trials involving gamma-retroviral vectors. However, as this is the first description of clonal dominance induced by a SIN vector, this observation and its molecular analysis will be closely evaluated by Paul-Ehrlich-Institut scientists and other European experts at the European Medicines Agency (EMEA).
There are currently no authorized clinical studies using lentiviral vectors or SIN vectors in Germany. There are two current studies using gamma-retroviral vectors. Occurrence of clonal cell dominance has not been reported.
According to the Wiley gene therapy clinical trial database, cells modified by lentiviral vectors are currently used in two other clinical trials in Europe and one additional trial in Europe has been ended. These studies comprise the treatment of patients suffering from Wiskott-Aldrich Syndrome (UK), X-linked cerebral adrenoleukodystrophy (France) and Parkinson´s disease (France). Clonal cell dominance has not been reported for any of the three.
Wiskott-Aldrich syndrome and adrenoleukodystrophy are inherited disorders resulting from a single gene defect. Wiskott-Aldrich syndrome is a rare X-linked recessive disease characterized by eczema, thrombocytopenia (low platelet count), immune deficiency, and bloody diarrhea (secondary to the thrombocytopenia). Adrenoleukodystrophy is a rare, inherited disorder that leads to progressive brain damage and eventually death. Parkinson´s disease is a degenerative brain disorder.
The gene therapy medicinal product was administered to the patient in June 2007. The patient suffered from a severe form of β-thalassemia, and had been dependent on transfusions for many years. To date, the biological and clinical evolution of the patient has been favourable. The patient has not required any red blood cell transfusion for the past 11 months and has now stable spontaneous haemoglobin levels above 9.5 gram per decilitre (g/d). Patients suffering from the disease show levels between 6-8 g/d while normal values range between 11-16 g/d.
What risk has this gene therapy shown?
Clonal cell dominance has been observed in one of the patients administered with a gene therapy medicinal product. Clonal cell dominance is a biological anomaly that preceded malignant disease in two earlier gene therapy clinical trials. Clonal cell dominance results from an increased expression of a cellular gene which is known to enhance cell growth (proliferation) and found altered in benign and malignant tumours. In the French patient, increased expression of a particular gene has been found. The most recent analysis performed in May 2009 showed that the proportion of circulating cells derived from the relative dominant clone had been stable for the past five months. An outgrowth of one of the clones might indicate a transition to lymphoproliferative disease or tumour growth.
Clonal cell dominance has been observed in one of the patients administered with a gene therapy medicinal product. Clonal cell dominance is a biological anomaly that preceded malignant disease in two earlier gene therapy clinical trials. Clonal cell dominance results from an increased expression of a cellular gene which is known to enhance cell growth (proliferation) and found altered in benign and malignant tumours. In the French patient, increased expression of a particular gene has been found. The most recent analysis performed in May 2009 showed that the proportion of circulating cells derived from the relative dominant clone had been stable for the past five months. An outgrowth of one of the clones might indicate a transition to lymphoproliferative disease or tumour growth.
Sickle Cell Anemia is an inherited disorder of the red blood cells. The red cells need to be flexible in order to pass small blood vessels. The cells' flexibility is decreased, which results in a risk of various acute and chronic complications, several of which are potentially lethal because the sickle-shaped red blood cells restrict blood flow. The disease occurs because of a single mutation in the haemoglobin gene (an oxygen transport protein in the red blood cells).
β-Thalassemia Major is an inherited disorder of the red blood cells. The genetic defect results in abnormal haemoglobin molecules, thus causing anaemia. Haemoglobin consists of four protein chains (2 alpha chains and 2 beta chains). The disease occurs because of a mutation in the β-haemoglobin gene.
The red blood cells contain haemoglobin. Haemoglobin is the oxygen transport in these cells. Haemoglobin consists of four protein chains (two alpha chains and two beta chains). Sickle cell anaemia occurs because of a single mutation in the haemoglobin gene. β-Thalassemia Major occurs because of a mutation in the β-haemoglobin gene.
The lentiviral vector is used to modify, outside of the body (ex vivo), autologous bone marrow cells from patients with sickle cell anemia or β-Thalassemia Major. Bone marrow cells contain haematopoietic stem cells. The vector adds a functional copy (without mutations) of the β-haemoglobin gene to the genome of the patient’s hematopoietic stem cells (HSCs). As the gene becomes integrated into the genome of the HSCs, it is transferred to all cells of the patients blood system that differentiate from the HSCs, including the red blood cells. The modification allows the stem cells to differentiate into other haematopoietic cells including red blood cells. As the modification involves the transfer of a functional, normal haemoglobin gene, the red blood cells derived from the modified stem cells now function normally in the treated patients if the treatment is successful.
Retroviruses comprise several groups, two of which are the gamma-retroviruses and the lentiviruses. Lentiviruses are known to induce acquired immunodeficiency syndromes in infected hosts. Their most prominent member is the human immunodeficiency virus HIV-1. They differ from the group of gamma-retroviruses in a number of accessory genes that contribute to their pathogenicity. Gamma-retroviruses and lentiviruses can infect human cells by inserting their genomic nucleic acid into the cell genome, which is termed 'integration' or 'insertion'.
Gamma-retroviral and lentiviral vectors are two types of retroviral vectors. The vectors are derived from the genome of gamma-retroviruses and lentiviruses, respectively, by removing the viral genes. Owing to the modifications, retroviral vectors are replication-incompetent and, therefore, unable to spread from treated to untreated cells. They cannot cause an infection and they cannot spread an infection from a treated patient to other persons. As mentioned above, a unique property of retroviral vectors is their ability to stably insert a therapeutic gene into the genome of the cell. Therefore, by treating stem cells, the therapeutic gene will also be transferred to cells that have been differentiated from the stem cell.
Retroviral vector particles are prepared from the culture media of special mammalian cells, which are known as packaging cells. As their genome, the vector particles contain the expression vector. The expression vector is also known as the therapeutic gene and is basically a piece of nucleic acid. Once integrated in the genome of cells treated with the retroviral vector particles, the therapeutic gene/expression vector is used by the cell to produce as a gene product a protein encoded by the expression vector.
In these types of gene therapy clinical trials, the patients‘ hematopoietic stem cells (HSCs) are removed and then genetically modified by lentiviral vector particles transferring a therapeutic gene. The therapeutic gene is a piece of nucleic acid which inserts randomly into the genome of each individual cell. The therapeutic gene then mediates expression of the wanted functional therapeutic protein.
The insertion of the therapeutic gene into the cell genome (also called 'chromosomal integration') leads to many different HSCs being treated with the vector particles which differ from each other by the site of integration of the therapeutic gene in their genome. Depending on the site of integration, the activity of neighbouring genes can be influenced and may thus result in slightly differing properties of each HSC. HSCs carrying vectors which induce a higher expression of a neighbouring cellular oncogene will survive better in the patient. A certain HSC or a hematopoietic cell derived from that HSC (here granulocytes and erythroblasts) may proliferate more than other cells, due to the site of vector integration. This cell will then overgrow the other cells and consequently represent with time a higher proportion of cells in the patient’s blood (clonal cell dominance). Under certain circumstances this can initially even be beneficial for the patient, e.g., when these cells also express the therapeutic protein. Nevertheless, finally, the clonal cell dominance may result in the development of a benign, or even a malignant tumour, at a later time point because additional genetic mutations occur in the dominant cells.
SIN vectors differ from conventional retroviral expression vectors in what is known as long terminal repeat (LTR) regions. The LTRs mediate expression of the therapeutic gene. Expression of the therapeutic gene in the cells leads to the production of the therapeutic protein within the cell. LTRs can also increase the expression of cell genes in the vicinity of the insertion site of a therapeutic gene/expression vector. If the expression of cellular oncogenes, known as proto-oncogenes, is increased due to vector insertion, the respective cells produce large amounts of onco-proteins and, therefore, show increased growth (proliferation) compared with normal cells and they may eventually transform into a benign or malignant tumour cell. This is called 'insertional oncogenesis' and the result is cancer or a related lymphoproliferative disease.
Retroviral SIN vectors contain self-inactivating (SIN) LTRs rather than full LTRs, but self-inactivating (SIN) LTRs. SIN vectors cannot enhance the expression of genes in their vicinity or at least show less enhancement. Genetic insulators have been found to shield genes near an inserted vector from overexpression.
This view is supported by recent results from non-clinical studies performed in mouse models which compare the risk of insertional oncogenesis induced by SIN or non-SIN retroviral vectors. These studies suggest that the risk of insertional oncogenesis is, in general, lower with lentiviral SIN vectors than with gamma-retroviral SIN vectors. The lentiviral SIN vector used to modify the cells administered to the patient in the trial discussed above contained an additional safety element. Reason for the alleged discrepancy between the outcome of the non-clinical studies and the occurrence of a clonal cell dominance in the trial await further molecular analysis.
Updated: 03.06.2009
