Delivery and In Vivo Toxicity

Delivery has long been the major issue for gene therapy. A search of one of the major gene therapy clinical trial databases (http://www.wiley.co.uk/ genetherapy/clinical/) shows that most gene transfer studies aim to treat cancer using direct injection of viral or naked DNA preparations (Edelstein et al. 2004). The goal of these trials is to either increase the sensitivity of tumour cells to apoptosis or deliver tumour antigens to promote an anti-cancer immune response. Another major mode of gene transfer investigated to date is the ex vivo transduction and subsequent reinfusion of autologous cells into patients. This delivery approach using haematopoietic stem/progenitor cells [with the caveat of observed toxicity in certain X-linked severe combined immune deficiency syndrome (SCID-X1) patients] was successful in treating SCID-X1 (Hacein-Bey-Abina et al. 2002; Cavazzana-Calvo et al. 2004; Gaspar et al. 2004) and adenosine deaminase deficient (ADA)-SCID (Bordignon et al. 1995; Aiuti et al. 2002a,b) patients. It has also been applied clinically to potentially generate human immunodeficiency virus (HIV)-protected cells (Kohn et al. 1999; Kang et al. 2002; Amado et al. 2004). Tempering gene therapy successes to date, a patient death after direct injection of an adenoviral preparation and the development of lymphoproliferative disease/leukaemia in three of the patients treated in the SCID-X1 clinical trial have highlighted some of the potential risks of this new form of treatment (discussed later in this section).

The ex vivo transduction approach is illustrated in Fig. 3. The patient receives granulocyte colony stimulating factor (G-CSF) to mobilise haematopoietic stem cells from the bone marrow into the peripheral blood (Hubel and Engert 2003). The protocols for this procedure were pioneered in the bone marrow transplantation setting, and a basic protocol involves the systemic administration of 10 pg/kg of G-CSF followed by blood collection (apheresis). Apheresis is performed as an outpatient procedure and nucleated (predomi-

Fig. 3 Flow diagram of the out-patient procedure for the ex vivo transduction and reinfusion of genetically modified cells. Prior to apheresis the patient receives granulocyte colony stimulating factor to mobilise stem cells from the bone marrow into the peripheral blood system. After mobilization, the cells in peripheral blood are apheresed (1) and the stem cells separated by the CD34+ surface marker (2). The stem cells are then cultured with recombinant virus that encodes the gene therapy. The recombinant virus illustrated contains a simple transfer vector composed of a long terminal repeat (LTR) (hatched) driving expression of a therapeutic gene (cross-hatched). In the final step, gene-modified cells are re-infused into the patient

Fig. 3 Flow diagram of the out-patient procedure for the ex vivo transduction and reinfusion of genetically modified cells. Prior to apheresis the patient receives granulocyte colony stimulating factor to mobilise stem cells from the bone marrow into the peripheral blood system. After mobilization, the cells in peripheral blood are apheresed (1) and the stem cells separated by the CD34+ surface marker (2). The stem cells are then cultured with recombinant virus that encodes the gene therapy. The recombinant virus illustrated contains a simple transfer vector composed of a long terminal repeat (LTR) (hatched) driving expression of a therapeutic gene (cross-hatched). In the final step, gene-modified cells are re-infused into the patient nantly mononuclear) cells from the equivalent of one to several blood volumes can be collected. Following this, the haematopoietic stem/progenitor cells are collected, generally using a CD34 antibody/bead complex and the cells cultured with cytokines to drive them into cycle. They are then transduced, harvested and reinfused to the patient. Reinfusing 1x106 CD34+ progenitor cells per kilogram is enough cells to rescue a person after bone marrow ablation (Chao and Blume 1990). In gene-therapy settings, larger doses of cells ranging from 2x106 to 5x106 cells/kg can be achieved to improve engraftment with gene-modified cells (Cavazzana-Calvo et al. 2000; Bordignon et al. 1995; Amado et al. 2004; Gaspar et al. 2004). For most applications, ex vivo transduction has utilised retroviral vectors encoding the therapeutic gene; this requires the cells be induced into cycle prior to exposure to the recombinant virus (Nolta and Kohn 1990). The induction of cell cycling does not appear to compromise stem/progenitor cell pluripotency—gene-modified cells of myeloid and lymphoid lineages have been reported in the peripheral blood system using this protocol (Kobari et al. 2000; Amado et al. 2004). In our own studies using this method, we have observed cell marking and gene expression greater than 3 years post infusion in T lymphocytes and monocytes (Amado et al. 2004).

Important aspects of future research in this field will include the search for the most appropriate stem/progenitor cells for a given application and the use of other retroviral vectors, such as lentiviral vectors that do not require the cycling of cells for transduction (Uchida et al. 1988; Naldini 1998; Douglas et al. 2001).

The occurrence of two cases of leukaemia (and a third with lymphoprolif-erative disease at the time of writing) in SCID-X1 children receiving retroviral gene therapy has been discussed within the field since early 2003. The theoretical occurrence of a mutagenic event caused by the integration of a transgene into or near to an oncogene was known; however, it was thought to be negligible using retroviral-incompetent vectors, given the data showing no leukaemia developing in mice, large animals and approximately 1,800 patients that had received retrovirus-modified cells. There was a previous single report of leukaemia development in mice. A retroviral vector containing the nerve growth factor receptor (NGFr) gene was shown to integrate within the ecotropic viral integration site-1 (Evi-1) in haematopoietic progenitor cells, giving rise to a single leukaemic clone within one of the five primary transplanted mice. Bone marrow was collected from primary transplanted mice, pooled and then re-transplanted into secondary mice. All 10 secondary transplant mice developed haematopoietic disorders within 22 weeks. In these mice the development of leukaemia appeared to be due to the combination of NGFr and Evi-1 (Li et al. 2002). In the SCID-X1 trial, the two children who presented with leukaemia were the youngest, while the recent third child was 8 months of age at the time of infusion. SCID-X1 is an immune deficiency caused by the absence of a gene located on the X chromosome encoding interleukin (IL)-2yc, an essential component of the IL-2, -4, -7,-9,-15 and -21 receptors and therefore essential to the maturation of T cells during thymopoiesis (Buckley 2004). This clinical work is considered the first major success for gene therapy with all patients developing Tcells and9outof11alargenumberofTcellswithdiverseTcellrepertoirethat was curative (Cavazzana-Calvo et al. 2000; Hacein-Bey-Abina et al. 2002). For the first two children affected by the leukaemic serious adverse event, the gene therapy procedure was directly implicated in the development of leukaemia. The malignant cells from both patients were characterised by an insertion of the retrovirus bearing the y common chain complementary (c)DNA in or near to the LMO2 gene locus, a gene known to be involved in T cell leukaemia in mice and humans (Hacein-Bey-Abina et al. 2003). However, this finding alone could not fully explain the development of leukaemia, as the other patients in retroviral-based clinical trials had not developed leukaemia after retroviral gene transfer, and the probability of each patient bearing at least one LMO2 insertion was high. Additional contributing factors have been summarised as (1) the transgene itself (Dave et al. 2004), (2) the age of the children (relatively immature haematopoietic system), (3) the massive proliferation of T cells in response to the introduction of the y common gene in these cells and (4) the disease itself. Up to the recent report on the third child, these events did not preclude the continuation of SCID-X1 trials, although there was a requirement for additional patient screening and the FDA evaluated it on a case-by-case basis (UK clinical trials were also successful and did not actually stop) (Berns et al. 2004; Gaspar et al. 2004).

Direct administration of vector to the patient is conceptually and logistically simpler; the vector can be introduced to the site (e.g. cancer) or systemically introduced. However, targeting gene delivery vectors to appropriate body sites is not a trivial undertaking and there has been very limited success to date. This is reflected in the majority of in vivo procedures being dependent on localising the delivery vehicle to the site of action (e.g. a tumour) by direct injection.

Adenoviral vectors have been used extensively for this purpose and their use was subject to considerable debate following the death of an adolescent in Philadelphia who was enrolled in a gene therapy trial for the treatment of partial ornithine transcarboxylase deficiency. This condition, due to the inborn error of urea synthesis (Lindgren et al. 1984; McCullough et al. 2000), leads to the build-up of ammonia causing death if not controlled. The patient received 6X1011 particles/kg, (the highest dose in the clinical trial) by direct injection into the right hepatic artery (Raper et al. 2001). Within 18 h, clinical signs of an adverse reaction were apparent and this led to systemic inflammatory response syndrome and ultimate failure of the lungs, resulting in death (Raper et al. 2003). In addition to clinical documentation that was found to be deficient, relevant issues included the relationship between dose of vector and toxicity—this did not appear to be linear and had substantial subject-to-subject variability. The NIH report (2002) inter alia, stated that deciphering the reasons why one patient reacted so violently has yet to be determined but will be important to the future of this form of gene therapy.

In 2003, a drug license was granted in China for a recombinant Ad-53 gene therapy for squamous cell carcinoma of the head and neck (SCCHN: Pearson and Jia 2004). SCCHN is a common cancer and, despite recent advances in treatment, still presents a major clinical challenge. This is the world's first commercially available gene therapy and we review here the important points associated with this product as an example of a directly injected gene therapy. p53 is a critical modulator of the cellular response to exogenous and endogenous stress and has a central role as a tumour suppressor. Inactivation of one or more components of the p53 network is a common event in human tumours (for review see Gasco and Crook 2003). There are several biotechnology firms in phase II or later clinical development based on a similar principle of delivering a gene directly to the tumour to restore or initiate apoptotic processes. In data directly related to the approved gene therapy, adenovirus encoding the p53 gene was administered weekly by direct injection of 1x1012 viral particles into the SCCHN. In combination with radiotherapy, 64% of late stage tumours completely regressed and 32% showed partial regression. (These data are from the SiBiono Web site www.Sibio.com, and to date they have not appeared in the peer-reviewed literature.)

As for classical small molecules, toxicity is also an issue for gene therapy; it is being addressed both in pre-clinical models and by the close monitoring of patients throughout the clinical trial period and in follow-up (Nyberg et al. 2004).

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