key: cord-0946871-ffxxb91i
authors: Schenk‐Braat, Ellen A. M.; Kaptein, Leonie C. M.; Hallemeesch, Marcella M.; Bangma, Chris H.; Hoeben, Rob C.
title: Gene therapy in The Netherlands: highlights from the Low Countries
date: 2007-08-23
journal: J Gene Med
DOI: 10.1002/jgm.1094
sha: f52befa9337648ce0f79a9e0b664c50adc30f7de
doc_id: 946871
cord_uid: ffxxb91i

Gene therapy is an active research area in The Netherlands and Dutch scientists involved in fundamental and clinical gene therapy research significantly contribute to the progresses made in this field. This ranges from the establishment of the 293, 911 and PER.C6 cell lines, which are used worldwide for the production of replication‐defective adenoviral vectors, to the development of targeted viral vectors and T lymphocytes as well as of non‐viral vectors. Several milestones have been achieved in Dutch clinical gene therapy trials, including the first treatment worldwide of patients with adenosine deaminase deficiency with genetically corrected hematopoietic stem cells in collaboration with French and British scientists. Until now, about 230 patients with various diseases have been treated with viral and non‐viral gene therapy in this country. Ongoing and upcoming Dutch clinical trials focus on the translation of new developments in gene therapy research, including the restoration of genetic defects other than SCID, and the use of oncolytic adenoviruses and targeted T cells for the treatment of cancer. The growing commercial interest in Dutch clinical gene therapy is reflected by the involvement of two Dutch companies in ongoing trials as well as the participation of Dutch clinical centres in large phase III international multicenter immuno‐gene therapy trials on prostate cancer sponsored by an American company. Translational gene therapy research in The Netherlands is boosted at a governmental level by the Dutch Ministry of Health via a dedicated funding programme. This paper presents an overview on milestones in Dutch basic gene therapy research as well as on past, present and future clinical gene therapy trials in The Netherlands. Copyright © 2007 John Wiley & Sons, Ltd.

The bulb fields are important eye catchers in The Netherlands. Besides their visible attractiveness they have been of large economical importance. For this reason agricultural sciences traditionally flourished in The Netherlands. This included microbiology (or better bacteriology) to study and prevent contagious infections in plants. It was this setting that led Martinus Willem Beijerinck in 1898 to discover that a prevalent mosaic disease of tobacco, which was at that time an important crop, was caused by a filterable agent, that he called 'contagium vivum fluidum'. He coined the term 'virus' for this agent, that now is known as tobacco mosaic virus [1] . This was the start of the virology research tradition in The Netherlands. Up to today, virology is still a main research area in the biological sciences. The fundamental branch of Dutch gene therapy research was born out of this virology field. Progressions made by gene therapy scientists in The Netherlands have had a significant impact on the translation of gene therapy into the clinic both at a national and international level. The expertise of Dutch gene therapy scientists is also recognized at an international level, as demonstrated by the fact that various Dutch research groups participate in several of the pre-clinical and clinical-oriented European gene therapy projects supported by the Framework Programmes of the European Union (Table 1) . This paper presents a brief overview on some milestones in Dutch basic gene therapy research as well as on past, present and future clinical gene therapy trials in The Netherlands.

Basic gene therapy research was initiated in the lab of Alex van der Eb, who chose adenoviruses as a research model in the mid-1960s. Adenoviruses were first identified by Rowe and colleagues in 1953 when they tried to establish cell lines from adenoidal tissue and tonsils [2] . A transmissible agent was found responsible for degeneration of the cultures. More than 50 serotypes of human adenoviruses (HAdV) have been described to date, which have been classified in six 'species', A through F (formerly called 'subgroups'). The evolution and classification of adenoviruses has recently been reviewed [3] . Adenoviruses attracted much attention with the discovery by Trentin et al. [4] and Huebner et al. [5] that certain serotypes (i.e. serotypes 12 and 18 of species A) can induce tumors in newborn hamsters, whereas others (i.e. serotypes 2 and 5 of species C) are not or only very weakly oncogenic. These observations triggered Van der Eb in Leiden to use adenoviruses as a model for animal studies on cellular transformation in vitro and tumor induction in vivo. This led to the observation that the difference in oncogenicity correlates with the capacity of serotype-12, but not serotype-5, to down-regulate antigen presentation by MHC class I [6, 7] . This illustrated how important the immune system is for preventing tumor growth.

As such, adenoviruses have played an important role in fundamental biomedical research. They provided the model systems for studying the organization of eukaryotic genes and the regulation of their expression, as well as for studying the mechanisms of DNA replication in mammalian cells [8] . The latter studies were performed predominantly in the lab of John Sussenbach and Peter Van der Vliet in Utrecht. Such studies not only led to detailed insight in many cellular and viral processes, but also yielded various broadly applicable tools and techniques.

The 'Graham & van der Eb' paper describing the calcium technique for efficient transfer of naked adenovirus DNA into cultured cells [9] has been cited more than 8500 times and this cheap and efficient technique for DNA transfer is routine in virtually all cell biology laboratories. This technique was used by Frank Graham in Alex van der Eb's lab to show that human cells, too, can be transformed and immortalized with naked adenovirus DNA. This is exemplified by the '293' cell line obtained after transformation of embryonal kidney cells with sheared adenovirus serotype-5 DNA [10] . Later, the Leiden group generated the '911' cell line, by transfer of plasmids carrying defined adenovirus type 5 sequences into human embryonal retinoblasts [11] . The observation that such adenovirus-transformed cells can be used for propagating viruses with defects in their Early Region 1 (E1) gene [12] facilitated the development and use of replication-defective adenovirus vectors. The relatively easy generation of E1-deleted replication-defective vectors made these vectors a prime choice for many gene therapists.

Although very useful, both the 293 and the 911 cell lines have the same intrinsic problem. During propagation of replication-defective E1-deleted adenovirus vectors replication-competent adenoviruses (RCA) are being generated with high frequency. RCA are reverted vectors that re-acquire the E1 region while losing the transgene cassette. This is the result of recombination between homologous sequences integrated in the helper cells and in the vector backbone [13] . In an attempt to solve the problem the Leiden group, in collaboration with scientists at Introgene (now Crucell), generated the PER.C6 cell line [14] . This line was established after transfer of a minimal portion of the adenovirus serotype 5 genome (bp 457-3510) into human embryonal retinoblasts, again using the calcium technique. In the process GLP conditions were used and the cell line, and the vectors produced on it, were extensively characterized [14, 15] . This cell system has been used for the production of clinical-grade adenovirus-vector batches [16, 17] . The use of this cell line in combination with matched E1deleted vectors that do not have sequence homology largely solved the issue of RCA generation by homologous recombination.

Also scientists from other fields embraced the gene therapy approach. While working as a PhD student in Alex van der Eb's group in Leiden, Dinko Valerio in 1983 was the first to report cloning of the human adenosine deaminase (ADA) cDNA and the corresponding gene [18] . He embraced the retroviral vector technology and eventually used these vectors to transfer the ADA cDNA into hematopoietic stem cells of three ADA patients in a study performed with French and British scientists [19] . This represented the first study in which hematopoietic stem cells of ADA patients were modified. In addition, Valerio founded the company IntroGene, now called Crucell. Crucell has been one of the key players in the adenoviral-vector field and pioneered the use of fibermodified and adenovirus serotype 35 derived vectors to overcome the paucity of the adenovirus receptor CAR and circumvent pre-existing immunity, respectively [20] .

The relation between retroviral vectors and cancer was studied in several other labs. Anton Berns in Amsterdam examined how murine leukaemia viruses (MLV) induced tumors upon infection in newborn mice. He recognized that the retroviruses found in tumors often had integrated into common sites. In these sites the proviruses disturbed the expression of nearby genes [21, 22] . The commonality of the integration sites pointed to the association of the nearby genes with the induction of the leukemic state. Berns' analyses of common integration sites led to the identification of several genes that can be involved in leukaemia. The use of MLV infection in transgenic animals further delineated the pathways in virus-induced leukaemia [23, 24] . The relevance of these observations to gene therapy became all too apparent with the development of leukaemia in four infants born with X-linked severe combined immunodeficiency (SCID) gene who were successfully treated with an infusion of autologous hematopoietic stem cells transduced with a defective retroviral vector [25] . This has been attributed to deregulation of expression of the LMO-2 gene by the integrated vector provirus.

In Amsterdam the use of adeno-associated vectors (AAV) has been thoroughly exploited. The company Amsterdam Molecular Therapeutics (AMT) was founded to generate and produce state-of-the-art AAV for clinical studies. This greatly facilitated scientists to move from the lab to the clinic and the operations of AMT boosted translational gene therapy research in the Netherlands.

To date, research on viral vectors has significantly expanded in The Netherlands. Various labs in Rotterdam, Amsterdam, Groningen, and Leiden are exploring diverse targeting strategies to increase the selectivity of adenoviruses as oncolytic agents. Transductional targeting is studied by Van Beusechem and collaborators, who replaced the adenovirus fiber with sequences of the spike protein of a reovirus [26] , and by Hoeben and collaborators who developed the adenovirus minor capsid protein IX as an anchor for targeting ligands [27] . Transcriptional targeting of the vectors to glioblastoma cells is employed by Sillevis Smitt and collaborators [28] , to cholangiocarcinoma cells in Bosma's group [29] , and to prostate cancer cells by Kraaij and collaborators [30] . Optimized delivery methods and enhanced-efficiency vectors are being evaluated in cell culture, spheroid cell aggregates [31] , tumor explants [32] , tissue slices [33] , and in small-animal models [34] . These approaches are not limited to adenovirus vectors. Also RNA viruses such as reoviruses, alphaviruses and coronaviruses are being evaluated for use as in anticancer strategies. Kranenburg and collaborators linked the capacity of reovirus to induce tumor-cell-specific cell death to the propensity of reovirus to sensitize ras-transformed cells to apoptosis [35] . Wilschut pioneered the use of alphaviruses and used these for eradication of pre-established tumors in an immunization protocol [36] . In a collaborative project Rottier's and Gerritsen's groups redirected the mouse hepatitis coronavirus to human tumor cells using a monoclonal-antibody dependent targeting strategy. This led to efficient syncytium formation in the tumor cells, which may eventually enhance the antitumor efficiency of this approach [37] .

In addition, retroviral vectors are used in studies aiming at targeting T lymphocytes to tumors. In these studies adoptive transfer of autologous T lymphocytes that have been genetically modified to express antigenspecific receptors may provide tumor-specific immunity to cancer patients [38] [39] [40] . This approach is being clinically evaluated in Rotterdam, and other studies will be initiated soon in Amsterdam and Leiden. In Utrecht a strategy to manage Graft-versus-Host disease after donor lymphocyte infusions has been developed. Several of these approaches have been brought to clinical evaluation (see below). In Nijmegen, van den Berg and collaborators have developed vectors modified to express genes specifically in response to inflammatory signals for fine-tuned gene therapy in rheumatoid arthritis [41] .

Because synthetic and non-viral vectors may be preferable over viral vectors in particular gene therapy applications, new vector systems are being developed. In Groningen, the use of so-called virosomes is being evaluated by Wilschut and collaborators. Virosomes are virus-like particles, consisting of reconstituted virus envelopes, lacking any genetic material of the native virus. These studies provide insight into how macromolecules can be transferred across the cytoplasmic membrane and how the macromolecules can be released from the endosomes to the cytoplasm [36] . In Utrecht, Hennink and collaborators are exploring the use of cationic polymer based gene delivery systems. The approach here is two-sided: on the one hand new polymers are generated and characterized, and on the other hand cell biology is used to pinpoint which intracellular processes Table 2 ). limit efficient gene transfer [42] . The Lambin group in Maastricht is taking a radically different approach and has evaluated the use of recombinant anaerobic clostridial spores as highly selective gene delivery vectors [43] . Ingenuity is also evident in the recent use of a tattoo device for intradermal DNA vaccination at the Dutch Cancer Institute in Amsterdam. This procedure led to robust T cell responses and eradication of pre-established subcutaneous tumors and may be broadly applicable [44] .

An overview of completed, ongoing and future clinical gene therapy trials for the treatment of various diseases in The Netherlands is presented in Tables 2-4. In total, 14 trials have been completed (Table 2 ), 6 trials are ongoing (Table 3) , and 13 protocols will be started in the near future (Table 4 ). Dutch scientists have been in the front line in the field of clinical gene therapy as well.

As mentioned earlier, the first clinical trial worldwide on the treatment of patients with ADA deficiency with autologous stem cells genetically modified with a retroviral vector was conducted by collaborating Dutch, French and British scientists [19] . Susanne Osanto and collaborators in Leiden were amongst the first in Europe to treat patients with cancer with IL-2 gene-modified allogeneic melanoma cells that express high levels of HLA-A1 and -A2 [45] . Furthermore, Chris Bangma in Rotterdam performed a clinical study on adenovirusmediated suicide gene therapy for prostate cancer on an outpatient basis [46] . Various gene therapy concepts developed by Dutch research groups are translated into the clinic. An example is the Rotterdam trial (Table 3 , protocol IM 97-014) studying T lymphocytes retargeted to carbonic anhydrase IX as a treatment for metastasized renal cell carcinoma. This protein is overexpressed on these tumor cells. In addition, in Amsterdam AAVmediated gene therapy for patients with lipoprotein lipase deficiency is being tested ( Table 3 , protocol IM 05-001) and exon skipping by an antisense oligoribonucleotide to restore dystrophin production in Duchenne muscular dystrophy patients is being clinically evaluated in Leiden (Table 3 , no protocol ID).

Most of the Dutch clinical gene therapy trials are phase I studies aimed at the assessment of safety. Table 2 ). * This trial will be conducted in collaboration with the Dutch company Amsterdam Molecular Therapeutics (Amsterdam). The results from these trials confirm the general observations that gene therapy is well tolerated without major complications. In the Rotterdam trial on T lymphocytes retargeted to renal cell carcinoma described above (Table 3 , protocol IM 97-014), liver toxicity was encountered in the first three patients. The trial was allowed to continue according to an amended protocol [47] and two patients have now been included without side effects. In accordance to trials conducted in other countries, clear clinical efficacy of gene therapy has not been demonstrated yet in Dutch trials. In the two randomized and placebo-controlled phase III trials on gene therapy for brain tumors ( Table 2 , protocols IM 95-004/95-015) and for critical limb ischemia in diabetes mellitus, efficacy could not be established [48] or the clinical effect was only minor [49] . The first results reported for the Leiden trial studying suicide gene therapy in patients with a loosened hip prosthesis (Table 3 , protocol IM 03-001) are promising. In this trial, interface tissue around the implant is destroyed by suicide gene therapy prior to fixation of the prosthesis with cement. Ongoing and upcoming clinical trials now focus on the translation of new developments in gene therapy research aimed at improvement of efficacy and safety of this treatment modality. The first Dutch trials on oncolytic adenovirus therapy for cancer will be carried out in Amsterdam and Rotterdam (Table 4) . Furthermore, the restoration of genetic defects other than SCID is addressed, including genetic lipoprotein lipase deficiency, Duchenne muscular dystrophy, Leber congenital amaurosis causing blindness and Crigler Najjar syndrome causing irreversible brain damage due to hyperbilirubinemia (Tables 3 and 4 ). The Dutch Ministry of Health has committed to gene therapy trials by approving a 15.6 M¤ funding the 'Translational Gene Therapy Research Programme' that has been developed and is executed by the funding agency ZonMw. The programme aims to improve the translation of pre-clinical gene therapy research to the clinic (www.zonmw.nl/genetherapy). So far, 11 phase I/II trials described in Tables 3  and 4 have been supported by this programme. ZonMw stimulates the cooperation of researchers and commercial enterprises. Besides investigator-initiated clinical studies, commercial interest in clinical gene therapy in The Netherlands is increasing. The phase I/II clinical study in Leiden involving the suicide gene therapy approach for prosthesis refixation has been sponsored by Innovata Ltd. (Ruddington, UK). Currently, Amsterdam Molecular Therapeutics is participating in the Amsterdam trial on lipoprotein lipase deficiency (Table 3 , protocol IM 05-001). Another Dutch company, Prosensa B.V. based in Leiden, is involved in the Leiden trial on Duchenne muscular dystrophy (Table 3 , no protocol ID). The Dutch drug development company ORCA Therapeutics B.V. in Amsterdam is currently preparing its lead product, an oncolytic adenovirus-expressing tumor suppressor p53, for clinical evaluation in cancer patients. Another company, Arthrogen B.V. (Amsterdam), is developing AAV-based gene therapy for rheumatoid arthritis for future clinical application. Furthermore, at least four Dutch clinical centres will participate in large phase III international multicenter immuno-gene therapy trials on prostate cancer sponsored by Cell Genesys, Inc. (San Fransisco, USA) ( Table 4 , protocols IM 06-001/06-003/06-010/06 011).

Dutch scientists involved in fundamental and clinical gene therapy research are significantly contributing to the progress made in this research area, including the translation of innovative developments into the clinic. The latter process has been greatly stimulated by the Translational Gene Therapy Research Programme supported by ZonMw. Many of the Dutch academic research centers have now made long-term commitment to clinical research involving gene therapy, which has resulted in the establishment of various gene transfer facilities. The national regulatory conundrum has precipitated into the establishment of the Gene Therapy Office, which coordinates the assessment of the clinical protocols by the various regulatory bodies. This should aid the applicants and should warrant efficient communication between and amongst the pertinent regulatory bodies and the applicants (see paper by Bleijs et al., this issue). The atmosphere surrounding Dutch gene therapy may also be evident from the fact that several active Dutch scientists have accepted invitations to serve on review panels and advisory boards that are involved in the regulation of clinical gene therapy. Also the Dutch Society of Gene Therapy has a formal role in the evaluation of the Gene Therapy Office. This tight network of interrelations ensures efficient communication between the Dutch gene therapy community and bodies that should facilitate safe and efficient progress in this powerful field.

Beijerinck's work on tobacco mosaic virus: historical context and legacy

Isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture

Genetic content and evolution of adenoviruses

The quest for human cancer viruses

Oncogenic effects in hamsters of human adenovirus types 12 and 18

Tumorigenicity of cells transformed by adenovirus type 12 by evasion of T-cell immunity

Expression of class I major histocompatibility antigens switched off by highly oncogenic adenovirus 12 in transformed rat cells

DNA Replication and Human Disease, DePamphilis ML

A new technique for the assay of infectivity of human adenovirus 5 DNA

Characteristics of a human cell line transformed by DNA from human adenovirus type 5

Characterization of 911: a new helper cell line for the titration and propagation of early region 1-deleted adenoviral vectors

Malignant transformation of human embryo retinoblasts by cloned adenovirus 12 DNA

Who's afraid of replication-competent adenoviruses?

New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses

Safety of recombinant adenoviruses produced on adenovirus-transformed human cells

Tumorigenicity assessments of Per.C6 cells and of an Ad5-vectored HIV-1 vaccine produced on this continuous cell line

Approaches to the release of a master cell bank of PER.C6 cells; a novel cell substrate for the manufacture of human vaccines

Isolation of cDNA clones for human adenosine deaminase

Bone marrow gene transfer in three patients with adenosine deaminase deficiency

Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease

Murine leukemia virusinduced T-cell lymphomagenesis: integration of proviruses in a distinct chromosomal region

Involvement of c-myc in MuLV-induced T cell lymphomas in mice: frequency and mechanisms of activation

Tumorigenesis in transgenic mice: identification and characterization of synergizing oncogenes

High-throughput retroviral tagging to identify components of specific signaling pathways in cancer

Good news for gene therapy

Genetic targeting of adenovirus vectors using a reovirus sigma1-based attachment protein

Spacers increase the accessibility of peptide ligands linked to the carboxyl terminus of adenovirus minor capsid protein IX

Increased glia-specific transgene expression with glial fibrillary acidic protein promoters containing multiple enhancer elements

Selection of tumour specific promoters for adenoviral gene therapy of cholangiocarcinoma

A small chimeric promoter for high prostate-specific transgene expression from adenoviral vectors

The organotypic multicellular spheroid is a relevant three-dimensional model to study adenovirus replication and penetration in human tumors in vitro

Gene therapy for esophageal carcinoma: the use of an explant model to test adenoviral vectors ex vivo

An ex vivo human model system to evaluate specificity of replicating and nonreplicating gene therapy agents

Locoregional delivery of adenoviral vectors

Sensitization to apoptosis underlies KrasD12-dependent oncolysis of murine C26 colorectal carcinoma cells by reovirus T3D

Virosomes for antigen and DNA delivery

Targeting nonhuman coronaviruses to human cancer cells using a bispecific single-chain antibody

Targeting selfantigens through allogeneic TCR gene transfer

Reprogramming of virus-specific T cells into leukemia-reactive T cells using T cell receptor gene transfer

Single chain Ig/gamma gene-redirected human T lymphocytes produce cytokines, specifically lyse tumor cells, and recycle lytic capacity

An inflammationinducible adenoviral expression system for local treatment of the arthritic joint

Cellular uptake of cationic polymer-DNA complexes via caveolae plays a pivotal role in gene transfection in COS-7 cells

Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo

A rapid and potent DNA vaccination strategy defined by in vivo monitoring of antigen expression

Vaccination of melanoma patients with an allogeneic, genetically modified interleukin 2-producing melanoma cell line

Virus specific immune responses after human neoadjuvant adenovirus-mediated suicide gene therapy for prostate cancer

Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience

A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme

Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia: a double-blind randomized trial

Treatment of relapsed malignant glioma with an adenoviral vector containing the herpes simplex thymidine kinase gene followed by ganciclovir

A phase 1-2 clinical trial of gene therapy for recurrent glioblastoma multiforme by tumor transduction with the herpes simplex thymidine kinase gene followed by ganciclovir. GLI328 European-Canadian Study Group

Virus-directed enzyme prodrug therapy: intratumoral administration of a replicationdeficient adenovirus encoding nitroreductase to patients with resectable liver cancer

Safety of intravenous administration of a canarypox virus encoding the human wild-type p53 gene in colorectal cancer patients

Tumoral and immunologic response after vaccination of melanoma patients with an ALVAC virus encoding MAGE antigens recognized by T cells

Immunogenicity, including vitiligo, and feasibility of vaccination with autologous GM-CSF-transduced tumor cells in metastatic melanoma patients

Intratumoral administration of canarypox virus expression human IL-2 gene (ALVA-IL-2) in cancer patients: results of a phase I study

Biological activity and safety of adenoviral vector-expressed wild-type p53 after intratumoral injection in melanoma and breast cancer patients with p53-overexpressing tumors

A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease

PET for evaluation of differential myocardial perfusion dynamics after VEGF gene therapy and laser therapy in end-stage coronary artery disease

The authors would like to thank Drs. Marjolein van Mierlo and G. A. P. Hospers for their support on the inventory of Dutch clinical gene therapy trials. All clinical gene therapy investigators from universities and industry who have contributed to this paper are acknowledged.