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Gene Therapy (2007) 14, 1555–1563& 2007 Nature Publishing Group All rights reserved 0969-7128/07 $30.00 REVIEWProgress and Prospects: Gene Therapy ClinicalTrials (Part 2) This is the second part of a review summarizing progress transcarbamylase deficiency, a -antitrypsin deficiency, haemo- and prospects in gene therapy clinical research. Twenty key diseases/strategies are succinctly described and commented Eric Alton, Stefano Ferrari and Uta Griesenbach on by leaders in the field. This part includes clinical trials for skin diseases, neurological disorders, HIV/AIDS, ornithine Keywords: clinical trials; skin diseases; Huntington’s disease; Alzheimer’s disease; HIV/AIDS; OCT; hemophilia; ADA-SCID;cancer  Gene therapy clinical trials for cancer are moving  Lentiviral vectors will move from preclinical safety rapidly from phase I to III and the first anti-cancer assessment studies to clinical trials.
gene therapy drug has been licensed in China.
 Studies to evaluate the genotoxicity of any gene  Some serious adverse events following gene therapy transfer vector will become a fundamental feature of have occurred, but progress has been made in understanding and overcoming these problems.
 Stem cells from a range of different tissues/organs  Clinical trials involving genetic modification of stem will become the target cells for many gene therapy cells other than bone-marrow derived (for example skin precursor cells) have been carried out.
 Ex vivo transduction of stem cells with integrating retroviruses ensures high and persistent level of genetransfer and is a promising strategy for severaldiseases.
transduction of epidermal stem cells using oncoretroviralvectors succeeded in fully correcting the genetic defects in keratinocytes obtained from patients with X-linked Inserm, University of Nice-Sophia Antipolis, Nice, France ichthyosis, lamellar ichthyosis, xeroderma pigmentosum, For a number of severe and untreatable diseases affecting junctional and dystrophic epidermolysis bullosa (JEB and the skin, the establishment of the phenotype/genotype relationship has made the development of therapeutic However, despite the encouraging results of the pre- approaches based on cutaneous gene transfer attractive.
clinical studies, evidence of clinical efficacy has been Indeed, the skin is accessible to direct in vivo gene targeting obtained so far only for JEB, a severe recessive skin and, with respect to ex vivo gene transfer, there is the blistering condition due to defects in genes for the capacity to reconstruct transplantable epithelia by tissue keratinocyte adhesion molecule laminin The small size engineering using keratinocyte cell cultures enriched in of the transgene required to cure the genetic defect in stem cells which give rise to a self-renewing tissue. While patients with an abnormal expression of the beta3 chain of the in vivo approaches turned out to be inefficient, laminin 5, and the restoration of the adhesion properties ofthe transduced JEB keratinocytes facilitated the implemen-tation of a pilot phase-I clinical trial on a single adult patient.
Correspondence: Dr U Griesenbach, Department of Gene Therapy, This pioneering work took advantage of the fact that: Emmanuel kaye Building, NHLI, Imperial College, Manresa Road,London, UK.
E-mail: (1) the recombinant retrovirus targeted almost 100% of Received 24 August 2007; accepted 24 August 2007 (2) the selected patient expressed low levels of the the striatal neurons in various experimental models mutant protein, which prevents an immune response mimicking HD and is, to date, the only gene therapy tested in patients. Adenoviraland lentiviral vectors (3) the grafted areas of the body had non-healing lesions were used to deliver CNTF into the striatum of rodent due to the absence of epithelial stem cells able to and primate HD models and showed neuronal survival compete with the transplanted epithelia in the and prevented behavioural deterioration. The only phase I gene therapy trial currently performed in HD patientsused encapsulated genetically-engineered BHK cells After nearly 2 years of follow-up the study has over-expressing CNTF.BHK cells were placed into provided proof-of-principle for the feasibility and safety polymer capsules formed by a semi-permeable mem- of a transduction/transplantation strategy that might be brane and introduced, via stereotactic neurosurgery, into applicable to other genetic skin disorders. However, the the right lateral ventricle of six HD patients. Capsule successful treatment of JEB by gene therapy needs to be membranes contain pores, which allow the release of confirmed by clinical trials with a larger number of CNTF and the entry of oxygen and nutrients, but prevent patients to establish statistical significance in terms of the the penetration of large proteins and host cells, and thus definition of the clinical parameters required for inclusion protects the cells from a host immune reaction. This of candidates, and the constellation of genetic mutations strategy allows the use of xenogenic engineered cells and treatable without induction of immune responses. Due to prevents proliferation of cells outside the capsule. The the extreme rarity of JEB patients, a consortium of trial lasted 2 years with a replacement of capsules every European groups is currently preparing a trans-national 6 months. The procedure proved to be safe. However, recruitment for clinical studies to be carried out in the only 11 out of the 24 retrieved capsules still released next years. However, for approval of these trials, safety quantifiable amounts of CNTF after 6 months. Primate concerns for the potential genotoxicity of the retroviral models had shown that implantation of 4 capsules was vectors must be met, notably, the development of self- required to achieve correction of the phenotype. The inactivating vectors safer than the first generation clinical trial, however, was primarily a safety trial and oncoretroviral backbone used in the pilot assay.
only 1 capsule was implanted per patient. It was, Despite these significant advances, the question therefore, not surprising that there was no clear clinical remains as to whether a similar ex vivo approach can be benefit in the patients. Nevertheless, surrogate marker extended to other genetic skin disorders with a medical (long latency reflex and somatosensory evoked poten- and societal impact larger than JEB, including dominant tials) improved in three out of six patients. A phase II inherited conditions. While the need for vectors present- trial allowing assessment of the therapeutic potential of ing the best compromise in terms of capacity to CNTF gene therapy to the brain in HD patients is accommodate large DNA fragments, transfer efficiency, currently waiting for more efficient delivery vectors.
safety, and biodistribution, undoubtedly remains a major Gene therapy is a source of hope for the cure or the challenge, the immune reaction in patients lacking improvement of HD but in vivo experimentation in expression of a gene product raises questions which at animal models are still required to meet the challenge of A-C Bachoud-Le´vi1,2,3 and M Peschanski41INSERM, U841, NPI, Creteil, France; 2ENS, DEC, Paris, France; 3AP-HP, Hospital Henri-Mondor, Sce de 1Department of Neurosciences, University of Neurologic, Creteil, France; 4INSERM/UEVE, U861, California, San Diego, La Jolla, CA; 2Veterans Administration Medical Center, San Diego, CA Huntington’s disease (HD) is a monogenic dominant- The nervous system growth factor Nerve Growth Factor negative neurodegenerative disease caused by the (NGF) prevents the death of, and stimulates the function expansion of CAG repeats in the IT5 gene on Chromo- of, cholinergic neurons in the rodent and primate central some 4 encoding huntingtin. HD occurs in adulthood nervous system. Further, NGF administration improves (30–50 years of age) and is fatal within 15–20 years of learning and memory in lesioned and aged rats, and diagnosis. Since identification of the HD’s gene muta- improves neuronal function and prevents cell death in a tion, the use of gene therapy has been envisaged. Down- regulation of the mutant IT5 allele while leaving the potent effects of NGF on cholinergic systems in the brain normal allele intact, is effective in in vitro models. Intra- are of clear relevance to patterns of neuronal degenera- striatal injection of interference RNA directed against tion in the most common human neurodegenerative mutant human huntingtin reduced protein expression disorder, Alzheimer’s disease (AD). However, to test the and improved behavioural and pathological abnormal- hypothesis that NGF will reduce neuronal degeneration ities in transgenic However, numerous technical and slow cognitive decline in AD, a satisfactory method issues have to be solved before embarking on a human of NGF delivery to the brain is required.
trial involving the delivery of RNAi to the brain.
In 2001 we initiated a trial of ex vivo NGF gene Gene therapy using neurotrophic factors such as NGF, delivery in early stage AD. To restrict NGF delivery to BDNF or CNTF (please define abbreviations) has also the basal forebrain in a localized and targeted manner, been postulated for the treatment of HD. CNTF deserves genetically modified cells served as biological mini- special attention. The protein reduced neuronal death of pumps to provide therapeutic levels of NGF to basal Gene Therapy
Gene therapy clinical trials (Part 2)E Alton et al forebrain neurons. Experiments in rodents and primates progressive and tragic course of this common human confirmed that grafts of NGF-expressing autologous fibroblasts were equally effective compared to intraven-tricular NGF protein infusions in preventing cholinergic Conflict of Interest Statement: AB and MHT have a neuronal degeneration and stimulating cell function.
financial interest in Ceregene, Inc.
Additional dose escalation studies confirmed the safetyand persistence of NGF gene expression over timeperiods of at least one year in the non-human primate brain. Based on the extensive pattern of efficacy andsafety demonstrated after ex vivo NGF gene delivery, a Phase I trial of ex vivo NGF gene delivery was initiated Department of Pathology, Jefferson Medical College, in 8 subjects diagnosed with early AD.Fibroblasts obtained from skin biopsies were genetically modified The challenge in HIV/AIDS gene delivery has been to using Moloney leukemia virus (MLV) retroviral vectors devise strategies to transduce sufficient HIV-susceptible to express NGF, and these autologous cells were cells durably enough to provide adequate antiretroviral stereotactically implanted adjacent to the Nucleus basalis protection. Recent clinical gene transfer studies give of Meynert (NBM) region of the brain. This brain region reason for optimism that this goal is feasible.
(of approximately 1 cm length and 3 mm width) contains Transgenes. Many transgenes protect HIV-susceptible degenerating cholinergic cell bodies in AD, and can be cells from HIV infection and/or replication in vitro. These practically targeted using techniques of ex vivo gene include genes encoding proteins that inactivate or inhibit delivery, yet this compact nucleus extends axonal HIV-1 proteins, or that make the cellular environment projections throughout the entire cortex, allowing NGF less hospitable to HIV-1 infection or replication.
to influence diverse cortical regions by targeting of the Untranslated RNA–antisense, ribozymes, decoys and interfering RNA–similarly may target viral or cellular Surgery for stereotaxic injections of cells was initially functions needed for HIV-1 infection and replication.
conducted in patients that were awake but sedated.
Many transgenes confer potent anti-lentiviral protection.
However, two patients abruptly moved during the Their specific structures and functions are probably less surgical session causing brain hemorrages. To mitigate critical than the means by which they are delivered, the this risk, all other subjects underwent surgery under cells to which they are delivered and how long they deep sedation or general anesthesia, and no further surgical adverse effects occurred. With follow-up now Therapeutic strategies. Clinical gene transfer studies for available for up to 5 years, no adverse events related to HIV/AIDS generally target hematopoietic stem cells either NGF or viral vector delivery have occurred. PET (HSC) or peripheral blood T cells (therapeutic immuni- scans in bilaterally-treated subjects indicated a signifi- zation is a separate topic.) Recent human studies cant increase in metabolic activity throughout the cortex, involving HSC and T cell transduction have begun to consistent with potential widespread modulation of offer hope as clinically feasible strategies.
cortical activity by NGF effects on the Nucleus basalis.
(a) T cells. Recent early phase clinical studies report Cognitive testing suggested potential slowing of cogni- that ex vivo gene delivery to stimulated blood T cells tive decline, although reliable conclusions in this respect using lentiviral or retroviral vectors, followed by expan- cannot be drawn from a small sample of subjects lacking sion and reinfusion, has so far been safe.In most placebo controls or blinded assessments. In addition, patients percentages of circulating lymphocytes (PBL) histological analysis of the brain of one of the subjects, carrying the transgene decline, often becoming unde- who died 5 weeks following gene delivery, demonstrated tectable by 6 months. However, gene-marked blood cells sprouting of cholinergic axons from the Nucleus basalis persist at low levels in some subjects.As most into NGF secreting grafts, a classic ‘trophic effect’ of the patients are simultaneously treated with highly active sort previously observed in rodent and primate studies.
antiretroviral pharmacotherapy (HAART), the therapeu- This anatomical response to NGF clearly established that tic effectiveness of transplanting gene-modified PBL is degenerating cholinergic neurons in the AD brain are difficult to assess, but potentially promising data are reported.Since this approach targets mature circulating As gene delivery technology advanced in the period cells, rather than progenitors, it probably is safer than following the initiation of the above Phase I trial, HSC gene delivery. However, as PBL have finite life preclinical studies were repeated using AAV-NGF gene expectancies, repeated transplants would probably be delivery. AAV-NGF injections in the brains of rodents necessary to maintain therapeutic effect.
and primates reproduced the full spectrum of biological (b) HSC. Most early studies of gene transfer for HIV/ efficacy observed in previous animal studies, with no AIDS involved transduction of HSC.This approach evidence of nervous system toxicity. Based on these offers the potential advantage of transducing progenitors findings, a second Phase 1 trial of AAV-NGF gene of several populations of HIV-susceptible cells. Ex vivo delivery was recently conducted in early and mid-stage stimulation and transduction are needed because oncor- AD, sponsored by Ceregene, As the phase I AAV- etroviral and lentiviral vectors transduce resting HSC NGF trial reaches a close, a multi-centre phase II sham poorly. Transduced cells are then reimplanted. HSC gene surgery controlled, double-blinded trial in AD is transfer promises a permanent supply of HIV-resistant planned. The phase II trial will explore the potential derivative cells of multiple lineages. Without pre- effect size of NGF gene delivery on cognitive decline in transplant conditioning engraftment is inefficient, yet AD, advancing a program that, if successful, will provide the most effective conditioning–bone marrow ablation–is a potential opportunity to fundamentally alter the impracticable as routine AIDS therapy. Results have been Gene Therapy
mixed: levels of gene-modified blood cells generally through 6 cohorts with evidence of modest gene transfer become low or undetectable within months of transplan- (by in situ hybridization of liver biopsies) and some tation. There is, however, evidence that gene-modified, toxicity.The second research subject in the highest HIV-resistant mature cells persist and have a survival projected dose developed a very different response to compared to their unmodified cousins.
therapy than the previous 17 subjects that led to severe Reports of tumors after human HSC gene delivery with systemic inflammation and lethal multi-organ failure.
oncoretroviral vectors mandate proceeding cautiously.
Subsequent studies suggested this was caused by an Strategies to increase percentages of transgene-positive exaggerated and unexplained innate immune response blood cells. Clinical T cell and HSC gene transfer trials suggest that for HIV/AIDS gene therapy to be clinically OTC deficiency remains an important model for the useful percentages of transduced cells must be increased.
evaluation of novel liver-directed gene therapies. Child- Thus, strategies employing in vivo selection are being ren born with a complete deficiency of OTC suffer severe considered. Gene transfer of methylguanine methyl- morbidity and premature mortality despite state-of-the- transferase (MGMT) followed by nitrosourea treatment art pharmacologic, dietary and supportive clinical treat- (BCNU) shows promise in selecting for gene-modified ments. The relative success of liver allograft transplants cells, especially as substrates less toxic than BCNU are in this disease indicates that hepatocyte gene transfer identified.Other approaches to selection and HSC should be curative. Promising new vectors based on transduction used in human and animal studies have adeno-associated viruses have shown tremendous pro- mise in mouse models of OTC deficiency.This vector Conclusions and future prospects. Recent clinical experi- platform is capable of very efficient targeting of ments using gene transfer to treat HIV/AIDS have hepatocytes in vivo without apparent activation of innate emphasized T cell-directed gene delivery, ex vivo expan- immunity and long-term transgene expression. Trans- sion and reinfusion. Improvements in HSC-directed gene gene-specific T cell responses appear to be blunted in the transfer also offer promise. These human studies suggest context of AAV delivered to the liver, which will help that progress is being made, in terms of both improved reduce toxicity and improve efficacy when treating persistence of gene-modified cells and antiretroviral patients with deletions, frame shift or premature stop mutations in the OTC gene. One concern that requiresadditional investigation prior to embarking on clinicaltrials is the oncogenicity of the vector and the activationof T cells to the input AAV capsids.
Gene Therapy Program, Department of Pathologyand Laboratory Medicine, University of Pennsylvania, The Children’s Hospital of Philadelphia, Philadelphia, The liver is an important target for gene therapy because of the central role it plays in a variety of metabolic Hemophilia is the X-linked bleeding disorder caused by pathways and in the production of serum proteins. The mutations in the genes encoding Factor IX (an enzyme) or first clinical trial of gene therapy for the liver was based Factor VIII (cofactor for the enzyme), both required for on transplantation of ex vivo modified homologous normal blood coagulation. Despite many advances, hepatocytes in patients with familial hypercholestero- attempts to manage a lifelong disease by intravenous lemia. The subsequent development of viral vectors infusion of a protein with a relatively short half-life capable of targeting cells in vivo suggested a more present major obstacles. Moreover, the cost of clotting practical and efficient approach for genetically modify- factor concentrates (typically $50 000-$100 000/year for ing the liver in which the vector is simply infused into an adult with severe disease) has been prohibitive, in fact, the circulation gaining access to hepatocytes through the out of reach, for the majority of the world’s hemophilia population. These factors have fuelled interest in devel- The first demonstration of correction of a liver oping alternate therapeutic approaches such as gene metabolic defect in an animal model following in vivo transfer. At the outset, hemophilia was deemed an vector delivery used a recombinant adenovirus infused appealing target for gene transfer, not only because of into newborn mice with a deficiency of ornithine the relatively large population of affected individuals, but transcarbamylase (OTC).A deficiency of this enzyme also because the therapeutic gene does not need to be in humans leads to a syndrome of repeated episodes of tightly regulated, and even modest increases in circulat- life threatening hyperammonemia. The mouse models ing levels of clotting factor (e.g. from o1% to B5%) result have a partial defect of OTC and a similar phenotype. We in a substantial improvement in symptoms, effectively began to develop the adenovirus platform for evaluation converting severe disease to a mild phenotype.
in research subjects with OTC deficiency. Prior to a Multiple strategies were pursued, in phase I/II trials consideration in humans, we developed improved involving a total of 41 patients with severe hemophilia A or generations of the vector to attenuate further expression B. These trials were sponsored by biotechnology companies of viral genes and diminish the associated toxicity. A including Transkaryotic Therapies (ex vivo transduction of phase I study was initiated in which adults with a partial autologous fibroblasts by a plasmid expressing a truncated deficiency of OTC received a third generation adenoviral Factor VIII molecule [B-domain deleted, BDD])Chiron vector expressing OTC via infusion into the hepatic Corporation (intravenous infusion of a retroviral vector artery. The phase I dose escalation study progressed expressing BDD F.VIII),Avigen, Inc. (AAV-mediated gene Gene Therapy
Gene therapy clinical trials (Part 2)E Alton et al transfer into skeletal muscle [first trial] and liver [second disorder characterised by immunological defects and trial] of a Factor IX minigene),and GenStar Corporation organ/systemic toxicity caused by the accumulation of (intravenous infusion of a gutted adenoviral vector purine metabolites. In the absence of an HLA-identical sibling donor, hematopoietic stem cell (HSC) transplan- All of these trials were first-in-class, i.e. the first tation from alternative donors is restricted by high instance of a particular vector being used in a particular morbidity and mortality, while enzyme replacement target tissue, and thus yielded important safety data for therapy (PEG-ADA) often fails to sustain long-term the approaches under investigation. None, however, immunity. Since September 1990, over 35 ADA-SCID resulted in long-term expression of the clotting factor at patients have been enrolled in phase I/II gene therapy therapeutic levels. Each approach encountered a critical trials in 6 different centres worldwide. Gene therapy was issue, either in terms of safety or efficacy, that required based on infusions of autologous peripheral blood further laboratory or clinical investigation. In the wake of lymphocytes (PBL) or HSC transduced ex vivo with these disappointing initial results, more rigorous analysis of pre-clinical data in the hemophilic dog model has Long-term follow up of children receiving PBL gene supported continued pursuit of some strategies and therapy has demonstrated long-term persistence of gene modification or abandoning of others. Circulating factor corrected T cells 412 years after the last infusion, levels in the range of 5–25% have now been achieved in without adverse However, all patients were hemophilic dogs or non-human primates using three maintained on PEG-ADA therapy, abrogating the poten- distinct approaches: a retroviral vector can be infused into tial selective growth advantage for gene corrected cells neonatal dogs, where hepatocytes are still rapidly and preventing the full evaluation of gene therapy dividing, and yield high-level clotting factor expression; efficacy. Discontinuation of PEG-ADA in one patient AAV vectors can be delivered to skeletal muscle via an treated with engineered PBL led to in vivo selection for intravascular route and can transduce a large number of transduced T cells with improvement of immune func- muscle fibers yielding therapeutic levels;or AAV vector tions, but not complete metabolic detoxification.
can be delivered to liver via the hepatic artery, the portal The cumulative experience from pilot HSC gene vein, or in the case of AAV-8 vectors, intravenously, to therapy studies showed an inadequate level of engraft- yield therapeutic levels of clotting factor ment of transduced cells, supporting the need for The retroviral approach will require either very young patients’ pre-conditioning in order to achieve long-term subjects (in utero or neonatal, where hepatocytes are engraftment of multipotent HSC and full correction of rapidly dividing), or some pharmacologic or surgical ADA-SCID phenotype. For this purpose, a gene therapy intervention to induce hepatocyte replication in older protocol, initiated in 2000, combined infusion of retro- subjects. This approach is not actively being pursued for virally transduced bone marrow CD34+ cells with low- hemophilia. AAV transduction of skeletal muscle is being dose busulfan conditioning.In the first two patients actively investigated in clinical trials for the muscular reported, long-term engraftment of multilineage HSC, dystrophies, and pursuit of this approach for hemophilia differentiating in both myeloid and lymphoid trans- will likely depend on development of intravascular duced cells, resulted in sustained ADA activity and (rather than intramuscular) delivery techniques that are efficient systemic detoxification. Gene therapy led to an likely to be pursued first in a setting of muscle disorders.
improvement of cellular and humoral responses in the Delivery of AAV vector to the liver for hemophilia has absence of enzyme replacement therapy, with proven shown the most promising results to date, having clinical benefit.The initial results have now been achieved circulating levels of B10–12% in one clinical extended in a larger cohort of patients (at present 12 study, although the duration of expression was only patients have been treated), and none of them showed weeks, as opposed to years in hemophilic dogs.The adverse events related to gene transfer.
etiology of the shortened duration of expression that Engraftment of ADA-carrying cells and sustained occurred in humans but not in animal models appears to biochemical and immunological correction was recently be the presence of memory CD8+ T cells to AAV capsid, obtained in a patient treated in the UK using melphalan found in humans because they are the only natural hosts as a pre-conditioning drug.An important limitation for for wild-type AAV-2 infection. Two trials of AAV in liver, the use of autologous transduced HSC may be repre- reflecting two possible solutions to this dilemma (tran- sented by the low number of stem cells availablein the sient immunosuppression to block the immune response bone marrow or by pre-existing chromosomal altera- to capsid until it is cleared from the cell or the use of an tionsin progenitor cells. Another critical issue is the alternate AAV serotype [AAV-8] to which humans are not potential risk of insertional mutagenesis related to the use naturally exposed) have been proposed and should be of retroviral vectors. Our analyses of a large collection of integration sites in ADA-SCID patients treated with genetherapy showed no clonal expansion and the absence ofin vivo skewing of the integration profil In summary, results of the latest clinical trials have provided evidence for safety and efficacy of gene therapyfor ADA-SCID, establishing as a paradigm for other A Aiuti1, F Cattaneo1 and MG Roncarolo1,2 genetic diseases, the use of reduced intensity condition- 1Pediatric Clinical Research Unit, San Raffaele Telethon ing in facilitating the engraftment of gene corrected HSC.
Institute for Gene Therapy (HSR-TIGET); 2Vita-Salute An extended follow up of gene therapy treated patients will be required to confirm these results and to compare Severe Combined Immunodeficiency (SCID) due to the the outcome of gene therapy with other treatment, in lack of adenosine deaminase (ADA) is a rare, fatal, order to obtain its recognition as an approved therapy.
Gene Therapy
transferring genes to T cells have been safe to date, thisstrategy is being implemented with caution. Finally, the in vivo expansion and persistence of adoptively trans- Center for Cell and Gene Therapy, Baylor College ferred immune cells can be increased by prior lympho- of Medicine, Texas Children’s Hospital, and The depletion of the patient with drugs, radiation or monoclonal antibodies, thereby removing unwanted Despite the success of ‘passive’ immunotherapy with inhibitory T regulatory cells, and favoring homeostatic monoclonal antibodies, treatment of cancer by active expansion of the infused anti-tumor T cells.
immunization or by adoptive transfer of a cellularimmune response has proven problematic. Most tumorsexpress weak self-antigens to which the host is tolerant, and they lack the co-stimulatory molecules necessary forfull recruitment of cellular immunity. Many tumors also employ active immune evasion strategies.Gene trans- Centre for Molecular Oncology, Barts and the London fer is being used to overcome these obstacles and the Queen Mary’s School of Medicine and Dentistry combination of cellular and gene therapy is now Oncolytic viruses multiply selectively within cancers, producing consistent rather than anecdotal success in causing cell death, with released mature viral particles infecting neighbouring cells. The tumour suppressor and Increasing antigenic stimulation from tumor cells and their cell defence mechanisms that viruses subvert are the microenvironment: Gene transfer can be used to increase same as those lost in carcinogenesis. Some viruses are the ‘visibility’ of tumors to the immune system by in vivo intrinsically tumour-selective in their replication, whilst or ex vivo modification of malignant cells, using genes the large DNA viruses require deletion of key viral genes encoding immunostimulatory cytokines (e.g. TNF-a, IFN-a), or co-stimulatory molecules (e.g. CD40 ligand).
Although anecdotal reports of virus-induced anti- Both approaches augment in vivo tumor antigen pre- tumour responses appeared over 100 years ago, the first sentation and help to recruit tumor-specific T cells and B formal clinical trials tested the E1B-55K deleted adeno- cells. Alternatively, professional Antigen Presenting Cells virus dl1520 (Onyx-015), which was hypothesised to (APCs) can be modified using tumor-peptides, recombi- replicate selectively in p53-negative cells. At least 15 nant protein, tumor lysates, RNA, DNA, or viral vectors, clinical trials using dl1520 have been completed and a to promote the activation of effector T cells in derivative, H101, is licensed in China. However, single Recently the FDA recommended approval for the first agent activity was poor, with minimal systemic efficacy genetically modified Dendritic Cell (DC) vaccine (for and the mechanism of activity was unrelated to p53 recurrent prostate cancer), opening the door for future status: in addition to inhibition of p53, E1B 55K promotes new agents of this type. Meanwhile current early phase the export of late viral mRNA from the nucleus. Tumour clinical studies are optimizing the immunostimulatory cells have altered mechanisms for export of these RNA efficacy of DC vaccines, by augmenting expression of species and thus do not rely on E1B 55K. Nonetheless, costimulatory molecules (such as CD40 ligand), enhan- doses of up to 10particles were safely administered by cing their secretion of immunostimulatory cytokines, or a variety of routes. Clinical trials of second generation adenoviral mutants, such as dl922-947 or D24, have not Modification of effector T cells: Sustained tumor elimina- yet commenced. Three clinical trials of transcriptionally- tion usually requires the presence and persistence of regulated adenoviruses, with the Prostate-specific Anti- large numbers of effector T cells. While active vaccination gen (PSA) promoter/enhancer elements driving E1 attempts to recruit these cells in vivo, the inhibitory expression, have been completed in hormone-refractory environment produced by cancer cells impedes this prostate cancer. CV706 and CG7870 were administered process. An alternative is to prepare effector T cells intratumourally and intravenously respectively with no ex vivo and adoptively transfer them to the patient.
significant toxicities and some transient PSA responses.
Tumor specificity can be induced through the selection of All Herpes simplex viruses tested in clinical trials to subpopulations expressing the appropriate native T cell date are deleted in one or both copies of the ICP34.5 receptor, or by transfer and expression of tumor specific gene, which inhibits the interferon/protein kinase R receptors derived from antibodies or from the ab TCR of response to viral infection. 1716 has been safely given by tumor specific T cell clones. For example, Morgan and direct injection in melanoma and glioma at doses of 105 colleagues expressed the a and b chains of an anti- pfu, whilst G207, which is also deleted in ICP6 MART-1 TCR in T cells from 15 patients with melanoma, (ribonucleotide reductase), has been given by intra- tumoural injection at doses of up to 3 Â 109 pfu in glioma We can improve the functionality as well as the with no dose limiting toxicities. OncoVexGMÀCSF is based specificity of adoptively transferred T cells. We can upon a clinical HSV1 isolate, rather than laboratory increase homing to tumor sites by expressing chemokine strains, and is deleted in both ICP34.5 and ICP47 (to receptors specific for the molecules the tumors produce, increase MHC class I presentation) and encodes GM-CSF.
while T cell expansion and persistence in the hostile Doses of up to 108 pfu mlÀ1 were given by direct tumor microenvironment can be increased by expressing intratumoural injection to cutaneous metastases or transgenes that confer resistance to apoptosis, senes- melanoma, with evidence of local, but not distant, cence, or inhibitory cytokines, or that provide autocrine T cell Any genetic strategy that enhances the A tk-negative Vaccinia virus mutant encoding GM- survival of T cells, however, runs the risk of producing CSF was tested in preliminary clinical trials in 1999, with Gene Therapy
Gene therapy clinical trials (Part 2)E Alton et al evidence of local responses in melanoma metastases tribution of the transferred gene compared to retroviral injected with up to 2 Â 107 pfu twice weeklyThere is vectors, while others have suggested that the immune also now renewed interest in Vaccinia, but no trial has response associated with expression of a suicide gene is an important contributor to the anticancer response. The Of the naturally occurring oncolytic viruses, New- efficacy of intratumoral administration of a replication- castle Disease Virus (NDV) and Reovirus have been defective adenovirus bearing Hstk may soon be known studied in phase I/II trials. NDV is a single-stranded from results of a large phase III clinical trial for human paramyxovirus that, like ICP34.5À HSV mutants, exploits malignant glioma about to be completed in Europe.
defects in the interferon response pathway to achieve Refinements will undoubtedly be required. Oncolytic tumour selectivity. In initial studies, intravenous doses of viruses delivering suicide genes may help address issues 1.2 Â 1011 pfu mÀ2 were associated with dose-limiting relating to the inefficiency of vector biodistribution and diarrhoea and flu-like symptoms. However, using an suicide gene delivery. Determination of whether the intra-patient dose-escalation desensitisation regime, the observed inflammatory response, particularly in the initial same doses could be administered with reduced toxi- phases, is beneficial by setting up an adaptive immune cityIn the two trials, objective responses were seen in response, blank or is detrimental by its elimination of six patients. Reovirus, a double stranded RNA virus, vector and transgene is also of importance. Promising replicates in cells with deregulated Ras signalling. Direct avenues of research include the identification of combina- intra-tumour injections of up to 109 pfu were given in tions of therapies that synergize with suicide gene therapy glioma safely, but without objective rPhase II (e.g. radiation, chemotherapy) or combinations based on studies in conjunction with radiotherapy in patients with new prodrug-activation paradigms. Imaging analysis of cutaneous metastases are on-going (K Harrington, gene transfer may provide useful correlates of gene therapy responses in humans. Finally, tumor distribution In summary, several oncolytic viruses have been tested of prodrug and the use of mutants or species variants of in clinical trials, with impressive safety records. All can the suicide gene that bioactivate the prodrug with higher induce anti-tumour responses after direct injection, but catalytic efficiency also need to be explored. Ultimately, systemic activity remains a challenge. Strategies for the the future of suicide gene therapy for cancer will depend immediate future will include arming viruses with on a successful phase III trial. The road to such a trial is transgenes and combining with chemo- and/or radio- expensive and fraught with potential roadblocks. How- therapy. For example, a phase II study of OncoVexGMÀCSF ever, the plight of patients with advanced cancers requires with chemoradiotherapy in locally advanced head and that such a journey be undertaken through carefully neck cancers is on-going. However, in the longer term, designed studies that maximize the scientific knowledge gaining a fuller understanding of host cell factors that gained and help establish which cancers, and which determine viral sensitivity as well as manipulating the patients, are likely to most benefit from this therapeutic immune responses to both virus and cancer cell will be essential if these agents are to become standard therapiesfor cancer.
1 Del Rio M, Gache Y, Jorcano JL, Meneguzzi G, Larcher F. Current approaches and perspectives in human keratinocyte-based genetherapies. Gene Ther 2004; 11 (Suppl 1): S57–S63.
1The Ohio State University Medical Center, Columbus, 2 Mavilio F, Pellegrini G, Ferrari S, Di Nunzio F, Di Iorio E, Recchia A et al. Correction of junctional epidermolysis bullosa by Suicide gene therapy, also known as prodrug-activation transplantation of genetically modified epidermal stem cells. Nat or gene-directed enzyme prodrug therapy, is a widely studied cancer gene therapy strategy, with numerous 3 Harper SQ, Staber PD, He X, Eliason SL, Martins IH, Mao Q et al.
publications providing evidence of significant effects in RNA interference improves motor and neuropathological animal models of cancer.Translation into human abnormalities in a Huntington’s disease mouse model. Proc Natl clinical trials has included herpes simplex thymidine Acad Sci USA 2005; 102: 5820–5825.
kinase (Hstk) gene transfer to induce susceptibility to 4 Rodriguez-Lebron E, Denovan-Wright EM, Nash K, Lewin AS, acyclovir, ganciclovir or valacyclovir, bacterial cytosine Mandel RJ. Intrastriatal rAAV-mediated delivery of anti-hun- deaminase (cd) gene transfer to induce susceptibility to tingtin shRNAs induces partial reversal of disease progression 5-fluorocytosine, and mammalian cytochrome P450 2B in R6/1 Huntington’s disease transgenic mice. Mol Ther 2005; (CYP2B) gene transferto enhance chemosensitivity to cyclophosphamide and ifosfamide, among others.
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Gene Therapy
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Gene Therapy



Protein phosphatase assay This protocol describes the standard strategy for measuring Ser/Thr protein phosphatase (PPase) activity in our laboratory using an artificial substrate (ex. Fzy-S50), a recombinant protein kinase (ex. Cdk) and [γ-32P]-ATP. This protocol can be modified/utilized to measure various PPase activity of your interest by changing substrate and kinase. 1, Purification o

Mental Health America of Franklin County 2323 W. Fifth Ave. Suite 160, Columbus, OH 43204 Telephone: (614) 221-1441 Fax: (614) 221-1491 Medications/Treatments: General Guidelines A Guide For Families, Friends, Board And Care Homes, Caregivers And Patients Prescription medications are helpful in reducing symptoms in people suffering with a mental illness. As with any medi

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