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Published online 8 July 2009 |
Nature
460,
164-169
(2009)
| doi:10.1038/460164a
Corrected online: 9 July 2009
News Feature
Human genetics: One gene, twenty years
When the cystic fibrosis gene was found in 1989, therapy seemed around the corner. Two decades on, biologists still have a long way to go, finds Helen Pearson.
During the day, Lap-Chee Tsui and Francis Collins were attending a gene-mapping workshop. At night they were scrutinizing the pages churning out of a fax machine they had set up in a dorm room.
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Title-: The Risk of gene therapy must be weighted carefully against risk, benefit, Toxicology, death and Existing treatment protocol and must be approved by state ethical committee Gene therapy is emerging as one of the important set backs in many diseases including in cardiovascular atheromatous, ischemic diseases, cancers, and leukemia in the rich and well developed countries with very high GDP. One of such a set back was infants born with X linked severe combined immunodeficiency Syndromes (SCID) resulting from mutation of ?c gene. In April 2002, a 18 months old infant was diagnosed to have a rare immune disorder, SCID, caused by a defective gene on X chromosome[1A]. This gene encodes the common ?c chain, an essential component of cytokine receptors, all of which are considered necessary for developments of T cells and NK cells lymphocytes. Without ?c chain (responsible for TCR), mature functioning T cells and NK cells are completely absent, whereas B cells are present in normal or in increased number at patient?s blood. So SCID is fatal during their first year of life because of severe recurrent infections. As a result of this much of child?s short duration of life was spent inside a sterile plastic bubble- a barrier for potential life threatening infections. A collaboration of scientists from London, Paris and Milan used retroviral vectors to introduce a normal functioning copy of the defective ?c gene in bone marrow stem cells taken & grafted as auto-logos stem cell with the hope that after re-infusion of engineered stem cell, they would engraft -& re constitute normal immune system. Integration and expression of ?c transgene & development of normal lymphocyte subgroup and their function in vitro were then analyzed for a period of 2.5 years after gene transfer done successfully. No immediate adverse reaction resulted from the procedure. The numbers of T cells and their in vitro response to several antigens were found normal and the therapy was declared successful [1].This success created an enthusiasm and 10 infants with SCID subsequently were treated with gene therapy with infusion of autologus heamopiotic stem cells transduced with normal functional ?c gene in a defective retroviral vector. All gene therapy in practical requires three elements 1) A vehicle for gene delivery 2) a therapeutic functional gene called transgene 3) a relevant target cells in which the gene is delivered. Retrovirus were the first virus adopted for use as vectors, owing to the simplicity of their genome and their capacity to stably integrate their genome in host human chromosome, The retroviral vector was constituted in several steps. The structural gene required for viral replication is deleted to render the vector non-replicating. After insertion of transgene of interest in to viral backbone, the recombinant retrovirus contain now the transgene, regulatory sequences(Promoter, enhancer) and packaging signals but it lacks the actual structural gene to produce the complete viron. It requires a helper cell (target cell) to produce the infectious viral particle. The choices of Target cell s for some diseases are limited as for haemopoietic stem cells as in haemoglobinopathy and cells of respiratory tract in case of lung diseases of cystic fibrosis. For other diseases, such as plasma protein deficiency (like hemophilia or erythropoietin responsive anemias) there may be latitude in the choice of target cells as long as transgenic products are secreted into circulation. In March 2000 Mark Kay[2] of Stanford university had a phase I clinical trial using adeno-associated virus vector(AAV) expressing the coagulation factor IX in haemophilia B patients. Recombinant AAV vectors are considered to be safest for use in gene therapy, and the virus do not cause disease naturally and rarely integrates its genome in to human genome. One patient in his study had 50% reduction in need of exogenous administration of factor IX while 2nd patient required 80% reduction [2]. Adeno viruses are also used for cardiovascular gene transfer. Adenoviruses are double stranded liner DNA and their wild type causes a self limited respiratory tract infection. This wild type adenovirus genome is 36Kda DNA molecule, and is divided into 100 map units. Majority of adenoviral vectors are derived from serotypes 2&5. These vectors are constructed by deletion of E1 region of the genome that normally encodes E1A and E1B motif. Without E1 region the virus can not replicate. This region is then replaced by the transgene of interest up to 7.5Kb size. Because the E1 deletion renders the replication inconsistent, adenoviral vectors are to be propagated in a helper cell line that express E1 protein in transfection. Adeno-associated virus (AAV) is a defective human parvovirus that is not able to replicate unless a helper virus such as adenovirus is present. Wild type AAV integrates in a site specific manner in 27 Kb regions of human chromosome. The AAV genome is flanked by 145bP inverted terminal repeats that contain the sequences required for packing integration and DNA replication. The coding region contains two ORF. Either of these ORFs can be replaced with trans gene and regulatory element to construct an AAV vectors. The ORF can accept only a trans gene of 4-5Kb thus limits the size of the trans gene insert. Propagation of AAV vectors requires AAV rep & cap proteins and five other adenoviral proteins E1A, E1B,E2A,E4,&VA. Clinical trials with cardiovascular gene therapy had been performed in vascular proliferative diseases and in angiogenesis so far. In a study done by Mann MG et al[3] cell cycle blockade by ex-vivo gene therapy of experimental vein grafts with a dominant negative transcription decoy E2F that led to inhibit the normal hyperplasia and subsequent accelerated atherosclerosis that leads to human CABG graft failure. This hypothesis was tested in a prospective randomized control trial to human to investigate the safety and biologic efficacy of intra-operative gene therapy in patient?s receiving CABG. Patients undergoing intra inguinal bypass were randomized to decoy oligo-deoxynucleotides ,scrambled oligodeoxynucleotides or no treatment. Oligonucleotides were delivered to grafts intra-operatively by ex-vivo pressure mediated transfection. The investigators found that E2F decoy treatment reduced proliferating cell nuclear antigen and c-myc mRNA concentration. But 12 months later there were some complications in the treatment group defined as fever, graft occlusions, revision or severe lesions [3] Several protocols had been identified to promote angiogenesis in myocardial ischemia or peripheral limb ischemia. The studies so far have been phase I trial with adenoviral vectors encoding VEGF, bFGF,aFGF or FGF4 genes by direct injections in to the heart or in limb muscle or by direct coronary artery infusion. In a phase I study, to treat myocardial ischemia recombinant VEGF or aFGF was injected directly in LIMA or LAD of patients undergoing coronary by pass surgery [5]. Patients with peripheral ischemia have been treated with direct injections of plasmid DNA encoding VEGF[4]. These phase I studies were promising. The treatment to date has been found safe with no subsequent toxicities. Phase II studies where double blind randomization & measurement of clinical efficacy, dose calculation not yet performed. Since 1999-2003 at least 27 phase I or Phase II trial were given in UK for head neck cancer, liver cancer, ovarian prostate cancer with nitro-reductase gene, FgF gene were used for peripheral arterial occlusion diseases, ICP34.5 gene for high grade gliomas, Head and neck cancer, melanoma, breast cancer, NHL, mesothelioma, HSVtk gene for metastatic melanoma and CML, HPV E6,E7 gene for cervical cancer & CIN gradeIII, Mel3 for melanoma, Hiv1 env gene for HIV, Oncofetal antigen 574 for advanced colorectal cancer, where adenovirus vector , HSV vector or retroviral vectors or vaccine virus vector were used. Adenovirus is the most popular virus vector in the clinical setting because of their ability to transducer a broad range of delivering in non-dividing cells also, they replicate episomally, they do not insert their genome to host immune cells & do not cause disruption of human cellular genome. The problem of using of adenovirus is that they are most immunogenic of all viral vectors. The advantages of using retroviral vector is their ability to integrate into genome and maintain expression of that gene for long period but disadvantages are tumor oncogenesis & development of leukemia. The enthusiasm of gene therapy halted when gene therapy resulted leukemia and death. About 3 years after treatment with transgenic retroviral gene in X linked SCID in 10 boys, 2 boys resulted T cell ALL[6]. The explanation was that both the patients in whom leukemia developed, the retrovirus carrying the ?c gene had inserted itself near Lmo2-an oncogene for childhood ALL, that was activated as a result of translocation in ALL. Dave etal[7] explained why ALL developed after gene therapy with SCID. He explained that insertion of ?c gene vector near LMO2 represent a double hit hypothesis meaning that cell linage might be only one mutation away from overt ?c gene leukemia development [6,8]. We authors think that insertion of retrovirus vector carrying ?c transgene resulted modification of cellular genome within the autologus stem cell itself which may presented as additional risk. The risks associated with human gene transplantation are many [9]. They can be summarized as follows* The vectors are potentially capable of propagating themselves recombining with other viruses or carrying out complex programme. ** The gene transfer functions on basis of genetic information rather than on chemical structure. *** Many agents acts simultaneously as delivery devices though their vectors as well as pharmacological agents through their trans gene **** Gene transfer agents that stably modify a persons tissue can involve probability of subtle toxic properties becoming manifested over long years***** much of the toxicity related to gene therapy is mediated through immune system ****** Immune over reaction may result death particularly if auto immunity results[9]. So several methodological issues must be considered when testing safety of gene transfer in Phase-1 trial. In animal model result may not mimic the human model result. Animal model may often fail to predict the toxicity. The Toxicological property of gene transfer trial may be expressed in human model* Person prior exposer to similar viruses of vector can influence gene transfer. Ethical committee must consider the fact that gene transfer trial express participant to the possibility of serious unforeseeable & latent harm including death. So such trial should aim to maximize beneficiaries, enroll people who have exhausted all over treatment options. References 1)Kale Ralph, Kelvin Harrington, Hardew Pandha ? Recent development & current status of Gene therapy using viral vectors in United Kingdom? BMJ 329:9th oct; 839-42:2004 2) Kay MA, Manno.C.S, Ragni MV , Larson PJ Etal ? Evidence for gene transfer & expression of factor IX in hemophilia B patients treated with AAV vectors ? Nature genetics 24;257-61;2000 3)Mann MJ, White More AD Donaldson MC etal ? Ex vivo gene therapy of human vascular grafts with E2F decoy: The PREVENT single center randomized controlled trial ? Lancet 354;1493-98;1996 4)Isner JM, Pleezek A, Schnifeld R ? Clinical evidence of angio genesis after arterial gene transfer of Ph VEGF 165 in patients with ischemic limb ? Lancet 348;370-74;1996 5)Tsurums Y, Takeshita S, Chen D ? Direct Intramuscular gene transfer of naked DNA encoding Vascular endothelial growth factors augments Collateral development & tissue perfusion? Circulation 94;3281-90;1996 6)Hacein Bey, Abine. S, Vonkalle. Etal? LMO2 associated clonal proliferation in two patients after gene therapy for SCID? Science 3024159;2003 7)Berns Antoni ? Good news for gene therapy ? New Eng. J. Med 350;16;1679-80;2004 8)Mc Cormeck MP, Rabbit TH? Activation of T cell ncogene LMO2 after gene therapy for X linked severe combined Immunodeficiency? New. Eng. J.Med 350;913-22;2004 9)Jonathan Kimmelmen ? Recent development in gene transfer: risk and ethics? BMJ 330;79-82;2005 Strictly Copy Righted material © to professor Pranab kumar Bhattacharya as per IPR copy right Rules . Authors 1) Professor Pranab Kr Bhattacharya MD(Path) Cal, FIC path (Ind.), Professor, Dept. of pathology, In charge of Histopathology Unit, in charge of Cytogenetics, Ex-In charge of 24 hours Ronald Ross Malaria clinic, Technical Supervisor In charge of Blood Bank, VCCTC Institute of Post Graduate Medical Education& Research (IPGMER) 244A AJC Bose Road, K0lkata-700020, West Bengal , India* Mr. Rupak Bhattacharya BSc(cal), MSc(JU))** Mr. Ritwik Bhattacharya B.Com(cal)7/51 Purbapalli Po= Sodepur ,Dist.- 24 parganas(North),West Bengal ,Pin 700110, India ***Miss Upasana Bhattacharya Dr. Pijus kanti Roy MS(cal) Dr. Anindya Chakraborty MBBS(cal)