Viruses and Gene Therapy Viruses Used Most gene therapy trials which utilize viral vectors use either adenovirus or retroviruses. We have discussed the retroviral life cycle in detail previously (e.g. HIV) which results in viral gene integration into the host The adenoviral life cycle does not result in gene integration into the host chromosome Adenoviruses: Introduction Adenoviruses are double-stranded DNA viruses. They have icosahedral capsids with twelve vertices and seven surface proteins. They are composed of 252 capsomers: 240 "hexons" + 12 "pentons" at vertices of icosahedron (2-3-5 symmetry). The virion is non-enveloped, spherical and about seventy to ninety nm in size. The genome encodes about thirty proteins. Both strands of adenovirus DNA encode genes. Covalently linked protein at 5? ends primes replication Transcription occurs in three stages -- immediate early, early and late Background on Adenovirus Adenoviruses are a frequent cause of acute upper respiratory tract (URT) infections, i.e. "colds". They were first isolated in 1953 by investigators trying to establish cell-lines from adenoidal tissue of children removed during tonsillectomy and from military recruits with febrile illness. By 1962, some Adenoviruses were shown to cause tumors in rodents, but this has never been observed in humans. Some characteristic features of Adenoviruses are: Widespread in nature, infecting birds, many mammals and man. There are 2 genera, Aviadenovirus (avian) and Mastadenovirus (mammalian) Can undergo latent infection in lymphoid tissues, becoming reactivated some time later (but not integrated) Adenoviral Disease Symptoms Adenovirus is responsible for five percent of acute respiratory childhood illness and ten percent of infantile gasteroenteritis. Incubation period is 5-8 days Spread mostly via respiratory tract secretions Symptoms are like the common cold Transmission is Ingestion, Fecal-Oral Route, Respiration (through respiratory droplets), Contact/Hand-to-eye transfer, Venereal Generally, this is a well-tolerated virus in humans Thus, there has been considerable interest in developing Adenoviruses as defective vectors to carry and express foreign genes for therapeutic purposes Adenoviral Replication Replication of all Adenoviruses is similar and occurs in the nucleus Replication is divided into EARLY and LATE phases Late phase is defined as beginning with the onset of DNA replication Events in Adenoviral Infections Late Imm Early and Early Adenoviral Uptake and Penetration Uptake of the adenovirus particle is a two stage process involving an initial interaction of the fibre protein with a range of cellular receptors, which include the MHC class I molecule & the coxsackievirus-adenovirus receptor. The penton base protein then binds to the integrin family of cell surface heterodimers allowing internalization via receptor-mediated endocytosis. Most cells express primary receptors for the adenovirus fibre coat protein, however internalization is more selective. Penetration involves phagocytosis into phagocytic vacuoles, after which the toxic activity of the pentons is responsible for rupture of the phagocytic membrane and release of the particle into the cytoplasm of the host cell. Adenoviral Uncoating Uncoating follows an ordered sequence first the pentons are removed, releasing a spherical, partially uncoated particle into the cytoplasm. The core migrates to the nucleus where the DNA enters through nuclear pores, whereupon it is converted into a virus DNA-cell histone complex. initial Early Transcripts The first mRNA/protein to be made (~1h after infection) is E1A. This protein is a trans-acting transcriptional regulatory factor whose precise mode of action is not known (not a DNA-binding "transcription factor") but is necessary for transcriptional activation of early genes. EIA can transform primary cells in culture E1A can interact with the Rb protein to promote cell cycle progression E1A also inhibits MHC class I expression which may facilitate tumorigenesis The second protein made is E1B. E1B can facilitate the transformation of cells by E1A E1B can interact with the p53 product to promote cell cycle progression Late Transcription At the onset of DNA replication, the pattern of transcription changes radically from the early to the late genes. There is cis-acting control of this switch, i.e. only newly replicated DNA is used for late gene transcription, but the mechanism controlling this is not understood. The late genes are transcribed from the major late promoter; at least 13 species of mRNA are produced by alternative splicing. Assembly occurs in the nucleus, but begins in the cytoplasm when individual monomers form into hexon and penton capsomers. Empty, immature capsids are assembled from these protomers in the nucleus, where the core is formed from genomic DNA + associated core proteins. Late Transcription Cont. Although host cell macromolecular synthesis ceases earlier in the infection, infected cells remain intact and do not lyse Virus particles tend to accumulate in the nucleus and are visible in the microscope as inclusion bodies These are thought to be the basis of latent infections - reactivation is caused by accidental lysis of infected cells, releasing virus particles from the nuclei - effectively a re-infection Transcriptional Map of Adenovirus The E1 and E3 genes are often replaced to make gene therapy vectors Late genes are structural genes Early genes are Needed for replication What is Gene Therapy? Gene therapy is the insertion of genes into an individual's cells and tissues to treat a disease (hereditary diseases in particular). Gene therapy typically aims to supplement a defective mutant allele with a functional one. Although the technology is still in its infancy, it has been used with some success. It can also be used to deliver a therapeutic protein to a cancerous area Background on Gene Therapy In the 1980s, advances in molecular biology had already enabled human genes to be sequenced and cloned. Scientists looking for a method of easily producing proteins, such as the protein deficient in diabetics ? insulin, investigated introducing human genes to bacterial DNA. The modified bacteria then produce the corresponding protein, which can be harvested and injected in people who cannot produce it naturally. Scientists took the logical step of trying to introduce genes straight into human cells, focusing on diseases caused by single-gene defects, such as cystic fibrosis, hemophilia, muscular dystrophy and sickle cell anemia. However, this has been much harder than modifying simple bacteria, primarily because of the problems involved in carrying large sections of DNA and delivering it to the right site on the genome. Types of Gene Therapy In theory it is possible to transform either somatic cells (most cells of the body) or cells of the germline (such as stem cells, sperm and eggs). All gene therapy so far in people has been directed at somatic cells Germline engineering in humans remains a highly controversial prospect. For the introduced gene to be transmitted normally to offspring, it needs not only to inserted into the cell, but also to be incorporated into the chromosomes by genetic recombination. Somatic gene therapy can be broadly split in to two categories: ex vivo (where cells are modified outside the body and then transplanted back in again) and in vivo (where genes are changed in cells still in the body.) Vectors in Gene Therapy Viruses can be used to deliver new genes into people Viruses will direct the cells in the host to produce the new proteins In addition to the instructions producing the components of the virus itself, viruses can carry additional genes containing instructions for creating other kinds of proteins. In theory, if we insert a gene that is missing from a patient in a virus, and infect that patient with the virus, the virus will spread the missing gene in all the infected cells of the patient. The missing gene is now replaced and the disease is cured. Viruses prominently used as vectors in gene therapy: retroviruses, adenoviruses and adeno-associated viruses Pox viruses or vaccinia viruses Herpes viruses Numbers of Clinical Trials Worldwide, in 2005 there were 261 clinical trials using retroviral vectors for gene therapy There were 260 trials using adenovirus as a vector for gene therapy There were 27 trials with AAV as a vector for gene therapy Ex vivo (in vitro) The ex vivo approach was the first to be put in to practice. In 1990 trials were run designed to treat children with an inherited immune deficiency, as well as children or adults with high serum cholesterol. Cells were removed from the patients body and incubated with vectors that inserted copies of the genes. Most gene-therapy vectors are viruses, although there are techniques for delivering DNA directly as well. After modification, the cells are transplanted back in to the patient where they will hopefully replicate and produce functional descendants for the life of the transplanted individual. This technique is best used for diseases where the desired cells can be extracted easily, such as the blood or liver In vivo For in vivo techniques the challenge of inserting the genes is even greater. The vector carriers have a difficult task to complete: they must deliver the genes to enough cells for results to be achieved and they have to remain undetected by the body's immune system. Much hope has been placed in viruses to deliver the DNA. After all, this is what viruses do naturally ? insert their genes into cells so that their hosts can reproduce them. Through millions of years of evolution viruses have developed very sophisticated ways of doing this. In vivo cont. Retroviruses reproduce by integrating their RNA into the host's DNA, so they carry the prospect of incorporating new genes into chromosomes, so that cells that divide will pass the genes to their progeny. Scientists have removed certain crucial genes from the viral genome, so that they cannot damage the host (viral oncogenes). Retroviruses only infect dividing cells which limits the use of these vectors to rapidly dividing cell types (cancers) Adenoviruses Adenoviruses are larger, DNA-based viruses, which can carry more genes than retroviruses. A problem affecting all virus-based vectors is recognition by the immune system. Antibodies can neutralize common viruses To avoid this, adenoviral vectors utilize unusual serotypes of retroviral and other recombination-based approaches, the virus The unpredictablity of where the new DNA inserts into the chromsomes of transfected cells raises issues as well Adenoviruses cont. If the gene is inserted in a bad place ? for example within the sequence of an important gene, or within non-coding (intron) regions that the cell will never translate to produce protein ? then the new gene would not be properly expressed and the cell could be made worse or even cancerous. Adenoviruses have an advantage in that they do not integrate into the host genome. Also, adenoviruses can infect both dividing and non-dividing cells Gene Therapy Vectors The fact that viruses can infect many cell types and can then express their genes in those cell types make the idea of using viruses to deliver gene therapy attractive The challenge is to create a virus that you can delete some viral gene and replace it with a cellular gene and still have a functional virus In some cases, you can insert the gene of interest into a necessary structural gene, and then replace the structural gene by infection with a helper virus in vitro This allows you to make virions that are whole and infectious in the first round, but that cannot replicate to make a second round of infection in vivo This may be okay if you can get a high primary infection and the integration of the gene of interest Adenoviruses Remember, adenoviruses are viruses that carry their genetic material in the form of DNA. The genetic material of the adenoviruses is not incorporated into the host cells genetic material. The DNA molecule is left free in the nucleus of the host cell, and the instructions in this extra DNA molecule are transcribed just like any other gene. The only difference is that these extra genes are not replicated when the cell is about to undergo cell division. So the descendants of that cell will not have the extra gene. This means that treatment with the adenovirus will require regular doses to add the missing gene every time new cells are produced without the gene. You can probably guess why this may be a problem Adenovirus Vectors subgroup C serotypes 2 or 5 are predominantly used as vectors. they replicate as episomal elements in the nucleus of the host cell & consequently there is no risk of insertional mutagenesis. The wild type adenovirus genome is approximately 35 kb of which up to 30 kb can be replaced with foreign DNA There are four early transcriptional units (E1, E2, E3 & E4), which have regulatory functions, & a late transcript, which codes for structural proteins. Progenitor vectors have either the E1 or E3 gene inactivated, with the missing gene being supplied in trans either by a helper virus, plasmid or integrated into a helper cell genome (human fetal kidney cells, line 293 E1 deleted adenoviruses lose oncogenic potential and increase the amount of DNA that can be cloned. They are replication deficient also Second generation vectors additionally use an E2a temperature sensitive mutant or an E4 deletion Genes most often replaced in adenoviral vectors Replacing early genes and helper virus rescue Helper virus to provide E1 genes, but helper is defective too Some of the Problems Adenoviral vectors are very efficient at transducing target cells in vitro & in vivo, & can be produced at high titres (>1011/ml). There have been some problems with adenoviral vectors Transgene expression in vivo from progenitor vectors tends to be transient. Following intravenous injection, 90% of the administered vector is degraded in the liver by a non-immune mediated mechanism. Thereafter, an MHC class I restricted immune response occurs, using CD8+ CTLs to eliminate virus infected cells & CD4+ cells to secrete IFN-alpha which results in anti-adenoviral antibody. Alteration of the adenoviral vector can remove some CTL epitopes, however the epitopes recognized differ with the host MHC haplotype. The remaining vectors, in those cells that are not destroyed, have their promoter inactivated & persisting antibody prevents subsequent administration of the vector. Length of Treatment With adenoviruses, the transiently expressed gene is usually expressed at highest levels 3-4 days post infection Expression is usually undetectable by 1-2 weeks post infection This requires multiple dosings with adenoviral vectors Multiple dosings however act like multiple vaccination boosters and eventually the immune response is strong enough to clear the infection immediately Considerations of Adenoviral Gene Therapy Approaches Approaches to avoid the immune response involving transient immunosupressive therapies have been successful in prolonging transgene expression & achieving secondary gene transfer. A less interventionist method has been to induce oral tolerance by feeding the host UV inactivated vector. However, it is desirable to manipulate the vector rather than the host. Although only replication deficient vectors are used, viral proteins are expressed at a very low level which are presented to the immune system. The development of vectors containing fewer genes, culminating in the "gutless" vectors which contain no viral coding sequences, has resulted in prolonged in vivo transgene expression in liver tissue. The initial delivery of large amounts of DNA packaged within adenovirus proteins, the majority of which will be degraded & presented to the immune system may still cause problems for clinical trials. Moreover the human population is heterogeneous with respect to MHC haplotype & a proportion of the population will have been already exposed to the adenoviral strain Some Adenoviral therapy applications may be okay for short term solutions-like this example to encourage new blood vessel growth in cardiovascular disease Adeno-associated viruses Adeno-associated viruses, from the parvovirus family, are small viruses with a genome of single stranded DNA. There are a few disadvantages to using AAV, mainly the small amount of DNA it can carry and the difficulty in producing it. This type of virus is being used, however, because it is non-pathogenic (most people carry this harmless virus). In contrast to adenoviruses, most people treated with AAV will not build an immune response to remove the virus or the cells that have been successfully treated with it. Several trials with AAV are on-going or in preparation, mainly trying to treat muscle and eye diseases, the two tissues where the virus seems particularly useful. Retroviruses The fact that retroviruses integrate their genome into the host is the reason for their biggest advantage and disadvantage Advantage is that the change to the host cell will be permanent because it has been incorporated into the chromosome Disadvantage is that you cannot control the site of integration Thus, this could lead to inactivation of critical genes, incorporation of viral promoters near oncogenes etc The basic idea in retroviral therapy is also to cut out a non-essential viral gene and replace it with gene of interest (similar to what we already discussed) First Gene Therapy Trial- ADA in 1990 Adenosine deaminase deficiency (ADA) is a genetic disorder that is life threatening ADA deficiency has been identified as the metabolic basis for 20-30% of cases with recessively inherited severe combined immunodeficiency (SCID). Affected infants are subject to recurrent chronic viral, fungal, protozoal, and bacterial infections and frequently present with persistent diarrhea, failure to thrive and candidiasis. Severely affected cases present neonatally with no detectable lymphocytes in peripheral blood or bone marrow and both cell-mediated and humoral immunity is defective. T cells are generally absent, agammaglobulinaemia is the rule and lymphocyte proliferative and specific antibody responses are lacking. The age of onset has varied from birth to 5 years of age, but is usually within the first 2 years of life. ADA is toxic to lymphocytes Adenosine deaminase catalyses the deamination of adenosine and 2'-deoxyadenosine to inosine or 2'-deoxyinosine respectively In patients homozygous for ADA deficiency, 2'-deoxyadenosine accumulating during the rapid turnover of cells rich in DNA is converted back to dATP, either by adenosine kinase or deoxycytidine kinase Many hypotheses have been advanced to explain the specific toxicity to the immune system in ADA deficiency The apparently selective accumulation of dATP in thymocytes and peripheral blood B cells, with resultant inhibition of ribonucleotide reductase and DNA synthesis is probably the principal mechanism. ADA and Gene Therapy A level of ADA that is even 5% of normal can provide protection from lethality and the SCID phenotype Thus, this was a perfect disease to try gene therapy Patient?s T cells were manipulated in vitro with retroviral vectors that supplied normal ADA and were then expanded in vitro with growth factors Cells were then reinfused into patients with relative success Patients were not ?cured?, but this allowed them to have some functional lymphocytes and reduced the numbers of infections in these patients Cancer Trials By far the most common type of gene therapy trial is related to cancer therapy This is delivered by intratumoral injection of the viral vector to target the infection to tumor cell areas Some approaches in cancer trials include Adenoviral delivery used to restore normal p53 to tumors To overproduce IL-2 or other cytokines in tumors to stimulate immune responses To deliver tumor antigens to cancers to speed immune recognition Angiogenesis inhibition in tumors Delivery of toxic genes Another approach is just to inject the tumors with infectious regular adenoviruses to try to facilitate adenoviral-mediated lysis of the solid tumors to help debulk the tumor Future of adenoviral mediated gene therapy It would be great if for tumor therapy, you could target the adenovirus to the tumor only, and not normal cells (especially if delivering toxic genes) Thus current efforts are trying to modulate the fiber proteins of adenovirus to make them ?specific? for certain receptors on certain types of tumors Other approaches are to use native adenovirus structural proteins to deliver gene therapy, but to replace the ubiquitous adenoviral promoters with tissue-specific promoters that will only be active in the specific cell type you are interested in Why is it still experimental? The Food and Drug Administration (FDA) has not yet approved any human gene therapy product for sale. The first trial for ADA was in 1990. In 1999, gene therapy suffered a major setback with the death of 18-year-old Jesse Gelsinger. Jesse was participating in a gene therapy trial for ornithine transcarboxylase deficiency (OTCD). Another major blow came in January 2003, when the FDA placed a temporary halt on all gene therapy trials using retroviral vectors in blood stem cells. FDA took this action after it learned that a second child treated in a French gene therapy trial had developed a leukemia-like condition. Both this child and another who had developed a similar condition in August 2002 had been successfully treated by gene therapy for X-linked severe combined immunodeficiency disease (X-SCID), also known as "bubble baby syndrome." How these setbacks have changed the face of gene therapy OTC deficiency causes ammonia to build up in the blood. In severe cases, babies go into comas and die within days of birth Researchers at U Penn had developed adenoviral vectors that could replace OTC They wanted to test them in sick babies, but the ethicists decided that parents of dying babies cannot provide ?informed consent? So they required the trial to be performed in patients with limited OTC deficiency These patients are maintained by low protein diets and lots of medications Jesse Gelsinger volunteered even though his disease was managable He knew he would not be cured. The trial was designed to test safety for treatment of babies in comas to save them until diet and medicines could be employed He said he wanted to be a ?hero? Jesse Gelsinger cont. The virus used in his trial had caused problems in 2 monkeys previously (undisclosed), but had been successfully given to 16 other patients-1 other at the high dose Jesse would receive Jesse died 4 days after infusion from multi organ failure due to an overwhelming immune response to the adenovirus Why he had that response is still unknown This created a HUGE uproar about whether scientists were being ethical in reporting side effects, etc It has totally changed the way informed consents and all clinical trials are done Far more animal data is now needed to ask for a trial and safety trials are more rigorous All ?adverse? effects must be reported to national databases for all physicians to see It has limited the enthusiasm of biotech companies to participate It has certainly limited public enthusiasm for gene therapy However, gene therapy provides the best hope for cures for some diseases Recent Clinical Trial Tragedy In March, 2006 in London, 6 men volunteered to be administered with a new drug called TGN1412 which was being explored as a treatment for leukemia Tests in primates had yeilded excellent safety data with only minimal gland swelling The informed consent documents were 15 pages long and all 6 patients read and signed within 30 minutes following consultation with physicians All 6 men were injected with the drug simultaneously (somewhat unusual procedure) and within the hour, all 6 were experiencing life-threatening complications and all required life support-4 lapsed into comas Men were paid about $3,500 for what should have been a two-week trial. They were to stay for three nights, and then attend 11 follow up days. ?It worries me that you could earn a living from being a participant,? said Ray Noble, a medical ethicist at University College London. ?It might blind people to the obvious potential pitfalls of participating in too many trials.? Others say that without offering financial incentives it would be extremely difficult to get subjects to participate in Phase 1 tests. It's a fine line. How do we attract people to do something for which there is not much reward? Phases of Clinical Trials Human testing of experimental drugs is done in phases. In Phase 1, the drug is given to a small number of people, usually healthy volunteers (although sometimes in disease patients), to see if it appears basically safe. Phase 2 is done to further establish safety and an appropriate dose, though this can give hints of effectiveness as well. Phase 3 testing involves a wider group of people who have the illness or disease the drug targets, and is meant to determine the treatment's effectiveness. Phase 3 trials are easier to find participants for because these often involve people with serious disease and few options What are some of the ethical considerations for using gene therapy? --Some Questions to Consider from the NIH... What is normal and what is a disability or disorder, and who decides? Are disabilities diseases? Do they need to be cured or prevented? Does searching for a cure demean the lives of individuals presently affected by disabilities? Is somatic gene therapy (which is done in the adult cells of persons known to have the disease) more or less ethical than germline gene therapy (which is done in egg and sperm cells and prevents the trait from being passed on to further generations)? In cases of somatic gene therapy, the procedure may have to be repeated in future generations. Preliminary attempts at gene therapy are exorbitantly expensive. Who will have access to these therapies? Who will pay for their use? If informed consent is appropriate, is it fair to deprive people willing to take the risk because some people are uncomfortable with it?
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