Retrovirus in Gene Manipulation and Vaccines
by
Robert Gorter, MD, PhD, et al.
May 15th, 2020
“Of great concern is that by the use of retroviruses, gene and chromosomal alterations can seduce the striving scientist and the billionaire philanthropist alike to improve the human race according to their standards and world views.”
A retrovirus is a type of RNA virus that inserts a copy of its genome into the DNA of a host cell that it invades, thus changing permanently the genome of that cell. It is a naturally occurring gene manipulation that could also have an evolutionary role over time. Once inside the host cell’s cytoplasm, the virus uses its reverse transcriptase enzyme to produce DNA from its RNA genome, the reverse of the usual pattern, thus retro (backward). The new DNA is then incorporated into the host cell genome by an integrase enzyme, at which point the retroviral DNA is referred to as a provirus. The host cell then treats the viral DNA as part of its genome, transcribing and translating the viral genes along with the cell’s genes, producing the proteins required to assemble new copies of the virus.
Although retroviruses have different subfamilies, they have three basic groups. The oncoretroviruses (oncogenic retroviruses), the lentiviruses (slow retroviruses), and the spumaviruses (foamy viruses). The oncoretroviruses can cause cancer in several species, the lentiviruses able to cause severe immunodeficiency Like HIV) and death in humans and other animals, and the spumaviruses being benign and not linked to any disease in humans or animals yet.
Many retroviruses cause serious diseases in humans, other mammals, and birds. Human retroviruses include HIV-1 and HIV-2, the cause of the disease of AIDS. Also, the Human T-lymphotropic virus (HTLV-1 and HTLV-2) causes lethal disease in humans. The murine leukemia viruses (MLVs) cause cancer in mouse hosts. Retroviruses are valuable research tools in molecular biology, and they have been used successfully in gene delivery systems.
Gene therapy
Gammaretroviral and lentiviral vectors for gene therapy have been developed that mediate stable genetic modification of treated cells by chromosomal integration of the transferred vector genomes. This technology is of use, not only for research purposes but also for clinical gene therapy aiming at the long-term correction of genetic defects, e.g., in stem and progenitor cells. Retroviral vector particles with tropism for various target cells have been designed. Gammaretroviral and lentiviral vectors have so far been used in more than 300 clinical trials, addressing treatment options for various diseases. Retroviral mutations can be developed to make transgenic mouse models to study various cancers and their metastatic models.
Summary
Retroviral vector-mediated gene transfer has been central to the development of gene therapy. Retroviruses have several distinct advantages over other vectors, especially when permanent gene transfer is the preferred outcome. The most important advantage that retroviral vectors offer is their ability to transform their single-stranded RNA genome into a double-stranded DNA molecule that stably integrates into the target cell genome. This means that retroviral vectors can be used to permanently modify the host cell nuclear genome. Recently, retroviral vector-mediated gene transfer, as well as the broader gene therapy field, has been re-invigorated with the development of a new class of retroviral vectors which are derived from lentiviruses. These have the unique ability amongst retroviruses of being able to infect non-cycling cells. Vectors derived from lentiviruses have provided a quantum leap in technology and seemingly offer the means to achieve significant levels of gene transfer in vivo.
The ability of retroviruses to integrate into the host cell chromosome also raises the possibility of insertional mutagenesis and oncogene activation. Both these phenomena are well known in the interactions of certain types of wild-type retroviruses with their hosts. However, until recently they had not been observed in replication-defective retroviral vector-mediated gene transfer, either in animal models or in clinical trials. This has meant the potential disadvantages of retroviral mediated gene therapy have, until recently, been seen as largely, if not entirely, hypothetical. The recent clinical trial of γc mediated gene therapy for X-linked severe combined immunodeficiency (X-SCID) has proven the potential of retroviral mediated gene transfer for the treatment of inherited metabolic disease. However, it has also illustrated the potential dangers involved, with 2 out of 10 patients developing T cell leukemia as a consequence of the treatment. A considered review of retroviral induced pathogenesis suggests these events were qualitative, if not quantitatively, predictable. Besides, the probability of such events can be greatly reduced by relatively simple vector modifications, such as the use of self-inactivating vectors and vectors derived from non-oncogenic retroviruses. However, these approaches remain to be fully developed and validated. This review also suggests that, in all likelihood, there are no other major retroviral pathogenetic mechanisms that are of general relevance to replication-defective retroviral vectors. These are important conclusions as they suggest that, by careful design and engineering of retroviral vectors, we can continue to use this gene transfer technology with confidence.
Conclusion
Retroviral mediated gene transfer remains an extremely attractive option for gene therapy when the stable and permanent genetic modification of the target cell is optimal. However, we must take greater care, and utilize more resources, for the pro-active, rather than reactive, refinement and testing of the basic technology that is used for gene therapy and the adoption of improved vector systems if adverse events are to be minimized. (Genet Vaccines Ther. 2004; 2: 9. Published online 2004 Aug 13. doi: 10.1186/1479-0556-2-9)
Summary
Replication-defective retroviral vectors have been used for more than 30 years as a tool for efficient and stable insertion of therapeutic transgenes in human cells. Patients suffering from severe genetic diseases have been successfully treated by transplantation of autologous hematopoietic stem-progenitor cells (HSPCs) transduced with retroviral vectors, and the first of this class of therapies, Strimvelis, has recently received market authorization in Europe. Some clinical trials, however, resulted in severe adverse events caused by vector-induced proto-oncogene activation, which showed that retroviral vectors may retain a genotoxic potential associated with proviral integration in the human genome. The adverse events sparked a renewed interest in the biology of retroviruses, which led in a few years to a remarkable understanding of the molecular mechanisms underlying retroviral integration site selection within mammalian genomes. This review summarizes the current knowledge on retrovirus-host interactions at the genomic level, and the peculiar mechanisms by which different retroviruses, and their related gene transfer vectors, integrated in, and interact with, the human genome. This knowledge provides the basis for the development of safer and more efficacious retroviral vectors for human gene therapy. (Mol Ther Methods Clin Dev. 2017 Oct 5;8:31-41. DOI: 10.1016/j.omtm.2017.10.001. eCollection 2018 Mar 16.)
Summary
Gene transfer vectors derived from oncoretroviruses or lentiviruses are the most robust and reliable tools to stably integrate therapeutic transgenes in human cells for clinical applications. Integration of these vectors in the genome may, however, have undesired effects caused by the insertional deregulation of gene expression at the transcriptional or post-transcriptional level. The occurrence of severe adverse events in several clinical trials involving the transplantation of stem cells genetically corrected with retroviral vectors showed that insertional mutagenesis is not just a theoretical event and that retroviral transgenesis is associated with a finite risk of genotoxicity. In addressing these issues, the gene therapy community offered a spectacular example of how scientific knowledge and technology can be put to work to understand the causes of unpredicted side effects, design new vectors, and develop tools and models to predict their safety and efficacy. As an added benefit, these efforts brought new basic knowledge on virus-host interactions and the biology and dynamics of human somatic stem cells. This review summarizes the current knowledge on the interactions between retroviruses and the human genome and addresses the impact of target site selection on the safety of retroviral vector-mediated gene therapy. (Hum Gene Ther. 2013 Feb; 24(2):119-31. DOI: 10.1089/hum.2012.203.)
Summary
Retroviral DNA integration into the host cell genome is an essential feature of the retroviral life cycle. The ability to integrate their DNA into the DNA of infected cells also makes retroviruses attractive vectors for the delivery of therapeutic genes into the genome of cells carrying adverse mutations in their cellular DNA. The sequencing of the entire human genome has enabled the identification of integration site preferences of both replication-competent retroviruses and retroviral vectors. These results, together with the unfortunate outcome of a gene therapy trial, in which integration of a retroviral vector in the vicinity of a protooncogene was associated with the development of leukemia, have stimulated efforts to elucidate the molecular mechanism underlying integration site selection by retroviral vectors, as well as the development of methods to direct integration to specific DNA sequences and chromosomal regions. This review outlines our current knowledge of the mechanism of integration site selection by retroviruses in vitro, in cultured cells, and in vivo; the outcome of several of the more recent gene therapy trials, which employed these vectors; and the efforts of several laboratories to develop vectors that integrate at predetermined sites in the human genome. (Hum Gene Ther. 2008 Jun;19(6):557-68. DOI: 10.1089/hum.2007.148.)
Clinical applications
Retroviral vector technology is the method used most commonly for gene transfer in gene therapy. However, although several clinical trials are underway, success has been limited because of problems transducing sufficient numbers of target cells. There are two general approaches: namely, in vivo gene therapy, in which genes are delivered directly to target cells; and the more usual ex vivo therapy, in which the target cells are genetically modified outside the body and then transplanted. Retroviral vectors have been used to introduce a drug susceptibility or “suicide” gene, such as herpes simplex thymidine kinase (TK), to target cells.14 When the patient is treated with a particular drug, such as gancyclovir, the target cells containing TK are killed selectively. The suicide gene approach has been used with some success for the treatment of recurrent malignancy. For example, when T cells, which have been previously harvested, are administered to patients with melanoma on the development of a recurrence, they are effective in destroying malignant cells. Unfortunately, the T cells can go on to damage normal tissue. Therefore, the harvested T cells are retrovirally transduced with a suicide gene, so that they can be removed after they have eliminated the recurrent tumor.
A more corrective approach has been used to restore apoptotic pathways in tumor cells by introducing apoptosis-related genes, such as p53 and BCL-xs, via retroviral vectors. However, experimentally both p53 and BCL-xs were unable to induce apoptosis in certain tumors, and it may be that a more complex set of signals is necessary to trigger this pathway. At present, the experimental focus has shifted to Fas ligand stimulated apoptosis. One success with the corrective approach has been in the treatment of the human genetic disease adenosine deaminase (ADA) deficiency, which results in children having severe combined immunodeficiency. The biochemical defect is a buildup of deoxyadenosine—an ADA substrate—which is converted to a toxic compound that disables T cells. Patients can be treated by retroviral mediated gene therapy to reintroduce T cells corrected for the defect, as well as by enzyme replacement. Although this treatment appears to overcome the disease, it has been difficult to separate the effectiveness of the gene therapy from the enzyme treatment.
Retroviral vectors have also been used in alternative efforts to enhance the body’s immune response to tumors; this treatment involves reinjecting tumor cells that have been UV irradiated, genetically manipulated, or admixed with non-specific adjuvants. Combinations of these strategies are underway, but to date, success has been limited.
Discussion and Warning
The ability to manipulate gene expression in the mouse embryo has rendered it the organism of choice for genetic studies, but the difficulty of physically manipulating the mammalian embryo limits the combination of these approaches to study the development of valuable gene manipulation and chromosomal altering. The use of retroviral vectors to alter gene expression has proven to be a valuable alternative to transgenic approaches. Many of the genetic manipulations that have proven so powerful in the mouse can be mimicked with retroviral vectors in higher mammals and humans. The use of retroviruses to alter gene expression has several advantages stemming from the comparative ease with which different expression patterns may be generated with the same virus. Many genes are reused in different tissues and at different times to mediate and guard development. By varying the injection protocol, a single high titer virus stock may be used to address the role of a gene in many different developmental decisions and applications.
Of great concern is that gene and chromosomal alterations can seduce the striving scientist and the billionaire philanthropist alike to improve the human race according to their standards and world views.