Progress and Potential for Gene-Based Medicines Mark R. Dyer, Paul L. Herrling  Molecular Therapy  Volume 1, Issue 3, Pages 213-224 (March 2000) DOI: 10.1006/mthe.2000.0044 Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 1 Evolution of therapeutic discovery. Discovery of new therapies during the 20th century can be divided into several historical phases. In the early 1900s there was a strong emphasis on synthetic chemistry. One of the key advances in pharmaceutical discovery was the ability to synthesize new and original chemical compounds at a time when biological knowledge about the diseases was beginning to accumulate. This phase of chemical predominance lasted until the late 1970s. In the early 1980s biological sciences had advanced to the point where knowledge of disease mechanisms began to define molecular targets for therapeutic discovery efforts. With the advent of molecular biology and recombinant DNA technology and its widespread application during the 1980s it became possible to begin isolating and sequencing genes important in diseases. One result of the advance in genetic engineering was the development of recombinant protein therapies. The ability to manipulate genes and engineer them, coupled with concurrent advances in cell biology, resulted in the origins of gene- and cell-based medicines during the early 1990s. Although not discussed in this review, the medical potential for artificial tissues and organs is also an area of active research. At the start of the 21st century it is apparent that in the coming decade multimodal therapies will be available to the patient, including new chemical compounds, recombinant proteins, and gene, cell, and organ therapies. Molecular Therapy 2000 1, 213-224DOI: (10.1006/mthe.2000.0044) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 2 Retroviral and adenoviral vectors. Retroviruses based on Moloney murine leukemia virus (MoMLV) were engineered as the first vectors for gene therapy and have been used extensively in clinical trials. Advantages of this vector class include highly efficient gene transduction of target cells and lack of viral antigens expressed on gene-transduced cells, thus minimizing patient immune responses to gene therapy. The therapeutic use of retroviral vectors is limited by the requirement for gene transfer into actively dividing cells. Attempts to generate vectors based on nonhuman lentiviruses (a genus of Retroviridae) may overcome this limitation. Adenoviral vectors offer unparalleled high-efficiency gene transfer into both dividing and quiescent target cell populations and are thus potentially attractive for in vivo gene therapy procedures. A major drawback of these vectors is their antigenicity and generation of strong immune responses in humans. Molecular Therapy 2000 1, 213-224DOI: (10.1006/mthe.2000.0044) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 3 Synthetic vectors. The ability to generate synthetic vectors offers an attractive alternative to viral vectors in terms of safety since there would be no risk of replication-competent viral particles and potentially less immunogenicity. The field is at an early stage and limited knowledge of the mechanism of action of these vectors is currently a barrier in achieving high-efficiency gene transfer in target cells. Molecular Therapy 2000 1, 213-224DOI: (10.1006/mthe.2000.0044) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 4 Hematopoietic stem cells and their progeny. By focusing on hematopoietic stem cells it should be possible to repopulate the blood system with gene-modified progeny cells which confer a therapeutic benefit. Hematopoietic stem cells are defined by the presence of CD34 and Thy-1 (also known as CD90) cell-surface proteins. These cell-surface markers are important in the isolation and purification of stem cell populations for gene and cell therapy applications. Molecular Therapy 2000 1, 213-224DOI: (10.1006/mthe.2000.0044) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 5 Gene-modified hematopoietic stem cells as therapy for HIV infection. The ability to purify human hematopoietic stem cell populations via their CD34 marker and gene transduce them ex vivo with retroviral vectors carrying therapeutic genes inhibiting HIV-1 replication (e.g., genes for transdominant REV mutants) offers a novel therapeutic approach to treat HIV infection. Repopulation of the patients immune systems with gene-modified CD4+ cells should restore immune function and decrease viral load. The approach is being evaluated in Phase I clinical trials and will offer an alternative gene-based approach to treat HIV infection that will complement standard anti-retroviral therapies based on chemical entities. Molecular Therapy 2000 1, 213-224DOI: (10.1006/mthe.2000.0044) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 6 Ligand-mediated control of therapeutic gene expression in gene therapy. Controlling therapeutic gene expression in vivo is an important goal for successful gene therapy. Such gene regulation systems will be useful for regulating levels of therapeutic proteins or eliminating gene-modified cells. Regulation is achieved by binding of an orally active low-molecular-weight ligand to a chimeric transactivating protein that is expressed tissue specifically or only in cells harboring the gene therapy vector. The complex of the ligand and the chimeric protein is then able to bind vector-encoded DNA elements controlling therapeutic or transgene expression. In the absence of the ligand the chimeric regulating protein is unable to interact with its target DNA sequence. Molecular Therapy 2000 1, 213-224DOI: (10.1006/mthe.2000.0044) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 7 Imbalance between human organ transplants and transplantation waiting lists. The problem of insufficiency of donor organs for transplantation is a major healthcare issue in terms of both life span and quality for afflicted individuals and healthcare costs of maintaining patients waiting for a suitable donor organ. The gap is increasing with time and xenotransplantation offers a means to satisfy the growing medical need. Molecular Therapy 2000 1, 213-224DOI: (10.1006/mthe.2000.0044) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 8 Immunological barriers in allo- and xenotransplantation. The immunological barriers in allotransplantation (i.e., transplantation within a given species such as humans) involve predominantly cell-mediated processes leading to donor organ rejection. This process is managed in the clinical setting with effective immunosuppressive therapy (e.g., cyclosporin in combination with adjunctive pharmacotherapy). In xenotransplantation (i.e., transplantation from one species to another such as pig-to-human transplants) additional humoral immune mechanisms involving preformed antibodies in the transplant recipient and activation of the complement cascade are responsible for hyperacute rejection and organ loss. Molecular Therapy 2000 1, 213-224DOI: (10.1006/mthe.2000.0044) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 9 The hyperacute rejection mechanism. Human serum contains preformed xenoreactive antibodies directed against terminal Gal-α1,3-Gal carbohydrate structures present on pig endothelium. Binding of these antibodies to transplanted pig tissue leads to complement and endothelial cell activation and the subsequent and rapid hyperacute rejection of the transplanted pig organ. Researchers aim to overcome hyperacute rejection by engineering transgenic pigs with organs expressing human complement-inhibiting proteins such as decay-accelerating factor (DAF; also called CD 55), membrane cofactor protein (MCP; also called CD46), and CD59. Additional strategies to maintain functioning xeno-organs resistant to hyperacute rejection include blocking the human natural antibodies and deleting the Gal-α1,3-Gal epitope from the transgenic pig herds. Molecular Therapy 2000 1, 213-224DOI: (10.1006/mthe.2000.0044) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 10 Producing transgenic pigs for xenotransplantation. By using transgenic technologies it is possible to insert human genes for complement-regulating proteins into the pig genome. Transgenic pig organs expressing human complement inhibitors have been evaluated in preclinical transplantation models using nonhuman primates. In these studies the transgenic organs expressing human DAF have significantly increased survival times versus nontransgenic organ controls. Molecular Therapy 2000 1, 213-224DOI: (10.1006/mthe.2000.0044) Copyright © 2000 American Society for Gene Therapy Terms and Conditions