Progress and Potential for Gene-Based Medicines - Cell Press

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doi:10.1006/mthe.2000.0044, available online at http://www.idealibrary.com on IDEAL

Progress and Potential for Gene-Based Medicines Mark R. Dyer and Paul L. Herrling1 Novartis Pharma AG, CH-4002 Basel, Switzerland

During the past decade researchers have explored the potential of gene-based medicines to extend current treatments employing chemical entities and proteins. However, progress has been slower than was originally predicted due to our limited knowledge of the genetic components of major diseases, the complexity of developing active biological agents as therapies, and the stringent and time-consuming tests necessary to ensure safety prior to introduction of these novel modalities in the clinic. In spite of the present technology challenges and clinical setbacks in gene therapy it is anticipated that gene-based medicines will find their niche in disease prevention and management strategies in the coming decade, extending the repertoire of medicines available to satisfy key unmet medical needs. Additionally, progress in xenotransplantation research is creating the opportunity to use gene-modified porcine organs for human transplantation. This innovative approach aims to address the current insufficiency of human donor organs for clinical transplantation.

GENOMICS AND OPPORTUNITIES MEDICINES

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GENE-BASED

The medical potential of gene-based therapies is gaining momentum with the progress of the Human Genome Project and the genomics field overall (1–3). The recent publication of the sequence of human chromosome 22 (4) is a major milestone en route to a completed genome sequence within the next 3 years. With increasing knowledge of the genetic components in major diseases such as Alzheimer’s disease (5), Parkinson’s disease (6, 7), diabetes (8), asthma (9), rheumatoid arthritis (10), and chronic myelogenous leukemia (11) it will soon be possible to identify underlying genetic defects and key disease targets (12). This in turn will result in an increased number of therapeutic products that address the cause, onset, or progression of the specific malady as opposed to merely treating its symptoms. The broad application of recombinant DNA technology and gene cloning (which started in the 1980s) and the resulting exponential increase in genomics data during the 1990s have enabled researchers to define some disease-causing genetic factors and to begin exploring the potential of “biological therapies” based on engineered genes, cells, and gene-modified organs. Although these gene- and cell-based therapies are a constituent of today’s pharmaceutical and biotechnology companies’ R & D pipelines, it is important to appreciate that they will complement but not necessarily substitute for medicines incorporating chemical entities and therapeutic proteins (Fig. 1). In many cases the gene- and cell-based medicines available to the patient will comprise biologically active components supported by chemical and/or proteinaceous agents that are necessary for their successful and safe applications in humans. 1 To whom correspondence should be addressed. Fax: ⫹41 61 324 2141. E-mail: [email protected].

MOLECULAR THERAPY Vol. 1, No. 3, March 2000 Copyright © The American Society of Gene Therapy

GENE DELIVERY SYSTEMS The ultimate goal for gene therapy is the genomic replacement in a site-specific manner of a disease-causing gene or allele with its “healthy” counterpart. Such a medical strategy envisages a corrected gene surrounded by appropriate regulating sequences and expressing its product in a physiologically relevant manner. Such an elegant molecular approach to human medicine is a long-term goal. More realistically, research is ongoing for development of gene therapy vectors to deliver and express genes at therapeutically meaningful levels in a controlled and/or sustainable manner (13). The first-generation gene therapy vectors have harnessed the innate ability of viral systems to deliver genetic information to human cells. Researchers are also exploring the potential of nonviral synthetic vectors and hybrid synthetic–viral systems as safer alternatives for gene delivery. A further approach uses human stem cells as a means to introduce the therapeutic genes into specific human cell populations where the therapeutic product is required. In spite of dedicated efforts throughout the past decade, the gene therapy field is still limited by the absence of effective gene delivery systems, which can target therapeutic genes in vivo to the tissue or organ systems where the therapy is needed. The safety profile of gene therapy vectors is another area that needs to be further improved with high priority.

VIRAL VECTORS

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GENE THERAPY

By engineering viral genomes and deleting genes involved in viral pathogenesis, replication, and those deemed “nonessential,” it has been possible to generate replication-incompetent vectors which can carry therapeutic genes for ex vivo and in vivo gene therapy procedures. Different vectors based on both DNA and RNA

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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.

viruses are being explored to meet diverse therapeutic needs. Each viral system has its advantages and disadvantages when one is considering clinical applications (Table 1). Some of the difficulties associated with viral vectors include limitations on the size of therapeutic gene payloads, which currently precludes using large genomic sequences containing important gene regulatory sequences; low gene transduction efficiencies for certain vector types; poor vector selectivity for target cells in vivo; random integration in the human genome and possibility of insertional mutagenesis; difficulty in achievement of durable and therapeutically relevant gene expression levels; and the immunogenic and safety aspects of viral vectors and transgenes used in clinical studies.

RNA VIRAL VECTORS In the historical context of the gene therapy field, retroviral vectors (Fig. 2) derived from mouse Moloney leukemia virus (MoMLV) were the first developed for gene therapy applications (14). A useful feature of retroviruses is their ability to integrate the proviral genome into human genomic DNA, thus conferring potential for longterm therapeutic gene expression. Although these firstgeneration viral vectors have the major advantage of an ability to gene transduce cells to high efficiency without promoting expression of viral antigens on gene-trans-

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duced cells (thus limiting host immune reactions), there are limitations dictated by the need for target cells to be actively dividing. Hence, their therapeutic applications in vivo are limited. To circumvent this barrier researchers have been exploring other RNA viral vectors based on lentiviruses such as human immunodeficiency virus (HIV) and nonhuman lentiviruses such as bovine immunodeficiency virus (BIV), simian immunodeficiency virus (SIV), and the ovine lentivirus Maedi visna (15, 16). An advantage of this vector class is that it can efficiently gene transduce several types of nondividing cells, thus increasing the number of potential disease indications to which in vivo gene therapy can be applied. A major safety concern arises in using lentiviral vectors based on HIV. This problem may be significantly diminished by the development of novel systems based on other Retroviridae that are not pathogenic to humans.

DNA VIRAL VECTORS DNA viral vectors based on adenoviruses (Fig. 2) and adeno-associated viruses (AAV) are also being evaluated in gene therapy approaches (17). Adenoviral vectors have an unparalleled capacity to gene transduce a broad range of nondividing cells in vivo and hence could potentially be applied to treat diseases of several organ systems. HowMOLECULAR THERAPY Vol. 1, No. 3, March 2000 Copyright © The American Society of Gene Therapy

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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. 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.

ever, adenoviral vectors have several limitations that need to be overcome before they can be administered as therapeutic agents. Unlike retroviruses, which integrate their proviral DNA into the human genome, adenoviral vector genomes take up an epichromosomal location in their target cells resulting in shorter-term expression of the therapeutic gene and the potential need for repeated administrations. To achieve optimal results with this system MOLECULAR THERAPY Vol. 1, No. 3, March 2000 Copyright © The American Society of Gene Therapy

research activities are ongoing to improve adenoviral vectors including engineering of their capsid and fiber proteins for improved cell-specific targeting in vivo. A major difficulty exists through human contact with wild-type adenoviruses, the causative agent of the common cold, and the resultant development of immunity. Hence, researchers are modifying adenoviral vector surface proteins such that they are no longer strongly immunogenic in

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REVIEW TABLE 1 Advantages

Disadvantages

Retroviral vectors

Integration into the human genome Potential for long-term therapeutic gene expression Random integration Potential for insertional mutagenesis Gene transduction High-efficiency gene transduction Need for dividing target cells Limits tissue specificity and in vivo applications Safety issues No expression of viral proteins on gene-transduced cells Potential for replication-competent retrovirus Low immunogenicity (RCR)

Lentiviral vectors

Integration into the human genome Potential for long-term therapeutic gene expression Potential for insertional mutagenesis of human genes Gene transduction High-efficiency gene transduction HIV vectors are not able to transduce all Transduces several nondividing cell types nondividing cells (e.g., hepatocytes) Broader applications in vivo/ex vivo Safety issues HIV-based systems not attractive in the clinic Limiting knowledge of nonhuman lentiviruses may slow development of vectors with clinical applicability

Adenoviral vectors

Nonintegration into the human genome No insertional inactivation of human genes Transient therapeutic gene expression for early generation vectors; gutless vectors may overcome this barrier Gene transduction High-efficiency gene transduction Transduction of dividing/nondividing cells Broader in vivo applications Safety issues Immunogenicity in humans

AAV vectors

Integration into the human genome Potential for long-term therapeutic gene expression Potential for insertional mutagenesis of human genes Gene transduction Medium-efficiency gene transduction Difficult to produce high-titer AAV vector Transduces dividing/nondividing cells supernatants for clinical studies Safety issues Expectation that vector-related immunogenicity will be low due to minimal viral sequences

humans. The development of gutless adenoviral vectors where the majority of viral structural genes are deleted is a move toward less immunogenic vectors. Adenoviral vectors are being pursued in a number of disease indications including cancer, hemophilia, and cardiovascular diseases and clinical trials have been initiated. Unlike adenoviral vectors, AAV vectors integrate their DNA into the target genome. However, the progression to clinical trials of gene therapies based on AAV vectors has been slow and one of the main difficulties has been production of hightiter, purified vector supernatants for clinical studies. AAV vectors are currently being used in clinical trails for hemophilia (18).

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NONVIRAL SYNTHETIC VECTORS

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HYBRID VECTORS

Synthetic vectors (19, 20) offer a possible alternative to viral vectors. Because they lack viral components they are less likely to trigger an immune response in the patient and thus have better safety profiles (Fig. 3). Early-generation synthetic vectors have included cationic lipid or cationic polymer complexes of expression plasmids. Research is still at an early stage and the mechanism of uptake of these vectors is poorly understood making it difficult to optimize them for in vivo gene delivery. Other research activities to improve synthetic vector capabilities include the use of specific ligands based on peptides or MOLECULAR THERAPY Vol. 1, No. 3, March 2000 Copyright © The American Society of Gene Therapy

REVIEW carbohydrates for vector targeting in vivo, blocking of nonspecific binding to cell surfaces using protective steric polymers, condensing DNA into small stable colloids for production of the vectors, improvement of cell penetration and cytoplasmic delivery, as well as effective nuclear import of the cargo DNA once inside the target cells. Due to the current difficulties in developing effective synthetic vectors capable of high-efficiency gene transfer, researchers are constructing hybrid viral–synthetic vectors systems, e.g., synthetic components such as cationic lipids combined with viral vectors or proteins (21). In this vector class some of the beneficial features of viral systems are retained, while keeping potentially harmful viral components to a necessary minimum. Proof-of-concept studies for nonviral synthetic vectors are ongoing in the respiratory disease area. The respiratory tract offers an attractive model system in which to test gene therapies since there are fewer problems associated with targeted delivery and vector clearance than with systemic gene delivery. The lung is accessible by direct aerosolized delivery, thus enabling gene therapy vectors to direct their therapeutic genes to cells and tissues involved in the disease process. A further important issue is the fact that synthetic vectors can be engineered to be less immunogenic than their viral counterparts and so there should be decreased risk of exacerbating the ongoing inflammatory conditions that characterize major lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). Synthetic vectors have been used in clinical trials to deliver the CFTR gene encoding a functional chloride channel into cystic fibrosis patients (22). As key genes involved in other diseases of the lung are defined by genomics and other disciplines, potential clinical applications will increase.

CELL-BASED DELIVERY

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THERAPEUTIC GENES

Although cell therapies have been used in medicine for several decades (e.g., blood transfusions), the use of cells manipulated ex vivo with therapeutic genes and then reintroduced into patients offers a new strategy by which to deliver gene-based medicines. The advantages of ex vivo genetic manipulation of cells include the ability to engineer highly purified cell populations and optimize the conditions for high-efficiency gene transfer outside the body. This obviates the issues of in vivo targeting of vectors and circulating host antibody responses. Furthermore, safety profiles of ex vivo modified cells can be stringently assessed prior to administration to the patient. Of particular interest in the biomedical community are human stem cells. This cell class gives rise to various cell lineages in particular organ systems. In theory, the genemodified stem cell should continually repopulate differentiated cell pools and thus confers long-term therapeutic benefit. This has the advantage that frequent reinfusions with gene-modified cells should be unnecessary. Furthermore, the choice of stem cell for gene modification enMOLECULAR THERAPY Vol. 1, No. 3, March 2000 Copyright © The American Society of Gene Therapy

ables subsets of tissues to be targeted with a therapeutic gene(s). At present, human hematopoietic stem cells (Fig. 4), mesenchymal stem cells, neuronal stem cells, and those of embryonic origin are sufficiently well characterized and are the focus of pharmaceutical discovery efforts (23). The panel of stem cells available to gene therapists will increase as researchers are better able to define and successfully isolate and culture stem cells from other organs systems or define pluripotent stem cells and appropriate factors which can be used to drive their differentiation along distinct cell lineage pathways. Issues currently being addressed include the development of vectors for efficient stem cell gene transduction or transfection, expression and regulation of therapeutic genes during lineage progression from stem cells to differentiated cells, control of stem cell growth, and expansion ex vivo and engraftment and differentiation in vivo. In addition to exploring autologous stem cell transplants (i.e., ex vivo gene modification of a patient’s own stem cells), researchers are working to achieve allotransplantation of stem cells across MHC barriers existing between individuals. Hematopoietic stem cell therapy is currently being used in clinical trials to transduce genes such as the RevM10 gene, which inhibits HIV-1 replication (24), into the immune system of HIV-infected individuals (Fig. 5). This approach offers potential for HIV-resistant immune cells and concomitant decrease in vivo of viral load and therapy-resistant HIV strains. In addition to specifically addressing the pressing medical need in HIV infection and AIDS, these studies are enabling researchers to obtain proof-of-concept data for cell-based gene therapy and further develop effective and safe applications in humans. Such pioneering studies also help define the regulatory framework that will ensure the appropriateness of preclinical studies used in supporting entry of these approaches into the clinical environment.

REGULATING THERAPEUTIC GENE EXPRESSION

IN

VIVO

Controlling therapeutic gene expression will be necessary for both the clinical efficacy and safety of all gene therapies. Controlled expression can be used to turn therapeutic genes on and off, regulate therapeutic protein levels, or eliminate gene-modified cells in case of adverse events. Regulation can be achieved via dose-dependent ligand binding and activation of chimeric transcription factor proteins, which then interact with DNA elements intrinsic to the gene therapy vector construct and regulate the therapeutic gene (Fig. 6). This concept has been proven in animal models using a bacterial operon/tetracycline repressor, nuclear hormone receptors, and a chemically induced protein dimerization strategy (25, 26). All of these systems are amenable to use of orally active low-molecular-weight chemical entities to regulate the therapeutic gene.

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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.

LOCALIZED THERAPY USING GENE-BASED APPROACHES Most of today’s therapies are based on chemical entities and proteins. They reach their site of action in the body via the blood stream after oral or parenteral administration, and hence systemic side effects are an important consideration in developing effective and safe medicines. One major advantage of gene therapy is its ability to specifically introduce one or more therapeutic genes directly into the tissue or organ system where the beneficial effect is needed, either by vector targeting or tissue-restricted therapeutic gene expression, hence minimizing undesirable effects in other parts of the body. The example cited above of HIV gene therapy vectors targeting

hematopoietic stem cells and their progeny illustrates this point. Further developments in vector targeting and tissue-restricted expression of the therapeutic genes they carry will enable localized gene therapy to be realized following in vivo administration.

PROGRESS IN CLINICAL TRIALS OF GENE THERAPIES

AND

SAFETY ASPECTS

During 1999 there were circa 300 clinical gene therapy protocols registered with the Office of Biotechnology Activities (OBA) (27). A majority of these clinical trials are directed at diseases that are life threatening and for which

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. 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.


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REVIEW currently available therapies are not highly effective, such as HIV infection and certain cancers. Gene therapy trials are also ongoing for monogenic disorders with low incidence in the population. These are being addressed through our knowledge of the defective genes in these rare inherited diseases. Clinical trials which do not address immediate therapeutic benefits to the individuals enrolled, such as gene-marking studies, are performed in order to test specific hypotheses concerning in vivo vector functioning and targeting or efficiency and fate of genemodified cells in humans. Following completion of the Human Genome Project and the increasing data on the genetic components involved in disease causation, it is reasonable to expect that gene-based medicines will provide prophylactic and therapeutic treatments for a number of major diseases. Recent patient fatalities have been reported in gene therapy clinical studies, including the death of an 18year-old patient receiving high-dose adenoviral gene therapy for an inherited deficiency in ornithine transcarbamylase (an enzyme involved in the urea cycle and ammonia metabolism) (28). Such fatalities underscore the risks involved in pioneering fundamentally new approaches in medicine. They also highlight the absolute requirement for comprehensive regulatory guidelines and stringent review procedures for clinical protocols. Clearly, the decision to move into clinical trials should be founded on comprehensive efficacy and safety data from preclinical studies. Hence, there remains a pressing need for further development of suitable animal models of gene therapy in which issues such as safety can be adequately addressed at the preclinical stage (29).

XENOTRANSPLANTATION Satisfying the Medical Need in Organ Transplantation With the development of effective immunosuppressive therapy in the mid-1980s human-to-human organ transplants (allotransplants) have become a widely accepted approach for treatment of kidney, heart, liver, and lung failure. However, the success of allotransplantation in the clinical setting has created its own problems in terms of the imbalance in demand and supply of donor organs. With expansion in disease indications benefiting from organ replacement therapy the gap between need and availability of human donor organs is increasing on a yearly basis (Fig. 7). As one viable alternative researchers have been exploring xenotransplantation, the transplanting of organs from one species to another (30). The pig appears to be the most suitable animal donor for human transplants since organs from these two species share many physiological and metabolic features, and pig organ size is compatible with human anatomy. Since the pig has been domesticated for many thousands of years it can also be postulated that humans have acquired resistance to many porcine pathogens during the course of history, while nonhuman primates do not have this advantage

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and so have been excluded as organ donors for xenotransplantation (31). Since immunological mechanisms preclude successful cross-species organ transplants, scientists are using genetic engineering as a means to modify porcine organs to express human proteins in an attempt to prevent organ rejection. Xenotransplantation offers several potential advantages over human-to-human allotransplants. These include the unlimited supply of donor organs allowing surgeons to elect the exact timing of the transplantation procedure with both positive impact on the quality of donor organs (shorter ischemic time) and lowering of healthcare costs (reduction in numbers of patients on waiting lists for donor organs). A further opportunity is the ability for researchers to stringently monitor and eliminate pathogenic organisms from the porcine donor herds prior to use of the organs in humans.

OVERCOMING HYPERACUTE REJECTION MECHANISMS USING GENE-MODIFIED XENO-ORGANS In human allotransplantation the major issue is the transplant recipient’s cell-mediated immune defenses, which recognize the donor organ as nonself. This process can be successfully controlled by immunosuppressive drugs (e.g., cyclosporin). In xenotransplantation additional humoral immune defense mechanisms in the transplant recipient must be overcome if the procedure is to be successful (Fig. 8). A “wild-type” xeno-organ transplanted into another species will undergo rapid attack by the recipient’s immune system, a process termed hyperacute rejection. Complement activation plays a key role in this process leading to irreparable damage to the donor organ and its destruction within minutes (Fig. 9). Gene-based approaches which inhibit the complement cascade are being used to postpone or inhibit onset of hyperacute rejection. To achieve this goal transgenic technologies are being applied in order to gene modify porcine organs so that they express human complement-inhibiting proteins on their surface, e.g., human DAF (decay-accelerating factor, also called CD55) (Fig. 10). Transplantation of genemodified porcine organs into nonhuman primate models has demonstrated that hyperacute rejection can be overcome with significant increases in graft survival (32). Work is also ongoing to produce second-generation transgenic pigs using transgenic and nuclear transfer technologies to express or delete additional protein and carbohydrate antigens (e.g., deletion of the Gal-␣1,3-Gal carbohydrate epitope on porcine endothelium that is recognized by human xeno-reactive antibodies). This will further contribute to survival and proper functioning of the xeno-organs in the human transplant recipient. As is the case with other gene- and cell-based medicines, the xeno-organ product available to the patient will be a multicomponent therapy. As an adjunct to gene-modified xeno-organs, the product is likely to include pharmacotherapy for effective immunosuppression, removal of patients’ preformed xeno-reactive antibodies, and management of acute and chronic transplant rejection episodes. MOLECULAR THERAPY Vol. 1, No. 3, March 2000 Copyright © The American Society of Gene Therapy

REVIEW SAFETY ISSUES RETROVIRUSES

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PORCINE ENDOGENOUS

One of the major issues in xenotransplantation prior to commencement of clinical trials is the rigorous evaluation of the potential for zoonosis, the ability of microorganisms innocuous in pigs to become pathogenic in humans after pig-to-human organ transplantation. While it has been possible to identify and eliminate many potential pathogens from transgenic herds by husbandry of specified pathogen-free animals, there is one remaining potential risk, so-called porcine endogenous retroviruses (PERVs). As is the case with other mammalian species, the pig genome is interspersed with retroviral sequences. PERVs are c-type retroviruses that can infect human cell lines in vitro, an observation that has led to concerns that there will be viral transmission in the clinical setting once transgenic pig organs are transplanted into immunosuppressed patients. Porcine cells, tissues, and proteins have already been used as therapeutics in several hundred patients worldwide (e.g., porcine skin grafting in burn patients and porcine pancreatic islet cell transplants in diabetics). Thus, it is possible to retrospectively study the potential for PERV transmission in these individuals using highly sensitive molecular diagnostic tools such as the polymerase chain reaction and specific antibody tests. In 160 patients receiving porcine tissues, no evidence of active PERV transmission was detected (33). While the aforementioned results go a long way to allay concerns regarding zoonosis in pig-to-human transplants, this is still an area of active concern and research (34). As we gain further understanding of PERV variants and their loci within the pig genome it should be possible to eliminate certain PERV sequences both by conventional breeding methodologies and by homologous recombination followed by cloning technologies. Use of standard retroviral therapies will also provide a strategy to manage PERV replication in those rare cases where it may occur. Firm understanding of the biology of PERVs coupled with extensive monitoring of donor pig herds and patients receiving xenotransplanted organs will minimize the safety risks associated with this new approach in transplantation.

ETHICAL CONSIDERATIONS IN USING GENE-BASED MEDICINES AND SECURITY OF GENETIC INFORMATION Current gene therapy protocols have addressed gene modifications in somatic cells and have not attempted to modify our genetic heritage by targeting germ-line cells. In fact, regulatory guidelines require stringent testing of patients receiving gene therapy in order to confirm that germ-line and other nontarget tissues have not been gene modified inadvertently. Gene modification of nontarget tissues appears to be a rare event (35) and potential for gene modification of nontarget tissues will remain an important area for testing as in vivo vector technology improves and gene transfer efficiencies increase. The foMOLECULAR THERAPY Vol. 1, No. 3, March 2000 Copyright © The American Society of Gene Therapy

cus on somatic gene therapy is highly appropriate and no change in current policy should be undertaken which would promote germ-line gene therapy in humans. Targeting germ-line DNA would change genetic information passed on to future generations and as such is undesirable since therapeutic benefits of genetic medicine should be conferred on individuals actually diagnosed as being at significant risk from the disease. Also, developments of new therapies that are not gene based may replace gene therapy as a standard treatment for a given disease thus making gene modification unnecessary. A further important consideration is one of informed consent of patients who elect to receive gene-based medicines: this would clearly not be an active decision by future generations inheriting laboratory-engineered genetic modifications to their DNA. For these reasons, we are strongly opposed to any form of germ-line gene therapy in humans. With regard to the need to monitor and document safety aspects of gene therapy the FDA has formally published guidelines for researchers, and regulatory bodies in other countries require similar safety testing. The major focus has been directed at patients receiving retroviral gene therapy, due to concerns about the potential for replication-competent retroviruses (RCR) in clinical grade vector supernatants (36). Similarly, the progress of xenotransplantation into clinical trials will require testing of patient tissue samples pre- and posttransplantation. These tests on biopsy materials from people receiving genebased medicines will generate large amounts of confidential biomedical data that need to be archived in secure databases and information management systems. Medical databases are also a feature of the genomics and molecular diagnostics fields and data concerning disease predisposition place us in a privileged position to address therapeutic needs tailored to the individual, provided we have appropriate preventatives or therapeutics in our medical repertoire. However, the accumulation of the individual’s genetic data and their use in predicting disease susceptibilities are areas of heated debate and need to be regulated to the highest standards of integrity. These data should provide a resource solely for the purpose of biomedical research and development of superior therapies and must in no way be used to discriminate against people in their private and professional lives. The elaboration of appropriate laws for genetic data protection should ensure sensible usage of this information with resultant health benefits (37).

CONCLUSIONS

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OUTLOOK

FOR THE

FIELD

The progress of gene-based medicines has been slower than was expected in the early 1990s. This is due to several factors: ● The task of pioneering new therapeutic modalities based on active biological systems such as genes and cells is highly complex, occurring at the leading edge of biomedical knowledge. ● Incomplete understanding of the genes involved in

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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. 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.

FIG. 9. The hyperacute rejection mechanism. Human serum contains preformed xenoreactive antibodies directed against terminal Gal-␣1,3-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-␣1,3-Gal epitope from the transgenic pig herds. 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.

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REVIEW disease causation limits our ability to generate effective genetic therapies, especially in major diseases with multiple interacting genetic and environmental components. ● Need exists for stringent and time-consuming safety studies and the elaboration of new regulatory frameworks to control the applications of gene-based medicines and ensure safety to the patient and the population at large. ● High costs are involved in R & D of gene-based medicines and issues of intellectual property and commercial rights. ● Resistance to genetic engineering has been voiced in several countries. In spite of the technological issues and clinical setbacks experienced at present, it is anticipated that gene-based medicines will find their niche in disease prevention and management and augment the new chemical entities and recombinant protein therapies that are now in development. This in turn will offer significant opportunities to effectively target the causative factors for major diseases afflicting mankind. ACKNOWLEDGMENTS The authors thank Dr. Ernst Boehnlein, Dr. Rao Movva, and Mr David Moon for their expert review and critical appraisal of the manuscript.

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