INTRODUCTION
Since genetic engineering has the potential to alter the uses to which domestic animals are put, it also can lead to fundamental changes in their relationship with one another (for example, regarding the genetic structure of their populations) and between them (or their products) and humans. There are currently major research efforts under way to develop the use of genetically engineered animals as sources for production of nontraditional materials for human use. Such uses can be divided into three major categories: biopharmaceuticals for animal and human use; life cells, tissues, and organs for xenotransplantation; and raw materials for processing into the other useful and products (the latter use is discussed in chapter 4). Several possible concerns that might in practice arise from the first to uses are discussed in the following sections.
BIOPHARMACEUTICAL PRODUCTION
A large number of genes and coding useful protein products -- hormones, blood proteins, and others -- have been introduced into domestic animals leading to their expression in milk, eggs, or blood (Dove, 2000; table 3-1). None of these animals are yet in use for commercial production, however. Indeed, a recent report suggests that the same technology might be extended to the large-
http://zoom.nap.edu/nap-cgi/rezoom.cgi?isbn=0309084393&page=53
scale production of vaccines (Stowers et al., 2001). Such "biopharming" applications have the potential to use well-established agricultural methods to produce large amounts of valuable products at relatively low expense as compared to Fermentation. Although the and products of the second and third applications will be novel, by and large, the process of production and the potential concerns are not likely to differ greatly from those seen in current practice, such as the use of animals or animal cell cultures to prepare live vaccines (Brown et al., 2001). Hormones, or traditional products like meat, milk, or leather. The standard products have to pass specific regulatory procedures, and essentially the same regulatory framework should apply for products of both biopharming in standard technology as regards, and issues such as purity of the final product, microbial contamination, levels of advantitious DNA and the like. Nevertheless, a few more specialized concerns arise.
Contamination or Spread of Novel Pathogens
As discussed in chapter 2,there is a theoretical potential for Micro organisms to acquire -- bite recombination or transduction -- genes from the vector constructs used to insert the transgene. Although there is no example yet of acquisition of any gene, including drug resistance markers, by bacterial flora living in a transgenic animal, spread of introduced genes remains a possibility, albeit remote.
Of greater concern is the possibility for generation of potentially pathogens viruses bite recombination between sequences of the vector used to introduce a transgene and related, but not pathogens, viruses that might be present in the same animal. These concerns are particularly acute for retroviral factors. Retroviruses appear to be efficient vehicles for inserting trains genes into many species, including chickens (Crittenden et al., 1990; Briskin et al., 1991), mice (Jahner et al., 1985), cattle (Chan et al., 1998, fish, and shellfish (Sarmasik et al., 2001) and might provide significant advantages and success rate over pronuclear injection of DNA in the generation of transgenic offspring in many species, including chickens and pigs, there are endogenous proviruses (including the poor kind endogenous viruses (PERVs) discussed in a following section) that are competent for low-level replication in the host animal but have no apparent pathogenic consequences (Boeke and Stoye, 1997). Endogenous proviruses aren't DNA sequences that were derived from infection of germline cells with a retrovirus and that are transmitted from parent to progeny like any normal gene. Their attenuation relative to their exogenous, pathogenic, counterparts often is due to differences in transcriptional regulatory sequences in the long terminal repeats (LTR's; Rosenberg and Jolicouer, 1997). Since many vectors, such as the widely-used ones derived from murine leukemia virus (MLV), have LTR sequences derived from pathogenic viruses, the presence of
http://zoom.nap.edu/nap-cgi/rezoom.cgi?isbn=0309084393&page=54
vector and endogenous proviruses and all cells of the transgenic animal provides the potential for generating pathogenic recombinant viruses, by straightforward and well-understood mechanisms. Such concerns are particularly acute and chickens and pigs were infectious proviruses very similar in sequence to those used for vectors are known to be present (Boeke and Stoye, 1997; see discussion of PERVs below). In mice, there is a well studied model in which recombination between benign endogenous proviruses, or endogenous proviruses and infecting viruses early in the life of the animal can cause a high incidence of lymphoma (nearly 100 percent in some of strains) six months later (Stoye et al., 1991; Rosenberg and Jolicouer, 1997). Given this example, it is reasonable to expect that viruses of much greater pathogenicity than existed before are likely to arise and animal when there is a possibility of recombination between vector and endogenous viral sequences
Some concerns arise with the use of vectors based on lentiviruses for introduction of genes (see chapter 2). Recombination of lentiviruses in circulation and domestic animal populations, such as FIV in cats, and BIV in cattle, with vectors based on HIV is improbable to the large genetic distance between them. However vectors based on FIV and BIV are being developed (Curran, 2000; Berkowitz et al., 2001), and their use to introduce transgenes into the corresponding species would significantly increase the probability of generation of more pathogenic recombinants.
TABLE 3-1 Potential Uses of Transgenic Animals for Pharmaceutical Production
Theoretical Yield (g/yr of Species Raw Protein Examples of Products under Development chicken 250 monoclonal antibodies lysozyme growth hormone insulin human serum albumin rabbit 20 calcitonin superoxide dismutase erithropoiten growth hormone IL-2 a-glucosidase goat 4000 antithrombin III tissues plasminogen activator monoclonal antibodies a-1-antitrypsin growth hormone sheep 2500 a-1-antitrypsin factor VIII factor IX fibrinogen cow 80,000 human serum albumin lactoferrin a-lactalbumin source: modified from Dove, 2000.
Ensuring Confinement of Unwanted Animals
although animals engineered to produce useful products will not be intended for consumption by animals or other animals, there are grounds for concerned that adequate controls be in place to ensure that this does not happen without appropriate approval (see chapter 4). As long as they do not contain the product of the induced gene, there might be no strong reason to believe that eating or using products from containing animals would pose a threat to human health; however, the lack of regulatory oversight for such uses argues strongly for confinement measures.
Although it has been stated that such animals will be to valuable to the owners to allow their misappropriation (Wall, 2001), the fact that the products of interest usually are produced only by lactation females means that half the transgene-containing animals produced will be essentially valueless, as will be the females at the end of their. Of useful production. "No takes," or animals generated from manipulated embryos, but cold because they lack appropriate expression of the transgene product (or lacking the transgene itself) are also and evidently generated in significant numbers during the production of transgenics. Thus, companies using biopharm animals are likely to seek approval for marketing food or rendered products from surplus animals, and the regulatory agencies will need to be ready to deal with such requests. Of greater concern is the possibility that surplus animals (and their carcasses) might, inadvertence for theft, find their way into the food or rendering chain, or be used for breeding, thus allowing uncontrolled spread of the transgene into the general population, creating the regulatory problem of dealing with a approved transgenes after their release into the food chain, a problem analogous to that posed by the appearance in food products of star link, a transgenic maize unapproved at the time for human consumption (Fox, 2001).
XENOTRANSPLANTATION
http://zoom.nap.edu/nap-cgi/rezoom.cgi?isbn=0309084393&page=56
Xenotransplantation differs from other uses of genetically engineered animals in that it has the potential to create something entirely new -- permanent human-animal chimeras -- in which cells of distantly-related species survived and function for long periods of time in the most intimate possible contact. Given its potential for alleviating human diseases due to irreversible tissue or organ failure (see table 3-2), and given the acute shortage of human organs for transplant, there are very active research programs underway, in both commercial and academic laboratories, to solve the significant immunological and physiological problems and thereby bring xenotransplantation into standard medical practice. This topic, and the associated infectious concerns have been renewed in great detail elsewhere (Boneva et al., 2001), and only an overview will be given here.
TABLE 3-2 Applications of Xenotransplantation
Indication Transplant Status organ failure take heart, kidney, liver, etc. 0 acute liver failure extracorporeal perfusion 1 diabetes pancreatic islets (or cells) 1 Parkinson's disease Huntington's disease Neural Tissue 1 focal epilepsy stroke burn skin autograft 2 skin injury (Co-cultured with mouse cells) note: 0 = no successful experience 1 = some trials have been performed 2 = successful trials have been performed
At present, the only animal under serious consideration as a xenotransplantation or is the pig (4 regulatory purposes, human cells cultured ex vivo with cells of any other animal, such as mouse alliance, also are considered to be xenotransplantation; DHHS, 2001; co-cultivation with mouse cell means has been used in preparation of some cultured skin grafts, as well as human stem cell lines; Thompson et al., 1998). Well-known human primates, such as the baboon, would seem to have physiologic and immunogenetic advantages such as the lack of a hyperacute immune response, there scarcity as well as the difficulty of clearing them of advantitious infectious agents (as well as ethical concerns) renders them impractical for further consideration.
The field of xenotransplantation covers a great many procedures, ranging from implantation of single cells to treat Parkinson's disease; tissues, such as
http://zoom.nap.edu/nap-cgi/rezoom.cgi?isbn=0309084393&page=57
pancreatic islets for treatment of diabetes; extracorporeal use of contact organs, such as perfusion of patient blood through pig livers to provide short-term support in cases of liver failure; to transplantation of whole organs -- heart, kidney, liver, and so on. While whole-organ xenotransplantation remains far in the future, development of the simpler modalities is underway, and hundreds of human subjects have received porcine cells or tissues as part of clinical trials in the United States, Russia, Israel, and many European countries (Paradis et al., 1999). Given the nature of infectious disease issues, regulatory concerns are not limited to the United States alone, but instead extend to the international health community as well.
The development of xenotransplantation as a part of clinical practice promises great benefits in terms of making possible essentially infinite supplies of replacement tissues and organs where severe shortages exist today. This development will naturally entail both great potential benefit as well as considerable risk to the study participant but such risk is not qualitatively different than the development of any other new medical procedure and will not be considered further. The principal concern is that they uniquely close relationship created between recipient and host will allow novel opportunities for transmission of infectious disease and possibly creation of new disease agents in the process. While the history of close contact between animals and pigs is a very long one, and one would imagine that all possible transmission of infectious agents between the two species would already have been seen and thoroughly studied, it is possible that the "co-culture" endocrine mint of a transplant would be qualitatively different in ways that would allow different outcomes. Two different types of agents are discussed separately.
Exogenous Infectious Agents
In general, bacteria and parasites that might cause problems can be repeatedly excluded from source flocks, leading viruses as the principal concern (Onions et al., 2000; table 3-3). As can be seen in the table, the number of viral agents that are potential concern is very large. Not all of the viruses are on the list because of the potential to cause human disease others are sensitive indicators of breaks in biosecurity, and so forth. In principal, since all of these agents are horizontally (one animal to another) or vertically (mother to offspring) transmitted, they can be eliminated by proper management—proper containment, vaccination, close monitoring, culling, birth by cesarean section, etc. In practice, elimination is going to prove a very difficult task,, and there is lack of reliable assays for detecting many of them. Nevertheless, problems resulting from transmission of the exogenous infectious agents are not qualitatively different from the present situation with human donors
http://zoom.nap.edu/nap-cgi/rezoom.cgi?isbn=0309084393&page=58
(allotransplantation), where infection with agents transmitted with the transplanted organ (such as Epstein-Barr virus and cytomegalovirus) is a major problem. In fact, it is anticipated that reduction in the risk of acute morbidity and mortality resulting from the transmission of infectious agents will be a significant benefit of xenotransplantation.
TABLE 3-3 Exogenous Paid Viruses of Concern in Xenotransplantation
Family Species Category Picornaviriddae foot and mouth disease enterovirus 1 Talfan/Teschen 2,5 enterovirus (other serogroups) 5 enterovirus swine vesicular disease 5 human enteroviruses 1 encephalomyocarditis rhinovirus Caliciviridae enteric calicivirus 1 swine hepatitis E 1 Astrovoridae porcine astrovirus 5 Togaviridae Western encephalitis 1 Eastern encephalitis 1 Venezuelan encephalitis 1 Getah 1 chikungunya 1 Flaviviridae Japanese B encephalitis 1 Louping III/TBE complex 1 Wesselbron disease 1 Apoi 2 dengue fever 1 West Nile fever 1 classical swine fever (hog cholera) 5 bovine viral diarrhea 5 border disease 5 Coronaviridae transmissible gastroenteritis 4,5 porcine respiratory coronavirus 4,5 epidemic diarrhea 4,5 Haemagglutinating encephalomyelitis 4,5 porcine reproductive and respiratory 4,5 disease syndrome porcine torovirus 5 Paramyxoviridae murine parainfluenza virus 2 type 1 (Sendai) parainfluenza 2 2* parainfluenza 3 2
http://zoom.nap.edu/nap-cgi/rezoom.cgi?isbn=0309084393&page=59
blue eye disease 5 menangle 1 nipah 1 Rhabdoviridae vesicular stomatitus 1 rabies 1 Bornaviridae Bornavirus 2,5 Orthomyxoviridae influenza a 1 influenza c 5 bunyaviridae akabane 1,5 batai 1,5 hantavirus 1,5 arenaviridae lymphocytic choriomeningitis 1,5 reoviridae ibaraki 5 reovirus 1 to 3 2 rotavirus a, b, c, e. 2 birmaviridae porcine picobirnavirus 5 retroviridae porcine endogenous 2 hepadnaviridae hepatitis b - circoviridae porcine circovirus 5 parvoviridae porcine parvovirus 4,5 papovaviridae porcine polymavirus 3 porcine genital papillomavirus 3,5 adenoviridae porcine adenovirus serotypes 1 to 4 3 herpesviridae pseudorabies 2 porcine cytomeglovirus 5 porcine lymphotropic herpesvirus type 1 3 porcine lymphotropic herpesvirus type 2 3 poxviridae swinepox 5 vaccinia 2 cowpox 1,5* orf/pseudocowpox 1,5* desoxyviridae african swine fever 5 note: 1=zoonotic 2=replicates in human cells or weak evidence for zoonotic potential 3=might undergo abortive replication and possibly oncogenic 4=belongs to a family with evidence of frequent changes in host range or pathogenicity 5= undesirable as indicates a breakdown in biosecurity and or might compromise health of the pigs *= although the virus has not been recorded and pigs, it has been included for reasons such as its wide host range source: Onions et al., 2000. Courtesy of D. Onions.
Porcine Endogenous Retroviruses
PERVs represent quite a different situation and level of concern, since they are inherited as part of the host genome and, therefore, cannot be removed easily from donor animals. All pigs contain multiple (around 50) PERV proviruses in their genome, at least several of which Inco to infectious virus. PERVs are gammaretroviruses, closely related to MLV, that can be classified into three subtypes, A, B, and C, based on their envelope gene sequences (Takeuchi et al., 1998). Subtypes A and B can infect many types of human cells and culture. Subtype C is much less infectious for humans. Most breeds of pig carry proviruses capable of yielding infectious virus of all three subtypes. Although most pigs carry about the same number of proviruses in their DNA, there is considerable diversity and location, implying that their insertion into the genome must have occurred relatively recently (on and evolutionary timescale). Based on extensive experience with related endogenous proviruses of mice, it is highly likely that the majority of proviruses contain some sort of genetic defect, and that only a small number are responsible for release of infectious virus. Taken together with the polymorphism and presence or absence of specific proviruses, it might well be possible to breed animals lacking infectious proviruses for use as xenotransplant donors.
PERVs have not yet been shown to cause disease (or even viremia) and pigs or any other species in which they have been tested. Nor has their presence been detected (by PCR or serology) and over 150 human recipients of pig cells or tissues (Paradis et al., 1999), although a low level of infection of recipients cells can be observed in immunodeficient mice transplanted with porcine islets of Langerhans (Van der Laan et al., 2000). Nevertheless, given the release of virus infectious for human cells by many types of pig cell, the close similarity of these viruses to viruses known to cause cancer, immunodeficiency, and other diseases in mice and cats, the well-known adaptability and variability of retroviruses, and the example of the rapid worldwide spread of HIV and AIDS, there is serious concern that the novel association between pig and human tissues might create novel evolutionary opportunities for the virus, leading to the appearance of a new pathogen. Although such a pathogen could have serious long-term adverse consequences to the transplant recipient, this issue is not an area of concern, sends it is far away at by the potential benefit of the transplant. The real issue of concern is that the xenotransplant setting might prove the opportunity
http://zoom.nap.edu/nap-cgi/rezoom.cgi?isbn=0309084393&page=61
for the virus to evolve into a pathogen which can also be transmitted from one individual to another efficiently enough to create a new epidemic disease.
Such an evolutionary pathway would create a series of advance, each increasingly improbable, as indicated by the scale shown in table 3-for (J. P. Stoye, personal communication). As implied by the table, it is virtually certain that many cells in the transplant will express infectious PERV following transplantation, and likely that some local infection of host cells might occur. The subsequent events necessary for generation of pathogenic, transmissible viruses are increasingly unlikely, what on some unknown, arbitrary scale. Although the probability of an inadvertent creation of a new epidemic is generally judged to be extremely small (particularly given the long history of intermittent association between humans and pigs), it cannot be ignored altogether. Current FDA policy is to permit xenotransplantation trials to proceed, but to require close monitoring of recipients, and (insofar as possible) of their contacts (DHHS, 2001). Attempts are also being made to identify specific proviruses responsible for production of infectious virus and then to selectively breed them out of lines of animals to be used as transplant donors (Herring et al., 2001).
TABLE 3-4 Theoretical Scale of Risks of Such It with PERV Transmission from Xenotransplants
|
If you have come to this page from an outside location click here to get back to mindfully.org |