Rv G_GENETIC AND CELLULAR THERAPIES TREVI- Hotere ue aR — MEDIC fate MATICNE STAMICE thereby reducing the target load and demand on the ribozyme. DNA Vaccines Nucleic acid (NA) vaccines involve the direct injection ‘of genes (in the form of naked, plasmid DNA or DNA delivered by another vector system) expressing viral pro: ‘gins into a patient to produce a sustained antigenic stim. ulus and so generate an ongoing immune response. DNA vaccines, which have now been in use about 10. years, provoked initial interest because af the relatively simple way in which they could be prepared to deliver a range -of protein antigens for immunisation, In animal studies these vaccines stimulate both humoral and cell-mediated immune mechanisms (similar to what occurs with live attenuated vaccines) without integrating into host DNA, Thus, they are an alternative but safer approach to live viral vaccines and are better than inactivated (clead) vac- tines in the breadth of the immune response they elicit. Routes: of administration are flexible, e.g., parenteral, mucosal or the gene gun, which delivers tiny amounts af DNA-coated gold or tungsten beads. DNA vaccines are presently being assessed in AIDS, malaria and" variety of cancers. Infectious agents that are difficult or dangerous to produce by conventional culture techniques fe.g., rabies virus) could also be better developed through recombi- ant DNA means. Genetic manipulation would also be useful to reduce the likelihood of reversion to wildetype strains fe.g, poliomyelitis) or to increase the antigenicity of a particular component cesived from the invfecting organism, Its evident from results now emerging that DNA vac- ines are safe, but this is offset by" their low potency, which reduces theif effectiveness. Ta correct this, it will be necessary to increase the efficiency with which the DIA is being delivered andar enhance the antigenicity of the protein produced. DNA vaccines also pose a reg Ulatory challenge since they lie somewhere between a sonventional vaccine (which has as one of its purifying steps the removal of any nucleic acids} and the traditional gene transfer vector. Hence, for the purpose of this dis- cussion, they have been grouped under “related gene therapies.” Issucs that remain unresolved include the unequivocal demonstration that integration into ONA does nat occur, and even if it does, what are the conse- ‘quences? Anti-DMA antibodies 10 injected DNA are knowin to develop in animal studies, but their develop. ment and potential for autoimmune disease in. humans remains unknown. “SSTEM CELLS Stem cells are of two classes: (1) embryonic—found within the embryo’s inner cell mass iFigure 6.8) and (2) adult—found within dilferentiated tissues such as the bone marraw. Stem cells have become topical as poten- tial novel ways in which to repair or replace damaged tissues, or even organs. For this, stems cells have two important properties: (1) self-renewal, with the capacity to make mare stem cells and (2) ability to give rise to dif. ferent progeny when exposed to the appropriate signals. In doing this, a progenitor cell is first formed. This is the precursor to the specialised cell (called a differentiated cell). Stem cells have varying patential to dilfere into other cells: (1) Unipotent stern cells develop into one Cell type, (2) Multipotent stem cells can form multiple cells types, (3) Pluripotent stem cells can differentiate into most, if not all, of the adult cell types in the body. (4) Totipotent stem cells will form all cell types, both adult and others, such as the placenta, Embryonic Stem Cells (ES Cells) In the: embryos blastocyst stage before implantation (about day 5-7 embryo), the inner cell mass contains all the cells that will make up the fetus. Some of these cells are pluripotential because they will give rise to all types of somatic ces 28 well as the germ cells. When these pluripotential stem cells are grown in vitro, they are Called embryonic stem cells or €8 cells (see Figure 6. When maintained under appropriate culture conditions, embryonic stem cells can be cultured indefinitely in an undifferentiated state. When differentiated, embryonic stem cells give rise tothe three major cell lineages (encia- dermal, mesodermal and ectodermal). Mouse embryonic stem cells were isolated in 1981, and the human equive ‘lenis were found in 1998, There are a number of potential applications. for embryonic stem cells: 1: Research—understanding disease pathogenesis: Trans- nic mice are useful animal mores to study human dlisardess (see also Chapter 5). They are produced by microinjection of DNA into the pronucleus of a fer- tlised oocyte. Although the gene of interest is nat inserted into its coereet position in the genome, it still remains possible to add new genes that can function in vivo. Thus, expression of the mutant vansgene will produce the clinical phenotype. An extensiom af this i the transgenic mouse, which has been created by gene knockout. This process involves homologous recombi ration between an introduced mutant gene and the corresponding wildtype gene. Now gene function can be inhibited or the effect of a specific mutation observed. Embryoni¢ stem cells have been critical for devel oping knockout transgenics. Sinee embryonic stem cells are totipotential, they can be genetically manip- Ulated and then reintroduced into the blastocyte of a developing mouse ta produce a chimavra. Foreign DNA that has hecome integrated into the germline of 1636 GENETIC AND CELLULAR THERAPIES Egg Fertilised Blastocyst Zygote fe Sperm fig. 6.8 Sources of stem calls Embryo Cord blood Adult Embryonic stem ces are derived from the 8-7 day embi known as the blastocyst The outer layer of cells depicted as circles is the wophoblast, which will go on to form the placenta, The cells at the bottom forming the inne¢ cel! mass ate the embryonic the chimaera will enable the gené to be trans: rmitted to progeny. Appropriate matings will produce homozygotes containing the transgene. Embryonic stem cells allow a gene to be targeted to its appropri- ate locus and replace the normal wild-type counter- part by homologous recombination, Using this approach, a better understanding of genetic inheri- tance or disease pathogenesis becomes possible. 2. Human transplantation of tissues or organs: The pluripotential and immortal qualities of embryonic stem cells make them ideal candidates for use in trans- plantation to repair damaged tissues oF replace tissues that have undergone degenerative changes. There is now evidence in mouse work that embryonic stem cells might prove useful in conditions such as Parkinson's disease, myocardial infarction and spinal cord injuries. Significant technical challenges. are needed befare the promises of the embryonic stem cells are achieved in humans. These challenges include growing large numbers of pure stem cells for the cell type required, I: remains to be determined ‘what would be the best type of cell to use in particu- lar situations, i.e., what degree of diflerentiated embryonic stem cell is needed far various scenarios? Another important consideration with embryonic stem cells is the question of rejection since the source of the tissue is not normally the recipients. This could be addressed by anti-rejection treatment similar to what is alzeady used in allografts. An alternative way in which the rejection of embryonic stem cells might be controlled is through therapeutic cloning (somatic 164 ils. Cord blood stem cells obtained from the placenta at bith and various tissues in the adi wide the source of adult coll nuclear transfer or SCNT, which is discussed further below. A comparison of embryonic stem cells and adult stem cells is given in Table 6.13. A controversial aspect of embryonic stem cells concerns their source, which presently comes from excess embryos obtained during in vitro ferilisation procedures (dis- cussed further in Chapter 10), While these excess embryos would eventually be destroyer, their use for human research has provoked controversy. In response to this, governments have in some cases limited access to embryonic stem cell lines. Far example, in the USA, only 15 lines created before 9 PM EDT on August 9, 2001, cary be used for research that is funded through the NIH (see hutpststemcells.nih.gowiregisteyindex.asp). Cell lines produced after the above date are available, but NIH research money cannat be used for experimentation with these lines, The regulatory framework for human embey- onic stem cell research is very much in flux with the US position described abave flanked by countries such as the UK and Singapore that allow therapeutic cloning (see cloning on page 166), and others such as Germany and Italy that do not allow any embryo research, including the production of embryonic stem cells, Adult Stem Cells Although embryonic stem is have the greatest poten tial to differentiate into various cell types, the disadvan- tages mentioned earlier as well as the ethical and moral questions raised about their source of derivation make adult stem cells attractive alternatives for transplantation6 GENETIC AND CELLULAR THERAPIES Table 6.13 Comparing properties of embryenic sid adull stem cells Property Embryonic dem call Source Apart from technical considerations, complicated by ethical, legal and social eves Immunogeniciry Rejection a concern if source i another hurnan, Le, am allograft (so immunosuppression likely to be requlied) ‘Amsigeniciy unresolved. A way around this is therapeutic cloning to abvain the embryonic stem cell fears the patient Abily te form mutiple fissues Le, plasticity capacity to diferent Production Although these stern eells ate pluripotentsl and immoral there remains a probiem with the selection and ‘expansion of pure populations of required calls yes, Telomerase ‘Telomerase, the enzyme restoring the telomeric ends of chromosomes fe Gone expression and tumour formation Convo! of ge Thece is ho dispute that embryonic stem cells have the 0 any cell type ng cell division, # found in high Teveis in immortal cells such as the embryanic stem cells xpressich may nat be tightly regulated. In mice, teratoma can cevolop alongside various aah sem cell Limited only by technical issues, Not relevant fautslogous, 40. sourced fram. ppaient. Becomes an issue if insufficient cells produced by the patient or the problem is acute and an allogeneic source is necded. ‘Theve stl debate about the plasicity of adult stem cell Nat proven beyond doubt that adult, sem cells are as versatile in ths panicular role Eisier to grow almost indefinitely ia eultoré, but ia teams of a mass market, adult sem cells laferioe te erabeyonce stem cell Telomorase fevels in haematopoietic stm cls ae insufficient to maistan the flomere length This would be a concern for longstesn growth Tumours noe reported. dlifferetiated cell ypes. Risk of tumour Formation in suman cell lines not krown, Success in elisa! tvialst0 date provided their potential to differentiate into different cells and tissue types (described as plasticity! can be proven Therefore, the debate centres around the degree af plas- ‘icity possible, with claims that adult stem cells can dif ferentiate into a wide range of tissues. Others are more sceptical, wanting to know ifthe adult stem cell is actu- ally changing its function, or if this apparent plasticity is, due to a coexistent or itinerant stem cell that has been carried along with, say, 2 haematopoietic stem cell that now appears to be producing a brain cell. Another expla- ration ofthe adult stem cell's apparent plasticity involves, cell fusion between something like a haematopoietic, stem cell and the host target cell, thereby making it appear as though the haematapoietic stem cell has dlif- ferentiated into a distinct cell The traditional hierarchical model for haemnatapaiesis hhas, at the top, a self-renewing pluripotential stem cell, Below this is the committed progenitor cell, The next Fevel is the fineage restricted precutsor cell. fit is proven that bone marrow (adult) stem cells can differentiate into tissues such as blood, bone, muscle, neural cells and so. oon, the hierarchical model cannot be correct. If $0, alte: native models proposed include (1) The marrow has a number of dlistinet stem cells 4o explain plasticity. Which predominates depends on various external stimuli. (2) Pract of principle as boen shown in animal stodes, but data from human work re stil very preliminary. ‘Already sucoessful--e-g., Bone marsow and cord blood tansplanis— although not ecessarily showing a pluriperential stem cell let as disfinet fran haematopoietic stem call ‘There are the equivalent of embryonic stem cells in bone marrow, which allows differentiation inte all lineages. (3) Haematopoietie stem cells differentiate into the expected blood cells until there is a stress injury of somo external stimulus, which allows some of these stem cells to form other lineages. (4) The final mechanism is dedifferentia tion followed by rediferentiation based on the environ ‘mental stimulus to which the adult stem cell is being exposed. The latter is the likely mechanism to explain Dolly: the sheep's origin fram an adult differentiated mammary gland cell. Potential uses of adult stern cells are comparable to what has been proposed for embryonic stem cells: 1, Research—understanding dedifferentiation and vedif- ferentiation: The complex controt of cellular differen- tiation is not understood, and so the option to have a model (the adult stem cell) to explore the molecular and cellular contiols would provide invaluable basic scientific knowledge, as well as possible therapeutic options te induce cells to change their primary differ- entiation pathway. 2. Therapeutic: Some evidence is already available fram mouse models that adult neurogenic stem cells can bbe used in the tweatment of Parkinson's disease. Like 1656 _GENETIC AND CELLULAR THERAPIES the embryonic: stem cells, this would also open up the options for stem cells to be used in discovering new drugs as well as testing drugs for toxicity, Stem cell therapy for ischaemic heart disease is another focus of clinical research. As better treatments are seducing the acute mortality associated with coronary artery disease, the number of survivors developing canges- tive cardiac failure is increasing. Conventional drug regimens are not effective with end-stage heart failure, and there are inadequate numbers of cardiac trans- plants available, Stem cells have been tried, and are showing same promise (Box 6.4). Telomerase activity in adult stem cells is less compared to the level in immontalised embryonic stem cells (see Table 6.13). However, genetic manipulation provides the oppor- tunity to add the telomerase gene to adult ster cells, prior to their use. In theary, this would extend the life of these stem cells. CLONING In molecular medicine, “cloning” is a frequently used ‘word with many different meanings. DIVA can be cloned, cells can be cloned, montizygotic twins are examples of clones, Dally the sheep proved that whale animals could be cloned. There are now unsubstantiated claims that human clones have been produced, in the context of cel- lular therapy, cloning refers to asexual reproduiction brought about by the process known as. somatic cell nuclear transfer (SCNT). In 1992, Dolly the sheep pro- vided the proof that DNA fram a differentiated tissue cell (marmary gland coll) could be taken and reprogrammed to produce a cleaned animal copy. The process invalved the removal of the nucleus (i.e, DNA) from the mammary gland cell. An egg from a donor sheep was ext enucleated, and the nucleus from the mammary sland cell inserted into that egg, which was returned to @ surrogate mother by the usual in vita fertilisation tech. niques (Figuee 6.9), The genetic composition of the clone in this ease is vi twally identical to the mother, and there is no patemal Contribution. “Virtually identical” is used because the recipient enucleated egg still has within its cytoplasm mitochondrial DNA, which contributes about 1% of the total DIVA in the clone. This process is called SCNT, and although it has produced Dolly the sheep as well asa host of other animals, it is very inefficient and error prong; for example, it took 277 attempts to.get Dolly and many of these produced malformed fetuses, Its assumed that the inefficiency of the procedure partially reflects the development of abnormalities. As indicated earlier, Dally’s genetic constitution excluded any paternal genes, and this is relevant because, as discussed in Chapters 4 and 7, regions of the genomé are imprinted, and in these regions, either the maternal or the paternally inherited gene is critieal to normal development. $0 it is not 166 for: entirely surprising that SCNT is neither efficient nor reli- able enough ta be used for human reproductive cloning, What has been described above is usually called reproductive clor In contrast is” therapeutic sctoning—another form of SCNT but the purpose naw is ta produce embryonic stem cells for research or therapy: Table 6.14). Two key issues remain unresolved. The first is the utility of embryonic stem cells Compared to adult ster cells, issue was discussed above. The second is the creation or use of embryos for research, and not Fertility purposes. There is a division of opinion hese, with same claiming that the latter is justified because of the downsveam potential of embryonic stem cells, Others are less convinced of this argument particularly when balanced by the spectre of cteating embryos and then destroying them for the purpose of research or perhaps the creation of “spare parts.” This is not an easy debate, but a key priority will be to conduct high-quality and eth. ically based research to answer some of the issues raised about the various types of stem cells and their potential for teating human diseases,