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InternalMed_Harrison. ES Cell Regulation It is important to identify genes involved in the regulation of stem cell function and to examine the effects of altered gene expression on ES and other stem cells. For example, core networks of TFs such as Pou5f1 (Oct4), Nanog, and Sox2, govern key gene regulatory pathways/networks for the maintenance of self-renewal and pluripotency of mouse and human ES cells. These TF networks are modulated by specific external factors through signal transduction pathways, such as leukemia inhibitory factor (Lif)/Stat3, mitogenactivated protein kinase 1/3 (Mapk1/3), the transforming growth factor β (TGFβ) superfamily, and Wnt/glycogen synthase kinase 3 beta (Gsk3b). Inhibitors of Mapk1/3 and Gsk3b signaling enhance the derivation of ES cells and help maintain ES cells in full pluripotency (“ground” or “naive state”). Recent data also indicate that 20–25 nucleotide RNAs, called microRNAs (miRNAs), play an important role in regulating stem cell function by repressing the


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InternalMed_Harrison. pluripotency (“ground” or “naive state”). Recent data also indicate that 20–25 nucleotide RNAs, called microRNAs (miRNAs), play an important role in regulating stem cell function by repressing the translation of their target genes. For example, it has been shown that miR-21 regulates cell cycle progression in ES cells and miR-128 prevents the differentiation of hematopoietic progenitor cells. These types of analyses should provide molecular clues about the function of stem cells and lead to a more effective means to manipulate stem cells for future therapeutic use.


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InternalMed_Harrison. Hematopoietic Stem Cells would ensue. In the blood, mature cells have variable average life spans, ranging David T. Scadden, Dan L. Longo from 7 h for mature neutrophils to a few months for red blood cells to All of the cell types in the peripheral blood and some cells in every tissue of the body are derived from hematopoietic (hemo: blood; poiesis: creation) stem cells. If the hematopoietic stem cell is damaged and can no longer function (e.g., due to a nuclear accident), a person would survive 2–4 weeks in the absence of extraordinary support measures. With the clinical use of hematopoietic stem cells, tens of thousands of lives are saved each year (Chap. 139e). Stem cells produce hundreds of billions of blood cells daily from a stem cell pool that is estimated to be only in the tens of thousands. How stem cells do this, how they persist for many decades despite the production demands, and how they may be better used in clinical care are important issues in medicine.


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InternalMed_Harrison. The study of blood cell production has become a paradigm for how other tissues may be organized and regulated. Basic research in hematopoiesis includes defining stepwise molecular changes accompanying functional changes in maturing cells, aggregating cells into functional subgroups, and demonstrating hematopoietic stem cell regulation by a specialized microenvironment; these concepts are worked out in hematology, but they offer models for other tissues. Moreover, these concepts may not be restricted to normal tissue function but extend to malignancy. Stem cells are rare cells among a heterogeneous population of cell types, and their behavior is assessed mainly in experimental animal models involving reconstitution of hematopoiesis. Thus, much of what we know about stem cells is imprecise and based on inferences from genetically manipulated animals.


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InternalMed_Harrison. All stem cell types have two cardinal functions: self-renewal and differentiation (Fig. 89e-1). Stem cells exist to generate, maintain, and repair tissues. They function successfully if they can replace a wide variety of shorter-lived mature cells over prolonged periods. The process of self-renewal (see below) assures that a stem cell population can be sustained over time. Without self-renewal, the stem cell pool would become exhausted and tissue maintenance would not be possible. The process of differentiation leads to production of the effectors of tissue function: mature cells. Without proper differentiation, the integrity of


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InternalMed_Harrison. FIGURE 89e-1 Signature characteristics of the stem cell. Stem cells have two essential features: the capacity to differentiate into a variety of mature cell types and the capacity for self-renewal. Intrinsic factors associated with self-renewal include expression of Bmi-1, Gfi-1, PTEN, STAT5, Tel/Atv6, p21, p18, MCL-1, Mel-18, RAE28, and HoxB4. Extrinsic signals for self-renewal include Notch, Wnt, SHH, and Tie2/Ang-1. Based mainly on murine studies, hematopoietic stem cells express the following cell surface molecules: CD34, Thy-1 (CD90), c-Kit receptor (CD117), CD133, CD164, and c-Mpl (CD110, also known as the thrombopoietin receptor).


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InternalMed_Harrison. many years for memory lymphocytes. However, the stem cell pool is the central, durable source of all blood and immune cells, maintaining a capacity to produce a broad range of cells from a single cell source, yet keeping itself vigorous over decades of life. As an individual stem cell divides, it has the capacity to accomplish one of three division outcomes: two stem cells, two cells destined for differentiation, or one stem cell and one differentiating cell. The former two outcomes are the result of symmetric cell division, whereas the latter indicates a different outcome for the two daughter cells—an event termed asymmetric cell division. The relative balance for these types of outcomes may change during development and under particular kinds of demands on the stem cell pool.


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InternalMed_Harrison. During development, blood cells are produced at different sites. Initially, the yolk sac provides oxygen-carrying red blood cells, and then the placenta and several sites of intraembryonic blood cell production become involved. These intraembryonic sites engage in sequential order, moving from the genital ridge at a site where the aorta, gonadal tissue, and mesonephros are emerging to the fetal liver and then, in the second trimester, to the bone marrow and spleen. As the location of stem cells changes, the cells they produce also change. The yolk sac provides red cells expressing embryonic hemoglobins while intraembryonic sites of hematopoiesis generate red cells, platelets, and the cells of innate immunity. The production of the cells of adaptive immunity occurs when the bone marrow is colonized and the thymus forms. Stem cell proliferation remains high, even in the bone marrow, until shortly after birth, when it appears to dramatically decline. The cells in the bone marrow are


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InternalMed_Harrison. is colonized and the thymus forms. Stem cell proliferation remains high, even in the bone marrow, until shortly after birth, when it appears to dramatically decline. The cells in the bone marrow are thought to arrive by the bloodborne transit of cells from the fetal liver after calcification of the long bones has begun. The presence of stem cells in the circulation is not unique to a time window in development; however, hematopoietic stem cells appear to circulate throughout life. The time that cells spend freely circulating appears to be brief (measured in minutes in the mouse), but the cells that do circulate are functional and can be used for transplantation. The number of stem cells that circulate can be increased in a number of ways to facilitate harvest and transfer to the same or a different host.


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InternalMed_Harrison. Cells entering and exiting the bone marrow do so through a series of molecular interactions. Circulating stem cells (through CD162 and CD44) engage the lectins (carbohydrate binding proteins) Pand E-selectin on the endothelial surface to slow the movement of the cells to a rolling phenotype. Stem cell integrins are then activated and accomplish firm adhesion between the stem cell and vessel wall, with a particularly important role for stem cell VCAM-1 engaging endothelial VLA-4. The chemokine CXCL12 (SDF1) interacting with stem cell CXCR4 receptors and ionic calcium interacting with the calcium sensing receptor appear to be important in the process of stem cells getting from the circulation to where they engraft in the bone marrow. This is particularly true in the developmental move from fetal liver to bone marrow.


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InternalMed_Harrison. However, the role for CXCR4 in adults appears to be more related to retention of stem cells in the bone marrow rather than the process of getting them there. Interrupting that retention process through either specific molecular blockers of the CXCR4/CXCL12 interaction, cleavage of CXCL12, or downregulation of the CXCR4 receptor can all result in the release of stem cells into the circulation. This process is an increasingly important aspect of recovering stem cells for therapeutic use as it has permitted the harvesting process to be done by leukapheresis rather than bone marrow punctures in the operating room. Granulocyte colony-stimulating factor and plerixafor, a macrocyclic compound that can block CXCR4, are both used clinically to mobilize marrow hematopoietic stem cells for transplant. Refining our knowledge of how stem cells get into and out of the bone marrow may improve our ability to obtain stem cells and make them more efficient at finding their way to the specific sites for


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InternalMed_Harrison. Refining our knowledge of how stem cells get into and out of the bone marrow may improve our ability to obtain stem cells and make them more efficient at finding their way to the specific sites for blood cell production, the so-called stem cell niche.


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InternalMed_Harrison. The concept of a specialized microenvironment, or stem cell niche, was first proposed to explain why cells derived from the bone marrow of one animal could be used in transplantation and again be found in the bone marrow of the recipient. This niche is more than just a housing site for stem cells, however. It is an anatomic location where regulatory signals are provided that allow the stem cells to thrive, to expand if needed, and to provide varying amounts of descendant daughter cells. In addition, unregulated growth of stem cells may be problematic based on their undifferentiated state and self-renewal capacity. Thus, the niche must also regulate the number of stem cells produced. In this manner, the niche has the dual function of serving as a site of nurture but imposing limits for stem cells: in effect, acting as both a nutritive and constraining home.


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InternalMed_Harrison. The niche for blood stem cells changes with each of the sites of blood production during development, but for most of human life it is located in the bone marrow. Within the bone marrow, the perivascular space particularly in regions of trabecular bone serves as a niche. The mesenchymal and endothelial cells of the marrow microvessels produce kit ligand and CXCL12, both known to be important for hematopoietic stem cells. Other cell types, such as sympathetic neurons, nonmyelinating Schwann cells, macrophages, osteoclasts, and osteoblasts, have been shown to regulate stem cells, but it is unclear whether their effects are direct or indirect. Extracellular matrix proteins like osteopontin also affect stem cell function. The endosteal region is particularly important for transplanted cells, suggesting that there may be distinctive features of that region that are yet to be defined that are important mediators of stem cell engraftment. The functioning of the niche as a supportive context


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InternalMed_Harrison. suggesting that there may be distinctive features of that region that are yet to be defined that are important mediators of stem cell engraftment. The functioning of the niche as a supportive context for stem cells is of obvious importance for maintaining hematopoiesis and in transplantation. An active area of study involves determining whether the niche is altered in disease and whether drugs can modify niche function to improve transplantation or normal stem cell function in hematologic disease.


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InternalMed_Harrison. In the absence of disease, one never runs out of hematopoietic stem cells. Indeed, serial transplantation studies in mice suggest that sufficient stem cells are present to reconstitute several animals in succession, with each animal having normal blood cell production. The fact that allogeneic stem cell transplant recipients also never run out of blood cells in their life span, which can extend for decades, argues that even the limiting numbers of stem cells provided to them are sufficient. How stem cells respond to different conditions to increase or decrease their mature cell production remains poorly understood. Clearly, negative feedback mechanisms affect the level of production of most of the cells, leading to the normal tightly regulated blood cell counts. However, many of the regulatory mechanisms that govern production of more mature progenitor cells do not apply or apply differently to stem cells. Similarly, most of the molecules shown to be able to change the size of the


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InternalMed_Harrison. mechanisms that govern production of more mature progenitor cells do not apply or apply differently to stem cells. Similarly, most of the molecules shown to be able to change the size of the stem cell pool have little effect on more mature blood cells. For example, the growth factor erythropoietin, which stimulates red blood cell production from more mature precursor cells, has no effect on stem cells. Similarly, granulocyte colony-stimulating factor drives the rapid proliferation of granulocyte precursors but has little or no effect on the cell cycling of stem cells. Rather, it changes the location of stem cells by indirect means, altering molecules such as CXCL12 that tether stem cells to their niche. Molecules shown to be important for altering the proliferation, self-renewal, or survival of stem cells, such as cyclin-dependent kinase inhibitors, transcription factors like Bmi-1, or microRNA-processing enzymes like Dicer, have little or different effects on progenitor cells.


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InternalMed_Harrison. survival of stem cells, such as cyclin-dependent kinase inhibitors, transcription factors like Bmi-1, or microRNA-processing enzymes like Dicer, have little or different effects on progenitor cells. Hematopoietic stem cells have governing mechanisms that are distinct from the cells they generate.


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InternalMed_Harrison. Hematopoietic stem cells sit at the base of a branching hierarchy of cells culminating in the many mature cell types that compose the blood and immune system (Fig. 89e-2). The maturation steps leading to terminally differentiated and functional blood cells take place both as a consequence of intrinsic changes in gene expression and niche-directed and cytokine-directed changes in the cells. Our knowledge of the details remains incomplete. As stem cells mature to progenitors, precursors, and, finally, mature effector cells, they undergo a series of functional changes. These include the obvious acquisition of functions defining mature blood cells, such as phagocytic capacity or hemoglobin synthesis. They also include the progressive loss of plasticity (i.e., the ability to become other cell types). For example, the myeloid progenitor can make all cells in the myeloid series but none in the lymphoid series. As common myeloid progenitors mature, they become precursors for either monocytes


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InternalMed_Harrison. types). For example, the myeloid progenitor can make all cells in the myeloid series but none in the lymphoid series. As common myeloid progenitors mature, they become precursors for either monocytes and granulocytes or erythrocytes and megakaryocytes, but not both. Some amount of reversibility of this process may exist early in the differentiation cascade, but that is lost beyond a distinct stage in normal physiologic conditions. With genetic interventions, however, blood cells, like other somatic cells, can be reprogrammed to become a variety of cell types.


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InternalMed_Harrison. As cells differentiate, they may also lose proliferative capacity (Fig. 89e-3). Mature granulocytes are incapable of proliferation and only increase in number by increased production from precursors. The exceptions to the rule are some resident macrophages, which appear capable of proliferation, and lymphoid cells. Lymphoid cells retain the capacity to proliferate but have linked their proliferation to the recognition of particular proteins or peptides by specific antigen receptors on their surface. Like many tissues with short-lived mature cells such as the skin and intestine, blood cell proliferation is largely accomplished by a more immature progenitor population. In general, cells within the highly proliferative progenitor cell compartment are also relatively short-lived, making their way through the differentiation process in a defined molecular program involving the sequential activation of particular sets of genes. For any particular cell type, the differentiation program is


InternalMed_Harrison

InternalMed_Harrison. way through the differentiation process in a defined molecular program involving the sequential activation of particular sets of genes. For any particular cell type, the differentiation program is difficult to speed up. The time it takes for hematopoietic progenitors to become mature cells is ~10–14 days in humans, evident clinically by the interval between cytotoxic chemotherapy and blood count recovery in patients.


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InternalMed_Harrison. Although hematopoietic stem cells are generally thought to have the capacity to form all cells of the blood, it is becoming clear that individual stem cells may not be equal in their differentiation potential. That is, some stem cells are “biased” to become mature cells of a particular type. In addition, the general concept of cells having a binary choice of lymphoid or myeloid differentiation is not entirely accurate. A cell population with limited myeloid (monocyte and granulocyte) and lymphoid potential is now added to the commitment steps stem cells may undergo.


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InternalMed_Harrison. The hematopoietic stem cell must balance its three potential fates: apoptosis, self-renewal, and differentiation. The proliferation of cells is generally not associated with the ability to undergo a self-renewing division except among memory T and B cells and among stem cells. Self-renewal capacity gives way to differentiation as the only option after cell division when cells leave the stem cell compartment, until they have the opportunity to become memory lymphocytes. In addition to this self-renewing capacity, stem cells have an additional feature characterizing their proliferation machinery. Stem cells in many mature adult tissues may be heterogeneous with some being deeply quiescent, serving as a deep reserve, whereas others are more proliferative and replenish the short-lived progenitor population. In the hematopoietic system, stem cells are generally cytokine-resistant, remaining dormant even when cytokines drive bone marrow progenitors to proliferation rates measured in hours.


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InternalMed_Harrison. population. In the hematopoietic system, stem cells are generally cytokine-resistant, remaining dormant even when cytokines drive bone marrow progenitors to proliferation rates measured in hours. Stem cells, in contrast, are thought to divide at far longer intervals, measured in months to years, for the most quiescent cells. This quiescence is difficult to overcome in vitro, limiting the ability to effectively expand human hematopoietic stem


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InternalMed_Harrison. Multipotent Progenitor IKAROS PU1 IL7 IL7 IL7 IL7 IL7 IL4 IL2 IL15 IL5 FLT-3 Ligand FLT-3 Ligand M-CSF G-CSF IL3, SCF EPOEPO TPO IL3, SCF TPO GM-CSF SCF TPO TPO Hox, Pbx1, SCL, GATA2, NOTCH Common Lymphoid Progenitor B Cell Progenitor T Cell Progenitor NK Cell Progenitor Monocyte Progenitor Granulocyte Monocyte Progenitor Granulocyte Progenitor Erythrocyte Progenitor Megakaryocyte Progenitor Megakaryocyte Erythroid Progenitor Common Myeloid Progenitor T/NK Cell Progenitor LEF1, E2A, EBF, PAX-5 NOTCH1 NOTCH1 Aiolos, PAX-5, AML-1 E2A, NOTCH1, GATA3 IKAROS, NOTCH,CBF1 Id2, Ets-1 RelB, ICSBP, ld2 Egn1, Myb GATA1 C/EBP˜C/EBP°Fli-1 AML-1 GATA1, FOG NF-E2, SCL Rbtn2


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InternalMed_Harrison. FIGURE 89e-2 Hierarchy of hematopoietic differentiation. Stem cells are multipotent cells that are the source of all descendant cells and have the capacity to provide either long-term (measured in years) or short-term (measured in months) cell production. Progenitor cells have a more limited spectrum of cells they can produce and are generally a short-lived, highly proliferative population also known as transient amplifying cells. Precursor cells are cells committed to a single blood cell lineage but with a continued ability to proliferate; they do not have all the features of a fully mature cell. Mature cells are the terminally differentiated product of the differentiation process and are the effector cells of specific activities of the blood and immune system. Progress through the pathways is mediated by alterations in gene expression. The regulation of the differentiation by soluble factors and cell-cell communications within the bone marrow niche are still being defined. The


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InternalMed_Harrison. pathways is mediated by alterations in gene expression. The regulation of the differentiation by soluble factors and cell-cell communications within the bone marrow niche are still being defined. The transcription factors that characterize particular cell transitions are illustrated on the arrows; the soluble factors that contribute to the differentiation process are in blue. This picture is a simplification of the process. Active research is revealing multiple discrete cell types in the maturation of B cells and T cells and has identified cells that are biased toward one lineage or another (rather than uncommitted) in their differentiation. EPO, erythropoi- etin; RBC, red blood cell; SCF, stem cell factor; TPO, thrombopoietin.


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InternalMed_Harrison. cells. The process may be controlled by particularly high levels of cyclin-dependent kinase inhibitors like p57 or CDKN1c that restrict entry of stem cells into the cell cycle, blocking the G1-S transition. Exogenous signals from the niche also appear to enforce quiescence, including the activation of the tyrosine kinase receptor Tie2 on stem cells by angiopoietin 1 on niche cells.


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InternalMed_Harrison. The regulation of stem cell proliferation also appears to change with age. In mice, the cyclin-dependent kinase inhibitor p16INK4a accumulates in stem cells in older animals and is associated with a change in five different stem cell functions, including cell cycling. Lowering expression of p16INK4a in older animals improves stem cell cycling and capacity to reconstitute hematopoiesis in adoptive hosts, making them similar to younger animals. Mature cell numbers are unaffected. Therefore, molecular events governing the specific functions of stem cells are being gradually made clear and offer the potential of new approaches to changing stem cell function for therapy. One critical stem cell function that remains poorly defined is the molecular regulation of self-renewal.


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InternalMed_Harrison. For medicine, self-renewal is perhaps the most important function of stem cells because it is critical in regulating the number of stem cells. Stem cell number is a key limiting parameter for both autologous and allogeneic stem cell transplantation. Were we to have the ability to use fewer stem cells or expand limited numbers of stem cells ex vivo, it might be possible to reduce the morbidity and expense of stem cell harvests and enable use of other stem cell sources. Specifically, umbilical cord blood is a rich source of stem cells. However, the volume of cord blood units is extremely small, and therefore, the total number of hematopoietic stem cells that can be obtained in any single cord blood unit is generally only sufficient to transplant an individual of <40 kg. This limitation restricts what would otherwise be an extremely promising source of stem cells. Two features of cord blood stem cells are particularly important. (1) They are derived from a diversity of individuals that


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InternalMed_Harrison. what would otherwise be an extremely promising source of stem cells. Two features of cord blood stem cells are particularly important. (1) They are derived from a diversity of individuals that far exceeds the adult donor pool and therefore can overcome the majority of immunologic cross-matching obstacles. (2) Cord blood stem cells have a large number of T cells associated with them, but (paradoxically) they appear to be associated with a lower


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InternalMed_Harrison. FIGURE 89e-3 Relative function of cells in the hematopoietic hierarchy. The boxes represent distinct functional features of cells in the myeloid (upper box) versus lymphoid (lower box) lineages. incidence of graft-versus-host disease when compared with similarly mismatched stem cells from other sources. If stem cell expansion by self-renewal could be achieved, the number of cells available might be sufficient for use in larger adults. An alternative approach to this problem is to improve the efficiency of engraftment of donor stem cells. Graft engineering is exploring methods of adding cell components that may enhance engraftment. Furthermore, at least some data suggest that depletion of host NK (natural killer) cells may lower the number of stem cells necessary to reconstitute hematopoiesis.


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InternalMed_Harrison. Some limited understanding of self-renewal exists and, intriguingly, implicates gene products that are associated with the chromatin state, a high-order organization of chromosomal DNA that influences transcription. These include members of the polycomb family, a group of zinc finger–containing transcriptional regulators that interact with the chromatin structure, contributing to the accessibility of groups of genes for transcription. One member, Bmi-1, is important in enabling hematopoietic stem cell self-renewal through modification of cell cycle regulators such as the cyclin-dependent kinase inhibitors. In the absence of Bmi-1 or of the transcriptional regulator, Gfi-1, hematopoietic stem cells decline in number and function. In contrast, dysregulation of Bmi-1 has been associated with leukemia; it may promote leukemic stem cell self-renewal when it is overexpressed. Other transcription regulators have also been associated with self-renewal, particularly homeobox, or “hox,” genes.


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InternalMed_Harrison. leukemia; it may promote leukemic stem cell self-renewal when it is overexpressed. Other transcription regulators have also been associated with self-renewal, particularly homeobox, or “hox,” genes. These transcription factors are named for their ability to govern large numbers of genes, including those determining body patterning in invertebrates. HoxB4 is capable of inducing extensive self-renewal of stem cells through its DNA-binding motif. Other members of the hox family of genes have been noted to affect normal stem cells, but they are also associated with leukemia. External signals that may influence the relative self-renewal versus differentiation outcomes of stem cell cycling include specific Wnt ligands. Intracellular signal transducing intermediates are also implicated in regulating self-renewal. They include PTEN, an inhibitor of the AKT pathway, and STAT5, both of which are downstream of activated growth factor receptors and necessary for normal stem cell functions


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InternalMed_Harrison. regulating self-renewal. They include PTEN, an inhibitor of the AKT pathway, and STAT5, both of which are downstream of activated growth factor receptors and necessary for normal stem cell functions including self-renewal, at least in mouse models. The connections between these molecules remain to be defined, and their role in physiologic regulation of stem cell self-renewal is still poorly understood.


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InternalMed_Harrison. The relationship of stem cells to cancer is an important evolving dimension of adult stem cell biology. Cancer may share principles of organization with normal tissues. Cancer cells are heterogeneous even within a given patient and may have a hierarchical organization of cells with a base of stem-like cells capable of the signature stem cell features: self-renewal and differentiation. These stem-like cells might be the basis for perpetuation of the tumor and represent a slowly dividing, rare population with distinct regulatory mechanisms, including a relationship with a specialized microenvironment. A subpopulation of self-renewing cells has been defined for some, but not all, cancers. A more sophisticated understanding of the stem cell organization of cancers may lead to improved strategies for developing new therapies for the many common and difficult-to-treat types of malignancies that have been relatively refractory to interventions aimed at dividing cells.


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InternalMed_Harrison. Does the concept of cancer stem cells provide insight into the cellular origin of cancer? The fact that some cells within a cancer have stem cell–like properties does not necessarily mean that the cancer arose in the stem cell itself. Rather, more mature cells could have acquired the self-renewal characteristics of stem cells. Any single genetic event is unlikely to be sufficient to enable full transformation of a normal cell to a frankly malignant one. Rather, cancer is a multistep process, and for the multiple steps to accumulate, the cell of origin must be able to persist for prolonged periods. It must also be able to generate large numbers of daughter cells. The normal stem cell has these properties and, by virtue of its having intrinsic self-renewal capability, may be more readily converted to a malignant phenotype. This hypothesis has been tested experimentally in the hematopoietic system. Taking advantage of the cell-surface markers that distinguish hematopoietic cells of


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InternalMed_Harrison. converted to a malignant phenotype. This hypothesis has been tested experimentally in the hematopoietic system. Taking advantage of the cell-surface markers that distinguish hematopoietic cells of varying maturity, stem cells, progenitors, precursors, and mature cells can be isolated. Powerful transforming gene constructs were placed in these cells, and it was found that the cell with the greatest potential to produce a malignancy was dependent on the transforming gene. In some cases, it was the stem cell, but in others, the progenitor cell functioned to initiate and perpetuate the cancer. This shows that cells can acquire stem cell–like properties in malignancy.


InternalMed_Harrison

InternalMed_Harrison. WHAT ELSE CAN HEMATOPOIETIC STEM CELLS DO? Some experimental data have suggested that hematopoietic stem cells or other cells mobilized into the circulation by the same factors that mobilize hematopoietic stem cells are capable of playing a role in healing the vascular and tissue damage associated with stroke and myocardial infarction. These data are controversial, and the applicability of a stem cell approach to nonhematopoietic conditions remains experimental. However, reprogramming technology offers the potential for using the readily obtained hematopoietic stem cell as a source for cells with other capabilities.


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InternalMed_Harrison. The stem cell, therefore, represents a true dual-edged sword. It has tremendous healing capacity and is essential for life. Uncontrolled, it can threaten the life it maintains. Understanding how stem cells function, the signals that modify their behavior, and the tissue niches that modulate stem cell responses to injury and disease are critical for more effectively developing stem cell–based medicine. That aspect of medicine will include the use of the stem cells and the use of drugs to target stem cells to enhance repair of damaged tissues. It will also include the careful balance of interventions to control stem cells where they may be dysfunctional or malignant.


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InternalMed_Harrison. Applications of Stem Cell Biology in Clinical Medicine John A. Kessler Damage to an organ initiates a series of events that lead to the recon-struction of the damaged tissue, including proliferation, differentiation, 90e stem cells Undifferentiated stem cells Erythropoietin Dopaminergic neurons Erythrocytes Hematopoietic stem cells Into striatum Into heart Intravenous FIGURE 90e-1 Strategies for transplantation of stem cells. 1. Undifferentiated or partially differentiated stem cells may be injected directly into the target organ or intravenously. 2. Stem cells may be differentiated ex vivo before injection into the target organ. 3. Growth factors or other drugs may be injected to stimulate endogenous stem cell populations.


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InternalMed_Harrison. and migration of various cell types; release of cytokines and chemokines; and remodeling of the extracellular matrix. Endogenous stem and progenitor cells are among the cell populations that are involved in these injury responses. In normal steady-state conditions, an equilibrium is maintained in which endogenous stem cells intrinsic to the tissue replenish dying cells. After tissue injury, stem cells in organs such as the liver and skin have a remarkable ability to regenerate the organ, whereas other stem cell populations, such as those in the heart and brain, have a much more limited capability for self-repair. In rare circumstances, circulating stem cells may contribute to regenerative responses by migrating into a tissue and differentiating into organ-specific cell types. The goal of stem cell therapies is to promote cell replacement in organs that are damaged beyond their ability to self-repair.


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InternalMed_Harrison. At least three different therapeutic concepts for cell replacement can be envisaged (Fig. 90e-1). One therapeutic approach involves direct administration of stem cells. The cells may be injected directly into the damaged organ, where they can differentiate into the desired cell type. Alternatively, stem cells may be injected systemically since they have the capacity to home in on damaged tissues by following gradients of cytokines and chemokines released by the diseased organ. A second approach involves transplantation of differentiated cells derived from stem cells. For example, pancreatic islet cells can be generated from stem cells before transplantation into diabetic patients, 90e-1 and cardiomyocytes can be generated to treat ischemic heart disease. A third approach involves stimulation of endogenous stem cells to facilitate repair. This goal might be accomplished by administration of appropriate growth factors and drugs that amplify the number of endogenous stem/progenitor cells


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InternalMed_Harrison. of endogenous stem cells to facilitate repair. This goal might be accomplished by administration of appropriate growth factors and drugs that amplify the number of endogenous stem/progenitor cells and/or direct them to differentiate into the desired cell types. Therapeutic stimulation of precursor cells is already a clinical reality in the hematopoietic system, where factors such as erythropoietin, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor are used to increase production of specific blood elements. In addition to these strategies for cell replacement, a number of other approaches could involve stem cells for ex vivo or in situ generation of tissues, a process termed tissue engineering (Chap. 92e). Stem cells are also excellent candidates as vehicles for cellular gene therapy (Chap. 91e). Finally, transplanted stem cells may exert paracrine effects on damaged tissues without the differentiation and replacement of lost cells.


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InternalMed_Harrison. Stem cell transplantation is not a new concept but rather is already part of established medical practice. Hematopoietic stem cells (Chap. 89e) are responsible for the long-term repopulation of all blood elements in recipients of bone marrow transplants, and hematopoietic stem cell transplantation is the gold standard against which other stem cell transplantation therapies will be measured. Transplantation of differentiated cells is also a clinical reality, and donated organs and tissues are often used to replace damaged tissues. However, the need for transplantable tissues and organs far outweighs the available supply, and organ transplantation has limited potential for some tissues, such as the brain. Stem cells offer the possibility of a renewable source of replacement cells for virtually all organs.


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InternalMed_Harrison. A variety of different types of stem cells (Chap. 88) could be used in regenerative strategies, including embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, umbilical-cord blood stem cells (USCs), organ-specific somatic stem cells (e.g., neural stem cells for treatment of the brain), and somatic stem cells that generate cell types specific for the target organ rather than the donor organ (e.g., bone marrow mesenchymal stem cells or CD34+ hematopoietic stem cells for cardiac repair). Although each cell type has potential advantages and disadvantages, there are a number of generic problems in developing any of these cell types into a useful and reliable clinical tool.


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InternalMed_Harrison. Embryonic Stem Cells Embryonic stem cells have the potential to generate all the cell types in the body; thus, in theory, there are no restrictions on the organs that could be regenerated. ES cells can self-renew endlessly, so that a single cell line with carefully characterized traits potentially could generate almost limitless numbers of cells. In the absence of moral or ethical constraints (see “Ethical Issues,” below), unused human blastocysts from fertility clinics could be used to derive new ES cell lines that are matched immunologically with potential transplant recipients. Alternatively, somatic cell nuclear transfer (“therapeutic cloning”) could be used to create ES cell lines that are genetically identical to those of the patient, although this endeavor has been technically refractory for human cells. However, human ES cells are difficult to culture and grow slowly. Techniques for differentiating them into specific cell types are just beginning


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InternalMed_Harrison. CHAPTER 90e Applications of Stem Cell Biology in Clinical Medicine to be developed. Cells tend to develop abnormal karyotypes and other abnormalities with increased time in culture, and ES cells have the potential to form teratomas if all cells are not committed to the desired cell types before transplantation. Further, human ES cells are ethically controversial and, on these grounds, would be unacceptable to some patients and physicians despite their therapeutic potential. Nevertheless, there have been limited clinical trials of ES-derived cells in a number of disorders, including macular degeneration, myopia, and spinal cord injury.


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InternalMed_Harrison. Induced Pluripotent Stem Cells The field of stem cell biology was transformed by the discovery that adult somatic cells can be converted (“reprogrammed”) into pluripotent cells through the overexpression of four transcription factors normally expressed in pluripotent cells (Chap. 88). These iPS cells share most properties with ES cells, although there are distinct differences in gene expression between ES and iPS cells. The initial use of viruses to insert the transcription factors into somatic cells made the resulting cells unsuitable for clinical use. However, a number of strategies have since been developed to circumvent this problem, including the insertion of modified mRNAs, proteins, or microRNAs rather than cDNAs; the use of non-integrating viruses such as Sendai virus; the insertion of transposons with the programming factors, followed by their subsequent removal; and the use of floxed viral constructs, followed by treatment with Cre recombinase to excise those constructs. The


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InternalMed_Harrison. of transposons with the programming factors, followed by their subsequent removal; and the use of floxed viral constructs, followed by treatment with Cre recombinase to excise those constructs. The safety of iPS cells in humans remains to be demonstrated, but clinical trials in macular degeneration and other disorders are planned. Potential advantages of iPS cells are that somatic cells from patients would generate pluripotent cells genetically identical to those of the patient and that these cells are not subject to the same ethical constraints as ES cells. It is not clear whether the differences in gene expression between ES and iPS cells will have any impact on their potential clinical utility, and studies of both cell types will be essential to resolve this issue.


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InternalMed_Harrison. Umbilical-Cord Stem Cells Umbilical-cord blood stem/progenitor cells (USCs) are widely and readily available. These cells appear to be associated with less graft-versus-host disease than are some other cell types, such as marrow stem cells. They have less human leukocyte antigen restriction than adult marrow stem cells and are less likely to be contaminated with herpesvirus. However, it is unclear how many different cell types can be generated from USCs, and methods for differentiating these cells into nonhematopoietic phenotypes are largely lacking. Nevertheless, there are ongoing clinical trials of these cells in dozens of disorders, including cirrhosis, cardiopathies, multiple sclerosis, burns, stroke, autism, and critical limb ischemia.


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InternalMed_Harrison. Organ-Specific Multipotent Stem Cells Organ-specific multipotent stem cells have the advantage of already being somewhat specialized so that the inducement of desired cell types may be easier. Cells potentially could be obtained from the patient and amplified in cell culture, circumventing the problems associated with immune rejection. Stem cells are relatively easy to harvest from some tissues, such as bone marrow and blood, but are difficult to harvest from other tissues, such as heart and brain. Moreover, these populations of cells are more limited in potentiality than are pluripotent ES or iPS cells, and they may be difficult to obtain in large quantities from many organs. Therefore, substantial efforts have been devoted to developing techniques for using more easily obtainable stem cell populations, such as bone marrow mesenchymal stem cells (MSCs), CD34+ hematopoietic stem cells (HSCs), cardiac mesenchymal cells, and adipose-derived stem cells (ASCs), for use in regenerative


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InternalMed_Harrison. cell populations, such as bone marrow mesenchymal stem cells (MSCs), CD34+ hematopoietic stem cells (HSCs), cardiac mesenchymal cells, and adipose-derived stem cells (ASCs), for use in regenerative strategies. Tissue culture evidence suggests that these stem cell populations may be able to generate differentiated cell types unrelated to their organ source (including myocytes, chondrocytes, tendon cells, osteoblasts, cardiomyocytes, adipocytes, hepatocytes, and neurons) in a process known as transdifferentiation. However, it is still unclear whether these stem cells are capable of generating differentiated cell types that integrate into organs, survive, and function after transplantation in vivo. A number of early studies of MSCs transplanted into heart, liver, and other organs suggested that the cells had differentiated into organ-specific cell types with beneficial effects in animal models of disease. Unfortunately, subsequent studies revealed that the stem cells had simply fused


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InternalMed_Harrison. that the cells had differentiated into organ-specific cell types with beneficial effects in animal models of disease. Unfortunately, subsequent studies revealed that the stem cells had simply fused with cells resident in the organs and that the observed beneficial effects were due to paracrine release of trophic and anti-inflammatory cytokines. Further studies will be necessary to determine whether transdifferentiation of MSCs, ASCs, or other stem cell populations occurs at a high enough frequency to make these cells useful for stem cell replacement therapy. Despite the remaining issues, clinical trials of MSCs, autologous HSCs, USCs, and ASCs are being performed in many disorders, including ischemic cardiac disease, cardiomyopathy, diabetes, stroke, cirrhosis, and muscular dystrophy.


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InternalMed_Harrison. Regardless of the source of the stem cells used in regenerative strategies, a number of generic problems must be overcome for the development of successful clinical applications. These problems include the devising of methods to reliably generate large numbers of specific cell types, to minimize the risk of tumor formation or proliferation of inappropriate cell types, to ensure the viability and function of the engrafted cells, to overcome immune rejection when autografts are not used, and to facilitate revascularization of regenerated tissue. Each organ system will also pose tissue-specific problems for stem cell therapies.


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InternalMed_Harrison. DISEASE-SPECIFIC APPLICATIONS OF STEM CELLS Ischemic Heart Disease and Cardiomyocyte Regeneration Because of the high prevalence of ischemic heart disease, extensive efforts have been devoted to the development of strategies for stem cell replacement of cardiomyocytes. Historically, the adult heart has been viewed as a terminally differentiated organ without the capacity for regeneration. However, recent studies have demonstrated that the heart has the capacity for low levels of cardiomyocyte regeneration (Chap. 265e). This regeneration appears to be accomplished by cardiac stem cells resident in the heart and possibly also by cells originating in the bone marrow. The heart might be an ideal source of stem cells for therapeutic use, but techniques for isolating, characterizing, and amplifying large numbers of these cells have not yet been perfected. For successful myocardial repair, stem cell therapy must deliver cells either systemically or locally, and the cells must survive,


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InternalMed_Harrison. amplifying large numbers of these cells have not yet been perfected. For successful myocardial repair, stem cell therapy must deliver cells either systemically or locally, and the cells must survive, engraft, and differentiate into functional cardiomyocytes that couple mechanically and electrically with the recipient myocardium. The optimal method for cell delivery is not clear, and various experimental and clinical studies have successfully employed intramyocardial, transendocardial, intravenous, intracoronary, and retrograde coronary venous injections. In experimental myocardial infarction, functional improvements have been achieved after transplantation of a variety of different cell types, including ES cells, HSCs, MSCs, USCs, and ASCs. Early studies suggested that each of these cell types might have the potential to engraft and generate cardiomyocytes. However, most investigators have found that the generation of new cardiomyocytes by these cells is at best a rare event and that


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InternalMed_Harrison. types might have the potential to engraft and generate cardiomyocytes. However, most investigators have found that the generation of new cardiomyocytes by these cells is at best a rare event and that graft survival over long periods is poor. The preponderance of evidence suggests that the observed beneficial effects of most experimental therapies were not derived from direct stem cell generation of cardiomyocytes but rather from indirect effects of the stem cells on resident cells. It is not clear whether these effects reflect the release of soluble trophic factors, the induction of angiogenesis, the release of anti-inflammatory cytokines, or another mechanism. A wide variety of cell delivery methods, cell types, and cell doses have been used in a progressively enlarging series of clinical trials, but the fate of the cells and the mechanisms by which they alter cardiac function are still open questions. In aggregate, however, these studies have shown a small but measurable improvement


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InternalMed_Harrison. trials, but the fate of the cells and the mechanisms by which they alter cardiac function are still open questions. In aggregate, however, these studies have shown a small but measurable improvement in cardiac function and, in some cases, reduction in infarct size. In short, the available evidence suggests that the beneficial clinical impact reflects an indirect effect of the transplanted cells rather than genuine cell replacement.


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InternalMed_Harrison. Diabetes Successes with islet cell and pancreas transplantation have provided proof of concept for cell-based therapies for type 1 diabetes. However, the demand for donor pancreases far exceeds the number available, and maintenance of long-term graft survival is a problem. The search for a renewable source of stem cells capable of regenerating pancreatic islets has therefore been intensive. Pancreatic beta cell turnover occurs even in the normal pancreas, although the source of the new beta cells remains controversial. This persistent turnover suggests that, in principle, it should be possible to develop strategies for reconstituting the beta cell population in diabetics. Attempts to devise techniques for promoting endogenous regenerative processes by using combinations of growth factors, drugs, and gene therapy have failed thus far, but this remains a potentially viable approach. A number of different cell types are candidates for use in stem cell replacement strategies, including


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InternalMed_Harrison. drugs, and gene therapy have failed thus far, but this remains a potentially viable approach. A number of different cell types are candidates for use in stem cell replacement strategies, including iPS cells, ES cells, hepatic progenitor cells, pancreatic ductal progenitor cells, and MSCs. Successful therapy will depend on the development of a source of cells that can be amplified to produce large numbers of progeny with the ability to synthesize, store, and release insulin when it is required, primarily in response to changes in the ambient level of glucose. The proliferative capacity of the replacement cells must be tightly regulated to avoid excessive expansion of beta cell numbers and the consequent development of hyperinsulinemia/hypoglycemia; moreover, the cells must withstand immune rejection. Although it has been reported that ES and iPS cells can be differentiated into cells that produce insulin, these cells have a low content of insulin and a high rate of apoptosis and


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InternalMed_Harrison. immune rejection. Although it has been reported that ES and iPS cells can be differentiated into cells that produce insulin, these cells have a low content of insulin and a high rate of apoptosis and generally lack the capacity to normalize blood glucose levels in diabetic animals. Thus, ES and iPS cells have not yet been useful for the large-scale production of differentiated islet cells. During embryogenesis, the pancreas, liver, and gastrointestinal tract are all derived from the anterior endoderm, and transdifferentiation of pancreas to liver and vice versa has been observed in a number of pathologic conditions. There is also substantial evidence that multipotential stem cells reside within gastric glands and intestinal crypts. These observations suggest that hepatic, pancreatic, and/or gastrointestinal precursor cells may be reasonable candidates for cell-based therapy for diabetes, although it is unclear whether insulin-producing cells derived from pancreatic stem cells or liver


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InternalMed_Harrison. gastrointestinal precursor cells may be reasonable candidates for cell-based therapy for diabetes, although it is unclear whether insulin-producing cells derived from pancreatic stem cells or liver progenitors can be expanded in vitro to clinically useful numbers. MSCs and neural stem cells both reportedly have the capacity to generate insulin-producing cells, but there is no convincing evidence that either cell type will be clinically useful. Clinical trials of MSCs, USCs, HSCs, and ASCs in both type 1 and type 2 diabetes are ongoing.


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InternalMed_Harrison. Nervous System Substantial progress has been made in the development of methodologies for generating neural cells from different stem cell populations. Human ES or iPS cells can be induced to generate cells with the properties of neural stem cells, and these cells in turn give rise to neurons, oligodendroglia, and astrocytes. Reasonably large numbers of these cells can be transplanted into the rodent brain with formation of appropriate cell types and no tumor formation. Multipotent stem cells present in the adult brain also can be easily amplified in number and used to generate all the major neural cell types, but the need for invasive procedures to obtain autologous cells is a major limitation. Fetal neural stem cells derived from miscarriages or abortions are an alternative but raise ethical concerns. Nevertheless, clinical trials of fetal neural stem cells have commenced in amyotrophic lateral sclerosis (ALS), stroke, and several other disorders. Transdifferentiation of MSCs and


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InternalMed_Harrison. ethical concerns. Nevertheless, clinical trials of fetal neural stem cells have commenced in amyotrophic lateral sclerosis (ALS), stroke, and several other disorders. Transdifferentiation of MSCs and ASCs into neural stem cells, and vice versa, has been reported by numerous investigators, and clinical trials of such cells have begun for a number of neurologic diseases. Clinical trials of a conditionally immortalized human cell line and of USCs in stroke are also in progress. Because of the incapacitating nature of neural disorders and the limited endogenous repair capacity of the nervous system, clinical trials of stem cells in neurologic disorders have been particularly numerous, including trials in spinal cord injury, multiple sclerosis, epilepsy, Alzheimer’s disease, ALS, acute and chronic stroke, numerous genetic disorders, traumatic brain injury, Parkinson’s disease, and others. In diseases such as ALS, possible benefits are more likely to be due to indirect trophic effects than


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InternalMed_Harrison. stroke, numerous genetic disorders, traumatic brain injury, Parkinson’s disease, and others. In diseases such as ALS, possible benefits are more likely to be due to indirect trophic effects than to neuron replacement. In Parkinson’s disease, the major motor features of the disorder result from the loss of a single cell population: dopaminergic neurons within the substantia nigra; this circumstance suggests that cell replacement should be relatively straightforward. However, two clinical trials of fetal nigral transplantation failed to meet their primary endpoint and were complicated by the development of dyskinesia. Transplantation of stem cell–derived dopamine-producing cells offers a number of 90e-3 potential advantages over the fetal transplants, including the ability of stem cells to migrate and disperse within tissue, the potential for engineering regulatable release of dopamine, and the ability to engineer cells to produce factors that will enhance cell survival. Nevertheless,


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InternalMed_Harrison. migrate and disperse within tissue, the potential for engineering regulatable release of dopamine, and the ability to engineer cells to produce factors that will enhance cell survival. Nevertheless, the experience with fetal transplants points out the difficulties that may be encountered.


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InternalMed_Harrison. At least some of the neurologic dysfunction after spinal cord injury reflects demyelination, and both ES cells and MSCs can facilitate remyelination after experimental spinal cord injury (SCI). Clinical trials of MSCs in this disorder have commenced in a number of countries, and SCI was the first disorder targeted for the clinical use of ES cells. However, the ES cell trial in SCI was terminated early for nonmedical reasons. At present, no population of transplanted stem cells has been shown to have the capacity to generate neurons that extend axons over long distances to form synaptic connections (as would be necessary for replacement of upper motor neurons in ALS, stroke, or other disorders). For many injuries, including SCI, the balance between scar formation and tissue repair/regeneration may prove to be an important consideration. For example, it may ultimately prove necessary to limit scar formation so that axons can reestablish connections.


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InternalMed_Harrison. Liver Liver transplantation is currently the only successful treatment for end-stage liver diseases, but the shortage of liver grafts limits its application. Clinical trials of hepatocyte transplantation demonstrate its potential as a substitute for organ transplantation, but this approach is limited by the paucity of available cells. Potential sources of stem cells for regenerative strategies include endogenous liver stem cells (such as oval cells), ES cells, MSCs, and USCs. Although a series of studies in humans as well as animals suggested that transplanted MSCs and HSCs can generate hepatocytes, fusion of the transplanted cells with endogenous liver cells, giving the erroneous appearance of new hepatocytes, appears to be the underlying event in most circumstances. The available evidence suggests that transplanted HSCs and MSCs can generate hepatocyte-like cells in the liver only at a very low frequency, but there are beneficial consequences presumably related to indirect paracrine


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InternalMed_Harrison. suggests that transplanted HSCs and MSCs can generate hepatocyte-like cells in the liver only at a very low frequency, but there are beneficial consequences presumably related to indirect paracrine effects. ES cells can be differentiated into hepatocytes and transplanted in animal models of liver failure without the formation of teratomas. Clinical trials are in progress in cirrhosis with numerous cell types, including MSCs, USCs, HSCs, and ASCs.


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InternalMed_Harrison. Other Organ Systems and the Future The use of stem cells in regenerative strategies has been studied for many other organ systems and cell types, including skin, eye, cartilage, bone, kidney, lung, endometrium, vascular endothelium, smooth muscle, and striated muscle, and clinical trials in these and other organs are ongoing. In fact, the potential for stem cell regeneration of damaged organs and tissues is virtually limitless. However, numerous obstacles must be overcome before stem cell therapies can become a widespread clinical reality. Only HSCs have been adequately characterized by surface markers so that they can be unambiguously identified, a prerequisite for reliable clinical applications. The pathways for differentiating stem cells into specific cellular phenotypes are largely unknown, and the ability to control the migration of transplanted cells or predict the response of the cells to the environment of diseased organs is presently limited. Some strategies may employ the


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InternalMed_Harrison. unknown, and the ability to control the migration of transplanted cells or predict the response of the cells to the environment of diseased organs is presently limited. Some strategies may employ the coadministration of scaffolding, artificial extracellular matrix, and/or growth factors to orchestrate differentiation of stem cells and their organization into appropriate constituents of the organ. There is currently no way to image stem cells in vivo after transplantation into humans, and it will be necessary to develop techniques to do so. Fortunately, stem cells can be engineered before transplantation to contain a contrast agent that may make their in vivo imaging feasible. The potential for tumor formation and the problems associated with immune rejection are impediments, and it will also be necessary to develop techniques for ensuring vascularization of regenerated tissues. There already are many strategies for supporting cell replacement, including coadministration of vasoactive


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InternalMed_Harrison. be necessary to develop techniques for ensuring vascularization of regenerated tissues. There already are many strategies for supporting cell replacement, including coadministration of vasoactive endothelial growth factor to foster vascularization of the transplant. Some strategies also include the genetic engineering of stem cells with an inducible suicide gene so that the cells can be easily eradicated in


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InternalMed_Harrison. CHAPTER 90e Applications of Stem Cell Biology in Clinical Medicine the event of tumor formation or another complication. The potential for stem cell therapies to revolutionize medical care is extraordinary, and disorders such as myocardial infarction, diabetes, and Parkinson’s disease, among many others, are potentially curable by such therapies. However, stem cell–based therapies are still at a very early stage of development, and perfection of techniques for clinical transplantation of predictable, well-characterized cells is going to be a difficult and lengthy undertaking.


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InternalMed_Harrison. Stem cell therapies raise ethical and socially contentious issues that must be addressed in parallel with the scientific and medical opportunities. Society has great diversity with respect to religious beliefs, concepts of individual rights, tolerance for uncertainty and risk, and boundaries for how scientific interventions should be used to alter the outcome of disease. In the United States, the federal government has authorized research using existing human ES cell lines but still restricts the use of federal funds for developing new human ES cell lines. Ongoing studies of existing lines have indicated that they develop abnormalities with time in culture and that they may be contaminated with mouse proteins. These findings highlight the need to develop new human ES cell lines. The development of iPS cell technology may lessen the need for deriving new ES cell lines, but it is still not clear whether the differences in gene expression by ES and iPS cells are important for potential


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InternalMed_Harrison. of iPS cell technology may lessen the need for deriving new ES cell lines, but it is still not clear whether the differences in gene expression by ES and iPS cells are important for potential clinical use.


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InternalMed_Harrison. In considering ethical issues associated with the use of stem cells, it is helpful to draw from experience with other scientific advances, such as organ transplantation, recombinant DNA technology, implantation of mechanical devices, neuroscience and cognitive research, in vitro fertilization, and prenatal genetic testing. These and other precedents have pointed to the importance of understanding and testing fundamental biology in the laboratory setting and in animal models before applying new techniques in carefully controlled clinical trials. When these trials occur, they must include full informed consent and careful oversight by external review groups.


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InternalMed_Harrison. Ultimately, there will be medical interventions that are scientifically feasible but ethically or socially unacceptable to some members of a society. Stem cell research raises fundamentally difficult questions about the definition of human life, and it has raised deep fears about the ability to balance issues of justice and safety with the needs of critically ill patients. Health care providers and experts with backgrounds in ethics, law, and sociology must help guard against the premature or inappropriate application of stem cell therapies and the inappropriate involvement of vulnerable population groups. However, these therapies offer important new strategies for the treatment of otherwise irreversible disorders. An open dialogue among the scientific community, physicians, patients and their advocates, lawmakers, and the lay population is critically important to raise and address important ethical issues and balance the benefits and risks associated with stem cell transfer.


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InternalMed_Harrison. by many factors, including an intense desire to develop therapies for 91e-1 Gene Therapy in Clinical Medicine hitherto untreatable diseases, lack of understanding of risks, and, in some cases, undisclosed financial conflicts of interest. After a teenager Katherine A. High died of complications related to vector infusion, the field underwent a


InternalMed_Harrison

InternalMed_Harrison. Gene transfer is a novel area of therapeutics in which the active agent is a nucleic acid sequence rather than a protein or small molecule. Because delivery of naked DNA or RNA to a cell is an inefficient process, most gene transfer is carried out using a vector, or gene delivery vehicle. These vehicles have generally been engineered from viruses by deleting some or all of the viral genome and replacing it with the therapeutic gene of interest under the control of a suitable promoter (Table 91e-1). Gene transfer strategies can thus be described in terms of three essential elements: (1) a vector; (2) a gene to be delivered, sometimes called the transgene; and (3) a physiologically relevant target cell to which the DNA or RNA is delivered. The series of steps in which the vector and donated DNA enter the target cell and express the transgene is referred to as transduction. Gene delivery can take place in vivo, in which the vector is directly injected into the patient, or, in the case of


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InternalMed_Harrison. DNA enter the target cell and express the transgene is referred to as transduction. Gene delivery can take place in vivo, in which the vector is directly injected into the patient, or, in the case of hematopoietic and some other target cells, ex vivo, with removal of the target cells from the patient, followed by return of the gene-modified autologous cells to the patient after manipulation in the laboratory. The latter approach effectively combines gene transfer techniques with cellular therapies (Chap. 90e).


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InternalMed_Harrison. Gene transfer is one of the most powerful concepts in modern molecular medicine and has the potential to address a host of diseases for which there are currently no available treatments. Clinical trials of gene therapy have been under way since 1990; a recent landmark in the field was the licensing, in 2012, of the first gene therapy product approved in Europe or the United States (see below). Given that vector-mediated gene therapy is arguably one of the most complex therapeutics yet developed, consisting of both a nucleic acid and a protein component, this time course from first clinical trial to licensed product is noteworthy for being similar to that seen with other novel classes of therapeutics, including monoclonal antibodies or bone marrow transplantation. Over 5000 subjects have been enrolled in gene transfer studies, and serious adverse events have been rare. Some of the initial trials were characterized by an overabundance of optimism and a failure to be appropriately


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InternalMed_Harrison. have been enrolled in gene transfer studies, and serious adverse events have been rare. Some of the initial trials were characterized by an overabundance of optimism and a failure to be appropriately critical of preclinical studies in animals; in addition, it was in some contexts not fully appreciated that animal studies are only a partial guide to safety profiles of products in humans (e.g., insertional mutagenesis). Initial exuberance was driven retrenchment; continued efforts led to a more nuanced understanding of the risks and benefits of these new therapies and more sophisticated selection of disease targets. Currently, gene therapies are being developed for a wide variety of disease entities (Fig. 91e-1).


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InternalMed_Harrison. Gene transfer strategies for genetic disease generally involve gene addition therapy, an approach characterized by transfer of the missing gene to a physiologically relevant target cell. However, other strategies are possible, including supplying a gene that achieves a similar biologic effect through an alternative pathway (e.g., factor VIIa for hemophilia A); supplying an antisense oligonucleotide to splice out a mutant exon if the sequence is not critical to the function of the protein (as has been done with the dystrophin gene in Duchenne’s muscular dystrophy); or downregulating a harmful effect through a small interfering RNA (siRNA). Two distinct strategies are used to achieve long-term gene expression: one is to transduce stem cells with an integrating vector, so that all progeny cells will carry the donated gene; and the other is to transduce long-lived cells, such as skeletal muscle or neurons. In the case of long-lived cells, integration into the target cell genome is


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InternalMed_Harrison. cells will carry the donated gene; and the other is to transduce long-lived cells, such as skeletal muscle or neurons. In the case of long-lived cells, integration into the target cell genome is unnecessary. Instead, because the cells are nondividing, the donated DNA, if stabilized in an episomal form, will give rise to expression for the life of the cell. This approach thus avoids problems related to integration and insertional mutagenesis.


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InternalMed_Harrison. IMMUNODEFICIENCY DISORDERS: PROOF OF PRINCIPLE Early attempts to effect gene replacement into hematopoietic stem cells (HSCs) were stymied by the relatively low transduction efficiency of retroviral vectors, which require dividing target cells for integration. Because HSCs are normally quiescent, they are a formidable transduction target. However, identification of cytokines that induced cell division without promoting differentiation of stem cells, along with technical improvements in the isolation and transduction of HSCs, led to modest but real gains in transduction efficiency. The first convincing therapeutic effect from gene transfer occurred with X-linked severe combined immunodeficiency disease (SCID), which results from mutations in the gene (IL2RG) encoding the γc subunit of cytokine receptors required for normal development of Immune responses to vector Theoretical risk of insertional mutagenesis (occurred in multiple cases)


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InternalMed_Harrison. Immune responses to vector Theoretical risk of insertional mutagenesis (occurred in multiple cases) Elicits few inflammatory responses, nonpathogenic 8.5 kb In need of a stable packaging system DNA RNA Large packaging Limited immune capacity with responses against persistent gene the vector transfer Residual cytotox-Transduced gene icity with neuron expression is specificity transient Abbreviations: AAV, adeno-associated virus; HSV, herpes simplex virus; SV, sarcoma virus. vector, so that errant clones can be quickly ablated, or using “insulator” elements in the cassette, which can limit the activation of genes surrounding the insertion site. Lentiviral vectors, which can efficiently transduce nondividing target cells, are also likely to be safer than retroviral vectors, based on patterns of integration; the field is thus gradually moving toward these to replace retroviral vectors.


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InternalMed_Harrison. More clear-cut success has been achieved in a gene therapy trial for another form of SCID, adenosine deaminase (ADA) deficiency (Chap. 374). ADASCID is clinically similar to X-linked SCID, although it can be treated by enzyme replacement therapy with a pegylated form of the enzyme (PEG-ADA), which leads to immune reconstitution but not always to normal T cell counts. Enzyme replacement therapy is expensive (annual costs: $200,000–$300,000 in U.S. dollars). The initial trials of gene therapy for


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InternalMed_Harrison. U.S. dollars). The initial trials of gene therapy for Number of trials (year 2013) ADA-SCID were unsuccessful, but modifications of FIGURE 91e-1 Indications in gene therapy clinical trials. The bar graph classifies this protocol to include the use of HSCs rather than clinical gene transfer studies by disease. A majority of trials have addressed cancer, T cells as the target for transduction; discontinuation with monogenic disorders, infectious diseases, and cardiovascular diseases the next of PEG-ADA at the time of vector infusion, so that the largest categories. (Adapted from SL Ginn et al: J Gene Med 15:65-77, 2013. Published transduced cells have a proliferative advantage over online in Wiley Online.)


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InternalMed_Harrison. T and natural killer (NK) cells (Chap. 374). Affected infants present in the first few months of life with overwhelming infections and/or failure to thrive. In this disorder, it was recognized that the transduced cells, even if few in number, would have a proliferative advantage compared to the nontransduced cells, which lack receptors for the cytokines required for lymphocyte development and maturation. Complete reconstitution of the immune system, including documented responses to standard childhood vaccinations, clearing of infections, and remarkable gains in growth occurred in most of the treated children. However, among 20 children treated in two separate trials, five eventually developed a syndrome similar to T cell acute lymphocytic leukemia, with splenomegaly, rising white counts, and the emergence of a single clone of T cells. Molecular studies revealed that, in most of these children, the retroviral vector had integrated within a gene, LMO-2 (LIM only-2), which encodes a


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InternalMed_Harrison. and the emergence of a single clone of T cells. Molecular studies revealed that, in most of these children, the retroviral vector had integrated within a gene, LMO-2 (LIM only-2), which encodes a component of a transcription factor complex involved in hematopoietic development. The retroviral long terminal repeat increases the expression of LMO-2, resulting in T cell leukemia.


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InternalMed_Harrison. The X-linked SCID studies were a watershed event in the evolution of gene therapy. They demonstrated conclusively that gene therapy could cure disease; of the 20 children eventually treated in these trials, 18 achieved correction of the immunodeficiency disorder. Unfortunately, 5 of the 20 patients later developed a leukemia-like disorder, and one died of this complication; the rest are alive and free of complications at time periods ranging up to 14 years after initial treatment. These studies demonstrated that insertional mutagenesis leading to cancer was more than a theoretical possibility (Table 91e-2). As a result of the experience in these trials, all protocols using integrating vectors in hematopoietic cells must include a plan for monitoring sites of insertion and clonal proliferation. Strategies to overcome this complication have included using a “suicide” gene cassette in the Gene silencing – repression of promoter


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InternalMed_Harrison. Gene silencing – repression of promoter Phenotoxicity – complications arising from overexpression or ectopic expression of the transgene Immunotoxicity – harmful immune response to either the vector or transgene Risks of horizontal transmission – shedding of infectious vector into environment


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InternalMed_Harrison. Risks of horizontal transmission – shedding of infectious vector into environment Risks of vertical transmission – germline transmission of donated DNA the nontransduced; and the use of a mild conditioning regimen to facilitate engraftment of the transduced cells have led to success without the complications seen in the X-linked SCID trials. There have been no complications in the 10 children treated on the Milan protocol, with a median follow-up of >8 years. ADA-SCID, then, is an example where gene therapy has changed therapeutic options for patients. For those with a human leukocyte antigen (HLA)-identical sibling, bone marrow transplantation is still the best treatment option, but this includes only a minority of those affected. For those without an HLA-identical match, gene therapy has comparable efficacy to PEG-ADA, does not require repetitive injections, and does not run the risk of neutralizing antibodies to the bovine enzyme.


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InternalMed_Harrison. NEURODEGENERATIVE DISEASES: EXTENSION OF PRINCIPLE


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InternalMed_Harrison. The SCID trials gave support to the hypothesis that gene transfer into HSCs could be used to treat any disease for which allogeneic bone marrow transplantation was therapeutic. Moreover, the use of genetically modified autologous cells carried several advantages including no risk of graft-versus-host disease, guaranteed availability of a “donor” (unless the disease itself damages the stem cell population of the patient), and low likelihood of failure of engraftment. Cartier and Aubourg capitalized on this realization to conduct the first trial of lentiviral vector transduction of HSCs for a neurodegenerative disorder, X-linked adrenoleukodystrophy (ALD). X-linked ALD is a fatal demyelinating disease of the central nervous system caused by mutations in the gene encoding an adenosine triphosphate–binding cassette transporter. Deficiency of this protein leads to accumulation of verylong-chain fatty acids in oligodendrocytes and microglia, disrupting myelin maintenance by these cells.


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InternalMed_Harrison. cassette transporter. Deficiency of this protein leads to accumulation of verylong-chain fatty acids in oligodendrocytes and microglia, disrupting myelin maintenance by these cells. Affected boys present with clinical and neuroradiographic evidence of disease at age 6–8 and usually die before adolescence. Following lentiviral transduction of autologous HSCs in young boys with the disease, dramatic stabilization of disease occurred, demonstrating that stem cell transduction could work for neurodegenerative as well as immunologic disorders. Investigators in Milan carried this observation one step further to develop a treatment for another neurodegenerative disorder that has previously responded poorly to bone marrow transplantation. Metachromatic leukodystrophy is a lysosomal storage disorder caused by mutations in the gene encoding arylsulfatase A (ARSA). The late infantile form of the disease is characterized by progressive motor and cognitive impairment, and death within a few years


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InternalMed_Harrison. caused by mutations in the gene encoding arylsulfatase A (ARSA). The late infantile form of the disease is characterized by progressive motor and cognitive impairment, and death within a few years of onset, due to accumulation of the ARSA substrate sulfatide in oligodendrocytes, microglia, and some neurons. Recognizing that endogenous levels of production of ARSA were too low to provide cross-correction by allogeneic transplant, Naldini and colleagues engineered a lentiviral vector that directed supraphysiologic levels of ARSA expression in transduced cells. Transduction of autologous HSCs from children born with the disease, at a point when they were still presymptomatic, led to preservation and continued acquisition of motor and cognitive milestones at time periods as long as 32 months after affected siblings had begun to lose milestones. These results illustrate that the ability to engineer levels of expression can allow gene therapy approaches to succeed where allogeneic bone


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InternalMed_Harrison. after affected siblings had begun to lose milestones. These results illustrate that the ability to engineer levels of expression can allow gene therapy approaches to succeed where allogeneic bone marrow transplantation cannot. It is likely that a similar approach will be used in other neurodegenerative conditions.


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InternalMed_Harrison. Transduction of HSCs to treat the hemoglobinopathies is an obvious extension of studies already conducted but represents a higher hurdle in terms of the extent of transduction required to achieve a therapeutic effect. Trials are now under way for thalassemia and for a number of other hematologic disorders, including Wiskott-Aldrich syndrome, and chronic granulomatous disease. LONG-TERM EXPRESSION IN GENETIC DISEASE: IN VIVO GENE TRANSFER WITH RECOMBINANT ADENO-ASSOCIATED VIRAL VECTORS


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InternalMed_Harrison. LONG-TERM EXPRESSION IN GENETIC DISEASE: IN VIVO GENE TRANSFER WITH RECOMBINANT ADENO-ASSOCIATED VIRAL VECTORS Recombinant adeno-associated viral (AAV) vectors have emerged as attractive gene delivery vehicles for genetic disease. Engineered from a small replication-defective DNA virus, they are devoid of viral coding sequences and trigger very little immune response in experimental animals. They are capable of transducing nondividing target cells, and the donated DNA is stabilized primarily in an episomal form, thus minimizing risks arising from insertional mutagenesis. Because the vector has a tropism for certain long-lived cell types, such as skeletal muscle, the central nervous system (CNS), and hepatocytes, long-term expression can be achieved even in the absence of integration.


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InternalMed_Harrison. These features of AAV were used to develop the first licensed gene therapy product in Europe, an AAV vector for treatment of the autosomal recessive disorder lipoprotein lipase (LPL) deficiency. This rare disorder (1–2/million) is due to loss-of-function mutations in the gene encoding LPL, an enzyme normally produced in skeletal muscle and required for the catabolism of triglyceride-rich lipoproteins and chylomicrons. Affected individuals have lipemic serum and may have eruptive xanthomas, hepatosplenomegaly, and in some cases, recurrent bouts of acute pancreatitis. Clinical trials demonstrated the safety of intramuscular injection of AAV-LPL and its efficacy in reducing frequency of pancreatitis episodes in affected individuals, leading to drug approval in Europe. Additional clinical trials currently under way that use AAV vectors in the setting of genetic disease include those for muscular dystrophies, α1 antitrypsin deficiency, Parkinson’s disease, Batten’s disease, hemophilia B,


InternalMed_Harrison

InternalMed_Harrison. currently under way that use AAV vectors in the setting of genetic disease include those for muscular dystrophies, α1 antitrypsin deficiency, Parkinson’s disease, Batten’s disease, hemophilia B, and several forms of congenital blindness.


InternalMed_Harrison

InternalMed_Harrison. Hemophilia (Chap. 78) has long been considered a promising disease model for gene transfer, because the gene product does not require precise regulation of expression and biologically active clotting factors can be synthesized in a variety of tissue types, permitting latitude in the choice of target tissue. Moreover, raising circulating factor levels from <1% (levels seen in those severely affected) into the range of 5% greatly improves the phenotype of the disease. Preclinical studies with recombinant AAV vectors infused into skeletal muscle or liver have resulted in long-term (>5 years) expression of factor VIII or factor IX in the hemophilic dog model. Administration to skeletal muscle of an AAV vector expressing factor IX in patients with hemophilia B was safe and resulted in long-term expression as measured on muscle biopsy, but circulating levels never rose to >1% for sustained periods, and a large number of IM injections (>80–100) was required to access a large muscle mass.


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InternalMed_Harrison. expression as measured on muscle biopsy, but circulating levels never rose to >1% for sustained periods, and a large number of IM injections (>80–100) was required to access a large muscle mass. Intravascular vector delivery has been used to access large areas of skeletal muscle in animal models of hemophilia and will likely be tested for this and other disorders in upcoming trials.


InternalMed_Harrison

InternalMed_Harrison. The first trial of an AAV vector expressing factor IX delivered to the liver in humans with hemophilia B resulted in therapeutic circulating levels at the highest dose tested, but expression at these levels (>5%) lasted for only 6–10 weeks before declining to baseline (<1%). A memory T cell response to viral capsid, present in humans but not 91e-3 in other animal species (which are not natural hosts for the virus), likely led to the loss of expression (Table 91e-2). In response to these findings, a second trial included a short course of prednisolone, to be administered if factor IX levels began to decline. This approach resulted in long-term expression of factor IX, in the range of 2–5%, in men with severe hemophilia B. Current efforts are focused on expanding these trials, and extending the approach to hemophilia A.


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InternalMed_Harrison. A logical conclusion from the early experience with AAV in liver in the hemophilia trial was that avoidance of immune responses was key to long-term expression. Thus immunoprivileged sites such as the retina began to attract substantial interest as therapeutic targets. This inference has been elegantly confirmed in the setting of the retinal degenerative disease Leber’s congenital amaurosis (LCA). Characterized by early-onset blindness, LCA is not currently treatable and is caused by mutations in several different genes; ~15% of cases of LCA are due to a mutation in a gene, RPE65, encoding a retinal pigment epithelial-associated 65-kDa protein. In dogs with a null mutation in RPE65, sight was restored after subretinal injection of an AAV vector expressing RPE65. Transgene expression appears to be stable, with the first animals treated >10 years ago continuing to manifest electroretinal and behavioral evidence of visual function. As is the case for X-linked SCID, gene transfer must


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InternalMed_Harrison. to be stable, with the first animals treated >10 years ago continuing to manifest electroretinal and behavioral evidence of visual function. As is the case for X-linked SCID, gene transfer must occur relatively early in life to achieve optimal correction of the genetic disease, although the exact limitations imposed by age have not yet been defined. AAV-RPE65 trials carried out in both the United States and the United Kingdom have shown restoration of visual and retinal function in over 30 subjects, with the most marked improvement occurring in the younger subjects. Trials for other inherited retinal degenerative disorders such as choroideremia are under way, as are studies for certain complex acquired disorders such as age-related macular degeneration, which affects several million people worldwide. The neovascularization that occurs in age-related macular degeneration can be inhibited by expression of vascular endothelial growth factor (VEGF) inhibitors such as angiostatin or


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InternalMed_Harrison. people worldwide. The neovascularization that occurs in age-related macular degeneration can be inhibited by expression of vascular endothelial growth factor (VEGF) inhibitors such as angiostatin or through the use of RNA interference (RNAi)-mediated knockdown of VEGF. Early-phase trials of siRNAs that target VEGF RNA are under way, but these require repeated intravitreal injection of the siRNAs; an AAV vector–mediated approach, which would allow long-term inhibition of the biological effects of VEGF through a soluble VEGF receptor, is now in clinical testing.


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InternalMed_Harrison. The majority of clinical gene transfer experience has been in subjects with cancer (Fig. 91e-1). As a general rule, a feature that distinguishes gene therapies from conventional cancer therapeutics is that the former are less toxic, in some cases because they are delivered locally (e.g., intratumoral injections), and in other cases because they are targeted specifically to elements of the tumor (immunotherapies, antiangiogenic approaches). Because cancer is a disease of aging, and many elderly are frail, the development of therapeutics with milder side effects is an important goal.


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InternalMed_Harrison. Cancer gene therapies can be divided into local and systemic approaches (Table 91e-3). Some of the earliest cancer gene therapy trials focused on local delivery of a prodrug or a suicide gene that would increase sensitivity of tumor cells to cytotoxic drugs. A frequently used strategy has been intratumoral injection of an adenoviral vector expressing the thymidine kinase (TK) gene. Cells that take up and express the TK gene can be killed after the administration of ganciclovir, which is phosphorylated to a toxic nucleoside by TK. Because cell division is required for the toxic nucleoside to affect cell viability, this strategy was initially used in aggressive brain tumors (glioblastoma multiforme) where the cycling tumor cells were affected but the nondividing normal neurons were not. More recently, this approach has been explored for locally recurrent prostate, breast, and colon tumors, among others.


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InternalMed_Harrison. Another local approach uses adenoviral-mediated expression of the tumor suppressor p53, which is mutated in a wide variety of cancers. This strategy has resulted in complete and partial responses in squamous cell carcinoma of the head and neck, esophageal cancer, and non-small-cell lung cancer after direct intratumoral injection of the vector. Response rates (~15%) are comparable to those of other single agents. The use of oncolytic viruses that selectively replicate in tumor cells but not in normal cells has also shown promise in squamous cell carcinoma of the head and neck and in other solid tumors. This approach is based on the observation that deletion of certain viral genes abolishes their ability to replicate in normal cells but not in tumor cells. An advantage of this strategy is that the replicating vector can proliferate and spread within the tumor, facilitating eventual tumor clearance. However, physical limitations to viral spread, including fibrosis, intermixed normal


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InternalMed_Harrison. is that the replicating vector can proliferate and spread within the tumor, facilitating eventual tumor clearance. However, physical limitations to viral spread, including fibrosis, intermixed normal cells, basement membranes, and necrotic areas within the tumor, may limit clinical efficacy. Oncolytic viruses are licensed and available in some countries but not in the United States.


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InternalMed_Harrison. Because metastatic disease rather than uncontrolled growth of the primary tumor is the source of mortality for most cancers, there has been considerable interest in developing systemic gene therapy approaches. One strategy has been to promote more efficient recognition of tumor cells by the immune system. Approaches have included transduction of tumor cells with immune-enhancing genes encoding cytokines, chemokines, or co-stimulatory molecules; and ex vivo manipulation of dendritic cells to enhance the presentation of tumor antigens. Recently, considerable success has been achieved using lentiviral transduction of autologous lymphocytes with a cDNA encoding a chimeric antigen receptor (CAR). The CAR moiety consists of a tumor antigen-binding domain (e.g., an antibody to the B cell antigen CD19) fused to an intracellular signaling domain that allows T cell activation. The transduced lymphocytes can then recognize and destroy cells bearing the antigen. This CAR–T cell approach has


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InternalMed_Harrison. CD19) fused to an intracellular signaling domain that allows T cell activation. The transduced lymphocytes can then recognize and destroy cells bearing the antigen. This CAR–T cell approach has proven extraordinarily successful in the setting of refractory chronic lymphocytic leukemia and pre-B-cell acute lymphoblastic leukemia. Infusion of gene-modified T cells engineered to recognize the B cell antigen CD19 has resulted in >1000-fold expansion in vivo, trafficking of the T cells to the bone marrow, and complete remission in a subset of patients who had failed multiple chemotherapy regimens. The cells persist as memory CAR+ T cells, providing ongoing antitumor functionality. Some patients experience a delayed tumor lysis syndrome requiring intensive medical management. This approach also causes an on-target toxicity, leading to B cell aplasia that necessitates lifelong IgG infusions. Current results indicate that long-lasting remissions can be achieved and the strategy can


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InternalMed_Harrison. also causes an on-target toxicity, leading to B cell aplasia that necessitates lifelong IgG infusions. Current results indicate that long-lasting remissions can be achieved and the strategy can theoretically be extended to other tumor types if a tumor antigen can be identified.


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InternalMed_Harrison. Gene transfer strategies have also been developed for inhibiting tumor angiogenesis. These have included constitutive expression of angiogenesis inhibitors such as angiostatin and endostatin; use of siRNA to reduce levels of VEGF or VEGF receptor; and combined approaches in which autologous T cells are genetically modified to recognize antigens specific to tumor vasculature. These studies are still in early-phase testing.


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InternalMed_Harrison. Another novel systemic approach is the use of gene transfer to protect normal cells from the toxicities of chemotherapy. The most extensively studied of these approaches has been transduction of hematopoietic cells with genes encoding resistance to chemotherapeutic agents, including the multidrug resistance gene MDRI or the gene encoding O6-methylguanine DNA methyltransferase (MGMT). Ex vivo transduction of hematopoietic cells, followed by autologous transplantation, is being investigated as a strategy for allowing administration of higher doses of chemotherapy than would otherwise be tolerated.


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InternalMed_Harrison. The third major category addressed by gene transfer studies is cardiovascular disease. Initial experience was in trials designed to increase blood flow to either skeletal (critical limb ischemia) or cardiac muscle (angina/myocardial ischemia). First-line treatment for both of these groups includes mechanical revascularization or medical management, but a subset of patients are not candidates for or fail these approaches. These patients formed the first cohorts for evaluation of gene transfer to achieve therapeutic angiogenesis. The major transgene used has been VEGF, attractive because of its specificity for endothelial cells; other transgenes have included fibroblast growth factor (FGF) and hypoxia-inducible factor 1, α subunit (HIF-1α). The design of most of the trials has included direct IM (or myocardial) injection of either a plasmid or an adenoviral vector expressing the transgene. Both of these vectors are likely to result in only short-term expression of VEGF, which may be


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InternalMed_Harrison. IM (or myocardial) injection of either a plasmid or an adenoviral vector expressing the transgene. Both of these vectors are likely to result in only short-term expression of VEGF, which may be adequate because there is no need for continued transgene expression once the new vessels have formed. Direct injection favors local expression, which should help to avoid systemic effects such as retinal neovascularization or new vessel formation in a nascent tumor. Initial trials of adeno-VEGF or plasmid-VEGF injection resulted in improvement over baseline in angiographically detectable vasculature, but no change in amputation frequency or cardiovascular mortality. Studies using different routes of administration or different transgenes are currently under way.


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InternalMed_Harrison. More recent studies have used AAV vectors to develop a therapeutic approach for individuals with refractory congestive heart failure. In preclinical studies, a vector encoding sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) demonstrated positive left ventricular inotropic effects in a swine model of volume-overloaded heart failure. Results of a phase II study in which vector was infused via the coronary arteries in patients with congestive heart failure demonstrated safety and some indications of efficacy; larger studies are now planned.


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InternalMed_Harrison. This chapter has focused on gene addition therapy, in which a normal gene is transferred to a target tissue to drive expression of a gene product with therapeutic effects. Another powerful technique under development is genome editing, in which a mutation is corrected in situ, generating a wild-type copy under the control of the endogenous regulatory signals. This approach makes use of novel reagents including zinc finger nucleases, TALENs and CRISPR, which introduce double-stranded breaks into the DNA near the site of the mutation and then rely on a donated repair sequence and cellular mechanisms for repair of double-strand breaks to reconstitute a functioning gene. Another strategy recently introduced into clinical trials is the use of siRNAs or short hairpin RNAs as transgenes to knock down expression of deleterious genes (e.g., mutant huntingtin in Huntington’s disease or genes of the hepatitis C genome in infected individuals).