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Lysosomal Disorders of the BrainRecent Advances in Molecular and Cellular Pathogenesis and Treatment$

Frances Platt and Steven Walkley

Print publication date: 2004

Print ISBN-13: 9780198508786

Published to Oxford Scholarship Online: September 2009

DOI: 10.1093/acprof:oso/9780198508786.001.0001

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Cell-mediated delivery systems

Cell-mediated delivery systems

(p.339) Chapter 14 Cell-mediated delivery systems
Lysosomal Disorders of the Brain

Kostantin Dobrenis

Oxford University Press

Abstract and Keywords

Diseases that involve the central nervous system (CNS) pose one of the most difficult challenges in human therapy. Cell-mediated therapy (CMT) is a uniquely complex and powerful approach that offers an unparalleled advantage. This chapter reviews work in the area of CMT with a bias towards understanding the critical mechanisms that underlie successful CMT for CNS storage disease. It highlights the advantages offered by employing cells as the therapeutic agent.

Keywords:   central nervous system, cell-mediated therapy, lysosomal storage disorders, CMT


Diseases that involve the central nervous system (CNS) pose one of the most difficult challenges in human therapy. Cell-mediated therapy (CMT) is a uniquely complex and powerful approach that offers an unparalleled advantage. The use of a cell as the therapeutic agent provides a multifaceted tool that can be utilized to deal with the multiple hurdles of CNS treatment and to achieve efficacious treatment by incorporating several complementary mechanisms. While the promise of ‘stem cells’ for therapy has drawn much attention in recent years, there is already an extensive record of varied cell-mediated approaches tested in the context of CNS storage diseases. In vitro studies have helped us recognize the ability to obtain ‘cross-correction’ through secretion and uptake from cells expressing lysosomal enzyme to enzyme-deficient cells, and pointed to other potentially valuable mechanisms (Chapter 13). Many in vivo studies on animal models (Chapter 11) have been performed utilizing approaches that include the use of haematopoietic cells that can migrate into the CNS or the introduction of other cell types directly into the CNS. Genetic manipulation has been used to improve the corrective power of cells to be delivered (Chapter 16), and in vivo gene replacement has revealed the importance of transduced resident cells cross-correcting their neighbours. Finally, the outcome of therapeutic studies has also contributed to our understanding of pathogenetic mechanisms, and hence provided additional ideas on how to use CMT effectively. Below, the work in this area is reviewed with a bias towards understanding the critical mechanisms that underlie successful CMT for CNS storage disease and highlights the advantages offered by employing cells as the therapeutic agent.

Basic principles of cell-mediated therapy

A cell-mediated approach to therapy may address storage disorders in more than one fundamental way. The first is to replace or compensate for defective cell populations with normal equivalents. If this is done early enough, or if there is sufficient delivery of healthy populations, normal tissue or organ function might be recovered. Examples of this approach include bone marrow transplantation (BMT) and the use of neural progenitor cells.

A second way to obtain therapeutic benefit can be through ‘cross-correction’. The nature of storage diseases makes them particularly amenable to this. In this scenario, deficient cells acquire lysosomal enzyme from cells that produce active enzyme (Chapter 13). This primarily relies on two cellular mechanisms: the ability of cells to secrete functional soluble lysosomal enzymes and the existence of natural vesicular endocytic pathways that can deliver (p.340) extracellular constituents to lysosomal compartments. In this way, ‘donor’ or ‘supply’ cells releasing enzyme can be utilized in situ as a chronic provider of extracellular enzyme for subsequent capture. Greater levels of secretion, which can be genetically engineered, will produce a higher extracellular concentration and result in proportionately greater enzyme uptake by neighbouring deficient cells through constitutive fluid-phase endocytosis. In addition, through diffusion, greater local secretion may provide effective concentrations of extracellular enzyme to more distal, deficient cells.

The type of endocytic mechanism involved will also play a significant role, essentially as discussed in the context of enzyme replacement therapy (ERT) (see Chapter 13). While enzymes will be taken up by fluid-phase endocytosis and in part delivered to lysosomes, far superior results are obtained if the target cells express surface receptors, or acceptors, able to recognize the secreted enzyme. Through capture by high-affinity binding sites on the plasma membrane, the efficiency of enzyme uptake is enhanced, and therapeutic efficacy may be achieved with extracellular concentrations that are insufficient to be effective using fluid-phase endocytosis. Cell-mediated therapy is unlikely to produce the high extracellular enzyme concentrations obtained, at least initially, after enzyme infusion in ERT. Thus, the extent of enzyme replacement achieved through fluid-phase endocytosis may be quite limited for CMT. Conversely, lower extracellular concentrations would not probably saturate receptor-mediated pathways and hence be fully subject to improved uptake by this mechanism, arguing that this may be a critical component of successful CMT.

Thirdly, CMT may produce improvement through indirect depletion of storage substrate, a mechanism that has not been adequately studied. Cells expressing lysosomal enzymes could take up and catabolize circulating extracellular substrate. This would then reduce the burden on those enzyme-deficient cells that are accumulating substrate, at least in part, through endocytosis. The potential impact of this mechanism would depend on the specific disease, as significant levels of extracellular substrate are not always present.

Finally, the mechanisms employed in CMT must be considered with respect to anatomic variables. For example, if cross-correction is pursued, deficient cells must have reasonable access to the enzyme. The extent of cellular involvement, organ cytoarchitecture, and fluid dynamics can all influence this need. Thus, appropriate placement and distribution of supply cells can become a critical factor. This is especially the case for treating the CNS which also imposes a barrier to circulating enzyme, as discussed below.

The challenges of the CNS and how CMT can address them

Successful therapy must effectively address a number of challenges posed by the CNS. Introduction of the therapeutic tool must deal with the blood–brain barrier (BBB) which prevents the entry of lysosomal enzymes and DNA. The BBB is created by formation of tight junctions between and low rates of transcytosis in the specialized endothelial cells of the CNS (Bradbury 1993; Janzer 1993; Staddon and Rubin 1996). Beyond this, additional barriers are present. Perivascular spaces are patrolled by phagocytes that can sequester foreign compounds (Kida et al. 1993) and by cells that can activate an immune response (reviewed in Dobrenis 1998). Furthermore, layers of basal laminae, macromolecular meshes of protein, lie between the vasculature and CNS parenchyma (Wolburg and Risau 1995). After this, the glia limitan, a sheath made up of the end feet of astroglia and microglia, forms another physical barrier (Lassmann et al. 1991). Finally, solutes beyond the endothelial barrier are also subject to an outward bulk fluid flow through contiguous spaces. Interstitial fluid from the (p.341) CNS parenchyma moves partly into the ventricular system and perivascular spaces, and subsequently solutes are carried to the subarachnoid spaces from which soluble constituents return to the bloodstream (Brightman 1965; Wood 1980; Rennels et al. 1985; Cserr 1988; Rattazzi and Dobrenis 1991 a). Thus, multiple barriers exist that limit and regulate the entry of elements from the blood circulation. While methods to temporarily open the BBB have been used to introduce enzymes or gene constructs (see Chapters 13 and 16), they do not sufficiently address the other barriers. On the other hand, appropriate cell types offer the ability to not only cross the endothelial barrier from the blood, but to actively migrate into the parenchyma and take up residence within the milieu of target diseased cells. As the CNS is extensively invested in vasculature, this route is actually attractive as it can facilitate widespread delivery of cells to address the global involvement present in CNS storage disease. As will be detailed later, cells of haematopoietic origin provide these capabilities. Some of the same hurdles are faced with intraventricular or intra-subarachnoid injections, which result in limited parenchymal entry of macromolecules unless attempts to alter fluid flow are made (Rattazzi and Dobrenis 1991a, 1991b; Ghodsi et al. 1999). Once again, successful results with cells introduced into the ventricular system (e.g. Snyder et al. 1995) suggest migratory capability, and appropriate extrinsic cues allow a cell-mediated approach to be a more effective delivery mechanism also through this route.

The compendium of barriers could be largely circumvented by directly injecting therapeutic agents into the CNS parenchyma. It is also fortuitous that foreign agents are better tolerated immunologically within the CNS relative to most other tissues and organs. Nevertheless, this invasive approach must contend with cellular destruction and BBB compromise, and subsequent deleterious cellular responses that can include inciting a greater immune response. These consequences might be argued acceptable and manageable if the therapeutic strategy is worthy. Focal introduction is a reasonable approach in diseases such as Parkinson's where sites of pathology are relatively contained, but appears inadequate in the face of global CNS involvement seen in storage diseases. However, even with this method of introduction, the active migration of injected cellular agents compared to macromolecular agents can offer an improved penumbra of therapeutic targeting.

Once extracellular enzyme is made available, one is faced with issues regarding the endocytic behaviour of neural cells. For neurones, a key target for therapy, endocytic pathways leading to the lysosomal compartment are poorly defined but likely to be complex and to arise only from specific membrane regions, given the polarized nature and functions of neurones (Broadwell et al. 1980; Sudhof and Jahn 1991; Parton et al. 1992; Yin and Yang 1992; Kelly 1993; Nixon and Cataldo 1995; Overly and Hollenbeck 1996; Buckley et al. 2000). It is also evident that constitutive fluid-phase endocytosis leading to accumulation of extracellular compounds in neurones is very low as compared to other cell types (Klatzo and Miquel 1960; Rattazzi et al. 1987; Ohata et al. 1990; Dobrenis et al. 1992; Matteoli et al. 1996; Kyttala et al. 1998; Rattazzi and Dobrenis 2001). The potential for appropriately localized and chronic supply offered by CMT may help in dealing with these problems. However, CMT, like ERT, will be most effective if suitable acceptors/receptor systems for neural uptake are better defined and appropriately utilized.

The additional potential of CMT to substitute normal cells for affected cell populations may be particularly useful in diseases involving severe compromise of cells, such as oligodendrocytes in Krabbe disease or where neuronal death is rampant as in the neuronal ceroid lipofuscinoses. Early intervention can permit normal neural progenitors to partake in ongoing developmental pathways fostering delivery to appropriate anatomic locations and (p.342) to compete with endogenous equivalents for survival during normal programmed cell death. Growing recognition that the adult mammalian CNS harbours considerable turnover of neural populations, including some neuronal types, from intrinsic stem or progenitor cells indicates that permissive conditions also exist post development for neural replacement. The challenge that the CNS presents for this approach lies in the extreme complexity of cellular specialization and interactions found in the nervous system, and the difficulty in substantiating that genuine integration of exogenous populations has been obtained.

Cross-correction in a dish

Early seminal and follow-up studies laid what we can now view as a cornerstone in CMT for storage diseases: the concept of cellular metabolic cross-correction (Fratantoni et al. 1968; Neufeld and Fratantoni 1970; Bach et al. 1972; Hickman and Neufeld 1972; Kresse and Neufeld 1972; von Figura and Kress 1972, 1974; Kaplan et al. 1977; Neufeld et al. 1977; Rome et al. 1979; Vladutiu and Rattazzi 1979; Neufeld 1991) (Chapter 13). Studies primarily using fibroblasts demonstrated that upon co-culturing with enzyme-expressing cells, activity became detectable within enzyme-deficient cells and could lead to catabolism of storage substrates, provided that the protein was not an integral or tightly bound membrane component. That secretion and subsequent endocytosis was involved was supported further by the observation that incubation with conditioned medium from donor cell cultures could also correct enzyme-deficient cells. Uptake of several enzymes was found to depend heavily on receptor-mediated endocytosis obtained through recognition of secreted enzyme-bearing phosphohexosyl moieties by plasma membrane mannose-6-phosphate (M6P) receptor. The critical importance of this receptor to realizing effective enzyme delivery for numerous cell types is now widely accepted (Chapter 6). Subsequent studies demonstrated that similar enzyme transfer phenomena could occur with other cell types as donors, such as peripheral macrophages or lymphocytes (Jessup and Dean 1982; McNamara et al. 1985; Olsen et al. 1993), pertinent to BMT as a therapeutic approach. More recent research has confirmed that transfer of enzymes is possible also between initially deficient fibroblasts when transgenes are inserted to convert deficient cells into supply cells (Taylor and Wolfe 1994; Francesco et al. 1997; Sangalli et al. 1998; Sun et al. 1999; Sena-Esteves et al. 2000; Teixeira et al. 2001).

A series of early studies revealed that efficient transfer of lysosomal enzymes to fibroblasts could be obtained by another mechanism referred to as ‘contact-dependent’ or ‘direct’ enzyme transfer. This transfer, largely explored using lymphocytes as donors and fibroblasts as recipients, was not inhibited by the addition of exogenous competing M6P to block recep-tor-mediated uptake, and the magnitude of transfer could not be replicated by using only conditioned medium from donor cells or when donor and recipient cells were physically separated by a membrane permeable to soluble enzyme (Olsen et al. 1981, 1982, 1983, 1986; Abraham et al. 1985). The full details of this mechanism are lacking, but secretion and endocytosis, possibly fluid-phase, are probably still responsible, as membrane-bound lysosomal enzymes and cytosolic enzymes were not found to be transferred in these studies (Olsen et al. 1983). The apparently high transfer efficiency may have been achieved through the benefit of close proximity (Olsen et al. 1986; Abraham et al. 1988), yielding enhanced extracellular enzyme concentrations within the micro-environment between cells. Indeed, the total amount of secreted enzyme that can be measured in recovered culture medium from the type of lymphocytes used is very low-to-undetectable yet ‘direct’ transfer substantial (Olsen et al. 1982, 1983, 1993; Sangalli et al. 1998). In a case where secreted enzyme was measurable (p.343) in 2-day direct co-cultures, the total amount of aspartylglucosaminidase transferred to deficient fibroblasts was three times that found in the extracellular fluid (Enomaa et al. 1995). Two additional observations provide further insight. Studies on direct transfer of acid α-D-mannosidase, which produced significant reduction of mannose-terminal oligosaccharides in deficient fibroblasts (Abraham et al. 1985), revealed that cell contact heightened the synthesis of enzyme by donor cells (Olsen et al. 1988). Second, microscopy analyses on β-glucuronidase transfer suggested that cell contact may have enhanced the rate of pinocytosis in recipient fibroblasts (Olsen et al. 1986). Collectively, the studies on direct transfer suggest added merit to employing cells to deliver enzyme within a target organ and argue that the specific cellular distribution and localization in the target tissue might play a significant role in the efficacy of CMT. This is not to say that diffusion of secreted enzyme followed by M6P receptor-mediated uptake is unimportant, nor that this and direct transfer are self-exclusive, as plasma cells (Olsen et al. 1993) and macrophages (McNamara et al. 1985) have been shown to correct fibroblasts through both mechanisms.

In comparison to fibroblasts, cross-correction studies on neural cells have been limited, given the greater difficulty of culturing them and the relative scarcity of suitable neural cell lines. Nevertheless, experiments suggest that the three major CNS lineages, neurones, astrocytes, and oligodendrocytes, are not so fundamentally different and are amenable to uptake of cell-secreted enzyme and able to serve as supply cells. For example, primary neuronal cell cultures have been shown to internalize β-hexosaminidase A (Flax et al. 1998), α-L-iduronidase (Stewart et al. 1997), aspartylglucosaminidase (Kyttala et al. 1998), and β-glucuronidase (Kosuga et al. 2001) either following co-culture with or exposure to conditioned medium from enzyme-secreting cells. The majority of uptake was compatible with M6P receptor-mediated endocytosis assessed in the latter two studies. Using neural cells from a Tay–Sachs mouse model, evidence for reduction of ganglioside storage was also provided (Flax et al. 1998). Transfer of human enzyme to rodent neurone-like cell lines has also been demonstrated (Enomaa et al. 1995; Stewart et al. 1997; Kyttala et al. 1998). The use of neuronal cell lines facilitates in vitro testing, but results must be interpreted cautiously, given most lines generate poorly differentiated neurons. Dramatic differences in the extent of enzyme uptake have been found, at least for α-L-iduronidase and aspartylglucosaminidase, between primary neurons and Neuro2a (Stewart et al. 1997) or PC12 and N18 (Kyttala et al. 1998) cell lines. Examples of enzyme transfer to astroglial and oligodendroglial lineage cells have been reported for arylsulfatase A (Sangalli et al. 1998; Matzner et al. 2000a, 2001; Muschol et al. 2002), aspartylglucosaminidase (Enomaa et al. 1995), α-L-iduronidase (Stewart et al. 1997), galactocerebrosidase (Luddi et al. 2001), and β-hexosaminidase A (Flax et al. 1998) and included evidence supporting lysosomal substrate catabolism (Flax et al. 1998; Matzner et al. 2000a, 2001). In addition, co-culture with secreting fibroblasts was found to correct an abnormal morphologic phenotype displayed by oligodendrocytes cultured from a mouse model of Krabbe disease (Luddi et al. 2001). Uptake of secreted arylsulfatase A and α-L-iduronidase by astrocyte-like cell lines 17−/− A1 (Matzner et al. 2001) and C6 (Stewart et al. 1997), respectively, was largely inhibited by excess free M6P, suggesting receptor-mediated endocytosis.

Relevant to strategies involving replacement of diseased neural cells with enzyme-express-ing equivalents, co-culture studies have tested the capacity of multipotent neural progenitor, astroglial lineage, and oligodendroglial lineage cells to transfer enzyme. Neural stem cells, including ones from human telencephalon, produced measurable reduction of ganglioside storage in Tay–Sachs disease brain cells after 10 days of co-culture without direct contact (p.344) (Flax et al. 1998). Neural progenitors overexpressing galactocerebosidase led to almost complete cross-correction of substrate-loaded fibroblasts from twitcher mice after 3 days in co-culture (Torchiana et al. 1998). Galactocerebosidase transfer has also been demonstrated from astroglia and oligodendroglial progenitors to their respective counterparts, obtained from twitcher mice, in 4-day co-culture studies (Luddi et al. 2001).

In summary, the cell-culture studies demonstrate the principle of cross-correction, yielding evidence of both enzyme transfer to and depletion of storage in diseased cells. In the absence of the many uncontrolled variables faced in vivo, they are able to reveal and quantify specific mechanisms that support the potential of the CMT approach. However, it is difficult to accurately extrapolate these findings to outcome in vivo, given inevitable extrinsic differences. Biologically active factors absent from the culture system may well modulate phenomena underlying enzyme transfer. In addition, cell-culture studies do not take into account fluid volume and flow dynamics of intact tissue. The typically large extracellular volume to cell ratio in culture may lead to some underestimation of potential long-distance enzyme transfer by diffusion, while the relatively stagnant fluid flow in vitro may favour overestimation of transfer between cells in closer proximity than possible in vivo. It should also be noted that co-culture studies have rarely used less than a 1:1 ratio of supply-to-demand cells and typically required arguably unrealistic ratios to obtain meaningful cross-correction. Only in rare cases, lower ratios did result in substantial impact by using transgenic ‘hyper-secreting’ cell lines as donors for neural cells (e.g. Luddi et al. 2001). Thus in addition to establishing principle, culture studies argue the importance of maximizing supply-cell number, enzyme release, and efficiency of uptake.

Secretion studies in vitro

Variables of secretion

While few examples of mammalian cells specialized to actively release lysosomal enzymes exist (Holtzman 1989), numerous cell types examined in culture studies appear to release or leak at least modest amounts. Studies with skin fibroblasts have led to a view that this release is the consequence of a default exocytic pathway. Partially processed yet active enzymes from Golgi and subsequent endosomal compartments diverge from their lysosomal destination and are released to the extracellular fluid. This is supported by the common finding of intermediate rather than lysosomally mature forms in extracellular fluids and fortunately for cross-correction, this can include enzyme bearing M6P moieties. However, release of enzymes from the lysosome proper can also occur even from fibroblastic and other cells not commonly viewed as secretory, following stimuli eliciting increases in intracellular Ca++ (Koenig et al. 1978; Rodriguez et al. 1997). Several cell types derived from the haematopoeitic system, and so relevant to BMT-mediated therapy, may possess specialized ‘secretory’ lysosomal compartments also subject to regulated exocytosis (Griffiths 1996; Claus et al. 1998). The relatively high release of enzyme that can be obtained from macrophages (Schnyder and Bagglioni 1978) is subject to Ca++ levels (Schneider et al. 1978; Ho and Klempner 1985; Griffiths 1996; Rodriguez et al. 1997) and may involve direct lysosome extrusion or premature phagosome–lysosome fusion (Schnyder and Bagglioni 1978; Griffiths 1996; Holtzman 1989).

Reported values for secretion of different enzymes or by different cell types widely vary. (In very general terms, the amount released in a period of 24 hr often corresponds to 10% (p.345) or less of that found within the cells.) It can be argued that experimental diversity, varying cell lysis, dissimilar reuptake, and varied instability of extracellular enzymes all contribute to exaggeration of actual secretion differences. Nonetheless, sufficiently controlled studies indicate that genuine heterogeneity in this regard does exist between cell types. Differing rates of enzyme production affecting levels released and alternative compartmental modes of release, as discussed above, no doubt contribute to this. The details of intracellular trafficking pathways may also play a role. For example, lymphocytes which release lower amounts of several lysosomal enzymes than fibroblasts (Olsen et al. 1982, 1983, 1993; Sangalli et al. 1998) also appear to rely to a greater extent on M6P-independent pathways for lysosomal delivery (DiCioccio and Miller 1991; Glickman and Kornfeld 1993) than fibroblasts (Ludwig et al. 1994; Dittmer et al. 1999). In the genetic absence of M6P-mediated trafficking, additional differences become apparent in the use of alternate pathways and release of cathepsin D between thymocytes, hepatocytes, and fibroblasts (Dittmer et al. 1999). Differences in trafficking also exist between varied lysosomal enzymes (see Chapter 6) and this could contribute to a ranging enzyme secretion profile for an individual cell type. Furthermore, studies with a macrophage cell line suggest that differential release may relate to the differential distribution of lysosomal enzymes in endosomal–lysosomal compartments (Claus et al. 1998). Finally, despite the ‘housekeeping’ view of lysosomal enzymes, studies show that extrinsic conditions can substantially affect secretion. For example, B-cell maturation (Olsen et al. 1993) or macrophage activation can lead to enhanced release of lysosomal enzymes (e.g. Davies et al. 1974; Schnyder and Bagglioni 1978; Dean et al. 1979; Jessup and Dean 1980; Petanceska et al. 1996; Liuzzo et al. 1999b). In summary, these observations indicate that the potential of CMT for storage disorders cannot be generalized in that the choice of supply cell type, the particular enzyme involved, and the ambient disease-induced conditions can all have deterministic influence on the level of secretion, and thus each disease may demand appropriate modification of strategic elements. Studies with cells particularly relevant to the CNS further support this, as described next.

Secretion by microglial and neural cells

Microglia, the parenchymal macrophages of the CNS, and several related brain macrophages found in perivascular, meningeal, and ventricular regions, have been shown to arise from donor cells in BMT studies. As potential enzyme donors, these cells are promising since they are relatively rich in lysosomes and lysosomal enzymes (e.g. Rio-Hortega 1932; Davidoff and Galabov 1976; Ling 1976, 1977; Peters et al. 1991; Banati et al. 1993; Nakajima and Kohsaka 1993; Petanceska et al. 1996) and are commonly viewed as highly secretory, releasing a wide variety of compounds (Dobrenis 1998). Most importantly, reported studies have verified that microglia have the capacity to release a number of acid glycosidases (Dobrenis et al. 1994, 1996) and cathepsins (Petanceska et al. 1996; Liuzzo et al. 1999a,b; Muschol et al. 2002). Little is known about the oligosaccharide structure of microglial secreted enzymes, but cathepsin D released by a murine microglial line was phosphate poor (Muschol et al. 2002).

As discussed for other cells, the amount of activity accumulating in culture medium of microglia/brain macrophages can widely differ between enzymes. For example, in cultures of purified cat microglia (Dobrenis 1998), enzyme activity accumulating in the extracellular fluid was found to be strikingly substantial for α-mannosidase and minimal for β-hexosaminidase and β-galactosidase (Dobrenis et al. 1994). In 24 h incubations, these amounted (p.346) to 300, 40, and 4 nmol substrate cleaved/h/1 × 106 cells. This difference was not simply due to differences in substrate-specific kinetics. Taken as a percentage of intracellular activity of the respective enzymes, values corresponded to 30% for α-mannosidase and 1% for β-hexosaminidase and β-galactosidase, implying differences in relative secretion (Dobrenis et al. 1996). Beyond providing another example of enzyme-specific variance, the significance of these differences is made clearer when the potential demand of target cells is considered. The β-hexosaminidase activity measured by standard assay methods is prominently higher in many cells and tissues, compared to α-mannosidase, β-galactosidase, and others, and this appears to be true also for neurones and macroglia (Raghavan et al. 1972; Bradel and Sloan 1988; Hirayama et al. 2001). Thus, with supply and demand considered together, the findings argue that there would be far greater potential for effective cross-correction in α-mannosidosis than for GM2 gangliosidosis in particular. Indeed, in vivo findings support this as will be discussed later. There is also evidence for relevant differences between microglia from different animal species. For example, comparison of mouse and cat microglia showed that for β-hexosaminidase, the ratio of extracellular to intracellular activity after 24 h was dramatically different. In contrast to near zero for the cat, it was over 10% for mouse microglia, and the absolute extracellular activity measured was almost two orders of magnitude higher in mouse cultures (Dobrenis et al. 1996). Further studies revealed that the apparent differences in release between cat α-mannosidase, β-galactosidase, and β-hexosaminidase, and between mouse and cat β-hexosaminidase may not simply be due to rates of secretion. Instead, assessments on enzyme in the culture medium suggest that the differences can at least in part be explained by differences in stability of the lysosomal enzymes at 37 °C in the physiological neutral pH of extracellular fluid (Dobrenis et al. 1996). This raises the point that xenogeneic selection of lysosomal enzymes may offer advantages to transgenic CMT strategies. The apparent release of murine compared to human β-galactosidase was also strikingly higher when the two were transgenically expressed in fibroblasts. The reason for this remained unclear and did not appear related to mRNA levels, but stability in the medium was not investigated (Sena-Esteves et al. 2000). Stability can also prove to be the same as demonstrated for rat versus human glucuronidase secreted by fibroblasts (Taylor and Wolfe 1994), again underscoring that enzymes must be weighed on an individual basis.

Considering the fact that microglia are highly responsive to pathologic conditions, transforming into activated macrophages (reviews, Moore and Thanos 1996; Streit et al. 1999), the possibility that enzyme release may be both significantly and differentially affected by ambient disease conditions should not be ignored. Upregulation of some agents active on microglia were noted in the CNS of a Sandhoff mouse model (Wada et al. 2000). Initial culture studies with physiological factors that may be elevated under disease conditions, granulocyte–macrophage colony stimulating factor and macrophage colony stimulating factor (M-CSF), have shown significant effects can transpire. Selective alterations in intracellular and extracellular levels of α-mannosidase, β-glucuronidase, and β-hexosaminidase were found in cultures of murine primary microglia and immortalized microglia. The most striking was a 100-fold increase in secreted α-D-mannosidase activity following M-CSF treatment (Earley et al. 1999). Other studies also show modulation by relevant factors. Intracellular increases in acid phosphatase levels in primary rodent microglia were found following M-CSF-mediated activation (Sawada et al. 1990) and neurotrophin stimulation (Nakajima et al. 1998). Similarly, intracellular and secreted levels of cathepsin S increased with basic fibroblast growth factor treatment of a murine microglial line (Liuzzo et al. 1999a). Microglial activation can also lead to reduction of (p.347) intracellular levels, as demonstrated for β-hexosaminidase (Beccari et al. 1997) and for cathepsins S, B, and L (Liuzzo et al. 1999b) in microglial cell lines exposed to lipopolysaccharide (LPS) and to LPS or selected cytokines, respectively. This does not necessarily mean that secretion is reduced, as in the latter case, the same cytokines enhanced cathepsin S secreted levels (Liuzzo et al. 1999b). We are far from fully understanding the concert of cellular mechanisms that underlie such differential changes in microglia, but they probably operate on more than just the transcriptional level (Liuzzo et al. 1999b) and hence will also be relevant to enzymes expressed by inserted transgenes with foreign promoters.

Culture studies have shown that secretion of acid hydrolases is detectable from normal neuroectodermal lineage cells (Lacorraza et al. 1996; Taylor and Wolfe 1997a; Flax et al. 1998; Heuer et al. 2001; Luddi et al. 2001; Buchet et al. 2002; Fu et al. 2002). The data are too limited to comment reliably on how neural cells compare to non-neural cells in this capacity. Nevertheless, the evidence supports the idea that cross-correction can also add to the benefits obtained through transplantation of neural cells or in vivo gene replacement targeted at neural cells.

Enhancement of secretion

Secretion of lysosomal enzymes has been enhanced through genetic manipulation, commonly by ‘overexpression’. Increased intracellular levels in a given cell type attained through extranumerary genes in normal and/or strong promotor-driven genes in mutant cells produce greater extracellular accumulation and often do so in a roughly proportional manner—more expression yields more secretion. Overexpression of enzymes normally bearing M6P do not appear to overwhelm the receptor, and other lysosomal enzymes are not by default also secreted at higher levels. While overexpression may result in some alterations in post-translational modifications, a significant portion of secreted enzyme retains M6P moieties (Ioannou et al. 1992; Francesco et al. 1997; Huang et al. 1997; Stewart et al. 1997; Matsuura et al. 1998; Sun et al. 1999; Sena-Esteves et al. 2000; Muschol et al. 2002). Transgenic expression has resulted in a wide range of enhanced intracellular enzyme values, from matching equivalent normal values to even 100-fold enhancements, and similarly varied improvements in secretion have been reported. For example, in studies relevant to CNS therapy, deficient bone marrow cells transduced in vitro produced extracellular α-L-iduronidase activity 10-fold greater than normal cells (Fairbairn et al. 1996), while ones transduced with a α-galactosidase A gene at best matched that secreted by normal cells (Takenaka et al. 2000). MPS VII fibroblasts transduced with a double-copy vector secreted up to 10 times the normal amount of glucuronidase (Wolfe et al. 1995), and 30-fold more acid sphingomyelinase secretion was obtained when mouse bone-derived mesenchymal stem cells expressing normal murine enzyme were transduced with DNA encoding the human enzyme (Jin et al. 2002). A striking elevation of secreted activity, approximately 100-fold greater than normal, was obtained with human β-glucuronidase expression in amniotic epithelial cells (AECs) derived from normal rats (Kosuga et al. 2001). Neural cells have also been transduced with appropriate constructs, yielding replacement or enhancement of enzyme expression (Torchiana et al. 1998; Enomaa et al. 1995; Luddi et al. 2001). When secretion was analysed in transduced neural progenitor cells from MPS VII mice, it was found to be double that of normal cells, and secretion continued following differentiation into progeny that included neurones (Heuer et al. 2001). Expression of human β-hexosaminidase α-subunit in murine progenitor cells yielded similar or better results in one (p.348) subclone (Lacorazza et al. 1996). Transduction of primary brain cell cultures from MPS VII (Taylor and Wolfe 1997a) or from MPS IIIB (Fu et al. 2002) mice yielded secretion similar to analogous cultures from normal mice. Secretion by neurones of overexpressed palmitoyl protein thioesterase, deficient in infantile neuronal ceroid lipofuscinosis, has also been reported (Heinonen et al. 2000). Notably, transduction of normal human neural progenitor cells with an additional human glucuronidase copy resulted in almost 100-fold increase in secretion, if cells were switched to media promoting differentiation of the cells (Buchet et al. 2002). Thus, significant elevation of secreted enzyme by these methods bring us closer to achieving enzyme replacement with realistic numbers of donor cells in CMT. Based on results of secretion of transgenic overexpressed glucuronidase, it was speculated that 2 × 109 cells would be sufficient to attain corrective enzyme levels in a young child (Taylor and Wolfe 1994). It is also important to point out that it is possible to obtain normal or enhanced levels of secretion with transgene expression even when corresponding mutant protein subunits are present (e.g. Taylor and Wolfe 1994; Teixera et al. 2001), further qualifying the utility of the technique as an in vivo and ex vivo CMT strategy. In addition, in the case of an enzyme produced by two subunits coded by different genes, such as β-hexosaminidase A, over-expression of one gene can fortuitously be accompanied by upregulation of the second (Lacorazza et al. 1996; Martino et al. 2002), although the ability to cross-correct is greatly heightened by concomitant overexpression of the second gene (Guidotti et al 1999).

Given that many enzymes are shuttled to lysosomes via M6P receptors, another approach to enhance secretion is through engineering reduction in phosphate-bearing residues, simulating the condition found in I-cell disease where many enzymes are uncontrollably released. This was done by transgenic expression of a mutant form of arylsulfatase A lacking M6P residues and produced approximately twice as much secretion as that obtained with the normal enzyme when expressed in an otherwise enzyme-deficient astrocytoma cell line. However, this sacrifices the benefit provided by M6P receptor-mediated endocytosis. Indeed, the secreted mutant enzyme was four-fold less effective than wild type in cross-correcting deficient astrocytoma cells (Matzner et al. 2001). Finally, CMT strategies employing in vitro or ex vivo gene insertion can benefit from selection techniques geared to isolate favourable stable subpopulations. For example, fluorescent substrate-based vital cell sorting in vitro, or following a period of in vivo graft survival, has been used to select optimal transduced subpopulations for subsequent transplantation (Lorinez et al. 1999).

Uptake and storage depletion

Our understanding of available receptor pathways in neural cells for effective uptake is relatively limited yet critically important to pursue, particularly for neurones which have relatively low rates of fluid-phase endocytosis leading to lysosomes. Several studies do indicate that neurones can express M6P receptor (Lesniak et al. 1988; Nielsen and Gammeltoft 1990; Nielsen et al. 1991; Couce et al. 1992; Dore et al. 1997; Stewart et al. 1997; Kyttala et al. 1998), and cell-culture studies have reported evidence of M6P-mediated uptake in primary neuronal cultures of β-glucuronidase (Kosuga et al. 2001) and aspartylglucosaminidase (Kyttala et al. 1998). Little-to-no enzyme uptake was observed when competed with free M6P sugar. However, these and other studies (Nielsen and Gammeltoft 1990; Nielsen et al. 1991; Dore et al. 1997) reporting binding to and endocytosis of ligands by the M6P/IGFII receptor have used cultures of relatively immature neurones. It remains unclear the extent to (p.349) which mature neurones express the receptor on the surface. Studies have indicated that levels decline with development and may be highly downregulated in the adult (Ocrant et al. 1988; Stewart et al. 1997; Walter et al. 1999), though contrasting evidence has also been reported for some neuronal populations (Couce et al. 1992; Kyttala et al. 1998).

Astrocytes also can express M6P receptor (Ocrant et al. 1988; Stewart et al. 1997) and this may be low or restricted to specific subpopulations in the adult brain (Ocrant et al. 1988; Couce et al. 1992; Walter et al. 1999). Upregulation of the receptor in astrocytes has been found in lesioned brain and whether similar events may occur in storage disease may be worth investigating (Walter et al. 1999). Culture studies show that substantial uptake of lysosomal enzymes via M6P moieties can be obtained in astrocytes or in astroglial-like cell lines (Kiess et al. 1989; Kessler et al. 1992; Stewart et al. 1997; Muschol et al. 2002), and provide evidence for subsequent lysosomal localization (Hill et al. 1985; Matzner et al. 2001). Available receptor for enzyme uptake in oligodendrocytes is probably low at best (Luddi et al. 2001).

The mannose receptor is expressed by astrocytes as well as by microglia and can mediate efficient internalization of mannosylated ligands in these cells (Burudi et al. 1999; Linehan et al. 1999; Marzolo et al. 1999). Expression of the receptor is subject to modulation by numerous agents such as cytokines that may be elevated under pathologic conditions and hence may prove to contribute differentially in varied storage diseases. If enzyme-donor cells like microglia secrete phosphate-poor enzyme as shown in some cases, it is conceivable that deficient microglia and astrocytes will still effectively capture it (Matzner et al. 2001; Muschol et al. 2002).

A greater understanding of the availability of these receptors and other possible glycosyl receptors (Naoi et al. 1987; Jenkins et al. 1988) that are available in CNS cells for enzyme uptake will be valuable, as will further exploration of novel approaches to enhance uptake as shown with incorporation of tetanus toxin C fragment or TAT peptides (Dobrenis et al. 1992; Xia et al. 2001). In doing so, the actual extent of lysosomal delivery should not be ignored. While there is considerable evidence that M6P receptors will send extracellular enzyme to storage-laden lysosomes, quantitative assessments of enzyme replacement typically measure total cellular activity and do not provide a direct evaluation of activity localized to lysosomes. Given the relative unfamiliarity with such pathways in neurones and the complexity of neuronal vesicular trafficking (Sudhof and Jahn 1991; Parton et al. 1992; Kelly 1993; Nixon and Cataldo 1995; Overly and Hollenbeck 1996; Nakata et al. 1998; Buckley et al. 2000), it remains important to verify the degree to which internalized enzymes accumulate in the lysosomal compartment. A sobering and surprising example comes from recent cross-correction studies with Tay–Sachs fibroblasts. Despite the majority of uptake accounted for by M6P receptor mediation, only a small proportion of internalized enzyme was localized to lysosomal fractions, and degradation of exogenously loaded radioactive ganglioside was undetectable (Martino et al. 2002). This raises another point. Cell-culture studies have often employed normal cells and/or cells exogenously loaded with ‘storage’ substrate as test subjects. We cannot reliably conclude that uptake and storage depletion will be similarly achieved in diseased neural cells. Alteration in endocytic membrane traffic using lipid probes has been reported for a number of sphingolipid storage disorders (Pagano et al. 2000; Marks and Pagano 2002), and the presence of some substrates may affect the formation of lysosomes and their intravesicular environment (Schmid et al. 1999). Therefore, in vitro studies using appropriate target cells with intrinsic storage may be imperative, as enzyme internalization and fate of exogenously loaded substrates may be altered.

(p.350) Delivery of cells to the CNS

A significant challenge for CMT is adequate delivery of cells to the CNS. For storage diseases, BMT has been employed extensively and its relevance to the CNS stems from the realization that haematopoietic cells contribute to the macrophage population of the CNS. Intraparenchymal and intraventricular injections have also been used to directly deliver cell suspensions into the CNS, and a major issue here is achieving adequately widespread dispersion of cells. (See Table 14.1 for summary of approaches and cells used in disease models.) In comparison to BMT, these latter approaches are still in their infancy for storage diseases despite the common use of grafting as an experimental tool in basic neuroscience studies and its application in other selected human pathologies such as Parkinson's disease. In particular, the wealth of information on oligodendroglial developmental biology, the array of well-characterized heritable disorders of and traumatic insults to myelin, and well-defined criteria of evaluation have led to considerable CMT research on myelin pathology (Franklin and Blakemore 1998; Billinghurst et al. 1998; Brustle et al. 1999). Relevant to storage disease, the twitcher mouse, a model of Krabbe disease that primarily affects white matter, received intraparenchymal injections of fetal brain cells at 7 days of age into forebrain or cerebellum. Donor-derived allogeneic oligodendrocytes were later identified by in situ hybridization and said to be considerably widespread with enrichment in white matter. Of tissue sections analysed, 6% of oligodendrocytes were found to originate from injected cells while no such glia were detected in transplanted normal littermates, suggesting potential disease-dependent determination of cell fate (Huppes et al. 1992). Availability of neural stem cell lines and experience with other myelin pathologies will probably yield significant advances in treating this disease (Billinghurst et al. 1998). Intra-neocortical injections of glucuronidase-expressing fibroblasts into adult MPS VII mice in contrast produced minimal migration. While cell survival was evident even at 5 months post-transplant, the fibroblasts persisted as focal grafts which became consolidated over time and surrounded by astrocytes (Taylor and Wolfe 1997b). Given experience with adult astroglial responses in other grafting scenarios, it is possible that these glia impeded migration. Transplantation of rat AECs over-expressing human β-glucuronidase was tested in young adult normal and MPS VII mice (Kosuga et al. 2001). Amniotic epithelial cells, a1 relatively accessible human resource, are attractive due to reduced expression of histocompatibility markers and evidence of neural cell potentiality. Following injection into the striatum, cells survived for at least 30 weeks. Some migrant cells identified based on a vital fluorescent tag, were evident in neighbouring regions by 9 weeks but not in contralateral cortex. The most remarkable dispersion of cells injected into CNS was obtained with murine neural progenitor cell lines introduced into the lateral ventricles of fetal or newborn mice (Snyder et al. 1995; Lacorazza et al. 1996). In MPS VII recipients, cells identified by transgenic lacZ expression were widely evident including within the olfactory bulb, cerebral cortex, sub-cortical regions, and midbrain, and persisted at least 8 months, suggesting permanent integration (Snyder et al. 1995). While studies in models of storage disease are few, they at least verify that survival and migration of foreign cells can be attained in relevant pathologies. Taken together with studies on normal recipients and unrelated disorders, it is evident that age, site of introduction, and donor cell type are important determinants of cell dispersion, a critical factor for addressing the global involvement of CNS in storage disease. Future research will have to further explore these variables and how extrinsic conditions instigated by individual storage disorders may affect migration, survival, and differentiation.


Table 14.1 Examples of cell types tested for central nervous system (CNS) correction in animal models of storage disease*

Cell type

Delivery to CNS

Key findings

Model and reference

Haematopoietic lineage bone marrow cells

From periphery following irradiation and BMT

Widespread CNS delivery of microglia/macrophages Long-lived within CNS

Significant reduction of neural storage

MPS I dog (Shull et al. 1988)

Significant neurologic improvement

α-Fucosidosis dog (Taylor et al. 1992)

Direct evidence of active enzyme in neurones (Walkley)

α-Mannosidosis cat (Walkley et al. 1994a)

Definitive evidence of donor-derived brain macrophages

Twitcher mouse (globoid cell leukodystropy)

Improved myelination and lifespan

(Hoogerbrugge et al. 1988; Suzuki et al. 1988; Wu et al. 2000)

Some locomotor improvement

Oligodendroglial storage persists

No (BMT on adult) or limited reduction of neuronal storage (BMT on neonate)

MPS VII mouse (Birkenmeier et al. 1991; Sands et al. 1993)

Perivascular and meningeal storage reduction, some with detectable enzyme activity

Extended lifespan and better mobility

No neuronal enzyme activity or storage reduction

Sandhoff GM2 gangliosidosis cat (Walkley et al. 1994b, 1996)

Many putative donor-derived macrophages/microglia

No neurologic improvement

No neuronal enzyme activity or storage reduction

Sandhoff GM2 gangliosidosis mouse

Putative donor-derived macrophages/microglia present

(Norflus et al. 1998; Oya et al. 2000; Wada et al. 2000)

Delayed neurologic symptoms (attributed to microglial turnover) and increased lifespan

No CNS neuronal improvement

GM1 gangliosidosis dog (O'Brien et al. 1990; Haskins et al. 1991; Walkley et al. 1996)

Some motor and behavioural improvement

Haematopoietic lineage bone marrow cells (overexpressing)

From periphery following irradiation and BMT

Some immunodetectable enzyme in neurones

Galactosialidosis mouse (Leimig et al. 2002)

Delayed Purkinje cell degeneration

Disappearance of tremor and ataxia up to 10 months after BMT

Definitive evidence of donor-derived cells in CNS

Metachromatic leukodystrophy mouse

Minimal improvement in CNS storage

(Matzner et al. 2000b, 2002)

Modest improvement in neuromotor behaviour

Fetal liver cells

In utero injection into fetal liver

Donor-derived perivascular cells detected but only in older animals

MPS VII (‘kit’-genetically myeloablated) mouse (Barker et al. 2001)

Slight reduction in neuronal and glial storage

Peripheral macrophages

Intravenous injection

Some perivascular and parenchymal localization by 24 h

Newborn MPS VII mouse (Freeman et al. 1999)

Enzyme activity still present after 3 weeks

No cross-correction

Bone marrow mesenchymal stem cells (overexpressing)

Intraparenchymal injection

Distance-dependent Purkinje cell rescue after cerebellar injection

3-week-old Niemann–Pick A/B (Jin et al. 2002)

Improved motor behaviour

Amniotic epithelial cells

Intraparenchymal injection

Cells survive ≥30 weeks

Adult MPS VII mouse (Kosuga et al. 2001)

Migration into neighbouring regions

Ipsilateral storage clearance

Fibroblasts (overexpressing)

Intraparenchymal injection

5-month survival

Adult MPS VII mouse (Taylor and Wolfe 1997b)

Mostly proximal cross-correction

Fetal brain cells

Intraparenchymal injection

Migration into white matter

1-week-old Twitcher (Krabbe disease) mouse (Huppes et al. 1992)

Oligodendroglial replacement

Extended lifespan combined with haematopoeitic transplant; motor deficit persisted

Neural progenitor cell lines

Intraventricular injection

Long-term cell survival

Newborn MPS VII mouse (Snyder et al. 1995)

Reach multiple CNS regions

Evidence for cross-correction of neurones

Resident neural cells

Transduced in situ by focal gene delivery

Enzyme replacement and storage depletion of non-transduced cells

Adult MPS VII mouse (Ghodsi et al. 1998; Brooks et al. 2002; Sferra et al. 2000)

Cross-correction highly distance (100s μm to > 1 mm) and time dependent (weeks to months)

Adult metachromatic leukodystrophy mouse (Consiglio et al. 2001)

Improved learning/memory behaviour (Brooks; Consiglio)

4-week-old MPS IIIB mouse (Fu et al. 2002)

Distal cross-correction through axonal retrograde transport of secreted enzyme

Adult MPS VII mouse (Passini et al. 2002)

Multifocal injection-enhanced multi-region cross-correction

Newborn (Frisella et al. 2001) or adult (Skorupa et al. 1999; Bosch et al. 2000) MPS VII mouse

Improved spatial learning behaviour (Frisella)

(*) Other cell types being developed for use include oligodendroglia, astroglia, and human neural progenitor cells. See text for details.

(p.352) (p.353) (p.354)

(p.355) Studies employing BMT have demonstrated that unparalleled widespread delivery of donor cells to the CNS can be obtained by this approach (Walkley et al. 1996; Dobrenis 1998). Combined with autologous transplantation and appropriate ex vivo genetic manipulation, this has the potential to address the CNS in a relatively safe manner while also treating peripheral organs. The principle limitation and considerable variable is the total number of donor-derived cells that circumvent the endothelial barrier and the extent to which they enter the parenchyma. The cell types in question are primarily microglia, a term often restricted to those cells that under normal conditions reside within the parenchyma-proper, and their cousins, ‘brain macrophages’ which are normally found in perivascular, meningeal, subarachnoid, and intraventricular sites.

Microglia constitute a significant proportion of cells in the adult mammalian CNS, in the range of 5–20% (Lawson et al. 1990; Peters et al. 1991; McKanna 1993). Basic studies on the origin of microglia and brain macrophages indicate that these cells are of haematopoietic lineage, probably related to the myelomonocytic lineage as are many other tissue macrophages (Kennedy and Abkowitz 1998; also see reviews Dobrenis 1998; Cuadros and Navascues 1998). Use of embryonic animal chimeras argue that microglial progenitors enter the rudimentary CNS during fetal development (Cuadros et al. 1992, 1993; Kurz and Christ 1998; Alliot et al. 1999). Immunohistochemical studies with human fetuses also point to early invasion (Andjelkovic et al. 1998; Rezaie et al. 1999). While this fetal population, through subsequent proliferation and differentiation, may account for the bulk of microglial cells in the postnatal animal (DeGroot et al. 1992; Alliot et al. 1999), the resident population in the adult is sustained in part through turnover by newly invading haematopoietic cells (Lawson et al. 1992). Existence of turnover in the human brain is supported by post-mortem studies on individuals that had received sex-mismatched bone marrow transplants (Unger et al. 1993; Krivit et al. 1995b). BMT studies carried out in rodents and employing identifiable allogenetic markers consistently show significant replacement of meningeal and non-parenchymal, perivascular macrophage populations in postnatal or adult animals, in the vicinity of 30% or more in 3 months (e.g. Lassmann et al. 1991; DeGroot et al. 1992; Hickey et al. 1992; Kennedy and Abkowitz 1997; Hickey and Kimura 1988). In contrast, the same and other studies show the rate of turnover of (parenchymal) microglia to be very slow and perhaps only involve subpopulations (DeGroot et al. 1992; Kennedy and Abkowitz 1998; Ono et al. 1999; review Dobrenis 1998). Even after extended periods of time such as 1 year, detectable donor-derived microglia are relatively rare, often representing 1–10% of the estimated population, with variations partly attributed to use of different detection methods (Priller et al. 2001). As these studies typically involve irradiation of recipients, it has been argued that even low estimates may over-represent what actually occurs naturally, an issue difficult to resolve. Nevertheless, as conducted, BMT can lead to delivery of cells to the CNS from the periphery. Fortuitously, it also appears that depending on the particulars of pathologic conditions at hand, invasion can become heightened (review Walkley et al. 1996). For example, recent comparative BMT studies have clearly demonstrated that greater numbers of donor-derived parenchymal ramified microglia arise after ischemia, axotomy, or autoimmune disease than in healthy animals (Flugel et al. 2001; Priller et al. 2001). Similarly, significantly higher numbers were found for animal models of Krabbe disease (Wu et al. 2000) and for Niemann–Pick types A and B (Miranda et al. 1997) when compared to transplanted normal mice. Thus pathology, including that arising in storage diseases, can enhance the extent of microglial/macrophage invasion.

(p.356) Experiments on the twitcher mouse provided some of the earliest compelling evidence that macrophages in the CNS were haematopoietically derived and that BMT could be used to obtain CNS improvements in a storage disease (Hoogerbrugge et al. 1988; Suzuki et al. 1988). The origin of invading macrophage populations was confirmed using histocompatibility markers (Hoogerbrugge et al. 1988) and again more recently with GFP-expressing cells (Wu et al. 2000). Virtually all BMT studies on storage disease have, when examined, found evidence consistent with cellular invasion of CNS. In most cases, assessments of invasion have been indirectly estimated on the basis of enzyme assays on brain homogenates or relied on semi-quantitative histochemical techniques providing approximate measures. However, a few have employed polymerase chain reaction-based analyses to detect donor-specific DNA in extracts of CNS tissue and yielded positive evidence (Krall et al. 1994; Learish et al. 1996; Matzner et al. 2000b). When quantified, donor cell numbers were estimated to represent 0.1–0.3% of brain cells 7–12 months after BMT of arylsulfatase A-deficient mice (Matzner et al. 2000b), and 0.02 and 0.04% in brain and spinal cord, respectively, of normal mice 6–8 months after receiving bone marrow cells carrying a human glucocerebrosidase gene (Krall et al. 1994). A time course study in the latter verified previous views that CNS infiltration is a slow process.

In as much as separate studies, often with different techniques, can be compared, it does appear that storage diseases differ in permitting invasion and persistence of donor cells in the CNS (reviews Walkley et al. 1996; Dobrenis 1998). BMT studies on the fucosidosis dog model (Taylor et al. 1986, 1992) and GM2 gangliosidosis cat model (Walkley et al. 1994b, 1995, 1996) reported particularly high parenchymal invasion of putative haematopoieticderived cells. Most studies with storage diseases, similar to BMT studies with normal recipients, detect relatively few donor cells in CNS, and the majority are found to be perivascular or meningeal macrophages (e.g., Birkenmeier et al. 1991; Sands et al. 1993; Krall et al. 1994; Hahn et al. 1998). Thus, pathologic conditions produced by different storage diseases may dictate both the quantity of cell delivery and the extent of migration into CNS parenchyma. One cannot exclude the possibility that differences also lie between species, as invasion did appear greater in the cat (Walkley et al. 1994b, 1995, 1996) than in the mouse model of Sandhoff disease (Oya et al. 2000). This again may relate to disease severity, as the same defect in different species can produce distinct phenotypes owing to other species-specific metabolic differences (Sango et al. 1995; Phaneuf et al. 1996) or may indicate fundamental distinctness in microglial/macrophage trafficking. Another point is that though widespread seeding is typically seen, different CNS regions can vary in the concentration of donor cell numbers, as demonstrated in BMT studies on murine GM2 gangliosidosis (Oya et al. 2000). Further insights into how regional differences in pathology or into the underlying turnover demands of the resident microglia (Lawson et al. 1990; Oya et al. 2000; Priller et al. 2001) may conspire to affect these numbers could help better address the specific limitations of individual storage diseases.

Finally, do variables in the BMT protocol itself substantially affect the delivery of cells to the CNS in storage diseases? These variables include irradiative conditioning, immunologic aspects, and recipient age, but the data in the context of storage diseases are minimal. In most cases, to deplete the original haematopoietic system, experimental animals have received total body irradiation, unlike most human patients undergoing BMT, but total lymphoid irradiation has also led to successful engraftment and evidence supporting substantial brain infiltration (Taylor et al. 1986, 1988, 1992). With total body irradiation, increasing dosage levels seem to increase brain delivery, but this may simply parallel the extent of blood cell (p.357) engraftment that is obtained (Birkenmeier et al. 1991; Sands et al. 1993; Miranda et al. 1997). Doses above that necessary to achieve maximal blood engraftment did not appear to further increase numbers of cells found in brain, at least when early transplants were performed (Miranda et al. 1997). It is worth noting that experiments with MPS VII mice did reveal cells in the brain, primarily meningeal or perivascular and some parenchymal, even in the absence of preparative radiation (Soper et al. 1999, 2001) and despite low-level chimerism in bone marrow and peripheral blood cells (Soper et al. 2001). How immunologic conditions in recipients may play a role deserves more attention, as one may well expect that histocompatibility differences together with disease state could modulate the extent of CNS invasion (Matsushima et al. 1994) since microglia are both strongly subject to and effectors of the immune system (Gehrmann et al. 1995). With regard to age, there is general acceptance that given the often infantile or juvenile clinical onset and progressive nature of these diseases, the earlier the intervention the better, as invasion of brain is also protracted relative to other organs. However, the growing view that the majority of microglial progenitors have entered the mammalian brain prenatally suggests that neonatal, juvenile, and young adult transplants may not produce very dramatic differences in ultimate numbers of donor-derived microglia. Indeed, using detection by in situ hybridization, a study with normal mice transplanted at various postnatal ages and analysed at several post-transplant periods, suggested only a limited fraction of microglia could ever be replaced, and this was not different when BMT was carried out in neonates versus young adults (DeGroot et al. 1992). On the other hand, experiments with MPS VII mice revealed histochemical evidence of some donor-derived cells in the CNS following neonatal transplant (Sands et al. 1993) but none with adult recipients (Birkenmeier et al. 1991). Similar results were obtained with in situ hybridization techniques applied to newborn versus adult sex-mismatched transplants of sphingomyelinase-deficient mice (Miranda et al. 1997). As radiation and other conditioning regimens may affect the newborn more than the adult (Sands et al., 1993; Miranda et al. 1997), different results with normal and disease animals may in part relate to how the diseased versus normal newborn animal responds to such treatments. More studies with sensitive, quantitative, and consistent methods are needed to achieve a reliable perspective on the variable of postnatal age of transplant. Nevertheless, the speculation remains that in utero transplants should ultimately provide a far greater boost to seeding the CNS with donor-derived cells. Although initial studies with MPS VII mice have been somewhat disappointing (Barker et al. 2001; Casal and Wolfe 2001), further refinement of methodologies to improve competitive cell engraftment and clarification of fetal origin of microglial progenitors is still pending.

Results of BMT experiments have spurred interest in understanding and exploring further the potential of macrophage populations. A question that arises is whether instead of BMT one could employ selected cells of this microglial/macrophage lineage(s) as vehicles to treat the CNS. Classic studies have shown that tagged monocytes introduced into the blood will enter the CNS taking on microglial characteristics (Ling et al. 1980), and more recent studies suggest that it is a mature subpopulation of monocytes that enter the CNS (Kennedy and Abkowitz 1998), although the number of cells found in both cases was very low. To explore the potential of peripherally injected cells in a therapeutic setting, stimulated peritoneal or bone marrow macrophages were intravenously injected into MPS VII mice. Following neonatal injections, numerous cells as identified by a histochemical stain for β-glucuronidase, were found in the brain, but were relatively rare in adult recipients (Freeman et al. 1999). Microglial populations introduced into the circulation can also enter the CNS (p.358) (Dobrenis et al. 1998, 2000; Suzuki et al. 2001) and may ultimately prove more advantageous than peripheral macrophages (Imai et al. 1997).

While microglia and macrophages have received much attention, lymphocytes also traverse the brain even under normal circumstances (Hickey et al. 1991) but have been largely ignored as potential CMT mediators in the CNS. The number of lymphocytes in the CNS is normally low and yet following BMT in normal animals donor-derived lymphocytes may outnumber donor-derived microglia in the parenchyma, at least in the short term (Hickey et al. 1992). This number can become significant under the right pathologic condition as shown under autoimmune conditions (Lassmann et al. 1993). Given studies of direct enzyme transfer involving lymphocytes, further evaluation of their presence in storage disease CNS may be worthwhile in understanding whether they may contribute to cross-correction (Bou-Gharios et al. 1993a, 1993b). Furthermore, like monocytes and macrophages, when T cells are intravenously injected they can subsequently be found in CNS. This occurs rapidly, by 3 h, but the number is low. One per 2 mm2 area in cryostat sections of spinal cord was found following intravenous injection of 5 × 105 cells in rats (Hickey et al. 1991). If the cells recognize local antigen they appear to persist (or continue to return). A clever therapeutic use was demonstrated with peripheral myelin-specific T cells that were engineered to over-secrete NGF and shown to target to sciatic nerve and locally release the NGF, a potential tool for treatment of peripheral neurodegenerative disease (Kramer et al. 1995).

Evidence of cross-correction within the CNS

As predicted by principle and by demonstration in cell culture, it is now clear that cross-correction can also take place within the CNS. Results of directly injected supply cell populations, of resident cells transduced in vivo and of BMT-derived invading cells all confirm that enzyme transfer to deficient cells in situ is possible and can yield a therapeutic outcome (see also Table 14.1).

Results from cells injected into the central nervous system

Direct parenchymal introduction of supply cells was applied in the neocortex of adult MPS VII mice by injection of 2–3 × 105 over-secreting fibroblasts (Taylor and Wolfe 1997b). Otherwise absent β-glucuronidase activity was detectable in the thalamus at levels of 1–4% of normal, despite the fact that the fibroblasts formed largely consolidated grafts and showed little cellular migration. Some distal cells were noted, including a substantial number associated with the ventricles, but most of these probably arose by dispersion during injection, as they were detectable by 24 h. Thus, it is possible that some thalamic enzyme activity arose from relatively nearby cells, particularly as it was highest in the first few days following injection. Importantly, by histologic examination 1 month after implant, most neurones and glia appeared to be free of storage within a 2 mm distance from the grafts, providing strong evidence of cross-correction mediated presumably by enzyme release and subsequent uptake beyond the immediate vicinity of donor cells.

Injection of one million over-secreting AECs into MPS VII adult mouse striatum resulted in findings similar in concept but with apparently greater impact (Kosuga et al. 2001). Evidence suggested migration of cells, tagged with a fluorescent marker, into overlying cortex by 9 weeks but not into contralateral cortex. Brain tissue was divided into quandrants, and homogenates were quantitatively assayed for β-glucuronidase activity. The (p.359) quarter containing the injection site had enzyme activity corresponding to 100% that of normal levels, the ipsilateral non-injected quarter had 50% of normal, the proximal contralateral had 20%, and the distal contralateral had 10%. The remarkably high values in all cases probably relate to the fact that very high levels of overexpression (up to 900-fold above normal) were achieved in the AECs by transduction with human β-glucuronidase gene, as evaluated in initial cell-culture studies. The substantial activity in the contralateral brain was probably the product of cumulative distribution of high levels of secretion, which had also been demonstrated in vitro. These results appropriately contrasted with those obtained using non-transduced AEC which produced much lower values in all quandrants. Histologic examination showed clearance of storage vacuoles in ipsilateral cortex and some reduction in contralateral cortex, even though histochemical staining for β-glucuronidase did not reveal positive neural cells in the latter. Presumably, sufficient enzyme was taken up to have an impact on storage but was below the level of histochemical detection. This study suggests that remarkable correction can occur with modestly disseminated over-secreting cells and that with very high levels of secretion a penumbra of correction can be obtained well beyond the vicinity of donor cells. Conversely, extensive migration of cells obtained by intraventricular injection of neural progenitor cells into the newborn may afford widespread cross-correction even in the absence of overexpression, as suggested by experiments on MPS VII mice (Snyder et al. 1995).

Injection of 50,000 mesenchymal stem cells overexpressing acid sphingomyelinase into the cerebellum of 3-week old deficient mice produced more localized but nonetheless important results (Jin et al. 2002). Purkinje neurones are prominently affected and lost in several storage disorders and have previously proven difficult to rescue. However, in this study, Purkinje cell survival was significantly above that of untreated animals up to the 24 weeks examined, and the neurones that persisted demonstrated reduced storage and histochemical evidence of enzyme activity. Furthermore, a decreasing gradient of survival with distance from the site of injection was revealed in sagittal sections taken up to 300 microns away. As mesenchymal cells obtained from bone marrrow have been shown to have multi-potent differentiative capacity including generation of neuronal phenotypes, the possibility that the transplanted cells gave rise to new Purkinje cells could not be fully dismissed, although collectively the results were more compatible with cross-correction.

Results from resident cells transduced in situ

Studies on gene replacement directed at the CNS have been reviewed elsewhere (Chapter 16), but several points are relevant in the present context. First, transduction of resident neural cells can also lead to cross-correction of deficient cells. Significant enzyme uptake and/or depletion of storage vacuoles can be detected where transduced cells are rare or absent. Second and related, a single site of transduction does not produce global improvement, but clearance of storage vacuoles is observed at considerable distance, often 1–2 mm away or sometimes more (Ghodsi et al. 1998; Bosch et al. 2000; Sferra et al. 2000; Fu et al. 2002). Third, when assessed over time (and provided gene expression is sustained), distal cross-correction is found to be a slow process, with improvements in some sites only evident weeks to months after initial transduction (Consiglio et al. 2001; Brooks et al. 2002). This delay may arise from temporally dependent migration of transduced donor cells or indicate that equilibration of interstitial fluid concentration of enzyme (Bosch et al. 2000) in this setting takes longer than predicted by CSF turnover rates. Perhaps the simplest explanation, supported by (p.360) findings in MPS VII mice where disappearance of storage vacuoles is evident distal to detectable enzyme (Bosch et al. 2000; Frisella et al. 2001), is that there is a spatial margin in which a low level of enzyme replacement is achieved that minimally overcomes the rate of storage such that overt clearance of vacuoles becomes evident only after extended periods of time. Both the temporal and spatial limitations argue the fourth point, the predicted importance of providing widespread sources of enzyme to treat these global diseases. It is not surprising that far broader correction has been obtained with multi-focal than with single-site injections of gene constructs. However, these studies also suggest that enzyme contributed from multiple sites of origin may overlap to achieve effective cross-correction at distances not otherwise possible (Skorupa et al. 1999; Bosch et al. 2000). Fifth, recent work more carefully examining the anatomic patterns of enzyme distribution relative to transduced cells intimates that retrograde axonal transport may underlie much of the cross-correction of distal neuronal cell bodies (Passini et al. 2002). This serves to remind us that a diffusion gradient of enzyme through a complex structure will not be a simple function of radial distance. Therefore, educated selection of injection sites may yield significantly more effective results. Finally, the collective observations from gene replacement studies make it clear that cell-mediated cross-correction is an integral part of gene therapy today.

Results from bone marrow transplantation

BMT as a therapy for storage disorders provides a large body of work from which to evaluate approaches involving cell-mediated correction. Indeed, BMT studies on different animal models can offer insight into critical mechanisms that underlie success through analysis of the wide spectrum of results that have been obtained. For example, highly successful reduction of neuronal and macroglial storage has been reported for feline α-mannosidosis (Walkley et al. 1994a), canine fucosidosis (Taylor et al. 1992), and canine MPS type I (Shull et al. 1988), which without treatment display high degree of substrate accumulation. The twitcher mouse (globoid cell leukodystrophy) was intermediate, with elimination of globoid macrophages and evidence of some remyelination, but oligodendrocytes still continued to display considerable storage (Hoogerbrugge et al. 1988; Suzuki et al. 1988). MPS VII mouse showed no neuronal improvement (Birkenmeier et al. 1991), unless BMT was performed neonatally which resulted in some modest storage reduction (Sands et al. 1993). Some decrease in glial and neuronal storage was detected after in utero haematopoietic transplants using fetal liver cells (Barker et al. 2001). Canine and feline GM1 gangliosidosis, as well as feline and murine (Sandhoff variant) GM2 gangliosidosis (Haskins et al. 1991; Walkley et al. 1994b, 1996; Norflus et al. 1998), all marked by severe neuronal storage showed no detectable improvement in CNS neurones. Syngeneic transplants with bone marrow cells transduced to overexpress arylsulfatase A produced little-to-no impact on CNS storage in metachromatic leukodystrophy model mice (Matzner et al. 2002).

Interpretation of BMT findings in the CNS is confounded more than for other CMT approaches, as BMT simultaneously effects variable impact on numerous non-CNS tissues and organs. In as much as the peripheral changes can influence neuropathology and its clinical assessment, distinguishing between mechanistic contributions dependent on donor-derived microglia or macrophages and those from outside the CNS is problematic. Nevertheless, findings to date have not made a case for correlation between peripheral improvements and correction of CNS in terms of neural enzyme replacement or histopathology. There are ample examples of significant therapy of visceral organs with (p.361) little-to-no CNS improvement (Birkenmeier et al. 1991; Walkley et al., 1994b; Takenaka et al. 2000; Matzner et al. 2002), and those with marked CNS changes are not necessarily exceptional in their non-CNS findings (reviews Haskins et al. 1991; Walkley et al. 1996). As discussed earlier, the blood-brain barrier prevents entry of lysosomal enzymes from the circulation. Despite debate on radiative damage to the barrier and its possible disease-related leakiness (Biswas et al. 2001), CNS improvement seems unrelated to enzyme serum levels following BMT (Matzner et al. 2000b) and can also be obtained following in utero haematopoietic cell transplants in the absence of irradiation (Barker et al. 2001). In the face of many concomitant and uncertain variables presented by BMT and disease, it is appropriate to still keep an open mind to extravasated enzyme making some contribution, but the consensus is that CNS improvement is predominantly dependent on immigration of donor-derived cell populations. Studies that have shown reduction in storage of resident neural cells also consistently find evidence supporting the infiltration of bone marrow-derived microglial/macrophage cells in the CNS (Taylor et al. 1986, 1992; Shull et al. 1987, 1988; Breider et al. 1989; Walkley et al. 1994a; Leimig et al. 2002). Furthermore, a distinctive result of BMT is that when many CNS regions are examined, storage depletion is found throughout along with widespread seeding of putative or confirmed donor microglia (Shull et al. 1988; Taylor et al. 1992; Walkley et al. 1994a; Leimig et al. 2002). Strong support of the idea that macrophage lineage cells play a key role comes from comparison of BMT results on galactosialidosis mice to results obtained when the disease mice were cross-bred with transgenic mice that carried a gene for the missing enzyme under the control of the CSF-1 receptor promoter. The resulting hybrid mouse, which expressed enzyme only in macrophage populations and did not undergo preparative irradiation and BMT, was similar to and in some ways more “ corrected” than the transplanted disease mouse. Purkinje cells which die in the disease and are not rescued by postnatal BMT were partially rescued possibly due to early enzyme delivery from expressing microglia and perivascular macrophages present (Hahn et al. 1998).

Bone marrow transplantation typically leads to the appearance of enzyme activity in the CNS, and the levels determined biochemically in homogenates of recipient CNS can be quite significant. While this is often used as a measure of ‘enzyme replacement’, clearly a portion of this represents activity within immigrated donor cells. In BMT experiments on galactosialidosis that showed some CNS improvement, immunocytochemistry revealed enzyme-positive cells that indeed included macrophages but also some that were neurones, supporting the idea that enzyme transfer had occurred (Leimig et al. 2002). In some instances, suitable histochemical substrates have been available allowing in situ testing for neural localization of active enzyme, and these provide further instructive results. In MPS VII experiments where reduction in neuronal storage was minimal at best, β-glucuronidase activity in neurones was essentially absent (Sands et al. 1993). On the other hand, striking positive neurones were demonstrated in BMT studies on feline α-mannosidosis where CNS neuronal correction of storage was exceptionally effective. The study confirmed the presence of enzyme activity in neurones throughout regions examined and again provided evidence suggesting the presence of donor cells, largely perivascular but also in parenchymal locations (Walkley et al. 1994a). This finding spurred studies on microglial secretion, as discussed earlier, that support a link between local donor-derived cells and correction of CNS cells. In purified cultures of primary feline microglia, exceptional amounts of α-mannosidase with retention of activity were found to be released, more than any other acid glycosidase evaluated (Dobrenis et al. 1994, 1996). Relatively minor amounts of extracellular β-hexosaminidase and β-galactosidase accumulated in the same cultures (Dobrenis et al. 1994, 1996). These results correlated with the success of (p.362) BMT on feline α-mannosidosis (Walkley et al. 1994a) and the fact that BMT on the feline disease models deficient in the latter two enzymes failed to show histopathologic improvement in CNS neurones (Haskins et al. 1991; Walkley et al. 1994b, 1996; Walkley and Dobrenis 1995). Indeed, this may resolve what were initially surprising findings in the feline GM2 gangliosidosis BMT studies, where using a histochemical substrate for β-hexosaminidase an enormous number of positive putative donor-derived perivascular macrophages and parenchymal microglia were present and yet no activity could be detected in neurones (Walkley et al. 1994b, 1996; Walkley and Dobrenis 1995). Although secretion studies extended to murine microglia showed more extracellular β-hexosaminidase activity than for feline cells (Dobrenis et al. 1996), enzyme activity by histochemistry was not detected in neurones following BMT in a mouse model of GM2 gangliosidosis (Norflus et al. 1998). One might speculate this is due to a combination of insufficient numbers of donor-derived cells and still not enough secretion to attain adequate extracellular enzyme. The possibility cannot be dismissed that GM2 gangliosidosis neurones are simply more refractory to uptake, but this was not evident in enzyme replacement studies with the analogous feline disease neurones in culture (Dobrenis et al. 1992).

The impact of enhancing secretion was tested in arylsulfatase A-deficient mice by transducing donor bone marrow cells with a mutant gene that produced active enzyme in a form that was poorly retained by cells, resulting in the doubling of secretion levels assessed in vitro (Matzner et al. 2001). This was not more effective in reducing CNS storage or in improving on neurologic behavioural tests than BMT using the native form of the enzyme (Matzner et al. 2000b, 2002). Indeed, although enhanced serum levels were compatible with enhanced secretion, enzyme activity in CNS and other tissues was considerably reduced. Interpretation is confounded by the fact that the mutant enzyme was secreted more due to lack of two of three glycosylation sites, resulting in the abrogation of binding to M6P receptors. At least, this argued that doubling secretion did not outweigh the advantage of M6P receptor endocytosis. Interestingly, when using the mutant versus normal gene construct, enzyme levels in CNS were proportionally less reduced than in liver or spleen. This could relate to the finding that in contrast to other cell types, normal macrophages and possibly microglia release arylsulfatase A in a form that is already poor in M6P moieties (Muschol et al. 2002). Therefore, if enzyme delivery to CNS is more dependent on colonizing macrophages/microglia compared to other tissues which can acquire enzyme from the circulation (which could also arise from non-macrophage cells and normally bear M6P residues), it makes sense that the CNS values were reduced less than those of other organs when using the mutant enzyme. Furthermore, limited uptake by CNS target cells may help explain the finding that BMT studies with normal arylsulfatase A did result in substantial total enzyme activity in the CNS, up to 33% of normal because of immigrated cells, yet had minor impact on neuropathology (Matzner et al. 2000b, 2002). Collectively, BMT studies examining enzyme activity and its distribution in the CNS argue the quantity of locally provided enzyme is important. However, there is a clear need to better understand what uptake mechanisms are available in each case as well as determining the enzyme activity needs of the target cells involved.

A wholly unanswered question in BMT studies is the relevance of location of immigrated cells relative to the parenchyma. As discussed earlier, beyond the endothelial barrier, there are additional potential impediments for enzyme delivery to parenchymal cells, and yet the bone marrow-derived cells are primarily found in perivascular spaces. From this location, secreted enzyme must still get past basal laminae and might be directed outward to the subarachnoid (p.363) by bulk fluid flow. The perivascular cell population itself would have ample opportunity to sequester much of the released enzyme. So, how much enzyme reaches neurones from this location and are they in fact critically dependent on intraparenchymal donor cells? Does the glia limitan formed by glial end-feet also block entry from perivascular sites or provide an opportunity for selected glia to endocytose extracellular enzyme from perivascular spaces? Resolving these questions and modifying strategies accordingly is a significant challenge, but it may allow substantial improvements in therapy even in the absence of enhancing donor cell number.

In addition to providing a source of enzyme, the BMT approach is after all a cell replacement strategy. The direct substitution of members of the microglial/brain macrophage population with ‘normal’ cells may thus contribute to CNS improvement independent of enzyme transfer. Neurologic improvements seen in α-fucosidosis dog (Taylor et al. 1992) and the twitcher mouse (Hoogerbrugge et al. 1988; Suzuki et al. 1988), disease models that both demonstrate large numbers of storage laden macrophages, may well derive from replacement of these undesirable populations with new bone marrow-derived cells. Furthermore, neuronal viability and function may be influenced by the ‘activation’ state of microglia, the importance of which has been recognized in numerous other CNS pathologies. For storage diseases, a strong case was made in explaining BMT results on a Sandhoff mouse model that demonstrated significant behavioural improvements but neither enzyme transfer to neurons or improvement of CNS storage (Norflus et al. 1998). Studies suggested that neuronal compromise and death was in large part instigated by neurotoxic compounds arising from microglia which were activated in part due to their own enzyme deficiency. Partial replacement of this activated population by normal bone marrow-derived cells helped alleviate this element and slow neurologic deterioration (Wada et al. 2000).

In summary, the complexity of BMT involving multiple organ effects, replacement of numerous cell types, and radiative and immunologic variables provide a puzzling array of potential mechanisms that may impact on CNS therapy. Nevertheless, most data indicate that donor-derived brain macrophages and microglia play a key role, both as local enzyme providers and possibly through their substitution of deleterious counterparts. This lineage of cells represents a powerful tool for CMT by providing a convenient route to the CNS that can result in widespread seeding and therapy, if appropriately maximized. The profound success of BMT in some animal models, where it also provides multi-region correction in the CNS, supports this idea.

Neurologic outcome and human therapy

The field of cellular implantation in CNS for storage diseases is still young and behavioural studies limited, hence a picture on neuroclinical improvements has not emerged. However, there is some evidence of impact even with relatively small cell numbers. Injection of 50,000 overexpressing mesenchymal stem cells into hippocampus and cerebellum of a mouse model for Niemann–Pick type A and B did result in improved motor behaviour (Jin et al. 2002). Partial oligodendroglial cell replacement in twitcher mice was said not to result in gross improvement of motor behaviour but did significantly enhance survival time when coupled with BMT (Huppes et al. 1992). Intraparenchymal gene therapy in MPS VII (Frisella et al. 2001; Brooks et al. 2002) and metachromatic leukodystrophy (Consiglio et al. 2001) mice yielded significant cross-correction via transduced cells and treated mice showed reduction of learning deficits. BMT studies have been quite extensive and like the histopathologic (p.364) findings also variable in neurologic outcome (Haskins et al. 1991; Walkley et al. 1996). For example, treated α-fucosidosis dogs (Taylor et al. 1992) and α-mannosidosis cats (Walkley et al. 1994a) developed almost no behavioural or motor signs. On the other hand, GM2 gangliosidosis and GM1 gangliosidosis in domestic animals showed no neurologic benefit (O'Brien et al. 1990; Haskins et al. 1991; Walkley et al. 1994b, 1996), while a GM2 gangliosidosis mouse did show significant improvements in behavioural motor tests (Norflus et al. 1998). BMT with ex vivo transduced stem cells for overexpression resulted in only modest improvement in neuromotor behaviour of metachromatic leukodystrophy mice (Matzner et al. 2002), but in significant delay of onset of tremor and cerebellar ataxia in galactosialidosis mice (Leimig et al. 2002). Underlying reasons for the different outcomes based on detailed analyses have been discussed earlier. BMT in humans has also resulted in highly variable outcomes, but they are more difficult to interpret reliably.

Several hundred patients with storage diseases have undergone haematopoietic stem cell transplants, largely by heterologous BMT, but also utilizing cord blood, mobilized stem cells, and fetal liver cells. As early intervention is viewed as particularly critical, some attempts at in utero transplants have been made but have been disappointing. Heterologous BMT continues to be a high-risk procedure despite technical improvements in preparative regimens and post-transplant management, and autologous approaches and strategies for immune tolerance are being pursued. Nonetheless, BMT has been able to improve the course of neuropathology in several forms of storage disease with mental retardation (Walkley et al. 1996; Krivit et al. 1999; Krivit 2002). Many Hurler patients (MPS IH) have undergone BMT. Patients often show stabilization of IQ and neuropsychologic profile, or increases if transplanted before 2 years, and significant improvement in storage-induced anatomic changes in CNS assessed by magnetic resonance imaging (MRI) (Whitley et al. 1993; Hoogerbrugge et al. 1995; Krivit et al. 1995a; Shapiro et al. 1995; Vellodi et al. 1997; Krivit et al. 1999; Neufeld and Muenzer 2001; Grewal et al. 2002). Recall that a canine model of this disease also was effectively treated by BMT (Shull et al. 1987, 1988). BMT on Hunter disease (MPS II) patients has in some cases produced measurable improvements but is mostly ineffective particularly in more severe forms and with late transplant (Shapiro et al. 1995; Vellodi et al. 1999; Peters and Krivit 2000; Neufeld and Muenzer 2001; Seto et al. 2001; Takahashi et al. 2001). MPS III disease typically evokes severe retardation and behavioural abnormalities, and neurologic decline appears to continue following BMT (Vellodi et al. 1992; Hoogerbrugge et al. 1995; Shapiro et al. 1995; Neufeld and Muenzer 2001). BMT in globoid cell leukodystrophy, which was partially successful in mice, has resulted in gains in intellect, cognition, and some other criteria, particularly in late onset forms (Hoogerbrugge et al. 1995; Shapiro et al 1995; Krivit et al. 1995a, 1998, 1999; Wenger et al. 2001). Results with metachromatic leukodystrophy are mixed and most promising for late onset forms (Hoogerbrugge et al. 1995; Krivit et al. 1995a, 1998, 1999; Shapiro et al. 1995; Kapaun et al. 1999; Von Figura et al. 2001). While very few transplants of α-mannosidosis and α-fucosidosis patients have been reported, they are worth noting given that BMT on the respective animal models was particularly effective on the CNS. A 7-year-old boy with α-mannosidosis receiving BMT died 18 weeks later, and no significant decrease in neuronal storage was evident (Will et al. 1987). However, a report on a boy transplanted at 22 months of age (Wall et al. 1998) indicated increases in IQ, language and social skills, and stabilization in other neurologic parameters. Overall, the rate of development was less than normal. A boy with α-fucosidosis transplanted at 8 months showed mild neurodevelopmental delay 18 months later and improvement by MRI (Vellodi et al. 1995). Another patient, transplanted at the onset of neurologic symptoms, has shown improved psychomotor development (p.365) (Miano et al. 2001). Two patients with GM2 gangliosidosis underwent BMT after their first year of life already showing neurologic signs (Hoogerbrugge et al. 1995). One with Tay–Sachs briefly showed some improvement followed by further deterioration and death 6 months later. One with Sandhoff disease only survived 1 month following transplant.

Findings from the above and transplants in other diseases have led to the opinion that BMT should not be performed in patients already fallen below a set developmental quotient, and clearly intervention at pre-symptomatic stages is most desirable. Furthermore, it generally appears that the procedure is most likely to alter the course of the neurologic decline in diseases that have milder CNS involvement. The simple conclusion is that BMT in humans has a limited impact on the CNS, sufficient only to address relatively minor deficiencies and largely unable to reverse pre-existing storage and cytopathology. However, the fact that it does have some positive effect, and consideration of findings in animal studies and on microglia, provides hope. It is reasonable to expect that further work on isolation and stable manipulation of human haematopoietic cells to over-secrete and research on ways to maximize CNS colonization will lead to significant improvements in human therapy. Also, more basic studies on secretion and uptake in cell culture with human, rather than animal, microglia and target neural cells, and on human microglial dynamics in vivo might prove insightful.

Studies on animal models have raised some important options for human therapy involving haematopoietic cell transplantation. BMT has been combined with other therapeutic modalities. MPS VII mice received weekly injections of β-glucuronidase beginning at birth and ending at 7 weeks (Sands et al. 1997). This was immediately followed by syngeneic BMT. The protocol produced CNS results similar to or better than mice receiving enzyme injections throughout their lifespan. In both cases, there was some long-term albeit limited reduction in neuronal storage, possibly due to enzyme entry through an incomplete BBB in the immature mice and greater neuronal requirement for enzyme during the developmental period. The combined therapy was more effective than ERT alone in reducing storage in meninges and retinal pigment epithelium, and produced higher total enzyme levels in brain, all presumably related to invaded bone marrow-derived cells. Detailed comparison to results from previous studies with BMT alone was not made, but the combined therapy was quite similar to BMT performed neonatally (Sands et al. 1993). The practical points are the following. The combined approach delays the use of the preparative regimens for BMT, which have significant deleterious effects on, for example, cerebellar development if applied perinatally (Sands et al. 1993). Furthermore, potentially productive early intervention with ERT in humans would allow more time for identification of suitable BMT donor or preparation and integration of transduced autologous haematopoietic cells. Bone marrow transplantation in a mouse model of Sandhoff disease also receiving N-butyldeoxynojirimycin to inhibit ganglioside synthesis, reducing the storage burden, resulted in greater efficacy than either treatment alone (Jeyakumar et al. 2001). Deterioration of neurologic function assessed by three motor-based behaviours was delayed or slowed more in mice with the combination therapy. This clearly complementary approach appears amenable to human application (see Chapter 15), offers similar temporal advantages as the ERT/BMT combination and may have additive or even synergistic benefit (Jeyakumar et al. 2001). Finally, as noted earlier, implantation of normal neural cells into Twitcher mice to give rise to new oligodendrocytes significantly extended survival of recipients when combined with BMT (Huppes et al. 1992). This kind of strategy is being further pursued (Wenger et al. 2000) and may provide neurologic benefit for Krabbe or other human diseases, particularly if limited anatomic sites of pathology most critical to function can be identified.

(p.366) Anticipated advances and aspirations

Despite the rarity of lysosomal storage diseases in the human population, the history of sustained efforts towards treatment has led to recognition of several factors that if maximized could lead to success. In the context of CMT, these include deposition of ample and well-dispersed cells, adequate secretion level, and efficient uptake of released enzyme. The effectiveness of focal cell injections and BMT could be bolstered with a better understanding of requirements for cell migration. Early fetal replacement of endogenous haematopoietic lineages could increase macrophage/microglial population of the CNS, potentially attaining a donor population that corresponds to 10% or more of the native CNS population. While such initial studies for storage disease have been disappointing, improvements in engraftment and proper identification of microglial precursors which may stem from progenitors to the haematopoietic system itself (Cuadros et al. 1992; Alliot et al. 1999) will be essential before final judgment can be passed. Early introduction of neural stem or progenitor cells is highly promising in regard to cell number, and ventricular or generative zone introduction coupled with permissive developmental pathways of migration appear to produce extensive dissemination (Snyder et al. 1995). A conclusive appreciation of the soundness of neural integration may be the greatest challenge here. In all cases, strides in tackling the immunologic challenge presented by CMT are expected to continue through autologous ex vivo approaches and improved understanding of immune tolerance.

Beyond cellular seeding, further steps to improve enzyme delivery are clear. In many cases, the amount of secretion and its sustained operation through stable gene integration has not been maximized. Ensuring that the gene product is adequately endowed with appropriate moieties for receptor-mediated uptake will secure significant enhancement. Indeed for the many, if not most, cells within the CNS that see low levels of extracellular enzyme, glycosyl receptor systems may well not be saturated. This suggests that even minor improvement in secretion levels coupled with high-uptake enzyme forms could yield disproportionately greater improvement. As classical enzyme receptors on neural cells may be limited, exploring more novel acceptor and receptor systems is also worthwhile (Dobrenis et al. 1992; Xia et al. 2001). In sum, there are several rational and feasible means to attain improved cross-correction. Given the notable examples of significant CNS correction already obtained, yet with methods not fully optimized, it is reasonable to expect considerable success in the near future. This may be even closer than we think if one considers cross-correction in light of the chronic progressive nature of the relevant diseases. In terms of storage, CMT outcome evaluated over the long term is after all the consequence of an ever-widening cumulative process determined by the daily balance between substrate load and enzyme activity. Therefore, minor improvements in enzyme delivery may yield profound improvements over time. The question then becomes whether this is rapid enough to halt the functional neurophysiological deficits that arise in storage diseases and underscores the importance of better understanding the pathogenetic cascades involved (see Chapter 12).

Hidden within failures and successes lay clues to additional mechanisms that we suspect but know little about. The interactions between donor and recipient cells, and the factors determining the fate of extracellular enzyme within the complex fabric of the CNS are probably complicated. It is only recent studies that begin to better dissect this, such as ones showing that delivery of enzyme to distal neuronal lysosomes might be realized more through intracellular transport along axonal pathways than through simple diffusion (Passini et al. 2002). Another case in point is the potential contribution of so-called ‘direct enzyme’ (p.367) transfer found to be very efficient in cell culture and independent of M6P-mediated uptake which may be limited in the adult CNS. Further investigation of this using relevant cell types and with appropriate in vivo experiments (Bou-Gharios et al. 1993b) could be valuable. A concept rarely addressed (Desnick et al. 1979) is that of indirect storage depletion. In diseases where storage substrates can be detected in the cerebrospinal fluid as well as the blood, to what extent might donor cells reduce the substrate load on enzyme-deficient cells by simply reducing extracellular substrate? It is possible that this ‘sink’ effect can help explain those instances where storage depletion has been reported in neurones without detectable enzyme uptake. It is also tempting to speculate that this played some role in the particularly effective result obtained with BMT on feline α-mannosidosis (Walkley et al. 1994a). Extracellular substrate loads of mannose-terminal oligosaccharides found in fluids including CSF (Warren et al. 1988) might have been efficiently reduced by donor-derived macrophages and microglia, given that these cell types express the mannose receptor (Linehan et al. 1999; Marzolo et al. 1999). Selection of appropriate cell types and transgene expression or amplification of appropriate receptors aimed at sequestering substrate might be worth exploring as another strategy for CMT. A greater understanding of the contribution of soluble agents such as toxic metabolites or disease-induced deleterious cytokines to compromise and loss of CNS cells is important, particularly to neural cell replacement strategies that could be challenged by their non-replaced cellular neighbours. Indeed, arguably the greatest merit for using neural cell replacement lies in diseases like the neuronal ceroid lipofuscinoses where cell death is a prominent feature. The use of neural cell progenitors appears exciting, and initial transplantation of progenitors derived from human tissue are encouraging (e.g. Flax et al. 1998; Buchet et al. 2002). Given technical and ethical issues associated with derivation of human cells, studies revealing the unexpected fate and generation of neural cells from primitive cells present in adult bone marrow (e.g. Brazelton et al. 2000; Mezey et al. 2000) opens important new and practical avenues of pursuit for human therapy (Koc et al. 1999; Mezey et al. 2003).

One can be considerably confident that dramatic new results of therapeutic success in mouse models will mark the next few years. Nevertheless, there is still a long bridge to cross to equivalent success with human disease. While many of the principles we have and continue to discover through animal studies are likely to apply to humans, the details and specific needs can be expected to be different. The solidification of CMT strategies for human trial merit the need to progress beyond development in transgenic mice, and ironically return to the larger naturally existing domestic animal models that were once the predominant test subjects. These stepping-stone models will allow consideration of the extent that developmental differences or the simple reality of having to address a larger brain pose additional challenge to adequate cell and enzyme delivery. Furthermore, the models will provide opportunity to assess more complex behaviours allowing superior evaluation of treatment benefit. This may be particularly critical to validating the more heroic approaches such as neural cell replacement.

The nature of the CMT strategy requires the consideration of more variables than other therapeutic modalities posed at storage diseases, but it also offers the potential to address more obstacles to success. The cell is more than just a pool of enzyme. Through intrinsic or manipulative genetic programming, it can respond appropriately to environmental cues to reach sites difficult to otherwise access and to perform multiple functions that may include debris removal, cell replacement, substrate removal, supply of neurotrophic factors, abrogate toxic elements, and even mediate gene delivery (Lynch et al. 1999). Based on advances that (p.368) have already been made, one can anticipate that CMT will play a central role in overcoming the devastating consequences of human storage diseases that affect the CNS.


There is now substantial evidence that CMT can effect significant improvement in CNS pathology in lysosomal storage diseases. Much of this can be attributed to cross-correction where cells release enzymes for uptake by deficient cells, and the impact of the mechanism can be enhanced through gene overexpression and use of receptor-mediated uptake systems. Studies with animal models of disease support these concepts. Direct implantation of cells and use of BMT to deliver microglial/brain macrophage precursors to the CNS have both resulted in cross-correction. The mechanism has also proved an important component of in vivo gene replacement in neural cells, by dramatically extending correction to distal cells through enzyme secretion from transduced cells. Other mechanisms in CMT may also be involved such as indirect storage depletion by donor cells or direct cell–cell contact. BMT has been extremely successful in some animal disease models. One of the benefits of the approach is that it results in extremely widespread delivery of cells to the CNS, important in treating the global nature of the diseases. Cell-mediated therapy is also a tool by which to directly replace enzyme-deficient populations. This may underlie some of the success of BMT which can replace deleterious brain macrophages. The integrative potential of neural progenitor cells appears exciting and can be coupled with enzyme overexpression, but much more basic research is needed on this approach. In all its manifestations, CMT offers the potential to effect a permanent cure through stable cellular population of the CNS. Bone marrow transplantation has also yielded a positive neurologic impact in some storage disease patients. Ongoing efforts to successfully employ autologous transplantation incorporating ex vivo gene overexpression and/or earlier intervention will in principle increase the effectiveness of haematopoietic cell therapy and could extend it to diseases as yet untreatable. Specific use of macrophage/microglial lineage cells and bone marrow stromal cells also merits further investigation, as well as various combination therapies that could incorporate substrate deprivation or direct implantation of neural progenitor cells in critical sites of pathology.


The author's research has been supported in part by funds from the March of Dimes and the Kirby Foundation.

This chapter is dedicated to the memory of my father-in-law, Joseph M. Horris, who recently lost his personal battle with disease.


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