Alzheimers Disease

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1. Introduction

As the most common cause of senile dementia, Alzheimer's disease (AD) affects millions of people worldwide. Clinically, patients experience progressive cognitive impairment leading to dementia and ultimately death. The neuropathological hallmarks of AD are senile neuritic plaques (NPs) and neurofibrillary tangles (NFTs). Neuritic plaques are extracellular aggregations of protein, including the fibrillar peptide, p-amyloid. NFTs are neuronal inclusions of filamentous structures containing hyperphosphorylated forms of the microtubule-associ-ated protein tau. Although these tissue and cellular abnormalities are well described, the role of each in producing neuronal demise is still actively debated.

AD is divided into 2 types based on age of symptom onset, early (before 60 years) and late (after 60 years). Early-onset Alzheimer's disease (EOAD) is primarily an inheritable form of the disease. Genetic linkage studies of several families exhibiting EOAD identified a locus on the long arm of chromosome 21 near the amyloid precursor protein (APP) gene (80). Further studies revealed that mutations in APP gene increased the production of p-amyloid (a major constituent of senile plaques) (81). The APP linkage represents <1% of early onset cases suggesting other genetic loci exist. Two loci, one on chromosome 14q and the other on chromosome 1 containing the presenilin (PS)-1 and- 2 genes, have also been identified in linkage studies (82,83). Many mutations in PS-1 have been identified in EOAD cases, which account for >50% of all EOAD cases. The function of the PSs is currently emerging and appears to involve signal transduction (84-86).

A genetic locus on chromosome 19q is implicated in late-onset Alzheimer's disease (LOAD) (87,88). The apolipoprot-ein E gene is located in this region, but no mutations have been found in AD; however, 1 of 3 isoforms of ApoE, E4, is a significant risk factor for LOAD (81). Other potential LOAD-linked genes have been identified (89-95) and further study is needed to substantiate the importance of their linkage. The vast majority of LOAD cases are sporadic with no identified genetic component. Studies using microarray gene expression profile analysis are beginning to identify altered gene expression in LOAD compared with normally aged brains (P. Coleman, personal communication, 2003). Such analyses will lead to further studies characterizing the role of candidate genes in AD, possibly allowing for a genetic screen to identify the best therapeutic strategy.

2. Putative Mechanisms Underlying AD

Based on the neuropathological findings of AD, NPs, and NFTs, extensive research efforts have focused on their role in the pathogenesis of AD. Many hypotheses are under examination: the acetylcholine hypothesis (96,97), the amyloid cascade hypothesis (98-100), the neuroinflammatory hypothesis (101), energy metabolism hypothesis (102), and the oxidative stress hypothesis (103). A comprehensive review of the research behind each hypothesis is beyond the scope of this discussion; instead, each is discussed generally as a potential target area for gene therapy.

3. Potential Gene-based Therapies for AD

One of the affected brain regions is the basal forebrain cholin-ergic complex as cholinergic deficiency correlates with both the magnitude of pathological severity and degree of dementia (96,104-107). One therapeutic approach is to augment cholin-ergic function by increasing activity of the biosynthetic enzyme for acetylcholine, choline acetyltransferase (ChAT) to the affected area. Nerve growth factor (NGF) up-regulates expression of ChAT (108), and promotes survival and maintenance of the septohippocampal pathway that is a major pathway for memory and learning (109). Studies reporting that NGF increases p-amyloid production in vitro contrast with in vivo studies, suggesting that NGF delivery does not increase plaque formation in primates (110). Grafting of fibroblasts genetically modified to produce NGF via a Moloney murine leukemia virus vector promotes restoration and survival of the septohippocampal pathway (111-115). Figure 5 depicts an example of the bioactivity of these transduced grafts in vivo. NGF clearly has therapeutic potential but further studies will be needed to evaluate its efficacy in ameliorating symptoms of AD.

Amyloid-containing plaques may be a cause of AD or a byproduct of the disease process. Identified mutations in both the APP and the PS genes correlate with increased production of p-amyloid and potentially plaques. PSs are transmembrane proteins localized predominantly in endosomes and Golgi ap-parati and are believed to promote the aggregation of p-amy-loid by increasing the activity of 7-secretase, the enzyme responsible for liberating the Ap 1-42 fragment from APP (86,116). Gene products that may modify the activity of por 7-secretase or proteins capable of disrupting p-amyloid aggregation may potentially slow progression of the disease.

Other areas of therapeutic interest for AD are inflammation and oxidative stress. Many mediators of inflammation have been detected in the brain of AD brain [reviewed in (101)]. Whether inflammation is activated by the production of plaques and tangles or inflammation initiates production of plaques and tangles, which in turn propagates an inflammatory response is not clear. It is believed that the inflammatory response of glia that leads to neuronal demise (101). Epidemiol-ogical studies with antioxidants and nonsteroidal anti-inflammatory drugs suggest that decreasing free radicals and inhibiting inflammatory processes may confer protection against AD and/or slow the rate of cognitive decline seen in AD [discussed in (103)]. The delivery of gene products capable of scavenging free radicals and blocking inflammatory processes may also prove an effective therapeutic approach.

Recent publications have highlighted the potential of active and passive Ap-based immunotherapies in the treatment of AD (117-119). Numerous investigative teams have reported diminution in AD-like pathology (i.e., Ap deposition) and behavioral improvements in different animal models of the disease as a result of Ap peptide immunization or via the administration of Ap-specific antibodies. The biological

Figure 5 Neurite penetration into implanted primary fibroblast cells that have been transduced with a murine retrovirus vector expressing nerve growth factor. (A) depicts an NGF-secreting autologous fibroblast graft located in a region of intact Nucleus Basalis of Meynart (NBM) showing penetration by acetylcholinesterase (AChE)-positive neurites 1 month after grafting in cynomolgous monkey. Arrow and ''g'' indicate graft. In contrast, an implanted NBM graft initially transduced with a control P-galactosidase-expressing retrovirus vector (B) exhibits no penetration of AChE-positive neurites, although sprouting at the edge of the graft potentially due to astrocytic NGF secretion is apparent. These studies and many others suggested that NGF-expressing autologous fibroblast grafts might represent a viable approach to promote regeneration of cholinergic neuronal pathways targeted by Alzheimer's disease. Early-phase human clinical trials are in progress to determine the safety of this therapeutic methodology. (From Ref. 114, © 1994 Elsevier Science B.V.)

Figure 5 Neurite penetration into implanted primary fibroblast cells that have been transduced with a murine retrovirus vector expressing nerve growth factor. (A) depicts an NGF-secreting autologous fibroblast graft located in a region of intact Nucleus Basalis of Meynart (NBM) showing penetration by acetylcholinesterase (AChE)-positive neurites 1 month after grafting in cynomolgous monkey. Arrow and ''g'' indicate graft. In contrast, an implanted NBM graft initially transduced with a control P-galactosidase-expressing retrovirus vector (B) exhibits no penetration of AChE-positive neurites, although sprouting at the edge of the graft potentially due to astrocytic NGF secretion is apparent. These studies and many others suggested that NGF-expressing autologous fibroblast grafts might represent a viable approach to promote regeneration of cholinergic neuronal pathways targeted by Alzheimer's disease. Early-phase human clinical trials are in progress to determine the safety of this therapeutic methodology. (From Ref. 114, © 1994 Elsevier Science B.V.)

mechanism(s) by which these therapies act are still subject to interpretation. However, 2 nonmutually exclusive hypotheses have emerged: (1) Ap-specific antibodies whether raised intrinsically or passively administered act at the blood-brain barrier (BBB) to alter the equilibrium of soluble Ap1-42 concentrations to favor its clearance from the brain. This is posited to prevent additional Ap peptide deposition and perhaps promote aggregate dissolution. (2) Ap-specific antibodies cross a compromised BBB and bind to Ap within the brain parenchyma where antibody-Ap complexes are postulated to be bound by complement, and recognized by microglia, which dissolve existing amyloid deposits. Both mechanisms of vaccine action can lead to possibly harmful side effects, with the most feared being systemic autoimmune disease and CNS inflammation as illuminated by recent findings from a Elan Pharmaceuticals peptide vaccine phase II clinical trial conducted in Europe [commentary in (120,121)]. Although presently untested, gene transfer technology due to its inherent versatility may allow for regulated antigen presentation and even codelivery of immunomodulatory gene products that could lead to safer and more efficacious vaccines for AD.

C. Lysosomal Storage Diseases

1. Introduction

Lysosomal storage diseases (LSDs) are a diverse group of greater than 40 disorders that originate from a deficiency of a lysosomal enzyme, resulting in accumulation of lysosomal proteins and cellular dysfunction. Many of the LSDs exhibit moderate to severe deleterious effects on somatic tissues as well as the CNS. These degenerative disorders differ from many of the neurodegenerative diseases described in that the pathogenic mechanism responsible for most LSDs is already known (i.e., lysosomal enzyme deficiency, defects in cofac-tors or transport proteins). There are examples of LSDs, however, for which the missing lysosomal function/protein activity has not been identified [e.g., a subset of the ceroid lipofuscinosis diseases; reviewed by (122)]. Depending the type of LSD, enzyme replacement by direct infusion of the missing enzyme into peripheral tissues has been an extremely successful strategy (123). This is no more evident than in the case of Gaucher disease, which results from a deficiency of glucocerebrosidase. Loss of glucocerebrosidase leads to accumulation of glucocerebroside, a byproduct of sphingolipid degradation, in macrophages resulting in spleen and liver enlargement, bone malformation, and pulmonary dysfunction (123). Infusion of recombinant glucocerebrosidase leads to significant correction of peripheral tissue disease (124). A similar approach has proven successful in the treatment of Fabry disease, another sphingolipid disorder that primarily affects kidney, heart, and skin [reviewed by (125)]. In clinical trials, intravenous delivery of a-galactosidase A was found to be safe and led to marked reductions in plasma glycosphin-golipid levels and microvascular endothelial deposits in major organs (126,127).

Other LSDs exhibit both peripheral and CNS involvement. The CNS component of the mucopolysaccharidosis (MPS) storage disorders and other LSDs has proven a more difficult task to correct using standard enzyme replacement therapy. This difficulty lies in the inability of peripherally infused lyso-somal enzymes to traverse the blood-brain barrier. Development of methodologies to effectively deliver and distribute the deficient enzyme throughout the brain would represent a major advance in therapies for LSDs. To that end, the implementation of gene-based technology in this endeavor has been enthusiastically pursued and has demonstrated initial promise.

2. Potential Gene-based Therapies for Correcting CNS Dysfunction Caused by LSDs

Numerous mouse models for the various LSDs have been developed and have proven extremely useful in assessing novel therapies. Gusmps mice exhibiting the p-glucuronidase enzyme deficiency associated with Sly disease undergo progressive lysosomal accumulation of non-egraded glycosami-noglycans in multiple organs including the brain. The Sands laboratory demonstrated that AAV vector-mediated delivery of p-glucuronidase into the anterior cortex and hippocampus of newborn MPS VII mice led to a reduction in glycosaminog-lycan deposition and concomitant improvement in cognitive function as measured in the Morris Water Maze paradigm (128). Although transgene expression levels were generally greater than normal p-glucuronidase levels, particularly at the injection site, no overt toxicity was observed. In addition, these results suggested that a single point source of continuous enzyme expression produced lysosomal storage correction at distal sites. Several other groups have also demonstrated the utility of AAV vector-mediated p-glucuronidase delivery in gusmps mice (129-131).

Davidson and colleagues have used recombinant viral vectors based on feline immunodeficiency virus (FIV) to deliver the p-glucuronidase gene to the brains of MPS VII mice to determine if restoration of this enzyme diminished preesta-blished lysosomal accumulations and corrected associated CNS deficits (132). FIV vector-mediated bilateral delivery of p-glucuronidase via the striatum led to bilateral correction of protein deposits and a reversal of spatial learning and memory impairments. The effect FIV vector-mediated expression of p-glucuronidase on lysosomal storage is illustrated in Fig. 6. Perhaps shedding light on potential mechanisms of action, gene expression profiling indicated significant increases in genes associated with neuronal plasticity mediation. An interesting extension of these studies was performed recently by Elliger and colleagues (133). The coding sequences for the IgK secretion and HIV-1 TAT uptake signals were engineered into the p-glucuronidase open reading frame in order to effect therapeutic benefit in more distal organs. This modified transgene was delivered intrathecally via an AAV recombinant vector to newborn gusmps mice. Treated mice were found to be more active, exhibited less stunted growth, and did not show evidence for abnormal storage deposits in the brain or liver, or in tissues not harboring AAV vector genomes.

The Twitcher mouse, which harbors a genetic disruption in the galactocerebrosidase locus, serves as an informative murine model for human globoid cell leukodystrophy (Krabbe disease). Intraventricular infusion of a recombinant adenovi-rus vector expressing galactocerebrosidase to newborn Twitcher mice led to marked reduction in lysosomal storage pathology (134). However, unlike in the case of gene delivery to MPS VII mice, treatment of Twitcher mice with preesta-blished disease had no significant effect on disease pathology. These results indicate that timing of interventions for Krabbe disease is crucial, and/or adenovirus vectors, due to their inherent immunogenicity and reduced duration of expression, are not as useful for treating this LSD. It is interesting to note that there exist autosomal recessive forms of Krabbe disease that have been identified in dogs and rhesus monkeys (135). As other gene transfer vector approaches are applied to this disease, these higher order mammalian models will become invaluable in the stepwise progression toward clinical application to the human form of Krabbe disease.

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