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The article discusses the complexity of neuropathology in lysosomal storage diseases (LSD) and blood-brain barrier (BBB), with particular focus on the developments in therapeutics targeting the central nervous system (CNS) of LSDs. It discusses LSD neuropathy, which has been attributed to diseases in CNS and peripheral nervous system (PNS). It suggests that the discovery of adeno-associated viral (AAV9) trans-BBB neutropism provides promise for LSD treatment through systemic vector delivery.

The primary pathology in mucopolysaccharidosis (MPS) IIIB is lysosomal storage of heparan sulfate (HS) glycosaminoglycans, leading to complex neuropathology and dysfunction, for which the detailed mechanisms remain unclear. Using antibodies that recognize specific HS glycoforms, we demonstrate differential cell-specific and domain-specific lysosomal HS-GAG distribution in MPS IIIB mouse brain. We also describe a novel neuron-specific brain HS epitope with broad, non-specific increase in the expression in all neurons in MPS IIIB mouse brain, including cerebellar granule neurons, which do not exhibit lysosomal storage pathology. This suggests that biosynthesis of certain HS glycoforms is enhanced throughout the CNS of MPS IIIB mice. Such a conclusion is further supported by demonstration of increased expression of multiple genes encoding enzymes essential in HS biosynthesis, including HS sulfotransferases and epimerases, as well as FGFs, for which HS serves as a co-receptor, in MPS IIIB brain. These data suggest that lysosomal storage of HS may lead to the increase in HS biosyntheses, which may contribute to the neuropathology of MPS IIIB by exacerbating the lysosomal HS storage. [ABSTRACT FROM AUTHOR]

The linear DNA genomes of recombinant adeno-associated virus (rAAV) gene delivery vectors are acted upon by multiple DNA repair and recombination pathways upon release into the host nucleus, resulting in circularization, concatemer formation, or chromosomal integration. We have compared the fates of single-strand rAAV (ssAAV) and self-complementary AAV (scAAV) genomes in cell lines deficient in each of three signaling factors, ATM, ATR, and DNA-PKCS, orchestrating major DNA double-strand break (DSB) repair pathways. In cells deficient in ATM, transduction as scored by green fluorescent protein (GFP) expression is increased relative to that in wild-type (wt) cells by 2.6-fold for ssAAV and 6.6-fold for scAAV vectors, arguing against a mechanism related to second-strand synthesis. The augmented transduction is not reflected in Southern blots of nuclear vector DNA, suggesting that interactions with ATM lead to silencing in normal cells. The additional functional genomes in ATM_/_ cells remain linear, and the number of circularized genomes is not affected by the mutation, consistent with compartmentalization of genomes into different DNA repair pathways. A similar effect is observed in ATR-deficient cells but is specific for ssAAV vector. Conversely, a large decrease in transduction is observed in cells deficient in DNA-PKCS, which is involved in DSB repair by nonhomologous end joining rather than homologous recombination. The mutations also have differential effects on chromosomal integration of ssAAV versus scAAV vector genomes. Integration of ssAAV was specifically reduced in ATM_/_ cells, while scAAV integration was more profoundly inhibited in DNA-PKCS _/_ cells. Taken together, the results suggest that productive rAAV genome circularization is mediated primarily by nonhomologous end joining. [ABSTRACT FROM AUTHOR]

Numerous preclinical studies have demonstrated the efficacy of recombinant adeno-associated virus (rAAV) gene delivery vectors, and recent clinical trials have shown promising results. However, the efficiency of these vectors, in terms of the number of genome-containing particles required for transduction, is hindered by the need to convert the single-stranded DNA (ssDNA) genome into double-stranded DNA (dsDNA) prior to expression. This step can be entirely circumvented through the use of self-complementary vectors, which package an inverted repeat genome that can fold into dsDNA without the requirement for DNA synthesis or base-pairing between multiple vector genomes. The important trade-off for this efficiency is the loss of half the coding capacity of the vector, though small protein-coding genes (up to 55 kd), and any currently available RNA-based therapy, can be accommodated. The increases in efficiency gained with self-complementary AAV (scAAV) vectors have ranged from modest to stunning, depending on the tissue, cell type, and route of administration. Along with the construction and physical properties of self-complementary vectors, the basis of the varying responses in multiple tissues including liver, muscle, and central nervous system (CNS) will be explored in this review.Molecular Therapy (2008) 16 10, 1648–1656 doi:10.1038/mt.2008.171 [ABSTRACT FROM AUTHOR]

The driving interest in adeno-associated virus (AAV) has been its potential as a gene delivery vector. The early observation that AAV can establish a latent infection by integrating into the host chromosome has been central to this interest. However, chromosomal integration is a two-edged sword, imparting on one hand the ability to maintain the therapeutic gene in progeny cells, and on the other hand, the risk of mutations that are deleterious to the host. A clearer understanding of the mechanism and efficiency of AAV integration, in terms of contributing viral and host-cell factors and circumstances, will provide a context in which to evaluate these potential benefits and risks. Research to date suggests that AAV integration in any context is inefficient, and that the persistence of AAV gene delivery vectors in tissues is largely attributable to episomal genomes. [ABSTRACT FROM AUTHOR]

Adenovirus (Ad) has been extensively studied as a eukaryotic viral vector. As these vectors have evolved from first-generation vectors to vectors that contain either very few or no viral genes ("gutless" Ad), significant reductions in the host innate immune response upon infection have been observed. Regardless of these vector improvements an unknown amount of toxicity has been associated with the virion structural proteins. Here we demonstrate the ability to generate high particle numbers (10[sup 11] to 10[sup 12]) of Ad empty virions based on a modification of Cre/lox gutless Ad vectors. Using a battery of analyses (electron microscopy, atomic force microscopy, confocal images, and competition assays) we characterized this reagent and determined that it (i) makes intact virion particles, (ii) competes for receptor binding with wild-type Ad, and (iii) enters the cell proficiently, demonstrating an ability to carry out essential steps of viral entry. To further study the biological impact of these Ad empty virions on infected cells, we carried out DNA microarray analysis. Compared to that for recombinant Ad, the number of mRNAs modulated upon infection was significantly reduced but the expression signatures were similar. This reagent provides a valuable tool for studies of Ad in that researchers can examine the effect of infection in the presence of the virion capsid alone. [ABSTRACT FROM AUTHOR]

Recentstudies have shown that wild-type and recombinant adeno-associated virus (AAV and rAAV) genomes persist in human tissue predominantly as double-stranded (ds) circular episomes derived from input linear single-stranded virion DNA. Using self-complementary recombinant AAV (scAAV) vectors, we generated intermediates that directly transition to ds circular episomes. The scAAV genome ends are palindromic hairpin-structured terminal repeats, resembling a double-stranded break repair intermediate. Utilizing this substrate, we found cellular DNA recombination and repair factors to be essential for generating circular episomal products. To identify the specific cellular proteins involved, the scAAV circularization-dependent vector was used as a reporter in 19 mammalian DNA repair-deficient cell lines. The results show that RecQ helicase family members (BLM and WRN), Mre11 and NBS1 of the Mre11-Rad50-Nbs1 (MRN) complex, and ATM are required for efficient scAAV genome circularization. We further demonstrated that the scAAV genome requires ATM and DNA-PKCS, but not NBS1, to efficiently convert to a circular form in nondividing cells in vivo using transgenic mice. These studies identify specific pathways involved for further elucidating viral and cellular mechanisms of DNA maintenance important to the viral life cycle and vector utilizations. [ABSTRACT FROM AUTHOR]

An abstract of the article "Defined Vector Substrates Used To Probe rAAV DNA Integration," by Douglas M. McCarty, Vivian Choi, Alda Fernandes and R. J. Samulski is presented.

An abstract of the article "Facilitation of AAV Genome Circularization by Host Cell Factors Involved in DNA Replication and Double-Strand Break Repair," by Vivian W. Choi, Douglas M. McCarty and Richard J. Samulski is presented.

Hairpin DNA ends are evolutionarily conserved intermediates in DNA recombination. The hairpin structures present on the ends of the adeno-associated virus (AAV) genome are substrates for recombination that give rise to persistent circular and concatemeric DNA episomes through intramolecular and intermolecular recombination, respectively. We have developed circularization-dependent and orientation-specific self-complementary AAV (scAAV) vectors as a reporter system to examine recombination events involving distinct hairpin structures, i.e., closed versus open hairpins. The results suggest that intramolecular recombination (circularization) is far more efficient than intermolecular recombination (concatemerization). Among all possible combinations of terminal repeats (TRs) involved in intermolecular recombination, the closed-closed TR structures are twice as efficient as the open-open TR substrates for recombination. In addition, both intramolecular recombination and intermolecular recombination exhibit the common dependency on specific DNA polymerases and topoisomerases. The circularization-dependent and orientation-specific scAAV vectors can serve as an efficient and controlled system for the delivery of DNA structures that mimic mammalian recombination intermediates and should be useful in assaying recombination in different experimental settings as well as elucidating the molecular mechanism of recombinant AAV genome persistence. [ABSTRACT FROM AUTHOR]

Mucopolysaccharidosis (MPS) IIIA is a neuropathic lysosomal storage disease caused by deficiency in N-sulfoglucosamine sulfohydrolase (SGSH). Genome-wide gene expression microarrays in MPS IIIA mice detected broad molecular abnormalities (greater than or equal to twofold, false discovery rate ≤10) in numerous transcripts (314) in the brain and blood (397). Importantly, 22 dysregulated blood transcripts are known to be enriched in the brain and linked to broad neuronal functions. To target the root cause, we used a self-complementary AAVrh74 vector to deliver the human SGSH gene into 4-6 weeks old MPS IIIA mice by an intravenous injection. The treatment resulted in global central nervous system (CNS) and widespread somatic restoration of SGSH activity, clearance of CNS and somatic glycosaminoglycan storage, improved behavior performance, and significantly extended survival. The scAAVrh74-hSGSH treatment also led to the correction of the majority of the transcriptional abnormalities in the brain (95.9%) and blood (97.7%), of which 182 and 290 transcripts were normalized in the brain and blood, respectively. These results demonstrate that a single systemic scAAVrh74-hSGSH delivery mediated efficient restoration of SGSH activity and resulted in a near complete correction of MPS IIIA molecular pathology. This study also demonstrates that blood transcriptional profiles reflect the biopathological status of MPS IIIA, and also respond well to effective treatments. [ABSTRACT FROM AUTHOR]

To date, little is known regarding the etiology and disease mechanisms of Alzheimer's disease (AD). There is a general urgency for novel approaches to advance AD research. In this study, we analyzed blood RNA from female patients with advanced AD and matched healthy controls using genome-wide gene expression microarrays. Our data showed significant alterations in 3,944 genes (≥⃒2-fold, FDR ≤1%) in AD whole blood, including 2,932 genes that are involved in broad biological functions. Importantly, we observed abnormal transcripts of numerous tissue-specific genes in AD blood involving virtually all tissues, especially the brain. Of altered genes, 157 are known to be essential in neurological functions, such as neuronal plasticity, synaptic transmission and neurogenesis. More importantly, 205 dysregulated genes in AD blood have been linked to neurological disease, including AD/dementia and Parkinson's disease, and 43 are known to be the causative genes of 42 inherited mental retardation and neurodegenerative diseases. The detected transcriptional abnormalities also support robust inflammation, profound extracellular matrix impairments, broad metabolic dysfunction, aberrant oxidative stress, DNA damage, and cell death. While the mechanisms are currently unclear, this study demonstrates strong blood-brain correlations in AD. The blood transcriptional profiles reflect the complex neuropathological status in AD, including neuropathological changes and broad somatic impairments. The majority of genes altered in AD blood have not previously been linked to AD. We believe that blood genome-wide transcriptional profiling may provide a powerful and minimally invasive tool for the identification of novel targets beyond Aβ and tauopathy for AD research. [ABSTRACT FROM AUTHOR]

Mucopolysaccharidosis (MPS) IIIB is a devastating neuropathic lysosomal storage disease with complex pathology. This study identifies molecular signatures in peripheral blood that may be relevant to MPS IIIB pathogenesis using a mouse model. Genome-wide gene expression microarrays on pooled RNAs showed dysregulation of 2,802 transcripts in blood from MPS IIIB mice, reflecting pathological complexity of MPS IIIB, encompassing virtually all previously reported and as yet unexplored disease aspects. Importantly, many of the dysregulated genes are reported to be tissue-specific. Further analyses of multiple genes linked to major pathways of neurodegeneration demonstrated a strong brain-blood correlation in amyloidosis and synucleinopathy in MPS IIIB. We also detected prion protein (Prnp) deposition in the CNS and Prnp dysregulation in the blood in MPS IIIB mice, suggesting the involvement of Prnp aggregation in neuropathology. Systemic delivery of trans-BBB-neurotropic rAAV9-hNAGLU vector mediated not only efficient restoration of functional α-N-acetylglucosaminidase and clearance of lysosomal storage pathology in the central nervous system (CNS) and periphery, but also the correction of impaired neurodegenerative molecular pathways in the brain and blood. Our data suggest that molecular changes in blood may reflect pathological status in the CNS and provide a useful tool for identifying potential CNS-specific biomarkers for MPS IIIB and possibly other neurological diseases. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated virus (rAAV) vectors have gained an extensive record of safety and efficacy in animal models of human disease. Infrequent reports of genotoxicity have been limited to specific vectors associated with excess hepatocellular carcinomas (HCC) in mice. In order to understand potential mechanisms of genotoxicity, and identify patterns of insertion that could promote tumor formation, we compared a self-complementary AAV (scAAV) vector designed to promote insertional activation (scAAV-CBA-null) to a conventional scAAV-CMV-GFP vector. HCC-prone C3H/HeJ mice and severe combined immunodeficiency (SCID) mice were infected with vector plus secondary treatments including partial hepatectomy (HPX) and camptothecin (CPT) to determine the effects of cell cycling and DNA damage on tumor incidence. Infection with either vector led to a significant increase in HCC incidence in male C3H/HeJ mice. Partial HPX after infection reduced HCC incidence in the cytomegalovirus-green fluorescent protein (CMV-GFP)-infected mice, but not in the cognate chicken β-actin (CBA)-null infected group. Tumors from CBA-null infected, hepatectomized mice were more likely to contain significant levels of vector DNA than tumors from the corresponding CMV-GFP-infected group. Most CBA-null vector insertions recovered from tumors were associated with known proto-oncogenes or tumor suppressors. Specific patterns of insertion suggested read-through transcription, enhancer effects, and disruption of tumor suppressors as likely mechanisms for genotoxicity. [ABSTRACT FROM AUTHOR]

The lysosomal storage pathology in Mucopolysaccharidosis (MPS) IIIB manifests in cells of virtually all organs. However, it is the profound role of the neurological pathology that leads to morbidity and mortality in this disease, and has been the major challenge to developing therapies. To date, MPS IIIB neuropathologic and therapeutic studies have focused predominantly on changes in the central nervous system (CNS), especially in the brain, and little is known about the disease pathology in the peripheral nervous system (PNS). This study demonstrates characteristic lysosomal storage pathology in dorsal root ganglia affecting neurons, satellite cells (glia) and Schwann cells. Lysosomal storage lesions were also observed in the myoenteric plexus and submucosal plexus, involving enteric neurons with enteric glial activation. Further, MPS IIIB mice developed progressive impairments in sensory functions, with significantly reduced response to pain stimulation that became detectable at 4--5 months of age as the disease progressed. These data demonstrate that MPS IIIB neuropathology manifests not only in the entire CNS but also the PNS, likely affecting both afferent and efferent neural signal transduction. This study also suggests that therapeutic development for MPS IIIB may benefit from targeting the entire nervous system. [ABSTRACT FROM AUTHOR]

The greatest challenge in developing therapies for mucopolysaccharidosis (MPS) IIIB is to achieve efficient central nervous system (CNS) delivery across the blood-brain barrier (BBB). In this study, we used the novel ability of adeno-associated virus serotype 9 (AAV9) to cross the BBB from the vasculature to achieve long-term global CNS, and widespread somatic restoration of α-N-acetylglucosaminidase (NAGLU) activity. A single intravenous (IV) injection of rAAV9-CMV-hNAGLU, without extraneous treatment to disrupt the BBB, restored NAGLU activity to normal or above normal levels in adult MPS IIIB mice, leading to the correction of lysosomal storage pathology in the CNS and periphery, and correction of astrocytosis and neurodegeneration. The IV delivered rAAV9 vector also transduced abundant neurons in the myenteric and submucosal plexus, suggesting peripheral nervous system (PNS) targeting. While CNS entry did not depend on osmotic disruption of the BBB, it was significantly enhanced by pretreatment with an IV infusion of mannitol. Most important, we demonstrate that a single systemic rAAV9-NAGLU gene delivery provides long-term (>18 months) neurological benefits in MPS IIIB mice, resulting in significant improvement in behavioral performance, and extension of survival. These data suggest promising clinical potential using the trans-BBB neurotropic rAAV9 vector for treating MPS IIIB and other neurogenetic diseases. [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB) is a great obstacle to the development of therapies for many neurological diseases, especially those globally involving central nervous system (CNS). Mannitol is a known osmotic BBB disruption reagent, which can facilitate the temporary opening of the BBB and enable CNS entry of peripherally delivered therapeutic materials, including rAAV vectors, within a limited time frame. We have optimized the timing of vector infusion, following mannitol pretreatment, to achieve maximum entry of rAAV into the CNS of adult wildtype mice (6-8 months of age). At times ranging from 5-20 minutes (at 1 minute intervals, n≥4/group) after an IV infusion of mannitol (1-2mg/g bw), animals received an IV injection of 1×1011 genome-containing particles of self-complementary rAAV2 viral vector (scAAV2-CMV-GFP) expressing green fluorescent protein (GFP). Four weeks after the injection, vibratome sections (50mm) of whole brain and spinal cord were collected and evaluated using immunofluorescence to detected GFP expression. Vector transgene expression was observed in both neuronal and non-neuronal cells throughout the brain and spinal cord. We found that optimal transduction was observed in the CNS when the vector was IV injected 8 minutes after the administration of mannitol, with GFP expression detected in 1.2-1.5×106 brain cells. This was approximately 10-fold higher than the CNS transduction observed when the vector was injected at either 5 or 10 minutes after mannitol pretreatment, and 2000-fold higher than when the vector was given at 20 minutes post-mannitol infusion. We believe that this optimized injection regimen can be applied to therapeutic protocols for a broad range of CNS diseases, with the potential of greatly improving the global CNS delivery of viral vectors.Molecular Therapy (2006) 13, S191–S191; doi: 10.1016/j.ymthe.2006.08.564 [ABSTRACT FROM AUTHOR]

Background: Recently, using a mouse model of mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease with severe neurological deterioration, we showed that MPS IIIB neuropathology is accompanied by a robust neuroinflammatory response of unknown consequence. This study was to assess whether MPS IIIB lymphocytes are pathogenic. Methods: Lymphocytes from MPS IIIB mice were adoptively transferred to naïve wild-type mice. The recipient animals were then evaluated for signs of disease and inflammation in the central nervous system. Results: Our results show for the first time, that lymphocytes isolated from MPS IIIB mice caused a mild paralytic disease when they were injected systemically into naïve wild-type mice. This disease is characterized by mild tail and lower trunk weakness with delayed weight gain. The MPS IIIB lymphocytes also trigger neuroinflammation within the CNS of recipient mice characterized by an increase in transcripts of IL2, IL4, IL5, IL17, TNFα, IFNα and Ifi30, and intraparenchymal lymphocyte infiltration. Conclusions: Our data suggest that an autoimmune response directed at CNS components contributes to MPS IIIB neuropathology independent of lysosomal storage pathology. Adoptive transfer of purified T-cells will be needed in future studies to identify specific effector T-cells in MPS IIIB neuroimmune pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Intrathecal (IT) gene transfer is an attractive approach for targeting spinal mechanisms of nociception but the duration of gene expression achieved by reported methods is short (up to two weeks) impairing their utility in the chronic pain setting. The overall goal of this study was to develop IT gene transfer yielding true long-term transgene expression defined as ≥ 3 mo following a single vector administration. We defined "IT" administration as atraumatic injection into the lumbar cerebrospinal fluid (CSF) modeling a lumbar puncture. Our studies focused on recombinant adeno-associated virus (rAAV), one of the most promising vector types for clinical use. Results: Conventional single stranded rAAV2 vectors performed poorly after IT delivery in rats. Pseudotyping of rAAV with capsids of serotypes 1, 3, and 5 was tested alone or in combination with a modification of the inverted terminal repeat. The former alters vector tropism and the latter allows packaging of self-complementary rAAV (sc-rAAV) vectors. Combining both types of modification led to the identification of sc-rAAV2/l as a vector that performed superiorly in the IT space. IT delivery of 3 × 10e9 sc-rAAV2/l particles per animal led to stable expression of enhanced green fluorescent protein (EGFP) for ≥ 3 mo detectable by Western blotting, quantitative PCR, and in a blinded study by confocal microscopy. Expression was strongest in the cauda equina and the lower sections of the spinal cord and only minimal in the forebrain. Microscopic examination of the SC fixed in situ with intact nerve roots and meninges revealed strong EGFP fluorescence in the nerve roots. Conclusion: sc-rAAVl mediates stable IT transgene expression for ≥ 3 mo. Our findings support the underlying hypothesis that IT target cells for gene transfer lack the machinery for efficient conversion of the single-stranded rAAV genome into double-stranded DNA and favor uptake of serotype 1 vectors over 2. Experiments presented here will provide a rational basis for utilizing IT rAAV gene transfer in basic and translational studies on chronic pain. [ABSTRACT FROM AUTHOR]

The blood–brain barrier is the main obstacle to efficient delivery of therapeutic reagents, including viral vectors, into the central nervous system (CNS) for treating global CNS diseases. In this study, the effects of mannitol infusions on global brain gene expression of a novel AAV vector were examined after intravenous (iv) or intracisternal injection. Initially, a self-complementary adeno-associated virus serotype 2 vector (scAAV) was compared to traditional single-stranded AAV2 vector for reporter gene expression in the brain of adult mice with or without pretreatment of an iv mannitol infusion. One to two months postinjection, analysis of vector-transduced green fluorescent protein (GFP) expression in the brain revealed that vector delivery to the CNS via iv injection required pretreatment with mannitol. This expression was observed only when scAAV vectors were used. Using these conditions, transgene expression was observed in various neurons and glial cells throughout the brain. The peripherally administered scAAV vectors also transduced the cells in multiple somatic tissues with efficient expression in liver (20–30% of hepatocytes), but was less efficient in other somatic tissues. Intracisternal injection of scAAV vector produced a broad and intense transgene expression in both neurons and glial cells in the CNS of injected mice ranging from the olfactory area to the brain stem and spinal cord. More than 50% of the Purkinje cells in the cerebellum expressed GFP. Intravenous infusion of mannitol before intracisternal injection of the scAAV vector enhanced the dispersion of the vector in the CNS. Further optimization of these steps combining peripheral and intracisternal scAAV gene delivery should facilitate the development of treatments for global CNS diseases, especially diseases involving both the somatic system and the CNS, such as lysosomal storage disorders. [Copyright &y& Elsevier]

An abstract of the article "Optimization of AAV Serotype and Promoter for Increased Distribution of Expression in Both Somatic and Central Nervous Systems of Mice," by Haiyan Fu, Joseph Muenzer and colleagues is presented.