Evaluating the state of the science for adeno-associated virus integration: An integrated perspective.

On August 18, 2021, the American Society of Gene and Cell Therapy (ASGCT) hosted a virtual roundtable on adeno-associated virus (AAV) integration, featuring leading experts in preclinical and clinical AAV gene therapy, to further contextualize and understand this phenomenon. Recombinant AAV (rAAV) vectors are used to develop therapies for many conditions given their ability to transduce multiple cell types, resulting in long-term expression of transgenes. Although most rAAV DNA typically remains episomal, some rAAV DNA becomes integrated into genomic DNA at a low frequency, and rAAV insertional mutagenesis has been shown to lead to tumorigenesis in neonatal mice. Currently, the risk of rAAV-mediated oncogenesis in humans is theoretical because no confirmed genotoxic events have been reported to date. However, because insertional mutagenesis has been reported in a small number of murine studies, there is a need to characterize this genotoxicity to inform research, regulatory needs, and patient care. The purpose of this white paper is to review the evidence of rAAV-related host genome integration in animal models and possible risks of insertional mutagenesis in patients. In addition, technical considerations, regulatory guidance, and bioethics are discussed.

Targeting Root Cause by Systemic scAAV9-hIDS Gene Delivery: Functional Correction and Reversal of Severe MPS II in Mice.

No treatment is available to address the neurological need and reversibility of MPS II. We developed a scAAV9-hIDS vector to deliver the human iduronate-2-sulfatase gene and test it in mouse model. We treated MPS II mice at different disease stages with an intravenous injection of scAAV9-mCMV-hIDS at different doses. The treatments led to rapid and persistent restoration of IDS activity and the reduction of glycosaminoglycans (GAG) throughout the CNS and somatic tissues in all cohorts. Importantly, the vector treatment at up to age 6 months improved behavior performance in the Morris water maze and normalized the survival. Notably, vector treatment at age 9 months also resulted in persistent rIDS expression and GAG clearance in MPS II mice, and the majority of these animals survived within the normal range of lifespan. Notably, the vector delivery did not result in any observable adverse events or detectable systemic toxicity in any treated animal groups. We believe that we have developed a safe and effective gene therapy for treating MPS II, which led to recent IND approval for a phase 1/2 clinical trial in MPS II patients, further supporting the extended potential of the demonstrated systemic rAAV9 gene delivery platform for broad disease targets.

Differential Prevalence of Antibodies Against Adeno-Associated Virus in Healthy Children and Patients with Mucopolysaccharidosis III: Perspective for AAV-Mediated Gene Therapy.

Recombinant adeno-associated virus (AAV) vectors are promising gene therapy tools. However, pre-existing antibodies (Abs) to many useful AAV serotypes pose a critical challenge for the translation of gene therapies. As part of AAV gene therapy program for treating mucopolysaccharidosis (MPS) III patients, the seroprevalence profiles of AAV1-9 and rh74 were investigated in MPS IIIA/IIIB patients and in healthy children. Using enzyme-linked immunosorbent assay for αAAV-IgG, significantly higher seroprevalence was observed for AAV1 and AAVrh74 in 2- to 7-year-old MPS III patients than in healthy controls. Seroprevalence for the majority of tested AAV serotypes appears to peak before 8 years of age in MPS III subjects, with the exception of increases in αAAV8 and αAAV9 Abs in 8- to 19-year-old MPS IIIA patients. In contrast, significant increases in seroprevalence were observed for virtually all tested AAV serotypes in 8- to 15-year-old healthy children compared to 2- to 7-year-olds. Co-prevalence and Ab level correlation results followed the previously established divergence-based clade positions of AAV1-9. Interestingly, the individuals positive for αAAVrh74-Abs showed the lowest co-prevalence with Abs for AAV1-9 (22-40%). However, all or nearly all (77-100%) of subjects who were seropositive for any of serotypes 1-9 were also positive for αAAVrh74-IgG. Notably, the majority (78%) of αAAV seropositive individuals were also Ab-positive for one to five of the tested AAV serotypes, mostly with low levels of αAAV-Abs (1:50-100), while a minority (22%) were seropositive for six or more AAV serotypes, mostly with high levels of αAAV-IgG for multiple serotypes. In general, the highest IgG levels were reactive to AAV2, AAV3, and AAVrh74. The data illustrate the complex seroprevalence profiles of AAV1-9 and rh74 in MPS patients and healthy children, indicating the potential association of AAV seroprevalence with age and disease conditions. The broad co-prevalence of Abs for different AAV serotypes reinforces the challenge of pre-existing αAAV-Abs for translating AAV gene therapy to clinical applications, regardless of the vector serotype.

Serum global metabolomics profiling reveals profound metabolic impairments in patients with MPS IIIA and MPS IIIB.

The monogenic defects in specific lysosomal enzymes in mucopolysaccharidosis (MPS) III lead to lysosomal storage of glycosaminoglycans and complex CNS and somatic pathology, for which the detailed mechanisms remain unclear. In this study, serum samples from patients with MPS IIIA (age 2-9 yr) and MPS IIIB (2-13 yr) and healthy controls (age 2-9 yr) were assayed by global metabolomics profiling of 658 metabolites using mass spectrometry. Significant alterations were detected in 423 metabolites in all MPS III patients, of which 366 (86.5%) decreased and 57 (13.5%) increased. Similar profiles were observed when analyzing data from MPS IIIA and MPS IIIB samples separately, with only limited age variations in 36 metabolites. The observed metabolic disturbances in MPS III patients involve virtually all major pathways of amino acid (101/150), peptide (17/21), carbohydrate (19/23), lipid (221/325), nucleotide (15/25), energy (8/9), vitamins and co-factors (8/21), and xenobiotics (34/84) metabolism. Notably, detected serum metabolite decreases involved all key amino acids, all major neurotransmitter pathways, and broad neuroprotective compounds. The elevated metabolites are predominantly lipid derivatives, and also include cysteine metabolites and a fibrinogen peptide fragment, consistent with the status of oxidative stress and inflammation in MPS III. This study demonstrates that the lysosomal glycosaminoglycans storage triggers profound metabolic disturbances in patients with MPS III disorders, leading to severe functional depression of virtually all metabolic pathways, which emerge early during the disease progression. Serum global metabolomics profiling may provide an important and minimally invasive tool for better understanding the disease mechanisms and identification of potential biomarkers for MPS III.

Effective Depletion of Pre-existing Anti-AAV Antibodies Requires Broad Immune Targeting.

Pre-existing antibodies (Abs) to AAV pose a critical challenge for the translation of gene therapies. No effective approach is available to overcome pre-existing Abs. Given the complexity of Ab production, overcoming pre-existing Abs will require broad immune targeting. We generated a mouse model of pre-existing AAV9 Abs to test multiple immunosuppressants, including bortezomib, rapamycin, and prednisolone, individually or in combination. We identified an effective approach combining rapamycin and prednisolone, reducing serum AAV9 Abs by 70%-80% at 4 weeks and 85%-93% at 8 weeks of treatment. The rapamycin plus prednisolone treatment resulted in significant decreases in the frequency of B cells, plasma cells, and IgG-secreting and AAV9-specific Ab-producing plasma cells in bone marrow. The rapamycin plus prednisolone treatment also significantly reduced frequencies of IgD-IgG+ class-switched/FAS+CL7+ germinal center B cells, and of activated CD4+ T cells expressing PD1 and GL7, in spleen. These data suggest that rapamycin plus prednisolone has selective inhibitory effects on both T helper type 2 support of B cell activation in spleen and on bone marrow plasma cell survival, leading to effective AAV9 Abs depletion. This promising immunomodulation approach is highly translatable, and it poses minimal risk in the context of therapeutic benefits promised by gene therapy for severe monogenetic diseases, with a single or possibly a few treatments over a lifetime.

Near-Complete Correction of Profound Metabolomic Impairments Corresponding to Functional Benefit in MPS IIIB Mice after IV rAAV9-hNAGLU Gene Delivery.

Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease with complex CNS and somatic pathology due to a deficiency in α-N-acetylglucosaminidase (NAGLU). Using global metabolic profiling by mass spectrometry targeting 361 metabolites, this study detected significant decreases in 225 and increases in six metabolites in serum samples from 7-month-old MPS IIIB mice, compared to wild-type (WT) mice. The metabolic disturbances involve virtually all major pathways of amino acid, peptide (58/102), carbohydrate (18/28), lipid (111/139), nucleotide (12/24), energy (2/9), vitamin and cofactor (11/16), and xenobiotic (11/28) metabolism. Notably, the reduced metabolites included eight essential amino acids, vitamins (C, E, B2, and B6), and neurotransmitters (serotonin, glutamate, aspartate, tryptophan, and N-acetyltyrosine). The metabolic impairments appear to emerge early during disease progression before the age of 2 months. Importantly, the restoration of NAGLU activity with an intravenous (i.v.) injection of rAAV9-hNAGLU vector led to near-complete correction of all serum metabolite abnormalities, with 201 (87%) metabolites normalized and 30 (13%) over-corrected. While the mechanisms are unclear, our data demonstrate that the lack of NAGLU activity triggers profound functional metabolic disturbances in MPS IIIB. These metabolic impairments respond well to a systemic rAAV9-hNAGLU gene delivery, supporting the surrogate biomarker potential of serum metabolomic profiles for MPS IIIB therapies.

Crossing the blood-brain-barrier with viral vectors.

The abundant vasculature of the CNS provides a compelling route of administration for the delivery of gene therapy vectors if the limitations imposed by the blood-brain-barrier (BBB) can be overcome. There are two general approaches to transporting viral vectors across the BBB: either by transient disruption of brain microvasculature endothelial tight junctions, or through the use of receptor-mediated transcytosis. Advances in BBB disruption have led to pre-clinical success for both global and localized gene delivery, while therapies based on receptor-mediated transcytosis have recently advanced to phase I clinical trials in humans. Both approaches show long term promise for treating a wide range of CNS diseases.

Functional correction of neurological and somatic disorders at later stages of disease in MPS IIIA mice by systemic scAAV9-hSGSH gene delivery.

The reversibility of neuropathic lysosomal storage diseases, including MPS IIIA, is a major goal in therapeutic development, due to typically late diagnoses and a large population of untreated patients. We used self-complementary adeno-associated virus (scAAV) serotype 9 vector expressing human N-sulfoglucosamine sulfohydrolase (SGSH) to test the efficacy of treatment at later stages of the disease. We treated MPS IIIA mice at 1, 2, 3, 6, and 9 months of age with an intravenous injection of scAAV9-U1a-hSGSH vector, leading to restoration of SGSH activity and reduction of glycosaminoglycans (GAG) throughout the central nervous system (CNS) and somatic tissues at a dose of 5E12 vg/kg. Treatment up to 3 months age improved learning ability in the Morris water maze at 7.5 months, and lifespan was normalized. In mice treated at 6 months age, behavioral performance was impaired at 7.5 months, but did not decline further when retested at 12 months, and lifespan was increased, but not normalized. Treatment at 9 months did not increase life-span, though the GAG storage pathology in the CNS was improved. The study suggests that there is potential for gene therapy intervention in MPS IIIA at intermediate stages of the disease, and extends the clinical relevance of our systemic scAAV9-hSGSH gene delivery approach.

A GLP-Compliant Toxicology and Biodistribution Study: Systemic Delivery of an rAAV9 Vector for the Treatment of Mucopolysaccharidosis IIIB.

No treatment is currently available for mucopolysaccharidosis (MPS) IIIB, a neuropathic lysosomal storage disease due to defect in α-N-acetylglucosaminidase (NAGLU). In preparation for a clinical trial, we performed an IND-enabling GLP-toxicology study to assess systemic rAAV9-CMV-hNAGLU gene delivery in WT C57BL/6 mice at 1 × 10(14) vg/kg and 2 × 10(14) vg/kg (n = 30/group, M:F = 1:1), and non-GLP testing in MPS IIIB mice at 2 × 10(14) vg/kg. Importantly, no adverse clinical signs or chronic toxicity were observed through the 6 month study duration. The rAAV9-mediated rNAGLU expression was rapid and persistent in virtually all tested CNS and somatic tissues. However, acute liver toxicity occurred in 33% (5/15) WT males in the 2 × 10(14) vg/kg cohort, which was dose-dependent, sex-associated, and genotype-specific, likely due to hepatic rNAGLU overexpression. Interestingly, a significant dose response was observed only in the brain and spinal cord, whereas in the liver at 24 weeks postinfection (pi), NAGLU activity was reduced to endogenous levels in the high dose cohort but remained at supranormal levels in the low dose group. The possibility of rAAV9 germline transmission appears to be minimal. The vector delivery resulted in transient T-cell responses and characteristic acute antibody responses to both AAV9 and rNAGLU in all rAAV9-treated animals, with no detectable impacts on tissue transgene expression. This study demonstrates a generally safe and effective profile, and may have identified the upper dosing limit of rAAV9-CMV-hNAGLU via systemic delivery for the treatment of MPS IIIB.

Broad functional correction of molecular impairments by systemic delivery of scAAVrh74-hSGSH gene delivery in MPS IIIA mice.

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.

Blood genome-wide transcriptional profiles reflect broad molecular impairments and strong blood-brain links in Alzheimer's disease.

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 Aß and tauopathy for AD research.

Feasibility and safety of systemic rAAV9-hNAGLU delivery for treating mucopolysaccharidosis IIIB: toxicology, biodistribution, and immunological assessments in primates.

No treatment is currently available for mucopolysaccharidosis (MPS) IIIB, a neuropathic lysosomal storage disease caused by autosomal recessive defect in α-N-acetylglucosaminidase (NAGLU). In anticipation of a clinical gene therapy treatment for MPS IIIB in humans, we tested the rAAV9-CMV-hNAGLU vector administration to cynomolgus monkeys (n=8) at 1E13 vg/kg or 2E13 vg/kg via intravenous injection. No adverse events or detectable toxicity occurred over a 6-month period. Gene delivery resulted in persistent global central nervous system and broad somatic transduction, with NAGLU activity detected at 2.9-12-fold above endogenous levels in somatic tissues and 1.3-3-fold above endogenous levels in the brain. Secreted rNAGLU was detected in serum. Low levels of preexisting anti-AAV9 antibodies (Abs) did not diminish vector transduction. Importantly, high-level preexisting anti-AAV9 Abs lead to reduced transduction in liver and other somatic tissues, but had no detectable impact on transgene expression in the brain. Enzyme-linked immunoabsorbent assay showed Ab responses to both AAV9 and rNAGLU in treated animals. Serum anti-hNAGLU Abs, but not anti-AAV9 Abs, correlated with the loss of circulating rNAGLU enzyme. However, serum Abs did not affect tissue rNAGLU activity levels. Weekly or monthly peripheral blood interferon-γ enzyme-linked immunospot assays detected a CD4(+) T-cell (Th-1) response to rNAGLU only at 4 weeks postinjection in one treated subject, without observable correlation to tissue transduction levels. The treatment did not result in detectable CTL responses to either AAV9 or rNAGLU. Our data demonstrate an effective and safe profile for systemic rAAV9-hNAGLU vector delivery in nonhuman primates, supporting its clinical potential in humans.

Amyloidosis, synucleinopathy, and prion encephalopathy in a neuropathic lysosomal storage disease: the CNS-biomarker potential of peripheral blood.

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.

Peripheral nervous system neuropathology and progressive sensory impairments in a mouse model of Mucopolysaccharidosis IIIB.

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.

Patterns of scAAV vector insertion associated with oncogenic events in a mouse model for genotoxicity.

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 ß-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.

Correction of neurological disease of mucopolysaccharidosis IIIB in adult mice by rAAV9 trans-blood-brain barrier gene delivery.

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.

Differential distribution of heparan sulfate glycoforms and elevated expression of heparan sulfate biosynthetic enzyme genes in the brain of mucopolysaccharidosis IIIB mice.

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.

Mucopolysaccharidosis IIIB, a lysosomal storage disease, triggers a pathogenic CNS autoimmune response.

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, TNFalpha, IFNalpha 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.

Restoration of central nervous system alpha-N-acetylglucosaminidase activity and therapeutic benefits in mucopolysaccharidosis IIIB mice by a single intracisternal recombinant adeno-associated viral type 2 vector delivery.

BACKGROUND: Finding efficient central nervous system (CNS) delivery approaches has been the major challenge facing therapeutic development for treating diseases with global neurological manifestation, such as mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease, caused by autosomal recessive defect of alpha-N-acetylglucosaminidase (NaGlu). Previously, we developed an approach, intracisternal (i.c.) injection, to deliver recombinant adeno-associated viral (rAAV) vector to the CNS of mice, leading to a widespread periventricular distribution of transduction. METHODS: In the present study, we delivered rAAV2 vector expressing human NaGlu into the CNS of MPS IIIB mice by an i.c. injection approach, to test its therapeutic efficacy and feasibility for treating the neurological manifestation of the disease. RESULTS: We demonstrated significant functional neurological benefits of a single i.c. vector infusion in adult MPS IIIB mice. The treatment slowed the disease progression by mediating widespread recombinant NaGlu expression in the CNS, resulting in the reduction of brain lysosomal storage pathology, significantly improved cognitive function and prolonged survival. However, persisting motor function deficits suggested that pathology in areas outside the CNS contributes to the MPS IIIB behavioral phenotype. The therapeutic benefit of i.c. rAAV2 delivery was dose-dependent and could be attribute solely to the CNS transduction because the procedure did not lead to detectable transduction in somatic tissues. CONCLUSIONS: A single IC rAAV2 gene delivery is functionally beneficial for treating the CNS disease of MPS IIIB in mice. It is immediately clinically translatable, with the potential of improving the quality of life for patients with MPS IIIB.