Pedro E. Cruz

Controlling neuropathic pain by adeno-associated virus driven production of the anti-inflammatory cytokine, interleukin-10

Despite many decades of drug development, effective therapies for neuropathic pain remain elusive. The recent recognition of spinal cord glia and glial pro-inflammatory cytokines as important contributors to neuropathic pain suggests an alternative therapeutic strategy; that is, targeting glial activation or its downstream consequences. While several glial-selective drugs have been successful in controlling neuropathic pain in animal models, none are optimal for human use. Thus the aim of the present studies was to explore a novel approach for controlling neuropathic pain. Here, an adeno-associated viral (serotype II; AAV2) vector was created that encodes the anti-inflammatory cytokine, interleukin-10 (IL-10). This anti-inflammatory cytokine is known to suppress the production of pro-inflammatory cytokines. Upon intrathecal administration, this novel AAV2-IL-10 vector was successful in transiently preventing and reversing neuropathic pain. Intrathecal administration of an AAV2 vector encoding beta-galactosidase revealed that AAV2 preferentially infects meningeal cells surrounding the CSF space. Taken together, these data provide initial support that intrathecal gene therapy to drive the production of IL-10 may prove to be an efficacious treatment for neuropathic pain.

IL-10 Alters Immunoproteostasis in APP Mice, Increasing Plaque Burden and Worsening Cognitive Behavior

Anti-inflammatory strategies are proposed to have beneficial effects in Alzheimers disease. To explore how anti-inflammatory cytokine signaling affects Aβ pathology, we investigated the effects of adeno-associated virus (AAV2/1)-mediated expression of Interleukin (IL)-10 in the brains of APP transgenic mouse models. IL-10 expression resulted in increased Aβ accumulation and impaired memory in APP mice. A focused transcriptome analysis revealed changes consistent with enhanced IL-10 signaling and increased ApoE expression in IL-10-expressing APP mice. ApoE protein was selectively increased in the plaque-associated insoluble cellular fraction, likely because of direct interaction with aggregated Aβ in the IL-10-expressing APP mice. Ex vivo studies also show that IL-10 and ApoE can individually impair glial Aβ phagocytosis. Our observations that IL-10 has an unexpected negative effect on Aβ proteostasis and cognition in APP mouse models demonstrate the complex interplay between innate immunity and proteostasis in neurodegenerative diseases, an interaction we call immunoproteostasis.

Repeated intrathecal injections of plasmid DNA encoding interleukin-10 produce prolonged reversal of neuropathic pain

&NA; Neuropathic pain is a major clinical problem unresolved by available therapeutics. Spinal cord glia play a pivotal role in neuropathic pain, via the release of proinflammatory cytokines. Anti‐inflammatory cytokines, like interleukin‐10 (IL‐10), suppress proinflammatory cytokines. Thus, IL‐10 may provide a means for controlling glial amplification of pain. We recently documented that intrathecal IL‐10 protein resolves neuropathic pain, albeit briefly (˜2–3 h), given its short half‐life. Intrathecal gene therapy using viruses encoding IL‐10 can also resolve neuropathic pain, but for only ˜2 weeks. Here, we report a novel approach that dramatically increases the efficacy of intrathecal IL‐10 gene therapy. Repeated intrathecal delivery of plasmid DNA vectors encoding IL‐10 (pDNA‐IL‐10) abolished neuropathic pain for greater than 40 days. Naked pDNA‐IL‐10 reversed chronic constriction injury (CCI)‐induced allodynia both shortly after nerve injury as well as 2 months later. This supports that spinal proinflammatory cytokines are important in both the initiation and maintenance of neuropathic pain. Importantly, pDNA‐IL‐10 gene therapy reversed mechanical allodynia induced by CCI, returning rats to normal pain responsiveness, without additional analgesia. Together, these data suggest that intrathecal IL‐10 gene therapy may provide a novel approach for prolonged clinical pain control.

IL-10 Suppresses Chemokines, Inflammation, and Fibrosis in a Model of Chronic Renal Disease

IL-10 is a pluripotent cytokine that plays a pivotal role in the regulation of immune and inflammatory responses. Whereas short-term administration of IL-10 has shown benefit in acute glomerulonephritis, no studies have addressed the potential benefits of IL-10 in chronic renal disease. Chronically elevated blood levels of IL-10 in rats were achieved by administration of a recombinant adeno-associated virus serotype 1 IL-10 (rAAV1-IL-10) vector. Control rats were given a similar dose of rAAV1-GFP. Four weeks after injection, IL-10 levels in serum were measured by ELISA, and chronic renal disease was induced by a 5/6 nephrectomy (n = 6 in each group). Eight weeks later, rats were killed and renal tissue was obtained for RNA, protein, and immunohistochemical analysis. Serum levels of IL-10 were 12-fold greater in the rAAV1-IL-10 group by 4 wk after rAAV1-IL-10 administration (345 +/- 169 versus 28 +/- 15 pg/ml; P = 0.001), and levels were maintained throughout the experiment. rAAV1-IL-10 treatment resulted in less proteinuria (P < 0.05), lower serum creatinine (P < 0.05), and higher creatinine clearances (P < 0.01) compared with rAAV1-GFP-treated rats. Renal interstitial infiltration was significantly attenuated by rAAV1-IL-10 administration as assessed by numbers of CD4+, CD8+, monocyte-macrophages (ED-1+) and dendritic (OX-62+) cells (P < 0.05), and this correlated with reductions in the renal expression of monocyte (renal monocyte chemoattractant protein-1 mRNA and protein) and T cell (RANTES mRNA) chemokines. rAAV1-IL-10 administration decreased mRNA levels of IFN-gamma and IL-2 in the kidney. The reduction in inflammatory cells was associated with a significant reduction in glomerulosclerosis and interstitial fibrosis. It is concluded that IL-10 blocks inflammation and improves renal function in this model of chronic renal disease. The feasibility of long-term overexpression of a gene using the AAV serotype 1 vector system in a model of renal disease is also demonstrated.

In vivo post-transcriptional gene silencing of alpha-1 antitrypsin by adeno-associated virus vectors expressing siRNA

α-1 Antitrypsin (AAT) deficiency is one of the most common genetic diseases in North America, with a carrier frequency of approximately 4% in the US population. Homozygosity for the most common mutation (Glu342Lys, PI*Z) leads to the synthesis of a mutant protein, which accumulates and polymerizes within hepatocytes rather than being efficiently secreted. This lack of secretion causes severe serum deficiency predisposing to chronic lung disease. Twelve to fifteen percent of patients with PI*ZZ also develop liver disease, which can be severe, even in infancy. This is thought to be due to toxic effects of the accumulated mutant Z-AAT within the hepatocyte. Thus, an approach to reduce AAT-deficient liver disease will likely require some mechanism to decrease the amount of Z-AAT within hepatocytes. In this report, we describe studies of small-interfering RNAs (siRNAs) designed to downregulate endogenous AAT within hepatocytes. Three different siRNA sequences were identified and cloned into a recombinant adeno-associated virus (rAAV) backbone, either singly or as a trifunctional (3X) construct. Each had activity independently, but the levels of AAT expression in cell culture models showed the greatest decrease with the 3X construct, resulting in levels that were five-fold lower than controls. The rAAV-3X-siRNA was then packaged into AAV8 capsids and used in vivo to transduce the livers of human Z-AAT overexpressing transgenic mice. Those studies showed a decrease in total human AAT, a clearing of Z-AAT accumulation by immunohistochemistry, and a decrease in monomer Z-AAT within the liver within 3 weeks after vector injection. The rAAV8-3X-siRNA vector may hold promise as a potential therapy for patients with AAT liver disease.

Comparison of Different Bioreactor Systems for the Production of High Titer Retroviral Vectors

Improved, human‐based packaging cell lines allow the production of high‐titer, RCR‐free retroviral vectors. The utility of these cell lines for the production of clinical grade vectors critically depends on the definition of optimal conditions for scaled‐up cultures. In this work, a clone derived from the TE Fly GALV packaging cell (Duisit et al. Hum. Gene Ther. 1999, 10 , 189) that produces high titers of a lacZ containing retroviral vector with a Gibbon Ape Leukemia Virus envelope glycoprotein was used. This clone can produce (2−5) × 106 PFU cm−3 in small scale cultures and has been evaluated for growth and vector production in different reactor systems. The performances of fixed bed reactors [CellCube (Costar) and Celligen (New Brunswick)] and stirred tank reactors [microcarriers and clump cultures] were compared. The cells showed a higher apparent growth rate in the fixed bed reactor systems than in the suspension systems, probably as a result of the fact that aggregation and/or formation of clumps led to a reduced viability and reduced growth of cells in the interior of the clumps. As a consequence, the final cell density and number were in average 3‐ to 7‐fold higher in the fixed bed systems in comparison to the suspension culture systems. The average titers obtained ranged from 0.5 to 2.1 × 107 PFU cm−3 for the fixed bed and microcarrier systems, while the clump cultures produced only (2−5) × 105 PFU cm−3. The differences in titers reflect cell densities as well as specific viral vector production rates, with the immobilization and microcarrier systems exhibiting an at least 10‐fold higher production rate in comparison to the clump cultures. A partial optimization of the culture conditions in the Celligen fixed bed reactor, consisting of a 9‐fold reduction of the seeding cell density, led to a 5‐fold increased vector production rate accompanied by an average titer of 3 × 107 PFU cm−3 (maximum titer (4−5) × 107 PFU cm−3) in the fixed bed reactor. The performance evaluation results using mathematical models indicated that the fixed bed bioreactor has a higher potential for retroviral vector production because of both the higher reactor productivity and the lower sensitivity of productivity in relation to the changes in final retrovirus titer in the range of 3 × 106 to 15 × 106 PFU cm−3.

Type I interferon induction of the Cdk-inhibitor p21WAF1 is accompanied by ordered G1 arrest, differentiation and apoptosis of the Daudi B-cell line.

We show, in this study, that type I IFN induction of the cyclin-dependent kinase (cdk) inhibitor p21WAF1 in the human Burkitt lymphoma B cell-line Daudi and ensuing cell cycle arrest correlate with the terminal differentiation of these cells, and is ultimately followed by apoptosis and cell death. The expression of p21WAF1 paralleled the onset of G1 arrest and the reduction of surface IgM expression which was used as a marker of the differentiation response, and the IFN treated cells acquired a typical plasma cell-like morphology. The type II IFN IFNγ, which does not inhibit the growth of Daudi cells, did not induce the expression of p21WAF1, nor affect the expression of surface IgM. The induction of p21WAF1 which paralleled the inhibition of the phosphorylation of the retinoblastoma protein, pRB, was preceded by the strong reduction in c-myc levels. We propose that the coupled down-regulation of c-myc and induction of p21WAF1 may be crucial to the induction of differentiation and G1 arrest in Daudi cells by type I IFN. Growth arrest and differentiation was followed by apoptosis and cell death, and was accompanied by the induction of the activity of the apoptotic ICE-family protease CPP32. G1 arrest and differentiation followed by apoptotic cell death are characteristics of terminal differentiation. Thus, our data suggest that the induction of p21WAF1 and G1 arrest mediated by type I IFN in Daudi cells is part of terminal differentiation response in these cells, highlighting a role for type I IFN as B cell terminal differentiation factors.

Sirtuin 1 Functionally and Physically Interacts with Disruptor of Telomeric Silencing-1 to Regulate α-ENaC Transcription in Collecting Duct

Aldosterone increases renal tubular Na+ absorption in large part by increasing transcription of the epithelial Na+ channel α-subunit (α-ENaC) expressed in the apical membrane of collecting duct principal cells. We recently reported that a complex containing the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) associates with and represses the α-ENaC promoter in mouse inner medullary collecting duct mIMCD3 cells, and that aldosterone acts to disrupt this complex and its inhibitory effects (Zhang, W., Xia, X., Reisenauer, M. R., Rieg, T., Lang, F., Kuhl, D., Vallon, V., and Kone, B. C. (2007) J. Clin. Invest. 117, 773–783). Here we demonstrate that the NAD+-dependent deacetylase sirtuin 1 (Sirt1) functionally and physically interacts with Dot1 to enhance the distributive activity of Dot1 on H3K79 methylation and thereby represses α-ENaC transcription in mIMCD3 cells. Sirt1 overexpression inhibited basal α-ENaC mRNA expression and α-ENaC promoter activity, surprisingly in a deacetylase-independent manner. The ability of Sirt1 to inhibit α-ENaC transcription was retained in a truncated Sirt1 construct expressing only its N-terminal domain. Conversely, Sirt1 knockdown enhanced α-ENaC mRNA levels and α-ENaC promoter activity, and inhibited global H3K79 methylation, particularly H3K79 trimethylation, in chromatin associated with the α-ENaC promoter. Sirt1 and Dot1 co-immunoprecipitated from mIMCD3 cells and colocalized in the nucleus. Sirt1 immunoprecipitated from chromatin associated with regions of the α-ENaC promoter known to associate with Dot1. Aldosterone inhibited Sirt1 association at two of these regions, as well as Sirt1 mRNA expression, in a coordinate manner with induction of α-ENaC transcription. Overexpressed Sirt1 inhibited aldosterone induction of α-ENaC transcription independent of effects on mineralocorticoid receptor trans-activation. These data identify Sirt1 as a novel modulator of α-ENaC, Dot1, and the aldosterone signaling pathway.

Capsid serotype and timing of injection determines AAV transduction in the neonatal mice brain.

Adeno-associated virus (AAV) mediated gene expression is a powerful tool for gene therapy and preclinical studies. A comprehensive analysis of CNS cell type tropism, expression levels and biodistribution of different capsid serotypes has not yet been undertaken in neonatal rodents. Our previous studies show that intracerebroventricular injection with AAV2/1 on neonatal day P0 results in widespread CNS expression but the biodistribution is limited if injected beyond neonatal day P1. To extend these observations we explored the effect of timing of injection on tropism and biodistribution of six commonly used pseudotyped AAVs delivered in the cerebral ventricles of neonatal mice. We demonstrate that AAV2/8 and 2/9 resulted in the most widespread biodistribution in the brain. Most serotypes showed varying biodistribution depending on the day of injection. Injection on neonatal day P0 resulted in mostly neuronal transduction, whereas administration in later periods of development (24–84 hours postnatal) resulted in more non-neuronal transduction. AAV2/5 showed widespread transduction of astrocytes irrespective of the time of injection. None of the serotypes tested showed any microglial transduction. This study demonstrates that both capsid serotype and timing of injection influence the regional and cell-type distribution of AAV in neonatal rodents, and emphasizes the utility of pseudotyped AAV vectors for translational gene therapy paradigms.

Conformational templating of α-synuclein aggregates in neuronal-glial cultures

BackgroundGenetic studies have established a causative role for α-synuclein (αS) in Parkinson’s disease (PD), and the presence of αS aggregates in the form of Lewy body (LB) and Lewy neurite (LN) protein inclusions are defining pathological features of PD. Recent data has established that extracellular αS aggregates can induce intracellular αS pathologies supporting the hypothesis that αS pathology can spread via a “prion-like” self-templating mechanism.ResultsHere we investigated the potential for conformational templating of αS intracellular aggregates by seeding using recombinant wild-type and PD-linked mutant (A53T and E46K) αS in primary mixed neuronal-glial cultures. We find that wild-type and A53T αS fibrils predominantly seed flame-like inclusions in both neurons and astrocytes of mixed primary cultures; whereas the structurally distinct E46K fibrils seed punctate, rounded inclusions. Notably, these differences in seeded inclusion formation in these cultures reflect differences in inclusion pathology seen in transgenic mice expressing the A53T or E46K αS mutants. We further show that the inclusion morphology is dictated primarily by the seed applied rather than the form of αS expressed. We also provide initial evidence that αS inclusion pathology can be passaged in primary astrocyte cultures.ConclusionThese studies establish for the first time that αS aggregation in cultured cells can occur by a morphological self-templating mechanism.