Single Systemic Administration of a Gene Therapy Leading to Disease Treatment in Metachromatic Leukodystrophy Arsa Knock-Out Mice.

Metachromatic leukodystrophy (MLD) is a rare, inherited, demyelinating lysosomal storage disorder caused by mutations in the arylsulfatase-A gene (ARSA). In patients, levels of functional ARSA enzyme are diminished and lead to deleterious accumulation of sulfatides. Herein, we demonstrate that intravenous administration of HSC15/ARSA restored the endogenous murine biodistribution of the corresponding enzyme, and overexpression of ARSA corrected disease biomarkers and ameliorated motor deficits in Arsa KO mice of either sex. In treated Arsa KO mice, when compared with intravenously administered AAV9/ARSA, significant increases in brain ARSA activity, transcript levels, and vector genomes were observed with HSC15/ARSA Durability of transgene expression was established in neonate and adult mice out to 12 and 52 weeks, respectively. Levels and correlation between changes in biomarkers and ARSA activity required to achieve functional motor benefit was also defined. Finally, we demonstrated blood-nerve, blood-spinal and blood-brain barrier crossing as well as the presence of circulating ARSA enzyme activity in the serum of healthy nonhuman primates of either sex. Together, these findings support the use of intravenous delivery of HSC15/ARSA-mediated gene therapy for the treatment of MLD.SIGNIFICANCE STATEMENT Herein, we describe the method of gene therapy adeno-associated virus (AAV) capsid and route of administration selection leading to an efficacious gene therapy in a mouse model of metachromatic leukodystrophy. We demonstrate the therapeutic outcome of a new naturally derived clade F AAV capsid (AAVHSC15) in a disease model and the importance of triangulating multiple end points to increase the translation into higher species via ARSA enzyme activity and biodistribution profile (with a focus on the CNS) with that of a key clinically relevant biomarker.

Natural variations in AAVHSC16 significantly reduce liver tropism and maintain broad distribution to periphery and CNS.

Adeno-associated viruses derived from human hematopoietic stem cells (AAVHSCs) are naturally occurring AAVs. Fifteen AAVHSCs have demonstrated broad biodistribution while displaying differences in transduction. We examine the structure-function relationships of these natural amino acid variations on cellular binding. We demonstrate that AAVHSC16 is the only AAVHSC that does not preferentially bind to terminal galactose. AAVHSC16 contains two unique amino acids, 501I and 706C, compared with other AAVHSCs. Through mutagenesis, we determined that residue 501 contributes to the lack of galactose binding. Structural analysis revealed that residue 501 is in proximity to the galactose binding pocket, hence confirming its functional role in galactose binding. Biodistribution analysis of AAVHSC16 indicated significantly less liver tropism in mice and non-human primates compared with other clade F members, likely associated with overall binding differences observed in vitro. AAVHSC16 maintained robust tropism to other key tissues in the peripheral and central nervous systems after intravenous injection, including to the brain, heart, and gastrocnemius. Importantly, AAVHSC16 did not induce elevated liver enzyme levels in non-human primates after intravenous injection at high doses. The unique glycan binding and tropism of AAVHSC16 makes this naturally occurring capsid an attractive candidate for therapies requiring less liver tropism while maintaining broad biodistribution.

Multiplexed, quantitative, and targeted metabolite profiling by LC-MS/MRM.

Targeted metabolomics, which focuses on a subset of known metabolites representative of biologically relevant metabolic pathways, is a valuable tool to discover biomarkers and link disease phenotypes to underlying mechanisms or therapeutic modes of action. A key advantage of targeted metabolomics, compared to discovery metabolomics, is its immediate readiness for extracting biological information derived from known metabolites and quantitative measurements. However, simultaneously analyzing hundreds of endogenous metabolites presents a challenge due to their diverse chemical structures and properties. Here we report a method which combines different chromatographic separation conditions, optimal ionization polarities, and the most sensitive triple-quadrupole MS-based data acquisition mode, multiple reaction monitoring (MRM), to quantitatively profile 205 endogenous metabolites in 10 min.

A role for PACE4 in osteoarthritis pain: evidence from human genetic association and null mutant phenotype.

OBJECTIVES: The aim of this study was to assess if genetic variation in the PACE4 (paired amino acid converting enzyme 4) gene Pcsk6 influences the risk for symptomatic knee osteoarthritis (OA). METHODS: Ten PCSK6 single nucleotide polymorphisms were tested for association in a discovery cohort of radiographic knee OA (n=156 asymptomatic and 600 symptomatic cases). Meta-analysis of the minor allele at rs900414 was performed in three additional independent cohorts (total n=674 asymptomatic and 2068 symptomatic). Pcsk6 knockout mice and wild-type C57BL/6 mice were compared in a battery of algesiometric assays, including hypersensitivity in response to intraplantar substance P, pain behaviours in response to intrathecal substance P and pain behaviour in the abdominal constriction test. RESULTS: In the discovery cohort of radiographic knee OA, an intronic single nucleotide polymorphism at rs900414 was significantly associated with symptomatic OA. Replication in three additional cohorts confirmed that the minor allele at rs900414 was consistently increased among asymptomatic compared to symptomatic radiographic knee OA cases in all four cohorts. A fixed-effects meta-analysis yielded an OR=1.35 (95% CI 1.17 to 1.56; p=4.3×10(-5) and no significant between-study heterogeneity). Studies in mice revealed that Pcsk6 knockout mice were significantly protected against pain in a battery of algesiometric assays. CONCLUSIONS: These results suggest that a variant in PCSK6 is strongly associated with protection against pain in knee OA, offering some insight as to why, in the presence of the same structural damage, some individuals develop chronic pain and others are protected. Studies in Pcsk6 null mutant mice further implicate PACE4 in pain.

siRNA off-target effects can be reduced at concentrations that match their individual potency.

Small interfering RNAs (siRNAs) are routinely used to reduce mRNA levels for a specific gene with the goal of studying its function. Several studies have demonstrated that siRNAs are not always specific and can have many off-target effects. The 3' UTRs of off-target mRNAs are often enriched in sequences that are complementary to the seed-region of the siRNA. We demonstrate that siRNA off-targets can be significantly reduced when cells are treated with a dose of siRNA that is relatively low (e.g. 1 nM), but sufficient to effectively silence the intended target. The reduction in off-targets was demonstrated for both modified and unmodified siRNAs that targeted either STAT3 or hexokinase II. Low concentrations reduced silencing of transcripts with complementarity to the seed region of the siRNA. Similarly, off-targets that were not complementary to the siRNA were reduced at lower doses, including up-regulated genes that are involved in immune response. Importantly, the unintended induction of caspase activity following treatment with a siRNA that targeted hexokinase II was also shown to be a concentration-dependent off-target effect. We conclude that off-targets and their related phenotypic effects can be reduced for certain siRNA that potently silence their intended target at low concentrations.

Relation of increased prebeta-1 high-density lipoprotein levels to risk of coronary heart disease.

Preß-1 high-density lipoprotein (HDL) plays a key role in reverse cholesterol transport by promoting cholesterol efflux. Our aims were (1) to test previous associations between preß-1 HDL and coronary heart disease (CHD) and (2) to investigate whether preß-1 HDL levels also are associated with risk of myocardial infarction (MI). Plasma preß-1 HDL was measured by an ultrafiltration-isotope dilution technique in 1,255 subjects recruited from the University of California-San Francisco Lipid and Cardiovascular Clinics and collaborating cardiologists. Preß-1 HDL was significantly and positively associated with CHD and MI even after adjustment for established risk factors. Inclusion of preß-1 HDL in a multivariable model for CHD led to a modest improvement in reclassification of subjects (net reclassification index 0.15, p = 0.01; integrated discrimination improvement 0.003, p = 0.2). In contrast, incorporation of preß-1 HDL into a risk model of MI alone significantly improved reclassification of subjects (net reclassification index 0.21, p = 0.008; integrated discrimination improvement 0.01, p = 0.02), suggesting that preß-1 HDL has more discriminatory power for MI than for CHD in our study population. In conclusion, these results confirm previous associations between preß-1 HDL and CHD in a large well-characterized clinical cohort. Also, this is the first study in which preß-1 HDL was identified as a novel and independent predictor of MI above and beyond traditional CHD risk factors.

Power in the phenotypic extremes: a simulation study of power in discovery and replication of rare variants.

Next-generation sequencing technologies are making it possible to study the role of rare variants in human disease. Many studies balance statistical power with cost-effectiveness by (a) sampling from phenotypic extremes and (b) utilizing a two-stage design. Two-stage designs include a broad-based discovery phase and selection of a subset of potential causal genes/variants to be further examined in independent samples. We evaluate three parameters: first, the gain in statistical power due to extreme sampling to discover causal variants; second, the informativeness of initial (Phase I) association statistics to select genes/variants for follow-up; third, the impact of extreme and random sampling in (Phase 2) replication. We present a quantitative method to select individuals from the phenotypic extremes of a binary trait, and simulate disease association studies under a variety of sample sizes and sampling schemes. First, we find that while studies sampling from extremes have excellent power to discover rare variants, they have limited power to associate them to phenotype­suggesting high false-negative rates for upcoming studies. Second, consistent with previous studies, we find that the effect sizes estimated in these studies are expected to be systematically larger compared with the overall population effect size; in a well-cited lipids study, we estimate the reported effect to be twofold larger. Third, replication studies require large samples from the general population to have sufficient power; extreme sampling could reduce the required sample size as much as fourfold. Our observations offer practical guidance for the design and interpretation of studies that utilize extreme sampling.

High-throughput and multiplexed LC/MS/MRM method for targeted metabolomics.

Target-based metabolomics, focused on a subset of metabolites representative of key pathways, is a valuable tool for assessing metabolic changes resulting from genetic mutation, altered gene expression, and protein dysfunction in a given disease state or as a consequence of an environmental perturbation, such as a pharmaceutical. However, simultaneously analyzing hundreds of endogenous metabolites presents a challenge because of their diverse chemical structures and properties. In this study, we report a high-throughput, sensitive, and reproducible method for target-based metabolomics studies. It combines different separation conditions, optimal ionization polarities, and the most sensitive triple-quadrupole MS-based data acquisition mode (MRM). In 10 min, 205 endogenous metabolites, divided into three subgroups (amino acids, sugar and nucleic acids, and organic acids), are sequentially analyzed on a LC/MS/MRM system. Low picogram sensitivity is achieved for more than half of the metabolites. A 3-4 order of linearity and assay coefficient of variation less than 15% are observed for approximately 80% of the metabolites. In summary, we have established a multiplex LC/MS/MRM method for quantitatively profiling hundreds of known metabolites from complex biological samples. The methodology is generally applicable and easily expandable to include more endogenous or drug metabolites.

Identification of DIO2 as a new susceptibility locus for symptomatic osteoarthritis.

Osteoarthritis [MIM 165720] is a common late-onset articular joint disease for which no pharmaceutical intervention is available to attenuate the cartilage degeneration. To identify a new osteoarthritis susceptibility locus, a genome-wide linkage scan and combined linkage association analysis were applied to 179 affected siblings and four trios with generalized osteoarthritis (The GARP study). We tested, for confirmation by association, 1478 subjects who required joint replacement and 734 controls in a UK population. Additional replication was tested in 1582 population-based females from the Rotterdam study that contained 94 cases with defined hip osteoarthritis and in 267 Japanese females with symptomatic hip osteoarthritis and 465 controls. Suggested evidence for linkage in the GARP study was observed on chromosome 14q32.11 (log of odds = 3.03, P = 1.9 x 10(-4)). Genotyping tagging single-nucleotide polymorphisms covering three important candidate genes revealed a common coding variant (rs225014; Thr92Ala) in the iodothyronine-deiodinase enzyme type 2 (D2) gene (DIO2 [MIM 601413]) which significantly explained the linkage signal (P = 0.006). Confirmation and replication by association in the additional osteoarthritis studies indicated a common DIO2 haplotype, exclusively containing the minor allele of rs225014 and common allele of rs12885300, with a combined recessive odds ratio of 1.79, 95% confidence interval (CI) 1.37-2.34 with P = 2.02 x 10(-5) in female cases with advanced/symptomatic hip osteoarthritis. The gene product of this DIO2 converts intracellular pro-hormone-3,3',5,5'-tetraiodothyronine (T4) into the active thyroid hormone 3,3',5-triiodothyronine (T3) thereby regulating intracellular levels of active T3 in target tissues such as the growth plate. Our results indicate a new susceptibility gene (DIO2) conferring risk to osteoarthritis.

Rapid identification of somatic mutations in colorectal and breast cancer tissues using mismatch repair detection (MRD).

Mismatch repair detection (MRD) was used to screen 93 matched tumor-normal sample pairs and 22 cell lines for somatic mutations in 30 cancer relevant genes. Using a starting amount of only 150 ng of genomic DNA, we screened 102 kb of sequence for somatic mutations in colon and breast cancer. A total of 152 somatic mutations were discovered, encompassing previously reported mutations, such as BRAF V600E and KRAS G12S, G12V, and G13D, as well as novel mutations, including some in genes in which somatic mutations have not previously been reported, such as MAP2K1 and MAP2K2. The distribution of mutations ranged widely within and across tumor types. The functional significance of many of these mutations is not understood, with patterns of selection only evident in KRAS and BRAF in colon cancer. These results present a novel approach to high-throughput mutation screening using small amounts of starting material and reveal a mutation spectrum across 30 genes in a large cohort of breast and colorectal cancers.

Application of pooled genotyping to scan candidate regions for association with HDL cholesterol levels.

Association studies are used to identify genetic determinants of complex human traits of medical interest. With the large number of validated single nucleotide polymorphisms (SNPs) currently available, two limiting factors in association studies are genotyping capability and costs. Pooled DNA genotyping has been proposed as an efficient means of screening SNPs for allele frequency differences in case-control studies and for prioritising them for subsequent individual genotyping analysis. Here, we apply quantitative pooled genotyping followed by individual genotyping and replication to identify associations with human serum high-density lipoprotein (HDL) cholesterol levels. The DNA from individuals with low and high HDL cholesterol levels was pooled separately, each pool was amplified by polymerase chain reaction in triplicate and each amplified product was separately hybridised to a high-density oligonucleotide array. Allele frequency differences between case and control groups with low and high HDL cholesterol levels were estimated for 7,283 SNPs distributed across 71 candidate gene regions spanning a total of 17.1 megabases. A novel method was developed to take advantage of independently derived haplotype map information to improve the pooled estimates of allele frequency differences. A subset of SNPs with the largest estimated allele frequency differences between low and high HDL cholesterol groups was chosen for individual genotyping in the study population, as well as in a separate replication population. Four SNPs in a single haplotype block within the cholesteryl ester transfer protein (CETP) gene interval were significantly associated with HDL cholesterol levels in both populations. Our study is among the first to demonstrate the application of pooled genotyping followed by confirmation with individual genotyping to identify genetic determinants of a complex trait.

Emerging strategies and applications of pharmacogenomics.

The rapid pace of genomic science advancements, including the completion of the human genome sequence, the extensive cataloguing of genetic variation and the acceleration of technologies to assess such variation, combined with clinical programmes with rich phenotypic data, serve as the foundation for the design and execution of pharmacogenomic studies which have an impact on the pharmaceutical pipeline from early discovery through to the marketplace. The authors discuss the required infrastructure to support pharmacogenomic studies and provide insight into the strategies and practical application to influence decision making in the pharmaceutical setting. Further, the influence of pharmacogenomics is currently affecting patient care in the oncology area and is highlighted as evident impact in the marketplace.

Correlation of polymorphic variation in the promoter region of the interleukin-1 beta gene with secretion of interleukin-1 beta protein.

OBJECTIVE: Significant variation in interleukin-1 beta (IL-1 beta) protein secretion between subjects has been observed when using a lipopolysaccharide (LPS)/ATP-mediated ex vivo blood stimulation assay. To explore the potential relationships between genetic polymorphisms in the IL1B cytokine gene and cellular responses to inflammatory stimuli such as LPS, we investigated the hypothesis that polymorphisms within the promoter and exon 5 of the IL1B gene contribute to the observed differences in IL-1 beta protein secretion. METHODS: The IL1B gene polymorphisms C-511T, T-31C, and C3954T were tested for association with LPS-induced secretion of IL-1 beta protein as measured by an ex vivo blood stimulation assay. Samples from 2 independent study populations (n = 31 and n = 25) were available for use in the ex vivo assay after consent was obtained to analyze the DNA. RESULTS: A specific haplotype, composed of the T allele at -511 and the C allele at -31, was significantly associated with a 2-3-fold increase in LPS-induced IL-1 beta protein secretion. This association was observed in both of the independent study populations (P = 0.0084 and P = 0.0017). CONCLUSION: These data suggest that polymorphisms within the promoter region of the IL1B gene contribute to observed differences in LPS-induced IL-1 beta protein secretion.

Association of the interleukin-1 gene cluster with radiographic signs of osteoarthritis of the hip.

OBJECTIVE: To study the role of the interleukin-1 beta gene (IL1B) and the IL-1 receptor antagonist gene (IL1RN) in relation to the occurrence of radiographic osteoarthritis (ROA) in the hip, knee, and hand and disc degeneration of the spine. METHODS: The study population consisted of a random sample of 886 subjects (ages 55-65 years) from a population-based cohort (the Rotterdam study). Two polymorphisms within IL1B (3953C>T and -511C>T) and one within IL1RN (the variable-number tandem repeat [VNTR]) were analyzed and used in an association study of the occurrence of ROA. Haplotyping and simultaneous logistic regression analysis were performed to investigate whether the associations observed were independent. RESULTS: Associations with a predisposition for hip ROA were observed for heterozygous and homozygous carriers of the rare IL1B allele -511T (crude odds ratio [OR] 1.8, 95% confidence interval [95% CI] 1.0-3.4 and OR 2.9, 95% CI 1.4-6.3, respectively) and of the IL1RN VNTR allele 2 (crude OR 2.0, 95% CI 1.1-3.4 and OR 3.3, 95% CI 1.4-7.8, respectively). An additive effect was observed for carriers of risk alleles of both polymorphisms, with a significant linear-by-linear association (P = 0.00022). CONCLUSION: Our findings suggest that the IL-1 gene cluster polymorphisms may play a significant role in the pathogenesis of OA of the hip.

Association between single-nucleotide polymorphisms in the endothelial lipase (LIPG) gene and high-density lipoprotein cholesterol levels.

Endothelial lipase (LIPG) is the latest addition to the triglyceride lipase family of genes that includes pancreatic lipase (PL), hepatic lipase (HL), and lipoprotein lipase (LPL). These lipolytic enzymes demonstrate both triglyceride lipase as well as phospholipase activities and are integrally involved in lipid absorption, transport, and metabolism. Several studies have demonstrated that LIPG is important for affecting lipid levels in mice but the data in humans is less complete. To more thoroughly characterize the LIPG gene, we resequenced it from an ethnically diverse population. Thirteen novel single-nucleotide polymorphisms (SNPs) were identified and seven others confirmed. High linkage disequilibrium was found among these SNPs spanning the length of the transcript, allowing interrogation of the entire gene for functional variation. Subjects with either high or low HDL cholesterol were used to investigate its association with LIPG gene variation. Associations were found with the most significant being the intronic variants C+42T/In5 and T+2864C/In8 (P=0.007 and 0.004, respectively). A trend for an association of the same SNPs with fewer myocardial infarctions (P=0.03) was also observed but was not significant after correction for multiple testing. The results of this study provide data linking variation in the human LIPG gene with HDL cholesterol levels as well as further evidence in support of LIPG as a potential target for therapeutic intervention.

The serotonin transporter polymorphism, 5HTTLPR, is associated with a faster response time to sertraline in an elderly population with major depressive disorder.

RATIONALE: A common polymorphism (5HTTLPR) within the promoter region of the serotonin transporter gene (LSC6A4) has been shown to influence response time as well as overall response to selective serotonin reuptake inhibitors (SSRIs) in subjects with major depressive disorder. We hypothesized that a similar effect in response time to sertraline would be observed and that no effect on response time would be seen in a placebo arm. OBJECTIVES: We tested the hypothesis that subjects homozygous for the long allele at the 5HTTLPR polymorphism would respond more rapidly to sertraline than subjects carrying one or two copies of the short allele. METHODS: HAM-D and CGI-I responses to sertraline and placebo were measured weekly in the context of an 8-week, placebo-controlled study in elderly depressed subjects. Genotyping of the 5HTTLPR polymorphism was performed to test for correlations with response at each week in the sertraline and placebo groups ( n=206). RESULTS: Subjects homozygous for the long allele of 5HTTLPR showed a significant increase in response at week 1 and week 2, as assessed by the CGI-I scale compared with subjects carrying one or two copies of the short allele ( P=0.01 at both weeks). No significant difference was observed in the placebo group. CONCLUSIONS: These results suggest that genetic variation in the serotonin transporter gene effects the response time to sertraline and provides complementing evidence to previous reports that this polymorphism affects response time to other SSRIs.