Sander van Deventer

Adenosine triphosphate-binding cassette transporter genes up-regulation in untreated hepatocellular carcinoma is mediated by cellular microRNAs†‡

Adenosine triphosphate (ATP)‐binding cassette (ABC) transporters are drug efflux pumps responsible for the multidrug resistance phenotype causing hepatocellular carcinoma (HCC) treatment failure. Here we studied the expression of 15 ABC transporters relevant for multidrug resistance in 19 paired HCC patient samples (16 untreated, 3 treated by chemotherapeutics). Twelve ABC transporters showed up‐regulation in HCC compared with adjacent healthy liver. These include ABCA2, ABCB1, ABCB6, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC10, ABCC11, ABCC12, and ABCE1. The expression profile and function of some of these transporters have not been associated with HCC thus far. Because cellular microRNAs (miRNAs) are involved in posttranscriptional gene silencing, we hypothesized that regulation of ABC expression in HCC might be mediated by miRNAs. To study this, miRNAs were profiled and dysregulation of 90 miRNAs was shown in HCC compared with healthy liver, including up‐regulation of 11 and down‐regulation of 79. miRNA target sites in ABC genes were bioinformatically predicted and experimentally verified in vitro using luciferase reporter assays. In total, 13 cellular miRNAs were confirmed that target ABCA1, ABCC1, ABCC5, ABCC10, and ABCE1 genes and mediate changes in gene expression. Correlation analysis between ABC and miRNA expression in individual patients revealed an inverse relationship, providing an indication for miRNA regulation of ABC genes in HCC. Conclusion: Up‐regulation of ABC transporters in HCC occurs prior to chemotherapeutic treatment and is associated with miRNA down‐regulation. Up‐regulation of five ABC genes appears to be mediated by 13 cellular miRNAs in HCC patient samples. miRNA‐based gene therapy may be a novel and promising way to affect the ABC profile and overcome clinical multidrug resistance. (Hepatology 2012)

Neutralizing antibodies against adeno-associated viruses in inflammatory bowel disease patients: implications for gene therapy.

Background: Inflammatory bowel diseases (IBDs) are comprised of two major disorders: Crohns disease (CD) and ulcerative colitis (UC). No curative treatment options are available, but gene therapy may offer an alternative therapeutic approach. For this a safe and reliable vector is needed. The adeno‐associated viruses (AAV) have attracted considerable interest as gene therapy vectors. However, neutralizing antibodies (nAbs) made in response to wildtype AAV have been associated with a partial to complete block of transduction in case of reexposure. Therefore, and in order to define AAV vector candidates to treat IBD patients, we characterized preexisting humoral responses to AAV in this population. Methods: We measured circulating antibodies against AAV serotypes 1, 2, 3, 4, 5, 6, and 8 using a previously established virus neutralization assay. In all, 100 healthy donors and 200 IBD patients serum samples (101 CD and 99 UC) were analyzed. Results: A significant difference was detected in the prevalence of nAbs for AAV types 1, 5, 6, and 8 between the healthy donors and the patient population. Furthermore, various disease phenotypic characteristics correlated with the prevalence of nAbs to all the serotypes studied. Conclusions: Our study establishes a foundation for the development of an AAV‐based gene therapy approach as a novel treatment for IBD. Furthermore, we show a relationship between disease phenotype in IBD patients and the humoral immune response to AAV.

The future of inflammatory bowel disease management: Combining progress in trial design with advances in targeted therapy

Anti-tumour necrosis factor antagonists have appreciably improved patient outcomes in Crohns disease, shifting the goals of treatment from control of symptoms to clinical remission (Crohns disease activity index <150) combined with mucosal healing - the new concept of deep remission. Achieving deep remission brings clinically meaningful benefits, including reduced hospitalization and reduced need for surgery. Aspects such as the dose, timing and intensification of anti-tumour necrosis factor therapy affect the likelihood of achieving deep remission, but definitive evidence on long-term benefits and the risk/benefit profile of treatment intensification is needed. A consequence of the success of anti-tumour necrosis factor therapies has been a change in the disease characteristics of the patient population entering clinical trials. Therefore, new clinical study paradigms, such as cluster randomization and therapeutic strategy trials, are needed. High placebo response rates and the ethics of testing emerging agents against placebo in an era of effective therapies are challenges to traditional randomized controlled trials. Overcoming these challenges will not only help to optimize anti-tumour necrosis factor therapy, but also advance development of emerging treatments for Crohns disease.

Apolipoprotein B Knockdown by AAV-delivered shRNA Lowers Plasma Cholesterol in Mice

Serum low-density lipoprotein cholesterol (LDL-C) levels are proportionate to the risk of atherosclerotic cardiovascular disease. In order to reduce serum total cholesterol and LDL-C levels in mice, RNA interference (RNAi) was used to inhibit expression of the structural protein of LDL-C, apolipoprotein B100 (ApoB). We developed and screened 19 short hairpin RNAs (shRNAs) targeting conserved sequences in human, mouse, and macaque ApoB mRNAs (shApoB) and subsequently narrowed our focus to one candidate for in vivo testing. Self-complementary adeno-associated virus serotype 8 (scAAV8) was used for long-term transduction of murine liver with shApoB. A strong dose-dependent knockdown of ApoB mRNA and protein was observed, which correlated with a reduction in total cholesterol levels, without obvious signs of toxicity. Furthermore, shApoB was found to specifically reduce LDL-C in diet-induced dyslipidemic mice, whereas high-density lipoprotein cholesterol (HDL-C) remained unaffected. Finally, elevated lipid accumulation was shown in murine liver transduced with shApoB, a known phenotypic side effect of lowering ApoB levels. These results demonstrate a robust dose-dependent knockdown of ApoB by AAV-delivered shRNA in murine liver, thus providing an excellent candidate for development of RNAi-based gene therapy for the treatment of hypercholesterolemia.

Design, Characterization, and Lead Selection of Therapeutic miRNAs Targeting Huntingtin for Development of Gene Therapy for Huntington's Disease

Huntingtons disease (HD) is a neurodegenerative disorder caused by accumulation of CAG expansions in the huntingtin (HTT) gene. Hence, decreasing the expression of mutated HTT (mtHTT) is the most upstream approach for treatment of HD. We have developed HTT gene-silencing approaches based on expression cassette-optimized artificial miRNAs (miHTTs). In the first approach, total silencing of wild-type and mtHTT was achieved by targeting exon 1. In the second approach, allele-specific silencing was induced by targeting the heterozygous single-nucleotide polymorphism (SNP) rs362331 in exon 50 or rs362307 in exon 67 linked to mtHTT. The miHTT expression cassette was optimized by embedding anti-HTT target sequences in ten pri-miRNA scaffolds and their HTT knockdown efficacy, allele selectivity, passenger strand activity, and processing patterns were analyzed in vitro. Furthermore, three scaffolds expressing miH12 targeting exon 1 were incorporated in an adeno-associated viral serotype 5 (AAV5) vector and their HTT knock-down efficiency and pre-miHTT processing were compared in the humanized transgenic Hu128/21 HD mouse model. Our data demonstrate strong allele-selective silencing of mtHTT by miSNP50 targeting rs362331 and total HTT silencing by miH12 both in vitro and in vivo. Ultimately, we show that HTT knock-down efficiency and guide strand processing can be enhanced by using different cellular pri-miRNA scaffolds.

Tolerability, pharmacokinetics and pharmacodynamics of TA-8995, a selective cholesteryl ester transfer protein (CETP) inhibitor, in healthy subjects.

AIMSnTwo double-blind, randomized studies were conducted to assess the tolerability, pharmacokinetics and pharmacodynamics of oral TA-8995, a new cholesteryl ester transfer protein (CETP) inhibitor, in healthy subjects.nnnMETHODSnStudy 1: Subjects received single doses of TA-8995 or placebo (fasted). Doses were 5, 10, 25, 50 (fed/fasted), 100 and 150u2009mg (Caucasian males, 18-55u2009years), 25u2009mg (Caucasian males, > 65u2009years and Caucasian females, 18-55u2009years), 25, 50, 100 and 150u2009mg (Japanese males, 18-55u2009years). Study 2: Caucasian males (18-55u2009years) received 1, 2.5, 10 or 25u2009mg once daily TA-8995 or placebo for 21-28u2009days. Blood and urine for pharmacokinetics and/or pharmacodynamics were collected. Tolerability was assessed by adverse events, vital signs, electrocardiograms and laboratory safety tests.nnnRESULTSnPeak TA-8995 concentrations occurred approximately 4u2009h post-dose. Mean half-lives ranged from 81 to 166u2009h, without an obvious dose relationship. Exposure increased less than proportionally to dose. TA-8995 was not excreted in urine. Following 2.5 to 25u2009mg once daily dosing, TA-8995 demonstrated nearly complete inhibition of CETP activity (92-99%), increased high density lipoprotein-cholesterol (HDL-C) by 96 to 140% and decreased low density liporotein-cholesterol (LDL-C) by 40% to 53%. There were dose-related increases in apolipoproteins A-1 and E, HDL2-C and HDL3-C, and decreases in apolipoprotein B and lipoprotein A. There was no evidence of significant effects of age, gender, ethnicity or food on pharmacokinetics or pharmacodynamics. All doses were well tolerated.nnnCONCLUSIONSnTA-8995 is a potent CETP inhibitor and warrants further investigation.

Mir‐142‐3p target sequences reduce transgene‐directed immunogenicity following intramuscular adeno‐associated virus 1 vector‐mediated gene delivery

Muscle represents an important tissue target for adeno‐associated virus (AAV) vector‐mediated gene transfer in muscular, metabolic or blood‐related genetic disorders. However, several studies have demonstrated the appearance of immune responses against the transgene product after intramuscular AAV vector delivery that resulted in a limited efficacy of the treatment. Use of microRNAs that are specifically expressed in antigen‐presenting cells (APCs) is a promising approach for avoiding those immune responses. Cellular mir‐142‐3p, which is APC‐specific, is able to repress the translation of its target cellular transcripts by binding to a specific target sequences.

Embedding siRNA sequences targeting Apolipoprotein B100 in shRNA and miRNA scaffolds results in differential processing and in vivo efficacy

Overexpression of short hairpin RNA (shRNA) often causes cytotoxicity and using microRNA (miRNA) scaffolds can circumvent this problem. In this study, identically predicted small interfering RNA (siRNA) sequences targeting apolipoprotein B100 (siApoB) were embedded in shRNA (shApoB) or miRNA (miApoB) scaffolds and a direct comparison of the processing and long-term in vivo efficacy was performed. Next generation sequencing of small RNAs originating from shApoB- or miApoB-transfected cells revealed substantial differences in processing, resulting in different siApoB length, 5′ and 3′ cleavage sites and abundance of the guide or passenger strands. Murine liver transduction with adeno-associated virus (AAV) vectors expressing shApoB or miApoB resulted in high levels of siApoB expression associated with strong decrease of plasma ApoB protein and cholesterol. Expression of miApoB from the liver-specific LP1 promoter was restricted to the liver, while the H1 promoter-expressed shApoB was ectopically present. Delivery of 1 × 1011 genome copies AAV-shApoB or AAV-miApoB led to a gradual loss of ApoB and plasma cholesterol inhibition, which was circumvented by delivering a 20-fold lower vector dose. In conclusion, incorporating identical siRNA sequences in shRNA or miRNA scaffolds results in differential processing patterns and in vivo efficacy that may have serious consequences for future RNAi-based therapeutics.

Optimization and comparison of knockdown efficacy between polymerase II expressed shRNA and artificial miRNA targeting luciferase and Apolipoprotein B100

BackgroundControlling and limiting the expression of short hairpin RNA (shRNA) by using constitutive or tissue-specific polymerase II (pol II) expression can be a promising strategy to avoid RNAi toxicity. However, to date detailed studies on requirements for effective pol II shRNA expression and processing are not available. We investigated the optimal structural configuration of shRNA molecules, namely: hairpin location, stem length and termination signal required for effective pol II expression and compared it with an alternative strategy of avoiding toxicity by using artificial microRNA (miRNA) scaffolds.ResultsHighly effective shRNAs targeting luciferase (shLuc) or Apolipoprotein B100 (shApoB1 and shApoB2) were placed under the control of the pol II CMV promoter and expressed at +5 or +6 nucleotides (nt) with reference to the transcription start site (TSS). Different transcription termination signals (TTS), namely minimal polyadenylation (pA), poly T (T5) and U1 were also used. All pol II- expressed shRNA variants induced mild inhibition of Luciferase reporters carrying specific targets and none of them showed comparable efficacy to their polymerase III-expressed H1-shRNA controls, regardless of hairpin position and termination signal used. Extending hairpin stem length from 20 basepairs (bp) to 21, 25 or 29 bp yielded only slight improvement in the overall efficacy. When shLuc, shApoB1 and shApoB2 were placed in an artificial miRNA scaffold, two out of three were as potent as the H1-shRNA controls. Quantification of small interfering RNA (siRNA) molecules showed that the artificial miRNA constructs expressed less molecules than H1-shRNAs and that CMV-shRNA expressed the lowest amount of siRNA molecules suggesting that RNAi processing in this case is least effective. Furthermore, CMV-miApoB1 and CMV-miApoB2 were as effective as the corresponding H1-shApoB1 and H1-shApoB2 in inhibiting endogenous ApoB mRNA.ConclusionOur results demonstrate that artificial miRNA have a better efficacy profile than shRNA expressed either from H1 or CMV promoter and will be used in the future for RNAi therapeutic development.

Murine CD4+CD25- cells activated in vitro with PMA/ionomycin and anti-CD3 acquire regulatory function and ameliorate experimental colitis in vivo

BackgroundInduced regulatory T (iTreg) lymphocytes show promise for application in the treatment of allergic, autoimmune and inflammatory disorders. iTreg cells demonstrate advantages over natural Treg (nTreg) cells in terms of increased number of starting population and greater potential to proliferate. Different activation methods to generate iTreg cells result in iTreg cells that are heterogeneous in phenotype and mechanisms of suppression. Therefore it is of interest to explore new techniques to generate iTreg cells and to determine their physiological relevance.MethodsUsing phorbol myristate acetate (PMA)/ionomycin and anti-CD3 activation of CD4+CD25- cells we generated in vitro functional CD4+CD25+ iTreg (TregPMA) cells. Functionality of the generated TregPMA cells was tested in vivo in a mouse model of inflammatory bowel disease (IBD) - CD45RB transfer colitis model.ResultsTregPMA cells expressed regulatory markers and proved to ameliorate the disease phenotype in murine CD45RB transfer colitis model. The body weight loss and disease activity scores for TregPMA treated mice were reduced when compared to diseased control group. Histological assessment of colon sections confirmed amelioration of the disease phenotype. Additionally, cytokine analysis showed decreased levels of proinflammatory colonic and plasma IL-6, colonic IL-1 β and higher levels of colonic IL-17 when compared to diseased control group.ConclusionsThis study identifies a new method to generate in vitro iTreg cells (TregPMA cells) which physiological efficacy has been demonstrated in vivo.