Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells

Stable expression of transgenes is essential in both therapeutic and research applications. Traditionally, transgene integration has been accomplished via viral vectors in a semi-random fashion, but with inherent integration site biases linked to the type of virus used. The randomly integrated transgenes may undergo silencing and more concerningly, can also lead to dysregulation of endogenous genes. Gene dysregulation can lead to malignant transformation of cells and has unfortunately given rise to cases of leukaemia in gene therapy trials. Genomic safe harbour (GSH) loci have been proposed as safe sites for transgene integration. To date, a number of sites in the human genome have been used for directed integration; however none of these pass scrutiny as bona fide GSH. Here, we conducted a computational analysis to identify 25 putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.

Regulacje bezpiecznej przystani (safe harbour) w obszarze cen transferowych

Transakcje o charakterze nieskomplikowanym i rutynowym nie powodują istotnego ryzyka zaniżenia dochodu i mogą korzystać z uproszczeń typu safe harbour. W Polsce dotyczą one usług o niskiej wartości dodanej oraz pożyczek, kredytów i emisji obligacji. Transakcje tego typu stanowią relatywnie niewielki odsetek transakcji usługowych, względnie finansowych, zaraportowanych za 2019 r. Transakcje finansowe objęte safe harbour stanowiły 0,96% zaraportowanych w TPR-C (w zakresie podatku dochodowego od osób prawnych) transakcji finansowych, a transakcje korzystające z safe harbour usługowego – 3,92% ogółu transakcji usługowych wykazanych w TPR-C za 2019 r. W dalszych pracach analitycznych warto rozważyć poszerzenie stosowania uproszczeń safe harbour.

Safe harbour: Should we be worried? An analysis of Clause 20 of the Harmful Digital Communications Bill, and its application to the law of defamation

<p>Liability of internet intermediaries for content created by third parties is a contentious area of defamation law. Recently, the law in New Zealand has begun to depart from English law, and move closer to strict liability. Parliament has responded with a ‘safe harbour’ in clause 20 of the Harmful Digital Communications Bill, which will provide online content hosts with conditional immunity from liability for content created by others. The author supports the creation of a legislative safe harbour for internet intermediaries, but highlights a number of deficiencies with clause 20 as currently drafted. This paper analyses the existing law, including possible defences, and clause 20. It also looks to other jurisdictions’ safe harbours, and concludes with recommendations on how clause 20 ought to be improved.</p>

Safe harbour: Should we be worried? An analysis of Clause 20 of the Harmful Digital Communications Bill, and its application to the law of defamation

<p>Liability of internet intermediaries for content created by third parties is a contentious area of defamation law. Recently, the law in New Zealand has begun to depart from English law, and move closer to strict liability. Parliament has responded with a ‘safe harbour’ in clause 20 of the Harmful Digital Communications Bill, which will provide online content hosts with conditional immunity from liability for content created by others. The author supports the creation of a legislative safe harbour for internet intermediaries, but highlights a number of deficiencies with clause 20 as currently drafted. This paper analyses the existing law, including possible defences, and clause 20. It also looks to other jurisdictions’ safe harbours, and concludes with recommendations on how clause 20 ought to be improved.</p>

Mānī on the Margins

Mānī (216–ca. 277 AD), the founder of Manichaeism, was brought up in a Jewish-Christian community at the end of the Arsacid dynasty. After several private revelations, he established his own religion, which he and his disciples propagated in the newly established Sasanian Iran. Spreading east along the Silk Road, Manichaeism arrived in China in 694, where it remained basically a religio licita until 843. After the Huichang persecution (843–845), Manichaeans found a relatively safe harbour in the southeastern regions, especially in present-day Zhejiang and Fujian provinces, where they survived for centuries, as reports from the Song, Yuan, and Ming dynasties attest. In this paper, after summarizing the main events before the Huichang persecution, I give an overview of what the historical sources recording various manifestations of southeastern Manichaeism tell us about the latest form of Manichaeism, which uniquely survived in this peripheral region at a time, when it had completely disappeared from other parts of the world. This overview differs from other similar endeavours in that it also incorporates some new developments that were inspired by the discovery of a new corpus of texts from Xiapu and Pingnan counties, where it was the figure of Lin Deng (1003‒1059), who played a pivotal role to preserve the Manichaean heritage of the Tang.

Back to the Origins of the Initial Public Offerings Price Range: Underwriter-Funds Network and Information Production Timeline

Although the initial price range in U.S. Initial Public Offerings (IPOs) is constrained by SEC regulations, a non-negligible percentage of IPO price ranges falls outside the &lsquo;safe harbour&rsquo;. We investigate how the price range - which sends the very first signals on the IPO quality to the market - is set in the due diligence phase, with special attention to unexplored networking patterns between underwriters and institutional investors. By making use of a Mixture Model applied to 1,246 US firms listed between 2004 and 2016, we show that underwriters that are centrally positioned in their network of regular investors are more likely to set a price range that is compliant with SEC guidelines. We argue that the flexibility resulting from being safe harbour-compliant allows underwriters to preserve their reputation for fair dealing with issuers by exploiting a dumping ground proviso or quid pro quo agreements with their network funds. Despite information produced by network funds in the due diligence step having no significant effect on the width of the price range, in our study, we provide evidence that the range does serve as a proxy of the uncertainty of the listing firms.

Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour

Background: Diseases such as hypertrophic cardiomyopathy (HCM) can lead to severe outcomes including sudden death. The generation of human induced pluripotent stem cell (hiPSC) reporter lines can be useful for disease modelling and drug screening by providing physiologically relevant in vitro models of disease. The AAVS1 locus is cited as a safe harbour that is permissive for stable transgene expression, and hence is favoured for creating gene targeted reporter lines. Methods: We generated hiPSC reporters using a plasmid-based CRISPR/Cas9 nickase strategy. The first intron of PPP1R12C, the AAVS1 locus, was targeted with constructs expressing a genetically encoded calcium indicator (R-GECO1.0) or HOXA9-T2A-mScarlet reporter under the control of a pCAG or inducible pTRE promoter, respectively. Transgene expression was compared between clones before, during and/or after directed differentiation to mesodermal lineages. Results: Successful targeting to AAVS1 was confirmed by PCR and sequencing. Of 24 hiPSC clones targeted with pCAG-R-GECO1.0, only 20 expressed the transgene and in these, the percentage of positive cells ranged from 0% to 99.5%. Differentiation of a subset of clones produced cardiomyocytes, wherein the percentage of cells positive for R-GECO1.0 ranged from 2.1% to 93.1%. In the highest expressing R-GECO1.0 clones, transgene silencing occurred during cardiomyocyte differentiation causing a decrease in expression from 98.93% to 1.3%. In HOXA9-T2A-mScarlet hiPSC reporter lines directed towards mesoderm lineages, doxycycline induced a peak in transgene expression after two days but this reduced by up to ten-thousand-fold over the next 8-10 days. Nevertheless, for R-GECO1.0 lines differentiated into cardiomyocytes, transgene expression was rescued by continuous puromycin drug selection, which allowed the Ca2+ responses associated with HCM to be investigated in vitro using single cell analysis. Conclusions: Targeted knock-ins to AAVS1 can be used to create reporter lines but variability between clones and transgene silencing requires careful attention by researchers seeking robust reporter gene expression.

From Liability Trap to the World’s Safest Harbour: Lessons from China, India, Japan, South Korea, Indonesia, and Malaysia

This chapter compares the intermediary liability rules of six major Asian countries and highlights how there seems to be confusion on their nature, although the commentators of all countries describe their respective rules as ‘safe harbours’ resembling section 512 of the US Digital Millennium Copyright Act. The chapter describes how China and South Korea inadvertently created a liability-imposing rule instead of a liability-exempting rule. Further, the chapter reviews India and Japan’s statutes that set out liability-exempting regimes closely resembling the EU e-Commerce Directive. India’s 2011 Intermediary Guidelines generate a strong cloud of obligations on intermediaries that threatens to convert the whole system into a liability-imposing one. However, that threat had an impact on the jurisprudence with the 2013 Shreya Singhal decision making the Indian system one of the world’s safest harbours. This chapter further discusses the importance of distinguishing between a liability-imposing rule and a liability-exempting one in the light of other Asian examples. Indonesia’s safe harbour draft regulation announced in December 2016 seems to move towards the dangerous model of both China and South Korea. Malaysia’s copyright notice and takedown appears to follow the US model closely but has a structure that allows the same misunderstanding made by South Korean regulators.