Toward more effective gene delivery

A report on the symposium 'In vivo barriers to gene delivery', Cold Spring Harbor, USA, 26-29 November 2007

Neoproterozoic to early Paleozoic extensional and compressional history of East Laurentian margin sequences: The Moine Supergroup, Scottish Caledonides

Neoproterozoic siliciclastic-dominated sequences are widespread along the eastern margin of Laurentia and are related to rifting associated with the breakout of Laurentia from the supercontinent Rodinia. Detrital zircons from the Moine Supergroup, NW Scotland, yield Archean to early Neoproterozoic U-Pb ages, consistent with derivation from the Grenville-Sveconorwegian orogen and environs and accumulation post–1000 Ma. U-Pb zircon ages for felsic and associated mafic intrusions confirm a widespread pulse of extension-related magmatism at around 870 Ma. Pegmatites yielding U-Pb zircon ages between 830 Ma and 745 Ma constrain a series of deformation and metamorphic pulses related to Knoydartian orogenesis of the host Moinerocks. Additional U-Pb zircon and monazite data, and 40Ar/39Ar ages for pegmatites and host gneisses indicate high-grade metamorphic events at ca. 458–446 Ma and ca. 426 Maduring the Caledonian orogenic cycle.The presence of early Neoproterozoic silici clastic sedimentation and deformation in the Moine and equivalent successions around the North Atlantic and their absence along strike in eastern North America reflect contrasting Laurentian paleogeography during the breakup of Rodinia. The North Atlantic realm occupied an external location on the margin of Laurentia, and this region acted as a locus for accumulation of detritus (Moine Supergroup and equivalents) derived from the Grenville-Sveconorwegian orogenic welt, which developed as a consequence of collisional assembly of Rodinia. Neoproterozoic orogenic activity corresponds with theinferred development of convergent platemargin activity along the periphery of the supercontinent. In contrast in eastern North America, which lay within the internal parts of Rodinia, sedimentation did not commence until the mid-Neoproterozoic (ca. 760 Ma) during initial stages of supercontinent fragmentation. In the North Atlantic region, this time frame corresponds to a second pulse of extension represented by units such as the Dalradian Supergroup, which unconformably overlies the predeformed Moine succession

Increasing the density of nanomedicines improves their ultrasound-mediated delivery to tumours

AbstractNanomedicines have provided fresh impetus in the fight against cancer due to their selectivity and power. However, these agents are limited when delivered intravenously due to their rapid clearance from the bloodstream and poor passage from the bloodstream into target tumours. Here we describe a novel stealthing strategy which addresses both these limitations and thereby demonstrate that both the passive and mechanically-mediated tumour accumulation of the model nanomedicine adenovirus (Ad) can be substantially enhanced. In our strategy gold nanoparticles were thoroughly modified with 2kDa polyethyleneglycol (PEG) and then linked to Ad via a single reduction-cleavable 5kDa PEG. The resulting Ad–gold–PEG construct was compared to non-modified Ad or conventionally stealthed Ad–poly[N-(2-hydroxypropyl)methacrylamide] (Ad–PHPMA). Notably, although Ad–gold–PEG was of similar size and surface charge to Ad–PHPMA the increase in density, resulting from the inclusion of the gold nanoparticles, provided a substantial enhancement of ultrasound-mediated transport. In an in vitro tumour mimicking phantom, the level and distance of Ad–gold–PEG transport was shown to be substantially greater than achieved with Ad–PHPMA. In in vivo studies 0.1% of an unmodified Ad dose was shown to accumulate in tumours, whereas over 12% of the injected dose was recovered from the tumours of mice treated with Ad–gold–PEG and ultrasound. Ultimately, a significant increase in anti-tumour efficacy resulted from this strategy. This stealthing and density-increasing technology could ultimately enhance clinical utility of intravenously delivered nanoscale medicines including viruses, liposomes and antibodies

Aminooxy-BCN glycoconjugates

NMR spectroscopy data for aminooxybicyclononyne derivatives and glycan conjugates and protein mass spectrometry data for protein ligation reaction

MicroRNA Controlled Adenovirus Mediates Anti-Cancer Efficacy without Affecting Endogenous MicroRNA Activity

MicroRNAs are small non-coding RNA molecules that regulate mRNA translation and stability by binding to complementary sequences usually within the 3′ un-translated region (UTR). We have previously shown that the hepatic toxicity caused by wild-type Adenovirus 5 (Ad5WT) in mice can be prevented by incorporating 4 binding sites for the liver-specific microRNA, mir122, into the 3′ UTR of E1A mRNA. This virus, termed Ad5mir122, is a promising virotherapy candidate and causes no obvious liver pathology. Herein we show that Ad5mir122 maintains wild-type lytic activity in cancer cells not expressing mir122 and assess any effects of possible mir122 depletion in host cells. Repeat administration of 2×1010 viral particles of Admir122 to HepG2 tumour bearing mice showed significant anti-cancer efficacy. RT-QPCR showed that E1A mRNA was down-regulated 29-fold in liver when compared to Ad5WT. Western blot for E1A confirmed that all protein variants were knocked down. RT-QPCR for mature mir122 in infected livers showed that quantity of mir122 remained unaffected. Genome wide mRNA microarray profiling of infected livers showed that although the transcript level of >3900 different mRNAs changed more than 2-fold following Ad5WT infection, less than 600 were changed by Ad5mir122. These were then filtered to select mRNAs that were only altered by Ad5mir122 and the remaining 21 mRNAs were compared to predicted mir122 targets. No mir122 target mRNAs were affected by Ad5 mir122. These results demonstrate that the exploitation of microRNA regulation to control virus replication does not necessarily affect the level of the microRNA or the endogenous mRNA targets

Active Adenoviral Vascular Penetration by Targeted Formation of Heterocellular Endothelial–epithelial Syncytia

The endothelium imposes a structural barrier to the extravasation of systemically delivered oncolytic adenovirus (Ad). Here, we introduced a transendothelial route of delivery in order to increase tumor accumulation of virus particles (vp) beyond that resulting from convection-dependent extravasation alone. This was achieved by engineering an Ad encoding a syncytium-forming protein, gibbon ape leukemia virus (GALV) fusogenic membrane glycoprotein (FMG). The expression of GALV was regulated by a hybrid viral enhancer-human promoter construct comprising the human cytomegalovirus (CMV) immediate-early enhancer and the minimal human endothelial receptor tyrosine kinase promoter (“eTie1”). Endothelial cell-selectivity of the resulting Ad-eTie1-GALV vector was demonstrated by measuring GALV mRNA transcript levels. Furthermore, Ad-eTie1-GALV selectively induced fusion between infected endothelial cells and uninfected epithelial cells in vitro and in vivo, allowing transendothelial virus penetration. Heterofusion of infected endothelium to human embryonic kidney 293 (HEK 293) cells, in mixed in vitro cultures or in murine xenograft models, permitted fusion-dependent transactivation of the replication-deficient Ad-eTie1-GALV, due to enabled access to viral E1 proteins derived from the HEK 293 cytoplasm. These data provide evidence to support our proposed use of GALV to promote Ad penetration through tumor-associated vasculature, an approach that may substantially improve the efficiency of systemic delivery of oncolytic viruses to disseminated tumors

Use of Tissue-Specific MicroRNA to Control Pathology of Wild-Type Adenovirus without Attenuation of Its Ability to Kill Cancer Cells

Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines; however, uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild-type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus adenovirus, we have engineered a hepatocyte-safe wild-type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice. To do this, we have included binding sites for hepatocyte-selective microRNA mir-122 within the 39 UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of mir-122 binding sites caused up to 80-fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild-type Ad5 (5610 10 viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild-type virus retained full activity within cancer cells and provided a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral disease

Liver specific microRNA control of adenovirus serotype five

MicroRNAs are small non-coding RNA molecules that regulate mRNA translation by binding to complementary sequences usually within the 3’ un-translated region (UTR). By inserting four perfectly complementary binding sites for the hepatic specific microRNA mir122 into the 3’ UTR of adenovirus wild type 5 (Ad5 WT) E1A mRNA I show that the acute liver toxicity caused by Ad5 WT in mice can be significantly reduced. This virus, termed Ad5-mir122, is a promising virotherapy candidate and causes no obvious liver pathology whilst maintaining Ad5 WT replication in mir122 negative cells.Data shows that repeat intravenous administration of Ad5-mir122 (2x1010vp) to HepG2 tumour bearing mice mediated significant anti-cancer efficacy. RT-QPCR for E1A mRNA demonstrated a 29-fold reduction when compared to Ad5 WT in murine liver whilst western blot confirmed that all E1A protein variants were knocked down. Viral genomic replication was also reduced in mouse liver by 25-fold compared to Ad5 WT. This control of virus activity reduced alanine and aspartate transaminase release by &gt;15-fold and histological analysis showed little to no pathology in Ad5-mir122 infected livers.Measurement of mature mir122 levels in Ad5-mir122 infected livers by RT-QPCR showed that the quantity of mir122 remained unaffected at therapeutic doses. Complete genome mRNA array profiling of infected livers showed that the transcript levels of &gt;3900 different mRNAs were changed more than 2-fold following Ad5 WT infection whilst less than 600 were changed by Ad5-mir122. A non-replicating control adenovirus vector altered &gt;550 mRNAs. No known mir122 target mRNAs were affected following infection with Ad5-mir122. Western blot analysis of a known mir122 regulated target (Aldolase A) confirmed these results, demonstrating no change in protein level despite infection with Ad5-mir122. These data combined demonstrate that the exploitation of microRNA mir122 regulation to control adenovirus replication is a safe method of control and does not alter the endogenous level or activity of the microRNA or its endogenous mRNA targets. Ad5-mir122 is a potent anti-cancer agent that replicates to wild-type levels in microRNA mir122 negative cells but is specifically and safely attenuated in hepatocytes.</p

Liver specific microRNA control of adenovirus serotype five

MicroRNAs are small non-coding RNA molecules that regulate mRNA translation by binding to complementary sequences usually within the 3’ un-translated region (UTR). By inserting four perfectly complementary binding sites for the hepatic specific microRNA mir122 into the 3’ UTR of adenovirus wild type 5 (Ad5 WT) E1A mRNA I show that the acute liver toxicity caused by Ad5 WT in mice can be significantly reduced. This virus, termed Ad5-mir122, is a promising virotherapy candidate and causes no obvious liver pathology whilst maintaining Ad5 WT replication in mir122 negative cells.Data shows that repeat intravenous administration of Ad5-mir122 (2x1010vp) to HepG2 tumour bearing mice mediated significant anti-cancer efficacy. RT-QPCR for E1A mRNA demonstrated a 29-fold reduction when compared to Ad5 WT in murine liver whilst western blot confirmed that all E1A protein variants were knocked down. Viral genomic replication was also reduced in mouse liver by 25-fold compared to Ad5 WT. This control of virus activity reduced alanine and aspartate transaminase release by >15-fold and histological analysis showed little to no pathology in Ad5-mir122 infected livers.Measurement of mature mir122 levels in Ad5-mir122 infected livers by RT-QPCR showed that the quantity of mir122 remained unaffected at therapeutic doses. Complete genome mRNA array profiling of infected livers showed that the transcript levels of >3900 different mRNAs were changed more than 2-fold following Ad5 WT infection whilst less than 600 were changed by Ad5-mir122. A non-replicating control adenovirus vector altered >550 mRNAs. No known mir122 target mRNAs were affected following infection with Ad5-mir122. Western blot analysis of a known mir122 regulated target (Aldolase A) confirmed these results, demonstrating no change in protein level despite infection with Ad5-mir122. These data combined demonstrate that the exploitation of microRNA mir122 regulation to control adenovirus replication is a safe method of control and does not alter the endogenous level or activity of the microRNA or its endogenous mRNA targets. Ad5-mir122 is a potent anti-cancer agent that replicates to wild-type levels in microRNA mir122 negative cells but is specifically and safely attenuated in hepatocytes.This thesis is not currently available in ORA