Evaluierung von Methoden zum Coating von Implantatmaterialien mit derivatisierten Biopolymeren im Hinblick auf den Einsatz im "Drug targeting"

Das Ziel dieser Untersuchung ist ein ``Drug Targeting System'' zu entwickeln, bei welchem ein Implantattyp universell einsetzbar ist. Ein unabhängiges Ladekonzept, wobei Dosis und Art der Medikation individuell justierbar sind, soll dem Rechnung tragen. Biokompatible, bioabbaubare Polymerbeads können mit einer großen Anzahl an unterschiedlichen Therapeutika beladen werden. Das Implantat soll mit einer Polymerschicht überzogen werden, welche für das umliegende Gewebe optimiert ist. Weiters soll diese Schicht spezifische Bindestellen für die medikamentbeladenen abbaubaren Nanopartikel besitzen, welche durch chemische Derivatisierung eingebracht werden. Es soll ein Methode zur Quantifizierung solcher Bindestellen entwickelt werden. Verschiedene Ansätze müssen evaluiert werden im Hinblick auf Anzahl, Stabilität und Spezifität der immobilisierten Bindestellen.Our studies aim to establish an all-purpose system, in which one type of implant can be used for every patient, due to an independent loading concept, whereas drug dose and type are individually adaptable. Biocompatible, biodegradable polymer beads can be charged with a large number of different therapeutics. The implant itself will be camouflaged with a polymer layer, that should be optimised for tissues located in the implant environment. Furthermore this layer will be derivatised chemically to contain specific binding pockets for the drug loaded biodegradable nano particles. A method for quantification of such binding sites is needed. Different approaches have to be evaluated regarding amount, stability and specificity of the immobilised binding sites

Cleave and Rescue, a novel selfish genetic element and general strategy for gene drive

There is great interest in being able to spread beneficial traits throughout wild populations in ways that are self-sustaining. Here, we describe a chromosomal selfish genetic element, CleaveR [Cleave and Rescue (ClvR)], able to achieve this goal. ClvR comprises two linked chromosomal components. One, germline-expressed Cas9 and guide RNAs (gRNAs)—the Cleaver—cleaves and thereby disrupts endogenous copies of a gene whose product is essential. The other, a recoded version of the essential gene resistant to cleavage and gene conversion with cleaved copies—the Rescue—provides essential gene function. ClvRenhances its transmission, and that of linked genes, by creating conditions in which progeny lacking ClvR die because they have no functional copies of the essential gene. In contrast, those who inherit ClvR survive, resulting in an increase in ClvR frequency. ClvR is predicted to spread to fixation under diverse conditions. To test these predictions, we generated a ClvR element in Drosophila melanogaster. ClvR^(tko) is located on chromosome 3 and uses Cas9 and four gRNAs to disrupt melanogaster technical knockout (tko), an X-linked essential gene. Rescue activity is provided by tko from Drosophila virilis. ClvR^(tko) results in germline and maternal carryover-dependent inactivation of melanogaster tko(>99% per generation); lethality caused by this loss is rescued by the virilis transgene; ClvR^(tko) activities are robust to genetic diversity in strains from five continents; and uncleavable but functional melanogaster tko alleles were not observed. Finally, ClvR^(tko) spreads to transgene fixation. The simplicity of ClvR suggests it may be useful for altering populations in diverse species

Split versions of Cleave and Rescue selfish genetic elements for measured self limiting gene drive

Gene drive elements promote the spread of linked traits, providing methods for changing the composition or fate of wild populations. Drive mechanisms that are self-limiting are attractive because they allow control over the duration and extent of trait spread in time and space, and are reversible through natural selection as drive wanes. Self-sustaining Cleave and Rescue (ClvR) elements include a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene, a tightly linked recoded version of the essential gene resistant to cleavage (the Rescue), and a Cargo. ClvR spreads by creating loss-of-function (LOF) conditions in which those without ClvR die because they lack functional copies of the essential gene. We use modeling to show that when the Rescue-Cargo and one or both components required for LOF allele creation (Cas9 and gRNA) reside at different locations (split ClvR), drive of Rescue-Cargo is self-limiting due to a progressive decrease in Cas9 frequency, and thus opportunities for creation of LOF alleles, as spread occurs. Importantly, drive strength and duration can be extended in a measured manner—which is still self-limiting—by moving the two components close enough to each other that they experience some degree of linkage. With linkage, Cas9 transiently experiences drive by hitchhiking with Rescue-Cargo until linkage disequilibrium between the two disappears, a function of recombination frequency and number of generations, creating a novel point of control. We implement split ClvR in Drosophila, with key elements on different chromosomes. Cargo/Rescue/gRNAs spreads to high frequency in a Cas9-dependent manner, while the frequency of Cas9 decreases. These observations show that measured, transient drive, coupled with a loss of future drive potential, can be achieved using the simple toolkit that make up ClvR elements—Cas9 and gRNAs and a Rescue/Cargo

Gene drive and resilience through renewal with next generation Cleave and Rescue selfish genetic elements

Gene drive-based strategies for modifying populations face the problem that genes encoding cargo and the drive mechanism are subject to separation, mutational inactivation, and loss of efficacy. Resilience, an ability to respond to these eventualities in ways that restore population modification with functional genes, is needed for long-term success. Here, we show that resilience can be achieved through cycles of population modification with “Cleave and Rescue” (ClvR) selfish genetic elements. ClvR comprises a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene and a recoded version of the essential gene resistant to cleavage. ClvR spreads by creating conditions in which those lacking ClvR die because they lack functional versions of the essential gene. Cycles of modification can, in principle, be carried out if two ClvR elements targeting different essential genes are located at the same genomic position, and one of them, ClvR^(n+1), carries a Rescue transgene from an earlier element, ClvR^n. ClvR^(n+1) should spread within a population of ClvR^n, while also bringing about a decrease in its frequency. To test this hypothesis, we first show that multiple ClvRs, each targeting a different essential gene, function when located at a common chromosomal position in Drosophila. We then show that when several of these also carry the Rescue from a different ClvR, they spread to transgene fixation in populations fixed for the latter and at its expense. Therefore, genetic modifications of populations can be overwritten with new content, providing an ongoing point of control

Gene drive that results in addiction to a temperature-sensitive version of an essential gene triggers population collapse in Drosophila

One strategy for population suppression seeks to use gene drive to spread genes that confer conditional lethality or sterility, providing a way of combining population modification with suppression. Stimuli of potential interest could be introduced by humans, such as an otherwise benign virus or chemical, or occur naturally on a seasonal basis, such as a change in temperature. Cleave and Rescue (ClvR) selfish genetic elements use Cas9 and guide RNAs (gRNAs) to disrupt endogenous versions of an essential gene while also including a Rescue version of the essential gene resistant to disruption. ClvR spreads by creating loss-of-function alleles of the essential gene that select against those lacking it, resulting in populations in which the Rescue provides the only source of essential gene function. As a consequence, if function of the Rescue, a kind of Trojan horse now omnipresent in a population, is condition dependent, so too will be the survival of that population. To test this idea, we created a ClvR in Drosophila in which Rescue activity of an essential gene, dribble, requires splicing of a temperature-sensitive intein (TS-ClvR^(dbe)). This element spreads to transgene fixation at 23 °C, but when populations now dependent on Ts-ClvR^(dbe) are shifted to 29 °C, death and sterility result in a rapid population crash. These results show that conditional population elimination can be achieved. A similar logic, in which Rescue activity is conditional, could also be used in homing-based drive and to bring about suppression and/or killing of specific individuals in response to other stimuli

Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs

A gene drive method of particular interest for population suppression utilizes homing endonuclease genes (HEGs), wherein a site-specific, nuclease-encoding cassette is copied, in the germline, into a target gene whose loss of function results in loss of viability or fertility in homozygous, but not heterozygous, progeny. Earlier work in Drosophila and mosquitoes utilized HEGs consisting of Cas9 and a single guide RNA (gRNA) that together target a specific gene for cleavage. Homing was observed, but resistant alleles immune to cleavage, while retaining wild-type gene function, were also created through nonhomologous end joining. Such alleles prevent drive and population suppression. Targeting a gene for cleavage at multiple positions has been suggested as a strategy to prevent the appearance of resistant alleles. To test this hypothesis, we generated two suppression HEGs in Drosophila melanogaster targeting genes required for embryonic viability or fertility, using a HEG consisting of CRISPR/Cas9 and gRNAs designed to cleave each gene at four positions. Rates of target locus cleavage were very high, and multiplexing of gRNAs prevented resistant allele formation. However, germline homing rates were modest, and the HEG cassette was unstable during homing events, resulting in frequent partial copying of HEGs that lacked gRNAs, a dominant marker gene, or Cas9. Finally, in drive experiments, the HEGs failed to spread due to the high fitness load induced in offspring as a result of maternal carryover of Cas9/gRNA complex activity. Alternative design principles are proposed that may mitigate these problems in future gene drive engineering

Morphological and Transcriptomic Analysis of a Beetle Chemosensory System Reveals a Gnathal Olfactory Center

OR gene tissue expression and their chromosomal localization. a Venn diagram showing the number of ORs expressed (RPKM ≥ 0.5) in the different body parts: antennae, legs, mouthparts (as piece of the head capsule anterior of the antennae), heads (the whole head capsule including mouthparts but excluding the antennae), and bodies (excluding head and legs). b Venn diagram comparing our results (yellow, green) with data from Engsontia et al. [115] (blue, red). Number of expressed ORs, defined by RPKM ≥ 0.5 (yellow), by RT-PCR (blue), not expressed RPKM < 0.5 (green), or with no RT-PCR amplicon (red). ORs of the brown group were not previously tested by Engsontia et al. c Chromosomal localization of T. castaneum ORs. Based on the Georgia GA-2 strain genome assembly 3.0 [81], only chromosomal linkage groups containing an IR or SNMP are depicted. Gene clusters are indicated by a number referring to the chromosome and a letter conveys the relative position on the chromosome. The number of genes within this cluster is indicated in the square brackets. (PDF 277 kb

The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species

The Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control