Adeno-associated viruses (AAV) are among the foremost vectors for
gene therapy. A number of monoclonal antibodies against several serotypes of AAV have previously been prepared. Many are ...neutralizing, and the predominant mechanisms have been reported as the inhibition of binding to extracellular glycan receptors or interference with some post-entry step. The identification of a protein receptor and recent structural characterization of its interactions with AAV compel reconsideration of this tenet. AAVs can be divided into two families based on which domain of the receptor is strongly bound. Neighboring domains, unseen in the high-resolution electron microscopy structures have now been located by electron tomography, pointing away from the virus. The epitopes of neutralizing antibodies, previously characterized, are now compared to the distinct protein receptor footprints of the two families of AAV. Comparative structural analysis suggests that antibody interference with protein receptor binding might be the more prevalent mechanism than interference with glycan attachment. Limited competitive binding assays give some support to the hypothesis that inhibition of binding to the protein receptor has been an overlooked mechanism of neutralization. More extensive testing is warranted.
Human gene therapy has advanced from twentieth-century conception to twenty-first-century reality. The recombinant Adeno-Associated Virus (rAAV) is a major gene therapy vector. Research continues to ...improve rAAV safety and efficacy using a variety of AAV capsid modification strategies. Significant factors influencing rAAV transduction efficiency include neutralizing antibodies, attachment factor interactions and receptor binding. Advances in understanding the molecular interactions during rAAV cell entry combined with improved capsid modulation strategies will help guide the design and engineering of safer and more efficient rAAV gene therapy vectors.
Adeno-associated virus (AAV) is a small ssDNA satellite virus of high interest (in recombinant form) as a safe and effective gene therapy vector. AAV's human cell entry receptor (AAVR) contains ...polycystic kidney disease (PKD) domains bound by AAV. Seeking understanding of the spectrum of interactions, goat AAVGo.1 is investigated, because its host is the species most distant from human with reciprocal cross-species cell susceptibility. The structure of AAVGo.1, solved by cryo-EM to 2.9 Å resolution, is most similar to AAV5. Through ELISA (enzyme-linked immunosorbent assay) studies, it is shown that AAVGo.1 binds to human AAVR more strongly than do AAV2 or AAV5, and that it joins AAV5 in a class that binds exclusively to PKD domain 1 (PKD1), in contrast to other AAVs that interact primarily with PKD2. The AAVGo.1 cryo-EM structure of a complex with a PKD12 fragment of AAVR at 2.4 Å resolution shows PKD1 bound with minimal change in virus structure. There are only minor conformational adaptations in AAVR, but there is a near-rigid rotation of PKD1 with maximal displacement of the receptor domain by ~1 Å compared to PKD1 bound to AAV5. AAVGo.1 joins AAV5 as the second member of an emerging class of AAVs whose mode of receptor-binding is completely different from other AAVs, typified by AAV2.
Adeno-associated virus (AAV) is a small ssDNA satellite parvovirus. As a recombinant vector with a protein shell encapsidating a transgene, recombinant AAV (rAAV) is a leading delivery vehicle for gene therapy, with two FDA-approved treatments and 150 clinical trials for 30 diseases. The human entry receptor AAVR has five PKD domains. To date, all serotypes, except AAV5, have interacted primarily with the second PKD domain, PKD2. Goat is the AAV host most distant from human with cross-species cell infectivity. AAVGo.1 is similar in structure to AAV5, the two forming a class with a distinct mode of receptor-binding. Within the two classes, binding interactions are mostly conserved, giving an indication of the latitude available in modulating delivery vectors.
Evolutionary life history theory seeks to explain how reproductive and survival traits are shaped by selection through allocations of an individual's resources to competing life functions. Although ...life-history traits evolve rapidly, little is known about the genetic and cellular mechanisms that control and couple these tradeoffs. Here, we find that two laboratory-adapted strains of C. elegans descended from a single common ancestor that lived in the 1950s have differences in a number of life-history traits, including reproductive timing, lifespan, dauer formation, growth rate, and offspring number. We identified a quantitative trait locus (QTL) of large effect that controls 24%-75% of the total trait variance in reproductive timing at various timepoints. Using CRISPR/Cas9-induced genome editing, we show this QTL is due in part to a 60 bp deletion in the 3' end of the nurf-1 gene, which is orthologous to the human gene encoding the BPTF component of the NURF chromatin remodeling complex. Besides reproduction, nurf-1 also regulates growth rate, lifespan, and dauer formation. The fitness consequences of this deletion are environment specific-it increases fitness in the growth conditions where it was fixed but decreases fitness in alternative laboratory growth conditions. We propose that chromatin remodeling, acting through nurf-1, is a pleiotropic regulator of life history trade-offs underlying the evolution of multiple traits across different species.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology ...is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry.
Most biological traits and common diseases have a strong but complex genetic basis, controlled by large numbers of genetic variants with small contributions to a trait or disease risk. The ...effect-size of most genetic variants is not absolute and is instead dependent upon multiple factors such as the age and genetic background of an organism. In order to understand the mechanistic basis of these changes, we characterized heritable trait differences between two domesticated strains of C. elegans. We previously identified a major effect locus, caused in part by a mutation in a component of the NURF chromatin remodeling complex, that regulates reproductive output in an age-dependent manner. The effect-size of this locus changes from positive to negative over the course of an animal's reproductive lifespan. Here, we use a previously published macroscale model of the egg-laying rate in C. elegans to show that time-dependent effect-size is explained by an unequal use of sperm combined with negative feedback between sperm and ovulation rate. We validate key predictions of this model with controlled mating experiments and quantification of oogenesis and sperm use. Incorporation of this model into QTL mapping allows us to identify and partition new QTLs into specific aspects of the egg-laying process. Finally, we show how epistasis between two genetic variants is predicted by this modeling as a consequence of the unequal use of sperm. This work demonstrates how modeling of multicellular communication systems can improve our ability to predict and understand the role of genetic variation on a complex phenotype. Negative autoregulatory feedback loops, common in transcriptional regulation, could play an important role in modifying genetic architecture in other traits.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The standard reference
strain, N2, has evolved marked behavioral changes in social feeding behavior since its isolation from the wild. We show that the causal, laboratory-derived mutations in two ...genes, npr-1 and glb-5, confer large fitness advantages in standard laboratory conditions. Using environmental manipulations that suppress social/solitary behavior differences, we show the fitness advantages of the derived alleles remained unchanged, suggesting selection on these alleles acted through pleiotropic traits. Transcriptomics, developmental timing, and food consumption assays showed that N2 animals mature faster, produce more sperm, and consume more food than a strain containing ancestral alleles of these genes regardless of behavioral strategies. Our data suggest that the pleiotropic effects of glb-5 and npr-1 are a consequence of changes to O
-sensing neurons that regulate both aerotaxis and energy homeostasis. Our results demonstrate how pleiotropy can lead to profound behavioral changes in a popular laboratory model.
The Caenorhabditis elegans somatic gonadal precursors (SGPs) are multipotent progenitors that give rise to all somatic tissues of the adult reproductive system. The hunchback and Ikaros-like gene ...ehn-3 is expressed specifically in SGPs and is required for their development into differentiated tissues of the somatic gonad. To find novel genes involved in SGP development, we used a weak allele of ehn-3 as the basis for a reverse genetic screen. Feeding RNAi was used to screen ∼2400 clones consisting of transcription factors, signaling components, and chromatin factors. The screen identified five members of the C. elegans SWI/SNF chromatin remodeling complex as genetic enhancers of ehn-3. We characterized alleles of 10 SWI/SNF genes and found that SWI/SNF subunits are required for viability and gonadogenesis. Two conserved SWI/SNF complexes, PBAF and BAF, are defined by their unique array of accessory subunits around a common enzymatic core that includes a catalytic Swi2/Snf2-type ATPase. Tissue-specific RNAi experiments suggest that C. elegans PBAF and BAF complexes control different processes during somatic gonadal development: PBRM-1, a signature subunit of PBAF, is important for normal SGP development, whereas LET-526, the distinguishing subunit of BAF, is required for development of a differentiated cell type, the distal tip cell (DTC). We found that the SWSN-4 ATPase subunit is required for SGP and DTC development. Finally, we provide evidence that C. elegans PBAF subunits and hnd-1/dHand are important for the cell fate decision between SGPs and their differentiated sisters, the head mesodermal cells.
Here we provide evidence for a C2H2 zinc finger gene family with similarity to Ikaros and hunchback. The founding member of this family is Caenorhabditis elegans ehn-3, which has important and poorly ...understood functions in somatic gonad development. We examined the expression and function of four additional hunchback/Ikaros-like (HIL) genes in C. elegans reproductive system development. Two genes, ehn-3 and R08E3.4, are expressed in somatic gonadal precursors (SGPs) and have overlapping functions in their development. In ehn-3; R08E3.4 double mutants, we find defects in the generation of distal tip cells, anchor cells, and spermatheca; three of the five tissues derived from the SGPs. We provide in vivo evidence that C. elegans HIL proteins have functionally distinct zinc finger domains, with specificity residing in the N-terminal set of four zinc fingers and a likely protein–protein interaction domain provided by the C-terminal pair of zinc fingers. In addition, we find that a chimeric human Ikaros protein containing the N-terminal zinc fingers of EHN-3 functions in C. elegans. Together, these results lend support to the idea that the C. elegans HIL genes and Ikaros have similar functional domains. We propose that hunchback, Ikaros, and the HIL genes arose from a common ancestor that was present prior to the divergence of protostomes and deuterostomes.
Glioma-associated oncogene (GLI) transcription factors function downstream of the hedgehog signal transduction pathway to regulate the development of many animals. Although the nematode ...Caenorhabditis elegans lacks a hedgehog pathway, it does have a GLI protein that represses male development in favor of hermaphrodite development. As we discuss here, recent findings implicate two conserved transcription-repressor complexes in the repression of male-specific genes. This research indicates a possible conserved role for these complexes in either GLI-directed gene repression or sex determination.
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