Genetic characterization of human immunodeficiency virus type 1 in elite controllers: lack of gross genetic defects or common amino acid changes

Despite reports of viral genetic defects in persons who control human immunodeficiency virus type 1 (HIV-1) in the absence of antiviral therapy, the extent to which such defects contribute to the long-term containment of viremia is not known. Most previous studies examining for such defects have involved small numbers of subjects, primarily focused on subjects expressing HLA-B57, or have examined single viral genes, and they have focused on cellular proviral DNA rather than plasma viral RNA sequences. Here, we attempted viral sequencing from 95 HIV-1 elite controllers (EC) who maintained plasma viral loads of <50 RNA copies/ml in the absence of therapy, the majority of whom did not express HLA-B57. HIV-1 gene fragments were obtained from 94% (89/95) of the EC, and plasma viral sequences were obtained from 78% (61/78), the latter indicating the presence of replicating virus in the majority of EC. Of 63 persons for whom nef was sequenced, only three cases of nef deletions were identified, and gross genetic defects were rarely observed in other HIV-1 coding genes. In a codon-by-codon comparison between EC and persons with progressive infection, correcting for HLA bias and coevolving secondary mutations, a significant difference was observed at only three codons in Gag, all three of which represented the historic population consensus amino acid at the time of infection. These results indicate that the spontaneous control of HIV replication is not attributable to shared viral genetic defects or shared viral polymorphisms.     

In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses

Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications.A large number of inherited or acquired diseases remain promising targets for human gene therapy. One vector that has shown outstanding potential thus far in numerous preclinical and clinical evaluations is based on nonpathogenic adeno-associated virus (AAV). A unique asset among various properties that make AAV especially attractive over its competitors, such as adenoviral or lentiviral vectors, is the availability of a vast number of natural isolates which differ significantly in their properties (24). We and others have shown previously that the function of an AAV vector particle is determined mainly by the capsid protein and that viral Rep proteins and genomic packaging elements are largely interchangeable (24, 27, 85). Paradoxically, the ever-increasing repertoire of naturally occurring and synthetically generated AAV capsid sequences (>300 to date) is currently creating a dilemma for the rational selection of the optimal serotype for a given application. The importance of finding the ideal capsid for efficient and safe gene transfer has been exemplified in many preclinical studies, as well as in a clinical trial using the AAV type 2 (AAV-2) prototype in human liver tissue (36, 47). In one previous study, the treatment of patients with severe hemophilia B with recombinant AAV-2 expressing human factor IX (hFIX) resulted in mildly elevated, yet therapeutic, levels of this blood coagulation factor. However, expression was short lived, and the hFIX decline was accompanied by a transient asymptomatic increase of liver transaminases, due to a T-cell immune response against the AAV-2 capsid (47). Also, preexisting neutralizing anti-AAV-2 antibodies (frequent in humans) in these individuals likely inhibited the linear vector dose response previously observed in animals.We and others have suggested previously that the use of novel AAV serotypes, in particular, nonhuman isolates, will help to overcome some of these problems (19, 24, 63). Important examples are AAV-8 and AAV-9, which can transduce mouse liver far better than AAV-2, albeit the difference in dogs or primates is less clear (17, 52, 54, 75). The potential for the complete transduction of liver tissue and perhaps other tissues makes these two non-AAV-2 serotypes also particularly interesting for therapeutic RNA interference (RNAi) (28). We recently demonstrated the feasibility of efficiently and persistently suppressing hepatitis B virus with RNAi from a double-stranded AAV-8 vector (28). On the other hand, a potential drawback of AAV-8 and AAV-9 is their lack of specific tissue tropism (34, 52). The resulting frequent vector dissemination into all organs, including the brain, even from low peripheral doses in mice or monkeys (52, 54) is a particular concern for RNAi therapies in which control over vector biodistribution and the limitation of off-target effects will be imperative for the success of the approach (28).In order to overcome the constraints of wild-type AAV serotypes, numerous groups have recently begun to develop novel strategies to engineer “designer” AAVs tailored for the therapeutic transduction of clinically relevant organs (reviewed in detail in references 9, 12, 35, 41, 51, and 85). Briefly, the variety of strategies can be grouped into indirect or chemical approaches and direct physical modification strategies. In the indirect approaches, specific molecules (e.g., bispecific antibodies [6] or avidin-coupled ligands [4]) are allowed to react with the viral surface (biotinylated in the case of avidin [4]), as well as a cellular receptor, forming a conjugate ideally able to retarget the capsid to a refractory cell type. Yet, numerous pharmacological problems, such as concerns about in vivo complex stability and difficulties in upscaling complex manufacturing, continue to prevent the broad adaptation of these approaches. Alternative, more powerful strategies rely on the direct physical modification of the AAV capsid protein and gene. Early examples include the generation of mosaic AAV capsids via the mixing of helper plasmids carrying capsid genes from distinct serotypes, such as AAV-1 and AAV-2 (30) and pairwise combinations of AAV-1 through AAV-5 (62). Similar mosaics were generated previously via a marker rescue approach, yielding AAV-2/AAV-3 recombinants with unique properties (8). A related strategy is the rational creation of chimeric virions via domain swapping among multiple parental serotypes, involving either entire capsid loops or parts thereof or individual residues. Notable examples include AAV-1/AAV-2 chimeras with improved tropism in muscle tissue (31), with one of these chimeras presently being studied in a phase I clinical trial for the treatment of Duchenne muscular dystrophy (85). Most recently, our own group described a battery of unique chimeras comprising elements from serotypes 2 and 8, which were exploited to identify capsid subdomains responsible for efficient AAV transduction in murine liver tissue in vivo (64).A special type of chimeric capsids are those containing foreign proteins or peptides inserted into various positions of the virion shell. The methods and strategies used are widely diverse, and again, we refer to comprehensive reviews (12, 35, 41). Noteworthy here are approaches to fuse targeting ligands to the N termini of AAV capsid proteins (ideally, VP2 [45, 83]), or more powerful, to insert short peptides (up to 14 amino acids [21], typically 7) into exposed regions of the assembled virion. This strategy is referred to as viral display, in analogy to phage display, and has already been used extensively to retarget AAV-2 virions to a multitude of refractory or hard-to-infect cell types, such as vascular endothelial, smooth muscle, and pancreatic islet cells (43, 55, 77, 81, 82) and various tumor lines (22, 58, 65, 66). It has particularly benefited from comprehensive mutational analyses by various groups (e.g., references 21, 33, 56, and 83) that have resulted in the identification of prominent locations within the AAV-2 capsid tolerating peptide insertion. Most notable is the heparin binding domain (HBD), consisting of a total of four arginine (R) residues and one lysine residue, with R585 and R588 representing the most crucial components (37, 56). Numerous groups have now consistently shown that the insertion of 7-mer peptides into this region not only is frequently well tolerated and efficiently mediates virus retargeting, but also provides the extra benefit that the endogenous AAV-2 tropism can be abolished, thus enhancing target specificity (e.g., reference 21).In addition to identifying sites for vector engineering, some of the mutational AAV studies directly yielded novel capsid variants with potential benefits for clinical use. A remarkable case was a recent study by Lochrie et al. (42) in which a set of 127 AAV-2 variants with point or insertion mutations were generated and screened for multiple properties. Several capsids were isolated which differed from the wild-type AAV-2 capsid in having better in vitro transduction efficiencies (albeit being equally efficient in vivo) or, clinically most relevant, higher-level resistance to individual or pooled human antisera. Nonetheless, the limitations of the approach also became clear, most notably, the extreme effort required to generate and manually screen a large number of mutants, which in fact prevented the interesting analyses of all possible combinations of beneficial point mutations in further capsids.Indeed, the factors of time and labor are the main reasons why an increasing number of groups have recently begun to develop novel means for AAV vector evolution that no longer rely on the rational modification of the AAV-2 capsid. Instead, the new combinatorial methodologies allow for the far more efficient creation and selection of interesting candidates in a library-based high-throughput format. Thus far, two different strategies have been reported, both principally expanding on previously developed techniques. One is the use of viral display libraries, in which random 7-mer peptides are inserted into the AAV-2 HBD (at amino acid 587 or 588), yielding between 4 × 10⁶ and 1.7 × 10⁸ capsids potentially exposing new ligands on their surfaces (50, 58, 76). Subsequent iterative selection on diverse cell types refractory to the wild type, e.g., coronary artery endothelial cells, cardiomyoblasts, and carcinoma, leukemia, and megakaryocytic cell lines, led to enrichment with peptide mutants with increased target specificities and efficacies (48, 50, 58, 76). The second library type, independently described by two groups in 2006, relies on error-prone PCR amplification of the AAV-2 capsid gene (46, 59). Similar to the methods in earlier mutational studies, this approach resulted in the identification of AAV-2 point mutations (usually up to two per capsid) which yielded mutants that differed from the wild type in having mildly enhanced efficacies in vitro and/or improved transduction efficiencies in the presence of neutralizing anti-AAV-2 antibodies either generated in rabbits or preexisting in individual human sera.Here, for the first time, we introduce the technology of DNA family shuffling into the realm of AAV vector evolution. The basic concept of this technology is the in vitro recombination of related parental genes with >50% homology, which are first fragmented and then reassembled based on partial homology, resulting in libraries of chimeric genes. Iterative amplification under pressure can then yield hybrids not only combining parental assets, but also ideally exhibiting novel and synergistic properties (70, 71). DNA family shuffling has been used extensively in recent years to evolve and improve all types of proteins, from markers and enzymes to vaccines (e.g., references 10, 13-15, and 39). Importantly, a set of reports also suggested its power to enhance viral gene therapy vectors by creating retro- or lentiviruses with improved stability or efficacy compared to that of the parental wild types (57, 61, 69). Here, we describe the novel use of DNA family shuffling for the highly efficient molecular interbreeding of eight multispecies AAVs to create chimeric capsids and, moreover, document its compatibility and synergism with existing AAV vector evolution technology.     

Accelerated prion replication in, but prolonged survival times of, prion-infected CXCR3-/- mice

Prion diseases have a significant inflammatory component. Glia activation, which is associated with increased production of cytokines and chemokines, may play an important role in disease development. Among the chemokines upregulated highly and early upregulated during scrapie infections are ligands of CXCR3. To gain more insight into the role of CXCR3 in a prion model, CXCR3-deficient (CXCR3^−/−) mice were infected intracerebrally with scrapie strain 139A and characterized in comparison to similarly infected wild-type controls. CXCR3^−/− mice showed significantly prolonged survival times of up to 30 days on average. Surprisingly, however, they displayed accelerated accumulation of misfolded proteinase K-resistant prion protein PrP^Sc and 20 times higher infectious prion titers than wild-type mice at the asymptomatic stage of the disease, indicating that these PrP isoforms may not be critical determinants of survival times. As demonstrated by immunohistochemistry, Western blotting, and gene expression analysis, CXCR3-deficient animals develop an excessive astrocytosis. However, microglia activation is reduced. Quantitative analysis of gliosis-associated gene expression alterations demonstrated reduced mRNA levels for a number of proinflammatory factors in CXCR3^−/− compared to wild-type mice, indicating a weaker inflammatory response in the knockout mice. Taken together, this murine prion model identifies CXCR3 as disease-modifying host factor and indicates that inflammatory glial responses may act in concert with PrP^Sc in disease development. Moreover, the results indicate that targeting CXCR3 for treatment of prion infections could prolong survival times, but the results also raise the concern that impairment of microglial migration by ablation or inhibition of CXCR3 could result in increased accumulation of misfolded PrP^Sc.     

Normalization of microarray data using a spatial mixed model analysis which includes splines

MOTIVATION: Microarray experiments with thousands of genes on a slide and multiple slides used in any experimental set represent a large body of data with many sources of variation. The identification of such sources of variation within microarray experimental sets is critical for correct deciphering of desired gene expression differences. RESULTS: We describe new methods for the normalization using spatial mixed models which include splines and analysis of two-colour spotted microarrays for within slide variation and for a series of slides. The model typically explains 45-85% of the variation on a slide with only approximately 1% of the total degrees of freedom. The results from our methods compare favourably with those from intensity dependent normalization loess methods where we accounted for twice as much uncontrolled and unwanted variation on the slides. We have also developed an index for each EST that combines the various measures of the differential response into a single value that researchers can use to rapidly assess the genes of interest.     

Assembly of the giant protein projectin during myofibrillogenesis in <Emphasis Type="Italic">Drosophila</Emphasis> indirect flight muscles

Background  

Projectin is a giant modular protein of Drosophila muscles and a key component of the elastic connecting filaments (C-filaments), which are involved in stretch activation in insect Indirect Flight Muscles. It is comparable in its structure to titin, which has been implicated as a scaffold during vertebrate myofibrillogenesis.     

Fish can encode order in their spatial map

Animals must often orient through areas that are larger than their perceptual range. The blind Mexican cave fish, Astyanax fasciatus, depends on detecting self-induced near-field wave perturbations by objects via the use of its lateral line organ. Its perceptual range (less than or equal to 0.05 m) is greatly exceeded by its ecological ranging requirements (ca. 30 m). Although known to possess a spatial map of its environment, it is not known how this fish links places (or the area over which the perceptual range extends) together. Using the blind cave fish's propensity to accelerate when faced with objects or environments that are recognizably different, I used a behavioural assay to test whether fishes can learn and remember the order of a landmark sequence. I show, to my knowledge for the first time, that blind Mexican cave fish can encode order in their spatial map. The ability to represent the order in which a series of places are spatially linked is a powerful tool for animals that must orient beyond the limit of their perceptual range. The resulting spatial map would be analogous to a jigsaw puzzle, where each piece represents a place whose size is constrained by the animal's perceptual range.     

S-adenosylmethionine (SAMe) protects against acute alcohol induced hepatotoxicity in mice small star, filled

Although S-Adenosylmethionine (SAMe) has beneficial effects in many hepatic disorders, the effects of SAMe on acute alcohol-induced liver injury are unknown. In the present study, we investigated effects of SAMe on liver injury in mice induced by acute alcohol administration. Male C57BL/6 mice received ethanol (5 g/kg BW) by gavage every 12 hrs for a total of 3 doses. SAMe (5 mg/kg BW) was administrated i.p. once a day for three days before ethanol administration. Subsequent serum ALT level, hepatic lipid peroxidation, enzymatic activity of CYP2E1 and hepatic mitochondrial glutathione levels were measured colorimetrically. Intracellular SAMe concentration was measured by high-performance liquid chromatography (HPLC). Histopathological changes were assessed by H&E staining. Our results showed that acute ethanol administration caused prominent microvesicular steatosis with mild necrosis and an elevation of serum ALT activity. SAMe treatment significantly attenuated the liver injury. In association with the hepatocyte injury, acute alcohol administration induced significant decreases in both hepatic SAMe and mitochondrial GSH levels along with enhanced lipid peroxidation. SAMe treatment attenuated hepatic SAMe and mitochondrial GSH depletion and lipid peroxidation following acute alcohol exposure. These results demonstrate that SAMe protects against the liver injury and attenuates the mitochondrial GSH depletion caused by acute alcohol administration. SAMe may prove to be an effective therapeutic agent in many toxin-induced liver injuries including those induced by alcohol.     

Modification of the N-terminus of peptidomimetic protein tyrosine phosphatase 1B (PTP1B) inhibitors: identification of analogues with cellular activity

Low molecular weight peptidomimetic compounds based on O-malonyl tyrosine and O-carboxymethyl salicylic acid are potent inhibitors of PTP1B. Modifications of the N-terminal Boc-Phe moiety were undertaken in an effort to improve physical chemical properties and to achieve cellular activity. Although Phe ultimately proved to be the optimal N-terminal amino acid, several viable replacements for the Boc group were identified, two of which afforded analogues that were effective at enhancing the insulin-stimulated uptake of 2-deoxyglucose by L6 myocytes.     

Action of gonadotropin-releasing hormone II on the baboon ovary

The content, binding affinity, and bioactivity of chicken II GnRH (GnRH II) and a stable analogue of GnRH II (GnRH II analogue) in the baboon ovary were studied. Although mammalian GnRH is rapidly degraded by baboon ovarian extracts, we designed a GnRH II analogue that is stable to ovarian enzymatic degradation. This analogue binds to the ovarian membranes with high affinity (41 +/- 3 nM), having 20-fold the affinity of a potent mammalian GnRH analogue. The bioactivity of GnRH II and this GnRH II analogue on the regulation of ovarian progesterone release was compared with that for a potent mammalian GnRH analogue using a baboon granulosa cell culture system. Both GnRH II and GnRH II analogue produced significant inhibition of progesterone release from the granulosa cells (P < 0.03 and P < 0.005, respectively), with a greater reduction observed using the GnRH II analogue. After 24 h in culture, this GnRH II analogue produced a 59% +/- 5% inhibition of progesterone with a concentration as low as 1 nM. Maximal inhibition of 75% +/- 1% was attained with 10 nM GnRH II analogue. The endogenous GnRH II content in the baboon ovary was 5-14 pmoles/g protein. The release of endogenous GnRH II from granulosa cells was observed throughout the 48 h in culture. These studies demonstrated the presence of high enzymatic activity for the degradation of mammalian GnRH in the ovary, whereas this GnRH II analogue was stable. High-affinity binding sites for this GnRH II analogue were also found. GnRH II and this GnRH II analogue can regulate progesterone production from baboon granulosa cells, suggesting that GnRH II is a potent regulator of ovarian function.     

Parathyroid hormone-related protein treatment of pregnant rats delays the increase in connexin 43 and oxytocin receptor expression in the myometrium

Myometrial quiescence during pregnancy is maintained by progesterone, which suppresses the expression of labor-associated genes such as connexin 43 (Cx43) and the oxytocin receptor (OTR). Parathyroid hormone-related protein (PTHrP) is a smooth muscle relaxant that inhibits myometrial contractions and therefore may act in synergy with progesterone to maintain myometrial quiescence during late pregnancy. We investigated the possibility that PTHrP, like progesterone, could act to suppress the expression of labor-associated genes. Pregnant rats were treated starting on Day 19 with daily i.p. injections of 100 microg/kg PTHrP (human synthetic fragment 1-34). On Day 22 of gestation, there was a significant reduction in the expression of Cx43 (mRNA and protein) and OTR (mRNA) in the myometrium of PTHrP-treated animals, whereas on Day 23 (labor) the expression of both Cx43 and OTR was unchanged by PTHrP treatment. Treatment of pregnant rats with PTHrP did not affect the time of delivery, concentrations of progesterone in maternal plasma, or levels of c-fos, fra-2, or parathyroid hormone/PTHrP receptor mRNA on any gestational day. Because PTHrP treatment delayed the dramatic increase in the expression of Cx43 and OTR, it may be an important factor in the maintenance of the quiescent state of the myometrium at a time when the concentrations of progesterone in maternal circulation decrease. PTHrP treatment did not prevent the increase in Cx43 and OTR gene expression on Day 23 or the timing of labor, suggesting that the effects of PTHrP signaling are overridden with the onset of labor.