Day: 03/04/2011

Gene therapy for HIV scores early success

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// // A method that specifically targets a single gene may have just had its very first success in the clinic!

Patients with the human immunodeficiency virus (HIV) were the first to receive the experimental gene therapy, which boosted their immune-cell counts.

The study was a phase 1 safety trial that tested enzymes known as ‘zinc finger nucleases,’ which are small proteins that can be designed to bind to and edit specific DNA sequences. The ones used here were developed by Sangamo BioSciences.

For the 6 men with HIV who participated, the standard regimen of highly active antiretroviral drugs (HAART) were keeping the virus at bay, but their immune-cell counts remained dangerously low.

  1. The researchers obtained samples of immune cells affected by HIV – known as CD4+T cells.
  2. Then they used Sangamo’s enzyme to disrupt the CCR5 gene, which encodes a protein that HIV uses to enter CD4+ cells.
  3. Then the engineered cells were then infused back into the patients.

The immune-cell counts rose for 5 of the 6 patients receiving the therapy. Researchers suspect that if they follow this procedure several times in a given patient, a high percentage of resistant cells could be established.

(Interesting fact: A small percentage of people have a natural mutation in the CCR5 gene and are actually resistant to most types of HIV infection.)

“Based on these very encouraging findings, we have recently extended our clinical studies to HIV-infected subjects who are not on HAART, or for whom HAART is no longer effective,” says Sangamo’s Dale Ando.

The method could one day replace the more common technique of inserting modified genes into the genome; there, researchers have less control over the gene in question. But the technique still has a relatively low efficiency and might have off-target effects.

source: smart planet

Artificial cells can inactivate deadly human viruses

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When viruses evolve, drugs made to kill them can become powerless. However, scientists at Weill Cornell Medical College have figured out how attract viruses with artificial cells…and use them to deactivate deadly viruses.

In the lab, two emerging henipaviruses didn’t stand a chance. Scientists from the National Institute of Standards and Technology and the Weill Cornell Medical College reported the results in a paper published in PLOS One.

The fake cells are called honey pot protocells. The honey pot cells were built with silica material and shaped like a regular cell, all part of the elaborate system designed to trick the viruses to come near. NIST materials scientist David LaVan said in a statement:

“The lure, the irresistibly sweet bait that you can use to capture something.”

As it turns out, the artificial cells were pretty amazing decoys. In experiments, the artificial cells got rid of the active viruses.

The researchers think the artificial cells will help them understand viruses better (and understand how viruses attack cells).

But eventually, the scientists hope the artificial cells could be used to develop an entirely new class of antiviral drugs. The honey pots go after the infection method (not the viruses), so the new drugs wouldn’t have the same drug-resistant problems drugs currently on the market might have.

source: smart planet

Microbial Communities of the Upper Respiratory Tract and Otitis Media in Children

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Streptococcus pneumoniae asymptomatically colonizes the upper respiratory tract of children and is a frequent cause of otitis media. Patterns of microbial colonization likely influence S. pneumoniae colonization and otitis media susceptibility. This study compared microbial communities in children with and without otitis media. Nasal swabs and clinical and demographic data were collected in a cross-sectional study of Philadelphia, PA, children (6 to 78 months) (n = 108) during the 2008-2009 winter respiratory virus season. Swabs were cultured for S. pneumoniae. DNA was extracted from the swabs; 16S rRNA gene hypervariable regions (V1 and V2) were PCR amplified and sequenced by Roche/454 Life Sciences pyrosequencing. Microbial communities were described using the Shannon diversity and evenness indices. Principal component analysis (PCA) was used to group microbial community taxa into four factors representing correlated taxa. Of 108 children, 47 (44%) were colonized by S. pneumoniae, and 25 (23%) were diagnosed with otitis media. Microbial communities with S. pneumoniae were significantly less diverse and less even. Two PCA factors were associated with a decreased risk of pneumococcal colonization and otitis media, as follows: one factor included potentially protective flora (Corynebacterium and Dolosigranulum), and the other factor included Propionibacterium, Lactococcus, and Staphylococcus. The remaining two PCA factors were associated with an increased risk of otitis media. One factor included Haemophilus, and the final factor included Actinomyces, Rothia, Neisseria, and Veillonella. Generally, these taxa are not considered otitis media pathogens but may be important in the causal pathway. Increased understanding of upper respiratory tract microbial communities will contribute to the development of otitis media treatment and prevention strategies.

IMPORTANCE Otitis media (middle ear infection) is the most common reason for pediatric sick visits in the United States. Streptococcus pneumoniae is a leading otitis media pathogen. S. pneumoniae must colonize the upper respiratory tract and compete with a complex community of nonpathogenic bacteria before infecting the middle ear. We compared microbial communities in the upper respiratory tract of children who had otitis media and those who did not. Members of the normal flora, i.e., Corynebacterium and Dolosigranulum, were protective for S. pneumoniae colonization and otitis media. As expected, the genera Haemophilus was associated with otitis media. Surprisingly, Actinomyces, Rothia, Neisseria, and Veillonella were associated with an increased risk of otitis media. These bacteria are not otitis media pathogens but may be associated with antibiotic use or involved in the causal pathway to disease. Increased understanding of upper respiratory tract microbial communities will lead to new ways to prevent middle ear infections, including probiotics.

 

source: journal of American society of microbiology

A differential proteome screening system for post-translational modification–dependent transcription factor interactions

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Post-translational modifications (PTMs) of transcription factors alter interactions with co-regulators and epigenetic modifiers. For example, members of the C/EBP transcription factor family are extensively methylated on arginine and lysine residues in short, conserved, modular domains, implying modification-dependent cofactor docking. Here we describe array peptide screening (APS), a systematic and differential approach to detect PTM-dependent interactions in the human proteome using chemically synthesized, biotinylated peptides coupled to fluorophore-labeled streptavidin. Peptides with and without a modified residue are applied in parallel to bacterial expression libraries in an arrayed format. Interactions are detected and quantified by laser scanning to reveal proteins that differentially bind to nonmodified or modified peptides. We have previously used this method to investigate the effect of arginine methylation of C/EBPβ peptides. The method enables determination of PTM-dependent transcription factor interactions, quantification of relative binding affinities and rapid protein classification, all independently of the transactivation potential of peptides or cellular abundance of interactors. The protocol provides a cost-effective alternative to mass spectrometric approaches and takes 3–4 d to complete.

source: nature genetics

Genome structural variation discovery and genotyping

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Comparisons of human genomes show that more base pairs are altered as a result of structural variation — including copy number variation — than as a result of point mutations. Here we review advances and challenges in the discovery and genotyping of structural variation. The recent application of massively parallel sequencing methods has complemented microarray-based methods and has led to an exponential increase in the discovery of smaller structural-variation events. Some global discovery biases remain, but the integration of experimental and computational approaches is proving fruitful for accurate characterization of the copy, content and structure of variable regions. We argue that the long-term goal should be routine, cost-effective and high quality de novo assembly of human genomes to comprehensively assess all classes of structural variation.

source: nature genetics

Analysis of multiple compound–protein interactions reveals novel bioactive molecules

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The discovery of novel bioactive molecules advances our systems-level understanding of biological processes and is crucial for innovation in drug development. For this purpose, the emerging field of chemical genomics is currently focused on accumulating large assay data sets describing compound–protein interactions (CPIs). Although new target proteins for known drugs have recently been identified through mining of CPI databases, using these resources to identify novel ligands remains unexplored. Herein, we demonstrate that machine learning of multiple CPIs can not only assess drug polypharmacology but can also efficiently identify novel bioactive scaffold-hopping compounds. Through a machine-learning technique that uses multiple CPIs, we have successfully identified novel lead compounds for two pharmaceutically important protein families, G-protein-coupled receptors and protein kinases. These novel compounds were not identified by existing computational ligand-screening methods in comparative studies. The results of this study indicate that data derived from chemical genomics can be highly useful for exploring chemical space, and this systems biology perspective could accelerate drug discovery processes.

source: Nature molecular system biology

Geography lesson in dry AMD

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American researchers have identified an RNA-based mechanism in the retina that could be responsible for triggering the blindness associated with advanced dry age-related macular degeneration.1 The team is developing inhibitors and ultimately hopes to submit an IND.

Unlike wet AMD, which results from scarring caused by leaky blood vessels and thus can be treated with a variety of angiogenesis inhibitors, dry AMD is initially characterized by the buildup of extracellular debris beneath the retina. Over time, those deposits cause significant atrophy of the retinal pigment epithelial (RPE) layer—a condition known as geographic atrophy—and can ultimately advance to permanent blindness.

Although difficult to predict, dry AMD can turn into wet AMD at any time during disease progression. There are no medical or surgical treatments for dry AMD.

To find potential targets for geographic atrophy, Jayakrishna Ambati and colleagues at the University of Kentucky College of Medicine initially looked at levels of proteins and nucleic acids in eye samples from dry AMD patients. Ambati is vice chair of ophthalmology and visual sciences at the college.

The researchers found that levels of the microRNA-processing enzyme dicer 1 ribonuclease type III (DICER1) were significantly lower in patient eyes than in non-AMD control eyes (p=0.0036). DICER1 levels were unchanged in the RPE layer of human eyes with other retinal diseases, suggesting that low DICER1 could be a specific marker of geographic atrophy.

The team next generated Dicer1 knockout mice to recapitulate the geographic atrophy phenotype in animals. All the Dicer1 knockouts showed degeneration of the RPE layer compared with wild-type littermate controls.

Subsequent studies in human RPE cells and mice revealed the specific mechanism by which low DICER1 levels led to degeneration of the RPE cells or layer, respectively. The decrease in DICER1 caused the accumulation of cytotoxic Alu RNA molecules in the RPE layer, where they caused degeneration of tissues making up the retina and macula. Alu RNAs are retrotransposon sequences that exist throughout the human genome but do not code for proteins.

In cultured human RPE cells with low DICER1 levels, antisense oligonucleotides targeting Alu RNA significantly blocked RPE cellular degeneration compared with control oligonucleotides (p<0.05).

The findings were published in Nature.

Ambati told SciBX that he now plans to explore “molecular platforms for Alu inhibition, including the Alu antisense therapy described in the paper. Once we settle on a lead candidate, we plan to proceed to GMP manufacture and FDA IND filing.”

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Targeting Alu

Although the University of Kentucky researchers initially plan to test Alu inhibitors as monotherapy for dry AMD, the heterogeneous nature of the disease makes it unlikely that targeting only Alu RNA would be sufficient to stave off blindness.

“A cocktail or combination therapy will likely result in the ideal therapeutic strategy.”

Samir Patel
Ophthotech Corp.

“Chronic diseases such as dry AMD are very heterogeneous and progress through a series of early and late stages. The advanced stage of dry AMD is characterized as either geographic atrophy or conversion to neovascular disease,” said Samir Patel, cofounder, president and CEO of Ophthotech Corp. “As a result, different molecular mechanisms may predominate at different times, and a single therapeutic target may not be useful for all stages. A cocktail or combination therapy will likely result in the ideal therapeutic strategy.”

He added: “It is important to use preclinical animal models, together with human histopathology and genetic studies, if possible, to better understand the role of Alu RNA in the overall pathogenesis and progression of dry AMD and geographic atrophy,” he continued. “This information should give us a better idea of how best to pair an Alu RNA–targeting strategy with other complementary mechanisms in dry AMD.”

Ophthotech’s ARC1905, a selective inhibitor of complement component 5, is in Phase IIa testing to treat advanced dry AMD with geographic atrophy. The company is targeting the complement system “because there is strong evidence that excessive inflammation is important in AMD and that local inflammation and activation of the complement cascade drives formation of crystalline deposits beneath the retina that are the hallmark of dry AMD,” said Patel.

Ryo Kubota, president and CEO of Acucela Inc., noted that it’s still unclear what causes the initial decreases of DICER1 levels that lead to high amounts of Alu RNA. “Our research suggests that the underlying pathology of AMD can be traced to oxidative stress caused by toxic byproducts of the visual cycle, which negatively impact human RPE cells,” said Kubota.

Acucela’s ACU-4429, a small molecule that targets the retinal pigment epithelium–specific protein 65 kDa (RPE65) to modulate the visual cycle, is in Phase II testing to treat dry AMD.

Ambati thinks targeting Alu RNA might have advantages over the other strategies. “Compared with complement inhibitors, which modulate the immune system, targeting Alu RNA could have less potential to increase the patient’s risk of infection. And compared with visual cycle inhibitors, an anti–Alu RNA approach might have lower rates of nontolerance by avoiding the negative effects visual cycle inhibitors can have on night vision,” he said.

source: sciBX