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Focus Feature on CRISPR Gene Editing: What’s new with CRISPR?
October 2019
by Jeffrey Bouley  |  Email the author
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Thanks to a trio of recent news items crossing my desk, I am reminded once again that CRISPR gene editing is not only a very new area, but also one that is undergoing dramatic and rapid evolution, much like gene-sequencing technologies did in the wake of the Human Genome Project.
 
With one story, we get a reminder that CRISPR isn’t just for DNA anymore, but is an RNA-modifying technology as well; with another, we discover previously unknown behavior of the Cas9 and Cpf1 enzymes that may have implications when developing therapies with CRISPR; and finally, in a third bit of news, we learn that integrating CRISPR/Cas9 technology with a metabolic engineering platform might accelerate the discovery of innovative antibiotics.
 
So, with that introduction, let’s take a look at just a few of the many and varied new faces of CRISPR.
 
New CRISPR platform expands RNA-editing capabilities
As noted in a recent news release from the Massachusetts Institute of Technology (MIT), CRISPR-based tools have revolutionized our ability to target disease-linked genetic mutations. CRISPR technology comprises a growing family of tools that can manipulate genes and their expression, including by targeting DNA with the enzymes Cas9 and Cas12 and targeting RNA with the enzyme Cas13.
 
This collection offers different strategies for tackling mutations, notes MIT, but not just with gene editing. Targeting disease-linked mutations in RNA, which is relatively short-lived, would avoid making permanent changes to the genome. In addition, some cell types, such as neurons, are difficult to edit using CRISPR/Cas9-mediated editing, and new strategies are needed to treat devastating diseases that affect the brain.
 
McGovern Institute Investigator and Broad Institute of MIT and Harvard core member Dr. Feng Zhang and his team have now developed one such strategy, called RESCUE (RNA Editing for Specific C to U Exchange), described recently in a paper in the journal Science.
 
Zhang and his team made use of a deactivated Cas13 to guide RESCUE to targeted cytosine bases on RNA transcripts and used a novel, evolved, programmable enzyme to convert unwanted cytosine into uridine—thereby directing a change in the RNA instructions. RESCUE builds on REPAIR, a technology developed by Zhang’s team that changes adenine bases into inosine in RNA.
 
What sets RESCUE apart in the CRISPR scene is that it allows expansion into the realm of targeting modifiable positions in proteins—such as phosphorylation, glycosylation and methylation sites—for what the researchers say is the first time. Such sites act as on/off switches for protein activity and are notably found in signaling molecules and cancer-linked pathways.
 
“To treat the diversity of genetic changes that cause disease, we need an array of precise technologies to choose from. By developing this new enzyme and combining it with the programmability and precision of CRISPR, we were able to fill a critical gap in the toolbox,” said Zhang, the James and Patricia Poitras Professor of Neuroscience at MIT. Zhang also has appointments in MIT’s departments of Brain and Cognitive Sciences and Biological Engineering.
 
The REPAIR platform that preceded RESCUE used the RNA-targeting CRISPR/Cas13 to direct the active domain of an RNA editor, ADAR2, to specific RNA transcripts where it could convert the nucleotide base adenine to inosine, or letters A to I. Since no natural editors exist with alternative activities, Zhang and colleagues took the REPAIR fusion, and evolved it in the lab until it could change cytosine to uridine, or C to U.
 
The team used the new platform on human cells and demonstrated that they could target natural RNAs in the cell as well as 24 clinically relevant mutations in synthetic RNAs. They then further optimized RESCUE to reduce off-target editing while minimally disrupting on-target editing.
 
Targeting RNA instead of DNA is important, the researchers explain, because it is reversible.
 
Says MIT: “Thus, RESCUE could be deployed transiently in situations where a modification may be desirable temporarily.”
The team showed, specifically, that RESCUE can target specific sites in the RNA encoding β-catenin that are known to be phosphorylated on the protein product, leading to a temporary increase in β-catenin activation and cell growth.
 
“If such a change was made permanently, it could predispose cells to uncontrolled cell growth and cancer,” MIT notes, “but by using RESCUE, transient cell growth could potentially stimulate wound healing in response to acute injuries.”
 
The researchers also targeted a pathogenic gene variant, APOE4, which has consistently emerged as a genetic risk factor for the development of late-onset Alzheimer’s disease.
 
The Zhang lab plans to share the RESCUE system broadly, as they have with previously developed CRISPR tools. The technology will be freely available for academic research through the non-profit plasmid repository Addgene.
 
Some ‘surprising’ behavior with Cas9 and Cpf1
In a recent Nature Microbiology paper, Dr. Brett Robb of New England Biolabs and Dr. Becky Xu Hua Fu of Stanford University investigated how programmable Cas enzymes interact with their targets, particularly in the case of small deletions or mismatches, by screening different gRNAs and examining the consequences of Cpf1 and Cas9 activities.
 
In reflecting on their findings, they say they were surprised to find that one can confer potent nickase activity (just cutting one DNA strand as opposed to the more traditional double-stranded cut) of these two molecules on specific classes of mismatched targets, without any protein engineering, by simply designing a more targeted gRNA.
 
Because nicking and double-stranded cleavage of DNA can be used to induce repair and replacement mechanisms in vivo, this research suggests a dual capability of CRISPR-Cas nucleases to initiate genetic change through both types of interaction, which reportedly is a previously unknown behavior of Cas nucleases.
 
In the case of companies looking to develop human therapies with CRISPR, their research could give researchers access to a guide-specific nicking repertoire, which can impact the selection of gRNA targets to avoid off-target consequences during genome editing, according to a spokesperson.
 
Noted the authors of the paper in their discussion of the research results: “This study also highlights the fact that a fraction of Cas9’s observed activities in vivo, either in native systems or in engineering applications, may reflect nicking rather than cleavage activities, with the balance presumably dependent on in-vivo conditions as well as the sequences of gRNAs and targets. Of particular importance in this regard, observations that nicking activities can provide advantages in genome editing indicate that such conditions could prove advantageous. We note that the consequences of nicking depend in each system on the kinetic balances between nick ligation, single-stranded exonuclease and endonuclease activities that might extend or convert nicks, other modes of DNA repair, and DNA replication/division rates.”
 
Accelerating the discovery of innovative antibiotics
French company Deinove says it has strengthened its expertise in the genetic engineering of rare and varied microorganisms to accelerate the discovery and optimization of innovative antibiotic structures with a new tech integration. Specifically, the integration of CRISPR/Cas9 technology into the company’s metabolic engineering platform “opens up promising avenues for the identification, characterization and optimization of new clusters of genes with antibiotic activities.”
 
In recent years, Deinove has set up a high-throughput genetic engineering platform specifically dedicated to rare microorganisms and says it has “thus demonstrated its ability to adapt genetic tools to poorly described organisms.” After developing a platform dedicated to the identification of novel antibiotic structures produced by rare bacteria (the AGIR program), Deinove wanted to strengthen its expertise in genetic engineering with integration of CRISPR/Cas9 technology.
 
The objective for Deinove is to be able to directly manipulate the strains producing antimicrobial activities or to transfer these activities into phylogenetically close frames. This has been successfully achieved by the company, it says, in part by making the Streptomyces chassis an effective producer of a pharmaceutical intermediate initially produced by Microbacterium arborescens.
“Our expertise in the genetic engineering of a variety of microorganisms—unusual for some—is unique, and the integration of CRISPR-Cas9 extends the possibilities of our platform,” said Dr. Georges Gaudriault, scientific director of Deinove. “We continue to structure the various technological bricks of the AGIR platform to be able to drastically accelerate the identification and optimization of new antibiotic structures. This technology is an additional asset in our race against the clock in the face of rising antimicrobial resistance.”
 

 
Q&A: Using CRISPR to simplify cancer immunotherapy
Refuge Biotech designs intelligent cell therapies with its CRISPR technology
 
By Mel J. Yeates
 
Refuge Biotechnologies Inc. has a secret weapon in their proprietary receptor-dCas platform, which uses a unique gene engineering approach based on precision CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi).
 
Refuge’s CRISPR interference technology is founded on the research of Stanley Qi of Stanford University, the scientific co-founder of Refuge Biotech.
 
Instead of cutting out pieces of DNA, Refuge can modify multiple gene expressions in varying degrees. By connecting ligand-specific receptors to dCas, Refuge Biotech enables cells to sense their surroundings and conditionally activate or repress multiple genes when they encounter specific external antigens.
 
DDNews spoke with Dr. Bing Wang, Refuge Biotech’s co-founder and CEO, to learn more about Refuge’s CRISPR technology and the company’s plans for it.
 
DDNews: How is Refuge’s CRISPR interference technology different from other CRISPR editing technologies?
 
Bing Wang, Ph.D.: The Refuge platform is unique in that it uses CRISPR interference/activation to facilitate inducible gene modulation of multiple genes simultaneously with cell therapies such as CAR-T. This approach is very different to standard gene-editing technology, such as standard CRISPR, TALENs and Zinc fingers, in that there is no cutting of the genome.
By being able to control gene modulation, the Refuge technology can titrate the level of up- or down-regulation, which provides an analogue response compared to an all-or-none effect from editing technologies. This will be an important tool for combination therapies, as you may not want to completely knock out endogenous genes, especially if there are permanent changes to the genome after tumor cells are cleared.
 
Because the Refuge platform does not cut genetic material, additional sgRNAs can be added for a multiplexed gene modulation. It is also important to note that the addition of more sgRNAs is done simultaneously and not sequentially, thereby providing better efficiency for manufacturing and avoiding translocation and genotoxicity risks.
 
DDNews: Can you tell us more about Refuge’s receptor-dCas platform? What kind of applications does this platform lend itself toward?
 
Wang: The CRISPR is “deactivated” to mute the cleavage site while maintaining all the gene-targeting specificity through a short guide RNA (sgRNA). In other words, our therapeutic cells can still pinpoint specific genes, while not making permanent cuts to the DNA. At the same time, a transcription regulator such as a KRAB complex, which down-regulates gene expression, or a VPR complex, which up-regulates gene expression, is combined to the deactivated CRISPR. This structure is tethered to the cell surface so that it is only released in response to tumor antigen engagement through activation of a CAR [chimeric antigen receptor] on the T cell surface. On release, the structure travels to the cell nucleus guided by sgRNA to prompt specific gene modulation, which is where multiplexed modulation comes in with the addition of multiple sgRNAs.
 
The synthetic biology approach means that we are able to combine multiple therapeutic mechanisms into a single therapy for the benefit of patients with cancer. It is known that while the genomic landscape in tumor biology is very complex, there are still some patterns that can be discerned amongst different tumor types. This means that we would be able to apply this strategy across the entire field of cancer treatment, rather than looking for one single pathway or mechanism to treat a particular cancer. It would be possible to tailor combinations of cell therapy and genetic modulation for treating different cancers.
 
DDNews: What is the company’s current pipeline status? Are there any targets or assets you are particularly excited about?
 
Wang: Refuge’s main lead pipeline asset is a HER2 CAR plus PD-1 knockdown system for various solid tumor indications, including breast, ovarian and gastric cancers. Discussions are currently moving forward with the FDA on this product line, with an expected IND filing in the next 12 to 18 months [and] Phase 1 trials beginning shortly thereafter.
 
Refuge is also developing a HER2-based CAR with additional checkpoint target down-regulation including simultaneous PD-1 and TIM-1 knockdown for GBM [glioblastoma multiforme], and we expect our timeline to be closely behind the lead indications I mentioned before. In addition, the Refuge team is developing various additional pipeline assets focused on other CAR targets, with modulation of different genetic target combinations derived through intensive bioinformatics analysis, for other oncology indications that will be disclosed in due course.
 
DDNews: How was the receptor-dCas technology used in creating the pipeline drug candidates?
 
Wang: The receptor-dCas technology effectively is a synthetic signal transducer, allowing the activation of a receptor by extracellular stimuli to directly influence the expression of particular therapeutic gene targets. Our platform aims to leverage existing cancer biology to create a pipeline of candidates that combine multiple compatible treatment modalities, such as HER2 CAR and knockdown of the PD-1 gene, as well as other gene and CAR targets.
 
DDNews: Does Refuge have a primary focus on oncological therapies, or does the company plan to move in other directions as well?
 
Wang: Our primary application is within the oncology space today; however, cell therapies are moving far beyond just oncology into infectious diseases, rare diseases, cardiovascular diseases and others. Outside of oncology, we also expect that the Refuge platform will be applicable to these diseases. In addition, our platform is not restricted to immune cells, so we are able to target multiple different cell types with the same synthetic biology strategy.
 
DDNews: Is there anything else that you would like to tell DDNews? What do you find most exciting about the receptor-dCas technology?
 
Wang: It’s generally agreed upon that the future of oncology treatment will not be a single “wonder” drug that is effective at treating many types of cancer, but rather combination therapies that can address the complexities of the disease. However, there are many downsides to giving patients, who most of the time are already fairly unwell, various different monotherapies that all have their own toxicity profiles. By effectively being able to combine multiple cancer treatment modalities together, the Refuge platform will be able to provide the benefits of many treatments, while also achieving a better safety profile, to treat patients with various cancers and other diseases.
 

Q&A: Reliability vs. versatility in CRISPR workflows
Exploring how reagents impact and support CRISPR research
 
By Kelsey Kaustinen
 
As CRISPR becomes more popular and familiar in labs, the targets and materials for such work become more varied. According to Ross Whittaker—who is senior manager of product management, genome modulation and editing, cell biology at Thermo Fisher Scientific—one way to segment the market is looking at the reagents used for CRISPR experiments, which generally break down into do-it-yourself (DIY) solutions and manufactured reagents. He gave us some insight into these two different approaches and how companies are applying them within the industry.
 
DDNews: What kinds of experiments are manufactured CRISPR reagents best suited for? Are these best for large or small labs?
 
Ross Whittaker: DIY solutions primarily use DNA plasmids to deliver the Cas9 enzyme and the targeting guide RNA (gRNA). These plasmids are readily available from Addgene and are routinely shared amongst labs. Manufactured reagent solutions utilize a purified protein and a chemically synthesized or synthetic gRNA, such as Thermo Fisher Scientific’s TrueCut Cas9 and TrueGuide Synthetic gRNAs.
 
We have found in general that the use of the manufactured reagents will provide much higher editing efficiency, often reaching 90 percent or higher, when compared to the DIY plasmid-based solutions. All labs, regardless of size, face the same challenges when creating cell models using genome-editing approaches; this involves the downstream work of identifying, validating and expanding edited clones. Typically, when we map the workflow for creating an edited cell line, effort and expense is consumed by these downstream tasks. By maximizing the editing efficiency using manufactured reagents, we can reduce the number of clones. This in turn reduces the cost, time and efforts included in creating edited cell lines, while at the same time increasing the overall success rate of acquiring an edited clone.
 
Additionally, as the scientific community is becoming more sophisticated about the use of the tools, there is more concern about off-target editing effects. Using a number of different approaches, such as sequencing in-silico predicted off-target sites, or using the available unbiased whole-genome approaches, such as GuideSeq or TegSeq, we and others have found that the number of off-target editing events that occur using purified Cas9 proteins and synthetic gRNAs is significantly reduced compared to using plasmid-based or DIY approaches.
 
DDNews: Are there limitations to manufactured reagents in terms of the type or size of experiments they can be used for?
 
Whittaker: There really are no limitations when it comes to manufactured reagents, as they are very scalable. By applying manufacturing principles, we are able to scale to the point that scientists can apply the technology to hundreds or thousands of genes in a single experiment. We are now able to support single targeted edits through unbiased, whole-genome CRISPR-based screens with product lines such as our LentiArray CRISPR libraries.
 
DDNews: What kind of versatility does a more “DIY” route provide in terms of CRISPR workflow? What are some of the drawbacks?
 
Whittaker: One of the features that users like about some of the DIY options is the ability to use a fluorescent marker to “enrich” for cells that have been transfected with the editing reagent via FACS [fluorescence activated cell sorting], which can help increase the number of edited cells in your cell line population. The other advantage of the DIY approach is that, since it has been so widely distributed, most scientists can readily obtain a CRISPR plasmid from a colleague and start playing around with the technology in the lab. Combined, these two features have helped the DIY approach stay very popular, particularly in academic labs.
 
The main drawbacks of the DIY approach, beyond needing to spend time cloning the plasmids for each experiment, really comes from the performance. In general, the DIY approach has much lower efficiency than using manufactured CRISPR reagents; this ultimately means that the user is going to end up screening a lot more clones. The process may be tolerable if you are creating one or two cell lines a year. However, as users adopt the technology, they want to start making lots of cell lines, and soon the low efficiency of the DIY approach really starts resulting in a significant bottleneck. Users end up spending more time generating cell lines than doing their experiments using the cell lines. The other major drawback is that off-target editing events occur at rates higher than what is observed using manufactured CRISPR reagents (purified Cas9 protein and synthetic gRNAs).
 
DDNews: What are some indications or experiment types in which you see CRISPR being applied now that it hadn’t yet been used in five years ago?
 
Whittaker: The manufactured CRISPR reagents are allowing the field to start applying the technology to a broader set of cell types, such as immune cells, iPSC cells and primary cells. The higher editing efficiency is allowing scientists to utilize CRISPR technology in cells where it is difficult or even impossible to isolate clones. Since we can drive the editing efficiency so high—greater than 90-percent editing efficiency, in many cases—we are able to use edited pools of cells instead of needing to isolate clones. Not only does this accelerate research programs, but this drive for higher efficiency is the key to the speed of which we are seeing CRISPR move into translational research. In the cell therapy field, where you don’t have time to isolate and grow up clones, the technology can be used to modify a high enough percentage of the cells in a given population that a therapeutic effect can be achieved.
 

Engineering “hairpins” increases CRISPR accuracy
 
DURHAM, N.C.—Biomedical engineers at Duke University have developed a method for improving the accuracy of the CRISPR genome-editing technology by what they say is an average of 50-fold. They believe it can be easily translated to any of the editing technology’s continually expanding formats.
 
The approach adds a short tail to the guide RNA which is used to identify a sequence of DNA for editing. This added tail folds back and binds onto itself, creating a “lock” that can only be undone by the targeted DNA sequence.
 
The study appeared online on April 15 in the journal Nature Biotechnology.
 
“CRISPR is generally incredibly accurate, but there are examples that have shown off-target activity, so there’s been broad interest across the field in increasing specificity,” said Dr. Charles Gersbach, the Rooney Family Associate Professor of Biomedical Engineering at Duke. “But the solutions proposed thus far cannot be easily translated between different CRISPR systems.”
 
Scientists in the field have spent years looking for new CRISPR systems with desirable properties and are constantly adding to the CRISPR arsenal. For example, some systems are smaller and better able to fit inside of a viral vector to deliver to human cells for gene therapy. But no matter their individual abilities, all have produced unwanted genetic edits at times.
 
A universal property of CRISPR systems is their use of RNA molecules as guides that home in on the targeted DNA sequence in the genome. Once a guide RNA finds its complementary genetic sequence, the Cas9 enzyme acts as the scissors that make the cut in the DNA, facilitating changes to the genome sequence. But because each homing sequence is only 20 nucleotides long and the human genome contains about three billion base pairs, there’s a lot to sort through, and the CRISPR can sometimes make mistakes with sequences one or two base pairs short of perfection.
 
One way to improve CRISPR’s accuracy is to require two Cas9 molecules to bind onto opposite sides of the same DNA sequence for a complete cut to be made. While this approach works, it adds more parts to the system, increasing its complexity and making it harder to deliver.
 
Another approach has been to genetically engineer the Cas9 protein to make it less energetic, so it’s less likely to jump the gun and make a mistake. While this has also shown promising results, this type of protein engineering is laborious and such efforts are specific to each CRISPR system.
 
“It seems like there’s a new CRISPR system being discovered almost every week that has some kind of unique property that makes it useful for a specific application,” said Gersbach. “Doing extensive re-engineering every time we find a new CRISPR protein to make it more accurate is not a straightforward solution.”
 
“We’re focused on a solution that doesn’t add more parts and is general to any kind of CRISPR system,” added Dewran Kocak, a Ph.D. student working in Gersbach’s laboratory and the leader of this project. “What’s common to all CRISPR systems is the guide RNA, and these short RNAs are much easier to engineer.”
 
Gersbach and Kocak’s solution is to extend the guide RNA by as many as 20 nucleotides in such a way that it folds back onto itself and binds onto the end of the original guide RNA, forming a hairpin shape. This creates a sort of lock that is very difficult to displace if even a single base pair is incorrect in a DNA sequence being scrutinized for a potential cut. But because the guide RNA would prefer to bind to DNA rather than itself, the correct combination of DNA is still able to break the lock.
 
While Kocak and Gersbach showed that this method can increase the accuracy of cuts being made in human cells by an average of 50-fold across five different CRISPR systems derived from four different bacterial strains, in one case that improvement rose to over 200-fold.
 
Moving forward, the researchers hope to see just how many different CRISPR variants this approach could work with, as well as complete an in-depth characterization of exactly how the locking mechanism works to see if there are differences across CRISPR variants. And because these experiments were conducted in cultured cells, the researchers are eager to see how well this approach might increase CRISPR accuracy within an actual animal model of disease.
 

New libraries for CRISPRa and CRISPRi screens
 
MOUNTAIN VIEW, Calif.—Earlier this year, Cellecta Inc. announced the launch of what it said were the first commercially available dual-sgRNA libraries designed for CRISPR activation (CRISPRa) and CRISPR interference/repression (CRISPRi) genetic screens. These new pooled libraries enhance activation or repression of genes to produce more robust results from genetic screens.
With the modified CRISPRa and CRISPRi systems, the standard CRISPR gene knockout capacity has been re-engineered to modulate gene activity. These variations on the standard CRISPR system extend the types of genetic screening possible. For example, CRISPRa can be used to screen for genes that change phenotypes when activated, rather than disrupted. These “gain-of-function” screens are not possible with the standard CRISPR knockout system.
 
“We found several examples where targeting more than one sgRNA to the same promoter enhanced expression levels of the target gene using the CRISPRa system, even when one sgRNA by itself had no detectable effect,” explained Donato Tedesco, director of research and development at Cellecta. “As a result, it made sense to build a library where each construct has more than one sgRNA targeting each gene. We expected this to increase the overall level of effectiveness for the library and indeed, this is what we saw when we compared the overall expression levels of our single-sgRNA CRISPRa library with the dual-sgRNA version.”
 
Over the past few years, many research laboratories have taken advantage of single-sgRNA libraries to perform CRISPRa, CRISPRi and standard CRISPR knockout screens to identify potential therapeutic targets responsible for controlling growth and differentiation, regulating disease development, or other biological responses. The development of the new dual-sgRNA CRISPRa and CRISPRi libraries extends the range of tools available for functional genetic screening and accelerates the identification of novel targets for therapeutics and biomarker analysis.
 

Cardea Bio and Nanosens Innovations to combine
 
LA JOLLA, Calif.—In early September, the boards of Cardea Bio and Nanosens Innovations authorized their respective companies to proceed with a proposed business combination. After the closing, Nanosens would become a subsidiary of Cardea, with the Nanosens brand representing a series of products leveraging the CRISPR-Chip technology. The closing of the proposed transaction is subject to stockholder approval, definitive documents and customary closing conditions.
 
“The CRISPR-Chip has generated impressive levels of interest from companies both large and small, who realize the disruptive potential CRISPR-Chip has across many markets,” said Dr. Francie Barron, vice president of Cardea’s Innovation Partnership Program. “With our Innovation Partnership Program, Cardea can finally offer the combined solution and work as one big team.”
 
Continued Cardea CEO (and Nanosens co-founder) Michael Heltzen: “To show the world how aligned and well-integrated the two teams already are from having worked closely together for so long, we decided to move up and launch our Early Access Program for the Genome Sensor today at AGBT, on the very day of the announcement of the proposed combination with of the two companies.”
 
The Genome Sensor product is just the first of more products using the CRISPR-Chip technology that Cardea will offer to the research market under the Nanosens brand, with additional products planned for other markets by Cardea’s Innovation Partners. With this launch, Cardea is paving the product development pathway for its partners, ensuring them a quicker time-to-market.
 
“In today’s society, we can get almost any information we need immediately via our smartphones and the internet, but really important precision health questions still take days or weeks,” noted Heltzen. “Cardea’s long term mission is to provide those answers immediately via FDA-approved systems, and with the launch of our Early Access Program we’ve taken a key step towards realizing that vision.”
 
Code: E101936

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