Funding, Deals & Partnerships: BIOLOGICS & MEDICAL DEVICES; BioMed e-Series; Medicine and Life Sciences Scientific Journal – http://PharmaceuticalIntelligence.com
Computationally designed “self”-peptide could be used to better target drugs to tumors, to ensure pacemakers are not rejected, and to enhance medical imaging technologies
Researchers have created a molecule that helps nanoparticles evade immune attack and could improve drug delivery.
By Dan Cossins | February 21, 2013
A macrophage at work in a mouse, stretching itself to gobble up two smaller particlesFLICKR, MAGNARAMA synthetic molecule attached to nanoparticles acts like a passport, convincing immune cells to let the particles pass unimpeded through the body, according to a study published today (February 21) in Science. The computationally designed “self”-peptide could be used to better target drugs to tumors, to ensure pacemakers are not rejected, and to enhance medical imaging technologies.
“It’s the first molecule that can be attached to anything to attenuate the innate immune system, which is currently limiting us from delivering therapeutic particles and implanting devices,” saidDennis Discher, a professor of biophysical engineering at the University of Pennsylvania and a coauthor of the study.
“This is really interesting work,” said Joseph DeSimone, a chemical engineer at the University of North Carolina, Chapel Hill, who was not involved in the research, in an e-mail to The Scientist. “[It] strongly validates the idea of using biological evasion strategies.”
Macrophages recognize, engulf, and clear out foreign invaders, whether they’re microbes entering through a wound or a drug-loaded nanoparticle injected to target disease. Previously, researchers have attempted to escape this response by coating nanoparticles with polymer “brushes” to physically block the adhesion of blood proteins that alert macrophages to the particles’ presence. But these brushes can only delay the macrophage-signaling proteins for so long, and they can hinder uptake by the diseased cells being targeted.
With that in mind, Discher and colleagues tried instead to find a way to convince macrophages that nanoparticles are part of the body. Their previous research had shown that a membrane protein called CD47, which binds to macrophages in humans, signals “self” to the immune system, so that particles with this protein are not attacked.
Examining the architecture of the bond between CD47 and its macrophage receptor, SIRPα, the researchers were able to design a synthetic self-peptide with a similarly snug fit. “This is the key, literally, to unlocking innate immune pacification,” said Discher.
When they chemically synthesized the 21-amino-acid self-peptide and attached it to nanobeads as small as viruses in mice genetically engineered to have human-like SIRPα receptors, the researchers showed that beads with the self-peptide stayed in the blood of for longer than beads with no peptide: 30 minutes after being injected with equal numbers of each type, there were 4 times as many beads with the peptide attached than without. The results demonstrate that the synthetic molecule can reduce the rate at which phagocytes clear the beads from the body, said Discher.
Then, in mice with human lung cancer, the researchers injected fluorescently dyed beads with and without the peptide, and saw that the “self”-beads got through the macrophage-filled spleen and liver and accumulated in greater numbers in the tumor, providing a brighter signal under when imaged. In fact, the self-beads provided a signal from the tumor as strong as beads coated with human CD47.
Finally, to see whether the biological evasion strategy can be successfully combined with targeting, the researchers loaded an anticancer drug into self-beads also coated with antibodies that target cancer cells. Sure enough, these antibody-coated self-beads consistently shrank tumors more than antibody-coated beads lacking the peptide. This confirmed that when antibodies draw the attention of the macrophage, the self-peptides inhibit the macrophage’s response, acting as a “don’t-eat-me” signal, said Discher.
The results demonstrate that the synthetic peptide can provide therapeutic nanoparticles with extra time in the body—time that improves drug delivery. Furthermore, the relative simplicity of the peptide means it can be easily synthesized, making it an attractive component for use in a variety of future applications.
“The findings are “compelling” and “the technology merits moving forward,” Omid Farokhzad, director of the Laboratory of Nanomedicine and Biomaterials at Brigham and Women’s Hospital, part of Harvard Medical School, said in an e-mail to The Scientist.
A crucial next step is to test the efficacy of synthetic self-peptides in humans, Farokhzad added. “The truly relevant test is looking at human pharmacokinetics to see circulating half-life advantages of nanoparticles and their effect on therapeutic outcome.”
P.L. Rodriguez et al., “Minimal ‘self’ peptides that inhibit phagocytic clearance and enhance delivery of nanoparticles,” Science, 339: 971-74, 2013.
We introduced the main aspects of the anatomy of the skin, the advantages and disadvantages of TDDS and the main factors that affect the efficacy of a TDDS and their different types. In this followup, will try to dig a little bit deeper and analyze some examples of TDDS already made it to public use. The first TDD patch to be introduced to the US market was scopolamine in 1979 (1a,1b) for prevention of nausea and vomiting associated with motion sickness and recovery from anesthesia and surgery. But the TDDS that revolutionized the transdermal market was the nicotine patch, which was first introduced in 1991 as a treatment for smoking cessation (1c). Since then there has been development of a number of different patches, including a testosterone patch for hypogonadism in males and combination patches of estradiol and norethindrone or levonorgestrel for menopausal symptoms. Figure 1 shows the global sales of TDDS products by segments.
However, there are many disease applications that are treated with peptide or protein preparations (ranging from 900 Da molecular mass to > 150,000 Da molecular mass), usually by means of injection, as they cannot be delivered via topical application at present. Dermal and transdermal delivery of large molecules such as peptides, proteins, and DNA has remained a significant challenge.
Several attempts have been made to develop topical formulations for macromolecules using a wide variety of tools such as using delivery enhancers, delivery vehicles, and different penetration methods. For instance, the chemical enhancers such as alcohols, fatty acids, surfactants, and physical enhancers such as microneedles, ultrasonic waves and low electrical current (iontophoresis) methods have been examined to improve topical delivery of macromolecules. These techniques however, suffer from different obstacles, ranging from inverse correlation between size and transdermal transport up to variably due to solvent ions, cargo charge and pH. Poorly water-soluble peptides and proteins, which can be more readily solubilized by the dual water/oil formulation may offset some of these disadvantages.
The majority of topically applied peptides and proteins cannot enter the circulation in the skin as there is no basal-to-apical transport of such molecules through the vascular endothelium, and as such they must travel in the lymphatics in order ultimately to reach the circulation.
In a recent paper, Dr. Gregory Russell-Jones and colleagues review the use of a microemulsion system to effectively deliver proteins through the skin (2).
Water-in-Oil microemulsions:
Microemulsions are nanosized, clear, thermodynamically stable, isotropic liquid mixtures of oil, water and surfactant, frequently in combination with a co-surfactant. These droplets can ‘hide’ water-soluble molecules within a continuous oil phase and therefore enable the use of water-soluble therapeutic drugs for different diseases, that otherwise cannot be achieved by the transdermal route.
Microemulsion system may have the potential advantages in delivery because of their:
High solubilization capacity
Ease of preparation,
Transparency,
Thermodynamic stability,
High diffusion and absorption rates
Previous work, both in small animals and humans, has utilized microemulsions containing small hydrophobic molecules, or small ‘model’ hydrophilic molecules. The validity of these models in measuring lateral movement of topically applied material is rather questionable. Whereas only few of the studies evaluated the efficacy of microemulsions as transdermal drug delivery systems and were shown for desmopressin, cyclosporine and folate analogue methotrexate ( ref 2). More notably are the advances in insulin delivery.
Diabetes for instance , is the most common endocrine disorder and by the year 2010, is estimated that more than 200 million people worldwide will have DM and 300 million will subsequently have the disease by 2025 (7). DM patients suffer from a defect in insulin secretion, insulin action, or both and therefore require a constant external administration of insulin to keep their sugar levels under control. Insulin is most commonly being administrated using a pen, a syringe, an automated pump and more recently using a patch to ensure a pain-free approach. Some of these patcesh are being evaluated in clinical trials.
As a different approach, the authors have evaluated the use of microemulsion to delivery different type of peptides such as IGF-1, GHRP-6 and Insulin in an obese mice model. Among the studies that were conducted they evaluated the effect of increasing the dose of topically administered insulin formulated in a water-in-oil microemulsion which was compared with subcutaneously administered insulin. It was possible to increase the dose of topically administered insulin from 10 to 100 µg as there was no reduction in serum glucose seen at this dose. By contrast, it was not possible to increase the dose of subcutaneously administered insulin owing to the potential of death through induction of hypoglycemia (2). These are very encouraging results!!!
The authors also noted changes in weight loss/gain of the mice upon treatment depending on the initial weight and which was consistent with the known anabolic effect of insulin. Presumably the greater effect seen with the topical insulin is due to the depot-like effect of this route of administration, leading to a longer stimulation of both adipocytes and muscle cells.
An exciting area of potential development is weight control management. The results using insulin, IGF-I and GHRP-6 given topically are particularly intriguing. Whether these results can be replicated in humans and whether the use of these drugs for potential treatment of obesity will be commercially viable will be particularly interesting to observe.
Summary:
Effective peptide and protein delivery to the skin has received much attention in the pharmaceutical industry, with many companies developing a variety of delivery devices to force peptides and proteins into and across the epithelium of the skin. Despite these many attempts, effective delivery of high-molecular-mass compounds has at best been poor. The water-in-oil microemulsion system may overcome the water-impermeable barrier of the epidermis and allows for effective delivery of highly water-soluble molecules such as peptides and proteins following topical application.
2. Gregory Russell-Jones and Roy Himes. “Water-in-oil microemulsions for effective transdermal delivery of proteins”. Expert Opin. Drug Delivery 2011 Invited review – 8, 537-546.
I recently read this beautiful paper by Fredrik Nederberg from the IBM Almaden Research Center and A*STAR Institute of Bioengineeringtitled “Biodegradable nanostructures with selective lysis of microbial membranes” (http://www.nature.com/nchem/journal/v3/n5/full/nchem.1012.html)
This paper gained a lot of attention as it merged as an innovation in nanotechnology and antibacterial therapeutics and therefore I have decided to introduce it here to the audience.
“Bacteria are increasingly resistant to conventional antibiotics and, as a result, macromolecular peptide-based antimicrobial agents are now receiving a significant level of attention. Most conventional antibiotics (such as ciprofloxacin, doxycycline and ceftazidime) do not physically damage the cell wall, but penetrate into the target microorganism and act on specific targets (for example, causing the breakage of double-stranded DNA due to inhibition of DNA gyrase, blockage of cell division and triggering of intrinsic autolysins). Bacterial morphology is preserved and, as a consequence, the bacteria can easily develop resistance. In contrast, many cationic peptides (for example, magainins, alamethicin, protegrins and defensins) do not have a specific target in the microbes, and instead interact with the microbial membranes through an electrostatic interaction, causing damage to the membranes by forming pores in them3. It is the physical nature of this action that prevents the microbes from developing resistance to the peptides. Indeed, it has been proven that cationic antimicrobial peptides can overcome bacterial resistance”.
“Most antimicrobial peptides have cationic and amphiphilic features, and their antimicrobial activities largely depend on the formation of facially amphiphilic a-helical or b-sheet-like tubular structures when interacting with negatively charged cell walls, followed by diffusion through the cell walls and insertion into the lipophilic domain of the cell membrane after recruiting additional
peptide monomers. The disintegration of the cell membrane eventually leads to cell death. Over the last two decades, efforts have been made to design peptides with a variety of structures, but there has been limited success in clinical settings, and only a few cationic synthetic peptides have entered phase III clinical trials. This is largely due to the cytotoxicity (for example, haemolysis) resulting from their cationic nature, their short half-lives in vivo (they are labile to proteases) and their high manufacturing costs”
A number of cationic polymers thatmimic the facially amphiphilic structure and antimicrobial functionalities of peptides have been proposed as a better approach, because they can be prepared more easily and their synthesis can be more readily scaled up compared with peptides.
The authors Yang, Hedrick and their co-workers have developed a polymer-based peptide alternative which avoids all of these problems. The polymer incorporates three key components: a non-polar hydrophobic head and tail, which drives the polymer to self-assemble into a nanoparticle; a positively charged block that selectively interacts with the bacterial cell membrane; and a carbonate backbone that slowly breaks down inside the cell, ensuring good biocompatibility. “The starting materials of our synthesis are inexpensive, and the synthesis of the antimicrobial nanoparticles is simple and can be scaled up easily for future clinical application.
“Polycarbonates are attractive biomaterials because of their biocompatibility, biodegradability, low inherent toxicity and tunable mechanical properties”
In general, Preclinical results confirm that the nanoparticles can efficiently kill fungi and multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), even at low concentrations. The nanoparticles also showed insignificant activity against red blood cells, and no significant toxicity was observed during the in vivo studies in mice, even at concentrations well above their effective dose.
In more specific, the authors evaluated the minimal inhibitory concentrations (MICs) of the polymers against Gram-positive bacteria such as Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), and the fungus Cryptococcus neoformans. MIC is an important parameter commonly used to evaluate the activity of new antimicrobial agents, and is generally defined as the minimum concentration of an antimicrobial agent at which no visible growth of microbes is observed
Some of the MIC were evaluated against conventional antimicrobial agents that are used in clinical settings to treat infections caused by these microbes, such as vancomycin for S. aureus, MRSA and E. faecalis, and amphotericin B for C. neoformans. When compared with these conventional antimicrobial agents, the polymers demonstrated comparable antimicrobial activities against all the microbes except for E. faecalis. This is important, because vancomycin-resistant E. faecalis, and S. aureus, as well as amphotericin B-resistant C. neoformans have been reported, and the resistant strains of these microbes are not susceptible to conventional antimicrobial agents. This suggests that there is an urgent need to develop safe and efficient macromolecular antimicrobial agents.
The hypothesis was that the cationic micelles can interact easily with the negatively charged cell wall by means of an electrostatic interaction, and the steric hindrance imposed by the mass of micelles in the cell wall and the hydrogen-binding/electrostatic interaction between the cationic micelle and the cell wall may inhibit cell wall synthesis and/or damage the cell wall, resulting in cell lysis. In addition, the micelles may easily permeate the cytoplasmic membrane of the organisms due to the relatively large volume of the micelle, destabilizing the membrane as a result of electroporation and/or the sinking raft model, leading to cell death.
Haemolysis is a major harmful side effect of many cationic antimicrobial peptides and polymers. The haemolysis of mouse red blood cells was evaluated after incubation with polymers 1 and 3 at various concentrations. Although the polymers disrupt microbial walls/membranes efficiently, they do not damage red blood cell membranes.
Toxicity tests in vivo showed that the micelles do not cause significant acute damage to liver and kidney functions, nor do they interfere with the electrolyte balance in the blood. Importantly, these parameters remain unchanged, even at 14 days post-injection.
In addition, no mouse treated with the polymer died, and no colour change was observed in the serum samples and urine of the mice treated with the polymer when compared with the control group. These findings demonstrate that the polymer did not induce significant toxicity to the mice during the period of testing. Nonetheless, preclinical studies should be conducted in the future to further evaluate potential long-term toxicity of the antimicrobial polymers before clinical applications.
In summary, the authors have designed and synthesized novel biodegradable, cationic and amphiphilic polycarbonates that can easily self-assemble into cationic micellar nanoparticles by direct dissolution in water. The cationic nanoparticles formed from the polymers, with optimal compositions, can efficiently kill Gram positive bacteria, MRSA and fungi, even at low concentrations. Importantly, they have no significant haemolytic activity over a wide range of concentrations, and cause no obvious acute toxicity to the major organs and the electrolyte balance in the blood of mice at a concentration well above the MICs. These antimicrobial polycarbonate nanoparticles could be promising as antimicrobial drugs for the decolonization of MRSA and for the treatment of various infectious diseases, including MRSA associated infections.
The data presented by the authors is very promising and open a new door to antimicrobial therapy. Several questions and new avenues comes in minds:
Can these polymers be proven for there efficiency in specific disease animal models?
Can these NPs or similar approach can be applied to gram-negative bacteria?
Can these polycarbonate affect massive biofilms?!
Looking forward to reading more news and results from this research group.
This post is in the second part of the reviews that focuses on the current status of drug delivery to bone and the issues facing this field. The first part can be accessed here
Annual treatment costs for musculoskeletal diseases in the US are roughly 7.7% (~ $849 billion) of total gross domestic product. Such disorders are the main cause of physical disability in US. Almost half of all chronic conditions in people can be attributed to bone and joint disorders. In addition there is increasing ageing population and associated increases in osteoporosis and other diseases, rising incidences of degenerative intervertebral disk diseases and numbers of revision orthopedic arthroplasty surgeries, and increases in spinal fusions. All these factors contribute towards the increasing requirement of bone regeneration and reconstruction methods and products. Delivery of therapeutic grade products to bone has various challenges. Parenteral administration limits the efficient delivery of drugs to the required site of injury and local delivery methods are often expensive and invasive. The theme issue of Advance Drug Delivery reviews focuses on the current status of drug delivery to bone and the issues facing this field. Here is the second part of these reviews and research articles.
An aging population in the developing world has led to an increase in musculoskeletal diseases such as osteoporosis and bone metastases. Left untreated many bone diseases cause debilitating pain and in the case of cancer, death. Many potential drugs are effective in treating diseases but result in side effects preventing their efficacy in the clinic. Bone, however, provides a unique environment of inorganic solids, which can be exploited in order to effectively target drugs to diseased tissue. By integration of bone targeting moieties to drug-carrying water-soluble polymers, the payload to diseased area can be increased while side effects decreased. The realization of clinically relevant bone targeted polymer therapeutics depends on (1) understanding bone targeting moiety interactions, (2) development of controlled drug delivery systems, as well as (3) understanding drug interactions. The latter makes it possible to develop bone targeted synergistic drug delivery systems.
Rheumatoid arthritis (RA) is a chronic autoimmune disease that is considered to be one of the major public health problems worldwide. The development of therapies that target tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and co-stimulatory pathways that regulate the immune system have revolutionized the care of patients with RA. Despite these advances, many patients continue to experience symptomatic and functional impairment. To address this issue, more recent therapies that have been developed are designed to target intracellular signaling pathways involved in immunoregulation. Though this approach has been encouraging, there have been major challenges with respect to off-target organ side effects and systemic toxicities related to the widespread distribution of these signaling pathways in multiple cell types and tissues. These limitations have led to an increasing interest in the development of strategies for the macromolecularization of anti-rheumatic drugs, which could target them to the inflamed joints. This approach enhances the efficacy of the therapeutic agent with respect to synovial inflammation, while markedly reducing non-target organ adverse side effects. In this manuscript, we provide a comprehensive overview of the rational design and optimization of macromolecular prodrugs for treatment of RA. The superior and the sustained efficacy of the prodrug may be partially attributed to their Extravasation through Leaky Vasculature and subsequent Inflammatory cell-mediated Sequestration (ELVIS) in the arthritic joints. This biologic process provides a plausible mechanism, by which macromolecular prodrugs preferentially target arthritic joints and illustrates the potential benefits of applying this therapeutic strategy to the treatment of other inflammatory diseases.
Peptides are attractive as novel therapeutic reagents, since they are flexible in adopting and mimicking the local structural features of proteins. Versatile capabilities to perform organic synthetic manipulations are another unique feature of peptides compared to protein-based medicines, such as antibodies. On the other hand, a disadvantage of using a peptide for a therapeutic purpose is its low stability and/or high level of aggregation. During the past two decades, numerous peptides were developed for the treatment of bone diseases, and some peptides have already been used for local applications to repair bone defects in the clinic. However, very few peptides have the ability to form bone themselves. We herein summarize the effects of the therapeutic peptides on bone loss and/or local bone defects, including the results from basic studies. We also herein describe some possible methods for overcoming the obstacles associated with using therapeutic peptide candidates.
Biomaterial scaffolds have been extensively used to deliver growth factors to induce new bone formation. The pharmacokinetics of growth factor delivery has been a critical regulator of their clinical success. This review will focus on the surface interactions that control the non-covalent incorporation of growth factors into scaffolds and the mechanisms that control growth factor release from clinically relevant biomaterials. We will focus on the delivery of recombinant human bone morphogenetic protein-2 from materials currently used in the clinical practice, but also suggest how general mechanisms that control growth factor incorporation and release delineated with this growth factor could extend to other systems. A better understanding of the changing mechanisms that control growth factor release during the different stages of preclinical development could instruct the development of future scaffolds for currently untreatable injuries and diseases.
Complications in treatment of large bone defects using bone grafting still remain. Our understanding of the endogenous bone regeneration cascade has inspired the exploration of a wide variety of growth factors (GFs) in an effort to mimic the natural signaling that controls bone healing. Biomaterial-based delivery of single exogenous GFs has shown therapeutic efficacy, and this likely relates to its ability to recruit and promote replication of cells involved in tissue development and the healing process. However, as the natural bone healing cascade involves the action of multiple factors, each acting in a specific spatiotemporal pattern, strategies aiming to mimic the critical aspects of this process will likely benefit from the usage of multiple therapeutic agents. This article reviews the current status of approaches to deliver single GFs, as well as ongoing efforts to develop sophisticated delivery platforms to deliver multiple lineage-directing morphogens (multiple GFs) during bone healing.
Over the past several decades, recombinant human bone morphogenetic proteins (rhBMPs) have been the most extensively studied and widely used osteoinductive agents for clinical bone repair. Since rhBMP-2 and rhBMP-7 were cleared by the U.S. Food and Drug Administration for certain clinical uses, millions of patients worldwide have been treated with rhBMPs for various musculoskeletal disorders. Current clinical applications include treatment of long bone fracture non-unions, spinal surgeries, and oral maxillofacial surgeries. Considering the growing number of recent publications related to clincal research of rhBMPs, there exists enormous promise for these proteins to be used in bone regenerative medicine. The authors take this opportunity to review the rhBMP literature paying specific attention to the current applications of rhBMPs in bone repair and spine surgery. The prospective future of rhBMPs delivered in combination with tissue engineered scaffolds is also reviewed.
The controlled delivery of growth factors and cells within biomaterial carriers can enhance and accelerate functional bone formation. The carrier system can be designed with pre-programmed release kinetics to deliver bioactive molecules in a localized, spatiotemporal manner most similar to the natural wound healing process. The carrier can also act as an extracellular matrix-mimicking substrate for promoting osteoprogenitor cellular infiltration and proliferation for integrative tissue repair. This review discusses the role of various regenerative factors involved in bone healing and their appropriate combinations with different delivery systems for augmenting bone regeneration. The general requirements of protein, cell and gene therapy are described, with elaboration on how the selection of materials, configurations and processing affects growth factor and cell delivery and regenerative efficacy in both in vitroand in vivoapplications for bone tissue engineering.
Reconstruction of complex craniofacial deformities is a clinical challenge in situations of injury, congenital defects or disease. The use of cell-based therapies represents one of the most advanced methods for enhancing the regenerative response for craniofacial wound healing. Both somatic and stem cells have been adopted in the treatment of complex osseous defects and advances have been made in finding the most adequate scaffold for the delivery of cell therapies in human regenerative medicine. As an example of such approaches for clinical application for craniofacial regeneration, Ixmyelocel-T or bone repair cells are a source of bone marrow derived stem and progenitor cells. They are produced through the use of single pass perfusion bioreactors for CD90+ mesenchymal stem cells and CD14+ monocyte/macrophage progenitor cells. The application of ixmyelocel-T has shown potential in the regeneration of muscular, vascular, nervous and osseous tissue. The purpose of this manuscript is to highlight cell therapies used to repair bony and soft tissue defects in the oral and craniofacial complex. The field at this point remains at an early stage, however this review will provide insights into the progress being made using cell therapies for eventual development into clinical practice.
Bone formation and regeneration therapies continue to require optimization and improvement because many skeletal disorders remain undertreated. Clinical solutions to nonunion fractures and osteoporotic vertebral compression fractures, for example, remain suboptimal and better therapeutic approaches must be created. The widespread use of recombinant human bone morphogenetic proteins (rhBMPs) for spine fusion was recently questioned by a series of reports in a special issue of The Spine Journal, which elucidated the side effects and complications of direct rhBMP treatments. Gene therapy – both direct (in vivo) and cell-mediated (ex vivo) – has long been studied extensively to provide much needed improvements in bone regeneration. In this article, we review recent advances in gene therapy research whose aims are in vivo or ex vivo bone regeneration or formation. We examine appropriate vectors, safety issues, and rates of bone formation. The use of animal models and their relevance for translation of research results to the clinical setting are also discussed in order to provide the reader with a critical view. Finally, we elucidate the main challenges and hurdles faced by gene therapy aimed at bone regeneration as well as expected future trends in this field.
Gene delivery to bone is useful both as an experimental tool and as a potential therapeutic strategy. Among its advantages over protein delivery are the potential for directed, sustained and regulated expression of authentically processed, nascent proteins. Although no clinical trials have been initiated, there is a substantial pre-clinical literature documenting the successful transfer of genes to bone, and their intraosseous expression. Recombinant vectors derived from adenovirus, retrovirus and lentivirus, as well as non-viral vectors, have been used for this purpose. Both ex vivoand in vivostrategies, including gene-activated matrices, have been explored. Ex vivodelivery has often employed mesenchymal stem cells (MSCs), partly because of their ability to differentiate into osteoblasts. MSCs also have the potential to home to bone after systemic administration, which could serve as a useful way to deliver transgenes in a disseminated fashion for the treatment of diseases affecting the whole skeleton, such as osteoporosis orosteogenesis imperfecta. Local delivery of osteogenic transgenes, particularly those encoding bone morphogenetic proteins, has shown great promise in a number of applications where it is necessary to regenerate bone. These include healing large segmental defects in long bones and the cranium, as well as spinal fusion and treating avascular necrosis.
RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing technique developed with dramatically increasing utility for both scientific and therapeutic purposes. Short interfering RNA (siRNA) is currently exploited to regulate protein expression relevant to many therapeutic applications, and commonly used as a tool for elucidating disease-associated genes. Osteoporosis and their associated osteoporotic fragility fractures in both men and women are rapidly becoming a global healthcare crisis as average life expectancy increases worldwide. New therapeutics are needed for this increasing patient population. This review describes the diversity of molecular targets suitable for RNAi-based gene knock down in osteoclasts to control osteoclast-mediated excessive bone resorption. We identify strategies for developing targeted siRNA delivery and efficient gene silencing, and describe opportunities and challenges of introducing siRNA as a therapeutic approach to hard and connective tissue disorders.
A new method for looking at how proteins fold inside mammal cells could one day lead to better flu vaccines, among other practical applications, say Cornell researchers.
The method, described online in the Proceedings of the National Academy of Sciences July 16, allows researchers to take snapshots of the cell’s protein-making machinery — called ribosomes — in various stages of protein production. The scientists then pieced together the snapshots to reconstruct how proteins fold during their synthesis.
Proteins are made up of long chains of amino acids called polypeptides, and folding gives each protein its characteristic structure, which determines its function. Though researchers have used synthetic and purified proteins to study protein folding, this study looks at proteins from their inception, providing a truer picture for how partially synthesized polypeptides can fold in cells.
Proteins fold so quickly — in microseconds — that it has been a longtime mystery just how polypeptide chains fold to create the protein’s structure.
“The speed is very fast, so it’s very hard to capture certain steps, but our approach can look at protein folding at the same time as it is being synthesized by the ribosomes,” said Shu-Bing Qian, assistant professor of nutritional sciences and the corresponding author on the paper. Yan Han, a postdoctoral associate in Qian’s lab, is the paper’s first author.
In a nutshell, messenger RNA (mRNA) carries the coding information for proteins from the DNA to ribosomes, which translate those codes into chains of amino acids that make up proteins. Previously, other researchers had developed a technique to localize the exact position of the ribosomes on the mRNA. Qian and colleagues further advanced this technique to selectively enrich only a certain portion of the protein-making machinery, basically taking snapshots of different stages of the protein synthesis process.
“Like a magnifier, we enrich a small pool from the bigger ocean and then paint a picture from early to late stages of the process,” Qian said.
In the paper, the researchers also describe applying this technique to better understanding a protein called hemagglutinin (HA), located on the surface of the influenza A virus; HA’s structure (folding) allows it to infect the cell.
Flu vaccines are based on antibodies that recognize such proteins as HA. But viruses have high mutation rates to escape antibody detection. Often, flu vaccines lose their effectiveness because surface proteins on the virus mutate. HA, for example, has the highest mutation rate of the flu virus’ surface proteins.
The researchers proved that their technique can identify how the folding process changes when HA mutates.
“If people know the folding picture of how a mutation changes, it will be helpful for designing a better vaccine,” Qian said.
“Folding is a very fundamental issue in biology,” Qian added. “It’s been a long-term mystery how the cell achieves this folding successfully, with such speed and with such a great success rate.”
The research was funded by the National Institute of Allergy and Infectious Diseases Division of Intramural Research, National Institutes of Health Grant, Ellison Medical Foundation Grant and U.S. Department of Defense Exploration-Hypothesis Development Award.
2012pharmaceutical
Amandeep Kaur
Aashir Awan, Phd
Abhisar Anand
Adina Hazan
Alan F. Kaul, PharmD., MS, MBA, FCCP
alexcrystal6
anamikasarkar
apreconasia
aviralvatsa
David Orchard-Webb, PhD
danutdaagmailcom
Demet Sag, Ph.D., CRA, GCP
Dror Nir
dsmolyar
Ethan Coomber
evelinacohn
FrasonFrancis
Gail S Thornton
Irina Robu
jayzmit48
jdpmd
jshertok
kellyperlman
Ed Kislauskis
larryhbern
Madison Davis
marzankhan
megbaker58
ofermar2020
Dr. Pati
pkandala
ritusaxena
Rick Mandahl
sjwilliamspa
Srinivas Sriram
stuartlpbi
Dr. Sudipta Saha
tildabarliya
zraviv06
zs22