Gene Editing by creation of a complement without transcription error
Larry H. Bernstein, MD, FCAP, Curator
LPBI
Nanoparticle-Based Artificial Transcription Factor
NanoScript: A Nanoparticle-Based Artificial Transcription Factor for Effective Gene Regulation
Transcription factor (TF) proteins are master regulators of transcriptional activity and gene expression. TF-based gene regulation is a promising approach for many biological applications; however, several limitations hinder the full potential of TFs. Herein, we developed an artificial, nanoparticle-based transcription factor, termed NanoScript, which is designed to mimic the structure and function of TFs. NanoScript was constructed by tethering functional peptides and small molecules called synthetic transcription factors, which mimic the individual TF domains, onto gold nanoparticles. We demonstrate that NanoScript localizes within the nucleus and initiates transcription of a reporter plasmid by over 15-fold. Moreover, NanoScript can effectively transcribe targeted genes on endogenous DNA in a nonviral manner. Because NanoScript is a functional replica of TF proteins and a tunable gene-regulating platform, it has great potential for various stem cell applications.
HIGHLIGHTS
- Transcription Factors (TF) are proteins that regulate transcription and gene expression
- NanoScript is an versatile, nanoparticle-based platform that mimics TF structure and biological function
- NanoScript is stable in physiological environments and localizes within the nucleus
- NanoScript initiates targeted gene expression by over 15-fold to 30 fold, which would be critical for stem cell differentiation and cellular reprogramming
- NanoScript transcribes endogenous genes on native DNA in a non-viral manner
Transcription factor (TF) proteins are master regulators of transcriptional activity and gene expression. TF-based gene regulation is an essential approach for many biological applications such as stem cell differentiation and cellular programming, however, several limitations hinder the full potential of TFs.
To address this challenge, researchers in Prof. KiBum Lee’s group (Sahishnu Patel and Perry Yin) developed an artificial, nanoparticle-based transcription factor, termed NanoScript, which is designed to mimic the structure and function of TFs. NanoScript was constructed by tethering functional peptides and small molecules called synthetic transcription factors, which mimic the individual TF domains, onto gold nanoparticles. They demonstrated that NanoScript localizes within the nucleus and initiates transcription of a targeted gene with high efficiency. Moreover, NanoScript can effectively transcribe targeted genes on endogenous DNA in a non-viral manner.
NanoScript is a functional replica of TF proteins and a tunable gene-regulating platform. NanoScript has two attractive features that make this the perfect platform for stem cell-based application. First, because gene regulation by NanoScript is non-viral, it serves as an attractive alternative to current differentiation methods that use viral vectors. Second, by simply rearranging the sequence of one molecule on NanoScript, NanoScript can target any differentiation-specific genes and induce differentiation, and thus has excellent prospect for applications in stem cell biology and cellular reprogramming.
This work was recently published in ACS Nano (DOI: 10.1021/nn501589f) and was selected to be highlighted by the following magazine (C&EN) and blogs (Nanowerk and UCSD).
A highly robust, efficient nanoparticle-based platform to advance stem cell therapeutics |
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| (Nanowerk News) Associate Professor Ki-Bum Lee has developed patent-pending technology that may overcome one of the critical barriers to harnessing the full therapeutic potential of stem cells. | |
| One of the major challenges facing researchers interested in regenerating cells and growing new tissue to treat debilitating injuries and diseases such as Parkinson’s disease, heart disease, and spinal cord trauma, is creating an easy, effective, and non-toxic methodology to control differentiation into specific cell lineages. Lee and colleagues at Rutgers and Kyoto University in Japan have invented a platform they call NanoScript, an important breakthrough for researchers in the area of gene expression. Gene expression is the way information encoded in a gene is used to direct the assembly of a protein molecule, which is integral to the process of tissue development through stem cell therapeutics. | |
| Stem cells hold great promise for a wide range of medical therapeutics as they have the ability to grow tissue throughout the body. In many tissues, stem cells have an almost limitless ability to divide and replenish other cells, serving as an internal repair system. | |
Schematic representation of NanoScript’s design and function. (a) By assembling individual STF molecules, including the DBD (DNA-binding domain), AD (activation domain), and NLS (nuclear localization signal), onto a single 10 nm gold nanoparticle, we have developed the NanoScript platform to replicate the structure and function of TFs. This NanoScript penetrates the cell membrane and enters the nucleus through the nuclear receptor with the help of the NLS peptide. Once in the nucleus, NanoScript interacts with DNA to initiate transcriptional activity and induce gene expression. (b) When comparing the structure of NanoScript to representative TF proteins, the three essential domains are effectively replicated. The linker domain (LD) fuses the multidomain protein together and is replicated by the gold nanoparticle (AuNP). (c) The DBD binds to complementary DNA sequences, while the AD recruits transcriptional machinery components such as RNA polymerase II (RNA Pol II), mediator complex, and general transcription factors (GTFs). The synergistic function of the DBD and AD moieties on NanoScript initiates transcriptional activity and expression of targeted genes. (d) The AuNPs are monodisperse and uniform. The NanoScript constructs are shown to effectively localize within the nucleus, which is important because transcriptional activity occurs only in the nucleus. (Reprinted with permission y American Chemical Society) (click on image to enlarge) Read more: Using nanotechnology to regulate gene expression at the transcriptional level |
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| Transcription factor (TF) proteins are master regulators of gene expression. TF proteins play a pivotal role in regulating stem cell differentiation. Although some have tried to make synthetic molecules that perform the functions of natural transcription factors, NanoScript is the first nanomaterial TF protein that can interact with endogenous DNA. | |
| ACS Nano, a publication of the American Chemical Society (ACS), has published Lee’s research on NanoScript (“NanoScript: A Nanoparticle-Based Artificial Transcription Factor for Effective Gene Regulation”). The research is supported by a grant from the National Institutes of Health (NIH). | |
| “Our motivation was to develop a highly robust, efficient nanoparticle-based platform that can regulate gene expression and eventually stem cell differentiation,” said Lee, who leads a Rutgers research group primarily focused on developing and integrating nanotechnology with chemical biology to modulate signaling pathways in cancer and stem cells. “Because NanoScript is a functional replica of TF proteins and a tunable gene-regulating platform, it has great potential to do exactly that. The field of stem cell biology now has another platform to regulate differentiation while the field of nanotechnology has demonstrated for the first time that we can regulate gene expression at the transcriptional level.” | |
| NanoScript was constructed by tethering functional peptides and small molecules called synthetic transcription factors, which mimic the individual TF domains, onto gold nanoparticles. | |
| “NanoScript localizes within the nucleus and initiates transcription of a reporter plasmid by up to 30-fold,” said Sahishnu Patel, Rutgers Chemistry graduate student and co-author of the ACS Nano publication. “NanoScript can effectively transcribe targeted genes on endogenous DNA in a nonviral manner.” | |
| Lee said the next step for his research is to study what happens to the gold nanoparticles after NanoScript is utilized, to ensure no toxic effects arise, and to ensure the effectiveness of NanoScript over long periods of time. | |
| “Due to the unique tunable properties of NanoScript, we are highly confident this platform not only will serve as a desirable alternative to conventional gene-regulating methods,” Lee said, “but also has direct employment for applications involving gene manipulation such as stem cell differentiation, cancer therapy, and cellular reprogramming. Our research will continue to evaluate the long-term implications for the technology.” | |
| Lee, originally from South Korea, joined the Rutgers faculty in 2008 and has earned many honors including the NIH Director’s New Innovator Award. Lee received his Ph.D. in Chemistry from Northwestern University where he studied with Professor Chad. A. Mirkin, a pioneer in the coupling of nanotechnology and biomolecules. Lee completed his postdoctoral training at The Scripps Research Institute with Professor Peter G. Schultz. Lee has served as a Visiting Scholar at both Princeton University and UCLA Medical School. | |
| The primary interest of Lee’s group is to develop and integrate nanotechnologies and chemical functional genomics to modulate signaling pathways in mammalian cells towards specific cell lineages or behaviors. He has published more than 50 articles and filed for 17 corresponding patents. |
| Source: Rutgers University |
Read more: A highly robust, efficient nanoparticle-based platform to advance stem cell therapeutics
Nanoparticle-based transcription factor mimics
http://nanomedicine.ucsd.edu/blog/article/nanoparticle-based-transcription-factor-mimics
Biologists have been enhancing expression of specific genes with plasmids and viruses for decades, which has been essential to uncovering the function of numerous genes and the relationships among the proteins they encode. However, tools that allow enhancement of expression of endogenous genes at the transcriptional level could be a powerful complement to these strategies. Many chemical biologists have made enormous progress developing molecular tools for this purpose; recent work by a group at Rutgers suggests how nanotechnology might allow application of this strategy in living organisms, and perhaps one day in patients.
In a paper published in ACS Nano, researchers led by KiBum Lee synthesized gold nanoparticles bearing synthetic or shortened versions of the three essential components of transcription factors (TFs), the proteins that “turn on” expression of specific genes in cells. Specifically, polyamides previously designed to bind to a specific promoter sequence, transactivation peptides, and nuclear localization peptides were conjugated to the nanoparticle surface. These nanoparticles enhanced expression of both a reporter plasmid (by ~15-fold) and several endogenous genes (by up to 65%). This enhancement is much greater than that possible using previous constructs lacking nuclear localization sequences; the team incorporated a high proportion of those peptides to ensure efficient delivery to the nucleus.
These nanoparticles offer an alternative to delivering protein TFs, which remains extremely challenging despite considerable effort towards the development of delivery systems that transport cargo into cells. Among other barriers to the use of native TFs, incorporating them into polymeric or lipid-based carriers often alters their shape, which would likely reduce their function.
While the group suggests future generations of these nanoparticles might one day be used to treat diseases caused by defects in TF genes, many questions remain. First, the duration of gene expression enhancement is not known; the study only assesses effects at 48 h post-administration. Further, whether gold is the best material for the core remains unclear, as its non-biodegradability means the particles would likely accumulate in the liver over time; synthetic TFs with biodegradable cores might also be considered.
Patel S et al., NanoScript: a nanoparticle-based artificial transcription factor for effective gene regulation,ACS Nano 2014; published online Sep 3.
http://www.wtec.org/bem/docs/BEM-FinalReport-Web.pdf
Biocompatibility and Toxicity of Nanobiomaterials
“Biocompatibility and Toxicity of Nanobiomaterials” is an annual special issue published in “Journal of Nanomaterials.”
http://www.hindawi.com/journals/jnm/toxicity.nanobiomaterials/
Porous Ti6Al4V Scaffold Directly Fabricated by Sintering: Preparation and In Vivo Experiment
Xuesong Zhang, Guoquan Zheng, Jiaqi Wang, Yonggang Zhang, Guoqiang Zhang, Zhongli Li, and Yan Wang
Department of Orthopaedics, Chinese People’s Liberation Army General Hospital, Beijing 100853, China AcademicEditor:XiaomingLi
The interface between the implant and host bone plays a key role in maintaining primary and long-term stability of the implants. Surface modification of implant can enhance bone in growth and increase bone formation to create firm osseo integration between the implant and host bone and reduce the risk of implant losing. This paper mainly focuses on the fabricating of 3-dimensiona interconnected porous titanium by sintering of Ti6Al4V powders, which could be processed to the surface of the implant shaft and was integrated with bone morphogenetic proteins (BMPs). The structure and mechanical property of porous Ti6Al4V was observed and tested. Implant shaft with surface of porous titanium was implanted into the femoral medullary cavity of dog after combining with BMPs. The results showed that the structure and elastic modulus of 3D interconnected porous titanium was similar to cancellous bone; porous titanium combined with BMP was found to have large amount of fibrous tissue with fibroblastic cells; bone formation was significantly greater in 6 weeks postoperatively than in 3 weeks after operation. Porous titanium fabricated by powders sintering and combined with BMPs could induce tissue formation and increase bone formation to create firm osseo integration between the implant and host bone.
Journal of Materials Chemistry B Issue 39, 2013
Nanomaterials research has in part been focused on their use in biomedical applications for more than several decades. However, in recent years this field has been developing to a much more advanced stage by carefully controlling the size, shape, and surface-modification of nanoparticles. This review provides an overview of two classes of nanoparticles, namely iron oxide and NaLnF4, and synthesis methods, characterization techniques, study of biocompatibility, toxicity behavior, and applications of iron oxide nanoparticles and NaLnF4nanoparticles as contrast agents in magnetic resonance imaging. Their optical properties will only briefly be mentioned. Iron oxide nanoparticles show a saturation of magnetization at low field, therefore, the focus will be MLnF4 (Ln = Dy3+, Ho3+, and Gd3+) paramagnetic nanoparticles as alternative contrast agents which can sustain their magnetization at high field. The reason is that more potent contrast agents are needed at magnetic fields higher than 7 T, where most animal MRI is being done these days. Furthermore we observe that the extent of cytotoxicity is not fully understood at present, in part because it is dependent on the size, capping materials, dose of nanoparticles, and surface chemistry, and thus needs optimization of the multidimensional phenomenon. Therefore, it needs further careful investigation before being used in clinical applications.
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Silvia S. Halacheva, Tony J. Freemont and Brian R. Saunders
citations…
DOI: 10.1039/c4tb00327f
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HAMLET interacts with lipid membranes and perturbs their structure and integrity
HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal …. of the alternative complement pathway preserves photoreceptors after retinal injury ….. Life-long in vivo cell-lineage tracing shows that no oogenesis originates from …. ananoparticle-based artificial transcription factor for effective gene regulation …
Authors: Ann-Kristin Mossberg, Maja Puchades, Øyvind Halskau, Anne Baumann, Ingela Lanekoff, Yinxia Chao, Aurora Martinez, Catharina Svanborg, & Roger Karlsson
www.regenerativemedicine.net/NewsletterArchives.asp?qEmpID…
Summary:
Background – Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure.
Methodology/Principal Findings – We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLAall-Ala). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles.
Conclusions/Significance – The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.
Source: Public Library of Science ONE; 5(2) (02/23/10)
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