Stem Cell Clinic

IGIB finds a protein with better precision in gene-editing – The Hindu

Researchers at the Delhi-based Institute of Genomics and Integrative Biology (CSIR-IGIB) have discovered a protein variant from a different species of bacteria that can edit the DNA with very high precision. In the tool now commonly used for editing disease-causing mutations in DNA (CRISPR-Cas9), the Cas9 protein behaves like a molecular scissors that cuts the DNA at a specific location and inserts a foreign piece of DNA to correct the mutation that causes the disease.

In addition to binding to the intended target on the DNA, the commonly used Cas9 protein from Strepotococcus pyogenes bacteria (SpCas9) and its engineered derivative tend to potentially bind to DNA at multiple unintended sites thereby leading to unnecessary alterations in the DNA.

The researchers found their new Cas9 protein, which binds and cuts the DNA, was able to correct sickle cell anaemia mutation in patient-derived stem cells. The protein (FnCas9) used by the researchers to edit the DNA is derived from a bacterium Francisella novicida.

The Cas9 protein is supposed to bind to the DNA only when there is a perfect match between the DNA and the protein, thus reducing the chances of the protein binding at non-target sites on the DNA. But even when three mismatches exist between the protein and the DNA, the currently used SpCas9 protein binds and cleaves the DNA. In contrast, the team led by Debojyoti Chakraborty from IGIB found the new FnCas9 protein showed negligible binding when there exists more than one mismatch in the target DNA. The results were published in the journal Proceedings of the National Academy of Sciences (PNAS).

The high specificity of the new FnCas9 protein arises due to reduced affinity to bind to DNA when there is even a single mismatch. And when there is more than one mismatch, complete absence of binding of the protein to the DNA is seen in many cases, says Dr. Chakraborty.

If the Cas9 protein remains bound to DNA at mismatched locations for a long time, there is a possibility that it might cut the DNA at these locations. Also, if it remains bound to DNA, the protein might block the transcription (which is the first step in gene expression) at that location. And if Cas9 is bound at multiple unintended sites then the transcription machinery gets stalled and the expression of genes at these locations might be altered, Dr. Chakraborty explains.

In nature, DNA often gets damaged and is routinely repaired through one of the two pathways. In the case of the homology-directed repair (HDR) pathway, which is relatively less error-prone, matching sequences are used to repair the DNA. The FnCas9 protein was found to increase the HDR repair rate fourfold compared to the widely used SpCas9, says Deepanjan Paul from CSIR-IGIB and one of the first authors of the paper.

The researchers tested the precision of binding and cleavage at the desired sites on the DNA using mouse cell lines (embryonic stem cells and brain cells), human kidney cell lines and induced pluripotent stem cells (iPSc). In the case of human iPS cells, the FnCas9 protein was found to bind to the DNA at the specific site, cut and repair the sickle cell anaemia mutation.

The correction process is the same for any disease-causing mutation and so our FnCas9 protein should theoretically correct any mutation in the DNA. The efficiency might vary, so we must test it for each disorder, says Dr. Chakraborty.

The efficiency of any Cas9 protein delivery as well the ability to correct mutations is generally low in the case of iPS cells. The efficiency of correction is about 1.6%. Though the efficiency to correct mutations is low in iPS cells, the corrected cells can be isolated, multiplied and converted (differentiated) into haematopoietic stem cells. Once differentiated into haematopoietic stem cells, they can be transfused into patients.

Differentiating iPS cells into haematopoietic stem cells is not trivial. Plenty of experimental work is under way to make it efficient for clinical translation, says Dr. Chakraborty.

Recalling how he started working on FnCas9 protein for genome editing, Dr. Chakraborty recalls that he was looking for a Cas9 protein which can target RNA instead of DNA. There was one study that reported that FnCas9 could potentially target viral RNA. We were not able to target RNA using FnCas9 proteins. So we started to investigate whether it can target DNA as well since it was not known if FnCas9 can be used for precise gene correction. We found that not only does it target the DNA but does so with very high specificity, he says.

We are now proceeding for preclinical studies to establish the efficacy of FnCas9 protein for genome-wide binding and targeting using patient-derived cells and mouse models, he says.

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IGIB finds a protein with better precision in gene-editing - The Hindu

Skin-Derived Heart Cells Help Uncover the Genetic Foundations of Cardiac Function – Technology Networks

Genome-wide association studies have uncovered more than 500 genetic variants linked to heart function, everything from heart rate to irregular rhythms that can lead to stroke, heart failure or other complications. But since most of these variations fall into areas of the genome that dont encode proteins, exactly how they influence heart function has remained unclear.

By examining heart cells derived from the skin samples of seven family members, researchers at University of California San Diego School of Medicine have now discovered that many of these genetic variations influence heart function because they affect the binding of a protein called NKX2-5.

NKX2-5 is a transcription factor, meaning it helps turn on and off genes in this case, genes involved in heart development. To do this, NKX2-5 must bind to non-coding regions of the genome. Thats where genetic variation comes in.

NKX2-5 binds to many different places in the genome near heart genes, so it makes sense that variation in the factor itself or the DNA to which it binds would affect that function, said senior author Kelly A. Frazer, PhD, professor of pediatrics and director of the Institute for Genomic Medicine at UC San Diego School of Medicine. As a result, we are finding that multiple heart-related traits can share a common mechanism in this case, differential binding of NKX2-5 due to DNA variants.

The study started with skin samples from seven people from three generations of a single family. The researchers converted the skin cells into induced pluripotent stem cells (iPSCs) as an intermediary. Like all stem cells, iPSCs can both self-renew, making more iPSCs, and differentiate into a specialized cell type. With the right cocktail of molecules and growth factors, the researchers directed iPSCs into becoming heart cells.

These heart cells actually beat in the laboratory dish, and still bear the genetic and molecular features of the individuals from which they were derived.

Frazer and team conducted a genome-wide analysis of these patient-derived heart cells. They determined that NKX2-5 can bind approximately 38,000 sites in the genome. Of those, 1,941 genetic variants affected NKX2-5 binding. The researchers investigated the role of those variants in heart gene function and heart-related traits. One of the genetic variants was associated with the SCN5A gene, which encodes the main channel through which sodium is transported in heart cells.

Since related individuals tend to share similar genetic variants, the team was able to validate their findings by analyzing the same variants in multiple samples.

People typically need a large number of samples to detect the effects of common DNA variants, so we were surprised that we were able to identify with high confidence these effects on NKX2-5 binding at so many sites across the genome with just few people, said first author Paola Benaglio, PhD, a postdoctoral researcher in Frazers lab.

Yet, she said, this finding may just be the tip of the iceberg.

There are probably a lot more genetic variants in the genome involved with NKX2-5 as well as with other important cardiac transcription factors, Frazer said. We identified almost 2,000 in this study, but thats probably only a fraction of what really exists because we were only looking at seven people in a single family and only at one transcription factor. There are probably many more variants in gene regulation sites across the entire population.

Not only does the team plan to further investigate cardiovascular genetics, but they also have their sights set on other organ systems.

We are now expanding this same model system to look at many different transcription factors, across different tissue types, such as pancreas and retina epithelia, and scaling it up to include more families, Benaglio said.

Reference

Benalgio, P. et al. (2019)Allele-specific NKX2-5 binding underlies multiple genetic associations with human electrocardiographic traits. Nature Genetics. DOI:https://doi.org/10.1038/s41588-019-0499-3

This article has been republished from the following materials. Note: material may have been edited for length and content. For further information, please contact the cited source.

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Skin-Derived Heart Cells Help Uncover the Genetic Foundations of Cardiac Function - Technology Networks

Novel Cell Sorting and Separation Market: Focus on Acoustophoresis, Buoyancy-activated, Dielectrophoresis, Magnetophoretics, Microfluidics,…

NEW YORK, Oct. 1, 2019 /PRNewswire/ --

INTRODUCTIONAdvances in the fields of cell biology and regenerative medicine have led to the development of various cell-based therapies, which, developers claim, possess the potential to treat a variety of clinical conditions. In 2018, it was reported that there were more than 1,000 clinical trials of such therapies, being conducted across the globe by over 900 industry players. Moreover, the total investment in the aforementioned clinical research efforts was estimated to be around USD 13 billion. Given the recent breakthroughs in clinical testing and the discovery of a variety of diagnostic biomarkers, the isolation of one or multiple cell types from a heterogenous population has not only become simpler, but also an integral part of modern clinical R&D. The applications of cell separation technologies are vast, starting from basic research to biological therapy development and manufacturing. However, conventional cell sorting techniques, including adherence-based sorting, membrane filtration-based sorting, and fluorescence- and magnetic-based sorting, are limited by exorbitant operational costs, time-consuming procedures, and the need for complex biochemical labels. As a result, the use of such techniques has, so far, been restricted in the more niche and emerging application areas.

Read the full report: https://www.reportlinker.com/p05817189/?utm_source=PRN

Over the years, extensive research in the field of flow cytometry has enabled the development of a variety of novel technologies that are capable of efficiently isolating cells from tissue samples and / or heterogenous cell populations. In fact, since 2014, over 3,000 patents were reported to have been filed / granted related to such advanced techniques, indicating the rapid pace of innovation in this domain. Developers of the aforementioned technologies claim that these new techniques offer numerous benefits, including fast and precise cell sorting, reduced sample requirement, improved portability, reduced risk to cell viability, and negligible need for expensive biochemical / magnetic labels. Moreover, they have been shown to be compatible for use across a myriad of applications, including research studies (bacteriology, immunology, stem cell research, and viral titering and infectivity), biomedical diagnostics (circulating tumor cell detection, in vitro fertilization, and non-invasive prenatal diagnosis), biological therapy-related process operations (bio-banking, drug discovery, sample preparation, single cell sequencing, and tumor cell characterization), and cell-based therapeutics (B- or T-cell immunotherapies). Consequently, these techniques have captured the interest of several stakeholders in the biopharmaceutical industry. It is also worth highlighting that stakeholders in this domain have received significant support from both private and public investors.

SCOPE OF THE REPORTThe 'Novel Cell Sorting and Separation Market: Focus on Acoustophoresis, Buoyancy-activated, Dielectrophoresis, Magnetophoretics, Microfluidics, Optoelectronics, Photoacoustics, Traceless Affinity and Other Technologies, 2019-2030' report features an extensive study of the current landscape and future outlook of the growing market for novel cell sorting and separation technologies (beyond conventional methods). The study presents detailed analyses of cell sorters, cell isolation kits, and affiliated consumables and reagents, that are based on the aforementioned technologies.

Amongst other elements, the report features: A detailed assessment of the current market landscape, featuring a comprehensive list of over 220 innovative cell sorters, cell isolation kits, and affiliated consumables and reagents, along with information on their respective specifications (such as size, weight, cell flow rate, cell sort rate, cell analysis rate, cell purity and viability, process time, and operating temperature and pressure), cell sorting technology (acoustophoresis, buoyancy-activated, dielectrophoresis, magnetophoretics, microfluidics, optoelectronics, photoacoustics, traceless affinity, and others), type of cell (animal cells, cancer cells, immune cells, microbial cells, red blood cells / platelets, stem cells, and others), cell separation approach (positive selection, negative selection and depletion), basis for separation (cell morphology and physiology, cell size and density, surface biomarkers, surface charge and adhesion, and others), and end use / application (research studies, biomedical diagnostics, biological therapy-related process operations, and cell-based therapeutics). An insightful company competitiveness analysis, taking into consideration the supplier power (based on size of employee base and experience in this segment of the industry) and portfolio-related parameters, such as number of products offered, number of target cells, end use(s) / application(s), and key product specification(s). Comprehensive profiles of key industry players (shortlisted on the basis of company competitiveness analysis scores) that are currently offering novel cell sorters / consumables and cell isolation kits, featuring an overview of the company, its financial information (if available), and a detailed description of its proprietary product(s). Each profile also includes a list of recent developments, highlighting the key achievements, partnership activity, and the likely strategies that may be adopted by these players to fuel growth in the foreseen future. An in-depth analysis of the patents that have been filed / granted related to novel cell sorting and separation technologies, since 2014. It highlights the key trends associated with these patents, across patent type, regional applicability, CPC classification, emerging focus areas, leading industry players (in terms of number of patents filed / granted), and current intellectual property-related benchmarks and valuation. A detailed publication analysis of more than 200 peer-reviewed, scientific articles that have been published since 2014, highlighting the research focus within the industry. It also highlights the key trends observed across the publications, including information on innovative technologies, potential application areas, target disease indications, type of cell, and analysis based on various relevant parameters, such as year of publication, and most popular journals (in terms of number of articles published in the given time period) within this domain. An analysis of the partnerships that have been established in the domain, in the period 2014-Q1 2019, covering R&D collaborations, licensing agreements, distribution agreements, mergers / acquisitions, asset purchase agreements, product development agreements, product utilization agreements, and other relevant deals. An analysis of the investments made at various stages of development, such as seed financing, venture capital financing, debt financing, grants / awards, capital raised from IPOs and subsequent offerings, by companies that are engaged in this field. An analysis to estimate the likely demand for novel cell sorting products and solutions across key application areas, including research studies, clinical diagnostics, cell-based therapeutics, and other applications, in different global regions for the period 2019-2030.

One of the key objectives of the report was to understand the primary growth drivers and estimate the future size of the novel cell sorting and separation market. Based on multiple parameters, such as potential application areas, likely adoption rate and expected pricing, we have provided an informed estimate on the likely evolution of the market, over the period 2019-2030. In addition, we have provided the likely distribution of the current and forecasted opportunity across [A] potential application areas (research studies, clinical diagnostics, cell-based therapeutics, and other applications), [B] end users (academic institutes, clinical testing labs, hospitals, and commercial organizations), [C] type of offering (cell sorters, and consumables and isolation kits), [D] cell sorting technology (buoyancy-activated, magnetophoretics, microfluidics, optoelectronics, and other advanced technologies), [E] type of cell (adult stem cells, CAR-T cells, circulating fetal cells, circulating tumor cells, dendritic cells, embryonic stem cells, insect cells, induced pluripotent stem cells, microbial cells, sperm cells, TCR cells, TILs, and tumor cells / cancer cells), [F] size of cell (< 5 m, 5-10 m, 10-15 m, 15-25 m, and > 25 m), and [G] key geographical regions (North America, Europe and Asia-Pacific). In order to account for the uncertainties associated with some of the key parameters and to add robustness to our model, we have provided three market forecast scenarios portraying the conservative, base and optimistic tracks of the industry's evolution.

The opinions and insights presented in this study were also influenced by discussions conducted with multiple stakeholders in this domain. The report features detailed transcripts of interviews held with the following individuals (in alphabetical order of organization names): John Younger (Co-founder and Chief Technology Officer, Akadeum Life Sciences) Sean Hart (Chief Executive Officer and Chief Scientific Officer, LumaCyte) Soohee Cho (Product Manager, Namocell)

All actual figures have been sourced and analyzed from publicly available information forums and primary research discussions. Financial figures mentioned in this report are in USD, unless otherwise specified.

RESEARCH METHODOLOGYThe data presented in this report has been gathered via secondary and primary research. For all our projects, we conduct interviews with experts in the area (academia, industry and other associations) to solicit their opinions on emerging trends in the market. This is primarily useful for us to draw out our own opinion on how the market will evolve across different regions and technology segments. Where possible, the available data has been checked for accuracy from multiple sources of information.

The secondary sources of information include Annual reports Investor presentations SEC filings Industry databases News releases from company websites Government policy documents Industry analysts' views

While the focus has been on forecasting the market till 2030, the report also provides our independent view on various non-commercial trends emerging in the industry. This opinion is solely based on our knowledge, research and understanding of the relevant market gathered from various secondary and primary sources of information.

CHAPTER OUTLINESChapter 2 is an executive summary of the insights captured in our research. It offers a high-level view on the likely evolution of novel cell sorting and separation market in the mid-long term.

Chapter 3 is an introductory chapter that presents a general overview of cell sorting and separation, including a brief history of development, and information on the basic components and principle of operation of a cell sorter. Further, it features a detailed discussion on conventional cell separation techniques and the various challenges associated with their use across different application areas. The chapter also includes detailed sections on the novel cell sorting and separation technologies (such as acoustophoresis, buoyancy-activated, dielectrophoresis, magnetophoretics, microfluidics, optoelectronics, photoacoustics, traceless affinity, and others), highlighting their advantages and challenges. Further, it features a discussion on the key growth drivers and roadblocks related to modern cell sorting technologies, as well as upcoming trends that the field is expected to witness in the coming years.

Chapter 4 includes information on more than 220 innovative cell sorters, cell isolation kits, and affiliated consumables and reagents, along with details on their respective specifications (such as size, weight, cell flow rate, cell sort rate, cell analysis rate, cell purity and viability, process time, and operating temperature and pressure), cell sorting technology (acoustophoresis, buoyancy-activated, dielectrophoresis, magnetophoretics, microfluidics, optoelectronics, photoacoustics, traceless affinity, and others), type of cell (animal cells, cancer cells, microbial cells, red blood cells / platelets, stem cells, and others), cell separation approach (positive selection, negative selection and depletion), basis for separation (cell morphology and physiology, cell size and density, surface biomarkers, surface charge and adhesion, and others), and end use / application (research studies, biomedical diagnostics, biological therapy-related process operations, and cell-based therapeutics). The chapter also highlights the contributions of various companies engaged in this domain, presenting detailed analyses, based on their year of establishment, size of employee base, geographical presence, and type of offering.

Chapter 5 features an insightful competitiveness analysis of the companies engaged in novel cell sorting and separation domain, based on various parameters, such as number of products offered, number of target cells, end use(s) / application(s), and key product specification(s). In the chapter, stakeholder entities have been plotted on a 2X2 matrix, featuring a company's Supplier Power (based on size of employee base and experience in this segment of the industry) and Company Competitiveness as the two axes.

Chapter 6 includes elaborate profiles of key industry players (shortlisted on the basis of company competitiveness analysis scores) that are offering novel cell sorters / sorting technologies; each profile features an overview of the company, its financial information (if available), and a detailed description of its proprietary product(s). Each profile also includes a list of recent developments, highlighting the key achievements, partnership activity, and the likely strategies that may be adopted by these players to fuel growth, in the foreseen future.

Chapter 7 includes detailed profiles of key industry players (shortlisted on the basis of company competitiveness analysis scores) that are offering novel consumables and cell isolation kits; each profile features an overview of the company, its financial information (if available), and a detailed description of its proprietary product(s). Each profile also includes a list of recent developments, highlighting the key achievements, partnership activity, and the likely strategies that may be adopted by these players to fuel growth, in the foreseen future.

Chapter 8 provides an in-depth patent analysis to provide an overview of how the industry is evolving from the R&D perspective. For this analysis, we considered those patents that have been filed / granted related to novel cell sorting and separation technologies, since 2014. The analysis also highlights the key trends associated with these patents, across patent type, regional applicability, CPC classification, emerging focus areas, leading industry players (in terms of number of patents filed / granted), and current intellectual property-related benchmarks and valuation.

Chapter 9 presents a detailed publication analysis of more than 200 peer-reviewed, scientific articles that have been published since 2014, highlighting the research focus within the industry. It also highlights the key trends observed across the publications, including information on innovative technologies, potential application areas, target disease indications, type of cell, and analyses based on various relevant parameters, such as year of publication, and most popular journals (in terms of number of articles published in the given time period) within this domain.

Chapter 10 features an elaborate analysis and discussion of partnerships / collaborations that have been established in this domain in the period 2014-Q1 2019. It includes a brief description of various types of partnership models (such as R&D collaborations, licensing agreements, distribution agreements, mergers / acquisitions, asset purchase agreements, product development agreements, product utilization agreements, and others) that have been employed by stakeholders within this domain. It also consists of a schematic representation showcasing the players that have established the maximum number of alliances related to novel cell sorting and separation technologies. Furthermore, we have provided a world map representation of all the deals inked in this field, highlighting those that have been established within and across different continents.

Chapter 11 provides information on funding instances and investments that have been made within the novel cell sorting and separation domain. The chapter includes details on various types of investments (such as seed financing, venture capital financing, debt financing, grants, capital raised from IPOs and subsequent offerings) received by companies in the period 2014-Q1 2019, highlighting the growing interest of the venture capital community and other strategic investors in this domain.

Chapter 12 provides an overview of the demand for novel cell sorting products and solutions across key application areas, including research studies, clinical diagnostics, cell-based therapeutics, and other applications, in the contemporary market. In order to estimate the aforementioned demand, we considered the number of ongoing / completed research studies, diagnostic tests and cell-based therapies under development across different geographies. We also estimated the likely adoption of such products and solutions across key application areas, over the period 2019-2030.

Chapter 13 features a comprehensive market forecast, highlighting the future potential of novel cell sorting and separation market till 2030, based on multiple parameters, such as potential application areas, likely adoption rate and expected pricing. In addition, we estimated the likely distribution of the current and forecasted opportunity across [A] potential application areas (research studies, clinical diagnostics, cell-based therapeutics, and other applications), [B] end users (academic institutes, clinical testing labs, hospitals, and commercial organizations), [C] type of offering (cell sorters, and consumables and isolation kits), [D] cell sorting technology (buoyancy-activated, magnetophoretics, microfluidics, optoelectronics, and other advanced technologies), [E] type of cell (adult stem cells, CAR-T cells, circulating fetal cells, circulating tumor cells, dendritic cells, embryonic stem cells, insect cells, induced pluripotent stem cells, microbial cells, sperm cells, TCR cells, TILs, and tumor cells / cancer cells), [F] size of cell (< 5 m, 5-10 m, 10-15 m, 15-25 m, and > 25 m), and [G] key geographical regions (North America, Europe and Asia-Pacific). We adopted a combination of top-down and bottom up approaches, backed by robust data and credible inputs from primary research, to estimate the likely size of the market, both in terms of value (USD billion) and volume (number of products).

Chapter 14 is a collection of executive insights of the discussions that were held with various key stakeholders in this market. The chapter provides a brief overview of the companies and details of interviews held with John Younger (Co-founder and Chief Technology Officer, Akadeum Life Sciences), Sean Hart (Chief Executive Officer and Chief Scientific Officer, LumaCyte), and Soohee Cho (Product Manager, Namocell).

Chapter 15 is a summary of the overall report. In this chapter, we have provided a list of key takeaways from the report, and expressed our independent opinion related to the research and analysis described in the previous chapters.

Chapter 16 is an appendix, which provides tabulated data and numbers for all the figures provided in the report.

Chapter 17 is an appendix, which contains the list of companies and organizations mentioned in the report.

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