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Research On Global Stem Cell Cartilage Regeneration Market (impact of COVID-19) with Top Players: Anika Therapeutics,Zimmer Biomet and Others – 3rd…

Global Stem Cell Cartilage Regeneration Market: Trends Estimates High Demand by 2027

The Stem Cell Cartilage Regeneration Market 2020 report includes the market strategy, market orientation, expert opinion and knowledgeable information. The Stem Cell Cartilage Regeneration Industry Report is an in-depth study analyzing the current state of the Stem Cell Cartilage Regeneration Market. It provides a brief overview of the market focusing on definitions, classifications, product specifications, manufacturing processes, cost structures, market segmentation, end-use applications and industry chain analysis. The study on Stem Cell Cartilage Regeneration Market provides analysis of market covering the industry trends, recent developments in the market and competitive landscape.

It takes into account the CAGR, value, volume, revenue, production, consumption, sales, manufacturing cost, prices, and other key factors related to the global Stem Cell Cartilage Regeneration market. All findings and data on the global Stem Cell Cartilage Regeneration market provided in the report are calculated, gathered, and verified using advanced and reliable primary and secondary research sources. The regional analysis offered in the report will help you to identify key opportunities of the global Stem Cell Cartilage Regeneration market available in different regions and countries.

The final report will add the analysis of the Impact of Covid-19 in this report Stem Cell Cartilage Regeneration industry.

Some of The Companies Competing in The Stem Cell Cartilage Regeneration Market are: Anika Therapeutics,Zimmer Biomet,BioTissue Technologies,DePuy (Johnson & Johnson),Genzyme,CellGenix,EMD Serono,Sanofi Aventis,Smith & Nephew.

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The report scrutinizes different business approaches and frameworks that pave the way for success in businesses. The report used Porters five techniques for analyzing the Stem Cell Cartilage Regeneration Market; it also offers the examination of the global market. To make the report more potent and easy to understand, it consists of info graphics and diagrams. Furthermore, it has different policies and improvement plans which are presented in summary. It analyzes the technical barriers, other issues, and cost-effectiveness affecting the market.

Global Stem Cell Cartilage Regeneration Market Research Report 2020 carries in-depth case studies on the various countries which are involved in the Stem Cell Cartilage Regeneration market. The report is segmented according to usage wherever applicable and the report offers all this information for all major countries and associations. It offers an analysis of the technical barriers, other issues, and cost-effectiveness affecting the market. Important contents analyzed and discussed in the report include market size, operation situation, and current & future development trends of the market, market segments, business development, and consumption tendencies. Moreover, the report includes the list of major companies/competitors and their competition data that helps the user to determine their current position in the market and take corrective measures to maintain or increase their share holds.

What questions does the Stem Cell Cartilage Regeneration market report answer pertaining to the regional reach of the industry?

The report claims to split the regional scope of the Stem Cell Cartilage Regeneration market into North America, Europe, Asia-Pacific, South America & Middle East and Africa. Which among these regions has been touted to amass the largest market share over the anticipated duration

How do the sales figures look at present how does the sales scenario look for the future?

Considering the present scenario, how much revenue will each region attain by the end of the forecast period?

How much is the market share that each of these regions has accumulated presently

How much is the growth rate that each topography will depict over the predicted timeline

A short overview of the Stem Cell Cartilage Regeneration market scope:

Global market remuneration

Overall projected growth rate

Industry trends

Competitive scope

Product range

Application landscape

Supplier analysis

Marketing channel trends Now and later

Sales channel evaluation

Market Competition Trend

Market Concentration Rate

Reasons to Read this Report

This report provides pin-point analysis for changing competitive dynamics

It provides a forward looking perspective on different factors driving or restraining market growth

It provides a six-year forecast assessed on the basis of how the market is predicted to grow

It helps in understanding the key product segments and their future

It provides pin point analysis of changing competition dynamics and keeps you ahead of competitors

It helps in making informed business decisions by having complete insights of market and by making in-depth analysis of market segments

TABLE OF CONTENT:

Chapter 1:Stem Cell Cartilage Regeneration Market Overview

Chapter 2: Global Economic Impact on Industry

Chapter 3:Stem Cell Cartilage Regeneration Market Competition by Manufacturers

Chapter 4: Global Production, Revenue (Value) by Region

Chapter 5: Global Supply (Production), Consumption, Export, Import by Regions

Chapter 6: Global Production, Revenue (Value), Price Trend by Type

Chapter 7: Global Market Analysis by Application

Chapter 8: Manufacturing Cost Analysis

Chapter 9: Industrial Chain, Sourcing Strategy and Downstream Buyers

Chapter 10: Marketing Strategy Analysis, Distributors/Traders

Chapter 11: Stem Cell Cartilage Regeneration Market Effect Factors Analysis

Chapter 12: GlobalStem Cell Cartilage Regeneration Market Forecast to 2027

Method of Research:

The predictions made in the report are derived with the use of accurate research methodologies as well as assumptions. The markets estimated growth rate in the forecast period has been calculated on the basis of various parameters that form the Porters Five Force Model. Data analysts have utilized the SWOT-based method, which helps list out all the risks, primary opportunities, strengths and weaknesses of the market. With the support of a dedicated and dynamic team, the report provides trusted information that has been accumulated with the use of the latest methodologies.

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Research On Global Stem Cell Cartilage Regeneration Market (impact of COVID-19) with Top Players: Anika Therapeutics,Zimmer Biomet and Others - 3rd...

Covid-19 Impact on Musculoskeletal Disorder Stem Cell Therapy Market Report with Capacity and Share by Manufacturers, Forecast Report 2020 – 3rd Watch…

This report studies the Musculoskeletal Disorder Stem Cell Therapy market status and outlook of Global and major regions, from angles of players, countries, product types and end industries; this report analyzes the top players in global market, and splits the Musculoskeletal Disorder Stem Cell Therapy market by product type and applications/end industries.

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Detailed TOC of Global Musculoskeletal Disorder Stem Cell Therapy Market 2019 by Company, Regions, Type and Application, Forecast to 2024: Table of Contents

1 Musculoskeletal Disorder Stem Cell Therapy Market Overview

1.1 Product Overview and Scope of Musculoskeletal Disorder Stem Cell Therapy

1.2 Classification of Musculoskeletal Disorder Stem Cell Therapy by Types

1.2.1 Global Musculoskeletal Disorder Stem Cell Therapy Revenue Comparison by Types (2019-2024)

1.2.2 Global Musculoskeletal Disorder Stem Cell Therapy Revenue Market Share by Types in 2018

1.3 Global Musculoskeletal Disorder Stem Cell Therapy Market by Application

1.3.1 Global Musculoskeletal Disorder Stem Cell Therapy Market Size and Market Share Comparison by Applications (2014-2024)

1.4 Global Musculoskeletal Disorder Stem Cell Therapy Market by Regions

1.4.1 Global Musculoskeletal Disorder Stem Cell Therapy Market Size (Million USD) Comparison by Regions (2014-2024)

1.4.1 North America (USA, Canada and Mexico) Musculoskeletal Disorder Stem Cell Therapy Status and Prospect (2014-2024)

1.4.2 Europe (Germany, France, UK, Russia and Italy) Musculoskeletal Disorder Stem Cell Therapy Status and Prospect (2014-2024)

1.4.3 Asia-Pacific (China, Japan, Korea, India and Southeast Asia) Musculoskeletal Disorder Stem Cell Therapy Status and Prospect (2014-2024)

1.4.4 South America (Brazil, Argentina, Colombia) Musculoskeletal Disorder Stem Cell Therapy Status and Prospect (2014-2024)

1.4.5 Middle East and Africa (Saudi Arabia, UAE, Egypt, Nigeria and South Africa) Musculoskeletal Disorder Stem Cell Therapy Status and Prospect (2014-2024)

1.5 Global Market Size of Musculoskeletal Disorder Stem Cell Therapy (2014-2024)

2 Manufacturers Profiles

2.1 Company 1

2.1.1 Business Overview

2.1.2 Musculoskeletal Disorder Stem Cell Therapy Type and Applications

2.1.2.1 Product A

2.1.2.2 Product B

2.1.3 Musculoskeletal Disorder Stem Cell Therapy Revenue, Gross Margin and Market Share (2017-2018)

2.2 Company 2

2.2.1 Business Overview

2.2.2 Musculoskeletal Disorder Stem Cell Therapy Type and Applications

2.2.2.1 Product A

2.2.2.2 Product B

2.2.3 Musculoskeletal Disorder Stem Cell Therapy Revenue, Gross Margin and Market Share (2017-2018)

3 Global Musculoskeletal Disorder Stem Cell Therapy Market Competition, by Players

3.1 Global Musculoskeletal Disorder Stem Cell Therapy Revenue and Share by Players (2014-2019)

3.2 Market Concentration Rate

3.2.1 Top 5 Musculoskeletal Disorder Stem Cell Therapy Players Market Share

3.2.2 Top 10 Musculoskeletal Disorder Stem Cell Therapy Players Market Share

3.3 Market Competition Trend

4 Global Musculoskeletal Disorder Stem Cell Therapy Market Size by Regions

4.1 Global Musculoskeletal Disorder Stem Cell Therapy Revenue and Market Share by Regions

4.2 North America Musculoskeletal Disorder Stem Cell Therapy Revenue and Growth Rate (2014-2019)

4.3 Europe Musculoskeletal Disorder Stem Cell Therapy Revenue and Growth Rate (2014-2019)

4.4 Asia-Pacific Musculoskeletal Disorder Stem Cell Therapy Revenue and Growth Rate (2014-2019)

4.5 South America Musculoskeletal Disorder Stem Cell Therapy Revenue and Growth Rate (2014-2019)

4.6 Middle East and Africa Musculoskeletal Disorder Stem Cell Therapy Revenue and Growth Rate (2014-2019)

and continued

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Covid-19 Impact on Musculoskeletal Disorder Stem Cell Therapy Market Report with Capacity and Share by Manufacturers, Forecast Report 2020 - 3rd Watch...

Global Negative Pressure Wound Therapy Market Segmentation, Trends, Growth, Competitive Analysis and Geographical Outlook till 2024 – Cole of Duty

Global Negative Pressure Wound Therapy Market report gives a comprehensive and detail picture of the present and upcoming market Opportunities that is been completed by investigating the effect by buyers, new entrants, Negative Pressure Wound Therapy industry competitors and suppliers available in the Negative Pressure Wound Therapy market. The goal of this report is to incorporate both authentic and future trends for Negative Pressure Wound Therapy supply, Market size, costs, exchanging, competition and value chain. The top to bottom information and data on what the business sectors definition, arrangements, applications, and commitment are covered and furthermore clarifies with the drivers and restraints of the market which is gotten from SWOT analysis.

This research essentially examines the market size, current trends and growth status of the Negative Pressure Wound Therapy market, as well as financing Opportunities, government policy, drivers, restraints, Opportunities, supply chain, and ambitious landscape. Technological innovation and rise will additionally upgrade the presentation of the product, making it all the more generally utilized in downstream applications. Moreover, Porters Five Forces Analysis (potential entrants, suppliers, substitutes, buyers, industry competitors) provides vital data for knowing the Negative Pressure Wound Therapy market.

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Major Players Of Negative Pressure Wound Therapy Market

Acelity (KCI), Smith & Nephew, Cardinal Health, Devon, Medela, Triage Meditech, WuHan VSD, Talley Group

This report covers the Types as well as Application data for Negative Pressure Wound Therapy Market along with the country level information for the period of 2015-2024

Market Segmented By Types and By its Applications:

Type: Conventional NPWT Devices Disposable NPWT Devices

Application: Hospitals Clinics Homecare

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Global Negative Pressure Wound Therapy Market Scope and Features

Global Negative Pressure Wound Therapy Industry Introduction and Overview Includes Negative Pressure Wound Therapy market Definition, Market Scope and Market Size Estimation and region-wise Negative Pressure Wound Therapy Value and Growth Rate history from 2015-2024, Negative Pressure Wound Therapy market dynamics:Drivers, Limitations, challenges that are faced, emerging countries of Negative Pressure Wound Therapy , Industry News and Policies by Regions.

Industry Chain Analysis To describe upstream raw material suppliers and cost structure of Negative Pressure Wound Therapy , major players of Negative Pressure Wound Therapy with company profile, Negative Pressure Wound Therapy manufacturing base and market share, manufacturing cost structure analysis, Market Channel Analysis and major downstream buyers of Negative Pressure Wound Therapy .

Global Negative Pressure Wound Therapy Market Analysis by Product Type and Application It gives Negative Pressure Wound Therapy market share, value, status, production, Negative Pressure Wound Therapy Value and Growth Rate analysis by type from 2015 to 2019. Although downstream market overview, Negative Pressure Wound Therapy consumption,Market Share, growth rate, by an application (2015-2019).

Regional Analysis This segment of report covers the analysis of Negative Pressure Wound Therapy production, consumption,import, export, Negative Pressure Wound Therapy market value, revenue, market share and growth rate, market status and SWOT analysis, Negative Pressure Wound Therapy price and gross margin analysis by regions.

Competitive Landscape, Trends And Opportunities: It includes the provides competitive situation and market concentration status of major players of Negative Pressure Wound Therapy with basic information i.e company profile, Product Introduction, Market share, Value, Price, Gross Margin 2015-2019E

Negative Pressure Wound Therapy Industry Analysis and Forecast by Region Includes Market Value and Consumption Forecast (2015-2024) of Negative Pressure Wound Therapy market Of the following region and sub-regions including the North America, Europe(Germany, UK, France, Italy, Spain, Russia, Poland), China, Japan,Southeast Asia (Malaysia, Singapore, Philippines, Indonesia, Thailand, Vietnam) Middle East and Africa(Saudi Arabia, United Arab Emirates, Turkey, Egypt, South Africa, Nigeria), India, South America(Brazil, Mexico, Colombia)

Do you want any other requirement or customize the report, Do Inquiry Here: https://www.globalmarketers.biz/report/medicine/global-gene-therapy-market-2019-by-company,-regions,-type-and-application,-forecast-to-2024/130926#inquiry_before_buying

1 Negative Pressure Wound Therapy Introduction and Market Overview

2 Industry Chain Analysis

3 Global Negative Pressure Wound Therapy Value (US$ Mn) and Market Share, Production , Value (US$ Mn) , Growth Rate and Average Price (US$/Ton) analysis by Type (2015-2020E)

4 Negative Pressure Wound Therapy Consumption, Market Share and Growth Rate (%) by Application (2015-2020E) by Application

5 Global Negative Pressure Wound Therapy Production, Value (US$ Mn) by Region (2015-2020E)

6 Global Negative Pressure Wound Therapy Production (K Units), Consumption (K Units), Export (%), Import (%) by Regions (2015-2020E)

7 Global Negative Pressure Wound Therapy Market Status by Regions

8 Competitive Landscape Analysis

9 Global Negative Pressure Wound Therapy Market Analysis and Forecast by Type and Application

10 Negative Pressure Wound Therapy Market Analysis and Forecast by Region

11 New Project Feasibility Analysis

12 Research Finding and Conclusion 13 Appendix 13.1 Methodology 13.2 Research Data Source

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Global Negative Pressure Wound Therapy Market Segmentation, Trends, Growth, Competitive Analysis and Geographical Outlook till 2024 - Cole of Duty

Bone Wax Market Size to Represent 3.5% CAGR By 2023 | COVID-19 Impact Analysis, Competitive Outlook, Share Estimation and Industry Insights – Press…

Bone Wax Research Report: By Product (Absorbable Bone Wax, Non-Absorbable Bone Wax), by Material (Natural Bone Wax, Synthetic Bone Wax), by application (Neurosurgery, Orthopedic Surgery, Orthopedic Surgery, Others), by End-User Forecast Till 2023

Bone Wax Market Analysis

The globalBone Wax Marketsize is likely to grow at a favourable 3.5% CAGR between 2018- 2023, according to the new report by Market Research Future (MRFR). Bone wax, simply put, is a sterile mixture of isopropyl palmitate and beeswax. This wax-softening agent is used for controlling bleeding from the surface of the bone during surgical operations. Bone wax is used right away after this is taken out of the package. It helps in stopping the bleeding of bone via blocking the holes, thereby causing instant bone hemostasis. This is used in those procedures, which include cutting through bones such as maxillofacial surgeries, oral surgeries, orthopedic, neurosurgery, thoracic, and sternotomy. Non-absorbable bone wax and absorbable bone wax are the two types of bone wax that are widely used in ambulatory surgical centers, specialty clinics, hospitals, and others.

Various factors are propelling the global bone wax market growth. According to the new MRFR report, such factors include increasing use of absorbable bone wax products, increasing geriatric population, and rising prevalence of accidental fracture & orthopedic diseases.

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On the contrary, the development of alternatives, certain side effects, stringent government regulations, and the grave effect of the most recent COVID-19 outbreak are factors that may limit the global bone wax market growth over the forecast period.

Bone Wax Market Segmentation

The MRFR report gives an inclusive segmental analysis of the global bone wax market report based on end user, application, material, and product.

By product, the global bone wax market is segmented into non-absorbable bone wax and absorbable bone wax.

By material, the global bone wax market is segmented into synthetic bone wax and natural bone wax.

By applications, the global bone wax market is segmented into oral surgery, thoracic surgery, orthopedic surgery, neurosurgery, and others. Of these, orthopedic surgery will have a major share in the market over the forecast period.

By end users, the global bone wax market is segmented into ambulatory surgical centers, specialty clinics, hospitals, and others.

Bone Wax Market Regional Analysis

Based on the region, the global bone wax market report covers the growth opportunities and recent trends across the Americas, Europe, the Asia Pacific (APAC), & the Middle East and Africa. Of these, the Americas will spearhead the market over the forecast period. Increasing R&D activities, a growing number of surgeries, and well-developed healthcare are adding to the global bone wax market growth in the region.

The global bone wax market in Europe is predicted to hold the second-largest share over the forecast period. Increasing expenditure on the healthcare sector, rising prevalence of injuries and accidents, and growing cases of orthopedic disease are adding to the global bone wax market growth in the region.

The global bone wax market in the APAC region is predicted to grow at a fast pace over the forecast period. Rising government expenditure on the healthcare sector, growing geriatric population, increasing incidence of bone diseases, and continuously developing economies are adding to the global wax market growth in the region. China has the maximum share in the region.

Browse Detailed TOC with COVID-19 Impact Analysis at:https://www.marketresearchfuture.com/reports/bone-wax-market-6463

The global bone wax market in the MEA is predicted to have the smallest share over the forecast period. Meanwhile, a good share will be held by the Eastern region for the increasing initiatives by the government for the healthcare sector, rising cases of bone disorders, and the presence of a well-developed healthcare sector.

Bone Wax Market Key Players

Eminent players profiled in the global bone wax market report include GPC Medical Ltd., WNDW Medical Inc., CP Medical Corp., Bentley Healthcare Pvt. Ltd., Futura Surgicare Pvt. Ltd., Medline Industries, Inc., Medtronic Plc, Baxter International, Ethicon, Inc., Aesculap, Inc., Wound Management Technologies, Inc, and ABYRX, Inc.

Browse More Healthcare Research Reports at:

Gene Therapy Marketsize is expected to exhibit a robust CAGR of 40.7% over the forecast period

Wearable Medical Devices Marketvaluation can surge to USD 27,200 million by 2023

NOTE: Our team of researchers are studying Covid19 and its impact on various industry verticals and wherever required we will be considering covid19 footprints for a better analysis of markets and industries. Cordially get in touch for more details.

About Market Research Future:

At Market Research Future (MRFR), we enable our customers to unravel the complexity of various industries through our Cooked Research Report (CRR), Half-Cooked Research Reports (HCRR), Raw Research Reports (3R), Continuous-Feed Research (CFR), and Market Research & Consulting Services.

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Bone Wax Market Size to Represent 3.5% CAGR By 2023 | COVID-19 Impact Analysis, Competitive Outlook, Share Estimation and Industry Insights - Press...

Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects – Science…

INTRODUCTION

Fusing a deaminase with the Cas9 nickase (nCas9) forms cytosine base editors (CBEs), which enable programmable conversion of cytidine-to-thymidine (C-to-T) mutations within a specific region of the genomic DNA without causing double-stranded breaks (13). CBEs have displayed substantially higher editing efficiency than the conventional Cas9 endonuclease-mediated homology-directed repair method for installing point mutations (4, 5). In addition, recent protein engineering efforts have improved their product purities and efficiencies (6, 7), greatly expanded the genome targeting scope (8), and minimized the undesirable RNA off-target effects (911). CBEs are important genetic tools and could potentially correct more than 5000 pathogenic single-nucleotide polymorphisms (SNPs) associated with human-inherited diseases caused by T-to-C (or G-to-A) mutations (3, 12, 13).

The presence of multiple targets within the CBEs activity window [e.g., the editing window of BE4max is approximately from positions 4 to 8 of the protospacer, counting the protospacer adjacent motif (PAM) as positions 21 to 23] can introduce unwanted bystander editing, resulting in deleterious multiC-to-T conversions (14). Earlier studies have shown that the activity window size can be narrowed using strategies such as modulating the catalytic activity of deaminase (15), using more rigid linkers between Cas9 and deaminase, or deleting nonessential deaminase sequences (16, 17). These approaches can systematically enhance precision for position-dependent single-nucleotide editing irrespective of nearby sequence contexts, although the genome targeting scope might be compromised because of the requirement that the target nucleotide needs to be placed at a specific position relative to an available PAM. Alternatively, sequence context-specific CBE can avoid bystander editing without sacrificing the activity window size (3). The engineered APOBEC3A (A3A) enzyme preferentially deaminates in the TCR motif (target C underlined), which has been exploited for more precise base editing, and the resulting eA3A-BE3 base editor exhibited high on-target precision with minimized bystander editing (18). However, in the most challenging case, when editable Cs are located consecutively within the activity window, especially in the case of CC dinucleotides when a bystander C is located right upstream of the target C, the existing CBEs nonselectively edit both of the Cs. Nearly 38% of the human pathogenic SNPs that are caused by T-to-C disease point mutations lie in the context of CC, followed by AC (29%), GC (21%), and TC (13%) (see data file S1) (1, 12), necessitating the development of new CBEs that can precisely discriminate between the target and bystander Cs.

Various APOBEC enzymes in vertebrates mediate defense against infections from retroviruses or retrotransposons by deaminating C to U in the viral complementary DNA (cDNA) (19, 20), suggesting that these cytosine deaminases could have unique preferences for particular sequence motifs to distinguish DNA sequences from the native host (2123). In this study, we identified human APOBEC3G (A3G) as a candidate for developing sequence-specific BEs in multiple C contexts. We characterized and engineered A3G-BE variants to efficiently edit a single C at various endogenous sites in human embryonic kidney293T (HEK293T) cells. By introducing mutations that improve catalytic activity, solubility, and overall protein scaffold, we obtained and characterized three novel variants (A3G-BE4.4, A3G-BE5.13, and A3G-BE5.14) that exhibit high editing efficiencies and precision in the context of the CC motif. A3G-BE variants have broader activity windows than BE4max that could expand the targeting scope for precision base editing. We also demonstrated that these variants could efficiently and precisely generate or correct mutated alleles associated with the known pathogenic phenotypes, illuminating A3G-BEs potential application in treating human genetic diseases. Last, we performed whole-genome sequencing (WGS) by using the most recently developed genome-wide off-target analysis by two-cell embryo injection (GOTI) method to detect DNA off-targets and used RNA sequencing (RNA-seq) to examine the RNA off-targets of cells treated with A3G-BE5.13. Our results showed that the most active A3G-BE5.13 induces baseline levels of the genome- and transcriptome-wide off-target mutations, suggesting high editing fidelity for future clinical applications.

Previous studies have demonstrated that A3G predominantly deaminates the third C in the 5-CCC-3 motif of a single-stranded DNA (ssDNA) substrate (24). To test whether this motif preference could be preserved when A3G is fused to nCas9 as A3G-BE, we replaced the rAPOBEC1 deaminase domain of BE4max with the full-length, human codon-optimized A3G to construct A3G-BE2.1 (6). Since it has been reported that the N-terminal domain (NTD) could mediate aggregation of A3G monomers to impede A3Gs mobility (25) and because the C-terminal domain (CTD) of A3G is sufficient for deamination activity in vitro (26, 27), we therefore truncated the NTD of A3G to construct A3G-BE4.4, which only contains the CTD of A3G (Fig. 1A). HEK293T cells were then transfected with plasmids expressing BE4max, A3G-BE2.1, and A3G-BE4.4 with single-guide RNAs (sgRNAs) targeting EMX1 #1 and FANCF #a3 sites, which contain dinucleotide Cs (C5 and C6 of EMX1 #1 and C6 and C7 of FANCF #a3) within the canonical BE4max activity window. We extracted the genomic DNA after 72 hours and amplified the target regions for high-throughput sequencing (HTS). Analysis of the C-to-T editing efficiencies of the dinucleotide Cs showed that A3G-BE2.1 and A3G-BE4.4 edited 21 to 42% of the cognate Cs (C6 of EMX1 #1 and C7 of FANCF #a3) but only 1 to 3% of the bystander Cs (C5 of EMX1 #1 and C6 of FANCF #a3), while BE4max edited 47 to 62% of both the cognate and bystander Cs without obvious selectivity (Fig. 1B). No significant difference was observed between A3G-BE2.1 and A3G-BE4.4 for editing efficiencies of the cognate Cs, suggesting that the CTD itself adequately determines the enzymatic activity and sequence specificity of A3G.

(A) Schematic showing the protein architecture of base editors. BE4max is used to replace the rAPOBEC1 with either full-length (NTD + CTD) or CTD-only human A3G to construct A3G-BE2.1 or A3G-BE4.4, respectively. Linkers between functional domains are shown as horizontal blue lines. NLS, nuclear localization signal; UGI, uracil glycosylase inhibitor. (B) C-to-T editing efficiency and specificity of A3G-BE2.1 and A3G-BE4.4 at EMX1 #1 and FANCF #a3 sites bearing the CC motif (red). (C) Nine endogenous sites of HEK293T bearing either CC or CCC motif (red) within the canonical BE4max activity window. Each PAM and the sequence motif identifying the nucleotides at +1 and 2 positions from the target C (underlined) are shown. (D) C-to-T editing efficiency and specificity of BE4max and A3G-BE4.4 at the endogenous sites listed in (C). Bar figures of (B) and (D) show means and error bars representing SD of n = 2 and n = 3 independent biological replicates performed on different days, respectively. Statistical significance shown on top of each bar using two-tailed Students t test compares to editing efficiency of the preceding bystander C of the same BE. For example, t test was performed between the BE4max editing efficiencies of C8 and C9 at DMD #1 site. ns (not significant), *P < 0.05, ***P < 0.001, ****P < 0.0001.

Because the wild-type A3G in nature preferentially deaminates in the C2C1C0A+1 sequences of the HIV-1 genome (28), we next examined whether nucleotides at positions 2 and +1 of the cognate C0 also affect the base editing efficiency and specificity. We tested BE4max and A3G-BE4.4 at nine different loci containing the dinucleotide Cs motif with different combinations of nucleotides placed at the 2 and +1 positions (N2C1C0D+1, where D denotes A, T, and G) (Fig. 1C). HTS analysis confirmed that A3G-BE4.4 showed selective editing of the cognate Cs across all the sites. At six of the nine sites, A3G-BE4.4 reached at least 79% of the editing efficiencies of the cognate Cs of those of BE4max (Fig. 1D). Notably, at DMD #1 site, which contains the ACCA motif, similar to the native CCCA, and harbors the cognate C9 outside the canonical BE4max activity window, A3G-BE4.4 induced threefold higher editing of the cognate C9 compared to BE4max. However, although being selective, A3G-BE4.4 displayed very low cognate C editing efficiencies with only 13, 6, and 3% C-to-T conversion rates at the remaining three PPP1R12C #a3, BCS1L #1, and EMX1 #a18 sites, respectively. These results may have occurred because the wild-type A3G disfavors deamination of certain motifs such as GCC, suggesting that the motif-dependent deamination activity of A3G could influence the efficiency of the selective base editing (29). We then quantified the specificity by dividing the editing efficiency of the cognate C by that of bystander C (cognate-to-bystander editing ratio). Across the nine sites, A3G-BE4.4 recorded the editing ratios ranging from 11 to 290, while BE4max achieved a maximum ratio of 6 at EMX1 #a18 and less than 2 at all other sites (fig. S1A). Non-T by-products generated by A3G-BE4.4 averaged slightly higher than BE4max in most of the sites (fig. S1B), consistent with previous observations that generally lower product purity is generated by editing of a single C versus multiple Cs (6). A3G-BE4.4 also showed significantly fewer indels than BE4max at three of the nine sites (HEK3 #1, HEK4 #a1, and EMX1 #a3), supporting an earlier study suggesting that single-nucleotide and multiple base editing have no significant correlation in terms of indel generation (fig. S1C) (18). Together, these results indicated that A3G-BE4.4 has sufficient editing efficiency to precisely edit the second C in the sequence context of 5-CC-3 dinucleotides.

Given the relatively low base editing efficiencies of A3G-BE4.4 for cognate Cs observed from the PPP1R12C #a3, BCS1L #1, and EMX1 #a18 sites, we envisioned that the wild-type A3G-CTD activity could be further improved. We devised three subsets of mutations that could be introduced into the A3G-CTD of A3G-BE4.4 based on different possible functional effects, including set A (P200A + N236A + P247K + Q318K + Q322K) to improve catalytic activity, set B (partial replacement of A3Gs loop 3 with A3As, that is H248N + K249L + H250L + G251C + F252G + L253F + E254Y) to increase ssDNA binding affinity, and set C (L234K + C243A + F310K + C321A + C356A) to enhance protein solubility (Fig. 2A and fig. S2A) (27, 30, 31). We first introduced set A to A3G-BE4.4 to construct A3G-BE5.1 and introduced sets B and C mutations to A3G-BE5.1 to construct A3G-BE5.3 and 5.4, respectively (fig. S2B and table S1). To further maximize A3Gs potential deamination activity, two additional mutations, T311A + R320L, were introduced to A3G-BE5.3 to construct A3G-BE5.10 (fig. S2B and table S1) (27, 31). We tested A3G-BE4.4, A3G-BE5.1, A3G-BE5.3, A3G-BE5.4, and A3G-BE5.10 at EMX1 #1 and FANCF #a3; all of the further improved mutants showed substantially higher editing efficiency than A3G-BE4.4 did on both the cognate Cs and the bystander Cs (Fig. 2B and fig. S2C). Notably, when the loop 3 of A3G was partially replaced with A3As by set B mutations, A3G-BE5.3 and A3G-BE5.10 exhibited substantial loss of the motif preference, and both Cs were efficiently edited. Structural alignment of the wild-type A3A, wild-type A3G, and the A3G containing the set A mutations, among which P247K lies in loop 3, showed that loop 3 of the wild-type A3A, as well as the A3G with set A mutations, exhibits greater proximity to the ssDNA substrate, suggesting that the observed increase in the editing efficiency and relaxation of the sequence specificity might be partly due to the stronger nonspecific binding to the ssDNA substrate (fig. S2D).

(A) Set of residue mutations of A3G for improving catalytic activity (set A), ssDNA binding (set B), and protein solubility (set C) listed on each row. Counting of the residue number starts with the first residue of the original full-length A3G. (B) Screening of A3G-BE mutants at EMX1 #1 site to determine variants with enhanced editing efficiency and retained sequence specificity. C-to-T editing efficiencies are represented as bidirectional bars with values for the cognate C6 (blue) on the right and the bystander C5 (red) on the left. (C) An enlarged view of the interactions of Tyr315 (green sticks) with the ssDNA substrate (yellow sticks). The hydrogen bond between the 5 phosphate group of the DNA backbone and the hydroxyl group of Tyr315, and the interaction between the rings of the target cytidine (dC0) and Tyr315 are represented as dashed lines. (D) C-to-T editing efficiency and specificity of A3G-BE5.13 and A3G-BE5.14 at three endogenous sites previously poorly edited by A3G-BE4.4. Panels (B) and (D) show means and error bars representing SD of n = 3 independent biological replicates performed on different days. For (D), statistical significance shown on top of each bar using two-tailed Students t test compares to editing efficiency of the preceding bystander C of the same BE. ns (not significant), **P < 0.01, ***P < 0.001, ****P < 0.0001.

We hypothesized that modulating the nonspecific binding to DNA could restore the sequence specificity. Using structure-guided analysis, Tyr315 of A3G was identified as a key residue that interacts with both the DNA backbone and the target C (Fig. 2C). We speculated that changing Tyr315 to Phe, which lacks only the hydroxyl group from Tyr, could remove the hydrogen bond with the 5 phosphate group of ssDNA while maintaining the - interaction with the target C. We introduced Y315F to A3G-BE5.1, A3G-BE5.3, A3G-BE5.4, and A3G-BE5.10 to construct A3G-BE5.12, A3G-BE5.13, A3G-BE5.14, and A3G-BE6.11, respectively (fig. S2B and table S1). Y315W (to provide steric hindrance) and Y315L (to remove both the hydrogen bond and the - interaction) were also introduced into A3G-BE5.10, resulting in A3G-BE6.16 and A3G-BE6.17, respectively. Additional mutations to further reduce the nonspecific binding, including N244Q, S286A, and R313A, were introduced into A3G-BE6.11 to construct A3G-BE6.18, A3G-BE6.19, and A3G-BE6.20, respectively. Last, we reverted the replacement of the A3Gs loop 3 with A3As from A3G-BE6.11 to construct A3G-BE6.21 (fig. S2B and table S1). Testing all the above variants at EMX1 #1 and FANCF #a3 showed that A3G-BE6.11 induced higher selectivity than A3G-BE5.10 by moderately reducing editing of the bystander Cs. At the same time, A3G-BE6.16 and A3G-BE6.17 displayed markedly reduced editing efficiencies of the cognate Cs, even below those of A3G-BE4.4 (Fig. 2B and fig. S2C). Although all A3G-BE6.18, A3G-BE6.19, A3G-BE6.20, and A3G-BE6.21 showed improved editing ratios of the cognate to bystander Cs compared with A3G-BE6.11, their cognate C editing efficiencies did not outperform A3G-BE4.4. Nevertheless, A3G-BE5.13 and A3G-BE5.14, both of which contain Y315F, exhibited greater cognate C editing efficiency than A3G-BE4.4 did and demonstrated appreciable restoration of the sequence specificity (Fig. 2B and fig. S2C).

We further tested A3G-BE5.13 and A3G-BE5.14 at the PPP1R12C #a3, BCS1L #1, and EMX1 #a18 sites at which the editing efficiencies of A3G-BE4.4 were previously low (Fig. 1D). HTS analysis showed that both A3G-BE5.13 and A3G-BE5.14 gained superior editing efficiency for the cognate Cs as compared to A3G-BE4.4 (Fig. 2D). Moreover, bystander editing of A3G-BE5.13 and A3G-BE5.14 remained substantially lower than that of BE4max, resulting in significant improvement of base editing efficiency while maintaining the specificity. Together, these results suggested that through rational engineering, A3G-BE5.13 and A3G-BE5.14 overcame the low editing drawbacks of A3G-BE4.4 on discrete sequence contexts.

To comprehensively understand the capability of sequence-specific base editing of A3G-BE5.13 and A3G-BE5.14, we tested them at eight other endogenous sites with the dinucleotide Cs motif positioned across the whole protospacer. HTS analysis confirmed that all A3G-BE4.4, A3G-BE5.13, and A3G-BE5.14 selectively edited the second C within the CC motifs across all the sites. The cognate-to-bystander editing ratios were calculated to be up to 186 (A3G-BE5.14 at EMX1 #c16 site), while BE4max either nonselectively edited both Cs or failed to perform outside its canonical activity window (Fig. 3A and fig. S3A). At BCS1L #6 and RNF2 #2 sites, which contained the cognate Cs at positions 12 and 15 of the protospacers, respectively, highly efficient and selective editing for the cognate Cs were only observed when using A3G-BE5.13 and A3G-BE5.14, while A3G-BE4.4 and BE4max did not yield efficient C-to-T editing (Fig. 3A). Notably, at both BCS1L #6 and RNF2 #2 sites, the single C located at the fifth position was not efficiently edited by all A3G-BE variants, probably due to lack of the CC dinucleotide sequence context. Both A3G-BE5.13 and A3G-BE5.14 displayed efficient editing up to C15 of RNF2 #2 but not C18 of FANCF #2 (Fig. 3A). For the two cognates Cs existing in EMX1 #b1 (C7 and C15) and FANCF #2 (C6 and C10) sites, A3G-BE4.4 efficiently edited only the ones residing closer to the 5 end (C7 of EMX1 #b1 and C6 of FANCF #2), indicating a possible narrower window size compared with A3G-BE5.13 and A3G-BE5.14. The lowest cognate-to-bystander editing ratios for all three A3G-BEs occurred at EMX1 #b1, which bears three consecutive Cs of the CCCA motif, suggesting that the requirement for single-nucleotide editing within more than two consecutive Cs might need to be more stringent. We did not find a consistent trend in the product purity following the treatment of all BEs, which might be due to the discrepancies among distinct properties of BEs that have different activity windows, deamination activities, and sequence specificities (fig. S3B) (6). We also observed indels being generated with varying frequencies across the sites without apparent correlation among BEs (fig. S3C).

(A) Heat maps are showing average C-to-T editing efficiencies of n = 3 independent biological replicates of BE4max, A3G-BE4.4, A3G-BE5.13, and A3G-BE5.14 at eight endogenous sites containing the preferential CC or CCC motif across the whole region within the protospacers. The cognate Cs predicted to be preferentially editable by A3G-BEs are indicated by the black triangles. (B) Average C-to-T base editing frequencies at each protospacer position from the six poly-C endogenous sites shown in fig. S4. Bidirectional arrows in between vertical dashed lines show the base-editable ranges within the protospacer region by the indicated A3G-BEs (C) Schematic representation of the activity window sizes of A3G-BE4.4, A3G-BE5.13, and A3G-BE5.14, with NGG PAM shown as positions 21 to 23. Standard, light, and near-transparent green represent the predicted relative base editing activity within the approximate regions of the protospacer.

To determine the sizes of the activity window of A3G-BEs, we tested A3G-BE4.4, A3G-BE5.13, and A3G-BE5.14 at six endogenous genomic sites, which contain consecutive Cs within the protospacer, and analyzed their C-to-T editing efficiencies. For all the tested sites, A3G-BE4.4, A3G-BE5.13, and A3G-BE5.14 revealed consistent and broad base editing activity window but differed mainly in their relative editing efficiencies, for which A3G-BE5.13 showed the highest followed by A3G-BE5.14 and A3G-BE4.4 (fig. S4). We observed that A3G-BE4.4 displayed comparatively lower editing efficiencies around positions 8 to 15 compared with those in positions 5 to 7 at four sites (VEGF #2, EMX1 PolyC #1, EMX1 PolyC #1, and HEK4 PolyC #1), suggesting that editing toward the 3 end of the protospacer, although targetable, could have lower editing efficiency. Next, we compared the average editing frequencies of Cs at each protospacer position from all the six sites. We found that the activity windows of A3G-BE4.4, A3G-BE5.13, and A3G-BE5.14 span from positions 5 to 15, 3 to 15, and 4 to 15 of the protospacer, respectively (Fig. 3, B and C). Together, these data indicated that A3G-BEs enable sequence-specific editing with broadened targeting ranges.

Given that the preferential motif of A3G extends to three consecutive Cs, C2C1C0, we hypothesized to test whether the sequence specificity could be maintained when the middle C, the 1 position of the target, is altered to other nucleotides. To assess this possibility, we selected five endogenous sites that contained a T or A at the 1 position (C2TC0 or C2AC0 motifs) and, now, counting editing of the C at 2 position to be the bystander incidence (fig. S5A). We transfected HEK293T with BE4max, A3G-BE4.4, A3G-BE5.13, and A3G-BE5.14 with sgRNAs targeted to the selected sites and performed HTS. After quantifying the C-to-T editing efficiencies, we found that, compared to BE4max, A3G-BEs indeed displayed significantly higher editing of the cognate Cs over bystander Cs within these altered sequence contexts (fig. S5B). A3G-BE5.14, among other A3G-BEs, exhibited the highest specificities (up to 89 cognate-to-bystander editing ratio) at four of the five sites (fig. S5B). While A3G-BE5.13 and A3G-BE5.14 have comparable or higher cognate C editing efficiency than BE4max, A3G-BE4.4 editing efficiencies of the cognate Cs were below 9% at four of the five sites, indicating that the absence of C at the 1 position might restrain A3G-BE4.4 from efficient editing. In addition, we observed relatively higher bystander C2 editing from A3G-BE5.13 at HEK3 #b1 and HEK3 #b2 sites, which contained T immediately upstream of the bystander C2. Since C and T are structurally similar compared to the other two nucleotides, we speculated that this sequence context might be more prone to bystander editing. These findings indicated that A3G-BEs could selectively edit a target C in the CTC and CAC motifs and therefore can further expand the targeting scope for precision base editing in broader sequence contexts.

To test A3G-BEs in disease-relevant contexts, we sought to precisely generate SNPs of reported human pathogenic diseases (32). Three genetic variants caused by C-to-T (or G-to-A) substitution in which the wild-type sequences lie within the preferential 5-CC-3 motif of A3G-BEs were selected, including cystic fibrosis (model 1), hypertonic myopathy (model 2), and transthyretin amyloidosis (model 3) (Fig. 4A). Individual sgRNAs targeted to these disease-associated sites were constructed and cotransfected into HEK293T with BE4max, A3G-BE4.4, A3G-BE5.13, and A3G-BE5.14. Genomic DNA was harvested after 72 hours and prepared for HTS to quantify the percentage of alleles perfectly modeled and of those that were imperfectly modified because of bystander editing. Direct comparison with BE4max of the modified allele frequencies demonstrated that A3G-BEs induced a substantially higher proportion of perfectly modified alleles for all three models (Fig. 4B). Despite the previous observations in which A3G-BE5.13 displayed more relaxed base-editing sequence specificity among other selected A3G-BEs, it achieved the highest percentage here of the perfectly modified alleles for hypertonic myopathy (model 2) (36%). For transthyretin amyloidosis (model 3), in which the target C lies at position 11 of the protospacer, all A3G-BEs produced the desired allele with high efficiencies (>35%), while BE4max failed to edit the target C (<0.1%) because of its inability to edit outside its activity window (fig. S6A). As a result, A3G-BE5.14 accomplished 613-fold higher correct modeling of transthyretin amyloidosis than BE4max did, highlighting the advantage of precise editing with an expanded activity window. Similarly, for cystic fibrosis (model 1), all A3G-BEs induced more than 50% of the perfectly modified alleles, while BE4max averaged 0.6%.

(A) Sequences of the protospacers and PAMs (blue) for model 1 (cystic fibrosis), model 2 (hypertonic myopathy), and model 3 (transthyretin amyloidosis). Position of the disease-relevant C>T (or G>A) point mutations are red and indicated by black triangles shown with the nucleotide numbers within the disease-associated genes. (B) Percent of alleles modified to the indicated genotypes following the treatment of BE4max and A3G-BEs for generating the three models presented in (A). (C) Sequences of the protospacers and PAMs (blue) for correction 1 (hereditary pyropoikilocytosis), correction 2 (cystic fibrosis), and correction 3 (holocarboxylase synthetase deficiency), bearing T>C (or A>G) point mutations for which the positions are indicated with black triangles showing the nucleotide numbers within the disease-associated genes. (D) Percent of alleles modified to the indicated genotypes following the treatment of BE4max and A3G-BEs for correcting the three disease-associated variants presented in (C). Panels (B) and (D) show means and error bars representing SD of n = 3 independent biological replicates performed on different days. Statistical significance shown on top of each bar using two-tailed Students t test compares to the percentages of perfectly generated/corrected alleles by BE4max. ns (not significant), *P < 0.05, ****P < 0.0001.

Next, to examine the therapeutic applicability of A3G-BEs, we selected three reported human pathogenic SNPs caused by T>C (or A>G) mutations, which can be preferentially targeted by A3G-BEs, including hereditary pyropoikilocytosis (correction 1), cystic fibrosis (correction 2), and holocarboxylase synthetase deficiency (correction 3) (Fig. 4C) (32). We generated three HEK293T lines containing 200 base pair (bp) of each disease-relevant sequence integrated into the genome (see Materials and Methods). Codelivery of the BEs and sgRNAs targeted to the disease-associated sites and analysis of the HTS data to quantify the perfectly corrected alleles verified that all A3G-BEs significantly outperformed BE4max by a minimum of threefold in corrections 1 and 2. In addition, A3G-BE4.4 exclusively induced more than 50% of perfectly corrected alleles among other BEs and accomplished 6496-fold higher correction than BE4max in correction 3 (Fig. 4D). Correction 3, in which the protospacer contained two motifs preferred by A3G-BEs, CC and CTC, interfered with the precise single C-to-T editing by A3G-BE5.13 and A3G-BE5.14 and resulted in substantial dual C editing due to their wide activity window sizes and high efficiencies (fig. S6A). Collectively, these comparisons indicated that A3G-BEs have higher targeting precision than BE4max for reversing pathogenic SNPs within their preferred sequence contexts.

We further investigated the editing efficiency of A3G-BEs in therapeutically more relevant cell types, including the induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs). We nucleofected iPSC and ESI-017 hESC lines with BE4max, A3G-BE4.4, A3G-BE5.13, and A3G-BE5.14 with sgRNA targeting the hypertonic myopathy (model 2)associated site and performed clonal expansion of the successfully nucleofected cells for 10 to 14 days before analysis. In the iPSCs, analysis of the sequencing chromatograms revealed that A3G-BEs more efficiently edited the cognate C7 than the bystander C6, which were 10, 46, and 34% at C7 and 2, 15, and 5% at C6 by A3G-BE4.4, A3G-BE5.13, and A3G-BE5.14, respectively. In contrast, BE4max nonselectively edited both Cs, 39 and 50% at C7 and C6, respectively (fig. S6B). The observed trend was consistent with the ESI-017 hESCs (fig. S6C), indicating the utility of A3G-BEs to serve as important tools to precisely model genetic variants in clinically relevant cell types.

Several CBEs were reported to generate genome- and transcriptome-wide off-target editing, which became a major concern for their clinical uses (9, 10, 33, 34). We then examined the propensity of A3G-BEs to cause deamination on off-target loci by performing orthogonal R-loop assay (35). Briefly, the nuclease-dead SaCas9 (dSaCas9) sgRNA complex creates an R-loop, recapitulation of a stochastic ssDNA exposure in the genome, at a DNA locus unassociated with the on-target site. Base editing mediated by cytosine deaminase in the off-target R-loop independently of SpCas9 nickase and its sgRNA is detected via targeted HTS (fig. S7A). We assessed six off-target loci (Sa #1 to #6 sites) by cotransfecting SpCas9-derived CBE (BE4max or A3G-BEs), on-target SpCas9 sgRNA, dSaCas9, and off-target dSaCas9 sgRNA into HEK293T (table S2). For the on-target editing at EMX1 #1 site, specificities and efficiencies of all CBEs exhibited consistent results with our previous observations without the dSaCas9 system (fig. S7B). We then quantified the editing activities of 18 cytosines within those six off-target loci. We found that A3G-BEs show substantially reduced off-target editing compared with BE4max, except at those cytosines lying within the 5-CC-3 motif, e.g., C10 and C15 at Sa #2, C11 at Sa #5, and C8 at Sa #6 sites (fig. S7C). A3G-BE4.4 showed no significant off-target editing at 10 of the 18 cytosines. A3G-BE5.13 induced higher off-target mutations than both A3G-BE4.4 and A3G-BE5.14 at all cytosines but still significantly lower than BE4max at 11 of the 18 cytosines. Together, these results suggested that A3G-BEs generally exhibit lower propensities to cause Cas9/sgRNA-independent off-target mutations. We then selected A3G-BE5.13, the most active variant among the three selected ones, for further whole-genome off-target characterization.

To comprehensively understand the capability of A3G-BE5.13 to generate Cas9/sgRNA-independent DNA off-target mutations, we performed WGS using the most recently established GOTI method (33). A blastomere of two-cell embryos derived from Ai9 (CAG-LoxP-Stop-LoxP-tdTomato) mice was injected with Cre mRNA, A3G-BE5.13 mRNA, and sgRNA. At embryonic day 14.5 (E14.5), progeny cells were FACS (fluorescence-activated cell sorting)sorted on the basis of tdTomato expression, and WGS was separately performed for the resulting two cell populations with (tdTomato+) and without (tdTomato) the tdTomato expression (Fig. 5A) (33). Using the WGS data obtained from the tdTomato sample as the reference, single nucleotide variants (SNVs) for the tdTomato+ sample were called via three different algorithms, and the overlapping SNVs detected from all the three algorithms were counted as the true off-target variants. Notably, we detected only 17 and 24 SNVs per embryo in each replicate from those treated by A3G-BE5.13, similar to the spontaneous mutation rate found from embryos delivered with Cre alone, as compared to the average of 283 SNVs per embryo by BE3 as previously detected (Fig. 5B and fig. S8A) (33). The mutation patterns of A3G-BE5.13 only showed a slight bias toward C-to-T or G-to-A compared with BE3 (Fig. 5C). We also tested the on-target Tyr-C site used in the GOTI experiments, which harbors both C3C4 and C4TC6 motifs. The WGS results showed that the editing only happened at the C6 in the C4TC6 motif, which is consistent with our previous data that A3G-BEs could selectively edit a target C in the CTC motif. (fig. S8B). Collectively, these data indicated that A3G-BE5.13 induces minimum DNA off-target SNVs across the genome while maintains highly efficient and selective editing at the on-target position.

(A) Scheme of the experimental workflow of GOTI. (B) Comparison of the total number of detected DNA off-target SNVs using the GOTI method. The number of SNVs identified in Cre-, BE3-, and A3G-BE5.13treated embryos were 14 12 (SD; n = 2), 283 32 (SD; n = 6), and 20 5 (SD; n = 2), respectively. (C) Distribution of DNA mutation types in each group. (D) Scheme of the experimental workflow of identifying transcriptome-wide off-target SNVs through RNA-seq. (E) Comparison of the total number of detected RNA off-target SNVs. The number of SNVs identified in nCas9-, BE4max-, A3G-BE5.13treated cells were 2669 712 (SD; n = 2), 198,688 37,775 (SD; n = 2), and 1410 39 (SD; n = 2), respectively. (F) Distribution of RNA mutation types in each group. For (C) and (F), the number in each cell indicates the percentage of a certain type of mutation among all mutations. For (B) and (E), each data point represents independent biological replicates performed on different days.

Last, we characterized the transcriptome-wide off-target effect of A3G-BE5.13. We transfected HEK293T with sgRNA and nCas9, BE4max, or A3G-BE5.13 encoded in plasmid as cotranslational P2A fusion to green fluorescent protein (GFP). After 48 hours, we sorted cells with the top 5% GFP signal to isolate the high-expression population (Fig. 5D). We first confirmed the robust on-target efficiency of DNA editing by BE4max and A3G-BE5.13 in these cells using HTS (fig. S8C). We then performed RNA-seq and analyzed the sequencing data to call SNVs in each replicate sample according to the method described previously (10). Our results showed that the engineered A3G-BE5.13 did not induce significant RNA SNVs as compared to the control treated by the nCas9 (Fig. 5E). However, BE4max caused a substantial amount of off-target mutations, in line with the previous studies of the wild-type rAPOBEC1-based CBEs (911). Distribution of mutation types of the detected SNVs of A3G-BE5.13 was similar to that of the nCas9 control, indicating a minimum disturbance on the transcriptome despite the high expression of intracellular A3G-BE5.13 proteins (Fig. 5F). These results further demonstrate that the A3G-BEs developed in this study are with high precision and markedly reduced RNA editing activity (9, 10) and indicate that A3G-BE5.13 could serve as a promising CBE variant with high fidelity and minimum risk of off-target effects.

Here, we developed and characterized three new base editors using the A3G deaminase that is capable of recognizing the unique natural motif of CCCA. A3G-BE4.4 displays considerable editing efficiency and selectivity when the target motif lies within around positions 5 to 11 of the protospacer. In most of the sites, A3G-BE4.4 exhibited remarkable sequence specificity by discriminating between two consecutive Cs. However, we also observed that A3G-BE4.4 editing efficiency was poor at certain sites, probably due to the presented motifs being disfavored by the wild-type A3G and its naturally moderate catalytic activity, which could be improved by our engineered A3G-BE5.13 and A3G-BE5.14 variants (36). Both A3G-BE5.13 and A3G-BE5.14 displayed high efficiency across broader activity windows, from positions 4 to 15, with slightly relaxed CC selectivity. An initial screening of these three A3G-BEs could be conducted to determine which one performs the best for the selective editing of a single desired C.

We estimated the scope of base-editable disease variants that could be corrected by using A3G-BEs. Among the total of 1515 pathogenic SNPs identified within the BEable-GPS (Base Editable prediction of Global Pathogenic-related SNVs) entries (12), 61% (929 of 1515) were found to lie within the CC or CNC sequence context preferred by A3G-BEs (18). We then identified 540 human pathogenic SNPs that could be precisely correctable by our A3G-BEs, occupying 36% of the total number (see data file S1). Manual filtering was conducted to ensure that neighboring bystander Cs within the activity window did not exist along with the target motif of A3G-BEs. This indicates that our engineered A3G-BEs greatly expand the number of precisely targetable genetic variants for potential therapeutic applications.

WGS and RNA-seq analysis suggested that our A3G-BEs variants induce minimum levels of both DNA and RNA off-target SNVs. A3Gs intrinsically high sequence specificity could reduce the probability of deaminating Cs other than its preferential motif. Our orthogonal R-loop assay showed that A3G-BEs exhibit a greater propensity to edit cytosines lying within the CC motif (fig. S7C). Apart from this reason, an earlier study indicated that mutations in the conserved zinc-coordinating, or catalytic, residues of either the NTD or CTD of the full-length A3G nearly abolished its capability to edit RNA and demonstrated that both domains are essential for optimal RNA editing (37). We speculate that the high fidelity of our engineered A3G-BEs could be due to the lack the NTD so that their ability to cause mutations in the transcriptome might be impaired (Fig. 5, D to F). These findings greatly mitigate the concerns about the off-target issues associated with A3G-BEs, showing great potential for their future therapeutic applications.

It is imperative that we develop genome editing tools that have the ability to produce anticipated results with the highest probability with minimum errors. Bystander editing is a major factor giving rise to imprecision, a limitation that should be improved for future clinical usage. Our engineered A3G-BEs here that recognize a specific CC motif could offer a toolkit to precisely edit a target C. These toolkits, if expanded, could allow versatile and precise editing of single nucleotides from various other distinct motifs. We envision that the continued development of novel base editing technology could facilitate the precise conversion of cytosines and treatment of human genetic diseases.

HEK293T cells (American Type Culture Collection, CRL-3216) were cultured in the T-75 flask (Corning) using high-glucose Dulbeccos modified Eagles medium (DMEM) with GlutaMAX and sodium pyruvate (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1 penicillin-streptomycin (Thermo Fisher Scientific) at 37C with 5% CO2. Upon reaching 80 to 90% confluency, cells were dissociated using TrypLE Express (Life Technologies) and passaged at a ratio of 1:3. Cells were verified mycoplasma-free using a mycoplasma detection kit (abm). ESI-017 hESCs (ESI BIO, CVCL_B854) and iPSCs (Coriell Institute, AICS-0058-067) were maintained in mTeSR1 (STEMCELL Technologies) in tissue culture dish coated with Matrigel (1:200; Corning). Dispase (STEMCELL Technologies) was used for routine passage. To perform nucleofection, a single-cell suspension was prepared using Accutase (Innovative Cell Technologies). The pluripotency of those cells was confirmed via staining of Oct4, Sox2, and Nanog. Both ESI-017 and iPSC lines were routinely tested for mycoplasma contamination and found negative.

A3G-BE2.1 was constructed by amplifying the BE4max plasmid (Addgene) outside the rAPOBEC1 region and In-Fusion cloning (Takara) with the synthesized human codon-optimized A3G fragment (Integrated DNA Technologies). Deletion of the NTD of A3G to construct A3G-BE4.4 was performed by polymerase chain reaction (PCR) amplification of A3G-BE2.1 outside the NTD region using Q5 High-Fidelity 2X Master Mix (New England Biolabs) and recloning the linearized fragment. Sets of mutations introduced into A3G-BE variants for enhancing editing efficienciesincluding A3G-BE5.1, A3G-BE5.3, A3G-BE5.4, and A3G-BE5.10were constructed using gBlocks (Integrated DNA Technologies) that contain the desired mutations and cloned with the remaining backbone of the A3G-BE4.4 plasmid. Other variants for introducing individual mutations, including Y315F, were constructed by site-directed mutagenesis using the general PCR method. Gibson assembly was used to attach P2A-GFP fragment to the C-terminal ends of nCas9, BE4max, and A3G-BE5.13 for the RNA-seq experiment that requires sorting of the transfected cells with the top 5% GFP signal. Similarly, the P2A-PuroR fragment was attached to the C-terminal ends of BE4max, A3G-BE4.4, A3G-BE5.13, and A3G-BE5.14 through Gibson assembly to select puromycin-resistant cells after nucleofection of iPSCs and hESCs. All assembled constructs were transformed into Stellar competent cells (Takara). Plasmids were extracted using either the QIAprep Spin Miniprep Kit (Qiagen) or the ZymoPURE II Plasmid Midiprep Kit (Zymo Research), and concentrations were measured using NanoDrop One (Thermo Fisher Scientific). sgRNAs were constructed by using the previous method (38). Briefly, a pair of primers for top and bottom strands encoding the 20-bp target sequence were 5 phosphorylated using T4 polynucleotide kinase (New England Biolabs) and annealed by heating the oligos to 95C and cooling down to room temperature at 5C/min1. The mixture was diluted 1:25 using water and ligated into a sgRNA expression vector using T4 DNA ligase (New England Biolabs) and BsaIHF v2 (New England Biolabs) following the manufacturers instructions.

The HEK293T stable cell line was constructed by cloning a 200-bp fragment of disease-associated gene upstream of an EF1 promoter to drive the expression of the puromycin-resistant gene in a lentiviral vector. The single-base mutation of a disease-associated gene was inserted by PCR and In-Fusion cloning (Takara). The lentiviral vector was transfected into HEK293T cells in a 24-well plate (Olympus) at 80 to 90% confluency. For each well, 288 ng of the plasmid containing the vector of interest, 72 ng of pMD2.G, and 144 ng of psPAX2 were transfected using 1.0 l of Lipofectamine 2000 and 25 l of Opti-MEM I reduced serum medium (Life Technologies). Viral supernatant was harvested 48 hours after transfection, filtered with a 0.45-m polyvinylidene difluoride filter (Millipore), and then serially diluted to add into a 24-well plate cultured with 5 104 HEK293T cells per well. After 24 hours, cells transduced with lentivirus were split into new plate wells supplemented with puromycin (3 g/ml1). Seventy-two hours after the puromycin selection, cells were harvested from the well with the fewest surviving colonies to ensure single-copy integration and were then further cultured for expansion.

Transfection and extraction of the genomic DNA were adopted from the previous method (7). Briefly, HEK293T cells were counted using Countess II FL (Thermo Fisher Scientific) and plated into a poly-d-lysinecoated 48-well plate (Corning) under 250 l of the cell culture medium with a density of 4.5 104 cells per well. After ~16 hours, cells were transfected using 1.2 l of Lipofectamine 2000 (Thermo Fisher Scientific) with 750 ng of base editor, plasmid and 250 ng of sgRNA plasmid per well following the manufacturers protocol. For orthogonal R-loop assay, 300 ng of BE, 300 ng of dSaCas9, 200 ng of SpCas9 sgRNA, and 200 ng of SaCas9 sgRNA plasmids were cotransfected per well using 1.2 l of Lipofectamine 2000. After incubation at 37C for 72 hours, the medium was aspirated and incubated under 100 l of lysis buffer [10 mM tris-HCl (pH 7.5), 0.05% SDS, and proteinase K (25 g/ml1) (Fisher BioReagents)] for 1 hour at 37C. The lysed mixture was heat inactivated at 80C for 30 min and stored at 4C until use. For preparing RNA-seq samples, 7.5 106 cells were seeded in 10-cm culture dish and transfected after 20 hours with 22.5 g of base editor P2A-GFP expression plasmid and 7.5 g of EMX1 #1targeting sgRNA plasmid mixed with 90 g of PEI MAX (Polysciences) in 1.0 ml of Opti-MEM I. The mixture was incubated for 30 min in room temperature and applied to the cells dropwise before cell sorting after 48 hours.

The HTS library was prepared using two rounds of PCR. For the first round, a 200-bp DNA fragment of the target region was amplified in a total volume of 25 l mixed with 12.5 l of the Q5 High-Fidelity 2X Master Mix, 1 l of the extracted genomic DNA, and a pair of primers (see the Supplementary Materials). Successful amplification of individual samples was checked using 1% agarose gel. For the second round, combinations of different Illumina indexes were attached at each 5 and 3 end of the first PCR products using the same total PCR volume. The PCR products were combined and column purified using a QIAquick PCR Purification kit (Qiagen) and further gel extracted to remove nonspecific amplifications. The final mixture of the library was quantified using the Qubit dsDNA HS Assay Kit (Life Technologies) and prepared for loading into a 150-cycle MiSeq reagent kit v3 (Illumina) according to the manufacturers protocol.

FASTQ files were generated by demultiplexing total sequencing reads using the MiSeq Reporter or Illuminas bcl2fastq 2.17 software. CRISPResso2 (available in GitHub; https://github.com/pinellolab/CRISPResso2) was used with the batch mode function to quantify the base editing conversion rates, indel frequencies, and product purities of the aligned reads (39). Heat maps displaying average base editing frequencies at each nucleotide position of three independent biological replicates were generated by running the CRISPResso2 analysis.

The use and care of animals followed the guidelines of the Biomedical Research Ethics Committee of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. GOTI experiments were performed according to the previous method (33). Briefly, mRNA of A3G-BE5.13 or Cre was generated by attaching the T7 promoter to the coding region through PCR amplification and using its purified PCR product as the template for in vitro transcription (IVT) using the mMESSAGE mMACHINE T7 ULTRA Kit (Invitrogen). Similarly, for sgRNA, the T7 promoter was attached, and the MEGAshortscript T7 Transcription Kit (Invitrogen) was used for IVT. mRNA and sgRNA products were purified using the MEGAclear Transcription Clean-Up Kit (Invitrogen). Fertilized embryos were obtained from C57BL/6 females (4 weeks old) mated to heterozygous Ai9 males (JAX strain 007909). A3G-BE5.13 mRNA (50 ng/l), Cre mRNA (2 ng/l), and sgRNA (50 ng/l) were mixed and injected using a FemtoJet microinjector (Eppendorf) into the cytoplasm of one blastomere of the two-cell embryo in a droplet of Hepes-CZB (Chatot-Ziomek-Bavister) medium containing cytochalasin B (5 g/ml). The embryos were incubated at 37C with 5% CO2 under KSOM (Potassium simplex optimized medium) medium for 2 hours and transferred into oviducts of ICR (Institute for Cancer Research) females at 0.5 days post coitum.

WGS and data analysis were performed according to the previous method (33). Briefly, at E14.5, prepared fetal tissues were dissociated using trypsin-EDTA (0.05%) and homogenized by passing through pipette tips multiple times. Cells were centrifuged, and the resulting pellet was resuspended in DMEM supplemented with 10% FBS before filtering through a 40-m cell strainer. tdTomato and tdTomato+ cells were isolated through FACS, and their genomic DNA were each extracted using the DNeasy Blood and Tissue Kit (Qiagen). WGS was performed at mean coverages of 50 by Illumina HiSeq X Ten. Burrows-Wheeler Aligner (version 0.7.12) was used to map qualified sequencing reads to the reference genome (mm10), and then the mapped BAM files were sorted and marked using Picard tools (version 2.3.0). SNVs were called from three algorithms, Mutect2 (version 3.5), LoFreq (version 2.1.2), and Strelka (version 2.7.1) with default parameters, separately (4042). Using the tdTomato sample from the same embryo as the reference, only variants shown to be mutated in the tdTomato+ at the same coordinate were counted within the mapped BAM file. SNVs overlapping from all the three algorithms were considered as the true variants.

Forty-eight hours after transfection, HEK293T cells cultured in 10-cm dish were washed with phosphate-buffered saline (Thermo Fisher Scientific) and dissociated by TrypLE Express. Cells were centrifuged, and the resulting pellet was resuspended in 5 ml of normal culture medium. Cells (0.5 to 0.7 106) with the top 5% GFP signal were sorted using SH800S cell sorter (Sony). Approximately a quarter of the sorted cells were collected in separate tubes for genomic DNA extraction and HTS analysis of the on-target base editing. For the remaining cells, the RNeasy Plus Mini Kit (Qiagen) was used to purify the total RNA. RNA library preparations and sequencing reactions were conducted at GENEWIZ LLC. (South Plainfield, NJ, USA). RNA samples were quantified using Qubit 2.0 fluorometer (Life Technologies), and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies). Sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following the manufacturers instructions (New England Biolabs). Briefly, mRNAs were enriched with Oligo(dT) beads and were fragmented for 15 min at 94C. First- and second-strand cDNAs were subsequently synthesized. cDNA fragments were end-repaired and adenylated at 3 ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies) and quantified by using Qubit 2.0 fluorometer and by quantitative PCR (Kapa Biosystems). The sequencing libraries were clustered on one lane of a flowcell and loaded on the Illumina HiSeq 4000 to be sequenced using a 2 150-bp paired-end configuration.

RNA-seq data analysis was carried out using the previous method (10). Qualified reads obtained from FastQC (version 0.11.3) and Trimmomatic (version 0.36) were aligned to the reference genome (Ensembl GRCh38) using STAR (version 2.5.2b) in two-pass mode with default parameters (43). Picard tools (version 2.3.0) were applied to sort and mark duplicates of the mapped BAM files. The refined BAM files were subject to split reads that spanned splice junctions, local realignment, base recalibration, and variant calling with SplitNCigarReads, IndelRealigner, BaseRecalibrator, and HaplotypeCaller tools from GATK (version 3.5), respectively (44). Clusters of more than four SNVs identified within a 35-bp window were filtered to maintain high-confidence variants, and found variants with base quality of >25, mapping quality score of >20, Fisher strand values of >30.0, qual by depth values of <2.0, and sequencing depth of >20 were counted.

For nucleofection of iPSCs and hESCs, cells were detached by using Accutase. For each reaction, 1.0 106 cells were resuspended in 82 l of P3 Primary Cell Nucleofector Solution and 18 l of supplement 1 using the P3 Primary Cell 4D-Nucleofector X Kit L (Lonza). Three micrograms of base editor P2A-PuroR expression plasmid and 1 g of sgRNA plasmid were added in the single-cell suspension and mixed well. The single-cell suspension was then transferred into a Nucleocuvette. Nucleofection was carried out in 4D-Nucleofector X Unit (Lonza) using code CB200, and cells were immediately plated on a Matrigel-coated 35-mm dish in mTeSR supplemented with 1 CloneR (STEMCELL Technologies). After 24 hours, puromycin (1.0 g/ml1) was supplemented into the medium for 1 day selection, and the surviving colonies were expanded for 10 to 14 days until extraction of the genome using the DNeasy Blood and Tissue Kit (Qiagen). The target region was PCR amplified using 30 cycles and sent for Sanger sequencing. EditR (baseeditr.com) was used to quantify the mutation peaks of Sanger chromatograms for analyzing the base conversion.

Bioinformatic analysis of pathogenic SNPs obtained from the BEable-GPS database (https://picb.ac.cn/rnomics/BEable-GPS/) was performed by finding correctable pathogenic SNPs that contain the target C located within the activity window of positions 4 to 8 of the protospacer, with NGG PAM positioned 21 to 23 (12). We then manually filtered the list on the basis of the sequence contexts containing the CC and/or CNC motif preferred by A3G-BEs. We counted precisely correctable pathogenic SNPs by manually filtering each disease on the basis of whether another base-editable bystander C was present within the activity window. For example, variant NM_012203.1(GRHPR): c.84-2A>G (protospacer; 5-TCACAGCCGCGGGGAAAGGG-3), in which the target C lies in the CC context but has a nearby bystander C lying in a CAC context potentially editable by A3G-BEs was removed from counting. The summarized list of SNPs can be found in data file S1.

Three biologically independent replicates performed on different days were used to calculate means and SD unless stated otherwise. All bar plots and figures except for heat maps were generated using Prism 8 (GraphPad). P values were calculated using Prism 8 by performing two-tailed Students t test, with a statistical significance level represented on each figure as ns (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Acknowledgments: We thank D. Zhangs NABLab (Rice University) and G. Baos laboratory (Rice University) for providing the usage of the MiSeq Sequencing System. Funding: This work was supported by the Robert A. Welch Foundation (C-1952 to X.G. and C-1559 to A.B.K.), the NIH grant (HL151545 to X.G.), the Rice University Creative Ventures Fund (to X.G. and A.B.K.), the NSF grants (CHE-1664218 to A.B.K. and PHY-1427654 to the Center for Theoretical Biological Physics), the National Natural Science Foundation of China (31922048 to E.Z.), and the Agricultural Science and Technology Innovation Program (to E.Z.). Author contributions: S.L., N.D., and X.G. designed the study. S.L. and N.D. constructed plasmids, performed FACS, and prepared the HTS library. S.L. performed transfection, HTS, and HTS data analysis. S.L. and Q.Y. maintained HEK293T cells and created disease-associated stable cell lines. Y.S., T.Y., and E.Z. performed GOTI, WGS, and software analysis of the off-target SNVs. J.L. and I.B.H. helped with RNA-seq sample preparation. S.L. and L.L. performed nucleofection and clonal expansion of iPSCs and ESI-017 hESCs. N.D. performed the analysis of pathogenic SNPs statistics. S.L. and J.Y. performed statistical analysis. S.L., N.D., Q.W., and A.B.K. provided structural insights into A3G. All authors wrote and edited the manuscript. Competing interests: S.L., N.D., and X.G. are inventors on a pending provisional patent application submitted by the William Marsh Rice University related to this work. The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. FASTQ files containing HTS reads have been deposited in the National Center for Biotechnology Information, NIH Sequencing Read Archive and are available with accession number PRJNA623461. Additional data related to this paper may be requested from the authors.

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Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects - Science...

Biotech Firms to Track and Monitor Patients Injected with Stem Cells or Gene Therapy Treatments – The Korea Bizwire

The new business entities should stop the supply and sale of human cells once they are verified to be harmful to human bodies. (Yonhap)

SEOUL, July 16 (Korea Bizwire) New government regulations will require South Korean biotech companies to track and monitor patients who are injected with stem cells or gene therapy treatments.

According to the Ministry of Food and Drug Safety on Wednesday, the Advanced Regenerative Bio Act, enacted to strengthen the safety management of advanced regenerative drugs as well as to facilitate their commercialization through rapid clinical studies, will take effect on Aug. 28.

Under this law, a new type of business called human cell management will be born, which will clear the way for professional management of human and animal cells and tissues in addition to animal organs.

During the process of operation, the new business entities should stop the supply and sale of human cells once they are verified to be harmful to human bodies.

In such cases, all of the human cells concerned should be withdrawn and disposed of.

The law will make it mandatory for biotech companies to register stem cell and gene therapy treatment injection data with the Korea Institute of Drug Safety & Risk Management.

They will also be obliged to report unusual cases and perform long-term tracking and monitoring of the patients.

Advanced regenerative medicine refers to the medical technology of curing, replacing or regenerating damaged cells or issues using human cells.

Kevin Lee (kevinlee@koreabizwire.com)

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Biotech Firms to Track and Monitor Patients Injected with Stem Cells or Gene Therapy Treatments - The Korea Bizwire

Platelet Rich Plasma and Stem Cell Alopecia Treatment Market Segmentation, Analysis by Recent Trends, Development by Regions to 2025 – 3rd Watch News

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15-month-old son of former Red Wing Kyle Quincey battling brain cancer – MLive.com

Kyle and Rachel Quinceys lives changed forever in late March when their 15-month-old son was diagnosed with brain cancer.

Two surgeries later, following many days of chemotherapy, Alx will continue radiation treatment in a Philadelphia hospital for another month while his parents wait for positive results.

They were encouraged by the second operation in June, in which 99 percent of the tumor was removed. But the fight is far from over.

It was really hard at the start, but very quickly it becomes your new normal, Quincey said. Really only one option, just being positive and grinding it out day by day.

Quincey played 10-plus years in the NHL, including five full seasons and parts of three others as a defenseman for the Detroit Red Wings. He retired from hockey in 2019.

Alx Quincey became ill in March, requiring multiple trips to the hospital. They suspected it was an ear infection or just a cold or the flu and administered antibiotics. On the third visit, on March 30, an emergency room nurse suggested a CAT scan.

Thats the first time we saw the mass, Quincey said in an interview with MLive. Things werent going well for about a month. He started showing symptoms and it took us about three weeks to get that CAT scan. You take the normal precautions first.

Axl was diagnosed with ependymoma. It is a type of tumor that can form in the brain or spinal cord. It begins in the ependymal cells in the brain and spinal cord that line the passageways where the fluid that nourishes your brain flows, according to the Mayo Clinics web site. Ependymoma can occur at any age, but most often occurs in young children.

Alx Quincey, the 14-month-old son of former Red Wings defenseman Kyle Quincey, has had two surgeries to remove a brain tumor. (courtesy of Kyle Quincey)

The initial surgery April 2 in Denver removed about one-third of the tumor. That was followed by two months of chemotherapy.

The first (procedure) was kind of immediate, to relieve all the symptoms, the pressure he was in a lot of discomfort for three weeks before we could figure out what it was, Quincey said. It was blocking all of his spinal fluid from draining.

Took a while to recover from that. He started chemo when he became well enough to do so and the whole plan was to get the second surgery. We found out we needed every cell of this tumor gone or it will just come back, so it was very important to find a surgeon we felt comfortable with, being aggressive enough to get every single cell out and also being safe enough to not damage the brain stem and all the other nerves.

The Quinceys have another son, 2 year-old Stone. Family life has been significantly disrupted.

Because of the COVID-19 restrictions, it was a long time before the two boys were together or even his mom got to come home because she was living in the hospital for so long, Quincey said. It was very hard, and then I wasnt allowed in the hospital at the same time because only one parent is allowed at a time.

After much research, the Quinceys decided on Childrens Hospital of Philadelphia and Dr. Jay Storm for the second surgery, which took 22 hours.

Hes a miracle worker, Quincy said. He scraped the tumor off the nerves, making sure he didnt damage any of the nerves of the brain stem.

Alx just completed day seven of 30 days of radiation therapy, which he undergoes Monday through Friday.

Hes under anesthesia every morning, Quincey said. It takes three hours (but) the actual session is probably seconds. It takes a long time to get it in the perfect spot because theyre actually hitting millimeters from the brain stem. Its very precise. Most of the time its just getting him sedated and comfortable and in the right spot.

Alx Quincey, the 14-month-old son of former Red Wings defenseman Kyle Quincey, is undergoing radiation treatments for a brain tumor. (courtesy of Kyle Quincey)

The next step, Quincey said, is pretty much just cross your fingers.

The surgery was a success, Quincey said. Hes confident in saying he got 99 percent out. Theres a little mark on the MRI theyre not 100 percent sure about; the radiation is focusing on that spot now.

Quincey is grateful to Flyers goaltender Brian Elliott for allowing them to stay at his home while he was away. If all goes well, the family will return home to Denver late next month. Then they will wait a couple of weeks before taking Alx for another MRI.

At that MRI were really hoping we get some good news and they say cancer-free, Quincey said. If not, we just take it from there and we keep the fight going. Its kind of one step at a time. When we got the diagnosis there were a lot of hoops to jump through and weve jumped through a few of them, not really thinking about what the next step is because this is kind of the last step they gave us in the first protocol.

Whatever news they receive next month, Alx will continue to be closely monitored for some time.

He has an MRI every three months until hes 5 and then every six months until hes 26, Quincey said. Hes not out of the woods, really ever, because this is such an aggressive tumor. But we feel we got a really good team and a good plan and were just executing it right now.

The Red Wings drafted Quincey in the fourth round in 2003 out of London (OHL). He played three seasons for AHL Grand Rapids, where he met Rachel, who is from Luther, Mich., in 2006. They were married 10 years later in Detroit, following Quinceys final season with the Red Wings.

After playing for three teams over the next two years (New Jersey, Columbus and Minnesota), Quincey spent the 2018-19 season in Finland with HIFK Helsinki before retiring at age 33.

My body just wasnt up for it, Quincey said, adding, I couldnt imagine being gone for this.

The Quinceys are in the process of starting a foundation to benefit childhood cancer. Haley Fowler, the wife of friend Paul Stastny, who plays for Vegas, contacted a company that produces t-shirts and crew necks that say, Team Ax.

We saw such a huge response, me and Rachel decided to start Team Ax Foundation, with proceeds to help childhood cancer, Quincey said.

We wanted to use our platform and all of our contacts to do good. We kind of found our calling, or the calling found us. Its just coming into fruition now.

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Stem Cell Antibody Market Dynamics Analysis to Grow at CAGR with Major Companies and Forecast 2021 Bulletin Line – Bulletin Line

Global Stem Cell Antibody market study presents an in-depth scenario which is segmented according to manufacturers, product type, applications, and regions. This segmentation will provide deep-dive analysis of the Stem Cell Antibody industry for identifying the growth opportunities, development trends and factors limiting the growth of the market. This report offers forecast market information based on past and present Stem Cell Antibody industry situations and growth aspects. All the key regions covered in Stem Cell Antibody report are North America, Europe, Asia-Pacific, South America, Middle East and Africa. The Stem Cell Antibody market share and market outlook of each region from 2020-2027 are presented in this report. A deep study of Stem Cell Antibody market dynamics will help the market aspirants in identifying the business opportunities which will lead to accumulation of revenue. This segment can effectively determine the Stem Cell Antibody risk and key market driving forces.

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Initially, the report presents the Stem Cell Antibody market overview covering product description, market analysis, market dynamics, opportunities and market share. Secondly, global report conducts a qualitative analysis to present the key manufacturers profile, Stem Cell Antibody market share, market size, sales volume, gross margin analysis.

The Stem Cell Antibody report is segmented to provide a clear and precise view of the global Stem Cell Antibody market statistics and market estimates. Stem Cell Antibody report Data represented in the form of graphs, charts, and figures will show the Stem Cell Antibody growth rate, volume, target consumer analysis. This report presents the crucial data to all Stem Cell Antibody industry aspirants which will facilitate useful business decisions.

Segment by Type, the Stem Cell Antibody market is segmented into Primary Antibodies Secondary Antibodies

Segment by Application, the Stem Cell Antibody market is segmented into Proteomics Drug Development Genomics

Regional and Country-level Analysis The Stem Cell Antibody market is analysed and market size information is provided by regions (countries). The key regions covered in the Stem Cell Antibody market report are North America, Europe, Asia Pacific, Latin America, Middle East and Africa. It also covers key regions (countries), viz, U.S., Canada, Germany, France, U.K., Italy, Russia, China, Japan, South Korea, India, Australia, Taiwan, Indonesia, Thailand, Malaysia, Philippines, Vietnam, Mexico, Brazil, Turkey, Saudi Arabia, U.A.E, etc. The report includes country-wise and region-wise market size for the period 2015-2026. It also includes market size and forecast by Type, and by Application segment in terms of sales and revenue for the period 2015-2026. Competitive Landscape and Stem Cell Antibody Market Share Analysis Stem Cell Antibody market competitive landscape provides details and data information by players. The report offers comprehensive analysis and accurate statistics on revenue by the player for the period 2015-2020. It also offers detailed analysis supported by reliable statistics on revenue (global and regional level) by players for the period 2015-2020. Details included are company description, major business, company total revenue and the sales, revenue generated in Stem Cell Antibody business, the date to enter into the Stem Cell Antibody market, Stem Cell Antibody product introduction, recent developments, etc.

The major vendors covered: Thermo Fisher Scientific, Inc. (U.S.) Merck Group (Germany), Abcam plc (U.K.) Becton, Dickinson and Company (U.S.) Bio-Rad Laboratories, Inc. (U.S.) Cell Signaling Technology, Inc. (U.S.) Agilent Technologies, Inc. (U.S.) F. Hoffmann-La Roche Ltd (Switzerland) Danaher Corporation (U.S.) GenScript (U.S.), PerkinElmer, Inc. (U.S.) Lonza (Switzerland), and BioLegend, Inc. (U.S.)

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The Stem Cell Antibody report cover following data points:

Part 1: This part enlists the global Stem Cell Antibody market overview, covering the basic market introduction, market analysis by type, applications, and regions. The major Stem Cell Antibody producing regions include North America, Europe, Asia-Pacific, Middle-East, and Africa. Stem Cell Antibody industry states and outlook (2020-2027) is presented in this part. In addition, Stem Cell Antibody market dynamics stating the opportunities, market risk, and key driving forces are studied.

Part 2: This part covers Stem Cell Antibody manufacturers profile based on their business overview, product type, and application. Also, the sales volume, Stem Cell Antibody product price, gross margin analysis, and Stem Cell Antibody market share of each player is profiled in this report.

Part 3 and Part 4: This part presents the Stem Cell Antibody competition based on sales, revenue, and market share of each manufacturer. Part 4 covers the Stem Cell Antibody market scenario based on regions. Region-wise Stem Cell Antibody sales and growth (2015-2019) is studied in this report.

Part 5 and Part 6: These two sections cover the North America and Europes Stem Cell Antibody industry by countries. Under this the Stem Cell Antibody revenue, market share of the countries like USA, Canada, and Mexico is provided. Under Europe Stem Cell Antibody report includes, the countries like Germany, UK, France, Russia, Italy, Russia and their sales and growth is covered.

Part 7, Part 8 and Part 9: These 3 sections covers Stem Cell Antibody sales revenue and growth for the regions like Asia-Pacific, South America, Middle East & Africa. Under these regions Stem Cell Antibody report covered, the countries like China, Japan, Korea, India, Brazil, Columbia, Argentina, Egypt, Saudi Arabia, Nigeria and South Africa. The sales and growth in these regions are presented in this Stem Cell Antibody industry report.

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Part 10 and Part 11: This part depicts the Stem Cell Antibody market share, revenue, sales by product type and application. The Stem Cell Antibody sales growth seen during 2012-2020 is covered in this report.

Part 12 and Part 13: This part provides forecast information related to Stem Cell Antibody market (2020-2027) for each region. The sales channels including direct and indirect Stem Cell Antibody marketing, traders, distributors, and future trends are presented in this report.

Part 14 and Part 15: These parts present Stem Cell Antibody market key research findings and conclusion, research methodology, and data sources are covered.

Thus, Global Stem Cell Antibody report is a complete blend covering all the vital market aspects.

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Stem Cell Antibody Market Dynamics Analysis to Grow at CAGR with Major Companies and Forecast 2021 Bulletin Line - Bulletin Line

Cell Therapy Processing Market is Booming Worldwide to Show Significant Growth by 2026 Cell Therapies Pty Ltd,Invitrx Inc.,Lonza Ltd,Merck & Co.,…

Cell therapy is the administration of living cells to replace a missing cell type or to offer a continuous source of a necessary factor to achieve a truly meaningful therapeutic outcome. There are different forms of cell therapy, ranging from transplantation of cells derived from an individual patient or from another donor. The manufacturing process of cell therapy requires the use of different products such as cell lines and instruments. These cell therapies are used for the treatment of various diseases such as cardiovascular disease and neurological disorders.

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Key Players:

Cell Therapies Pty Ltd,Invitrx Inc.,Lonza Ltd,Merck & Co., Inc. (FloDesign Sonics),NantWorks, LLC,Neurogeneration, Inc.,Novartis AG,Plasticell Ltd.,Regeneus Ltd,StemGenex, Inc.

Increase in the incidence of cardiovascular diseases, rise in the demand for chimeric antigen receptor (CAR) T cell therapy, increase in the R&D for the advancement in the research associated with cell therapy, increase in the potential of cell therapies in the treatment of diseases associated with lungs using stem cell therapies, and rise in understanding of the role of stem cells in inducing development of functional lung cells from both embryonic stem cells (ESCs) & induced pluripotent stem (iPS) cells are the key factors that fuel the growth of the cell therapy processing market.

Moreover, increase in a number of clinical studies relating to the development of cell therapy processing, rise in adoption of regenerative drug, introduction of novel technologies for cell therapy processing, increase in government investments for cell-based research, increase in number of GMP-certified production facilities, large number of oncology-oriented cell-based therapy clinical trials, and rise in the development of allogeneic cell therapy are other factors that augment the growth of the market. However, high-costs associated with the cell therapies, and bottlenecks experienced by manufacturers during commercialization of cell therapies are expected to hinder the growth of the market.

The cell therapy processing market is segmented into offering type, application, and region. By type, the market is categorized into products, services, and software. The application covered in the segment include cardiovascular devices, bone repair, neurological disorders, skeletal muscle repair, cancer, and others. On the basis of region, the market is analyzed across North America (U.S., Canada, and Mexico), Europe (Germany, France, UK, Italy, Spain, and rest of Europe), Asia-Pacific (Japan, China, India, and rest of Asia-Pacific), and LAMEA (Latin America, Middle East, and Africa).

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KEY MARKET SEGMENTS

By Offering Type Products Services Software

By Application Cardiovascular Devices Bone Repair Neurological Disorders Skeletal Muscle Repair Cancer Others

By Region

North America o U.S. o Canada o Mexico Europe o Germany o France o UK

Key question and answered in the report include:

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Cell Therapy Processing Market is Booming Worldwide to Show Significant Growth by 2026 Cell Therapies Pty Ltd,Invitrx Inc.,Lonza Ltd,Merck & Co.,...