Category Archives: Gene Medicine

A new technique called sci-Space, combined with data from other technologies, could lead to four-dimensional atlases of gene expression across diverse cells during embryonic development of mammals.

Such atlases would map how the gene transcripts in individual cells reflect the passage of time, cell lineages, cell migration, and location on the developing embryo. They would also help illuminate the spatial regulation of gene expression.

Mammalian embryonic development is a remarkable phenomenon: a fertilized egg divides repeatedly and turns, in a matter of weeks or months, into a complex organism capable of a myriad of physiological processes and composed of a variety of cells, tissues, organs, anatomical structures.

A better understanding of how mammals form before birth -- particularly the prenatal spatial patterns of gene expression at a single-cell level during embryonic development -- could advance biomedical and veterinary research on a variety of conditions. These range from inherited disorders to congenital malformations and developmental delays. Understanding how organs originate might also assist future regenerative medicine efforts.

An international team led by scientists at UW Medicine, Howard Hughes Medical Institute and the Brotman Baty Institute for Precision Medicine in Seattle demonstrated the proof-of-concept of their sci-Space technique in mouse embryos.

Their results are published in the July 2 edition of Science. The lead authors are Sanjay R. Srivatsan of the Department of Genome Sciences at the University of Washington School of Medicine, and Mary C. Regier of the UW Department of Bioengineering.

The senior authors are Jay Shendure, UW Medicine professor of genome sciences, and director of the Brotman Baty Institute, and an investigator at the Allan Discovery Center for Cell Lineage Tracing; Kelly R. Stevens, UW assistant professor of bioengineering; and Cole Trapnell, associate professor of genome sciences. Regier and Stevens are also investigators at the UW Medicine Institute for Stem Cell and Regenerative Medicine Research.

The researchers observed the orchestration of genes in 120,000 cell nuclei. All the body's somatic cells contain the same DNA code. The researchers captured information on which genes were turned on or off in these nuclei as mouse embryos took shape. The scientists also investigated how cells' locations in an embryo affected which genes were activated during development.

This technique builds on previous work in which these scientists and other groups developed ways of conducting whole-organism profiling of gene expression and DNA-code accessibility, in thousands of single cells, during embryonic development. They did so to track the emergence and trajectory of various cell types.

How cells are organized spatially - what physical positions they take as an embryo forms - is critical to normal development. Misplacements, disruptions, or cells not showing at the right time in the right spot can cause serious problems or even prenatal death.

However, gaining knowledge on spatial patterns of gene expression has been technically difficult. It has been unwieldy to assay gene transcripts of individual cells over wide swaths of the embryo. This limited the scientific understanding of how spatial organization influences gene expression and, consequently, why which cell types form where, or how neighboring groups of cells influence each other's future roles.

The scientists on the present study had earlier developed a method to label cell nuclei, a technique they called sci-Plex. They then went on to index single-cell RNA sequencing, with a method called sci-RNA-sequencing.

Now, with sci-Space, by analyzing spatial coordinates and cell gene transcripts the scientists identified thousands of genes whose expression was anatomically patterned. For example, certain genetic profiles emerged in neurons in the brain and spinal cord and others in cardiac muscle cells in the heart.

The scientists also used spatial and gene profile information to annotate subtypes of cells. For example, while both blood vessel cells and heart muscle might both express the gene for a particular growth factor, only the heart muscle cells produced certain growth factor receptors.

The researchers also observed that cell types varied greatly in the extent of their spatial patterning of gene expression. For example, connective tissue progenitor cells showed a relatively large proportion of spatially restricted gene expression. This observation suggests that subtypes of these cells behave in a position-dependent manner throughout the body.

To measure the power of spatial position on a cell type's gene transcript profile, the researchers also calculated the physical distance between cells and the angular distance of their gene expression profiles.

"For many cell types, as the physical distance between cells increased, so did the angular distance between their transcriptomes," the researchers noted in their paper. However, they added that this trend varied considerably. It was most pronounced in certain brain and spinal cord cells.

The genetic transcript profiles of some other cell types were highly influenced by their position in the developing embryo. Among these are certain cartilage cells, which become part of the scaffolding for bones of the head and face.

The researchers also studied gene expression dynamics that took place as part of brain cell differentiation and migration during mouse embryonic development. The researchers examined how various brain cell trajectories were anatomically distributed. The researchers did so by using the Allen Institute's Anatomical Reference Brain Atlas as a guide.

"Cells from each trajectory overwhelmingly occupied distinct brain regions," the researchers noted. They also observed gradients of developmental maturity in different regions of the brain. These gradients revealed both known and new patterns of migration.

In the future, the researchers hope sci-Space will be further applied to serial sections that span the entire mouse embryo and that cover many points of time.

Reference:Srivatsan SR, Regier MC, Barkan E, et al. Embryo-scale, single-cell spatial transcriptomics. Science. 2021;373(6550):111-117. doi:10.1126/science.abb9536

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

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Cellular Model of a Developing Mouse Is Built With Spatial Resolution - Technology Networks

As the cell and gene revolution heats up, contract manufacturer AGC Biologics is getting ahead of the curve with plans for its second commercial plant in Colorado.

Angling to bolster cell and gene production, AGC has clinched a deal for a commercial Novartis Gene Therapies factory in Longmont, Colorado. Located just 16 miles from AGCs 20,000-liter mammalian facility in Boulder, the new plant is expected to add significant additional capacity, AGC said in a release.

The move comes shortly after AGC charted an expansion at its cell and gene site in Milan, Italy, which it snared last July in its buyout of Italian CTG biotech Molecular Medicine (MolMed).

AGC hasnt divulged the Novartis plants price. The company didnt say how big it expects the Longmont workforce to be, but it will [aim] to hire a significant percentage of Novartis staff there.

RELATED:Novavax enlists AGC Biologics to manufacture adjuvant for COVID-19 shot

The 622,000-square-foot factory comes equipped with offices and production space across six buildings. It sits on a 229-acre campus located 40 miles north of Denver, AGC said.

Last June,AGC got its hands on its Boulder plant through similar means, picking up the commercial facility from AstraZeneca. That facility came equipped with two 20,000-liter stainless steel bioreactors, plusspace to add four more in the future. That same month, AGC tied up with Novavax to scale up and produce the Matrix-M adjuvant for its late-stage COVID-19 vaccine candidate,NVX-CoV2373.

Meanwhile, AGC has invested heavily in cell and gene therapiessince acquiring MolMed in 2020. With the addition of two MolMed commercial plants in Italy, AGC became one of the very few CDMOs to include both plasmid production and end-to-end cell and gene therapy services in its manufacturing repertoire, the company noted last year.

RELATED:AGC plots $194.5M, capacity-doubling upgrade to Copenhagen biologics site

At the time, AGC specifically highlighted MolMeds manufacturing know-how in genetically modified cells and viral vectors, or the engineered viruses used to deliver the cutting-edge medicines. That component, which is also used in AstraZeneca and Johnson & Johnsons recombinant COVID-19 vaccines, is already in shortage, with the bottleneck expected to tighten even more unless regulators, biopharmas and contractors move fast to address production shortfalls, GlobalDatasaid in a recent report.

And in March, AGC blueprinted an upgrade to its factory in Milan, sketching a capacity boost and the introduction of viral vector suspension capabilities. The expanded facilities should startfull operations in 2022, AGC has said.

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AGC Biologics expands further in Colorado with purchase of Novartis Gene Therapies plant - FiercePharma

SAN RAFAEL, Calif., July 2, 2021 /PRNewswire/ --BioMarin Pharmaceutical Inc.(NASDAQ: BMRN) today announced three oral presentations and nine poster presentations related to valoctocogene roxaparvovec, an investigational gene therapy for the treatment of adults with severe hemophilia A, at the International Society on Thrombosis and Haemostasis (ISTH) 2021 Virtual Congress being held July 17-21, 2021. Notably, these presentations will include highlights from the Phase 3 GENEr8-1 trial, the largest gene therapy trial in Hemophilia A, and five years of clinical follow-up from the Phase 1/2 study, both of which continue to demonstrate prolonged hemostatic efficacy without the need for other treatment for hemophilia A.

"We are proud of the consistent and dramatic bleed control results to date, based on both long-term extension studies of at least five years, and the largest and most definitive gene therapy study in Hemophilia A. We look forward to the scientific presentations of the growing body of evidence for valoctocogene roxaparvovec and ensuing discussions at this important meeting," saidHank Fuchs, M.D., President, Worldwide Research and Development at BioMarin.

BioMarin's presentations at ISTH include:

Platform Presentations

Efficacy and Safety of Valoctocogene Roxaparvovec Adeno-associated Virus Gene Transfer for Severe Hemophilia A: Results from the Phase 3 GENEr8-1 TrialProfessor Margareth C. Ozelo, Hematology and Transfusion Medicine,Internal Medicine Department - School of Medical Sciences of UNICAMP,University of Campinas-UNICAMPMonday, July 19, 2021, 10-11 AM EDT

Hemostatic Response is Maintained for up to 5 Years Following Treatment with Valoctocogene Roxaparvovec, an AAV5-hFVIII-SQ Gene Therapy for Severe Hemophilia AProfessor Michael Laffan, faculty of Medicine, Department of Immunology and Inflammation at Imperial College London, Director of the Hammersmith Hospital Haemophilia CentreWednesday, July 21, 2021, 10-11 AM EDT

Investigation of Early Outcomes Following Adeno-associated Viral Gene Therapy in a Canine Hemophilia ModelDr. Paul Batty, Department of Pathology and Molecular Medicine, Queen's UniversityWednesday, July 21, 2021, 1-2 PM EDT

Poster Presentations

Poster #

Title and Authors

LPB0022

Global seroprevalence of pre-existing immunity against various AAV serotypes in people with haemophilia A

Klamroth R, Hayes G, Andreeva T, Suzuki T, Hardesty B, Shima M, Pollock T, Slev P, Oldenburg J, Ozelo M, Castet S, Mahlangu J, Peyvandi F, Kazmi R, Leavitt A, Callaghan M, Pan-Petesch B, Quon D, Li M, Wong WY.

PB0663

A savvy approach in clinical trial recruitment for the SAAVY (Seroprevalence of AAV AntibodY) study in the era of COVID-19: Designing for a prospective, observational study in the United States during a global pandemic

Valentino L, Vaghela M, Lauw M, Dela Cerda G, Jones M, Hinds D, Newman V, Leal-Padinas F, Rotellini D, Schafer K, Pipe S.

PB0488

Exploring the level of congruence between patient- and physician-reported anxiety and depression in persons with haemophilia A

Burke T, Shaikh A, Pedra G, Hawes C, Camp C, O'Hara J.

PB0468

Examination and validation of a patient-centric joint metric: "PROBLEM JOINT"; empirical evidence from the CHESS Paediatrics dataset

Burke T, Rodriguez-Santana I, O'Hara J, Chowdary P, Curtis R, Khair K, McLlaughlin P, Noone D, O'Mahoney B, Pasi J, Skinner M.

PB0452

Real-world clinical and patient-centric outcomes in people with haemophilia A in France: Combined findings from the CHESS and CHESS II studies

Shaikh A, Burke T, Hawes C, Duport G, O'Hara J, Camp C.

PB0487

Real-world clinical and patient-centric outcomes in people with haemophilia A in Germany: Combined findings from the CHESS and CHESS II studies

Shaikh A, Burke T, Hawes C,Becker T, Brandt S, O'Hara J, Camp C.

PB0464

Real-world clinical and patient-centric outcomes in people with haemophilia A in Italy: Combined findings from the CHESS and CHESS II studies

Shaikh A, Burke T, Hawes C, Lupi A, O'Hara J, Camp C.

PB0456

Real-world clinical and patient-centric outcomes in people with haemophilia A in Spain: Combined findings from the CHESS and CHESS II studies

Shaikh A, Burke T, Hawes C, O'Hara J, Camp C.

PB0479

Real-world clinical and patient-centric outcomes in people with haemophilia A in the United Kingdom: Combined findings from the CHESS and CHESS II studies

Shaikh A, Burke T, Hawes C, McKeown W, Morgan D, O'Hara J, Camp C.

Founded in 1969, the ISTH is the leading worldwide not-for-profit organization dedicated to advancing the understanding, prevention, diagnosis and treatment of thrombotic and bleeding disorders. The ISTH is an international professional membership organization with more than 7,700 clinicians, researchers and educators working together to improve the lives of patients in more than 110 countries around the world. Among its highly regarded activities and initiatives are education and standardization programs, research activities, meetings and congresses, peer-reviewed publications, expert committees and World Thrombosis Day on 13 October.

Regulatory Status

BioMarin resubmitted a Marketing Authorization Application (MAA) to the European Medicines Agency (EMA) on June 25, 2021. In May 2021, the EMA granted the Company's request for accelerated assessment. Accelerated assessment potentially reduces the time frame for the EMA Committee for Medicinal Products for Human Use (CHMP) and Committee for Advanced Therapies (CAT) to review a MAA for an Advanced Therapy Medicinal Product (ATMP). A CHMP opinion is anticipated in the first half of 2022.

The MAA submission includes safety and efficacy data from the 134 subjects enrolled in the Phase 3 GENEr8-1 study, all of whom have been followed for at least one year after treatment with valoctocogene roxaparvovec, as well as four and three years of follow-up from the 6e13 vg/kg and 4e13 vg/kg dose cohorts, respectively, in the ongoing Phase 1/2 dose escalation study.

In the United States, BioMarin intends to submit two-year follow-up safety and efficacy data on all study participants from the Phase 3 GENEr8-1 study to support the benefit/risk assessment of valoctocogene roxaparvovec, as previously requested by the Food and Drug Administration (FDA). BioMarin is targeting a Biologics License Application (BLA) resubmission in the second quarter of 2022, assuming favorable study results, followed by an expected six-month review by the FDA.

The FDA granted Regenerative Medicine Advanced Therapy (RMAT) designation to valoctocogene roxaparvovec inMarch 2021. RMAT is an expedited program intended to facilitate development and review of regenerative medicine therapies, such as valoctocogene roxaparvovec, that are intended to address an unmet medical need in patients with serious conditions. The RMAT designation is complementary to Breakthrough Therapy Designation, which the Company received in 2017.

In addition to the RMAT Designation and Breakthrough Therapy Designation, BioMarin's valoctocogene roxaparvovec also has received orphan drug designation from the FDA and EMA for the treatment of severe hemophilia A.The Orphan Drug Designation program is intended to advance the evaluation and development of products that demonstrate promise for the diagnosis and/or treatment of rare diseases or conditions.

Robust Clinical Program

BioMarin has multiple clinical studies underway in its comprehensive gene therapy program for the treatment of hemophilia A. In addition to the global Phase 3 study GENEr8-1 and the ongoing Phase 1/2 dose escalation study, the Company is actively enrolling participants in a Phase 3b, single arm, open-label study to evaluate the efficacy and safety of valoctocogene roxaparvovec at a dose of 6e13 vg/kg with prophylactic corticosteroids in people with hemophilia A. The Company is also running a Phase 1/2 Study with the 6e13 vg/kg dose of valoctocogene roxaparvovec in people with hemophilia A with pre-existing AAV5 antibodies, as well as another Phase 1/2 Study with the 6e13 vg/kg dose of valoctocogene roxaparvovec in people with hemophilia A with active or prior FVIII inhibitors.

About Hemophilia A

People living with hemophilia A lack sufficient functioning Factor VIII protein to help their blood clot and are at risk for painful and/or potentially life-threatening bleeds from even modest injuries. Additionally, people with the most severe form of hemophilia A (FVIII levels <1%) often experience painful, spontaneous bleeds into their muscles or joints. Individuals with the most severe form of hemophilia A make up approximately 45 to 50 percent of the hemophilia A population. People with hemophilia A with moderate (FVIII 1-5%) or mild (FVIII 5-40%) disease show a much-reduced propensity to bleed. The standard of care for adults with severe hemophilia A is a prophylactic regimen of replacement Factor VIII infusions administered intravenously up to two to three times per week or 100 to 150 infusions per year. Despite these regimens, many people continue to experience breakthrough bleeds, resulting in progressive and debilitating joint damage, which can have a major impact on their quality of life.

Hemophilia A, also called Factor VIII deficiency or classic hemophilia, is an X-linked genetic disorder caused by missing or defective Factor VIII, a clotting protein. Although it is passed down from parents to children, about 1/3 of cases are caused by a spontaneous mutation, a new mutation that was not inherited. Approximately 1 in 10,000 people have Hemophilia A.

About BioMarin

BioMarin is a global biotechnology company that develops and commercializes innovative therapies for patients with serious and life-threatening rare and ultra-rare genetic diseases. The company's portfolio consists of six commercialized products and multiple clinical and pre-clinical product candidates. For additional information, please visitwww.biomarin.com. Information on BioMarin's website is not incorporated by reference into this press release.

Forward Looking Statement

This press release contains forward-looking statements about the business prospects of BioMarin Pharmaceutical Inc., including without limitation, statements about: (i) the development of BioMarin's valoctocogene roxaparvovec program generally, (ii) the impact of valoctocogene roxaparvovec gene therapy for treating patients with severe hemophilia A, (iii) the anticipated timing of a CHMP opinion in the first half of 2022, (iv) our plans in the U.S. to submit two-year follow-up safety and efficacy data on all study participants from the GENEr8-1 study in response to FDA's request for these data to support their benefit-risk assessment of valoctocogene roxaparvovec, (v) our target Biologics License Application (BLA) submission date in the second quarter of 2022, assuming favorable study results, followed by an expected six-month review procedure by the FDA, and (vi) the potential approval and commercialization of valoctocogene roxaparvovec for the treatment of severe hemophilia A, including timing of such approval decisions.

These forward-looking statements are predictions and involve risks and uncertainties such that actual results may differ materially from these statements. These risks and uncertainties include, among others: results and timing of current and planned preclinical studies and clinical trials of valoctocogene roxaparvovec, including final analysis of the above interim data; any potential adverse events observed in the continuing monitoring of the patients in the Phase 1/2 trial; the content and timing of decisions by the FDA, the European Commission and other regulatory authorities, including the potential impact of the COVID-19 pandemic on the regulatory authorities' abilities to issue such decisions and the timing of such decisions; the content and timing of decisions by local and central ethics committees regarding the clinical trials; BioMarin's ability to successfully manufacture valoctocogene roxaparvovec; and those other risks detailed from time to time under the caption "Risk Factors" and elsewhere in BioMarin's Securities and Exchange Commission (SEC) filings, including BioMarin's Quarterly Report on Form 10-Q for the quarter endedMarch 31, 2021, and future filings and reports by BioMarin. BioMarin undertakes no duty or obligation to update any forward-looking statements contained in this press release as a result of new information, future events or changes in its expectations.

BioMarin is a registered trademark of BioMarin Pharmaceutical Inc.

Contacts:

Investors

Media

Traci McCarty

Debra Charlesworth

BioMarin Pharmaceutical Inc.

BioMarin Pharmaceutical Inc.

(415) 455-7558

(415) 455-7451

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BioMarin Announces 12 Presentations at the International Society on Thrombosis and Haemostasis (ISTH) 2021 Virtual Congress - BioSpace

Collaboration combines Apellis expertise in complement, a complex biological system, with Beams proprietary base editing platform

Companies will collaborate on six research programs directed to tissues modulated by the complement system, including the eye, liver, and brain

WALTHAM, Mass. and CAMBRIDGE, Mass., June 30, 2021 (GLOBE NEWSWIRE) -- Apellis Pharmaceuticals, Inc. (Nasdaq: APLS) and Beam Therapeutics Inc. (Nasdaq: BEAM) today announced an exclusive five-year research collaboration focused on the use of Beams proprietary base editing technology to discover new treatments for complement-driven diseases. The companies will collaborate on six research programs focused on C3 and other complement targets in the eye, liver, and brain.

Beam has pioneered base editing, which holds significant promise as a best-in-class technology for precision gene editing. This collaboration builds on our deep scientific expertise in complement and, together with our growing pipeline, positions Apellis for long-term leadership in the complement field, said Cedric Francois, M.D., Ph.D., co-founder and chief executive officer, Apellis. Apellis and Beam share a vision for advancing transformative medicines for patients, which is critically important as we embark on a highly innovative effort to modulate complement and discover new treatments across a wide range of debilitating diseases.

Base editing represents a potential new class of precision genetic medicine that uses a chemical reaction designed to create precise, predictable, and efficient single base changes at targeted genomic sequences without making double-stranded breaks in the DNA. Editing key elements of the complement pathway in target organs has the potential to alter the complement cascade and durably address diseases driven by abnormal complement activity.

Apellis has established itself as a leader in complement with the advancement of compelling targeted C3 therapies, said John Evans, chief executive officer, Beam. This collaboration allows us to combine our proprietary technologies and capabilities in base editing with Apellis expertise in targeting the complement pathway to develop new medicines for diseases driven by complement biology. This also represents an important strategic initiative to explore opportunities that expand the application of base editing to address more biologically complex diseases for patients in need of new treatment options.

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Under the terms of the collaboration agreement, Beam will apply its base editing technology and conduct preclinical research on up to six base editing programs that target specific genes within the complement system in various organs, including the eye, liver, and brain. Apellis will have exclusive rights to license each of the six programs and will assume responsibility for subsequent development. Beam may elect to enter a 50-50 U.S. co-development and co-commercialization agreement with Apellis with respect to one program licensed under the collaboration.

As part of the collaboration, Beam will receive a total of $75 million in upfront and near-term milestones from Apellis $50 million upon signing and an additional $25 million payment on the one-year anniversary of the contract execution date. After exercise of the opt-in license rights for each of the up to six programs, Beam will be eligible to receive development, regulatory, and sales milestones from Apellis, as well as royalty payments on sales. The collaboration has an initial term of five years and may be extended up to two years on a per year and program-by-program basis.

About Apellis Apellis Pharmaceuticals, Inc. is a global biopharmaceutical company that is committed to leveraging courageous science, creativity, and compassion to deliver life-changing therapies. Leaders in targeted C3 therapies, we aim to develop transformative therapies for a broad range of debilitating diseases that are driven by excessive activation of the complement cascade, including those within hematology, ophthalmology, nephrology, and neurology. For more information, please visit https://www.apellis.com.

About Beam TherapeuticsBeam Therapeutics (Nasdaq: BEAM) is a biotechnology company committed to establishing the leading, fully integrated platform for precision genetic medicines. To achieve this vision, Beam has assembled a platform that includes a suite of gene editing and delivery technologies and is in the process of building internal manufacturing capabilities. Beams suite of gene editing technologies is anchored by base editing, a proprietary technology that enables precise, predictable and efficient single base changes, at targeted genomic sequences, without making double-stranded breaks in the DNA. This enables a wide range of potential therapeutic editing strategies that Beam is using to advance a diversified portfolio of base editing programs. Beam is a values-driven organization committed to its people, cutting-edge science, and a vision of providing life-long cures to patients suffering from serious diseases.

Apellis Forward-Looking Statement Statements in this press release about future expectations, plans and prospects, as well as any other statements regarding matters that are not historical facts, may constitute forward-looking statements within the meaning of The Private Securities Litigation Reform Act of 1995. These statements include, but are not limited to, statements in respect of the expected closing of the exchanges. The words anticipate, believe, continue, could, estimate, expect, intend, may, plan, potential, predict, project, should, target, will, would and similar expressions are intended to identify forward-looking statements, although not all forward-looking statements contain these identifying words. Actual results may differ materially from those indicated by such forward-looking statements as a result of various important factors, including: whether the research collaboration will result in programs that are licensed by Apellis; whether any product candidates that arise from these programs or are otherwise developed by Apellis will advance into clinical trials or through the clinical trial process on a timely basis or at all; whether the results of clinical trials of the companys product candidates will warrant submissions for regulatory approval or regulatory approval; whether any products that receive regulatory approval will be successfully distributed and marketed; and other factors discussed in the Risk Factors section of Apellis Quarterly Report on Form 10-Q with the Securities and Exchange Commission on April 28, 2021 and the risks described in other filings that Apellis may make with the Securities and Exchange Commission. Any forward-looking statements contained in this press release speak only as of the date hereof, and Apellis specifically disclaims any obligation to update any forward-looking statement, whether as a result of new information, future events or otherwise.

Beam Forward-Looking Statement This press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Investors are cautioned not to place undue reliance on these forward-looking statements, including, but not limited to, statements related to: the therapeutic applications and potential of our technology, including our ability to develop life-long, curative, precision genetic medicines for patients through base editing. Each forward-looking statement is subject to risks and uncertainties that could cause actual results to differ materially from those expressed or implied in such statement, including, without limitation, risks and uncertainties related to: our ability to raise additional funding, which may not be available; our ability to obtain, maintain and enforce patent and other intellectual property protection for our platform technology; the potential impact of the COVID-19 pandemic; risks related to competitive products; and the other risks and uncertainties identified under the heading Risk Factors in our Annual Report on Form 10-K for the year ended December 31, 2020, our Quarterly Report on Form 10-Q for the quarter ended March 31, 2021, and in any subsequent filings with the Securities and Exchange Commission. These forward-looking statements (except as otherwise noted) speak only as of the date of this press release. Factors or events that could cause our actual results to differ may emerge from time to time, and it is not possible for us to predict all of them. We undertake no obligation to update any forward-looking statement, whether as a result of new information, future developments or otherwise, except as may be required by applicable law.

Contacts:

Apellis:

Media:Lissa Pavlukmedia@apellis.com617.977.6764

Investors: Argot Partners apellis@argotpartners.com212.600.1902

Beam Therapeutics:

Media:Dan Budwick1ABdan@1abmedia.com

Investors:Chelcie ListerTHRUST Strategic Communicationschelcie@thrustsc.com

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Apellis and Beam Therapeutics Enter Exclusive Research Collaboration to Apply Base Editing to Discover Novel Therapies for Complement-Driven Diseases...

London, July 01, 2021 (GLOBE NEWSWIRE) -- Roots Analysis has announced the addition of Cell Therapy Manufacturing Market (4th Edition), 2021-2030 report to its list of offerings.

Owing to the complex manufacturing processes, requirement of advanced production facilities and the growing demand for cell therapy products, developers are actively outsourcing certain manufacturing operations, in addition to expanding their in-house capabilities.

To order this 620 page report, which features 210+ figures and 280+ tables, please visit https://www.rootsanalysis.com/reports/view_document/cell-therapy-manufacturing/285.html

Key Market Insights

Around 200 organizations claim to be engaged in cell therapy manufacturingThe market landscape is dominated by industry players, which constitute 65% of the total number of stakeholders. Amongst these, over 25% companies are large firms.

280+ production facilities dedicated to cell therapies have been established worldwideNorth America has emerged as the manufacturing hub for cell therapies, with the presence of nearly 45% of the manufacturing facilities; this is followed by Europe (31%). Other emerging regions include China, Japan, South Korea and Australia.

90 cell therapy manufacturers are focused on immune cell and stem cell therapiesMost of the players in this domain are focused on manufacturing of T cell therapies, primarily CAR-T therapies, while the stem cell therapy manufacturers are primarily engaged in the production of adult stem cells and mesenchymal stem cell therapies

Presently, more than 70 companies carry out manufacturing at all scales of operation. Nearly 45% players have the required capabilities for commercial scale manufacturing. It is worth noting that all the industry players manufacture cell therapies required for clinical purposes.

35+ companies offer automated and closed systems to cell therapy developers More than 60 automated and closed systems are being used for cell therapy manufacturing. Organizations that are presently offering customized automated solutions for cell therapy processes / manufacturing are Fraunhofer Institute for Manufacturing Engineering and Automation IPA (Germany), KMC Systems (US), RoosterBio (US) and Mayo Clinic Center for Regenerative Medicine (US).

Several partnerships were established in this domain, during the period 2016-2021More than 180 deals have been inked during the given time period. A large proportion (34%) of the partnerships were related to manufacturing of cell therapies, followed by acquisitions (17%) and licensing agreements (14%).

Expansion activity in this domain has grown at a CAGR of 59%, between 2016 and 2021More than 75 facility expansions were reported during the given time period. Over 80% instances were related to the establishment of new facilities, followed by those involving the expansion of existing facilities (17%).

Role of big pharma players in this industry has evolved over the last few years; their initiatives increased at a CAGR of 41% during the period 2016-2020Several big pharma players have undertaken various initiatives focused on cell therapy manufacturing. Gilead sciences, Takeda Pharmaceutical and Novartis are some of the prominent big pharma players in this domain.

The currently available global cell therapy manufacturing capacity is estimated to be over 1.88 billion sq. ft. of dedicated cleanroom areaThe maximum (48%) installed capacity (in terms of cleanroom area) belongs to companies based in North America (48%); the region has higher number of players having multiple production facilities. This is followed by Asia Pacific (29%) and Europe (23%).

The demand for cell therapies is anticipated to grow at a CAGR of 22%, during 2021-2030Presently, the clinical demand for stem cell and CAR-T cell-based products is the highest; this trend is unlikely to change in the foreseen future as well. On the other hand, the demand for tumor cell, NK cell and dendritic cell therapies is expected to grow at a relatively faster pace, over the next decade.

By 2030, the market for commercial scale cell therapy manufacturing is likely to grow at an annualized rate of 31.5%Currently, North America and Europe capture more than 70% share of the overall market. Specifically, the cell therapy manufacturing market in Asia Pacific is driven by countries, such as China, Japan, South Korea, India and Singapore. It is worth noting that the current market in Asia Pacific is primarily driven by the clinical demand for cell therapies.

To request a sample copy / brochure of this report, please visit https://www.rootsanalysis.com/reports/view_document/cell-therapy-manufacturing/285.html

Key Questions Answered

The USD 14.5 billion (by 2030) financial opportunity associated with cell therapy manufacturing market has been analyzed across the following segments:

The report also features inputs from eminent industry stakeholders, according to whom, the manufacturing of cell therapies is largely being outsourced due to exorbitant costs associated with the setting-up of in-house expertise. The report includes detailed transcripts of discussions held with the following experts:

The research includes profiles of key players (industry and non-industry; listed below), featuring a brief overview company / organization, information on its manufacturing facilities, service portfolio, recent partnerships and an informed future outlook.

For additional details, please visithttps://www.rootsanalysis.com/reports/view_document/cell-therapy-manufacturing/285.html or email sales@rootsanalysis.com

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The cell therapy manufacturing market is projected to reach USD 14.5 billion by 2030, growing at - GlobeNewswire

Basel, 02 July 2021 - Roche (SIX: RO, ROG; OTCQX: RHHBY) today announced that new data from its haemophilia A clinical programme will be presented at the virtual International Society on Thrombosis and Haemostasis (ISTH) 2021 Congress, from 17-21 July 2021. Data will include the final analysis from the phase IIIb STASEY study of Hemlibra (emicizumab) and updated data from the phase I/II study of SPK-8011, an AAV-based gene therapy in development by Spark Therapeutics (a member of the Roche Group).1,2

Were excited to present new data from our haemophilia A programme at the ISTH 2021 Congress, said Levi Garraway, M.D., Ph.D., Roches Chief Medical Officer and Head of Global Product Development. These data reinforce our continued commitment to developing transformational therapies for the haemophilia A community and advancing understanding of the long-term efficacy and safety profile of Hemlibra.

Haemophilia A is a serious, inherited bleeding disorder in which a persons blood doesn't clot properly, as they either lack or do not have enough of a clotting protein called factor VIII. This can lead to uncontrolled bleeding, either spontaneously or after minor trauma. These bleeds can present a significant health concern as they often cause pain and can lead to chronic swelling, deformity, reduced mobility, and long-term joint damage.3 The development of factor VIII inhibitors can be a significant challenge in the treatment of people with haemophilia A as they bind to and block the efficacy of replacement factor VIII.4

The STASEY study is one of the largest open-label studies primarily assessing the safety and tolerability of a medicine for adults and adolescents with haemophilia A with factor VIII inhibitors. Final data from the STASEY study, evaluating the safety and tolerability of Hemlibra prophylaxis in adults and adolescents with haemophilia A with factor VIII inhibitors, will be presented at the congress.1 These results confirm the favourable safety profile of Hemlibra, as previously demonstrated in the phase III HAVEN clinical trials.1,5,6,7

Spark Therapeutics will share updated data from the ongoing phase I/II clinical trial of SPK-8011, an investigational AAV-based gene therapy developed for the treatment of haemophilia A. These data demonstrate that hepatocyte expression of factor VIII can be stable and durable for up to four years following vector administration, with an acceptable safety profile.2

We are looking forward to sharing data on our investigational gene therapy, SPK-8011, which is being evaluated in the largest phase I/II gene therapy trial in haemophilia A to date, and which reinforces Sparks mission to bring a novel gene therapy option to persons with haemophilia A, said Gallia Levy, M.D., Ph.D., Chief Medical Officer, Spark Therapeutics.

SPK-8011 data presentationUpdated results from Sparks ongoing phase I/II study of investigational SPK-8011 will be shared as an oral presentation during the meeting. These data highlight the safety profile and durability of SPK-8011 in 18 participants, up to four years following vector administration with SPK-8011 in four dose cohorts, ranging from 5x1011 vg/kg to 21012 vg/kg, with results showing a 93% reduction in annualised bleed rate (ABR) and a 97% reduction in annualised infusion rate.2

Key Hemlibra and haemophilia data presentationsFinal data from the STASEY study presented at the congress demonstrate that Hemlibra is effective, with ABRs consistent with observations reported from the pivotal HAVEN studies.1,5,6,7 Additionally, no new safety signals were identified in adults and adolescents with haemophilia A with factor VIII inhibitors, consistent with previous safety observations.1

Roche will also present a retrospective analysis comparing ABR and Hemlibra concentrations among obese and non-obese adults with haemophilia A from pooled data from the HAVEN 1, 3, and 4 studies.8 These data suggest that body weight does not significantly impact the efficacy of Hemlibra, regardless of dosing regimen, demonstrating that Hemlibra offers an effective, well tolerated treatment, with flexible dosing options, in obese and non-obese people with haemophilia A.

Additionally, an analysis of data from the 2017 CHESS PAEDs (Cost of Haemophilia across Europe: a Socioeconomic Survey in the Paediatric Population) study, examining the association between physical activity levels and bleed rates in children with haemophilia A, will be presented.9 Results from this analysis demonstrate the potential treatment needs and clinical burden in physically active children with moderate and severe haemophilia A receiving factor VIII replacement therapy.

Key abstracts from Roche and Spark that will be presented at ISTH can be found in the table below.

Follow Roche and Spark on Twitter via @Roche and @Spark_tx respectively, and keep up to date with ISTH 2021 Congress news and updates by using the hashtag #ISTH2021.

Virtual Meeting Room 3

Virtual Meeting Room 3

Virtual Meeting Room 8

About Hemlibra (emicizumab)Hemlibra is approved for routine prophylaxis of bleeding episodes in people with haemophilia A with and without factor VIII inhibitors in over 100 countries worldwide for those with inhibitors and over 80 countries for those without inhibitors, in adults and children, ages newborn and older. Hemlibra is a bispecific factor IXa- and factor X-directed antibody. It is designed to bring together factor IXa and factor X, proteins involved in the natural coagulation cascade, and restore the blood clotting process for people with haemophilia A. Hemlibra is a prophylactic (preventative) treatment that can be administered by an injection of a ready-to-use solution under the skin (subcutaneously) once-weekly, every two weeks or every four weeks (after an initial once-weekly dose for the first four weeks). Hemlibra was created by Chugai Pharmaceutical Co., Ltd. and is being co-developed globally by Chugai, Roche and Genentech. It is marketed in the United States by Genentech as Hemlibra (emicizumab-kxwh), with kxwh as the suffix designated in accordance with Nonproprietary Naming of Biological Products Guidance for Industry issued by the US Food and Drug Administration.

About SPK-8011 for haemophilia AInvestigational SPK-8011, a novel bio-engineered adeno-associated viral (AAV) vector utilizing the AAV-LK03 capsid, also referred to as Spark200, contains a codon-optimized human factor VIII gene under the control of a liver-specific promoter. The Food and Drug Administration (FDA) granted orphan-disease designation and breakthrough therapy designation in the U.S., while the European Commission has granted orphan designation to SPK-8011.

About haemophilia AHaemophilia A is an inherited, serious disorder in which a persons blood does not clot properly, leading to uncontrolled and often spontaneous bleeding. Haemophilia A affects around 900,000 people worldwide,10,11 approximately 35-39% of whom have a severe form of the disorder.11 People with haemophilia A either lack or do not have enough of a clotting protein called factor VIII. In a healthy person, when a bleed occurs, factor VIII brings together the clotting factors IXa and X, which is a critical step in the formation of a blood clot to help stop bleeding. Depending on the severity of their disorder, people with haemophilia A can bleed frequently, especially into their joints or muscles.10 These bleeds can present a significant health concern as they often cause pain and can lead to chronic swelling, deformity, reduced mobility, and long-term joint damage.3 A serious complication of treatment is the development of inhibitors to factor VIII replacement therapies.12 Inhibitors are antibodies developed by the bodys immune system that bind to and block the efficacy of replacement factor VIII,13 making it difficult, if not impossible, to obtain a level of factor VIII sufficient to control bleeding.

About Roche and Spark Therapeutics gene therapy research in haemophilia AWe believe gene therapy has the potential to revolutionise medicine and improve the lives of patients with genetic and other serious diseases. Pairing Roches long-standing commitment to developing medicines in haemophilia with Spark Therapeutics proven gene therapy expertise brings together the best team of collaborators researching gene therapies in haemophilia A.

It is our aligned objective to develop gene therapies for haemophilia A that, with the lowest effective dose and the optimal immunomodulatory regimen, demonstrate safety, predictability, efficacy, and durability for patients.

About Spark TherapeuticsAt Spark Therapeutics, a fully integrated, commercial company committed to discovering, developing and delivering gene therapies, we challenge the inevitability of genetic diseases, including blindness, haemophilia, lysosomal storage disorders and neurodegenerative diseases. We currently have four programs in clinical trials. At Spark, a member of the Roche Group, we see the path to a world where no life is limited by genetic disease. For more information, visit http://www.sparktx.com, and follow us on Twitter and LinkedIn.

About Roche in haematologyRoche has been developing medicines for people with malignant and non-malignant blood diseases for over 20 years; our experience and knowledge in this therapeutic area runs deep. Today, we are investing more than ever in our effort to bring innovative treatment options to patients across a wide range of haematologic diseases. Our approved medicines include MabThera/Rituxan (rituximab), Gazyva/Gazyvaro (obinutuzumab), Polivy (polatuzumab vedotin), Venclexta/Venclyxto (venetoclax) in collaboration with AbbVie, and Hemlibra (emicizumab). Our pipeline of investigational haematology medicines includes T-cell engaging bispecific antibodies, glofitamab and mosunetuzumab, targeting both CD20 and CD3, and cevostamab, targeting FcRH5 and CD3; Tecentriq (atezolizumab), a monoclonal antibody designed to bind with PD-L1; and crovalimab, an anti-C5 antibody engineered to optimise complement inhibition. Our scientific expertise, combined with the breadth of our portfolio and pipeline, also provides a unique opportunity to develop combination regimens that aim to improve the lives of patients even further.

About RocheRoche is a global pioneer in pharmaceuticals and diagnostics focused on advancing science to improve peoples lives. The combined strengths of pharmaceuticals and diagnostics, as well as growing capabilities in the area of data-driven medical insights help Roche deliver truly personalised healthcare. Roche is working with partners across the healthcare sector to provide the best care for each person.

Roche is the worlds largest biotech company, with truly differentiated medicines in oncology, immunology, infectious diseases, ophthalmology and diseases of the central nervous system. Roche is also the world leader in in vitro diagnostics and tissue-based cancer diagnostics, and a frontrunner in diabetes management. In recent years, Roche has invested in genomic profiling and real-world data partnerships and has become an industry-leading partner for medical insights.

Founded in 1896, Roche continues to search for better ways to prevent, diagnose and treat diseases and make a sustainable contribution to society. The company also aims to improve patient access to medical innovations by working with all relevant stakeholders. More than thirty medicines developed by Roche are included in the World Health Organization Model Lists of Essential Medicines, among them life-saving antibiotics, antimalarials and cancer medicines. Moreover, for the twelfth consecutive year, Roche has been recognised as one of the most sustainable companies in the Pharmaceuticals Industry by the Dow Jones Sustainability Indices (DJSI).

The Roche Group, headquartered in Basel, Switzerland, is active in over 100 countries and in 2020 employed more than 100,000 people worldwide. In 2020, Roche invested CHF 12.2 billion in R&D and posted sales of CHF 58.3 billion. Genentech, in the United States, is a wholly owned member of the Roche Group. Roche is the majority shareholder in Chugai Pharmaceutical, Japan. For more information, please visit http://www.roche.com.

All trademarks used or mentioned in this release are protected by law.

References[1] Jimnez-Yuste J, et al. Final Analysis of the STASEY Trial: A Single-arm, Multicenter, Open-label, Phase III Clinical Trial Evaluating the Safety and Tolerability of Emicizumab Prophylaxis in Persons with Hemophilia A (PwHA) with factor (F)VIII Inhibitors. Presented at: International Society on Thrombosis and Haemostasis (ISTH) Congress; 2021 Jul 17-21; Philadelphia, PA, USA. Abstract PB0521.[2] George LA, et al. Phase I/II trial of SPK-8011: Stable and Durable FVIII Expression After AAV Gene Transfer for Hemophilia A. Presented at: International Society on Thrombosis and Haemostasis (ISTH) Congress; 2021 Jul 17-21; Philadelphia, PA, USA. Abstract OC 67.2.[3] Franchini M, et al. Haemophilia A in the third millennium. Blood Rev. 2013; 179-84.[4] Shannon L. Meeks at al. Hemophilia and inhibitors: current treatment options and potential new therapeutic approaches. Hematology Am Soc Hematol Educ Program 2016 (1): 657662. [5] Oldenburg J, et al. Emicizumab Prophylaxis in Hemophilia A with Inhibitors. N Engl J Med. 2017; 377:809-818.[6] Young G, et al. Emicizumab prophylaxis provides flexible and effective bleed control in children with hemophilia A with inhibitors: results from the HAVEN 2 study. Blood. 2018; 132 (Supplement 1): 632.[7] Pipe S, et al. Emicizumab subcutaneous dosing every 4 weeks is safe and efficacious in the control of bleeding in persons with haemophilia A with and without inhibitors: Results from the phase 3 HAVEN 4 study. Presented at: WFH World Congress; 2018 May 20-24; Glasgow, Scotland, UK. Abstract M-LBMED01-005 (854).[8] Recht M, et al. Emicizumab in Obese Adults with Hemophilia A Pooled Data from Three Phase III Studies (HAVEN 1, 3 and 4). Presented at: International Society on Thrombosis and Haemostasis (ISTH) Congress; 2021 Jul 17-21 Philadelphia, PA, USA. Abstract PB0495.[9] Ofori-Asenso et al. Association of Physical Activity with Bleeding Frequency in Children with Hemophilia A: a CHESS PAEDs Study Analysis. Presented at: International Society on Thrombosis and Haemostasis (ISTH) Congress; 2021 Jul 17-21; Philadelphia, PA, USA. Abstract PB0512.[10] Srivastava, A, Santagostino, E, Dougall, A, et al. WFH Guidelines for the Management of Hemophilia, 3rd edition. Haemophilia. 2020: 26 (Suppl 6): 1158.[11] Iorio A et al. Establishing the Prevalence and Prevalence at Birth of Hemophilia in Males. Ann Intern Med. 2019 Oct 15;171(8):540-546.[12] Gomez K, et al. Key issues in inhibitor management in patients with haemophilia. Blood Transfus. 2014; 12:s319-s329.[13] Whelan SF, et al. Distinct characteristics of antibody responses against factor VIII in healthy individuals and in different cohorts of haemophilia A patients. Blood. 2013; 121:1039-48.

Roche Group Media RelationsPhone: +41 61 688 8888 / e-mail: media.relations@roche.com

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Top officials from the US Food and Drug Administration (FDA) exhort the benefits of master protocols and hope the momentum of using these protocols continues in the post-COVID-19 era. Officials also say the pandemic has not dampened the enthusiasm for gene therapy development as the agency continues to receive a healthy number of investigational new drug applications (INDs) for these therapies.These were some of the learnings imparted by agency officials in discussing the effects of the COVID-19 pandemic at a 1 July virtual town hall convened by the Drug Information Association during its annual meeting. A good part of the one-hour town hall addressed the use of master protocols as the way forward for clinical trials because of their ability to study large, diverse patient populations with efficacy rates mirroring real world use.The use of these protocols was strongly supported by FDA Acting Commissioner Janet Woodcock, Patrizia Cavazzoni, the director of the Center for Drug Evaluation and Research (CDER), and Peter Marks, the director of the Center for Biologics Evaluation and Research (CBER).COVID turbo charges master protocolsCavazzoni said that before the pandemic, sponsors had very limited experience with master protocols, yet COVID-19 changed things as more sponsors were adopting this design.Before the pandemic, protocols from platform trials were very limited, and the COVID experience was a learning curve and gave a boost to CDER to evaluate the trials, said Cavazzoni. I think it has been a very important experience because it has turbo charged the utilization of master protocols.She added that while FDAs new guidance on master protocols is still labeled a COVID-19 guidance she said that I have every expectation and hope that this will be an approach that will be used to a much greater extent in the post-COVID arena.In May, FDA released a guidance addressing how master protocols can be used in developing drugs to treat or prevent COVID-19. (RELATED: FDA issues new COVID-19 master protocol guidance, Regulatory Focus 17 May 2021).Woodcock: master protocols more complicatedWoodcock observed that that while master protocols are more complicated and may take longer to set up then conventional trials, they can evaluate multiple agents efficiently once theyre up and running. What we found is that the master protocols from discovery to active trials to repurposed trials are able to perform very well. and can impart actionable data.Woodcock said that a recent FDA study showed that a disproportionate amount of the adequately powered and randomized clinical trial data generated for therapeutics emanate from master protocols. She noted that many of the obstacles to getting master protocols going that were there before the pandemic still remain, and we have to overcome that.Woodcock has long advocated for the benefit of master protocols. In 2017, she co-authored a New England Journal of Medicine piece advocating for the use of platform trials for efficient generation of evidence in precision medicine. (RELATED: FDA officials: master protocols needed for precision medicine, Regulatory Focus, 7 July 2017)Strong correlations between clinical trial and real-world data Marks elaborated on the robust data emanating from COVID-19 trials and attributes this to the strong correlation between clinical trials data and real-world data, with trial populations largely mirroring the overall population.He said that COVID-19 mRNA vaccine trials are showing 94 to 95% efficacy in preventing the cirus, which mirrors the 94 to 97% efficacy in the real world.The strong correlation between clinical trial efficacy and real-world efficacy, he said is something that should be fostered and something to move forward with after the pandemic.The better evidence we can collect and the more robust it is the better it will be. That is the reason we are seeing such great correlations between the real-world effectiveness of the mRNA vaccines and the clinical trials, said Marks.Woodcock: pushing for research out of the ivory towerWoodcock said that because of these benefits and efficiencies of studying drugs in large patient populations, she will be pushing for larger trials run out of community-based sites.She said that we need to have master protocols that are run by the community because there are so many remaining questions about the treatment policy for most diseases. What should you start with? Which disease should you use? Which doses should you use and how can you personalize this? This should be answered by expert opinions and not data. We need to get clinical research out of the ivory tower and into the communities of everyone who gets medical care.She cited some of the inefficiencies of cancer trials, noting that only about 8% of patients with cancer are actually enrolled in trials, although the treatments in these trials could help most cancer patients.As part of the effort to move more clinical trials out of academic medical centers and boost community enrollment, Cavazzoni said that FDA is working on a guidance on decentralized trials for remote data assessment targeting community-based sites.Robust gene therapy development despite pandemicIn other areas, the pandemic has not dampened interest in new cell and gene therapy development, said Marks. He said that the number of cell and gene therapy INDs submitted to the agency increased by 20% this past year, showing that there is a tremendous amount of work going on.He said that we predicted that by 2025 we would approve between five and ten gene therapies a year. I dont think we will be that far off despite the pandemic slowing things down a little bit. There are still a healthy number of applications.Currently, some sponsors are experiencing speed bumps with their data as some toxicity issues have emerged, but the agency is working with sponsors to help resolve these issues, said Marks.Additionally, Marks noted that many clinical trial sponsors experienced significant disruption of their programs during the height of the pandemic, when many patients could not travel. We will have to work with them on a case-by-case basis and salvage what we can, said Marks.DIA Annual Meeting

2021Regulatory Affairs Professionals Society.

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DIA: Woodcock, other top officials tout benefits of master protocols, want momentum to continue post-COVID - Regulatory Focus

Newswise BUFFALO, N.Y. Two Roswell Park Comprehensive Cancer Center experts were invited to present new insights on treatment of gastroesophageal cancers during the European Society for Medical Oncology (ESMO) World Congress on Gastrointestinal Cancer 2021. In their talks, both presented July 1, the Roswell Park physician-researchers highlighted easily adoptable methods that may help other clinicians to provide care supporting improved patient outcomes.

Sarbajit Mukherjee, MD, MS, Assistant Professor of Oncology in the Department of Medicine, shared findings of a study showing a significant association between inflammation, cell proliferation and outcomes in patients with gastroesophageal cancer who received immunotherapy (Abstract SO-5).

Immune checkpoint inhibitors (ICI) have changed the landscape of cancer treatment in recent years, yet very few patients respond to this therapy, notes Dr. Mukherjee. So it is of utmost importance that we pursue the possibilities further to see which patients can benefit most from immunotherapy.

Earlier research from Dr. Mukherjee and colleagues shows that obese patients respond better to immune checkpoint inhibitor (ICI) therapy, compared to nonobese patients. They hypothesized that obesity leads to inflammation, which can be reversed by ICI, and that obesity is associated with better treatment response to ICI.

To test this hypothesis, the team here examined the gene expression profile of the tumors from metastatic gastroesophageal cancer patients. Overweight patients with a body-mass index (BMI) of 25 or more represented 61 percent of the study cohort.

We found that the inflammation status of the tumor was independently associated with outcomes, regardless of obesity, he reports. The novelty of our work lies in the use of a unique gene-expression profile to determine the inflammation status of the tumor, which can be used as a biomarker for ICI therapy.

The researchers used a standard FDA-approved test to assess gene expression, which suggests that this approach can be adopted broadly. Such tests can help preselect patients who are likely to respond to immune checkpoint inhibitors and avoid unnecessary toxicity in others, Dr. Mukherjee says, noting that further study to better understand the role of these mechanisms in response to ICI therapy is needed.

In another study, spearheaded by Dr. Mukherjees mentee, Lei Deng, MD, Hematology/Oncology Fellow at Roswell Park, researchers explored the prognostic and predictive role of preoperative chemotherapy sensitivity in gastric adenocarcinoma (Abstract SO-7).

Using the National Cancer Database, the researchers identified 2,952 patients with gastric adenocarcinoma diagnosed between 2006 to 2017. The data revealed that, among these patients, sensitivity to preoperative chemotherapy is not only associated with survival, but also that sensitivity can predict benefit from postoperative chemotherapy.

The team used a novel approach, defining sensitivity to treatment based on stage change before and after preoperative chemotherapy and surgery. Sensitivity was defined as very sensitive (no residual disease at time of surgery after treatment), sensitive (lower stage after treatment) or refractory (no stage change or more advanced disease after treatment).

In this study, patients with sensitive disease were shown to have a significant survival benefit from postoperative chemotherapy. Postoperative chemotherapy improved overall survival in sensitive patients with a 5-year survival rate of 73.9% compared to 65% among those who did not receive this treatment. No improvement with postoperative chemotherapy was observed among very sensitive or refractory patients.

These findings suggest that sensitivity to preoperative chemotherapy is prognostic and can predict benefit from postoperative chemotherapy in this patient population, but validation is required.

While this work is at an early stage, if our findings are validated in prospective studies, this approach may help better select patients who should receive postoperative chemotherapy and avoid unnecessary toxicity in those who do not need these treatments, notes Dr. Deng. The simple sensitivity definition utilized in this study will also enable rapid clinical adoption.

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An online version of this release is available on our website.

Roswell Park Comprehensive Cancer Center is a community united by the drive to eliminate cancers grip on humanity by unlocking its secrets through personalized approaches and unleashing the healing power of hope. Founded by Dr. Roswell Park in 1898, it is the only National Cancer Institute-designated comprehensive cancer center in Upstate New York. Learn more at http://www.roswellpark.org, or contact us at 1-800-ROSWELL (1-800-767-9355) or [emailprotected].

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Intermittent fasting is an umbrella term for diets that restrict food intake to certain time windows. These diets can include fasting for several hours, or even days, at a time.

The dietary practice has become increasingly popular in recent years as a way to lose weight and improve health. The reason for its popularity may be that people consider it easier to maintain than some other diets.

Findings from studies show that intermittent fasting could help reduce weight, blood pressure, insulin sensitivity, and cholesterol.

However, so far, studies investigating the dietary practice in humans have found that although it is safe and effective, it is no more effective than other diets that restrict calorie intake.

A major challenge for researchers is being able to distinguish between the health and weight loss benefits specific to fasting and other diets.

Scientists from the University of Bath in the United Kingdom recently headed an international collaboration between research institutions in the U.K., Switzerland, and Taiwan to conduct a study investigating the specific effects of intermittent fasting.

Echoing previous research, the teams findings suggest that alternate-day fasting and daily energy restriction are similarly effective for weight loss.

However, while weight loss from daily energy restriction mostly came from reducing body fat, for those who were fasting, just half of the total weight loss came from body fat. The other half came from fat-free mass.

The researchers published their findings in Science Translational Medicine.

The scientists recruited 36 lean, healthy adults in the U.K. between 2015 and 2018 for the study and monitored their baseline diet and physical activity for 4 weeks. They then randomly allocated the participants to one of three groups of 12.

The participants in the first group, the energy restriction group, consumed 75% of their normal energy intake each day.

The second group used two methods of weight loss: fasting and energy restriction. They fasted on alternate days and consumed 150% of their regular calorie intake on their eating days.

The third group did not face any energy restriction. They fasted on alternate days and consumed 200% of their regular calorie intake on their eating days.

The fasting groups consumed no energy-providing nutrients during their fasting periods. This ensured that their dietary interventions were standardized and allowed enough time for fasting-related bodily functions to activate.

The participants underwent various lab tests before and after the 3-week intervention. The researchers also monitored the participants diet and physical activity levels throughout and extracted fat tissue samples from some individuals.

Those on energy restriction diets lost an average of 1.91 kilograms (kg) at the end of the study period. Meanwhile, those fasting with energy restriction lost an average of 1.60 kg, and those fasting without energy restriction lost an average of 0.52 kg.

To explain their results, the researchers say that the difference in body mass between the energy restriction groups may be partly due to a reduction in physical activity, and thus energy lost from heat production, in those who fasted.

They did not observe decreased physical activity among those who fasted without energy restriction, however.

The researchers also noted that all of the groups lost similar levels of visceral fat over the study period. Visceral fat is fat that the body stores around the abdomen, and it is linked to type 2 diabetes and heart disease.

No short-term changes in metabolic health such as blood sugar levels, cholesterol, and blood pressure or fat tissue gene expression occurred among the study participants. This, say the authors, may be because the participants were not overweight at the start of the study.

The researchers conclude that reduced physical activity during calorie-restricted fasting may limit weight loss and that people should include physical activity as part of alternate-day fasting diets to get the best weight loss results.

They note, however, that they cannot completely explain weight loss from fat-free mass in fasting diets, as no participants chose to provide skeletal muscle samples. Another limitation, they explain, is that their dietary intervention only lasted 3 weeks.

Many people believe that diets based on fasting are especially effective for weight loss or that these diets have particular metabolic health benefits even if you dont lose weight, senior study author Prof. James Betts commented on the teams results.

But intermittent fasting is no magic bullet, and the findings of our experiment suggest that there is nothing special about fasting when compared with more traditional, standard diets people might follow, he continued.

Most significantly, if you are following a fasting diet, it is worth thinking about whether prolonged fasting periods [are] actually making it harder to maintain muscle mass and physical activity levels, which are known to be very important factors for long-term health.

Prof. James Betts

Though intentional calorie restriction is not always feasible long term, eating nutrient-dense foods those high in fiber, vitamins, minerals, adequate protein, and healthy fats will often fill you up better than foods that are not nutrient-dense, Kristin Kirkpatrick, MS, RDN, who was not involved in the study, told Medical News Today. The more you feel full, the less you eat. If you eat without limit, you may not be as successful as someone not fasting but restricting calories.

Given this, I am not that surprised about the findings. However, its important that the benefits of intermittent fasting are well-documented in the data. You just need to pay attention to nutrition as well, she added.

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Abstract

Understanding the evolutionary stability and possible context dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While synthetic auxotrophy previously prevented Escherichia coli from exhibiting detectable escape from batch cultures, its long-term effectiveness is unknown. Here, we report automated continuous evolution of a synthetic auxotroph while supplying a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution, triplicate populations exhibit no observable escape and exhibit normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction reveals the contribution of three genes to increased fitness at low BipA concentrations. Based on its evolutionary stability, we introduce the progenitor strain directly to mammalian cell culture and observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on time scales and in contexts that enable diverse applications.

New safeguards are needed for the deliberate release of engineered microbes into the environment, which has promise for applications in agriculture, environmental remediation, and medicine (1). Genetically encoded biocontainment strategies enable attenuation of engineered live bacteria for diverse biomedical applications (24), including as potential vaccines (510), diagnostics (11), and therapeutics (1215). Auxotrophy, which is the inability of an organism to synthesize a compound needed for its growth, is an existing strategy for containment. However, foundational studies of auxotrophic pathogens demonstrated proliferation in relevant biological fluids (16) and reversion to prototrophy upon serial passaging (17, 18). Modern genome engineering strategies can prevent auxotrophic reversion, and auxotrophy has been a key component of microbial therapies that have reached advanced clinical trials. However, the ability for auxotrophs to access required metabolites within many host microenvironments, and after leaving the host, remains unaddressed. Auxotrophy may not be effective in scenarios where engineered living bacteria encounter metabolites from dead host cells (19) or invade host cells (20). Growth of double auxotrophs is supported in vivo by neoplastic tissue (13). Auxotrophy may also be insufficient for tight control of cell proliferation in environments rich with microbial sources of cross-feeding (21), such as gut, oral, skin, and vaginal microbiomes. Given that most naturally occurring microorganisms are auxotrophs (22), it is also unlikely that auxotrophy will limit the spread of an engineered microbe once it leaves the body and enters the environment.

Synthetic auxotrophy may overcome these hurdles by requiring provision of a synthetic molecule for survival of the engineered bacteria. This strategy was first implemented successfully in Escherichia coli by engineering essential proteins to depend on incorporation of a nonstandard amino acid (nsAA) (23, 24). We previously engineered E. coli strains for dependence on the nsAA biphenylalanine (BipA) by computer-aided redesign of essential enzymes in conjunction with expression of orthogonal translation machinery for BipA incorporation (23). Among several synthetic auxotrophs originally constructed, one strain harbored three redesigned, nsAA-dependent genesadenylate kinase (adk.d6), tyrosyl-tRNA synthetase (tyrS.d8), and BipA-dependent aminoacyl-tRNA synthetasefor aminoacylation of BipA (BipARS.d6). This BipA-dependent strain, dubbed DEP, exhibited undetectable escape throughout 14 days of monitoring at an assay detection limit of 2.2 1012 escapees per colony-forming unit (CFU) (23). Although this strain demonstrates effective biocontainment in 1-liter batch experiments, its precise escape frequency and long-term stability remained unexplored.

Here, we perform the first study of evolutionary stability of a synthetic auxotroph with the aid of automated continuous evolution. Continuous evolution better emulates scenarios where biocontainment may be needed by fostering greater genetic variability within a population. We posited that decreasing BipA concentrations would add selective pressure for adaptation or for escape, either of which would be enlightening. Adaptive laboratory evolution of DEP may improve its fitness in relevant growth contexts, as previously demonstrated for its nonauxotrophic but recoded ancestor, C321.A (25). We report that DEP maintains its inability to grow in the absence of synthetic nutrient, even after three parallel 100-day chemostat trials. In addition, we find evidence of adaptation, with evolved DEP isolates requiring 10-fold lower BipA concentration to achieve optimal growth than ancestral DEP (0.5 M rather than 5 M). We resequence evolved populations and perform allelic reconstruction in ancestral DEP using multiplex automatable genome engineering (MAGE), identifying alleles that partially restore the adaptive phenotype. Last, we advance this technology toward host-microbe coculture applications, demonstrating direct mixed culture of DEP and mammalian cells without the need for physical barriers or complex fluidics.

To perform continuous evolution of E. coli, we constructed custom chemostats for parallelized and automated culturing (Fig. 1A). Our design and construction were based on the eVOLVER system (26), an open-source, do-it-yourself automated culturing platform (figs. S1 to S4). By decreasing BipA concentration over time in our chemostats, we provide an initial mild selection for escape and steadily increase its stringency. This design is analogous to a morbidostat, where a lethal drug is introduced dynamically at sublethal concentrations to study microbial drug resistance (27), but with synthetic auxotrophy providing selective pressure. Our working algorithm for automated adjustment of BipA concentration as a function of turbidity is shown in Fig. 1B, and a representative image of our hardware is shown in Fig. 1C (see also fig. S5).

(A) Illustration of a smart sleeve connected to separate nonpermissive media and biphenylalanine (BipA; structure shown in blue) feed lines for automated adjustment of BipA concentration based on growth rate. Pumps and optics are integrated with Arduino controller hardware and Python software based on the eVOLVER do-it-yourself automated culturing framework. (B) Working algorithm for maintenance of cultures in continuous evolution mode. Criteria for lowering the BipA concentration are based on the difference in time elapsing between OD peaks (tpeak OD). Smaller time elapsed between OD peaks is indicative of higher growth rates, triggering decrease in BipA concentration when below a threshold value. (C) Representative configuration of hardware for parallelized evolution in triplicate, with three empty sleeves shown. Photo credit: Michael Napolitano, Harvard Medical School.

Our long-term culturing experiments featured two phases. The first phase included one chemostat (N = 1) that was inoculated with DEP for an 11-day incubation, with an initial concentration of BipA of 100 M and automated adjustment based on growth rate (Fig. 2A). Because we observed no colony formation when the outgrowth from this population was plated on nonpermissive media, we then began a second phase in replicate. We used our population grown for 11 days to inoculate three chemostats in parallel (N = 3) where BipA supply decreased automatically over the following 90 days from 100 M to nearly 100 nM. One controller provided identical BipA concentrations to all three vials at any given time. To determine whether the decrease in BipA supply was due to escape from dependence on BipA, we periodically performed escape assays. We continued to observe no escape, including when we seeded liter-scale cultures and plated the associated outgrowth on nonpermissive media. Evolved isolates were obtained after this procedure (fig. S6), and their growth was characterized across BipA concentrations (Fig. 2B and fig. S7). At 0.5 to 1 M BipA, we observed growth of all evolved isolates and no growth of the ancestral DEP strain.

(A) Timeline for continuous evolution, with detection limits for escape frequency assays shown in parentheses. (B) Doubling times of progenitor and evolved synthetic auxotrophs as a function of BipA concentration, normalized to the doubling time of DEP at 100 M BipA. Error bars represent the SD across technical triplicates within the same experiment.

To identify the causal alleles contributing to decreased BipA requirement of all three evolved isolates, we performed whole-genome sequencing and mutational analysis. We expected that mutations in auxotrophic markers or orthogonal translation machinery associated with aminoacylation of BipA would be observed. However, no variants were detected in the plasmid-expressed orthogonal translation machinery (aminoacyl-tRNA synthetase and tRNA) reference sequence. Instead, in all three evolved isolates, variants were observed in three nonessential genes, all of which are implicated in molecular transport: acrB, emrD, and trkH (Fig. 3A). AcrB and EmrD are biochemically and structurally well-characterized multidrug efflux proteins (28), and TrkH is a potassium ion transporter (29). These exact mutations have no precedent in the literature to our knowledge. Because they are missense mutations or in-frame deletions, it is unclear whether they cause loss of function or altered function (table S1). Because permissive media contain four artificial targets of efflux (BipA, l-arabinose, chloramphenicol, and SDS), mutations that confer a selective advantage during continuous evolution could disable BipA/l-arabinose efflux, improve chloramphenicol/SDS efflux, or affect transport of these or other species more indirectly. Given the strong selective pressure enforced by decreasing BipA concentration, we hypothesize that mutations observed are more likely to affect BipA transport. We also observed mutations in all evolved populations to the 23S ribosomal RNA (rRNA) gene rrlA (table S2). 23S rRNA mutations have been found to enhance tolerance for D-amino acids (30) and -amino acids (31). However, 23S rRNA mutations could also be related to increased tolerance of chloramphenicol (32).

(A) List of alleles identified through next-generation sequencing. Sequencing results originally obtained during the project identified this EmrD allele as a 33-bp deletion, which was then reconstructed in the experiment shown in (B). However, resequencing performed at the end of the project identified the allele as a 39-bp deletion and was confirmed by Sanger sequencing. A repetitive GGCGCG nucleotide sequence corresponding to G323-A324 and G336-A337 creates ambiguity about the precise positional numbering of the deletion. However, the three possible 13amino acid deletions (323335, 324336, and 325337) result in the same final protein sequence. (B) Effect of reconstructed allele in DEP progenitor on doubling time as a function of BipA concentration, normalized to the doubling time of DEP at 100 M BipA. Error bars represent the SD across technical triplicates within the same experiment.

To learn how identified transporter alleles may contribute to increased growth rates at low BipA concentration, we performed allelic reconstruction in the progenitor DEP strain using MAGE (33). Among four mutants that we generated in DEP, we observed growth of all mutants at 2 M BipA, a condition in which progenitor DEP could not grow (Fig. 3B and fig. S8). Furthermore, only emrD mutants exhibited near-normal growth at 1 M BipA. To investigate possible differential sensitivity of strains that contain reconstructed alleles to other media components of interest (SDS, l-arabinose, tris buffer, and chloramphenicol), we varied the concentration of these components and measured doubling times (fig. S9). We observed no significant deviation in doubling time from DEP in any of these cases. These results collectively suggest that observed transporter alleles are linked to BipA utilization.

The unobservable escape of DEP even after 100 days of evolution encouraged us to explore the possibility of an improved in vitro model for host-microbe interactions. In vitro models allow direct visualization and measurement of cells and effectors during processes such as pathogenesis (34). They are more relevant than animal studies for several human cell-specific interactions due to biological differences across animal types (35, 36). A nonpathogenic E. coli strain engineered to express heterologous proteins could be particularly useful for studying or identifying virulence factors and disease progression. However, an obstacle associated with coculture of microbial and mammalian cells is microbial takeover of the population. Approaches used to address this are bacteriostatic antibiotics (37), semipermeable Transwell membranes (3840), microcarrier beads (41), microfluidic cell trapping (42), peristaltic microfluidic flow (43, 44), and microfluidic perfusion (45). However, the use of a well-characterized synthetic auxotroph capable of limited persistence could offer a superior alternative for spatiotemporal control of microbial growth, especially for studying longer duration phenomena such as chronic infection or wound healing. Our study demonstrates how temporal control can be achieved by removal of BipA; we anticipate that spatial control could be achieved by patterning BipA onto a variety of solid surfaces with limited diffusion, such as a skin patch.

We investigated mammalian cell culture health, growth, and morphology after simple transient exposure to a hypermutator variant of DEP that we engineered by inactivating mutS during allelic reconstruction (DEP*). The use of DEP* rather than DEP is yet another form of a stress test to increase opportunity for escape under coculture conditions. We directly cocultured adherent human cell line human embryonic kidney (HEK) 293T with either no bacteria, nonauxotrophic E. coli DH5, or DEP* overnight (24 hours). HEK293T cells were cultured in selection media that allow only growth of desired but not contaminant strains while selecting for bacterial plasmid maintenance. After coculture, we washed cells and replenished cells with media varying in inclusion of BipA and/or an antibiotic cocktail (penicillin/streptomycin/amphotericin B). We continued incubation and imaged cells at days 2, 4, and 7 after initial coincubation. HEK293T cells contain a copy of mCherry integrated into the AAVS1 locus, and they appear red. DH5 and DEP* were transformed with Clover green fluorescent protein before coculture and appear green.

Compared to the control culture where bacteria were not added (Fig. 4A), HEK293T cells cocultured with DH5 display visible bacterial lawns with no attached human cells in the absence of the antibiotic cocktail at all days of observation (Fig. 4B). In the presence of antibiotic, cocultures containing DH5 sharply transition from bacterial overgrowth to apparent bacterial elimination (Fig. 4C). In contrast, cells cocultured with DEP* in the absence of BipA exhibited similar morphology to the control at all days of observation and no detectable bacteria by fluorescence microscopy on day 7, without the need for antibiotics to achieve bacterial clearance (Fig. 4D). Thus, DEP* addition was not detrimental to HEK293T cells in the absence of BipA, and DEP* remains biocontained and cannot survive because of cross-feeding. Clearance of bacterial cells from human cells appears to occur faster for DEP* when not provided BipA (Fig. 4D) than for DH5 when provided with the antibiotic cocktail (Fig. 4C).

Bacteria were added to HEK293T cell cultures and coincubated for 24 hours before washing and replenishing media. HEK293T cells express mCherry, whereas bacterial cells express Clover green protein marker. Images were taken at days 2, 4, and 7 after coincubation. (A) Untreated HEK293T cells. (B) HEK293T with commercial E. coli DH5 in the absence of antibiotic cocktail. (C) HEK293T with DH5 in presence of antibiotic cocktail. (D) HEK293T and DEP* (mismatch repair inactivated to create hypermutator phenotype) in the absence of BipA. (E) HEK293T cells and DEP* in the presence of BipA. (F) HEK293T and DEP* in the absence of BipA until day 2 [identical at this point to condition in (D)], and then 100 M BipA was added to this condition daily until day 7.

To learn how the synthetic auxotroph behaves when supplied its essential nutrient in these coculture settings, we tested DEP* cocultures with continual resupply of 100 M BipA. Here, DEP* proliferates and in turn decreases proliferation and viability of HEK293T cells (Fig. 4E). A bacterial lawn begins to form on day 2, and at later times, human cell debris is overtaken by DEP*. This demonstrates that DEP* is fully capable of taking over the coculture if supplied with BipA. Replicates for these experiments can be found in figs. S10 to S12.

Given that DEP* grows in cocultures when BipA is provided, we sought to understand whether it could be rescued by readdition of BipA after multiple days of withholding. The possible time scale of reemergence influences applications where the duration of bacterial activity would need to be prolonged and/or repeated via limited BipA introduction while remaining contained. We find that coculturing DEP* with HEK293T cells for 2 days in the absence of BipA followed by the addition of BipA at day 2 does not rescue the DEP* growth (Fig. 4F and fig. S13). Human cells still grow and look morphologically similar to untreated cells, and bacteria are not visible. To look at analogous questions for nonauxotrophic E. coli, we removed antibiotics after 2 days of coculturing and do not observe bacterial rescue (fig. S13). We also investigated whether bacterial clearance could be delayed by the addition of antibiotic after some growth of DH5. DH5 cells grown in the absence of the antibiotic cocktail for 2 days before addition of the cocktail and maintenance to day 7 result in bacterial lawns (fig. S13, A and D). This demonstrates that antibiotic cocktails ordinarily used in mammalian cell culture maintenance can become ineffective beyond a certain amount of nonauxotrophic bacterial growth, whereas synthetic auxotrophy is subject to fewer and different constraints.

To further investigate the persistence of progenitor DEP and its evolved descendants, we performed BipA readdition studies in Lennox lysogeny broth (LB-Lennox) monoculture. Within 7 hours of BipA removal, DEP cell populations that are harvested from midexponential or stationary phases can be reactivated upon delayed BipA addition with unperturbed growth kinetics after a highly tunable lag phase (fig. S14). Further studies are ongoing to investigate the amount of time after which BipA reintroduction can recover growth of synthetic auxotrophs under different contexts.

We have shown that synthetic auxotrophy can exhibit long-term stability and function in unique contexts, enabling reliable control of microbial proliferation. Recent work has also shown that the escape rate and fitness of multiple synthetic auxotrophs can be improved by increasing the specificity of nsAA incorporation machinery (46). Collectively, these engineering and characterization efforts advance synthetic auxotrophy as a powerful safeguard for basic and applied research when using engineered microbes.

Cultures for general culturing, growth rate assays, biocontainment escape assays, MAGE, and fluorescent protein assays were prepared in LB-Lennox medium [bacto tryptone (10 g/liter), sodium chloride (5 g/liter), and yeast extract (5 g/liter)] supplemented with chloramphenicol (15 g/ml), 0.2% (w/v) l-arabinose, 20 mM tris-HCl buffer, 0.005% SDS, and variable concentration of L-4,4-biphenylalanine (BipA). Unless otherwise indicated, all cultures were grown in 96-well deep plates in 300 l of culture volumes at 34C and 400 rpm. The above media are permissive for growth of the synthetic auxotroph. Nonpermissive media are identically formulated as permissive media except for BipA, which is not included.

Construction of appropriate fluidics and chambers followed the eVOLVER framework (26) (figs. S1 and S2). The following components were included: (i) fluidics and chambers (reactor vial, inlet and outlet lines, filters, pumps, stirrers, and inlet and outlet reservoirs); (ii) light source and detector (LED and photodiode); (iii) controller hardware (circuit and microprocessors); and (iv) controller software (Arduino for controlling tasks, Raspberry Pi for computing tasks, and Python code for programming tasks) (full build of materials included in table S3). Briefly, our apparatus consisted of a custom smart sleeve (fig. S3), with the following modifications: Each vial was constructed without temperature control and was supplied by two media pumps (one for permissive media and another for nonpermissive media) and connected to one waste pump. All pumps were RP-Q1 from Takasago Fluidics, each driven off a standard N power MOSFET (metal oxide semiconductor field-effect transistor) with an Arduino controlling the gate. Like the eVOLVER system, we installed a stirring fan underneath each sleeve that consisted of magnets attached to a computer fan. By including a small stir bar within each reactor vial, we enabled efficient mixing of 1-ml working volumes. To enable automated measurement of turbidity [optical density (OD)], we used a 605-nm LED (LO Q976-PS-25) and an OPT101P-J photodiode detector. We mounted the LED and detector on custom printed circuit boards mounted to the vial sleeve to enable easier construction and better control of ambient light leakage into the light path (fig. S4). To monitor turbidity within each vial and to control pump arrays in response, we constructed printed circuit board designs in Gerber format as is standard for circuit fabrication. We attached an Arduino Mega microcontroller with an analog-digital converter and directed it using a PyMata script (47).

Chemostats were operated by automated maintenance of culture OD within a specified parameter range within exponential growth phase (20 to 80% of dynamic range) depending on linearity of photodiode measurements. Constant fixed dilutions of permissive media were used to decrease OD until desired equilibrium of cell growth and dilution rates. This resulted in a sawtooth curve (27), where time between peaks is recorded as a proxy for growth rate. Our program gradually decreased the ratio of permissive to nonpermissive media as step functions, with a specified number of dilution cycles allowed to elapse before the next decrease to provide time for acclimation. Time between OD peaks lengthened as strain fitness decreased. Once a threshold difference between ancestral peak-to-peak time and current peak-to-peak time was passed, the ratio of permissive to nonpermissive media remained fixed. This allowed cells to evolve until peak-to-peak time returns to ancestral values, which initiated the next phase of decrease in BipA concentration. To assess the quality of our continuous evolution process, we paused chemostat trials on a weekly basis for strain storage, strain evaluation, chemostat cleaning, and investigation of contamination.

Growth assays were performed by plate reader with blanking as previously described (25). Overnight cultures were supplemented with different BipA concentrations depending on the strain. The DEP progenitor strain was grown in permissive media containing 100 M BipA, and evolved DEP strains DEP.e3, DEP.e4, and DEP.e5 were grown in permissive media containing 1 M BipA. Saturated overnight cultures were washed twice in LB and resuspended in LB. Resuspended cultures were diluted 100-fold into three 150-l volumes of permissive media. BipA concentrations used in this assay were 0, 0.001, 0.01, 0.1, 0.5, 1, 10, and 100 M. Cultures were incubated in a flat-bottom 96-well plate (34C, 300 rpm). Kinetic growth (OD600) was monitored in a Biotek Eon H1 microplate spectrophotometer reader at 5-min intervals for 48 hours. The doubling times across technical replicates were calculated as previously indicated. We refer to these as technical replicates because although triplicate overnight cultures were used to seed triplicate experiment cultures, the overnight cultures were most often seeded from one glycerol stock.

Escape assays were performed as previously described with minor adjustments to decrease the lower detection limit for final evolved populations (23, 46). Strains were grown in permissive media and harvested in late exponential phase. Cells were washed twice with LB and resuspended in LB. Viable CFU were calculated from the mean and SEM of three technical replicates of 10-fold serial dilutions on permissive media. Twelve technical replicates were plated on noble agar combined with nonpermissive media in 500-cm2 BioAssay Dishes (Thermo Fisher Scientific 240835) and monitored daily for 4 days. If synthetic auxotrophs exhibited escape frequencies above the detection limit (lawns) on nonpermissive media, escape frequencies were calculated from additional platings at lower density. The SEM across technical replicates of the cumulative escape frequency was calculated as previously indicated.

Genomic DNA was obtained from evolved populations and ancestral clone using the Wizard Genomic DNA purification kit (Promega). Sequencing libraries were prepared as described in Baym et al. (48). Sequencing was performed using a NextSeq instrument, producing 75base pair (bp), paired-end reads. Resulting data were aligned to the E. coli C321.delA nonauxotrophic but recoded reference sequence (GenBank no. CP006698.1) and the sequence of the plasmid encoding nsAA incorporation machinery. The Millstone software suite was used to identify variants, provide measures of sequencing confidence, and predict their likelihood of altering gene function (49). Genomic variants of low confidence, low sequence coverage, or presence in the ancestral strain were discarded, prioritizing variants observed in three nonessential genes that encode membrane proteins: acrB, emrD, and trkH.

Subsequent genomic sequencing was performed on genomic DNA extracted from the evolved populations and ancestral clone using the DNeasy Blood and Tissue Kit (Qiagen). Genomic DNA was then sent to the Microbial Genome Sequencing Center (MiGS) in Pittsburgh, PA. Variants were identified through the variant calling service from MiGS.

MAGE (33) was used to inactivate the endogenous mutS gene in the DEP strain. Overnight cultures were diluted 100-fold into 3 ml of LB containing chloramphenicol, BipA, l-arabinose, and tris-HCl buffer and grown at 34C until midlog. The genome-integrated lambda Red cassette in this C321.A-derived strain was induced in a shaking water bath (42C, 300 rpm, 15 min), followed by cooling the culture tube on ice for at least 2 min. The cells were made electrocompetent at 4C by pelleting 1 ml of culture (8000 rcf, 30 s) and washing thrice with 1 ml of ice-cold 10% glycerol. Electrocompetent pellets were resuspended in 50 l of dH2O containing the desired DNA; for MAGE oligonucleotides, 5 M of each oligonucleotide was used. Allele-specific colony polymerase chain reaction (PCR) was used to identify desired colonies resulting from MAGE as previously described (50). Oligonucleotides used for MAGE and for allele-specific colony PCR are included in table S4.

This assay was performed using a similar protocol as described in the Measurement of doubling times section. The cultures for DEP and its single mutants were grown overnight in 100 M BipA. Then, cultures were diluted 100 in the media specified. Those conditions include standard media conditions and single component changes: 0% SDS, 0.01% SDS, 0.02% (w/v) arabinose, 0 mM tris-HCl, and chloramphenicol (30 g/ml). The cultures were grown in triplicate for each condition and in a SpectraMax i3 plate reader, shaking at 34C for 24 hours. The OD600 was measured about every 5 min. The doubling times were then calculated as previously described.

HEK293T cells containing one copy of mCherry marker (red) integrated into the AAVS1 locus were grown at 40 to 50% confluency in DMEM (Dulbeccos modified Eagles medium) high-glucose medium (Thermo Fisher Scientific, catalog no. 11965175) with 10% inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, catalog no. 10082147), 100 MEM NEAA (nonessential amino acids; Thermo Fisher Scientific, catalog no. 11140050), and 100 diluted anti-anti cocktail [antibiotic-antimycotic: penicillin (10,000 U/ml), streptomycin (10,000 g/ml), and Gibco amphotericin B (25 g/ml); Thermo Fisher Scientific, catalog no. 15240112). Commercially acquired E. coli DH5 bacteria were used as control to the E. coli DEP mutS or DEP* strain. A plasmid containing Clover (green marker) containing a UAA stop codon compatible with the biocontained strain DEP, and under the selection marker ampicillin was transformed into both DH5 and DEP* strains to visualize them with the mammalian cells (red). BipA-dependent auxotroph DEP* bacteria were grown to an OD of 0.6 in LB medium supplemented with 1% l-arabinose, 100 M BipA, carbenicillin (100 g/ml), and chloramphenicol (25 g/ml) and then washed three times with 1 phosphate-buffered saline (PBS). DEP* culture conditions with l-arabinose, carbenicillin, and chloramphenicol supplements did slightly affect HEK293T early cell growth compared to untreated cells, although insufficient to affect conclusions drawn from these experiments. DH5 strain was grown to an OD of 0.6 with carbenicillin (100 g/ml). The pellet of 10-ml bacterial cell culture was resuspended in mammalian cell medium as described above without any antibiotics and anti-anti, and split equally among all conditions and their replicates. Auxotroph bacteria are added to HEK293T cells plated in pretreated 12-well plates in 2 ml of mammalian cell medium. The coculture is incubated overnight before the medium that contains the bacterial cells is removed. HEK293T cells were washed three times with 1x PBS (Thermo Fisher Scientific, catalog no. 10010023) and replenished with fresh media as conditions indicate. Media were replaced and added fresh to all conditions daily for 7 days. Imaging of cells was done with the inverted microscope Nikon Eclipse TS100 at days 2, 4, and 7 after initial coculture at 200 magnification.

Conditions:

Control: HEK293T grown in regular 10% FBS media with anti-anti and NEAA as described above.

DH5: HEK293T cells cocultured with this strain in mammalian cell media supplemented with carbenicillin (100 g/ml) to maintain plasmid during growth and absence of anti-anti.

DH5; anti-anti (antibiotic cocktail): HEK293T cells cocultured with this strain in mammalian cell media supplemented with carbenicillin (100 g/ml) to maintain plasmid during growth and presence of anti-anti cocktail.

DH5; anti-anti after day 2: HEK293T cells cocultured with this strain in mammalian cell media supplemented with carbenicillin (100 g/ml) to maintain plasmid during growth and absence of anti-anti cocktail. At 48 hours, anti-anti added and maintained to day 7.

DH5; anti-anti; no anti-anti after day 2: HEK293T cells cocultured with this strain in mammalian cell media supplemented with carbenicillin (100 g/ml) to maintain plasmid during growth and presence of anti-anti until day 2. After day 2, no anti-anti added and maintained to day 7.

DEP*: HEK293T cells cocultured with the biocontained strain in media supplemented with l-arabinose, chloramphenicol (25 g/ml), and carbenicillin (100 g/ml) to maintain bacteria and green marker. No bipA or anti-anti was added.

DEP*; bipA: HEK293T cells cocultured with the biocontained strain in media supplemented with l-arabinose, chloramphenicol (25 g/ml), and carbenicillin (100 g/ml) to maintain bacteria and green marker. One hundred micromolar bipA and no anti-anti added.

DEP*; bipA after day 2: HEK293T cells cocultured with the biocontained strain in media supplemented with l-arabinose, chloramphenicol (25 g/ml), and carbenicillin (100 g/ml) to maintain bacteria and green marker. No bipA or anti-anti added. At 48 hours, bipA at 100 M concentration added and maintained to day 7.

DEP*; anti-anti: HEK293T cells cocultured with the biocontained strain in media supplemented with anti-anti, l-arabinose, chloramphenicol (25 g/ml), and carbenicillin (100 g/ml) to maintain bacteria and green marker. No bipA added.

DEP*; bipA; anti-anti: HEK293T cells cocultured with the biocontained strain in media supplemented with anti-anti, l-arabinose, chloramphenicol (25 g/ml), and carbenicillin (100 g/ml) to maintain bacteria and green marker. One hundred micromolar bipA added.

Persistence was evaluated by two kinds of assays: plate reader and colony count. For the plate reader case, DEP, DEP.e3, DEP.e4, and DEP.e5 cultures were grown overnight in permissible media conditions with 100 M BipA. For cells harvested at midexponential phase, the cultures were diluted 100 and grown to that state. Both stationary-phase and midexponential-phase cultures were then washed twice with LB media and resuspended in the original volume of nonpermissible media containing all specified media components except BipA. The resuspended cultures were then diluted 100 into nonpermissible media in triplicate for each time point to be tested. The specified concentration of BipA was then added back to those cultures at the specified time points. Typically, the BipA readdition occurred at 10 or 5 M concentrations and at hourly or daily intervals. The cultures were then incubated with shaking in SpectraMax i3 plate readers in a flat, clear-bottom 96-well plate with breathable and optically transparent seal for an upward of 84 hours at 34C. Approximately every 5 min, the OD600 was measured to determine cell growth kinetics.

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