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CRISPR technology offers the promise to cure human genetic diseases with gene editing. This promise became a reality when the worlds first CRISPR therapy was approved by regulators to treat patients with sickle cell disease and beta-thalassemia last year.

American biopharma Vertex Pharmaceuticals CASGEVY works by turning on the BCL11A gene, which codes for fetal hemoglobin. While this form of hemoglobin is produced before a baby is born, the body begins to deactivate the gene after birth. As both sickle cell disease and beta-thalassemia are blood disorders that affect hemoglobin, by switching on the gene responsible for fetal hemoglobin production, CASGEVY presents a curative, one-time treatment for patients.

As CASGEVYs clearance is a significant milestone, the technology has come a long way. CRISPR/Cas9 was first used as a gene-editing tool in 2012. Over the years, the technology exploded in popularity thanks to its potential for making gene editing faster, cheaper, and easier than ever before.

CRISPR is short for clustered regularly interspaced short palindromic repeats. The term makes reference to a series of repetitive patterns found in the DNA of bacteria that form the basis of a primitive immune system, defending them from viral invaders by cutting their DNA.

Using this natural process as a basis, scientists developed a gene-editing tool called CRISPR/Cas that can cut a specific DNA sequence by simply providing it with an RNA template of the target sequence. This allows scientists to add, delete, or replace elements within the target DNA sequence. Slicing a specific part of a genes DNA sequence with the help of the Cas9 enzyme, aids in DNA repair.

This system represented a big leap from previous gene-editing technologies, which required designing and making a custom DNA-cutting enzyme for each target sequence rather than simply providing an RNA guide, which is much simpler to synthesize.

CRISPR gene editing has already changed the way scientists do research, allowing a wide range of applications across multiple fields. Here are some of the diseases that scientists aim to tackle using CRISPR/Cas technology, testing its possibilities and limits as a medical tool.

Cancer is a complex, multifactorial disease, and a cure remains elusive. There are hundreds of different types of cancer, each with a unique mutation signature. CRISPR technology is a game-changer for cancer research and treatment as it can be used for many things, including screening for cancer drivers, identifying genes and proteins that can be targeted by cancer drugs, cancer diagnostics, and as a treatment.

China spearheaded the first in-human clinical trials using CRISPR/Cas9 as a cancer treatment. The study tested the use of CRISPR to modify immune T cells extracted from a patient with late-stage lung cancer. The gene-editing technology was used to remove the gene that encodes for a protein called PD-1 that some tumor cells can bind to to block the immune response against cancer. This protein found on the surface of immune cells is the target of some cancer drugs termed checkpoint inhibitors.

CRISPR technology has also been applied to improve the efficacy and safety profiles of cancer immunotherapy, such as CAR-T cell and natural killer cell therapies. In the U.S., CRISPR Therapeutics is one of the leading companies in this space, developing off-the-shelf, gene-edited T cell therapies using CRISPR, with two candidates targeting CD19 and CD70 proteins in clinical trials.

In 2022, the FDA granted Orphan Drug designation to Intellia Therapeutics CRISPR/Cas9-gene-edited T cell therapy for acute myeloid leukemia (AML). Currently, Vor BioPharmas VOR33 is undergoing phase 2 trials to treat AML, and the CRISPR trial is one to watch, according to a report published by Clinical Trials Arena earlier this year.

However, CRISPR technology still has limitations, including variable efficiency in the genome-editing process and off-target effects. Some experts have recommended that the long-term safety of the approach remain under review. Others have suggested using more precise gene-editing approaches such as base editing, an offshoot of CRISPR that hit the clinic in the U.S. last year.

There are several ways CRISPR could help us in the fight against AIDS. One is using CRISPR to cut the viral DNA that the HIV virus inserts within the DNA of immune cells. This approach could be used to attack the virus in its hidden, inactive form, which is what makes it impossible for most therapies to completely get rid of the virus.

The first ever patient with HIV was dosed with a CRISPR-based gene-editing therapy in a phase 1/2 trial led by Excision Biotherapeutics and researchers at the Lewis Katz School of Medicine at Temple University in Philadelphia back in 2022.

The decision to move the therapy to the clinic was bolstered by the success of an analog of the drug EBT-101 called EBT-001 in rhesus macaques infected with simian immunodeficiency virus (SIV). In a phase 1/2 study, EBT-101 was found to be safe.

Another approach could make us resistant to HIV infections. A small percentage of the worlds population is born with a natural resistance to HIV, thanks to a mutation in a gene known as CCR5, which encodes for a protein on the surface of immune cells that HIV uses as an entry point to infect the cells. The mutation changes the structure of the protein so that the virus is no longer able to bind to it.

This approach was used in a highly controversial case in China in 2018, where human embryos were genetically edited to make them resistant to HIV infections. The experiment caused outrage among the scientific community, with some studies pointing out that the CRISPR babies might be at a higher risk of dying younger.

The general consensus seems to be that more research is needed before this approach can be used in humans, especially as recent studies have pointed out this practice can have a high risk of unintended genetic edits in embryos.

Cystic fibrosis is a genetic disease that causes severe respiratory problems. Cystic fibrosis can be caused by multiple different mutations in the target gene CFTR more than 700 of which have been identified making it difficult to develop a drug for each mutation. With CRISPR technology, mutations that cause cystic fibrosis can be individually edited.

In 2020, researchers in the Netherlands used base editing to repair CFTR mutations in vitro in the cells of people with cystic fibrosis without creating damage elsewhere in their genetic code. Moreover, aiming to strike again with yet another win is the duo Vertex Pharmaceuticals and CRISPR Therapeutics, which have collaborated to develop a CRISPR-based medicine for cystic fibrosis. However, it might be a while until it enters the clinic as it is currently in the research phase.

Duchenne muscular dystrophy is caused by mutations in the DMD gene, which encodes for a protein necessary for the contraction of muscles. Children born with this disease experience progressive muscle degeneration, and existing treatments are limited to a fraction of patients with the condition.

Research in mice has shown CRISPR technology could be used to fix the multiple genetic mutations behind the disease. In 2018, a group of researchers in the U.S. used CRISPR to cut at 12 strategic mutation hotspots covering the majority of the estimated 3,000 different mutations that cause this muscular disease. Following this study, Exonics Therapeutics was spun out to further develop this approach, which was then acquired by Vertex Pharmaceuticals for approximately $1 billion to accelerate drug development for the disorder. Currently, Vertex is in the research stage, and is on a mission to restore dystrophin protein expression by targeting mutations in the dystrophin gene.

However, a CRISPR trial run by the Boston non-profit Cure Rare Disease targeting a rare DMD mutation resulted in the death of a patient owing to toxicity back in November 2022. Further research is needed to ensure the safety of the drug to treat the disease.

Huntingtons disease is a neurodegenerative condition with a strong genetic component. The disease is caused by an abnormal repetition of a certain DNA sequence within the huntingtin gene. The higher the number of copies, the earlier the disease will manifest itself.

Treating Huntingtons can be tricky, as any off-target effects of CRISPR in the brain could have very dangerous consequences. To reduce the risk, scientists are looking at ways to tweak the genome-editing tool to make it safer.

In 2018, researchers at the Childrens Hospital of Philadelphia revealed a version of CRISPR/Cas9 that includes a self-destruct button. A group of Polish researchers opted instead for pairing CRISPR/Cas9 with an enzyme called nickase to make the gene editing more precise.

More recently, researchers at the University of Illinois Urbana-Champaign used CRISPR/Cas13, instead of Cas9, to target and cut mRNA that codes for the mutant proteins responsible for Huntingtons disease. This technique silences mutant genes while avoiding changes to the cells DNA, thereby minimizing permanent off-target mutations because RNA molecules are transient and degrade after a few hours.

In addition, a 2023 study published in Nature went on to prove that treatment of Huntingtons disease in mice delayed disease progression and that it protected certain neurons from cell death in the mice.

With CASGEVYs go-ahead to treat transfusion-dependent beta-thalassemia and sickle cell disease in patients aged 12 and older, this hints that CRISPR-based medicines could even be a curative therapy to treat other blood disorders like hemophilia.

Hemophilia is caused by mutations that impair the activity of proteins that are required for blood clotting. Although Intellia severed its partnership with multinational biopharma Regeneron to advance its CRISPR candidate for hemophilia B a drug that was recently cleared by the FDA to enter the clinic the latter will take the drug ahead on its own.

As hemophilia B is caused by mutations in the F9 gene, which encodes a clotting protein called factor IX (FIX), Regenerons drug candidate uses CRISPR/Cas9 gene editing to place a copy of the F9 gene in cells in order to get the taps running for FIX production.

The two biopharmas will continue their collaboration in developing their CRISPR candidate to treat hemophilia A, which manifests as excessive bleeding because of a deficit of factor VIII. The therapy is currently in the research phase.

While healthcare companies were creating polymerase chain reaction (PCR) tests to screen for COVID-19 in the wake of the pandemic, CRISPR was also being put to use for speedy screening. A study conducted by researchers in China in 2023, found that the CRISPR-SARS-CoV-2 test had a comparable performance with RT-PCR, but it did have several advantages like short assay time, low cost, and no requirement for expensive equipment, over RT-PCRs.

To add to that, the gene editing tool could fight COVID-19 and other viral infections.

For instance, scientists at Stanford University developed a method to program a version of the gene editing technology known as CRISPR/Cas13a to cut and destroy the genetic material of the virus behind COVID-19 to stop it from infecting lung cells. This approach, termed PAC-MAN, helped reduce the amount of virus in solution by more than 90 percent.

Another research group at the Georgia Institute of Technology used a similar approach to destroy the virus before it enters the cell. The method was tested in live animals, improving the symptoms of hamsters infected with COVID-19. The treatment also worked on mice infected with influenza, and the researchers believe it could be effective against 99 percent of all existing influenza strains.

As European, U.S., and U.K. regulators have given their stamp of approval for the first-ever CRISPR-based drug to treat patients, who is to say we wont see another CRISPR-drug hitting this milestone in the near future.

And apart from the diseases mentioned, CRISPR is also being studied to treat other conditions like vision and hearing loss. In blindness caused by mutations, CRISPR gene editing could eliminate mutated genes in the DNA and replace them with normal versions of the genes. Researchers have also demonstrated how getting rid of the mutations in the Atp2b2 and Tmc1 genes helped partially restore hearing.

However, one of the biggest challenges to turn CRISPR research into real cures is the many unknowns regarding the potential risks of CRISPR therapy. Some scientists are concerned about possible off-target effects as well as immune reactions to the gene-editing tool. But as research progresses, scientists are proposing and testing a wide range of approaches to tweak and improve CRISPR in order to increase its efficacy and safety.

Hopes are high that CRISPR technology will soon provide a way to address complex diseases such as cancer and AIDS, and even target genes associated with mental health disorders.

New technologies related to CRISPR research:

This article was originally published in June 2018, and has since been updated by Roohi Mariam Peter.

Read more here:
Seven diseases that CRISPR technology could cure - Labiotech.eu

The global genetic analysis market was evaluated at USD 10.55 billion in 2023 and is expected to attain around USD 23.60 billion by 2033, growing at a CAGR of 8.39% from 2024 to 2033. The increasing demand for genetic testing services is driving growth within the genetic analysis market.

Market Overview

The genetic analysis market is experiencing significant transformation due to advances in genetic technology, which are fundamentally changing perceptions and practices within the healthcare industry. At the heart of this transformation lies the process of genetic analysis, which involves the examination of DNA samples to identify mutations that may influence disease susceptibility or treatment response. This analysis is pivotal for understanding the structure and function of genes, with techniques such as gene cloning playing a crucial role in isolating and replicating specific genes for detailed examination.

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One notable aspect of genetic analysis is its diverse clinical applications. It serves as a diagnostic tool, aiding in the confirmation of diagnoses in symptomatic individuals, while also facilitating the monitoring of disease prognosis and treatment response. Additionally, genetic analysis enables predictive or predisposition testing, allowing for the identification of individuals at risk of developing certain diseases before symptoms manifest.

The emergence of predictive genetic testing is creating new market opportunities, as it enables proactive disease prevention strategies and early interventions. As perceptions regarding genetic testing continue to evolve, the market for genetic analysis is expected to witness sustained growth, driven by its potential to revolutionize patient care and improve health outcomes.

Key Insights

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North America to sustain its position in the upcoming years with the U.S. being largest contributor

In 2023, North America emerged as the dominant force in the genetic analysis market, particularly in the United States. The US showcased a robust infrastructure with 200 laboratories actively conducting 37,124 clinical tests, underscoring the region's significant investment and adoption of genetic analysis technologies. Notably, 29 laboratories specialized in whole exome sequencing (WES), while 17 laboratories focused on whole genome sequencing (WGS), indicating a wide array of genetic testing capabilities available within the country.

The United States exhibits a proactive approach towards healthcare, as evidenced by mandatory newborn screening programs targeting a specific set of genetic diseases. Although the exact set of diseases screened may vary from state to state, the emphasis remains on conditions where early diagnosis is crucial for effective treatment or prevention strategies. This regulatory framework underscores the importance placed on leveraging genetic analysis for proactive healthcare management and disease prevention initiatives.

Beyond clinical applications, genetic analysis in North America extends to ecological and environmental contexts. The presence of invasive species such as Phragmites australis subsp. australis poses ecological challenges across multiple regions. The co-occurrence of this invasive subspecies with native counterparts and instances of hybridization necessitates precise differentiation methods for effective management strategies. Genetic analysis plays a pivotal role in distinguishing between phragmites subspecies or haplotypes, facilitating targeted management efforts to mitigate ecological harm and preserve native ecosystems.

Asia Pacific to witness lucrative opportunities in the upcoming years

Asia Pacific emerges as a pivotal region poised for substantial growth in the genetic analysis sector, driven by dynamic developments in genetic counselling and genome mapping initiatives. Forecasts indicate that Asia Pacific will experience the fastest growth rate in the genetic analysis market during the forecast period, underscoring the region's significance in shaping the future of genetic healthcare services.

A recent milestone in the region's genetic counselling landscape is the establishment of the Professional Society of Genetic Counsellors in Asia (PSGCA). Formed as a special interest group of the Asia Pacific Society of Human Genetics, PSGCA aims to spearhead the advancement and integration of the genetic counselling profession across Asia. With a vision to become the premier organization driving genetic counselling mainstream adoption in the region, PSGCA endeavors to ensure equitable access to genetic counselling services for individuals. Its mission centers on elevating standards of practice, curriculum, research, and continuing education to promote quality genetic counselling services throughout Asia.

The rapid evolution of genetic and genomic technologies has significantly transformed healthcare services in low- and middle-income countries (LMICs) across the Asia-Pacific region. Initially focused on population-based disease prevention strategies, genetic services have transitioned towards clinic-based and therapeutics-oriented approaches. Notably, the region's genetic diversity, exemplified by populous and genetically varied countries such as China, India, Japan, and Indonesia, positions them as prime candidates for genome mapping research endeavors.

How the genetic analysis market in Asia Pacific

Report Highlights

By Product

The reagents & kits segment asserted dominance in the genetic analysis market in 2023. DNA reagents play a pivotal role in various DNA-related processes and techniques, including sequencing, synthesis, cloning, and mutagenesis. These products encompass a diverse range, such as plasmids, buffers, labeling technology, columns, and comprehensive test kits utilized in DNA testing, including direct-to-consumer (DTC) genetic tests. While offering accessible information about the scientific basis of tests, the usage of DTC genetic tests carries inherent risks due to the absence of personalized guidance concerning the results.

The instruments segment emerged as the fastest-growing sector within the genetic analysis market. Core laboratory instruments constitute essential tools in genetic engineering research, facilitating precise and reliable experimentation. Polymerase Chain Reaction (PCR) machines, also known as thermal cyclers, stand as indispensable equipment in genetic engineering labs, enabling the amplification of specific DNA segments crucial for detailed analysis.

By Test

In 2023, the disease diagnostic testing segment emerged as the dominant force in the genetic analysis market. This segment specializes in identifying whether individuals harbor specific genetic diseases by detecting alterations in particular genes. While these tests excel at pinpointing gene mutations, they often fall short in determining disease severity or age of onset. Thousands of diseases stem from mutations in a single gene, making diagnostic testing pivotal in confirming or ruling out genetic diseases and chromosomal abnormalities. Frequently utilized during pregnancy or when symptomatic, diagnostic genetic testing offers crucial insights for accurate diagnosis and timely intervention.

The prenatal and newborn testing segment emerged as the fastest-growing sector in the genetic analysis market during the forecast period. Prenatal genetic testing provides prospective parents with vital information regarding potential genetic disorders in the fetus. Prenatal screening tests assess the likelihood of fetal aneuploidy and select disorders, while prenatal diagnostic tests definitively ascertain the presence of specific disorders. These tests, conducted on fetal or placental cells obtained through procedures like amniocentesis or chorionic villus sampling (CVS), play a pivotal role in informed decision-making during pregnancy.

Newborn screening, a subset of prenatal and newborn testing, comprises a set of laboratory tests performed on newborns to detect known genetic diseases. Typically conducted via a heel prick within the first few days of life, newborn screening enables early identification and intervention for treatable genetic conditions, thereby improving health outcomes. As the demand for early detection and preventive measures rises, the prenatal and newborn testing segment is poised for continued growth, bolstering the comprehensive landscape of genetic analysis.

By Technology

In 2023, the real-time PCR system segment emerged as the dominant force in the genetic analysis market. Real-time PCR (RT-PCR) systems offer unparalleled capabilities for quantitative genotyping and detection of single nucleotide polymorphisms (SNPs), allelic discrimination, and genetic variations even in samples with minimal mutation carriers. Multiplex PCR systems, a subset of RT-PCR, are gaining prominence, particularly in plant/microbe associations, where standard PCR methods prove inadequate. Multiplex RT-PCR facilitates the identification of multiple genes through the utilization of fluorochromes and analysis of melting curves, providing enhanced accuracy and efficiency in genetic analysis.

The next-generation sequencing (NGS) segment emerged as the fastest-growing sector in the genetic analysis market. NGS technology revolutionizes DNA sequencing and RNA sequencing and variant/mutation detection by enabling high-throughput sequencing of hundreds to thousands of genes or whole genomes within a short timeframe. The sequence variants/mutations detected by NGS hold profound implications for disease diagnosis, prognosis, therapeutic decision-making, and patient follow-up, paving the way for personalized precision medicine initiatives.

By Application

In 2023, the infectious diseases segment asserted dominance in the genetic analysis market, offering molecular genetic tests capable of identifying common viruses or bacteria responsible for respiratory infections and infectious diarrhea. These tests, conducted on samples collected from the nose and throat or a single stool sample, facilitate rapid and accurate diagnosis, enabling timely treatment and containment of infectious outbreaks.

The genetic diseases segment emerged as the fastest-growing sector in the genetic analysis market during the forecast period. The extent to which genes contribute to diseases varies, presenting opportunities for advancements in understanding genetic mechanisms underlying various conditions. This progress facilitates the development of early diagnostic tests, novel treatments, and preventive interventions to mitigate disease onset or severity.

By End Use

In 2023, the research & development laboratories segment emerged as the dominant force in the genetic analysis market, actively driving advancements in genetic disease study and testing technology. These laboratories are pivotal in enhancing clinical patient care by conducting rigorous research and development activities aimed at improving test strategies and introducing novel genetic tests. Board-certified directors and genetic counsellors collaborate closely with laboratory supervisors and technologists to ensure the delivery of accurate and reliable results within stipulated timelines. With a focus on meeting stringent validation standards, approved tests undergo thorough evaluations of methodology and clinical utility. Research programs within these laboratories leverage collective expertise to propel the field of genetics and genetic testing forward.

The diagnostic centers segment is poised for significant growth in the genetic analysis market during the forecast period. Diagnostic centers offer a comprehensive range of testing services crucial for diagnosing diverse medical conditions. By providing accurate and informed diagnoses, diagnostic centers enable physicians to develop effective treatment plans, ultimately enhancing patient outcomes. Leveraging advanced diagnostic technologies and techniques, these centers play a vital role in identifying underlying causes of diseases, monitoring disease progression, and devising personalized treatment approaches. Collaborating with healthcare providers like primary care physicians, specialists, and hospitals, diagnostic centers ensure accurate and timely diagnoses across a spectrum of medical conditions, reinforcing their indispensable role in modern healthcare delivery.

Market Dynamics

Driver: Advances in Genetic Sequencing and Gene Therapy

Significant strides in genetic sequencing, human genome analysis, and medical genetics have revolutionized disease understanding, diagnostic accuracy, and drug development targets. A pivotal breakthrough in medical genetics is the emergence of gene therapy, which involves modifying or replacing genes to treat or prevent diseases. Already applied successfully in treating conditions like inherited blindness and severe combined immunodeficiency (SCID), gene therapy is poised to expand its impact further.

Future projections indicate that gene therapy will play an increasingly vital role in medical genetics, offering treatments for previously untreatable diseases. This trajectory is expected to fuel the growth of the genetic analysis market, as the demand for advanced genetic testing and analysis escalates to support the development and implementation of gene therapy treatments.

Restraint: Privacy Concerns in Genetic Analysis

Privacy concerns poses a major challenge in the genetic analysis domain due to the inherent uniqueness of genomic data, hindering true anonymization efforts. Additionally, security measures are crucial to restrict access to data based on authorized clearance levels, safeguarding against unauthorized breaches. Confidentiality emerges as a key ethical consideration, dictating the responsible sharing of genetic data. These privacy concerns, among others, including consent and data ownership, serve as significant restraints in the genetic analysis market. Addressing these challenges effectively is essential to ensure ethical practices and foster trust among stakeholders, thereby mitigating the barriers to market growth.

Opportunity: Integration of Artificial Intelligence in Genetic Analysis

The integration of artificial intelligence (AI) is revolutionizing clinical genetics, offering unprecedented opportunities for advancement. AI algorithms possess the capability to analyse vast volumes of genetic data rapidly and accurately, facilitating more precise diagnoses and tailored treatment plans. Furthermore, AI empowers predictive analysis of disease risk, enabling the development of proactive disease prevention strategies. In genetic engineering and gene therapy research, AI serves as a powerful tool, aiding in hypothesis generation and experimental techniques. Leveraging AI, researchers can detect hereditary and gene-related disorders with greater efficiency.

Moreover, AI-driven developments hold immense promise for rational drug discovery and design, ultimately impacting humanity's well-being. As AI and machine learning (ML) technologies continue to drive innovation in drug development, genetics emerges as a prime beneficiary, with AI expected to influence every facet of the human experience. This presents a compelling opportunity for the genetic analysis market to capitalize on AI-driven advancements and propel transformative growth.

Recent Developments

Key Players in the Clinical Trials Market

Segments Covered in the Report

By Product

By Test

By Technology

By Application

By End-use

By Geography

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Genetic Analysis Market Size to Attain Around USD 23.60 BN by 2033 - BioSpace

Genes affected by germline structural variation could conceivably influence cancer risk.

Researchers at Baylor College of Medicines Dan L Duncan Comprehensive Cancer Center and Human Genome Sequencing Center investigated the extent to which forms of genetic variation called germline or inherited structural variation (SV) influence gene expression in human cancers.

Structural variation is one type of genomic variation and can be beneficial, neutral or, if it affects functionally relevant regions of the genome, can seriously affect gene function and contribute to disease, including cancer, said corresponding author Dr. Chad Creighton, professor ofmedicineand co-director of cancer bioinformatics at theDan L Duncan Comprehensive Cancer Centerat Baylor.

Structural variations are larger differences in the genome that occur when a piece of DNA is duplicated, deleted, or switched around, which can impact genetic instructions encoded in DNA and affect the expression of nearby genes. Previous studies led by the researchers have shown that structural variations occurring in specific cell types, like breast cells, can strongly influence gene expression in ways that contribute to transforming a healthy breast cell into a cancer cell.

Its known that germline structural variation also can contribute to the molecular profile of cancers, Creighton said. Here we study the extent of its contribution. The study is published in Cell Reports Medicine.

The researchers worked with data developed by the Pan-Cancer Analysis of Whole Genomes consortium, which includes whole genome sequencing data from 2,658 cancers across 38 tumor types involving 20 major tissues of origin. The team integrated these data with RNA data to identify genes whose expression was associated with nearby germline structural variations.

We found most of the genes associated with germline structural variations would not necessarily have specific roles in cancer, but for some genes, the expression variation might be associated with other conditions, Creighton said.

At the same time, several genes affected by germline structural variation could conceivably contribute to cancer, for instance if these genes have an established cancer association or an association with patient survival.

This study shows that germline structural variation would represent a normal class of genetic variation passed down through generations and may play a significant role in cancer development. The researchers propose that the subset of genes with cancer-relevant associations arising in this study would represent strong candidates for further investigation on their value in genetic testing.

Fengju Chen, Yiqun Zhang and Fritz J. Sedlazeck also contributed to this work.

This study was supported by the National Institutes of Health grant P30CA125123.

By Ana Mara Rodrguez, Ph.D.

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Genetic variation passed down through generations may influence cancer development - Baylor College of Medicine | BCM

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Healing the Heart: The Role of Stem Cells in Cardiac Care - State Times

Patient characteristics

The baseline characteristics of the study population are presented in Table1. A total of 8764 patients were included, from which 7725 (88%) received rATG, and 1039 (12%) received PTCy as GVHD prophylaxis.

Overall, the majority of patients were transplanted for acute leukemia (58%), myelodysplastic syndrome (MDS) (19.7%), myeloproliferative neoplasm (MPN) (9.7%) or lymphoma (9%). A high proportion of patients had a low/intermediate Disease Risk Index (DRI, 72.1%), and myeloablative conditioning (MAC) was more frequently performed (53.3%) than reduced intensity conditioning (RIC).

Patients in the rATG group were older, with a median age of 58.6 years (IQR (48.1, 65.4)) vs. 53 years in the PTCy group (IQR 38.6, 62.3) (p<0.01), with a similar proportion of males (57.3% in rATG vs. 58.9% in PTCy, p=0.33), along with a significantly lower use of TBI (14.5% vs. 24.7%, p<0.01) and lower use of MAC (52% vs. 62.3%, p<0.01). Also, the disease relapse index was lower and the year of transplant was more recent in the PTCy group (Table1). The remaining parameters were balanced between the two groups. Median follow up was 2.1 years in both arms. More detailed information is given in Table1.

Univariate outcomes are shown in Figs.1, 2and Table2. The results of the multivariate analyses are summarized in Table3. The P-values and hazard ratios (HR) presented in the following results section are derived from the multivariate analysis.

A NRM; B Overall survival, C Relapse incidence, D Progression-free survival and E GVHD-free relapse-free survival. Cumulative incidences are shown.

A Acute GVHD grades IIIV; B Acute GVHD grades IIIIV, C Chronic GVHD all grades and D Extensive chronic GVHD - Cumulative incidences are shown.

Patients receiving PTCy had a significantly lower NRM as compared to patients receiving rATG (2y incidence: 12.4% vs. 16.1%; HR: 0.72 [95% CI 0.550.94], p=0.016). Similarly, OS and PFS showed a statistically significant and clinically meaningful benefit for PTCy arm, with a higher OS (2y incidence: 73.9% vs. 65.1%; HR: 0.82 [95% CI 0.720.92], p=0.001), and a higher PFS (2y incidence: 64.9% vs. 57.2%; HR: 0.83 [95% CI 0.740.93], p<0.001). RI was lower in the PTCy arm (2y incidence: 22.8% vs. 26.6%; HR: 0.87 [95% CI 0.751.00], p=0.046).

The causes of death are given in Table4. No major differences between the two groups were apparent. Relapse of the underlying malignancy was the most frequent cause of death, accounting for ~50% of total deaths in both arms, followed by NRM causes: infections ~18%, GVHD~16% and other alloSCT-related causes ~8% of total deaths. Secondary malignancies contributed to approximately 1% of total deaths.

Overall chronic GVHD was lower in the PTCy group (2y incidence: PTCy 28.4% vs. rATG 31.4%; HR: 0.77 [95% CI 0.630.95], p=0.012). Extensive chronic GVHD was also reduced in patients receiving PTCy vs. rATG: (2y incidence: 11.9% vs. 13.5%; HR: 0.75 [95% CI 0.620.91], p=0.004).

The incidence of acute GVHD grades II-IV in patients receiving PTCy, compared to those receiving ATG was not statistically significant: (100d incidence: 24.1% vs. 26.5%; HR: 0.85 [95% CI 0.691.04], p=0.11). Similarly, for severe acute GVHD grades IIIIV (100d incidence: 8.7% vs. 9.7%; HR: 0.76 [95% CI 0.551.05], p=0.091).

GRFS was significantly higher in the PTCy arm compared to the rATG arm (2y incidence: 51% vs. 45%; HR: 0.86 [95% CI 0.750.99], p=0.035).

The EBMT Database does not contain data on graft failure/rejection. To get insight into the initial grafts success and any subsequent requirement for additional transplantation procedures, we investigated neutrophil recovery after the first alloSCT as well as the incidence of a second alloSCT. The median incidence of neutrophil recovery at days +30 and +60 in the ATG vs. PTCy groups was: d+30 ATG 96% (IC95% 95.596.4) vs. PTCy 91% (8992.7) and d+60 ATG 97.9% (97.698.2) vs. PTCy 97.4% (96.298.3). The median incidence of a second alloSCT at 2 years was 4.3% (3.84.8) in the ATG group and 3.2% (2.24.6) in the PTCy group.

Continue reading here:
ATG or post-transplant cyclophosphamide to prevent GVHD in matched unrelated stem cell transplantation? | Leukemia - Nature.com

While ex vivo CD34-selected allogeneic hematopoietic stem cell transplants (HCTs) are promising treatments for patients with hematologic and myeloid malignancies, they can be limited by delayed immune recovery and increased risk of death not caused by relapse.

A late-breaking abstract presented at the 2024 Transplantation and Cellular Therapy Tandem Meetings investigated a new approach to allogeneic HCT. Investigators of the phase 2 PRAISE-IR study (NCT04872595) explored using a model-based approach to determine the optimal dose of antithymocyte globulin (ATG), which is used to prevent graft-vs-host disease after transplant. Previous studies suggested high ATG exposure might contribute to nonrelapse mortality.

According to Michael Scordo, MD, the model successfully achieved a low posttransplant ATG exposure, and immune reconstitution by day 100 was achieved in 69% of patients, meeting the studys primary end point. Further, the 2-year rates of nonrelapse mortality and relapse were 9% and 13%, respectively, and relapse-free survival and overall survival rates were high at 78% and 86%, respectively.

These findings suggest that using a model to determine the ATG dose for ex vivo CD34-selected allogeneic HCT can lead to improved immune reconstitution and excellent survival outcomes. This approach may help reduce nonrelapse mortality previously observed in other trials and improve the safety and effectiveness of this type of transplant.

In an interview with Targeted OncologyTM, Scordo, bone marrow transplant specialist and cellular therapist at Memorial Sloan Kettering Cancer Center in New York, New York, discussed the findings from this study and their implications for the allogeneic HCT treatment landscape.

Targeted Oncology: What was the rationale or inspiration for the study you presented at the Tandem Meetings?

Scordo: Ex vivo CD34-selected [allogeneic] transplant is one of the many methods of reducing graft-vs-host disease. It uses a myeloablative conditioning platform and integrates ATG, antithymocyte globulin, into that platform to help reduce the risk of rejection. This has been well studied over the years, but 1 of the downsides of this approach is the delayed immune recovery, particularly the T-cell immune recovery that occurs after [allogeneic] transplant with this approach. What we did based on a recent publication that we have from last year was we used a different dosing of ATG to ensure that the T-cell immune recovery after [allogeneic] transplant using ex vivo CD34 selection would be improved.

What are some of the unmet needs in this space?

There are many methods to reduce graft-vs-host disease after transplant CD34 selection. Many of the other methods including posttransplant cyclophosphamide [PTCy], which has now become a standard of care, are out there and should be used in the appropriate setting. In matched donor transplants, ex vivo CD34 selection is one of the methods of being able to use an ablative or intensive conditioning regimen with very low rates of particularly chronic graft-vs-host disease. We saw this as an opportunity to improve on this platform significantly, using a novel approach but a simple approach.

What were the goals of this study?

The primary end point of the study was the ability to improve the CD34 T-cell immune recovery by day 100 after transplant. This was a sort of a validated predictor in other studies. We had key secondary end points that included nonrelapse mortality, relapse rates, progression-free, and overall survival. With the primary end point, we exceeded that end point. With our trial, about 70% of our 56 patients achieved this appropriate immune recovery by day 100, which was significantly higher than our historical numbers had shown.

What were some of the other findings?

Aside from achieving the primary end point, we saw very low rates of nonrelapse mortality at 2 years, estimated at 8%, which is much lower than some of the previously published data using this platform in the last couple of years. [We also saw] low relapse rates [of] about 12% at 2 years and very favorable progression-free and overall survival, which was 80% and 87%, respectively, at 2 years.

What are some of the takeaways?

I look at this as a simple but novel approach to improving on a platform. We have existing platforms that work well, but we can improve them doing well. To community oncologists, I would say that for patients with myeloid malignancies, there are many different types of transplants that can be done safely and effectively. The appropriate choice of a platform really depends on many factors. We can improve on all these platforms individually, including PTCy. [For] ex vivo CD34 selection, I look at this as a method of just improving on what we have already shown to be an effective platform, being able to use dose-intensive chemotherapy or total body radiation to achieve maximal disease control but making the platform safe and tolerable.

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New Allo-HCT Approach Boosts Immune Response, Survival - Targeted Oncology

Scientists reversed some signs of immune aging in mice with a new treatment that could one day potentially be used in humans.

The new immunotherapy works by disrupting a natural process by which the immune system becomes biased towards making one type of cell as it ages.

The mouse study is an "important" proof-of-concept, but it's currently difficult to gauge the significance of the findings, Dr. Janko . Nikolich-Zugich, a professor of immunobiology at the University of Arizona who was not involved in the research, told Live Science in an email. More work is needed to see how well the therapy shifts the immune system into a more youthful, effective state.

All blood cells, including immune cells and the red blood cells that carry oxygen around the body, start life as hematopoietic stem cells (HSC) in the blood and bone marrow, the spongy tissue found within certain bones. HSCs fall into two main categories: those destined to become so-called myeloid cells and those that will develop into lymphoid cells.

Myeloid cells include red blood cells and immune cells belonging to our broadly reactive first line of defense against pathogens, including cells called macrophages that trigger inflammation. Lymphoid cells include cells that develop a memory of germs, such as T and B cells.

Related: 'If you don't have inflammation, then you'll die': How scientists are reprogramming the body's natural superpower

As we age, the HSCs slated to become myeloid cells gradually increase in number and eventually outnumber the lymphoid stem cells. This means we can't respond to infections as well when we're older as when we're young, and we're more likely to experience chronic inflammation triggered by increasing levels of myeloid cells that trigger inflammation.

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In the new study, published Wednesday (March 27) in the journal Nature, scientists developed an antibody-based therapy that selectively targets and destroys the myeloid HSCs, thus restoring the balance of the two cell types and making the blood more "youthful." The antibodies latch onto the targeted cells and flag them to be destroyed by the immune system.

The authors injected the therapy into mice aged 18 to 24 months, or roughly the equivalent of being between 56 and 69 years old as a human.

They then extracted HSCs from the mice after treatment and analyzed them, revealing the rodents had a smaller percentage of the myeloid HSCs than untreated mice of the same age.

This effect lasted for two months. Compared with untreated mice, the treated mice also produced more naive T cells and mature B cells. These cells can go on to form memory cells, which are directly involved in the immune attack; in the case of the B cells, they can form antibody-producing plasma cells.

"Not only did we see a shift toward cells involved in adaptive immunity, but we also observed a dampening in the levels of inflammatory proteins in the treated animals," Dr. Jason Ross, lead study author and postdoctoral researcher at Stanford University, said in a statement. Specifically, the researchers saw that the levels of one proinflammatory protein fell in the treated mice. This protein, called IL-1beta, is mainly made by myeloid cells.

Eight weeks post-treatment, the researchers vaccinated the mice against a virus they'd never been exposed to before. The mice that had received the immunotherapy had more apt immune responses to vaccination than the untreated mice, producing more T cells against the germ.

"We believe that this study represents the first steps in applying this strategy in humans," Ross said. However, other experts have cautioned against jumping to conclusions.

Nikolich-Zugich noted that, although the researchers measured changes in the numbers of naive T cells in the mice, they didn't look at the function of the organ that makes them: the thymus. The team also saw reductions only in IL-1beta and not other inflammatory proteins. They also didn't test whether the mice's baseline immunity to new infections could be improved with this therapy, without vaccination, he said.

Furthermore, the study didn't consider potential long-term side effects of the treatment, such as anemia, or a deficiency in red blood cells, said Dr. Ilaria Bellantuono, a professor in musculoskeletal aging at the University of Sheffield in the U.K. who was not involved in the research.

Although an "interesting" study, more work is needed to understand whether it can bring "meaningful changes" in the immune system, Bellantuono told Live Science in an email, whether that of mice or humans.

Ever wonder why some people build muscle more easily than others or why freckles come out in the sun? Send us your questions about how the human body works to community@livescience.com with the subject line "Health Desk Q," and you may see your question answered on the website!

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New immunotherapy could make blood more 'youthful,' mouse study hints - Livescience.com

Theres a universal fact for women. If they live long enough, their capacity to bring forth children will end, and they will become menopausal. Menopause can be when the thermostat becomes their most prized possession.

But not all women have hot flashes. Some go through this period wondering why they have no symptoms. The best advice for them is, Enjoy the smooth sailing!

Other women endure needless suffering. There are treatments, and these women should see their doctors.

The medical journal The Lancet has urged women to become educated about hormone replacement therapy. Menopause should not be considered a disease. It is a natural process. Be cautious with commercial interests of pharmaceutical companies propaganda. Seek information from a medical specialist.

The authors of The Lancet report stress they are not opposed to HRT, as it can be effective in treating hot flashes, vaginal dryness, and genital urinary symptoms. Many years ago, HRT was often used by women to control menopausal symptoms. The standard treatment involved the hormones estrogen and progestin, a synthetic form of progesterone.

But a large and widely publicized study called the Womens Health Initiative identified problems with HRT. Doctors and patients concluded HRT was dangerous, and this misconception lingers today. The study had significant shortcomings, however, and subsequent studies have more nuanced conclusions. For women under 60, or for those less than a decade out of menopause, the benefits of HRT in fighting debilitating symptoms outweighed the risk. There was one other caution. Those using HRT should not have a family history of stroke, breast cancer, or coronary heart disease.

Which women suffer the most from menopause? Its those who are affected by severe symptoms. Imagine a stalwart high school principal. She has handled the tough job for years. But with the onset of menopause, the slightest provocation has her bursting into tears behind closed doors. For the first time, she feels incapable of the task. If she meets the criteria mentioned above, then she is a textbook case for HRT. Within a week, her problem would be history.

Menopause is not just one event or one symptom, such as hot flashes. A gradual decrease in the production of estrogen influences organs such as the vagina and urinary bladder. Its these organs that women are loath to discuss with their family doctor, to say nothing of their partners.

It may come as a shock to younger people to know that seniors have sexual relations. But menopause can make vaginal tissues thinner and more easily irritated. Past columns have tried to explain this with a touch of eloquence, noting that its hard for females to sing with a sore throat. Put plainly, its hard for menopausal and post-menopausal women to enjoy sex with an inflamed vagina (atrophic vaginitis). Sometimes neither the woman nor her partner knows whats causing the severe pain. Unfortunately, many women suffer silently.

Those who ask for help will find there are good remedies. Something as simple as an estrogen cream can resolve an irritated vagina within two weeks. Other consequences of menopause, like the accelerated loss of bone density, may also be treated with HRT.

Sometimes problems are missed because a vaginal examination is not done during a check-up. Or patients dont mention issues to the doctor.

The comedian Joan Rivers made a joke about news that having a dog makes you 10 years younger. My first thought was to rescue two more, she said, before adding, but I dont want to go through menopause again.

Today, women can and should get their symptoms treated.

Dr. W. Gifford-Jones, aka Ken Walker, is a graduate of the University of Toronto and Harvard Medical School. You can reach him online at his website, docgiff.com, or via email at contact-us@ docgiff.com. Follow him and his daughter on Instagram @docgiff and @diana_gifford_jones.

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The Doctor Game: What women suffer most from menopause? - The Westerly Sun

One of the most significant challenges in treating HIV is the virus ability to integrate its genome into the hosts DNA. This means that lifelong antiretroviral therapy is essential as latent HIV can reactivate from reservoirs as soon as treatment ends.

One potential technique being developed to address this problem is the use of gene editing technology to cut out and incapacitate HIV from infected cells. Currently, there is a Phase I/II Clinical Trial underway in people with HIV-1 (the most common strain of HIV)

Now, new research from another team shows that gene editing can be used to eliminate all traces of the HIV virus from infected cells in the laboratory.

The research is being presented early ahead of the European Congress of Clinical Microbiology and Infectious Diseases, which will be held from 27-30 April in Barcelona, Spain. Its been carried out by scientists from the Amsterdam Medical University in the Netherlands, and the Paul Ehrlich Institute in Germany, and has not yet been submitted for peer review.

Our aim is to develop a robust and safe combinatorial CRISPR-Cas regimen, striving for an inclusive HIV cure for all that can inactivate diverse HIV strains across various cellular contexts, they write in a conference abstract submitted ahead of ECCMID.

CRISPR-Cas gene editing technology acts like molecular scissors to cut DNA and either delete unwanted genes or introduce new genetic material, while guidance RNA (gRNA) tells CRISPR-Cas exactly where to cut at designated spots on the genome.

In this research, the authors used 2 gRNAs that target conserved parts of the viral genome this means they remain the same or conserved across all known HIV strains. This genetic sequence does not have a match in human genes, to prevent the system going off target and causing mutations elsewhere in the human genome.

The hope is to one day provide a broad-spectrum therapy capable of combating multiple HIV variants effectively. But before this dream can become a reality, the researchers had to address a number of issues with getting the CRISPR-Cas reagents into the right cells.

To delivered CRISPR components into cells in the body a viral vector, containing genes that code for the CRISPR-Cas proteins and gRNA, is used. This is the vehicle that delivers into the host cell the instructions to make all necessary components, but these instructions need to be kept as simple and short as possible.

Another issue is making sure the viral vector enters HIV reservoir cells specifically cells that express the receptors CD4+ and CD32a+ on their surface.

They found that in one system, saCas9, the vector size was minimised, which enhanced its delivery to HIV-infected cells. They also included proteins that target the CD4+ and CD32a+ receptors specifically in the vector.

This system showed outstanding antiviral performance, managing to completely inactivate HIV with a single guide RNA (gRNA) and excise (cut out) the viral DNA with two gRNAs in cells in the lab.

We have developed an efficient combinatorial CRISPR-attack on the HIV virus in various cells and the locations where it can be hidden in reservoirs and demonstrated that therapeutics can be specifically delivered to the cells of interest, the authors write.

These findings represent a pivotal advancement towards designing a cure strategy.

But the researchers stress that, while these preliminary findings are very encouraging, it is premature to declare that there is a functional HIV cure on the horizon.

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How CRISPR-Cas genome editing could be used to cure HIV - Cosmos

CRISPR gene editing technology is beginning to deliver on a promise to quickly create crops with traits that withstand a changing climate, resist aggressive pests and reinvigorate healthy soils, according to experts at the South by Southwest event in Austin earlier this month.

Companies exploring CRISPR to make climate-friendly foods and medicines are enjoying some tailwinds:

At the same time, startups and researchers are taking on investment partnerships with larger organizations to commercialize CRISPR innovations. Bayer has a project with Pairwise to create a corn crop that is more resilient to environmental factors. In 2011, The Gates Foundation gave a $10.3 million grant to the International Rice Research Institute (IRRI) and has re-invested more than $16 million to the organization in 2023 to create climate resistant rice varieties.

The past 200 years of industrialized agriculture have increased yields and eased shipping with large, durable produce often to the detriment of the soil, the planet and taste.

"We think with gene editing you wont have to make that choice," said Tom Adams, CEO of Pairwise. The startup is producing the first CRISPR consumer product by editing out the wasabi-like spiciness of a mustard green to make it more palatable to eaters.

Pairwise sold the green at a New York grocer earlier this year and is seeking to partner with companies to sell to consumers. The companys main focus is developing business-to-business markets by selling ingredient crops or seeds to big agricultural companies or seed banks.

Traditionally, farmers mated or cross-pollinated organisms to augment their desired characteristics. It could take decades to cultivate a plant to the desired enhancement for human consumption.

In the 1970s, scientists began genetically modifying organisms (GMOs) by cultivating foreign DNA in a bacteria or virus and then inducing those cells to add their modified DNA into a plant or animal. The modified DNA would typically offer resistance to pests or diseases.

CRISPR opens up new possibilities to modify crops by knocking out or enhancing genes that are already present. "Its more precise, and more accurate and more intuitive than breeding," said Elena Del Pup, a plant genetics researcher at Wageningen University in the Netherlands. "[It] allows us to make very specific edits."

"The hope and the promise of [CRISPR] is that by making a few simple edits, you confer a highly valuable disease resistance trait onto a crop," said Vipula Shukla, senior program officer at the Bill and Melinda Gates Foundation.

If European Union states eventually accept the recent parliamentary vote, they would exempt plants with CRISPR edits from GMO labeling requirements.

The EU has been notoriously strict on GMOs, requiring labeling under consumer "right to know" rules since 1997. Every GMO product must receive EU authorization and a risk assessment.

In the United States, the FDA began requiring clear labeling on consumer products containing GMOs in 2022. In 2018, the USDA decided that CRISPR-edited foods do not need to be regulated or labeled as genetically edited because these modifications could have been done with traditional breeding alone.

Experts think the new EU vote that exempts CRISPR from these rules indicates a willingness to embrace new tools to address the challenges of providing enough food for a growing population facing climate change.

Heres how advocates foresee CRISPR helping the food system become more resilient to climate change.

In agriculture, maximizing yield remains a top priority. Crops that produce more food and use less fertilizer, water and pesticides also decrease embedded emissions.

Pairwise, in collaboration with Bayer, is editing corn that yields more kernels per ear. Another edited corn grows to 6 feet rather than the conventional 9 feet tall.

"The advantage is that it's much sturdier," said Adams. "So if there's a big wind it doesn't get blown over." It also makes applying insecticides, fungicides and herbicides easier.

To engineer the next generation of climate-efficient plants, scientists need to find specific genes in them, such as for controlling water usage or nitrogen fixation.

"One of the biggest limitations [for CRISPR] is our relatively limited knowledge of the biology of the organisms that were trying to edit," Shukla said. "You can't apply CRISPR to a gene if you don't know what the gene does."

Farmers and researchers are field-testing a strain of CRISPR-edited rice designed to resist bacterial blights, which can kill 75 percent of a crop. Rice blight is a particular problem in India and Africa.

Since 2011, The Gates Foundation has been funding field trials of CRISPR rice in India. It has engaged in similar field tests of a virus-resistant corn in Mexico since 2015. "The Gates Foundation wants to come in at a point where there's a testable hypothesis," Shukla said. "We're focusing on developing and delivering these innovations to people."

The foundation looks for preliminary laboratory results or small scale, proven field testing. It then funds a larger scale pilot in real-world conditions in developing countries.

"I don't personally have a lot of faith that we're going to reverse climate change," Adams said. "So, I think we probably should be investing in adapting to it."

Farmers need plants that can survive temperature extremes, including higher nighttime temperatures, as well as erratic rainfall patterns. CRISPR can help native plants adapt to their changing environment by enhancing their genes.

"One of the consequences of climate change is having to move crops into places they havent been before because it's warmer or wetter or drier," Shukla said. "And crops are not adapted to those pests [in the new locations]. We have the ability with gene editing to confer traits that make those crops more tolerant to pests and diseases that they haven't experienced before."

The Gates Foundation is looking at genes for heat tolerance as its next target for research and investment, according to Shukla.

CRISPR technology may also diversify the genetic composition of current crops and domesticate new crops. That could help address the damage done by industrial, monoculture farming practices, in which a single crop species dominates a field or farm, depleting the soil of its nutrients.

"Wild relatives of plants contain traits that can be super-valuable for agriculture," Shukla said. "But we haven't had a way through crossing or other methods to bring those traits into the agricultural system."

If Pairwises mild mustard green becomes a hit, it might offer an incentive for farmers to plant a new leafy green alongside their kale, lettuce and spinach adding to biodiversity.

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Why Bayer and the Gates Foundation are using CRISPR to reduce food's climate impact - GreenBiz

Leadership change simultaneous to the Eclipse Cell Engineering platform spinout asEditCo Bio

REDWOOD CITY, Calif., March 27, 2024 /PRNewswire/ -- Synthego Corporation, a leading provider of genome engineering solutions, announced that Paul Dabrowski will step down as Chief Executive Officer, effective immediately. Craig Christianson has been appointed Chief Executive Officer following an extensive search process. Mr. Dabrowski, a co-founder of the company, will continue his role as a Board Director and advisor. Additionally, the company announces the divestiture of the Eclipse Cell Engineering platform as EditCo Bio, Inc., enablingSynthego's unique focus on therapeutic applications of CRISPR.

"Founding and growing Synthego the past 12 years has been the privilege of a lifetime," said Dabrowski. "Our team has transformed the CRISPR landscape by staying true to our values and providing everyone, from individual scientists to the world's leading biotechnology companies, with unprecedented access to advanced genome engineering. I'm confident Craig is an ideal fit to further our mission by building a robust commercial engine leveraging Synthego's platform - in addition to his impeccable track record, he embodies Synthego's culture of innovation and excellence. As the world enters the era of CRISPR based therapeutics, Synthego is now focused to be the premier supplier to hundreds of programs entering the clinic."

Christianson has a track record of spearheading global commercial strategies, business development and operations to build global life sciences and other businesses. He joins Synthego from Water Street Healthcare Partners, preceded by 12 years with global biotechnology company Promega Corporation where he led commercial operations, accelerating their growth to $700M+ in annual sales through profit-driven strategies and successful digital transformation.

"I am honored to join this pioneering organization which plays an important role in the impact CRISPR has on life science research and clinical development," said Christianson. "Paul is a visionary who has built a foundation upon which Synthego will become the best partner for clients in terms of co-development and regulatory compliance for the advancement of therapies and, ultimately, human health."

Christianson's appointment, along with the spinout of EditCo Bio, previously operating as Synthego's Eclipse platform, reinforces Synthego's commitment to provide CRISPR therapeutic developers with best-in-class guide RNAs. With its state-of-the-art GMP facility and extensive experience of producing leading products, Synthego is uniquely positioned to address escalating clinical requirements and changing regulatory frameworks. Bolstered by the FDA approval of the first CRISPR-based therapy, Synthego is more dedicated than ever to accelerating life-saving technologies for improved human health in its next chapter.

For more information, click here.

About Synthego:Synthego is a genome engineering company that enables the acceleration of life science research and development in the pursuit of improved human health. Based on a foundation of engineering and chemistry, Synthego leverages automation and machine learning to synthesize high-quality CRISPR reagents for science at scale. Synthego's mission is to enable agile life science research and development from discovery through clinical trials by providing scientists with comprehensive CRISPR solutions for each phase coupled with full technical and regulatory support from industry-leading experts. With its technologies cited in hundreds of peer-reviewed publications and utilized by thousands of commercial and academic researchers and therapeutic drug developers, Synthego is at the forefront of innovation, enabling the next generation of medicines by delivering genome editing at an unprecedented scale.

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Synthego Announces CEO Transition to Focus on Enabling CRISPR Therapeutics - PR Newswire

The new Integrated Classifier Pipeline system uses genetic fingerprints to identify unintended bystander CRISPR edits. A confocal microscope image of an early blastoderm-stage nucleus in aDrosophila(fruit fly) embryo uses colorful fluorescent markers to highlight the homothorax gene undergoing transcription from two separate parental chromosomes (two distinct signal clusters). Credit: Bier Lab, UC San Diego

The ICP system can cleanly establish whether a given individual insect has inherited specific genetic components of the CRISPR machinery from either their mothers or fathers since maternal versus paternal transmission result in totally different fingerprints, said Bier, a professor in the UC San Diego School of Biological Sciences.

The ICP can help untangle complex biological issues that arise in determining the mechanisms behind CRISPR. While developed in insects, ICP carries vast potential for human applications.

There are many parallel applications of ICP for analyzing and following CRISPR editing outcomes in humans following gene therapy or during tumor progression, said study first author Li. This transformative flexible analysis platform has many possible impactful uses to ensure safe application of cutting-edge next-generation health technologies.

ICP also offers help in tracking inheritance across generations in gene drive systems, which are new technologies designed to spread CRISPR edits in applications such as stopping the transmission of malaria and protecting agricultural crops against pest destruction. For example, researchers could select a single mosquito from the field where a gene-drive test is being conducted and use ICP analysis to determine whether that individual had inherited the genetic construct from its mother or its father, and whether it had inherited a defective element lacking the defining visible markers of that genetic element.

The CRISPR editing system can be more than 90 percent accurate, said Bier, but since it edits over and over again it will eventually make a mistake. The bottom line is that the ICP system can give you a very high-resolution picture of what can go wrong.

In addition to Li and Bier, coauthors included Lang You and Anita Hermann. Prior Bier lab member Kosman also made important intellectual contributions to this project.

Funding for the study was provided primarily by an award from the Bill and Melinda Gates Foundation.

Competing interest disclosure: Bier has equity interest in two companies he co-founded: Agragene Inc. and Synbal Inc., which may potentially benefit from the research results. He also serves on Synbals board of directors and the scientific advisory boards for both companies.

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New Genetic Analysis Tool Tracks Risks Tied to CRISPR Edits - University of California San Diego

ICP: an integrated pipeline for classifying CRISPR/Cas9 induced mutant alleles

We developed an integrated bioinformatic tool ICP (Integrated Classifier Pipeline), to parse complex DSB repair outcomes induced by CRISPR/Cas9 and automatically call for experimental errors generated during NGS library preparation and sequencing: 1) a Nucleotide Position Classifier (NPClassifier), and 2) a Single Allele-resolution Classifier (SAClassifier). We employed these two complementary sequence analysis modules in tandem to enable in-depth interpretation of deep sequencing data at single allele resolution (Fig.1ac, see Methods section for detailed description of ICP tools). In line with the unique DNA signatures generated by distinct DSB repair pathways, we categorized the repair products into four major categories. Alleles with a deletion only on the PAM-distal side (PAM-proximal side was protected by Cas9 protein after cleavage), a common category, were termed as PEPPR class mutations (PAM-End Proximal Protected Repair, PEPPR)41,42. While single strand cleavage by the Cas9 RuvC domain can also nick the non-complementary strand at locations beyond the canonical site between the 6th and 7th nucleotide upstream of the PAM sequence, we restrict our analysis here to the majority cases wherein Cas9 cleavage generates blunt DSB ends to simplify the robust classification scheme developed in this study43,44,45. Mutant alleles judged to be generated by directly annealing 2bp microhomology sequences spanning the gRNA cleavage site were assigned into MMEJ class (again acknowledging that such alleles can also be generated with 1bp microhomology sequence, which however, are not readily amenable to the semi-automated analysis we developed)46,47,48, while pure deletion alleles not belonging to either the PEPPR or MMEJ categories were classified as DELET class mutations. Remaining alleles that include insertions-only and indels (deletion plus insertion) were categorized as insertion class (INSRT) mutations (Fig.1b).

The process of DSB repair pattern profiling consists of preparing a NGS library (a), classifying the resulting parsed alleles (b) and displaying processed alleles by rank order and class of mutations (c). a NGS library preparation: Genomic DNA from F1 test flies carrying both Cas9 and gRNA expressing cassettes either maternally (dark blue bars) or paternally (red bars, or progeny from other designated crosses) are subjected for targeted PCR amplification with primers containing Illumina compatible adapters at the 5 terminal to detect somatic indels. The gray rectangle represents a short region of genomic DNA containing a Cas9/gRNA target: purple circle depicts Cas9 protein and sky-blue line is gRNA. b Classification: Raw NGS data are subjected to the NPClassifier to parse alleles into specific primary categories required for building allelic dictionaries used by the SAClassifier. Four major indel groups are categorized: PEPPR (PAM-End Proximal Protected Repair, sky-blue), MMEJ (Microhomology Mediated End-Joining, dark pink), DELET (deletion, any deletions do not belong to PEPPR and MMEJ, orange) and INSRT (insertion, including the alleles only with inserted nucleotides or had deletions and insertions, purple). The 24-nt short PEPPR, MMEJ and DELET dictionaries are used for a more accurate classification and error calling by binning together all alleles with the same seed region that match primary allelic entries in the SAClassifier dictionaries. c DSB repair pattern visualization: intuitive rendering of the processed raw sequence data as an output of rank ordered classes of alleles. Allelic classes derived from NGS sequencing of individual flies or mosquitoes are displayed by their ranked frequency (allele landscape) and repair pattern fingerprints (color-coded by categories).

Briefly, raw reads generated from deep sequencing were subjected to a preliminary categorization using the NPClassifier, which recognizes the relative positions of editing start- and end-points flanking Cas9 cleavage site and then generates a collection of priori alleles for each category. These primary outputs (MMEJ and DELET) were used for building full-length standard comprehensive dictionaries listing all observed mutations and derived 24-nt short dictionaries (with the same seed region flanking the Cas9 cleavage site) as inputs of the SAClassifier. In addition, a synthetic PEPPR dictionary was built by iteratively increasing the length of deletions by a single nucleotide distal to the PAM site, excluding alleles belonging to the MMEJ category. By fishing the raw reads with 24-nt dictionaries, we were able to automatically recognize reads that also contained experimentally generated errors (e.g., from PCR amplification), which usually are located outside of the narrow 24-nt short dictionary window, thereby assigning such composite alleles to correctly matched root alleles (Fig.1b). These dual iteratively employed ICP classification tools provide a robust and precise classification of CRISPR/Cas9 induced DSB repair outcomes. Next, we developed an evocative user-friendly interface to visualize processed allelic category information in the form of rank ordered allelic landscape plots and repair pattern fingerprints (color-coded DSB repair categories), both of which are sorted by read frequency (Fig.1c). These intuitively accessible data outputs are far more informative and discriminating than the unprocessed primary DNA sequence reads (e.g., compare the seemingly idiosyncratic raw lesions depicted in Fig.2a to the obviously unique processed and concordant replicate patterns shown Fig.2b, c). The ICP was thus employed to visualize results in all the following experiments.

a Examples of the top five somatic indels from individual flies derived from split-drive crosses in which the Cas9 transgene is inherited either maternally (Maternal-S, left) or paternally (Paternal-S, right), but separately from a cassette carryingthe gRNAtransmittedby the other parent. Purple stars indicate the color codes for mutation categories (dark pink: MMEJ, sky-blue: PEPPR, orange: DELET, purple: INSRT) and dark green star indicates the separate raw sequence color coded for the four nucleotides A, T, G, and C. The red bar indicates Paternal-S crosses while dark blue bar represents Maternal-S crosses. b Landscapes of top 50 alleles ranked by reads ratio. All six sequenced individual flies are plotted together, with dark blue lines plotting the data from Maternal-S crosses and the red lines from Paternal-S crosses. The y-axis presents the fraction of reads for a given allele and the x-axis depicts the top 50 alleles according to rank order by read frequency. c DSB repair fingerprints for three representative sequenced individual flies from each cross. The x-axis is the same as depicted in panel b. Both panels show the top 50 ranked alleles. d. Bar plots of Class Fraction for top 50 alleles. Color codes for classes are as in panels a and c. Correlation analysis of two out of three replicates from Maternal-S cross (e) or Paternal-S (f) cross. r2 values and p-values are indicated. Source data for panels b, d, e and f are provided as a Source Data file.

Since DSB repair outcomes have been found to vary considerably as a function of Cas9 or gRNA source and level49,50, we employed the ICP platform to parse somatic indels generated by co-expressing Cas9 and gRNAs in somatic cells of fruit flies (Drosophila melanogaster) and mosquitoes (Anopheles stephensi) in various configurations associated with gene-drive systems. We first applied ICP analysis to a split gene-drive system inserted into the Drosophila pale (ple)gene that is designed to detect copying of a gene cassette in somatic cells. This element, referred to as a CopyCatcher (pleCC), carries a gRNA targeting the first intron of Drosophila ple locus49. In this current study, we make use of low-level ectopic somatic Cas9 expression (which is substantial and broad for vasa-Cas9) to analyze DSB repair patterns across diverse cell types in F1 progeny carrying both Cas9 and gRNAs51,52,53. Because cells actively undergoing meiosis make up only a small fraction of dividing cells in an adult fly, the mutational effects of Cas9/gRNA cleavage in such F1 individuals largely reflect the somatic action of these nuclease complexes. We thus conducted several alternative crossingschemes to assess the somatic mutagenic activity of vasa-Cas9 and gRNA components when transmitted to F1 individuals in various configurations from their F0 parents: 1) Maternal Split (Maternal-S, females carrying vasa-Cas9 crossed with males carrying pleCC); 2) Paternal Split (Paternal-S, males carrying vasa-Cas9 crossed with females carrying pleCC); and 3) Maternal Full (Maternal-F, females carrying both the pleCC and vasa-Cas9 transgenes); or Paternal Full (Paternal-F, males carrying both the pleCC and vasa-Cas9 transgenes)49. Comparative ICP analysis revealed several striking and consistent differences between the prevalent somatic mutations generated in individual progeny in each of these different crossing schemes. In the case of Paternal-S crosses, the resulting mutations were dominated by PEPPR alleles (4 out of top 5 alleles in Fig.2a, Fig. S1a, and 70% of the top 50 alleles as rendered in rank ordered allelic landscapes and color coded DSB repair fingerprints in Fig.2c). In contrast, Maternal-S crosses primarily generated MMEJ and INSRT indels (4 out of top 5 alleles were MMEJ, and at least 50% of the top 50 alleles were INSRT mutations, Fig.2a, c, Supplementary Fig. S1a). These differences were also evident in the steeper allelic landscape curves that were generated from the Maternal-S versus Paternal-S crosses (Fig.2b) as characterized by the initial portion of the curve depicting the 5 most frequent alleles (i.e., the dark blue lines in Fig.2b are all above the red lines for the 5 most frequent alleles). We further quantified differences in allelic profiles between crosses by bar plots displaying the summed proportions of the different allelic classes (summing the percentages of all alleles from each category) which we termed as Class Fraction (Fig.2d). This analysis revealed that INSRT alleles were generated at a significantly higher frequency in Maternal-S crosses, while the PEPPR class dominated among the top 50 alleles in the reciprocal Paternal-S crosses (Fig.2d).

A striking feature of the highly divergent DSB repair signatures generated from maternally versus paternally inherited Cas9 sources was the remarkable reproducibility of their DSB repair fingerprints observed across three individual replicates from each cross (Fig.2e, f). We performed a correlation analysis within replicates by extracting 23 common alleles across all six sequenced flies and plotted the resulting allelic profiles together relative to an arbitrarily chosen Paternal-S replicate as reference (bold red line, Supplementary Fig. S1b). We observed that the frequency distributions of these 23 common alleles were much more similar to each other within intra-cross comparisons than between inter-crosses (Supplementary Fig. S1b). This trend was also revealed by higher correlation coefficients for intra-cross comparisons than for inter-cross comparisons based on allelic read ratios (Supplementary Fig. S1cg). Conspicuous defining differences between the Maternal-S and Paternal-S fingerprints were also evident based on the Class Fraction index (Fig.2d). In summary, a variety of differing statistical measurements all underscore the robust consistent similarities shared among allele profiles generated from individual replicates of same cross and clearly distinctive DSB repair pattern fingerprints generated by maternal versus paternal Cas9 inheritance.

We extended our ICP analysis of mutant allele profiles generated in the ple locus to the more extreme Maternal-F (dark blue lines) and Paternal-F (red lines) cross schemes to assess the role of inheritance patterns when both the source of vasa-Cas9 and gRNA originated from a single parent49. Again, we observed highly dominant alleles in the Maternal-F crosses, clearly evident in allelic landscapes, that deviated markedly from those produced by the Paternal-F crosses, which produced more evenly distributed spectra of alleles spread across a broad range of allelic frequencies (Fig.3a, b). As expected based on these large differences, the repair pattern fingerprints generated from different crosses produced clearly distinguishable patterns of mutation classes, which was particularly evident when considering the Class Fraction (Fig.3e). Cumulatively, these data suggest that the developmental timing and/or levels of Cas9 expression (maternal, early zygotic, or late zygotic) are likely to play a key role in determining which particular DSB repair pathway or sub-pathway is engaged in resolving DSBs.

ad Unique DSB repair signatures obtained using different Cas9 sources are displayed with the top 20 alleles (landscapes and DSB repair pattern fingerprints). NGS sequencing was performed on pools of 20 adults. a vasa-Cas9 inserted in the X chromosome and the pleCC element carrying the gRNA were both carried by either female or male parents, mimicking a full-drive configuration (Maternal-F and Paternal-F crosses with vasa-Cas9). b vasa-Cas9 split crosses wherein the Cas9 transgene was transmitted either maternally (Maternal-S) or paternally (Paternal-S) and the pleCC gRNA bearing cassette was carried by the other parent. Same Maternal-S versus Paternal-S crosses as in panel b, but using either actin-Cas9 (c) or nanos-Cas9 (d) sources. e Class Fraction Index for crosses in panels ad. Bars are shaded according to allelic class color codes. f UMAP embedding for visualizing a common set of 59 alleles shared between the four split crosses with actin-Cas9 and vasa-Cas9. Dots represent single alleles, and the colors indicate the allelic category. g Distribution of top 20 alleles generated from single flies derived from across between parents carrying theSpo11 gRNA and vasa-Cas9elements (Paternal-S cross: red lines and Maternal-S cross: dark blue lines). The top plot shows the allelic landscape for the top 20 alleles from all six sequenced single flies and the bottom shows three examples of the classification fingerprints (with all allelic classes condensed into single rows) color coded for the allele categories. h Class Fraction Index for Spo11 gRNA crosses. i, j Correlation analysis between two replicates from each cross. Dark blue is Maternal-S and red is for Paternal-S. r2 values and p-values are indicated. Source data are provided as a Source Data file.

Previous studies have shown that the relative frequencies of NHEJ versus HDR events depend on the source of Cas9 both in terms of timing and level of expression49,50,54. We thus wondered whether ICP analysis would similarly reveal distinct DSB repair outcomes for two additional Cas9 sources (actin-Cas9 and nanos-Cas9, expressing level of Cas9: actin-Cas9>vasa-Cas9>nanos-Cas9) inserted at the same locus with vasa-Cas9 (Fig.3c, d)49.

As was observed for the vasa-Cas9 source, the actin-Cas9 and nanos-Cas9 sources both generated differing allelic landscapes and repair pattern fingerprints when transmitted maternally versus paternally, which also were readily distinguishable from each other (Fig.3bd). Mirroring results with the vasa-Cas9 source, significant differences between the proportions of PEPPR versus MMEJ class among the top 20 alleles were observed in Maternal-S versus Paternal-S crosses for actin-Cas9. For the nanos-Cas9 source, both the MMEJ and INSRT categories were particularly reduced in Paternal-S crosses, although this latter sex-based difference was not as dramatic as for the other Cas9 sources (presumably due to its more germline restricted expression, Fig.3d)55,56. Overall, the general trend once again indicated that maternally inherited Cas9 sources biased somatic DSB repair outcomes in favor of MMEJ and INSRT classes over PEPPR alleles, while paternal transmission of Cas9 generated mutant alleles dominated by PEPPR class alleles (Fig.3e).

Based on the overall similarities of the DSB repair outcomes observed for actin-Cas9 and vasa-Cas9 crosses, we extracted a set of 59 shared alleles that appeared in all sequenced samples and performed UMAP (Uniform Manifold Approximation and Projection) analysis to cluster these common alleles, condensing them into 5 distinct clouds (Fig.3f). Clouds 1, 2, 3, and 4 were dominated by alternative subsets of PEPPR alleles distinguished primarily by the length of deletion (the average deletion sizes were 24bp, 40bp, 31bp for PEPPR Mini, Midi-I and Midi-II cluster, and it was longer than 55bp for PEPPR Maxi cluster), while cloud 5 was predominantly comprised of MMEJ alleles. We reviewed raw sequences for the few trans-cloud assigned alleles and discovered that some of these alleles could be interpreted as having been generated from a second round of repair using one of the core alleles from the same cloud as a repair template. For example, we inferred that allele 58 was actually a PEPPR deletion with several nucleotides potentially having been back-filled. This result is consistent with the previous report that alleles with insertions or complex repair outcomes would be generated from several rounds of synthesis following the generation of a primary deletion event57,58. Assessing the impact of such potential complexities, which we ignore here for simplicity, will require additional future scrutiny. The remainder of these alleles, such as allele 44, could be accounted for variability in the exact Cas9 cleavage site (between the 6th and 7th nucleotidescounting from the PAMside), with an extra nucleotide being deleted on the PAM-proximal side of the gRNA cleavage site (Fig.3f)43,59,60. Since both of these outcomes were rare, we hypothesized second-order origins for such outlier alleles further validate the robust nature of our ICP platform in recognizing core primary categories of DNA repair outcomes. We also analyzed the common 59 alleles by plotting their read frequencies and observed that the differences between the allelic landscapes for the two reciprocal crosses per each Cas9 source mirrored the trend in Fig.3ad described above (Supplementary Fig. S2a, b). Cumulatively, these concordant findings support a key role for theparental origin of Cas9 servingas a major determinant of the DSB repair outcome.

Another obvious determinant of DSB repair outcome is the local genomic DNA context. We assessed the general applicability of theICP by employing it to classify alleles generated by gRNAs targeting four other loci: prosalpha2 (pros2), Rab11, Spo11 and Rab5 using the vasa-Cas9 source61. Paralleling our findings from the ple locus, we observed divergent allelic profiles between Paternal-S and Maternal-S crosses with distinct dominant mutation categories based on the specific target site. For example, the predominant allelic classes generated at the Spo11, pros2 and Rab11 loci were PEPPR and INSRT alleles, while PEPPR and MMEJ alleles were most prevalent for the Rab5 targets (Fig.3g, h, Supplementary Figs. S36). Among these four targets, Spo11 displayed the greatest divergence in the prevalence of top alleles generated from Maternal-S and Paternal-S crosses (reminiscent of the fine distinctions parsed for the ple locus, Fig.3g). We nonetheless still observed high correlation coefficients between two replicates within the same cross and significantly lower correlation coefficients associated with inter-cross comparisons between maternal versus paternal Cas9 inheritance (averaged r2=0.33, Fig.3i, j, Supplementary Fig. S3). We also observed distinctive sex-specific DSB repair patterns for Cas9 transmission at the pros2 and Rab11 gRNAs targeting sites (Supplementary Figs. S4 and S5), although these differences were less pronounced than for ple and Spo11 gRNAs, while for Rab5, the allelic patterns were similar for both maternal and paternal crosses (Supplementary Fig. S6, see Supplementary Discussion Section). In summary, these data support the broad utility of the ICP pipeline to deliver unique discernable locus-specific fingerprints associated with distinct parental inheritance patterns of Cas9 that generalize to other genomic targets.

Given the strong Cas9 inheritance-dependent distinctions observed for allelic profiles resulting from maternal versus paternal Cas9/gRNA-induced DSBs in Drosophila, we wondered whether similar DSB repair pattern fingerprints could be discerned in mosquitoes carrying a linked full gene-drive in which the Cas9 and gRNA transgenes are carried together in a single cassette62,63,64,65. We examined this possibility using the transgenic An. stephensi Reckh drive,which is inserted into the kynurenine hydroxylase (kh) locus63. Because of the Cas9 and gRNA linkage, the Reckh drive behaves as the Maternal-F and Paternal-F cross configurations described above in which all CRISPR components are carried by a single parental sex63.

Consistent with our observations in flies, the Reckh Maternal-F crosses generated a high proportion of indels that were dominated to a remarkable extent by single mutant alleles with read percentages exceeding 85% for each of the three single mosquitoes sequenced, followed by a long distributed tail of lower frequency alleles. The highly biased nature of the replicate allelic distributions is readily revealed by a virtual step-function in their rank-ordered allelic landscapes (Fig.4a). In striking contrast, over 50% alleles recovered from the Paternal-F crosses were wild-type (WT), which presumably reflects alleles that either remained uncut or DSB ends that were rejoined accurately without further editing. The highly predominant WT allele was followed by a very shallow tail distribution of low frequency mutant alleles in the paternal rank-ordered allelic landscapes (Fig.4a). This dramatic difference in allelic profiles between Maternal-F versus Paternal-F crosses was also clearly displayed by the class-tally bars color coded for the different fractions of each class (black = WT) located beneath each landscape (Fig.4a). Here, the Class Fraction Index measure indicated that Maternal-F crosses generated a greater proportion of INSRT alleles in the first two samples, while Paternal-F crosses produced a high frequency of PEPPR alleles (Fig.4b). As in the case of allelic profiles recovered at the ple and Spo11 loci in flies, common sets of highly correlated mutant DSB repair fingerprints were observed across all three replicates of the Paternal-F Reckh crosses (Supplementary Fig. S7). A similar comparison of allelic distributions in the maternal crosses was precluded by virtue of the single highly dominant alleles and corresponding paucity of lower frequency events, the nature of which varied greatly between replicates. We conclude that the high-resolution performance of the ICP platform in Drosophila can be generalized to other insects such as An. stephensi to robustly discern sex-dependent CRISPR transmission patterns resulting in distinct DSB repair outcomes.

a Rank-ordered landscapes of the top 50 alleles generated from NGS analysis of single mosquitoes. Colored bars with red dots indicate mutated alleles, and black bars with black dots indicate an unmutated WT allele. Middle panels: allelic class fingerprints color coded as in previous figures. Bottom bars: fraction of each allelic class, including WT (black), PEPPR (sky-blue), MMEJ (deep pink), DELET (orange) and INSRT (purple). Numbers indicate the percentage of the corresponding class. b Class Fraction Index for single mosquito sequencing data in panel a. c Developmental time-points for sample collections. d Kinetics of Cas9 mutagenesis generated by the Reckh gRNA. Lines represent the summed fraction of mutant alleles at each time-point. Dark-blue lines indicate maternal (Maternal-F) crosses and red lines paternal (Paternal-F) crosses. e DSB repair fingerprints at different timepoints. Samples were collected at the time points shown in panel c and 20 eggs, larvae, pupae or adults were pooled together for genomic DNA extraction and deep sequencing. The far left and far right panels indicate the Class percentages including WT alleles (black), displaying the proportion of each class at single time-points. Source data are provided as a Source Data file.

Given the dramatic differences we observed in the frequency and nature of somatic alleles generated in maternal versus paternal-sourced Cas9 in both flies and mosquitoes, we wondered whether the developmental timing of Cas9/gRNA expression (maternal=early? and paternal=late?) was the key determinant for these highly reproducible DSB repair fingerprints. We tested this hypothesis by assessing whether DSB repair fingerprints varied as a function of developmental progression using a series of narrowly timed sample collections of F1 mosquitoes produced from crosses of Reckh parents to WT and assayed DSB repair spectra using the ICP pipeline at 12 different developmental stages (Fig.4c. Note: as homozygous Reckh transgenic mosquitoes were crossed to WT, all F1 progeny carried one Reckh allele and one WT receiver allele, the latter of which was amplified for DSB repair analysis). We tracked a diminishing proportion of WT (presumably uncut) alleles and a corresponding increase in mutant alleles of various classes at each of the time points (Fig.4d). Strikingly, nearly half of the target alleles were edited in embryos by 30minutes post-oviposition for both the Maternal-F and Paternal-F Reckh crosses, which corresponds to early pre-blastoderm stages prior to the maternal-to-zygotic transition, suggesting a very early activity of Cas9 in mosquito embryos driven either by maternally inherited Cas9/gRNA complexes or potentially by very early zygotic expression of the Cas9 and gRNA components (Fig.4d)66. We also observed similarly frequent indels being generated as early as 30min in flies expressing Cas9 (either maternally or paternally) with a gRNA targeting the pros2 locus, although the dynamics of Cas9 production are distinct in these two organisms (Supplementary Fig. S8a). Following this initial surge in target cleavage, we observed divergent trajectories in the accumulation of mutant alleles between maternal versus paternal lineages. As an overall trend, mutant alleles accumulated progressively in the Maternal-F lineage until virtually no WT alleles remained, while in Paternal-F lineage, even at the endpoint of adulthood, approximately 60% of WT alleles persisted, in line with our single time point experiments (Fig.4a, d, Supplementary Fig. S8b). As observed in the final distributions of adult alleles, progeny from Maternal-F crosses tended to be enriched for INSRT alleles over the entire developmental time course, while PEPPR alleles were more common in Paternal-F crosses with pronounced accumulation of such alleles during later stages (Fig.4e). A finer scale analysis of the categories of mutant alleles generated over time revealed dynamic patterns of prevalent alleles during mosquito developmental stages (Fig.4e). For example, the proportion of MMEJ alleles peaked at the 2-hour and 4-hour time points (Fig.4e). Similarly, a split-drive expressing a gRNA targeting the Drosophila pros2 locus generated distinct temporal profiles of cleavage patterns in crosses from female versus male parents carrying the drive element (Supplementary Fig. S9).

One unexpected feature of the developmental variations in allelic composition we observed was that the proportion of WT alleles increased at certain time points (e.g., 1-hour in maternal cross and 12-hour - day 1=24h in paternal cross). These temporal fluctuations were also observed in flies expressing Cas9 and a pros2 gRNA at two hours after oviposition (Supplementary Figs. S8a and S9), revealing that this phenomenon might reflect a generally relevant form of clonal selection for WT cells during pre-blastoderm stages. The latter clonal selection might arise if mutant cells experienced negative selection at certain development stages. In the case of paternal transmission, one strong line of evidence supporting this WT clonal selection hypothesis is that in adults, the Reckh element is transmitted to over 99% of F1 progeny, indicating that nearly all target alleles in the germline must be WT. This high frequency of paternal germline transmission is also consistent with the high prevalence of WT alleles tallied at 12h in embryos derived from the paternal crosses (Fig.4e, see Supplementary Discussion Section for more in-depth consideration of this point). We analyzed the developmental distributions of 21 common alleles that were generated at all time-points (Supplementary Fig. S10ae). Most of these common alleles belonged to the PEPPR class, while only five were INSRT alleles, despite the INSRT class overall being the most prevalent for both crosses, again suggesting that INSRT alleles have a higher diversity than other mutation categories (Supplementary Fig. S10a). Overall, this analysis is in line with our previous observation that Maternal-F crosses produced more INSRT alleles while Paternal-F crosses generated a preponderance of PEPPR alleles (Supplementary Fig. S10b).

Given the strong influence of maternal versus paternal origin of Cas9 on the resulting distributions of alleles characterized above by ICP analysis, we wondered whether such allelic signatures could be exploited for lineage tracing in randomly mating multi-generational population cages. We first examined ICP outputs from a controlled crossing scheme carried out over three generations with pleCC and Reckh gRNAs to derive allelic fingerprints distinguishing parents of origin by identifying both somatic alleles in the F1 generation as well as assessment of which of those alleles might be transmitted through the germline to non-fluorescent progeny (i.e., those not inheriting the pleCC or Reckh element) at the F2 generation (Fig.5ad, Supplementary Fig. S11). As anticipated, in both pleCC and Reckh Maternal-F crosses, single dominant somatic alleles were observed in the F1 generation, with the top single allele representing more than 50% of all alleles (Fig.5a, c). Furthermore, all such predominant somatic mutant alleles, which precluded gene-cassette copying of the pleCC or Reckh drive elements in those F1 individuals, were transmitted faithfully through the germline to non-fluorescent F2 progeny with approximately 50% frequency. Furthermore, we observed marked differences in the other half of total reads in F2 progeny depending on the origin of Cas9/gRNA complexes. Thus, a distribution of multiple diverse low frequency mutations were generated when crossing F1 pleCC+ or Reckh+ females with WT males (presumably derived from F1 drive females having deposited Cas9/gRNA complexes maternally that then acted on the paternally sourced WT allele somatically in F2 individuals). In the reciprocal male cross, however, approximately 50% of all alleles remained WT (Fig.5b, d, Supplementary Fig. S12af). These findings support the hypothesis that the top somatic indels derived from maternal Cas9 sources were generated at very early developmental stages (possibly at the point of fertilization or shortly thereafter during the first somatic cell division), resulting in a single mutant allele being initially produced and then transmitted to every descendent cell including all germline progenitor cells49. With the paternal-sourced Cas9 and gRNA, arrays of variable somatic mutations were recovered with the most prominent alleles accounting for fewer than 10% of the total alleles in F1 progeny (Fig.5b). Accordingly, paternally generated F1 somatic alleles were more randomly transmitted via the germline of individuals that failed to copy the gene cassette for either the pleCC or Reckh elements. As a result of this diversity of somatic F1 alleles, only occasionally were the most prevalent alleles also transmitted through germline (e.g., individuals 1, 4 and 5 in Fig.5b, Supplementary Fig. S12gl).

Primary DNA sequences of top single alleles and their percentages of the total alleles from six individual sequenced flies derived from ple gRNA Maternal-F (a) and Paternal-F (b) crosses. Gray bars indicate the location of the gRNA protospacer and red arrowheads are the associated PAM sites. The first row depicts the reference sequence covering the expected DSB cleavage site. Colored squares in the right column indicate the class to which a given allele belongs to. The tables shown on the right of each allele show its frequency among all reads. Left columns of the table indicate frequencies of the somatic allele, and the right columns are the top germline mutant allele frequency obtained by sequencing F2 non-fluorescence progeny derived from same F1 individuals whose top somatic allele is displayed in the left column (excluding WT alleles). Colored dots indicate different alleles with the same color shared between two columns indicating that the same allele appeared as both top 1 somatic and germline indels from the same F0 founders. c, d Allele profiles generated by Reckh parents and progeny generated with the same crossing scheme as for the pleCC. c Tabulation of the Maternal-F cross. d Tabulation of the Paternal-F cross. e Crossing scheme forthe Reckh cage trials. Three individual cages were seeded with 10 homozygous Reckh females, 90 WT females and 100 WT males for the maternally initiated lineage, while the paternally initiated cages were seeded with 10 homozygous Reckh males, 90 WT males and 100 WT females. At each of the following three generations, 10 Reckh+ females and 10 Reckh+ males were randomly collected for single mosquito deep sequencing. f Biased inheritance of Reckh was observed in the maternally seeded cages at generations 2 and 3, but not for the paternally seeded cages. Pink bars denote the fraction of sequenced individual mosquitoes inheriting Reckh from female parents, and cyan colored bars represent Reckh inheritance from the males. Source data are provided as a Source Data file.

The Reckh element in mosquitoes performed similarly to the fly pleCC, however, Reckh F1 individuals displayed less frequent zygotic cleavage and a corresponding reduction in the diversity of resulting somatically generated mutations (>50% WT alleles remained, Paternal-F cross). Consistent with this limited number and array of somatic mutations in the F1 generation from Paternal-F cross, NHEJ mutations were only rarely transmitted to the F2 generation, probably due to more germline-restricted expression of vasa-Cas9 in mosquitoes as compared to flies (Fig.5c, d). These results again suggest that cleavage and repair events were generated later during development in paternal crosses resulting in a stochastic transmission of F1 somatic alleles to the germline, which were largely uncorrelated with the most prevalent allele present somatically in the F1 parent49. Taken together, these highly divergent sex-dependent DSB repair signatures suggested that such genetic fingerprints could be used to track parental history in the context of randomly mating multi-generation population cages.

Based on the highly dominant mutant indels (Maternal-F) versus WT (Paternal-F) alleles generated by Reckh genetic element described above, we evaluated inheritance patterns of indels in multi-generational cages initiated by a 5% introduction of Reckh into WT populations either through maternal or paternal lineages in the F0 generation (Fig.5e). We randomly selected at least 20 fluorescence marker-positive mosquitoes (10 females and 10 males) for NGS analysis at generations 2 and 3, when the Reckh allele was still present at relatively low frequencies in the population and random mating was more likely to have taken place between Reckh/+ heterozygous and WT mosquitoes. Thus, we envisioned that the source of Reckh allele could be tracked back to a male versus female parent of origin by examining whether a dominant WT allele was present (inherited paternally) or not (inherited maternally) (Fig.5e, f). Following this reasoning, we inferred a strong bias for progeny inheriting the Reckh element from a Reckh+ males mating with WT females during generations 2 and 3 than the reverse (i.e., female transmission of Reckh alleles) in the maternally seeded lineage. Indeed, in one maternally seeded replicate (cage 2, generation 3), 100% of the progeny had inherited the Reckh element from their fathers (Fig.5f). In contrast to the striking sex-specific transmission bias observed in maternally seeded cages, progeny from paternally seeded cages displayed more evenly distributed stochastic parental inheritance patterns (Fig.5f). These highly reproducible parent of origin signatures demonstrate the utility of ICP in allelic lineage tracking, which could be of great potential utility in evaluating alternative initial release strategies for gene-drive mosquitoes as well as post-release surveillance of gene-drives as they spread through wild target populations (see Discussion).

Another important challenge for deciphering DSB repair outcomes is to track both NHEJ and gene-cassette mediated HDRevents within the same sample. Such a comprehensive genetic detection tool could have broad impactful applications (see Discussion). For example, one important and non-trivial application is to follow the progress of gene-drives in a marker free fashion as they spread through insect populations. Such dual tracking capability would address the potential concern that mutations eliminating a dominant marker for the gene-drive element could evade phenotype-based assessments of the drive process. Accordingly, we devised a three-step short-amplicon based deep sequencing (200400bp) strategy based on tightly linked colony-specific nucleotide polymorphisms distinguishing donor versus receiver chromosomes to detect copying of two CopyCatcher elements, pleCC and hthCC, from their chromosomes of origin (donor chromosome) to WT homologous (receiver chromosome) targets (Fig.6a)49. Notably, this strategy only amplified the inserted gene cassette on the donor chromosome and or the cassette if it copied onto the receiver chromosome. Thus, the measured allelic frequencies indicate the relative proportions of gene cassettes copied to the receiver chromosome versus those residing on the donor chromosome (Fig.6b displays the inferred somatic HDR frequency quantified from the three-step NGS sequencing protocol as well as Indels quantified by our standard 2-step NGS sequencing protocol - see Methods section for additional details).

a Scheme for tracking gene-drive copying using NGS. Gray bars: genomic DNA, pink oval: Cas9 protein, sky-blue line: gRNA, colored asterisks: polymorphisms. Color coded rectangles represent four nucleotides. Four possible recombinants listed are generated by resolving Holliday junctions at different sites marked with black crosses. b NGS sequencing-based quantification of somatic HDR generated by pleCC in F1 progeny. Areas delineated by dotted lines indicate patches of cells in which somatic HDR copying events have taken place either under bright field (upper) or RFP fluorescent filed (middle). Bottom bars are the summary of the inferred frequency for the somatic HDR (orange), indels (green) and WT alleles (black) derived from the deep sequencing data using the same samples photographed above. More than three flies from each cross were imaged and used for analysis. Scale bars indicate 200 pixels. c Somatic HDR profile with ple gRNA. The red line is for Maternal-F cross and dark blue line for the Paternal-F cross. d Diagram of the hthCC. Black double arrow: recoded hth cDNA, blue rectangles: exon 1, light green rectangles: exons 2-14, and colored lines underneath represent probes used for detection. e In situ images with embryos laid from hthCC-vasa-Cas9 females crossed with WT males. Blue=exon 1, green=WT exons 2-14, red=recoded cDNA for exons 2-14. Insets are magnified single nuclei indicated by colored arrows. This experiment has been repeated at least three times. Scale bars stand for 10m. f Temporal profiles for somatic HDR-mediated copying of the hthCC element assessed by NGS as described for the pleCC in panels c and f. Y-axis tabulates the percentage of HDR at a given time point. Table at the bottom quantifies the HDR fraction at given time points for both the Paternal-F and Maternal-F crosses. Source data are provided as a Source Data file.

In our first set of experiments, we analyzed editing outcomes by examining F1 progeny derived from Maternal-S and Paternal-S pleCC crosses. We compared the rates of somatic HDR measured by NGS analysis to those evaluated by image-based phenotypes associated with copying of the CopyCatcher element. As summarized previously, CopyCatchers such as the pleCC are designed to permit quantification of concordant homozygous mutant clonal phenotypes (e.g., pale patches of thoracic cuticle and embedded sectors ofcolorless bristles), with underlying DsRed+ fluorescent cell phenotypes49. Individual flies in which imaging-based analysis had been conducted were then subject toseparate NGS HDR-fingerprinting and INDELs-fingerprinting resulting in a comprehensive quantification of HDR, NHEJ, and WT alleles within the same sample (Fig.6b, libraries for HDR-fingerprinting and INDELs-fingerprinting were prepared from the same individual fly, but with different DNA preparation and sequencing protocols as detailed description in Methods). For these experiments, F1 flies were genotyped and those carrying both Cas9 and pleCC gRNA were used for NGS analysis (data shown here are the inferred frequencies of somatic HDR, NHEJ events, and WT alleles). This dual integrated analysis revealed that HDR in the Maternal-S crosses resulted in ~15% somatic HDR-mediated cassette copying events on average based on sequencing, and that such cassette copying was yet more frequent in Paternal-S crosses, producing ~25% somatic HDR. The nearly two-fold greater HDR-mediated copying efficiency detected by sequencing in Paternal-S crosses mirrors phenotypic outcomes wherein maternally inherited Cas9 similarly results in a lower frequency of cassette copying detected by fluorescence image analysis in somatic cells than for paternally inherited Cas9 (Fig.6b)49.

Our genetic analysis of stage-dependent differences in DSB repair pathway activity in this study is consistent with a commonly held view in the gene-drive field based on a variety of indirect genetic transmission data that HDR-mediated cassette copying does not occur efficiently during early embryonic stages50,51,63,67,68,69,70. This inference, however, has not yet been verified experimentally. We thus sought to provide direct evidence supporting this key supposition using NGS-based HDR-fingerprinting to track the somatic HDR events across a range of developmental stages in both Maternal-F and Paternal-F crosses in which the Cas9 and gRNA transgenes are transmitted together either maternally or paternally using our validated NGS sequencing protocol. Notably, we collected samples at 9 timepoints and pooled 20 F1 progeny together for pooled sequencing to prime the developmental profile of somatic HDR with pleCC (samples were thus collected without genotyping since it is impractical to genotype individual embryos and young larvae). Because of the limitations imposed by embryo pooling we were unable to use the same samples collected here for also quantifying the generation of somatic NHEJ alleles (i.e., only half of the F1 progeny carried the vasa-Cas9 transgene on the X chromosome and those embryos lacking this transgene were not suitable for generating mutations - note that such an analysis was possible in the case of the viable Reckh drive shown in Fig.4e as well as for a viable split-drive allele inserted into the essential prosalpha2 locus shown in Supplementary Fig. S9). Indeed, NGS analysis detected only very rare examples of somatic HDR events in early embryos derived from both crosses (Fig.6c). Notably, HDR in the Paternal-F cross detected by this sequencing protocol increased substantially to 35.9% during adult stages, a period coinciding with the temporal peak of the pale expression profile (note that in this experiment we employed the actin-Cas9 rather than vasa-Cas9 source, which has higher level of Cas9 expression in somatic cells and generates a correspondingly higher frequency of somatic HDR)49.

We extended our sequencing-based strategy to quantify somatic HDR using a second CopyCatcher element (hthCC) designed specifically to identify even rare copying events in early blastoderm-stage embryos. The hthCC is inserted into the homothorax (hth) gene and was engineered to visualize HDR-mediated copying of the gene cassette by fluorescence in situ hybridization (FISH) using discriminating fluorescent RNA probes complementary to specific endogenous versus recoded cDNA sequences (Fig.6d, e). In this system, copying of the transgene from the donor chromosome to the receiver chromosome would be indicated by the presence of two nuclear dots of red fluorescence detected by the hth recoded cDNA-specific probe (indicating two copies of recoded hth cDNA). In contrast, cells in which no copying occurred should contain only a single nuclear red dot signal (from the donor allele). Such in situ analysis detected no clear case of gene cassette copying in any of the ~5000 blastoderm stage cells examined across ~500 embryos (with the caveat that some mitotic nuclei generate ambiguous signals depending on their orientation). This qualified negative result assessed by in situ analysis was consistent with the very low estimates of HDR frequency during the same early blastoderm-stage developmental window based on NGS analysis in staged time-course experiments, although the latter sequencing method did detect very low levels of somatic HDR at ~3hours after egg laying from the Paternal-F crosses (and no copying until day three of larvae with the maternal cross Fig.6df). The very low levels of somatic HDR observed in early embryos for the hthCC construct either by in situ hybridization or by NGS sequencing parallel the results summarized above for the pleCC element (Fig.6c, f). The maximal somatic HDR frequency observed for the hthCC Maternal-F crosses (0.06% at day 3 after egg laying) was somewhat lower than that for the similar cross for pleCC (0.35% at adult stage), consistent with the predominance of single mutant alleles being generated at very early stages following fertilization in Maternal-F crosses. In contrast to the exceedingly rare copying of the hthCC element detected in early embryos for either the Maternal-F or Paternal-F crosses, the same element frequently copied to the homologous chromosome during later developmental stages in Paternal-F crosses as assessed by NGS sequencing. The hthCC elementagain copied with somewhat lower efficiency than the pleCC element (e.g., 15.2% for hthCC versus 35.9% for pleCC tabulated in adults), presumably reflecting differing genomic cleavage rates or gene conversion efficiencies generated by their respective gRNAs (including total cleavage levels and temporal features). In aggregate, these two examples of quantitative analysis of copying frequencies based on both NGS and in situ analysis demonstrate that ICP and NGS-based quantification of gene conversion events can be successfully integrated for a comprehensive analysis of DSB repair outcomes, including both NHEJ and HDR events as a function of developmental stage. These powerful tools also could be applied for following gene-drive spread through freely mating populations in a marker-free manner as well as for a variety of other applications including gene therapy (see Discussion).

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Developmental progression of DNA double-strand break repair deciphered by a single-allele resolution mutation ... - Nature.com

Co-author Steven Gray, Ph.D., is Associate Professor of Pediatrics, Molecular Biology, Neurology, and in the Eugene McDermott Center for Human Growth and Development at UTSouthwestern.

DALLAS March27, 2024 A gene therapy developed by researchers at UTSouthwestern Medical Center for a rare disease called giant axonal neuropathy (GAN) was well tolerated in pediatric patients and showed clear benefits, a new study reports. Findings from the phase one clinical trial, published in the New England Journal of Medicine, could offer hope for patients with this rare condition and a host of other neurological diseases.

This trial was the first of its kind, for any disease, using an approach to broadly deliver a therapeutic gene to the brain and spinal cord by an intrathecal injection, said co-author Steven Gray, Ph.D., Associate Professor of Pediatrics, Molecular Biology, Neurology, and in the Eugene McDermott Center for Human Growth and Development at UTSouthwestern. Even with the relatively few patients in the study, there were clear and statistically significant benefits demonstrated in patients that persisted for years.

Dr. Gray developed this gene therapy with co-author Rachel Bailey, Ph.D., Assistant Professor in the Center for Alzheimers and Neurodegenerative Diseases and of Pediatrics at UTSW.Dr. Gray is an Investigator in thePeter ODonnell Jr. Brain Institute.

GAN is extraordinarily rare, affecting only about 75 known families worldwide. The disease is caused by mutations in a gene that codes for a protein called gigaxonin. Without normal gigaxonin, axons the long extensions of nerve cells swell and eventually degenerate, leading to cell death. The disease is progressive, typically starting within the first few years of a childs life with symptoms including clumsiness and muscle weakness. Patients later lose the ability to walk and feel sensations in their arms and legs, and many gradually lose hearing and sight and die by young adulthood.

In the clinical trial conducted at the National Institutes of Health (NIH), Drs. Gray and Bailey worked with colleagues from the National Institute of Neurological Disorders and Stroke (NINDS) to administer the therapy to 14 GAN patients from 6 to 14 years old. Using a technique they developed to package the gene for gigaxonin into a virus called adeno-associated virus 9 (AAV-9), the researchers injected it into the intrathecal space between the spinal cord and the thin, strong membrane that protects it. Tested for the first time for any disease, this approach enabled the virus to infect nerve cells in the spinal cord and brain to produce gigaxonin in nerve cells, allowing them to heal the cells axons, which grow throughout the body.

Amanda Grube, 14, one of the trial's participants, has seen improvement in her diaphragm and other muscles associated with breathing, her mother says. However, many of Amanda's other functions, including her mobility, did not benefit. (Photo credit: McKee family)

After one injection, the researchers followed the patients over a median of nearly six years to determine whether the treatment was safe and effective. Only one serious adverse event was linked to the treatment fever and vomiting that resolved in two days suggesting it was safe. Over time, some patients showed significant recovery on an assessment of motor function. Other measurements revealed that several of the patients improved in how their nerves transmitted electrical signals.

One of the trials participants, 14-year-old Amanda Grube, has experienced improvement in her diaphragm and other muscles associated with breathing, according to her mother, Katherine McKee. However, many of Amandas other functions did not benefit including her mobility.

Thats why I hope theres more to come from the research that can help patients even more,Mrs. McKee said. Amanda has dreams and ambitions. She wants to work with animals, save the homeless, and design clothes for people with disabilities.

Dr. Gray said that in many ways, the study offers a road map to carry out similar types of clinical trials. The findings have broader implications because this study established a general gene therapy treatment approach that is already being applied to dozens more diseases, he said.

Although the phase one results are promising, Dr. Gray said he and other researchers will continue to fine-tune the treatment to improve results in future GAN clinical trials. He is also using this method for delivering gene therapies to treat other neurological diseases at UTSW, where he is Director of the Translational Gene Therapy Core, and at Childrens Health. Work in theGray Labhas already led to clinical trials for diseases including CLN1 Batten disease, CLN5 Batten disease, CLN7 Batten disease, GM2 gangliosidosis, spastic paraplegia type 50, and Rett syndrome.

The GAN study was funded by the National Institute of Neurological Disorders and Stroke (NINDS), Division of Intramural Research, NIH; Hannahs Hope Fund; Taysha Gene Therapies; and Bamboo Therapeutics-Pfizer.

Drs. Bailey and Gray are entitled to royalties from Taysha Gene Therapies. Dr. Gray has also consulted for Taysha and serves as Chief Scientific Adviser.

About UTSouthwestern Medical Center

UTSouthwestern, one of the nations premier academic medical centers, integrates pioneering biomedical research with exceptional clinical care and education. The institutions faculty members have received six Nobel Prizes and include 25 members of the National Academy of Sciences, 21 members of the National Academy of Medicine, and 13 Howard Hughes Medical Institute Investigators. The full-time faculty of more than 3,100 is responsible for groundbreaking medical advances and is committed to translating science-driven research quickly to new clinical treatments. UTSouthwestern physicians provide care in more than 80 specialties to more than 120,000 hospitalized patients, more than 360,000 emergency room cases, and oversee nearly 5 million outpatient visits a year.

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Gene therapy offers hope for giant axonal neuropathy patients - UT Southwestern

According to latest study, the global advanced therapy medicinal products CDMO Market size was valued at USD 6.10 billion in 2023 and is projected to reach USD 34.53 billion by 2033, growing at a CAGR of 18.93% from 2024 to 2033.

Key Takeaways:

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owing to risingclinical trialsfor advanced therapy medicinal products and the increasing awareness among researchers about the benefits of advanced therapies, driving the advanced therapy medicinal products (ATMP) CDMO market growth. Tissue engineering has greatly benefited in recent years from technological development. The damaged tissues and organ function are replaced or restored using this technique. Similarly, gene and cell therapy are attracting a lot of patients for the treatment of rare diseases, whose incidence is rising globally.

With rising demand for robust disease treatment therapies, key players have focused their efforts to ramp up research and development for effective gene therapies that target the cause of disorder at a genomic level. According to ASGCT, the number of cell and gene therapies in the U.S. pipeline programs (phase I-III trials) increased from 483 in 2021 to 529 in 2022. Furthermore, the FDA delivers constant support for innovations in the gene therapy field via a number of policies with regard to product manufacturing. In January 2020, the agency released six final guidelines on the manufacturing and clinical development of safe & efficient gene therapy products.

Moreover, awareness about ATMP treatment options is being driven by initiatives aimed at informing the public about the benefits of these products, which, in turn, is leading to increased adoption of advanced therapies and fueling market growth for CDMOs. For instance, Alliance for Regenerative Medicine Foundation for Cell and Gene Medicine prioritizes activities for increasing public awareness through educational programs, underlining the clinical & societal benefits of regenerative medicine.

Increasing clinical trial activity along with new product launches generates growth opportunities for the market. As of 2022, there are 1451 ATMPs in preclinical stages and 535 are being studied in Phase 1 to 3 studies. Since August 2020, EMA has approved six of these additional ATMPs, and five more will be approved by 2023. In the UK, there were approximately 168 advanced therapy medicinal product trials underway in 2021, up from the 154 studies reported the year before, which is a 9% increase. 2021 saw a 32% increase in phase 1 trials, indicating a significant shift from experimental medicines to first-in-human studies.

On the other hand, key players are undertaking various strategic initiatives to introduce novel products, which is expected to propel market growth. For instance, in March 2021, CureVac N.V. signed a partnership agreement with Celonic Group, engaged in the manufacture of CVnCoV, CureVacs mRNA-based COVID-19 vaccine candidate. CureVac's COVID-19 vaccine candidate is manufactured at Celonic's commercial manufacturing unit for ATMPs and biologics in Heidelberg, Germany. Under the terms of the commercial supply agreement, the Celonic facility could produce over 100 million doses of CVnCoV.

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Advanced Therapy Medicinal Products CDMO Market Trends

Segments Insights:

Product Insights

The gene therapy segment held the largest share of over 49.11% in 2023. Increase in financial support and rise in number of clinical trials for gene therapies are driving demand for gene therapy segment. In 2020, in the first three quarters, gene therapies attracted financing of over USD 12 billion globally, with around 370 clinical trials underway. Additionally, in mid-2022, approximately 2,000 gene therapies were in development, targeting several therapeutic areas, such as neurological, cancer, cardiovascular, blood, and infectious diseases.

The cell therapy segment is expected to show lucrative growth over the forecast period. The field of cellular therapeutics is constantly advancing with inclusion of new cell types, which, in turn, provides ample opportunities for companies to enhance their market positions. Furthermore, the market is attracting new entrants due to high unmet demand for cell therapy manufacturing, the recent approval of advanced therapies, and proven effectiveness of these products.

Indication Insights

The oncology segment accounted for the largest revenue share in 2023. The segments dominance is attributed to disease burden, strategic initiatives undertaken by key players, and availability of advanced therapies used for treating various cancer indications. In January 2021, around 18,000 to 19,000 patients and 124,000 patients were estimated to be potential patients for treating cancer using cell & gene therapy products Kymriah (Novartis AG) and Yescarta (Gilead Sciences, Inc.), respectively. Furthermore, a publication on PubMed reports that as of the conclusion of the first quarter of 2023, there have been over 100 distinct gene, cell, and RNA therapies approved globally, along with an additional 3,700-plus in various stages of clinical and preclinical development.

The cardiology segment is estimated to register the fastest CAGR over the forecast period. This is attributed to the increasing prevalence of cardiovascular diseases and research collaboration for development of advanced therapies. For instance, in October 2023, Cleveland Clinic administered a novel gene therapy to the first patient globally as part of a clinical trial, aiming to deliver a functional gene to combat the primary cause of hypertrophic cardiomyopathy (HCM). Similarly, in February 2021, Trizell GmbH entered into partnership with Catalent, Inc. for development of phase 1 cell therapy to treat micro- and macroangiopathy. Trizell's medication is an Advanced Therapy Medicinal Product (ATMP) that employs regulatory macrophagesa platform technology developed in Germany.

Phase Insights

The phase I segment dominated the market in 2023 due to growing R&D activities and increasing number of human trials for advanced therapies. Phase 1 helps ensure the safety levels of a drug at different doses and dosage forms administered to a small number of patients. This phase is mainly conducted to determine the highest dose a patient can take without any adverse effects. Around 70% of drugs in phase 1 move to the next phase.

The phase II segment has been anticipated to show lucrative growth over the forecast period. Phase II clinical studies comprise the largest number of developing ATMPs, due to the high clearance rate of phase I clinical studies. According to data published by Alliance for Regenerative Medicine, as of June 2022, more than 2,093 clinical trials are ongoing globally, out of which 1,117 are under phase II clinical trials accounting for 53%. Thus, the increase in number of products in phase II is driving the segment.

Regional Insights

North America dominated the overall market share of 49.11% in 2023. This can be attributed to increasing outsourcing activities and rising awareness about advanced therapy. North America has consistently been a leader in R&D for advanced treatments, and it is anticipated that it will keep this position during the forecast period. Recent approvals of products such as Kymriah and Yescarta have propelled investments in the regional market. Moreover, in March 2021, the U.S. FDA approved Abecma, the first approval of CAR-T cells to fight against cancer. Similarly, in December 2023, Casgevy and Lyfgenia, the initial cell-based gene therapies for sickle cell disease (SCD) in patients aged 12 and above, received approval from the U.S. Food and Drug Administration, marking a significant milestone.

The U.S. accounted for the largest share of the global market in the North America region in 2023. The U.S. maintains dominance in this sector due to the presence of a robust and highly advanced biopharmaceutical industry with a considerable focus on research and development. Additionally, the continuous presence of numerous pharmaceutical and biotechnology companies, along with academic and research institutions, generates a sustained demand for rigorous safety testing, further reinforcing the country's leadership in the field.

The Asia Pacific region is expected to grow at the fastest CAGR over the forecast period due to the increasing demand for novel ATMPs and rising R&D activities to develop novel therapies. Moreover, the market growth is driven by continuously expanding CDMO Cell Therapy in the country, a number of domestic players have collaborated with biotech companies from other countries involved in mesenchymal stem cell research and therapy development. In addition, in September 2022 Takara Bio, Inc. launched CDMO Cell Therapy for gene therapy products using siTCR technology for its genetically modified T-cell therapy products.

China accounted for the largest share of the global market in the Asia Pacific region in 2023 due to its strategic focus on advancing research and development capabilities, particularly in the pharmaceutical and biotechnology sectors. Additionally, with a rapidly growing biopharmaceutical industry and supportive government initiatives, China has become a key market for advanced therapy medicinal products (CDMO) market.

Recent Developments

Key Companies & Market Share Insights

Some of the key players operating in the market include AGC Biologics,WuXi Advanced Therapies and Celonic

Minaris Regenerative Medicine and BlueReg are some of the emerging market players in the global market.

Key Advanced Therapy Medicinal Products CDMO Companies:

Segments Covered in the Report

This report forecasts revenue growth at country levels and provides an analysis of the latest industry trends in each of the sub-segments from 2021 to 2033. For this study, Nova one advisor, Inc. has segmented the Advanced Therapy Medicinal Products CDMO market.

By Product

By Phase

By Indication

By Region

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Advanced Therapy Medicinal Products CDMO Industry is Rising Rapidly - BioSpace

The Care Quality Commission (CQC) has registered Gender Plus Hormone Clinic to provide hormone treatments to 16 and 17-year-old children.

This paves the way for other private clinics to be registered, which would offer controversial medical treatments with lifelong consequences to vulnerable teenagers. The decision of the CQC to license a private clinic, creates a significant risk of a two tier approach, with less protection for those who seek help from the private sector. This further risks undermining the work of the Cass review for NHSE practice.

I want the court to set aside the registration by the CQC of Gender Plus Hormone Clinic to provide hormone treatment for teenagers. I also hope that this litigation will prevent the registration of other private clinics providing this controversial treatment. I want to ensure that those under 18 years old, do not suffer irreversible, lifelong harms both physical and psychological, from taking a controversial hormonal treatment which is not evidenced as safe or effective.

Why I am asking for this Judicial Review

I was in the NHS for nearly 40 years and I am now a psychotherapist in private practice. I have worked with people who present with issues around their gender identity for over 20 years. In my clinical experience of working with children and young people, I have not, to date, encountered a 16 to 17-year-old who I would have assessed to be sufficiently fully informed and psychologically ready to make such a life changing, potentially harmful decision. They are in the process of development from child to adult which involves significant mental and physical adjustments. Many of the young people with gender dysphoria/incongruence have no clear understanding of their underlying motivations to take cross, sex, hormones. However they are usually very aware of the discomfort they experience, and often hold a strong belief that the medication will help them feel better. They hope a change to their physical body will bring about a comfort in their mind. Some also receive strong messages from certain groups that medication is the answer to their difficulties which creates an urgent pressure on them and those around them for a solution. As a result, they are rarely able to give a full, in-depth psychological consideration to the implications and consequences of commencing a physical treatment, which is known to have serious, harmful side-effects, and, as yet has a very low level evidence base for it's efficacy and safety.

Under its current registration by the CQC, Gender Plus Hormone Clinic (GHPC) is not prevented from providing GnRH analogues (blockers) for the purpose of suspending puberty. There are some 16-year-olds who have not reached pubertal maturation. Further, the GPHC has said that it would prescribe puberty blockers alongside oestrogen therapy to achieve feminising effects. The NICE report (National Institute of Clinical Excellence) and the Cass review both state that this treatment model is not proven.

There is also considerable risk of complications due to this powerful medication. There are many known side-effects, including blood clots, gallstones, vaginal atrophy and male pattern baldness for females and potential loss of fertility, amongst many others.

The evidence base

The Cass review was commissioned by the NHS to provide a comprehensive review of the appropriate treatment for children and young people with gender dysphoria. The Cass Review sought advice from the National Institute for Health and Care Excellence (NICE) which conducted two separate evidence reviews.

Neither of them has found sufficient evidence to support the use of either puberty blockers or cross sex hormones as safe and effective.

In her interim report published in February 2022, Dr Cass has emphasised the gaps in the "evidence base regarding hormone treatment" (Para 1.41). Although some of her observations related specifically to puberty blockers, she also addressed cross-sex, hormones, and hormone treatment more generally. She said, among other things:

"The Review is not able to provide definitive advice on the use of puberty blockers and feminising/masculinising hormones at this stage, due to gaps in the evidence base; however, recommendations will be developed as our research programme progresses.

The lack of available high-level evidence was reflected in the recent NICE review into the use of puberty blockers and feminising/masculinising hormones commissioned by NHS England, with the evidence being too inconclusive to form the basis of a policy position(para 5.21)

At present we have the least information for the largest group of patients birth- registered females first presenting in early teens(para 5.11).

Your help:

I need your help to ensure that the registration of GPHC is cancelled and the other private clinics are unable to prescribe this controversial treatment to children under 18. We should not be careless or look away from the potential harms this medical treatment might cause to childrens previously healthy bodies.

Please support me with the legal fees required to mount a judicial review and challenge the CQC decision. I was the original claimant who started the Kiera Bell JR with Mrs A and our application on that occasion was successful in providing further scrutiny and attention in this area of paediatric healthcare. That judicial review potentially helped prevent irreversible harms to much younger children too as it led to a much wider scrutiny of the model of treatment in the GIDS.

I have assembled an expert legal team and will be lodging my claim with the High Court in the next few days. Please join me in seeking to protect vulnerable young people and share this crowdfunder link. I know these cases keep coming but we need to protect the next generation.

My X (twitter) handle is @sueevansprotect

Thank you very much.

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PROTECT TEENAGERS FROM HARMFUL AND IRREVERSIBLE MEDICAL TREATMENT - CrowdJustice

Stressing heart cells to study disease - AIP.ORG  American Institute of Physics

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Stressing heart cells to study disease - AIP.ORG - American Institute of Physics

How the genetic battle of the sexes plays out in species that can switch sex  Phys.org

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How the genetic battle of the sexes plays out in species that can switch sex - Phys.org

Mating Study Unlocks the Genetic Code of Attraction  Neuroscience News

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Mating Study Unlocks the Genetic Code of Attraction - Neuroscience News

Allogeneic hematopoietic stem-cell transplantation for patients with Richter transformation: a retrospective study on ...  Nature.com

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Allogeneic hematopoietic stem-cell transplantation for patients with Richter transformation: a retrospective study on ... - Nature.com

Cryonics: advancements, ethics, and skepticism  Kevin MD

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Cryonics: advancements, ethics, and skepticism - Kevin MD

Linux kernel 4.14 gets a life extension, thanks to OpenELA  The Register

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Linux kernel 4.14 gets a life extension, thanks to OpenELA - The Register

Puberty blocker clinic accepted 20k donation from 'sugar daddy'  The Times

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Puberty blocker clinic accepted 20k donation from 'sugar daddy' - The Times

CRISPR Therapeutics AG (CRSP) Outperforms Broader Market: What You Need to Know  Yahoo Finance

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CRISPR Therapeutics AG (CRSP) Outperforms Broader Market: What You Need to Know - Yahoo Finance

Engineering the Microbiome: CRISPR Leads the Way  The Scientist

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Engineering the Microbiome: CRISPR Leads the Way - The Scientist