Although many have never heard of it, β-thalassemia is one of the most common autosomal recessive diseases globally and is a serious blood disorder. Scientists have estimated that about 1.5% of the global population carries mutations associated with β-thalassemia, with more than 60,000 new cases diagnosed every year (1). This blood disorder reduces the production of hemoglobin, the iron-containing protein in red blood cells that plays an essential role in delivering oxygen throughout the body. Scientists and researchers, therefore, seek to produce a gene therapy to treat β-thalassemia and correct the underlying genetic imbalance that causes the condition.

One promising approach involves using clustered regularly interspaced short palindromic repeats (CRISPR) to correct the mutated gene. However, scientists are still working to improve and validate CRISPR’s editing efficiency. Furthermore, CRISPR’s ability to successfully correct mutation associated with -thalassemia is still uncertain. Researchers, therefore, need to pair CRISPR editing protocols with a quality control tool, such as droplet digital polymerase chain reaction (ddPCR), that accurately detects the presence of successful CRISPR edits.

When correcting the mutations with β-thalassemia, scientists do not have a straightforward path toward addressing this condition at the genetic level. Adult hemoglobin is composed of two globin subunits, α-globin and β-globin, which must be expressed in equal numbers for hemoglobin to develop correctly. People with β-thalassemia have a genetic mutation in the gene for β-globin, 

, leading to the gene’s downregulation. With unequal quantities of α-globin and β-globin circulating, the free α-globin forms toxic precipitates that impair the development of red blood cells and kill mature red blood cells. As a result, patients can experience a wide range of severe symptoms, including an increased risk of developing blood clots, weakness, and fatigue, which often lead to early death.

Some research suggests that deleting the α-globin gene, 

, may improve outcomes. Alternatively, introducing a healthy 

 gene via a lentiviral vector improves patients’ clinical outcomes, but only if these patients already express some β-globin (2). A research group based in France and Italy recently combined these two approaches: they used CRISPR to delete one copy of 

 in hopes of restoring the balance between the two hemoglobin subunits (3).

Performing this kind of dual edit is a complex task. Researchers must first design a guide RNA (gRNA) to locate the gene to be edited. Once the gRNA identifies the correct site containing the 

 gene, Cas9 must perform the cut and facilitate the insertion of the 

 gene previously occupied. A dual edit approach only works if CRISPR correctly removes 

 in the same spot. Assessing the success of this technique requires rigorous quality control to ensure CRISPR performs the correct edits. This is where ddPCR technology comes into the equation.

While researchers commonly use quantitative polymerase chain reaction (qPCR) to assess gene-editing success, this technique can have drawbacks, especially with regards to quantification. qPCR can only estimate the transgene copy numbers by relying on a standard curve of serial dilutions to interpret samples. Therefore, the results are less sensitive and can not measure down to one gene per cell. In contrast, ddPCR is well-suited to the task, as it delivers absolute nucleic acid quantification without the aid of a standard curve. ddPCR technology is a highly sensitive tool designed to detect and quantify rare genetic variants, and it can be used to detect outcomes of CRISPR editing. For example, researchers have used ddPCR assays to detect CRISPR edits via homology-directed repair and nonhomologous end-joining (4).

ddPCR technology works by partitioning a sample into approximately 20,000-nL-sized droplets and running a separate PCR reaction in each one. Each droplet contains one or a few nucleic acid strands. If a droplet includes a strand with the target genetic sequence, that DNA will amplify, and the droplet will emit a strong fluorescent signal. On the other hand, if a droplet does not contain the target sequence, the droplet will only emit a weak fluorescence signal. By counting the strongly vs. weakly fluorescent droplets, scientists can detect specific sequences with exacting sensitivity and measure the concentration of the target sequence in the original sample.

Compared to next-generation sequencing, ddPCR technology is fast, inexpensive, and not labor-intensive, and it can detect rare events without being limited by reading depth (5). ddPCR technology is also more sensitive and accurate than qPCR. The France-Italy research group used ddPCR assays to assess the success of their dual editing approach for treating β-thalassemia (3). The researchers first used ddPCR technology to quantify 

 copy number, which correlated with -thalassemia severity. They also used the technology to detect the successful insertion of 

. In human umbilical cord blood-derived erythroid progenitor cells, the team showed robust insertion of the 

 gene, confirming on-target integration of the gene at 0.8 copies per cell.

The team could not have detected this integration using qPCR. Because of the inherent variability in how qPCR results are measured, the technique cannot detect gene copies at concentrations lower than two or three copies per cell. Without ddPCR technology, these researchers would not have shown that their CRISPR strategy has potential for future clinical testing.

Nearly 40 clinical trials are currently investigating whether CRISPR can be used to treat genetic diseases, and regulatory agencies might approve the first CRISPR-based gene therapy in less than a decade (6). But given the continued challenge of developing a reliable CRISPR editing protocol, biopharmaceutical companies developing CRISPR therapies must take care to ensure their therapies are safe and effective by using tools like ddPCR technology to provide the confidence they need. For example, ddPCR assays can be designed to detect any CRISPR edits by using probes that span the junction between the native genome and the donor sequence. Researchers and biopharmaceutical manufacturers can screen out cell lines containing unsuccessful edits before they even reach patients by analyzing cells lines for specific CRISPR edits. This, in turn, will increase the chance of clinical success for CRISPR-based gene therapies and open the door to a new generation of treatments for difficult-to-treat genetic diseases, such as -thalassemia.

 5 (11) (2010).
2. S. Mettananda, et al., 

 125 (24) 3694–3701 (2015).
3. G. Pavani, et al., 

 5 (5) 1137–1153 (2021).
4. Y. Miyaoka, et al., 

 1768, 349–362 (2018).
5. C. Peng, et al., 

 11 (2020).
6. US National Library of Medicine, “NIH,” 

Marwan Alsarraj is the Biopharma Segment manager, Digital Biology Group, at Bio-Rad.

Articles

Features

One get obtain a clearer assessment of gene-editing outcomes through more exacting analytical tools.



Using ddPCR to Assess the Success of CRISPR Gene Therapies

Modeling approaches have been gaining in popularity in the biopharma industry with companies seeking ways to maintain drug development costs. Preclinical studies are one area where modeling techniques are applied, but what is the feasibility of going to purely modeling techniques for preclinical studies? Will the industry opt to combine a modeling approach with traditional animal studies?

Animal studies have been the go-to approach in the biopharmaceutical industry for testing new biotherapeutics in the preclinical phase. According to Lorna Ewart, PhD, chief scientific officer at Emulate, a US-based provider of next-generation 

 models, the advantage of using animals in preclinical drug development is that they provide a dynamic environment to assess drug response across multiple organ systems. Scientists can, therefore, study the relationship between pharmacodynamics (PD) and pharmacokinetics (PK) required to understand efficacy and safety. Many modern biologic drugs, however, are designed against humanized targets, Ewart notes which may reduce the usefulness of the animal model as the pharmacology won’t be assessed.

“As genetic and physiological differences exist across species, animal models are not likely to give an accurate picture of a drug’s pharmacological and pharmacokinetic properties,” Ewart states. In addition, animal models of disease are regularly cited as being poor surrogates of the human condition and thus generate pharmacological data that may not accurately translate to human outcomes, Ewart explains—pointing out that Alzheimer’s models are a good example of inaccurately translated human outcomes from animal studies.

Sheng Guo, vice-president, data science and bioinformatics, at Crown Bioscience, a contract research organization and a JSR Life Sciences company, says that by providing an effective human substitute, murine animal models offer distinct advantages in preclinical drug development. The similarity in human and animal model 

 microenvironments allows for PK/PD studies to produce results closely representative of those expected in humans. Murine models also offer comparability between treated and untreated subjects and are often repeatable, which Guo says is important during preclinical drug development.

“However, these studies can be costly and can present many challenges when trying to screen large cohorts, especially for multi-factor screening. Ensuring studies modeled 

 are completed within often tight timelines can also be difficult, as some animal models may need to be revived, which can take up to three months,” Guo explains.

Moreover, the differing growth rates of tumors mean that some may take many weeks to reach the size needed for these preclinical studies.

“These potential cons need to be carefully considered and balanced against the pros when deciding whether to commence with a study to be modeled 

Using a modeling approach in preclinical studies can be beneficial where traditional animal studies may be challenging.

Such models will often be based on historical data from 

 models that have been accumulated over many years, emphasizes Guo. These models, therefore, have the potential to save money and time, as the 

 studies have already been conducted, Guo points out.

“The use of carefully curated large data sets is essential when using an 

 model approach to gain the greatest insight into preclinical studies. Implementing well-established mathematical modeling methods that are readily applicable can further shorten time frames when conducting these 

Meanwhile, Ewart states that a modeling approach using organ-on-a-chip technology enables scientists to overcome the major limitation of the animal model because the organ-on-a-chip model can be created using human cells. As a result, human-relevant pharmacology can be explored.

“Because the organ-on-a-chip models are also dynamic, the relationship between pharmacology and pharmacokinetics can be explored over time. In addition, the organ-on-a-chip technology has shown that cells remain functional and viable for longer periods of time, enabling researchers to study longitudinal drug responses,” Ewart says.

As models such as this become more mature, the endpoints that scientists can measure are becoming more relevant to those endpoints performed in the clinic, thus enhancing the translational value of the model, Ewart adds.

Particular modeling strategies would make more sense for use in the preclinical stage of biologic drug development. For instance, models built using human cells would be preferable, according to Ewart.

For biologic drugs in the preclinical stage of development, strategies should combine empirical data analysis, statistical modeling, and computational simulations in the design and analysis of murine clinical trials to produce studies that are more rational and powerful than 

 models alone, adds Guo. According to Guo, these modeling strategies should use a comprehensive and diverse data collection that could include:

 studies using similar and related biologic drugs

 studies using validated and well-characterized cell lines

Fluorescence-activated cell sorting data.

“Ideally, modeling strategies should be based on methods with prior successful applications where there is an established framework and guidelines on the design, analysis, and application of the resulting murine clinical trial. By applying these modeling strategies carefully, insight can be gained into the mechanism of action of biologic drugs, and their efficacy can be predicted,” states Guo.

It is unlikely that modeling will completely substitute 

 studies, says Guo. However, modeling is a powerful approach that can be used to complement preclinical murine models to help them achieve their full potential.

“Modeling can be used to improve murine model selection, experimental design, data analysis, result interpretation, and biomarker discovery. As a result, more effective clinical studies that are frequently biomarker-guided in nature can be achieved,” Guo states.

Using modeling methods has made it possible to establish empirical quantitative relationships between mouse number and measurement accuracy for categorical and continuous efficacy endpoints, Guo adds.

“Studying a drug’s efficacy is not currently subject to regulatory guidelines, and it is feasible that models could be used exclusively,” states Ewart. “However, assessment of a drug’s safety is a regulatory requirement with strict guidelines in place. The regulatory community still advocates for animal data to make a risk assessment and is less likely to support drug progression on modeling alone.”

Ewart also points out that, until more models are subject to rigorous evaluation processes and amass data across a broad range of therapeutic modalities, the scenario that will yield better preclinical results is one that incorporates data sets from both animal studies and modeling methods.

Guo concludes that modeling, if applied correctly, can be a useful approach to understanding biologic drugs at the preclinical stage; however, the combination of modeling and murine studies together can be much more powerful.

“As well as the improvements in experimental design and analysis of data, this approach can offer faster biologic drug development, allowing critical drugs to be brought to patients in shorter time frames,” Guo says.

Feliza Mirasol is the science editor for 

One can use modeling methods to generate data and shorten development timelines in preclinical studies.

Adopting a Modeling Approach for Preclinical Studies

Astellas Pharma and GO Therapeutics announced that Xyphos Biosciences, a wholly owned subsidiary of Astellas, and GO have entered into a strategic research collaboration and license agreement to develop novel immuno-oncology therapeutics. Xyphos is paying $20.5 million in upfront cash, but milestone and contingency payments could increase the total to as much as $783 million.

According to an Astellas press release from June 1, 2022, the two companies will collaborate exclusively to identify antibodies with high affinity to two different glycoprotein targets for use in a range of therapeutic modalities. GO will work toward discovering high-affinity antibodies against the two targets, while Astellas will be responsible for research activities, clinical development, and commercialization of any derived therapeutics.

“We are excited to collaborate with Astellas to develop a new class of immuno-oncology therapeutics," said Constantine Theodoropulos, co-founder and CEO, GO Therapeutics, in the press release. “The combination of GO’s targets and antibodies and Astellas’ ACCEL [advanced cellular control through engineered ligands] technology promises to create a new generation of cancer treatments that have a greater therapeutic index. This will enable oncologists to increase the efficacy of antibody-based immunotherapies for solid tumors with less damage to healthy tissues.”

"At Astellas, we have positioned immuno-oncology as one of the primary focuses of our R&D strategy,” said Naoki Okamura, chief strategy office, Astellas, in the press release. “We believe that this collaboration will bring synergies between the two companies' cutting-edge research and will ultimately lead to the development of new therapeutics for patients with great unmet medical needs.”

News

Astellas and GO Therapeutics will work together to develop novel antibodies for immuno-oncology.

Astellas and GO Therapeutics Enter into Immuno-Oncology Research and License Agreement Worth Up to $783 Million

Thermo Fisher Scientific and Qatar Genome Program, a member of the Qatar Foundation, have announced a partnership to use custom genotyping arrays to accelerate genomic research and the clinical applications of predictive genomics in Qatar. The goal of this partnership is to work toward expanding the benefits of precision medicine across global Arab populations.

According to a May 23, 2022 press release, Thermo Fisher and Qatar Genome Program will develop an Axiom custom genotyping array for pan-Arab populations via whole genome sequencing data from 19 Arab countries. The array consists of approximately 800,000 variants and aims to drive research into conditions like diabetes, cardiovascular and metabolic diseases, autism, inherited genetic disorders, and cancer. The array is designed to be a cost-effective alternative to whole genome sequencing for Arab populations.

A partnership between the two entities first began in 2018 when they developed the first microarray specifically designed for the Qatari population, the Q Chip, which detects genetic alterations that increase individuals' risk of various diseases. Per the terms of this agreement, the organizations will continue to refine algorithms and define clinically actionable content to assess polygenic risk scores and clinically relevant variants, including those related to pharmacogenomics.

“Qatar’s national vision is to provide a high standard of living for our people, and that includes providing access to genomics data, technology and insights to improve population health across the country,” said Radja Badji, genome operations manager, Qatar Genome Program, in the press release. “Our collaboration with Thermo Fisher will not only help us advance precision medicine in Qatar but also among people of Arab identity worldwide.”

“Thermo Fisher’s Axiom microarray technology is designed to help accelerate precision medicine, and we are honored to partner with the Qatar Genome Program as they take bold steps to leverage the power of genomics to improve the standard of care for human health,” said Chad Carter, vice-president and general manager of microarray genetic solutions, Thermo Fisher Scientific, in the press release. “Together, we’re creating building blocks for implementing comprehensive precision medicine initiatives at scale for population health.”

Thermo Fisher Scientific and Qatar Genome Program will use custom genotyping arrays to accelerate genomic research in Qatar.

Thermo Fisher Scientific and Qatar Genome Program Partner to Advance Precision Medicine

Bio-Rad Laboratories announced the launch of the CFX Duet Real-Time PCR System on May 26, 2022. The CFX Duet is designed to help researchers develop singleplex and duplex quantitative polymerase chain reaction (qPCR) assays.

According to a company press release, the CFX Duet is a two-color system that is factory calibrated for common dyes, allowing for the precise quantification of up to two targets in genotyping and multiple gene expression analysis without the need for passive reference dies. The device features thermal gradient functionality to enable optimization in fewer runs.

An additional fluorescence resonance energy transfer (FRET) mode supports protein melt analysis for basic protein characterization. The device uses CFX Maestro Software for easy experimental setup and analysis, allowing it to deliver customized reports without exporting data to other programs.

“We are pleased to expand our portfolio of qPCR instruments with the CFX Duet System to help customers run singleplex and duplex assays with a software that matches the robust thermal capabilities of our premium CFX Opus formats.” said Steven Blakely, director for gene expression and software technology, Bio-Rad, in the press release. “The versatile CFX Duet System allows users to quickly get started with gene expression, genotyping, quantification and thermal gradient optimization, as well as protein characterization using FRET.”

New Technology

BioRad Laboratories CFX Duet System is designed to help researchers develop singleplex and duplex quantitative polymerase chain reaction assays.

Bio-Rad Launches CFX Duet Real-Time PCR System

Thermo Fisher Scientific is teaming up with biotech incubator LabShares Newton to provide support to the biotech ecosystem in Boston. Through the collaboration, the partners will provide instruments, lab equipment, and consumables to help early-stage life sciences companies accelerate drug discovery efforts, according to a May 19, 2022 company press release.

LabShares provides fully furnished lab space, services, and equipment to more than 25 biotech companies, which alleviates logistics constraints in bringing novel therapeutics to market. Under the agreement, Thermo Fisher will outfit the shared lab space with equipment, including ultra-low temperature freezers, cell culture incubators, microscopes, and polymerase chain reaction instruments.

Although investment in the biotech industry continues to climb, the high cost of bringing drugs through R&D to market is limiting for many early-stage companies. By offering alternative lab space and more tailored support and resources, LabShares and its partners aim to aid emerging biotech companies clear hurdles that previously hindered growth for smaller companies trying to break through to the market, according to the press release.

“Thermo Fisher Scientific is incredibly excited to partner with LabShares in support of the emerging life science and biotech companies in Newton,” said Angela N. Hokanson, vice-president, corporate accounts, Thermo Fisher Scientific, in the press release. “In line with our mission to enable our customers to make the world healthier, cleaner, and safer, we look forward to collaborating with LabShares and their members to innovate, validate, and commercialize the next generation of life-saving therapies.”

“With little lab space available in historic biotech hubs such as Cambridge [Mass.], emerging companies are looking to the suburbs for more accessible and affordable lab space,” said Jeff Behrens, CEO and founder, LabShares, in the press release. “Working with partners such as Thermo Fisher allows LabShares to provide a caliber of equipment that smaller companies may not otherwise have access to, empowering promising biotech startups to hit the ground running.”

Through the Thermo Fisher Scientific and LabShares Newton partnership, life science startups can accelerate early-stage discovery and development with shared lab spaces.

Thermo Fisher Scientific and LabShares Partner to Support Emerging Boston Biotechs

PerkinElmer has introduced its ultrarapid whole genome sequencing (urWGS) services through PerkinElmer Genomics, expanding the company’s portfolio of whole genome sequencing offerings. The urWGS offering provides physicians with comprehensive, meaningful results in five days to help inform clinical management and improve outcomes for critically ill patients in neonatal and pediatric intensive care units (NICUs and PICUs).

urWGS uses a dried blood spot sample to provide phenotype-driven analysis with a mean coverage of 40x a patient’s genome. According to a May 16, 2022 press release, studies suggest that up to 15% of disease-causing genetic variants may be found in the non-coding regions of the genome, which WGS helps identify. In addition to an analysis of the mitochondrial genome, copy number variation detection—which identifies deletions, duplications, other gene- and chromosomal-level events, spinal muscular atrophy, and a repeat disorders screen—the urWGS offering includes a StepOne comprehensive biochemical profile. The StepOne offering screens for more than 70 inherited conditions and disorders, including the recommended universal newborn screening panel as well as many others that may not be found in state-mandated programs.

Rapid WGS tests have been shown to reduce healthcare costs for patients in NICUs and PICUs (1). In addition, these testing services include the elimination of unnecessary tests and procedures and reduced length of hospital stays, according to PerkinElmer in its press release.

“Increasingly, whole genome sequencing is proving its value as a first-tier clinical test for many patients, especially in a NICU and PICU where timely clinical decisions are critical for timely intervention,” said Madhuri Hegde, senior vice-president and chief scientific officer, Global Lab Services, PerkinElmer, in the press release. “By making services such as this urWGS combined with StepOne available to more hospital systems and physicians, our hope is that we may help shorten the diagnostic odyssey for more newborns and their families. With PerkinElmer’s global leadership in newborn screening using dried blood spot card technology, we will continue to enhance urWGS with additional assays.”

PerkinElmer has expanded its genomics testing services with the launch of its ultrarapid whole genome sequencing.

PerkinElmer Introduces Ultrarapid Whole Genome Sequencing

On May 24, 2022, Lonza and Israel Biotech Fund (IBF), a venture fund investing in Israeli and Israeli-related biotech companies, announced that they have entered into a framework agreement. Under the agreement, Lonza will provide pre-investment due diligence support to IBF and offer IBF's portfolio companies tailored advice as well as flexibility and services for the development and manufacturing of biologics and small molecules. In return, IBF will provide Lonza with access to its portfolio companies and broad network in the Israeli biotech industry.

The two-year agreement aims to broaden the scope of services provided by Lonza to pharmaceutical companies in the Israeli ecosystem. It also gives Lonza an opportunity to support the unique needs of Israeli pharmaceutical companies looking to simplify and de-risk the development of their molecules.

Israel had more than 1750 life science companies active at the end of 2020, many of which were start-ups and small biotech companies. IBF, started in 2015, is dedicated to investing in and developing the biotech industry in Israel and has played a key role in the Israeli Biotech ecosystem. The organization has exposure to and familiarity with the market and key players in the Israeli pharma industry as well as most biotech companies and projects.

As part of the collaboration, Lonza will advise IBF in its due diligence review of candidate biotech targets and provide a tailored offering, advice, and services to IBF's portfolio companies that are targeting innovations in the healthcare sector and that are focused on developing therapeutic assets and disruptive platforms.

"We chose to collaborate with IBF, a leading investment fund in Israel, due to their unique investment strategy, broad network, and approach. We are excited to implement this framework agreement that will provide IBF and their portfolio companies with services and expertise across multiple modalities,” said Pnina Weitz, global head of Venture Capital Business Development and Relationship Management, Lonza, in a company press release. “IBF’s network will allow Lonza to benefit from these connections and offer services and expertise across multiple modalities. Lonza's customized and scalable solutions in the development and manufacture of both biologics and small molecules will allow these companies to leverage our global network and experience and focus on what they do best—developing innovative and transformative treatments."

"Lonza is a leading and reputative global manufacturer, and we are excited to collaborate with them. The unique business model offered by the collaboration agreement will provide Lonza with broad access to the Israeli market and allow our portfolio companies to leverage Lonza's expertise and innovative business models. Such companies will benefit from a streamlined development and manufacturing process, allowing them to focus on research and development and pre-clinical studies," said Ido Zairi, co-founder and managing partner, IBF, in the press release.

Lonza and Israel Biotech Fund form a two-year agreement to support accelerated development and manufacture of biologics and small molecules.

BioPharm International's BP Elements, June 2022