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Commensurate with technological improvements and the subsequent rise in genetic research and genetics-based tests and treatments, the term personalized medicine is being used more frequently. But what does it mean? This explainer outlines what personalized medicine is, its advantages and some concerns that have been raised in relation to it.

Also known as precision medicine, personalized medicine is a rapidly expanding field of practice that uses an individuals genetic profile to guide decisions about the prevention, diagnosis, and treatment of disease.

Although the concept of personalized medicine began in the 1990s, following advances in DNA sequencing technology, it pretty much remained just a concept and was rarely applied. Since then, continued advances have generated enormous amounts of new information. The discovery of genes, proteins, and pathways has enabled studies into the genetics underlying both rare and common diseases and aided the identification of new drug targets.

Whats good about personalized medicine

For a long time, the practice of medicine has largely been reactive, waiting for the onset of disease before treating or curing it. But were all unique in terms of genetic makeup, environment, and lifestyle factors. Our growing understanding of genetics and genomics the study of all of a persons genes and how they drive health, disease and treatment in individual people offers an opportunity to step away from a one size fits all approach based on broad population averages and adopt an individualized approach.

In addition to advances in the field of genomics, developments in the fields of science and technology play a crucial role in personalized medicine. For example, the development of high-resolution analytics, biotech research and chemistry, and the ability to decipher molecular structures, signaling pathways, and protein interactions that underpin the mechanisms of gene expression.

Personalized medicine is about more than prescribing the best drugs, although thats a large part of it. Proponents say it would shift medicines emphasis from reaction to prevention, better predict disease susceptibility and improve diagnosis, produce more effective drugs and reduce adverse side effects, and eliminate the inefficiency and cost of adopting a trial-and-error approach to healthcare.

Goetz & Schork

Weve already seen personalized medicine positively impact patient care in patients with diseases like breast cancer, melanoma, and cardiovascular disease. And the use of patient-derived cell and organoid avatars as disease models to identify beneficial treatments provides truly personalized medicine to individual patients. Then theres CRISPR, technology that allows genetic material to be added, removed, or altered at particular locations in the genome as a direct way of treating genetic and other conditions.

Concerns about personalized medicine

Despite its numerous benefits, the adoption of a personalized medicine approach raises several issues. For it to reach peak efficiency, a lot of genomic data must be collected from a large and diverse section of the population, and its critical that participants privacy and confidentiality are protected. Privacy issues extend to the collection, storage and sharing of that information.

Extensive changes to the healthcare system, including ethical changes, are likely needed to overcome the ethical obstacles of personalized medicine use, including knowledge gap and informed consent, privacy and confidentiality, and the availability of healthcare services. Social benefit versus science development and individual benefit needs to be considered and balanced. And there are concerns that data collected might be used unethically, such as insurance companies not offering some policies to people with a certain genetic predispositions.

Legally, a physician is negligent when they fail to follow generally accepted practice. In personalized medicine, when the interpretation of genetic information is at issue, there may be no generally accepted practice or standard. It begs the question, at what point does clinical genetic knowledge become a standard of practice?

Cost is another relevant factor. While the expense associated with large-scale DNA sequencing is decreasing, its still expensive. And drugs developed based on molecular or genetic variations are likely to be costly. Further, massive amounts of data require massive infrastructure changes, including changes to the mechanisms of data collection, storage and sharing, all of which require investment.

Will personalized medicine happen?

Personalized medicine is already happening in the form of things like CRISPR, mRNA vaccines and the large-scale genome sequencing of newborns. Its the kind of future that was envisioned when the Human Genome Project was first completed 20 years ago, and it certainly has its benefits.

However, the widespread adoption of personalized medicine may prove more difficult than first imagined. In addition to the concerns already mentioned, there needs to be a change in public attitudes and the way medical professionals, patients and health regulators view the approach. It may require a new approach to the way drugs are tested and a willingness to embrace risk.

Nonetheless, the potential benefits are so great and the march of technology and knowledge so inexorable that it is a near certainty that personalized medicine will continue to develop and become standard in healthcare systems at some point in the future it's just a matter of how fast the advances are made and how soon the aforementioned hurdles can be overcome as to exactly when this might be.

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Personalized medicine: The pros, cons and concerns - New Atlas

Abstract

The convergence of artificial intelligence (AI) and precision medicine promises to revolutionize health care. Precision medicine methods identify phenotypes of patients with lesscommon responses to treatment or unique healthcare needs. AI leverages sophisticated computation and inference to generate insights, enables the system to reason and learn, and empowers clinician decision making through augmented intelligence. Recent literature suggests that translational research exploring this convergence will help solve the most difficult challenges facing precision medicine, especially those in which nongenomic and genomic determinants, combined with information from patient symptoms, clinical history, and lifestyles, will facilitate personalized diagnosis and prognostication.

In a recent National Academy of Medicine report about the current and future state of artificial intelligence (AI) in health care, the authors noted unprecedented opportunities to augment the care of specialists and the assistance that AI provides in combating the realities of being human (including fatigue and inattention) and the risks of machine error. Importantly, the report notes that whereas care must be taken with the use of these technologies, much promise exists.1 The digitization of healthrelated data and the rapid uptake in technology are fueling transformation and progress in the development and use of AI in healthcare.2, 3, 4 However, multimodal data integration, security, federated learning (which requires fundamental advances in areas, such as privacy, largescale machine learning, and distributed optimization), model performance, and bias may pose challenges to the use of AI in health care.5

Three main principles for successful adoption of AI in health care include data and security, analytics and insights, and shared expertise. Data and security equate to full transparency and trust in how AI systems are trained and in the data and knowledge used to train them. As humans and AI systems increasingly work together, it is essential that we trust the output of these systems.

Analytics and insights equate to purpose and people where augmented intelligence and actionable insights support what humans do, not replace them. AI can combine input from multiple structured and unstructured sources, reason at a semantic level, and use these abilities in computer vision, reading comprehension, conversational systems, and multimodal applications to help health professionals make more informed decisions (e.g., a physician making a diagnosis, a nurse creating a care plan, or a social services agency arranging services for an elderly citizen). Shared expertise equates to our complementary relationship with AI systems, which are trained by and are supporting human professionals, leading to workforce change, which leads to new skills. The ability to create cuttingedge AI models and build highquality business applications requires skilled experts with access to the latest hardware.

A vast amount of untapped data could have a great impact on our healthyet it exists outside medical systems.6 Our individual health is heavily influenced by our lifestyle, nutrition, our environment, and access to care. These behavioral and social determinants and other exogenous factors can now be tracked and measured by wearables and a range of medical devices. These factors account for about 60% of our determinants of health (behavioral, socioeconomical, physiological, and psychological data), our genes account for about 30%, and last our actual medical history accounts for a mere 10%.6 Over the course of our lifetimes, we will each generate the equivalent of over 300 million books of personal and healthrelated data that could unlock insights to a longer and healthier life.7

The phenomenon of big data can be described using the five Vs: volume, velocity, variety, veracity, and value. Volume refers to the vast amount of complex and heterogenous data, which makes data sets too large to store and analyze using traditional database technology. Velocity refers to the speed at which new data are generated and moves around. Variety refers to the different types of structured, semistructured, and unstructured data, such as social media conversations and voice recordings. Veracity refers to the certainty, accuracy, relevance, and predictive value of the data. Value refers to the conversion of data into business insights. Whereas the volume, variety, velocity, and veracity of data are contributing to the increasing complexity of data management and workloadscreating a greater need for advanced analytics to discover insightsmobile devices have made technology more consumable, creating user demand for interactive tools for visual analytics.

Big data analytics and AI are increasingly becoming omnipresent across the entire spectrum of health care, including the 5 Ps spanning: payer, provider, policy maker/government, patients, and product manufacturers. Up to 10% of global health care expenditure is due to fraud and abuse and AIbased tools help mitigate fraud, waste, and abuse in payer programs.8 Reliable identification of medical coding errors and incorrect claims positively impacts payers, providers, and governments by saving inordinate amounts of money, time, and efforts.9 As an example, IBM DataProbe, an AIbased business intelligence tool, was able to detect and recover US $41.5million in feeforservice payments over a 2year period in Medicaid fraud for Iowa Medicaid Enterprise.10 In the provider space, AI is used for evidencebased clinical decision support,11 detection of adverse events, and the usage of electronic health record (EHR) data to predict patients at risk for readmission.12 Healthcare policymakers and government use AIbased tools to control and predict infections and outbreaks. An example is FINDER, a machinelearned model for realtime detection of foodborne illness using anonymous and aggregated web search and location data.13 Another example is the integrated data hub and caremanagement solution using IBM Connect360 and IBM Watson Care Manager that Sonoma County, California government agencies used to transform health and healthcare for socially disadvantaged groups and other displaced individuals during a time of communitywide crisis.14 This solution enabled integration of siloed data and services into a unified citizen status view, identification of clinical and social determinants of health from structured and unstructured sources, construction of algorithms to match clients with services, and streamlining of care coordination during the 2017 and 2019 Sonoma County wildfires. With the advent of the global pandemic coronavirus disease 2019 (COVID19) in early 2020, such a model can be used to predict atrisk populations, and potentially provide additional risk information to clinicians caring for atrisk patients.15 The use of AI for patients and life sciences/healthcare products are addressed extensively in the sections that follow.

AI is not, however, the only datadriven field impacting health and health care. The field of precision medicine is providing an equal or even greater influence than AI on the direction of health care16 and has been doing so for more than a decade.17 Precision medicine aims to personalize care for every individual. This goal requires access to massive amounts of data, such as data collected through the United Kingdoms UK Biobank and the All of Us project, coupled with a receptive health care ecosystem willing to abandon the conventional approach to care in favor of a more highly individualized strategy. The convergence of these fields will likely accelerate the goals of personalized care and tightly couple AI to healthcare providers for the foreseeable future. In the sections that follow, we will briefly summarize the capabilities of existing AI technology, describe how precision medicine is evolving, and, through a series of examples, demonstrate the potentially transformative effect of AI on the rate and increasing breadth of application for precision medicine.

The past 10years have seen remarkable growth and acceptance of AI in a variety of domains and in particular by healthcare professionals. AI provides rich opportunities for designing intelligent products, creating novel services, and generating new business models. At the same time, the use of AI can introduce social and ethical challenges to security, privacy, and human rights.1

AI technologies in medicine exist in many forms, from the purely virtual (e.g., deeplearningbased health information management systems and active guidance of physicians in their treatment decisions) to cyberphysical (e.g., robots used to assist the attending surgeon and targeted nanorobots for drug delivery).18 The power of AI technologies to recognize sophisticated patterns and hidden structures has enabled many imagebased detection and diagnostic systems in healthcare to perform as well or better than clinicians, in some cases.19 AIenabled clinical decisionsupport systems may reduce diagnostic errors, augment intelligence to support decision making, and assist clinicians with EHR data extraction and documentation tasks.20 Emerging computational improvements in natural language processing (NLP), pattern identification, efficient search, prediction, and biasfree reasoning will lead to further capabilities in AI that address currently intractable problems.21, 22

Advances in the computational capability of AI have prompted concerns that AI technologies will eventually replace physicians. The term augmented intelligence, coined by W.R. Ashby in the 1950s,23 may be a more apt description of the future interplay among data, computation, and healthcare providers and perhaps a better definition for the abbreviation AI in healthcare. A version of augmented intelligence, described in the literature in Friedmans fundamental theorem of biomedical informatics,24 relates strongly to the role of AI in health care (depicted in Figure1). Consistent with Friedmans description of augmented intelligence, Langlotz at Stanford stated that Radiologists who use AI will replace radiologists who dont.25

A version of the Friedmans fundamental theorem of informatics describing the impact of augmented intelligence. The healthcare system with AI will be better than the healthcare system without it. AI, artificial intelligence.

An AI system exhibits four main characteristics that allow us to perceive it as cognitive: understanding, reasoning, learning, and empowering.26 An AI system understands by reading, processing, and interpreting the available structured and unstructured data at enormous scale and volume. An AI system reasons by understanding entities and relationships, drawing connections, proposing hypotheses, deriving inferences, and evaluating evidence that allows it to recognize and interpret the language of health and medicine. An AI system learns from human experts and realworld cases by collecting feedback, learning from outcomes at all levels and granularities of the system, and continuing to improve over time and experience. An AI system empowers and interacts clinicians and users by providing a more integrated experience in a variety of settings, combining dialog, visualization, collaboration, and delivering previously invisible data and knowledge into actionable insights. In contrast, humans excel at common sense, empathy, morals, and creativity.

Augmenting human capabilities with those provided by AI leads to actionable insights in areas such as oncology,27 imaging,28 and primary care.29 For example, a breast cancer predicting algorithm, trained on 38,444 mammogram images from 9,611 women, was the first to combine imaging and EHR data with associated health records. This algorithm was able to predict biopsy malignancy and differentiate between normal and abnormal screening results. The algorithm can be applied to assess breast cancer at a level comparable to radiologists, as well has having the potential to substantially reduce missed diagnoses of breast cancer.30 This combined machinelearning and deeplearning model trained on a dataset of linked mammograms and health records may assist radiologists in the detection of breast cancer as a second reader.

The field of precision medicine is similarly experiencing rapid growth. Precision medicine is perhaps best described as a health care movement involving what the National Research Council initially called the development of a New Taxonomy of human disease based on molecular biology, or a revolution in health care triggered by knowledge gained from sequencing the human genome.31 The field has since evolved to recognize how the intersection of multiomic data combined with medical history, social/behavioral determinants, and environmental knowledge precisely characterizes health states, disease states, and therapeutic options for affected individuals.32 For the remainder of this paper, we will use the term precision medicine to describe the health care philosophy and research agenda described above, and the term personalized care to reflect the impact of that philosophy on the individual receiving care.

Precision medicine offers healthcare providers the ability to discover and present information that either validates or alters the trajectory of a medical decision from one that is based on the evidence for the average patient, to one that is based upon individuals unique characteristics. It facilitates a clinicians delivery of care personalized for each patient. Precision medicine discovery empowers possibilities that would otherwise have been unrealized.

Advances in precision medicine manifest into tangible benefits, such as early detection of disease33 and designing personalized treatments are becoming more commonplace in health care.34 The power of precision medicine to personalize care is enabled by several data collection and analytics technologies. In particular, the convergence of highthroughput genotyping and global adoption of EHRs gives scientists an unprecedented opportunity to derive new phenotypes from realworld clinical and biomarker data. These phenotypes, combined with knowledge from the EHR, may validate the need for additional treatments or may improve diagnoses of disease variants.

Perhaps the most wellstudied impact of precision medicine on health care today is genotypeguided treatment. Clinicians have used genotype information as a guideline to help determine the correct dose of warfarin.35 The Clinical Pharmacogenetics Implementation Consortium published genotypebased drug guidelines to help clinicians optimize drug therapies with genetic test results.36 Genomic profiling of tumors can inform targeted therapy plans for patients with breast or lung cancer.34 Precision medicine, integrated into healthcare, has the potential to yield more precise diagnoses, predict disease risk before symptoms occur, and design customized treatment plans that maximize safety and efficiency. The trend toward enabling the use of precision medicine by establishing data repositories is not restricted to the United States; examples from Biobanks in many countries, such as the UK Biobank,37 BioBank Japan,38 and Australian Genomics Health Alliance39 demonstrate the power of changing attitudes toward precision medicine on a global scale.

Although there is much promise for AI and precision medicine, more work still needs to be done to test, validate, and change treatment practices. Researchers face challenges of adopting unified data formats (e.g., Fast Healthcare Interoperability Resources), obtaining sufficient and high quality labeled data for training algorithms, and addressing regulatory, privacy, and sociocultural requirements.

AI and precision medicine are converging to assist in solving the most complex problems in personalized care. Figure2 depicts five examples of personalized healthcare dogma that are inherently challenging but potentially amenable to progress using AI.40, 41, 42

Dimensions of synergy between AI and precision medicine. Both precision medicine and artificial intelligence (AI) techniques impact the goal of personalizing care in five ways: therapy planning using clincal, genomic or social and behavioral determinants of health, and risk prediction/diagnosis, using genomic or other variables.

Genomeinformed prescribing is perhaps one of the first areas to demonstrate the power of precision medicine at scale.43 However, the ability to make realtime recommendations hinges on developing machinelearning algorithms to predict which patients are likely to need a medication for which genomic information. The key to personalizing medications and dosages is to genotype those patients before that information is needed.44

This use case was among the earliest examples of the convergence between AI and precision medicine, as AI techniques have proven useful for efficient and highthroughput genome interpretation.45 As noted recently by Zou and colleagues,46 deep learning has been used to combine knowledge from the scientific literature with findings from sequencing to propose 3D protein configurations, identify transcription start sites, model regulatory elements, and predict gene expression from genotype data. These interpretations are foundational to identifying links among genomic variation and disease presentation, therapeutic success, and prognosis.

In medulloblastoma, the emergence of discrete molecular subgroups of the disease following AImediated analysis of hundreds of exomes, has facilitated the administration of the right treatment, at the right dosage, to the right cohort of pediatric patients.47 Although conventional treatment of this disease involved multimodal treatment, including surgery, chemotherapy, and whole brain radiation, precision genomics has enabled treatment of the wingless tumor subgroup, which is more common in children, with chemotherapy aloneobviating the need for radiation.48 Avoiding radiotherapy is particularly impactful for mitigating potential neurocognitive sequelae and secondary cancers from wholebrain radiation among disease survivors.49, 50

The initial successful paradigm of AI in imaging recognition has also given rise to radiogenomics. Radiogenomics, as a novel precision medicine research field, focuses on establishing associations between cancer imaging features and gene expression to predict a patients risk of developing toxicity following radiotherapy.51, 52, 53 For example, Chang et al. proposed a framework of multiple residual convolutional neural networks to noninvasively predict isocitrate dehydrogenase genotype in grades IIIV glioma using multiinstitutional magnetic resonance imaging datasets. Besides, AI has been used in discovering radiogenomic associations in breast cancer,52 liver cancer,54 and colorectal cancer.53 Currently, limited data availability remains the most formidable challenge for AI radiogenomics.51

Knowing the response to therapy can help clinicians choose the right treatment plan. AI demonstrates potential applications in this area. For example, McDonald et al. trained a support vector machine using patients gene expression data to predict their response to chemotherapy. Their data show encouraging outcomes across multiple drugs.55 Sadanandam et al. proposed approaches of discovering patterns in gene sequences or molecular signatures that are associated with better outcomes following nontraditional treatment. Their findings may assist clinicians in selecting a treatment that is most likely to be effective.56 Although tremendous progress has been made using AI techniques and genomics to predict treatment outcome, more prospective and retrospective clinical research and clinical studies still need to be conducted to generate the data that can then train the algorithms.

Incorporating environmental considerations into management plans require sufficient personal and environmental information, which may affect a patients risk for a poor outcome, knowledge about care alternatives, and conditions under which each alternative may be optimal.

One such example has been the challenge of identifying homelessness in some patients.57, 58, 59 These patients may require care in varying locations over a short period, requiring frequent reassessments of patient demographic data. Related issues, such as transportation, providing medications that require refrigeration, or using diagnostic modalities that require electricity (for monitoring), need to be modified accordingly.

Another environmental consideration is the availability of expertise in remote locations, including the availability of trained professionals at the point of need. AI has provided numerous examples of augmenting diagnostic capabilities in resourcepoor locations, which may translate into better patient classification and therefore more personalized therapy planning. Examples include the use of deep learning to identify patients with malaria60 and cervical cancer,61 as well as predicting infectious disease outbreaks,62 environmental toxin exposure,63 and allergen load.64

Finally, in addition to genomic considerations and social determinants of health, clinical factors are imperative to successful therapy planning. Age, comorbidities, and organ function in particular predicate treatment considerations and AI has emerged as a central pillar in stratifying patients for therapy. In one study, machine learning classifiers were used to analyze 30 comorbidities to identify critically ill patients who will require prolonged mechanical ventilation and tracheostomy placement.65 Other studies have used AI algorithms to analyze bedside monitored adverse events and other clinical parameters to predict organ dysfunction and failure.66, 67

Actress Angelina Jolies response to her inheritance of the BRCA gene illustrates the potential impact of more advanced genomic information on disease risk and prevention options.68 This case is not novel; the case of Woodie Guthrie and Huntingtons disease disclosed a similar conundrum for health care.69 Although the ethics of genetic testing without a clear cure continues to be debated, the broad availability of genetic information offered by nextgeneration sequencing and directtoconsumer testing renders personalized prevention and management of serious diseases a reality.70

Cardiovascular medicine is an area with a long history of embracing predictive modeling to assess patient risk.71 Recent work has uncovered methods to predict heart failure72 and other serious cardiac events in asymptomatic individuals.73 When combined with personalized prevention strategies,74, 75 these models may positively impact disease incidence and sequela. Complex diseases, such as cardiovascular disease, often involve the interplay among gender, genetic, lifestyle, and environmental factors. Integrating these attributes requires attention to the heterogeneity of the data.76 AI approaches that excel at discovering complex relationships among a large number of factors provide such opportunities. A study from Vanderbilt demonstrated early examples of combining EHR and genetic data, with positive results in cardiovascular disease prediction.77 AIenabled recognition of phenotype features through EHR or images and matching those features with genetic variants may allow faster genetic disease diagnosis.78 For example, accurate and fast diagnosis for seriously ill infants that have a suspected genetic disease can be attained by using rapid wholegenome sequencing and NLPenabled automated phenotyping.79

Automated speech analytics have benefited from improvements in the technical performance of NLP, understanding, and generation. Automated speech analytics may provide indicators for assessment and detection of earlystage dementia, minor cognitive impairment, Parkinsons disease, and other mental disorders.80, 81, 82, 83 Efforts also are underway to detect changes in mental health using smartphone sensors.84

AIassisted monitoring may also be used in realtime to assess the risk of intrapartum stress during labor, guiding the decision of cesarean section vs. normal vaginal deliveries, in an effort to decrease perinatal complications and stillbirths.85 This exemplifies realtime AIassisted monitoring of streaming data to reduce manual error associated with human interpretation of cardiotocography data during childbirth.

AI is also being used in the detection and characterization of polyps in colonoscopy.86 Wider adaptation of AI during endoscopy may lead to a higher rate of benign adenoma detection and reduction of cost and risk for unwarranted polypectomy.87 AImediated image analysis aimed at improving disease risk prediction and diagnosis will likely continue to increase in use for detection of diabetic retinopathy88 and metastasis in cancer,89 as well as for identification of benign melanoma.90 AIbased image analysis has become a part of a directtoconsumer diagnostic tool for anemia as well.91

The widespread use of home monitoring and wearable devices has long been accompanied by the expectation that collected data could help detect disease at an earlier state. Indeed, these advances have fueled new, noninvasive, wearable applications for monitoring and detecting specific health conditions, such as diabetes, epilepsy, pain management, Parkinsons disease, cardiovascular disease, sleep disorders, and obesity.92 Digital biomarkers are expected to facilitate remote disease monitoring outside of the physical confines of a hospital setting and can support decentralized clinical trials.93 Wearable tools that provide continuous multidimensional measurements of preselected biomarkers would enable the detection of minimum residual disease and monitor disease progression.94 In the field of cancer care, evolving technology using wearable devices continuously analyzes circulating tumor cells to screen for relapsed disease.95

We have observed increasing efforts to implement AI in precision medicine to perform tasks such as disease diagnosis, predicting risk, and treatment response. Although most of these studies showed promising experimental results, how AI improved health care is not fully demonstrated. In reality, the success of transforming an AI system to a realworld application not only depends on the accuracy but also relies on the capability of working accurately in a reliable, safe, and generalizable manner.5 For example, the difference among institutions in coding definitions, report formats, or cohort diversity, may result in a model trained using onesite data to not work well in another site (https://www.bmj.com/content/368/bmj.l6927). Here, we highlighted three main challenges that would impact the success of transitions to realworld healthcare.

Fairness and bias. The health data can be biased while building and processing the dataset (e.g., a lack of diverse sampling, missing values, and imputation methods; https://datasociety.net/library/fairnessinprecisionmedicine/). An AI model trained on the data might amplify the bias and make nonfavorable decisions toward a particular group of people characterized by age, gender, race, geographic, or economic level. Such unconscious bias may harm clinical applicability and health quality. Thus, it is crucial to detect and mitigate the bias in data and models. Some potential solutions include improving the diversity of the data, such as the All of Us program that aimed to recruit participants with diverse backgrounds. AI communities also proposed several techniques to fight against bias (https://arxiv.org/abs/1908.09635). IBM has developed an online toolkit (AI Fairness 360) that implemented a comprehensive set of fairness metrics to help researchers examine the bias among datasets and models, and algorithms to mitigate bias in classifiers (https://doi.org/10.1147/JRD.2019.2942287). However, fairness and protected attributes are closely related to the domain context and applications. More work is needed in biomedical research to define and explore the fairness and bias in AI models trained with historical patient data. To address the challenge, a collaborative effort that involves the AI and biomedical community is needed.

Socioenvironmental factors. The environmental factors and workflows where the AI model would be deployed may impact model performance and clinical efficacy. A recent prospective study carried out by Google Health evaluated an AI system for screening diabetic retinopathy in a real clinical environment. The AI system was developed to augment diabetic retinopathy screening by providing intime assessment, before this the process may take several weeks. Despite a specialistlevel accuracy (>90% sensitivity and specificity) achieved on retrospective patient data; however, the system has undergone unexpected challenges when applied to Thailand clinics (https://doi.org/10.1145/3313831.3376718). For example, the variety of conditions and workflows in clinics impaired the quality of the images that did not meet the system' high standards, resulting in a high rejection rate of images. The unstable internet connection restricted the processing speed of the AI models and caused a longer waiting time for the patients. Travel and travel costs may deter participants from remaining in the study. Such prospective studies highlighted the importance of validating the AI models in the clinical environment and considering an iteration loopthat collects users feedback as new input for learning and system improvement96 before applying the AI system widely. Of note, in healthcare, obtaining such feedback would take a long time at a high cost. It may take a longer time to evaluate a therapys effect and associated longterm health outcomes than what is required to validate whether a product is appealing to a customer. There is a need to explore other ways to facilitate creation of highperforming AI systems, for example, generating synthetic data that carries similar distributions and variances as the realworld data, or leveraging a simulated environment. Early examples by groups, such as Baowaly and colleagues,89 demonstrate much promise, but more AI research efforts are needed.

Data safety and privacy. Data is crucial to an AIdriven system. As AI and precision medicine are converging, data (e.g., genomics, medical history, behaviors, and social data that covers peoples daily lives) will be increasingly collected and integrated. Individuals concerns for data privacy are closely related to trust when they use AIenabled services. Building a safe and wellcontrolled ecosystem for data storage, management, and sharing is essential, requiring new technology adoptions, and collaborations, as well as the creation of new regulations and business models.

The training of AI methods and validation of AI models using large data sets prior to applying the methods to personal data may address many of the challenges facing precision medicine today. The cited examples reinforce the importance of another potential use of augmented intelligence, namely that of the role of technology in the hands of consumers to help communicate justintime risk or as an agent of behavior change. Although most studies to date are small and the data are limited, the ability to identify atrisk patients will translate into personalized care when identification is combined with strategies to notify and intervene. Researchers are actively pursuing the use of mobile apps, wearables, voice assistants, and other technology to create personspecific interfaces to intelligent systems. A review of these approaches is beyond the scope of this paper.

Active research in both AI and precision medicine is demonstrating a future where healthrelated tasks of both medical professionals and consumers are augmented with highly personalized medical diagnostic and therapeutic information. The synergy between these two forces and their impact on the healthcare system aligns with the ultimate goal of prevention and early detection of diseases affecting the individual, which could ultimately decrease the disease burden for the public at large, and, therefore, the cost of preventable health care for all.

This work was funded by a partnership between IBM Watson Health and Vanderbilt University Medical Center.

Drs. Weeraratne, Rhee, and Snowdon are employed by IBM Watson Health. All other authors declared no competing interests for this work.

The authors thank Karlis Draulis for his assistance with the figures.

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Precision Medicine, AI, and the Future of Personalized Health Care

BOSTON, Nov. 01, 2024 (GLOBE NEWSWIRE) -- Praxis Precision Medicines, Inc. (NASDAQ: PRAX), a clinical-stage biopharmaceutical company translating genetic insights into the development of therapies for central nervous system (CNS) disorders characterized by neuronal excitation-inhibition imbalance, today announced it will report its financial results from the third quarter ended September 30, 2024, before the financial markets open on Wednesday, November 6, 2024.

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Praxis Precision Medicines to Announce Third Quarter 2024 Financial Results and Host Conference Call on Wednesday, November 6, 2024

Fiocruz and GEMMABio announce partnership for the development of gene therapies  Fiocruz

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Fiocruz and GEMMABio announce partnership for the development of gene therapies - Fiocruz

The Johns Hopkins Center for Nanomedicine (CNM) brings together engineers, scientists, and clinicians working together under one roof on translation of novel drug and gene delivery technologies. The focus is to overcome major challenges to drug efficacy, including biological barriers to delivery, patient compliance, and toxicity. Furthermore, we educate and train the next generation of researchers to innovate at the interface of engineering, medicine, and the life sciences in the development of drug delivery technologies with potential for clinical implementation. We believe that collaboration and commitment to diversity are key in having the maximum impact on human health.

Nanomedicine is defined as the medical application of nanotechnology. In the CNM, we focus on harnessing nanotechnology to more effectively diagnose, treat, and prevent various diseases. Our entire bodies are exposed to the medicines that we take, which can lead to unpleasant side effects and minimize the amount of medicine that reaches the places where it is needed. Medications can be more efficiently delivered to the site of action using nanotechnology, resulting in improved outcomes with less medication.

We design our nanotechnology-based platforms for clinical translation. What this means is that we strive for innovative simplicity and the use of components that have a history of medical safety, so that our nanomedicines can be tested in clinical trials and developed into useful products. To this end, we often design our platforms to mimic nature or select our systems based on how they naturally distribute in the body. To date, our faculty have founded more than 10 start-up companies, resulting in several FDA-approved products and others being tested in clinical trials.

Focus areas: Glaucoma, age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, diabetic macular edema, uveitis, dry eye disease, corneal neovascularization, corneal graft rejection, thyroid eye disease, cataract surgery, glaucoma surgery, ocular trauma

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Focus areas: Preterm birth, intrauterine inflammation, infections,in vitrofertilization, reproductive tract cancers, contraception

Focus areas:Brain tumors, cerebral palsy, neonatal stroke, traumatic brain injury, autism spectrum disorders, Parkinsons disease, Alzheimers disease

The CNM team includes basic science and clinical faculty, research staff, postdoctoral fellows, graduate students, and undergraduates. Our goal is to train, mentor, and promote inclusive and supportive research environments.

In addition to a rigorous and broad training in unbiased experimental design, methods, data analysis, interpretation, and reporting, we strive to support our trainees in career and professional development. We proudly support diverse career goals, and our alumni have gone on to careers in academia, industry, regulatory, venture capital, consulting, science communication, policy, law, and medicine. We participate in numerous training programs that promote and develop trainees from low income and underrepresented groups in biomedical research.

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Center for Nanomedicine - Johns Hopkins Medicine

P T. 2012 Sep; 37(9): 512-517, 525.

Part 1: Emerging Concepts

The author is a Consultant Medical Writer living in New Jersey.

Nanoparticles may soon be used to transport diagnostic and therapeutic drugs to targeted sites not normally accessible, thereby improving treatment and reducing costs. Further research is still needed to establish the efficacy and safety of these nanomaterials.

This is the first in a series of three articles about nanomedicine. Part 2 will discuss the current and future clinical applications of nanomedicine. The third article in this series will focus on the regulatory and safety challenges presented by nanomedicine.

Nanomedicine, the application of nanotechnology to medicine, is currently at an early stage but it is expected to have a revolutionary impact on health care.1 Nanomedical research is heavily supported by public policy and investment, and is progressing rapidly.1,2 The continued development of nanomedicines has the potential to provide numerous benefits, including improved efficacy, bioavailability, doseresponse, targeting ability, personalization, and safety compared to conventional medicines.25 The most exciting concept in nanomedical research may be the design and development of multifunctional nanoparticle (NP) complexes that can simultaneously deliver diagnostic and therapeutic agents to targeted sites.5,6 These capabilities are unprecedented and represent tremendous progress toward improving patient diagnosis, treatment, and follow-up.6 However, despite these potential benefits, essential data regarding the pharmacokinetics, pharmacodynamics, and toxicity of many nanomaterials are currently lacking.5,7

Nanotechnology is a rapidly advancing field that is expected to have a revolutionary impact on many industries, including medicine.8,9 Nanotechnology has been made possible through the convergence of many scientific fields, including chemistry, biology, physics, mathematics, and engineering.1,2,9

A nanometer (nm) is one billionth of a meter, and the prefix nano- comes from the Greek word for dwarf.4,10 Nanotechnology provides scientists with new tools for the investigation, manipulation, and control of atoms, molecules, and submicroscopic objects, generally ranging from 1 to 100 nm.1,6 Nanotechnology allows scientists to take advantage of naturally occurring quantum effects at the nanoscale level that influence biological, physical, chemical, mechanical, and optical properties.6,10,11 These unique effects often give nanoscale materials desirable chemical, physical, and biological properties that differ from those of their larger, or bulk, counterparts.12

The convergence of nanotechnology and medicine has led to the interdisciplinary field of nanomedicine.6 Advances in genetics, proteomics, molecular and cellular biology, material science, and bioengineering have all contributed to this developing field, which deals with physiological processes on the nanoscale level.6,9 Many of the inner workings of a cell naturally occur on the nanoscale level, since the dimensions of many biologically significant molecules like water, glucose, antibodies, proteins, enzymes, receptors, and hemoglobin are already within the nanoscale range (see ).6,11 Many researchers are currently working on medical treatments, devices, and instruments that use nanotechnology to increase efficacy, safety, sensitivity, and personalization.11 Potentially beneficial properties of nanotherapeutics include improved bioavailability, reduced toxicity, greater dose response, and enhanced solubility compared with conventional medicines.2

This scale depicts the relative size of nanoscale, microscopic, and macroscopic objects. (Adapted from the National Cancer Institute.15)

The National Nanotechnology Initiative (NNI), a federal research and development program, defines nanotechnology as the science of materials and phenomena in the range of 1 to 100 nm in diameter.2,4,10 Many federal agencies, including the FDA and the Patent and Trademark Office (PTO), continue to use this definition.2 However, some experts say that this size limitation is artificial and misleading, since nanomaterials can have unique properties even in sizes up to several hundred nanometers.2

The National Institutes of Health (NIH) has presented an alternative definition of nanotechnology that doesnt rely on size; instead, it defines the field as (1) studies that use nanotechnology tools and concepts to study biology, (2) the engineering of biological molecules to have functions that differ from those that they have in nature, or (3) the manipulation of biological systems by methods more precise than standard molecular biological, synthetic, chemical, or biochemical approaches.2

Nanotechnology has the potential to be used in a wide range of products, including medicines, electronics, cosmetics, and foods.1,1315 According to the Project for Emerging Nanotechnologies at The Woodrow Wilson International Center for Scholars, more than 800 nanotechnology-based products are already on the market.9 Nanotechnology has been used in laptop computers, cell phones, digital cameras, water-filtration systems, and cosmetics.14,15 Nanotechnology research is also under way to improve the bioavailability of food nutrients and to develop food packaging that detects and prevents spoilage.14,16

Nanotechnology has also been applied to improve a number of medical products and processes;14,15 these include drugs, medical imaging, antimicrobial materials, medical devices, sunscreens, burn and wound dressings, dental-bonding agents, sunscreens, and protective coatings for eyeglasses.14,15 Nanotechnology has improved drug targeting and bioavailability, diagnostic imaging, biomarker detection sensitivity, and drug-delivery efficiency.16 Some nanomedicines that are currently on the market include doxorubicin HCl liposome injection (Doxil, Ortho Biotech) for ovarian cancer; daunorubicin citrate liposome injection (DaunoXome, Diatos) for advanced AIDS-related Kaposis sarcoma; and amphotericin B liposome injection (AmBisome, Gilead) for fungal infections.3,5 In addition, paints containing silver NPs, which have antimicrobial properties, are being used in indoor medical settings, such as in hospitals.17

Nanotechnology is a rapidly growing field. In 2008, nanotechnology was estimated to be a $10.5 billion industry in the U.S, mostly due to consumer product applications.17 It is estimated that the nanotechnology industry will grow to $1 trillion by 2015, representing an increase of about 100-fold in just 7 years.17

Nanomedicine has always been a major application for nanotechnology. 8 According to the National Science Foundation (NSF), by 2020, one-third of patents and start-up companies in the nanotechnology sector will involve biomedical applications. 8,18 The NSF also predicts that nearly half of future pharmaceuticals will have some nanotechnology components.4,18

The physical characteristics of NPs can differ in many ways that influence function.9 A discussion of several of these physical features follows.

NPs are inherently small, with at least one dimension in the range of 1 to 100 nm, although they can also be micrometer (m)-sized particles.6,9 NPs have novel structural, optical, and electronic properties that many larger molecules or bulk solids lack.9 They also have improved solubility, so they may be used to reinvestigate bulk drug counterparts that are known to have poor solubility.6 This property may provide the ability to convert insoluble or poorly soluble drugs into soluble aqueous suspensions, thus eliminating the need for toxic organic solvents.4 Another key benefit related to the small size of NPs is an increased bioavailability and circulation time.3 Studies have shown that particles under 200 nm have longer circulation times, compared with larger particles, irrespective of any surface modifications present.3

NPs come in a variety of shapes, including spheres, discs, hemispheres, cylinders, cones, tubes, and wires.6,9 NPs can also be hollow, porous, or solid.5 These characteristics of NPs can be selected on the basis of interactivity, loading capacity, and transport capabilities.6 For example, a hollow NP may be an attractive carrier for drug therapies or imaging contrast agents.6

One feature of NPs that gives them unique physical properties is a large surface area relative to size.2 As particle size decreases, total surface area increases exponentially ().2,11 An increase in surface area means that a greater proportion of atoms are located on the particle surface relative to the core.2 This phenomenon makes NPs more reactive compared with conventional larger molecules, or bulk solid counterparts.2 Increased surface area is also responsible for the enhanced water solubility and bioavailability that often occur with NPs.2

Illustration depicting the exponential increase in surface area that occurs with nanoscale materials. (Adapted from the National Technology Initiative.11)

The large surface area of NPs also allows them to be designed to include a broad range of surface characteristics, including conjugation with electrostatic charges or biomolecules.6 Such surface features can be strategically selected for targeting and other purposes and are therefore determined on that basis.9

If NPs are properly designed, their small size can enable them to cross physiological barriers to deliver drugs to sites that are not normally accessible by traditional means.6 For example, the increased permeability of an NP may allow it to transport cancer drugs into tumors by passing through neovessel pores that are less than 1 m in diameter.5 The increased permeability of NPs may also allow them to cross the bloodbrain barrier through the use of different uptake mechanisms.6

A wide variety of NPs and materials are used in nanomedicine, depending on the application.6 Among the most widely used are liposomes, polymers, quantum dots (QDs), iron oxide (IO) particles, and carbon nanotubes and nanoshells.6

A liposome is a spherical vesicle composed of a lipid bilayer membrane and an empty core that usually carries an aqueous solution.5 Liposomes are usually 90 to 150 nm in diameter and are thus slightly larger than conventional NPs.5 Liposomes are often designed to carry biomolecules (e.g., monoclonal antibodies, antigens) that are conjugated to the surface as ligands.5

Liposomes are often used in nanomedical research because they have many unique properties.5 The components of liposomes are similar to natural human cell membranes; thus, they confer liposomal drug delivery with several intrinsic benefits.5 Liposomes circulate in the bloodstream for an extended time, compared with non-liposomal drugs, providing a longer treatment effect. Liposomes also accumulate at the site of a tumor or infection, naturally locating and delivering higher drug levels to these targets.5 Liposomes can carry and deliver either hydrophilic or hydrophobic therapies, which can be stored in their empty cores.6 By using lipids of different fatty-acid-chain lengths, scientists can construct liposomes to be temperature-sensitive or pH-sensitive, thereby permitting the controlled release of their contents only when they are exposed to specific environmental conditions.5

In contrast to other materials, data on the safety and efficacy of many polymers already exist; therefore, polymer NPs are widely used in nanomedical research.3 Polymer NPs can be fabricated in a wide range of varieties and sizes, ranging from 10 nm to 1 m.3,5 Some polymer NPs can facilitate drug release for weeks and do not accumulate in the body.3,5,6 As such, polymeric NPs are considered promising carriers for numerous medications, including those used in cancer, cardiovascular disease, and diabetes treatments; bone-healing therapies; and vaccinations.3 Contrast agents can also be conjugated to the surface of polymeric NPs, allowing them to be used in diagnostic imaging.5

Biodegradable polymers are of particular interest, since they can be fully metabolized and removed from the body.6Poly-lactic-co-glycolic acid (PLGA) is an especially intriguing example of a biodegradable polymer, since relative proportions of polylactic acid (PLA) and polyglycolic acid (PGA) can be used to fine-tune the biodegradability of PLGA.6

Quantum dots (QDs) are semiconductor nanocrystals that range in size from 2 to 10 nm and usually consist of 10 to 50 atoms.4,5 Although QDs have been used in electronics and optics for 20 years, they have only recently been applied to nanomedical research.5 The most commonly used QDs for biomedical applications contain cadmium selenide (CdSe) or cadmium telluride (CdTe).4 QDs containing indium phosphide (InP) and indium arsenide (InAs) are also frequently used.4

QDs have unique optical and electronic properties, making them valuable as luminescent probes and giving them tremendous potential in many biomedical applications.4,5 QDs are intrinsically fluorescent and emit light over a broad range, from the near-ultraviolet (UV) to mid-infrared spectrum.9 They have size-dependent optical properties, extraordinary photostability, and surface properties that can be fine-tuned, which make them ideal for optical imaging.4 QDs have molar extinction coefficients that are 10 to 50 times larger than those of organic dyes, making them much brighter in in vivo conditions.5 They have long blood circulation times and can fluoresce for several months in vivo.5

QDs also have sufficient surface area to attach agents for simultaneous targeted drug delivery and in vivo imaging or for tissue engineering.4 Many uses of QDs for in vivo imaging have already been reported, including lymph node and angiogenic vessel mapping and cell subtype isolation.5 QDs are very efficient agents for cancer diagnosis in vivo, because the extremely small size of the QDs allows unimpeded access to systemic circulation and surface modifications can target them to neoplastic sites.4 Additional potential uses for QDs include image-guided surgery, light-activated therapies, and diagnostic tests.19

Surface coatings have been found to enhance the surface fine-tunability and increase the fluorescent yield of QDs.4 They may also reduce the adverse effects that can be elicited by QDs containing Cd, Se, and As, which are toxic materials.4 At present, the investigation of QDs is restricted to in vitro and animal studies because of toxicity concerns regarding these heavy metals.5,19 Novel methods to produce new generations of QDs in which toxic materials are reduced or absent are being pursued for future applications in humans.5

Superparamagnetic NPs, like iron oxide (SPIO) and magnetite, have been used for years as nontargeted contrast agents for magnetic resonance imaging (MRI).1,5,17 However, these NPs do have superparamagnetic properties that allow them to be directed in situ with the use of a magnetic field.17 They also have a long retention time in circulation, are usually biodegradable, and have low toxicity.5 They are therefore excellent candidates for producing imageable therapeutic nanodevices.5

In addition to possessing other desirable properties, SPIO NPs can also be functionalized (designed) to achieve specific tumor targeting.5 SPIO NPs are increasingly being used for the development of target-specific MRI contrast agents.5 To date, SPIO NPs have been used for many applications, such as the delivery of antibiotics and drugs with simultaneous enhancement of MRI contrast and for the separation of bacteria from biomolecules.17

Carbon nanotubes are composed of a distinct molecular form of carbon atoms that give them unusual thermal, mechanical, and electrical properties.5 For example, they are 100 times stronger than six times their weight in steel.5 Carbon nanotubes modified with polyethylene glycol (PEG) are surprisingly stable in vivo, with long circulation times and low uptake by the reticuloendothelial system (RES).5 Carbon nanotubes have been used for the delivery of imaging and therapeutic agents and in the transport of DNA molecules into cells.5 The nanoscale dimensions of single-walled and multiwalled carbon nanotubes, along with their electrocatalytic properties and high surface area, have compelled researchers to utilize them as nanoelectrodes.20

Carbon nanoshells are composed of a silica core that is covered by a thin metallic shell, usually composed of gold.5 Carbon nanoshells have an ability to scatter light, a feature that is useful for cancer imaging.5 However, their primary use continues to be in thermal ablation therapy.5 Alternatively, focused lasers have been useful for cancer thermotherapy, but they cannot discriminate between diseased and healthy tissue.1 However, when carbon nanoshells are used for targeting in thermal ablation therapy, thermal energy passes through healthy tissue without causing harm, killing only the targeted tumor cells.5 In mice, carbon nanoshells and near-infrared spectroscopy (NIRS) thermal ablation therapy completely eliminated colon carcinoma cell tumors in vivo.5

The aforementioned, and other, NPs are used to construct multifunctional NP complexes that mix and match different features, or functionalizations, in order to achieve an intended purpose.17 A multifunctional NP complex may be designed to include the following components ():3,5,21

surface ligands that target the attachment of NPs to specific locations (e.g., organs, cells, or tissues).

linker molecules that release the cargo carried by the NP at the target site in response to a remote trigger or specific environmental cues.

a core that encapsulates targeting or imaging cargo or has optical or magnetic properties (gold, SPIO) that can localize the NP at the target site.

one or more therapeutic or diagnostic cargoes that are encapsulated in the NP core or attached to its surface.

a coating, such as PEG, that improves biocompatibility and/or enhances bioavailability by increasing circulation times and slowing clearance from the body.

Diagram representing a multifunctional NP complex. The carrier particle, payload, and surface modifiers can be customized. PEG = polyethylene glycol. (Adapted from Ferrari M. Nat Rev Cancer 2005;5[3]:161171.21)

One of the most interesting capabilities in nanomedicine is the functionalization of NPs.7 Functionalization involves altering properties of an NP through chemical or physical modifications that are applied to achieve a desired effect.7 This process can provide local or directed delivery, prolong drug effects, facilitate transport into target cells, locate a tumor or area of infection, provide feedback regarding efficacy or drug delivery, or reduce blood flow shear effects.9 A discussion of the various approaches to functionalizing NPs follows.

NPs can be administered locally or can be actively targeted using cell-specific ligands, magnetic localization, and/or size-based selectivity.3 Many factors need to be considered when constructing targeted NPs, including size, biocompatibility, target affinity, avoidance of the RES, and stability in the blood, as well as the ability to facilitate controlled drug release.7

Magnetic polymer nanocomposites or magnetoliposomes grafted with drug molecules have great potential for targeted drug delivery.3 These NPs have potentially favorable biodistribution and pharmacokinetic profiles, which can be enhanced by the external application of a static magnetic field at the site of action.3 For example, in one study, MRI confirmed that magnetic NPs had migrated toward neodymium/iron/boron (NdFeB) magnets that had been placed outside the peritoneal cavity, above grafts of a human ovarian carcinoma.3

NPs can be engineered to incorporate a wide variety of chemotherapeutic agents that can be targeted directly and specifically to the tumor site for better efficacy and safety.4 NPs can also be filled with contrast agents for imaging purposes.6 In comparison to small-molecule contrast agents, multifunctional NP complexes or NPs used in diagnostic imaging have the advantage of a large surface area that allows targeting through surface modifications and the ability to simultaneously deliver therapeutic agents.1

One way in which NPs can be functionalized for specific applications is through surface conjugation.17 Nanoparticle surfaces can be conjugated with a wide range of diagnostic or therapeutic agents.1 Some candidate biomolecules for NP surface conjugation are cell-penetrating peptides (CPPs) that enhance intracellular delivery, fluorescent dyes for imaging, and agents for genetic therapy such as small inhibitory RNA (siRNA).7 Nanoparticle surfaces, conjugated with a targeting molecule that binds to highly expressed tumor cell receptors, can also facilitate the transport of imaging contrast agents that provide increased sensitivity and specificity, which aid in tumor detection.5

The surfaces of NPs can also be conjugated with drug therapies. 3 Surface conjugation with ligands that specifically bind to the target site can enhance the efficacy of NP drug-delivery systems while significantly reducing toxicity.4 In cancer treatment, tumor targeting can be achieved by conjugating a molecule or biomarker (such as a peptide, protein, or nucleic acid) that is known to bind to tumor cell receptors on the NP surface.5

NPs are generally cleared from circulation by immune system proteins called opsonins, which activate the immune complement system and mark the NPs for destruction by macrophages and other phagocytes.3 Neutral NPs are opsonized to a lesser extent than charged particles, and hydrophobic particles are cleared from circulation faster than hydrophilic particles.3 NPs can therefore be designed to be neutral or conjugated with hydrophilic polymers (such as PEG) to prolong circulation time.3 The bioavailability of liposomal NPs can also be increased by functionalizing them with a PEG coating in order to avoid uptake by the RES.5 Liposomes functionalized in this way are called stealth liposomes.5

NPs are often covered with a PEG coating as a general means of preventing opsonization, reducing RES uptake, enhancing biocompatibility, and/or increasing circulation time.5 SPIO NPs can also be made water-soluble if they are coated with a hydrophilic polymer (such as PEG or dextran), or they can be made amphophilic or hydrophobic if they are coated with aliphatic surfactants or liposomes to produce magnetoliposomes. 5 Lipid coatings can also improve the biocompatibility of other particles.3

NPs can also be designed so that they can be activated to release therapeutic or diagnostic cargo in response to a site-specific or remote trigger.3 Properties that can be used to functionalize NPs for controlled release include pH, temperature, magnetic field, enzymatic activity, or other features such as light or radiofrequency signals.6 NPs constructed with pH-responsive materials can be designed to trigger drug release at a target site upon detecting a change in pH.3 For example, the mildly acidic environment inside inflammatory and tumor tissues (pH 6.8) and cellular vesicles, such as endosomes (pH 5.56.0) and lysosomes (pH 4.55.0), can be exploited to trigger drug release.3

Thermally responsive linkers, consisting of nucleic acids, peptides, proteins, lipids, carbohydrates, or polymers, can also be used to attach one or more agents for controlled release from the NP.3 When the thermally responsive linker is exposed to a specific temperature or temperature range (the trigger temperature), the linker is disrupted and the agent is released.3 For example, DNA molecules with heat-labile hydrogen bonding between complementary strands can act as a heat-sensitive linker.5 An NP can also be designed to include several thermally responsive linkers that are designed to disrupt at different temperatures, enabling drug delivery to occur in a specific order over varied periods of time.3

The release of agents from NPs can also be achieved through the incorporation of bonds that degrade under other specific conditions at the target site.5 For example, tumor-specific processes may be exploited to break a bond and trigger the release of a therapeutic agent.5 Tumor site-specific conditions that could be used to trigger release might include abnormal oxygen levels, unique biomarkers, or exposure to proteolytic enzymes that are overexpressed in tumors.5

The tunability of NP properties is an important and powerful concept.20 NPs have a broad range of tunable biologic, optical, magnetic, electric, and mechanical features that differ dramatically from the same materials in larger forms because of modified quantum mechanics occurring at the nanoscale level.6 By changing the size of an NP, researchers can fine-tune many different properties of nanomaterials.11 For example, they can achieve different colors of fluorescence by changing the size of an NP, allowing a means of color coding or labeling during diagnostic imaging applications.11

Nanomedicines might someday provide answers to longstanding problems in medical research, ranging from poor drug solubility to a lack of target specificity for therapeutic compounds.2 Nanomedicine also has tremendous promise as a noninvasive tool for diagnostic imaging, tumor detection, and drug delivery because of the unique optical, magnetic, and structural properties of NPs that other tools do not possess.1

Nanomedicine presents new opportunities to improve the safety and efficacy of conventional therapeutics.5 Drugs with low bioavailability can now be targeted directly to the site required.3,5 The large surface area and greater reactivity of NPs may allow dose reduction of a drug, which can improve toxicity profiles and patient compliance.2,3 The large surface area of NPs can also increase the dissolution rate, saturation solubility, and intracellular uptake of drugs, improving in vivo performance.2,3 Combining encapsulation, release modalities, and surface modifications to improve therapeutic targeting or bioavailability could improve the efficacy of NP formulations several-fold compared with bulk counterparts.4 Targeted NPs can also transport large doses of therapeutic agents into malignant cells while sparing normal, healthy cells.4,5

One of the most exciting applications of nanomedicine is the use of multifunctional NP complexes for simultaneous non-invasive targeting, imaging, and treatment.1,4,5 Multifunctional NPs for cancer treatment can potentially include a variety of tumor targeting ligands as well as imaging and therapeutic agents that allow noninvasive monitoring and treatment.5 Multifunctional NPs that include fluorescent dyes can also provide in vivo imaging of biologic events during drug administration as well as potential diagnostic labels for the early detection and localization of tumors.7

Recent research efforts are also focused on developing magnetic NPs for the targeted delivery of various therapeutic or diagnostic agents.4 Interest in magnetic NP targeting applications is inspired by the possibility of detecting the particles by MRI and then correlating the results with histologic findings after treatment.3 Polymer/SPIO composites are the most common NPs used for theranostics (diagnostics).3 More than one cancer drug can also be incorporated on a polymer/IO conjugate backbone.3 The drugs can be released at the tumor site, allowing them to act together synergistically, potentially achieving higher efficacy.3 Because SPIO NPs generate heat when exposed to an alternating field, electromagnetic fields can also be applied externally for remote activation of SPIO NPs for thermal ablation therapy.5

Nanotechnologies have already transformed genetic and biological analysis through devices that examine molecular biomarkers.1 Compared with conventional modalities, these tests can be conducted more rapidly, reliably, and cost effectively via in vitro and in vivo diagnostic technologies that, for example, might use nanochips or QDs.1 Nanotechnologies can also produce diagnostic devices that are more sensitive and can detect earlier signs of metabolic imbalances, which can assist in the prevention of diseases like diabetes and obesity.20 The continued application of nanotechnologies to produce better and more cost-effective means of detecting molecular biomarkers will also open the way to the more routine practice of personalized medicine.1

Despite the benefits that nanomedicine has to offer, much research is still required to evaluate the safety and toxicity associated with many NPs.3 Much of nanomedical research has concentrated on drug delivery, with relatively few studies focusing on the pharmacokinetics or toxicity of NPs.7 Investigating NP pharmacokinetics, pharmacodynamics, and potential long-term toxicity in vivo is essential to monitoring the effects of NPs on patient populations.5 Validating every nanotherapeutic agent for safety and efficacy, whether drug, device, biologic, or combination product, presents an enormous challenge for researchers and the FDA, which is currently struggling to formulate testing criteria and accumulate safety data.2,3

Studies are also needed to assess the immunogenicity of NPs.20 Nanotherapeutics and diagnostics may present unexpected toxic effects because of increased reactivity compared with their bulk counterparts.2 The most frequently reported side effect after injection of a nanotherapeutic agent seems to be a hypersensitivity reaction, which may be caused by activation of the immune complement system.6 The main molecular mechanism for in vivo NP toxicity is thought to be the induction of oxidative stress through the formation of free radicals.3 In excess, free radicals can cause damage to lipids, protein, DNA, and other biological components through oxidation. Several authors have reported that intrinsic characteristics of NPs, such as aspect ratio and surface area, can be pro-oxidant and pro-inflammatory.7 However, the formation of free radicals in response to an NP can also have other causes, such as the reaction of phagocytic cells to foreign material, insufficient antioxidants, the presence of transition metals, environmental factors, and other intrinsic chemical or physical properties.3

Research to evaluate the size and surface properties of NPs may also help to identify the critical dimensions at which they tend to significantly accumulate in the body.20 NPs have an increased ability to cross biological barriers and therefore have the potential to accumulate in tissues and cells because of their small size.2 The possible tissue accumulation, storage, and slow clearance of these potentially free radicalproducing particles, as well as the prevalence of numerous phagocytes in the RES, may make organs such as the liver and spleen the main targets of oxidative stress.3,6

This lack of data about potential toxicity issues forces nanomedical research to focus predominantly on polymer NPs, for which safety and efficacy data already exist.3 In fact, several nanomedicines containing polymer NPs are already approved by FDA.3 Unlike other materials that may become toxic in NP form, the lipid NPs are also considered to be biocompatible and tolerable.3 Consequently, biodegradable, soluble, nontoxic NPs, such as polymers, liposomes, and IO particles, are much more desirable to use in nanomedicines than biopersistent components are.5 The use of NPs like carbon nanotubes, QDs, and some metallic nanocarriers that are not biodegradable might be more problematic.1,7 This characteristic need not discourage nanomedical research with these NPs but should reinforce efforts to identify additional biodegradable shapes, materials, and surface treatments.7

Although nanomedicine is still at an early stage of development, several drugs that utilize nanotechnology have been approved and marketed, and many others are being studied.2,3 Nanomedicines potentially offer a means of earlier diagnosis; more effective, safer, and personalized treatments; as well as reduced health care costs.1 Many experts agree that nanomedicine will create a paradigm shift that revolutionizes health care within the next 10 years.2,8 However, for significant progress to be made toward this goal, much more work is needed to establish testing criteria, validate efficacy, and accumulate safety data for various nanotherapeutic agents and materials.2,3

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WALTHAM, Mass., Oct. 04, 2024 (GLOBE NEWSWIRE) -- TScan Therapeutics, Inc. (Nasdaq: TCRX), a clinical-stage biotechnology company focused on the development of T cell receptor (TCR)-engineered T cell (TCR-T) therapies for the treatment of patients with cancer, today announced the acceptance of three abstracts for poster presentation at the upcoming Society for Immunotherapy of Cancer (SITC) 39th Annual Meeting being held November 6 – 10 in Houston, TX and virtually.

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TScan Therapeutics Announces Upcoming Presentations at the Society for Immunotherapy of Cancer 39th Annual Meeting

CHESTERBROOK, Pa., Oct. 04, 2024 (GLOBE NEWSWIRE) -- Trevena, Inc. (Nasdaq: TRVN), a biopharmaceutical company focused on the development and commercialization of novel medicines for patients with central nervous system (CNS) disorders, today announced that on October 4, 2024 the Company received notice that the Nasdaq Hearings Panel (the “Panel”) had determined to delist the Company’s common stock from The Nasdaq Stock Market LLC (“Nasdaq”) due to the Company’s failure to comply with the minimum stockholder’s equity requirement under Nasdaq Listing Rule 5550(b)(1) (the “Equity Standard Rule”). As previously disclosed, the Panel had provided the Company until October 2, 2024, to regain compliance with the Equity Standard Rule.

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Trevena Announces Receipt of Nasdaq Delisting Notification

BEDFORD, Mass., Sept. 12, 2024 (GLOBE NEWSWIRE) -- Ocular Therapeutix, Inc. (NASDAQ: OCUL, “Ocular”), a biopharmaceutical company committed to improving vision in the real world through the development and commercialization of innovative therapies for retinal diseases and other eye conditions, today announced that it has agreed to grant inducement awards to its newly appointed Director, IT Cybersecurity, Russell Isaacs. The awards were made as inducements material to Mr. Isaacs’ acceptance of employment with Ocular under Ocular’s 2019 Inducement Stock Incentive Plan in accordance with Nasdaq Listing Rule 5635(c)(4).

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Ocular Therapeutix™ Reports Inducement Grant Under Nasdaq Listing Rule 5635(c)(4)

Abstract

Tissue engineering and regenerative medicine (TERM) is an emerging field that has provided new therapeutic opportunities by delivering innovative solutions. The development of nontraditional therapies for previously unsolvable diseases and conditions has brought hope and excitement to countless individuals globally. Many regenerative medicine therapies have been developed and delivered to patients clinically. The technology platforms developed in regenerative medicine have been expanded to various medical areas; however, their applications in colorectal surgery remain limited. Applying TERM technologies to engineer biological tissue and organ substitutes may address the current therapeutic challenges and overcome some complications in colorectal surgery, such as inflammatory bowel diseases, short bowel syndrome, and diseases of motility and neuromuscular function. This review provides a comprehensive overview of TERM applications in colorectal surgery, highlighting the current state of the art, including preclinical and clinical studies, current challenges, and future perspectives. This article synthesizes the latest findings, providing a valuable resource for clinicians and researchers aiming to integrate TERM into colorectal surgical practice.

Keywords: Tissue engineering, Regenerative medicine, Colorectal surgery

Regenerative medicine encompasses a wide range of disciplines, including stem cell biology, biomedical engineering, biomaterials sciences, and gene therapy [1]. This field utilizes cells, biomaterials, and biological factors to develop therapeutic solutions to repair or replace damaged tissues and organs to restore normal function. The term regenerative medicine was coined and appeared in the literature as early as 1999; nonetheless, the field has existed for more than a century, with its history more closely intermingled with that of surgery than any other field in the health sciences [2, 3]. Early attempts at regenerative procedures, such as hip arthroplasty in the 1700s, underscore the long-standing interplay between regenerative efforts and surgical practice [4, 5].

Many regenerative medicine therapies have been developed and delivered to patients clinically. The technology platforms developed in regenerative medicine have been expanded to various medical areas; however, their applications in colorectal surgery are limited. Applying regenerative medicine technologies to engineer biological tissue and organ substitutes may address the current therapeutic challenges and overcome some complications in colorectal surgery, especially in inflammatory bowel diseases, short bowel syndrome (SBS), and diseases of motility and neuromuscular function [6]. To that end, this review provides an overview of the technological platforms available for regenerative medicine, followed by tissue stem cell biology and tissue engineering approaches relevant to organs and their clinical applications. It also covers relevant surgical studies aiming to alter underlying pathophysiology and replace damaged organs, and discusses potential future outlooks in the field, including major hurdles for clinical translation [1, 5].

Regenerative medicine uses innovative technologies and tools, which can be employed alone or in concert, to develop therapies for repairing or replacing damaged tissues and organs. Thus, the strategies can range from using biomolecules or cells to promote regeneration through changing the environment to generating ex vivo tissue or organ constructs for subsequent implantation in vivo. These fundamental components include technologies using scaffolds, cells and/or organoids, and biomolecules ().

Essential components of regenerative medicine. Tissue engineering and regenerative medicine is an exciting field that holds promise for colorectal surgery. The field makes use of scaffolds, cells, and biomoleculesalone or in combinationin order to restore tissue and organ function.

In tissue engineering and regenerative medicine (TERM), scaffolds are the crucial building blocks that provide the necessary structural support for cell attachment, proliferation, and differentiation, mimicking the extracellular matrix (ECM) [7]. The materials from which these scaffolds are fabricated can range from natural biomaterials to synthetic polymers, depending on the physical and functional characteristics of target tissues and organs. Numerous scaffold design parameters must be considered, depending on the properties of the tissue or organ one is trying to engineer or regenerate () [8]. For example, the preferred scaffolds for colorectal surgery applications may comprise degradable synthetic or naturally derived biomaterials that facilitate rapid tissue remodeling. These materials can be fabricated in various configurations to generate target tissue-like structures, such as sheets, tubes, or solid mass, to recapitulate the target tissue anatomy and function. Natural polymers such as collagen, hyaluronic acid, and chitosan have been widely used due to their biocompatibility and ability to promote cell adhesion, which is crucial for the regeneration of intestinal and colorectal tissues [927]. These materials are often combined with growth factors to enhance their regenerative potential. Synthetic polymers, such as polylactic acid, polyglycolic acid, polycaprolactone, and polyethylene glycol, offer controlled degradation rates and mechanical properties tailored to specific applications, such as intestinal anastomosis and rectal reconstruction. Composite materials combine natural and synthetic polymers and can optimize the mechanical properties and biological activity of scaffolds suitable for target tissue applications.

Design parameters to consider when designing a scaffold for tissue engineering and regenerative medicine. Adapted from Echeverria et al. [8], available under the Creative Commons License.

Cells are an essential component of tissue regeneration. Various cell types and sources, including autologous, allogeneic, and xenogeneic cells from preclinical and clinical sources, have been used in TERM research (). In addition to tissue and organ-derived somatic cells, stem cells have been used for many translational applications [2839]. Stem cells have attracted significant interest due to their capacity to differentiate into diverse cell types and their regenerative potential [3436, 39]. Embryonic stem cells (ESCs) are pluripotent cells derived from early-stage embryos, capable of differentiating into any cell type. However, their use is limited by ethical concerns and potential for teratoma formation [7]. Induced pluripotent stem cells (iPSCs) are generated by reprogramming somatic cells to a pluripotent state, offering an ethical alternative to ESCs with patient-specific applications. However, challenges remain in ensuring their safety and functionality. Adult stem cells include mesenchymal stem cells (MSCs) derived from bone marrow, adipose tissue, and umbilical cord, which are known for their multipotency and immunomodulatory properties. MSCs are particularly valued for their versatility and application in various regenerative medicine approaches, due to their immunomodulatory effects and differentiation capabilities. Additionally, most adult tissues contain a population of progenitor cells (such as intestinal or colonic epithelial stem cells) that are capable of dividing and regenerating, to some extent, their tissue of origin. All these cell types have been employed in TERM for a range of applications, each offering specific advantages and disadvantages depending on the application.

Cell sources in colorectal regenerative medicine with advantages and disadvantages

Organoids are 3-dimensional (3D) cell cultures that essentially function as miniaturized versions of organs that replicate some of the structure and function of their full-sized counterparts. Organoids are often formed from ESCs, iPSCs, or adult stem cells under specific culture conditions that promote self-organization into organ-like structures. They exhibit cellular diversity and spatial organization similar to native organs, making them valuable for studying organ development and disease [4045]. Organoids are used to model colorectal diseases such as cancer, inflammatory bowel disease (IBD), and congenital disorders, enabling the study of pathophysiology and drug responses in a controlled environment. Organoids derived from patient-specific cells can be used to test drug efficacy and toxicity, enabling personalized treatment approaches for colorectal diseases [46]. Organoids also hold potential for regenerative medicine, with ongoing research into their use for tissue repair, transplantation, and as cell building blocks for tissue engineering.

Biomolecules play essential roles in modulating cellular activities and enhancing tissue regeneration. The biomolecules used in TERM include growth factors, cytokines, and extracellular vesicles [4754]. Growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), have been used extensively. For example, VEGF stimulates the body to promote angiogenesis to enhance the neovascularization of engineered tissues for survival and maturation, and FGF has been used to support cell proliferation and differentiation in various tissues. Extracellular vesicles are exosomes and/or microvesicles secreted by cells into the extracellular environment. These vesicles can carry proteins, lipids, and RNAs, influencing a recipient cells behavior and promoting tissue repair and regeneration [7]. Extracellular vesicles have been used in many tissue applications, including wound healing and IBD, and as delivery vehicles for therapeutic agents for various disease processes.

These essential technological components constitute the foundation of most TERM therapies. Many TERM applications have been developed and benefited numerous patients in numerous medical areas; however, research on TERM in colorectal surgery is limited. This article provides an overview of the field's current state and how TERM technologies could be applied to colorectal diseases to develop innovative solutions for various colorectal diseases with limited treatment options, thereby improving patient care.

While many colorectal diseases are treated with conventional medical and surgical approaches with satisfactory outcomes, some pathologic conditions present challenges requiring alternative therapeutic solutions. TERM technologies may provide opportunities to overcome the current treatment limitations and improve patient care. The platform technologies utilized in other tissue applications may be adapted to develop treatment modalities specific to diseases in the colorectal field, such as SBS, loss of colon/rectum, motility disorders, incontinence, IBD, and anorectal or rectovaginal fistulas () [5559].

Summary of regenerative medicine applications in colorectal surgery

SBS is a severe and debilitating condition that arises due to significant loss of the functional small intestine. This can be a consequence of congenital anomalies, extensive surgical resections for diseases such as necrotizing enterocolitis or Crohn disease, or traumatic injuries. Patients with SBS suffer from malabsorption, chronic diarrhea, malnutrition, and a heavy reliance on parenteral nutrition, which leads to a significantly compromised quality of life [6062]. Moreover, long-term parenteral nutrition places patients at risk for numerous complications, such as sepsis, liver failure, and even death. Only select patients are candidates for bowel-lengthening surgery, and these generally provide only partial relief. Intestinal transplants have not been the cure they were hoped to be due to the high doses of immunosuppression required and the relatively poor overall and graft survival [63, 64]. As a result, tissue-engineered small intestine (TESI) has emerged as a promising therapeutic avenue [23, 6571].

TESI represents an innovative approach in regenerative medicine aimed at creating functional intestinal tissue constructs in vitro, which can be transplanted into patients to restore intestinal function and improve nutrient absorption [6, 23, 26, 6572]. The development of TESI involves using biodegradable scaffolds seeded with cells to form a bioengineered segment of the intestine that mimics the structure and function of the native bowel [23, 65, 68, 7375]. Specifically, TESI must be capable of performing absorption and peristalsis to be clinically effective () [13]. Cell sources have ranged from autologous progenitor cells such as intestinal epithelial stem cells to iPSCs and ESCs. Numerous scaffolds have been employed, such as nonwoven biodegradable materials (polyglycolic acid), polyglycerol sebacate, natural scaffolds (consisting of collagen and/or fibrin), decellularized material such as small intestinal submucosa, and even perfusion decellularized intestinal tissue [23, 24, 26, 46, 65, 66, 7679]. These constructs are first implanted into the omentum to achieve an adequate vascular supply before being placed in continuity with the intestine. This method was first reported by Grikscheit et al. [73], who demonstrated the feasibility and efficacy of TESI in animal models using biodegradable scaffolds seeded with autologous cells to construct small intestine segments, which were then implanted into animals with massive small bowel resections. They observed significant improvements in nutrient absorption and reduced dependence on parenteral nutrition, highlighting the potential therapeutic benefits of TESI for SBS patients. Deguchi et al. [7] reviewed advancements in TESI for pediatric surgery, emphasizing its potential to provide long-term solutions for children with SBS. They discussed the critical factors for successful TESI, including scaffold design, cell sourcing, vascularization, and integration with host tissue, and highlighted the ongoing research aimed at overcoming existing challenges. Gardner-Thorpe et al. [75] investigated the angiogenic potential of TESI by characterizing the microvasculature and angiogenic growth factors in engineered small intestine segments. Their findings underscored the importance of angiogenesis for the success of TESI, particularly for ensuring adequate vascularization to support the survival and function of the transplanted tissue.

(A) Native structure of the small intestine with its layers. (B) Goal of a tissue-engineered small intestine that accomplishes the minimum functions of absorption and peristalsis. The engineered intestine would consist of a scaffold containing smooth muscle cells and neuronal cells to promote peristalsis, with the inner layer lined with epithelial cells to promote absorption. Adapted from Boys et al. [13], available under the Creative Commons License.

The integration of TESI in clinical practice for SBS patients holds significant promise. By providing a functional bioengineered intestinal segment, TESI could potentially overcome the limitations of current surgical treatments, reduce the complications associated with long-term parenteral nutrition, and improve the overall quality of life for patients with SBS. While clinically relevant TESI has eluded tissue engineers to date, much progress has been made, and with continued research, a translational solution may emerge. Ongoing research is essential to address the challenges related to scaffold materials, cell viability, and long-term functionality of the engineered intestine. The future of SBS treatment lies in the continued development and refinement of TESI technologies, which could ultimately lead to a curative approach to this challenging condition.

Loss of the colon due to diseases such as IBD, colorectal cancer, or traumatic injury can lead to significant physiological disruptions, including issues with electrolyte balance, enterohepatic circulation, and water homeostasis, profoundly affecting patients' quality of life. Traditional surgical solutions, such as the creation of ileostomies, colostomies, or ileal pouches, often result in substantial lifestyle limitations and complications. Regenerative medicine, particularly through the development of tissue-engineered colon (TEC), offers innovative therapeutic approaches to address these challenges and restore normal function [9, 10, 8082]. Admittedly, the clinical need for TEC revolves more around significant quality-of-life issues than around life-threatening problems. That is, often, patients who have colon resections for cancer or IBD can be managed reasonably well with ileostomies or colostomies. Because of this, there has been less work on TEC than on TESI.

TEC, like TESI, involves creating bioengineered colonic tissue constructs that can be surgically implanted into patients to replace lost or damaged sections of the colon [9, 10, 8183]. This approach utilizes a scaffold seeded with cells to engineer tissue constructs that mimic the architecture and function of the native colon. Similar to TESI, scaffolds can be made from materials like polylactic acid and polycaprolactone or decellularized colon segments to provide structural support while allowing the gradual degradation of polymers or remodeling of tissue-derived decellularized colon segments as new tissue forms and integrates with native tissue. Intestinal stem cells and colonic organoid units are employed to regenerate the mucosal lining and create a functional colon [9, 10, 81, 82]. Trecartin and Grikscheit [84] emphasized the importance of stem and progenitor cells in tissue engineering for functional gastrointestinal regions, including the colon. They highlighted the critical factors in scaffold design, cell sourcing, and the role of progenitor cells in developing functional tissue constructs. For example, recent studies have discussed the importance of promoting angiogenesis to ensure the viability and functionality of the engineered colon tissue. Growth factors such as VEGF are being incorporated into scaffolds to promote vascularization.

Motility disorders in the colon, including conditions such as Hirschsprung disease and functional intestinal motility disorders, present significant clinical challenges. These conditions often result in severe gastrointestinal dysfunction, impacting patients' quality of life. Traditional treatments, including surgical interventions and pharmacotherapy, often provide limited relief and are associated with various complications [85]. Regenerative medicine offers innovative therapeutic approaches to address these challenges and restore normal motility [81, 8694].

The enteric nervous system (ENS) is a complex network of neurons and glial cells that regulate gastrointestinal motility, secretion, and blood flow. Disorders of the ENS, such as Hirschsprung disease, are characterized by the absence of ganglion cells in the distal bowel, leading to severe motility issues. Regenerative approaches aim to restore the functionality of the ENS through the use of stem cells and tissue engineering techniques [90]. Previous studies demonstrated the maintenance of intestinal smooth muscle cells by basic FGFs after implantation into the omentum, highlighting the potential for growth factors to support the restoration of motility [95, 96].

Stem cell therapy aims to repopulate aganglionic segments of the bowel with functional neurons, restoring normal motility. Stem and progenitor cells can be used to enhance neuronal density and functionality in affected bowel segments, improving motility and overall gastrointestinal function. ENS progenitor cells are necessary for re-establishing the ENS, which controls gut motility and function. This approach has shown promise in preclinical studies and is moving toward clinical applications [89, 97]. Recent studies have shown the successful differentiation of stem cells into functional neurons and their integration into the host ENS, improving motility in animal models [29]. Similarly, Pan et al. [86] demonstrated the successful transplantation of stem cells into animal models with motility disorders, showing improved gastrointestinal function. Despite many advances, hurdles remain before these therapies can be used clinically. For further detail, the reader is referred to the recent excellent review by Ohkura et al. [30], which describes the current state of the art, potential cell sources, and the challenges that still lie ahead.

Incontinence, which can result from surgery, trauma, childbirth, or congenital conditions, poses significant challenges to patient quality of life and daily functioning. Traditional treatments, such as surgical repairs and pharmacotherapy, often provide limited relief and can be associated with complications. Newer therapies are emerging, such as injectable aluminum potassium sulfate and tannic acid as a bulking agent (for incontinence) and/or sclerotherapy (for rectal prolapse), and have shown reasonable results [56, 57]. The physiology of the internal and external anal sphincters and continence are complex. These mechanisms involve numerous biochemical pathways and reflexes to maintain resting pressure and to relax for defecation, which have been recently reviewed by Kim et al. [98]. Although regenerative medicine has developed new promising therapeutic approaches to restore continence via engineered anal sphincters and cell therapy, because of the immense complexity of the sphincter mechanism, much work remains to be done before these therapies will become viable clinical treatments.

The development of engineered anal sphincters involves creating bioengineered sphincter tissue that can be implanted to restore normal function. This approach again utilizes scaffolds seeded with smooth muscle cells and often neurons to replicate the structure and function of the native anal sphincter. Hecker et al. [22] developed a 3D physiological model of the internal anal sphincter bioengineered in vitro from isolated smooth muscle cells. That study demonstrated the potential for creating functional sphincter tissue that mimics the physiological properties of the native sphincter. Somara et al. [21] successfully bioengineered an internal anal sphincter derived from isolated human internal anal sphincter smooth muscle cells. Smooth muscle cells were isolated and cultured to populate the scaffolds, creating a tissue construct that replicated the function of the native sphincter. Their findings highlighted the feasibility of using patient-specific cells for creating functional anal sphincter constructs. Raghavan et al. [17] demonstrated the successful implantation of a physiologically functional bioengineered mouse internal anal sphincter, demonstrating the feasibility of restoring normal anal function in vivo with an engineered anal sphincter.

Another approach to restoring continence is the use of cell therapy. This method involves transplanting stem cells or progenitor cells to regenerate the internal anal sphincter and restore continence. The goal of cell therapy is to regenerate the smooth muscle of the internal anal sphincter, thereby restoring its tone and contractile function, which are crucial for continence. When successful, cell therapy facilitates the integration of new cells within the existing sphincter tissue, ensuring functional restoration. For a detailed review of the current state of cell therapy for treating incontinence, readers can refer to the comprehensive review by Balaphas et al. [99]. In short, numerous cell types have been used, ranging from skeletal and smooth muscle derivatives (depending on if the internal or external anal sphincter is being treated), stem cells (adipose-derived, mesenchymal, and bone-marrow-derived), and ENS progenitor cells. Some of these approaches have been used in clinical trials [99].

IBD presents significant therapeutic challenges due to its chronic, relapsing nature and the complex interplay of genetic, environmental, and immunological factors. Current treatments, including immunosuppressive agents and biologics, often provide incomplete relief and can have significant side effects. Regenerative medicine, particularly through cell and biomolecule therapy, offers innovative approaches to modulate the immune response, repair damaged tissues, and restore normal bowel function.

MSCs have shown particular promise in IBD due to their immunomodulatory properties and ability to differentiate into various cell types. MSCs modulate the immune response by secreting anti-inflammatory cytokines and growth factors, thereby reducing inflammation in the gut. MSCs can be administered intravenously, directly into the affected bowel segment, or encapsulated in biomaterials to enhance their viability and therapeutic efficacy. Ko et al. [31] reviewed the efficacy and safety of MSC therapy for IBD, highlighting their potential to alleviate inflammation and promote tissue regeneration. MSC therapy has shown promise in reducing inflammation and promoting perianal fistula healing, but the ability of MSCs to treat systemic Crohn disease is unclear, with mixed results.

Exosomes and other extracellular vesicles that carry bioactive molecules are also being investigated as potential immunomodulatory therapeutics for IBD [47]. Exosomes can contain numerous biologically active components and may derive from multiple cell types. For details, the reader is referred to the excellent review by Ocansey et al. [47]. Some of these exosome therapies have shown promise in reducing inflammation and promoting tissue repair in preclinical models of IBD [47].

Anorectal fistulas and fissures present significant therapeutic challenges due to their chronic nature and the complexity of the affected tissues. Traditional surgical approaches, while often necessary, can be associated with high recurrence rates and significant morbidity. Newer surgical techniques have been developed, including cell-assisted lipotransfer and the transanal opening of the intersphincteric space, and these techniques have demonstrated promising results [58, 59]. In addition, regenerative medicine offers promising new approaches, mainly through cell therapy and regenerative wound dressings, gels, and matrices, to enhance healing and reduce recurrence.

MSCs, in particular, have shown promise due to their anti-inflammatory properties and ability to differentiate into various cell types. Cell therapy has been effective in promoting the healing of perianal fistulas associated with Crohn disease (as described above), showing reduced recurrence rates and improved quality of life. MSCs have shown potential in treating chronic anal fissures that do not respond to conventional treatments. MSCs modulate the immune response by secreting anti-inflammatory cytokines, reducing inflammation at the site of the fistula or fissure. Garca-Olmo et al. [100] reported the successful use of autologous stem cell transplantation for treating rectovaginal fistulas in patients with perianal Crohn disease. This pioneering study demonstrated the potential of cell-based therapies to promote healing in complex anorectal conditions. Pans et al. [101] conducted a long-term study on the efficacy and safety of stem cell therapy (Cx601) for complex perianal fistulas in Crohn disease patients, showing promising results for sustained fistula closure and reduced recurrence. Lastly, recent studies have demonstrated the effectiveness of adipose-derived MSCsa readily available and potent source of stem cells in treating complex perianal fistulas [32]. For a recent detailed review of cell therapies used in treating perianal and rectovaginal fistulas, the reader is directed to the superb review by Kent et al. [102].

Regenerative wound dressings, gels, and matrices are designed to create an optimal environment for healing by providing structural support, promoting cell migration, and delivering bioactive molecules. Wound dressings can be impregnated with growth factors, cytokines, and other bioactive molecules to promote tissue regeneration and reduce inflammation. Scaffolds and matrices provide a framework for cell attachment and proliferation, facilitating the regeneration of the damaged tissue. Lastly, these materials can promote wound healing by maintaining a moist environment, protecting the wound from infection, and reducing mechanical stress. Regenerative wound dressings can be used in conjunction with surgical procedures to promote healing and reduce the risk of recurrence. Bioactive gels and matrices can accelerate the healing of chronic anal fissures, improving patient outcomes and comfort. Finally, the use of fibrin glue in combination with cell therapy has shown synergistic effects, enhancing the overall healing process and reducing recovery time [103].

TERM is an emerging field that has led to new therapeutic opportunities by delivering innovative solutions. The development of nontraditional therapies for previously unsolvable diseases and conditions has brought hope and excitement to countless individuals globally. Despite the promise and potential of TERM, many scientific and technological challenges must be overcome before translation into the clinic. Here, we discuss these hurdles as well as the exciting prospects of TERM in colorectal surgery.

Tissues and organs are 3D structures, and as such, 3D scaffolds are needed to recreate them. While there are innumerable biomaterials, fabrication techniques, and methods for developing scaffolds, finding the ideal scaffold to provide the appropriate environment for the engineered tissue construct remains paramount. It is unlikely that a single scaffold will be suitable for all applications; thus, a scaffold often needs to be designed for each tissue application based on the tissue anatomy, characteristics, and function. For example, it is unlikely that a scaffold for colon tissue engineering would work well for engineering an anal sphincter and vice versa. Specifically, the scaffold should recapitulate the complex microarchitecture of the colorectal tissue, including the mucosal layer, submucosa, muscularis propria, and serosa. While an in-depth discussion of scaffolds for tissue engineering is outside the scope of this review, it is worth noting the general categories that scaffolds fall into [7].

The biomaterials used for scaffolds can be permanent or biodegradable, however, the majority used in regenerative medicine for colorectal surgery are degradable. Secondly, these materials can be synthetic (e.g., polylactic acid, commonly used in Vicryl sutures) or naturally derived (such as type I collagen). Synthetic polymers are easier to control, but natural materials may be more similar to the native environment the scaffolds try to recapitulate. Synthetic polymers may lack bioactivity, while natural polymers can have the disadvantage of poor mechanical strength and variability. Identifying materials that are biocompatible, biodegradable, and possess the mechanical properties needed to mimic native tissue is challenging.

Another type of scaffold is a decellularized scaffold where all the cells of the tissue of interest (e.g., a segment of the colon) are removed while preserving the native tissue ECM. This type of scaffold has the advantage of retaining the correct 3D structure as well as many of the environmental cues contained within the ECM [104]. Moreover, decellularization often results in a scaffold that could be surgically implanted. The ECM could be configured into a hydrogel for injection therapy or bioink that could be printed to generate an implantable tissue construct. Advanced 3D printing technologies offer precise control over scaffold architecture and composition, enabling the creation of patient-specific scaffolds, but these too can suffer from adequate mechanical strength [105]. Although numerous scaffold types and configuration options exist, there is no ideal scaffold for all applications. Instead, one must weigh the advantages and disadvantages of each material, scaffold fabrication technique, and scaffold size based on the organ or tissue to be regenerated.

As with scaffolds, selecting the proper cell type and source is critical for the success of regenerative medicine, as each has advantages and disadvantages. Depending on the target tissue and expected function, various cell sources and types are considered. While stem cells are attractive as a cell source for regenerative medicine due to their ability to differentiate into multiple cell types, limitations such as consistency in differentiation into the target lineage, the large expansion capacity of terminal cell types, cell banking, and biomanufacturing processes remain to be solved. Unlike ESCs, iPSCs are recognized as an attractive cell source because they contain the pluripotent potential of ESCs but can be made from a persons own somatic cells. However, the reprogramming process remains complex, and there are concerns about genetic stability and tumorigenicity (a tendency to form teratomas) [106]. Despite this, if large-scale production becomes available, iPSCs will likely be used for many colorectal disease applications, especially those requiring all 3 germ layers.

Adult stem or progenitor cells are another potential cell source for colorectal tissue engineering; however, these cells are typically limited to one germ layer. For example, colon epithelial organoids can be created from colon epithelial stem cells; however, they can only form the endodermal layer. Thus, to recreate an entire colonic tissue, one would need progenitor cells from each tissue. This may be possible in some cases, but often, depending on the tissue type, adults may not harbor enough progenitor cells for this to be practical. Harvesting these cells from patients can also be invasive and yield insufficient quantities for therapeutic use [107]. As noted above, no perfect cell source exists that could be used universally. In addition to acquiring a reliable cell source, another consideration is cellular function. For regenerative medicine therapy targeted at mitigating inflammatory conditions (such as IBD) or promoting healing, autologous MSCs may be a good source. These cells release significant levels of anti-inflammatory mediators, which may promote healing and regeneration [33]. Therefore, it is critical to identify an ideal cell source that provides sufficient numbers of reliable and functional cells for the target applications. Due to the challenges in procuring a reliable and reproducible cell source, investigations have been pursued to develop and establish a universal donor cell manufacturing and banking system that could be used for multiple tissue applications.

Establishing adequate vascularization to implanted engineered tissue constructs has been an unsolved challenge in TERM. Since its inception, tissue engineering has been plagued by the problem of delivering oxygen and nutrients to implanted cells within the construct. The diffusion of oxygen to implants is limited to approximately 1 mm3 without established vascularization. Numerous strategies have been employed to encourage vascularization. The earliest attempts were to place constructs in the omentum of animals [9, 10, 75, 108]. Other strategies have included the use of growth factors such as VEGF and, more recently, bioprinting to directly incorporate vascularization within a construct [109, 110]. While promoting angiogenesis (the growth of new blood vessels from existing ones) is crucial, creating a fully functional vascular network within the construct is more complex. One promising strategy involves using decellularized tissue and organ scaffolds that retain the microarchitecture of native tissue structure with intact vasculature, identical to normal tissue anatomy [24, 26, 104]. The decellularized tissue vasculature can then be recellularized with vascular endothelial cells to the vascular wall, allowing blood to perfuse without forming thrombi. More recently, perfusable tissue constructs containing a network of vascular channels have been bioprinted for eventual surgical implantation [111, 112]. Future work will need to continue incorporating ready-made vasculature for the desired tissue constructs so that tissues of a clinically relevant size can be engineered. Without tissues of a clinically relevant size, these therapies will have minimal benefit for patients and, as will be noted in the next section, will be difficult to implant surgically. Finally, for the long-term success of engineered tissues, engineered vascular networks must support the growth and maintenance of the engineered tissue and its vascular network while integrating seamlessly with the hosts circulatory system.

Developing a clinically relevant tissue construct for clinical use requires scale-up and streamlined manufacturing processes. This involves producing an immense number of target cellson the order of billionsmaking the selection of cell sources critically important. Careful considerations related to cell isolation, expansion, and differentiation must be made to maximize the production of target cells. Scaffold fabrication and preparation protocols must be developed and validated before creating a cell-seeded construct. In addition to scale-up in size and volume, all these technologies will require consistent and safe manufacturing. While this has historically been a relatively straightforward process for devices and materials, regenerative medicine technologies involving cells and biological factors combined with biomaterials increase the complexity of safely manufacturing tissue implants by several orders of magnitude. For this reason, scale-up and biomanufacturing have been identified as challenges that need to be addressed to accelerate the distribution of TERM therapies. Toward this goal, several societies have been founded to help explore and improve the technologies required for efficient and safe manufacturing of regenerative medicine technologies. For example, the Regenerative Medicine Manufacturing Society was formed to help address many of these issues and to develop pathways for US Food and Drug Administration (FDA) approval of regenerative medicine therapies [113]. However, navigating the regulatory landscape for the approval of complex tissue-engineered products can also be time-consuming and costly.

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Tissue engineering and regenerative medicine approaches in colorectal ...

Abstract

Genetics have undoubtedly become an integral part of biomedical science and clinical practice, with important implications in deciphering disease pathogenesis and progression, identifying diagnostic and prognostic markers, as well as designing better targeted treatments. The exponential growth of our understanding of different genetic concepts is paralleled by a growing list of genetic terminology that can easily intimidate the unfamiliar reader. Rendering genetics incomprehensible to the clinician however, defeats the very essence of genetic research: its utilization for combating disease and improving quality of life. Herein we attempt to correct this notion by presenting the basic genetic concepts along with their usefulness in the cardiology clinic. Bringing genetics closer to the clinician will enable its harmonious incorporation into clinical care, thus not only restoring our perception of its simple and elegant nature, but importantly ensuring the maximal benefit for our patients.

All inheritable traits of living organisms are determined by their genetic material, the genome, a long nucleic acid called deoxyribonucleic acid (DNA). The DNA consists of 3109 nucleotides. Each nucleotide is made up of a sugar (deoxyribose), a nitrogenous base (adenine (A), guanine (G), cytosine (C) or thymine (T)) and a phosphate group () [1,2]. The four nitrogenous bases are divided into two groups: purines (including A and G) have two joined heterocyclinc rings and pyrimides (including C and T) have a single heterocyclic ring. Successive sugar and phosphate residues are linked by covalent phosphodiester bonds, forming the backbone of the DNA molecule and a nitrogenous base is attached to each sugar. The stability of DNA is primarily dependent on the strong covalent bonds that connect the constituent atoms of its linear backbone, and also on a number of weak non-covalent bonds that exist. Meanwhile, because of the phosphate group charges present in each nucleotide, DNA is negatively charged and therefore highly soluble in water.

The chemical structure of a nucleotide including the phosphate (yellow), the sugar (deoxyribose in green) and adenine as the nitrogenous base (pink).

The DNA structure is a double helix, in which two DNA molecules are held together by weak hydrogen bonds [37]. Hydrogen bonding occurs between laterally opposed bases, of the two strands according to Watson-Crick rules: A specifically binds to T, and G to C. The two strands are therefore complementary [8]. As the phosphodiester bonds link carbon atoms number 3 and number 5 of successive sugar residues, the end of each DNA strand will have a terminal sugar residue where carbon atom number 5 is not linked to a neighboring sugar residue, and is therefore called 5 end. The other end of the molecule is similarly called 3 end. The two DNA strands are antiparallel because they always associate (anneal) in such a way that the 53 direction of one DNA strand is the opposite to that of its partner. To describe a DNA sequence, the sequence of bases of one strand only, are usually provided, and are provided in the 53 direction. This is the direction of DNA replication as well as transcription.

The human DNA is estimated to be approximately 2 m long. In order for it to fit in the 10 m nucleus of human cells it is imperative that it is tightly folded. The DNA double helix is therefore subjected to at least two levels of coiling: the first involving coiling around a central core of eight histone proteins, resulting in units called nucleosomes, which are connected by spacer DNA; and the second involving coiling of this string of nucleosomes into a chromatin fiber [3]. During the different phases of the cell cycle, the DNA varies in the extent of its condensation. For example, during interphase the chromatin fibers are organized into long loops, whereas in metaphase chromosomes, the DNA is compacted to about 1/10,000 of its stretched out length. In humans there are 24 different chromosomes, namely 122 autosomes, and sex chromosomes X and Y [9]. Since humans are diploid organisms, our DNA is found in two copies, one inherited from each parent, and is folded into 46 chromosomes. Among the major DNA sequence elements of each chromosome are: the centromeres (constriction site where sister chromatics are joined and chromosomes link to the mitotic spindle), the telomeres (structures capping the ends of chromosomes) and the origins of replication (where DNA replication begins). Chromatin is encountered in extended (euchromatin) or highly condensed (heterochromatin) states, which in turn affect the transcriptional status of the corresponding DNA regions (being active or inactive, respectively) [10]. Under the light microscope, these regions appear as light and dark bands of metaphase chromosomes ().

Diagrammatical representation of the human karyotype of haploid chromosome set with X and Y as the sex chromosome complement. The alternating light and dark bands are characteristic of each chromosome in standard G-banding karyotype, and they represent euchromatic and heterochromatic regions, respectively.

Genomic DNA contains coding as well as non-coding regions. The non-coding regions are involved in DNA folding, chromosome formation, chromatin organization within the nucleus, regulation of transcription and more [1114]. The coding regions are responsible for the transcription of RNA molecules and ultimately protein synthesis.

The genes are stretches of DNA that code for polypeptides. Specifically, genes contain regulatory and coding regions, which regulate their transcription or code for the polypeptide product, respectively. A key regulatory region is the promoter, where the transcription machinery binds for transcription to be initiated. Other possible regulatory regions include enhancers, which regulate gene expression in different tissues or cells, and can be found upstream or downstream of the coding region, as far as several thousand bases. The coding regions are represented by exons, whose size and number varies among different genes. Interspersed between gene exons, there are non-coding sequences named introns, which tend to make up the largest percentage of a gene. The human genome is estimated to contain approximately 20,000 different genes [15]. Interestingly, it is estimated that 80% of the human genome is expressed, yet only 2% is coding for proteins [16].

The central dogma of molecular biology was first stated in 1958 and re-stated in 1970 by Francis Crick [17,18]. According to this dogma there are three major classes of biopolymers: DNA, RNA and protein, and three classes of direct transfer of information that can occur between these biopolymers: general transfers, special transfers and unknown transfers. Of these, only the general transfers are believed to occur normally in most cells and they involve DNA replication to DNA, DNA transcription to mRNA and mRNA translation to proteins ().

The central dogma of molecular biology, as it currently applies in most cells (general transfers = black),or under specific conditions (in some viruses and in vitro: special transfers = grey).

The transfer of information from the DNA to the protein level is achieved step-wise and starts with the transcription of a gene to mRNA. Specifically, the transcription machinery (including RNA polymerase and a variety of transcription factors) binds to the gene promoter, the double helix opens in that location and a single strand primary mRNA molecule (hn-RNA), complementary to that gene sequence, is synthesized base by base () [19]. RNA as opposed to DNA, is a single strand nucleic acid containing ribose instead of deoxyribose and uracil instead of thymine. The heterogenous (hn)-RNA molecules go through a series of processing steps including a 5 cap, a poly-A (50250 adenine molecules and a 70kDa protein) tail at the 3 end and splicing, to remove the intronic sequences () [2023]. Alternative splicing can also occur, which removes certain exons and contributes to the diversity of proteins any single gene can produce [24].

During transcription the transcription machinery (including RNA polymerase and a variety of transcription factors) binds to the gene promoter, the double helix opens in that location and a single strand primary mRNA molecule (hn-RNA),complementary to that gene sequence, is synthesized base by base.

The promoter and enhancer elements of each gene are involved in gene transcription to precursor mRNA (hn-RNA) molecules which are then appropriately processed to give mature mRNA.

The mature mRNA molecules can be translated to proteins [25]. This process takes place in the cytoplasm with the aid of ribosomes, which are complexes of RNAs and proteins called ribonucleoproteins. The ribosomes are divided into two subunits: the smaller subunit binds to the mRNA, while the larger subunit binds to the tRNA which carries the amino acids. When a ribosome finishes reading a mRNA, these two subunits split apart. In particular, ribosomes bind mRNA and read through it as triplet codons, usually starting with an AUG triplet (initiation codon) downstream of the ribosome binding site. For each codon, the ribosome, with the aid of initiation and elongation factors, recruits a complementary tRNA molecule, which in turn carries a specific amino acid. Each codon codes for a specific amino acid as shown in . As the amino acids are linked into the growing peptide chain, they begin folding into the correct conformation. The translation process ends with the stop codons UAA, UGA or UAG. The nascent polypeptide chain is then released from the ribosome as a mature protein [25]. In some cases the new polypeptide chain requires additional processing to make a mature protein. Mature proteins in turn can be subjected to a range of post-translational modifications. Their ultimate roles in cell physiology can be highly variable including cytoarchitecture, enzymatic activity, intracellular signalling, transportation, communication etc.

Genetic variation refers to genetic difference between individuals within or between different populations. This variation is what renders each individual unique in its phenotypic characteristics. Genetic variation occurs on many different scales, ranging from gross alterations in the human karyotype to single nucleotide changes. These variations can be divided in polymorphisms and mutations.

Polymorphisms are defined as variants found in >1% of the general population [26]. Due to their high frequency they are considered unlikely to be causative of genetic disease. They can however, together with other genetic and environmental factors, affect disease predisposition, disease progression or response to treatments (e.g. [27]). Three common types of polymorphisms are the single nucleotide polymorphisms (SNPs), small insertions/deletions (indels) and the large-scale copy number polymorphisms (CNPs or CNVs). SNPs are single base changes that occur on average about every 1000 bases in the genome. Their distribution is not homogenous and they occur more frequently in non-coding regions where there is less selective pressure () [28,29]. Most SNPs are neutral; yet 35% are thought to have a functional role, i.e. affect the phenotype of the individual carrying them. Depending on their effect at the protein level, SNPs can be characterized as synonymous (coding for the same amino acid as the wild type DNA sequence) or non-synonymous (coding for a different amino acid than the wild type DNA sequence) [29]. Indels are small insertions or deletions ranging from 1 to 10,000 bp in length, although the majority involves only a few nucleotides [30,31]. They are considered the second most common form of variation in the human genome following SNPs, with over 3 million short indels listed in public databases. CNVs are variations in the number of copies of DNA regions. They can involve loss of one or both copies of a region of DNA, or the presence of more than two copies of this region. They can arise from DNA deletions, amplifications, inversions or insertions and their size can range from 1 kb (1,000 bases) to several megabases [32]. SNPs, indels and CNVs can either be inherited or arise de novo.

A Single-Nucleotide DNA Polymorphism (SNP) is defined as a single DNA variation detected when a single nucleotide in the genome (or other common sequence) is different between species or paired chromosomes in an individual. In this case there is a substitution of a C (Cytosine) in a T (Tymine) which causes the change of a G (Guanine) in a A (Adenine) in the complementary DNA strand.

Mutations on the other hand, are rare (by some defined as variations with <1% frequency in the general population, although there are many exceptions to this rule) changes in the DNA sequence that can change the resulting protein, impair or inhibit the expression of the gene, or leave both the gene function and protein levels/structure unaffected. Although a variety of definitions have been considered over the years, for most scientists mutation has become synonymous with disease. They can arise during DNA replication or as a result of DNA damage through environmental agents including sunlight, cigarette smoke and radiation. A variety of different types of mutations exist and the terminology used to describe them is based on their effect either on DNA structure, on protein product function, or on the fitness of the individual carrying them.

In terms of DNA structure modification, mutations can be categorized as:

A) point mutations in which a single nucleotide is changed for a different one (). These are divided into missense mutations (meaning that when translated this DNA sequence leads to the incorporation of a different amino acid into the produced protein, with possible implications in the protein function), nonsense mutations (where the new nucleotide changes the sequence so that a stop codon is formed earlier than in the normal sequence and therefore the produced protein is truncated), silent mutations (where the nucleotide change does not affect the amino acid in the corresponding position of the produced protein, and therefore the final protein product remains unaltered), and splice-site mutations (which affects the splice site invariant donor or acceptor dinucleotides (5GT or 3AG).

Different types of mutation and possible conseuquence on protein function:A) missense mutation; B) nonsense mutation; C) deletion; D) inversion.

B) insertions in which one or more nucleotides are inserted in the normal DNA sequence, therefore disrupting it. This can have a moderate or severe effect on the corresponding mutant protein product. For example it can affect the splicing or the reading frame ( frame-shift mutations), therefore leading an incorrect reading of all the downstream nucleotide triplets and consequently their translation to a significantly different and/or truncated amino acid sequence.

C) deletions in which one or more nucleotides are deleted from the normal DNA sequence (). As in the case of insertions this can lead to minor (e.g. single amino acid changes) or major protein defects (e.g. reading frame modifications with implications for the entire downstream amino acid sequence of the mutant protein). When larger chromosomal regions are deleted, multiple genes can be lost and/or previously distant DNA sequences can now be juxtaposed (such juxtapositions can lead, for example, to the production of abnormal proteins containing sequences from different genes that have now been merged or abnormal expression of otherwise normal proteins by deletions affecting their upstream regulatory regions).

D) amplifications leading to multiple copies of chromosomal regions and consequently to an increased number of copies of the genes located within them and increased levels of the corresponding proteins.

E) inversions involving the reversal of the orientation of a DNA segment, with variable implications for the protein product, similar to the ones described above ().

F) translocations where regions from non-homologous chromosomes are interchanged.

Mutations can affect the expression of a transcript and its corresponding protein, or modify the structure of the resulting protein therefore impairing its function [33]. Depending on their functional effect, mutations can be classified as dominant negative (the mutant gene product acts antagonistically to the wild-type allele), gain-of-function (the mutant gene product gains a new and abnormal function), and loss-of-function (the mutant gene product has less or no function). Loss-of-function mutations can be associated with haploinsufficiency, a common occurrence in the molecular cardiomyopathy setting.

Haploinsufficiency occurs when the gene product of one of the two alleles in an individual is lost due to a DNA deletion or to instability/degradation of the mutant protein. Other terms used to describe the effect of a mutation on the fitness of the carrier are: harmful or deleterious mutations (decreases the fitness of the carrier), beneficial or advantageous mutations (increases the fitness of the carrier), and lethal mutations (leading to the death of the individual carrying them).

In the field of cardiovascular genetics, when a new genetic variant is identified a common occurrence given the large number of genes and different variants thereof being screened it is crucial to first determine whether it represents a benign polymorphism or a pathogenic mutation. Identifying pathogenic mutations enables the characterization of the molecular mechanisms of pathogenesis, and more importantly for the clinical setting, it allows the development of genetic tests for mutation detection in other family members (including pre-symptomatically) as well as unrelated patients with similar phenotypes (see section on Cardiovascular genetics in clinical practice).

The Clinical Molecular Genetics Society1 and the American College of Medical Genetics2 have issued guidelines to facilitate the determination of the potential pathogenic role of a novel/unclassified variant (). The Human Gene Mutation Database,3 along with locus-specific or disease-specific mutation databases, are valuable resources for first deciphering whether a detected genetic variant represents a known mutation. The databases Online Mendelian Inheritance in Man,4 dbSNP5 and Ensembl,6 along with thorough searches of the literature via PubMED, Google Scholar, Scopus or the Web of Science, can also provide valuable information. From thereon carefully matched controls need to be included in the study populations, co-occurrence with known (in trans) deleterious mutations in the same gene needs to be ruled out, co-segregation with the disease in the family represents useful information, and occurrence of the novel variant concurrent with the incidence of a sporadic disease can be a strong indicator. Bioinformatically, it is important to determine if the unclassified variant leads to an animo acid change and how different the biophysical properties of the new amino acid are: the greater the difference, the higher the likelihood to possess a pathogenic role. Similarly, the more conserved a DNA region is across species, the greater an impact any variations therein are likely to have. A range of in silico analysis tools can also be used for the predication of a pathogenic effect (e.g. Align GVGD, Sorting Intolerant From Tolerant [SIFT], Polyphen, and Alamut) or the prediction of splice sites. One of the best means of determining pathogenicity, however, is the use of suitable functional assays and transgenic animal models [34,35].

From mutation to disease. A DNA mutation can cause qualitative or quantitative changes at the protein level, leading to either a dysfunctional/non-functional protein product and/or aberrant protein expression levels. Both mechanisms can in turn lead to CVD.

Once a mutation has been directly associated with a pathological phenotype a number of additional parameters need to be evaluated in order to maximize its value in the clinical setting. These parameters relate to the mode of inheritance of a mutation, which impacts directly the chances of detecting it in other family members of the patient, or his/her offspring. The categorization gonosomal or autosomal depends on whether the mutations are located on either of the sex chromosomes or not. For example a mutation on the Y chromosome will only affect males. The dominant or recessive nature relates to the need of one or both alleles, respectively, to carry the mutation for the pathogenic phenotype to develop. In hypertrophic cardiomyopathy (HCM) a number of cases have been reported with homozygosity for the pathogenic mutation. Nishi et al. first reported homozygosity for a MYH7 mutation in two brothers with HCM [36]. Homozygous mutations were also detected in MyBPC in HCM patients [37]. The patients who harbour homozygous mutations present with a more severe clinical phenotype than their heterozygous family members. These observations support the notion of a mutation dosage effect, in which a larger amount of the defective protein leads to a greater disruption of the sarcomere function and results in a more severe clinical outcome. For example, in our Egyptian HCM cohort, none of the mutation-positive patients were homozygous for the mutation detected (data not published) which might be explained either by the rarity of its occurrence in the specific cohort or due to technical limitations in the mutation screening method ().

Mutation screening by denaturing high performance liquid chromatography (dHPLC) using WAVE, Transgenomics. dHPLC can be used as an initial mutation screening method, being dependent on heteroduplex (wild type-mutant) formation, and variant profiles from the wild pattern are subsequently sequenced. Note however, that dHPLC is not capable of detecting homozygosity.

Importantly, a number of exceptions apply to the aforementioned inheritance mode rules, such as in the case of incomplete penetrance (a percentage of the individuals carrying the mutation fail to present the corresponding trait) where mutation carriers may not present with any symptoms even in the presence of a dominant mutation. Furthermore, the phenomena of variable expressivity (variations in a phenotype among individuals carrying a particular genotype) and epistasis (one gene is modified by one or several other genes, e.g. modifier genes) can lead to a range of pathological characteristics despite the presence of the same mutation. These parameters, potentially in combination with environmental factors, can often lead to significant clinical heterogeneity in most inherited CVDs, between unrelated individuals as well as family members carrying the same mutation () [38].

Role of genetic and environmental factors in determining the spectrum of the disease phenotype.(Strachan T, Read AP. Genes in pedigrees and populations in Human molecular genetics 3. 3rd ed. London; New York: Garland Press; 2004).

Another exception is this of compound heterozygotes (carriers of two different mutations on the two alleles of the same gene) or double heterozygotes (carriers of mutations in two different genes), which carry one copy of each mutation, yet they can develop the disease. Notably, the concomitant presence of multiple genetic defects contributing to the same disease is usually associated with a more severe clinical phenotype. For example, in HCM the presence of multiple pathogenic mutations could be included amongst the risk stratification criteria [39]. Multiple mutations have been observed in about 5% of HCM patients and they are usually associated with higher septal thickness and worse clinical outcomes, such as heart failure and sudden death [4043]. Double heterozygosity is commonly detected in the Myosin heavy chain (MYH7) and Myosin binding protein C (MyBPC) genes, probably because they represent the most commonly involved genes in the pathogenesis of HCM. Compound heterozygosity in MyBPC however, leading to the absence of a normal protein, has been reported to results in neonatal death in two independent cases, where the parents were each heterozygous for one of the mutations [44]. Similarly to HCM, double heterozygosity has been reported in other CVDs such as long QT, with a similar frequency of 5% [45].

Hereditary CVDs include a variety of different aspects and structures of the cardiovascular system such as inherited cardiomyopathies, arrhythmias, metabolic disorders affecting the heart, congenital heart diseases, as well as vascular disorders such as Marfan syndrome [4648]. Over the past two decades significant progress has been made towards the identification of the genetic basis of CVD, with tens of genes now known to be implicated in almost all of the different disorders. The magnitude of the role of genetics however, remains elusive. Although in some cases the pathogenesis appears to involve complex mechanisms and multifactorial (genetic and environmental) aetiology, multigenic inheritance (e.g. familial hypercholesterolemia: LDLR, APOB, ABCG5, ABCG8, ARH, PCSK9; hypertrophic cardiomyopathy: MYH7, TNNT2, TPM1, TNNI3, MYL2, MYBPC3, ACTC, MYL3) or even monogenic, also known as Mendelian, inheritance (e.g. Marfan syndrome: FNB1) has been described. Pinpointing the gene(s) and their specific mutations that lead to each pathological phenotype can give rise to valuable, complementary genetic diagnostic/prognostic tools for significantly improved clinical management of CVD patients and their families.

For example, the recently published consensus statement on the state of genetic testing for cardiomyopathies and channelopathies has elegantly presented the list of different genes which contribute by >5% to these inherited disorders. [49,50]. There are more than 50 distinct channelopathy/cardiomyopathy-associated genes with hundreds of mutations discovered to date. Each of these mutations/genes usually accounts for a small percentage of the reported cases, while in many cases the causative mutation/gene is never identified. For example, in channelopathies a mutation is found in <20% of short QT syndrome cases and up to 75% in long QT syndrome cases. An exceptional scenario is this of mutations in the cardiac ryanodine receptor (RYR2) gene in catecholaminergic polymorphic ventricular tachychardia, which account for up to 65% of affected patients [51]. In cardiomyopathies, positive genetic testing results range in frequency from <20% in restrictive cardiomyopathy to 60% in familial HCM. Despite the fact that two decades ago, HCM was termed a disease of the sarcomere involving at least 8 causative genes, the rate of mutation detection ranged in frequency from 2530% in MyBPC and MYH7 to 5% in TNNT2 and TNNTI3, and 1% in other sarcomeric genes [42,52]. Additionally, there are HCM phenocopies (same phenotype) associated with non-sarcomeric gene mutations and different modes of inheritance, which may on occasion be difficult to exclude from sarcomeric HCM based on clinical evaluation alone. Therefore, multiple genes need to be screened for a multigenic disease such as HCM.

Overall, our understanding of the genetic basis of CVD has been rapidly expanding over the years with important lessons learned both on monogenic as well as complex disease forms [53]. However, the true value of these findings lies in their translation to the clinical setting and their utilisation towards improved CVD diagnosis, prognosis and treatment. Along these lines, genetic testing is currently available for a number of CVDs in the form of clinical service in most Western countries, and increasingly in the developing world.

Genetic testing can serve three main goals in the clinical practice: first to determine the mode of inheritance of the specific disease in the specific family and identify if there is risk for other family members; second to organize the clinical assessment of unaffected family members through predictive genetic testing so as to distinguish those who are at risk for the disease and should have regular cardiac follow-up (mutation carriers) and those who are not (mutation non-carriers); third, following the establishment of distinct genotypephenotype correlations, the application of genetic testing in disease diagnosis, prognosis and personalized treatment (i.e. identification of the drugs to which each patient will respond best) [54].

The clinical value of genetic screening of a cardiovascular disease patient is therefore valuable initially at the diagnostic/prognostic/therapeutic level, provided the genotypephenotype associations have been established first. These associations vary considerably among different cardiovascular diseases, different genes and different mutations thereof. The relevance of genetic testing towards these three levels of clinical management is possibly best shown in the setting of the long QT syndrome [49,50]. It is critical to note however, that genetic testing in the cardiovascular disease setting cannot be the basis for clinical management of patients, but can serve a complementary role to the comprehensive clinical evaluation to better address the patient's and his/her family's needs.

Identifying the causative mutation of a proband further allows the genetic screening of its family members, a process of marked predictive power and therefore high importance in the cardiovascular clinic [55]. The significance of such pre-symptomatic genetic testing for the probands family members ranges from ensuring that unaffected mutation carriers receive regular clinical follow-up and prophylactic treatment (where available) to reassurance that clinically suspicious findings are unlikely to be indicative of the specific form of the disease in the absence of the specific family mutation (e.g. ) [56,57]. Importantly however, a negative genetic test result in the proband's family members cannot by itself exclude the presence of disease in general, since a large number of different genes and a variety of mutations thereof can contribute to the same or a different pathological cardiovascular phenotype and by chance, a family member could be a carrier of a different gene mutation.

Pedigree of an HCM positive family from the BA HCM Study. A pathogenic mutation in MYH7 exon 23 (Glu927Lys) was detected in the proband II-4. Echo screening of all siblings was undertaken, and sister II-10 was found to have an interventricular septal measurement of 14 mm. Genetic screening of all family members excluded HCM diagnosis for the sister (II-10).However, the symptom free and echo clear son of the proband, was positive for the mutation and therefore given a pre-symptomatic diagnosis of HCM at the age of12 years. Symbols in white represent unaffected individuals, in black are individuals with HCM based on clinical or genetic findings, and in blue are individuals who have not been screened by echo or genetic testing (unpublished data).

Genetic testing of children in the family has always posed an ethical concern, particularly for adult-onset diseases. Therefore pre-symptomatic testing of children should be extensively discussed with the family after a mutation has been identified in the proband, and in the context of the specific cardiovascular disease [58]. In cases where pre-symptomatic genetic screening and mutation identification has direct implications on the child's clinical follow-up, lifestyle adaptations and preventive treatments, it would be valuable to proceed with genetic testing, upon the parents approval. For example, for long QT syndrome and catecholaminergic polymorphic ventricular tachychardia, and occasionally in high risk HCM families, in which preventive measures or prophylactic therapy is advisable for asymptomatic mutation positive family members, genetic testing should be undertaken in early childhood, i.e. regardless of age. On the other hand, for late-onset and/or reduced penetrance diseases, it is reasonable to proceed with clinical monitoring as needed during childhood, leaving the genetic testing option open for when the individual reaches adulthood [49,50]. When a child has already presented with a CVD, the use of genetic testing is complementary to all other clinical tests, and especially valuable for identifying other family members at risk, since childhood-onset cases, even when presumed as sporadic, can often have a genetic aetiology. For example, approximately half of the presumed sporadic cases of childhood-onset hypertrophy have genetic causes [59].

Although the translation of molecular genetics to routine clinical practice is slow, a series of certified genetic testing centers (www.genetests.org) have been established, and guidelines have already been issued for a number of cardiovascular diseases such as HCM, dilated cardiomyopathy (DCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC) [60,61]. The consensus is that a minimum of three to four generation family history needs to be obtained, the relatives at risk need to be identified and directed for clinical screening, the potential genetic nature of the disease needs to be explained, and the possibility of genetic testing should be discussed where appropriate.

In order to follow these guidelines, cardiology clinics around the world need to ensure that cardiologists are provided with appropriate training in key genetic concepts, along with information on the latest developments in cardiovascular genetics and the best means to apply them in the clinic. Importantly, the close interaction between cardiologists, geneticists and genetic counsellors, especially in complex cases, will significantly expedite the benchside-to-bedside translation of the latest genetic discoveries and optimize the clinical care provided to the patient [62,63]. For example, when routine cardiovascular genetic screening fails to detect the causative mutations, screening can be extended to include broad gene panels and/or application of high throughput technologies. Similarly, in cases where new mutations are identified, targeted genetic tests can be designed, if needed, for screening family members at risk. Currently, different modes of cardiologist-geneticist interactions are being adopted in clinical settings around the world, a process that requires time, continuing education and to some extent, reorganization of health systems [64]. An example of such an evolving system of interdisciplinary interactions is that of the Egyptian National Genetic study of HCM ().

Combined clinical and genetic evaluation of CVD patients will allow for improved disease management and patient care.

In conclusion, cardiovascular genetic testing is valuable for improving the standards of care for CVD patients and their families at the diagnostic, prognostic and therapeutic level. Importantly, for healthcare systems worldwide, it further represents a cost-effective approach by enabling the timely identification of individuals at risk, ensuring regular follow-up only for the individuals at risk and early disease detection, as well as enabling, where possible, the use of disease preventative measures in order to minimize the environmental contributing factors [65]. To this end, clinical cardiovascular genetics is increasingly emphasized in undergraduate and postgraduate medical education and incorporated in cardiological clinics worldwide [54].

The tremendous technological advancements over the past decade have empowered the discovery of new biological concepts and the emergence of entirely new scientific fields. Among them, cardiac systems genetics a systems-based analysis of genetic variants considering all different levels spanning from their effect on the cardiac transcriptome, proteome, metabolome to organ physiology/pathophysiology (phenome) (). The global analysis of the downstream functional molecular and cellular implications of different genetic variants, will allow the meaningful integration of molecular and clinical data in a powerful way.

To fully unravel the intricate pathways regulating cardiac physiology and pathophysiology the global studies of the human genome will need to be extended to similar studies at the epigenome (chemical changes to the DNA and histone affecting the chromatin structure and function of the genome),transcriptome (the full set of transcripts produced from the human DNA), miRNome (the full set of microRNAs produced from the human DNA), proteome (the full set of proteins) and metabolome (full set of metabolites) levels.

Systems genetics will in turn, serve as an integral part of network medicine, an advanced form of molecular medicine, where perturbations, rather than individual molecules, are investigated as the underlying causes of complex diseases [66]. In cardiology, examples of important first steps in this direction are the identification of cardiac gene expression signatures related with response to left ventricular assist device implantation [67,68] and peripheral leukocyte expression signatures indicative of post-cardiac transplantation tissue rejection [69]. Parameters such as epigenetics and microRNAs are increasingly integrated in network medicine, adding new dimensions to the intricate mechanisms of cardiovascular disease (e.g. [70,71]). A likely next addition to network medicine, based on emerging new data [72,73], could be this of metagenomics the genomic investigation of micro-organisms inside the human body, and their effect on the global networks orchestrating human cardiac physiology/pathophysiology.

Cardiology is rapidly transformed with powerful new technologies expediting the acquisition of new knowledge and exciting new discoveries enriching our understanding of the intricate genotypephenotype correlations. The close interaction of cardiologists and geneticists is facilitating the transition of novel findings to clinical practice and vice versa. It is also enabling the rapid establishment of appropriate research strategies to address emerging clinical questions. Ultimately the convergence of the two disciplines promises to transform the way we perceive, manage and treat CVD.

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Molecular genetics made simple - PMC - National Center for ...

Branch of biology that studies biological systems at the molecular level

Molecular biology is a branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions.[1][2][3]

Though cells and other microscopic structures had been observed in living organisms as early as the 18th century, a detailed understanding of the mechanisms and interactions governing their behavior did not emerge until the 20th century, when technologies used in physics and chemistry had advanced sufficiently to permit their application in the biological sciences. The term 'molecular biology' was first used in 1945 by the English physicist William Astbury, who described it as an approach focused on discerning the underpinnings of biological phenomenai.e. uncovering the physical and chemical structures and properties of biological molecules, as well as their interactions with other molecules and how these interactions explain observations of so-called classical biology, which instead studies biological processes at larger scales and higher levels of organization.[4] In 1953, Francis Crick, James Watson, Rosalind Franklin, and their colleagues at the Medical Research Council Unit, Cavendish Laboratory, were the first to describe the double helix model for the chemical structure of deoxyribonucleic acid (DNA), which is often considered a landmark event for the nascent field because it provided a physico-chemical basis by which to understand the previously nebulous idea of nucleic acids as the primary substance of biological inheritance. They proposed this structure based on previous research done by Franklin, which was conveyed to them by Maurice Wilkins and Max Perutz.[5] Their work led to the discovery of DNA in other microorganisms, plants, and animals.[6]

The field of molecular biology includes techniques which enable scientists to learn about molecular processes.[7] These techniques are used to efficiently target new drugs, diagnose disease, and better understand cell physiology.[8] Some clinical research and medical therapies arising from molecular biology are covered under gene therapy, whereas the use of molecular biology or molecular cell biology in medicine is now referred to as molecular medicine.

Molecular biology sits at the intersection of biochemistry and genetics; as these scientific disciplines emerged and evolved in the 20th century, it became clear that they both sought to determine the molecular mechanisms which underlie vital cellular functions.[9][10] Advances in molecular biology have been closely related to the development of new technologies and their optimization.[11] Molecular biology has been elucidated by the work of many scientists, and thus the history of the field depends on an understanding of these scientists and their experiments.[citation needed]

The field of genetics arose from attempts to understand the set of rules underlying reproduction and heredity, and the nature of the hypothetical units of heredity known as genes. Gregor Mendel pioneered this work in 1866, when he first described the laws of inheritance he observed in his studies of mating crosses in pea plants.[12] One such law of genetic inheritance is the law of segregation, which states that diploid individuals with two alleles for a particular gene will pass one of these alleles to their offspring.[13] Because of his critical work, the study of genetic inheritance is commonly referred to as Mendelian genetics.[14]

A major milestone in molecular biology was the discovery of the structure of DNA. This work began in 1869 by Friedrich Miescher, a Swiss biochemist who first proposed a structure called nuclein, which we now know to be (deoxyribonucleic acid), or DNA.[15] He discovered this unique substance by studying the components of pus-filled bandages, and noting the unique properties of the "phosphorus-containing substances".[16] Another notable contributor to the DNA model was Phoebus Levene, who proposed the "polynucleotide model" of DNA in 1919 as a result of his biochemical experiments on yeast.[17] In 1950, Erwin Chargaff expanded on the work of Levene and elucidated a few critical properties of nucleic acids: first, the sequence of nucleic acids varies across species.[18] Second, the total concentration of purines (adenine and guanine) is always equal to the total concentration of pyrimidines (cysteine and thymine).[15] This is now known as Chargaff's rule. In 1953, James Watson and Francis Crick published the double helical structure of DNA,[19] based on the X-ray crystallography work done by Rosalind Franklin which was conveyed to them by Maurice Wilkins and Max Perutz.[5] Watson and Crick described the structure of DNA and conjectured about the implications of this unique structure for possible mechanisms of DNA replication.[19] Watson and Crick were awarded the Nobel Prize in Physiology or Medicine in 1962, along with Wilkins, for proposing a model of the structure of DNA.[6]

In 1961, it was demonstrated that when a gene encodes a protein, three sequential bases of a gene's DNA specify each successive amino acid of the protein.[20] Thus the genetic code is a triplet code, where each triplet (called a codon) specifies a particular amino acid. Furthermore, it was shown that the codons do not overlap with each other in the DNA sequence encoding a protein, and that each sequence is read from a fixed starting point.During 19621964, through the use of conditional lethal mutants of a bacterial virus,[21] fundamental advances were made in our understanding of the functions and interactions of the proteins employed in the machinery of DNA replication, DNA repair, DNA recombination, and in the assembly of molecular structures.[22]

In 1928, Frederick Griffith, encountered a virulence property in pneumococcus bacteria, which was killing lab rats. According to Mendel, prevalent at that time, gene transfer could occur only from parent to daughter cells. Griffith advanced another theory, stating that gene transfer occurring in member of same generation is known as horizontal gene transfer (HGT). This phenomenon is now referred to as genetic transformation.[23]

Griffith's experiment addressed the pneumococcus bacteria, which had two different strains, one virulent and smooth and one avirulent and rough. The smooth strain had glistering appearance owing to the presence of a type of specific polysaccharide a polymer of glucose and glucuronic acid capsule. Due to this polysaccharide layer of bacteria, a host's immune system cannot recognize the bacteria and it kills the host. The other, avirulent, rough strain lacks this polysaccharide capsule and has a dull, rough appearance.[citation needed]

Presence or absence of capsule in the strain, is known to be genetically determined. Smooth and rough strains occur in several different type such as S-I, S-II, S-III, etc. and R-I, R-II, R-III, etc. respectively. All this subtypes of S and R bacteria differ with each other in antigen type they produce.[6]

The AveryMacLeodMcCarty experiment was a landmark study conducted in 1944 that demonstrated that DNA, not protein as previously thought, carries genetic information in bacteria. Oswald Avery, Colin Munro MacLeod, and Maclyn McCarty used an extract from a strain of pneumococcus that could cause pneumonia in mice. They showed that genetic transformation in the bacteria could be accomplished by injecting them with purified DNA from the extract. They discovered that when they digested the DNA in the extract with DNase, transformation of harmless bacteria into virulent ones was lost. This provided strong evidence that DNA was the genetic material, challenging the prevailing belief that proteins were responsible. It laid the basis for the subsequent discovery of its structure by Watson and Crick.

Confirmation that DNA is the genetic material which is cause of infection came from the HersheyChase experiment. They used E.coli and bacteriophage for the experiment. This experiment is also known as blender experiment, as kitchen blender was used as a major piece of apparatus. Alfred Hershey and Martha Chase demonstrated that the DNA injected by a phage particle into a bacterium contains all information required to synthesize progeny phage particles. They used radioactivity to tag the bacteriophage's protein coat with radioactive sulphur and DNA with radioactive phosphorus, into two different test tubes respectively. After mixing bacteriophage and E.coli into the test tube, the incubation period starts in which phage transforms the genetic material in the E.coli cells. Then the mixture is blended or agitated, which separates the phage from E.coli cells. The whole mixture is centrifuged and the pellet which contains E.coli cells was checked and the supernatant was discarded. The E.coli cells showed radioactive phosphorus, which indicated that the transformed material was DNA not the protein coat.

The transformed DNA gets attached to the DNA of E.coli and radioactivity is only seen onto the bacteriophage's DNA. This mutated DNA can be passed to the next generation and the theory of Transduction came into existence. Transduction is a process in which the bacterial DNA carry the fragment of bacteriophages and pass it on the next generation. This is also a type of horizontal gene transfer.[6]

The Meselson-Stahl experiment was a landmark experiment in molecular biology that provided evidence for the semiconservative replication of DNA. Conducted in 1958 by Matthew Meselson and Franklin Stahl, the experiment involved growing E. coli bacteria in a medium containing heavy isotope of nitrogen (15N) for several generations. This caused all the newly synthesized bacterial DNA to be incorporated with the heavy isotope.

After allowing the bacteria to replicate in a medium containing normal nitrogen (14N), samples were taken at various time points. These samples were then subjected to centrifugation in a density gradient, which separated the DNA molecules based on their density.

The results showed that after one generation of replication in the 14N medium, the DNA formed a band of intermediate density between that of pure 15N DNA and pure 14N DNA. This supported the semiconservative DNA replication proposed by Watson and Crick, where each strand of the parental DNA molecule serves as a template for the synthesis of a new complementary strand, resulting in two daughter DNA molecules, each consisting of one parental and one newly synthesized strand.

The Meselson-Stahl experiment provided compelling evidence for the semiconservative replication of DNA, which is fundamental to the understanding of genetics and molecular biology.

In the early 2020s, molecular biology entered a golden age defined by both vertical and horizontal technical development. Vertically, novel technologies are allowing for real-time monitoring of biological processes at the atomic level.[24] Molecular biologists today have access to increasingly affordable sequencing data at increasingly higher depths, facilitating the development of novel genetic manipulation methods in new non-model organisms. Likewise, synthetic molecular biologists will drive the industrial production of small and macro molecules through the introduction of exogenous metabolic pathways in various prokaryotic and eukaryotic cell lines.[25]

Horizontally, sequencing data is becoming more affordable and used in many different scientific fields. This will drive the development of industries in developing nations and increase accessibility to individual researchers. Likewise, CRISPR-Cas9 gene editing experiments can now be conceived and implemented by individuals for under $10,000 in novel organisms, which will drive the development of industrial and medical applications.[26]

The following list describes a viewpoint on the interdisciplinary relationships between molecular biology and other related fields.[27]

While researchers practice techniques specific to molecular biology, it is common to combine these with methods from genetics and biochemistry. Much of molecular biology is quantitative, and recently a significant amount of work has been done using computer science techniques such as bioinformatics and computational biology. Molecular genetics, the study of gene structure and function, has been among the most prominent sub-fields of molecular biology since the early 2000s. Other branches of biology are informed by molecular biology, by either directly studying the interactions of molecules in their own right such as in cell biology and developmental biology, or indirectly, where molecular techniques are used to infer historical attributes of populations or species, as in fields in evolutionary biology such as population genetics and phylogenetics. There is also a long tradition of studying biomolecules "from the ground up", or molecularly, in biophysics.[30]

Molecular cloning is used to isolate and then transfer a DNA sequence of interest into a plasmid vector.[31] This recombinant DNA technology was first developed in the 1960s.[32] In this technique, a DNA sequence coding for a protein of interest is cloned using polymerase chain reaction (PCR), and/or restriction enzymes, into a plasmid (expression vector). The plasmid vector usually has at least 3 distinctive features: an origin of replication, a multiple cloning site (MCS), and a selective marker (usually antibiotic resistance). Additionally, upstream of the MCS are the promoter regions and the transcription start site, which regulate the expression of cloned gene.

This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells can be done by transformation via uptake of naked DNA, conjugation via cell-cell contact or by transduction via viral vector. Introducing DNA into eukaryotic cells, such as animal cells, by physical or chemical means is called transfection. Several different transfection techniques are available, such as calcium phosphate transfection, electroporation, microinjection and liposome transfection. The plasmid may be integrated into the genome, resulting in a stable transfection, or may remain independent of the genome and expressed temporarily, called a transient transfection.[33][34]

DNA coding for a protein of interest is now inside a cell, and the protein can now be expressed. A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interest at high levels. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied.[35]

Polymerase chain reaction (PCR) is an extremely versatile technique for copying DNA. In brief, PCR allows a specific DNA sequence to be copied or modified in predetermined ways. The reaction is extremely powerful and under perfect conditions could amplify one DNA molecule to become 1.07 billion molecules in less than two hours. PCR has many applications, including the study of gene expression, the detection of pathogenic microorganisms, the detection of genetic mutations, and the introduction of mutations to DNA.[36] The PCR technique can be used to introduce restriction enzyme sites to ends of DNA molecules, or to mutate particular bases of DNA, the latter is a method referred to as site-directed mutagenesis. PCR can also be used to determine whether a particular DNA fragment is found in a cDNA library. PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and, more recently, quantitative PCR which allow for quantitative measurement of DNA or RNA molecules.[37][38]

Gel electrophoresis is a technique which separates molecules by their size using an agarose or polyacrylamide gel.[39] This technique is one of the principal tools of molecular biology. The basic principle is that DNA fragments can be separated by applying an electric current across the gel - because the DNA backbone contains negatively charged phosphate groups, the DNA will migrate through the agarose gel towards the positive end of the current.[39] Proteins can also be separated on the basis of size using an SDS-PAGE gel, or on the basis of size and their electric charge by using what is known as a 2D gel electrophoresis.[40]

The Bradford assay is a molecular biology technique which enables the fast, accurate quantitation of protein molecules utilizing the unique properties of a dye called Coomassie Brilliant Blue G-250.[41] Coomassie Blue undergoes a visible color shift from reddish-brown to bright blue upon binding to protein.[41] In its unstable, cationic state, Coomassie Blue has a background wavelength of 465nm and gives off a reddish-brown color.[42] When Coomassie Blue binds to protein in an acidic solution, the background wavelength shifts to 595nm and the dye gives off a bright blue color.[42] Proteins in the assay bind Coomassie blue in about 2 minutes, and the protein-dye complex is stable for about an hour, although it is recommended that absorbance readings are taken within 5 to 20 minutes of reaction initiation.[41] The concentration of protein in the Bradford assay can then be measured using a visible light spectrophotometer, and therefore does not require extensive equipment.[42]

This method was developed in 1975 by Marion M. Bradford, and has enabled significantly faster, more accurate protein quantitation compared to previous methods: the Lowry procedure and the biuret assay.[41] Unlike the previous methods, the Bradford assay is not susceptible to interference by several non-protein molecules, including ethanol, sodium chloride, and magnesium chloride.[41] However, it is susceptible to influence by strong alkaline buffering agents, such as sodium dodecyl sulfate (SDS).[41]

The terms northern, western and eastern blotting are derived from what initially was a molecular biology joke that played on the term Southern blotting, after the technique described by Edwin Southern for the hybridisation of blotted DNA. Patricia Thomas, developer of the RNA blot which then became known as the northern blot, actually did not use the term.[43]

Named after its inventor, biologist Edwin Southern, the Southern blot is a method for probing for the presence of a specific DNA sequence within a DNA sample. DNA samples before or after restriction enzyme (restriction endonuclease) digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via capillary action. The membrane is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest.[44] Southern blotting is less commonly used in laboratory science due to the capacity of other techniques, such as PCR, to detect specific DNA sequences from DNA samples. These blots are still used for some applications, however, such as measuring transgene copy number in transgenic mice or in the engineering of gene knockout embryonic stem cell lines.[30]

The northern blot is used to study the presence of specific RNA molecules as relative comparison among a set of different samples of RNA. It is essentially a combination of denaturing RNA gel electrophoresis, and a blot. In this process RNA is separated based on size and is then transferred to a membrane that is then probed with a labeled complement of a sequence of interest. The results may be visualized through a variety of ways depending on the label used; however, most result in the revelation of bands representing the sizes of the RNA detected in sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed. The procedure is commonly used to study when and how much gene expression is occurring by measuring how much of that RNA is present in different samples, assuming that no post-transcriptional regulation occurs and that the levels of mRNA reflect proportional levels of the corresponding protein being produced. It is one of the most basic tools for determining at what time, and under what conditions, certain genes are expressed in living tissues.[45][46]

A western blot is a technique by which specific proteins can be detected from a mixture of proteins.[47] Western blots can be used to determine the size of isolated proteins, as well as to quantify their expression.[48] In western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE. The proteins in the gel are then transferred to a polyvinylidene fluoride (PVDF), nitrocellulose, nylon, or other support membrane. This membrane can then be probed with solutions of antibodies. Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including colored products, chemiluminescence, or autoradiography. Often, the antibodies are labeled with enzymes. When a chemiluminescent substrate is exposed to the enzyme it allows detection. Using western blotting techniques allows not only detection but also quantitative analysis. Analogous methods to western blotting can be used to directly stain specific proteins in live cells or tissue sections.[47][49]

The eastern blotting technique is used to detect post-translational modification of proteins. Proteins blotted on to the PVDF or nitrocellulose membrane are probed for modifications using specific substrates.[50]

A DNA microarray is a collection of spots attached to a solid support such as a microscope slide where each spot contains one or more single-stranded DNA oligonucleotide fragments. Arrays make it possible to put down large quantities of very small (100 micrometre diameter) spots on a single slide. Each spot has a DNA fragment molecule that is complementary to a single DNA sequence. A variation of this technique allows the gene expression of an organism at a particular stage in development to be qualified (expression profiling). In this technique the RNA in a tissue is isolated and converted to labeled complementary DNA (cDNA). This cDNA is then hybridized to the fragments on the array and visualization of the hybridization can be done. Since multiple arrays can be made with exactly the same position of fragments, they are particularly useful for comparing the gene expression of two different tissues, such as a healthy and cancerous tissue. Also, one can measure what genes are expressed and how that expression changes with time or with other factors.There are many different ways to fabricate microarrays; the most common are silicon chips, microscope slides with spots of ~100 micrometre diameter, custom arrays, and arrays with larger spots on porous membranes (macroarrays). There can be anywhere from 100 spots to more than 10,000 on a given array. Arrays can also be made with molecules other than DNA.[51][52][53][54]

Allele-specific oligonucleotide (ASO) is a technique that allows detection of single base mutations without the need for PCR or gel electrophoresis. Short (2025 nucleotides in length), labeled probes are exposed to the non-fragmented target DNA, hybridization occurs with high specificity due to the short length of the probes and even a single base change will hinder hybridization. The target DNA is then washed and the unhybridized probes are removed. The target DNA is then analyzed for the presence of the probe via radioactivity or fluorescence. In this experiment, as in most molecular biology techniques, a control must be used to ensure successful experimentation.[55][56]

In molecular biology, procedures and technologies are continually being developed and older technologies abandoned. For example, before the advent of DNA gel electrophoresis (agarose or polyacrylamide), the size of DNA molecules was typically determined by rate sedimentation in sucrose gradients, a slow and labor-intensive technique requiring expensive instrumentation; prior to sucrose gradients, viscometry was used. Aside from their historical interest, it is often worth knowing about older technology, as it is occasionally useful to solve another new problem for which the newer technique is inappropriate.[57]

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Abstract

Peripheral nerve injury (PNI) is a common neurological disorder and complete functional recovery is difficult to achieve. In recent years, bone marrow mesenchymal stem cells (BMSCs) have emerged as ideal seed cells for PNI treatment due to their strong differentiation potential and autologous transplantation ability. This review aims to summarize the molecular mechanisms by which BMSCs mediate nerve repair in PNI. The key mechanisms discussed include the differentiation of BMSCs into multiple types of nerve cells to promote repair of nerve injury. BMSCs also create a microenvironment suitable for neuronal survival and regeneration through the secretion of neurotrophic factors, extracellular matrix molecules, and adhesion molecules. Additionally, BMSCs release pro-angiogenic factors to promote the formation of new blood vessels. They modulate cytokine expression and regulate macrophage polarization, leading to immunomodulation. Furthermore, BMSCs synthesize and release proteins related to myelin sheath formation and axonal regeneration, thereby promoting neuronal repair and regeneration. Moreover, this review explores methods of applying BMSCs in PNI treatment, including direct cell transplantation into the injured neural tissue, implantation of BMSCs into nerve conduits providing support, and the application of genetically modified BMSCs, among others. These findings confirm the potential of BMSCs in treating PNI. However, with the development of this field, it is crucial to address issues related to BMSC therapy, including establishing standards for extracting, identifying, and cultivating BMSCs, as well as selecting application methods for BMSCs in PNI such as direct transplantation, tissue engineering, and genetic engineering. Addressing these issues will help translate current preclinical research results into clinical practice, providing new and effective treatment strategies for patients with PNI.

Keywords: Bone marrow mesenchymal stem cells, Peripheral nerve injury, Schwann cells, Myelin sheath, Tissue engineering

Core Tip: Bone marrow mesenchymal stem cells (BMSCs) have become ideal seed cells for the treatment of peripheral nerve injury (PNI) due to their strong differentiation potential and the possibility of autologous transplantation. In this review, we introduce the biological characteristics of BMSCs related to PNI, outline the current mechanisms by which BMSCs promote the regeneration and repair of PNI, and summarize the various application methods of BMSCs in PNI, confirming the potential of BMSCs in the treatment of PNI and providing great support for the development of new treatment strategies for nerve regeneration and repair in PNI.

Peripheral nerve injury (PNI) refers to damage that occurs to the peripheral nerve trunk or its branches due to direct or indirect trauma from external sources. It is characterized by sensory, motor, and autonomic dysfunction in the trunk or limbs, representing one of common neurological disorders in clinical practice[1]. PNI is a global issue, with an annual incidence rate of approximately 13/100000 to 23/100000 in developed countries[2-5]. While peripheral nerve axons can regenerate after injury, achieving complete functional recovery is often challenging in cases of proximal nerve injuries or large nerve defects[6]. Currently, autologous nerve transplantation is considered the gold standard for PNI repair[7]. However, even under ideal conditions, this approach does not fully restore impaired motor and sensory functions[8]. Additionally, it has significant drawbacks, such as prolonged surgical time, high economic costs, insufficient donor areas for reconstruction of long or multiple nerve defects, and potential donor site damage (painful neuroma, scarring, and sensory deficits)[9]. In recent years, several new methods for PNI repair have emerged, showing positive effects on restoring the continuity of injured neuroanatomy. However, their ability to restore nerve function is not ideal, and they all have varying degrees of limitations[10].

Tissue engineering is an emerging discipline in the field of biotechnology and has gained significant attention in PNI research. Previous studies have demonstrated that transplantation of Schwann cells (SCs) can promote nerve regeneration and accelerate nerve function recovery[11]. However, obtaining a large number of SCs in a short period is challenging, and it may cause irreversible damage to the donor area, thus limiting the clinical application of SCs transplantation[12]. Recent research has found that adult mesenchymal stem cells (MSCs) can also promote nerve regeneration and show potential for treating PNI, making them a more ideal alternative to SCs. Bone marrow MSCs (BMSCs) are one type of adult MSC with strong differentiation potential and advantages in autologous transplantation. Numerous studies have indicated that BMSCs can differentiate into nerve-like cells during the PNI treatment process and play a crucial role in nerve growth factor (NGF) secretion, endogenous stem cell migration and differentiation, and neovascularization[13-15]. These findings suggest that BMSCs effectively promote the repair of neurological deficits, which makes them ideal seed cells for PNI repair. Researchers are also striving to translate preclinical research findings into practical clinical applications for PNI patients. BMSCs can be applied to PNI therapy through a variety of techniques, such as cell transplantation, tissue engineering, gene engineering, and cell therapy, including the use of BMSC-derived exosomes. These approaches have the potential to improve the effectiveness of PNI regeneration and offer new hope for PNI patients.

Through literature search and analysis (Figure ), in this review, we present the biological properties of BMSCs associated with PNI. We summarize the current mechanisms by which BMSCs promote nerve regeneration and repair in PNI, as well as various application methods in PNI. Moreover, based on these findings, we identify the existing problems and limitations in order to deepen our understanding of BMSCs, optimize treatment strategies, address their shortcomings in clinical application in PNI, and promote their use in PNI clinical practice.

Flow chart of literature search and selection criteria. The initial search resulted in 344 articles. Out of 344 full-texts assessed, 251 articles were excluded. Thus, 93 articles that met the eligibility criteria were included.

BMSCs are a type of pluripotent stem cell that, under specific conditions, can differentiate not only into tissue cells from the mesodermal lineage, such as osteocytes, chondrocytes, and cardiomyocytes[16,17], but also undergo transdifferentiation across germ layers to form neurons, glial-like cells from the ectoderm, and hepatocytes, among others[18]. Silva et al[19] discovered that BMSCs express genes associated with both epithelial tissues and mesenchymal tissues, providing a theoretical basis for their multi-lineage differentiation potential at the gene level. Additionally, BMSCs possess self-renewal capacity. Tamir et al[20] found that approximately 90% of BMSCs are in the G0/G1 phase, which confirms their robust self-renewal capabilities.

BMSCs have no specific surface markers and generally exhibit low expression of major histocompatibility complex (MHC)-I molecules and do not express MHC-II molecules. They also do not express molecules required for T lymphocyte activation, such as Fas ligand and co-stimulatory molecules like B7-1, B7-2, and CD40 L[21]. This characteristic gives BMSCs low immunogenicity and strong immune-suppressive properties. Therefore, studies have shown that when co-cultured with allogeneic and xenogeneic T lymphocytes, BMSCs do not induce significant T cell proliferation but rather inhibit T cell proliferation[22]. In addition to being non-immunogenic, BMSCs are not targeted by CD8+ T cells, which allows them to evade cytotoxic T cell and natural killer cell killing, making them beneficial for successful autologous and allogeneic transplantations[23]. Furthermore, the antigenicity of BMSCs does not increase with their differentiation[24].

Indeed, it is evident that BMSCs possess the potential for multi-lineage differentiation and robust self-renewal capacity. Moreover, when transplanted into the body, they do not trigger significant rejection responses and can be allografted without causing immune rejection reactions[25,26]. The fact that BMSCs do not require the use of immunosuppressive drugs further adds to their appeal as seed cells for treating PNI, making them a promising candidate for potential applications in PNI therapy.

After PNI, if neurons have not died, their axons can undergo regeneration. SCs play a critical role in the repair of the peripheral nervous system. Following Wallerian degeneration of the peripheral nerve, SCs rapidly and massively proliferate, forming Bngner bands. They are involved not only in the formation, synthesis, and secretion of various NGFs but also in the synthesis and secretion of various extracellular matrix (ECM) components and other cell adhesion molecules. The above-mentioned NGFs, ECM, and cell adhesion molecules form gaps or tight junctions with adjacent axons, creating direct channels for the transfer of small molecules and information. These play an essential role in nerve injury regeneration and repair. Under specific conditions, BMSCs can differentiate into neural cells, including SC-like cells, and exert corresponding effects. In this section, we will explore the various functions of BMSCs in PNI repair and list the involved molecular mechanisms.

BMSCs are one of the most widely used sources of cells for nerve regeneration. After transplantation, they can differentiate into different cells, such as neurons, astrocytes, and SC-like cells, under the influence of different physiological microenvironments and express corresponding antigen markers. In vitro studies have found that BMSCs can be induced to differentiate into neural-like cells by antioxidants (such as dimethyl sulfoxide and -mercaptoethanol), cytokines [retinoic acid, basic fibroblast growth factor (bFGF), and epidermal growth factor], traditional Chinese medicine preparations (tetramethylpyrazine and baicalin), gene transfection, and other methods[27,28]. However, whether these induced neural-like cells possess the functional characteristics of normal neurons remains controversial. For instance, Hofstetter et al[29] successfully induced rat BMSCs to differentiate into neural cells using butylated hydroxyanisole but did not record the electrophysiological activity of mature neuronal cells. Some researchers believe that this phenomenon is not related to cell differentiation but rather cytotoxic changes[27]. On the other hand, other studies have shown successful induction of rat BMSCs into neural-like cells using a combination of bFGF, dimethyl sulfoxide, and butylated hydroxyanisole, with the capture of excitatory electrophysiological characteristics[27,28]. Wislet-Gendebien et al[30], through co-culturing, induced rat BMSCs to differentiate into neural cells that produced single action potentials and responded to neurotransmitters such as -aminobutyric acid, glycine, and glutamate. These findings suggest that BMSCs can differentiate into excitable neural-like cells in vitro.

In in vivo studies, Cuevas et al[31] injected 50000 bone MSCs (pre-labelled with bromodeoxyuridine BrdU) in 5 L of culture medium solution into the distal stump of transected sciatic nerve of the rats, and found that after 33 d of implantation, almost 5% of BrdU cells express Schwann cell-like phenotype. Dezawa et al[32] obtained GFP-expressing BMSCs (GFP-MSCs) by retroviral vectors, adjusted the concentration of GFP-MSCs to (1-2) 107 cells/mL, and then injected them into hollow fibres to make an artificial graft. The artificial graft was anastomosed to the cut end of the proximal nerve segment of the sciatic nerve in rats, and a large number of newly formed fibers were observed after 3 wk. They found that BMSCs had a myelination effect in regenerating nerve fibers through immunoelectron microscopy and confocal microscopy, indicating that BMSCs can differentiate into neuron-like cells and secrete a large amount of NGFs to induce axon growth. Additionally, BMSCs can directly transform into SCs to repair injured nerves, which has attracted considerable attention[33]. Furthermore, inflammatory cytokines such as tumor necrosis factor-alpha (TNF-) and interleukin (IL)-1 have been reported to affect the differentiation of MSCs, possibly driving MSCs toward specific cell phenotypes, such as astrocytes. Elevated levels of such pro-inflammatory cytokines can inhibit neuronal differentiation and promote the differentiation of BMSCs into astrocytes[34]. In conclusion, under specific conditions, BMSCs can differentiate into SCs and neural-like cells both in vitro and in vivo, facilitating nerve repair through cell replacement.

Neurotrophic factors have the function of promoting nerve growth and inducing cell differentiation into neural cells, and they can be used to induce the differentiation of BMSCs into neural cells. BMSCs can secrete a variety of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), NGF, vascular endothelial growth factor (VEGF), bFGF, and insulin-like growth factor (IGF)[14]. They upregulate the expression of VEGF receptor (VEGFR) and IGF1 receptor (IGF-1R) and promote the secretion of endogenous neurotrophic factors in the central nervous system. These neurotrophic factors are synthesized and retrogradely transported to nerve cells, transmitting information or paracrine signals to proximal and distal nerves. They bind to their specific receptors, such as NGF with NGF receptor A, BDNF with tyrosine receptor kinase B, and neurotrophin-3 (NT-3) and neurotrophin-4/5 with neurotrophic tyrosine receptor kinase 3. Activation or inhibition of signaling pathways such as PI3K/Akt, Ras-ERK, cAMP/PKA, and PLC--dependent pathways occurs, thereby promoting neuron survival, accelerating axonal and vascular growth, stimulating nerve fiber regeneration, preventing cell apoptosis, inducing SCs migration, proliferation, and myelination formation, and slowing down muscle atrophy, thus reversing the negative effects of PNI (such as preventing cell death caused by axonal injury)[5,35-37]. This improves the supportive microenvironment for neuron survival and regeneration[38] and exerts a neuroprotective effect on nerve cells[39]. Neuhuber et al[40] suggested that the neurotrophic factors produced by human BMSCs are essential for mediating axonal growth and functional recovery after spinal cord injury.

Wang et al[41] conducted a study and reported that using BMSCs transplantation in rats with PNI achieved results similar to autologous nerve transplantation, possibly due to the release of a large number of neurotrophic factors by BMSCs. Isele et al[42] found that the growth condition medium of BMSCs significantly reduced cross-cell-induced apoptosis in fetal rat hippocampal neurons, demonstrating a significant neuroprotective effect. During this process, they observed an increase in phosphorylation of MAPK/ERK and Akt. Blocking this protective effect occurred when using MAPK/ERK and PI3K/Akt specific inhibitors, suggesting that the neurotrophic factors secreted by BMSCs counteracted apoptosis stress response by activating these survival pathways and exerting a neuroprotective effect. They also discovered that stressed neuronal cells stimulated BMSCs to increase the secretion of trophic factors. In another study by Yang et al[43], they used BMSCs as support cells and injected them into a silk fibroin-based nerve conduit. This approach increased the expression of the SCs marker molecule S100 and enhanced the secretion of various neurotrophic growth factors such as BDNF, bFGF, and ciliary neurotrophic factor (CNTF). This, in turn, facilitated histological and functional recovery in rats with sciatic nerve injuries.

The ECM is a complex reticular structure composed of large molecules such as proteins and polysaccharides secreted by cells. It includes laminin, fibronectin, collagen, and other components. The ECM plays a crucial role in promoting cell proliferation and differentiation, supporting the transmission of important signals in the peripheral nervous system[44], which, together with neurotrophic factors and cell adhesion molecules, provides a favorable microenvironment for the survival of nerve cells and the formation of nerve connections[45-47]. Chen et al[48] mixed BMSCs cultured in vitro with gelatin and transplanted them into a 15 mm defect model of the rat sciatic nerve using silicone conduits. Compared to the gelatin-only control group, the experimental group showed improved walking behavior in rats, reduced atrophy of the gastrocnemius muscle, and decreased reduction in compound motor action potential amplitude, with a significant amount of regenerated axons observed. Both in vitro and in vivo, BMSCs synthesize and secrete various ECM components, including NGF, CNTF, BDNF, glial cell-derived neurotrophic factor (GDNF), as well as type I and type IV collagen, fibronectin, laminin, and other ECM molecules. After transplantation, both early and late stages of nerve regeneration are accompanied by high expression of neurotrophic factors. Wright et al[49] reported that BMSCs can stimulate neuronal development and mediate nerve regeneration by modulating the expression of ECM components such as chondroitin sulfate proteoglycans, myelin-associated glycoproteins, and Nogo-A.

Cell adhesion molecules are also critical for axon guidance, including integrins, neural cell adhesion molecules, and calcium-binding proteins such as N-cadherin. Among them, neural cell adhesion molecules may preferentially promote the growth of sensory axons[50]. BMSCs can express various factors related to cell adhesion, such as Ninjurins 1 and 2, Netrin 4, Robo 1, and Robo 4[51-53]. These factors are recognized as neuroregenerative factors and effectively promote axonal growth and cell migration. In summary, BMSCs improve the microenvironment for neuron survival and regeneration through paracrine secretion of neurotrophic factors, ECM factors, adhesion molecules, and various other mechanisms. By promoting the regeneration of damaged neurons, BMSCs contribute to the repair of neural functions.

After PNI occurs, the blood vessels within the nerves are damaged. Therefore, promoting vascular regeneration and restoring blood circulation are essential for the recovery of the normal neural tissue environment. Peripheral nerve regeneration is closely related to angiogenesis, which is a crucial process in the repair of peripheral nerves. VEGF is considered an effective factor for both angiogenesis and neuron generation, and it has long been recognized for its importance in promoting neuron survival and SCs proliferation. Popovich et al[54] reported that BMSCs can secrete various neuroprotective trophic factors such as BDNF, NGF, and VEGF in an autocrine and/or paracrine manner, which can upregulate the expression of these factors, thereby promoting local microvascular regeneration, nerve regeneration, and reconstruction, and ultimately facilitating the repair of injured cells. Induced SCs-like cells from BMSCs have been found to exhibit enhanced immunostaining for VEGF, suggesting that BMSCs may also promote blood vessel formation[55]. BMSCs can also increase the expression levels of endogenous VEGF and its receptor VEGFR2 in the ischemic penumbra, thereby promoting neovascularization[15]. Zurita and Vaquero[56] also observed that blood vessel wall cells in newly regenerated neural tissue at the site of spinal cord injury were differentiated from injected BMSCs. These studies indicate that BMSCs can promote angiogenesis through paracrine secretion of VEGF, and the newly formed blood vessels can, in turn, facilitate the repair of peripheral nerve injuries.

Myelination is another essential process in the regeneration of PNI, determining the quality and functional recovery of nerve regeneration[5,35,47]. Typically, myelination can be achieved by promoting endogenous repair mechanisms or providing an exogenous source of myelinating cells, leading to subsequent nerve function restoration[47]. In a study conducted by Kizilay et al[57], the systemic application of BMSCs was explored in a PNI compression model. Wistar albino rats were used, and the sciatic nerve was compressed for 5 min to create the model. Approximately 5 105 BMSCs were injected intravenously. The results showed that animals treated with BMSCs exhibited higher nerve conduction velocity, compound action potential, and axon numbers compared to the control group. In addition, myelin damage was less severe in the BMSC-treated group, suggesting that systemic application of BMSCs has a positive impact on both myelination and axon survival in the peripheral nerve compression model.

SCs and various types of adult stem cells (in the form of SCs-like cells) have the ability to form myelinating neuronal cells and regenerate nerves. During the regeneration process after PNI, intracellular cAMP levels are elevated when SCs or SCs-like cells further differentiate into myelin-forming cells. This leads to the synthesis and secretion of abundant myelin proteins, such as myelin basic protein, myelin protein zero, peripheral myelin protein 22 (PMP22), and other proteins that are crucial for myelin structure and function. This promotes remyelination during and after regeneration[5,47] and increased expression of IGF-1R and neurofilament type 1 and type 3 enhances axon alignment and myelination gene expression, resulting in increased myelin thickness and internodal length[35,50]. BMSCs also provide various cytokines and growth factors for nerve regeneration[58], including NGF, NT-3, VEGF, PMP22[59-62], and more. Zhao et al[63] also demonstrated that exosomes from BMSCs upregulate the expression of PMP22, VEGF, NGF receptors, and S100 protein, promoting increased neuronal length and axon diameter in the dorsal root ganglion. These protein factors play crucial roles in peripheral nerve regeneration. During the repair process, BMSCs not only directly affect SCs through their neurotrophic functions[64] but may also differentiate towards SCs directionally.

BMSCs, in addition to their ability to differentiate into neuron-like cells[65], also stimulate and induce axonal growth[66], and play an important role in maintaining the normal structure and function of myelin sheaths[67,68]. BMSCs can promote the repair of damaged nerves by regulating the expression of myelination-related genes. For instance, differentiation of BMSCs into SC-like cells can enhance the mRNA expression of myelin-associated factors, significantly increasing the number of myelinated axons, thereby promoting the functional recovery of the facial nerve[69]. In conclusion, MSCs promote myelination and axonal regeneration through various mechanisms, including the secretion of neurotrophic factors, direct interactions with neurons, and upregulation of genes involved in myelination. These combined effects contribute to enhanced axonal growth and improved functional recovery after PNI.

After PNI, various immune cells and cytokines are present, and the coordination of local inflammatory response is essential for the recovery of PNI. BMSCs possess significant immunomodulatory properties, which can promote neural tissue regeneration and alleviate inflammation, therefore making them valuable in PNI treatment. BMSCs can exert immunomodulatory effects by regulating the expression of various cytokines. IL-6 is a multifunctional cytokine produced by macrophages and fibroblasts during PNI[70]. IL-17 is produced by activated CD4+ T cells and can increase the production of pro-inflammatory cytokines and neutrophil chemoattractants, showing elevated levels after PNI[71]. Studies by Ge et al[72] found that BMSCs can secrete high levels of IL-6 to modulate the balance of CD4+ T cell subgroups, promote the proliferation and differentiation of T helper type 17 (Th17) cells that secrete IL-17, and subsequently stimulate prostaglandin E2 secretion. Elevated prostaglandin E2 levels then inhibit Th17 cell secretion of IL-17, achieving therapeutic effects for facial nerve injury. The increased expression of IL-10 protein is associated with regeneration of myelin protein. Research by Cui et al[73] revealed that IL-10-stimulated BMSCs can inhibit the expression of the pro-inflammatory cytokines TNF- and IL-1. Fan et al[74] suggested that this may be achieved by reducing the release of the pro-inflammatory cytokines IL-2, interferon-, and TNF- and increasing the secretion of IL-10 in lymphocyte supernatant and serum, thereby promoting neural regeneration.

BMSCs can modulate the polarization of macrophages, promoting their transition from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. This shift in macrophage polarization is crucial for controlling inflammation and establishing an environment for tissue repair and regeneration. Zhong et al[75] reported that BMSCs secrete GDNF, which converts the damaging M1 phenotype in microglia to the regenerative M2 phenotype, thereby suppressing neural inflammation. This process may be related to inhibiting the nuclear factor-kappaB signaling pathway and promoting the PI3K/AKT signaling pathway.

Another important aspect of MSC-mediated immune regulation is the release of extracellular vesicles (EVs), including apoptotic bodies, exosomes, microvesicles, etc.[76], which contain bioactive components. These EVs are considered an intriguing non-cellular therapy due to their low immunogenicity and ability to mediate cell-to-cell communication and modulate the function of recipient immune cells, contributing to the overall immunomodulatory effects of BMSCs. BMSCs EVs may exhibit similar anti-inflammatory functions as BMSCs themselves by decreasing the levels of inflammatory cytokines and enhancing anti-inflammatory responses. For instance, Schfer et al[77] found that BMSCs can release soluble mediators such as TNF- and IL-1 to alleviate inflammation after PNI. It is evident that BMSCs can exert their immunomodulatory effects through various mechanisms, including regulating the expression of various cytokines, regulating macrophage polarization, releasing EVs, and secreting soluble factors. These effects can help control inflammation, prevent autoimmune reactions, and create a more favorable environment for nerve repair and regeneration following PNI.

In summary, BMSCs play a crucial role in promoting PNI repair and regeneration through various mechanisms (Table ). First, BMSCs are able to differentiate into nerve cells (such as neurons and SCs) to replace damaged nerve cells and facilitate nerve regeneration. Second, they secrete neurotrophic factors, ECM molecules, and adhesion molecules, while also exerting immunomodulatory effects, creating a supportive microenvironment for the growth, differentiation, and survival of nerve cells. Third, BMSCs promote the formation of new blood vessels to ensure the necessary blood supply for the repair and accelerated regeneration of damaged nerves. Lastly, by synthesizing and releasing of proteins related to myelination and axon regeneration, BMSCs enhance the growth of myelinated axons and ultimately promote neuron regeneration. BMSCs utilize these different mechanisms to promote the repair and regeneration of damaged nerve cells and enhance the functional recovery after PNI. Utilizing these pathways can significantly enhance the therapeutic potential of BMSCs in PNI treatment.

Mechanisms of bone marrow mesenchymal stem cell therapy for peripheral nerve injury

The unique mechanisms of action of BMSC make them promising candidates for the treatment of PNI. In this section, we will explore the various application methods of MSCs in PNI treatment (Figure ), analyzing the advantages and disadvantages of each approach in order to comprehensively explore their potential in PNI treatment.

Application of bone marrow-derived mesenchymal stem cells in the treatment of peripheral nerve injury. Bone marrow-derived mesenchymal stem cells can be isolated from bone marrow, expanded in vitro, and directly transplanted into damaged nerve tissue. They can be loaded onto nerve conduits, which provide structural support, using tissue engineering techniques. Additionally, bone marrow-derived mesenchymal stem cells can be genetically modified with neurotrophic factors before being applied to the treatment of peripheral nerve injury to promote neuronal repair and regeneration. PNI: Peripheral nerve injury; MSC: Mesenchymal stem cell.

BMSCs have self-renewal and multi-lineage differentiation capabilities that make neuronal regeneration and nerve function recovery possible, rendering them one of the best choices for stem cell therapy in PNI treatment. Apart from their regenerative potential, BMSCs have been shown to migrate to the injury site and home to the injured area, exhibiting potential for targeted therapy[78,79]. Furthermore, BMSCs do not significantly stimulate the proliferation of T cells nor serve as a target for CD8+ T cells. Thus, when applied in autologous or allogeneic transplantation, they can evade the killing and clearance by immune cells in the body, further exerting their reparative effects. Cuevas et al[31] and Cuevas et al[80] cultured BMSCs from adult rats, labeled them with BrdU, and then injected them into the distal stump of the 5 mm-deficient sciatic nerve in rats. At 18 d and 33 d post-surgery, footprint analysis showed significant improvement in the motor function of the rat limbs compared to the control group injected with only culture medium. Immunofluorescence double-labeling showed that BrdU-labeled cells survived for at least 33 d after surgery, and nearly 5% of the cells expressed the S100 phenotype of SCs. In March 2004, they conducted a similar study on the long-term recovery of rat limbs 180 d after BMSC transplantation, finding that BMSCs continued to have a promoting effect on long-term recovery after surgery[80]. This experiment proves the great potential of BMSCs in peripheral nerve regeneration and lays the foundation for their application in the field of peripheral nerve regeneration. Wang et al[41] investigated the reparative effects of BMSCs by injecting them into the muscles after sciatic nerve injury in rats, and the results showed that the number of regenerating nerve fibers and spinal cord ventral horn neurons increased significantly, as well as a significant increase in regenerated myelin sheath thickness, which indicated that transplantation of BMSCs in PNI rats can achieve similar results as autologous nerve transplantation. Hu et al[81] transplanted BMSCs to repair a 50 mm midline nerve injury in monkeys and found that the healing process was similar to that of autologous transplantation, showing good functional and morphological outcomes. Another study found that when BMSCs were directly transplanted around the sciatic nerve stump, they induced axonal growth by differentiating into neuron-like cells and secreting neurotrophic factors[32]. They also differentiated into SCs to repair the injured nerves[33] and promoted remyelination of regenerating nerve fibers. From this, it can be seen that direct transplantation of BMSCs has played a positive role in repairing various PNI-damaged nerves. However, the invasive procedures required for obtaining BMSCs and the limited quantity of cells obtained, as well as the reduced proliferative and differentiation abilities with increasing patient age, have restricted the research and application of BMSCs in clinical settings.

Scaffold technology has become a hot topic in tissue engineering research in recent years, and nerve conduits are a type of artificial tubular scaffold. BMSCs can simulate the structure and function of the human nervous system when loaded onto nerve conduits and connecting on both sides of the nerve stump. Nerve conduits can be made from natural materials such as chitosan and collagen or synthetic materials such as polyglycolic acid and polylactic acid. Each material has its own characteristics, generally inducing nerve axon regeneration and preventing infiltration of surrounding tissues to interfere with nerve repair. By loading BMSCs onto nerve conduits, not only does it achieve the neurotrophic guidance function of the nerve conduit, but it also provides a space for BMSCs and nerve axon regeneration induction, which helps to promote the effects of BMSCs in promoting nerve growth and regulating the microenvironment of the injury site[82]. In the process of repairing injured nerves using tissue engineering methods, comparing the transplantation effects of nerve conduits with and without BMSCs, it was found that the number and diameter of nerve axons in the experimental group significantly increased, and the improvement of nerve function was significantly better than that in the control group[83].

Costa et al[84] inplanted BMSCs into poly(L-lactic acid) nerve conduit scaffolds for repairing facial nerve defects in rats. The results showed that BMSCs could successfully integrate into the conduit, survive within the nerve tissue, and maintain their phenotype for up to 6 wk. In another study, researchers loaded BMSCs into chitosan nerve conduits and observed cell survival and proliferation within the conduit for 8-16 wk, which effectively promoted the repair of an 8 mm nerve defect[85]. Subsequent research by this team demonstrated that BMSC-loaded chitosan nerve conduits not only accelerated the efficiency of nerve repair but also improved the quantity and quality of regenerated nerve fibers, achieving therapeutic effects comparable to autologous nerve transplantation[86]. The degradation products of nerve conduit materials often trigger local immune reactions, leading to an inflammatory state at the site of injury, which can affect the repair outcome. However, in a study by Hsu et al[87], researchers modified chitosan nerve conduits with laminin to enhance the adhesion capability of BMSCs within the conduit. They observed that BMSCs successfully inhibited the local inflammatory response caused by chitosan degradation, resulting in improved promotion of nerve repair. Other experimental studies have also used BMSCs implanted in nerve conduits made of different materials, such as fibroin gel conduits[88], polylactic-co-glycolic acid conduits with ECM gel[89], and polyglycolic acid conduits[90], to intervene in PNI animal models, and all achieved favorable results.

Although encouraging results have been obtained in animal experiments, further research is still needed to optimize the design of nerve conduits, determine the optimal combination of BMSCs and biomaterials[91], and assess the long-term safety and efficacy of nerve conduits in clinical settings[92]. By addressing these issues, the use of BMSCs in tissue engineering approaches may have a more significant impact on PNI treatment, providing new strategies to promote neural functional recovery and improve the quality of life for patients.

Gene-modified BMSCs have also gained increasing attention in tissue engineering research. In the field of neural repair tissue engineering, the main purpose of gene modification is to design target cells to overexpress growth factors, migration molecules, and adhesion molecules, as well as to inhibit the expression of defective genes. NT-3, NT-4, BDNF, NGF, CNTF, bFGF, and others are major neural growth factors suitable for peripheral nerve gene delivery, as they can provide a suitable microenvironment for the survival and axonal growth of BMSCs. In a study by Zhang et al[93] in 2015, BMSCs transfected with BDNF and CNTF were used for the treatment of rat sciatic nerve injuries. The results showed that BDNF- and CNTF-transfected BMSCs combined with nerve transplantation significantly improved the sciatic nerve function index, promoted the recovery of muscle activity, and increased the thickness of regenerating nerve myelin sheaths. This indicates that this approach is effective in promoting axonal growth and facilitates nerve repair in PNI. In another study[94], BDNF was successfully transfected into BMSCs using gene engineering technology, and the transfected BMSCs were combined with decellularized allogeneic nerve grafts to repair peripheral nerve defects. The results showed a significant improvement in the repair effectiveness of the nerve grafts and the morphology of the injured nerves. Gene-modified MSCs have multiple potentials in the treatment of PNI. However, since gene therapy is still in the experimental stage, its application in clinical settings requires addressing numerous challenges, such as the selection of diverse target genes, stable expression of target genes in the host, combination therapy with multiple genes, and ethical considerations.

Unlike the central nervous system, the peripheral nervous system has the ability for self-regeneration and repair after injury. However, this endogenous repair is limited, and extensive nerve damage cannot be fully repaired. Cell therapy is considered to be an important direction for future medical development, and in recent years, the field of PNI neural regeneration and repair has made vigorous progress, with enormous market potential and clinical application value. BMSCs have the advantages of abundant sources, easy and simple procurement, being easy to isolate and cultivate, and the potential for rapid expansion under certain conditions. Additionally, autologous BMSCs transplantation avoids ethical issues and immune rejection, offering broad prospects for PNI treatment. In this paper, we have reviewed the current biological characteristics of BMSCs related to PNI, summarized the mechanisms by which BMSCs promote PNI neural regeneration and repair, and explored various application methods of BMSCs in PNI, confirming the potential of BMSCs in treating PNI.

However, most research on BMSCs transplantation for PNI intervention is still in the pre-clinical stage and has not yet had significant implications for clinical practice, and there are also certain limitations, such as the lack of specific surface markers on BMSCs[21], which poses some difficulties in identifying cultured BMSCs, and the lack of standardized treatment regimens, where many times after BMSC transplantation, the survival rate is not high, and the proportion of differentiation into neurons is low, resulting in unsatisfactory nerve repair effects. There are also safety issues with BMSC transplantation, where inducers transplanted into the human body along with BMSCs can cause varying degrees of damage to the human body, and there is a possibility of BMSCs transforming into malignant tumors[95]. These issues that need to be resolved point to a certain direction for future research, such as establishing standardized procedures for the extraction, identification, and cultivation of BMSCs; further clarifying the therapeutic mechanisms of BMSCs; and observing the safety of BMSCs applications. The choice of BMSCs application methods in PNI, such as direct transplantation, tissue engineering, and gene engineering, also requires further investigation. In conclusion, BMSCs transplantation offers broad prospects for PNI treatment, but significant theoretical and experimental research are needed before its clinical application can be fully developed and perfected.

Xiong-Fei Zou, Department of Orthopedic Surgery, Peking Union Medical College Hospital, Beijing 100730, China.

Bao-Zhong Zhang, Department of Orthopedic Surgery, Peking Union Medical College Hospital, Beijing 100730, China. nc.hcmup@hzbgnahz.

Wen-Wei Qian, Department of Orthopedic Surgery, Peking Union Medical College Hospital, Beijing 100730, China.

Florence Mei Cheng, College of Nursing, The Ohio State University, Ohio, OH 43210, United States.

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BOCA RATON, Fla., Aug. 23, 2024 (GLOBE NEWSWIRE) -- Entero Therapeutics, Inc., (NASDAQ: ENTO), (“Entero Therapeutics” or the “Company”), a clinical-stage biopharmaceutical company specializing in the development of targeted, non-systemic therapies for gastrointestinal (GI) diseases, today announced that it received a letter from the Listing Qualifications Department of the Nasdaq Stock Market (“Nasdaq”) advising that, because the Company failed to timely file its Quarterly Report on Form 10-Q for the fiscal quarter ended June 30, 2024 (the “Form 10-Q”), the Company was not in compliance with Nasdaq Listing Rule 5250(c)(1) (the “Rule”). Nasdaq has informed the Company that it has until October 21, 2024, to submit a plan to regain compliance with the Rule. If Nasdaq approves the Company’s plan, it has the discretion to grant the Company an extension of up to 180 calendar days from the due date of the Form 10-Q (or until February 17, 2025) to regain compliance.

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Entero Therapeutics, Inc. Receives Nasdaq Notification Regarding Delayed Form 10-Q